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all protocols were approved by the institutional animal care and use committee of the mayo clinic . mice heterozygous for a loss of function mutation in the csf1 gene were obtained from the jackson laboratory ( bar harbor , me ; strain : b6 ; c3fe a / a - csf1/j , cat . the osteopetrosis ( csf1 ) mutation is a single base pair insertion in the coding region of the csf1 gene that generates a stop codon 21 base pair downstream of the insertion . detection of wild - type ( wt ) csf1 and heterozygous animals by sanger sequencing of polymerase chain reaction ( pcr ) products was performed by laragen ( culver city , ca ) . homozygous csf1 mice were distinguished from their normal littermates phenotypically at 1015 days of age by the absence of incisor eruption . due to the lack of teeth , csf1 pups were started on a liquid diet at 34 weeks of age and were weaned from the mother at 56 weeks of age . these mice were maintained until they were up to 6 months old by the use of a specialized wet diet ( bio - serv , frenchtown , nj ) . wt csf1 and csf1 mice were treated with a single injection of streptozotocin ( 160 mg / kg ) at 12 weeks of age to make them diabetic . the mice that did not become diabetic after 1 week received up to two further injections of streptozotocin spaced a week apart . using this regimen , all wt csf1 mice became diabetic ( defined as blood glucose > 250 mg / dl ) . mice weighing less than 20 g at 12 weeks old were not used in this study . starting 4 days after induction , blood glucose levels were measured daily . nj ) was injected once a day intraperitoneally when the glucose levels were over 500 mg / dl to keep the diabetic mice alive and but to still keep blood glucose levels between 400 and 600 mg / dl in order for complications of diabetes to develop . oxidative stress was measured by determining malondialdehyde ( mda ) levels in the plasma as previously described elsewhere using a commercial kit to detect thiobarbituric acid reactive substances ( oxi - tek ; zeptometrix , buffalo , ny ) . gastric emptying of solids ( cooked egg yolk ) was measured after an overnight fast using a [ c]-octanoic acid breath test as described previously elsewhere . csf1 mice required more training than wt csf1 mice due to their lack of teeth . however , by preparing the egg meal to be a soft consistency , the majority of animals could be trained to eat it within 5 minutes of being offered the meal . because csf1 mice do not have teeth , they were fed with less scrambled cooked egg yolk ( 100 mg ) than used for the csf1 mice ( 200 mg ) . if the mice did not eat > 50% of the egg meal after 3 weeks of training , they were removed from the study . four baseline values for ge half - time ( t1/2 ) were obtained before the delivery of streptozotocin . the stomach was cut along the lesser curvature , and the mucosal layer was removed by sharp dissection from the smooth muscle layer of the body and antrum for experiments . the antibodies used were a rat monoclonal raised against the mouse f4/80 epitope conjugated to alexa fluor 488 ( cat . mf48020 ; life technologies , grand island , ny ) , and a rat monoclonal raised against mouse kit , ack2 ( cat . freshly dissected tissues were incubated in f4/80 antibody alexa fluor 488 conjugate diluted to 0.4 g / ml with ca - containing hepes - buffered physiologic salt solution containing 135 mm nacl , 5 mm kcl , 2 mm cacl2 , 1.2 mm mgcl2 , 10 mm glucose , and 10 mm hepes , adjusted to ph 7.4 with tris and incubated at 37c for 1 hour in the dark . the tissue was then fixed in 4% paraformaldehyde in 0.1 m phosphate buffer . after the tissue was rinsed in 0.1 m phosphate - buffered saline ( pbs ) then water , the nuclei in the tissue were stained by incubating the tissue for 30 minutes at 4c with 0.3 m 46-diamidino-2-phenylindole dihydrochloride ( dapi ) ( molecular probes , eugene , or ) in water . after fixation by immersion in cold acetone for 15 minutes , icc were detected by use of an anti - kit antibody ( ack2 ) . the tissue was washed three times in 0.1 m pbs then blocked in 10% normal donkey serum ( nds ) in pbs and 0.3% triton overnight at 4c . the kit antibody ( 1.7 g / ml ) was applied by incubation in 5% nds / pbs/0.3% after rinsing in pbs , donkey anti - rat igg conjugated to fluorescein ( 3 g / ml ; chemicon , temecula , ca ) was added in 2.5% nds / pbs/0.3% triton and incubated overnight at 4c in the dark . after rinsing in pbs then water , nuclei in the tissue were stained by incubating the tissue for 30 minutes at 4c with 0.3 m 46-diamidino-2-phenylindole dihydrochloride ( dapi ) in water . labeled tissues were examined with a laser scanning confocal microscope using a 20 ( na 0.95 ) xlumplanfl objective ( olympus japan ) in fluoview ( olympus japan ) using the optimal confocal aperture to give a resolution of 0.994 0.994 1.13 m ( x y z ) . stacks of confocal images across the full thickness of the muscularis propria were collected from three nondiabetic csf1 mice ( 2022 weeks old ) , three diabetic csf1 mice ( 2022 weeks old ) , three nondiabetic csf1 mice ( 2022 weeks old ) , and three diabetic csf1 with normal ge and 3 diabetic csf1 with delayed ge mice ( 2 , 3 , and 4.6 weeks after onset of diabetes ) . six images from the gastric body and three from the antrum were taken for each animal . for quantification of the labeling , all of the confocal image stacks were flattened into projections using the fv10-asw viewer ( olympus ) . the flattened images were renumbered in random order and then assessed by scoring the integrity of the icc on a 10-cm visual analogue scale for network density . scoring was done in random order by two independent investigators blind to the image and tissue source . rna extracted from gastric muscularis propria of csf1 and wild - type csf1 mice were used for this real - time quantitative reverse - transcription polymerase chain reaction ( qrt - pcr ) . rna extraction was done with recoverall ( ambion , austin , tx ) according to the manufacturer s instructions . the superscript vilo cdna synthesis kit ( invitrogen , carlsbad , ca ) was used to generate cdna from extracted rna and qrt - pcr was performed on cdna using commercial primer sets and rt2sybr green / rox qrt - pcr master mix according to the manufacturer 's instructions ( sabiosciences , frederick , md ) . the data were normalized to the expression of actin , a housekeeping gene , by transforming the difference in threshold cycle ( ct ) to the ratio of the gene of interest to the housekeeping gene ( 2 ) , and expressed as the mean standard error of the mean ( sem ) for six animals of each group . the statistical methods used were unpaired t tests and one - way analysis of variance ( anova ) with post - test adjustments for multiple comparisons . all authors had access to the study data and reviewed and approved the final manuscript . mice heterozygous for a loss of function mutation in the csf1 gene were obtained from the jackson laboratory ( bar harbor , me ; strain : b6 ; c3fe a / a - csf1/j , cat . the osteopetrosis ( csf1 ) mutation is a single base pair insertion in the coding region of the csf1 gene that generates a stop codon 21 base pair downstream of the insertion . detection of wild - type ( wt ) csf1 and heterozygous animals by sanger sequencing of polymerase chain reaction ( pcr ) products was performed by laragen ( culver city , ca ) . homozygous csf1 mice were distinguished from their normal littermates phenotypically at 1015 days of age by the absence of incisor eruption . due to the lack of teeth , csf1 pups were started on a liquid diet at 34 weeks of age and were weaned from the mother at 56 weeks of age . these mice were maintained until they were up to 6 months old by the use of a specialized wet diet ( bio - serv , frenchtown , nj ) . wt csf1 and csf1 mice were treated with a single injection of streptozotocin ( 160 mg / kg ) at 12 weeks of age to make them diabetic . the mice that did not become diabetic after 1 week received up to two further injections of streptozotocin spaced a week apart . using this regimen , all wt csf1 mice became diabetic ( defined as blood glucose > 250 mg / dl ) . mice weighing less than 20 g at 12 weeks old were not used in this study . starting 4 days after induction , blood glucose levels were measured daily . bridgewater , nj ) was injected once a day intraperitoneally when the glucose levels were over 500 mg / dl to keep the diabetic mice alive and but to still keep blood glucose levels between 400 and 600 mg / dl in order for complications of diabetes to develop . oxidative stress was measured by determining malondialdehyde ( mda ) levels in the plasma as previously described elsewhere using a commercial kit to detect thiobarbituric acid reactive substances ( oxi - tek ; zeptometrix , buffalo , ny ) . gastric emptying of solids ( cooked egg yolk ) was measured after an overnight fast using a [ c]-octanoic acid breath test as described previously elsewhere . csf1 mice required more training than wt csf1 mice due to their lack of teeth . however , by preparing the egg meal to be a soft consistency , the majority of animals could be trained to eat it within 5 minutes of being offered the meal . because csf1 mice do not have teeth , they were fed with less scrambled cooked egg yolk ( 100 mg ) than used for the csf1 mice ( 200 mg ) . if the mice did not eat > 50% of the egg meal after 3 weeks of training , they were removed from the study . four baseline values for ge half - time ( t1/2 ) were obtained before the delivery of streptozotocin . after the onset of diabetes , the stomach was cut along the lesser curvature , and the mucosal layer was removed by sharp dissection from the smooth muscle layer of the body and antrum for experiments . the antibodies used were a rat monoclonal raised against the mouse f4/80 epitope conjugated to alexa fluor 488 ( cat . mf48020 ; life technologies , grand island , ny ) , and a rat monoclonal raised against mouse kit , ack2 ( cat . freshly dissected tissues were incubated in f4/80 antibody alexa fluor 488 conjugate diluted to 0.4 g / ml with ca - containing hepes - buffered physiologic salt solution containing 135 mm nacl , 5 mm kcl , 2 mm cacl2 , 1.2 mm mgcl2 , 10 mm glucose , and 10 mm hepes , adjusted to ph 7.4 with tris and incubated at 37c for 1 hour in the dark . the tissue was then fixed in 4% paraformaldehyde in 0.1 m phosphate buffer . after the tissue was rinsed in 0.1 m phosphate - buffered saline ( pbs ) then water , the nuclei in the tissue were stained by incubating the tissue for 30 minutes at 4c with 0.3 m 46-diamidino-2-phenylindole dihydrochloride ( dapi ) ( molecular probes , eugene , or ) in water . after fixation by immersion in cold acetone for 15 minutes , icc were detected by use of an anti - kit antibody ( ack2 ) . the tissue was washed three times in 0.1 m pbs then blocked in 10% normal donkey serum ( nds ) in pbs and 0.3% triton overnight at 4c . the kit antibody ( 1.7 g / ml ) was applied by incubation in 5% nds / pbs/0.3% after rinsing in pbs , donkey anti - rat igg conjugated to fluorescein ( 3 g / ml ; chemicon , temecula , ca ) was added in 2.5% nds / pbs/0.3% triton and incubated overnight at 4c in the dark . after rinsing in pbs then water , nuclei in the tissue were stained by incubating the tissue for 30 minutes at 4c with 0.3 m 46-diamidino-2-phenylindole dihydrochloride ( dapi ) in water . labeled tissues were examined with a laser scanning confocal microscope using a 20 ( na 0.95 ) xlumplanfl objective ( olympus japan ) in fluoview ( olympus japan ) using the optimal confocal aperture to give a resolution of 0.994 0.994 1.13 m ( x y z ) . stacks of confocal images across the full thickness of the muscularis propria were collected from three nondiabetic csf1 mice ( 2022 weeks old ) , three diabetic csf1 mice ( 2022 weeks old ) , three nondiabetic csf1 mice ( 2022 weeks old ) , and three diabetic csf1 with normal ge and 3 diabetic csf1 with delayed ge mice ( 2 , 3 , and 4.6 weeks after onset of diabetes ) . six images from the gastric body and three from the antrum were taken for each animal . for quantification of the labeling , all of the confocal image stacks were flattened into projections using the fv10-asw viewer ( olympus ) . the flattened images were renumbered in random order and then assessed by scoring the integrity of the icc on a 10-cm visual analogue scale for network density . scoring was done in random order by two independent investigators blind to the image and tissue source . rna extracted from gastric muscularis propria of csf1 and wild - type csf1 mice were used for this real - time quantitative reverse - transcription polymerase chain reaction ( qrt - pcr ) . rna extraction was done with recoverall ( ambion , austin , tx ) according to the manufacturer s instructions . the superscript vilo cdna synthesis kit ( invitrogen , carlsbad , ca ) was used to generate cdna from extracted rna and qrt - pcr was performed on cdna using commercial primer sets and rt2sybr green / rox qrt - pcr master mix according to the manufacturer 's instructions ( sabiosciences , frederick , md ) . the data were normalized to the expression of actin , a housekeeping gene , by transforming the difference in threshold cycle ( ct ) to the ratio of the gene of interest to the housekeeping gene ( 2 ) , and expressed as the mean standard error of the mean ( sem ) for six animals of each group . the statistical methods used were unpaired t tests and one - way analysis of variance ( anova ) with post - test adjustments for multiple comparisons . all authors had access to the study data and reviewed and approved the final manuscript . the gastric muscularis propria of wt csf1 mice was densely populated with macrophages as detected by f4/80 immunoreactivity ( figure 2a ) . these cells had a characteristic morphology with several flattened pseudopodia that branched from the main axis of the cell . however , no f4/80-positive macrophages were found in the muscle layers from the stomach of csf1 mice before or after the development of diabetes ( see figure 2b and c ) . before the development of diabetes , the csf1 mice were smaller in size when compared with the wt csf1 mice ( see figure 2d ; wt 25.11 3.72 , op / op 21.92 2.23 g ; p < .05 , unpaired t test ) . the blood glucose values in nondiabetic mice did not differ between the groups ( figure 3a ; wt 117 16 , op / op 106 18 mg / dl ; p = not statistically significant [ ns ] , one - way anova with tukey s post - test ) . after treatment with streptozotocin to induce diabetes , the diabetic mice had similar elevated nonfasting blood glucose levels regardless of whether they were wt csf1 or csf1 ( see figure 3a ; wt 410.89 62.79 , op / op 342.15 67.94 ; p = ns , one - way anova with tukey s post - test ) . both groups of mice received similar levels of insulin to maintain blood glucose < 600 mg / dl after the onset of diabetes ( wt 3.98 0.57 , op / op 5.77 1.55 unit / week ; p = ns , t test ) . normal ge values for csf1 mice had not been previously reported , so we determined the normal ranges for ge times in csf1 and wt csf1 mice in separate cohorts . the normal range was defined as between the 2.5th and 97.5th percentiles ( see figure 3b ; wt dashed lines , 53119 minutes , median 80 minutes , n = 9 ; csf1 solid lines , 3496 minutes , median 63 , n = 15 ) . in the nondiabetic mice in this study , the t1/2 values ( mean sem ) for ge were statistically significantly lower in csf1 mice compared with wt ( see figure 3b ; wt 86 4.5 minutes , n = 10 ; op / op 66.77 5.37 minutes , n = 15 ; p < .05 , one - way anova with tukey s post - test ) . despite severe diabetes , in csf1 mice the ge values remained in the normal range for up to 8 weeks after induction of diabetes and were not statistically significantly different from the values obtained before diabetes ( see figure 3b , op / op nondiabetic 66.77 3.42 ; op / op diabetic : 62.12 1.82 minutes , p = ns , one - way anova with tukey s post - test ) . in distinct contrast , 4 out of 13 wt mice developed delayed ge at a mean of 6.25 2.5 weeks after induction of diabetes as indicated by readings outside the normal range ( dashed lines ) for wt nondiabetic mice ( see figure 3b ) . this was statistically significantly different from the observation that no csf1 mice had delayed ge over the same period ( p = .03 , fisher s exact test ) , similar to the percentage of diabetic , nonobese diabetic ( nod ) mice that developed delayed ge in our previous studies . diabetic wt csf1 mice with delayed ge had longer t1/2 values than the nondelayed diabetic wt csf1 , nondiabetic wt csf1 , and both diabetic and nondiabetic csf1 mice ( see figure 3b , wt diabetic with delayed ge ; 154.9 12.17 minutes ; p < .05 , one - way anova with tukey s post - test ) . these data are consistent with an absence of macrophages resulting in resistance to development of delayed ge in the cohort studied . previous work using nod mice showed that oxidative stress is elevated in diabetic nod mice and further elevated in diabetic nod mice with delayed ge . mda levels greater than 80 nmol / ml were invariably associated with delayed ge . in nondiabetic mice the levels of oxidative stress as measured by plasma mda levels ( mean sem ) were not different between csf1 mice and wt csf1 littermates ( figure 4 ; wt nondiabetic 4.34 1.30 , op / op nondiabetic 10.03 5.11 nmol / ml ; n = 5 , p = ns , one - way anova with tukey s post - test ) . after the onset of diabetes mda levels were statistically significantly elevated in the csf1 compared with the csf1 littermates ( see figure 4 ; op / op diabetic with normal ge 75.8 9.1 nmol / ml , wt diabetic with normal ge 41.2 13.6 ; n = 5 , p < .05 , one - way anova with tukey s post - test ) . as previously reported for nod mice , development of delayed ge in wt , csf1 was accompanied by increased levels of mda ( see figure 4 ; wt diabetic with delayed ge 102.5 9.19 nmol / ml ; n = 5 , p < .05 , one - way anova with tukey s post - test ) . despite diabetic csf1 mice all having normal ge , the mda levels were as high as those found in diabetic wt csf1 mice with delayed ge ( see figure 4 ; n = 5 , p = ns , one - way anova with tukey s post - test ) . reduced kit expression and impairment of icc networks in gastric muscularis propria are associated with development of delayed ge and impairment of icc networks in diabetic mice . we measured the kit mrna levels by qrt - pcr in gastric muscularis propria ( figure 5 ) . although there was statistically significant variation in the levels of kit mrna from the tissues , there was no statistically significant difference in the kit mrna levels between csf1 and wt mice with normal ge whether diabetic or not diabetic ( see figure 5 ; wt nondiabetic 2.16 0.87 , op / op nondiabetic 4.23 2.45 fold change over actin ; n = 5 , p = ns , one - way anova with tukey post - test ) . diabetic wt csf1 mice with delayed ge had statistically significantly lower expression of kit mrna compared with diabetic wt csf1 mice with normal ge ( p = .03 , t test ) , but this was not statistically significant for multiple comparisons ( see figure 5 ; wt diabetic 2.12 0.76 ; wt diabetic with delayed ge 1.01 0.19 , fold change ; n = 5 , p = ns , one - way anova with tukey post - test ) . damage to icc networks is a common observation in mice and humans with delayed ge.5 , 6 , 9 to determine whether diabetic csf1 mice had disrupted icc networks even though ge was normal , we immunolabeled tissues for kit and examined the icc networks . there was no difference in density of kit - positive icc between gastric muscularis propria tissues from nondiabetic csf1 mice ( figure 6b ) and tissues from csf1 mice that had been diabetic for more than 8 weeks ( see figure 6d ) , as scored by two independent , blinded investigators ( see figure 6f ; op / op nondiabetic 6.06 0.37 , op / op diabetic 6.98 0.14 density ; n = 5 , p = ns , one - way anova with dunnett s post - test ) . no differences were also observed in icc density between the nondiabetic csf1 mice ( see figure 6b ) and the nondiabetic wt ( see figure 6a ) csf1 ( see figure 6f ; wt nondiabetic 6.61 0.32 ; n = 5 , p = ns , one - way anova with dunnett s post - test ) . however diabetic csf1 mice that developed delayed ge ( see figure 6e ) had reduced icc density when compared with the diabetic csf1 with normal ge ( see figure 6d and f ; wt diabetic with delayed ge 5.71 0.17 , wt diabetic with normal ge 6.81 0.04 density ; n = 5 , p < .005 , one - way anova with dunnett s post - test ) . our main and surprising finding was that diabetic csf1 mice do not develop delayed ge while their wt siblings do . we performed this study because we previously had reported that ho1-expressing , cd206-positive , alternatively activated macrophages protected diabetic nod mice from development of delayed ge and that numbers of cd206-positive macrophages correlate with icc numbers in humans with diabetic gastroparesis . we thus hypothesized that depletion of macrophages including , of course , m2 macrophages would lead to higher rates of delayed ge in diabetic mice . our findings support an alternative hypothesis that although m2 macrophages can protect gastric cell types from injury , macrophages likely m1 macrophages releasing injurious proinflammatory cytokines are required to damage these key cell types , including icc . the protection of the diabetic csf1 mice against developing delayed ge occurred despite levels of oxidative stress , elevated blood glucose , and dosing with insulin that were not different from the wt siblings used in this study . we also found that changes associated with delayed ge in diabetic mice and humans,5 , 14 namely , disruption of icc networks and reduced expression of kit , were not found in the long - term diabetic csf1 mice . thus , it appears that mechanisms that result in development of delayed ge in diabetic mice on the time scale we studied are mediated through a population of macrophages that are dependent on csf1 signaling . however this requires confirmation by testing the effect of populating the gastric muscularis propria of diabetic csf1 mice with macrophages . we used csf1 mice as we could not produce long - term depletion of macrophages in diabetic mice either by using clodronate liposomes as described by van rooijen et al or by treating with diphtheria toxin in mice that specifically express the diphtheria toxin receptor on cd11b - positive macrophages . we found that clodronate liposomes did not cause a sustained loss of macrophages , which was required for our experiments , and the mice treated with diphtheria toxin all died within 4 weeks ( data not shown ) . the csf1 mouse does not express macrophages in the lamina propria or the muscularis propria of the small intestine,11 , 20 and we found that it does not express gastric macrophages even when made diabetic . csf1 mice have significant developmental problems that include failure to develop teeth , skeletal abnormalities , and lower levels of blood monocytes . moreover , as elsewhere reported , they are smaller in size when compared with age - matched wt littermates . despite this , with careful handling , the mice survived the induction of diabetes and survived to the end of the 8 weeks of diabetes . the csf1 mice had low levels of plasma mda comparable with the wt csf1 littermates before induction of diabetes and had significantly higher levels after the onset of diabetes . our study indicates that high oxidative stress as indicated by oxidation of lipids and elevated mda levels is not sufficient to cause cellular injury without macrophages . a recent study has highlighted a role of resident macrophages in regulating intestinal contractility through an interaction with the enteric nervous system and the intestinal microbiome . it therefore appears that both resident and activated macrophages have previously unknown roles in modulating gastrointestinal motor function separate from their known role in immunity . wt csf1 mice behaved as observed in other studies on nod , c57bl6 , and balb / c mice ( unpublished data)6 , 16 in that diabetic gastroparesis was associated with higher levels of oxidative stress and that decreased kit expression is found in the diabetic mice that develop delayed ge . the time course for development of delayed ge ( 6.25 weeks after onset of diabetes ) was similar to that found in nod mice ( 5.5 weeks ) . thus , it appears that the streptozotocin - induced diabetic mouse model is useful for studying mechanisms that might underlie diabetic gastroparesis . csf1 mice did have slightly but significantly faster ge than wt siblings before and after induction of diabetes due to the inability of these animals to eat the full 0.2 g meal . however , a consequence of the faster ge in csf1 mice was that the upper threshold for normal ge was lower in mice after induction of diabetes compared with wt mice but even that lower threshold was not crossed with onset of diabetes . thus , csf1 mice represent a strain of mice that can be exploited to further investigate the role of macrophages in diabetes - induced tissue injury . our data strongly suggest that development of delayed ge in diabetes requires the presence of muscle - layer macrophages and that known mechanisms of injury in diabetes appear to converge on macrophages . in particular , m2 macrophages can protect gastric cells from injury , and m1 macrophages are required to damage these key cells , including icc . in fact , despite the absence of m2 macrophages , the absence of injurious m1 cells during diabetes in op / op mice results in no changes in icc and therefore in the absence of delay . targeting macrophages may therefore be an effective therapeutic option to prevent cellular damage and the development of delayed ge in diabetes .

we use urethane ( 1.5 g / kg , sigma - aldrich co. , st . luis , mo ) - prepared as 20% solution by diluting 20 g of urethane in 100 ml of phosphate buffered saline ( pbs ) i.e. the animal will receive 7.5 ml / kg of urethane solution intraperitonealy ( ip ) . because of its dose - response curve , the total dose of urethane is divided in to four applications , each separated by approximately 30 minutes . before the first two applications of urethane , animal is anesthetized with isoflurane administered at a concentration of 3 - 4% ( oxygen at 2 liters per minute ) and maintained at 2% ( oxygen at up to 2 liters per minute ) to prevent an animal from getting stressed by injection . during surgery , an additional administration of isofluorane ( 2% concentration for 2 minutes ) can be used if needed . shave the fur along the incision site ( dorsal side of the head ) to ensure that the fur will not contaminate the wound and that a sufficient area can be disinfected around the incision site . prepare surgical site antiseptically using organic iodine or chlorhexidine soap and antiseptics ( ethanol ) . if needed , probes can be painted with a fluorescent marker prepared antiseptically ( e.g. dye i , invitrogen co. ) , to later reveal the position of the probe in histological analysis . to do this we use a small and very soft paint brush and delicately touch the back of the silicon probe with the dye . this procedure should always be done over a microscope to make sure the probe is not being damaged or bent . always check to make sure that the dye is not blocking any of the channels . any residual dye left on the probe can be later removed with alcohol , followed by rinsing with water . silicon electrodes care : because of their small size , silicon probes are delicate and extremely susceptible to breaking even when using it with considerable care . thus , we recommend monitoring potential hazards at all times , such as : ( i ) keep the probes in an appropriately designated box ; ( ii ) prevent crashing or dropping probes when handling them ; ( iii ) make sure that the stereotaxic manipulators are well fixed to prevent any accidents ; ( iv ) when recording , surrounding tissue and/or blood can attach to the probes . therefore , it is important to be also careful when removing the probes after recording . using a constant flow of pbs or saline solution is strongly recommended ; ( v ) always clean the probes after recording . to clean the probes , it is strongly recommended to submerge them in a liquid enzymatic cleaner ( boston , bausch & lomb ) for a few minutes . always rinse the probes with distilled water and return them to their dedicated box . in our experience , adequate care of the silicon probes ensures good quality recordings for more than 10 experiments . if a channel ' gets noisy ' during use , it should be excluded from subsequent analyses . we have found that cross - talk between channels is minimal ; therefore a noisy channel usually has very little effect on a signal in other channels . administer lidocaine hcl 2% with epinephrine ( 2 - 4 mg / kg diluted to 0.5 % solution ) subcutaneously at the incision site before the incision is made to reduce the potential risk of the wound bleeding and for local anesthesia . make a 2 cm incision through the scalp midline and displace the skin laterally to expose the skull surface . clear the dorsal scalp from fascia by blunt dissection and control the bleeding by microcautery or by using bone wax . make two drilling holes to attach small screws in the occipital bone of the skull for animal grounding and reference . the reference screw must be in contact with dura . in order to reduce electrical noise during the recordings , it is recommended to keep the screws wet at all times . to do this , a small acrylic barrier surrounding the screws make a 1 - 2 mm diameter craniotomy in the coordinates corresponding to the cortical and/or subcortical area(s ) of interest . use compressed air to remove bone dust as needed during craniotomy ( drilling hole ) . make a second craniotomy if needed to insert the second probe . in this video , we are presenting a recording from two cortical areas simultaneously , therefore two craniotomies are made . build a small barrier of acrylic resin ( e.g. : lang dental manufacturing , co. , inc . , wheeting , il ) surrounding the two craniotomies in order to keep pbs on top of exposed dura , preventing it from drying . apply pbs constantly during the whole experiment . two needles ( 30 gauge ) bended on the tips ( forming a hook ) , may be used for this manipulation . attach the connector of the silicon probes to the stereotaxic manipulatiors . connectors are attached to a pole in order to have the shanks of the probes perfectly parallel to the stereotaxic manipulator . connect the reference screw to the silicon probe reference pin . to reduce electrical noise the ground screws have to be connected to : i ) the ground of the probe plug ; ii ) the chassis ground from the recording device ; and iii ) the stereotaxic frame . place the tip of the silicon probes just in contact with the pia . slowly introduce the silicon probes and make sure that the shanks of the silicon probes ( fig . if needed , add drops of pbs on the top of craniotomies to avoid drying of the tissue . because urethane anesthesia should only be used for terminal procedures , the animal is euthanized by an i.p . injection of at least 200mg / kg of sodium pentobarbital diluted to a concentration of no more than 60 mg / ml . this is followed by decapitation of the animal . after performing spike sorting ( discriminating spikes originating from different neurons ; fig 3b , c ; ) silicon probe recordings provide the ability to investigate neuronal population activity involved in , among other , the following processes : memory and decision making , plasticity , stimulus coding , spontaneous activity and effects of various drugs . a. example of a silicon probe with a single shank is superimposed on a montage of reconstructed neurons ( courtesy of s. sakata ) . b. example of a probe with a tetrode configuration at each of the 8 shanks . c. schematic of a rat skull . the green boxes represent the extent of the craniotomy over the right and left somatosensory cortex . two screws located over the cerebellum will be connected to ground and reference , respectively . a. image shows 500ms of a representative electrophysiological recording of local field potential ( lfp ) from one tetrode ( note : negative voltage is plotted up on the ordinate ) . insert shows - a waveform of a spike . b. two - dimensional views of unit clusters in feature space from 1 tetrode . each cluster represents spikes from a single unit ( neuron ) . c. average spike waveforms from representative units ( color coded ) at each of the four recording sites from a single tetrode . note the relatively constant energy values for each unit ; this indicates a stable recording over the duration of the displayed period . we use urethane ( 1.5 g / kg , sigma - aldrich co. , st . luis , mo ) - prepared as 20% solution by diluting 20 g of urethane in 100 ml of phosphate buffered saline ( pbs ) i.e. the animal will receive 7.5 ml / kg of urethane solution intraperitonealy ( ip ) . because of its dose - response curve , the total dose of urethane is divided in to four applications , each separated by approximately 30 minutes . before the first two applications of urethane , animal is anesthetized with isoflurane administered at a concentration of 3 - 4% ( oxygen at 2 liters per minute ) and maintained at 2% ( oxygen at up to 2 liters per minute ) to prevent an animal from getting stressed by injection . during surgery , an additional administration of isofluorane ( 2% concentration for 2 minutes ) can be used if needed . shave the fur along the incision site ( dorsal side of the head ) to ensure that the fur will not contaminate the wound and that a sufficient area can be disinfected around the incision site . prepare surgical site antiseptically using organic iodine or chlorhexidine soap and antiseptics ( ethanol ) . if needed , probes can be painted with a fluorescent marker prepared antiseptically ( e.g. dye i , invitrogen co. ) , to later reveal the position of the probe in histological analysis . to do this we use a small and very soft paint brush and delicately touch the back of the silicon probe with the dye . this procedure should always be done over a microscope to make sure the probe is not being damaged or bent . always check to make sure that the dye is not blocking any of the channels . any residual dye left on the probe can be later removed with alcohol , followed by rinsing with water . silicon electrodes care : because of their small size , silicon probes are delicate and extremely susceptible to breaking even when using it with considerable care . thus , we recommend monitoring potential hazards at all times , such as : ( i ) keep the probes in an appropriately designated box ; ( ii ) prevent crashing or dropping probes when handling them ; ( iii ) make sure that the stereotaxic manipulators are well fixed to prevent any accidents ; ( iv ) when recording , surrounding tissue and/or blood can attach to the probes . therefore , it is important to be also careful when removing the probes after recording . using a constant flow of pbs or saline solution is strongly recommended ; ( v ) always clean the probes after recording . to clean the probes , it is strongly recommended to submerge them in a liquid enzymatic cleaner ( boston , bausch & lomb ) for a few minutes . always rinse the probes with distilled water and return them to their dedicated box . in our experience , adequate care of the silicon probes ensures good quality recordings for more than 10 experiments . if a channel ' gets noisy ' during use , it should be excluded from subsequent analyses . we have found that cross - talk between channels is minimal ; therefore a noisy channel usually has very little effect on a signal in other channels . administer lidocaine hcl 2% with epinephrine ( 2 - 4 mg / kg diluted to 0.5 % solution ) subcutaneously at the incision site before the incision is made to reduce the potential risk of the wound bleeding and for local anesthesia . make a 2 cm incision through the scalp midline and displace the skin laterally to expose the skull surface . clear the dorsal scalp from fascia by blunt dissection and control the bleeding by microcautery or by using bone wax . make two drilling holes to attach small screws in the occipital bone of the skull for animal grounding and reference . the reference screw must be in contact with dura . in order to reduce electrical noise during the recordings , it is recommended to keep the screws wet at all times . to do this , a small acrylic barrier surrounding the screws make a 1 - 2 mm diameter craniotomy in the coordinates corresponding to the cortical and/or subcortical area(s ) of interest . use compressed air to remove bone dust as needed during craniotomy ( drilling hole ) . , we are presenting a recording from two cortical areas simultaneously , therefore two craniotomies are made . build a small barrier of acrylic resin ( e.g. : lang dental manufacturing , co. , inc . , wheeting , il ) surrounding the two craniotomies in order to keep pbs on top of exposed dura , preventing it from drying . two needles ( 30 gauge ) bended on the tips ( forming a hook ) , may be used for this manipulation . attach the connector of the silicon probes to the stereotaxic manipulatiors . connectors are attached to a pole in order to have the shanks of the probes perfectly parallel to the stereotaxic manipulator . connect the reference screw to the silicon probe reference pin . to reduce electrical noise the ground screws have to be connected to : i ) the ground of the probe plug ; ii ) the chassis ground from the recording device ; and iii ) the stereotaxic frame . slowly introduce the silicon probes and make sure that the shanks of the silicon probes ( fig . 1 & 2 ) can be inserted into the brain without resistance or bending . if needed , add drops of pbs on the top of craniotomies to avoid drying of the tissue . because urethane anesthesia should only be used for terminal procedures , the animal is euthanized by an i.p . injection of at least 200mg / kg of sodium pentobarbital diluted to a concentration of no more than 60 mg / ml . this is followed by decapitation of the animal . representative recordings of local field potentials and spiking activity are illustrated in figure 3a . after performing spike sorting ( discriminating spikes originating from different neurons ; fig 3b , c ; ) silicon probe recordings provide the ability to investigate neuronal population activity involved in , among other , the following processes : memory and decision making , plasticity , stimulus coding , spontaneous activity and effects of various drugs . a. example of a silicon probe with a single shank is superimposed on a montage of reconstructed neurons ( courtesy of s. sakata ) . b. example of a probe with a tetrode configuration at each of the 8 shanks . c. schematic of a rat skull . the green boxes represent the extent of the craniotomy over the right and left somatosensory cortex . two screws located over the cerebellum will be connected to ground and reference , respectively . a. image shows 500ms of a representative electrophysiological recording of local field potential ( lfp ) from one tetrode ( note : negative voltage is plotted up on the ordinate ) . insert shows - a waveform of a spike . b. two - dimensional views of unit clusters in feature space from 1 tetrode . each cluster represents spikes from a single unit ( neuron ) . c. average spike waveforms from representative units ( color coded ) at each of the four recording sites from a single tetrode . note the relatively constant energy values for each unit ; this indicates a stable recording over the duration of the displayed period . this paper demonstrates how to use the silicon electrode arrays to record from large population of neurons ( > 100 ) in multiple cortical areas simultaneously . in order to be successful in surgery and recording , the following issues should be considered : introduction of the probes to a desired area : when inserting the probes in brain tissue , it is possible to cause considerable damage . we recommend the following : ( i ) introduce the probes at a certain degree angle ( recommended to use a 10 degree angle ) . by doing this , dendritic damage of the recorded neurons can be reduced ; ( ii ) after the probes are inserted in the brain tissue , they are initially lowered at a faster rate ( approximately 50 to 100 microns per 10 - 30 sec ) until they get closer to the desired area of recording . when the probes are about 200 microns from the target area , the position is adjusted more slowly ( approximately 10 microns every 2 or 3 minutes ) . stabilization of recordings : when the probes are in the designated area and considerable activity is detected ( distinctive spikes in the majority of the channels ) , it is recommended to wait approximately 30 minutes before starting a recording . this will allow brain tissue to mechanically stabilize after the insertion of probes and ensure a more stable recording . brain pulsation : occasionally it is possible to see brain pulsation that can significantly reduce the quality of a recording . a small craniotomy ( just enough space to fit the probe ) can reduce brain pulsation in a recording area . if necessary , the cisterna magna can be punctured . silicon probe configuration : silicon probes can have a variety of shapes and recording site configurations . for example , they can vary in the number of shanks , the length and thickness of shanks , and in the arrangement of recording sites ( e.g. tetrode vs. linear configuration ; see : www.neuronexustech.com ) . the choice of probe used with a specific configuration depends on the scientific question needing to be answered . for example , if the objective is to record populations of neurons in multiple locations in one specific layer ( as presented in these recordings ) , the best choice is to use a probe with eight shanks and a tetrode configuration . this allows for recordings of multiple single units at each tetrode and sampling of eight locations across a 1.4 mm span . in another instance , if someone would like to study activity propagations across cortical layers , the best choice would be to use a probe with one shank with regularly distributed recording sites along that shank which allows recording in multiple cortical layers simultaneously .

it is estimated that 366 million people worldwide in the age of 2079 years were affected by diabetes in 2010 and the prevalence is expected to increase to 552 million people in 2030 . cardiovascular disease is the main cause of death among people with diabetes , accounting for 50% of all diabetic deaths . diabetes mellitus is per se a risk factor for developing cardiovascular disease , with ischemic heart disease being the most frequently found , but also diabetic neuropathy , specifically cardiovascular autonomic neuropathy , is considered a risk factor [ 36 ] . silent myocardial ischemia and infarction as well as sudden death are also more frequently found in patients where diabetes and cardiovascular autonomic neuropathy coexist and cardiovascular autonomic neuropathy is furthermore believed to be a contributing factor to the higher mortality and morbidity found in patients with diabetes mellitus following myocardial ischemia or infarction [ 37 ] . neurons that can contribute to the pathological picture of autonomic neuropathy are the afferent sensory nerves coming from the organ , the neurons involved in central processing of the nociceptive signal , and the efferent autonomic nerves leading to the organ and regulating its function . during myocardial ischemia , lactic acid is produced , causing a decrease in local ph ( decrease of epicardial ph from 7.35 to 6.98 after 5 min of coronary artery occlusion has been demonstrated ) , and release of inflammatory and/or algogenic ( i.e. , pain - causing ) mediators , such as bradykinin , adenosine , serotonin , prostaglandin e2 , and histamine . these metabolic components have been shown to activate / sensitize spinal and vagal afferents in the heart . the effect is ( 1 ) release of neuropeptides leading to local effects in the myocardium and ( 2 ) transmission of the nociceptive signal through the primary afferents ( via the dorsal root ganglia ( drgs ) ) to segments c8-t9 in the spinal cord [ 1115 ] or to the nucleus of the solitary tract in medulla oblongata . neurons can project to the brain giving rise to perception of anginal pain and associated feelings ( e.g. , nausea and anxiety ) and/or activate autonomic efferent fibers , leading to regulation of cardiovascular function . diabetes - induced alterations in cardiac sensory nerves could thus have pronounced effects on the way a patient responds to myocardial ischemia and infarction , including changes in perception of the pain often accompanying myocardial ischemia and infarction , and in the cardiac compensatory responses . diabetic neuropathy is thought to be caused by several hyperglycemia - induced changes : neurovascular dysfunction , lack of trophic support , and metabolic changes , such as an increased polyol pathway flux , increased production of advanced glycation end products ( ages ) , and oxidative stress . furthermore , alterations in [ ca]i homeostasis have been suggested to contribute to the pathogenesis of diabetic neuropathy [ 1618 ] . altered calcium channel activity has been shown in primary and secondary sensory neurons from diabetic rodents . very little is known of the consequences of diabetes on sensory nerve function of primary afferents innervating the heart . schultz found a decreased activity of cardiac vagal afferents in stz - diabetic rats compared to control rats after epicardial application of bradykinin and capsaicin and recently , diabetes - induced alterations in the spinal processing of nociceptive input from the heart were found in diabetic rats following cardiac stimulation . the purpose of the present study was to investigate the calcium activity of c8-t5 dorsal root ganglion ( drg ) neurons ( primary neuron ) in zucker diabetic fatty rats , a spontaneous animal model for type 2 diabetes , and the effect of stimulation with algogenic substances , known to play part during myocardial ischemia , as well as with capsaicin , the pungent ingredient in chili peppers , at ph 7.4 and 6.0 . to the best of our knowledge , this is the first study assessing the calcium activity in upper thoracic drg neurons from any type of diabetic rat . experiments were performed in male diabetic zucker diabetic fatty ( zdf ) rats as well as age - matched nondiabetic zdf rats . the zdf rat is a spontaneous animal model for type 2 diabetes in which the animals are homozygous for nonfunctional leptin receptors [ 22 , 23 ] . on a purina 5008 diet the animals were stabled in pairs and were fed with purina 5008 , an energy - rich fodder . once a week , the animals were weighed and blood glucose was measured from a tail vein using a glucometer ( onetouch ultra2 , lifescan ) . at the age of 1418 weeks , the rats were decapitated and c8-t5 drg 's were removed bilaterally and placed in hank 's buffered salt solution ( hbss ) . the ganglia were transferred to 5 ml papain solution ( 2 mg / ml , sigma - aldrich ) for 35 min at 37c . after centrifugation ( 2800 rpm , 2 min ) the supernatant was removed and the pellet was resuspended in 5 ml of a collagenase / dispase solution ( collagenase : 1.11 mg / ml , medinova ; dispase : 1.33 mg / ml , roche diagnostics gmbh ) . after centrifugation ( 2800 rpm , 2 min ) and removal of supernatant , the cells were triturated in 1 ml hbss ( by at least 10 passages through a fire - polished pasteur pipette ) and gently placed on 7 ml percoll ( 20% v / v , sigma - aldrich ) . after centrifugation ( 2800 rpm , 7 min ) , the percoll containing ganglia remnants was removed and the pellet resuspended in 1 ml complete f-12 medium . after centrifugation ( 2800 rpm , 3 min ) , the supernatant was removed leaving approximately 3060 l of fluid . the cells were resuspended in the remaining fluid and approximately 1020 l of the cell suspension was placed on each of three poly - d - lysine / laminin 12 mm coverslips ( becton dickinson cdc ) . the coverslips were placed at 37c for two hours to allow the cells to adhere to the coverslips and afterwards , 2.5 ml of complete f-12 medium was added to each well . the cells were transferred to pss , adjusted to ph 7.4 and loaded with the fluorescent ca - indicator fura-2 acetoxymethyl ester ( fura-2/am , 4.4 m ) dissolved in dmso and chremaphor el detergent ( 4 : 1 ) for 3060 min at 37c . after loading , the cells were transferred to fresh pss buffer and allowed to stand for approx . changes in intracellular calcium concentrations in neurons from c8-t5 drg 's were measured by fura-2 ratiometric imaging in the acutely dissociated culture . at the time of measurement , cells were round and without processes . the cells were illuminated alternately with light of 340 nm ( specific for ca - bound fura-2 ) and 380 nm ( specific for ca - free fura-2 ) , and light of 510 nm was collected via a cooled ccd camera ( hamamatsu ) . each coverslip was initially stimulated twice with 20 mm k for 10 s separated by 7 min wash with pss . the first depolarization was undertaken to ensure vitality of the neurons and saturation of the intracellular compartments and was excluded from further analysis . after washout of the second administration of k , neurons were stimulated for 10 s with one of the following solutions : ( 1 ) 0.3 m capsaicin , ( 2 ) 0.5 m capsaicin , ( 3 ) mixture of algogenic chemicals ( ac ) combined with 0.3 m capsaicin , ( 4 ) ac combined with 0.5 m capsaicin , ( 5 ) ac combined with 0.3 m capsaicin at ph 6.0 , or ( 6 ) ac combined with 0.5 m capsaicin at ph 6.0 . after stimulation with one of these solutions , the neurons were washed with pss for 8 min . the experiment was concluded with a third k stimulation to evaluate the continued viability of the neuron for the extent of the protocol . the mixture of algogenic chemicals contained bradykinin , serotonin , prostaglandin e2 , all in the concentration of 10 m , and adenosine ( 10 m ) . ph 6.0 was chosen for the two acidic protocols studies as drg neurons containing trpv1 and acid sensing ion channels ( asics ) have been demonstrated to react to this ph [ 2527 ] . prior to experiments , inclusion criteria for data analysis were defined : ( 1 ) labeling with fura-2 , ( 2 ) only neurons included , ( 3 ) neurons should stay within its marked area throughout the experiments , ( 4 ) cells responding to 20 mm k were included ( 20% increase from baseline ) , and ( 5 ) only neurons returning to baseline during washout period were included . to monitor the [ ca]i changes in single cells , specific areas of interest were chosen for cells and for background . for each time frame , the ratio of background - corrected fluorescence intensities detected at 510 nm and resulting from excitation at 340 and 380 nm was calculated : ( 1)r = i340i340(background)i380i380(background ) . this ratio is an arbitrary value and was used as a measure of the activity level and to record changes in the activity level of the neurons . baseline , peak value , and auc of light intensity ratios were measured for each response in chart 5 ( ad instruments ) . for k stimulations , auc was calculated from 0 s to 180 s after initiation of response ( auc0180 s ) ; for all other stimulations , auc060 s was calculated , where 0 s indicates beginning of rise in [ ca]i . the maximum increase from baseline , that is , the peak height , was calculated for all neurons , both in absolute terms ( ) , and relative as the percentage increase from baseline ( % - ) . for all stimulations except k , , % - and auc were furthermore compared to the k response by calculating the ratio of a given response and the preceding k response for each neuron . neuron diameter was measured and the neurons were classified as small ( s , < 30 m ) , medium ( m , 3039 m ) , or large ( l , 40 m ) , similar to the classification by lu et al . . to further analyze the distribution of neuron diameter in control and diabetic animals , the neurons were divided into bins , each of 5 m width . responsiveness was defined as 20% increase from baseline ( i.e. , % - 20% ) , after stimulation with any substance in the protocol . during analysis it was found that neurons stimulated with 0.3 and 0.5 m capsaicin elicited similar responses , and this was also seen for neurons stimulated with the combination of ac and either 0.3 or 0.5 m capsaicin . for subsequent analysis , 0.3 and 0.5 m capsaicin was therefore combined and henceforward is referred to as capsaicin and furthermore , the protocols of ac in combination with either 0.3 or 0.5 m capsaicin were combined and henceforward are referred to ac + capsaicin . the results are presented either as mean sem or as median ( 5th percentile ; 95th percentile ) , depending on normality of data . comparisons were made with student 's t - test or , when appropriate , the non - parametric mann - whitney rank sum test . in total , 8 diabetic zdf animals and 8 age - matched control zdf rats were used in this study . at the age of 7 weeks , the zdf diabetic rats had developed significantly higher blood glucose concentrations than control rats and this hyperglycemia persisted for the whole experimental period . at the time of experiment , all diabetic animals had an elevated blood glucose level of 27.5 4.5 mm compared to 6.2 0.5 mm in control animals ( p < 0.001 , n = 16 ) . the body weight was larger in diabetic animals from 7 weeks of age and this increased body weight persisted throughout week 14 after which body weight no longer differed between control and diabetic animals . at the time of experiment , body weight was 378.0 26.3 g for the diabetic animals and 365 23.9 g for the control animals ( p > 0.05 , n = 16 ) . the mean cell diameter of the recorded drg neurons was slightly smaller for diabetic than for control animals ( 25 m ( 18.0 ; 33.2 ) versus 26 m ( 19.0 ; 33.0 ) , p < 0.05 , n = 1124 ) . the distribution of small- , medium- , and large - sized neurons , though , did not differ between the two animal groups ; for neurons from diabetic animals , 82% were small - sized , 17% medium - sized , and 1% large - sized compared to 83% , 15% , and 2% , respectively , for neurons from control animals . the distribution based on neuron diameter was also not significantly different in control and diabetic animals ( figure 1 ) . when differentiating , however , between neurons responding to either of the capsaicin protocols ( capsaicin - responsive ) and neurons not responding to the tested capsaicin protocol ( capsaicin - unresponsive ) , analysis showed that the distribution of capsaicin - responsive neurons from diabetic animals was significantly different from the distribution of control animals ( p < 0.05 ) with a median neuron diameter in diabetic animals of 23 m ( 16.7 ; 30.0 ) compared to 25 m ( 18.9 ; 34.1 ) in control animals ( p < 0.01 ) . the baseline ca level did not differ between diabetic and control animals ( 0.58 ( 0.46 ; 0.80 ) versus 0.59 ( 0.47 ; 0.77 ) , p > 0.05 , n = 1127 ) . response to a given stimulus was elicited by an increase in intracellular ca - level and viewed as an increase in fluorescence intensity ratio , that is , a peak that slowly returned to baseline . an example of a capsaicin - responsive and - unresponsive neuron following stimulation with 20 mm k and a capsaicin - containing stimulus is depicted in figure 2 . neuron responses with and without shoulder were found among both groups of animals as well as among capsaicin - responsive and -unresponsive neurons and thus , presence or lack of shoulder in the response was not confined to single groups of neurons investigated . control as well as diabetic neurons responded to stimulation with 20 mm k by an increase in [ ca]i , assessed by an increase in 340/380 nm fluorescence ratio . the diabetic neurons showed an overall increased responsiveness to stimulation with 20 mm k compared to neurons from control animals ( table 1 ) . this was seen as an augmented auc , peak height ( ) , and % increase from baseline ( % - ) . the increased responsiveness was observed both in capsaicin - responsive and capsaicin - unresponsive neurons ( table 2 ) . for both control and diabetic animals it was seen that capsaicin - responsive neurons generally had a higher responsiveness to 20 mm k than capsaicin - unresponsive neurons ( table 2 ) . stimulation with capsaicin alone , 0.3 or 0.5 m ( no combination ) caused a rise in [ ca]i for approximately 23% of the neurons in both groups of animals . the neurons from diabetic animals exhibited an augmented response , assessed by a larger peak height , percentage increase from baseline , and auc ( figures 4(a)4(c ) and table 3 ) . capsaicin - responsive neurons stimulated with capsaicin in combination with the algogenic substances at either ph 7.4 or 6.0 were followed by increases in [ ca]i in neurons from both diabetic and control animals ( figures 4(d ) , 4(g ) , and 4(j ) ) . none of the stimulations gave rise to differences in peak height or auc between diabetic and control animals ( figures 4(d)4(k ) , table 3 ) . scatter plots of the capsaicin responses indicated that neurons responding to capsaicin - containing solutions might be divided in high- and low - responders ( figure 5 ) . stimulation with 0.5 m capsaicin in combination with the algogenic chemicals at ph 6.0 caused a significantly augmented ca response compared to any of the other stimulations . this was seen for neurons from diabetic animals ( ; 1.63 ( 0.16 ; 1.87 ) , auc ; 63.51 ( 2.87 ; 97.95 ) , p < 0.001 compared to every other stimulation of diabetic neurons , n = 63 ) as well as from control animals ( ; 1.7 ( 0.21 ; 2.37 ) ; auc ; 72.13 ( 1.58 ; 117.24 ) , p < 0.001 compared to every other stimulation of control neurons , n = 13 ) . the responses were similar in neurons from control and diabetic animals ( p > 0.05 ) . the major findings of the present study were the following : ( 1 ) baseline [ ca]i were unaltered in c8-t5 drg neurons from zdf diabetic compared to control animals , ( 2 ) k - induced and ( 3 ) capsaicin - induced depolarization caused augmented [ ca]i currents in neurons from diabetic animals . the current study showed no differences in baseline [ ca]i in c8-t5 drg neurons from diabetic and control animals . this is in accordance with several other studies performed on drg neurons from stz - diabetic mice and rats , db / db diabetic mice and bb / w rats [ 18 , 2932 ] , and on dorsal horn neurons from lumbar l6l7 spinal cord segments from stz - diabetic rats [ 19 , 33 ] . , however , have found an increased baseline [ ca]i in l4l6 drg neurons from stz - induced diabetic rats at the same time as an unaltered baseline in dh neurons , suggesting a differential effect by diabetes on primary and secondary neurons . the segmental location of the neurons investigated also appear to be affected differently by diabetes as neurons with longer axons are more susceptible to neuropathic changes than neurons with shorter axons . this is supported by reports of an increase in baseline [ ca]i in stz - diabetic drg neurons extracted from l4l6 levels where the neurons have longer axons because they innervate the hind paws [ 18 , 34 ] , while huang and colleagues , in the same study , found no baseline changes in c5-l3 drg neurons , which agrees with the results from the present study on c8-t5 drg neurons . the present study found an increased depolarization - induced [ ca]i current in drg neurons from zdf diabetic animals compared to control animals . the increase comprised an absolute and a percentage increase in amplitude from baseline [ ca]i and an increased auc(0180 s ) . hall et al . also found an increased elevation of [ ca]i in thoracic drg neurons ( d. 2040 m ) after stimulation with 50 mm kcl for 6 s in bb / w rats . most other studies with drg and dh neurons have not detected differences in the amplitude of [ ca]i elevation between diabetic and nondiabetic rats and mice [ 19 , 2931 , 3335 ] . an exception , however , is huang et al . who found a decreased amplitude of depolarization - induced [ ca]i current in small drg neurons from l4l6 in stz - diabetic animals ( 14-week duration ) . the decrease in amplitude which contrasts the findings of the present study could be explained by the increase in [ ca]i baseline that huang et al . found in the drg neurons from diabetic animals , which might have limited the potential change in [ ca]i in these neurons . the discrepancy between studies might also be related to the duration of diabetes since huang et al . investigated neurons from stz - diabetic rats with longer duration ( 814 weeks ) of diabetes than what was seen in other studies . the type of diabetes might also influence calcium homeostasis differently , since the present study and the study by hall et al . , both of which found increases in amplitude were performed in zdf and bb / w rats , respectively compared to the other studies in which stz - diabetic animals were employed [ 18 , 19 , 2931 , 3335 ] . there is a general agreement on a prolonged recovery following depolarization - induced [ ca]i currents in neurons from diabetic rats and mice , measured as recovery time , residual [ ca]i ( 60 or 90 s after depolarization ) , or , as in the present study , auc(0180 s ) [ 18 , 19 , this suggests that the diabetes - induced effect on recovery of [ ca]i is somewhat fundamental as the studies are based on both type 1 and type 2 diabetes , different rodent species , neuron sizes , segmental orientations of the neurons investigated , and on different parts of the nervous system ( drg or dh ) . the level of [ ca]i in the drg neurons is the sum of mechanisms leading to ca - entry into the intracellular space and mechanisms leading to removal of ca from the intracellular space . during stimulation , intracellular calcium level rises because calcium enters the cell via plasmalemmal voltage- and ligand - gated , and store - operated ca - channels , and also because calcium is released from the endoplasmatic reticulum [ 36 , 37 ] . kinetics of the recovery phase is affected by release of ca ions from the mitochondria . voltage - dependent calcium currents through l- and n - channels are enhanced in dorsal horn neurons of stz rats in vitro . this is in accordance with the study by hall et al . who found a decreased inhibitory effect of the opioid agonist dyn a in thoracic drgs from bb / w diabetic rats and an impaired inhibitory g - protein function in thoracic and lumbar drgs from bb / w and stz rats , thus contributing to an increased calcium influx . several mechanisms thus seem to contribute to the increased k - induced depolarization found in the present study , and since these changes are seen in different species , diabetes models , and at different drg levels , the impact of diabetes on neuronal calcium metabolism seems to be universal . the present study showed an increased elevation of [ ca]i following stimulation with capsaicin ( pooled results for 0.3 and 0.5 m ) and this agrees with studies by hall et al . and hong and wiley who found an increased peak [ ca]i in thoracic drg neurons ( 2040 m ) from bb / w rats and larger and longer - lasting inward currents in drg neurons from stz - diabetic rats , respectively , following application of 1 m capsaicin . the expression of trpv1 has been shown to be decreased in the hearts of stz - diabetic mice although strecker and colleagues reported unaltered capsaicin - induced cgrp release from human right atrium from subjects with diabetes . the findings of increased ca - response to k and capsaicin show that diabetes causes functional changes in these neurons . the augmented response measured in neurons from diabetic animals following stimulation with capsaicin suggests that the function or expression of trpv1 might be altered by diabetes . song et al . have found a decreased expression of membrane tvpr1 in heart which is supported by hong and wiley who found decreased trpv1 expression in small l1l6 drg neurons . they showed , however , in the same study that functional trpv1 was increased in neurons from diabetic animals and furthermore , the acid - evoked ( ph 5.0 ) transient , mediated by trpv1 , was increased in these neurons which is in accordance with the increased capsaicin response seen in the present study . the function of the pkc - induced phosphorylation , and thus sensitization , of trpv1 has been shown to be increased in lumbar drg neurons from stz diabetic rats . glutamate receptors ( glur ) colocalized with trpv1 on small diameter drg neurons have been demonstrated to contribute to the activity of the capsaicin - induced trpv1 sensitization and glur could possibly contribute to the finding in the present study that capsaicin - responsive neurons elicited larger ca responses than neurons from capsaicin - unresponsive neurons ( the difference was seen both in diabetic as well as control neurons ) . scatter plots of the responses to capsaicin - containing solutions indicated that the tested neurons might be either high- or low - responders . it can not be ruled out that diabetes has a differentiated effect on these two potentially different subpopulations of neurons . when 0.5 m capsaicin was applied in combination with ac at acidic ph 6.0 , the calcium response was larger ( amplitude and auc ) for neurons , regardless of diabetic state , compared to all other applied solutions . this is in accordance with other studies showing that the capsaicin - induced neurons activity can be amplified by lowering ph and by adding inflammatory substances , such as bradykinin 5-ht , pge2 , and adenosine [ 10 , 13 , 26 , 27 ] . the findings of increased ca - response to k and capsaicin show that diabetes causes functional changes in the investigated drg neurons . the augmented response measured in neurons from diabetic animals following stimulation with capsaicin suggests that the function or expression of trpv1 might be altered which is supported by other studies . this is , to the best of our knowledge , the first study to investigate calcium activity in drgs from zdf rats and the study shows that the diabetes - induced effects on peripheral neuron function have common characteristics with other diabetic animal models . the zdf rat thus seems to be a relevant model for studying diabetes - induced neuronal alterations in drg neurons . diabetic neuropathy is a very complex condition with multiple factors playing part in the pathophysiology . the present study suggests that diabetes induces changes in drg neuron function and this might be a contributing factor to the altered pain perception and the altered neuronal response by cardiac neurons seen in diabetic patients during myocardial ischemia and infarction .

sirenomelia is a rare and fatal congenital defect characterized by varying degrees of lower limb fusion , thoracolumbar spinal anomalies , sacrococcygeal agenesis , genitourinary , and anorectal atresia . the incidence of sirenomelia is 0.8 - 1 case/100,000 births with male to female ratio being 3:1 . the rarity of the case is obvious from the fact that many a gynecologist might not have come across a case of sirenomelia in their whole professional carrier . there is a strong association with maternal diabetes where relative risk is 1:200 - 250 and up to 22% of fetuses with this anomaly will have mothers with diabetes . we report two cases of sirenomelia where maternal drug abuse and overt diabetes may have been the cause of this rare anomaly . a 27-year - old unbooked g3p1l1a1 at 39 weeks 5 days of gestational age with previous one live vaginal birth and one first trimester spontaneous abortion was admitted in the labor room with pain in the abdomen . she had no history of prior antenatal care and belonged to a tribal community with lower socioeconomic status . she was otherwise healthy with no known history of genetic or congenital anomaly in her family . on examination , she was observed to be in the second stage of labor with cephalic presentation and regular fetal heart rate . the baby died within 30 min postbirth in spite of resuscitation attempts by neonatologist . on physical examination , the infant showed narrow chest , bilateral hypoplastic thumb , fused lower limbs with a single foot and 5 toes , absent external genitalia , imperforate anus and umbilical cord with single umbilical artery [ figure 1 ] . there were also prominent epicanthal folds , hypertelorism , downward curved nose , receding chin , low - set soft dysplastic ears and small slit - like mouth suggestive of potter 's facies [ figure 2 ] . photograph of the baby showing fusion of lower limbs , hypoplastic thumb , absent external genitalia and features of potter 's facies sirenomeliac baby with narrow chest and potter 's facies ( prominent infraorbital folds , small slit - like mouth , receding chin , downward curved nose , and low - set soft dysplastic ears ) a preterm baby weighing 1.6 kg was delivered vaginally at 34 weeks gestation by a 23-year - old primigravida with an unsupervised pregnancy . the apgar score was 3 at 1 and same at 5 min following which the baby was shifted to neonatal intensive care unit , but died 12 h postbirth due to severe respiratory distress . the new born baby had gross anomalies like narrow chest indicating lung hypoplasia , fused both lower limbs and feet with 10 toes , absence of external genitalia , imperforate anus and single umbilical artery [ figures 3 and 4 ] . examination of the fused lower limbs showed the presence of all thigh and leg bones thus classifying our patient as type i of stocker and heifetz classification . the infant also had features of potter 's facies including prominent infraorbital folds , small slit - like mouth , receding chin , downward curved nose , and low - set ears . ultrasonography revealed bilateral renal agenesis . on autopsy , there was an absence of both kidneys , ureters , urinary bladder , seminal vesicle , and urethra . the gastrointestinal system ended in a blind loop at the rectosigmoid area and was filled with meconium . examination of brain , heart , liver , adrenal glands , and pancreas revealed normal anatomy . sirenomeliac baby with fused lower limbs containing 10 toes , potter 's facies , narrow chest , and absent external genitalia photograph of baby showing imperforate anus a 27-year - old unbooked g3p1l1a1 at 39 weeks 5 days of gestational age with previous one live vaginal birth and one first trimester spontaneous abortion was admitted in the labor room with pain in the abdomen . she had no history of prior antenatal care and belonged to a tribal community with lower socioeconomic status . she was otherwise healthy with no known history of genetic or congenital anomaly in her family . on examination , she was observed to be in the second stage of labor with cephalic presentation and regular fetal heart rate . the baby died within 30 min postbirth in spite of resuscitation attempts by neonatologist . on physical examination , the infant showed narrow chest , bilateral hypoplastic thumb , fused lower limbs with a single foot and 5 toes , absent external genitalia , imperforate anus and umbilical cord with single umbilical artery [ figure 1 ] . there were also prominent epicanthal folds , hypertelorism , downward curved nose , receding chin , low - set soft dysplastic ears and small slit - like mouth suggestive of potter 's facies [ figure 2 ] . photograph of the baby showing fusion of lower limbs , hypoplastic thumb , absent external genitalia and features of potter 's facies sirenomeliac baby with narrow chest and potter 's facies ( prominent infraorbital folds , small slit - like mouth , receding chin , downward curved nose , and low - set soft dysplastic ears ) a preterm baby weighing 1.6 kg was delivered vaginally at 34 weeks gestation by a 23-year - old primigravida with an unsupervised pregnancy . the apgar score was 3 at 1 and same at 5 min following which the baby was shifted to neonatal intensive care unit , but died 12 h postbirth due to severe respiratory distress . the new born baby had gross anomalies like narrow chest indicating lung hypoplasia , fused both lower limbs and feet with 10 toes , absence of external genitalia , imperforate anus and single umbilical artery [ figures 3 and 4 ] . examination of the fused lower limbs showed the presence of all thigh and leg bones thus classifying our patient as type i of stocker and heifetz classification . the infant also had features of potter 's facies including prominent infraorbital folds , small slit - like mouth , receding chin , downward curved nose , and low - set ears . ultrasonography revealed bilateral renal agenesis . on autopsy , there was an absence of both kidneys , ureters , urinary bladder , seminal vesicle , and urethra . the gastrointestinal system ended in a blind loop at the rectosigmoid area and was filled with meconium . examination of brain , heart , liver , adrenal glands , and pancreas revealed normal anatomy . sirenomeliac baby with fused lower limbs containing 10 toes , potter 's facies , narrow chest , and absent external genitalia photograph of baby showing imperforate anus anomalies observed in sirenomelia are described as the most severe form of caudal regression syndrome . fusion of the lower extremities , presence of single umbilical and persistent vitelline artery are major features of sirenomelia . although the primary molecular defect resulting in sirenomelia remains unclear , two main pathogenic hypotheses namely the vascular steal hypothesis and the defective blastogenesis hypothesis are proposed . according to vascular steal hypothesis , fusion of the limbs results from a deficient blood flow and nutrient supply to the caudal mesoderm , which in turn results in agenesis of midline structures and subsequent abnormal approximation of both lower limb fields . however in defective blastogenesis hypothesis , the primary defect in development of caudal mesoderm is attributed to a teratogenic event during the gastrulation stage . such defect interferes with the formation of notochord , resulting in abnormal development of caudal structures . maternal diabetes , tobacco use , retinoic acid and heavy metal exposure are possible environmental factors . in our first case , there was history of tobacco use before and during pregnancy , while in the second case the mother had overt diabetes . sirenomelia is usually fatal within a day or two of birth because of complications associated with abnormal kidney and urinary bladder development and function . in literature approximately 300 cases are reported worldwide of which 14 are from india . in most of the cases , sirenomelia can be diagnosed as early as 13 weeks by using high resolution or color doppler sonography . the condition is usually incompatible with life due visceral abnormalities especially that of renal system . exceptional cases without renal agenesis have survived , the best example being tiffany yorks , a 13-year - old girl who was born with fused legs . over the years , she has undergone numerous operations to separate her lower extremities . the facial abnormality usually found in sirenomeliac infants known as potter 's facies , which includes large , low - set ears , prominent epicanthic fold , hypertelorism , flat nose and receding chin . in both of our cases , when features of potter 's facies are combined with oligamnios and pulmonary hypoplasia it is known as potter 's syndrome , which was present in our second case . in our first case , stocker and heifetz classified sirenomeliac infants from type i to type vii according to the presence or absence of bones within the lower limb . although we did not have radiographs to classify our case with certainty , nevertheless based on external examination , we suggest our first and second case belonged to type iv ( partially fused femurs and fused fibula ) and type i ( all thigh and leg bones are present ) , respectively . sirenomelia is a rare and lethal congenital anomaly . when diagnosed antenatally , termination should be offered . regular antenatal checkup with optimum maternal blood glucose level in preconceptional period and in first trimester should be maintained to prevent this anomaly .

assembly of separate phases within a single bulk grain is important in the production of structural and functional materials : it can determine the mechanical properties of steels and optimize thermoelectric performance . vertical nanocomposites in heteroepitaxial thin films enable combination of the properties of the component phases . in the field of complex transition - metal oxides , chemical control and optimization of properties has conventionally relied on the modification of single - phase structures by targeted substitutions . this has had significant impact in areas as diverse as high - temperature superconductivity , batteries , solid oxide fuel cells , magnetism , ferroelectricity , and multiferroics . the structures adopted by complex oxides allow fine - tuning of physical behavior by chemical substitution , for example , the use of charge reservoir layers in superconducting copper oxides to remotely control the electron count in the electronically active cuo2 planes . the abo3 cubic perovskite ba0.5sr0.5(co0.8fe0.2)o3 has small d co and fe cations disordered on the b - sites of an ao3 network defined by the larger ba and sr cations . it is a single - phase material because the similar charges and bonding chemistries of co and fe can be accommodated on a single site in the average structure , producing a combination of electronic ( b - site d electrons ) and oxide ion ( anion vacancies ) transport that makes it an effective solid oxide fuel cell ( sofc ) cathode . in this work , we explore the effect of incorporating the highly charged d mo cation onto the b - site of this system . the distinct chemistry of this cation frustrates the formation of a single perovskite phase , as it does not match those of co or fe sufficiently to form single ordered or disordered distributions . the system retains the perovskite structure by a structural and compositional phase separation , which occurs solely on the octahedral b - sites within a single ao3 matrix . this ao3 matrix is coherent throughout the micrometer - sized grains and templates the formation of two distinct perovskite phases . the dominant phase has no anion vacancies , but the endotaxial interfaces with the minority mixed ionic - electronic conductor ( miec ) phase give the nanocomposite unexpectedly good solid oxide fuel cell cathode performance . stoichiometric amounts of the starting materials were mixed together by planetary ball milling ( fritsch pulverisette 7 ) for 24 hours in 2-propanol with zro2 balls , followed by drying , grinding , pressing into pellets , calcining at 700 c for 6 hours , and finally heating to 900 c for 8 hours . the resulting pellets were hand - ground in an agate mortar and the powder was ball - milled further for 18 hours in 2-propanol . after being milled , the powder was dried , ground , pressed into pellets , and subsequently sintered in air at 1000 c for 10 hours ; the grinding / pellet pressing / heating step was repeated a total of four times to achieve phase purity and good homogeneity throughout . powder x - ray diffraction data were collected on a panalytical x - pert pro bragg brentano geometry laboratory x - ray diffractometer . synchrotron x - ray diffraction data were obtained on beamline i11 at diamond light source , u.k . , and time - of - flight neutron diffraction ( nd ) data were collected on the high - resolution powder diffractometer ( hrpd ) instrument at the isis facility , rutherford appleton laboratories . structural parameters were refined by the rietveld method , which is described in full in supporting information sections s3 and s4 . the transmission electron microscopy ( tem ) study was carried out on a jeol jem3010 ( jeol , lab6 filament , 300 kev ) , and energy - dispersive spectrometry ( eds ) data were collected on a jem2000fx ( jeol , w filament , 200 kev ) . high - angle annular dark field ( haadf ) images were collected on a jeol jem 2100fcs , with a schottky field emission gun operating in scanning tunneling electron microscopy ( stem ) mode with a ceos aberration - corrected probe . ac impedance measurements were recorded over the frequency range 1 mhz0.01 hz by use of a solartron 1260 frequency response analyzer ( fra ) with a modulation potential of 10 mv , over the temperature range 8731073 k in static air . the symmetrical cell was held for 90 min at each temperature to allow thermal equilibration , and measurements were made with zplot v.2.9b ( scribner associates ) every 50 c . the impedance arcs were modeled by use of equivalent circuit models ( ecm ) with zview v.3.2b ( scribner associates ) . all experimental procedures and analysis are described in detail in the supporting information . incorporation of mo on the b - site affords three distinct regions correlated with low , intermediate , and high mo contents within the ba0.5sr0.5(co0.8xfe0.2ymox+y)o3 phase field ( figure 2a ) defined by x - ray diffraction ( xrd ) ( details of the xrd analysis can be found in supporting information , section s3 ) . we have chosen to highlight three compositions that are characteristic of low ( 1 ) , intermediate ( 2 ) , and high ( 3 ) mo content . crystal structures of ( a ) single perovskite ( sp ) and ( b ) double perovskite ( dp ) . a - site cations are shown as orange spheres , and b - site cations are octahedrally coordinated by oxygen ( red spheres ) in the abo3 perovskite structure . the sp has a single b - site ( shown in light blue ) whereas the dp has an ordered arrangement of two different b - sites ( shown in dark blue and yellow ) . ( a ) b - site compositions studied in the ba0.5sr0.5(co0.8xfe0.2ymox+y)o3 phase field classified according to the phase assemblage observed by xrd . the sp region is shown in gray , sp / dp in blue , and sp / dp / bamoo4 in red . the m = 4 black dashed line corresponds to a co / fe ratio of 4 . the black square on the co / fe axis at m = 4 is the optimal composition for undoped ( ba , sr)(co , fe ) material ( bscf ) . large symbols are compositions 1 , 2 , and 3 for which rietveld refinements of xrd data and tem images are shown in panels b , c , and d , respectively . the two points marked with crosses correspond to the sp and dp component phases refined for 2 . ( i ) rietveld refinements of xrd data and ( ii ) df tem images of representative compositions in the three regions of the phase field are shown for ( b ) z = 0.125 ( 1 , sp ) , ( c ) z = 0.375 ( 2 , sp / dp ) , and ( d ) z = 0.45 ( 3 , sp / dp + bamoo4 ) at m = 4 . reflection markers refer to sp ( gray ) , dp ( blue ) , and bamoo4 ( red ) ; the inset shows the region where the double perovskite superstructure reflection is observed , ( a wider q - space range for the inset is given in supporting information figure s3 , showing the major bamoo4 reflections ) . tem was performed on [ 110 ] oriented grains , with solely the * superstructure reflections denoting the dp selected by the objective aperture . in the df tem images the darker regions are sp and the lighter regions are dp ; in panel b ( ii ) for 1 , dp domains as shown here were observed in three out of 12 crystals , with average domain size 510 nm over a region that is approximately 100 200 nm ; no dp reflections were detected by xrd . supporting information figures s7 and s9 show that the microstructure of 2 persists throughout the sp / dp region of the phase diagram . low mo contents [ x + y = z 0.15 for co / fe ratio ( 0.8 x)/(0.2 y ) = m 4 ; z < 0.1 for m 2 ] afford a region with a single perovskite ( sp ) phase isostructural with ba0.5sr0.5(co0.8fe0.2)o3 ( cell parameter ap 3.8 ) , with disordered substitution of mo onto the octahedral b - site together with co and fe [ 0.5 in ba0.5sr0.5(co0.8fe0.2)o3 ] . at the m = 4 co / fe ratio of the ba0.5sr0.5(co0.8fe0.2)o3 parent , the z = 0.125 material 1 adopts this sp structure ( figure 1a ) . at intermediate mo content ( into the region marked sp / dp on figure 2a ) , a second perovskite phase coexists with the sp , with a doubled perovskite ( dp ) unit cell ( figure 1b , cell parameter 2ap 7.6 ) containing two distinct octahedral b - sites . this symmetry change is produced by cation ordering due to the size and charge differences between the first transition series d [ 0.645 high - spin ( hs ) fe , 0.745 hs co ] and the second series d mo ( 0.59 ) cations . this phase assemblage is adopted at m = 4 by the z = 0.375 composition 2 ( figure 2c ) . further increase in mo content results in the third region with the expulsion of bamoo4 ( marked as sp / dp + bamoo4 ) coexisting with the sp / dp mixture , represented at m = 4 by the z = 0.45 composition 3 ( figure 2d ) . dark - field ( df ) imaging in the transmission electron microscope ( tem ) shows dp coexists with sp on the nanometer scale within individual grains in the sp / dp region of the phase diagram [ figure 2c(ii ) ] . the microstructure is increasingly dominated by dp domains in the sp / dp + bamoo4 region [ figure 2d(ii ) ] . selected area electron diffraction and df images in the sp region [ figure 2b(ii ) ] reveal coexistence of pure sp grains with grains that are majority sp but contain 510 nm domains of the dp phase that are too small to be observed by xrd [ figure 2b(i ) ] . the locations , compositions , cation distributions , and atomic - scale structures of the dp and sp intragrain components in the sp / dp material ba0.5sr0.5co0.5fe0.125mo0.375o3 ( 2 ) were evaluated by tem ( grain - specific , averaging over atomic columns and showing spatial relationships ) and diffraction ( bulk - sensitive xrd and neutron diffraction ( nd ) , which both provide long - range volume - weighted information ) . the individual grains ( figure 3a ) consist of an intergrowth of dp and sp phases with a range of domain sizes , some reaching the order of a hundred nanometers , while other areas have a complex arrangement of smaller domains of a few tens of nanometers in size ( figure 3b ) . fourier transforms ( ft ) of separate areas of a high - resolution transmission electron microscopy ( hrtem ) image of a single grain of 2 ( figure 3c ) show ( iii ) distinct sp and ( iv ) b - site cation ordered dp domains oriented endotaxially ( the fts of the two domains have the same relationship to the grain ) . the image shows coherent interfaces between the sp and dp components with the cation positions lying on a single lattice . intragrain phase separation in the m = 4 sp / dp material ba0.5sr0.5co0.5fe0.125mo0.375o3 ( 2 ) . ( a ) dark - field image based on the double perovskite superstructure reflections , showing dp ( light ) and sp ( dark ) domains of a range of sizes within a single grain . ( b ) higher magnification dark - field image showing an area where the sp and dp structures coexist on the nanometer scale . ( c ) ( i ) hrtem image of a [ 110 ] oriented area and ( ii ) its fourier - transformed ( ft ) diffraction pattern . the ft pattern is indexed as a double perovskite with the dp superstructure reflections indicated by white arrows . simulation of the sp and dp regions from an hrtem image of a single grain are shown in supporting information figure s14 . ( iii ) ft of a section ( indicated by a white circle ) of the area imaged in panel i. the ft diffraction pattern is indexed as a single perovskite ( sp ) , with the absence of superstructure spots indicated by white arrows . ( iv ) ft of a second section of the same image ( i ) , with the intense dp superstructure reflections emphasized by white arrows . the combination of images i , iii , and iv confirms the coexistence of single and double perovskite regions in individual crystallites of 2 . nd and xrd data ( figure 4 ) from 2 were refined simultaneously against a model of two discrete sp and dp phases ( for full details see supporting information section s4 ) : this is an approximation , as it averages over the interphase boundary volume between the two components . the nominal cation composition of 2 was confirmed by eds ( supporting information figure s2 ) and used to restrain the refinement , which produced a refined global b - site composition of co0.499(4)fe0.125(3)mo0.376(2 ) . as required by the endotaxial domain growth , the sp and dp have closely related ap and 2ap unit cell parameters with derived ap = 3.9861(2 ) and 3.9899(1 ) respectively , giving 0.095% mismatch strain . mo is accommodated in the composite by formation of the dp ( 70.4(1)% ; table 1 and figure 5a ) , which is based on the co / mo charge - driven b - site alternation of baco0.5mo0.5o3 . the refined dp composition of ba0.5sr0.5(co0.483(3)fe0.018(2))(mo0.453(1)fe0.047(1))o3.00(1 ) is thus enriched in mo over the global composition , with 96% and 90% occupancy of the two b - sites by co and mo , respectively . final observed ( black ) , calculated ( green ) , and difference ( brown ) profiles from combined rietveld refinement of powder diffraction data from ( a ) time - of - flight neutron diffraction ( backscattering bank ) and ( b ) laboratory x - rays on ba0.5sr0.5co0.5fe0.125mo0.375o3 ( 2 ) nominal composition . tick marks show the bragg reflections for sp in gray and dp in blue . reliability factors for the two perovskite phase model are ( a ) rwp 3.88% , rexp = 2.80% , = 1.92 , rbragg(dp ) = 1.97% , and rbragg(sp ) = 1.52% ; ( b ) rwp = 6.96% , rexp = 5.60% , = 1.54 , rbragg(dp ) = 3.10% , and rbragg(sp ) = 3.26% refined oxygen content was verified by iodometric titration ( supporting information section s6 ) . chemical composition and cation ordering in component phases of the m = 4 sp / dp material ba0.5sr0.5co0.5fe0.125mo0.375o3 ( 2 ) . ( a ) structure of the dp phase ba0.5sr0.5(co0.483(3)fe0.018(2))0.5(mo0.453(1)fe0.047(1))0.5o3.00(1 ) established from the combined refinements , viewed along the [ 110 ] direction , showing cation ordering on the octahedral b - sites , which produces separate columns of distinct composition [ mo - rich site ( yellow ) and mo - poor site ( dark blue ) ] in this orientation . the orientation shown is the same as that in the haadf image [ panel b ( ii ) ] . ( b ) intensity analysis of haadf image ( ii ) for a [ 110 ] oriented dp region of a single grain . ( i ) qstem multislice simulation of a vertical line scan [ indicated by the red arrow in ( ii ) and traversing a and b - sites , corresponding to the vertical direction in panel a ] according to the refined composition of the dp phase . a- and b - sites are labeled showing the cation ordering ; bmo and bco refer to the mo - rich and co - rich b - sites , respectively . the blue rectangles in ( ii ) indicate three lines of cations along which the variation of the intensity ( corresponding to the horizontal direction in panel a ) is shown in each case as indicated by the blue arrows to part ( iii ) . top and bottom rows correspond to b - sites ( where the alternation between co- and mo - rich sites is clear ) , whereas the middle row corresponds to a - sites ( homogeneous cation distribution ) . ( c ) sp phase ba0.5sr0.5co0.539(8)fe0.266(8)mo0.195(2)o2.81(3 ) established from the combined refinements , viewed along the same [ 110 ] direction . the orientation shown is the same as that in the haadf image in panel d ( ii ) . ( d ) intensity analysis of haadf image ( ii ) for a [ 110 ] oriented sp region of the same grain analyzed in panel b. ( i ) qstem multislice simulation of a vertical line scan [ indicated by the red arrow in ( ii ) , traversing a- and b - sites and corresponding to the vertical direction in panel c ] according to the refined composition of the sp phase . a- and b - sites are labeled , showing the absence of b cation ordering in contrast to panel b ( i ) . the absence of b - site order is shown in ( iii ) in the top and bottom line scans ( corresponding to the horizontal direction in panel c ) together with the homogeneous cation distribution in the middle a row . introduction of fe ( oxidation state determined by mssbauer spectroscopy ; supporting information section s5 and figure s12 ) into the 2+/6 + b - site array of the oxygen - stoichiometric dp respectively reduces and increases the charges of the mo- and co - based sublattices . in contrast to the mo - rich character of this phase , fe solubility in the dp is below that expected from the global composition , as it reduces the charge and composition imbalance between the b - sites and suppresses the cation order that defines this phase . fe - induced defects in the b - site ordering pattern are assimilated in the composite by the formation of the fe - rich , mo - deficient , b - site disordered sp phase ba0.5sr0.5co0.539(8)fe0.266(8)mo0.195(2)o2.81(3 ) . this sp phase is isostructural with the parent material ( figure 5c ) and , in contrast to the dp , contains anion vacancies . the refined o content and stability of mo in air allow the mean co / fe oxidation state in the sp to be calculated as + 3 , which with the mssbauer determination of the fe oxidation state as + 3 gives an sp co oxidation state of + 3 . the transition metal charges are co and fe in undoped ba0.5sr0.5(co0.8fe0.2)o3 , which has a much higher oxygen vacancy rate than the sp here . the reduced mo content minimizes the occurrence of unfavorable mo near - neighbor pairs in the disordered sp . the global refined composition of 2 is ba0.5sr0.5co0.499(4)fe0.125(3)mo0.376(2)o2.94(2 ) . oxygen content was verified by iodometric titration as o2.96(1 ) ( supporting information section s6 ) , in agreement with the refined composition and consistent with a thermogravimetric analysis study ( supporting information section s7 ) . the overall o content of 2 is thus higher than the 2.752.4 range found for ba0.5sr0.5(co0.8fe0.2)o3. ba0.5sr0.5co0.499(4)fe0.125(3)mo0.376(2)o2.94(2 ) consists of a nanoscale assembly of 70.4(1)% ba0.5sr0.5(co0.483(3)fe0.018(2))(mo0.453(1)fe0.047(1))o3.00(1 ) ( dp ) and 29.6(1)% ba0.5sr0.5co0.539(8)fe0.266(8)mo0.195(2)o2.81(3 ) ( sp ) . the observed sp / dp microstructure is persistent in the ba0.5sr0.5(co0.8xfe0.2ymox+y)o3 materials because of the charge - based frustration of mo - induced dp b - site order by fe , driving the intragrain compositional and structural segregation reflected in the refined compositions of the two component phases . fe substitution occurs preferentially on the mo site rather than the co site in the dp for charge balance reasons . this produces low fe solubility in the dp as high fe contents will favor sp formation , thus explaining the persistence of the dp / sp single sublattice phase separation in all the materials investigated , while the common ao3 network promotes intragrain coexistence , resulting in the associated microstructures . at the mo content of 2 , the different chemistries of the three b - site cations thus drive the formation of two phases that are compositionally and structurally distinct but are endotaxially related . the dp is expected to be a pure electronic conductor whereas the oxygen vacancies in the sp make it a mixed ionic - electronic conductor ( miec ) , demonstrating that distinct functions are generated by the phase separation . the observed potential curves along the atomic columns in high - angle annular dark field ( haadf ) images of different regions of a [ 110 ] oriented single grain of 2 agree with multislice simulations based on the refined crystallographic models of each constituent phase within the composite ( figure 5b , d ; details of the simulations are in supporting information section s8.1 ) . the haadf images show that the a sublattice is equivalent in dp and sp , whereas these regions differ in their b sublattice occupancy , in agreement with the diffraction work . the bulk diffraction methods further demonstrate the coherence of the ao3 sublattice over which the b - site compositional and site - ordering cation separation occurs through the refined a - o distances of 2.81863(3 ) ( sp ) and 2.82211(3 ) ( dp ) . the dp o position is not fixed by symmetry , so the observed bond length match is not simply a consequence of the unit cell dimensions . there is thus a single ba0.5sr0.5o3 network with constant , almost unstrained ( 0.12% ) a o distances spanning the coherent sp and dp phases defining each grain . the octahedral b - sites within this network are occupied in a spatially modulated manner by the three transition metal cations . the separation into two structurally matched phases of distinct composition observed here is distinct from the order disorder phenomena observed in materials such as relaxor ferroelectrics , which are compositionally homogeneous . this is demonstrated by the observation of two distinct sets of cell parameters in the mo - substituted materials studied here , which relaxes strain and leaves the different chemical bonding environments for the three cations achieved by the formation of two phases as the reason for the phase separation . the effect of this nanoscale single sublattice phase separation on the stability of 2 was then investigated ( full details in supporting information section s9 ) . the stability after aging for 120 hours at 750 c in air of ba0.5sr0.5(co0.8fe0.2)o3 and the mo - substituted ba0.5sr0.5(co0.8xfe0.2ymox+y)o3 phases at the constant co / fe ratio m = 4 is shown in figure 6a ( extended aging of 2 is shown in figure 6b ) . the cubic perovskite structure is retained for the sp / dp composite 2 , whereas there is significant decomposition of the sp materials ba0.5sr0.5(co0.8fe0.2)o3 and ba0.5sr0.5co0.7fe0.175mo0.125o3 ( 1 ) to a hexagonal perovskite . dc conductivity measurements in co / co2 atmospheres ( supporting information section s11 ) and x - ray analysis post - measurement ( figure 6b ) reveal that 2 is stable to 10 atm o2 and under 1600 ppm co2 at 700 c ; a reduction in lattice parameter was observed due to the loss of lattice oxygen , as observed for bscf under similar conditions , but no decomposition products were visible in the pxrd pattern . the sp / dp stabilization of the cubic structure extends to higher co / fe ratios ( e.g. , 4 , m = 10 , z = 0.45 ) . the b - site compositional and ordering segregation driven by mo substitution in the sp / dp region of the phase field affords sp domains stabilized locally by enhanced fe content over that of the global composition ( higher fe contents are known to stabilize pure cubic ba0.5sr0.5(co1xfex)o3 against the formation of the hexagonal phase ) together with co / mo - rich dp stabilized by cation ordering and the absence of anion vacancies . local compositions of the two component phases emerging from the b - site occupancy modulation within the coherent cubic ao3 network defining the entire grain give the observed stability against the hexagonal perovskite . . thermal stability of perovskite structures in sp and sp / dp regions of the ba0.5sr0.5(co0.8xfe0.2ymox+y)o3 system . ( a ) pxrd ( logarithmic intensity scale ) after aging at 750 c for 120 h in air for ba0.5sr0.5co0.8fe0.2o3 ( bscf , black , sp ) , ba0.5sr0.5co0.7fe0.175mo0.125o31 ( gray , sp ) , ba0.5sr0.5co0.5fe0.125mo0.375o32 ( blue , sp / dp ) , and ba0.5sr0.5co0.50fe0.05mo0.45o34 ( blue , sp / dp ) . the appearance of hexagonal perovskite decomposition products is indicated by asterisks in the sp materials bscf and 1 . ( b ) pxrd patterns ( linear intensity scale ) showing phase stability of 2 after long - term thermal aging : ba0.5sr0.5co0.5fe0.125mo0.375o32 was aged in air for 5 days ( blue ) and 10 days ( purple ) and aged in po2 = 10 atm in the presence of 1600 ppm co2 ( gray ) . ( inset ) superposition of the 10 days and 5 days pxrd patterns in the main figure over a narrower q range , showing that the 110 cubic perovskite bragg reflection common to the sp and dp phases is identical after aging in air . composition 2 was studied by powder x - ray thermodiffraction from 25 to 900 c ( figure 7 ) ; above this temperature , it reacts with the quartz capillary . the prevalence of the domain structure was observed throughout this temperature range with little variation in phase fraction or mo content of the sp phase ( figure 7b ) . lattice parameters of the sp and dp phases increase linearly within a large part of the measured temperature range ( figure 7a ) . a deviation in linearity of the thermal expansion was found at lower temperatures , which can be attributed to a slight change in oxygen composition upon heating from room temperature to 500 c , as has been observed previously for bscf . the lattice parameter difference between the phases remains nearly unaltered throughout the temperature range measured . this reflects the clamping of the lattice parameters due to the fixed ao3 matrix within which the b - site compositional exchange takes place . this almost parallel evolution is characterized by linear thermal expansion coefficients from 500 c that are very similar for the two phases ( 14.1 10 and 14.5 10 k for sp and dp phases , respectively ) , while being smaller than values reported previously for bscf in a similar temperature range ( ( 19.022.9 ) 10 k ) , suggesting an improved compatibility with typical ceria - based electrolytes sdc ( sm0.2ce0.8o1.9 , 11.4 10 k ) and gdc ( gd0.2ce0.8o1.9 , 12.21 10 k ) when compared to bscf . in parallel , the size of the sp and dp domains , as determined by analysis of sample broadening , showed little deviation within this temperature range ( figure 7c ) . moreover , the biphasic character at the synthetic temperature was confirmed ex situ with a quenching experiment from 1000 c , evidencing a mixture of 43(3)% sp and 57(3)% dp . the in situ experiment also showed a rearrangement of the domain structure from 500 to 800 c that may be correlated with the oxygen loss detected by thermogravimetric analysis ( tga ) occurring in this temperature range , provoking a different charge repartition in the ao3 network as the average formal oxidation states of transition metals are reduced . oxygen loss favors the expansion of oxygen - deficient sp domains , with a slight increase of their molybdenum content [ from 0.20(2 ) to 0.25(2 ) ] , while dp domains are therefore contracted . variation of the oxygen content is expected to have a dramatic effect on the domain structure as it is a compositional tool to influence the b - site chemical content in terms of electronic charges . the charge frustration maintains the observed phase separation over a wide temperature range , in contrast to the disappearance of this separation at the spinodal temperature seen , for example , in lead - based thermoelectric materials . microstructural and compositional features from rietveld refinements of variable - temperature synchrotron x - ray diffraction data for 2 , ba0.5sr0.5co0.5fe0.125mo0.375o3 ( m = 4 , z = 0.375 ) . ( a ) lattice parameter progression , expressed in terms of the single perovskite unit cell , ap . linear thermal expansion coefficients in the temperature range 500900 c ( solid line ) are given , and these are extrapolated to lower temperature to show a deviation from linearity ( dashed line ) . ( b ) sp phase fraction and mo content refined for the sp phase . dp odd refers solely to the supercell reflections for the dp , and as such represent the volume - weighted crystallite size of the dp in absence of any antiphase boundaries that may contribute to the dp subcell reflections . error bars shown are 3 times the standard deviation from the refinements ; in panel a , errors on the lattice parameters are on the fifth decimal place and therefore lie beneath the plotted symbols . symmetrical cells were prepared from selected compositions in the three regions of the phase field to evaluate their sofc cathode performance by measurement of the area - specific resistance ( asr ) ( figure 8a ; supporting information section s10 and table s11 ) . the single - phase sp parent material ba0.5sr0.5(co0.8fe0.2)o3 itself is an miec and effective sofc cathode that displays a range of asr values from 0.03 to 10 cm at 650 c , depending on processing conditions and electrolytes . the aim of this study is not to produce the lowest reported asr for ba0.5sr0.5(co0.8fe0.2)o3-related cathodes but rather to examine the effect of charge frustration on structure and properties ; here we measure an asr of 0.15 cm for the ba0.5sr0.5(co0.8fe0.2)o3 parent at 650 c with ce0.8sm0.2o2 electrolyte . mo is a component of heterogeneous catalyst systems involving dioxygen activation and may enhance the oxygen reduction performance of the ba0.5sr0.5(co1xfex)o3 family . physical properties and cathode performance in sp and sp / dp regions of the ba0.5sr0.5(co0.8xfe0.2ymox+y)o3 system . ( a ) asr of bscfm compositions measured in air by ac impedance of symmetrical cells represented on a logarithmic scale between 600 and 800 c . colors are selected according to the location in the phase field shown in figure 2a : black , bscf ; gray , sp ; blue , sp / dp ; red , sp / dp + bamoo4 . z = x + y is the mo content ; m is the co / fe ratio . ( b ) asr at constant co / fe ratio ( m = 4 ) with varying mo content z at 650 c , highlighting the anomalous nature of the z = 0.375 composition 2 ( blue diamond ) . the same symbols are used for each composition as in panel a. ( c ) dc conductivity in air for the compositions given in panel a with the same color scheme . the linear variation with temperature for 2 indicates that the changes in composition and microstructure on heating are minor , which is confirmed by rietveld refinement of variable - temperature synchrotron x - ray diffraction data in figure 7 and supporting information section s3.5 and by tga data in supporting information section s7 and figure s13 . asr values measured for mo - substituted materials range from 0.13 to 1.0 cm at 650 c . the lowest asr is observed for ba0.5sr0.5co0.5fe0.125mo0.375o3 ( 2 ) , which is located in the sp / dp region ( z = 0.375 ) and has the co / fe ratio m = 4 identified as optimal in the ba0.5sr0.5(co1xfex)o3 family . the asr of 2 is lower than that of ba0.5sr0.5(co0.8fe0.2)o3 between 650 and 800 c ( figure 8a ) . in order to separate compositional and porosity effects on electrode performance , we assessed the cathode microstructure by scanning electron microscopy ( sem ) imaging and determined the surface area by gravimetric methods . direct measurement of the pore volume by sorption measurements is challenging due to the low masses of electrode material used relative to the dense sdc electrolyte used in the symmetrical cell configuration . we achieved this by using the identical screen - printing process for the cathode but onto thin pt substrates supported on the electrolyte ; once removed from the electrolyte support , this permitted gravimetric determination of n2 sorption on the pt - supported cathode and , once the cathode is dissolved by use of 3 m hcl , the pt substrate itself ( figure 9a ) . the surface area of the cathode coating was calculated by point - by - point subtraction between data for the pt - supported cathode and for the bare pt foil [ figure 9b(ii ) ] to ensure that the observed weight difference could be solely ascribed to n2 uptake for the cathode . teller ( bet ) surface area was then calculated from the pt - subtracted data by fitting the dependence of n2 uptake with pressure to the bet equation in the low pressure range [ figure 9b(iii ) ] . for full details of the subtractive technique , bet calculations , and bet surface area results for the tested cathodes , see supporting information section s10.4 . ( a ) schematic of the preparation of pt - supported cathode and subsequent acid removal of the cathode , as used for bet measurements . ( b ) ( i ) scheme illustrating the subtractive protocol used to calculate the surface area of the cathodes . ( ii ) observed weight gain with n2 pressure for a cathode sample of 2 prepared by the method outlined in panel a and for the bare pt substrate . ( iii ) bet fitting ( blue line ) for the low - pressure region of n2 sorption data ( blue circles ) obtained from the difference between as - made pt supported cathode , 2 , and bare pt substrate . this subtractive technique allowed direct determination of the electrode pore volume across the series ( table 2 ) , giving a trend in agreement with the sem observations ( supporting information section s10.4 and figure s17 ) ; similar surface areas were observed for all the mo - substituted systems , which were systematically larger than those found for the ba0.5sr0.5(co0.8fe0.2)o3 parent under the processing conditions used . c is the bet constant and vm is the monolayer adsorbed gas quantity . evolution of the asr with increasing mo content z at the co / fe ratio m = 4 is nonmonotonic ( figure 8b ) , although the total dc conductivity dc ( figure 8c and supporting information section s11 ) decreases as expected from the reduction in d electron and oxygen vacancy concentration on substitution of d by d mo cations . this suggests that mo catalyzes the oxygen reduction reaction ( orr ) , consistent with its role in catalytic systems involving dioxygen activation . the asr of 2 ( z = 0.375 ) is lower than that of both sp [ z = 0.125 ( 1 ) ] and sp / dp ( z = 0.2 , 0.25 ) materials with reduced mo contents . it is also lower than that of both z = 0.45 ( 3 ) from the sp / dp / bamoo4 region and lower fe content m = 10 , z = 0.45 ( 4 ) in the sp / dp region . ac impedance arcs at 650 c [ figure 10a(i ) ] show that both sp / dp 2 and sp ba0.5sr0.5(co0.8fe0.2)o3 display similar impedance responses dominated by a gerischer - type contribution [ figure 10a(ii ) ] , which is colimited by oxygen surface exchange and bulk oxide transport . all the other higher asr ba0.5sr0.5(co0.8xfe0.2ymox+y)o3 materials display different responses associated with charge transfer ( electronic ) and mass transport ( chemical ) processes ( detailed discussion can be found in supporting information sections s10.6s10.9 ) . these comparisons suggest the superiority of the sp / dp architecture of 2 over the sp - only 1 ( despite a lower dc resistance for 1 ) , together with the improved performance of this architecture at 2 in comparison with , for example , 4 ( m = 10 , z = 0.45 ) . the relationship between mo content ( and related factors such as oxygen nonstoichiometry , sp phase fraction , and dc ) and asr implicates the observed sp / dp intragrain microstructure in the cathode performance . the mechanistic similarity between 2 and ba0.5sr0.5(co0.8fe0.2)o3 demonstrated by the impedance responses suggest that this sp / dp assembly can give cathode performance comparable to best - in - class single - phase materials , despite being compositionally dominated by an ionic insulator . the self - organization of the endotaxial composite confers higher stability than the single - phase parent and opens up the possibility of further optimization in this family of sofc cathode materials . ( a ) ( i ) electrochemical impedance spectroscopy ( eis ) arcs for the compositions given in figure 8a ( with the same color scheme ) at 650 c ( arcs have been normalized to 0 on the x - axis to remove the electrolyte contribution ) : symbols are measured data , and lines are from fitting of equivalent circuit models ( ecm ; supporting information section s10.6 and figure s19 ) . the same electrode processes are identified from the ecm for 2 and bscf , and differ from those occurring in 1 and 4 . composition 4 ( m = 10 ) is shown here , as it has lower asr than 3 ( m = 4 ) , which is shown in supporting information figure s19d . ( ii ) close - up of the eis arcs for 1 , 2 , and bscf shown in panel i. as each cathode was manufactured by the same protocol ( milling regime , ink processing , screen printing parameters , and thermal treatment ) and displays similar morphology and surface area ( supporting information section s10.4 ) , differences in the observed arcs and selected ecm are a reflection of differences in the electrochemical properties of each composition . hrtem and xrd studies show the phase assemblage and microstructure were unaffected by preparation of the symmetrical cells ( supporting information section s10.2 and figure s15 ) . the gerischer - type half - tear - drop shape is apparent in the ac impedance arcs of 2 and bscf as the dominant large - resistance arc . ( b ) schematic representation of oxygen ion ( red ) and electron ( black ) transport pathways available in a two - phase endotaxial composite grain of 2 . the absence of oxygen vacancies makes the dp ( blue ) a pure electronic conductor , whereas oxygen mobility is possible within the sp and along the sp / dp interfaces . the sp / dp / air triple phase boundary of 2 is highlighted as an orange dotted line ; this boundary , as well as the surface of the sp miec regions , is active in the orr . the microstructure of 2 can be approximated as a coherent endotaxial intergrowth of a pure electronic conductor ( dp , with no oxygen vacancy content but noninteger transition - metal valence : active in the orr in isolation only at the triple phase boundary with electrolyte and air ) and a miec ( sp , with vacancies for oxide transport and orr active at all interfaces with air ) ( figure 10b ) . the pathways for electronic conduction are throughout and between the dp and sp regions , whereas oxide ion transport is restricted to the interconnected sp phase regions and their surfaces , including interfaces with the dp . the microstructure of figure 10b creates a high - surface - area triple phase boundary for o formation where the electronically conducting , catalytically active dp meets the miec sp and air , with the single ao3 network interfaces between sp and dp assisting oxide transfer between them . the sp component of 2 is richer in fe than the global m = 4 composition , and thus expected to be a poorer cathode than m = 4 ba0.5sr0.5(co0.8fe0.2)o3 , on the basis of variation of ba0.5sr0.5(co1xfex)o3 performance with fe content . the presence of 70 wt % dp in 2 would be expected to lead to poor cathode performance due to reduced electronic conductivity and absence of oxygen vacancies . this is not the case , however : 30 wt % sp alone is not sufficient to account for the low asr value of 2 , as sp / dp ba0.5sr0.5(co0.8xfe0.2ymox+y)o3 materials such as m = 3 , z = 0.21 [ 63(2)% sp ] with higher sp content have higher asr ( figure 8a , b ) . this shows that the dp is not a spectator phase and that its high mo and co content contributes to the oxygen reduction performance of the nanocomposite 2 , whose structure favors synergic interaction of the dp and the oxide ion - conducting sp . the functional behavior of 2 suggests that the coherent ao3 network can integrate the properties of the distinct sp and dp regions to produce performance from the composite that is superior to that expected from the local component phases in isolation . composition 2 has superior cathode performance to the other sp / dp materials evaluated because its interplay of sp / dp ratio , domain sizes , phase compositions , and spatial locations best combines the distinct properties of sp and dp . it is stabilized by local separation into compositionally and structurally distinct domains of fe - rich sp and cation - ordered , anion vacancy - free mo - rich dp , which are spanned by a single ao3 network . this microstructure results from a heterogeneous distribution of the three electronically active cations over the b - sites . the perovskite structure adapts to the compositional complexity and contraindicated chemistry of the three b - site cations that frustrate the formation of a single phase by this reorganization correlated beyond the unit cell . the resulting nanoscale modulation of the b - site occupancy produces the compositionally and structurally distinct endotaxially related phases forming the composite . the matching of cation occupancy to the crystal chemical environment that is possible in the two - phase b - site separation within a single ao3 network increases the stability of the cubic perovskite with respect to competing structures over that of the single - phase parent material . the sp / dp composite performs well as an sofc cathode , exceeding expectations based on the local compositions of the constituent phases ( one of which is not capable of anion transport ) . the stability of the self - organized endotaxial composite is thus determined by the local phase compositions , but its properties surpass the sum of these local components in isolation because of the synergic integration of the functions of the two phases by the coherent interfaces of the composite . the chemical frustration produced by charge and bonding differences drives the self - assembly of compositionally and structurally ordered regions , spanned by a common network . this frustration - based approach is clearly well - suited to the diverse cation ordering patterns known in perovskites . the single sublattice phase separation and resulting property integration may also prove applicable to the many well - known families of complex oxides that feature separate structural networks .

retinoblastoma is a highly malignant tumor of the eye that grows relentlessly and almost always has a fatal outcome if untreated . both sexes are affected equally , and the tumor has no predilection for the right or left eye . it has an incidence varying from 1 in 3300 to 1 in 20,000 live births , and accounts for approximately 2.5 - 4% of all cancers diagnosed in children younger than 15 years . most tumors occur before the age of 2 years and are diagnosed upto the age of 5 years . approximately , 40% of all retinoblastomas are bilateral and 60% are unilateral . in the absence of a known family history of the disease , the early months of retinoblastoma growth in a child 's eye usually go undetected by both the family and the primary care physician . bilateral retinoblastoma is most often asymmetric and is usually diagnosed at an earlier age than the unilateral disease . the average age at diagnosis in bilateral disease is 12 months ; in unilateral disease , it is 24 months . a white reflex , i.e. , cat's - eye reflex or leukocoria in the pupil is the most common clinical sign ( 56% ) . transillumination , fluorescein angiography , ultrasonography , computed tomography , magnetic resonance imaging are routinely used in diagnosing retinoblastoma . early diagnosis and prompt treatment are important factors in a child 's survival and can often salvage useful vision in one or both eyes . however , unilateral retinoblastoma , unfortunately , is usually far advanced at the time of detection , and little normal retina is seen . in bilateral cases , the presentation is often strikingly asymmetric , with one eye involved by massive tumor and the opposite eye much less involved . in both of these situations , the modalities currently used to treat intraocular retinoblastoma include enucleation , laser photocoagulation , laser hyperthermia , cryotherapy , radioactive plaque brachytherapy , external beam radiotherapy ( ebr ) , and systemic chemotherapy . the approach to treatment depends on the size and extent of tumors , the sites of involvement , and the patient 's systemic involvement . several studies have shown that the most common cause of enucleation in pediatric population is retinoblastoma . the psychological effect of losing an eye may present greater difficulties for the patient than the physical disability . the patient loses an organ with an important sensory function and is also faced with a distortion of facial appearance . the associated psychological effect of these defects on the patient requires immediate management and rehabilitation intervention by a team of specialists . the pre - operative time spent addressing eye removal , as well as discussing life after losing an eye , will reap benefits in post - operative recovery and acceptance . increasing interest in this field has resulted in a better understanding of the problems associated with surgical treatment of retinoblastoma and presently , a multidisciplinary approach is considered a most effective approach to rehabilitate rather than just treat the patient . therefore , the combined efforts of the ophthalmologist , the plastic surgeon , psychologist , and the maxillofacial prosthodontist are essential to restore the patient 's quality of life . this paper presents a palliative approach to rehabilitate a 5-year - old girl with retinoblastoma , who has undergone enucleation of her left eye . a 5-year - old female patient reported to the department of prosthodontics with the complaint of unaesthetic facial appearance due to absence of left eye [ figure 1 ] . her parents gave history of retinoblastoma which was diagnosed at the age of 2 years , for which enucleation was done at 2.5 years of age . patient also received chemotherapy and radiotherapy for treatment of her malignancy . at the age of 3 years , a stock artificial eye was placed in the patient 's eye but , due to pain and irritation caused by artificial eye , patient used to remove the prosthesis and showed extreme resistance to wearing of the prosthesis . pre - operative photograph showing enucleated socket of left eye patient 's past experience with prosthesis precluded the use of stock ocular prosthesis . after careful examination of the area of the defect and treatment planning , it was planned to fabricate a custom ocular prosthesis for the patient . the procedure , including its maintenance and limitations , was explained to the patient / guardian to gain their co - operation . the procedure was initiated by the first making the impression of the socket with a thin mix of ophthalmic alginate ( opthalmic moldite , milton roy company , sarasota , fl , usa ) , then making a two - piece dental stone ( kalabhai karson pvt . mumbai , india ) cast from the impression by double pour technique [ figure 2 ] . the wax pattern was formed on the cast using carving wax mixed with sticky wax , and tried in the patient 's eye and checked for accuracy in the terms of retention , proper extension in all directions , scleral contour and convexity and finally positioning the iris , matching in size and color of contralateral eye , obtained from stock eye . the final wax pattern with iris button was evaluated in the patient 's socket and modified accordingly [ figure 3 ] . impression of the ocular defect ( a ) and cast obtained ( b ) final wax pattern try - in after making necessary modifications , the wax pattern was invested , flasked , and de - waxing was done [ figure 4 ] . the next step was to reproduce scleral shade of the normal eye . for this , shade tabs were prepared by mixing and matching different shades and proportions of tooth - colored acrylic ( sc 10 , pyrax , roorkee , india ) till the color of sclera of the other eye was replicated . to simulate the veins of the normal eye , veins were separated from the veined heat - cured poly - methyl methacrylate ( pmma ; trevalon , dentsply india pvt . ltd . , gurgaon , india ) and they were incorporated in the dough of the determined acrylic shade , followed by routine curing , finishing , and polishing . finally , a thin film of the sclera was removed and replaced by a clear film of transparent heat - cured pmmae resin ( trevalon , dentsply india pvt . ltd . ) to simulate corneal translucency . after processing , the properly finished and polished prosthesis was inserted in the socket [ figure 5 ] after being disinfected with 0.5% chlorhexidine and 70% isopropyl alcohol for 5 min and lubricated with an ophthalmic lubricant ( ecotears , intas pharmaceuticals limited , ahmedabad , india ) to maintain a tear film over the prosthesis and to improve eye movements . minor adjustments were made at the time of delivery as per the patient 's comfort and esthetics . instructions were given to the patient regarding proper care and handling of the prosthesis as well as the maintenance of socket hygiene and the need for regular recall appointments was emphasized . as in the early childhood , orbital volume increases in a linear fashion , and a change of prosthesis is required between 18 and 26 months following prosthesis placement in children . post - operative photograph showing prosthesis in lieu of enucleated eye in the follow - up period of 6 months , the patient was able to wear a cosmetically acceptable ocular prosthesis without any discomfort . after careful examination of the area of the defect and treatment planning , it was planned to fabricate a custom ocular prosthesis for the patient . the procedure , including its maintenance and limitations , was explained to the patient / guardian to gain their co - operation . the procedure was initiated by the first making the impression of the socket with a thin mix of ophthalmic alginate ( opthalmic moldite , milton roy company , sarasota , fl , usa ) , then making a two - piece dental stone ( kalabhai karson pvt . ltd . , mumbai , india ) cast from the impression by double pour technique [ figure 2 ] . the wax pattern was formed on the cast using carving wax mixed with sticky wax , and tried in the patient 's eye and checked for accuracy in the terms of retention , proper extension in all directions , scleral contour and convexity and finally positioning the iris , matching in size and color of contralateral eye , obtained from stock eye . the final wax pattern with iris button was evaluated in the patient 's socket and modified accordingly [ figure 3 ] . impression of the ocular defect ( a ) and cast obtained ( b ) final wax pattern try - in after making necessary modifications , the wax pattern was invested , flasked , and de - waxing was done [ figure 4 ] . for this , shade tabs were prepared by mixing and matching different shades and proportions of tooth - colored acrylic ( sc 10 , pyrax , roorkee , india ) till the color of sclera of the other eye was replicated . to simulate the veins of the normal eye , veins were separated from the veined heat - cured poly - methyl methacrylate ( pmma ; trevalon , dentsply india pvt . ltd . , gurgaon , india ) and they were incorporated in the dough of the determined acrylic shade , followed by routine curing , finishing , and polishing . finally , a thin film of the sclera was removed and replaced by a clear film of transparent heat - cured pmmae resin ( trevalon , dentsply india pvt . ltd . ) to simulate corneal translucency . after processing , the properly finished and polished prosthesis was inserted in the socket [ figure 5 ] after being disinfected with 0.5% chlorhexidine and 70% isopropyl alcohol for 5 min and lubricated with an ophthalmic lubricant ( ecotears , intas pharmaceuticals limited , ahmedabad , india ) to maintain a tear film over the prosthesis and to improve eye movements . minor adjustments were made at the time of delivery as per the patient 's comfort and esthetics . instructions were given to the patient regarding proper care and handling of the prosthesis as well as the maintenance of socket hygiene and the need for regular recall appointments was emphasized . as in the early childhood , orbital volume increases in a linear fashion , and a change of prosthesis is required between 18 and 26 months following prosthesis placement in children . post - operative photograph showing prosthesis in lieu of enucleated eye in the follow - up period of 6 months , the patient was able to wear a cosmetically acceptable ocular prosthesis without any discomfort . cancer is always a frightening diagnosis , and when it affects children , the situation is overwhelming . eye cancer in children , though rare , is the third leading cancer of childhood . retinoblastoma can be a particularly distressing form of cancer as it is potentially fatal and can result in loss of one or both eyes . depression , feelings of helplessness , hopelessness , and sadness can occur throughout the cycle of grieving and beyond . enucleation during infancy or congenital anophthalmos or severe microphthalmos can lead to severely underdeveloped bony orbital growth , cosmetic deformity , and facial asymmetry . placement of prosthesis has been proven to play a definite role in stimulating the orbital growth . the regular replacement or modification of prosthesis is important to give continuous stimulus for socket growth . the fundamental objective in restoring a congenital as well as acquired defect of eye with an ocular prosthesis is to enable the patient to cope better with the difficult process of rehabilitation after an enucleation or evisceration . a cosmetically acceptable prosthesis is that reproduces the color , form , and orientation of iris and allows the patient to return to accustomed lifestyle . they are available in standard sizes , shapes , and colors and require no special skills or materials for fabrication . however , in majority of cases of enucleation , custom ocular prosthesis is advantageous as there is improved adaptation to underlying tissues , increased mobility of the prosthesis , and excellent esthetics due to better match of the size and color of the iris and sclera . the repercussions of retinoblastoma , its treatment , and its outcome are felt throughout the life of the patient and his / her family , so providing the patient with a cosmetically pleasing custom ocular prosthesis is a palliative approach for rehabilitation of a pediatric patient with retinoblastoma . custom eyes have several advantages including better mobility , even distribution of pressure due to equal movement , thereby reducing the incidence of ulceration , improved fit , comfort , and adaptation , improved facial contours , and enhanced esthetics gained from the control over the size of the iris , pupil , and color of the iris and sclera . custom ocular prosthesis not only fulfills the esthetic and psychological demands but also stimulates bony orbital growth , thereby reaping long - term benefits of restoring facial symmetry and esthetics .

skin aging is a complex process induced by constant exposure to ultraviolet ( uv ) irradiation . uv generates reactive oxygen species leading to collagen deficiency and eventually skin wrinkling ( 1 , 2 ) . aging of the skin is characterized by irregular pigmentation , increased wrinkling , loss of elasticity , dryness and roughness ( 3 ) . recently , emulsions have established growing interest as a vehicle for the drug delivery to the body . the use of natural compounds in skin protection especially topical application of antioxidants indicates that they usefully decrease skin aging ( 4 ) . basil ( ocimum basilicum ) is an annual plant , with surprising medicinal properties and contains several antioxidant compounds . basil in traditional medicine , has been used for the treatment of coughs , headaches , constipation and fever ( 5 ) . skin aging is a complex process induced by constant exposure to ultraviolet ( uv ) irradiation . uv generates reactive oxygen species leading to collagen deficiency and eventually skin wrinkling ( 1 , 2 ) . aging of the skin is characterized by irregular pigmentation , increased wrinkling , loss of elasticity , dryness and roughness ( 3 ) . recently , emulsions have established growing interest as a vehicle for the drug delivery to the body . the use of natural compounds in skin protection especially topical application of antioxidants indicates that they usefully decrease skin aging ( 4 ) . basil ( ocimum basilicum ) is an annual plant , with surprising medicinal properties and contains several antioxidant compounds . basil in traditional medicine , has been used for the treatment of coughs , headaches , constipation and fever ( 5 ) . in this study a w / o emulsion containing extract of basil was formulated and its effects on different parameters related to skin aging was evaluated . basil leaves and flowers collected and identified at cholistan institute of desert studies of the islamia university of bahawalpur , pakistan ( voucher # oblf- 4 - 11 - 21 ) were shade dried and finely grounded . four hundreds gm of the powdered material was extracted at room temperature with 2000 ml of 95% ethanol for 48 hrs . then the glass beaker was sealed with aluminium foil and shaken for 10 min after every 12 hrs . it was filtered through a whatman # 01 filter paper , evaporated under reduced pressure at 40 c in a rotavapor ( eyela , co. ltd . japan ) till the concentrate was reduced to the one third of the initial volume of the solvent used and stored in freezer at 8 c in refrigerator till further analyses . the presence of phenolic compounds and flavonoids was tested according to the methods in the literature ( 6 ) . the antioxidant activity of the extract was determined using dpph ( 1,1-diphenil-2-picrylhydrazyl ) which is a stable free radical , and vitamin c as standard ( 7 ) . the antioxidant activity of basil extract was found to be 88% in comparison to the standard . different formulations containing different concentrations of parrafin oil ( merck , germany ) , abil em 90 ( franken chemicals germany ) and distilled water were prepared in this study . oil phase comprised of paraffin oil and surfactant ( abil em 90 ) heated up to 75c1c . aqueous phase comprising of water was heated to the same temperature and then treated with the extract . in the case of base no extract was added to the aqueous phase . w / o emulsions were prepared by addition of aqueous to the oily phases with continuous stirring first at 2000 rpm by the mechanical mixer ( euro - star , ika d 230 , germany ) for 15 min then 1000 rpm for 5 min , and thereafter 500 rpm for 5 minutes for complete homogenization by the mechanical mixer ( euro - star , ika d 230 , germany ) and finally the emulsion was cooled to room temperature . creams were divided into four parts and stability tests were performed at 8c0.1c in refrigerator ( dawlance , pakistan ) and at 25c1c , 40c1c and 40c1c in incubator with 75% relative humidity ( rh ) . electrical conductivity using conductivity meter ( wtw cond-197i , germany ) , and ph using ph meter ( wtw ph-197i , germany ) of formulations were noted at various time intervals for 8 weeks ( 8) . the formula which was found to be most stable among different formulations and was selected for further studies is given in table 1 . q.s.= quantity sufficient this study was approved by the board of advanced studies and research , and its ethical committee for in - vivo studies ( reference no 3715/ acad . ) , of the islamia university of bahawalpur and was conducted according to the guidelines of helsinki declaration . a total of 11 male volunteers with mean age of 48 years were selected for the study and consent forms were taken . every volunteer applied creams at night on cheeks for the period of 12 weeks and came for measurement on 2nd , 4th , 6th , 8th , 10th and 12th week in morning at 10 a.m. they were allowed to wash their faces with water and , sit to become accustomed with the environment for 30 minutes before taking any measurements . values for different parameters were taken in controlled room temperature of 251c and 452% relative humidity . skin microrelief parameters were evaluated using visio scan vc98 ( courage and khazaka , germany ) . the skin moisture was determined with a skin capacitance meter ( corneometer mpa 5 ) and transepidermal water loss ( tewl ) was determined by an evaporimeter ( tewameter mpa 5 , courage and khazaka , germany ) . the percentage of changes with respect to initial values / zero hour of volunteers for different parameters , taken at 2nd basil leaves and flowers collected and identified at cholistan institute of desert studies of the islamia university of bahawalpur , pakistan ( voucher # oblf- 4 - 11 - 21 ) were shade dried and finely grounded . four hundreds gm of the powdered material was extracted at room temperature with 2000 ml of 95% ethanol for 48 hrs . then the glass beaker was sealed with aluminium foil and shaken for 10 min after every 12 hrs . it was filtered through a whatman # 01 filter paper , evaporated under reduced pressure at 40 c in a rotavapor ( eyela , co. ltd . japan ) till the concentrate was reduced to the one third of the initial volume of the solvent used and stored in freezer at 8 c in refrigerator till further analyses . the presence of phenolic compounds and flavonoids was tested according to the methods in the literature ( 6 ) . the antioxidant activity of the extract was determined using dpph ( 1,1-diphenil-2-picrylhydrazyl ) which is a stable free radical , and vitamin c as standard ( 7 ) . the antioxidant activity of basil extract was found to be 88% in comparison to the standard . different formulations containing different concentrations of parrafin oil ( merck , germany ) , abil em 90 ( franken chemicals germany ) and distilled water were prepared in this study . oil phase comprised of paraffin oil and surfactant ( abil em 90 ) heated up to 75c1c . aqueous phase comprising of water was heated to the same temperature and then treated with the extract . in the case of base no extract was added to the aqueous phase . w / o emulsions were prepared by addition of aqueous to the oily phases with continuous stirring first at 2000 rpm by the mechanical mixer ( euro - star , ika d 230 , germany ) for 15 min then 1000 rpm for 5 min , and thereafter 500 rpm for 5 minutes for complete homogenization by the mechanical mixer ( euro - star , ika d 230 , germany ) and finally the emulsion was cooled to room temperature . creams were divided into four parts and stability tests were performed at 8c0.1c in refrigerator ( dawlance , pakistan ) and at 25c1c , 40c1c and 40c1c in incubator with 75% relative humidity ( rh ) . electrical conductivity using conductivity meter ( wtw cond-197i , germany ) , and ph using ph meter ( wtw ph-197i , germany ) of formulations were noted at various time intervals for 8 weeks ( 8) . the formula which was found to be most stable among different formulations and was selected for further studies is given in table 1 . q.s.= quantity sufficient this study was approved by the board of advanced studies and research , and its ethical committee for in - vivo studies ( reference no 3715/ acad . ) , of the islamia university of bahawalpur and was conducted according to the guidelines of helsinki declaration . a total of 11 male volunteers with mean age of 48 years were selected for the study and consent forms were taken . every volunteer applied creams at night on cheeks for the period of 12 weeks and came for measurement on 2nd , 4th , 6th , 8th , 10th and 12th week in morning at 10 a.m. they were allowed to wash their faces with water and , sit to become accustomed with the environment for 30 minutes before taking any measurements . values for different parameters were taken in controlled room temperature of 251c and 452% relative humidity . skin microrelief parameters were evaluated using visio scan vc98 ( courage and khazaka , germany ) . the skin moisture was determined with a skin capacitance meter ( corneometer mpa 5 ) and transepidermal water loss ( tewl ) was determined by an evaporimeter ( tewameter mpa 5 , courage and khazaka , germany ) . the percentage of changes with respect to initial values / zero hour of volunteers for different parameters , taken at 2nd basil leaves and flowers collected and identified at cholistan institute of desert studies of the islamia university of bahawalpur , pakistan ( voucher # oblf- 4 - 11 - 21 ) were shade dried and finely grounded . four hundreds gm of the powdered material was extracted at room temperature with 2000 ml of 95% ethanol for 48 hrs . then the glass beaker was sealed with aluminium foil and shaken for 10 min after every 12 hrs . it was filtered through a whatman # 01 filter paper , evaporated under reduced pressure at 40 c in a rotavapor ( eyela , co. ltd . japan ) till the concentrate was reduced to the one third of the initial volume of the solvent used and stored in freezer at 8 c in refrigerator till further analyses . the presence of phenolic compounds and flavonoids was tested according to the methods in the literature ( 6 ) . the antioxidant activity of the extract was determined using dpph ( 1,1-diphenil-2-picrylhydrazyl ) which is a stable free radical , and vitamin c as standard ( 7 ) . the antioxidant activity of basil extract was found to be 88% in comparison to the standard . different formulations containing different concentrations of parrafin oil ( merck , germany ) , abil em 90 ( franken chemicals germany ) and distilled water were prepared in this study . oil phase comprised of paraffin oil and surfactant ( abil em 90 ) heated up to 75c1c . aqueous phase comprising of water was heated to the same temperature and then treated with the extract . in the case of base no extract was added to the aqueous phase . w / o emulsions were prepared by addition of aqueous to the oily phases with continuous stirring first at 2000 rpm by the mechanical mixer ( euro - star , ika d 230 , germany ) for 15 min then 1000 rpm for 5 min , and thereafter 500 rpm for 5 minutes for complete homogenization by the mechanical mixer ( euro - star , ika d 230 , germany ) and finally the emulsion was cooled to room temperature . creams were divided into four parts and stability tests were performed at 8c0.1c in refrigerator ( dawlance , pakistan ) and at 25c1c , 40c1c and 40c1c in incubator with 75% relative humidity ( rh ) . electrical conductivity using conductivity meter ( wtw cond-197i , germany ) , and ph using ph meter ( wtw ph-197i , germany ) of formulations were noted at various time intervals for 8 weeks ( 8) . the formula which was found to be most stable among different formulations and was selected for further studies is given in table 1 . this study was approved by the board of advanced studies and research , and its ethical committee for in - vivo studies ( reference no 3715/ acad . ) , of the islamia university of bahawalpur and was conducted according to the guidelines of helsinki declaration . a total of 11 male volunteers with mean age of 48 years were selected for the study and consent forms were taken . every volunteer applied creams at night on cheeks for the period of 12 weeks and came for measurement on 2nd , 4th , 6th , 8th , 10th and 12th week in morning at 10 a.m. they were allowed to wash their faces with water and , sit to become accustomed with the environment for 30 minutes before taking any measurements . values for different parameters were taken in controlled room temperature of 251c and 452% relative humidity . skin microrelief parameters were evaluated using visio scan vc98 ( courage and khazaka , germany ) . the skin moisture was determined with a skin capacitance meter ( corneometer mpa 5 ) and transepidermal water loss ( tewl ) was determined by an evaporimeter ( tewameter mpa 5 , courage and khazaka , germany ) . the percentage of changes with respect to initial values / zero hour of volunteers for different parameters , taken at 2nd , 4th , 6th , 8th , 10th and 12th week were calculated . in this study , the ph of freshly prepared base and formulation were 5.59 and 5.68 respectively , which were within the range of skin ph ( 9 ) . by applying anova , it was found that the change in ph of different samples of base and formulation was not significant at different time intervals and temperature . the colors of freshly prepared base and active formulation were white and light green respectively . there was no change in color of any sample of base and formulation at different storage conditions . no change are in color may be attributed to different factors that related to emulsion stability including the components of oil phase , paraffin oil and abil- em90 which are colorless , transparent and non toxic liquids ( 10 ) . no electrical conductivity was found in any sample of base and formulation throughout the period of the study . this is because the emulsion is of w / o type and oil being the continuous phase contributes to no passage of current . the samples were stable at 8c , 25c , but slight phase separation in the sample of base occurred at 40c and 40c+75% rh on 56th day of observation whereas the formulation was stable . this may be due to the antimicrobial properties of basil which protects the emulsion from microbial contamination and degradation ( 11 ) . average ph values of base and formulation kept at 8c , 25c , 40c and 40c+75% rh for a period of 8 weeks . the skin moisture content and tewl were measured before application of creams ( 0 hour readings ) and then at 2nd , 4th , 6th , 8th , 10th and 12th week of study period . the percent of changes in the values for 11 volunteers were calculated and given in figures 1 and 2 respectively . percentage of change in skin moisture content after application of base and formulation percentage of change in tewl after application of base and formulation . in this study base improved the moisture content of the skin to some extent but there was regular increase after the application of formulation throughout the study period . the base showed irregular pattern in the values of tewl of skin but there was regular decrease in the skin tewl after application of the formulation throughout the study period . by applying anova two way analysis it was found that the base had insignificant and the formulation had significant ( p0.05 ) effects on moisture content and tewl with respect to time . by the use of paired sample t - test it was evident that significant differences in the moisture and tewl values were observed after application of formulation throughout the study period . the enhancement in the skin moisture content after application of formulation is due to flavonoids present in basil which implies that treatment with flavonoids result in increase of extracellular collagen . collagen synthesis ultimately leads to increased skin moisture and more elastic skin ( 12).tewl is the outward transmission of water through skin . the mechanism of tewl decline is not known but flavonoids intervene cutaneous blood flow which may contribute towards a better skin appearance ( 13 ) . the values for parameters volume and energy were taken by visioscan vc 98/ software sels 2000 before application of creams ( 0 hour readings ) and at 1st , 2nd and 3rd month of the study period . in this study the base had statistically insignificant ( p 0.05 ) effects at all reading intervals on the parameter volume which is the virtual amount of liquid ( mm ) needed to fill the depths in image . if the surface is smoother , less virtual liquid is required to fill the depths . the formulation also showed significant effects when paired sample t - test was applied for variation between the two creams . this shows skin smoothness as less rough skin would require less amount of virtual liquid ( 14 ) . energy parameter describes the level of hydration of the skin . in this study increase in the energy values for formulation percentage of changes are represented in figure 4 . by treatment with moisturizing and anti aging formulations , the energy values increased . which is supported by the values obtained for skin moisture by corneometer mpa 5 as highly hydrated elastic skin has higher energy values compared to the dry skin with less moisture ( 14 ) . the values of different sels parameters ser , sesc , sesm and sew measured by visioscan vc 98/ software sels 2000 before application of creams and at 1st , 2nd and 3rd months of the study period are given in table 3 . the percent changes in the values for 11 volunteers were calculated and given in figure 5 . percentage of change in mean vc 98 units of sels parameters after application of base and formulation . 1b ; base values after one month , 1f ; formulation values after one month , 2b ; base values after two months , 2f ; formulation values after two months , 3b ; base values after three months , 3f ; formulation values after three months sels parameters values ( meansd ) . ser ; skin roughness , sesc ; skin scaliness , sesm ; skin smoothness , sew ; skin wrinkles . sesm is the index of smoothness and is calculated from the mean width and depth of wrinkles . sesc is the index of scaliness of skin which shows the level of dryness of the skin . sew identifies aging including wrinkles and is calculated from the proportion of horizontal and vertical wrinkles ( 15 ) . in this study it was found that the base produced statistically insignificant ( p0.05 ) effects on the roughness parameter of skin and the formulation produced significant effects at all reading intervals when anova two ways analysis was performed . when paired sample t - test was applied , significant effects were observed for active formulation . gradual decrease in the values of roughness , scaliness , smoothness and wrinkles were observed for the formulation . the formulation showed decrease in mean values of skin smoothness in contrast to skin roughness which indicates that the formulation possess anti - aging properties . the smaller sesc value corresponds to higher skin moisture as treatment with moisturizing or antiaging formulations resulted in lower values for sesc ( 14 ) . lower values for the parameter sew indicate that there were less wrinkles present on the skin which indicates that the formulation reduced the fine wrinkles . this is directly related to the loss of collagen which has strong relation with transepidermal water loss . greater epidermal water loss leads to less water retained by the collagen and results in collagen degeneration ( 16 ) . the decrease in tewl as measured by tewameter support the development of collagen which ultimately lead to less wrinkles . the improvement in skin surface parameters can be attributed to the phenolic compounds and flavanoids present in basil which include quercetin , isoquercetin , kaempferol , caffeic acid , rosmarinic acid , rutin , catechin , ferulic acid , rutiniside and apigenin ( 17 ) . in this study , the ph of freshly prepared base and formulation were 5.59 and 5.68 respectively , which were within the range of skin ph ( 9 ) . by applying anova , it was found that the change in ph of different samples of base and formulation was not significant at different time intervals and temperature . the colors of freshly prepared base and active formulation were white and light green respectively . there was no change in color of any sample of base and formulation at different storage conditions . no change are in color may be attributed to different factors that related to emulsion stability including the components of oil phase , paraffin oil and abil- em90 which are colorless , transparent and non toxic liquids ( 10 ) . no electrical conductivity was found in any sample of base and formulation throughout the period of the study . this is because the emulsion is of w / o type and oil being the continuous phase contributes to no passage of current . the samples were stable at 8c , 25c , but slight phase separation in the sample of base occurred at 40c and 40c+75% rh on 56th day of observation whereas the formulation was stable . this may be due to the antimicrobial properties of basil which protects the emulsion from microbial contamination and degradation ( 11 ) . average ph values of base and formulation kept at 8c , 25c , 40c and 40c+75% rh for a period of 8 weeks . the skin moisture content and tewl were measured before application of creams ( 0 hour readings ) and then at 2nd , 4th , 6th , 8th , 10th and 12th week of study period . the percent of changes in the values for 11 volunteers were calculated and given in figures 1 and 2 respectively . percentage of change in skin moisture content after application of base and formulation percentage of change in tewl after application of base and formulation . in this study base improved the moisture content of the skin to some extent but there was regular increase after the application of formulation throughout the study period . the base showed irregular pattern in the values of tewl of skin but there was regular decrease in the skin tewl after application of the formulation throughout the study period . by applying anova two way analysis it was found that the base had insignificant and the formulation had significant ( p0.05 ) effects on moisture content and tewl with respect to time . by the use of paired sample t - test it was evident that significant differences in the moisture and tewl values were observed after application of formulation throughout the study period . the enhancement in the skin moisture content after application of formulation is due to flavonoids present in basil which implies that treatment with flavonoids result in increase of extracellular collagen . collagen synthesis ultimately leads to increased skin moisture and more elastic skin ( 12).tewl is the outward transmission of water through skin . the mechanism of tewl decline is not known but flavonoids intervene cutaneous blood flow which may contribute towards a better skin appearance ( 13 ) . the values for parameters volume and energy were taken by visioscan vc 98/ software sels 2000 before application of creams ( 0 hour readings ) and at 1st , 2nd and 3rd month of the study period . in this study the base had statistically insignificant ( p 0.05 ) effects at all reading intervals on the parameter volume which is the virtual amount of liquid ( mm ) needed to fill the depths in image . if the surface is smoother , less virtual liquid is required to fill the depths . the formulation also showed significant effects when paired sample t - test was applied for variation between the two creams . this shows skin smoothness as less rough skin would require less amount of virtual liquid ( 14 ) . energy parameter describes the level of hydration of the skin . in this study increase in the energy values for formulation percentage of changes are represented in figure 4 . by treatment with moisturizing and anti aging formulations , the energy values increased . which is supported by the values obtained for skin moisture by corneometer mpa 5 as highly hydrated elastic skin has higher energy values compared to the dry skin with less moisture ( 14 ) . the values of different sels parameters ser , sesc , sesm and sew measured by visioscan vc 98/ software sels 2000 before application of creams and at 1st , 2nd and 3rd months of the study period are given in table 3 . the percent changes in the values for 11 volunteers were calculated and given in figure 5 . percentage of change in mean vc 98 units of sels parameters after application of base and formulation . 1b ; base values after one month , 1f ; formulation values after one month , 2b ; base values after two months , 2f ; formulation values after two months , 3b ; base values after three months , 3f ; formulation values after three months sels parameters values ( meansd ) . ser ; skin roughness , sesc ; skin scaliness , sesm ; skin smoothness , sew ; skin wrinkles . sesm is the index of smoothness and is calculated from the mean width and depth of wrinkles . sesc is the index of scaliness of skin which shows the level of dryness of the skin . sew identifies aging including wrinkles and is calculated from the proportion of horizontal and vertical wrinkles ( 15 ) . in this study it was found that the base produced statistically insignificant ( p0.05 ) effects on the roughness parameter of skin and the formulation produced significant effects at all reading intervals when anova two ways analysis was performed . when paired sample t - test was applied , significant effects were observed for active formulation . gradual decrease in the values of roughness , scaliness , smoothness and wrinkles were observed for the formulation . the formulation showed decrease in mean values of skin smoothness in contrast to skin roughness which indicates that the formulation possess anti - aging properties . the smaller sesc value corresponds to higher skin moisture as treatment with moisturizing or antiaging formulations resulted in lower values for sesc ( 14 ) . lower values for the parameter sew indicate that there were less wrinkles present on the skin which indicates that the formulation reduced the fine wrinkles . this is directly related to the loss of collagen which has strong relation with transepidermal water loss . greater epidermal water loss leads to less water retained by the collagen and results in collagen degeneration ( 16 ) . the decrease in tewl as measured by tewameter support the development of collagen which ultimately lead to less wrinkles . the improvement in skin surface parameters can be attributed to the phenolic compounds and flavanoids present in basil which include quercetin , isoquercetin , kaempferol , caffeic acid , rosmarinic acid , rutin , catechin , ferulic acid , rutiniside and apigenin ( 17 ) . in this study , the ph of freshly prepared base and formulation were 5.59 and 5.68 respectively , which were within the range of skin ph ( 9 ) . by applying anova , it was found that the change in ph of different samples of base and formulation was not significant at different time intervals and temperature . the colors of freshly prepared base and active formulation were white and light green respectively . there was no change in color of any sample of base and formulation at different storage conditions . no change are in color may be attributed to different factors that related to emulsion stability including the components of oil phase , paraffin oil and abil- em90 which are colorless , transparent and non toxic liquids ( 10 ) . no electrical conductivity was found in any sample of base and formulation throughout the period of the study . this is because the emulsion is of w / o type and oil being the continuous phase contributes to no passage of current . the samples were stable at 8c , 25c , but slight phase separation in the sample of base occurred at 40c and 40c+75% rh on 56th day of observation whereas the formulation was stable . this may be due to the antimicrobial properties of basil which protects the emulsion from microbial contamination and degradation ( 11 ) . average ph values of base and formulation kept at 8c , 25c , 40c and 40c+75% rh for a period of 8 weeks . the skin moisture content and tewl were measured before application of creams ( 0 hour readings ) and then at 2nd , 4th , 6th , 8th , 10th and 12th week of study period . the percent of changes in the values for 11 volunteers were calculated and given in figures 1 and 2 respectively . percentage of change in skin moisture content after application of base and formulation percentage of change in tewl after application of base and formulation . in this study base improved the moisture content of the skin to some extent but there was regular increase after the application of formulation throughout the study period . the base showed irregular pattern in the values of tewl of skin but there was regular decrease in the skin tewl after application of the formulation throughout the study period . by applying anova two way analysis it was found that the base had insignificant and the formulation had significant ( p0.05 ) effects on moisture content and tewl with respect to time . by the use of paired sample t - test it was evident that significant differences in the moisture and tewl values were observed after application of formulation throughout the study period . the enhancement in the skin moisture content after application of formulation is due to flavonoids present in basil which implies that treatment with flavonoids result in increase of extracellular collagen . collagen synthesis ultimately leads to increased skin moisture and more elastic skin ( 12).tewl is the outward transmission of water through skin . the mechanism of tewl decline is not known but flavonoids intervene cutaneous blood flow which may contribute towards a better skin appearance ( 13 ) . the values for parameters volume and energy were taken by visioscan vc 98/ software sels 2000 before application of creams ( 0 hour readings ) and at 1st , 2nd and 3rd month of the study period . in this study the base had statistically insignificant ( p 0.05 ) effects at all reading intervals on the parameter volume which is the virtual amount of liquid ( mm ) needed to fill the depths in image . if the surface is smoother , less virtual liquid is required to fill the depths . the formulation also showed significant effects when paired sample t - test was applied for variation between the two creams . this shows skin smoothness as less rough skin would require less amount of virtual liquid ( 14 ) . energy parameter describes the level of hydration of the skin . in this study increase in the energy values for formulation percentage of changes are represented in figure 4 . by treatment with moisturizing and anti aging formulations which is supported by the values obtained for skin moisture by corneometer mpa 5 as highly hydrated elastic skin has higher energy values compared to the dry skin with less moisture ( 14 ) . the values of different sels parameters ser , sesc , sesm and sew measured by visioscan vc 98/ software sels 2000 before application of creams and at 1st , 2nd and 3rd months of the study period are given in table 3 . the percent changes in the values for 11 volunteers were calculated and given in figure 5 . percentage of change in mean vc 98 units of sels parameters after application of base and formulation . 1b ; base values after one month , 1f ; formulation values after one month , 2b ; base values after two months , 2f ; formulation values after two months , 3b ; base values after three months , 3f ; formulation values after three months sels parameters values ( meansd ) . ser ; skin roughness , sesc ; skin scaliness , sesm ; skin smoothness , sew ; skin wrinkles . sesm is the index of smoothness and is calculated from the mean width and depth of wrinkles . sesc is the index of scaliness of skin which shows the level of dryness of the skin . sew identifies aging including wrinkles and is calculated from the proportion of horizontal and vertical wrinkles ( 15 ) . in this study it was found that the base produced statistically insignificant ( p0.05 ) effects on the roughness parameter of skin and the formulation produced significant effects at all reading intervals when anova two ways analysis was performed . when paired sample t - test was applied , significant effects were observed for active formulation . gradual decrease in the values of roughness , scaliness , smoothness and wrinkles were observed for the formulation . the formulation showed decrease in mean values of skin smoothness in contrast to skin roughness which indicates that the formulation possess anti - aging properties . the smaller sesc value corresponds to higher skin moisture as treatment with moisturizing or antiaging formulations resulted in lower values for sesc ( 14 ) . lower values for the parameter sew indicate that there were less wrinkles present on the skin which indicates that the formulation reduced the fine wrinkles . this is directly related to the loss of collagen which has strong relation with transepidermal water loss . greater epidermal water loss leads to less water retained by the collagen and results in collagen degeneration ( 16 ) . the decrease in tewl as measured by tewameter support the development of collagen which ultimately lead to less wrinkles . the improvement in skin surface parameters can be attributed to the phenolic compounds and flavanoids present in basil which include quercetin , isoquercetin , kaempferol , caffeic acid , rosmarinic acid , rutin , catechin , ferulic acid , rutiniside and apigenin ( 17 ) . from the results of this study it appears that basil ( ocimum basilicum ) possesses potential antiaging properties when applied topically and stable topical emulsions containing basil extract can be formulated . the active formulation was found to have skin moisturizing effects as it increased skin moisture content . the decrease in volume , sels parameters and tewl showed that the formulation possess anti - wrinkle affects . this study also depicts that non invasive biophysical techniques are a valuable tool for assessment of anti aging effects of topical skin applications . furthermore the formulation showed no harmful effects and it can be used as cost effective topical anti - aging treatment . future studies are required to be conducted to un - reveal the anti - aging mechanism of basil constituents .

direct oral anticoagulants ( doacs ) , targeting activated coagulation proteases , have been recently introduced in therapeutics for stroke prevention in patients with nonvalvular atrial fibrillation , and prevention and treatment of venous thromboembolism ; they are currently in development for various other indications.1,2 doacs are an alternative to vitamin k antagonists ( vkas ) for mid- or long - term antithrombotic treatments . the main limitation of vkas is the fear of bleeding.3,4 the rate of major bleeding among long - term users of vkas is 1.5%5.2% per year , with a mortality rate from bleeding > 10% . in the recent past , > 40,000 patients included in the comparative arms of randomized controlled trials were administered vkas up to the conventional target of international normalized ratio ( inr ) between two and three , for at least 3 months . the rate of major bleeding , fatal bleeding , and intracranial hemorrhages in these patients were 4.64% , 0.52% , and 1.05% , respectively.5 in real life , it can be speculated that this risk is superior . vkas have several other limitations , including the need for laboratory monitoring , and in many patients , instability of anticoagulation , dietary and drug interactions , a slow onset of action , and a narrow therapeutic window . doacs that directly inhibit coagulation factor xa ( xabans ) or thrombin ( dabigatran ) have been developed to overcome these limitations . however , vkas will probably be present on the market still for long time , because : 1 ) they have been approved for use in a larger set of thrombotic conditions compared to doacs ; 2 ) they can be prescribed in patients with severe renal insufficiency ; and 3 ) dose adjustment using inr is possible when strong drug interference is anticipated . doacs have been developed without specific antidote on the basis of their relatively short half - life ( hours ) and on the assumption of improved safety compared to vkas . although doacs produced a modest reduction of major bleeding in randomized trials,1,6 which seems to be reproduced in real life,79 a rapid reversal strategy of the their anticoagulant effect is desirable.10 idarucizumab is the first marketed doac antidote targeting dabigatran . this review provides short insights on the target drug dabigatran and the mechanism of its action , current development , potential indications , and clinical utility of idarucizumab . dabigatran is the active metabolite of dabigatran etexilate ( pradaxa ; boehringer - ingelheim , ingelheim , germany).11 it is a small ( 471 da ) nonpeptidic inhibitor of thrombin ( factor iia ) , the key serine protease product of the common coagulation pathway of blood coagulation . dabigatran and its glucuronide metabolites bind thrombin reversibly with an affinity close to 700 pm , via a salt bridge that anchors the benzamidine moiety of dabigatran to a hydrophobic cavity in the active site of thrombin.11 dabigatran blocks thrombin s catalytic activity , but not the interaction of thrombin exosite with macromolecular substrates , including protease - activated receptors . after oral administration , dabigatran etexilate is rapidly hydrolyzed by nonspecific , ubiquitous esterases to the active metabolite.12 dabigatran etexilate , but not dabigatran , is a substrate for the efflux transporter p - glycoprotein . in healthy volunteers , peak plasma concentrations ( cmax ) of dabigatran and the anticoagulant effects dabigatran plasma concentrations decline in a biphasic manner characterized by a rapid distribution phase , followed by a prolonged terminal elimination phase , resulting in a mean plasma terminal half - life of 1214 hours , independent of dose . increased trough concentrations are observed in patients with moderate renal insufficiency ( creatinine clearance : < 50 ml / min ) and dabigatran is contraindicated in severe renal impairment ( creatinine clearance : < 30 ml / min ) . the pharmacodynamic profile is parallel to the pharmacokinetics,13 with a prolongation of the global coagulation tests ( prothrombin time or activated partial thromboplastin time [ aptt ] ) and thrombin time ( tt).13,14 dabigatran has been evaluated at fixed doses ( 110 or 150 mg bid ) without laboratory monitoring in large randomized clinical trials for the prevention of venous thromboembolism after total hip or knee replacement ( re - novate,15 re - model,16 re - mobilize),17 treatment of acute venous thromboembolism ( re - cover18 ) , and prevention of stroke and cardioembolism in nonvalvular atrial fibrillation ( re - ly19 ) before approval in these indications . idarucizumab is a humanized monoclonal antibody fab fragment designed for the reversal of dabigatran.20,21 a similar approach has been successfully used for the neutralization of other drugs ( digoxin , colchicine , and desipramine).22 monoclonal antibody obtained in mice immunized with dabigatran conjugated to carrier protein were selected , the fab fragment isolated , and a chimeric humanized fragment expressed in a mammalian cell line . idarucizumab binds in a 1:1 ratio to the small molecule dabigatran and its active glucuronide metabolites with high affinity ( 2 pm , ie , 350-fold higher affinity than thrombin ) , thereby preventing them from thrombin inhibition . the molecular mechanism of dabigatran capture into the cavity of the antibody binding site shares strikingly structural similarities with that of dabigatran binding to the active site of thrombin , raising the possibility that idarucizumab could mimic some of the effects of thrombin on its physiological substrates . this possibility was excluded in vitro and in vivo , and idarucizumab has no intrinsic anticoagulant or procoagulant effect and does not activate platelets.23 preclinical studies performed in rats and pigs pretreated with dabigatran have shown that idarucizumab not only normalizes prolonged thrombin - dependent clotting tests immediately after intravenous ( iv ) administration but also reduces excessive blood loss in a porcine trauma model.23 a pharmacokinetic study after iv bolus or 1-hour infusion of idarucizumab indicated that peak concentration ( cmax ) , obtained shortly after the end of administration , and total exposure increased proportionally with dose.24 the distribution volume at steady state was close to the blood volume ( 0.06 l / kg ) and idarucizumab was cleared from plasma with an initial half - life of about 45 minutes , mainly eliminated unchanged in the urine , as expected for a small protein ( 48 kda ) . preexisting antibodies cross - reacting with idarucizumab were observed in a small proportion of subjects randomized to placebo or test group , but there were no consistent antibody responses after idarucizumab administration . the treatment was well tolerated and no relevant adverse events were reported.24 a phase i trial performed on a randomized , double - blind , placebo - controlled design in healthy men , pretreated for 3 days with 220 mg dabigatran etexilate twice daily ( higher dose compared to therapeutic 150 mg bid ) , showed that idarucizumab reversed dabigatran - induced anticoagulation in a dose - dependent manner and was well tolerated , with no unexpected or clinically relevant safety concerns.25 similar results were obtained in the elderly and in subjects with renal impairment.26 importantly , this effect was immediate and sustained for at least 24 hours for idarucizumab doses equimolar to the body load of dabigatran , indicating that dabigatran redistribution from the tissues toward blood compartment was neutralized . as idarucizumab is much larger than dabigatran , the complex was cleared by the kidney at the same rate as the free protein . given these findings , a prospective cohort trial of idarucizumab was undertaken in patients treated with dabigatran with potential indications of urgent reversal ( reverse - ad clinicaltrials.gov number nct02104947).27 in the absence of an alternative to idarucizumab with high - quality evidence , it was unethical to randomly assign patients to receive a placebo . this study is continuing , but results of an interim analysis on the first 90 patients ( expected recruitment : 300 patients ) have been published.28 the primary objective of this study is to evaluate the safety and ability to reverse the anticoagulant effect of dabigatran . all patients were given 5 g of idarucizumab as two 50 ml iv infusions of 2.5 g at 15-minute intervals . this dose , which was not tested in phase ii studies , was chosen to be able to reverse the total body load of dabigatran that was associated with the 99th percentile of the dabigatran levels , measured in the re - ly trial.29 the study included two groups of patients : group a : overt , uncontrollable , or life - threatening hemorrhage ( n=51 ) ; and group b : patients who required surgery or other invasive procedures that could not be delayed for at least 8 hours and for which normal hemostasis was required ( n=39 ) . patient characteristics were representative of those included in clinical trials of dabigatran in fibrillation or venous thromboembolism and in clinical practice . the main result is that idarucizumab rapidly and completely reversed the anticoagulant effect of dabigatran in a majority of patients with elevated clotting times ( dilute tt or ecarin clotting time ) at baseline . a transient increase in total dabigatran in plasma , quasi - irreversibly bound to idarucizumab and without thrombin inhibitory effect , however , the rare patients with a baseline concentration above the 95th percentile ( > 600 ng / ml ) , corresponding to definite overdose,29 displayed a rebound within hours following the administration of idarucizumab.28 in contrast , the efficacy on clinical outcomes is difficult , if not impossible , to assess at this stage . in group a , this is due to the severity of the index event at enrollment and the confounding effect of intensive associated support therapy . in group b , a majority of patients underwent surgery or invasive procedures , with reports of normal intraoperative hemostasis , but the indications of surgery were heterogeneous and with variable risks of bleeding . a major limitation of the study , besides the lack of control group , was the small patient population . it is important to note that baseline concentrations of plasma dabigatran were relatively low ( median : 84 and 75 ng / ml ; first quartile [ q1 ] : 38 and 29 ng / ml , in groups a and b , respectively ) , corresponding to usual trough concentrations , in accordance with the time since the last intake of dabigatran being > 12 hours in two - thirds of patients.29 a judgment about the efficacy of an antidote given when the target drug is no longer present at a concentration relevant to bleeding is hazardous . thrombotic events were documented in five patients ( 5.5% ) , none of them receiving antithrombotic therapy at the time of event . these events occurred 226 days after administration , suggesting that they were not directly linked to idarucizumab . therefore , the results of interim analysis confirm that idarucizumab rapidly reverses the anticoagulant effect of dabigatran in the usual range of plasma concentrations , and there is no signal indicating a lack of effectiveness or unexpected adverse events . these results justify the continuation of the trial to this end . considering that the intermediate criterion ( reversion of anticoagulant effect ) has a strong rationale for being associated with improved hemostasis , idarucizumab has been approved in the us and europe under the name praxbind ( boehringer - ingelheim ) for the indications of reverse - ad.27 it is expected that this ultra - rapid approval would not make the enrollment in the reverse - ad28 clinical trial of patients with the same profile and indications more difficult . to date , idarucizumab is the only available antidote of dabigatran evaluated in a prospective trial . of note , idarucizumab is not a reversal agent for other licensed direct thrombin inhibitors ( bivalirudin and argatroban ) . idarucizumab is available as a ready - to - use solution in a package containing two 50 ml glass vials , each containing 2.5 g at a concentration of 50 mg / ml ( shelf life of 24 months at 2c8c ) . the first set of indications for rapid reversal of dabigatran is bleeding from critical sites ( intracranial , intraspinal , pericardial , intraocular , pulmonary , retroperitoneal , or intramuscular with compartment syndrome ) or persistent major bleeding despite local hemostatic measures ( gastrointestinal , gynecologic , or urologic ) . , the risk is consistently reduced by about one - half compared to vkas , irrespective of the type of doac , including dabigatran , and subgroups of patients.30 the incidence of fatal hemorrhage in the re - ly trial was 22/12,091 patients in the dabigatran arms ( annual rate : 0.09%/year vs 0.14%/year in the warfarin arm).31 the second set of indication is emergency surgery or other invasive procedure at high risk of bleeding.32 arbitrarily , in the reverse - ad trial,27 the maximal delay for patient inclusion between admission and intervention was 8 hours , and this is reproduced in the summary of product characteristics released in some countries . this should be mitigated for nonelective intervention that could be delayed for > 8 hours in patients with reduced drug clearance ( impairment of renal function , strong drug interference ) or evidence of overdose at admission . a potential indication is the reversal of dabigatran in patients with acute ischemic stroke before fibrinolytic therapy . as a majority of these patients have very low anticoagulant levels at admission , in contrast with those with hemorrhagic stroke , monitoring dabigatran concentration before reversal could be particularly valuable.33 these two examples raise the question of the utility of laboratory testing in critically ill patients . specific assays calibrated for each of the doacs , including dabigatran , have been developed and are now commercially available . they are equivalent to the reference method ( liquid chromatography - tandem mass spectrometry ) in a large range of concentrations > 50 ng / ml.34 they can be performed in a central laboratory in a rapid turnaround time , similar to that required for the measurement of inr used for the management of patients treated by vkas . when drug levels in plasma are low and unlikely to play a major role in bleeding , the administration of the antidote can be avoided . conversely , when higher drug levels are observed , the administration of the antidote is justified . a value of 50 ng / ml can be proposed for these thresholds , without definite proof of validity . finally , knowledge of drug concentration could help to adjust the dose of the antidote in patients with excessively high dabigatran levels . global coagulation tests ( prothrombin time or aptt ) are not reliable for monitoring doacs due to their low sensitivity , the lack of reagent standardization , a plateau effect of aptt at supratherapeutic concentrations , and alternative causes of prolongation in critically ill patients.13,14 however , if the drug concentration is unknown , as far as dabigatran is concerned , normal prothrombin time and aptt , provided these tests are performed with a sensitive reagent , indicate that the levels of dabigatran are probably low enough to perform an invasive act without additional risk due to the treatment . the tt is exquisitely sensitive to dabigatran , and samples may be unclottable at therapeutic levels as low as 50 ng / ml.35 a normal tt indicates an absence of dabigatran and could therefore be used to exclude the drug as the cause of hemorrhage . importantly , regular tt should not be confused with the dilute tt , which provides a measurable clotting time directly proportional to dabigatran concentrations over the therapeutic range . in reverse - ad , tt and dilute tt rapidly returned to the normal range after idarucizumab administration , indicating the complete absence of unbound dabigatran , but a rebound of tt and , to a lower extent , dilute tt was observed in the rare patients with overdose at baseline.28 therefore , although being a nonspecific test , tt can be of value for monitoring incomplete reversal or ongoing bleeding after idarucizumab . the current management of dabigatran - induced bleeding , trauma , or before an emergency invasive procedure is the matter of numerous , remarkably concordant , published guidelines or expert advices.3641 cessation of anticoagulant treatment and administration of activated charcoal , if intake is recent , are the first measures . supportive care is essential , with special attention to maintain renal perfusion and urine output , because dabigatran is mainly eliminated through the kidneys . because the half - life of dabigatran is short ( 1217 hours ) , a majority of bleeding episodes are managed by supportive methods alone . in mechanical methods to arrest bleeding , including compression , tamponade , surgery , and endoscopic or vascular embolization may be essential . however , if bleeding is life - threatening or is not controlled by these measures , an additional hemostatic agent should be considered . prothrombin complex concentrates ( pccs ) or activated prothrombin complex concentrate factor viii inhibitor bypassing activity ( feiba ; baxter international inc , deerfield , il , usa ) has been shown to improve dabigatran - induced coagulopathy in vitro and to be effective in animal models of bleeding , when given before or shortly after the induction of bleeding.4245 numerous case reports and short series indicate , with possible publication biases , that these agents are also effective in patients , and their use as first - line therapy in life - threatening hemorrhages and as second - line therapy in severe uncontrolled bleeding has been incorporated in all recommendations . however , these procoagulant agents have not been prospectively evaluated , and these recommendations are therefore of low level of evidence . in addition , before procoagulant agents are given to patients with a prothrombotic condition , the risk of thrombotic adverse events should be considered . intuitively , antidote availability is perceived as a major step toward the achievement of safer anticoagulant therapy . antidotes for doacs may reduce the fear of bleeding associated with oral anticoagulation and , as such , increase confidence to prescribe these drugs in relevant indications.46 however , it should be recalled that bleeding associated with vkas , for which a specific fast - acting antidote exists ( pccs ) and has been available for several years in europe and more recently in north america , remains a major concern . in the re - ly trial , the mortality associated with spontaneous intracerebral hemorrhage was ~50% , with no significant differences between the patients receiving dabigatran ( no antidote available at this time ) and those receiving warfarin ( pccs available).47 first , the mortality and morbidity due to a hemorrhage are , in part , attributable to underlying disorders ( vascular injury , complex coagulopathy , or other issues ) . for example , in a large randomized , placebo - controlled trial , treatment of spontaneous acute intracerebral hemorrhage with recombinant activated factor viia ( eptacog alfa or novoseven , novonordisk , bagsvaerd , denmark ) reduced growth of the hematoma but did not improve survival or functional outcome.48 added to the incompressible delay of patient referral , further in - hospital delay for access to treatment is frequent . in a recent multicenter observational study of medical practice , guideline - recommended administration of pccs and vitamin k was associated with reduced mortality in patients on vka therapy presenting with severe hemorrhage.49 particularly , in patients with intracerebral hemorrhage , a three - fold decrease in mortality was observed if vka reversal was initiated at the appropriate dose within 8 hours after admission . the management was largely heterogeneous between centers and only 38% of patients received the appropriate treatment , indicating the need for improved guideline adherence . finally , bleeding or an invasive act is associated with discontinuation of the anticoagulant for variable periods , which may be responsible for secondary thrombotic events . therefore , a perfect antidote is only a part , maybe not the leading one , of a global strategy for patient management in these critical conditions . idarucizumab constitutes a progressive step for the safety of patients treated by dabigatran but , as it will probably not address all patients who could benefit from it , it is far from being a panacea . emphasis should be put on good medical practice for the use of dabigatran itself : proper indications and respect of contraindications , appropriate dosing , regular control of renal function , avoiding unnecessary coadministration of interfering drugs , especially antiplatelet agents , and patient s education before prescribing the drug . the introduction of idarucizumab should be accompanied by a comprehensive effort to accelerate diagnostic procedures , improve in - hospital idarucizumab availability , as well as prepare and diffuse structured local protocols of pathways / algorithms for the management of patients admitted in overcrowded emergency departments.50 in this manner , the promise of idarucizumab will become a reality .

this was a prospective , open - label , multicenter , observational , postmarket study evaluating vyc-17.5l for the treatment of moderate to severe nlfs . assessments were conducted : ( 1 ) at baseline , ( 2 ) after initial treatment ( day 0 ) , ( 3 ) 2 weeks after initial treatment , ( 4 ) after top - up ( performed at day 14 , as needed ) , ( 5 ) 1 month after last treatment , ( 6 ) 9 months after last treatment , and ( 7 ) 12 months after last treatment . repeat treatment was offered to subjects at month 12 with a follow - up visit at 1 month ; however , this article will focus on the data collected from the initial and top - up treatments only . the study was registered at www.clinicaltrials.gov ( nct # 01680497 ) and approved by applicable human subjects institutional review boards . male and female subjects aged 18 years or older with moderate to severe nlfs were recruited at 3 european centers in germany and the netherlands between june 2012 and january 2013 . as this study was mainly descriptive in nature , a sample size power calculation was not performed . a conservative 15% dropout rate was assumed ; therefore , investigators aimed to enroll 70 subjects to retain at least 60 subjects . the vyc-17.5l package contained a syringe filled with 1 ml of product and two 30 g needles . to reflect real - world clinical practice , needle / cannula type , injected volume , injection technique , and massage after injection were at the investigators ' discretion and guided by the directions for use . fourteen days after the initial injection , subjects returned to the clinic for the first follow - up visit . the investigators subjectively assessed if optimum correction had been achieved or if top - up treatment was needed at this visit ; top - up treatment followed the same procedures as described for the initial treatment . follow - up visits occurred 14 days after the initial treatment , and 1 , 9 , and 12 months after the last treatment . investigators and subjects completed safety , effectiveness , and satisfaction questionnaires at all follow - up visits . the primary endpoint was to evaluate investigator - assessed mean change of nlf severity from baseline to month 12 using the validated 5-point photonumeric nlf severity scale ( nlfss ) ( data on file , allergan plc , 2012 ) . the nlfss scale is defined as 0 = none ( no wrinkle ) , 1 = mild ( shallow , just perceptible wrinkle ) , 2 = moderate ( moderately deep wrinkle ) , 3 = severe ( deep wrinkle , well - defined edges but not overlapping ) , and 4 = extreme ( very deep wrinkle , redundant fold , and overlapping skin ) . investigator - assessed mean change of nlf severity from baseline to all other follow - up visits were reported ( i.e. , after initial / top - up treatment , month 1 , and month 9 ) . investigators and subjects assessed satisfaction with the aesthetic outcome after the initial and top - up ( if performed ) treatments and at all follow - up visits on an 11-point scale with response options ranging from 5 ( definitely not ) to 5 ( definitely ) . the investigators were asked : ( 1 ) are you satisfied with the result ? , ( 2 ) was the result what you expected ? , and ( 3 ) do you think that the product gave a smooth finish ? mean change from baseline to month 1 , month 9 , and month 12 after last treatment was assessed for these subject - assessed satisfaction endpoints . at month 12 , subjects were asked to indicate whether they would like to be injected with the same product again and whether they would recommend the product to others . additionally , subjects indicated how long it took them to return to normal activities after receiving treatment ( < 24 hours , 13 days , 48 days , 914 days , or > 14 days ) . they were asked how they would rate the : ( 1 ) ease of injection , ( 2 ) extrusion force , and ( 3 ) moldability / malleability . response options ranged from 0 ( easy ) to 10 ( hard ) on an 11-point scale . statistical analyses were performed , and data appendices were created using sas version 9.2 ( cary , nc ) . comparisons from baseline to month 12 were performed using wilcoxon signed - rank test . all adverse events ( aes ) and serious adverse events ( saes ) judged to be more severe or more prolonged than routinely observed were recorded . after initial and top - up treatments , subjects reported pain , swelling , and bruising on an 11-point scale ranging from 0 ( none ) to 10 ( worst imaginable ) . male and female subjects aged 18 years or older with moderate to severe nlfs were recruited at 3 european centers in germany and the netherlands between june 2012 and january 2013 . as this study was mainly descriptive in nature , a sample size power calculation was not performed . a conservative 15% dropout rate was assumed ; therefore , investigators aimed to enroll 70 subjects to retain at least 60 subjects . the vyc-17.5l package contained a syringe filled with 1 ml of product and two 30 g needles . to reflect real - world clinical practice , needle / cannula type , injected volume , injection technique , and massage after injection were at the investigators ' discretion and guided by the directions for use . fourteen days after the initial injection , subjects returned to the clinic for the first follow - up visit . the investigators subjectively assessed if optimum correction had been achieved or if top - up treatment was needed at this visit ; top - up treatment followed the same procedures as described for the initial treatment . follow - up visits occurred 14 days after the initial treatment , and 1 , 9 , and 12 months after the last treatment . investigators and subjects completed safety , effectiveness , and satisfaction questionnaires at all follow - up visits . the primary endpoint was to evaluate investigator - assessed mean change of nlf severity from baseline to month 12 using the validated 5-point photonumeric nlf severity scale ( nlfss ) ( data on file , allergan plc , 2012 ) . the nlfss scale is defined as 0 = none ( no wrinkle ) , 1 = mild ( shallow , just perceptible wrinkle ) , 2 = moderate ( moderately deep wrinkle ) , 3 = severe ( deep wrinkle , well - defined edges but not overlapping ) , and 4 = extreme ( very deep wrinkle , redundant fold , and overlapping skin ) . investigator - assessed mean change of nlf severity from baseline to all other follow - up visits were reported ( i.e. , after initial / top - up treatment , month 1 , and month 9 ) . investigators and subjects assessed satisfaction with the aesthetic outcome after the initial and top - up ( if performed ) treatments and at all follow - up visits on an 11-point scale with response options ranging from 5 ( definitely not ) to 5 ( definitely ) . the investigators were asked : ( 1 ) are you satisfied with the result ? , subjects were asked : ( 1 ) do you feel attractive ? , ( 2 ) do you feel confident ? , and ( 3 ) do you feel comfortable in social situations ? mean change from baseline to month 1 , month 9 , and month 12 after last treatment was assessed for these subject - assessed satisfaction endpoints . at month 12 , subjects were asked to indicate whether they would like to be injected with the same product again and whether they would recommend the product to others . additionally , subjects indicated how long it took them to return to normal activities after receiving treatment ( < 24 hours , 13 days , 48 days , 914 days , or > 14 days ) . they were asked how they would rate the : ( 1 ) ease of injection , ( 2 ) extrusion force , and ( 3 ) moldability / malleability . response options ranged from 0 ( easy ) to 10 ( hard ) on an 11-point scale . statistical analyses were performed , and data appendices were created using sas version 9.2 ( cary , nc ) . all adverse events ( aes ) and serious adverse events ( saes ) judged to be more severe or more prolonged than routinely observed were recorded . after initial and top - up treatments , subjects reported pain , swelling , and bruising on an 11-point scale ranging from 0 ( none ) to 10 ( worst imaginable ) . investigators enrolled 70 subjects in the study ( 94% female , mean age : 49 10 , age range : 2671 ) ( table 2 ) . three subjects had major protocol deviations and 2 were lost to follow - up ( figure 1 ) ; these subjects were not included in the 12-month effectiveness or satisfaction endpoint analyses . all enrolled subjects were included in the safety analyses ( i.e. , any adverse event that occurred before loss to follow - up was included in the safety analyses ) . eleven subjects did not complete the 12-month questionnaire because of a clerical data collection error . seventy subjects received initial treatment at baseline , and 40% ( n = 28 ) received top - up treatment at day 14 . table 3 shows the treatment characteristics for both the right and left nlfs combined . injected volume to achieve optimum correction , including both initial and top - up treatment , ranged between 2.0 and 6.0 ml ( mean = 3.0 1.0 ml ) for both nlfs . at initial treatment , retrograde tunneling other mainly included the depot technique ( injection into one single plane ) , the tower technique ( injection through a perpendicular approach to the deep tissue plane with a gradual tapering of product deposition as the needle is withdrawn ) , and the vertical supraperiosteal depot technique ( small aliquots of filler injections deposited on the periosteum for maximum support ) . a majority of needles used for initial treatment were cannulas ( 73% ) ( 27 g 38 mm [ n = 7 ] , 27 g 25 mm [ n = 21 ] , and 25 g 50 mm [ n = 23 ] ) ; whereas for top - up treatment , standard 30 g needles were mainly used ( 93% ) . initial and top - up treatment characteristics from baseline to month 12 , there was significant improvement in mean nlf severity for the right ( mean change : 0.8 0.7 ) and left ( mean change : 0.8 0.8 ) nlfs ( p < .0001 for both nlfs ) as assessed by the investigator ( figure 2 ) . significant differences between baseline and each time point are shown ( p < .001 * * * ) . investigators reported significant improvement in mean nlf severity from baseline to all other time points assessed ( i.e. , after last treatment , month 1 , and month 9 ) ( figure 2 ) . subject - assessed mean nlfss scores were similar to the investigator - assessed results at all time points . in addition , investigator and subject - assessed responder rates were high at month 1 and month 9 ( table 4 ) . at month 12 , responder rates slightly decreased , but the majority of subjects still had 1 point improvement in their nlfs . investigator and subject - assessed responder rates ( n = 67 ) * investigators and subjects indicated high satisfaction with the treatment at all follow - up visits . mean investigator scores for the question are you satisfied with the aesthetic outcome ? ranged from 4.0 1.0 to 4.6 0.6 ( scores could range from 5 = definitely not to 5 = definitely ) . at baseline , most subjects felt somewhat attractive , confident , and comfortable in social situations , and these scores significantly increased from baseline to all follow - up visits ( figure 3 ) . were significantly higher at all follow - up visits except at month 9 . additionally , the majority of subjects ( 82.1% , n = 67 ) needed less than 24 hours to return to normal activities after receiving treatment . only 4 subjects indicated that they needed 4 to 8 days to return to normal activities . significant differences were assessed between baseline and each time point as appropriate ( p < .05 * , after initial treatment , the mean scores were as follows : ease of injection = 1.2 1.3 , extrusion force = 1.2 1.4 , and ease of molding / malleability = 1.1 1.1 ( scores could range from 0 = easy to 10 = hard ) . all questions at top - up treatment had a mean score of 0.9 0.5 . mean scores for subject - reported pain , swelling , and bruising were generally low after both initial and top - up treatments ( results could range from 0 = none to 10 = worst imaginable ) . pain was rated as 2.0 1.8 for both sides after initial treatment and decreased to 0.8 1.7 ( right ) and 0.7 1.5 ( left ) after day 14 . pain at top - up was slightly lower : 1.5 1.8 ( right ) and 1.6 1.8 ( left ) . swelling was rated as 1.7 1.6 for both sides after initial treatment and decreased to 1.5 2.2 ( right ) and 1.4 2.2 ( left ) after day 14 . swelling at top - up was similar to initial treatment : 1.8 1.4 ( right ) and 1.9 1.5 ( left ) . bruising was rated as 0.3 0.9 for both sides after initial treatment and slightly increased to 0.9 1.9 ( right ) and 0.5 1.3 ( left ) after day 14 . bruising at top - up was similar to initial treatment : 0.4 0.8 ( right ) and 0.5 0.9 ( left ) . one subject reported redness around the mouth , swelling , and decreased sensitivity 1 day after receiving 1 ml of the product . the second ae was described as swelling in the injection area that began 1 hour after receiving 2 ml of the product . this subject had been diagnosed with urticaria in the past and should have been excluded from participation in the study , but he / she had not informed the investigator of this diagnosis . investigators enrolled 70 subjects in the study ( 94% female , mean age : 49 10 , age range : 2671 ) ( table 2 ) . three subjects had major protocol deviations and 2 were lost to follow - up ( figure 1 ) ; these subjects were not included in the 12-month effectiveness or satisfaction endpoint analyses . all enrolled subjects were included in the safety analyses ( i.e. , any adverse event that occurred before loss to follow - up was included in the safety analyses ) . eleven subjects did not complete the 12-month questionnaire because of a clerical data collection error . seventy subjects received initial treatment at baseline , and 40% ( n = 28 ) received top - up treatment at day 14 . table 3 shows the treatment characteristics for both the right and left nlfs combined . injected volume to achieve optimum correction , including both initial and top - up treatment , ranged between 2.0 and 6.0 ml ( mean = 3.0 1.0 ml ) for both nlfs . at initial treatment , retrograde tunneling other mainly included the depot technique ( injection into one single plane ) , the tower technique ( injection through a perpendicular approach to the deep tissue plane with a gradual tapering of product deposition as the needle is withdrawn ) , and the vertical supraperiosteal depot technique ( small aliquots of filler injections deposited on the periosteum for maximum support ) . a majority of needles used for initial treatment were cannulas ( 73% ) ( 27 g 38 mm [ n = 7 ] , 27 g 25 mm [ n = 21 ] , and 25 g 50 mm [ n = 23 ] ) ; whereas for top - up treatment , standard 30 g needles were mainly used ( 93% ) . from baseline to month 12 , there was significant improvement in mean nlf severity for the right ( mean change : 0.8 0.7 ) and left ( mean change : 0.8 0.8 ) nlfs ( p < .0001 for both nlfs ) as assessed by the investigator ( figure 2 ) . significant differences between baseline and each time point are shown ( p < .001 * * * ) . investigators reported significant improvement in mean nlf severity from baseline to all other time points assessed ( i.e. , after last treatment , month 1 , and month 9 ) ( figure 2 ) . subject - assessed mean nlfss scores were similar to the investigator - assessed results at all time points . in addition , investigator and subject - assessed responder rates were high at month 1 and month 9 ( table 4 ) . at month 12 , responder rates slightly decreased , but the majority of subjects still had 1 point improvement in their nlfs . investigators and subjects indicated high satisfaction with the treatment at all follow - up visits . mean investigator scores for the question are you satisfied with the aesthetic outcome ? ranged from 4.0 1.0 to 4.6 0.6 ( scores could range from 5 = definitely not to 5 = definitely ) . at baseline , most subjects felt somewhat attractive , confident , and comfortable in social situations , and these scores significantly increased from baseline to all follow - up visits ( figure 3 ) . were significantly higher at all follow - up visits except at month 9 . additionally , the majority of subjects ( 82.1% , n = 67 ) needed less than 24 hours to return to normal activities after receiving treatment . only 4 subjects indicated that they needed 4 to 8 days to return to normal activities . significant differences were assessed between baseline and each time point as appropriate ( p < .05 * , p < .01 * * , p < .001 * * * ) . after initial treatment , the mean scores were as follows : ease of injection = 1.2 1.3 , extrusion force = 1.2 1.4 , and ease of molding / malleability = 1.1 1.1 ( scores could range from 0 = easy to 10 = hard ) . all questions at top - up treatment had a mean score of 0.9 0.5 . mean scores for subject - reported pain , swelling , and bruising were generally low after both initial and top - up treatments ( results could range from 0 = none to 10 = worst imaginable ) . pain was rated as 2.0 1.8 for both sides after initial treatment and decreased to 0.8 1.7 ( right ) and 0.7 1.5 ( left ) after day 14 . pain at top - up was slightly lower : 1.5 1.8 ( right ) and 1.6 1.8 ( left ) . swelling was rated as 1.7 1.6 for both sides after initial treatment and decreased to 1.5 2.2 ( right ) and 1.4 2.2 ( left ) after day 14 . swelling at top - up was similar to initial treatment : 1.8 1.4 ( right ) and 1.9 1.5 ( left ) . bruising was rated as 0.3 0.9 for both sides after initial treatment and slightly increased to 0.9 1.9 ( right ) and 0.5 1.3 ( left ) after day 14 . bruising at top - up was similar to initial treatment : 0.4 0.8 ( right ) and 0.5 0.9 ( left ) . one subject reported redness around the mouth , swelling , and decreased sensitivity 1 day after receiving 1 ml of the product . the second ae was described as swelling in the injection area that began 1 hour after receiving 2 ml of the product . this subject had been diagnosed with urticaria in the past and should have been excluded from participation in the study , but he / she had not informed the investigator of this diagnosis . this is the first postmarket study to evaluate vyc-17.5l for the treatment of moderate to severe nlfs . the primary endpoint analysis revealed that vyc-17.5l provides a long - lasting filling effect of nlfs up to 1 year after treatment . this sustained correction may be attributed to the crosslinking of low molecular weight ha used in the vyc-17.5l formulation , a manufacturing process that creates a gel with a high level of resistance to degradation . in addition , investigators indicated that vyc-17.5l was easy to use ( i.e. , ease of injection , extrusion force , moldability / malleability ) . this may be because the vyc-17.5l formulation also contains high molecular weight ha , which contributes to cohesivity and malleability . the combination of low and high molecular weight ha in vyc-17.5l produces a gel with rheological properties suited to treat facial wrinkles and folds such as nlfs . subject - assessed mean scores for feeling attractive , confident , and comfortable in social situations significantly increased at all time points after treatment compared with baseline . these results are consistent with previous research indicating that treatment with a dermal filler for correction of nlfs improves psychosocial outcomes , including quality of life and self - esteem . at all follow - up visits , the majority of subjects indicated that they would like to be injected with the product again and also would recommend the product to others . most subjects returned to normal activities within 24 hours after treatment , indicating that the treatment had only a minimal impact on their daily routine . results indicate that pain , swelling , and bruising after the treatments were generally low . the most likely reason for the low pain scores is that the vyc-17.5l formulation includes lidocaine . although subject assessment of pain , swelling , and bruising showed that expected injection responses did occur , only 2 events were considered abnormal and reported as aes . one patient , who had a history of urticaria that was not disclosed during screening , had swelling in the injection area ; and the second patient had redness around the mouth with swelling . this study assessed the safety and effectiveness of vyc-17.5l across multiple time points using numerous investigator and patient - assessed outcomes . although this study enrolled participants across a wide age range , only 6% were men and most were fitzpatrick skin type ii and iii . this study had a high subject retention rate ( 97% ) , with only 2.9% lost to follow - up ; however , satisfaction data from 11 subjects were lost due to a clerical error . as this was the first postmarket study to assess vyc-17.5l , an open - label design was used , which may introduce the possibility of certain biases . vyc-17.5l is highly effective in treating moderate to severe nlfs with results lasting up to 1 year . in addition , vyc-17.5l was well - tolerated by subjects , investigators rated the product as easy to use , and investigator and subjects were highly satisfied with the aesthetic outcome through 1 year . these results can be attributed to the proprietary formulation and manufacturing process of vyc-17.5l that provides the appropriate rheological properties needed to treat nlfs . as fillers are uniquely suited to treat specific indications and regions of the face , aesthetic clinicians can use the results of this study to help create tailored treatment algorithms and long - term treatment plans to attain optimal treatment outcomes for their patients .

patients with hemiparesis due to stroke tend to use the non - affected side of the body rather than the affected side1 . therefore , muscle weakness in the upper and lower extremities of the affected side increases , and asymmetric body alignment and weight shift can occur2 . improvement of independent walking ability is an important criterion for functional recovery , as well as one of the aims of treatment4 , 5 . the affected side lower extremity muscle weakness in hemiparetic patients plays an important role in the restriction of walking ability6 , 7 . muscle strengthening exercises for the lower extremities , especially strengthening the extensor muscle in the knee joint , is very effective in the improvement of balance during walking , walking speed , and walking distance8 . among the lower extremity muscles , the quadriceps femoris muscle is important for dynamic stability in a standing position , while the gastrocnemius muscle is very important in the generation of vertical ground reaction force for push - off during walking9 , 10 . therefore , muscle - strengthening exercises using body weight have typically been used to improve muscle strength in the lower extremities in clinical practice . among them , squatting is the most appropriate exercise method to strengthen the quadriceps femoris , making it ideal to strengthen lower extremity muscles for patients with functional problems due to damage to the neuromusculoskeletal system5 . tang et al . reported that the vastus medialis muscle improved after using closed kinetic chain ( ckc ) exercises , such as squatting11 . in particular , squatting performed at a knee joint angle between 45 and 60 showed the most effective result . furthermore , very few studies have been done on changes in muscle activity of the lower extremities in the squatting position in conjunction with changes in ankle joint angle in a variety of ground environments . the purpose of this study was to examine the effect of changes in ground tilt angle during squatting on the affected lower extremity muscle activity of hemiparetic patients , and the results of this study would be used as foundational data for rehabilitation programs for lower extremity muscle strengthening in hemiparetic patients . chronic hemiparetic patients ( n=15 ) with at least 6 months elapsed after being diagnosed with stroke participated in this study after giving informed consent . for the criteria to select the subjects , three main points were considered . the selected subjects had to be able to walk independently with assistive devices and had to have a score of more than 5 on the walk item of the functional independence measure ( fim ) , they had to be able to understand and perform the research instructions with a score of more than 25 on the mini - mental state examination ( mmse ) , and they had to have no orthopedic disease in both lower extremities ( table 1table 1 . general characteristics of the subjectscharacteristics(n=15)gendermale8 ( 53)female7 ( 47)age ( yr)67.1313.30height ( cm)161.475.32weight ( kg)59.679.06bmi ( kg / m)22.802.64stroke typeinfarction8 ( 53)hemorrhage7 ( 47)paretic sideleft9 ( 60)right6 ( 40)masgrade 00 ( 0)grade 112 ( 80)grade 1 + 3 ( 20)duration ( mo.)17.7312.83mmse - k27.122.81n ( % ) , meansd , bmi : body mass index , mas : modified ashworth scale , mmse - k : mini - mental state examination - korea ) . all of the protocols used in this study were approved by the university of daejeon . before participation , the procedures , risk , and benefits were explained to all the participants , who gave their informed consent . the participants rights were protected according to the guidelines of the university of daejeon . n ( % ) , meansd , bmi : body mass index , mas : modified ashworth scale , mmse - k : mini - mental state examination - korea to measure extensor muscle activity in the affected lower extremity , a qemg-4 ( lxm3204 , laxtha , daejeon , south korea ) , a 4-channel surface electromyogram ( semg ) system , was used . the collected data were then analyzed with the telescan 2.89 electromyogram software ( laxtha , daejeon , south korea ) . for the surface electrode , an ag / agcl electrode ( 3 m , st . paul , mn , usa ) with a sampling rate of 1024 hz was used . signals were processed through a band - pass filter of 20450 hz and a notch filter of 60 hz . for the semg electrode position , electrodes were attached to the vastus medialis , vastus lateralis , gastrocnemius medialis , and gastrocnemius lateralis with the proposed seniam project method ( surface electromyography for the non - invasive assessment of muscles , 2005 ) . during squatting , the semg signal was collected in five - second segments , and the measurement was done three times for each position . the semg data obtained in the first and last one second were removed , and only the data for the three seconds between the first and last seconds were transformed into root mean square ( rms ) values and recorded . the experiment was done at three positions ( 15 ankle joint plantar flexion , neutral , and 15 dorsiflexion ) and using our own manufactured quadriceps board . for squatting , the knee joint was positioned at 45 of flexion , using a goniometer while the legs were placed shoulder - width distance from each other and the hands were placed freely . to shift weight to the affected side during squatting , a posture mirror , on which a line was drawn , was used . a sufficient amount of practice was performed to ensure that the same experimental posture would be used during measurement . measurement in each experiment was done three times , and the order of the experiment was assigned randomly . to reduce muscle fatigue , spss version 18.0 for windows was used for statistical analysis . for general characteristics of the subject one - way repeated measures anova was used to compare the lower extremity muscle activity according to the ground tilt angle , and the post hoc bonferroni correction was applied . the muscle activity in the affected lower extremity during squatting according to the three positions ( 15 ankle joint plantar flexion , neutral , and 15 dorsiflexion ) is shown in table 2table 2 . comparison of electromyography activation according to ground tilt angle ( n=15)muscleground tilt angle15015vastus medialis51.8031.0648.2528.7946.7427.97vastus lateralis*62.6734.4554.8929.4154.7430.83gastrocnemius medialis21.147.0618.654.1419.4110.61gastrocnemius lateralis*29.099.4728.2110.5022.818.39mean ( % rms)sd , * p<0.05 . for the vastus medialis , the muscle activity during squatting was the highest at 51.80% for 15 plantar flexion and the lowest at 46.74% for 15 dorsiflexion . for the vastus lateralis , the highest muscle activity was 52.67% at 15 plantar flexion and the lowest was 54.74% at 15 dorsiflexion . for the gastrocnemius medialis , the highest muscle activity was 21.14% at 15 plantar flexion , and the lowest was 18.65% at 0 neutral position . for the gastrocnemius lateralis , the highest muscle activity was 29.09% at 15 plantar flexion and the lowest was 22.81% at 15 dorsiflexion . ckc exercises , like squatting , can cause co - contraction of the quadriceps femoris and hamstring and reduce anterior - posterior tibiofemoral translation . since squatting facilitates selective contraction of the vastus medialis compared with open kinetic chain ( okc ) exercise , it also has been used frequently for knee joint rehabilitation11 . the alignment and movement of the patella is determined by the amount and direction of force applied to the soft tissues around the knee joint ; in particular , the vastus medialis acts as a medial stabilizing muscle for the patella12 . contraction of the vastus medialis and rectus femoris can cause medial tilting and lateral rotation of the patella13 . the function of the patella is lateral tilt and rotation during knee extension from 0 to 20 in the ckc14 and lateral tilt and rotation during squatting from 0 to 9015 . furthermore , the patella is moved medially during the initial period of squatting between 0 and 20 and then laterally above a knee flexion angle of about 4516 . to determine the patella movement and the q - angle during knee joint movement , analysis was done using a three - dimensional motion analysis system , and the results showed that the in addition to the increase in q - angle , the patella was tilted and rotated laterally up to 45 of knee flexion15 . therefore , it caused increasing activation of the vastus lateralis , thereby producing lateral tilt of the patella . although measurement of the lateral tilt of the patella was not done in this study , a significant change in the vastus lateralis occurred in the above study . experiments with healthy subjects showed that when the ankle was moved , the lack of stability of the knee joint caused co - contraction of the vastus medialis and vastus lateralis11 . when a squat extension exercise was performed on flat however , when the ankle joint tilt was applied to the heel , the activation of both the quadriceps femoris and knee extension increased . knee extension and ankle plantar flexion were used at the same time to lift the center of gravity , and squatting might be facilitated by reducing fatigue of the quadriceps femoris through activation of the gastrocnemius16 . the results of the above study were similar to the measurement results of this study in that activation of the affected gastrocnemius showed the highest at 15 plantar flexion during squatting . however , cook et al . showed that the effect on the gastrocnemius was at the minimum during squatting at 25 to 30 on a descending ramp17 . however , the significant change in the affected gastrocnemius lateralis during squatting , performed when the ground tilt angle was 15 plantar flexion , was thought to be affected by the increase in activation of the vastus lateralis and the lateral tilting of the patella up to 45 of knee joint flexion in the q - angle analysis and during patella movement , as reported by wilson et al15 . based on the previous studies showing that changes in ground tilt angle during squatting can influence stability and lower extremity muscle activity , this study also compared the semg activation of the quadriceps femoris and gastrocnemius in hemiparetic patients . more activation of the vastus lateralis and gastrocnemius lateralis was found in the squatting position when the ankle joint was in the plantar flexion condition , while inhibition was found in the squatting position when the ankle joint was in the dorsiflexion condition . therefore , the results suggest that it is more effective for a stroke patient in the initial phase , as characterized by having the problem of spasticity of the ankle plantar flexor , to apply squatting on the ground in the dorsiflexion condition rather than on flat ground for the purpose of inhibition . on the other hand , it is more effective to apply squatting on the ground in the ankle joint plantar flexion condition rather than on flat ground to increase overall muscle strength of the lower extremities .

there were 37 men and 208 women ; overall mean age was 64.3 years ( range , 50 to 76 years ) . the mean follow - up period was 2.8 years ( range , 1 to 8 years ) ( table 1 ) . for all patients , arthroscopy was performed before the procedure for accurate selection of uka candidates . during the arthroscopic evaluations , patients with torn anterior cruciate ligaments or with articular cartilage defects higher than grade 3 or 4 ( outerbridge classification ) in the lateral compartment were excluded . patients with previous high tibial osteotomy , a femorotibial angle of more than valgus 15 or varus 10 , lateral unicompartmental replacement , and ligamentous laxity and instability were also excluded . all uka procedures were performed with oxford uni ( biomet ltd . , bridgent , uk ) , and were carried out by the two senior authors . postoperative complications were defined as implant - related problems , such as loosening of the components and soft tissue problems developed after uka , including cases that underwent revision operation . the range of motion and clinical outcomes , such as knee society score ( kss ) and hospital for special surgery ( hss ) knee scores , were evaluated at final follow - up . in a total of 246 cases , complications were developed in 24 cases ( 9.8% ) . they occurred in 5 men and 19 women , with a mean age of 65.2 years ( range , 51 to 76 years ) ( table 1 ) . intraoperative complications were seen in 6 cases , while postoperative complications occurred in 18 cases ( table 2 ) . no definite complications of infection or deep vein thrombosis were observed in any of the cases . intraoperative complications were developed in a total of 6 cases ( 1 male , 5 female ) . the following intraoperative complications were observed : fracture of the medial tibial condyle during operation ( 1 case ) , intercondylar eminence fracture ( 2 cases ) , rupture of the medial collateral ligament ( 1 case ) , tka conversion of large bony defect of tibial avascular necrosis ( 1 case ) , and widening of the peg hole and mismatch of the posterior femoral condyle cutting level leading to femoral component malposition ( 1 case ) . the malpositioning of the femoral component eventually caused loosening of the femoral component ( table 2 ) . these postoperative complications were observed from 2 months to 7 years , postoperatively ( mean duration , 24.6 months ) . the following postoperative complications were observed : aseptic loosening of the femoral component ( 4 cases ) , soft tissue impingement due to malalignment ( 1 case ) , polyethylene bearing dislocation ( 9 cases ) and suprapatellar bursitis ( 1 case ) , periprosthetic fracture ( 1 case ) , irritation of the medial joint line due to overhanging of the tibial components by more than 5 mm ( 1 case ) , and an unexplained cause ( 1 case ) . the most common complications were polyethylene bearing dislocation ( 9 cases ) and aseptic loosening of the femoral components ( 4 cases ) . revision procedures were as follows : change of femoral component ( 1 case ) , conversion to tka ( 8 cases ) , change of bearing components ( 7 cases ) , bursectomy of the suprapatellar bursa ( 1 case ) , and minimally invasive osteosynthesis ( 1 case ) . the mean time until reoperation was 22.3 months ( range , 2 to 106 months ) ( table 2 ) . polyethylene bearing dislocation was developed in 9 cases ( 3.6% ) , including three males and 6 females ( mean age , 61.4 years ; range , 54 to 70 years ) . this complication was developed most commonly at the average time of 3.7 months ( range , 2 to 7 months ) after surgery . dislocated bearings were replaced with bearing components that were about 2 - 3 mm larger . for the cases which were accompanied with component loosening , revision tka or uka was performed . after change of the bearing components , the tibiofemoral angle was increased from valgus 6.5 2.3 to valgus 8.5 2.7. in one case , the 3-mm dislocated bearing was replaced with an 8-mm thick bearing , after which the valgus angle was increased from 8.5 to 12.6 ( fig . the patient complained of mild posterolateral knee discomfort , but her knee function was good . the valgus angle was found to have decreased at follow - up , which was suspected to be due to adaptation to the ligament tension . interestingly , one posterior bearing dislocation recurred again after revision of the dislocation , but spontaneous reduction of the dislocated bearing was developed ( fig . that patient was very satisfied with the clinical results without any problems at the 2-year follow - up . in cases for which only a bearing change was performed , the radiological parameters such as preoperative average tibiofemoral angle , varus / valgus angle and flexion / extension angle ( femur ) , and varus / valgus angle and posterior tilt angle ( tibia ) were measured . in all cases , the average preoperative tibiofemoral angle was valgus 5.4 2.3. the tibiofemoral angle in the revision group was larger than that of the group without revision , but there were no significant differences between the two groups ( p = 0.730 ) ( table 3 ) . varus / valgus angle ( femur component ) was measured as valgus 6.5 4.0 ; flexion / extension angle ( femur component ) : flexion 5.1 5.2 ; varus / valgus angle ( tibia component ) : valgus 3.1 3.8 ; and posterior tilt ( tibia component ) angle : 6.5 4.7. there were no significant differences between these radiologic parameters of the normal and revision ( bearing change ) cases . in the follow - up radiologic evaluations , component loosening was found in 4 cases , 1 male and 3 female , all of which the loosening was developed in the femoral component . the mean age was 64.2 12.8 years ( range , 51 to 78 years ) . the mean time until revision surgery was 44.8 months ( range , 4 to 106 months ) . a revision of the femoral component was performed in 1 case , and tka conversion was performed in 3 cases . in this subgroup of femoral component loosening ( 4 cases ) , the varus / valgus angle ( femur ) was measured as valgus 9.1 2.7. in the case of conversion to tka , a 5-mm overhang of the medial compartment caused irritation of the medial aspect of the knee , resulting in cellulitis - like symptoms . we have experienced several cases of tibial component overhang , owing to the smaller posteromedial tibia size of the korean population . however , in this case , conservative treatment such as rest and intravenous antibiotics was performed , but the pain continued . it was supposed that the cause was irritation of the bursa after intra - articular bleeding from the uka procedure . one case of soft tissue impingement due to malalignment , and one case of unexplained severe pain also resulted in tka conversion ( fig . 5 ) . one periprosthetic fracture occurred in the distal femur , after which minimal invasive osteosynthesis was performed using the plate . at the final follow - ups , the overall hss score had improved from an average of 70 points preoperatively to a final score of 97 points . in the complication group , the mean kss ( pain ) and functional score of all the patients improved from 65 and 60 preoperatively to final scores of 87 and 82 , respectively . at the last follow - up , intraoperative complications were developed in a total of 6 cases ( 1 male , 5 female ) . the following intraoperative complications were observed : fracture of the medial tibial condyle during operation ( 1 case ) , intercondylar eminence fracture ( 2 cases ) , rupture of the medial collateral ligament ( 1 case ) , tka conversion of large bony defect of tibial avascular necrosis ( 1 case ) , and widening of the peg hole and mismatch of the posterior femoral condyle cutting level leading to femoral component malposition ( 1 case ) . the malpositioning of the femoral component eventually caused loosening of the femoral component ( table 2 ) . these postoperative complications were observed from 2 months to 7 years , postoperatively ( mean duration , 24.6 months ) . the following postoperative complications were observed : aseptic loosening of the femoral component ( 4 cases ) , soft tissue impingement due to malalignment ( 1 case ) , polyethylene bearing dislocation ( 9 cases ) and suprapatellar bursitis ( 1 case ) , periprosthetic fracture ( 1 case ) , irritation of the medial joint line due to overhanging of the tibial components by more than 5 mm ( 1 case ) , and an unexplained cause ( 1 case ) . the most common complications were polyethylene bearing dislocation ( 9 cases ) and aseptic loosening of the femoral components ( 4 cases ) . revision procedures were as follows : change of femoral component ( 1 case ) , conversion to tka ( 8 cases ) , change of bearing components ( 7 cases ) , bursectomy of the suprapatellar bursa ( 1 case ) , and minimally invasive osteosynthesis ( 1 case ) . the mean time until reoperation was 22.3 months ( range , 2 to 106 months ) ( table 2 ) . polyethylene bearing dislocation was developed in 9 cases ( 3.6% ) , including three males and 6 females ( mean age , 61.4 years ; range , 54 to 70 years ) . this complication was developed most commonly at the average time of 3.7 months ( range , 2 to 7 months ) after surgery . dislocated bearings were replaced with bearing components that were about 2 - 3 mm larger . for the cases which were accompanied with component loosening , revision tka or uka was performed . after change of the bearing components , the tibiofemoral angle was increased from valgus 6.5 2.3 to valgus 8.5 2.7. in one case , the 3-mm dislocated bearing was replaced with an 8-mm thick bearing , after which the valgus angle was increased from 8.5 to 12.6 ( fig . the patient complained of mild posterolateral knee discomfort , but her knee function was good . the valgus angle was found to have decreased at follow - up , which was suspected to be due to adaptation to the ligament tension . interestingly , one posterior bearing dislocation recurred again after revision of the dislocation , but spontaneous reduction of the dislocated bearing was developed ( fig . that patient was very satisfied with the clinical results without any problems at the 2-year follow - up . in cases for which only a bearing change was performed , the radiological parameters such as preoperative average tibiofemoral angle , varus / valgus angle and flexion / extension angle ( femur ) , and varus / valgus angle and posterior tilt angle ( tibia ) were measured . in all cases the average preoperative tibiofemoral angle was valgus 5.4 2.3. the tibiofemoral angle in the revision group was larger than that of the group without revision , but there were no significant differences between the two groups ( p = 0.730 ) ( table 3 ) . varus / valgus angle ( femur component ) was measured as valgus 6.5 4.0 ; flexion / extension angle ( femur component ) : flexion 5.1 5.2 ; varus / valgus angle ( tibia component ) : valgus 3.1 3.8 ; and posterior tilt ( tibia component ) angle : 6.5 4.7. there were no significant differences between these radiologic parameters of the normal and revision ( bearing change ) cases . in the follow - up radiologic evaluations , component loosening was found in 4 cases , 1 male and 3 female , all of which the loosening was developed in the femoral component . the mean age was 64.2 12.8 years ( range , 51 to 78 years ) . the mean time until revision surgery was 44.8 months ( range , 4 to 106 months ) . a revision of the femoral component was performed in 1 case , and tka conversion was performed in 3 cases . in this subgroup of femoral component loosening ( 4 cases ) , the varus / valgus angle ( femur ) was measured as valgus 9.1 2.7. in the case of conversion to tka , a 5-mm overhang of the medial compartment caused irritation of the medial aspect of the knee , resulting in cellulitis - like symptoms . we have experienced several cases of tibial component overhang , owing to the smaller posteromedial tibia size of the korean population . however , in this case , conservative treatment such as rest and intravenous antibiotics was performed , but the pain continued . it was supposed that the cause was irritation of the bursa after intra - articular bleeding from the uka procedure . one case of soft tissue impingement due to malalignment , and one case of unexplained severe pain also resulted in tka conversion ( fig . one periprosthetic fracture occurred in the distal femur , after which minimal invasive osteosynthesis was performed using the plate . at the final follow - ups , the overall hss score had improved from an average of 70 points preoperatively to a final score of 97 points . in the complication group , the mean kss ( pain ) and functional score of all the patients improved from 65 and 60 preoperatively to final scores of 87 and 82 , respectively . at the last follow - up , polyethylene bearing dislocation was developed in 9 cases ( 3.6% ) , including three males and 6 females ( mean age , 61.4 years ; range , 54 to 70 years ) . this complication was developed most commonly at the average time of 3.7 months ( range , 2 to 7 months ) after surgery . dislocated bearings were replaced with bearing components that were about 2 - 3 mm larger . for the cases which were accompanied with component loosening , revision tka or uka was performed . after change of the bearing components , the tibiofemoral angle was increased from valgus 6.5 2.3 to valgus 8.5 2.7. in one case , the 3-mm dislocated bearing was replaced with an 8-mm thick bearing , after which the valgus angle was increased from 8.5 to 12.6 ( fig . the patient complained of mild posterolateral knee discomfort , but her knee function was good . the valgus angle was found to have decreased at follow - up , which was suspected to be due to adaptation to the ligament tension . interestingly , one posterior bearing dislocation recurred again after revision of the dislocation , but spontaneous reduction of the dislocated bearing was developed ( fig . that patient was very satisfied with the clinical results without any problems at the 2-year follow - up . in cases for which only a bearing change was performed , the radiological parameters such as preoperative average tibiofemoral angle , varus / valgus angle and flexion / extension angle ( femur ) , and varus / valgus angle and posterior tilt angle ( tibia ) were measured . in all cases the average preoperative tibiofemoral angle was valgus 5.4 2.3. the tibiofemoral angle in the revision group was larger than that of the group without revision , but there were no significant differences between the two groups ( p = 0.730 ) ( table 3 ) . varus / valgus angle ( femur component ) was measured as valgus 6.5 4.0 ; flexion / extension angle ( femur component ) : flexion 5.1 5.2 ; varus / valgus angle ( tibia component ) : valgus 3.1 3.8 ; and posterior tilt ( tibia component ) angle : 6.5 4.7. there were no significant differences between these radiologic parameters of the normal and revision ( bearing change ) cases . in the follow - up radiologic evaluations , component loosening was found in 4 cases , 1 male and 3 female , all of which the loosening was developed in the femoral component . the mean age was 64.2 12.8 years ( range , 51 to 78 years ) . the mean time until revision surgery was 44.8 months ( range , 4 to 106 months ) . a revision of the femoral component was performed in 1 case , and tka conversion was performed in 3 cases . in this subgroup of femoral component loosening ( 4 cases ) , the varus / valgus angle ( femur ) was measured as valgus 9.1 2.7. in the case of conversion to tka , a 5-mm overhang of the medial compartment caused irritation of the medial aspect of the knee , resulting in cellulitis - like symptoms . we have experienced several cases of tibial component overhang , owing to the smaller posteromedial tibia size of the korean population . however , in this case , conservative treatment such as rest and intravenous antibiotics was performed , but the pain continued . it was supposed that the cause was irritation of the bursa after intra - articular bleeding from the uka procedure . one case of soft tissue impingement due to malalignment , and one case of unexplained severe pain also resulted in tka conversion ( fig . 5 ) . one periprosthetic fracture occurred in the distal femur , after which minimal invasive osteosynthesis was performed using the plate . at the final follow - ups , the overall hss score had improved from an average of 70 points preoperatively to a final score of 97 points . in the complication group , the mean kss ( pain ) and functional score of all the patients improved from 65 and 60 preoperatively to final scores of 87 and 82 , respectively . at the last follow - up , there have been several complications reported after performing uka , such as rupture of the medial or lateral collateral ligaments , dislocation of the polyethylene bearing , dissociation of the prosthesis , degenerative changes in the opposite compartment , and fracture of the medial proximal tibia.3,10,11,12,13,14 ) previous literature reported that the failure was caused by the aseptic loosening of the femoral or tibial prosthesis , dislocation or instability of the prosthesis , malalignment of the prosthesis , deep infection , periprosthetic fracture , abrasion of the polyethylene liner , the progression of arthritis , etc.5,7,8,9 ) uka is a demanding procedure that requires special experience , and includes a risk of early failure . lindstrand et al.15 ) reported that 8 revisions performed in 123 operated knees due to loosening , subsidence , or fracture . these revisions were performed within 1 year , and were mostly related to the operative technique used . in our series , berger et al.10 ) reported that only 2 patients ( 2 knees ) out of 62 cases ( mean follow - up of 12 years ) with well - fixed components underwent a revision to tka , at seven and eleven years , because of progression of patellofemoral arthritis . at the final follow - up , no radiographically loose component was found , and there was no evidence of periprosthetic osteolysis in that study . in our study , complications developed in 9.8% of the cases . change of initially planned uka would require longer surgical time , and increase the perioperative complications during the procedure . this means that having an accurate surgical technique is very important in the uka procedure . compared to the other reports , occurrence of suprapatellar bursitis and medial tibial plateau fracture were very unusual in our series . barrett and scott16 ) suggested that errors in patient selection were responsible for the failure in 31% , and also saldanha et al.17 ) also noted that 13 failures among 100 cases were due to poor patient selection . however , since arthroscopy was performed in all patients in our series and patients with higher than outerbridge grade 3 articular cartilage injury in the lateral or patellofemoral compartments were excluded , the possibility of having poor patient selection for the uka procedure was reduced . however , 1 patient developed suprapatellar bursitis in the drain site after the arthroscopic procedure . pandit et al.18 ) reported a ten - year survival rate of 96% , if all implant - related reoperations are considered failures and the incidence of implant - related reoperations was 2.9% during the mean follow - up of 5.6 years . the most common reason for further surgical intervention was progression of arthritis in the lateral compartment ( 0.9% ) , followed by dislocation of the bearing ( 0.6% ) , and revision for unexplained pain ( 0.6% ) . berger et al.10 ) reported that progressive patellofemoral arthritis was the primary mode of failure . the postoperative tibiofemoral angle has also been considered to be a very important factor determining the prognosis of the surgery , and the level of abrasion and instability of the prosthesis . tibial or femoral component malalignment has been found to lead to early failure.19 ) malalignment of the prosthesis or the generation of excessive tension during flexion or complete extension may limit the range of motion , possibly causing loosening of the prosthesis to develop over time . perkins and gunckle14 ) reported in a study with a 6-year follow - up period that the possibility of needing revision surgery was high when the postoperative tibiofemoral angle was larger than 3 varus , or larger than 7 valgus . in our small series with femoral component loosening , it was observed that valgus malalignment of the femoral components may eventually cause early femoral component loosening , but no statistical significance was obtained , owing to the small number of cases ( only 5 ) . fracture of the medial tibial condyle during insertion of the tibial component occurred in 1 case . this was due to the natural tendency to place the tibial tray too medial in the varus knee , in order to avoid impingement on the anterior cruciate ligament . however , this practice could lead to a metaphyseal fracture of the tibia due to decreased bone support beneath the tibial components . in our study , the most prevalent complication observed was polyethylene bearing dislocation . a bearing change can be performed for cases of simple bearing dislocation , but component revision or conversion to tka is needed when this is accompanied by femoral or tibial component loosening . usually in cases of bearing dislocation , the dislocated bearing was removed and a bearing approximately 2 - 3 mm larger was inserted . interestingly , in one case where a bearing of 3 mm in thickness was replaced with an 8-mm bearing , excessive valgus tibiofemoral angle was developed . generally , bearing change could be done by replacement with a 2 - 3 mm larger sized bearing . since lateral compartmental arthritis may develop if excessive valgus angles are induced , careful attention should be given . in our case , the 6-month follow - up revealed the valgus tibiofemoral angle to be reduced from 12 to 8 because of ligament adaptation . bearing dislocations have been associated with proximal tibial varus greater than 5 , excessive femoral component varus or valgus , and excessive postoperative tibial slope.20,21 ) in our series , the tibiofemoral angle increased from valgus 6.5 to valgus 8.5 after the bearing change . however , there were no significant differences between the several radiologic factors , such as the pre- and postoperative tibiofemoral angles , varus / valgus angles ( femur , tibia component ) flexion / extension angles ( femur component ) , and posterior tilt angles ( tibia component ) . compared to the normal group , the tibiofemoral angle showed no significant differences in the complication group ( p = 0.079 ) . we suspected that the cause of bearing dislocation was multifactorial , and that lifestyle , including activities such as squatting , was one of the most important factors in asian populations . emerson et al.11 ) reported that mobile bearings have the disadvantages of higher complication and early revision rates , resulting from bearing dislocation and impingement syndromes caused by suboptimal operation technique or instability . interestingly , spontaneous reduction was developed in 1 revision case in this study . after bearing change , it was suspected that lax capsular tension might have caused spontaneous reduction of the posterior dislocated bearing . one particular case , which was not observed in other studies , was the recurrent suprapatella bursitis developed after performing arthroscopy . even though several aspirations were performed , suprapatellar bursitis continued to progress , until open excision of the suprapatella bursitis was finally performed . generally , the size of the tibia of koreans is relatively smaller than caucasians . in one case , the protruded tibial component irritated the posteromedial area of the knee joint continuously , causing cellulitis - like symptoms and pain to develop . a previous study showed that oxford designs resulted in mediolateral overhang for all the comparative anteroposterior dimensions in asian populations.9 ) manufacturing of prostheses with an adequate size to fit to the bone structure of asian populations would be required to reduce associated complications . in a total of 246 cases , bearing dislocation was the most common complication in our series . for preventing these complications and improving clinical results , an accurate preoperative surgical plan and skillful surgical technique are required .

the detection and characterization of shiga toxin producing escherichia coli ( stec ) , salmonella spp . , and yersinia enterocolitica strains in human patients , animal reservoirs , and foods of animal origin intended for human consumption are relevant to public health . these organisms are transmitted through contaminated drinking water and food and can cause intestinal and extraintestinal clinical manifestations in humans [ 1 , 2 ] . stec is associated with hemorrhagic colitis and hemolytic uremic syndrome ( hus ) ; its pathogenicity is attributed to virulence factors that facilitate effective colonization of the human gastrointestinal tract and subsequent release of shiga toxins . argentina has the highest incidence of hus in the world , with e. coli o157:h7 as the primary etiological agent . previous studies in our country have been focused on the prevalence of stec in cattle and byproducts intended for human consumption . salmonella spp . can enter the food chain , thereby triggering either sporadic cases or outbreaks of human salmonellosis . foodborne outbreaks triggered by salmonella serotypes are of rare occurrence in our province ; however , s. panama , s. newport , s. sandiego , s. enteritidis , s. montevideo , s. anatum , and s. typhimurium strains have been isolated from sources of diverse origin . pathogenic strains usually carry a 72 kb virulence plasmid ( pyv ) bearing yada , yops , and virf genes and chromosomal virulence markers such as ail , yst , myfa , and inv genes . the assessment of virulence markers contributes to properly establish the pathogenic potential of the y. enterocolitica isolates . since y. enterocolitica is not routinely investigated in the clinical laboratories of argentina , a systematic epidemiological study of y. enterocolitica in environment , reservoirs , foods , and humans is still pending . for contributing to the knowledge of prevalence and distribution of these enteropathogens in patients , presumable animal reservoirs , and foods of our region , this study was aimed ( i ) to detect stec , salmonella spp . , and y. enterocolitica in human and animal feces and foods of animal origin intended for human consumption , ( ii ) to assess the pathogenic potential of y. enterocolitica strains through phenotypic and molecular virulence markers , ( iii ) to test the antimicrobial susceptibility of all strains , and ( iv ) to determine possible genetic relationships among isolates of each species by subtyping using pulsed field gel electrophoresis ( pfge ) . y. enterocolitica o:9 w1024 pyv ( belgium ) , y. enterocolitica mch700 b4 o:3 pyv ( canada ) , y. enterocolitica 29c-43 b4 o:3 pyv ( norway ) , and y. enterocolitica b2 o:9 pyv ( argentina ) were used as positive controls in phenotypic virulence assays , assessment of genetic virulence markers by pcr and comparison of dna profiles by pfge . escherichia coli o157:h7 edl933 sor-/glu-/e - hly+/eae+ , biotype c , producer of stx1 and stx2 , was employed as positive control in pcr targeting stec stx1/stx2 genes and salmonella braenderup h9812 was used as molecular size marker in pfge . y. enterocolitica b1a o:5 clo229 and y. enterocolitica b1a o:6,30 clo225 , local strains isolated from sausages , were utilized as positive controls in ystb gene pcr . these strains were maintained at 4c on trypticase soy agar slants ( tsa , merck , darmstadt , germany ) . for determining culture purity , isolations on mac conkey agar ( mc , merck ) , sorbitol mac conkey agar ( smac , merck ) , and salmonella shigella agar ( ss , merck ) for y. enterocolitica , e. coli o157:h7 , and s. braenderup , respectively , were made prior to each experiment . plates were incubated 48 h at 25c for y. enterocolitica and 24 h at 37c for e. coli o157:h7 and s. braenderup . a total of 453 samples from human and animal sources in san luis city and upcoming rural areas , argentina , were analyzed during the period june 2008november 2011 . they included 70 samples of human stools from patients with enterocolitis symptoms attending a local clinical laboratory ; 167 stool samples obtained from feedlot bovines ( n = 61 ) , grazing bovines ( n = 68 ) , porcine ( n = 20 ) , ovine ( n = 10 ) , goats ( n = 6 ) , and equines ( n = 2 ) ; and 216 samples of animal origin intended for human consumption such as chicken carcasses ( n = 80 ) , porcine skin and bones ( n = 10 ) , goat cheeses ( n = 30 ) , fresh sausages chorizos ( n = 90 ) , and six samples of three wild boars , tongues ( n = 3 ) , and tonsils ( n = 3 ) . samples of animal stools were randomly collected in regional cattle markets and farms immediately after defecation . samples of foods of animal origin intended for human consumption were purchased at five retail markets in san luis city . all samples were packed in individual sterile plastic bags and stored at 4c for up to 6 h before processing . the presence of total coliforms was investigated in samples of animal origin intended for human consumption , except tonsils and tongues of wild boars . decimal dilutions of each sample were prepared in 0.1% peptone water ph 7.2 ( pw , merck ) ; one milliliter of each dilution was seeded in violet - lactose - neutral red - bile agar ( vlrb , merck ) and incubated at 37c for 24 h. counts of characteristic colonies were performed and results were expressed as log10 cfu / g . samples of 25 g were homogenized in stomacher ( iul masticator , germany ) for 90 s and incubated in 225 ml of ec broth ( merck ) for 24 h at 37c . for avoiding false negative pcr results produced by interference of organic compounds and background microflora present in samples , each plate showed confluent growth in the initial streaking zone where bacteria were clustered and individual colonies in the final streaking zone where bacteria were isolated . then , dna was extracted from confluent growth for screening stx1/stx2 genes by pcr ; if amplified , five sorbitol nonfermenting and five fermenting colonies were randomly selected from smac plates and their dna were individually tested by pcr targeted to stx1 and stx2 genes . when positive results were observed , gram staining and biotyping of each colony was performed . sorbitol nonfermenting e. coli colonies were challenged against o157 antiserum ( national institute of infectious diseases - inei anlis-dr . carlos g. malbrn , buenos aires , argentina ) by a slide agglutination test . twenty five grams of each homogenized sample was enriched in 225 ml of lactose broth ( lb , merck ) at 37c for 24 h ; then , one - ml lb aliquots was transferred into two tubes containing 9 ml of tetrathionate broth ( merck ) and two tubes with 9 ml of rappaport - vassiliadis broth ( merck ) . one tube of each selective broth was incubated at 37c for 24 h and the other one was incubated at 42c for 24 h. isolations were done on ss agar ( merck ) for 24 h at 37c and suspect lactose nonfermenting colonies were examined by gram staining and biochemical assays . serotyping was performed in the national reference center for enterobacteria , inei - anlis , buenos aires , argentina . this species was searched by enrichment of 25-g homogenized sample in 225 ml of phosphate buffered saline ph 7.6 added with 1% sorbitol and 0.15% bile salts and incubated 21 days at 4c . isolations were made on mc agar for 48 h at 25c and presumptive yersinia colonies were examined by gram staining and identified by biochemical assays ( http://www.fda.gov/food/foodscienceresearch/laboratorymethods/ucm072633.htm , accessed may 2008 ) . e. carniel , national reference center of yersinia , institute pasteur , paris , france . each strain was suspended in an eppendorf tube containing 150 l of te 1x buffer ( 10 mm tris ( sigma aldrich , st . louis , mo , usa ) , 1 mm edta ( sigma ) , ph 8.0 ) added with 1% triton - x100 ( parafarm , buenos aires , argentina ) . suspensions were boiled 15 min and centrifuged at 10,000 rpm for 5 min ( sigma centrifuge model 3k30 , germany ) . each supernatant containing dna was transferred to other tube and stored at 20c before use . a 25 l volume of a pcr mix containing 1x pcr buffer , 0.1 mm dntp , 1.5 mm mgcl2 , 2 pmol/l of stx1 primers ( forward 5-gaagagtccgtgggattacg-3 and reverse 5-agcgatgcagctattaataa-3 ) for an amplicon of 130 bp , 0.4 pmol/l of stx2 primers ( forward 5-ttaaccacaccccaccgggcagt-3 and reverse 5-gctctggatgcatctctggt-3 ) for an amplicon of 346 bp , 0.02 u/l taq dna polymerase ( productos biolgicos , quilmes , argentina ) , 2 l dna template , and ultrapure water was prepared . cycling conditions consisted of an initial denaturation at 94c for 5 min followed by 30 cycles of 94c for 30 s , 58c for 30 s , and 72c for 30 s and a final extension of 72c for 3 min . the pcr products were electrophoresed in a 2% agarose gel ( productos biolgicos ) added with gelred acid gel stain ( biotium , hayward , ca , usa ) , 1.5 l stock solution/40 l gel , at 80 v for 40 min , and visualized in an uv transilluminator ( uvp , upland , ca , usa ) . for comparison , a 100-bp dna ladder ( productos biolgicos ) was used . each bacterial strain was isolated on mueller hinton agar ( mh , britania , buenos aires , argentina ) and incubated 24 h at 37c for salmonella and e. coli , or 48 h at 25c for yersinia strains . colonies of each strain were directly suspended in 4 ml of the suspension buffer ( 100 mm tris , 100 mm edta , ph 8.0 ) to od610 nm 1.0 . two hundred microliters of bacterial suspension were mixed with equal volume of 1% seakem gold agarose ( cambrex , rockland , me , usa ) and poured in molds to obtain plugs . plugs were treated for 20 h in a lysis solution ( 50 mmol tris - edta ( sigma ) , 1% sodium lauroyl sarcosine ( sigma ) , and proteinase k 0.1 mg / ml ( fluka chemie , buchs , switzerland ) ph 8.0 ) at 37c and then washed four times with te buffer ( 10 mm tris ( sigma ) , 1 mm edta ( sigma ) , ph 8.0 ) for 30 min at 37c . chromosomal dna contained in agarose plugs was digested with 10 u xbai ( fermentas , burlington , ontario , canada ) for two hours , according to manufacturer 's instructions . plugs were cut in approximately 1-mm - thick slices , placed in the chef - driii chamber ( bio - rad , hercules , ca , usa ) , and pfge was performed using an electric field of 6 v / cm at 14c , angle of 120 and switching times of 2.2 ( initial time ) to 63.8 s ( final time ) for salmonella and e. coli o157:h7 , and 1.8 s ( initial time ) to 20 s ( final time ) for y. enterocolitica , over 20 h [ 6 , 8 ] . migration of the dna fragments was achieved in a 1.0% pulsed - field agarose gel ( bio - rad ) submerged in 0.5x tbe buffer ( 45 mm tris - borate ( sigma ) and 1 mm edta ( sigma ) ) . the gels were stained with gel red acid gel stain ( biotium , hayward , ca , usa ) under the conditions suggested by the manufacturer , visualized in uv transilluminator , and photographed . these assays were performed according to us fda bacteriological analytical manual ( http://www.fda.gov/food/foodscienceresearch/laboratorymethods/ucm072633.htm , accessed september 2011 ) . each strain was inoculated into two tubes of methyl red - voges proskauer broth ( merck ) . after 18 to 24 h , the tubes were observed for bacterial autoagglutination the mr - vp tube incubated at 25c for 24 h should show turbidity from bacterial growth . the 37c mr - vp tube should show agglutination ( clumping ) of bacteria along walls and/or bottom of tube with clear supernatant fluid . isolates giving this result were presumptive positive for the virulence plasmid . any other pattern for autoagglutination at these two temperatures congo red - magnesium oxalate sodium agar ( cr - mox ) was composed by tsa added with 2.68 g / l sodium oxalate ( mallinckrodt ) , 4.067 g / l mgcl2h2o ( sigma ) , and 5 ml / l of 1% congo red dye ( baker ) , which allowed visualization of calcium - dependent growth and uptake of congo red dye on the same plate . decimal dilutions in physiologic saline were made to obtain 1,000 cells / ml . volumes of 0.1 ml of the appropriate dilution were spread - plated on cr - mox plates and incubated at 37c . presumptive plasmid - bearing y. enterocolitica showed scarce growth of pinpoint , round , convex , red , and opaque colonies ; whereas plasmidless y. enterocolitica exhibited abundant growth of large , irregular , flat , and translucent colonies . this test was performed on esculin agar ( 10 g polypeptone ( britania ) , 1 g esculin ( sigma ) , 1 g ferric ammonium citrate ( sigma ) , and 5 g agar ( britania ) in 1000 ml of distilled water ) . strains were inoculated , incubated at 25c , and observed up to 7 days after seeding . the development of dark pigment on the agar surface was indicative of lacking of virulence . strains were inoculated over the entire slant of pyrazinamide agar ( 30 g tsa ( merck ) , 1 g pyrazinecarboxamide ( sigma ) , and 0.2 m tris - maleate buffer ( sigma ) for 1000 ml ) , incubated at 25c for 48 h , and flooded with 1 ml of 1% freshly prepared ferrous ammonium sulfate . development of pink color within 15 min was positive test , indicating presence of pyrazinoic acid formed by the pyrazinamidase enzyme . dna extraction was performed as described . a nested - pcr targeted to y. enterocolitica yada gene five microliters of the template was used for the first pcr and 2 l of the product obtained in this step was used as template for the second pcr . the reaction mixture ( 50 ml ) contained 1x pcr buffer , 1.5 mm mgcl2 , 200 mmol / ml of each dntp , 1 u of taq dna - polymerase , and 0.1 mmol / ml of each primer ( productos biolgicos ) . the first primer pair integrated by the following oligonucleotides : yada1 5-taa gat cag tgt ctc tgc ggc a-3 and yada2 5-tag tta ttt gcg atc cct agc ac-3 was used under the following cycling conditions : initial denaturation at 95c for 3 min , then 40 cycles of denaturation at 95c for 30 s , annealing at 58c for 60 s , and extension at 72c for 90 s , followed by a final extension at 72c for 10 min for amplifying a 747 bp fragment . the second primer pair consisting of yada3 5-gcg ttg ttc tca tct cca tat gc-3 and yada4 5-ggc ttt cat gac caa tgg ata cac-3 was used under the following cycling conditions : initial denaturation at 95c for 3 min followed by 20 cycles of denaturation at 95c for 30 s , annealing at 62c for 60 s , and extension at 72c for 90 s , and a final extension at 72c for 10 min which amplified a 529 pb fragment . products were electrophoresed at 80 v for 40 min in a 1% agarose gel stained with gelred , visualized in an uv transilluminator ( uvp ) , and compared with a 100-bp ladder dna ( productos biolgicos ) . the reaction mixture ( 25 l ) contained pcr buffer 1x , 200 m of each dntp , 1.5 mm mgcl2 , 0.08 u/l of taq dna - polymerase , and 1 pmol/l of each primer ( productos biolgicos ) . it used the primer pair constituted by ystb - f 5gtacattaggccaagagacg 3 and ystb - r 5 gcaacatacctcacaacacc 3 for amplifying a 146 bp fragment . the pcr product was electrophoresed at 80 v for 40 min in a 2% agarose gel stained with gelred and visualized in an uv transiluminator ( uvp ) . the antimicrobial susceptibility of the isolates was addressed by the disk diffusion method on mueller hinton agar . the following antibiotic disks ( britania ) were used : ampicillin , 10 g ; cephalotin , 30 g ; aztreonam , 30 g ; erythromycin , 15 g ; colistin 10 g ; chloramphenicol , 30 g ; gentamicin , 10 g ; tetracycline , 30 g ; trimethoprim - sulfamethoxazole , 25 g ; ciprofloxacin , 5 g ; neomycin , 30 g ; furazolidone , 5 g ; nalidixic acid , 30 g ; cefotaxime , 30 g ; ceftriaxone , 30 g ; and phosphomycin , 50 g . zones of growth inhibition were evaluated according to clinical and laboratory standards institute ( clsi ) guidelines . statistical analysis of counts of coliforms related to type of sample was performed using chi - square test ( analytical software , tallahassee fl , usa ) . statistical calculations were based on confidence level equal or higher than 95% ( p 0.05 was considered statistically significant ) . confidence intervals for each percentage were calculated by using the formula : 95% ci = percentage ( 1.96 se ) , with se , standard error , calculated as se = percentage/(n of positive cases ) . the discrimination index ( di ) values of pfge were calculated by simpson 's diversity index . clustering of the patterns obtained by pfge was performed using statistica 6.0 software ( statsoft inc . , tulsa , ok , usa ) and the unweighted pair group method with arithmetic average ( upgma ) . counts of total coliforms in fresh sausages and chicken carcasses yielded 5.4 0.7 and 5.0 0.7 log10 cfu / g , respectively , being significantly higher ( p 0.05 ) than those observed for porcine skin and bones ( 3.9 0.6 log10 cfu / g ) and goat cheeses ( 0.7 0.2 log10 cfu / g ) ( table 1 ) . the detection frequencies of stec , salmonella serotypes , and yersinia species in 453 samples are shown in table 2 . the number of yersinia positive samples ( 9/453 , 2.0% , 95% ci , 0.73.3% ) was higher ( p 0.05 ) than those observed for stec ( 4/453 , 0.9% , 95% ci , 01.8% ) and salmonella spp . ( 3/453 , 0.7% , 95% ci , 01.5% ) . a sample was considered presumptive stec positive when only stx1/stx2 signals were detected by pcr on dna extracted from confluent growth on smac . thus , four samples were positive : one stool sample from a pediatric patient with diarrhea ( 1/70 , 1.4% , 95% ci , 04.2% ) yielded one e. coli o157:h7 strain by culture on smac which was characterized as stx2 by pcr and three samples of bovine stools ( 3/129 , 2.3% , 95% ci , 05.0% ) that amplified stx genes from dna extracted from confluent growth ( two samples were stx1/stx2and the third one was stx2 ) . individual stec colonies could not be isolated from these samples . on the other hand , three samples yielded four salmonella isolates corresponding to different serotypes : one s. typhimurium strain was obtained from stools of a symptomatic patient ( 1/70 , 1.4% , 95% ci , 04.2% ) and one s. newport and two s. gaminara strains were isolated from one tonsil and one tongue of wild boar among 216 samples of animal origin intended for human consumption ( 2/216 , 0.9% , 95% ci , 02.1% ) . in the small number of wild boar samples , the salmonella recovery was high ( 1/3 , 33% , 95% ci , 099% ) . furthermore , nine yersinia isolates were recovered from nine ( 9/216 , 4.2% , 95% ci , 1.56.9 ) samples of animal origin intended for human consumption . two isolates were classified as y. enterocolitica b1a o:12,25 - 12,26 , five as y. enterocolitica b1a o:7,8 - 8 - 8,19 , one as y. intermedia b6 o:17 , and other one as y. intermedia b4 o:40 ( table 2 ) . they were isolated from six chicken carcasses ( 6/80 , 7.5% , 95% ci , 1.513.5% ) and three porcine skin and bones ( 3/10 , 30% , 95% ci , 065% ) . two major clusters , a and b , with a 65% similarity were obtained . even though six s. newport isolates from tonsil and five s. gaminara isolates from tonsil and tongue were initially recovered from one wild boar , the analysis of their dna restriction profiles by pfge showed that all s. newport strains grouped in cluster a while all s. gaminara ones grouped in the genotype gtb1 within cluster b. since identical dna band patterns between isolates of the same serovar were observed , only three salmonella strains were reported ( table 2 ) . in dendrogram , s. typhimurium of human source was included in gtb2 within cluster b , showing 68% similarity with gtb1 . although fifteen yersinia isolates were originally recovered from nine positive samples , the analysis of dna restriction profiles observed by pfge allowed to conclude that some isolates were replicates of the same strain . therefore , bacterial isolates were grouped into two major clusters , a and b ( 63% similarity ) , according to yersinia species ( figure 2 ) . thus , cluster a comprised gta1 with five y. enterocolitica b1a o:12,25 - 12,26 strains isolated from chicken carcasses , gta2 with eight y. enterocolitica b1a o:7,8 - 8 - 8,19 isolates from chicken carcasses and porcine skin , and gta3 with the reference y. enterocolitica w1024 strain . cluster b included gtb1 consisting of y. intermedia b6 o:17 strain and gtb2 corresponding to y. intermedia b4 o:40 strain ( 86% similarity ) , both strains recovered from chicken carcasses . five y. enterocolitica b1a o:7,8 - 8 - 8,19 strains and two y. enterocolitica b1a o:12,25 - 12,26 strains isolated in this study produced negative results for calcium dependent growth and congo red binding at 37c showed pyrazinamidase activity , hydrolyzed esculin and autoagglutinated at 37c ( table 3 ) . when ystb gene was assayed , one y. enterocolitica b1a o:7,8 - 8 - 8,10 strain isolated from porcine skin and bones was positive . the human stec strain was susceptible to all antimicrobials assayed except ampicillin and rifampicin . similarly , salmonella strains were susceptible to all drugs except cephalotin . meanwhile , all y. enterocolitica isolates shared resistance to ampicillin , one y. enterocolitica b1a o:12,25 - 12,26 strain was susceptible to cephalotin , and two y. enterocolitica b1a o:7,8 - 8 - 8,10 strains showed resistance to both cephalotin and erythromycin . total coliforms are considered indicators of hygienic quality and their presence in foods may correlate with the presence of pathogenic bacteria . the low total coliform counts observed in porcine skin and bones , and goat cheeses might be attributed to the effects of thermal treatments applied to pig carcasses during slaughtering , and pasteurization and preservation of dairy products , respectively . no microbiological specifications for porcine skin and bones are included in the argentinean alimentary code ( aac , http://www.anmat.gov.ar/alimentos/normativas_alimentos_caa.asp , accessed november 2013 ) . on the other hand , values up to 500 total coliforms per gram at 45c are allowed for cheeses with 36 to 46% moisture ( aac ) . thus , low coliform counts for this food would be consistent with good practices of manufacture . in contrast , low microbiological quality of ingredients or poor hygiene could explain coliform counts higher than 10 mpn / g which is the maximal limit established by aac for fresh sausages . although no microbiological standards for chicken carcasses are addressed by aac , counts of coliforms in this work were higher than 2.7 log10 cfu / g observed by capita et al . in spain . clearly , contamination is possible at any stage of the production process , from defeathering , evisceration , and washing to storage by cooling or freezing . regarding the search of enteropathogens , the human e. coli o157:h7 strain was isolated by culture and characterized as stx2 by pcr . on the contrary , no stec strain could be isolated from positive stx1/stx2 cattle stools , probably because they were viable but noncultivable strains . concurrently , jure et al . identified the stx2 gene in seven samples of meat in argentina ; however , only one e. coli o157:h7 strain could be isolated . the low detection of stec from cattle in san luis contrasts with reports of 4 to 39% stec isolates recovered from calves by meichtri et al . in our country , who enriched stools and rectal swabs in tsb added with antibiotics and then performed screening of stx genes by conventional pcr in dna extracted from confluent bacterial growth on smac . similarly , sanz et al . recovered 44% stec from bovines for slaughtering in other argentina regions . a wide range of protocols have been described for detection or isolation of stec since that all serotypes can not be detected by one method . trypticase soy broth , e. coli broth , buffered peptone water , and brain heart infusion broth added with selective agents have been recommended for stec enrichment . in the present study , samples were enriched in ec broth without antibiotics which may be advisable when stressed or injured stec cells are cultured . in addition , a comparative study of enrichment protocols by vimont et al . showed that the initial level of e. coli o157 was not greatly influenced by the enrichment protocol tested , whereas the initial level of background microflora appeared to decrease when ec broth was used . the immunomagnetic separation ( ims ) can be used after enrichment and prior to plating for the selective concentration of stec cells , and it is well established for the detection of e. coli o157 in foods , yielding detection limits as low as 1 - 2 cfu/25 g . while ims was not used in this study , subsequent stec researches in our laboratory will include this procedure . otherwise , molecular methods such as conventional pcr and real - time pcr are very sensitive and provide results in shorter times than cultures . thus , the iso / ts 13136:2012 standard is based on the sample enrichment followed by a real - time pcr targeted to the detection of the stx and eae virulence genes , and the determination of o157 , o111 , o26 , o103 , and o145 stec serogroups in foods and animal foodstuffs . also , immunoassay - based methods such as an available eia for testing shiga toxins 1 and 2 have been used in the stec detection from human stools . differences in stec carriage have been observed between grass - fed and feedlot cattle ; in the present study , two positive stx1/stx2 samples corresponded to feedlot animal and the other one came from a grazing animal . although stec detection and/or recovery were negative in other samples studied here , ojo et al . demonstrated stec in feces of cattle ( 15.2% ) , sheep ( 10.7% ) , goats ( 7.5% ) , and pigs ( 5.6% ) as well as in beef ( 3.8% ) , goat - meat ( 1.7% ) , and pork ( 4.0% ) . the isolation of s. typhimurium from stools of a patient was consistent with studies reporting this one as the most frequently isolated serovar from humans in argentina since 2006 . we report the isolation of s. newport and s. gaminara from wild boars for the first time in our region . these salmonella serotypes have been previously isolated from clinical samples during an outbreak caused by consumption of unpasteurized orange juice in usa and recovered from patients with diarrhea in caribbean zone of colombia . environmental factors and seasonal variations as well as different supply sources of samples might have influenced in the low recovery of salmonella from animal samples in our study . the yersinia prevalence observed in this work was lower than 5.5% from pork and beef sausages and minced meat obtained by lucero estrada et al . who detected y. enterocolitica b1a ( o:5 and o:6,30 ) , b2 o:9 , and y. intermedia in our region . isolated 10% y. enterocolitica b1a o:5 , y. intermedia , and y. frederiksenii from 70 chicken carcasses in argentina . aac establishes no y. enterocolitica limits in relation to any of the foods here analyzed , but the absence of this pathogen is desirable . although y. enterocolitica was not detected in human and animal stools investigated in the present study , this microorganism has been isolated from human diarrheic feces and animal stools in our country . in this study , virulence phenotypic tests for y. enterocolitica b1a strains produced negative results excepting autoagglutination at 37c ; however , the yada gene was not detected by pcr . lack of correlation between y. enterocolitica phenotypic and genotypic virulence markers such as the above mentioned has been reported by zheng et al . . these authors found that some y. enterocolitica strains contain other unknown virulence markers that interact with each other and play an important role in the pathogenesis . in this regard , the chromosomal gene ystb had been strongly linked to the production of diarrhea by b1a strains . opportunely , among 115 y. enterocolitica isolates of pig origin analyzed by bonardi et al . , 75.7% corresponded to b1a with ystb as the most common virulence gene ( 72.4% ) . in our study , this gene was demonstrated in one y. enterocolitica b1a o:7,8 - 8 - 8,10 strain ( 1/7 , 14% , 95% ci , 042% ) isolated from porcine skin and bones . the presence of y. enterocolitica b1a and y. intermedia in chicken carcasses and porcine skin and bones could be the result of cross - contamination during processing of these products or carriage by slaughtered pigs , respectively . related to stec antimicrobial susceptibility , since antimicrobials can injure the bacterial membrane causing an acute release of preformed shiga toxin , the treatment of hus in patients is mostly supportive with adequate corporal fluid and electrolyte management , control of the haematological complications , antihypertensive and analgesic therapy , mechanical ventilation , and dialysis when necessary , avoiding antibiotic administration . in our region , stec strains isolated from patients with diarrhea have demonstrated in vitro susceptibility to antibiotics commonly used in the treatment of infections triggered by other enterobacteria . contrary to the antibiotic sensitivity demonstrated by our salmonella isolates , ibar et al . observed multidrug resistance in different salmonella serotypes isolated from porcine in argentina against antimicrobials commonly used in veterinary medicine . regarding y. enterocolitica antimicrobial susceptibility , our results matched those reported by lucero estrada et al . and bonardi et al . who observed resistance to cephalotin and ampicillin was observed in human , animal , and food samples in this region of argentina . the low number of stec found in this study , one e. coli o157:h7 from human stool , as compared to other works , might be attributed to the detection methods used . otherwise , the detection of stx1/stx2 genes in cattle stools highlights the risk of exposure to stec animal carriers and reinforces the requirement of the good practices of hygiene during slaughtering and meat processing . on the other hand , the high salmonella frequency observed in the small number of wild boar samples emphasizes the need of further studies in these animals whose byproducts are manufactured and marketed at retail . lastly , bio / serotypes and virulence traits characterizing our y. enterocolitica isolates were related to null or low pathogenicity for humans ; however , a wide field of knowledge remains unexplored about y. enterocolitica b1a virulence . our results suggest that a close microbiological monitoring might contribute to the knowledge of prevalence and distribution of these enteropathogens in patients , presumable animal reservoirs , and foods in our region , which would allow public health services to take preventive measures .

several factors should be coordinated with each other to make an appropriate condition for developing and improvement of ps with observing patient 's rights in all aspects ( 2 ) . this topic deserves attention because it is broadly a worthy goal and is a potentially important mediator for a range of outcomes . satisfied patients are statistically more compliant with their medical instructions , suggesting that satisfaction may be an important component in promoting health and well - being ( 3 - 5 ) . satisfaction may also directly affect the financial viability of an institution by influencing consumer choice in the future . moreover , it is a highly valuable factor for quality - improvement purposes which is gaining momentum in iran as well as other countries ( 6 ) . these factors have led to a proliferation of studies on ps over the last years ( 7 ) . although the field of emergency medicine is comparatively new in iran , it has not been neglected in the ps research . however , like the larger ps body of literature , many of the existing emergency department ( ed ) ps studies have serious methodological flaws , which has led to inconsistent and , at times , contradictory conclusions ( 7 ) . in iran , many studies have utilized questionnaires in order to measure ps ( 8 - 12 ) ; however , no specially designed scale for ed was found reporting satisfactory psychometric properties . studies that utilize such scales have the risk of acquiring limited or inconsistent data ( 7 , 13 ) . as a result , the present study aimed to develop and initially validate brief emergency department patient satisfaction scale ( bepss ) . study design and setting considering the body of literature , effective items in ps evaluation were extracted in a valid and reliable manner . all items aimed to measure a specific aspect of ps . the preliminary battery of items was then checked for content by a panel of experts including two hospital managers , two quality - improvement officers , one physician , and one psychometrics professional . the items were categorized into seven major domains of admission , nursing , physician care , environment , patient s family , waiting time , and general satisfaction . authors evaluated the content validity of the instrument in the final step of scale development as well as initial steps . reviews were used to evaluate if the instrument covers required aspects of ed patient satisfaction ( 7 , 11 ) . exploratory factor analysis ( efa ) was performed in order to identify underlying components of the instrument . a broad item analysis was conducted prior to efa ( 14 ) . in this step , items exclusion criteria were set as : ( a ) missing more than 15% of data ( b ) having inappropriate indices of skewness and kurtosis and ( c ) inappropriate cross - loadings in efa . each item was scored from four ( complete satisfaction ) to one ( complete dissatisfaction ) as the response option was provided in a 4-point scale of likert sort . cronbach s alpha coefficient was calculated to assess the internal consistency of each domain and total scale as a measure of reliability . initial validation : 301responders were consecutively recruited from two hospitals in tehran , summer of 2014 . all participants were given final approved questionnaire by the panel as well as demographic questions . demographics consisted of respondent ( patient / family ) , post - examination status ( released / hospitalized / else ) , age , sex , time of admission , delay before admission ( waiting time ) , and educational level . demographic questions were developed in line with the existing literature and on an exploratory basis . the verbal consent of all participants was obtained before administering the questionnaires in the emergency department . statistical analysis data entry and analyses were performed in a blinded manner by staff members who were not involved in the process of data collection . statistical analysis was performed using statistical package for the social sciences ( spss ) software ( version 21.0 ; spss inc , chicago , illinois ) . kaiser - meyer - olkin ( kmo ) measure was calculated to evaluate sampling adequacy . bartlett s test of sphericity was also performed . finally , one - way analysis of variance ( anova ) and independent t - test were used to evaluate total satisfaction among different groups derived from demographic characteristics . the minimum value of kmo measure for adequacy of data matrix for factorability was considered as 0.6 ( 15 ) . considering the body of literature in ps evaluation , 32 items were extracted but the final questionnaire approved by the panel consisted of 24 items . 301 full field questionnaires met the inclusion criteria and were entered in the validation analysis . since the minimum value of this measure for adequacy of data matrix for factorability is 0.6 , the data matrix had the required assumptions for factor analysis . four items ( 3 , 7 , 9 , and 19 in the 24-item version ) were discarded in this step because of inappropriate cross - loadings . another efa was performed on the remaining 20 items ( kmo=0.925 ) using varimax rotation and fixed number of five domains ( appendix 1 ) . these domains were respectively named as emergency department staff ( eds ) , emergency department environment ( ede ) , physician care satisfaction ( pcs ) , general patient satisfaction ( gps ) , and patient s family s satisfaction ( pfs ) . after rotation , these domains accounted for 16.1% , 15.1% , 14.5% , 13.6% , and 11.4% of the total variance . one - way anova and t - test detected no significant difference in satisfaction of patients differentiated by gender , education , post - examination status , and responding person ( p>0.05 ) . however , those patients who were visited with shorter waiting times were significantly more satisfied ( f=10.267 ; p<0.001 ) as predicted ( table 5 ) . finally , the overall satisfaction score was inversely associated with waiting time ( r=-0.295 , p<0.01 ) . the results of exploratory factor analysis ( rotated component matrix ) corresponding loadings are bolded , emergency department staff ( eds ) , emergency department environment ( ede ) , physician care satisfaction ( pcs ) , general patient satisfaction ( gps ) , and patient s family satisfaction ( pfs ) . n = number , emergency department staff ( eds ) , emergency department environment ( ede ) , physician care satisfaction ( pcs ) , general patient satisfaction ( gps ) , and patient s family satisfaction ( pfs ) . emergency department staff ( eds ) , emergency department environment ( ede ) , physician care satisfaction ( pcs ) , general patient satisfaction ( gps ) , and patient s family satisfaction ( pfs ) . brief emergency department patient satisfaction scale permission to use this measure is not required ; however , seek permission if any item is modified for use in research . for each item , the following response scale should be used : 1 = completely disagree , 2 = mildly disagree , 3 = mildly agree , 4 = completely agree . following this study , brief emergency department patient satisfaction scale ( bepss ) was developed to evaluate the ps . this scale involves 20 items that score 1 to 4 and are categorized in 5 domains ( eds , ede , pcs , gps , and pfs ) . the satisfaction of ed clients can not be achieved without research and an organized way of assessment in the field ( 17 ) . so , this topic has been considered by researchers for many years to find an appropriate worldwide scale , but the search still goes on ( 18 , 19 ) . variety of cultural effective factors in different countries and even different area may be one of the reasons of limitation for using the current questionnaires ( 20 , 21 ) . in this manner , thus , assessment of ps within emergency departments of iranian hospitals was in need of a reliable and valid instrument ( 22 ) . secondly , cross - cultural differences may play a central role in perception of healthcare quality from the viewpoint of patients ( 23 ) . using unrelated instruments such as inpatient / outpatient satisfaction tools also runs the risk of low face and content validity ( 24 ) . therefore , a specific scale for measuring ps in eds seems essential as emergency patients have complicated and specific situations . one of the problems associated with above - mentioned patients is a lack of time ( 25 ) . therefore , brevity should be considered quite noteworthy . additionally , adopting non - validated approaches of measurement measures of ps should adhere to basic principles of psychometric measurement ( 15 , 26 ) . a study analyzed 195 studies of ps and concluded that authors demonstrated a poor understanding of the importance of core measurement properties required if a measure is to assess satisfaction with confidence ( 26 ) . bepss made an effort to incorporate all effective factors of ps into an integrated measure in order to assess ps in emergency departments . it was illustrated that delay ( waiting time ) in ed is significantly associated with dissatisfaction of patients . two delay - related items ( 20 and 21 ) are present in bepss , which are loaded in the general patient satisfaction ( gps ) domain . periodic assessment of ps , as a critical indicator in healthcare quality , seems centrally important . strategies for quality - improvement purposes are made upon figures derived from the process of assessment . widening the target population on a national level could have strengthened the results of the study ; especially its generalizability . since the primary properties of the scale are very good , this tool calls for further validation across the country . it seems that bepss , as a newly developed instrument with highly satisfactory psychometric properties , can be used for the assessment of emergency department s patient satisfaction . all authors passed four criteria for authorship contribution based on recommendations of the international committee of medical journal editors .

vision serves as a vital function ensuring the feeling of control over the immediate surroundings and creating a sense of contact with the world . therefore , the fact does not render it peculiar that cases connected with vision disturbances and eyesight loss entail heightened anxiety and the fear for autonomy limitation [ 13 ] . statistical data prove that the risk of developing cataract , glaucoma , and other ophthalmological disorders increases with age [ 4 , 5 ] . cataract affects almost 20% of people between the ages of 65 and 74 and for 50% of those who are between 75 and 84 years of age becomes the main factor leading to blindness [ 6 , 7 ] . the risk of developing glaucoma , on the other hand , occurs in 2% population segment of forty - five - year - olds and older patients , increasing markedly in the case of people at an advanced age . due to the fact that initial stages of the disease development are difficult to diagnose and since the diagnosis itself may lack precision , a strong feeling of anxiety can arise [ 810 ] . low vision is one of the most important reasons of lowered quality of life , anxiety , and poor adaptation , especially in older patients who have two kinds of problems , relating to their health and age [ 1113 ] . the research projects on the patient groups focus predominantly on establishing the quality of life pattern and on examining changes both in the paradigm and in the vision - related functional status after the treatment . only a fraction of these researches dealt with such variables as depression and anxiety , expectations concerning the future , the feeling of loneliness , or self - esteem [ 15 , 16 ] . the importance of psychological variables such as strategies of coping or anxiety intensification factor has been duly underlined in glaucoma research due to the fact that the concealed development of the disease and the possibility of unfavourable prognosis may potentially lead to the development of the feeling of anxiety [ 8 , 17 ] . according to current research results both socioeconomic factors and psychological variables influence quality of life in elderly patients with vision disturbances [ 2 , 1820 ] . this study has been intended to constitute a probe into coping styles in cases of patients with vision disturbances to demonstrate interrelationships existing between the emerging pattern and quality of life assessment in cataract- and glaucoma - affected patients . the problem can be formulated in the following questions.what styles of coping do patients with vision disturbances adopt?what is the adaptive value of the styles of coping exhibited by elderly patients with vision disturbances who undergo cataract and glaucoma treatment ? what styles of coping do patients with vision disturbances adopt ? what is the adaptive value of the styles of coping exhibited by elderly patients with vision disturbances who undergo cataract and glaucoma treatment ? quality of life is defined as an individuals ' perception of their position in life in the context of the culture and value systems in which they live and in relation to their goals , expectations , standards , and concerns additionally , the levels of self - esteem and such clinical variables as anxiety , depression , and loneliness were also diagnosed . its goal was to detect the styles of coping most frequently assumed in this group of patients and to test their adaptive value . in light of the literature on the subject , task - oriented coping and adaptive flexibility figure as having adaptive value . therefore , it was postulated that h1 : moderate mobilization of different styles of coping showing clear tendency towards employing task - oriented coping has adaptive value and that h2 : coping based on emotion - oriented and avoidance - oriented coping has lesser adaptive value indicated by relatively low quality of life , low self - esteem , and a heightened level of anxiety , hopelessness , and loneliness . the sample consisted of 237 patients ( mean age : m = 67,8 ; sd = 9,5 ) including 188 cataract and 49 glaucoma patients . a time frame for the recruitment of the participants was one year , and they were consecutive patients , except those who refused to participate in this investigation due to their poor well - being or lack of motivation . since there were no major differences regarding quality of life or coping styles , both glaucoma patients and cataract patients were treated as a unit irrespective of their sex . coping styles were investigated by means of the coping inventory for stressful situations ( ciss ) by endler and parker . the inventory consists of three main scales : task - oriented coping ( toc ) , emotion - oriented coping ( eoc ) , and avoidance - oriented coping ( aoc ) , with the last one with two subscales : engagement in substitute activity ( esa ) and seeking for social relationships ( ssr ) . cronbach alpha for three main scales checked in a sample of elderly patients with low vision ( n = 50 , 25 male and 25 female ) is 0.81 , 0.78 , and 0.74 , respectively , and for subscales it is 0.60 and 0.67 . quality of life was measured with the help of the quality of life questionnaire by schalock and keith . the questionnaire consists of 40 items measuring the quality of life in four domains , each comprising 10 items : satisfaction , competence / productivity , empowerment / independence , and social belonging / community integration . the fifth domain was added to the present research : health related quality of life ( 5 items ) . the questionnaire is based on an interview containing 45 questions , each with 3 possible answers scored 1 , 2 , or 3from low to high quality of life . reliability of the quality of life questionnaire ( 40 items ) , checked in cataract sample ( n = 50 , 25f and 25 m ) , is cronbach alpha = 0.86 and for each scale 0.81 , 0.86 , 0.58 , 0.50 , and 0.79 , respectively . the following scales were used to assess other variables indicating psychological functioning of the patients : self - esteem scale ( ses ) by rosenberg , state - trait anxiety inventory ( stai ) by spielberger and reheiser , hopelessness scale ( hs ) by beck et al . , and loneliness scale revised ( ucla ls - r ) by russell et al . . in order to isolate subgroups of patients employing distinct styles of coping , cluster analysis based on k - means method and conducted on the standardized scores in three main scales of ciss by endler and parker was carried out . three groups of patients exhibiting different coping styles were thus singled out : patients exhibiting high mobilization for coping with emotion - oriented and avoidance - oriented coping prevalent ( n = 89 , including 68 cataract patients and 21 glaucoma subjects);patients exhibiting task - oriented coping ( n = 69 , including 59 cataract patients and 10 glaucoma subjects);patients exhibiting low mobilization for coping ( n = 79 , including 61 cataract patients and 18 glaucoma subjects ) . patients exhibiting high mobilization for coping with emotion - oriented and avoidance - oriented coping prevalent ( n = 89 , including 68 cataract patients and 21 glaucoma subjects ) ; patients exhibiting task - oriented coping ( n = 69 , including 59 cataract patients and 10 glaucoma subjects ) ; patients exhibiting low mobilization for coping ( n = 79 , including 61 cataract patients and 18 glaucoma subjects ) . according to ciss scales , all of the differences emerging between the groups were significant ( p < 0.001 ) ( cf . interestingly enough in all three groups three main coping strategies were used to a lesser or higher degree . in the first group , avoidance - oriented coping and emotion - oriented coping appear to be concurrent with each other . furthermore , the patients belonging to this group achieved comparatively high scores as far as task - oriented coping is concerned , and this suggests flexibility of coping . the second group demonstrated task - oriented coping combined with the inclination to search for and employ social support . in the third group , the division of thus delineated groups with respect to quality of life revealed several crucial differences ( table 2 ) . the second group , namely , patients characterized by task - oriented coping , evidently reveals higher quality of life in contrast to the other two groups . this fact pertains not only to the overall result achieved on the total quality of life scale , but also to the results obtained in other two domains , that is , empowerment / independence and social belonging / community integration . it is also worth noting that , as for variables describing quality of life , there were no significant differences between the first group of patients with high mobilization for coping and the third group of patients with low mobilization for coping . further analysis aims at exploring the adaptive value of the styles of coping , respectively . on the basis of all the results presented so far , it transpires that task orientation can hold a higher adaptive value than the generally heightened as well as decreased mobilization for coping . such an interpretation is plausible in light of higher quality of life in the majority of task - oriented patients in contrast to subjects belonging to the remaining two groups . but why is it the case ? in order to answer the question , the levels of anxiety ( measured by means of state - trait anxiety inventory ( stai ) ) , of hopelessness ( hopelessness scale ( hs ) ) , of loneliness ( ucla loneliness scale revised ( ls - r ) ) , and of self - esteem ( self - esteem scale ( ses ) ) were compared in the three selected groups . the juxtaposition of mean results in the scales measuring aforementioned clinical variables indicated that anxiety , hopelessness , and loneliness levels were markedly lower in the group of patients using mainly task - oriented coping ( table 3 ) . from a statistical point of view , there existed no significant differences between the first group ( with high mobilization and emotion - avoidance orientation ) and the third group ( with low mobilization for coping ) . for example , intensity of hopelessness ( hs ) reached a clinical level in both of the groups . the level of self - esteem was higher in the group of task - oriented patients in comparison with the two remaining groups : with high and low mobilization for coping ( there was no significant difference between the mean results in both groups ) . participants with high and low mobilization for coping had lower self - esteem than task - oriented patients and higher level of clinical variables such as anxiety , hopelessness , and loneliness . this implies that high mobilization for coping was not necessarily an adaptive orientation for patients with vision disturbances as well as the absence of mobilization in confrontation with stress . research results seem to emphasize the fact that not only mobilization embodying all the styles of coping which implies flexibility has adaptive value . for adaptive functioning , two factors appear to be decisive : the effort put into the process of coping and the apt guiding of the process towards task - oriented coping . moreover , the tendency towards searching for social support was also exhibited and it could be interpreted as task - oriented in the case of elderly persons with visual disturbances to keep and develop social support seems a big challenge for the elderly and the sick . the conducted studies once again attested to the adaptive value of task - oriented coping and in general an active life in old age [ 19 , 29 , 30 ] . patients adhering to this style exhibit higher quality of life and a lower level of anxiety , pessimism , and loneliness unlike subjects adhering to coping style based on emotions or avoidance . furthermore , it was discovered that , to a certain extent , task - oriented coping entails searching for and employing social support , which , on account of the patient age and vision disturbances involved , appears to show adaptive value [ 29 , 31 , 32 ] . this conclusion together with the interrelation between coping and its adaptive value , on the one hand , and self - evaluation , on the other hand , is not characteristic of persons with vision disturbances exclusively . however , the result suggesting that both high mobilization for coping and the absence of mobilization seem to be equally nonadaptive becomes distinctive for the examined group of patients . the former case is often accompanied by noneffective exploitation of personal resources and the latter by passivity and the adoption of a resigned attitude which can be moderated by negative affectivity or depression [ 16 , 35 ] ; low quality of life cooccurring with an increasing level of anxiety , pessimism , loneliness , and lowered self - esteem is often generated in either of the cases . most probably , not so much the effort engaged as coping directed towards the task - oriented style and pertinent mobilization of the support of close persons , appropriate groups , and relevant institutions which accords with positive self - evaluation and self - esteem proves decisive in the process of adaptation throughout illness . the ability of engaging oneself in substitute activities in place of disintegrated routine tasks appears to be equally important ( e.g. , concentrating on listening to the radio rather than watching television , involvement in gardening instead of reading papers , or going to the cinema ) . whenever the psychological corollary of disease symptoms developed due to cognitive functional disturbances especially those related to visual perception results in the loss of self - confidence , deterioration of social relations , poor performance of social or professional roles , the decline in the family status , and the development of negative emotions such as anxiety , tension , or lower self - esteem , the effectiveness of coping turns out to be vitally important [ 3 , 1517 , 3739 ] . however not all studies support the conclusion that visual impairment is necessarily linked to well - being or depression . one should remember also about individual differences not only in effectiveness of given coping style but also in temperament as well as in defining life domains which are important to the patients and which constitute their quality of life [ 41 , 42 ] . at the same time when faced with tense situations , they cope employing whatever methods are available or simply give up . the ability to retain self - esteem favours strengthening task - oriented coping and the ability to take advantage of social support , which , on its part , is conducive to lowering the anxiety , depression , and loneliness threshold . there are some limitations of this study ; first , this study , which is typical for questionnaire approach , based on self report data ( and not verified , e.g. , by objective observation ) , second , the research was conducted in one country ( poland ) , and , third , socioeconomic status of elderly people ( rather low in most cases ) could influence the results , especially assessment of quality of life , depression ( i.e. , hopelessness ) , or anxiety and loneliness . while we have distinguished a few groups of patients different in coping with stress related to their illnesses , one can conduct similar analysis taking temperamental features as a criterion for division of the groups , providing also a relationship between coping , quality of life , and temperament . in fact , empirical data suggest that patients differ also in their psychological functioning due to specific temperamental features it seems to be an interesting topic for further research , especially when such a study would be focused on how the patients face the illness as well as on more practical issue , namely , how to organize and propose better care for elderly people with vision disturbances . the conclusions to be drawn from the presented study are as follows.patients who follow the pattern of task - oriented coping and who take advantage of social support are characterized by higher quality of life , higher self - esteem , and comparatively lower level of anxiety , pessimism , and loneliness . this proves task - oriented coping to be of greater adaptive value than passivity in stress exposure situations and a generally heightened mobilization for coping in emotion - avoidance pattern , in particular.the results hint at the significance of social support and at the value of interpersonal relationships for quality of life in elderly persons with vision disturbances such as glaucoma or cataract , provided that task orientation is involved . the tendency towards searching for interpersonal interactions as a way of coping detected in the task - oriented group of patients can be plausibly accounted for . in the case of elderly persons coping with their illnesses , seeking and employing the support of other people are not exclusively of adaptive value , but they also appear to constitute a definite task enabling them to function on a daily basis.both demobilization and high mobilization for coping appear to be nonadaptive styles of functioning which entail lowered quality of life , lowered self - esteem , and increased level of pessimism , loneliness , and anxiety . patients who follow the pattern of task - oriented coping and who take advantage of social support are characterized by higher quality of life , higher self - esteem , and comparatively lower level of anxiety , pessimism , and loneliness . this proves task - oriented coping to be of greater adaptive value than passivity in stress exposure situations and a generally heightened mobilization for coping in emotion - avoidance pattern , in particular . the results hint at the significance of social support and at the value of interpersonal relationships for quality of life in elderly persons with vision disturbances such as glaucoma or cataract , provided that task orientation is involved . the tendency towards searching for interpersonal interactions as a way of coping detected in the task - oriented group of patients can be plausibly accounted for . in the case of elderly persons coping with their illnesses , seeking and employing the support of other people are not exclusively of adaptive value , but they also appear to constitute a definite task enabling them to function on a daily basis . both demobilization and high mobilization for coping appear to be nonadaptive styles of functioning which entail lowered quality of life , lowered self - esteem , and increased level of pessimism , loneliness , and anxiety .

oral lichen planus ( olp ) is a t - cell - mediated chronic inflammatory oral mucosal disease of unknown etiology , but it is believed to result from an abnormal t - cell - mediated immune response in which basal epithelial cells are recognized as foreign body because of changes in the antigenicity of their cell surface . the lymphocytic infiltrate in olp is composed almost exclusively of t - cells , and the majority of t - cells within the epithelium and adjacent to damaged basal keratinocytes are activated cd8 + lymphocytes . olp is diagnosed on the basis of the presence of wickham 's striae that produce either a lacelike pattern or radiating lines that can be faint or prominent , which presents a symmetrical and bilateral distribution on the buccal mucosa . they appear as white papules , plaques , erythema , erosions or blisters , affecting predominantly the buccal mucosa , tongue and gingivae . lesions are usually bilateral , and atrophic and erosive lesions are often sensitive or painful . alternative treatments include retinoids , ultraviolet phototherapy , steroid - sparing agents ( hydroxychloroquine , azathioprine , mycophenolate mofetil ) and pimecrolimus . although the above - mentioned drugs have shown positive results in the treatment of olp , resistance to treatment and a high risk of toxicities limit their use . deerfield , il , usa ) is an immunosuppressive agent currently available worldwide for the prevention of organ transplant rejection . in recent years , tacrolimus ointment 0.1% and cream 0.1% ( protopic ; fujisawa inc . ) have received fda approval for the treatment of atopic dermatitis in children , as has pyoderma gangrenosum because of its clinical benefits and safety profile . tacrolimus is 10100-times as potent as cyclosporine in its ability to inhibit il-2 mrna synthesis , and it inhibits mediator release from basophils and mast cells . it inhibits enzyme calcineurin phosphatase activity , resulting in decreased il-2 synthesis and secretion hence inhibiting t cell multiplication [ figure 1 ] . with such an insight , this investigation was performed to assess the efficacy of 0.1% tacrolimus ointment with oraguard - b paste for the treatment of patients with symptomatic olp . colgate oragard - b paste is a powerful reliever and mucoadhesive with a protective barrier to give your patients fast pain relief . this study was conducted at the department of oral medicine and radiology after receiving ethical clearance from the institute , with 150 patients participating in the study . a minimum data set ( patient age , gender , medical history and habits ) [ table 1 ] was documented by means of case history performa and clinical photographs of the affected site were taken . demographics of patients with oral lichen planus patients with clinical evidence of the lesion followed by histopathological confirmation were included in the study . patients on medication for other systemic diseases or with any history of renal , hepatobiliary or malignant disease , uncontrolled hypertension or recurrent acute infection , etc . , were excluded from the study group . the patients subjective assessment for symptoms of pain and burning was performed by means of a visual analogue scale ( vas ) [ table 2 ] . the pain vas is a unidimensional measure of pain intensity , which has been widely used in diverse adult populations . the pain vas is a continuous scale comprised of a horizontal ( hvas ) or vertical ( vvas ) line , usually 10 cm ( 100 mm ) in length , anchored by two verbal descriptors , one for each symptom extreme . instructions were given to the patient that vas consists of a 10 cm line on which 0 cm is no pain and 10 cm is pain as bad it could be . the patient is asked to mark the point along the line to know his best experience of pain score and then it is measured from no pain to the worst pain till the end of the scale . visual analogue scale ( vas ) ( 0 - 10 ) the extent and types of lichen planus were recorded by using a scoring system [ table 3 ] . in this study , patients were provided with 0.1% tacrolimus ointment with colgate oraguard - b paste for the study medication . patients were asked to use the medication over the symptomatic areas three times a day until resolution of the lesion . the wilcoxon signed - rank test was performed , which is a nonparametric statistical hypothesis test used when comparing two related samples , matched samples or repeated measurements on a single sample to assess whether their population mean ranks differ ( i.e. it is a paired difference test ) . a total of 150 patients were enrolled in the study , of whom 43 patients had erosive olp , 56 patients had reticular olp , 34 patients had atrophic olp and 17 patients had mixed lesions . the vas was used to assess the improvement in pain intensity among different forms of lichen planus by recording the pre- and posttreatment values [ table 2 and graph 1 ] . the demographic data were recorded along with type and site of lesion given in table 1 and clinical scoring in table 3 . pre- and post - treatment response of tacrolimus therapy in different types of oral lichen planus the clinical response of 150 patients with different types of olp was recorded following topical application of tacrolimus therapy for 2 years and 6 months . in 150 patients , 71 ( 47.33% ) patients had complete remission or complete reduction in size of lesion after tacrolimus therapy for the duration of 1325 months . sixty - five ( 43.33% ) patients had partial remission of lesion after tacrolimus therapy for the duration of 1528 months [ figure 2 ] . fourteen ( 9.33% ) patients had no response or recurrence after tacrolimus therapy for a duration of 46 months . ( 1a ) atrophic lichen planus ( pre - operative ) , ( 1b ) atrophic lichen planus ( post - operative ) , ( 2a ) mixed lichen planus ( pre - operative ) , ( 2b ) mixed lichen planus ( post - operative ) , ( 3a ) erosive lichen planus ( pre - operative ) , ( 3b ) erosive lichen planus ( post - operative ) ( 4a ) reticular lichen planus ( pre - operative ) , ( 4b ) reticular lichen planus ( post - operative ) the values of the vas , both pre- and posttreatment , for male and female patients in terms of mean standard deviation and wilcoxon signed - rank test to test the significant differences in pre- and postobservational scores are listed in table 4 . the pre- and post - vas values were compared and statistically highly significant difference ( p < 0.001% ) in mean vas and clinical scores was observed . topical tacrolimus is one of a new class of medications , the noncorticosteroid topical immunomodulators . the present study significantly extends previous reported findings regarding the role of 0.1% topical tacrolimus ointment in the management of symptomatic erosive / ulcerative olp by both increasing the number of treated patients and duration of therapy . tacrolimus ointment in 0.1% orabase paste appears to be an effective and promising alternative in the treatment of patients with olp . colgate oragard - b paste is a powerful reliever and mucoadhesive with a protective barrier to give fast pain relief to the patient . tacrolimus , also a calcineurin inhibitor , is a steroid - free topical immunosuppressive agent approved for the treatment of atopic dermatitis . adverse effects associated with the therapy were mild and transient ; they were limited only to a burning sensation . the reason for fewer adverse effects may be attributed to the fact that compounds having a mass unit greater than approximately 500 da and scarcely penetrate the epidermis or epithelium of normal skin mucosa . inflamed mucosa , due to increased permeability , allows penetration of molecules of higher molecular weight such as tacrolimus 823 da . once the inflammation ( and permeability ) decreases and the lesion improves due to the anti - inflammatory activity of topical tacrolimus , the penetration of the compound into the epithelium decreases , thereby limiting the potential side - effects of this particular regime . tacrolimus treatment is revealed as an effective and secure alternative due to its low systemic absorption and the low incidence and importance of its secondary effects . this study was designed to determine the safety of tacrolimus with colgate orabase in 150 patients for a period of 2 years and 6 months . in our study , topical tacrolimus was found to be safe and effective in all the 150 patients who participated in the study . in the present study , the demographic characteristics of the patients were taken on the basis of gender , age , type and site of olp [ table 1 ] , which were similar to a previously reported study . in the present study , patient 's assessment for the symptoms of pain and burning sensations was recorded by means of a vas . the intensity of pain and burning sensation was worst , scoring 910 by vas assessment during the first visit and , after 2 months of therapy , the pain and burning sensation was drasticallyreduced up to 12 [ table 2 ] . this is because tacrolimus blocks the activation of t - lymphocytes by targeting calcineurin , an important activator of t - lymphocyte , and colgate oragard - b paste , which is a powerful reliever and mucoadhesive , retains the contact of drug for a longer duration . in the present study , 47.33% ( 71 of 150 patients ) had complete resolution , including erosive , ulcerated and even the reticular form of olp . partial remission in the symptoms of burning and pain were observed in 43.33% ( 65 of 150 patients ) patients . in 9.33% ( 14 of 150 patients ) patients , there was recurrence of the lesion in erosive and atrophic type of olp , but the intensity of recurrence observed was mild and the patients were symptomless [ table 5 ] . few studies have shown the role of tacrolimus therapy in the treatment of symptomatic reticular , erosive and atrophic olp . clinical response of 150 patients with oral lichen planus following topical tacrolimus therapy for 2 years and 6 months in the present study , oraguard - b has been a promising base , showing good mucoadhesive properties through patients responses and is a powerful reliever with a protective barrier to give your patients fast pain relief . because the study was nonblinded and noncomparative , whether topical tacrolimus can be used as the first line of treatment or used in other concentrations needs to be assessed . in the present study , oraguard - b has showed promising base through patients responses due to its good mucoadhesive properties , protective barrier and powerful reliever to give patients fast relief from pain . the encouraging results of this study lead us to conclude that topical tacrolimus ointment 0.1% with oraguard - b paste is an effective treatment for symptomatic olp . however , the effect is temporary and , when topical tacrolimus is discontinued in a few patients , the lesion may flare again .

hyoscine - n - butyl bromide ( hbb ) is a widely used antispasmodic and anticholinergic drug belonging to the belladonna alkaloid class of semisynthetic quaternary ammonium compounds . these compounds exert a spasmolytic action on the smooth muscles of the bile - gastrointestinal tract,1 the genitourinary tract , the salivary glands ( xerostomia),2 and also on the visual apparatus at an ocular level - in particular , the irido - ciliary complex . as a consequence , they provoke collateral effects such as mydriasis and accommodation disturbances.3,4 we report the case of a 23-year - old woman who complained of accommodation disturbances and a diplopia upon oral administration of hbb . the patient ( 23 years old ) attended the department of sense organs , university of rome la sapienza , rome , italy 2 hours after administration of one pill of buscopan ( boehringer ingelheim , ingelheim , germany ) complaining about double vision in the primary position and cloudy vision in the left eye ( le ) more than in the right eye ( re ) , in addition to dry mouth . the patient was quickly sent to our ocular motility center , where she underwent an ophthalmologic examination 2 hours after drug administration . the examination included the following tests : best refraction test for far and near vision test of the anterior segment through slit - lamp biomicroscope fondus oculi test through indirect schepens ophthalmoscopy goldmann applanation tonometry . during each examination , ocular motility was analyzed through a corneal reflex test , cover test , diplopia trial , wirt test , worth test , and hess screen . laboratory examinations , radiological analyses , and neurologic and internistic visits were carried out in order to exclude the most frequent causes of diplopia in adulthood such as virus infection , hypertension , diabetes , toxic factors , and neurological or demyelinating diseases.57 the previous ophthalmologic visits in other medical centers had shown a natural visual acuity of 10/10 in both eyes and an ocular motility without any particular alteration . at our first check ( 2 hours after drug administration ) , the patient presented visual acuity in the re of 10/10 and in the le of 8/10 . in the last one , visual acuity reached 10/10 after the addition of + 1d spherical lens . in both eyes , examination of the anterior segment detected pupil anisocoria ( le > re).8 intraocular pressure and fundus examination were normal . the ocular motility tests detected a slight vertical deviation increasing in the sight downward and decreasing in an anomalous position . the hess screen revealed a slight deficit of the superior oblique muscle of the le , and the bielschowsky test was positive . at the second check , after 2 days , natural visual acuity was 10/10 in both eyes , without the need to correct the hypermetropia of the le detected in the previous check . with regard to ocular motility , the diplopia test with red filter on the re revealed a higher diplopia of the sight downward and to the right with white lower light . specifically , a hypofunction of the superior oblique muscle of the le was evident and a hyperfunction of the inferior rectus muscle of the re ( figure 1 ) . also present was a hyperfunction of the inferior oblique muscle of the le and a hypofunction of the superior rectus muscle of the re . the bielschowsky sign was positive , with consequent anomalous position of the head inclined to the right . the worth test highlighted a diplopia at near distance , which was compensated for by the patient with a modification of the head position . the laboratory analyses ( neurological and radiological ) excluded toxic factors and systemic neurological or demyelinating pathologies of adult diplopia ( either chronic or acute ) . at the third check , after 1 week , visual function was full and all alterations of ocular motility ( slight hypofunction of the superior oblique muscle of the le and slight hyperfunction of the inferior rectus of the re ) were not observed ( figure 2 ) . at the fourth check , carried out after 1 month , the clinical picture was as in the third check . hbb , an antispasmodic and anticholinergic drug belonging to the belladonna alkaloid class of semisynthetic compounds , as a quaternary ammonium derivative , does not involve the central nervous system , as it does not cross the hematoencephalic barrier.8,9 in the reported case , the anticholinergic effect acted at a peripheral level ( through blocking the locoregional ganglia and antimuscarinic activity ) , causing , in addition to oral drying , the ocular disturbances recorded at the first check : mydriasis , accommodation disorders,3 and , as a consequence , diplopia . because hbb , in general , does not act directly on the skeletal muscle tissue , most likely the observed temporary diplopia was not due to a direct action of the drug on the oculomotor muscles . it is possible that it could have been due to the cycloplegic effect on the accommodation of le , which was able to alter the mechanism of sensory - motor fusion and highlight a hypofunction of the superior oblique muscle of the le . the latter was evident either by the positivity of biesholwky sign or by the red filter diplopia trial . such a hypothesis is supported by the fact that even 1 month after the disappearance of symptoms and diplopia , the persistence of the functional alteration of the oculomotor muscles was not confirmed . it could be supposed that the le , in normal conditions , by exerting an accommodation power of + 1d , is able to maintain the function of the oculomotor muscles of both eyes in a balanced condition , allowing them to remain in axis in all sight positions . in the reported case , it is likely that hbb was the causal factor of the diplopia , due to a partial block of the accommodation . as a consequence , because a latent hypermetropia was present in the le , the fusional balance ( sensory and motor ) maintaining the orthophoria was probably broken . furthermore , the consequent diplopia of the patient was peculiar because instead of the most common horizontal deviation there was a transient , vertical deviation associated with a hypofunctionality of the superior oblique muscle of the le and a secondary hyperfunctionality of the inferior rectus muscle of the re .

two major vascular pathologies underlie brain damage in patients who have important abnormalities involving small size penetrating brain arteries and arterioles ; 1 ) thickening of the arterial media with encroachment on the arterial lumens and 2 ) obstruction of the origins of penetrating arteries by parent large intracranial artery intimal plaques . hypertension , diabetes and other as yet undetermined genetic and other factors may promote thickening of the media of penetrating arteries by fibrinoid deposition and hypertrophy of smooth muscle and other connective tissue elements . the arterial media can also contain foreign deposits as in amyloid angiopathy and genetically mediated conditions such as cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy ( cadasil ) . miller fisher , nearly a half century ago , analyzed the arterial pathology that caused small deep infarcts by carefully assessing serial sections of vessels at necropsy.1 he observed that the penetrating arteries that supplied the territory of lacunar infarcts showed a characteristic vascular pathology . these tiny vessels often contained focal enlargements and small hemorrhagic extravasations through the walls of these arteries . subintimal foam cells sometimes obliterated the lumens , and pink - staining fibrinoid material was present within the vessel walls . the arteries in some regions were often replaced by whorls , tangles , and wisps of connective tissue that obliterated the usual vascular layers . fisher called these processes segmental arterial disorganization , fibrinoid degeneration , and lipohyalinosis.1 figure 1 , given to me by fisher , shows a severely narrowed penetrating artery in a patient with severe hypertension . the media is hypertrophied and contains fibrinoid material . when fisher reviewed the charts of 114 patients who had lacunes at necropsy , all but three patients had hypertension defined by a prior history of high blood pressures , elevated blood pressure recorded on examination , or heart weight exceeding 400 g without another explanation.2 the onset of the clinical symptoms of brain infarction often developed at the onset or hypertension or during periods of an increase in blood pressure values . fisher attributed segmental arterial disorganization and lipohyalinosis to hypertension . at the time of his studies , performed nearly a half century ago , there was little effective treatment for hypertension and hypertensive crises and hypertensive encephalopathy were common . later epidemiological studies of patients with small deep infarcts has shown that the presence of hypertension , although very common , seemed no different than the frequency in patients with large artery extracranial and intracranial occlusive disease , raising doubt about the crucial importance of high blood pressure in causing the vascular changes that caused these small deep infarcts . the role of diabetes , hyperlipidemia , and aging in causing penetrating artery lesions remains uncertain . curiously , necropsy examination of patients with chronic small disease does not show evidence of thrombosis or past thrombotic occlusion of these diseased arteries.3 the other major pathology that directly involves the walls of penetrating arteries is infiltration of the coats of these vessels with materials foreign to vessels . the penetrating arteries , arterioles and capillaries in a hereditary condition dubbed cadasil contain a granular material in the media that extends into the adventitia . this disorder is caused by dominant mutations in the notch3 gene that encodes a transmembrane receptor predominantly expressed in vascular smooth muscle and pericyte cells in adults.4,5 this receptor is needed for the structural and functional integrity of small vessels - small arteries , arterioles and capillaries . periodic acid - schiff ( pas ) staining suggests the presence of glycoproteins , but staining for elastin and amyloid have been negative . figure 2 is a pas stained section that shows glycoprotein material within vessel walls in a cadasil . the endothelium may be absent and replaced by collagen fibers . at times abnormalities also found in hypertensive patients are also found , including duplication and splitting of the internal elastic lamina , adventitial fibrosis and hyaline change , and fibrosis and hypertrophy of the arterial media . another hereditary angiopathic condition , now known to be associated with mutations in a gene that encodes procollagen type iv alpha 1 ( col4a1 ) , has been identified . this genetic mutation affects small brain arteries as well as larger retinal and cerebral arteries.5,6 cerebral amyloid angiopathy ( caa ) affects small arteries and arterioles in the leptomeninges and cerebral cortex ; involved arteries are thickened by a acellular hyaline material that stains positively with periodic acid - schiff stains , and has an apple - green birefringence with polarized congo red stain.7,8 arteries and arterioles in the basal ganglia and deep cerebral white matter also show thickened media but these arteries do not contain amyloid . at times , caa predominantly affects persons older than 65 years and increases in frequency in the eighth and ninth decades . the other type of pathology that causes small deep infarcts involves the large arteries that give rise to penetrating artery branches rather than intrinsic disease of the branches themselves . fisher and caplan,9 fisher,10 and caplan11 described the vascular pathology in these branches , and dubbed the condition intracranial branch atheromatous disease . figure 3 contain cartoons that illustrate the location and mechanism of the pathology within the parent arteries . the orifices of the penetrating branches could be blocked by atheroma in the parent artery , atheroma could originate in the parent artery and extend into the branch ( so - called junctional atheromatous plaques ) , or microatheromas could arise at the origin of the branch itself . thrombus was sometimes superimposed on the atheromas and occasionally a microdissection developed in the parent artery and spread into the first millimeters of the branch.9 it is now possible to image intracerebral branch atheromatous disease using high resolution mri . plaques in the middle cerebral artery and basilar artery can be shown to impinge upon or occlude penetrating branches by mri techniques that show axial sections of the origins of branches from the parent arteries . the location within the parent artery is critical in blocking lenticulostriate , thalamostriate , and basilar artery branches.12 the major related pathology in the brain are small deep infarcts and degenerative abnormalities in the cerebral and cerebellar white matter . lacunar infarcts are small , discrete , often irregular lesions , ranging from 1 to 15 mm in size . the most common locations of these lacunar infarcts are the putamen and the pallidum , followed by the pons , thalamus , caudate nucleus , internal capsule , and corona radiata . these lesions are not found in the cerebral or cerebellar cortices . at necropsy patients with chronic penetrating artery disease the combination of multiple small deep infarcts and extensive white matter abnormalities has been referred to as binswanger disease,3,13,14 and the clinical findings are often classified as vascular dementia of the small artery type . grossly visible in the cerebral white matter are confluent areas of soft , puckered , and granular tissue . these areas are patchy and emphasize the occipital lobes and periventricular white matter , especially anteriorly and close to the surface of the ventricles.3,13,14 figure 5 is a myelin stained section from the cerebral hemispheres that shows a large area of myelin loss in the cerebral white matter . the white - matter abnormalities surrounding the ventricles may reduce the strength of the supporting tissue and allow mechanically more ventricular distension.3,13,14 the white - matter abnormalities are nearly always accompanied by some lacunes . microscopic examination shows myelin pallor . usually , the myelin pallor is not homogeneous , but islands of decreased myelination are surrounded by normal tissue . at times , the white - matter abnormalities are so severe that necrosis and cavitation occur . two major vascular pathologies underlie brain damage in patients who have important abnormalities involving small size penetrating brain arteries and arterioles ; 1 ) thickening of the arterial media with encroachment on the arterial lumens and 2 ) obstruction of the origins of penetrating arteries by parent large intracranial artery intimal plaques . hypertension , diabetes and other as yet undetermined genetic and other factors may promote thickening of the media of penetrating arteries by fibrinoid deposition and hypertrophy of smooth muscle and other connective tissue elements . the arterial media can also contain foreign deposits as in amyloid angiopathy and genetically mediated conditions such as cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy ( cadasil ) . miller fisher , nearly a half century ago , analyzed the arterial pathology that caused small deep infarcts by carefully assessing serial sections of vessels at necropsy.1 he observed that the penetrating arteries that supplied the territory of lacunar infarcts showed a characteristic vascular pathology . these tiny vessels often contained focal enlargements and small hemorrhagic extravasations through the walls of these arteries . subintimal foam cells sometimes obliterated the lumens , and pink - staining fibrinoid material was present within the vessel walls . the arteries in some regions were often replaced by whorls , tangles , and wisps of connective tissue that obliterated the usual vascular layers . fisher called these processes segmental arterial disorganization , fibrinoid degeneration , and lipohyalinosis.1 figure 1 , given to me by fisher , shows a severely narrowed penetrating artery in a patient with severe hypertension . the media is hypertrophied and contains fibrinoid material . when fisher reviewed the charts of 114 patients who had lacunes at necropsy , all but three patients had hypertension defined by a prior history of high blood pressures , elevated blood pressure recorded on examination , or heart weight exceeding 400 g without another explanation.2 the onset of the clinical symptoms of brain infarction often developed at the onset or hypertension or during periods of an increase in blood pressure values . fisher attributed segmental arterial disorganization and lipohyalinosis to hypertension . at the time of his studies , performed nearly a half century ago , there was little effective treatment for hypertension and hypertensive crises and hypertensive encephalopathy were common . later epidemiological studies of patients with small deep infarcts has shown that the presence of hypertension , although very common , seemed no different than the frequency in patients with large artery extracranial and intracranial occlusive disease , raising doubt about the crucial importance of high blood pressure in causing the vascular changes that caused these small deep infarcts . the role of diabetes , hyperlipidemia , and aging in causing penetrating artery lesions remains uncertain . curiously , necropsy examination of patients with chronic small disease does not show evidence of thrombosis or past thrombotic occlusion of these diseased arteries.3 the other major pathology that directly involves the walls of penetrating arteries is infiltration of the coats of these vessels with materials foreign to vessels . the penetrating arteries , arterioles and capillaries in a hereditary condition dubbed cadasil contain a granular material in the media that extends into the adventitia . this disorder is caused by dominant mutations in the notch3 gene that encodes a transmembrane receptor predominantly expressed in vascular smooth muscle and pericyte cells in adults.4,5 this receptor is needed for the structural and functional integrity of small vessels - small arteries , arterioles and capillaries . periodic acid - schiff ( pas ) staining suggests the presence of glycoproteins , but staining for elastin and amyloid have been negative . figure 2 is a pas stained section that shows glycoprotein material within vessel walls in a cadasil . the endothelium may be absent and replaced by collagen fibers . at times abnormalities also found in hypertensive patients are also found , including duplication and splitting of the internal elastic lamina , adventitial fibrosis and hyaline change , and fibrosis and hypertrophy of the arterial media . another hereditary angiopathic condition , now known to be associated with mutations in a gene that encodes procollagen type iv alpha 1 ( col4a1 ) , has been identified . this genetic mutation affects small brain arteries as well as larger retinal and cerebral arteries.5,6 cerebral amyloid angiopathy ( caa ) affects small arteries and arterioles in the leptomeninges and cerebral cortex ; involved arteries are thickened by a acellular hyaline material that stains positively with periodic acid - schiff stains , and has an apple - green birefringence with polarized congo red stain.7,8 arteries and arterioles in the basal ganglia and deep cerebral white matter also show thickened media but these arteries do not contain amyloid . at times , caa predominantly affects persons older than 65 years and increases in frequency in the eighth and ninth decades . the other type of pathology that causes small deep infarcts involves the large arteries that give rise to penetrating artery branches rather than intrinsic disease of the branches themselves . fisher and caplan,9 fisher,10 and caplan11 described the vascular pathology in these branches , and dubbed the condition intracranial branch atheromatous disease . figure 3 contain cartoons that illustrate the location and mechanism of the pathology within the parent arteries . the orifices of the penetrating branches could be blocked by atheroma in the parent artery , atheroma could originate in the parent artery and extend into the branch ( so - called junctional atheromatous plaques ) , or microatheromas could arise at the origin of the branch itself . thrombus was sometimes superimposed on the atheromas and occasionally a microdissection developed in the parent artery and spread into the first millimeters of the branch.9 it is now possible to image intracerebral branch atheromatous disease using high resolution mri . plaques in the middle cerebral artery and basilar artery can be shown to impinge upon or occlude penetrating branches by mri techniques that show axial sections of the origins of branches from the parent arteries . the location within the parent artery is critical in blocking lenticulostriate , thalamostriate , and basilar artery branches.12 the major related pathology in the brain are small deep infarcts and degenerative abnormalities in the cerebral and cerebellar white matter . lacunar infarcts are small , discrete , often irregular lesions , ranging from 1 to 15 mm in size . the most common locations of these lacunar infarcts are the putamen and the pallidum , followed by the pons , thalamus , caudate nucleus , internal capsule , and corona radiata . these lesions are not found in the cerebral or cerebellar cortices . at necropsy patients with chronic penetrating artery disease the combination of multiple small deep infarcts and extensive white matter abnormalities has been referred to as binswanger disease,3,13,14 and the clinical findings are often classified as vascular dementia of the small artery type . grossly visible in the cerebral white matter are confluent areas of soft , puckered , and granular tissue . these areas are patchy and emphasize the occipital lobes and periventricular white matter , especially anteriorly and close to the surface of the ventricles.3,13,14 figure 5 is a myelin stained section from the cerebral hemispheres that shows a large area of myelin loss in the cerebral white matter . the ventricles are enlarged , and the corpus callosum is usually small . the volume of white matter is reduced , but the cortex is generally spared . the white - matter abnormalities surrounding the ventricles may reduce the strength of the supporting tissue and allow mechanically more ventricular distension.3,13,14 the white - matter abnormalities are nearly always accompanied by some lacunes . microscopic examination shows myelin pallor . usually , the myelin pallor is not homogeneous , but islands of decreased myelination are surrounded by normal tissue . at times , the white - matter abnormalities are so severe that necrosis and cavitation occur . intrinsic disease of a penetrator ( lipohyalinosis , caa , cadasil or other occlusive diseases within the course of the penetrating artery ) causes a small infarct in the center of the penetrating artery supply . collateral circulatory capacity is very limited in the territory of these deep penetrators and occlusive changes in the adjoining penetrators can lead to infarcts that are larger than the territory of the acutely compromised penetrating vessel . blockage of the orifice of branch arteries by a plaque in a parent artery also causes ischemia but often the resulting infarction is larger than that resulting from intrinsic small artery disease . at times plaques can block multiple penetrators . tandem ischemia in the territory of multiple penetrators and hypertensive crises with recurrent white matter edema have been posited . g rosenberg and colleagues that attributes the degenerative white matter abnormalities to chronic leakage of fluid related to increased vascular permeability.15 venous occlusive changes may contribute to the edema . the abnormal components of the vascular walls of penetrating arteries compromise the continence of the vessels to components of the blood that is travelling through the vessels . matrix metalloproteins have an important role in understanding vascular permeability.15,16,17 matrix metalloproteinase-9 levels are significantly elevated in the white matter and cerebrospinal fluid of patients with chronic white matter disease pathology.16,17 matrix metalloproteinases disrupt the blood - brain barrier by degrading tight junction proteins found within blood vessels.17 these increased levels of metalloproteinases in thickened penetrating arteries could promote leakage of fluid from these blood vessels . brown and colleagues found arteriolar tortuosity and described gradual thickening of the walls of periventricular veins and venules with collagen among 186 brains studied at necropsy.18 some veins and venules were occluded and others had very narrow lumens . this chronic venous collagenosis was very extensive in some patients who had leukoaraiosis.18 decreased venous drainage can promote brain edema . support for this theory of pathogenesis is that in cadacil , white matter abnormalities precede brain infarcts .

occipital neuralgia ( on ) refers to a paroxysmal stabbing pain , with or without persistent aching between paroxysms , in the distributions of the greater or lesser occipital nerves . nervus intermedius neuralgia ( nin ) usually provokes a very intense and stabbing pain localized in the depth of the ear canal , which is an uncommon disorder that affects a sensory branch of the facial nerve . these two cranial nerve neuralgias are uncommon and usually occur in isolated and sporadic fashion . only one report described combined occurrence and familial clustering in a swiss family which sparked our interest in reporting the present familial presentation . with regard to familial clustering of cranial neuralgias , it is known that comparatively common trigeminal nerve neuralgia ( tng ) may have a positive family history in 12% cases indicating a possible autosomal dominant ( ad ) inheritance [ 10 , 16 ] . we present a chinese family with five cases of occipital and nervus intermedius neuralgia alone or in combination in three generations , suggestive of an x - linked dominant ( xld ) or an ad inheritance mode . an 89-year - old man presented with a history of episodes of a paroxysmal headache on the right occipital region , superior and posterior to the ear , over the past 40 years . he complained of abrupt onset , sharp , electric - like pain in the right occiput radiating toward the vertex accompanied by an intense and stabbing pain localized in the depth of the auditory canal . the pain in the depth of the right auditory canal did not appear during every episode but only when the occipital pain was severe . the pain attacks usually occurred at the time the patient felt fatigued or when he was afflicted by a cold with or without fever . during the latest episode , the occipital pain was accompanied by symptoms of a herpes zoster and/or herpes simplex reactivation in the territories of the right maxillary and auriculotemporal nerves . specifically , the skin was erythematous and small vesicles appeared on the right side of the face , around the right mouth angle and the right external auditory canal . the frequency of the pain attacks was increasing from once in every 1 or 2 years in the antecedent decade to two or three times per year in recent years . neurological examination revealed normal findings with no hypesthesia in the affected area , no cranial autonomic features , and no occipital skin lesions . pain was eased temporarily by local anesthetic nerve block with lidocaine . magnetic resonance image ( mri ) a diagnosis of on in combination with nin was established based on symptoms which fulfilled the international headache society ( ichd - ii ) criteria . in the antecedent decade , carbamazepine was used at a dosage of 0.3 g daily ( divided into three times ) during the pain attacks and the intensity and frequency of the pain attacks could be eased , but the episodic duration of pain attacks was not reduced . the index patient had a family history of on which had been diagnosed in four other family members . . 1 ) has on in combination with nin , and the two others ( ii . 1 and 2 in fig . 1 ) have on only . the two sons ( ii . 3 and 5 in fig . 1 ) are spared of on and nin , and none of their offsprings ( iii . 4 and 6 in fig . . 1 ) has on alone without combined nin , and the other grandson ( iii . 5 in fig . 1 ) and all the granddaughters are spared of on and nin . in the third generation , filled symbols indicate individuals with occipital neuralgia ; open symbols , unaffected status ; symbols with arrows point to individuals with neuralgia of nervus intermedius . the index patient is underlined . ages of the third and the fourth generations : iii.1 32 years , iii.2 37 years , iii.3 35 years , iii.4 30 years , iii.5 26 years , iii.6 26 years , iv.1 5 years , iv.2 8 years , iv.3 5 years pedigree of occipital and nervus intermedius neuralgia . filled symbols indicate individuals with occipital neuralgia ; open symbols , unaffected status ; symbols with arrows point to individuals with neuralgia of nervus intermedius . the index patient is underlined . ages of the third and the fourth generations : iii.1 32 years , iii.2 37 years , iii.3 35 years , iii.4 30 years , iii.5 26 years , iii.6 26 years , iv.1 5 years , iv.2 8 years , iv.3 5 years the two elder daughters ( ii . 1 and 2 in fig . 1 ) of the index patient developed abrupt - onset , sharp , electric - like pain in the distribution of the occipital nerve at the age of 50 and 55 , respectively . these two elder daughters never had ear canal pain prior to the onset of the electric - like pain in the distribution of the occipital nerve . 1 ) had the same symptoms like her father that began at the age of 43 with electric - like pain in the distribution of the occipital nerve and pain in the depth of the ear canal . the three daughters had these pain symptoms for 12 , 6 and 11 years , respectively with the same pain attack frequency : one episodic attack every 1 or 2 years . the pain attacks occurred predominantly at the time they felt fatigued and at the time they were afflicted by a cold with or without fever . the second daughter also had an intercostal neuralgia caused by herpes zoster recently at the age of 62 . the second daughter also had an mri examination with the result of normal cervical spine and brain image . based on the international headache society ( ichd - ii ) criteria , a diagnosis of on and/or nin was established . all women had an optimal response to carbamazepine treatment during the pain attack , which was similar to their father s . son of the index patient s second daughter was first affected at the age of 33 . he had two episodes of abrupt - onset , sharp , electric - like pain in the distribution of the occipital nerve in the past 4 years . the pain attacks occurred one time when he was very busy with limited sleep time , another time after heavy alcohol consumption . he also had an optimal response to carbamazepine treatment during the pain attack , which was similar to his grandfather s . the international headache society ( ichd - ii ) criteria for on includes : ( 1 ) paroxysmal stabbing pain , with or without persistent aching between paroxysms , in the distribution(s ) of the greater , lesser and/or third occipital nerves ; ( 2 ) tenderness over the affected nerve ; and ( 3 ) pain eased temporarily by local anaesthetic block of the nerve . our index patient and his third daughter fully fulfilled the current ihs criteria for on , and two other family members fulfilled the current ihs criteria for on except one criterion ; namely , a decrease in pain by anesthetic block . most ons are idiopathic , but in rare cases , they may be a result of cervical spine osteochondroma , cavernoma [ 4 , 5 ] , multiple sclerosis , schwannoma [ 2 , 8 ] , meningioma , and myelitis [ 3 , 9 ] . though mri examination was not conducted in all family members , the mri results of the index patient and his second daughter had excluded local pathologies thus indicating idiopathic on . our patient and his third daughter also had combined auditory canal pain with the presence of a trigger area in the posterior wall of the auditory canal which fitted the ichd - ii criteria for nin , thus a diagnosis of on in combination with nin was established in these two cases of this family . microvascular compression is an accepted cause of classic tng but little is known about the etiology of idiopathic on and nin , apart from two articles suggestive of microvascular compression . one article about the anatomy of occipital nerve in a postmortem study showed that occipital artery and occipital nerve crossed each other and an occipital nerve has indentation by the occipital artery at the crossing point in all specimens studied . this may be suggestive of microvascular compression in on , though direct evidence is lacking . a case report showed vascular compression of the nervus intermedius by the anterior inferior cerebellar artery , and the patient s pain was successfully managed with microvascular decompression . thus , on and nin may share the same pathology of microvascular compression with tgn . and immunological abnormalities or infection may function as triggers of this neuralgia , because the neuralgia usually recurred while they were feeling fatigued or had a common cold . this is in consistence with a recent report showing that on was secondary to respiratory tract infection . the most important finding in our cases is that the neuralgia occurred in every generation of a family . this is the second report of familial ons with only one previous report of a swiss family in which a man was affected in the first generation and all women but none of the men were affected in the second generation . in the current report , the same transmission mode was observed but across three generations . our cases combined with the recently reported familial cases suggest that familial on may be characterized by xld or ad transmission . the observation that affected individuals in the current family had optimal treatment responses to the sodium channel blocker carbamazepine leads to the speculation that familial neuralgia may be linked to a sodium channel mutation . for example , the gain - of - function mutation of one particular sodium channel , nav1.7 , causes inherited erythromelalgia , an ad inherited painful neuropathy , and paroxysmal extreme pain disorder . this nav1.7 mutation results in relatively large responses to small , subthreshold depolarizations , i.e. hyperexcitability of drg neurons . thus , we hypothesize that there may exist familial inherited hyperexcitability of drg neurons and that these neurons may be easily responsive to immunological imbalances . further reports of similar cases may help to improve our understanding of the nature of idiopathic neuralgias and to develop optimized therapeutic management .

over twenty unstable and expanded microsatellite repeats have been identified as the cause of human neurological disorders . these repeats , mostly consisting of trinucleotides or tetranucleotides , are considered dynamic mutations ; they possess the unusual characteristic that repeat tract length is variable . most microsatellite repeats show a normal range of relatively short and stable repeats and disease - causing longer tracts that are often unstable . a longer repeat is typically associated with more clinical problems , on top of earlier onset of symptoms . since mutation rate increases with repeat length , successive generations are faced with larger risks of developing more severe disease , a phenomenon called anticipation [ 1 , 2 ] . myotonic dystrophy type i ( dm1 ) is caused by an expanded ctg - repeat in the 3 utr of the dmpk gene that is quite unstable when transmitted to the next generation [ 35 ] . myotonic dystrophy type i is a multisystem disorder with patients showing not only muscle problems , but also cataract , cardiac anomalies , testicular atrophy , gastrointestinal , and endocrine abnormalities , as well as problems originating in the central nervous system . ongoing somatic expansion in dm1 patients is thought to contribute to disease progression . in addition to dm1 , many other trinucleotide repeat ( tnr ) diseases are highly debilitating . efforts are therefore aimed at understanding not only pathogenesis , but also mechanisms of tnr instability . thus far , replication , ( bidirectional ) transcription , and dna repair processes have been described to play a role in tnr instability mechanisms [ 79 ] . one of the major pathogenic models proposed to underlie dm1 is a toxic effect of the presence of expanded cug - containing transcripts . mutant dmpk mrnas are retained in the nucleus , accumulate in foci , and form complexes with regulatory proteins , thereby preventing these proteins from exerting their normal function . aberrant mirna metabolism has also been described in patients with expanded ctg - repeats [ 12 , 13 ] . recent evidence that bidirectional transcription and nonconventional rna translation are taking place at several tnr loci is complicating the traditional picture of rna toxicity . these findings point at a scenario where not just one single expanded rna transcript is responsible for disease development . moreover , chromatin dynamics are increasingly recognised to influence both tnr instability and gene expression at tnr loci and thereby probably disease outcome . tnrs can affect nucleosome positioning [ 16 , 17 ] , and ctg - repeats specifically have been identified as preferential location for nucleosome formation . ctg- and cag - repeats have been described to form a functional component of insulator elements , thereby influencing gene expression levels . at the dm1 locus , the ctg - repeat forms an insulator together with the two ctcf - binding sites ( ctcfbs ) that flank the repeat . long ctg - tracts were shown to induce condensation of dna at the dm1 locus , which could hinder access of gene regulators to this region . transcription of the six5 gene that neighbours dmpk was decreased in patient cells expressing expanded ctg - repeats . these findings together led to the proposal that the expanded ctg - repeat induces a transcriptionally repressive region . indeed , long ctg - repetitions were shown to induce heterochromatin formation , which can then spread into neighbouring regions . for instance , an expanded cgg - repeat is associated with cpg hypermethylation , heterochromatinisation , and silencing of the fmr1 gene , which is the cause of fragile x syndrome . dna methylation and histone modifications representative of silent chromatin have been observed around the expanded gaa - repeat in intron 1 of the frataxin gene that causes friedreich 's ataxia . although the chromatin context is now considered important for dm1 and other tnr loci [ 9 , 2325 ] , few comprehensive studies addressing multiple factors involved in or associated with chromatin remodelling simultaneously have been performed . recently , studies in mouse models for huntington 's disease suggested that the chromatin context of the transgene integration site determines cag - repeat instability and transcription levels . thus , these processes seem tightly linked , underscoring the need to understand chromatin dynamics at tnr loci . we therefore set out to study the consequences of ctg - repeat expansion for cpg methylation , ctcf - binding , chromatin conformation , and gene expression at the dm1 locus , making use of the transgenic mouse model previously generated in our laboratory . these mice carry a large human genomic transgene that encompasses the dmpk gene as well as the neighbouring genes dmwd and six5 . the transgene includes either a normal ctg - repeat of 20 trinucleotides or disease - associated expanded repeats , with the latter showing ctg - repeat instability patterns similar to dm1 patients ( strongly biased towards expansions , length- and age - dependent somatic instability , albeit showing smaller repeat length changes per instability event in mice ) [ 2830 ] . the transgene also encompasses important regulatory sequences such as the two ctcf - binding sites ( ctcfbs ) that flank the ctg - repeat in humans and the enhancer of the downstream six5 gene . since these mice display multiple characteristics seen in dm1 [ 27 , 28 , 32 , 33 ] , they are considered a valuable model to investigate mechanisms implicated in ctg - repeat instability and dm1 pathogenesis [ 34 , 35 ] . benefitting from this dm1 mouse model , we aimed to study the epigenetic consequences of the expanded ctg - repeat at the dm1 locus . we present evidence that expanded ctg - repeats induce cpg methylation and local heterochromatinisation and concurrent decreased transcription around the repeat , without affecting significantly ctcf binding at the dm1 locus . we also found binding of pcna around the ctg - repeat and propose that it might lie at the basis of ctg - repeat expansion - induced repressive changes in chromatin dynamics . mice used in this study harbour a transgene consisting of 45 kb of human genomic dna cloned from a dm1 patient and have been described previously ( crossbred to > 90% c57/bl6 background ) [ 27 , 28 , 31 ] . mice were genotyped by pcr amplification of tail dna using oligonucleotide primers dmhr8 ( 5-tgacgtggatgggcaaactg-3 ) , dmhr9 ( 5-agctttgcactttgcgaacc-3 ) , and dmm9 ( 5-gcttgtaactgatggctggg-3 ) , which amplify the endogenous murine dmpk ( dmhr8 and dmm9 ) and the human transgene dmpk ( dmhr8 and dmhr9 ) . ctg - repeat length was determined by pcr amplification of dna extracted from tail at weaning , with oligonucleotide primer 101 ( 5-cttcccaggcctgcagtttgcccatc-3 ) and primer 102 ( 5-gaacggggctcgaagggtcttgtagc-3 ) , as described before , followed by electrophoresis of pcr products on a large 0.8% ( w / v ) agarose gel . for the current paper , heterozygous mice with the following ctg - repeat lengths were used : dm20 : mice of the dm20 - 949 line that carry the normal unexpanded human allele , dm300 : mice of the dm300 - 328 line that currently have an average of 610 ctgs ( range : 545700 ) , and dmsxl : mice of the dm300 - 328 line that have undergone large expansions and now carry alleles with over 1000 ctgs . in this particular study , adult mice of 35 months of age were used for all experiments described in this study ( mean age did not differ among the different genotype groups , as tested with kruskal - wallis . dm20 : mean age : 4.4 months , dm300 : mean age : 4.3 months , and dmsxl : 4.1 months , p = 0.634 ) . we chose heart as a representative tissue for disease , which shows the highest dmpk expression levels in our mice . hearts were dissected , snap - frozen in liquid nitrogen , and stored at 80c until use . animals were housed and cared for according to guidelines by the french council on animal care methylation status of the sequences flanking the ctg - repeat was studied by bisulfite conversion of dna isolated from adult hearts ( extracted with qiagen dneasy blood and tissue kit , according to manufacturer 's instructions ) . 500 ng of dna was bisulfite - converted with qiagen 's epitect bisulfite kit . bisulfite converts an unmethylated cytosine ( c ) into a thymine ( t ) , while leaving methylated cs unchanged . subsequent pcr amplification and sequencing of the pcr product and comparison with the target ( genomic dna ) sequence then allow distinction between cs that were or were not methylated at the time of bisulfite conversion . primers were chosen so as not to contain any cpgs , such that dna templates can be amplified irrespective of their methylation status . methylation interference studies identified guanine nucleotides whose methylation prevents binding by ctcf , predominantly on the noncoding strand . as most dnase i - hypersensitive sites induced by ctcf binding are on this strand , we conclude that the status of this strand is most relevant for our studies . seminested pcr was performed with the following primers for ctcfbs1 : f ( forward ) : 5-tagtagtagtagtatttt-3 , r1 ( reverse ) : 5-tagtagtagtagtatttt-3 , and r2 ( for seminested pcr in combination with primer f ) : 5-ctttccctactcctatt-3. ctcfbs2 was amplified with f1 : 5-gttttgggtagatggagggtt-3 , r : 5-aatcacaaaccatttctttct-3 , and f2 : 5-ggttttaggtggggatagata-3. three parallel seminested pcr reactions were performed with 4 l of pcr product ( total reaction volume 25 l ) of the first amplification as input , with an annealing time 2c higher than the one used in the first pcr and 3 more cycles ( 30 and 33 cycles for subsequent pcr rounds ) , to obtain sufficient pcr product . products were subsequently cut out and snap - frozen in liquid n2 in columns of the millipore dna gel extraction kit . the snap - frozen agarose band was spun down and dna in the flow - through was precipitated using classical nacl and ethanol precipitation . these purified pcr products were used to subclone into pmosblue vector , using the pmosblue blunt - ended pcr cloning kit , according to manufacturer 's instructions ( ge healthcare ) . colony pcr was performed with primers t7 and u19 and correctly sized clones were sent for sequencing at the sequencing platform of cochin hospital in paris , france . sequences and cpg methylation of individual cpgs were subsequently analysed using biq analyser software . of each mouse , at least 10 clones were sequenced . an average percentage of methylation per cpg was calculated per mouse , of which an overall weighted average percentage methylation was calculated per cpg , per genotype . protein - dna interactions were cross - linked for 10 minutes at room temperature ( rt ) , while rotating . fixation was quenched by adding glycine to a final concentration of 0.125 m and rotating for 5 minutes at rt . samples were spun at ~470 g ( 2500 rpm in table top centrifuge ) for 10 minutes at 4c , with slow deceleration . the pellet was washed with cold pbs , for 10 minutes at 4c , setting the centrifuge to slowly decelerate . pellet was resuspended , vigorously vortexed in 150 l sds lysis buffer ( 1% sds , 10 mm edta , 50 mm tris , ph 8 , with phosstop phosphatase inhibitors ( roche ) and complete protease inhibitors ( roche ) ) , and left on ice for 15 minutes , with repeated vortexing . the tissue samples were then sonicated ( branson sonifier cell disruptor b15 ) in lysis buffer until macroscopically homogenised , while being kept cold . samples were spun down at 11000 rpm , for 10 minutes at 4c ( normal deceleration from here onwards ) . 100 l of supernatant was transferred to a separate tube and kept on ice . 100 l of sds lysis buffer was added to the pellet , in which the pellet was resuspended and vortexed . the supernatant fraction was again sonicated to shear the chromatin , obtaining fragments of 2001000 bp . the sheared chromatin was diluted 5 times with chip dilution buffer ( 0.01% sds , 1.1% triton x-100 , 1.2 mm edta , 16.7 mm tris - hcl ph 8.1 , and 167 mm nacl ) , supplemented with phosphatase and protease inhibitors . 40 l of dynal protein a dynabeads ( life technologies ) per immunoprecipitation ( ip ) reaction was incubated with antibody at rt for one hour , while rotating . the following antibodies were used for chip : rabbit anti - ctcf ( abcam ab70303 , 2 g per ip ) , rabbit anti - h3k9/14ac ( millipore 06 - 599 , 5 g ) , mouse anti - h3k27me ( abcam ab6002 , 2 g ) , rabbit anti - h3k9me3 ( millipore 07 - 442 , 3 g ) , and mouse anti - pcna ( santa cruz sc56 , 5 g ) . 200 l of 5x diluted chromatin was taken per ip and subjected to a further 2x dilution with chip dilution buffer supplemented with phosphatase and protease inhibitors and added to the antibody - covered dynabeads ( the abchip reaction ) . for each sample , a control ip was taken along , using rabbit or mouse igg ( santa cruz ) as appropriate . an aliquot of the same cross - linked and sheared chromatin was kept aside and purified in parallel , to be used as input chromatin control for pcr . antibody - covered beads were incubated with the chromatin overnight at 4c , while rotating . the following day , the beads were washed , using a dynal dynamag - spin magnet ( life technologies ) , for 4 minutes at 4c , while rotating , twice with chip dilution buffer , 1x with low salt immune complex washing buffer ( 0.1% sds , 1% triton x-100 , 2 mm edta , 20 mm tris - hcl ph 8.1 , and 150 mm nacl ) , 2x with high salt immune complex washing buffer ( 0.1% sds , 1% triton x-100 , 2 mm edta , 20 mm tris - hcl ph 8.1 , and 500 mm nacl ) , 1 time with licl immune complex buffer ( 0.25 m licl , 1% igepal - ca630 , 1% deoxycholic acid ( sodium salt ) , 1 mm edta , 10 mm tris ph 8.1 , millipore 20 - 156 ) , and twice in te buffer ( 10 mm tris - hcl , 1 mm edta ph 8.0 ) . beads were then taken up in 150 l complete elution buffer ( 20 mm tris - hcl ph 7.6 , 5 mm edta , 50 mm nacl , 1% sds , and 130 g / ml proteinase k ( life technologies ) ) , and incubated at 67c for at least 4 hours , while rotating , to elute the protein - dna complexes off the beads , reverse cross - links , and digest proteins simultaneously . eluates were then collected , and beads were rinsed with 75 l elution buffer ( 20 mm tris - hcl ph 7.6 , 5 mm edta , and 50 mm nacl ) . eluates were combined and purified with qiaquick dna purification columns ( qiagen 28106 ) , according to protocol , with the exception of the addition of 1 volume of isopropanol , in addition to 5 volumes of buffer pb in the first step to optimise isolation of small fragments . dna was eluted in two steps using twice 25 l elution buffer provided with the kit . relative abundance at target loci in the chiped dna was analysed using quantitative pcr , using power sybr green master mix ( applied biosystems ) in an ab7300 real - time pcr system ( applied biosystems ) . for all antibodies used , amplicons were measured at ctcf binding site 1 ( q - bs1 ) , ctcf binding site 2 ( q - bs2 ) , and the enhancer region ( q - enh ) , as indicated in figure 1 . primer sequences were as follows : q - bs1-forward ( f ) : 5-ctgccagttcacaaccgctc-3 , q - bs1-reverse ( r ) : 5-cgagccccgttcgccg-3 , q - bs2-f : 5-cgtccgtgttccatcctc-3 , q - bs2-r : 5-cgtccgtgttccatcctc-3 , q - enh - f : 5-ggaggcgtgtggaggcgg-3 , and q - enh - r : 5-tcccccaaccctgattcg-3. the following amplicons were used as positive or negative pcr controls for chip reactions : myc - f : 5-cttgttctattgcctttccgtttc-3 and myc - r : 5-aacccatccctactttctgacagtc-3 ( positive control for ctcf - chip ) , gapdh - f : 5-ataagcagggcgggaggc-3 and gapdh - r : 5-cgtctctggaacagggaggag-3 ( positive control for active histone mark , negative control for repressive histone modifications and ctcf ) , amylase - f : 5-ctccttgtacgggttggt-3 and amylase - r : 5-aatgatgtgcacagctgaa-3 ( negative control for active histone modification , positive control for pcna ) , and hoxd9-f : 5-tgctccggggctttggataa-3 and hoxd9-r : 5-ctctctgggtcctgcgatct-3 ( positive control for repressive histone modifications ) . a standard curve of serial dilutions of genomic dna a dissociation curve was run in every experiment to assess quality of the reaction and ensure absence of primer - dimer or other nonspecific pcr products . making use of the formula derived of the standard curve , quantities were calculated from the obtained quantification cycle ( cq ) values for each sample . quantities obtained in ip reactions ( antibody - chip abchip or igg - mock ip igg - ip ) were normalised by division by the quantity obtained in input chromatin ( non - iped ) samples ( enrichment ) . enrichments were normalised against the abchip enrichment value of the positive control amplicon , for each respective ctg - repeat length category , to correct for possible differences in chromatin density . all pairs of normalised igg- and abchip - enrichment values for a given genotype and amplicon were subjected to a mann - whitney u test to see if enrichment in abchip was statistically significantly larger than in igg - ip , according to expectations ( expected for positive control amplicons but not for negative control amplicons ) . the input - corrected enrichment values of all specific abchip samples were further subjected to statistical analysis using ibm spss statistics standard edition version 20 software , to analyse possible effects of increasing ctg - repeat length . snap - frozen adult hearts taken from dm20 , dm300 , and dmsxl mice were homogenized in trizol ( life technologies ) using a tissue lyser ( retsch mm400 , 2x 2,5 minutes , using 2 stainless steel beads ( qiagen ) ) . after chloroform extraction , the aqueous phase was mixed with 70% ethanol and transferred to a spin cartridge of the purelink rna mini kit ( ambion by life technologies ) . a purelink dnase step was inserted in the protocol , as recommended after binding of the rna to the column . rna concentrations were determined by absorbance at 260 nm using a nanodrop 1000 spectrophotometer ( thermofisher scientific ) and quality and absence of genomic dna were verified on agarose gel . different rna extraction methods have been compared , of which the one described here was found to give the highest yield . efficacy of the various methods to recover expanded rna was assessed by comparing the total rna recovery and dmpk expression levels between hemizygous and homozygous mice . cdna was synthesized from 0.40.6 g rna , using superscript ii reverse transcriptase ( life technologies ) when using random hexamer primers ( for six5 mrna quantification ) or using superscript iii reverse transcriptase ( life technologies ) when using strand - specific primers ( for dmpk sense and antisense mrna quantification ) , according to manufacturer 's instructions ( also see ) . . please refer to table 1 for primer sequences used for strand - specific rt . relative abundance of transcripts was analysed using power sybr green master mix ( applied biosystems ) in an ab7300 real - time pcr system ( applied biosystems ) . dmpk , six5 , and antisense transcript levels were calculated relative to 18s and endogenous murine dmpk mrna levels . we used standard curves of serial dilutions of a plasmid carrying the amplicon . reverse transcriptase efficiency for each gene and each primer set a dissociation curve was run in every experiment to assess quality of the reaction and ensure absence of primer - dimer or other nonspecific prcr products . reverse transcriptase was performed in duplo , followed by separate qpcr analyses on each cdna sample . all qpcr reactions were performed in triplicate and experiments ( from rt reaction to qpcr analysis ) were done twice . rna from hearts of 8 dm20 , 5 dm300 , and 6 dmsxl mice was used for these expression studies . averages of triplicate quantities obtained for each mouse were normalised against a control sample that was taken along in every qpcr experiment . the average expression level of the two parallel qrt - pcr experiments was subjected to statistical analyses . jonckheere terpstra test for trend ( ibm spss statistics standard edition version 20 software ) was performed to investigate whether expression levels change with increasing ctg - repeat length . differences between repeat length categories were further investigated by means of non - parametric mann - whitney pairwise comparisons . we studied cpg methylation around the ctg - repeat , since dna methylation can affect binding of transcription factors or attract chromatin - remodelling enzymes . previous methylation analyses had shown substantial cpg methylation in various tissues of dm300 mice , in both upstream and downstream regions flanking the ctg - repeat , while dm20 tissues were almost completely devoid of cpg methylation . cpg methylation analysis in dm1 patient tissues showed a clearly polarised pattern , with only methylated cs at and around ctcfbs1 and not at ctcfbs2 . we extended these observations by performing a detailed analysis of the cpg methylation pattern by bisulfite sequencing in individual mice carrying normal alleles with 20 ctgs ( dm20 ) , or mice expressing expanded alleles ( dm300 : 545700 repeats or dmsxl : 13001600 ctgs ) . we chose to study heart , which shows the highest dmpk expression levels in our mice . bisulfite sequencing was directed at the non - ctg strand , because a methylation interference assay indicated that ctcf showed stronger contacts on this strand . figure 2(b ) illustrates the results of 10 clones ( representing 10 different cells ) of four mice for each repeat length category . it shows that cpg methylation is very low in dm20 mice , more prominent in dm300 mice , and quite abundant in dmsxl mice . further expansion of the ctg - repeat from around 600 to ~1450 ctgs is associated with more pronounced cpg methylation . this trend is seen around both ctcfbs1 and ctcfbs2 , although less pronounced at ctcfbs2 . note that ctcfbs1 itself is relatively spared from cpg methylation , as compared to its flanking sequences . figure 2(c ) , illustrating the methylation pattern of each clone , shows that the cpg methylation pattern is variable in individual cells within a tissue . other mice that we analysed showed a similar pattern of distinct methylation patterns in individual cells ( data not shown ) . based on in vitro observations , it had been postulated that binding of ctcf is lost when cpgs in the ctcf recognition sequence are methylated or mutated . since we did not observe an all - or - nothing cpg methylation pattern , we investigated ctcf binding to the two ctcfbs flanking the ctg - repeat . we performed chip on chromatin preparations of adult heart , comparing the three repeat length categories , followed by qpcr to analyse quantities of immunoprecipitated dna . positions of amplicons at the dm1 locus are drawn in figure 1 . for chip analyses of histone modifications and ctcf binding we performed control experiments with both a positive and a negative control amplicon . details of statistical analyses can be found in supplementary tables 1 and 2 ( see supplementary material available online at http://dx.doi.org/10.1155/2013/567435 ) . we observed enrichment in ctcf - immunoprecipitated samples of all ctg - repeat length categories at ctcfbs1 , but no significant difference between mice carrying normal or expanded repeat ( figure 3 ) . although the enrichment appeared slightly lower in dmsxl as compared to dm20 and dm300 , this trend did not reach statistical significance . at ctcfbs2 , binding of ctcf seemed lower than binding at ctcfbs1 and enrichment was significant only in dmsxl mice , probably due to experimental variability . no significant trend across the categories was observed for ctcf enrichment at ctcfbs2 . at the enhancer region , no statistically significant enrichment was seen , showing , as expected , no ctcf binding in this region ( figure 3 ) . methylated cpgs may attract chromatin - remodelling enzymes ; thus we analysed chromatin remodelling in the presence of an expanded ctg - repeat in our mice . we performed chip with antibodies directed against histone modifications that represent actively transcribed ( h3k9/14ac ) or repressed ( h3k27me3 and h3k9me3 ) chromatin . enrichment for these histone modifications around ctcfbs1 , ctcfbs2 , and the enhancer region was studied by qpcr on chromatin immunoprecipitated dna . we observed statistically significant enrichment with an antibody directed against acetylated h3k9/14 ( h3k9/14ac ) around ctcfbs1 , ctcfbs2 , and to a lower extend at the enhancer region ( figure 4(a ) ) . at both ctcfbs1 and bs2 , jonckheere terpstra test for trend revealed a statistically significant decrease of h3k9/14ac enrichment across ctg - repeat length categories ( ctcfbs1 : z - score : 2.931 , p = 0.003 , ctcfbs2 : z - score : 2.996 , p = 0.003 , where a negative z - score indicates a descending trend ; thus a lower median enrichment was seen with increasing ctg - repeat length ) . hence , chromatin of mice with longer repeats was less enriched for the active histone modification than chromatin of control mice . enrichment at the enhancer region was low and did not show a trend across the different categories of mice . as a first investigation of possible heterochromatinisation , we performed chip with an antibody directed against the repressive histone mark trimethylated h3k27 ( h3k27me3 ) . we saw low but statistically significant enrichment at all amplicons and in all ctg - repeat length categories , as shown in figure 4(b ) . although enrichment appeared slightly higher in the dmsxl mice for the ctcfbs1 and ctcfbs2 amplicons , no statistically significant trend with increasing repeat length was seen . we next investigated another histone methylation mark representative of transcriptionally repressed chromatin : trimethylated h3k9 ( h3k9me3 , figure 4(c ) ) . dm20 and dm300 did not show statistically significant enrichment in the specific antibody chip reaction ( abchip ) versus igg - ip at ctcfbs1 and bs2 , whereas dmsxl did . this was confirmed by a statistically significant trend across categories ( ctcfbs1 : z - score : 3.084 , p = 0.002 , ctcfbs2 : z - score : 2.599 , p = 0.009 ) . no statistically significant enrichment nor a statistically significant trend for h3k9me3 enrichment was seen at the enhancer region ( figure 4(c ) ) . we investigated possible changes in expression levels at the dm1 locus , since chromatin remodelling is generally accompanied with changes in gene expression . sense dmpk transcript levels showed a sharp decrease between dm20 and mice with expanded repeats ( figure 5 ) . an overall statistically significant trend was observed across the repeat length categories for both reference genes ( see supplementary table 3 for results of jonckheere terpstra test for trend : normalised against 18s : z - score : 3.160 , p = 0.002 , dmpk : z - score : 2.332 , p = 0.02 ) . mann - whitney pairwise comparisons did not show a statistically significant decrease in dmpk mrna expression between dm300 and dmsxl ( medians and interquartile ranges are shown in supplementary table 3 , data of mann - whitney analysis not shown ) . the dmpk antisense transcript did not show a similar trend of changing expression across repeat length categories when antisense mrna levels were normalised against dmpk ( figure 5 ) ( dmpk : jonckheere terpstra test for trend z - score : 0 , p = 1 , supplementary table 3 ) . however , a statistically significant trend was observed when antisense transcript levels were normalised against 18s ( z - score : 2,107 , p = 0.035 ) . post hoc pairwise mann - whitney comparisons however did not show statistically significant differences between any of the repeat length categories , when correcting the p values for multiple comparisons ( data not shown ) . six5 expression levels are affected by increasing repeat length , as they show a significant decrease across the repeat length categories . the decrease is lower than that seen for dmpk sense transcripts , but consistent with both reference genes ( jonckheere terpstra test for trend : versus 18s : z - score : 2.332 , p = 0.02 , versus dmpk : z - score : 3.912 , p < 0.001 , supplementary table 3 ) . in addition to its role in replication , pcna recruits a variety of epigenetic regulators . loops of slipped - strand structures formed by expanded ctg - repeats could serve as loading sites for pcna and binding of pcna to the expanded ctg - repeat could be the beginning of the cascade of chromatin remodelling event [ 8 , 42 ] . we therefore investigated whether pcna binds near the ctg - repeats in our mice , by chip , followed by qpcr analysis . enrichments measured with the enhancer amplicon were modest compared to the positive amylase control and were significant only for dm300 mice ( p = 0.01 , igg versus pcna antibody , figure 6 ) . at both ctcfbs1 and 2 , we did not see statistically significant enrichment at dm20 , but enrichment was detected for dm300 and dmsxl , suggesting binding of pcna to expanded ctg - repeats . jonckheere terpstra test for trend did not reveal a statistically significant trend , indicating that similar pcna - binding was detected despite a longer repeat . at the enhancer region , on the contrary , we did not observe statistically significant enrichment , except for modest enrichment in dm300 . thus , these preliminary data seem to suggest that pcna specifically binds close to the amplicons at ctcfbs1 and 2 , but not the enhancer region . we here show that expanded ctg - repeats induce a locally repressed chromatin state and accompanying reduced sense gene transcription at the dm1 locus in adult transgenic mouse hearts . mice with expanded repeats showed substantial methylation at and around the ctcfbs , as opposed to dm20 , which showed very little cpg methylation . this cpg methylation is not seen at all cpgs nor in all individual cells , but overall dna methylation levels are higher with increasing repeat length . dm20 and dm300/sxl are independent transgenic lines and we can not exclude an influence of the transgene integration site sequences . however , the transgene is large ( 45 kb ) and contains the major regulatory sequences between dmpk and six5 [ 14 , 19 ] . all different lines obtained with different repeat lengths showed the same tissue - specific pattern of dmpk expression , which is also similar to the mouse dmpk gene and to the dmpk gene in human tissues [ 31 , 32 ] . in addition , expression levels correlate to copy number of the integrated transgene indicating that the surrounding mouse sequences have no or minimal impact on the transgene . we observed that the percentage of cells carrying a methyl group at a given cpg was substantial in dm300 and even higher in dmsxl , both at ctcfbs1 and bs2 . cpg methylation was more abundant at ctcfbs1 than at ctcfbs2 , confirming a polarised localisation of methylation at the dm1 locus as described before . this is in line with evidence pointing at a more important regulatory role for ctcfbs1 [ 19 , 43 ] . our observation that the ctcfbs1 recognition sequence itself was relatively spared from cpg methylation as opposed to surrounding sequences is also worth noting in this respect . cpg methylation around the dm1 ctg - repeat had previously been assessed by measuring the height of chromatogram peaks obtained for cytosine after sequencing of bisulfite - converted dna . most importantly , adult human dm1 samples never showed methylation at ctcfbs2 , whereas dm300 mice did , indicating that the mouse model does not fully mimic the human situation underlining limitations of animal models . however , one human foetus did show cpg methylation at and around ctcfbs2 , indicating individual variation among human patients . we demonstrate here that mice with around 1450 ctgs clearly show more methylation than mice with around 600 ctg with a strong bias 5 of the ctg - repeat . the variable dna methylation pattern found in our mice around the ctg - repeat resembles that observed around the sca7-cag - repeat in a transgenic mouse model for spinocerebellar ataxia 7 ( sca7 ) . a strong correlation between the severity of disease symptoms and level of dna methylation has been described at the cgg - repeat and promoter region of the fmr1 gene [ 44 , 45 ] . in addition , it was recently proposed that also variable methylation patterns in the promoter of the atxn2 gene explain considerable variation in anticipation , in the absence of intergenerational cag - repeat instability . different degrees of methylation of the atxn2 promoter could be related to age of onset in patients with sca2 sca3 , suggesting that gene dosage through this epigenetic mechanism is important for disease outcome . thus , these observations underline that cpg methylation is no all - or - nothing phenomenon at tnr loci and underscore the importance of careful examination of methylation status of individual cpgs . relevant mechanistic information might be missed when a more general approach is followed . in vitro studies have shown disrupted binding of ctcf to the ctcfbs upon mutation or methylation of the recognition sequence . since our data show that cpg methylation is no all - or - nothing phenomenon at the studied locus , it was unclear what to expect concerning ctcf binding . for the first time in mammalian tissues we show that ctcf still binds to ctcfbs1 despite the presence of an expanded ctg - repeat of up to ~1600 units ( figure 3 ) . we did not detect significant binding of ctcf to ctcfbs2 , which is consistent with in vitro binding assays that showed stronger binding of ctcf to site 1 . it was surprising to observe clear ctcf binding at ctcfbs1 in vivo despite abundant cpg methylation of the region . however , it is interesting to note that the ctcf recognition sequence is relatively spared from methylation when compared to the adjacent region . it is possible that the repeat in our mice is not large enough to induce full methylation of the binding site . alternatively , at the h19 locus , binding of ctcf has been demonstrated to prevent cpg methylation [ 47 , 48 ] . methylated cpgs are known to attract chromatin - remodelling enzymes [ 41 , 49 , 50 ] . in vitro , the nucleosome assembly of dna containing repeating ctg triplets showed that the efficiency of nucleosome formation increased with expanded triplet blocks [ 1618 ] , suggesting that such blocks may profoundly alter local chromatin structure and repress transcription through the creation of stable nucleosomes . we therefore explored possible chromatin rearrangement in mice with expanded ctg - repeats , as opposed to dm20 . we found chromatin remodelling indicative of a transcriptionally repressed state , close to the expanded ctg - repeat , in dm300 and dmsxl mice ( figure 4 ) . the enhancer region generally showed a different enrichment pattern for histone modifications than around the ctcfbs , suggesting that a local region of heterochromatin is formed close to the expanded ctg - repeat , within a euchromatin region . this has previously been demonstrated in patient cells : heterochromatin spreading was seen upon expansion of the ctg - repeat , as active hk4me3 was replaced by the repressive h3k9me3 mark . when the expanded ctg - repeat induced heterochromatinisation , adjacent genes were silenced by propagation of heterochromatin along the chromosome . we did see increased enrichment of h3k9me3 around very long ctg - repeats , but heterochromatinisation did not propagate to the enhancer region . prominent decreasing trends of h3k9/14ac enrichment at both ctcfbs were observed across increasing ctg - repeat length categories . a similar graded loss of acetylated h3 and h4 with increasing cgg - repeat length has been observed in fxs patient cells . considering that qpcr reactions for ctcfbs1 and bs2 were performed on the same chiped dna and that qpcr efficiencies were very similar as yet , we can not know whether this has a functional implication or whether it is linked to the seemingly more important regulatory role of ctcfbs1 . we chose to study one active histone modification only , as genome - wide histone modification maps show that the distribution of most histone marks recognised to be active is highly similar . fewer histone modifications associated with repressive chromatin have been described and little is known about their global linkage . our data show that h3k9me3 and h3k27me3 did not respond in the same way to the expanded ctg - repeat . in a study that investigated the epigenetic status of the euchromatic region of the human y chromosome , h3k9me3 and h3k9ac enrichment correlated with the expression status , whereas h3k27me3 enrichment did not . this suggests a mechanism where h3k9me3 and h3k9ac dominate over h3k27me modifications to determine expression status of the chromatin . consistent with chromatin changes representative of transcriptional repression , we saw lower sense dmpk and six5 expression in mice that carry an expanded ctg - repeat ( figure 5 ) . we did not detect a further decrease of dmpk and six5 transcription levels when comparing dm300 and dmsxl at 5 months of age , despite the 30% decrease previously observed at 2 months of age in the same transgenic mice . this could be due to the decreased transgene expression we observed with age ( data not shown ) . it is expressed in many tissues , with expression being the highest in heart , as is the case for the dmpk sense transcript , although it does not follow nor mirror the same expression profile . since the antisense transcript and six5 have overlapping promoter regions , it could be postulated that they are subject to similar regulatory factors . however , in the current study , six5 mrna levels decreased , whereas antisense transcript levels remained unaffected in the presence of an expanded ctg - repeat . interestingly , this finding demonstrates that regulation of dmpk antisense is independent although some regulatory sequences might be shared with dmpk and six5 . future research will likely shed more light on the role of bidirectional transcription in the dmpk gene and at other tnr loci . previous studies have demonstrated that pcna can be loaded onto dsdna - ssdna junctions in dna - loops or loops of slipped - strand structures formed by expanded ctg - repeats [ 8 , 42 ] . we therefore recognised in pcna a candidate molecule that might bind to the expanded ctg - repeat and then cause a cascade of chromatin - modifying events . we here show evidence that pcna indeed binds to or close to the expanded ctg - repeat ( figure 6 ) . pcna appears to bind to a similar extent to ctcfbs1 and bs2 amplicons , which is according to expectations , since both amplicons lie very close to the ctg - repeat . it is possible that more pcna was bound to the longer ctg - repeat in dmsxl mice , but that we can not detect this due to the size of our sheared fragments . by recruiting epigenetic regulators pcna is known to interact with dna methyltransferases dnmt1 , -3a and -3b , as well as with histone methyltransferases . these interactions cause h3k9 and h3k27 to become trimethylated , yielding a repressed chromatin environment . via another route , dnmt1 interacts with histone deacetylases ( hdacs ) , which also contributes to a repressed chromatin context . the observed enrichment pattern of histone modifications and hypermethylation around the expanded ctg - repeat in adult hearts of mice fit with this model . importantly , we detected enrichment for pcna in mice with expanded ctg - repeats , to a similar extent at both ctcfbs , but not at the enhancer region , suggesting that pcna binds to or very near the expanded ctg - repeat specifically . thus , multiple parallel pathways seem responsible for the establishment of a repressed chromatin status . the involvement of pcna needs to be confirmed and the precise order of events remains to be elucidated . we here presented evidence that expanded ctg - repeats induce cpg methylation and local heterochromatinisation close to the repeat . ctcf binding at the dm1 locus was not affected by the expansion of the ctg - repeat . we found that pcna binds in the vicinity of expanded ctg repeats and might be recruited to the expanded ctg - repeat . we propose that it could subsequently attract chromatin - remodelling enzymes that yield the repressive changes in chromatin dynamics . a better understanding of the precise cascade of processes induced by expanded tnrs , and , importantly , the starting point of these changes , will provide us with therapeutic targets to alleviate disease progression and limit further tnr expansion .

odontogenic infections are one of the most prevalent diseases worldwide and the principal reason for seeking dental care . dental prescriptions account for nearly 7% to 11% of all common antibiotic prescriptions . the commonest emergency odontogenic infections are periapical abscess ( 25% ) , pericoronitis ( 11% ) , and periodontal abscess ( 7% ) . odontogenic infections are mostly polymicrobial and frequently encountered odontopathogens are streptococci spp . , corynebacterium spp . and staphylococcus spp . , prevotella spp . [ 2 , 3 ] . therapeutic success in odontogenic infections is determined by the control of infection by surgical debridement and/or antimicrobial therapy which is indicated when there are clear signs of systemic involvement such as pyrexia or lymphadenopathy . the polymicrobial nature of odontogenic infections necessitates the use of antibiotics active against both aerobic and anaerobic bacteria . the antibiotics most commonly prescribed for acute dental abscesses are amoxicillin , penicillin , metronidazole , and erythromycin with clindamycin as an alternative in individuals allergic to the beta - lactam antibiotics . recently , published evidence suggests that penicillin and amoxicillin are being rendered increasingly less effective because of beta - lactamase producing bacteria . more than half of the gram - negative anaerobic bacilli ( including prevotella , porphyromonas , bacteroides , and fusobacterium spp . ) are capable of producing beta - lactamase leading to treatment failures in dental infections . studies have revealed the presence of beta - lactamase producing species in 7488% of patients with periodontitis . addition of a beta - lactamase inhibitor such as clavulanic acid to amoxicillin ( augmentin ) confers resistance to beta - lactamases thereby extending the antibiotic spectrum to anaerobes such as prevotella spp . and bacteroides spp . efficacy of amoxicillin / clavulanic acid in the treatment of acute periapical abscess has been established in several studies [ 2 , 4 ] . clindamycin is a broad - spectrum antibiotic with activity against aerobic , anaerobic bacteria including coverage against beta - lactamase producing pathogens . clinical trials have demonstrated the efficacy of clindamycin in treating odontogenic infections [ 711 ] . use of clindamycin in dental infections is based on careful patient selection in view of reported cases of pseudomembranous colitis ( a rare but serious consequence of clindamycin ) . despite published evidence evaluating different oral formulations of amoxicillin / clavulanic acid in dental infections , there is limited published data on the use of twice daily dosing of 875/125 mg in odontogenic infections . available evidence suggests that twice daily dosing with 875/125 mg amoxicillin / clavulanic acid results in a successful clinical outcome , better patient compliance , and less gastrointestinal upset , due to a reduction in the dose of clavulanic acid . the aim of the current study was to assess the clinical efficacy and safety of amoxicillin / clavulanic acid 875/125 mg twice daily versus clindamycin 150 mg four times daily , for 5 or 7 days in dental infections . aug117044 was a phase iv , randomised , parallel group , comparative , observer blind study to evaluate efficacy , safety , and tolerability of amoxicillin / clavulanic acid ( 875 mg/125 mg ) and clindamycin ( 150 mg ) in the treatment of acute odontogenic infection with or without abscess . the study was conducted in fifteen centres across four countries with four centres each in malaysia , philippines , and vietnam and three in thailand . the study protocol , the informed consent , and other information that required preapproval were reviewed and approved by a national , regional , or investigational centre ethics committee or institutional review board , in accordance with the international conference on harmonisation of technical requirements for registration of pharmaceuticals for human use ( ich ) good clinical practice ( gcp ) and applicable country - specific requirements . written informed consent was obtained from each subject prior to the performance of any study - specific procedures . the study was conducted in accordance with ich gcp and all applicable subject privacy requirements and the ethical principles that are outlined in the declaration of helsinki 2008 . a total of 472 subjects were randomised in a 1 : 1 ratio in each of the treatment arms . the study included a one - day screening period followed by a treatment period of five days that could be extended to seven days based on clinical response . eligible subjects were randomized on the day of their screening visit or within a day of screening . efficacy and safety evaluations were performed on day 2 , day 5 , and/or day 7 ( based on treatment duration ) ( figure 1 ) . study treatment included amoxicillin / clavulanic acid ( 875 mg/125 mg ) administered twice daily or clindamycin ( 150 mg ) administered four times daily along with meals for 5 or 7 days . clinical efficacy ( cure ( cure was defined as complete resolution of signs and symptoms of infection present at baseline such that no additional antimicrobial therapy was required ) , improvement ( improvement was defined as resolution of fever ( if present at baseline ) and > 70% reduction in swelling and pain and improvement in other signs and symptoms such that no additional antimicrobial therapy was required ) , and failure ( failure was defined as inability to improve the signs and symptoms of infection after seven days of therapy so that additional antimicrobial therapy was required ) ) of the study treatment was assessed based on the response shown by the subjects on the visual analogue scale ( vas ) scores of pain and swelling . since these clinical efficacy parameters were based on symptomatic relief , an optimal study design would have been a double blind design . however , the different dosage regimens and formulations of the study drugs presented practical challenges for a double blind design . based on these considerations , the study was designed to be an observer blind study with the investigator remaining blinded throughout the study period . an unblinded study team member was appointed for the study for drug dispensing and drug accountability and was also present during the clinic visits to ensure that the investigators remained blinded to treatment assignment . adherence to the study design requirements was essential and no protocol waivers or exemptions were allowed during the study . this study did not require an independent data safety monitoring board and no interim analysis was performed . inclusion criteria : the study enrolled subjects 18 years of age with a diagnosis of acute odontogenic infections ( periapical abscess , acute periodontitis , and pericoronitis ) that required antibiotic therapy . radiographic evidence of odontogenic infection and dental pain on mastication were mandatory diagnostic criteria for enrollment . exclusion criteria : subjects presenting with complicated odontogenic infections ( such as osteomyelitis , dentocutaneous and dentoalveolar fistula , and facial - space swelling ) or odontogenic infections secondary to traumatic injury or requiring hospitalisation , aggressive intravenous antimicrobial therapy , or local application of antimicrobials for the treatment of odontogenic infection were excluded . further , patients with other key exclusion criteria such as conditions prone to infective endocarditis and those treated with systemic antibiotics within two weeks before the study of drug administration or injectable long acting antibiotics administered four weeks prior to study of drug administration were also excluded from study . the primary objective of the study was the comparison of clinical efficacy of amoxicillin / clavulanic acid with clindamycin in subjects with acute odontogenic infection with or without abscess . this efficacy endpoint was based on the percentage of subjects achieving clinical success ( cure or improvement ) at the end of treatment ( day 5 or 7 ) . the secondary endpoints of the study included percentage of subjects achieving clinical success at day 5 and change in the vas score for pain and swelling from baseline to days 2 , 5 , and 7 . safety assessments included monitoring of adverse events ( aes ) and serious adverse events ( saes ) from the start of study treatment until the end of study treatment . the antibiotic susceptibility of bacterial isolates obtained from pus specimens was recorded at baseline . a sample size of 205 evaluable subjects in each of the treatment arms provided 90% power to assess noninferiority for the primary endpoint . this was based on a noninferiority margin of 10% , assuming clinical success response rate in the comparator arm ( clindamycin 150 mg ) of 90% and a one - sided = 2.5% . considering a 15% drop - out rate , a final sample size of 236 subjects in each study group thus total randomised subjects in the study were 472 for a 1 : 1 treatment allocation in each study group to get at least 205 evaluable subjects in each arm . the per - protocol ( pp ) population was a subset of intent - to - treat ( itt ) population that had a postbaseline clinical success response assessment and did not report major protocol deviation(s ) . however , those subjects who discontinued from the study without any postbaseline assessment and where the reason for discontinuation was documented as lack of efficacy or treatment failure were included in the pp population with clinical success outcome treated as clinical failure . for noninferiority analysis , the pp population using observed case ( oc ) method was treated as the primary dataset . the assessment of noninferiority for clinical success response was based on two - sided 95% confidence interval ( ci ) ; the upper limit of two - sided 95% ci of the difference of proportion between the two treatments of less than 0.10 ( 10% ) was set to conclude the noninferiority between the treatment arms . the analysis of the intent - to - treat - efficacy ( itt - e ) population ( all randomised subjects with at least one postbaseline assessment of clinical success response ) using the oc dataset was provided as a sensitivity analysis for the primary endpoint and was also used to evaluate secondary endpoints . the itt population ( all randomised subjects who received at least one dose of study medication ) was the safety dataset for the study . the investigator 's judgment was considered decisive for the assessment of clinical improvement in a subject . in the event that the main signs and symptoms were cured or improved ( complete resolution of fever and > 70% reduction in swelling and pain ) and there was no change or worsening from baseline in other signs and symptoms ( such as increased leucocyte count / tooth mobility / lymphadenopathy ) , the investigator 's opinion was sought as to whether additional antimicrobial therapy was required . subjects that required no additional antimicrobial therapy per the investigators judgment were considered a success while those requiring additional antimicrobials were deemed as the analysis based on the investigator 's judgment of clinical success or failure was considered as the primary analysis for testing of noninferiority between the treatment groups and the analysis excluding the investigator 's assessment for clinical success was presented as supportive analysis . for sensitivity analysis , all subjects with an assessment of cure or improvement ( complete resolution of fever , > 70% reduction in swelling and pain ) but with no change or worsening from baseline in other signs and symptoms ( increased leucocyte count / tooth mobility / lymphadenopathy ) were considered as clinical failures irrespective of the clinical judgment of investigator . any lft parameter which was out of reference range for that particular laboratory was considered an adverse event ( ae ) . however , ast , alt , and alkaline phosphatase values > 3 upper limit of reference range ( ulrr ) and total bilirubin > 1.5 ulrr were considered to be of potential clinical concern ( pcc ) as defined by sponsor . a total of 510 subjects were screened for a planned enrollment of 472 subjects . amongst the 472 randomised subjects , 235 ( 46.1% ) subjects were randomised to the amoxicillin / clavulanic acid arm and 237 ( 46.5% ) to the clindamycin arm . however , a total of 236 subjects received amoxicillin / clavulanic acid and 235 subjects received clindamycin ( one subject randomised to the amoxicillin / clavulanic acid arm did not receive any study drug and two subjects randomised to the clindamycin arm incorrectly received amoxicillin / clavulanic acid ) . a similar proportion of enrolled subjects completed the study in both the treatment arms ( 223 ( 94.9% ) in the amoxicillin / clavulanic acid arm and 229 ( 96.6% ) in the clindamycin arm ) . a total of 11 ( 4.7% ) subjects in the amoxicillin / clavulanic acid arm and 8 ( 3.4% ) subjects in the clindamycin arm discontinued before study completion and the main reasons for discontinuations were as follows : aes ( one and two in amoxicillin / clavulanic acid and clindamycin arms , resp . ) , protocol noncompliance ( one and four in amoxicillin / clavulanic acid and clindamycin arms , resp . ) , and meeting the withdrawal criteria ( nine and two in amoxicillin / clavulanic acid and clindamycin arms , resp . ) . subjects recruited in the study were south east asian in origin with similar age and sex distribution across both treatment arms . periapical abscess was the predominant odontogenic infection across both arms ( 56.8% and 54.9% subjects in the amoxicillin / clavulanic acid and clindamycin arms , resp . ) . there was no significant difference in baseline characteristics such as pain , swelling , radiographic evidence of dental infection , and medical history / preexisting conditions between the treatment arms ( table 1 ) . the primary efficacy analysis using the pp population demonstrated that the clinical efficacy of amoxicillin / clavulanic acid was noninferior to clindamycin , since the upper limit of two - sided 95% ci was within the protocol specified noninferiority margin of 10% . the percentage of subjects achieving clinical success using the primary analysis population was 88.2% ( 95% ci : 83.0% , 92.2% ) in the amoxicillin / clavulanic acid arm and 89.7% ( 95% ci : 84.6% , 93.5% ) in the clindamycin arm . the treatment difference between the treatment arms was 1.5% ( 95% ci : 4.7% , 7.7% ) using miettinen and nurminen method , 1.5% ( 95% ci : 4.9% , 8.0% ) using farrington and manning method , and 1.5% ( 95% ci : 4.5% , 7.6% ) using a two - sample proportion test . since the upper limit of the two - sided 95% ci for between - group percentages differences was less than the prespecified noninferiority margin of 10% , noninferiority of amoxicillin / clavulanic acid to clindamycin with respect to clinical success was demonstrated . this result was also corroborated by sensitivity analysis using the itt - e population ( table 2 ) . a slightly higher percentage of subjects achieved clinical success in the amoxicillin / clavulanic acid arm by day 5 [ 76.8% ( 95% ci : 70.7% , 82.2% ) ] than the clindamycin arm [ 69.1% ( 95% ci 62.7% , 75.0% ) ] possibly indicating a faster response in subjects receiving amoxicillin / clavulanic acid arm as compared to subjects who received clindamycin . the least square mean change in the vas score for pain ( using itt - e dataset with oc approach ) was maximum at day 7 ( 6.38 and 6.34 in the amoxicillin / clavulanic acid and clindamycin arms , resp . ) compared to day 5 ( 5.49 and 5.38 in the treatment arms , resp . ) and day 2 ( 3.34 and 3.07 , resp . ) and it was similar between the treatment arms at each time point . a summary of vas by visit and treatment arms demonstrated that higher mean percentage reduction in pain by day 2 was achieved in the amoxicillin / clavulanic acid arm ( 49.5% ) compared with ( 45.6% ) the clindamycin arm . similarly , a higher mean percentage reduction in swelling by day 2 was achieved in the amoxicillin / clavulanic acid arm ( 43.6% ) compared with the clindamycin arm ( 39.6% ) . pus specimens were obtained in 58 subjects who consented to microbiological sampling including two who were screen failures ( 25 in the amoxicillin / clavulanic acid arm and 31 in the clindamycin arm ; the 2 screen failures were excluded ) . a total of 61 isolates were obtained from 56 samples , 26 in the amoxicillin / clavulanic acid arm and 35 in the clindamycin arm . organisms isolated in both the treatment arms were similar and predominantly viridans streptococci group ( ( n = 24 ) including streptococcus oralis , streptococcus mitis , and streptococcus parasanguinis ) , enterobacter spp . clsi breakpoints are not uniformly available for all isolates for both study drugs which posed a challenge in providing meaningful interpretation of susceptibility data . a total of 243 treatment emergent aes ( teaes ) were reported in 123 subjects in the amoxicillin / clavulanic acid arm and 236 events were reported in 124 subjects in the clindamycin arm . the most frequently observed teaes with frequency 3% were abdominal discomfort , raised liver enzymes ( ast , alt , and bilirubin ) , diarrhoea , dizziness , headache , increased appetite , and somnolence ( table 4 ) . generally the incidence of teaes was similar between treatment arms , except for diarrhoea and headache which were reported in slightly more patients in the clindamycin arm . the incidence of drug related teaes in both the treatment arms was similar ( 165 aes reported in 93 ( 39.4% ) subjects and 171 aes reported in 97 ( 41.3% ) subjects in the amoxicillin / clavulanic acid and clindamycin arms , resp . ) . the most frequently reported drug related teaes were gastrointestinal disorders including abdominal discomfort , diarrhoea , nausea and vomiting , abnormal lfts , increased appetite , somnolence , dizziness , and headache . most of the related teaes were mild to moderate in intensity except for six events of severe intensity . these were elevated alt , headache , and vomiting reported in the amoxicillin / clavulanic acid arm and burning sensation , hypertension , and hypersomnia in the clindamycin arm . a total of 89 subjects in the amoxicillin / clavulanic acid arm and 76 subjects in the clindamycin arm had aes that remained ongoing at the end of the study . increased lft was the predominant ongoing ae and was present in 46 subjects in the amoxicillin / clavulanic acid arm and 40 subjects in clindamycin arm , respectively . the probable reason for the ongoing aes could be the short duration of the study ( 7 to 8 days ) and the lack of a planned follow - up visit after study treatment . no subjects in the study showed shift in alt and ast from normal at baseline ( with respect to local laboratory reference range ) to pcc range ( as defined in statistical analysis section ) at end of the study whereas three subjects each in both the study arms showed shift in total bilirubin to pcc range at the end of the study . however , since these subjects only had increased bilirubin with no increase in alt or ast , these were not of clinical concern . four subjects in the clindamycin arm with high lft parameter ( one subject each for alt and ast and two subjects for total bilirubin ) at baseline remained in the pcc range at the end of the study ( figure 2 ) . the primary treatment in acute odontogenic infections is surgical drainage while antibiotics are an adjunct in patients showing signs of systemic involvement . the polymicrobial component of odontogenic infection necessitates the use of antibiotics that are active against both aerobic and anaerobic bacteria and therefore are recommended . the current study was aimed at comparing the clinical efficacy of amoxicillin / clavulanic acid ( 875 mg/125 mg ) to clindamycin ( 150 mg ) in subjects with acute odontogenic infections . amoxicillin / clavulanic acid ( 875 mg/125 mg ) was administered twice daily for 57 days and was found to be noninferior or comparable to clindamycin ( 150 mg ) administered four times daily . the overall clinical success seen with amoxicillin / clavulanic acid in the study ( 88.2% ( 95% ci : 83.0% , 92.2% ) ) was similar to results seen in other published studies . success rates of 87% with amoxicillin / clavulanic acid 1 g twice daily and 96% with amoxicillin / clavulanic acid 625 mg thrice daily have been previously reported in other studies [ 13 , 14 ] . a higher percentage of pain and swelling reduction ( 49.5% and 43.6% ) was achieved in the amoxicillin / clavulanic acid arm compared to the clindamycin arm ( 45.6% and 39.6% ) after two days of treatment . in a similar study aimed at demonstrating possible differences in the severity of symptoms after the use of amoxicillin and amoxicillin / clavulanic acid in dental ailments , thus , these results indicate that amoxicillin / clavulanic acid given twice daily for the treatment of odontogenic infection serves as an appropriate treatment option with the potential advantage of an early clinical response . safety of the subjects , assessed throughout the study , showed that the overall incidences of teaes were similar across both the treatment arms . these were mainly events such as abdominal discomfort , diarrhoea , and raised liver enzymes . as per the available safety information of amoxicillin / clavulanic acid 875/125 mg , nausea and diarrhoea are commonly reported events . diarrhoea was seen in 8.1% of subjects in amoxicillin / clavulanic acid arm ( compared to 11.9% in the clindamycin arm ) and this is consistent with the known pharmacological effects of the drug and with the global prescribing information . raised liver enzymes are a known but uncommon side effect ( 1/1,000 to < 1/100 ) of amoxicillin / clavulanic acid . in the current study , a total of 34 subjects ( 14.4% ) in the amoxicillin / clavulanic acid arm had raised liver enzymes posttreatment compared to 33 subjects ( 14.04% ) in the clindamycin arm that were assessed by the investigator as related to the study drug . however , most of these events were of mild to moderate intensity and none of the subjects were considered to have any lft values that were of clinical concern . pseudomembranous colitis is a rare but serious side effect of both clindamycin and amoxicillin - clavulanic acid . however , literature evidence suggests that the incidence is particularly low when these antibiotics are given in outpatient care settings ( 6.7 cases/100,000 antibiotic exposures ) . in dental infections there is not much difference in the incidence of c. difficile colitis between the two drugs . in the current study one of the main limitations of the study was the use of an outcome measure based on a subjective score ( vas score ) to assess pain and swelling to derive the composite clinical outcome . however , to overcome the subjectivity of the vas score and to reflect real world practice , only subjects demonstrating > 70% reduction in pain and swelling were considered for calculating clinical success response . another limitation was not having a planned follow - up visit for the subjects after the end of the study visits . as a result , ongoing aes typically key laboratory parameters such as liver enzymes could not be followed until resolution ; however , most were mild and transient in nature . amoxicillin / clavulanic acid ( 875 mg/125 mg ) administered twice daily was found to be comparable to clindamycin ( 150 mg ) administered four times daily in achieving clinical success in acute odontogenic infections with or without abscess . it was also found to be well tolerated with a safety profile consistent with the known pharmacologic effects of amoxicillin / clavulanic acid and with that described in the global prescribing information .

, the prevalence of diabetes in the eastern mediterranean region is estimated to be between 5% to 15%.1 in bahrain , the number of diabetics is 150,0002 and the prevalence is 14%.3 in a 1997 population - based visual disability survey in bahrain using who standard methodology , 2426 cases were examined . the results indicated that dr is a major cause of disability , accounting for 6% of cases.4 an established protocol for screening dr is needed to improve the management of diabetic eye disease and to prevent blindness . therefore , the institution of an efficient screening program directed toward detecting patients at risk is an urgent priority . the main goal of this project was to develop an integrated screening program within bahrain 's healthcare system to provide quality dr screening for early detection and management through an organized system of primary , secondary , and tertiary facilities to provide optimum care for diabetics . we began with a pilot telemedicine project for a period of 1 year from january through december 2003 . a screening unit was established at the muharraq health center , bahrain , for this purpose . the aim was to study the technical feasibility as well as the value of introducing a telemedicine screening program . primary health care is provided by the government through 22 primary health care centers . in each governorate , there is one regional and several other health care centers . following the evaluation and success of the pilot project , the program was expanded to other regional health care centers to cover the total population of the five governorates . by 2008 , five units are now established at health care centers and one unit at the diabetic outpatient clinic at salmaniya medical complex ( smc ) , the main referral hospital . the cumulative results of the program are based on data obtained from january 2003 through december 2009 . the entire program is a collaborative effort between the ministry of health , the national committee for the prevention of blindness , and a non - governmental organization . diabetes specialist nurses recorded the data from each patient on a dr screening card [ figure 1 ] . the data in the pilot study included demographic information , family physician details , diabetes and ophthalmological history , visual acuity , and fundus examination results . the data from 2003 to 2009 included the distribution of dr and associated ophthalmic conditions among the diabetic patients screened . the eyes of each patient were dilated with 1% topical tropicamide to obtain a clear fundus photograph . diabetic screening card each unit is equipped with a canon cr6 45 nm non - mydriatic digital camera connected to a computer . single - field 45 fundus photos were obtained by a trained ophthalmic technician , who was instructed to recapture any images of poor quality at the same sitting . the single - field 45 photograph was centered on a point halfway between the temporal edge of the optic disc and the fovea . the images were transferred electronically through the internet to the reading center at the ophthalmology department at the smc . the photographs were categorized by an ophthalmologist as no diabetic retinopathy ( ndr ) and diabetic retinopathy ( dr ) . patients with retinopathy were requested to return for further evaluation and treatment as warranted . on examination , retinopathy cases were classified according to the international classification of dr : no retinopathy , mild dr , moderate dr , or severe dr , nonproliferative dr ( npdr ) , and proliferative dr.5 those who did not require treatment were referred back for further digital photography follow - up appointments at the individual health centers . the study received written approval and ethical approval from the ministry of health medical research committee . the pilot project included data of 736 diabetic patients ; 49% were women and 51% were men . the diabetic history indicated that 25 ( 4% ) of the patients had type i diabetes ; 654 ( 89% ) had type ii diabetes , and 7% had diet controlled diabetes . almost all [ 708 ( 96% ) ] of the patients were under primary care ; 2 ( 1% ) were under secondary care ; and 24 ( 3% ) were under shared care . the visual acuity distribution for each eye in the pilot project is shown in table 1 . approximately 20% of patients were visually impaired or blind in at least one eye ( 6/24 or worse ) . the vast majority ( 90% ) of the fundus photographs were of good quality for interpretation . visual acuity in the pilot project cases table 2 shows the distribution of the different retinopathy classifications for the pilot project in 2003 and the total for 20032009 . of the total number of diabetic patients screened , dr was detected in 25.8% of cases in 2003 and in 20.4% of the cases in 20032009 . concurrent ophthalmic conditions in the patients in the pilot project were 29 cases of cataract , 6 cases of advance optic nerve disc cupping , 4 cases of pale disc , 4 cases of vitreous hemorrhage , 3 cases of chorioretinal scar , 1 case of macular hole , and 1 case of central retinal vein occlusion . the ophthalmic conditions of the patients from 2003 to 2009 included 326 ( 1.9% ) cases of cataract , 225 ( 1.3% ) cases of advanced optic disc cupping , 64 ( 41.0% ) cases of vitreous hemorrhage , 54 ( 0.3% ) cases of chorioretinal scars , 197 ( 1.1% ) cases of drusen , 12 ( 0.07% ) cases of central retinal vein occlusion , 16 ( 0.09% ) cases of retinal pigment epithelial changes , and 13 ( 0.13% ) cases of myelinated nerve fibers . to justify the dr screening project , a pilot diabetic screening unit using digital fundus photographs to screen a large number of patients and sending images through the internet was initiated and the results of this pilot study project indicated the effectiveness and feasibility of moving forward with the complete dr screening project . this screening program is a suitable method for detecting and grading dr in a non - selected diabetic population so that laser treatment can be performed at an optimal time in all patients with diabetes.69 ophthalmologists and primary care physicians can share digital retinal images of their patients , which will improve patient treatment . using an ophthalmologist at a reference center to diagnose and grade the digital images saves valuable time . while in most previous studies79 trained readers interpreted the retinal image photographs , in the present study ophthalmologists interpreted the retinal image photographs . single fundus photographs , however , can serve as an effective screening tool for dr when they are used to identify patients with retinopathy for referral for more complete ophthalmic evaluation and management , and they can be used as a more cost - effective means of screening for dr , which has been documented by other studies.1012 however , the single fundus photographs are of limited value for the diagnosis of macular edema.12 in this project , the majority of patients with diabetes received their care in a primary care setting . most primary care physicians have neither the expertise nor the equipment necessary to accurately screen for dr . moreover , they are overloaded with high patient volume at their clinics . applying screening tools at the primary care setting will improve patient compliance and be useful for training family physicians to screen images at the same time to identify some other common eye abnormalities , which will facilitate the screening process and help to prevent blindness.67 information technology infrastructure at the ministry of heath where all the primary and secondary care center systems are connected by the internet , as well as the advances in technology instituted by the government at bahrain facilitated this telemedicine approach and can be implemented in other specialties , which is an excellent and feasible approach for screening and treating patients for many other disease modalities in bahrain . the percentage of patients with dr from all regions of bahrain was 20.4% , which is similar to that reported in many studies in which screening was performed with a digital camera.1012 the number of patients receiving this service at the health care centers is increasing , but still they do not represent the expected number of diabetic patients , and this can be improved through patient education and promotion of the program . digital photography was useful for creating a network of several screening centers organized around a central ophthalmological reading center . digital imaging is important for community screening of blinding eye diseases such as glaucoma , cataract , and dr . the data indicate that a screening program for dr is feasible and justified to prevent blindness in patients with diabetes . this project represents a collaborative effort between primary care physicians and ophthalmologists for effective screening and timely treatment . recommendations and future plans include grading , follow - up of cases by family physicians through established guidelines , the use of full - time trained personnel to operate the digital camera for sustainability of the program , clinical workshops for primary care physicians to reinforce concepts , and the establishment of additional regional screening units .

hemolytic uremic syndrome ( hus ) , a common cause of acute renal failure in children , is one of a group of disorders referred to as thrombotic microangiopathies . hus - related symptoms include fever , microangiopathic hemolytic anemia , thrombocytopenia and renal failure . prior association of hus resulting from preceding infection with veracytotoxin or shigella - like toxin - producing bacteria , in particular the enteric pathogen enterohemorrhagic escherichia coli ( ehec ) o157:h7 and other e. coli serotypes have been documented.13 ehec results in hemorrhagic bloody diarrhea . additionally , infections involving neuraminidase - producing bacteria , especially streptococcus pneumoniae , have also been documented as etiologies of atypical non - diarrheal hus.57 the pathophysiology involves neuraminidase cleavage of the n - acetylneuraminic ( sialic ) acid from glyco - proteins in the cell membranes of erythrocytes , platelets and glomerular endothelial cells with subsequent exposure of the thomsen - friedenreich antigen ( t antigen).5 anti - t igm antibodies , present in plasma of humans greater than six months of age,8 subsequently bind the exposed antigen on involved rbcs , platelets , and endothelial cells ( see fig . a two year old caucasian female presented with fever and respiratory difficulty including tachypnea , grunting , intercostal retractions , and a cough . a right - sided otitis media was also noted . initial x - ray results were significant for bilateral lobar pneumonia , which was consistent with the physical exam findings . her blood counts decreased over the first three days of hospitalization , as can be seen in table 1 . also present no reactivity was observed with the anti - a and anti - b typing reagents , which are of monoclonal origin and not human source material . her direct antiglobulin test was 2 + positive , due to erythrocyte - bound complement ( c3b , c3d ) components . the antibody screen , using both low - ionic strength saline and polyethylene glycol - potentiated tests , was negative , as was a short cold panel . when tested with several samples of human adult and cord serum , the patient s red blood cells revealed strong reactivity with adult sera only . the sample was sent to a reference laboratory ( immucorgamma , houston , tx ) for confirmation of polyagglutination and for classification of the particular polyagglutinable state . the patient s red cells were found to be reactive ( 4 + ) with arachis hypogaea ( peanut lectin ) and glycine soja ( soybean lectin ) and non - reactive with salvia sclarea and salvia horminum lectins ( see table 2 ) , most consistent with t - activation . blood culture was positive for s. pneumoniae and was identified as serotype 19a by the texas department of health . nasopharyngeal cultures were negative for adenoviruses , influenza virus types a and b , and parainfluenza types 1 , 2 , and 3 . fecal cultures were negative for e. coli o157:h7 and campylobacter spp . as well as other enteric pathogens . peripheral blood smear examination revealed schistocytes and thrombocytopenia consistent with microangiopathic hemolytic anemia ( fig . this was considered most likely hus because of acute renal failure ( as evidenced by increased serum creatinine and blood urea nitrogen ) and known association with pneumococcal infections and t - activation , although confirmation with adamts13 was not performed . the patient ultimately required mechanical ventilation to treat metabolic acidosis prior to transfer to an institution capable of pediatric dialysis . the patient s diagnoses upon transfer included thrombotic microangiopathy most likely hus with acute renal failure secondary to pneumococcal pneumonia and sepsis , acute respiratory distress syndrome , pneumothoraces , and pleural effusions ultimately requiring bilateral thoracostomy tube placement after transfer . upon transfer , the patient underwent 3 weeks of continuous ambulatory peritoneal dialysis ( capd ) followed by nighttime intermittent peritoneal dialysis ( nipd ) . the patient s urine output slowly improved . a renal biopsy performed some time later at the outside institution revealed acute tubular necrosis ( atn ) without evidence of cortical necrosis . considered was the possibility that atn secondary to sepsis was the primary disease process ; atn , however , is not typically associated with microangiopathic hemolytic anemia or t - activation . the patient was ultimately discharged on hospital day # 36 . at the one week follow - up appointment , the patient was instructed to avoid crowded places for 23 months and to receive the influenza vaccine in order to minimize the chances of acquiring any respiratory illness . the long - term renal prognosis for this patient appears to be good in light of her normal physical exam , normal blood pressure , and normal renal function , although careful follow - up is ongoing because of her prolonged atn and the presence of interstitial fibrosis on biopsy . this case is not meant to provide new information on the topic of t - activation , but is meant to be a review of a relatively uncommon clinical phenomenon . polyagglutinable rbcs are red blood cells agglutinated by a large proportion of human adult serum , regardless of blood group , and are usually non - reactive with their autologous serum or serum from cord blood samples.12 principle causes of polyagglutination are modification of rbc membrane structure by microbial enzymes , incomplete biosynthesis of rbc membrane - associated carbohydrates , and inheritance of uncommon haplotypes . several forms of polyagglutination have been described . those associated with microbial infection are t , tk , th , tx , and acquired b. tn syndrome is a typical cause of polyagglutination by incomplete biosynthesis , while those due to inheritance are cad , nor , hempas , va , and hemoglobin m - hyde park.13,14 differentiation can be made by use of seed extracts , known as lectins ( except for nor ) . reaction patterns of some of the polyagglutinable states with a lectin panel are depicted in table 2.12,13 primary populations affected by t - activation include children with necrotizing enterocolitis , ehec , and serious streptococcus pneumoniae infections . adult t - activation has been demonstrated in patients with sepsis , gastrointestinal disorders , as well as respiratory and wound infections . t antigen sites have been demonstrated on red cells , platelets , white blood cells , and tissue cells . in hus patients specifically , the t - antigen has been demonstrated to be present on the glomerular capillary loops and renal tubular epithelium.14,15 t antigens , normally hidden , are exposed when neuraminidases are released from bacterial organisms which then cleave the sialic acid residues covering the t - antigens ( fig . 2).8,13,16 t - activation can be identified by the red cells reactions with the lectins derived from peanuts , arachis hypogaea and their reactions with soybean lectin , glycine soja.13,17,18 ( table 2 ) . t - activation caused by bacterial infections may occur quickly in vivo , but is usually transient , resolving within weeks or months . additionally , previous research has revealed approximately 0.5% of healthy individuals may also be affected with this condition.16 anti - t , a naturally occurring antibody , reacts best at room temperature or colder , rather than at 37 c , and does not appear to activate complement . the antibody is predominately igm , which likely forms as a result of exposure to intestinal flora with bacterial structures that have antigenic similarity to red blood cell cryptantigens . t antibodies , found in all human adult sera , begin to develop between 3 and 6 months of age and are present at adult levels by two years of age.16 the concern of transfusing patients with t - activated rbcs is related to the possibility of anti - t antibodies present in plasma - containing blood products causing hemolysis . hemolysis in patients with t - activation has been reported , whether or not the patient has been transfused . some reports show that no hemolysis was observed after transfusion of anti - t containing products,15 while very severe hemolysis and death have been reported in other patients.8 post - transfusion hemolysis may be related or dependent on the cause of t - activation . in addition to hemolysis , patients with s. pneumoniae associated - hus with t - activation may be subject to renal damage from transfusion of anti - t containing blood products . however , transfusion may be required for the hemostatic factors provided in these components and may outweigh the risk of hemolysis associated with passive transfusion of anti - t.16 therefore , the prudent course of action would be to limit transfusions to products containing low titers of anti - t antibody . blood products and derivatives containing small amounts of igm include cryoprecipitate , purified albumin concentrates , and ivig . if rbc transfusion is indicated , washed rbcs or red cells suspended in 10% albumin or plasma protein fraction may decrease hemolytic complications . for selection of donor plasma , a minor crossmatch may be beneficial in identifying those units with the lowest anti - t titers.16 however , other authors have demonstrated that there is minimal to no risk of transfusion - associated hemolysis in patients with t - activation,19,20 making this a very controversial issue . polyagglutinable rbcs are red blood cells agglutinated by a large proportion of human adult serum , regardless of blood group , and are usually non - reactive with their autologous serum or serum from cord blood samples.12 principle causes of polyagglutination are modification of rbc membrane structure by microbial enzymes , incomplete biosynthesis of rbc membrane - associated carbohydrates , and inheritance of uncommon haplotypes . those associated with microbial infection are t , tk , th , tx , and acquired b. tn syndrome is a typical cause of polyagglutination by incomplete biosynthesis , while those due to inheritance are cad , nor , hempas , va , and hemoglobin m - hyde park.13,14 differentiation can be made by use of seed extracts , known as lectins ( except for nor ) . reaction patterns of some of the polyagglutinable states with a lectin panel are depicted in table 2.12,13 primary populations affected by t - activation include children with necrotizing enterocolitis , ehec , and serious streptococcus pneumoniae infections . adult t - activation has been demonstrated in patients with sepsis , gastrointestinal disorders , as well as respiratory and wound infections . t antigen sites have been demonstrated on red cells , platelets , white blood cells , and tissue cells . in hus patients specifically , the t - antigen has been demonstrated to be present on the glomerular capillary loops and renal tubular epithelium.14,15 t antigens , normally hidden , are exposed when neuraminidases are released from bacterial organisms which then cleave the sialic acid residues covering the t - antigens ( fig . 2).8,13,16 t - activation can be identified by the red cells reactions with the lectins derived from peanuts , arachis hypogaea and their reactions with soybean lectin , glycine soja.13,17,18 ( table 2 ) . t - activation caused by bacterial infections may occur quickly in vivo , but is usually transient , resolving within weeks or months . additionally , previous research has revealed approximately 0.5% of healthy individuals may also be affected with this condition.16 anti - t , a naturally occurring antibody , reacts best at room temperature or colder , rather than at 37 c , and does not appear to activate complement . the antibody is predominately igm , which likely forms as a result of exposure to intestinal flora with bacterial structures that have antigenic similarity to red blood cell cryptantigens . t antibodies , found in all human adult sera , begin to develop between 3 and 6 months of age and are present at adult levels by two years of age.16 the concern of transfusing patients with t - activated rbcs is related to the possibility of anti - t antibodies present in plasma - containing blood products causing hemolysis . hemolysis in patients with t - activation has been reported , whether or not the patient has been transfused . some reports show that no hemolysis was observed after transfusion of anti - t containing products,15 while very severe hemolysis and death have been reported in other patients.8 post - transfusion hemolysis may be related or dependent on the cause of t - activation . in addition to hemolysis , patients with s. pneumoniae associated - hus with t - activation may be subject to renal damage from transfusion of anti - t containing blood products . however , transfusion may be required for the hemostatic factors provided in these components and may outweigh the risk of hemolysis associated with passive transfusion of anti - t.16 therefore , the prudent course of action would be to limit transfusions to products containing low titers of anti - t antibody . blood products and derivatives containing small amounts of igm include cryoprecipitate , purified albumin concentrates , and ivig . if rbc transfusion is indicated , washed rbcs or red cells suspended in 10% albumin or plasma protein fraction may decrease hemolytic complications . for selection of donor plasma , a minor crossmatch may be beneficial in identifying those units with the lowest anti - t titers.16 however , other authors have demonstrated that there is minimal to no risk of transfusion - associated hemolysis in patients with t - activation,19,20 making this a very controversial issue .

transcatheter aortic valve implantation ( tavi ) to treat severe , calcific aortic stenosis ( as ) has been shown to be both feasible and beneficial in those at prohibitive or high risk for surgical aortic valve replacement ( savr ) risk.1 2 previous surgical series have frequently identified mitral regurgitation ( mr ) in association with as.35 combined surgical intervention is associated with increased mortality and morbidity.46 current guidelines therefore suggest careful multidisciplinary evaluation and that , in the absence of morphological abnormalities , surgery for non - severe mr is not required , as relief of left ventricular pressure by savr resolves matters . there is no specific guidance regarding the management of more significant mr and the authors recognised the lack of data in the field.7 the issue of how to approach combined as and mr prior to tavi is complicated by the lack of a feasible technique for combined intervention . there has been discord in observational assessments of the effects of significant mr upon outcome after tavi using both balloon - expandable and self - expanding systems.815 we sought to identify the prognostic impact of both significant mr and change in mr severity in patients undergoing tavi using the balloon - expandable edwards sapien / sapien xt bioprosthesis . we performed a retrospective analysis of 316 consecutive , successful implantations of the edwards bioprosthesis at st . multimodality assessments were performed as previously described and cases reviewed at a multidisciplinary team meeting.16 tavi was performed using transfemoral , transapical or transaortic routes . patients were prospectively enrolled onto a dedicated database of patient demographics , characteristics , procedural results and long - term outcome . this involved assessment of valve morphology , colour doppler and continuous - wave doppler of the regurgitant jet , vena contracta width , upstream venous flow , regurgitant volume and effective orifice area ( where available).17 all scans were performed by british society of echocardiography accredited echocardiographers with final adjudication by a consultant cardiologist specialising in cardiac imaging . , new york , usa ) and prism v.6 ( graphpad software , california , usa ) . univariate cox proportionate hazards modelling was performed using an unadjusted model for each covariate , and we then constructed a multivariate model using a forward elimination method and entry criteria of p0.05 . we analysed 316 patients who underwent tavi for severe as using either the edwards sapien or sapien xt bioprostheses . in total , 60/316 ( 19.0% ) patients were identified as having moderate - to - severe mr . patients with significant mr had higher perioperative risk according to logistic euroscore ( 28.716.6% vs 20.310.7% , p=0.004 ) and society of thoracic surgeons ( sts ) score ( 7.73.7 vs 5.73.1 , p<0.001 ) . they also had more severe dyspnoea ( new york heart association iv in 20.0% vs 7.4% , p=0.014 ) , poorer lvef ( 44.014.6% vs 50.411.8% , p=0.004 ) and more frequent significant ( grade 3 ) aortic or tricuspid regurgitation ( tr ) ( 28.3% vs 12.1% ( p=0.046 ) and 28.3% vs 8.9% ( p<0.001 ) , respectively ) . egfr , estimated glomerular filtration rate ; mr , mitral regurgitation ; sts , society of thoracic surgeons . efficacy of the tavi regardless of mr was manifest by the improvement in peak transaortic gradient at follow - up ( figure 1 ) . patients with significant preprocedural mr had higher rates of postprocedural haemofiltration ( 18.3% vs 7.0% , p=0.006 ) , although there was no difference in length of intensive care stay ( 3.56.8 days vs 3.210.2 days , p=0.840 ) . significant mr is denoted by orange , non - significant mr by blue . * p is non - significant ( ie , e>0.05 ) . meier analysis revealed that patients with significant preprocedural mr had higher all - cause mortality ( 28.3% vs 20.2% at 12 months ) and significantly divergent mortality plots ( log - rank p=0.023 ) ( see figure 2a ) . patients in whom mr deteriorated had a poorer outcome than those with an improvement in mr ( log - rank p=0.05 , see figure 2b ) . we found no difference between the demographics of these groups except a higher rate of pulmonary disease in those mr improved ( see online supplementary table s1 ) . ( a ) kaplan meier curve demonstrating the increased cumulative mortality of significant preprocedural mitral regurgitation ( mr ) . ( b ) kaplan meier curve demonstrating the effects of change in mr severity after transcatheter aortic valve implantation upon mortality . when we consider the overall study population , there was no difference in the distribution of mr severity between the pre - tavi , 30-day and 12-month time - points . however , only 47.1% of the 60 patients with significant preprocedural mr had grade 34 mr at 30 day ( mcnemar 's test p<0.001 ) , with no significant difference at 12 m. in those patients without baseline significant mr , there was no significant change in the distribution ( p=0.07 ) . thirty - seven had 30 day echo data available , and of these only 13 ( 35.1% ) had significant tr ( p<0.001 ) . in the 60 patient cohort with significant mr , the aetiology was organic in 83.3% of patients whose mr deteriorated and 72.4% in those in whom mr improved ( p<0.001 ) . patients undergoing tavi experienced an improvement in lvef ( p<0.001 ) , driven by an improvement in those patients without significant mr ( p<0.001 ) . multivariate cox proportional hazards modelling revealed that significant preprocedural mr had an independent association with mortality ( hr 4.94 ( 95% ci 2.07 to 11.76 ) , p<0.001 ) as did estimated glomerular filtration rate ( hr 0.97 ( 95% ci 0.95 to 0.99 ) , p=0.001 ) . present in 40 patients ( 12.7%)reached significance in the univariate analysis but not the multivariate . efficacy of the tavi regardless of mr was manifest by the improvement in peak transaortic gradient at follow - up ( figure 1 ) . patients with significant preprocedural mr had higher rates of postprocedural haemofiltration ( 18.3% vs 7.0% , p=0.006 ) , although there was no difference in length of intensive care stay ( 3.56.8 days vs 3.210.2 days , p=0.840 ) . significant mr is denoted by orange , non - significant mr by blue . * p is non - significant ( ie , e>0.05 ) . kaplan meier analysis revealed that patients with significant preprocedural mr had higher all - cause mortality ( 28.3% vs 20.2% at 12 months ) and significantly divergent mortality plots ( log - rank p=0.023 ) ( see figure 2a ) . patients in whom mr deteriorated had a poorer outcome than those with an improvement in mr ( log - rank p=0.05 , see figure 2b ) . we found no difference between the demographics of these groups except a higher rate of pulmonary disease in those mr improved ( see online supplementary table s1 ) . ( a ) kaplan meier curve demonstrating the increased cumulative mortality of significant preprocedural mitral regurgitation ( mr ) . ( b ) kaplan meier curve demonstrating the effects of change in mr severity after transcatheter aortic valve implantation upon mortality . when we consider the overall study population , there was no difference in the distribution of mr severity between the pre - tavi , 30-day and 12-month time - points . however , only 47.1% of the 60 patients with significant preprocedural mr had grade 34 mr at 30 day ( mcnemar 's test p<0.001 ) , with no significant difference at 12 m. in those patients without baseline significant mr , there was no significant change in the distribution ( p=0.07 ) . thirty - seven had 30 day echo data available , and of these only 13 ( 35.1% ) had significant tr ( p<0.001 ) . in the 60 patient cohort with significant mr , the aetiology was organic in 83.3% of patients whose mr deteriorated and 72.4% in those in whom mr improved ( p<0.001 ) . patients undergoing tavi experienced an improvement in lvef ( p<0.001 ) , driven by an improvement in those patients without significant mr ( p<0.001 ) . multivariate cox proportional hazards modelling revealed that significant preprocedural mr had an independent association with mortality ( hr 4.94 ( 95% ci 2.07 to 11.76 ) , p<0.001 ) as did estimated glomerular filtration rate ( hr 0.97 ( 95% ci 0.95 to 0.99 ) , p=0.001 ) . present in 40 patients ( 12.7%)reached significance in the univariate analysis but not the multivariate . our study confirms that preprocedural significant mr has an independent association with mortality after tavi using the edwards bioprosthesis but also that significant regurgitation also improves in many of these patients . given that improvement and that the mortality in the mr cohort is still reassuringly smaller than that of tavi candidates treated medically,1 tavi should not be denied to these patients on the basis of mr alone . significant mr at the time of savr is associated with an increased risk of adverse outcome4 5 and current guidelines recommend treatment of both conditions if the valvular haemodynamics suggest significant disease in both.7 concomitant mr with as is well described in the surgical literature and has also been noted in patients with tavi.4 5 9 12 18 functional mr is frequently seen in dilated cardiomyopathy and increases mortality.19 20 it is caused by distortion of the lv and papillary muscle geometry , displacing the mitral valve leaflets ( mvl ) below the plane of the mv annulus : when there is insufficient mvl area to compensate , the valve apparatus is rendered incompetent.2025 leaflet thickness and area increase in response to abnormal loading with increased tension during systole21 through greater expression of collagen and aminoglycosans rendering them less compliant.2628 recently , results from a large italian corevalve registry have suggested a higher 1-month mortality in the severe and moderate mr groups than those with mild or no mr ( 11% vs 9% vs 5% , respectively ) , extending to the 1-to-12-month time period ( 17% vs 12% vs 10% ) . the presence of greater - than - mild mr increased the risk of death by almost threefold , and 8.4% of patients with less - than - mild mr suffered a deterioration in their grade of mr.12 the design of the more intrusive corevalve prosthesis may cause greater interference of the anterior mvl.29 toggweiler and colleagues have previously noted an increase in 30-day mortality in patients with moderate severe mr receiving the edwards device ( adjusted hr 2.10 , p=0.02)however , mortality beyond 30 days was similar.8 an ' as - treated analysis of cohort a patients from the partner trial found that preprocedural mr had no effect upon mortality after balloon - expandable tavi but did adversely impact outcome after savr.9 severe mr was a contraindication to enrolment into the partner trial though in the subsequent analysis , seven patients ( 2.3% ) did have severe mr . in the primary analysis of the partner b cohort , there was a trend to favour tavi above medical therapy in patients with moderate severe mr ( pinteraction=0.09).1 recently , two studies analysing atrioventricular regurgitation and transapical tavi have shown a similar improvement in mr but no long - term effect upon mortality13 14though one noted a fourfold increase in risk of in - hospital death.14 sixty - seven per cent of patients in a study by hutter et al demonstrated an improvement in mr after tavi . while those with moderate severe mr had higher 12 m mortality ( 30.2% vs 21.2% ) , it did not reach statistical significance ( p=0.068).15 improvement in the degree of mr after tavi has been associated with the absence of pulmonary hypertension and atrial fibrillation,8 12 mean aortic valve gradient > 40 mm hg8 and functional mr.8 12 similarly after savr functional mr has been associated with improvement in the severity of mr.4 functional mr relies on an incompetent orifice and sufficient systolic gradient between the lv and left atrium . lv dysfunction and remodelling and increased lv afterload as seen in as can lead to these morphological and haemodynamic changes . lv wall stress and ejection pressure have been shown to improve after savr,30 which may explain some of the beneficial effects of aortic valve intervention . our own data are consistent with the literature and suggest that patients whose significant mr improved had higher rates of functional mr , suggesting that there may be a threshold lv pressure causing mr in an abnormal ventricle / valve unit that tavi may improve . given existing data detailed above , perhaps caution should be exhibited in patients with organic mr and sequelae such as atrial fibrillation and pulmonary hypertension . however , such a recommendation would require studies larger by orders of magnitude than those currently in the literature . the data for our study were gathered prospectively into a tavi patient database . however , the analyses were performed post hoc using definitions devised after the initiation of data collection and are limited by the available parameters . the study , like all registry data , is subject to the effects of possible confounders and bias in its design . the limited size of the study means that differences between patients with / without significant preprocedural mr or with / without improved mr may not have reached significance due to limited power . finally , the raw quantitative data used in the assessment of mr severity were not always available and so could not be included for analysis . previous data regarding the effects of preprocedural mr upon outcome after balloon - expandable tavi have been inconsistent . we have demonstrated that significant mr does have a significant effect upon mortality after tavi , but the magnitude of this change would appear to be less significant than the potential benefits of tavi . as such , mr should not present an absolute contraindication to tavi . given the deleterious effect of worsening of mr severity , we suggest that larger collaborative studies are necessary to identify factors associated with risk of deterioration . key messageswhat is already known on this subject?mitral regurgitation ( mr ) is often found in patients with calcific aortic stenosis and current guidelines suggest it can be left untreated at the time of surgical aortic valve replacement unless severe . mr has also been described in high - risk patients undergoing transcatheter aortic valve implantation ( tavi ) , but there is disagreement regarding its impact upon outcome.what might this study add?our study has found an independent association between significant ( ie , moderate severe ) mr and the risk of death after tavi , while confirming that there is also a significant improvement in the degree of mr seen after tavi . mortality rates seen in patients with significant mr are considerably lower than we would expect in patients from this cohort receiving only medical therapy . patients with significant mr who experience an improvement have higher rates of functional mr rather than organic disease and have lower mortality than those in whom mr worsens . we also found a reduction in significant tricuspid regurgitation.how might this impact on clinical practice?while clinicians assessing patients for tavi should be mindful of the increased risk of patients with significant mr , it should not be considered a contraindication . but careful examination of the severity and aetiology of the mr is necessary in preprocedural selection and planning . given the deleterious effects of worsening mr , further collaborative work is necessary to identify those at risk to improve safety for patients with mr undergoing tavi . mitral regurgitation ( mr ) is often found in patients with calcific aortic stenosis and current guidelines suggest it can be left untreated at the time of surgical aortic valve replacement unless severe . mr has also been described in high - risk patients undergoing transcatheter aortic valve implantation ( tavi ) , but there is disagreement regarding its impact upon outcome . significant ( ie , moderate severe ) mr and the risk of death after tavi , while confirming that there is also a significant improvement in the degree of mr seen after tavi . mortality rates seen in patients with significant mr are considerably lower than we would expect in patients from this cohort receiving only medical therapy . patients with significant mr who experience an improvement have higher rates of functional mr rather than organic disease and have lower mortality than those in whom mr worsens . while clinicians assessing patients for tavi should be mindful of the increased risk of patients with significant mr , it should not be considered a contraindication . but careful examination of the severity and aetiology of the mr is necessary in preprocedural selection and planning . given the deleterious effects of worsening mr , further collaborative work is necessary to identify those at risk to improve safety for patients with mr undergoing tavi .

potential is essential for survival following partial hepatic resection ( e.g. , for tumor removal or live - donor liver transplantation ) and from acute and chronic liver injury secondary to toxins , infections , immune dysfunction , metabolic diseases , or other causes [ 13 ] . nevertheless , liver diseases remain an important cause of morbidity and mortality , and inadequate hepatic regeneration likely contributes . based on these considerations , the mechanisms that regulate liver regeneration continue to be the subject of intense research , with hope that the knowledge gained will lead to novel strategies with which to improve the outcomes of many liver diseases . a number of studies have identified fatty liver as an important risk factor for impaired liver regeneration in humans and experimental animal models . in apparent distinction to those observations however , and still a lingering paradox in the literature , a number of reports suggest that the transient hepatocellular fat accumulation characteristic of early regeneration following partial hepatectomy ( ph ) in rodents is actually required for physiological liver regeneration . here , we review these data , propose a hypothesis for the seemingly dichotomous relationship of chronic versus acute hepatic fat accumulation on liver regeneration , and consider important areas for future research . the best characterized and most readily controlled experimental model for investigating the molecular , cellular , and physiologic mechanisms that control liver regeneration has been ph in rodents . in the most typically used version of this paradigm , ph , the anesthetized rodent is subjected to midventral laparotomy with sequential ligation and resection of the left and median hepatic lobes followed by closure of the surgical wounds and recovery . subsequent regeneration is assessed by analyses of hepatocellular proliferation , liver mass , gene and protein expression , and signaling events at serial time points after the surgery . studies using this experimental system show that regeneration after ph is precisely regulated in both its initiation and duration , terminating only when the original liver - to - body mass ratio is restored . furthermore , this response does not require recruitment or mobilization of either a resident or exogenous stem cell population . rather , all of the normally quiescent hepatocytes in the mature liver have the potential to proliferate in response to partial hepatic resection . pharmacological and genetic manipulations of animals subjected to ph have identified many signals , including cytokines ( e.g. , tumor necrosis factor ( tnf ) and interleukin 6 ( il6 ) ) , growth factors ( e.g. , hepatocyte growth factor ( hgf ) , epidermal growth factor - receptor ligands , and fibroblast growth factors ) , intracellular signaling events ( e.g. , wnt/-catenin ) , and transcription factors ( e.g. , nfb , stat3 , creb , c / ebp , ap1 , fxr , and lxr ) , that are regulated in response to ph and influence the subsequent hepatic regenerative response ( reviewed in [ 13 , 5 , 6 ] ) . those signals , some of which are initiated within minutes of surgical resection , promote restoration of normal hepatic mass , architecture , and function over the ensuing days , after which regeneration ceases . one advantage of the ph model of liver regeneration over others is the absence of injury to the remnant liver tissue following the ( surgically induced ) regenerative stimulus , which thereby minimizes potential confounders to interpretation of the functional specificity of induced signals for the regenerative response itself . nevertheless , experimental models of toxin - induced liver regeneration ( e.g. , carbon tetrachloride ( ccl4 ) , thioacetamide [ 7 , 8 ] ) have also been extensively used to investigate regenerative mechanisms , and the importance of several regulatory pathways identified in the ph model has been shown to be conserved in such paradigms [ 9 , 10 ] . in contrast , the hepatocellular proliferative response to primary mitogens does not depend upon tnf , hgf , nfb , and several other signals implicated as important in hepatic insufficiency - induced liver regeneration [ 1116 ] . despite the broad knowledge gained from almost a century of studies using these models , the nature and identities of the most proximal events that initiate hepatic regeneration as well as the distal events that terminate this process are still not known . liver mass is maintained or recovered in precise proportion to body mass , giving rise to the concept of an intrinsic regulator of liver : body mass ratio , that is , the hepatostat the liver plays critical roles in the regulation of systemic metabolism and extrahepatic energy consumption , which themselves are influenced by body mass . together , these considerations suggest that changes in intermediary metabolism in response to hepatic insufficiency could contribute essential signals for initiation of liver regeneration , and , conversely , that restoration of metabolic homeostasis after recovery of the normal liver : body mass ratio might provide signals that terminate this response . consistent with the liver 's central role in gluconeogenesis , rodents subjected to ph become hypoglycemic . extending this paradigm , other studies show that either intravenous or enteral dextrose supplementation suppresses both ph- [ 1824 ] and toxin- ( ccl4 , thioacetamide ; [ 25 , 26 ] and j. huang and d.a . similarly , dietary caloric restriction accelerates onset of hepatocellular proliferation in response to surgical- or toxin - induced hepatic insufficiency [ 27 , 28 ] . circulating insulin levels decline in response to ph - induced hypoglycemia ( and are augmented by dextrose supplementation ) , and , interestingly , hepatocellular proliferation in models of toxin - induced liver injury is accelerated and augmented in mice with streptozotocin - induced insulin - deficient diabetes [ 29 , 30 ] . nevertheless , it remains to be established if changes in insulin signaling mediate the effects of dextrose supplementation on liver regeneration in such models . somewhat paradoxically , studies have also shown that insulin supplementation reverses hepatic lobar atrophy in response to portacaval shunting in vivo and that insulin augments the activity of hepatocyte mitogens in cell culture . this latter observation is consistent with studies , including those noted above , demonstrating that differences exist between the signals that regulate hepatocellular proliferation in response to surgical- or toxin - induced loss of liver mass and those that determine mitogen - induced proliferation . dextrose - mediated inhibition of ph - induced liver regeneration is associated with disruption of many signaling events identified as important for regeneration . for example , provision of supplemental dextrose augments hepatic expression of the mitoinhibitory factors c / ebp , p21 , and p27 and suppresses expression of the proregenerative transcriptional regulator , foxm1 , in animals subjected to ph . consistent with these findings , induction of proregenerative signals , including il6 , transforming growth factor ( tgf ) , and hgf , is accelerated by caloric restriction in toxin- ( thioacetamide)-induced liver regeneration . together , these observations support a model in which the hypoglycemic response to hepatic insufficiency initiates the signals that promote liver regeneration . many of the regenerative signals that are disrupted by dextrose supplementation are also deranged in association with the impaired regenerative response observed in aged animals subjected to ph [ 31 , 32 ] . those changes in older mice appear to be mediated , at least in part , by age - dependent epigenetic effects [ 33 , 34 ] . together , these findings support a model in which the metabolic responses to hepatic insufficiency after ph ( e.g. , hypoglycemia ) activate a transcriptional network through pathways including epigenetic regulation . in contrast to ph - induced liver regeneration , toxin - induced regeneration is undiminished in old versus young animals . the observations noted above implicate hypoglycemia and subsequently induced alterations in systemic metabolism as modulators of physiological liver regeneration . this consideration has prompted further investigation of the metabolic response to hepatic insufficiency in experimental models of regeneration . recent reports have characterized the stereotypical decline in systemic lean and adipose tissue stores and the ensuing rise in circulating and hepatic free fatty acids and specific amino acids that occur in response to ph prior to the onset of regeneration [ 3638 ] . those analyses also show that specific alterations in metabolism , like the regenerative response itself , occur in proportion to the extent of hepatic insufficiency . for example , two - thirds ph results in a significantly greater loss of systemic adipose stores ( and a more robust hepatocellular proliferative response ) than does one - third hepatectomy . systemic fat depletion has also been observed in various models of toxin - induced liver regeneration [ 36 , 39 ] . these findings together suggest that catabolism of systemic adipose tissue might regulate the hepatic regenerative response to surgical and toxin - induced loss of liver mass . as noted above , it has long been recognized that the early regenerating liver transiently accumulates hepatocellular fat after ph [ 4043 ] . other work has demonstrated fat accumulation concomitant with cellular proliferation in primary hepatocyte culture , raising the possibility that fat accumulation might in turn regulate hepatocyte proliferation . furthermore , the patterns of hepatic mrna induction during early ph - stimulated liver regeneration suggest the existence of a conserved transcriptional program leading to regulated transient steatosis during the regenerative response [ 45 , 46 ] . increased de novo hepatic fatty acid production has been reported in regenerating liver , but mice with liver - specific disruption of fatty acid synthase expression ( i.e. , faskol mice , ) exhibit comparable hepatic fat accumulation ( and liver regeneration ) after ph compared to that in wild - type controls , strongly suggesting that de novo hepatic lipogenesis is not required for the development of such transient hepatic steatosis or the regenerative response . these and other findings point to systemic adipose tissue as the primary source of the lipid that accumulates in regenerating liver [ 39 , 40 ] . a number of experimental observations provide support for the possibility that the alterations in hepatic and systemic lipid metabolism discussed above are essential for normal liver regeneration . for example , older studies have noted increased dependency following ph of regenerating liver on -oxidation of fatty acids for energy production [ 22 , 48 ] . indeed , it has been speculated that the inhibitory effect of dextrose supplementation on liver regeneration might be secondary to the suppressive effect of such supplementation on the release of free fatty acids from systemic adipose stores , and infusion of an inhibitor of -oxidation , ( ( + ) -octanoylcarnitine ) , has been reported to impair regeneration . moreover , parenteral administration of lipid emulsions or of carnitine , which mediates uptake of acyl groups into mitochondria for -oxidation , has been reported to accelerate ph - induced regeneration , and dietary supplementation with palmitate and carnitine augments toxin- ( thioacetamide)-induced hepatocellular proliferation . however , questions about the role of alterations in -oxidation have been raised by analyses of ppar knockout mice in which -oxidation is dysregulated . some reports demonstrate normal ph - induced regeneration [ 37 , 50 ] , and others show impaired regeneration [ 51 , 52 ] in these animals . more recent studies have reported inhibition of liver regeneration by various experimental interventions that decrease hepatic fat accumulation after partial hepatectomy , including both pharmacological ( e.g. , clofibrate , leptin , or propranolol supplementation ) and genetic ( e.g. , liver - specific disruption of glucocorticoid receptor expression , ) strategies . those findings collectively imply a requisite role for hepatic steatosis in liver regeneration . however , as alluded to above , other findings raise questions about the specific function of hepatic fat accumulation during normal regeneration . for example , fat accumulation is suppressed but regeneration proceeds normally following ph in liver fatty acid binding protein ( l - fabp ) knockout mice . in addition , caveolin 1-null mice exhibit reduced hepatic steatosis after ph , with regeneration reported to be impaired in one study but not another . finally , regeneration proceeds normally in mice with intestine - specific deletion of the microsomal triglyceride transfer protein ( mtp - iko ) , which is essential for intestinal absorption of dietary fat and which exhibit decreased peripheral adipose tissue . importantly , hepatectomy - induced fat accumulation was reduced but not completely abrogated in l - fabp - null and mtp - iko mice , leading to speculation about the existence of a threshold of adaptive lipogenesis essential for regeneration but not influenced in those models . the role of mobilization of lipid from adipose tissue stores during ph- and toxin - induced liver regeneration has also been investigated by analyses of fatty liver dystrophy ( fld ) mice , which have a paucity of systemic adipose tissue as a result of global disruption of lpin1 expression . fld mice exhibit reduced hepatocellular triglyceride accumulation and proliferation with augmented hepatic p21 expression after ph compared to littermate controls . , these data support a model in which metabolism of systemic adipose tissue in response to hepatic insufficiency promotes initiation of hepatocellular proliferation . an important caveat to the analyses of fld mice is that the target gene of interest , lpin1 , is expressed in liver and muscle in addition to adipose tissue , and its expression is globally disrupted in fld mice . thus , lpin1 might have effects on hepatic steatosis and hepatocellular proliferation during liver regeneration dependent on its hepatic expression and independent of its effects on systemic adipose tissue stores . interestingly , hepatic lpin1 expression is induced after ph , and such induction is attenuated in liver - specific glucocorticoid receptor null mice , in which ( as noted above ) the metabolic and hepatocellular proliferative responses to ph are deranged ( and d. a. rudnick , unpublished observations ) . these findings , together with the known pleiotropic functions of the protein product of lpin1 ( lipin1 , ) suggest several potential alternative mechanisms to explain the impaired regenerative phenotype in fld mice ) . for example , lipin1 amplifies peroxisome proliferator - activated receptor gamma coactivator 1- ( pgc1- ) regulated transcription in hepatocytes to increase expression of genes encoding enzymes involved in fatty acid oxidation ( and known to be regulated during liver regeneration , [ 59 , 60 ] ) . lipin1 also stimulates peroxisome proliferator - activated receptor - ( ppar- ) dependent adipogenic gene expression in adipocytes . finally , lipin1 is a phosphatidic acid phosphatase enzyme , catalyzing the conversion of phosphatidic acid ( pa ) to diacylglycerol ( dag , ) . pa and dag also function as lipid second messengers in protein kinase c activation , which occurs during liver regeneration [ 6567 ] . thus , loss of the transcriptional or enzymatic activities of hepatic lipin1 might contribute to impaired regeneration in fld mice . a further consideration , not exclusive of those outlined above , is that alterations in hepatic and circulating pools of cholesterol might play a role in some phases of liver regeneration . this possibility has emerged from analyses in pcsk9 knockout mice , which exhibit decreased pools of circulating cholesterol , impaired regeneration , and hepatic necrosis after ph , all of which are reversed by high cholesterol feeding . additional support for this idea comes from demonstration that ligand - induced liver x receptor ( lxr ) activation in mice subjected to ph alters plasma and hepatic cholesterol pools and impairs liver regeneration . the data summarized above suggest that alterations in lipid metabolism that occur in response to hepatic insufficiency contribute to the initiation of both resection- and toxin - induced liver regeneration . although the specific mechanisms that couple changes in lipid metabolism to onset of hepatic regeneration have not been elucidated , several possibilities are worthy of consideration ( figure 1 ) . for example , lipids delivered from the periphery or synthesized de novo in response to partial hepatic resection or other liver injury might serve simply as the substrate for energy production or for membrane synthesis required for hepatocellular proliferation . another intriguing consideration is that lipid - derived metabolites might influence regenerative signaling pathways via transcriptional or epigenetic mechanisms . for example , the transcriptional activities of the nuclear steroid hormone transcription factors ppar , fxr , and lxr , which have each been implicated as important during ph - induced liver regeneration [ 51 , 52 , 69 , 71 ] , are regulated by binding to specific classes of phospholipid , bile acids , or oxysterols , respectively . in addition , certain fatty acids have been reported to influence the acetylation state of metabolic enzymes in hepatocytes and thus might also regulate epigenetic changes in regenerating liver . finally , hepatic insufficiency - induced alterations in lipid metabolism might affect physiologic liver regeneration via adipose - derived hormones . indeed , the influence of adipokines on regeneration has been suggested by studies showing inhibition of ph - induced liver regeneration in wildtype mice by leptin supplementation and impaired regeneration in adiponectin knockout mice [ 74 , 75 ] . the influence of adipose metabolism on liver regeneration has also been demonstrated by the recognized association between chronic hepatic steatosis and impaired regeneration in experimental animal models . leptin - deficient ( ob / ob ) [ 7679 ] and -resistant ( db / db ) [ 80 , 81 ] , diabetic kk - a(y ) , western and high - fructose diet - fed mice , and leptin - resistant obese zucker rats [ 85 , 86 ] , each of which exhibit hepatic steatosis , have all been reported to demonstrate impeded regeneration after ph or ccl4 administration . in contrast , liver regeneration is not impaired in models of mild hepatic steatosis , including orotic acid- and choline- deficient diet - fed rats , leading some investigators to speculate that the degree of steatosis is important in determining its effect on liver regeneration . consistent with that interpretation , liver regeneration is variably affected in animals administered a methionine - choline deficient ( mcd ) , a phenotype dependent on the magnitude of steatosis [ 86 , 8891 ] . despite these many studies linking chronic hepatic steatosis with impaired liver regeneration , the mechanisms responsible remain enigmatic . moreover , the basis for the differences in the influence of chronic and acute hepatic fat accumulation on regeneration is undefined . chronic steatosis has also been associated with adverse outcomes after major hepatic resection in humans . a recent meta - analysis showed that the risk of postoperative complications in patients with any degree of steatosis undergoing hepatectomy ( for neoplasm ) was double that of their nonsteatotic counterparts , and that those with excessive ( > 30% ) steatosis had an almost 3-fold increased risk of death . this analysis did not address whether impaired regeneration was the culprit ; however , a study of patients undergoing liver resection ( for living related liver donation ) showed reduced recovery of liver volume over the initial 3 months following surgery in patients with mild steatosis ( versus no steatosis , ) , and another study reported decreased recovery of liver function 612 months after hepatectomy in such patients . as enumerated above , extensive older and more recent analyses implicate alterations in adipose metabolism in response to surgical- or toxin - induced hepatic insufficiency as functionally important for initiation of normal liver regeneration . similarly , the mechanisms responsible for the inhibitory effect of chronic hepatic steatosis on liver regeneration in experimental model systems and humans undergoing hepatic resection remain to be established . it is tempting to speculate that the acute changes in systemic lipid metabolism that occur in response to hepatic insufficiency have specific , direct transcriptional , and epigenetic proregenerative effects , and that such events are modified or reversed in chronic fatty liver disease . future studies should investigate the functional relationships between these metabolic , genetic , and epigenetic alterations during normal liver regeneration and examine the influence of chronic hepatic steatosis on those relationships .

depression is one of the most common mental health problems and is a significant cause of morbidity . depression interferes with daily functioning and quality of life and places significant burden on the health care system.1 depression is also an important risk factor for suicide.2 many factors contribute to the etiology of depression , including stressful life events,3,4 medical treatments , nonpsychiatric illnesses , such as neurological conditions,5 stroke,6 diabetes,7 and cancer,8 and psychological factors . recent studies have demonstrated an association between air pollution and depressive symptoms in humans.918 gaseous ambient air pollutants such as ozone are known to behave as harmful agents.19 fine particulate matter are associated with increased mortality due to respiratory disease , heart disease , and stroke.20 ozone exposure is known to affect health19,2128 and is mainly a risk factor for respiratory conditions such as asthma.2931 several studies have also linked ambient ozone to nonrespiratory conditions such as cardiac disease,31,32 appendicitis,33 headaches and migraines,34 and skin conditions.3539 moreover , because of its oxidative properties , ozone may induce oxidative stress , affecting the central nervous system ( cns ) , which is a potential cause of depression.40 indeed , some studies have demonstrated links between ozone levels and depression9,10,16 and suicide attempts.11,40,41 a study conducted in edmonton , canada , suggested that emergency department ( ed ) visits for depression are associated with exposure to ambient air pollution.9 this time series study analyzed 15,556 ed visits for depression between 1992 and 2002 . in another canadian study , a hierarchical clusters design was used to examine 27,047 ed visits for depression in 6 canadian cities . results showed a correlation between ed visits for depression and ambient air pollution , most of which results from the combustion of fossil fuels , such as from motor vehicles.10 a study of 537 participants in seoul , korea , reported that an increase in ambient air pollution , specifically pm2.5 , no2 , and o3 , was associated with an increase in depressive symptoms among elderly adults . these results add to the growing body of literature , suggesting an association between ambient air pollution and emotional symptoms.16 suicide attempts as a result of depression have also been linked to air pollution exposure . in a study in vancouver , canada , ambient air pollution was associated with ed visits for suicide attempts.11 a study in taipei city , taiwan , examined 4,857 deaths by suicide , demonstrating a temporal relation between suicide and air pollution.41 results from several studies suggest that exposure to air pollutants may affect men and women differently.42 for example , sex hormones estradiol and testosterone are known to influence transportation and accumulation of toxic chemicals to the brain and other organs . it is not yet known how these sex differences might bear on depression and suicide risk.42 only limited number of studies have been reported on the relations between air pollution and depression , and most of these studies were done in a single city with small sample size . this might be as a result of the fact that depression is not a conventional disease that has been biologically linked to air pollution . the aim of this multicity study was to investigate the association between ambient air pollution , especially ozone exposure , and ed visits for depression during the period from april 2004 to december 2011 in nine urban areas across ontario , canada : algoma , halton , hamilton , london , ottawa , peel , toronto , windsor , and york . we also examined the relations between air pollution exposure and risk of suicide attempts among patients admitted to the ed for depression . we conducted this multicity study to see whether the results would corroborate past findings on air pollution and depression . a case - crossover ( cc ) design was used to evaluate the potential association between ed visits for depression and ambient air pollution.43 ed visits were used to represent health outcomes and were analyzed as separate individual cases . we have expressed air pollutant concentrations , temperature , and relative humidity as daily mean values . the nacrs contains data for all hospital and community - based ambulatory care such as day and night surgery , outpatient clinics , and ed visits , which are reported from ontario s hospitals and have been collected at the time of services in participating hospitals ( for more information , see canadian institute for health information website : www.cihi.ca ) . ed visits for depression were retrieved from the nacrs using the international classification of diseases , tenth revision ( icd-10 ) codes icd-10 : f32 ( mild depressive episode ) and icd-10 : f33 ( recurrent depressive disorder ) , during the period of april 2004 to december 2011 . icd-10 codes with prefix f ( mental and behavioral disorders ) were also retrieved for the second part of the study . the environmental data were obtained from environment canada ( see naps website : http://www.etc-cte.ec.gc.ca/napsdata ) . to obtain data on ambient air pollution , naps stations within 35 km of each patient the daily average values of ozone ( o3 ) , nitrogen dioxide ( no2 ) , fine particulate matter with a median aerodynamic diameter no greater than 2.5 m ( pm2.5 ) , and sulfur dioxide ( so2 ) were calculated by averaging over all the stations within this 35 km radius . data on carbon monoxide levels were eliminated from the study due to large amounts of missing data in most of the cities . hourly data for relative humidity and temperature for all cities were also retrieved from environment canada . we calculated the daily levels for these weather parameters by averaging hourly data over 24-hour periods . if more than one station was available for a patient , the daily values of all stations were averaged . first , a time - stratified cc design was applied.44 this method is an adaptation of the case control design in which cases act as their own controls on a set of predefined control days proximate to the time of admission to hospital . this method has become very popular in studies of air pollution as it avoids confounding by individual characteristics and longer time - varying covariates such as seasonal effects.45 a p - value less than 0.05 was considered statistically significant in all presented statistical results . results are reported as odds ratios ( ors ) and 95% confidence intervals ( cis ) associated with an increase in interquartile range ( iqr ) of the air pollutants , after controlling for daily temperature and humidity . ors for o3 , no2 , pm2.5 , and so2 were calculated separately for each city by using the proc phreg procedure in sas ( enterprise guide , version 4.2 ) . several different time lags for air pollutants and meteorological factors were modeled , from same - day exposure ( ie , 0 day lag ) to exposure 8 days before admission ( ie , 08 day lag ) . in order to reduce bias , the control periods were selected from the same day of the week as the case s hospital visit , in the same month and year ; for each case , three or four controls were selected . the second stage involved a meta - analysis of the estimates and their standard errors to achieve a pooled effect estimate . to test the assumption that individual effect sizes represent the same underlying parameter for all cities , both fixed- and random - effects models were calculated.46 meta - analysis and meta - regression were performed using r.47 the nacrs contains data for all hospital and community - based ambulatory care such as day and night surgery , outpatient clinics , and ed visits , which are reported from ontario s hospitals and have been collected at the time of services in participating hospitals ( for more information , see canadian institute for health information website : www.cihi.ca ) . ed visits for depression were retrieved from the nacrs using the international classification of diseases , tenth revision ( icd-10 ) codes icd-10 : f32 ( mild depressive episode ) and icd-10 : f33 ( recurrent depressive disorder ) , during the period of april 2004 to december 2011 . icd-10 codes with prefix f ( mental and behavioral disorders ) were also retrieved for the second part of the study . the environmental data were obtained from environment canada ( see naps website : http://www.etc-cte.ec.gc.ca/napsdata ) . to obtain data on ambient air pollution , naps stations within 35 km of each patient s the daily average values of ozone ( o3 ) , nitrogen dioxide ( no2 ) , fine particulate matter with a median aerodynamic diameter no greater than 2.5 m ( pm2.5 ) , and sulfur dioxide ( so2 ) were calculated by averaging over all the stations within this 35 km radius . data on carbon monoxide levels were eliminated from the study due to large amounts of missing data in most of the cities . hourly data for relative humidity and temperature for all cities were also retrieved from environment canada . we calculated the daily levels for these weather parameters by averaging hourly data over 24-hour periods . only stations within 100 km of each patient if more than one station was available for a patient , the daily values of all stations were averaged . first , a time - stratified cc design was applied.44 this method is an adaptation of the case control design in which cases act as their own controls on a set of predefined control days proximate to the time of admission to hospital . this method has become very popular in studies of air pollution as it avoids confounding by individual characteristics and longer time - varying covariates such as seasonal effects.45 a p - value less than 0.05 was considered statistically significant in all presented statistical results . results are reported as odds ratios ( ors ) and 95% confidence intervals ( cis ) associated with an increase in interquartile range ( iqr ) of the air pollutants , after controlling for daily temperature and humidity . ors for o3 , no2 , pm2.5 , and so2 were calculated separately for each city by using the proc phreg procedure in sas ( enterprise guide , version 4.2 ) . several different time lags for air pollutants and meteorological factors were modeled , from same - day exposure ( ie , 0 day lag ) to exposure 8 days before admission ( ie , 08 day lag ) . in order to reduce bias , the control periods were selected from the same day of the week as the case s hospital visit , in the same month and year ; for each case , three or four controls were selected . the second stage involved a meta - analysis of the estimates and their standard errors to achieve a pooled effect estimate . to test the assumption that individual effect sizes represent the same underlying parameter for all cities , both fixed- and random - effects models were calculated.46 meta - analysis and meta - regression were performed using r.47 figure 1 shows frequency ( counts ) of ed visits for depression by sex and age from 0 to 100 years . the patients aged 100 years and older are represented on the graph as 100 years ( ie , by one point ) . the highest frequency of ed visits was at the age of 17 years for females ( n = 1,803 ) and 43 years for males ( n = 1,182 ) . females made more ed visits for depression ( 58% ) than did males ( 42% ) . table 1 shows the frequency of visits by age group , sex , city , and season : cold months ( october march ) and warm months ( april september ) . table 2 shows the frequency of visits by type ( mild depressive episode or recurrent depressive episode ) . the majority ( 95% ) of visits were for mild depressive episode ( icd-10 code f32 ) versus recurrent depressive disorder ( icd-10 code f33 ) . descriptive statistics for relative humidity and temperature of each city are presented in table 3 . the results of pooling estimates ( fixed effects ) for depression across all cities were calculated for warm months ( april september ) and cold months ( october march ) , as well as collapsed across all months . ors and their 95% ci were calculated for a one unit increase in iqr = 75th percentile25th percentile of air pollutants . the iqr values are 9 ppb , 14.5 ppb , 7.12 g / m , and 2.5 ppb , respectively , for daily means of no2 , o3 , pm2.5 , and so2 . when all months were considered together , exposure to o3 was associated with increased risk of an ed visit for depression in both females and males at multiple time lags ( see table 5 ) . increased so2 was also associated with increased risk for females seven days after exposure , or = 1.013 ( 1.001 , 1.025 ) . for males , exposure to pm2.5 was associated with increased risk one day after exposure , or = 1.014 ( 1.000 , 1.028 ) . results for the warm months are presented in figure 2 for males ( left panel ) and females ( right panel ) . in contrast , when only cold months were considered , few significant effects emerged . during the cold months , exposure to o3 was associated with increased risk of ed visit for depression 2 , 3 , 5 , 6 , 7 , and 8 days after exposure for females only . random - effects estimates allow the possibility that the estimates from different cities may be estimates of different parameters . results of a q - statistic test indicated that for the majority of estimates ( 92% ) , the fixed - effects model was a good fit for the data . effects were calculated using random - effects meta - analysis ( data not shown ) , and patterns of results were similar to those obtained using fixed - effects analysis . in order to examine comorbidity of depression with suicide attempts , we identified patients who in addition to a primary diagnosis of depression had also attempted suicide . for this purpose , among all 118,602 visits for depression during the study period , we identified the patients whose secondary icd-10 code starts with x6 ( suicide by cause of poisoning ) , x7 ( suicide by cause of hanging , strangulation , suffocation , drowning , cutting , and/or piercing with sharp object ) , or x80 - 3 ( suicide by cause of jumping from a high place , jumping , or lying before a moving object ) . because of the small sample size of this study , we considered both genders for all the cities together ( n = 4,131 patients ; 67% females , 33% males ) . the highest frequency occurred at the age of 19 years among both females ( 4.63% ) and males ( 3.59% ) . analyses did not reveal a significant association between exposure to air pollutants and risk of suicide attempts . random - effects estimates allow the possibility that the estimates from different cities may be estimates of different parameters . results of a q - statistic test indicated that for the majority of estimates ( 92% ) , the fixed - effects model was a good fit for the data . effects were calculated using random - effects meta - analysis ( data not shown ) , and patterns of results were similar to those obtained using fixed - effects analysis . in order to examine comorbidity of depression with suicide attempts , we identified patients who in addition to a primary diagnosis of depression had also attempted suicide . for this purpose , among all 118,602 visits for depression during the study period , we identified the patients whose secondary icd-10 code starts with x6 ( suicide by cause of poisoning ) , x7 ( suicide by cause of hanging , strangulation , suffocation , drowning , cutting , and/or piercing with sharp object ) , or x80 - 3 ( suicide by cause of jumping from a high place , jumping , or lying before a moving object ) . because of the small sample size of this study , we considered both genders for all the cities together ( n = 4,131 patients ; 67% females , 33% males ) . the highest frequency occurred at the age of 19 years among both females ( 4.63% ) and males ( 3.59% ) . analyses did not reveal a significant association between exposure to air pollutants and risk of suicide attempts . this study investigated the effect of short - term exposure to ambient air pollution on ed visits for depression . to this end , we examined records from 118,602 ed visits for depression by patients across nine major urban areas in the province of ontario , canada . to our knowledge , this is among the largest multisite studies of ed visits for depression . multisite studies have the advantage of generating more reliable results and tend to be less vulnerable to bias when compared to small studies conducted at individual centers.48 overall , results indicated that short - term exposure to increased levels of several common pollutants is significantly associated with increased risk of an ed visit for depression . several studies suggest that outdoor air pollution , especially ozone , particulate matter , and carbon monoxide may have a significant impact on cns health and disease.4952 ozone , because of its oxidative properties , may induce oxidative stress in the cns , which is a potential cause of depression.40 several previous studies have linked ozone levels to depression9,10,16,53 and suicide attempts.11,40,41 in this study , higher ozone levels were associated with increased risk of hospital visits for depression , for both males and females , at multiple time points within the eight days immediately following exposure . the effect of ozone seemed more pronounced during the warm months ( april september ) than the cold months ( october march ) . these results are supported by previously published studies of the effects of ozone on depression.9,10,16 the levels of ozone are also higher in warm seasons . results of the current study also suggest an association between ed visits for depression and levels of pm2.5 , no2 , and so2 . there is a growing body of literature linking depression with exposure to air pollution . a large epidemiological study of 15,556 ed visits in edmonton , canada , found similar evidence for an association between ozone exposure and depression , reporting that higher daily ozone levels were associated with more ed visits for depression one day post exposure.9 this study also reported effects of increased levels of carbon monoxide , nitrogen oxide , sulfur dioxide , and airborne particulate matter on emergency visits for depression . similarly , a study of 537 participants in seoul , korea , reported that an increase in ambient air pollution , pm2.5 , no2 , and o3 may increase depressive symptoms among elderly adults . these results support the hypothesis that ambient air pollution is associated with emotional symptoms . in further support of this hypothesis , studies using postmortem sampling have identified particulate matter in the human brain , albeit of unknown composition and origin.54 some authors have suggested that ambient particulate matter , such as that originating from diesel exhaust , may have toxic effects on dopaminergic neurons in the brain . this neurotoxicity may be a result of oxidative stress and has been hypothesized to affect depressive moods.5557 other authors have suggested a link between air pollutant exposure and levels of serotonin,58 a neurotransmitter that has also been implicated in depression.59 rat studies have demonstrated a near immediate effect of exposure to ozone on metabolism of both dopamine and serotonin.60 it is not yet known whether ozone may contribute to imbalances in these neurotransmitters in humans . suicide attempts as a result of depression have also been linked to air pollution exposure.11,40,41 in this study , we examined data from patients who in addition to a primary diagnosis of depression had also attempted suicide . we did not find any significant associations between suicide attempt and exposure to the air pollutants studied . these results are in contrast to results of other studies linking ambient air pollution to suicide risk.11,40,41 a study from vancouver , canada , showed a potential link between the effects of ambient air pollution on ed visits for suicide attempts.11 a study in middle franconia of 1,008 suicides and 917 suicide attempts from 2004 to 2007 reported that days on which two or more suicides occurred had higher ambient ozone levels than those days on which one or zero suicides were observed.40 this study also reported a seasonal effect on suicide , with higher than usual suicide rates observed from july to september , when ozone levels are typically highest . a study of 4,857 deaths by suicide in taipei city , taiwan , assessed the temporal relation between suicide and air pollution.41 the authors reported that higher levels of gaseous air pollutants such as o3 and so2 were associated with an increased risk of suicide at longer time delays . their findings also suggested a seasonal pattern of increased suicide occurring in early summer . a seasonal variation in suicide with a peak in march april and august , respectively , and with a decrease in december january was also reported in belgium for the period 19791987.61 disparities in sampling strategies may contribute to the conflicting findings between studies . the present study examined the link between exposure to air pollutants and suicide risk only among patients with an ed visit for depression . the previously reported link between ambient air pollution and suicide may therefore be explained by the increased risk of depression conferred by exposure to pollutants . in addition , many studies reporting associations between air pollution exposure and suicide have not accounted for meteorological variables such as ambient temperature , which may explain the previously reported link between summer months and suicide risk.40 ozone levels tend to be higher on warmer days , and results of the present study suggest that the association between ozone levels and suicide is not statistically significant . some authors have suggested that seasonal trends in suicide may be due in part to higher ambient temperatures during the summer months , rather than due to high ozone levels . psychological factors , such as aggression and impulsivity , may be influenced by higher temperatures and may account for increases in suicide risk in the summer months.62 however , results of the present study indicated that even when meteorological factors were controlled for , associations between ambient air pollution and ed visits for depression were more pronounced during the summer months . this suggests that additional factors may be at play in the potentially complex relationship between season , air pollution , and depression . results of this study suggest that ed visits for depression are associated with short - term exposure to ambient air pollutants and that this pattern of association differs for warm vs. cold months in canada . the cases of depression assessed by this study were only those in which individuals sought ed treatment . presumably , these represent more severe depressive episodes , but future research assessing associations between pollutant exposure and depression in a community sample would lend more confidence to our conclusions . the results from this study agree with the recently reported conclusions from observation in rodents that short - term exposure to ozone or particulate matter activates the stress response axis and increases stress hormone levels.63,64 efforts to reduce ozone levels in the atmosphere may mitigate ed presentations to eds for depressions .

wound healing is a dynamic process involving many factors and cell types including soluble mediators , blood cells , fibroblasts , endothelial cells , and extracellular matrix . normal wound healing is divided into several sequential phases that overlap in space and time : homeostasis , inflammation , granulation tissue formation , and tissue remodeling . chronic wounds develop as a result of defective regulation of one or more of the complex molecular and biological events involved in proper healing [ 13 ] . diabetic foot ulcers and other similar chronic wounds impact approximately 6.5 m people and cost approximately $ 25b / year in the us alone . the critical need for a cure of chronic wounds is underlined by the continuous increase in type ii diabetes which accounts for 9095% of all diabetes . challenges in understanding these problematic events result from varying disease etiologies , existing comorbidities and , importantly , lack of animal models that mimic the characteristics of chronic wounds in humans . the importance of developing such models has been recognized by the national institute of general medical sciences call for proposals in this field . furthermore , difficulties with human tissue collection complicate chronic wound research ; for the most part , a clinician sees the patient when the wound is already at an advanced stage of chronicity and critical evidence of causality is already lost . although the series of events leading to development of chronic wounds remains unclear , toxic concentrations of reactive oxygen species ( ros ) and presence of biofilm - producing bacterial colonies are considered as key players [ 610 ] . the db / db mouse model of type ii diabetes has impaired healing with decreased levels of reduced glutathione ( gsh ) , which results in reduction of glutathione peroxidase ( gpx ) activity and an increase in ros . the latter promote leukocyte adhesion , thereby increasing immigration of inflammatory cells into the wound tissue . it has also been suggested that ros impair keratinocyte migration in vitro , potentially inhibiting reepithelialization . high levels of ros also lead to dna damage , gene dysregulation , cell death , and a hostile proteolytic environment . moreover , chronic wounds in humans are rich in microbiota , including anaerobic bacteria not revealed by culture that can form biofilms and cause excessive bioburden in these wounds [ 8 , 9 ] . staphylococcus aureus , enterococcus faecalis , pseudomonas aeruginosa , coagulase - negative staphylococci , and proteus species are among the most commonly cultured species in human chronic wounds . we hypothesize that manipulating specific redox parameters immediately after wounding will lead to development of chronic wounds in db / db mice and that restoring the antioxidant status will reverse chronicity and lead to proper healing . here we show that inhibition of the activity of gpx and catalase , two antioxidant enzymes , immediately after wounding generates chronic wounds containing spontaneously formed antibiotic - resistant polymicrobic bacterial biofilms . moreover , chronicity can be reversed by treatment with the antioxidants n - acetyl cysteine ( nac ) and -tocopherol ( -toc ) . this novel model for chronic wounds provides insight into the mechanisms involved in chronic wound development and may contribute to development of new therapeutics . c57bl/6 and db / db mice ( jackson laboratories ; bar harbor , me ) were housed in the ucr vivarium and used following protocols approved by the institutional animal care and use committee ( iacuc ) . briefly , mice were anesthetized with a single intraperitoneal injection of ketamine ( 80 mg / kg body weight)/xylazine ( 16 mg / kg body weight ) . full thickness 7 mm punch wounds ( excision of the skin and the underlying panniculus carnosus ) were made on the back of the mice . the animals were euthanized using carbon - dioxide at various time points and the wound tissue was collected for histology and protein analysis using a 10 mm punch ( wound bed + surrounding tissue ) . for protein analysis , zirconium oxide beads weighing approximately the same as the wound tissue were added to tissue in safe lock tubes , followed by addition of 10 ml of ripa buffer per mg of tissue . the supernatants were used fresh or aliquots prepared and stored at 80c for later use . total tissue superoxide dismutase ( sod ) activity was measured by using a commercially available kit ( cayman chemical , catalog number 706002 , ann arbor , usa ) that measures all three types of sod ( cu / zn- , mn- , and ec - sod ) . one unit of sod is defined as the amount of enzyme needed to cause 50% dismutation of the superoxide radical . briefly , xanthine oxidase and hypoxanthine generate superoxide radicals that are dismutated by sod and in the process tetrazolium salt are converted to a formazan dye that is read at 450 nm . the sod activity of the samples was calculated from the linear regression of a standard curve that was determined using the sod activity of bovine erythrocytes at various concentrations run under the same conditions . tissue hydrogen peroxide ( h2o2 ) levels were measured by using a commercially available kit ( cell technology inc . , catalog number floh 100 - 3 , mountain view , usa ) that utilizes a nonfluorescent detection reagent . the assay is based on the peroxidase - catalyzed oxidation by h2o2 of the nonfluorescent substrate 10-acetyl-3,7-dihydroxyphenoxazine to a fluorescent resorufin . 50 l of tissue extracts collected at different time points after wounding and normalized to protein concentration was mixed with 50 l of the reaction cocktail in an opaque 96-well assay plate . fluorescent intensities were measured at 530 nm ( excitation)/590 nm ( emission ) using a victor 2 ( fluorescence and absorbance ) microplate reader . the amounts of h2o2 in the supernatants were derived from a seven - point standard curve generated with known concentrations of h2o2 . tissue catalase activity was measured by using a commercially available kit ( cayman chemical , catalog number 707002 , ann arbor , usa ) . the enzyme assay for catalase is based on the peroxidatic function of catalase with methanol to produce formaldehyde in the presence of an optimal concentration of h2o2 . the formaldehyde produced was measured spectrophotometrically , with 4-amino-3-hydrazino-5-mercapto-1,2,4-triazole ( purpald ) as the chromogen , at 540 nm in a 96-well place . the catalase activity was expressed as nmol / min / ml of tissue extract . a commercially available kit ( cayman chemical , catalog number 703102 , ann arbor , usa ) was used to measure tissue glutathione peroxidase ( gpx ) activity . the activity was measured indirectly by a coupled reaction with glutathione reductase ( gr ) . gpx reduces h2o2 to h2o and in the process oxidized glutathione ( gssg ) is produced that in turn is recycled to its reduced state by gr and nadph . oxidation of nadph to nadp+ is accompanied by a decrease in absorbance at 340 nm . under conditions in which gpx activity is rate limiting , the rate of decrease in the absorbance measured at 340 nm , in a 96-well plate at 1 min interval for a total of 5 min using a victor 2 microplate reader , is directly proportional to the gpx activity of the sample . to generate chronic wounds in db / db mice we performed full thickness 7 mm diameter excision wounds on the dorsum of 6 - 7-month - old mice . twenty minutes prior to wounding , mice were treated once intraperitoneally ( ip ) with 3-amino-1,2,4-triazole ( atz ) ( aldrich chemistry ; st . louis , mo ) at 1 g / kg body weight , an inhibitor for catalase . immediately after wounding , they were treated once topically with the inhibitor for gpx , mercaptosuccinic acid ( msa ) , ( sigma lifesciences ; st . immediately after wounding , the wounds were covered with tegaderm ( 3 m ; st . paul , mn ) to prevent contamination and were kept covered for the duration of the experiments . in these mice it is easy to fully remove the hair from the back and hair grows very slowly ; hence we had no problems keeping the tegaderm in place . the tegaderm was removed periodically to take pictures of the wound and then immediately replaced . the wounds were fully chronic 20 days after wounding and remained open sometimes for more than 3 months , depending on the experiment . control db / db mice were treated exactly the same way but instead of inhibitors of the antioxidant enzymes they were treated with the vehicle ( pbs ) . to reverse chronicity , at 20 days , the antioxidant nac ( aldrich chemistry ( st . louis , mo ) ) was topically applied to the wound at 200 mg / kg and the tegaderm replaced . simultaneously , the mice were injected intraperitoneally with -toc , sigma lifesciences ( st . louis , mo ) at 50 mg / kg . this treatment continued with nac applied to the wound topically every day using an insulin syringe to deposit the solution under the tegaderm and over the wound and with -toc ip every other day for 20 days ( 40 days after wounding ) . at this point , the antioxidant treatment was stopped and the wounds went on to heal around 30 days after initiation of treatment with antioxidants ( 50 days after wounding ) . for the antioxidant controls , the mice were treated exactly the same but with vehicle rather than antioxidants . in some experiments , tissues were collected at various time points for detailed histological / histochemical , biochemical , and cellular / molecular evaluation , and in some cases the tissues were analyzed for type and level of bacterial infection / biofilm production . chronic wounds were successfully created in over 100 animals . to obtain the wound microbiome samples we used sterile cotton q - tips to swab the wound bed , including the surface of the wound , but yet minimizing disruption of the wound microenvironment to allow for longitudinal studies of the microbiome . the content of each swab was suspended in 1.0% w / v protease peptone and 20.0% v / v glycerol solution . wounded tissue for bacteria analysis was obtained using sterile scissors and suspended in 1.0% w / v protease peptone and 20.0% v / v glycerol solution . tissues were homogenized in the presence of zirconium oxide beads using a bullet blender at 4c . bacteria were cultured for 1618 h at 37c on tryptic soy agar plates ( bd difco , sparks , md ) , containing 5.0% v / v defibrinated sheep blood ( colorado serum company , denver , co ) , and 0.08% w / v congo red dye ( aldrich chemistry , st . colonies were differentiated and isolated based on size , hemolytic pattern , and congo red uptake . gram - negative rods were characterized using the api20e identification kit ( biomerieux , durham , nc ) and oxidase test ( fluka analytical , st . louis , mo ) . when required , the pseudomonas isolation agar culture test , 42c growth test in tryptic soy broth ( tsb ) ( bd difco , sparks , md ) , and motility test were used . gram positive cocci cultures were differentiated based on catalase activity and coagulation activity ( fluka analytical , st . louis , mo ) , 6.5% w / v nacl tolerance test , and hemolytic activity . , 35 l of the wound swabbed sample was seeded in 100 l of tsb and grown in humidified incubator at 37c in a 96-well polystyrene flat bottomed tissue culture plate under static condition . the plate was washed three times by slowly submerging the plate and gently flicking the inverted plate to remove the water . the wells were dried by tapping onto absorbent paper and then air dried at 65c for 30 minutes . excessive stain was removed by rinsing the plate with water and then air dried overnight . the optical density at 570 nm was then taken using the spectramax m2e microplate reader ( molecular device , sunnyvale , ca ) . samples that give an od of or above 0.125 were considered biofilm positive whereas od below 0.125 was considered biofilm negative . wound swab samples were resuspended in sterile luria broth ( lb ) to yield a 1 : 4 v / v ratio of sample - to - tsb solution . bacterial colonies were visually counted on trypticase soy agar plates containing 5% sheep red blood cells incubated at 37c overnight in a humidified incubator . wound swab samples ( containing bacteria ) that were seeded on flat bottomed tissue culture plates for 3 - 4 hr at 37c in a humidified incubator were challenged with antibiotic for 12 hr at various concentrations in tsb . optical density at 595 nm ( od 595 nm ) was used to quantify bacterial growth . the community minimal inhibitory concentration ( cmic ) is defined as the lowest concentration of antibiotic that resulted in 50% increase in od 595 nm compared to that before introduction of antibiotic . frozen sections of chronic wound tissues were stained using viagram red + bacterial gram - stain and viability kit ( life technologies , carlsbad , ca ) with modifications to the manufacturer 's protocol . briefly , the frozen tissue sections were washed in 1x pbs for 5 minutes at room temperature to remove the oct . sections were then incubated in wheat germ agglutinin ( wga ) conjugate stock solution for 5 minutes . the wga solution was drained off the slide followed by the addition of 2.5 l of the dapi / sytox green working solution for 10 minutes at room temperature . sections were mounted and visualized using a nikon microphot - fxa microscope with a nikon ds - fi1 digital camera . tissues collected were fixed in 4% paraformaldehyde for 4 hrs at room temperature and then processed as described in . briefly , samples were dehydrated in a series of ethanol for 20 min each followed by critical point drying of the tissues , using balzers cpd0202 and au / pd sputtering in the sputter coater cressington 108 auto . chronic wound swab samples were washed with 80% and 100% ethanol to eliminate low molecular weight components and then with 2 : 1 ( v / v ) chloroform : methanol to remove lipids and acetone in preparation for drying . total protein content of the dried swab sample was estimated by the lowry protein assay . total carbohydrate content of dried swab sample was estimated by colorimetric phenol - sulfuric acid assay , using gum arabic as the standard . for glycosyl composition analysis , dried swab sample was cleaved by trifluoroacetic acid hydrolysis and the resulting monosaccharides were derivatized by methanolysis , n - acetylation , and trimethylsilylation as described [ 21 , 22 ] with minor modifications . gas chromatography - flame ionization detection and gas chromatography - mass spectrometry were performed as previously described . dna was extracted using the dneasy blood and tissue kit ( qiagen , chatsworth , ca ) according to the manufacturer 's instructions . dna concentration was measured and purity confirmed by calculating the od 260/od 280 absorption ratio . equipped with an nlo interface for a femtosecond titanium - sapphire laser excitation source ( chameleon - ultra , coherent , incorporated , santa clara , ca ) for multiphoton excitation , an inverted zeiss lsm 510 nlo meta laser scanning microscope ( carl zeiss microscopy , llc , thornwood , ny ) for transmitted light and epifluorescence was used . the chameleon laser provided femtosecond pulses at a repetition rate of about 80 mhz , with the center frequency tunable from 690 to 1040 nm . the sample two - photon signals were epicollected and discriminated by the short pass 650 nm dichroic beam splitter . a meta detection module with signals sampled in a 394405 nm detection range ( lex = 800 nm ) was used to collect the shg images . each image presented in this work is 12 bit , 512 512 pixels representing 225 mm 225 mm field of view . analysis of variance ( anova ) was used to test significance of group differences between two or more groups . in experiments with only two groups , we used the student 's t - test . because the differences we observe are not small , we perform experiments in groups of three mice and then repeat the experiment in groups of three as many times as needed to be confident of the results . for the majority of the cases , 2 sets of experiments to a total of 6 animals were sufficient to achieve significant results . both db / db and c57bl/6 mice were wounded as described in the methods section . the c57bl/6 wounds closed in about 11 days whereas the db / db mouse wounds took up to 32 days to close , with flaky and crusty appearance ( figures 1(a ) and 1(b ) ) . we examined levels of oxidative stress in these wounds by measuring the detoxifying capacity of sod . sod activity was significantly elevated in db / db mice compared to c57bl/6 mice ( figure 1(c ) ) , as was the product of this detoxification process , h2o2 ( figure 1(d ) ) . however , catalase activity was significantly lower in db / db mice than that of the c57bl/6 ( figure 1(e ) ) whereas gpx activity was slightly lower very early and more so at 48 hrs ( figure 1(f ) ) . these results show an increase in oxidative stress very early after wounding in the db / db mouse wounds . the nonwounded skin of the db / db mice ( t = 0 ) already has exacerbated levels of oxidative stress ( figures 1(c ) and 1(d ) ) which correlates well with the impaired healing these mice exhibit . this led us to hypothesize that high oxidative stress levels in the wound tissue critically contribute to impaired healing and that exacerbated oxidative stress contributes to chronic wound development . has failed to proceed through an orderly and timely reparative process to produce anatomic and functional integrity or that has proceeded through the repair process without establishing a sustained anatomic and functional result [ 24 , 25 ] . in humans these wounds stay nonhealing for at least 3 months whereas in animals it has been difficult to establish how long wounds need to be impaired to be considered chronic . however , in general , wounds that do not close by the normative period of time and show minimalistic healing by 26 days have been considered chronic . to test our hypothesis we significantly increased oxidative stress in the db / db wounds by further inhibiting , at the time of wounding , both catalase and gpx activity , two potent antioxidant enzymes . the mice were wounded and treated as described in methods section under chronic wound model . 3-amino-1,2,4-triazole ( atz ) was chosen to inhibit catalase because this inhibitor binds specifically and covalently to the active center of the enzyme and inactivates it . mercaptosuccinic acid ( msa ) was chosen to inhibit gpx because the thiol moiety binds to the selenocysteine active site of this enzyme and inactivates it . atz has been shown to reduce catalase activity when injected intraperitoneally in rats and mice [ 2931 ] . in rats , the inhibition of catalase activity in liver and kidney was shown to return to normal levels by day 7 after one ip dose of inhibitors [ 29 , 30 ] . msa , also known as thiomalate , is the most potent of all mercaptans for inhibiting gpx . it has been shown to clear from the plasma in a matter of a few hours to a couple of days . the specific doses described in the methods section were chosen after an extensive literature search to determine what has worked in mice for effective inhibition of these enzymes without causing major side effects [ 3235 ] . moreover , the treatment with inhibitors affects the breakdown of h2o2 , a product of the oxidative burst generated by neutrophils that are present early after wounding . as a consequence , inhibition of the two antioxidant enzymes increases the strength of oxidative burst without affecting neutrophil activity . control c57bl/6 mice treated with either pbs or the inhibitors for the two antioxidant enzymes ( iae ) closed by day 12 and 20 , respectively ( figures 2(a ) and 2(b ) ) . the wounds in the iae treated mice took longer to close and initially appeared to be ulcerous but did not form exudate . db / db mice treated with pbs healed by 30 days and did not form exudate or become ulcerous ( figure 2(c ) ) . the wounds of the db / db treated with iae , however , became ulcerous , developed exudate , and remained open as long as 100 days and sometimes more ( figures 2(d ) and 2(e ) ) . the areas of the wounds were measured by removing the tegaderm , lightly cleaning the wound , and taking a picture before covering the wound again with tegaderm ( inset in figure 2(e ) shows a picture of such wounds ) . as is observed in humans with chronic wounds , the weight of db / db mice with chronic wounds steadily decreased by as much as 40% by day 40 after wounding ( figure 2(f ) ) . this was not primarily due to treatment with the inhibitors because db / db mice treated with the iae without wounding lost only weight until day 15 and then began to recover whereas the mice with chronic wounds continue to dramatically lose weight ( figure 2(f ) ) . microbial infection of wounds in db / db mice was first observed about 4 days after treatment with iae and persisted for at least 56 days in most animals ( figure 3(a ) ) ; the presence of biofilm - associated microbial infection was first seen about 15 days after wounding . bacterial isolation and characterization showed that the samples collected from the wounds are comprised of many bacterial species and contains both biofilm- and non - biofilm - producing species ( figure 3(b ) ) . interestingly , monospecies cultures revealed that s. epidermidis isolated from day 4 samples produces biofilm when grown independently from the community , suggesting that communal interactions may be an important regulator of biofilm production . as the infection progresses , the bacterial composition evolves from several species of nonbiofilm producers ( day 4 ) to progressively fewer species at day 15 that are biofilm producers . by e. cloacae ) and by day 56 the only bacterium remaining was e. cloacae which by that time had become biofilm producing ( figures 3(b ) and 3(c ) ) . furthermore , evaluation of chronic wound tissues to obtain the bacterial profile in the wound bed showed that the profile was closely similar to the bacterial species in the wound bed collected in the swab sample , with only minor differences ( e.g. , relative abundance among bacterial species ) . counts of colony forming units ( cfu ) from wound swab samples revealed that bacterial burden was at its highest at 20 days after wounding ( 1.4 0.1 10 cfu ) , thereby pushing the wound into a state of chronicity . at day 30 , there was a reproducible dramatic decrease in bacterial burden ( approximately 28x ) compared to day 20 that coincided with the disappearance of s. epidermidis and of pseudomonas sp . however , by day 40 , wound bioburden was dominated by e. cloacae and bioburden had recovered to a level comparable to day 20 and remained relatively unchanged thereafter ( figure 3(d ) ) . microbial communities living within biofilms are often resistant to antibiotics . quantifying the microbial community minimal inhibitory concentration ( cmic ) of amoxicillin required to inhibit the growth of the microbial community demonstrated that biofilm positive bacterial communities appearing at 15 days and after are more resistant to antibiotic killing , and biofilm negative bacterial communities collected at 4 days after wounding showed cmic of less than 15 g / ml and , by day 56 , these communities were more than 200 g / ml and the biofilm - forming bacteria . to determine whether normal skin microbiota can be the source for infection , we took skin swabs from unwounded c57bl/6 and db / db mice and cultured the skin microbial flora ( figure 3(f ) ) . the majority of the bacteria found on both c57bl/6 and db / db skins belonged to the firmicutes phylum , specifically staphylococcus sp . and streptococcus sp . we were also able to isolate bacteria from the proteobacteria phylum ( specifically bacillus pseudomonas and enterobacter ) ( figure 3(f ) ) . the bacterial species that colonized c57bl/6 and db / db wounds were similar but the db / db skin on average has higher bacterial density than c57bl/6 ( not shown ) . thus , skin microbiota of nonwounded skin in our model is very similar to that of humans and is a contributor to the biofilm found in chronic wounds . it is notable that the bacterial profile isolated from the skin of db / db mice was similar to that observed using near - full - length 16s rrna sequencing for bacterial species that colonized db/+ and db / db unwounded skin . in this model system , to reverse chronicity , we treated the animals with the antioxidant agents ( aoa ) nac and -toc . nac is produced by most cells in the body ; hence we applied it locally to the wound every day . -toc , on the other hand , is produced primarily by the liver and circulates throughout the body . therefore , we applied it ip [ 42 , 43 ] , simulating systemic delivery in humans . the treatment doses for -toc and nac were chosen after extensive literature searches [ 4446 ] . we found that healing improved dramatically by 30 days after aoa treatment ( 50 days after wounding ) as compared to the wounds treated with vehicle that can take up to 100 days to close ( compare figures 4(a ) and 4(b ) with figures 2(a)2(e ) ) . moreover , the weights of the db / db mice treated with aoa began to stabilize starting at 20 days after the initiation of treatment ( 40 days after wounding ) ( figure 4(c ) ) . these results , coupled with our finding that when the mice are treated only with inhibitors without wounding they do not lose weight past day 15 or so , indicate that the great loss of weight is due to the wounds and not to the treatment with iae ( compare figure 4(c ) with figure 2(f ) ) . the aoa were also able to restore the detoxifying capability of catalase and gpx to reduce oxidative stress . sod increased within 10 days after treatment with aoa ( 30 days after wounding ) ( figure 4(d ) ) . also , h2o2 levels decreased by day 10 after treatment compared to increasing h2o2 levels in chronic wounds ( figure 4(e ) ) and both catalase and gpx enzyme activity increased following treatment whereas they were approximately constant in chronic wounds ( figures 4(f ) and 4(g ) ) . to test whether the two aoa are needed together to reverse chronicity , we treated the chronic wounds individually with nac ( figure 5(a ) ) or -toc ( figure 5(b ) ) . visual examination of the wounds treated with single antioxidants shows little difference from those treated with both together ( compare figures 5(a ) and 5(b ) with figure 4(a ) ) . quantitatively , -toc was a close approximation to the two agents together for wound closure ; nac was slightly less effective ( figure 5(c ) ) . similarly , for weight loss , -toc closely matched combined agents and nac was slightly less efficient ( figure 5(d ) ) . . however , these measurements of macroscopic parameters are insufficient to adequately characterize reversal of chronicity . as shown in figure 6 , the structure of the wound tissue is immature when the mice are treated with the individual aoa but not when treated with both . histological examination of db / db chronic wounds shows a complete loss of tissue structure ; there is no epidermis and all that is present of the granulation tissue is a very thin band of undifferentiated tissue ( figure 6(a ) ; shown between the arrows ) . db / db nonchronic wounds after closure showed that the granulation tissue is poorly developed ( figure 6(b ) , first panel ) . chronic wounds treated with nac or -toc individually ( figure 6(b ) , panels 2 and 3 ) are much improved but their epidermis and granulation tissue are immature . in contrast , chronic wounds treated with both antioxidants ( figure 6(b ) , panel 4 ) show a well - developed granulation tissue and thinner epidermis containing rets . moreover , masson trichrome ( mt ) stain and second harmonic imaging microscopy ( shim ) for collagen deposition and structure revealed a similar pattern ( figure 6(c ) ) ; the overall deposition of fibrillar collagen in the wounds treated with both aoa was better than those of db / db nonchronic wounds and chronic wounds treated with either of aoa alone ( figure 6(c ) compares panel 4 with 13 ) . as shown by keratin 10 staining ( figure 6(d ) ) , the epidermis was well organized and thinner in the animals treated with both aoa ( panel 4 ) than in the nonchronic db / db and those treated with the aoa individually ( panels 13 ) . after 10 days of aoa treatment ( day 30 after wounding ) , biofilm - producing capacity of the chronic wound microbial flora is reduced by approximately 30% ( figure 7(a ) ) . by day 50 after wounding the microbial flora was considered to be negative for biofilm production ( od 570 nm < 0.125 ) . similarly , microbial profiling showed that by day 50 the overall biofilm - producing capacity of the individual bacterial species was reduced markedly ( figure 7(b ) ) . this is most noticeable by the drastic decrease in biofilm production by s. epidermidis at day 50 compared to day 20 and the loss of biofilm production by pseudomonas sp . at day 60 the use of aoa treatment affected the bacterial composition of the wounds , most notably by the reduction and then elimination of enterococcus sp . , the increasing prevalence of nonbiofilm producing e. cloacae , and the colonization by pseudomonas sp . cfu counts ( figure 7(d ) ) revealed a marked decrease in bacterial burden at 10 days of aoa treatment that , in contrast to non - aoa - treated wounds , remained relatively unchanged thereafter . to determine whether the reduction in biofilm production resulting from aoa treatment enhances the antimicrobial effect of amoxicillin , cmics of wounds treated with nac and -toc were evaluated ( figure 7(e ) ) . similar reduction was observed with other antibiotics , such as carbenicillin and gentamicin ( figure 7(e ) ) . these observations confirm that abating redox stress with nac + -toc alters the wound environment resulting in a bacterial phenotype that produces less biofilm and is more sensitive to antibiotics . to further confirm the presence of biofilm - forming bacteria in chronic wounds and the decrease in colonization upon aoa treatment , we performed both light microscopy with fluorescent stainings and scanning electron microscopy ( sem ) . for the light microscopy we stained frozen sections of chronic wound tissue ( iae treated ) at 20 days after wounding with a combination of sytox green , which has high affinity for nucleic acids , and wheat germ agglutinin ( wga ) conjugated with texas red which labels ecm molecules including biofilm . both gram - negative and gram - positive bacteria in the tissue appear as very small green specks because their nuclei are stained with the sytox green . they are associated and embedded in the red matrix stained by wga ( figures 8(a ) and 8(b ) ) . as previously reported , although sytox green and wga stain both host and bacteria , the size and morphology of the cells enabled distinguishing the presence of bacteria from host cells . this technique is now considered as one of the staining techniques for biofilms in tissue specimens . the sem pictures of chronic wounds ( iae treated ) show an abundance of bacteria at 20 days after wounding when compared to a normal db / db wound at 10 days ( figures 8(c ) and 8(d ) ) . we used 10 days for the db / db nonchronic wounds as control because biofilm is already seen by day 10 in wounds treated with iae . bacteria were embedded in a biofilm - like matrix ( stars ) covering the wound ( figure 8(c ) ) . upon treatment with aoa for 10 days ( 30 days after wounding ) , there was a reduction in the biofilm matrix seen in the wound ( compare figure 8(e ) with figure 8(f ) ) . furthermore , by 20 days of aoa treatment , there was further decrease in the presence of matrix and bacteria when compared to the increase in biofilm formation observed in the chronic wound ( compare figure 8(g ) with figure 8(h ) ) . following bacterial species identification and confirmation of presence of biofilms in the chronic wound , we evaluated the composition of the biofilm ( figure 8(i ) ) . total protein content of the dried biofilm in swab sample was 44.6% 15.1% ( w / w ) and total dna content was 1.55% 0.19074% ( w / w ) . total carbohydrate content of the dried biofilm in the swab sample was analyzed by colorimetric phenol - sulfuric acid assay was 1.98% 0.45% ( w / w ) and by gas chromatography analyzing glycosyl composition was 2.74% 1.46% ( w / w ) . the latter showed that n - acetylglucosaminyl ( glcnac ) , galactosyl , mannosyl , galacturonosyl , and glucuronosyl were the major glycosyl residues in the dried biofilm . neither iduronosyl nor n - acetylgalactosaminyl residues were detected in the biofilm . among the glycosyl residues , the relatively high amounts of n - acetylglucosaminyl and mannosyl residues were consistent with the presence of n - glycan type glycoproteins . we show here that we can generate chronic wounds in a diabetic mouse model of impaired healing by creating high levels of oxidative stress in the wound tissue using iae at wounding . we also show that chronicity of these wounds can be reversed by application of appropriate aoa , leading to healing . briefly , we find that h2o2 is elevated very early after wounding of these mice and that the antioxidant enzyme activity is significantly lower than in wounds of normal mice , suggesting an increase in oxidative stress in the wound tissue . accordingly , we developed a protocol in which the oxidative stress environment in these impaired wounds is further increased by inhibition of the two critical antioxidant enzymes , catalase and gpx . the ulcers remain open for months and develop a complex microbiota of biofilm - forming bacteria that are also found in chronic wounds in humans . chronicity was reversed by treatment of the chronic wounds with the antioxidant agents nac and -toc . under these conditions , the oxidative stress falls rapidly , the biofilm dismantles , and the wound heals . this , to the best of our knowledge , is the first chronic wound model in which biofilm develops spontaneously allowing for longitudinal assessment of the wound microbiome in its natural environment . these findings define a unique relationship between redox imbalance and complex biofilm development in wound chronicity , providing an opportunity to understand the mechanisms of action of both and potentially for the development of new therapies for humans . there are many similarities between the development of chronicity in these mice and similar chronic wounds in humans , especially nonpressure diabetic ulcers . for example , mice with chronic wounds lose considerable weight , as also occurs in many humans suffering from chronic wounds . also , a very complex microbiota spontaneously develops in these mice wounds , as it does in humans wounds . staphylococcus epidermidis and other coagulase - negative staphylococci exist as harmless or even beneficial commensal bacteria of the skin . however , in diabetic , elderly , and immobile individuals , these commensal bacteria can cause diseases and are responsible for a large percentage of infection in wounds that do not heal . in our animal model , microbial flora that colonizes the db / db mouse wounds is identified as s. epidermidis , enterococcus sp . , enterobacter cloacae , and pseudomonas sp . , and they exist as a complex and dynamic community . although commensal gram - negative bacteria e. cloacae are not reported from normal human skin , their presence in human wounds comes from gastrointestinal contamination of the skin [ 51 , 52 ] . others have shown that microbial flora cultured from patients with venous leg ulcers with or without clinical symptoms is polymicrobic [ 16 , 53 ] . furthermore , similar microbiota is found in combat wounds , chronic necrotizing skin diseases , and other chronic wounds [ 5456 ] . the establishment of selective microbial populations toward chronicity is probably due to both bacterial competition and cooperation in concert with the unique clinical and immunological phenotype and pathophysiology of the host wound tissue . in the diabetic mice of our study , chronic wounds evolve into essentially a monospecific infection dominated by biofilm - producing e. cloacae . in other species ( including humans ) , the specific biofilm producers may differ but the overall result is likely to be the same . from our observations , the bacterial ecology in our mouse model is polymicrobic and dynamic . the molecular factors and parameters that resulted in the dynamics observed in our mouse model are yet to be defined and require further investigation . our hypothesis is that due to competition and fitness , certain species will outcompete to predominate in the wounds and establish chronic infection . therefore , there are differences in the bacterial species that colonize the wound at days 20 and 30 . for reasons that we can not yet explain , the microbial ecology tends to change dramatically around day 30 , transitioning from a polymicrobic infection to a monospecific infection . it is clear from our histological sections that the granulation tissue of the chronic wounds is virtually nonexistent , making it essentially impossible to perform studies to identify inflammatory cells and/or blood vessels . in addition , we observed that the epidermis does not progress to close the wound and , in fact , the wound size increases . this is in agreement with our previously published findings in which cornea excision wounds in mice exposed to second - hand cigarette smoke increased in size . in that case , the smoke exposure created increased stress in the wound that led to inhibition of cornea epithelial migration to cover the wound . furthermore , the leading edge to the epithelium did not adhere to the underlying cornea stroma . we speculate that similar processes are occurring in the chronic wounds and we are currently investigating these possibilities . ros is known to play a critical role in wound healing [ 15 , 58 ] . under normal physiological conditions , generation of h2o2 this occurs in the presence of nicotinamide - adenine - dinucleotide ( nadh ) dependent oxidases ( noxs ) produced by resident endothelial cells and fibroblasts , followed by neutrophils and macrophages . for proper healing , a delicate balance needs to exist between their good and deleterious effects in the wound tissue . a balanced ros response will clean and inhibit infection while simultaneously triggering signaling mechanisms that stimulate healthy healing [ 15 , 60 , 61 ] . however , elevated levels of ros can cause a wide variety of tissue damage and lead to impaired healing . therefore , for a positive role in healing , ros needs to be in balance to be an effective antimicrobial and signaling agent but yet not to cause destructive effects to the tissue . if not , the high levels of ros or the reduced levels of antioxidant scavenger molecules such as the antioxidant enzymes catalase and gpx and antioxidant vitamins such as vite , c and d , will lead to chronic wounds the data presented here show that exacerbation of the levels of ros immediately after wounding by inhibiting catalase and gpx causes the wound to become populated with a polybacterial infection that then leads to selection for bacteria that form biofilm , causing wounds with impaired healing to become chronic . the question now is : how does the increase in ros levels lead to development of biofilm that puts the wound in a state that leads to chronicity ? to that end , we currently are investigating the differences in gene expression levels in the presence or absence of iae in both c57bl/6 and db / db mice ( unpublished data ) . the observed pattern of gene expression , coupled with the fact that some of them are related to changes in oxidative stress , inflammation , apoptosis , and mitochondrial functions that regulate oxidative stress , suggests that these processes are critical for development of biofilm - induced chronicity . we speculate that a variety of genes that are altered when ros is significantly increased favor the ability of the bacteria to produce virulence factors that allow them to form biofilm . we have been successful in creating the first chronic wound model in which biofilm forms naturally without the need of introducing bacteria grown ex vivo . in this model , excessive increase in ros at the time of wounding provides the skin microbiota with favorable conditions to grow and be able to form biofilms ( figure 9 ) . the restoration of the redox balance by application of aoa causes biofilm to disappear from the wounds , resulting in significant improvement in wound healing . others have proposed that the pathogenesis of chronic wounds is dependent on factors such as tissue hypoxia , bacterial colonization , ischemia - reperfusion injury , and an altered cellular and systemic stress response . we propose that the critical parameter for development of chronic wounds in diabetic humans , and perhaps others , is an excessively high level of reactive oxygen species in the local microenvironment of the wound very early after injury . in this microenvironment , normal skin bacteria that otherwise would not form biofilm colonize the wound and become biofilm producers .

in the midnight of august 6 , 2010 , leh in ladakh region of north india received a heavy downpour . the cloud burst occurred all of a sudden that caught everyone unawares . within a short span of about 2 h , it recorded a rainfall of 14 inches . there were flash floods , and the indus river and its tributaries and waterways were overflowing . as many as 234 people were killed , 800 were injured , and many went missing , perhaps washed away with the gorging rivers and waterways . the main telephone exchange and mobile network system ( bsnl ) , which was the lifeline in the far - flung parts of the region , was completely destroyed . leh airport was flooded and the runway was covered with debris , making it non - functional . road transport was badly disrupted as roads were washed away and blocked with debris at many places . the civil medical and health facilities were also severely affected , as the lone district civil hospital was flooded and filled with debris . the present case study is based on the authors own experience of managing a natural disaster caused by the flash floods . the data was collected from the records of the district civil administration , the civil hospital , and the army hospital , leh . it included data collection from the primary sources of the district collectorate , interviews with the district civil administration and army officials who organized rescue operations , restoration of communication , and transport , mass casualty management , and informal discussions with local residents . these strategies included : i ) response , rescue , and relief operations , ii ) mass casualty management , and iii ) rehabilitation . the rescue and relief work was led by the indian army , along with the state government of jammu and kashmir , central reserve police force ( crpf ) , and indo - tibetan border police ( itbp ) . the indian army activated the disaster management system immediately , which is always kept in full preparedness as per the standard army protocols and procedures . there were just two hospitals in the area : the government civil hospital ( snm hospital ) and army hospital . during the flash floods , although the national disaster management act(1 ) was in place , with the government civil hospital being under strain , the applicability of the act was hampered . the army hospital quickly responded through rescue and relief operations and mass casualty management . by dawn , the patients admitted in the civil hospital were evacuated to the army hospital , leh in army helicopters . the runway of leh airport was cleared up within a few hours after the disaster so that speedy inflow of supplies could be carried out along with the evacuation of the casualties requiring tertiary level healthcare to the army command hospital in chandigarh . the work to make the roads operational though the main mobile network was dysfunctional , the other mobile network ( airtel ) still worked with limited connectivity in the far - flung areas of the mountains . the army communication system was the main and the only channel of communication for managing and coordinating the rescue and relief operations . severely injured people were evacuated from distant locations by helicopters , directly landing on the helipad of the army hospital . in order to reinforce the medical staff , nurses were flown in from the super specialty army hospital ( research and referral ) , new delhi , to handle the flow of casualties by the third day following the disaster . the mortuary of the government civil hospital was still functional where all the dead bodies were taken , while the injured were handled by army hospital , leh . those who sustained fractures were evacuated to army command hospital , chandigarh , by the army 's helicopters , after first aid . healthcare staff from the government civil hospital joined the army hospital , leh to assist them . in the meanwhile , medical equipment and drugs were transferred from the flooded and damaged government civil hospital to one of the nearby buildings where they could receive the casualties . by the third day following the disaster , the operation theatre of the government civil hospital was made functional . admissions in the army hospital , leh the analysis of the data showed that majority of the people who lost their lives were mainly local residents ( 49.6% ) . among the dead , there were 10.3% foreign nationals as well [ table 2 ] . the age - wise analysis of the deaths showed that the majority of deaths were reported in the age group 2650 years , accounting for 44.4% of deaths , followed by 1125 year group with 22.2% deaths . number of deaths according to status of residence the gender analysis showed that 61.5% were males among the dead , and 38.5% were females . a further analysis showed that more females died in < 10 years and 50 years age group , being 62.5% and 57.1% , respectively [ table 3 ] . age and sex distribution of deaths victims who survived the disaster were admitted to the army hospital , leh . over 90% of them about 3% suffered from cold injuries and 6.7% as medical emergencies [ table 4 ] . these strategies included : i ) response , rescue , and relief operations , ii ) mass casualty management , and iii ) rehabilitation . the initial response was carried out immediately by the government of india . the rescue and relief work was led by the indian army , along with the state government of jammu and kashmir , central reserve police force ( crpf ) , and indo - tibetan border police ( itbp ) . the indian army activated the disaster management system immediately , which is always kept in full preparedness as per the standard army protocols and procedures . there were just two hospitals in the area : the government civil hospital ( snm hospital ) and army hospital . during the flash floods although the national disaster management act(1 ) was in place , with the government civil hospital being under strain , the applicability of the act was hampered . the army hospital quickly responded through rescue and relief operations and mass casualty management . by dawn , the patients admitted in the civil hospital were evacuated to the army hospital , leh in army helicopters . the runway of leh airport was cleared up within a few hours after the disaster so that speedy inflow of supplies could be carried out along with the evacuation of the casualties requiring tertiary level healthcare to the army command hospital in chandigarh . the work to make the roads operational though the main mobile network was dysfunctional , the other mobile network ( airtel ) still worked with limited connectivity in the far - flung areas of the mountains . the army communication system was the main and the only channel of communication for managing and coordinating the rescue and relief operations . severely injured people were evacuated from distant locations by helicopters , directly landing on the helipad of the army hospital . in order to reinforce the medical staff , nurses were flown in from the super specialty army hospital ( research and referral ) , new delhi , to handle the flow of casualties by the third day following the disaster . the mortuary of the government civil hospital was still functional where all the dead bodies were taken , while the injured were handled by army hospital , leh . those who sustained fractures were evacuated to army command hospital , chandigarh , by the army 's helicopters , after first aid . healthcare staff from the government civil hospital joined the army hospital , leh to assist them . in the meanwhile , medical equipment and drugs were transferred from the flooded and damaged government civil hospital to one of the nearby buildings where they could receive the casualties . by the third day following the disaster , the operation theatre of the government civil hospital was made functional . admissions in the army hospital , leh the analysis of the data showed that majority of the people who lost their lives were mainly local residents ( 49.6% ) . among the dead , there were 10.3% foreign nationals as well [ table 2 ] . the age - wise analysis of the deaths showed that the majority of deaths were reported in the age group 2650 years , accounting for 44.4% of deaths , followed by 1125 year group with 22.2% deaths . number of deaths according to status of residence the gender analysis showed that 61.5% were males among the dead , and 38.5% were females . a further analysis showed that more females died in < 10 years and 50 years age group , being 62.5% and 57.1% , respectively [ table 3 ] . age and sex distribution of deaths victims who survived the disaster were admitted to the army hospital , leh . over 90% of them suffered traumatic injuries , with nearly half of them being major traumatic injuries . about 3% suffered from cold injuries and 6.7% as medical emergencies [ table 4 ] . the families were transferred to tents provided by the indian army and government and non - government agencies . 100,000 as an ex - gratia to the next of kin of each of those killed , and relief to the injured . 100,000 each would be paid to the next of kin of the deceased from the chief minister 's relief fund of the state government . the army maintains an inventory of essential medicines and supplies in readiness as a part of routing emergency preparedness . the essential non - food items were airlifted to the affected areas . these included blankets , tents , gum boots , and clothes . gloves and masks were provided for the persons who were working to clear the debris from the roads and near the affected buildings . public health is seriously threatened in disasters , especially due to lack of water supply and sanitation . people having lost their homes and living in temporary shelters ( tents ) puts a great strain on water and sanitation facilities . the pumping station was washed away , thus disrupting water supply in the leh township . a large number of toilets became non - functional as they were filled with silt , as houses were built at the foothills of the himalayan mountains . temporary arrangements of deep trench latrines were made while the army engineers made field flush latrines for use by the troops . water was stagnant and there was the risk of contamination by mud or dead bodies buried in the debris , thus making the quality of drinking water questionable . therefore , water purification units were installed and established . the national disaster response force ( ndrf ) airlifted a water storage system ( emergency rescue unit ) , which could provide 11,000 l of pure water . further , super - chlorination was done at all the water points in the army establishments . to deal with fly menace in the entire area , anti - fly measures there was an impending high risk of food shortage and crisis of hunger and malnutrition . the majority of food supply came from the plains and low - lying areas in north india through the major transport routes leh srinagar and leh manali national highways . the local agricultural and vegetable cultivation has always been scanty due to extreme cold weather . government agencies , nongovernmental organizations , and the indian army immediately established food supply and distribution system in the affected areas from their food stores and airlifting food supplies from other parts of the country . there was an urgent need to prevent disease transmission due to contaminated drinking water sources and flies . there was also a need to rehabilitate people who suffered from crush injuries sustained during the disaster . the public health facilities , especially , the primary health centers and sub - health centers , were not adequately equipped and were poorly connected by roads to the main city of leh . due to difficult accessibility , it took many hours to move casualties from the far - flung areas , worsening the crisis and rescue and relief operations . the population would have a higher risk of mental health problems like post - traumatic stress disorder , deprivation , and depression . therefore , relief and rehabilitation would include increased awareness of the symptoms of post - traumatic stress disorder and its alleviation through education on developing coping mechanisms . although it would be too early to estimate the impact on economy , the economy of the region would be severely affected due to the disaster . the scanty local vegetable and grain cultivation the summer season is the peak tourist season in ladakh and that is when the natural disaster took place . a large number of people came from within india and other countries for trekking in the region . the disaster would have a long - term economic impact as it would take a long time to rebuild the infrastructure and also to build the confidence of the tourists . the floods put an immense pressure and an economic burden on the local people and would also influence their health - seeking behavior and health expenditure . the area has a high strategic importance , being at the line of control with china and pakistan . the civil administration is with the leh autonomous hill development council ( lahdc ) under the state government of jammu and kashmir . the families were transferred to tents provided by the indian army and government and non - government agencies . 100,000 as an ex - gratia to the next of kin of each of those killed , and relief to the injured . 100,000 each would be paid to the next of kin of the deceased from the chief minister 's relief fund of the state government . the army maintains an inventory of essential medicines and supplies in readiness as a part of routing emergency preparedness . the essential non - food items were airlifted to the affected areas . these included blankets , tents , gum boots , and clothes . gloves and masks were provided for the persons who were working to clear the debris from the roads and near the affected buildings . public health is seriously threatened in disasters , especially due to lack of water supply and sanitation . people having lost their homes and living in temporary shelters ( tents ) puts a great strain on water and sanitation facilities . the pumping station was washed away , thus disrupting water supply in the leh township . a large number of toilets became non - functional as they were filled with silt , as houses were built at the foothills of the himalayan mountains . temporary arrangements of deep trench latrines were made while the army engineers made field flush latrines for use by the troops . water was stagnant and there was the risk of contamination by mud or dead bodies buried in the debris , thus making the quality of drinking water questionable . therefore , water purification units were installed and established . the national disaster response force ( ndrf ) airlifted a water storage system ( emergency rescue unit ) , which could provide 11,000 l of pure water . further , super - chlorination was done at all the water points in the army establishments . to deal with fly menace in the entire area , anti - fly measures there was an impending high risk of food shortage and crisis of hunger and malnutrition . the majority of food supply came from the plains and low - lying areas in north india through the major transport routes leh srinagar and leh manali national highways . the local agricultural and vegetable cultivation has always been scanty due to extreme cold weather . government agencies , nongovernmental organizations , and the indian army immediately established food supply and distribution system in the affected areas from their food stores and airlifting food supplies from other parts of the country . there was an urgent need to prevent disease transmission due to contaminated drinking water sources and flies . there was also a need to rehabilitate people who suffered from crush injuries sustained during the disaster . the public health facilities , especially , the primary health centers and sub - health centers , were not adequately equipped and were poorly connected by roads to the main city of leh . due to difficult accessibility , it took many hours to move casualties from the far - flung areas , worsening the crisis and rescue and relief operations . the population would have a higher risk of mental health problems like post - traumatic stress disorder , deprivation , and depression . therefore , relief and rehabilitation would include increased awareness of the symptoms of post - traumatic stress disorder and its alleviation through education on developing coping mechanisms . although it would be too early to estimate the impact on economy , the economy of the region would be severely affected due to the disaster . the scanty local vegetable and grain cultivation the summer season is the peak tourist season in ladakh and that is when the natural disaster took place . a large number of people came from within india and other countries for trekking in the region . the disaster would have a long - term economic impact as it would take a long time to rebuild the infrastructure and also to build the confidence of the tourists . the floods put an immense pressure and an economic burden on the local people and would also influence their health - seeking behavior and health expenditure . the area has a high strategic importance , being at the line of control with china and pakistan . the civil administration is with the leh autonomous hill development council ( lahdc ) under the state government of jammu and kashmir . . however , disaster preparedness plans and protocols in the civil administration and public health systems could be very helpful in rescue and relief and in reducing casualties and adverse impact on the human life and socio economic conditions.(2 ) however , the health systems in india lack such disaster preparedness plans and training.(3 ) in the present case , presence of the indian army that has standard disaster management plans and protocols for planning , training , and regular drills of the army personnel , logistics and supply , transport , and communication made it possible to immediately mount search , rescue , and relief operations and mass casualty management . not only the disaster management plans were in readiness , but continuous and regular training and drills of the army personnel in rescue and relief operations , and logistics and communication , could effectively facilitate the disaster management operations . the army 's communication system served as an alternative communication channel as the public communication and mobile network was destroyed , and that enabled effective coordination of the disaster operations . emergency medical services and healthcare within few hours of the disaster was critical to minimize deaths and disabilities . preparedness of the army personnel , especially the medical corps , readiness of inventory of essential medicines and medical supplies , logistics and supply chain , and evacuation of patients as a part of disaster management protocols effectively launched the search , rescue , and relief operations and mass casualty reduction . continuous and regular training and drills of army personnel , health professionals , and the community in emergency rescue and relief operations are important measures . emergency drill is a usual practice in the army , which maintains the competence levels of the army personnel . similar training and drill in civil administration and public health systems in emergency protocols for rescue , relief , mass casualty management , and communication would prove very useful in effective disaster management to save lives and restore health of the people.(24 ) natural disasters not only cause a large - scale displacement of population and loss of life , but also result in loss of property and agricultural crops leading to severe economic burden.(36 ) in various studies,(3478 ) several shortcomings have been observed in disaster response , such as , delayed response , absence of early warning systems , lack of resources for mass evacuation , inadequate coordination among government departments , lack of standard operating procedures for rescue and relief , and lack of storage of essential medicines and supplies . the disaster management operations by the indian army in the natural disaster offered several lessons to learn . the key lessons were : response time is a critical attribute in effective disaster management . this was made possible as the army had disaster and emergency preparedness plans and protocols in place ; stocks of relief supplies and medicines as per standard lists were available ; and periodic training and drill of the army personnel and medical corps was undertaken as a routine . the disaster response could be immediately activated.there is an important lesson to be learned by the civil administration and the public health system to have disaster preparedness plans in readiness with material and designated rescue officers and workers.prompt activation of disaster management plan with proper command and coordination structure is critical . the indian army could effectively manage the disaster as it had standard disaster preparedness plans and training , and activated the system without any time lag . these included standard protocols for search , rescue , and evacuation and relief and rehabilitation . there are standard protocols for mass casualty management , inventory of essential medicines and medical supplies , and training of the army personnel.hospitals have always been an important link in the chain of disaster response and are assuming greater importance as advanced pre - hospital care capabilities lead to improved survival - to - hospital rate.(9 ) role of hospitals in disaster preparedness , especially in mass casualty management , is important . army hospital , leh emergency preparedness played a major role in casualty management and saving human lives while the civil district hospital had become dysfunctional due to damage caused by floods . the hospital was fully equipped with essential medicines and supplies , rescue and evacuation equipments , and command and communication systems.standard protocols and disaster preparedness plans need to be prepared for the civil administration and the health systems with focus on quick response teams inclusive of healthcare professionals , rescue personnel , fire - fighting squads , police detachments , ambulances , emergency care drugs , and equipments.(10 ) these teams should be trained in a manner so that they can be activated and deployed within an hour following the disaster . triage has to be the basic working principle for such teams.effective communication system is of paramount importance in coordination of rescue and relief operations . in the present case study , although the main network with the widest connectivity was extensively damaged and severely disrupted , the army 's communication system along with the other private mobile network tided over the crisis . it took over 10 days for reactivation of the main mobile network through satellite communication system . thus , it is crucial to establish the alternative communication system to handle such emergencies efficiently and effectively.(211)disaster management is a multidisciplinary activity involving a number of departments / agencies spanning across all sectors of development.(2 ) the national disaster management authority of india , set up under national disaster management act 2005,(1 ) has developed disaster preparedness and emergency protocols . it would be imperative for the civil administration at the state and district levels in india to develop their disaster management plans using these protocols and guidelines.health system 's readiness plays important role in prompt and effective mass casualty management.(2 ) being a mountainous region , the ladakh district has difficult access to healthcare , with only nine primary health centers and 31 health sub - centers.(12 ) there is a need for strengthening health systems with focus on health services and health facility network and capacity building . more than that , primary healthcare needs to be augmented to provide emergency healthcare so that more and more lives can be saved.(7)training is an integral part of capacity building , as trained personnel respond much better to different disasters and appreciate the need for preventive measures . training of healthcare professionals in disaster management holds the key in successful activation and implementation of any disaster management plan . the army has always had standard drills in all its establishments at regular intervals , which are periodically revised and updated . the civil administration and public health systems should regularly organize and conduct training of civil authorities and health professionals in order to be ready for action.(14)building confidence of the public to avoid panic situation is critical . community involvement and awareness generation , particularly that of the vulnerable segments of population and women , needs to be emphasized as necessary for sustainable disaster risk reduction . increased public awareness periodic mock drills and exercise in disaster management protocols in the general population can be very useful.(134 ) response time is a critical attribute in effective disaster management . this was made possible as the army had disaster and emergency preparedness plans and protocols in place ; stocks of relief supplies and medicines as per standard lists were available ; and periodic training and drill of the army personnel and medical corps was undertaken as a routine . the disaster response could be immediately activated . there is an important lesson to be learned by the civil administration and the public health system to have disaster preparedness plans in readiness with material and designated rescue officers and workers . the indian army could effectively manage the disaster as it had standard disaster preparedness plans and training , and activated the system without any time lag . these included standard protocols for search , rescue , and evacuation and relief and rehabilitation . there are standard protocols for mass casualty management , inventory of essential medicines and medical supplies , and training of the army personnel . hospitals have always been an important link in the chain of disaster response and are assuming greater importance as advanced pre - hospital care capabilities lead to improved survival - to - hospital rate.(9 ) role of hospitals in disaster preparedness , especially in mass casualty management , is important . army hospital , leh emergency preparedness played a major role in casualty management and saving human lives while the civil district hospital had become dysfunctional due to damage caused by floods . the hospital was fully equipped with essential medicines and supplies , rescue and evacuation equipments , and command and communication systems . standard protocols and disaster preparedness plans need to be prepared for the civil administration and the health systems with focus on quick response teams inclusive of healthcare professionals , rescue personnel , fire - fighting squads , police detachments , ambulances , emergency care drugs , and equipments.(10 ) these teams should be trained in a manner so that they can be activated and deployed within an hour following the disaster . effective communication system is of paramount importance in coordination of rescue and relief operations . in the present case study , although the main network with the widest connectivity was extensively damaged and severely disrupted , the army 's communication system along with the other private mobile network tided over the crisis . it took over 10 days for reactivation of the main mobile network through satellite communication system . thus , it is crucial to establish the alternative communication system to handle such emergencies efficiently and effectively.(211 ) disaster management is a multidisciplinary activity involving a number of departments / agencies spanning across all sectors of development.(2 ) the national disaster management authority of india , set up under national disaster management act 2005,(1 ) has developed disaster preparedness and emergency protocols . it would be imperative for the civil administration at the state and district levels in india to develop their disaster management plans using these protocols and guidelines . health system 's readiness plays important role in prompt and effective mass casualty management.(2 ) being a mountainous region , the ladakh district has difficult access to healthcare , with only nine primary health centers and 31 health sub - centers.(12 ) there is a need for strengthening health systems with focus on health services and health facility network and capacity building . more than that , primary healthcare needs to be augmented to provide emergency healthcare so that more and more lives can be saved.(7 ) training is an integral part of capacity building , as trained personnel respond much better to different disasters and appreciate the need for preventive measures . training of healthcare professionals in disaster management holds the key in successful activation and implementation of any disaster management plan . the army has always had standard drills in all its establishments at regular intervals , which are periodically revised and updated . the civil administration and public health systems should regularly organize and conduct training of civil authorities and health professionals in order to be ready for action.(14 ) building confidence of the public to avoid panic situation is critical . community involvement and awareness generation , particularly that of the vulnerable segments of population and women , needs to be emphasized as necessary for sustainable disaster risk reduction . increased public awareness is necessary to ensure an organized and calm approach to disaster management . periodic mock drills and exercise in disaster management protocols in the general population can be very useful.(134 ) natural disasters not only cause a large - scale displacement of population and loss of life , but also result in loss of property and agricultural crops leading to severe economic burden.(36 ) in various studies,(3478 ) several shortcomings have been observed in disaster response , such as , delayed response , absence of early warning systems , lack of resources for mass evacuation , inadequate coordination among government departments , lack of standard operating procedures for rescue and relief , and lack of storage of essential medicines and supplies . the disaster management operations by the indian army in the natural disaster offered several lessons to learn . the key lessons were : response time is a critical attribute in effective disaster management . this was made possible as the army had disaster and emergency preparedness plans and protocols in place ; stocks of relief supplies and medicines as per standard lists were available ; and periodic training and drill of the army personnel and medical corps was undertaken as a routine . the disaster response could be immediately activated.there is an important lesson to be learned by the civil administration and the public health system to have disaster preparedness plans in readiness with material and designated rescue officers and workers.prompt activation of disaster management plan with proper command and coordination structure is critical . the indian army could effectively manage the disaster as it had standard disaster preparedness plans and training , and activated the system without any time lag . these included standard protocols for search , rescue , and evacuation and relief and rehabilitation . there are standard protocols for mass casualty management , inventory of essential medicines and medical supplies , and training of the army personnel.hospitals have always been an important link in the chain of disaster response and are assuming greater importance as advanced pre - hospital care capabilities lead to improved survival - to - hospital rate.(9 ) role of hospitals in disaster preparedness , especially in mass casualty management , is important . army hospital , leh emergency preparedness played a major role in casualty management and saving human lives while the civil district hospital had become dysfunctional due to damage caused by floods . the hospital was fully equipped with essential medicines and supplies , rescue and evacuation equipments , and command and communication systems.standard protocols and disaster preparedness plans need to be prepared for the civil administration and the health systems with focus on quick response teams inclusive of healthcare professionals , rescue personnel , fire - fighting squads , police detachments , ambulances , emergency care drugs , and equipments.(10 ) these teams should be trained in a manner so that they can be activated and deployed within an hour following the disaster . triage has to be the basic working principle for such teams.effective communication system is of paramount importance in coordination of rescue and relief operations . in the present case study , although the main network with the widest connectivity was extensively damaged and severely disrupted , the army 's communication system along with the other private mobile network tided over the crisis . it took over 10 days for reactivation of the main mobile network through satellite communication system . thus , it is crucial to establish the alternative communication system to handle such emergencies efficiently and effectively.(211)disaster management is a multidisciplinary activity involving a number of departments / agencies spanning across all sectors of development.(2 ) the national disaster management authority of india , set up under national disaster management act 2005,(1 ) has developed disaster preparedness and emergency protocols . it would be imperative for the civil administration at the state and district levels in india to develop their disaster management plans using these protocols and guidelines.health system 's readiness plays important role in prompt and effective mass casualty management.(2 ) being a mountainous region , the ladakh district has difficult access to healthcare , with only nine primary health centers and 31 health sub - centers.(12 ) there is a need for strengthening health systems with focus on health services and health facility network and capacity building . more than that , primary healthcare needs to be augmented to provide emergency healthcare so that more and more lives can be saved.(7)training is an integral part of capacity building , as trained personnel respond much better to different disasters and appreciate the need for preventive measures . training of healthcare professionals in disaster management holds the key in successful activation and implementation of any disaster management plan . the army has always had standard drills in all its establishments at regular intervals , which are periodically revised and updated . the civil administration and public health systems should regularly organize and conduct training of civil authorities and health professionals in order to be ready for action.(14)building confidence of the public to avoid panic situation is critical . community involvement and awareness generation , particularly that of the vulnerable segments of population and women , needs to be emphasized as necessary for sustainable disaster risk reduction . increased public awareness is necessary to ensure an organized and calm approach to disaster management . periodic mock drills and exercise in disaster management protocols in the general population can be very useful.(134 ) response time is a critical attribute in effective disaster management . this was made possible as the army had disaster and emergency preparedness plans and protocols in place ; stocks of relief supplies and medicines as per standard lists were available ; and periodic training and drill of the army personnel and medical corps was undertaken as a routine . the disaster response could be immediately activated . there is an important lesson to be learned by the civil administration and the public health system to have disaster preparedness plans in readiness with material and designated rescue officers and workers . the indian army could effectively manage the disaster as it had standard disaster preparedness plans and training , and activated the system without any time lag . these included standard protocols for search , rescue , and evacuation and relief and rehabilitation . there are standard protocols for mass casualty management , inventory of essential medicines and medical supplies , and training of the army personnel . hospitals have always been an important link in the chain of disaster response and are assuming greater importance as advanced pre - hospital care capabilities lead to improved survival - to - hospital rate.(9 ) role of hospitals in disaster preparedness , especially in mass casualty management , is important . army hospital , leh emergency preparedness played a major role in casualty management and saving human lives while the civil district hospital had become dysfunctional due to damage caused by floods . the hospital was fully equipped with essential medicines and supplies , rescue and evacuation equipments , and command and communication systems . standard protocols and disaster preparedness plans need to be prepared for the civil administration and the health systems with focus on quick response teams inclusive of healthcare professionals , rescue personnel , fire - fighting squads , police detachments , ambulances , emergency care drugs , and equipments.(10 ) these teams should be trained in a manner so that they can be activated and deployed within an hour following the disaster . effective communication system is of paramount importance in coordination of rescue and relief operations . in the present case study , although the main network with the widest connectivity was extensively damaged and severely disrupted , the army 's communication system along with the other private mobile network tided over the crisis . it took over 10 days for reactivation of the main mobile network through satellite communication system . thus , it is crucial to establish the alternative communication system to handle such emergencies efficiently and effectively.(211 ) disaster management is a multidisciplinary activity involving a number of departments / agencies spanning across all sectors of development.(2 ) the national disaster management authority of india , set up under national disaster management act 2005,(1 ) has developed disaster preparedness and emergency protocols . it would be imperative for the civil administration at the state and district levels in india to develop their disaster management plans using these protocols and guidelines . health system 's readiness plays important role in prompt and effective mass casualty management.(2 ) being a mountainous region , the ladakh district has difficult access to healthcare , with only nine primary health centers and 31 health sub - centers.(12 ) there is a need for strengthening health systems with focus on health services and health facility network and capacity building . more than that , primary healthcare needs to be augmented to provide emergency healthcare so that more and more lives can be saved.(7 ) training is an integral part of capacity building , as trained personnel respond much better to different disasters and appreciate the need for preventive measures . training of healthcare professionals in disaster management holds the key in successful activation and implementation of any disaster management plan . the army has always had standard drills in all its establishments at regular intervals , which are periodically revised and updated . the civil administration and public health systems should regularly organize and conduct training of civil authorities and health professionals in order to be ready for action.(14 ) building confidence of the public to avoid panic situation is critical . community involvement and awareness generation , particularly that of the vulnerable segments of population and women , needs to be emphasized as necessary for sustainable disaster risk reduction . increased public awareness is necessary to ensure an organized and calm approach to disaster management . periodic mock drills and exercise in disaster management protocols in the general population can be very useful.(134 )

first , the widespread adoption of commercial ehr systems presents a barrier to researchers and investigators seeking to deploy or integrate new tools and interventions into clinical practice . second , despite their challenges , ehrs will remain the primary method of collecting / generating clinical data yet there is little to no input from the informatics community into their configuration , implementation , and support . for the field of informatics to remain relevant , successful tools and interventions that are developed for research need to be put into general clinical practice and integrated into the clinical workflow . this is one of the primary goals of the patient - centered outcomes research institute ( pcori),1 which was established to provide information to patients and clinicians on what treatments are most effective , and to more quickly push research findings into clinical practice . on the genomics front , the electronic medical records and genomics ( emerge ) network is looking to incorporate genome - wide association study results into clinical practice.2 unfortunately , these aims are in conflict with the actual landscape of health information technology today . as part of the american recovery and reinvestment act ( arra ) , the federal government allocated billions of dollars to speed up the adoption of electronic health records ( ehrs).3 and while there are over 750 certified ehr vendors,4 just six control over half the market.5 some 127 million people , over 40% of the usa population , are estimated to have an ehr in the systems of a single vendor epic.6 in pediatrics , of the 12 institutions on the us news and world report 's best children 's hospitals 201213 honor roll,7 10 of the 12 are customers of either epic or cerner . the adoption of ehrs was supposed to provide a single view of the patient 's record , bringing together data from previously siloed systems . in accomplishing this task , however , ehrs have evolved into a walled garden , forcing all interactions to be within the system and making the integration of outside data or tools all but impossible . to illustrate this point , my development team recently worked with a group that was looking to create a system for monitoring immunosuppressant medication adherence in patients with a kidney transplant.8 as inputs , the system took the sd of previous immunosuppressant laboratory values , self - reported medication adherence information collected via a web - based system , and medication adherence as reported by pill bottles that track when they are opened and closed.9 these data were combined into an adherence score and presented in a report , along with the patient 's risk stratification and recommended course of action . the total budget for the entire project , which was supported by an institutional grant , was us$100 000 . as long as it was stand - alone , creating a similar system for other transplant types or to spread it to other institutions required minimal effort . integrating such a tool into the ehr , however , proved to be almost impossible . there was no way to recreate the dynamic , self - reported survey tool or import the results , the ehr was not capable of computing sds for the laboratory values , and it lacked functionality for integrating the adherence data . in the end , most of the decision support and risk stratification was jettisoned , leaving only the calculation of the adherence score , which had to be manually entered into the ehr . as another example , my development team is halfway through a federal grant where we are working in collaboration with improvecarenow,10 a network of 36 centers focused on pediatric inflammatory bowel disease ( ibd ) that is aiming to build an ehr - linked registry . data are collected in discrete fields within the ehr during the clinic visit , extracted , and then uploaded into the registry . they could then be used for research , quality improvement , and clinical purposes such as pre - visit planning , population management , and patient engagement . an ehr - linked registry would achieve the long - cherished clinician goal of data in once, that is , data initially collected for clinical care and then reused for other purposes . the improvecarenow network is a particularly good candidate for such a project as their registry data are the same elements that should be collected as part of model ibd care . when we started the project , we approached the various ehr vendors represented in the network to ask them about creating and supporting a form that could be used by all of their customers . aside from epic , which quickly agreed to participate , the other ehr vendors have been very slow to respond . as a result , all of the non - epic centers are likely to have to build and support their own data collection forms and create their own custom extracts , which poses a barrier to adoption . since few institutions have ehr staff dedicated to handling research requests , this project must compete for priority against initiatives like the icd-10 conversion and phase 1 of meaningful use or the implementation of the ehr itself . once we do have the attention of the ehr staff , we must ensure that the form is built so that it : ( a ) easily integrates into the clinical workflow ( something frequently ignored by researchers ) ; ( b ) can be completed quickly ; ( c ) can , once completed , be pulled into a progress note or referral letter ( also overlooked ) ; and ( d ) has elements that are easily queried / extracted . if criteria a , b , and c are not met , then the clinicians will not use the form . if criterion d is unsatisfied , the enterprise reporting team will have trouble generating an extract . with ehrs , the easiest method of data collection is to typically create a text - based template where a clinician can fill in certain keywords . this leads most clinicians to believe they are capturing data discretely , but unless a significant amount of effort is undertaken to tag those keywords with a unique identifier , the result is stored as a blob of text . other data collection methods may store data discretely , but do not integrate into the clinical workflow . after the forms are created , we face a second set of issues in transferring the data back out . in this project , we have taken the decidedly low - tech approach of having each center work with their enterprise reporting team to generate a report , which will then be uploaded to the registry . two of the more sophisticated approaches to this problem , data transfer using the health level 7 ( hl7 ) continuity of care document ( ccd)11 or the integrating the healthcare enterprise ( ihe ) retrieve form for data capture ( rfd ) protocol,12 while appealing , are not suitable for our purposes . with the hl7 ccd , none of the condition - specific variables needed by improvecarenow are included in the specification . with rfd , an emerging standard that allows clinical trial forms to be embedded in the ehr and pre - populated with certain data , the data in the form are sent to a remote server . no data are retained in the ehr , however , which is a significant drawback when the data needed for research are also utilized for clinical care . in the end , far too much effort is spent working around the limitations of the ehr instead of developing better tools and interventions . for ehrs to be truly useful and not just an electronic version of a paper chart , their design must change . it must be easier to create data collection forms and other interfaces that are easily integrated into the clinical workflow yet are tied to underlying data elements that are common across the vendor 's system . customers should be able to author these forms , but there should be a mechanism that allows them to be vendor supported through system extensions and upgrades . at the same time , it needs to be easier to extract information from ehrs through common application programming interfaces ( apis ) . we need alternatives to the current approaches of a database query or an hl7 feed . in addition to making data sharing easier , this would also allow innovation in areas like safety monitoring and decision support , which require real - time access to production ehr data . while it may take some time , the winning ehr design of the future will be one that adopts a robust data model with a loosely coupled service - oriented architecture . this would allow easier access to the underlying data and an interface for the use of external services such as quality reporting and decision support . while the ideal case would be to have all ehrs utilize a common reference model with a corresponding set of end - user apis , that is an unlikely prospect . a more realistic and obtainable solution would be to have all of the ehr vendors publish their apis ( and create them if they do not already exist ) . having standardization across vendor apis for a given functional area ( demographics or medication orders , for instance ) would be optimal , but one could certainly imagine the emergence of a secondary market that would provide such standardized overlays to achieve the same result . having a published set of apis is the first step towards the establishment of an ecosystem of external services and applications . given the current environment , it is unrealistic to expect a completely wide - open marketplace like that of android . a more step - wise solution would be to establish a set of design patterns and an approval process for developers to deploy their tools ( ie , the apple approach ) . to some degree , this is the concept behind the substitutable medical apps , reusable technologies ( smart ) project,13 although unlike the current version of smart , the ability to write data would be required . there are definite limitations to the apple model , but even so , it would be a drastic improvement over what we have today . in this landscape , the vendor 's revenue stream would come from de - identified comparative effectiveness research or other analysis of their patient population . if vendors also adopted the apple philosophy toward external applications , they could establish a secondary revenue stream through licensing fees . without outside pressure from the marketplace , from patients , and from clinicians , however , vendors have no incentive or motivation to change . perhaps amia should convene a working group to liaise between the informatics community , the general public , and commercial ehr vendors , providing a forum for a more open and transparent dialogue . barring such a development , innovation in medical informatics will occur at the speed of the vendor 's upgrade cycle . while the informatics community needs to push for change in the design of ehrs , at the same time it needs to become more involved in their configuration and implementation . for years , the relationship between clinical operations and research at most institutions ranged anywhere from non - existent to hostile , to , in the best cases , something close to benign neglect . the implementation of enterprise - wide ehrs has now caused these worlds to come crashing together . the operational staff who are charged with implementing the ehr have the most control over the design and configuration of the final build . they are the ones who know the capabilities of the system and are the ones making recommendations on how to electronically capture the outputs of a clinical workflow . if we are to ever achieve the vision of a learning health system,14 where learning occurs with every patient encounter , we need to ensure that the clinical information systems are configured to allow this learning . otherwise , we should not be surprised when ehr data turn out to be less than optimal for quality improvement and research . we must leverage the vast numbers of staff who are tasked with maintaining the institutional ehr . expectations must be set that their role is not only to support clinical care , but also quality improvement and research . in a learning health system , the clinical informatics community has much to contribute here , particularly in the areas of clinical decision support , care process redesign , and workflow analysis / optimization . in the same way that other faculty and staff run laboratories and/or see patients , we should embrace ( and reward ) this service role . by leveraging our expertise in clinical terminology representation , structured data capture , interface design , etc , we can drive towards a more successful implementation and utilization of existing commercial ehrs . this type of collaboration is messy and requires a tremendous amount of effort at times , but given a world where government funding appears increasingly threatened , is likely to be the key to survival .

binding selectivity is one of the most important requirements for the development of a safe therapeutic ; in fact , large differences in binding affinity between the intended target and similar proteins are often sought in order to avoid unwanted and often unknown side effects . conversely , in some cases a compound with a specific promiscuous profile might be equally desirable for reasons of efficacy . yet , the identification and design of selective ( or promiscuous ) chemical tools is a difficult task , in particular when working on protein families with conserved folds and similar binding pocket residues . a typical example is provided by the protein kinase family , for which different assay panels have been developed in order to determine selectivity profiles of different ligands . the development of bromodomain ( brd ) chemical probes poses the same selectivity challenge due their conserved fold . brds are epigenetic mark readers that specifically recognize -n - lysine acetylation motifs ; 61 human brds have been found in 46 human nuclear and cytoplasmic proteins , and they are typically divided into eight families based on sequence and structural similarity ( figure 1a ) . with acetylation motifs often found in macromolecular complexes implicated in chromatin remodeling , dna repair and cell - cycle control , and especially on histones , brd inhibitors are finding broad application in medicine and basic biological research , in particular in oncology and inflammation . despite their sequence diversity , they all share a conserved fold ( figure 1b ) that renders the design of selective ligands a challenging task . selective probes can , however , lead to better preclinical target validation and profiling , and eventually to better clinical outcomes . ligands and ( a ) phylogenetic tree of the human bromodomain family ; in green are the brds included in the study . ( b ) conserved fold of human bromodomains ( pdb i d 2oss ) . depicted as red spheres is the conserved water network found in most brd binding pockets , and as blue sticks the conserved hydrogen bond donor , an asparagine residue in the majority of brds . a transparent surface shows the cavity in between the za and bc loops forming the acetyl - lysine binding pocket . two clefts , placed in between the wp residues ( conserved in subfamilies i and ii ) and the za and bc loops and exploited by many bromodomain inhibitors are indicated . engineering selectivity adds considerable complexity to the rational design of new drugs , as it involves the optimization of multiple binding affinities . different strategies have been adopted for selectivity design , such as shape and electrostatics complementarity , conformational selection , water displacement , and allosteric binding . yet computationally , the prediction of binding selectivity is a difficult task too , and not many generally applicable methodologies are available to the computational and medicinal chemistry communities . henrich et al . evaluated the performance of target - specific scoring functions , developed from known sets of binders , for three protein targets : thrombin , trypsin , and urokinase - type plasminogen activator . the quality of the predictions appeared to be system dependent and the authors discussed how inclusion of protein flexibility and conserved waters might result in better docking methods . thanks to advances in theory and computing , however , the prediction of binding affinities using physics - based biomolecular simulations is becoming increasingly feasible . all - atom simulations naturally take into account effects such as the dynamics of the binding pocket , the role of the solvent , and entropy changes upon binding , which are poorly captured with scoring - function - based approaches . in particular , free energy calculations based on molecular dynamics ( md ) and alchemical pathways could offer an alternative and more rigorous approach to the problem , and one that does not require the use of training sets . moreover , such calculations are becoming progressively more accessible to current levels of computational power . recent studies have shown the potential of relative binding free energy ( rbfe ) calculations for small - molecule lead optimization . rbfe calculations can also allow the evaluation of free energy changes upon protein mutation , but they become unfeasible when a large number of residues differ between the proteins . absolute binding free energy ( abfe ) calculations are a more general approach as they do not require knowledge of any affinity value in advance and , in principle , could be used to compare the affinity of any ligand for any protein target . such calculations could thus be employed as a tool to probe ligand selectivity across different binding pockets , in order to identify the scaffolds providing the desired selectivity profile . for instance , lin and co - workers used absolute free energy calculations to explain the specificity of gleevec for abl kinase versus similar homologous tyrosine kinases . more recently , we have shown how calculations based on standard implementations of alchemical transformations might be applied for the accurate affinity estimation of diverse , drug - like organic molecules . abfe calculations are computationally demanding and , despite the few examples mentioned above , their performance has had only limited testing against experimental data , with a focus on model systems . careful retrospective validation is therefore still needed before the methodology can be confidently applied in a prospective drug discovery scenario , with the ability to anticipate when these calculations might be the most suitable tool and where potential pitfalls might lie . here , we test the robustness of abfe calculations across different binding pockets , in order to assess the accuracy of the computational method against multiple proteins and thus evaluate its performance and potential utility for the study of ligand selectivity . we calculated the affinities for 36 complexes , involving 22 bromodomains and 3 ligands ( rvx - oh , rvx-208 , and bromosporine ; see figure 1c ) , without any information on the affinities or structures of the complexes . we compare the results with high - quality isothermal titration calorimetry ( itc ) data and discuss the performance of the calculations in relation to their potential application in drug design . protein conformations were taken from crystal structures as listed in tables 1 and 2 ( more information on the protein models is in table s1 ) . if multiple chains were present in the asymmetric unit , only monomer a would be kept for the calculations . missing atoms in the crystals were modeled with the what - if web interface and all organic molecules were removed from the system , whereas all crystallographic waters were retained . the binding poses for rvx-208 and rvx - oh were taken from a previous study , where the poses were identified via docking following the same procedure here used for bromosporine . the preferred binding pose for bromosporine was identified by docking the ligand in an apo x - ray structure of brd4(1 ) ( pdb i d 2oss ) and then performing free energy calculations on the clustered poses . for bromosporine and rvx-208 , the highest affinity pose ( as predicted by the calculations ) was assumed to be conserved and was modeled into the pockets of the other bromodomains by structural alignment to brd4(1 ) with pymol . for rvx - oh , however , two alternative poses were tested via free energy calculations for all bromodomains binding to this ligand . when placing ligands into the pockets , crystallographic water molecules within 1.4 of the organic molecule were removed in order to avoid steric clashes . all other crystallographically resolved water molecules were retained , so that the conserved network of water molecules at the bottom of the cavities was preserved in the calculations . after adding hydrogens with maestro ( v9.5 , schrdinger ) , the ligands were parametrized with the general amber force field ( gaff v1.7 ) using ambertools14 . restrained electrostatic potential ( resp ) charges ( hf/6 - 31g*//hf/6 - 31 g * ) for bromosporine were obtained with the pyred server using gaussian 09 ( rev . resp charges for the ligands rvx-208 and rvx - oh were obtained with a geometry optimization followed by a molecular electrostatic potential calculation , both at the hf/6 - 31 g * level of theory . the resp fit was done using antechamber in ambertools14 , while all qm calculations were performed in gaussian 09 ( revision d.01 ) . the pyred server was not employed in the latter cases due to its unavailability for a number of months in late 2015 . gromacs topologies and coordinate files were generated from the amber ones using acpype ( v.2014 - 08 - 27 rev . the amber99sb - ildn force field was used for the protein and the tip3p model for water molecules . the complexes were solvated in a dodecahedral box with periodic boundary conditions and a minimum distance between the solute and the box of 12 . sodium and chloride ions were added to neutralize the systems at the concentration of 0.15 m. itc data were taken from picaud et al . itc uncertainties are an estimate based on the standard deviation of the g values observed in the abrf - mirg02 inter - laboratory assessment . refers to rcsb protein data bank code for the protein structure used for the free energy calculations . pose indicates in which one of the two orientations the ligand is expected to bind given the experimental evidence , where h is the horizontal pose and v is the vertical pose . in square brackets are the 95% confidence intervals of the statistics based on percentile bootstrap . itc uncertainties are an estimate based on the standard deviation of the g values observed in the abrf - mirg02 inter - laboratory assessment . gcalc refers to the calculated free energy values with the initial bromosporine model ; gcalc refers to the model with modified torsional parameters for the benzensulfonamide group . in square brackets are the 95% confidence intervals of the statistics based on percentile bootstrap . the ligands were docked into an x - ray structure of brd4(1 ) ( pdb i d 2oss ) using moe ( chemical computing group , v2014.09 ) . all water and organic molecules were removed from the model , with the exception of five water molecules at the bottom of the binding pocket , which are conserved across most bromodomains . the ligands were initially drawn in 2d using marvin sketch ( chemaxon , v6.1.0 ) and a stochastic conformational search was performed in moe for the generation of multiple 3d conformers . the number of conformers generated was limited to one hundred and duplicates ones ( rmsd < 0.25 ) were removed . protein and ligands were parametrized using the amber10:eht force field option in moe . thus , the protein parameters were taken from amber ff99sb , while the ligand bonded parameters were obtained with 2d extended hckel theory , the vdw parameters were derived from gaff , and the charges from bond charge increments . the pharmacophore query was built based on the structure of bound acetyl - lysine , as found in a holo x - ray structure ( pdb i d 3uvw ) , and consisted of a hydrogen - bond acceptor site ( mimicking the acetyl oxygen ) and a nonpolar site ( mimicking the methyl moiety ) . the london g function was used for the initial scoring of the poses , and each binding pose was then minimized and rescored with the gbvi / wsa g function . duplicate poses were removed based on their hydrogen - bond and hydrophobic patterns using this option in moe . in addition , also poses with positive binding free energy as predicted by the gbvi / wsa g scoring function were removed . the remaining poses were clustered by rmsd with a 3.0 cutoff in order to reduce the number of poses to test via free energy calculations , while also retaining a diverse set of binding orientations ; in fact , similar binding orientations might interconvert during the simulations , resulting in almost equivalent ensembles . finally , the best scoring poses within each cluster were selected for free energy calculations . the parameters for the two different biaryl torsions present in bromosporine and the two rvx ligands were optimized by least - squares fitting to a qm torsion scan at the mp2/6 - 31 g * level of theory ( gaussian 09 ; rev . three torsion angles present in the benzensulfonamide moiety were also later reparametrized specifically for bromosporine using the program paramfit in ambertools14 . parameters were derived by fitting the mm energies to single - point energy calculations at the mp2/6 - 31 g * level of theory ( figure s2 ) performed with gaussian 09 ( rev . the parameter search was performed using the hybrid genetic algorithm with a mutation rate of 0.1 ; all other input parameters were used with their default values . a total of 514 conformations were generated by systematic variation of the dihedral angles for the fitting of 3 torsions ( 4 terms per torsion ) within the benzensulfonamide moiety . conformations with qm relative energies below 5 kcal / mol were assigned weights of 1.0 , conformations with energies between 5 and 10 kcal / mol were assigned weights of 0.5 , and conformations with energies above 10 kcal / mol were assigned weights of zero . only the force constant and the phase of the torsion were fit , while the periodicity was fixed . the parameters for all these terms are shown in figure s2 . in order to improve the fit of the ligands into the binding pockets of the structures used for the free energy calculations , a brief preliminary md simulation was run for each complex . 10 000 energy minimization steps were performed using a steepest descent algorithm ( the protein ligand structures obtained after this step were submitted to the csm - lig server for scoring ) . the system was subsequently simulated for 0.5 ns in the canonical ensemble and for 1 ns in the isothermal isobaric ensemble with harmonic position restraints applied to all solute heavy atoms with a force constant of 1000 kj mol nm . temperature was coupled using langevin dynamics at the target temperature of 298.15 k , while pressure was coupled using the berendsen weak coupling algorithm with a target pressure of 1 atm . a 15 ns unrestrained run was then performed in the npt ensemble with the parrinello the last frame of the run provided the starting system conformation for the free energy calculations . the last 5 ns of the unrestrained simulations were also used to obtain equilibrium values for the protein ligand restraints used during the free energy cycle as described in the next paragraph . all calculations were carried out in gromacs 5.0.4 ( all input files for the abfe calculations are available as supporting information ) . the ligand van der waals interactions were decoupled and the charges annihilated using a linear alchemical pathway with = 0.05 for the van der waals and = 0.1 for the coulombic transformations . for the addition of the ligand restraints , 12 nonuniformly distributed values were used ( 0.0 , 0.01 , 0.025 , 0.05 , 0.075 , 0.1 , 0.15 , 0.2 , 0.3 , 0.5 , 0.75 , 1.0 ) . a total of 42 windows for the complex simulations and 31 windows for the ligand simulations were therefore employed . for each window , 10 000 energy minimization steps were carried out using a steepest descent algorithm . the system was subsequently simulated for 0.5 ns in the canonical ensemble with harmonic position restraints applied to the solute heavy atoms with a force constant of 1000 kj mol nm . a 1 ns position restrained run in the isothermal isobaric ensemble was then performed using the berendsen weak coupling algorithm and with a target pressure of 1 atm . several 15 ns unrestrained production runs were performed for data collection using hamiltonian - exchange langevin dynamics in the npt ensemble with the parrinello a total of 3 million swaps between any state pair were attempted every 1000 time steps , following the gibbs sampling scheme proposed by chodera and shirts . the relative position and orientation of the bound ligand with respect to the protein was restrained by means of one distance , two angles , and three dihedral harmonic potentials with force constant of 10 kcal mol [ rad ] . the equilibrium distance and angles for the restraints were taken from the average values of the last 5 ns of the 15 ns preliminary md simulations . the analytical part of the contribution of this set of restraints to the free energy can be evaluated as described by boresch et al . the equation used to evaluate this contribution also includes a correction for the standard - state dependence of the binding free energy . a soft - core potential was employed for the van der waals interactions transformed . in all simulations , the particle mesh ewald ( pme ) algorithm was used for electrostatic interactions with a real space cutoff of 12 , a spline order of 6 , a relative tolerance of 10 , and a fourier spacing of 1.0 . the verlet cutoff scheme was used with a van der waals interaction cutoff of 12 and a buffer tolerance of 0.005 kj mol ps . the p - lincs constraint algorithm was used only on h - bonds . the gromacs long - range dispersion correction for energy and pressure was used , and an additional numerical long - range dispersion correction ( exp - lr ) was applied as described by shirts et al . to address the failure of the isotropic assumption of the analytical correction during simulations of protein no correction for box - size effects was applied as it has been shown that , contrary to charged ligands , such effects are negligible for neutral solutes . relative free energy calculations were performed in order to estimate the effect of the sulfonamide torsional parameters on the calculated binding free energies for bromosporine . the force constant and phase of the dihedrals were interpolated between the two models using 12 uniformly distributed values . the equilibration and production phases were carried out as per the protocol described above for absolute free energy calculations . no restraints have been used in this calculations , and all ligand atoms were interacting with the environment during the transformations . for the ligand in solution , five repeats were carried out , while for the protein ligand complexes simulations a single repeat was performed . free energy estimates were obtained with the implementation of the multiple bennet acceptance ratio ( mbar ) provided with the python package pymbar ( https://github.com/choderalab/pymbar ) and using the alchemical analysis tool ( https://github.com/mobleylab/alchemical-analysis ) . the data from each lambda state were subsampled in order to include only uncorrelated data - points by calculating the autocorrelation time and statistical inefficiency of the derivative of the potential energy with respect to lambda ( dhdl ) . only the dhdl components that were changing at a particular lambda state were used as the observable for the calculation of the autocorrelation times . the equilibrated region was determined by visualizing the forward and reverse convergence plots as suggested by klimovich et al . multiple such plots were generated by discarding data at increments of 1 ns , from a minimum of 1 ns to a maximum of 12 ns , and identifying the equilibration time producing a convergence plot with the least drift in free energy in any one direction . the uncertainty of the free energy estimate was taken from the mbar asymptotic variance - derived uncertainty as the square root of the statistical variance of the total free energy . for the free energy of decoupling the ligand from solution , the mean and sample standard deviation of multiple repeats were used . for bromosporine and rvx - oh , the five initial conformations of the ligand in solution were determined by the five docked poses ; for rvx-208 , which only had two poses returned by the docking and clustering procedure , another three snapshots were extracted from the simulations in order to provide three additional random starting conformations . the final binding free energy is the difference between the decoupling of the ligand from the solution and from the solvated complex ; the final uncertainty in the binding free energies is thus the root sum square of the uncertainties of ligand and complex calculations . the binding free energies for the bromosporine models with modified torsional parameters were obtained by adding the relative free energy values to the absolute free energy ones that were predicted with the original model . the final uncertainty was derived as the root sum square of the standard errors of the absolute and relative free energies . the 95% confidence intervals ( cis ) for the performance statistics ( mean unsigned error ( mue ) , root - mean - square error ( rmse ) , pearson product - moment correlation ( rp ) , spearman rank correlation ( rs ) ) were calculated with percentile bootstrap , after building 10 bootstrap samples by resampling the experimental and calculated free energies according to their mean and standard errors , assuming normality . throughout the text we report the 95% ci in square brackets immediately after the statistics . rvx-208 is an orally available small molecule that is currently being investigated in clinical trials for the treatment of atherosclerosis , diabetes , and cardiovascular diseases . it has been found to inhibit the bromo and extraterminal ( bet ) family of bromodomains . rvx-208 shows a slight selectivity ( from about 8- to 21-fold in affinity ) for the second ( bd2 ) versus the first bromodomains ( bd1 ) of the bet family . rvx - oh is the synthetic precursor of rvx-208 and also targets the bet bromodomain family ; however , it does not show the same preference for the second bromodomains . the compound in fact rearranges in the binding pockets of the first bet bromodomains and binds with a different pose as compared to rvx-208 , where it occupies the wp / za shelf ( see figure 1b for the bromodomain structure ) . in such a way , rvx - oh achieves similar affinities for both first and second bet bromodomains , abrogating the subtle selectivity observed for rvx-208 . the crystallographically observed binding poses for rvx-208 and rvx - oh in the binding pockets of a first and second bet bromodomains are shown in figure 2 . for ease of description , and consistently with the orientation of the figures in this text , we denote the binding orientation of rvx - oh in bd2 pockets as the vertical v - pose , and the binding orientation of rvx - oh in bd1 pockets as the horizontal h - pose , despite such naming not having any physical or biological meaning . note that it is not possible for rvx-208 to bind in the h - pose , due to the presence of an additional hydroxyethyl group that would clash with the protein . these ligands thus represent an interesting case scenario to test the ability of the calculations to identify both the binding modes and slight selectivity of a ligand for multiple proteins . in this study , the 7 bet brds ( out of 8) with at least a crystal structure available in the rcsb protein data bank ( pdb ) and affinity data determined by itc were considered . comparison of the x - ray structures of rvx-208 and rvx - oh in the first ( bd1 ) and second ( bd2 ) bet bromodomains binding pockets , and the corresponding docked structures . rvx-208 binds with the same pose to both bd1 and bd2 , while rvx - oh adopts two distinct binding orientations . the two ligands have been docked into an apo structure of brd4(1 ) ( pdb i d 2oss ) , and the docking poses best representing the crystal structures are shown . bd1s are represented by white cartoons , and bd2s by gray cartoons ; rvx-208 is highlighted in light blue , and rvx - oh in light green ; the conserved asn residue and network of waters are shown as reference points . the docking pose for rvx-208 was taken from a previous study , in which absolute free energy calculations were shown to be able to identify the crystallographic pose for this ligand in brd4(1 ) . similarly , the two docking poses for rvx - oh corresponding to the v- and h - poses were taken from the same study ; in this case , too , it was shown how the calculations identified the correct pose for brd4(1 ) . here , however , we were interested in assessing whether the calculations could predict the difference in its binding orientation between bd1s and bd2s ; therefore , we rescored both the v - pose and h - pose for all 7 brds considered . the two poses for rvx - oh , and the one for rvx-208 , were modeled into the structures of the other six bet bromodomains considered . in order to allow the binding pockets to relax around the ligands , a short ( 15 ns ) md simulation of the complexes given the two potential binding poses of rvx - oh for the bet bromodomains , the calculations were run starting from both orientations for all the seven bet bromodomains considered ( no structure is yet available for brdt(2 ) ) in order to test whether the computational protocol could reliably identify the correct pose for the first ( h - pose ) versus second ( v - pose ) bet bromodomains . table 1 summarizes the results of the calculations obtained for rvx - oh , and shows how the orientation that would be expected to be the more stable based on the crystallographic evidence was always predicted to have a more negative binding free energy value , i.e. a higher affinity , than the other competing orientation . for each bromodomain / rvx - oh complex we then took the higher affinity pose as being the most stable orientation , and compared the corresponding binding free energy value to the itc data . as previously mentioned , rvx-208 can bind only in one orientation ( v - pose ) , thus this exercise was not carried out for this ligand . however , it needs to be kept in mind that it is the retrospective nature of this study that has allowed us to confidently make such an assumption . in a prospective drug discovery scenario , it is likely that limited information will be available on the compound being investigated , so that one might need to be more careful when assuming a conserved pose across different proteins . table 1 and figure 3 summarize the correspondence of the computational predictions to the itc data for the two ligands together ( full breakdown of free energy terms in table s2 ) . it is possible to see how , in this case , the predictions reproduced experimental values in absolute terms extremely well . all of the predicted binding free energies were within 2 kcal / mol of the itc values , and about two - thirds were within 1 kcal / mol . this resulted in low mue and rmse : 0.81 [ 0.74 , 0.90 ] kcal / mol and 0.95 [ 0.87 , 1.05 ] kcal / mol , respectively . the correlation with itc values was particularly good too for absolute calculations , with a pearson correlation ( rp ) of 0.75 [ 0.67 , 0.80 ] and a spearman ( rs ) of 0.78 [ 0.64 , 0.85 ] . considering rvx-208 alone , the calculations achieved rp = 0.84 [ 0.76 , 0.90 ] and rs = 0.75 [ 0.71 , 0.93 ] . on the other hand , when considering rvx - oh only , lower correlation values with larger uncertainties were returned ( rp = 0.49 [ 0.27 , 0.66 ] and rs = 0.46 [ 0.14 , 0.71 ] ) despite the calculations performing well in terms of absolute errors for both ligands . this is expected given the small dynamic range of 1.3 kcal / mol when considering rvx - oh alone . scatter plot of calculated versus experimental affinities for the compounds rvx-208 and rvx - oh . the shaded gray areas indicate where the 1 and 2 kcal / mol error boundaries lie . on the right - hand side are the distributions of the calculated binding free energies for the two ligands , binding to bd1s ( light blue and light green ) and bd2s ( dark blue and dark green ) ; gaussian curves have been fitted to the data . if the predictions were grouped into first [ brd2(1 ) , brd3(1 ) , brd4(1 ) , brdt(1 ) ] and second [ brd2(2 ) , brd3(2 ) , brd4(2 ) ] bet bromodomains , it would be possible to anticipate the slight selectivity of rvx-208 for bd2s , as one can visually gather from figure 3 . in fact , the sample mean and standard deviation of rvx-208 binding free energies for bd1s were of 6.5 0.7 kcal / mol , while for bd2s of 8.6 1.1 kcal / mol . a two - tailed t test returns a p - value of 0.03 , which would suggest that the predicted difference in affinity between the two groups is unlikely to be caused by chance . contrariwise , rvx - oh was predicted to have an average binding free energy of 8.5 1.6 kcal / mol for bd1s and of 7.9 0.6 kcal / mol for bd2s , showing a larger overlap of their distributions and consequently returning a p - value of 0.57 when applying a two - tailed t test , correctly suggesting the probable lack of any preference for either the first or second bet bromodomains . for comparison , we used a recent machine learning scoring function to test whether similar results might have been achievable with a much cheaper computational method . more specifically , we used csm - lig , a machine - learning method that relies on graph - based signatures and showed a performance statistically comparable to rf - score and superior to 18 other common scoring functions . ligand structures were the docking poses modeled in all brds considered after minimization of the whole system in explicit solvent using gromacs . the scoring function identified the correct pose for rvx - oh five out of seven times , and returned more modest correlation with experimental affinities ( rp = 0.44 and rs = 0.35 ) . furthermore , the predictions systematically underestimated the binding for these two ligands ( table s5 ) , returning large values for the mue and rmse of 5.82 and 5.86 kcal / mol , respectively . figure s3 plots the predicted versus experimental binding free energies and shows how the scoring - based predictions deviated substantially from the line of identity . bromosporine ( figure 1c ) is a broad - spectrum bromodomain inhibitor that binds across all bromodomain families with a relatively wide range of affinities ( from 25 m to 10 nm ; from 6.2 to 10.7 kcal / mol in binding free energy terms ) . as affinities for a number of brds have been experimentally determined by itc ( table 2 ) , it is an ideal system to test the ability of free energy calculations to capture the affinity profile of a ligand across many similar binding pockets . as for rvx - oh and rvx-208 , all bromodomains with at least a crystal structure available in the pdb and affinity data determined by itc were considered , for a total of 22 bromosporine / brd pairs . at the outset of this study , no structure of bromosporine in complex with a bromodomain had been deposited in the pdb . we therefore docked the ligand in the binding pocket of brd4(1 ) in order to determine its likely binding pose . five different clusters of poses were identified and free energy calculations were performed on one pose from each cluster in order to determine the most stable orientation . these were distinct poses that did not interconvert within the simulations time scale . figure 4 provides an overview of these docking poses , ranked by their predicted affinity . in july 2015 the first bromosporine structure in complex with a human bromodomain ( brpf1b , family iv ) was released on the pdb ( pdb i d 5c7n ) , so that we were able to compare the docked poses to this experimentally resolved structure . figure 4a shows the superimposition of the docking pose with highest predicted affinity in brd4(1 ) and the x - ray structure of bromosporine in complex with brpf1b . the predicted pose resembled well the crystallographic orientation , with a rmsd of 2.3 , mainly due to a rotation of the ethyl carbamate side chain . the fact that bromosporine was shown to bind to brpf1b in the same fashion as predicted for brd4(1 ) also gave us confidence that the binding mode for this ligand was conserved across bromodomain families . the second pose ( figure 4c ) was close to the experimental structure as well ( rmsd of 3.4 ) , but with the sulfonamide group directed toward the solvent rather than making contacts with the protein . the remaining three poses ( figure 4d f ) were bound in substantially different orientations as compared to the first two docking poses and the crystallographic pose , as indicated by the larger rmsds of 4.6 , 5.0 , and 6.2 . the binding affinity of bromosporine for brd4(1 ) was , however , overestimated by over 1 kcal / mol : the calculated free energy of the most stable pose was predicted to be 11.3 0.3 kcal / mol , while the itc measure is of 9.7 0.1 kcal / mol . correspondingly , the docking poses c , d , and e are unexpectedly assigned large affinities while binding very differently from the x - ray pose . ( a ) comparison of the pose with highest predicted affinity to the x - ray structure of bromosporine in complex with brpf1b ( pdb - id 5c7n ) . ( b e ) the five diverse bromosporine poses suggested by docking , with their predicted binding affinity and rmsd with respect to the binding mode found in the crystal structure . after establishing the most likely binding orientation of bromosporine in brd pockets via docking , the ligand was modeled into the x - ray structures of the other 21 bromodomains , from 7 of the 8 bromodomain families . as for the rvx ligands , a short md simulation of the complexes was run before performing free energy calculations for all the bromosporine / brd pairs . the results of the calculations are summarized in table 2 ( full breakdown of free energy terms in table s3 ) and figure 5a . a reasonable overall agreement with experiment in terms of absolute values was achieved , with mue of 1.76 [ 1.66 , 1.90 ] kcal / mol and rmse of 2.13 [ 2.03 , 2.26 ] kcal / mol . roughly a third of the results were within 1 kcal / mol of the itc values , and two - thirds within 2 kcal / mol . however , this left another third of the results being off by at least 2 kcal / mol , which corresponds to about a 30-fold error in the dissociation constant . the correlation with experiment was reasonable too , but not as strong as for rvx - oh / rvx-208 , with rp = 0.48 [ 0.41 , 0.53 ] and rs = 0.50 [ 0.41 , 0.62 ] . the low number of samples from each bromodomain family hampered a meaningful statistical analysis , and in fact a one - way analysis of variance revealed that the error distribution of each subfamily did not differ in a statistically significant manner from any other subfamily . however , there seemed to be a pattern where calculations involving bromodomains from subfamilies ii , vii , and viii , returned better results than other subfamilies . ( b ) calculated binding free energies for the bromosporine model with optimized benzensulfonamide torsions . the shaded gray areas indicate where the 1 and 2 kcal / mol error boundaries lie . overall , in this set of predictions , the binding free energies are overestimated ; in fact , the mean signed error was of 1.29 [ 1.42 , 1.17 ] kcal / mol . as we were interested in identifying possible sources for this issue , we evaluated the effect of the charge model on the results by carrying out relative calculations where resp charges were transformed into am1-bcc ones ( data not shown ) . however , this change aggravated the overestimation issue , resulting in higher mue and rmse ( 2.15 and 2.54 kcal / mol , respectively ) and a more negative mean signed error ( 2.00 kcal / mol ) , while leaving the correlations almost unvaried ( rp = 0.46 and rs = 0.48 ) . we then focused on the parameters of the soft dihedrals present in the molecule , since such terms are known to have limited transferability across different molecules and might lead to inaccurate sampling of ligand conformations . in particular , chemical groups such as sulfonamides are especially challenging when considering that also quantum effects like the interaction of the nitrogen lone pair with antibonding orbitals involving the sulfur affect the torsional energy around the n s bond . for these reasons , we decided to optimize the torsional parameters that determine the ensemble of conformations explored by the benzensulfonamide moiety in bromosporine . these dihedral terms were optimized specifically for bromosporine using the program paramfit(43 ) where the target energies were obtained with single - point energy calculations at the mp2/6 - 31 g * level of theory . the parameters derived in such a way are specific for the bromosporine model we used , and provided a better set of parameters in terms of agreement with qm energies in vacuo ( figure s2 ) . in order to estimate the effect of the new set of torsional parameters on the affinity predictions , rbfe calculations were carried out . the predicted binding free energies for the optimized model were derived by adding the g obtained from the rbfe calculations , to the gcalc values obtained with abfe calculations for the initial model . table 2 and figure 5b summarize the results obtained for the bromosporine models with modified torsional parameters for the benzensulfonamide group ( full breakdown of the free energy terms in table s4 ) . it is possible to note how the optimization of the sulfonamide moiety had a positive effect on the absolute errors of the calculations , with mue decreasing to 1.54 [ 1.46 , 1.68 ] kcal / mol and rmse to 1.88 [ 1.78 , 2.02 ] kcal / mol . the percentage of predicted binding free energy values with error relative to itc of less than 1 kcal / mol also increased slightly , while the percentage of less than 2 kcal / mol remained constant . importantly , the strong overestimation problem was alleviated , so that the errors were distributed more symmetrically around zero , with a mean signed error of 0.53 [ 0.65 , 0.41 ] kcal / mol . on the other hand , unexpectedly , the correlation between calculated and experimental affinities slightly deteriorated ( rp = 0.43 [ 0.36 , 0.49 ] and rs = 0.46 [ 0.38 , 0.58 ] ) . we further proceeded to derive optimized parameters for other soft torsions found in bromosporine : the ones found in the ethyl carbamate side chain , and again the one between the two aromatic rings . however , new parameters for these dihedrals did not result in improved agreement with experiment in terms of absolute error or correlation ( data not shown ) . it thus appeared that the sulfonamide parameters only ( among the one tested ) were contributing toward the systematic overestimation of the binding free energies for bromosporine . nonetheless , the modification did not completely solve the overestimation issue , and the overall accuracy of the predictions for this ligand was still modest as compared to what achieved with the two rvx ligands , and what previously reported for the binding of 11 other compounds to brd4(1 ) . this suggests there probably are other factors contributing toward the errors in the predictions for this test case , which we have not yet been able to identify . as it was done for rvx - oh and rvx-208 , the abfe calculations were also compared to a computationally cheaper , yet state of the art , scoring function . in this case too , the csm - lig method returned lower correlation to experiment as compared to abfe , with rp = 0.22 and rs = 0.19 ( figure s3 ) . moreover , the predictions deviated substantially from the itc measurements , returning a mue of 5.38 kcal / mol and a rmse of 5.62 kcal / mol . however , contrary to the case in rvx - oh and rvx-208 , for bromosporine this approach overestimated binding , with most binding free energies being around 14 kcal / mol ( table s6 ) . as for the rvx compounds , none of these predictions was within 2 kcal / mol of the experimentally measured binding free energy . here , we presented two test cases in which the prediction of the different affinities for single ligands binding to multiple bromodomains was attempted . excellent agreement with experimental data was achieved for the compounds rvx - oh and rvx-208 , while the broad - spectrum inhibitor bromosporine proved to be more challenging . it is important to note how the quality of the ligand parameters might vary largely between different small molecules depending on the chemical groups present . the fact that reparameterization of the soft torsions in the benzensulfonamide moiety for the bromosporine model alleviated the systematic overestimation of binding affinities corroborates the hypothesis that transferability of dihedral parameters in small molecules can be an issue . parameterization approaches where some of the ligand parameters , such as charges and torsions , are derived on an ad hoc basis for the specific molecule at hand might provide more accurate organic molecule models for the study of dynamics and binding free energies . certainly , this is not the only issue with classical force field models ; nonetheless , it is one that can be readily tackled without modification of current functional forms . small - molecule force fields are also relatively young when compared to protein force fields , with much room for improvement . as demonstrated by the recent opls3 force field , little adjustments such as off - site charges and careful parametrization that takes advantage of increasing availability of experimental data can provide better models of protein guest systems , for which highly converged calculations can be obtained , will prove valuable for the parametrization of the next generations of small - molecule force fields . for bromosporine , even with the optimization of torsional parameters , reasonable but not excellent agreement with itc data was eventually obtained , in particular in terms of correlation . this was caused by large ( > 2 kcal / mol ) overestimation or underestimation of the binding free energy in a number of cases . the identification of the causes for such disagreement with itc proved challenging , as a large number of potential reasons can be conceived . the imprecision of the calculations alone can not justify the magnitude of such errors ( see si text t1 for a more detailed analysis of the uncertainty estimates , and si text t2 for a more comprehensive discussion on the possible sources of error ) . however , severe sampling issues , such as the failure to access relevant states ( e.g. , side - chain rotamers or loop motions ) , might contribute toward the errors . identifying specific sampling deficiencies on a case - by - case basis can , however , be both challenging and impractical , in particular with the prospect of being able to apply these calculations to large numbers of protein ligand pairs . the combination of free energy calculations with enhanced sampling techniques might thus be beneficial , as it does not require the identification of specific degrees of freedom that have been under - sampled . force field inaccuracies can also have a large effect on the calculations . aside from bonded terms , inaccuracies in the nonbonded parameters can cause over / under - estimation of interaction energies and consequently protein ligand affinities . while parameters such as the lennard - jones can be improved with larger sets of experimental data , effects such as polarization , which can also affect the results of the calculations across different proteins , will likely need a more substantial alteration of the current models . interactions have been shown to be important for the binding of certain ligands to bromodomains such as crebbp , and this is an example of a protein ligand interaction that is unlikely to be captured accurately by current classical force fields . these are only some of the challenges that might , currently , negatively affect the accuracy , reliability , and generality of binding free energy calculations . tautomerisation , changes of protonation states , and effects due to the presence of the buffer , are other issues that may arise and can also affect predicted protein ligand binding free energies . despite all the challenges , the results presented here are encouraging . in terms of absolute errors , the predictions achieved a rmse below the 1 kcal / mol mark for the rvx - oh / rvx-208 test case and below 2 kcal / mol for bromosporine . the ability to quantitatively reproduce binding free energies , rather than only correlate with them , has been a difficult task for decades and for most computational approaches . now , physics - based models of chemical and biological matter and computer simulations seem to be able to provide sufficient physical accuracy and degree of sampling in order to reproduce one of the most important thermodynamic quantities in biology and pharmacology . in fact , while it is important to start assessing the accuracy and precision of the calculations on specific systems , the test sets used so far for abfe calculations are limited in their size and diversity . as the computer power available continues to increase , along with algorithmic improvements , in the future it will be possible to expand such validation studies to larger benchmark sets , similarly to what has been only recently possible for rbfe calculations . with an expanded chemical and biological space used for testing , the results will provide a better picture of the capabilities and pitfalls of the methodology as well as its most fruitful fields of applicability . we also showed how rigorous calculations based on md , in these two test cases , outperformed a state - of - the - art machine learning scoring function , providing superior agreement to experiment both in terms of correlation and absolute error . nonetheless , it is important to realize that the difference in computational cost of the two approaches is vast . while , currently , abfe calculations need the use computer clusters for hours to days , scoring functions can return a prediction within seconds on a regular desktop machine . the calculations reported here also required considerable human time for their setup . the use of a few ad hoc in - house scripts aided the process ; however , ligand parametrization , generation of input files , setup of restraints , and the data analysis all involved some degree of human intervention . the lack of automated workflows might be partly due the method still being explored , so that strong consensus around a generally applicable standard protocol is missing . nonetheless , the human effort needed to familiarize and setup the calculations does constitute an entry barrier for new users and might prevent a widespread use . in fact , the development of automated and user - friendly software has contributed to the spread of rbfe calculations in only the past few years . credit must be given , however , to developers of freely available simulation software who keep improving the capabilities of these programs for free energy calculations . we are confident that , gradually , as interest in these calculations increases thanks to further developments and testing , workflows will become more and more automated and user - friendly . the reader who is already interested in learning how to run these calculations can find some additional considerations , and references to background information , in si text t3 . in our opinion , abfe calculations can have the highest potential at the hit discovery and lead development stages , complementing established techniques such as docking as well as emerging ones like rbfe calculations . the objective is not to have perfect predictions and replace experiments , but to shorten and reduce the cost of development cycles , and to promote the testing of novel and synthetically challenging hypotheses . during hit discovery , one can foresee virtual screening protocols where a number of techniques are employed hierarchically . docking will always be faster than abfe calculations , and could be used for the initial enrichment of binders within the library screened ; this could be followed by intermediate approaches in terms of accuracy and speed , such as implicit solvent calculations like molecular mechanics poisson boltzmann surface area ( mmpbsa ) ; finally , for the most accurate rescoring and ranking , abfe calculations may be used . at the lead development stage , such calculations could be employed to assess the likelihood of a ligand with a very different scaffold to retain binding , or to assess different ligands for their ability to hit the target selectively , as discussed in this study . in this work , we have shown how absolute binding free energy calculations based on molecular dynamics can be applied in order to computationally predict the affinity profile of a ligand across multiple proteins with no prior knowledge of the structure of the complexes or affinities for similar ligands . the prediction of binding affinities and the engineering of selectivity are both still major challenges in drug design ; however , with increasing computational power at our hands and with more rigorous and general computational approaches , these problems can start being tackled . it is here shown how a thorough simulation protocol can , in the cases considered , achieve good correlation and agreement with experimental values for drug - like ligands . it is , however , evident that the accuracy of the calculations is system dependent , and further investigation is needed in order to assess the effectiveness of such predictions in different case scenarios and on different protein and ligand systems . while the accuracy of these calculations for other protein families still needs to be established ,

traditionally , students learned in the classroom through interaction with the teachers , which required his physical presence and confidence in asking questions to clear his doubts . many students may not take part in the discussion openly due to peer pressure and fear of asking a question that may appear silly and unimportant . if the absorbing capacity of the student differs from the normal , he may find it difficult to follow the lectures , which are usually done quite fast within the time frame allotted for that particular lecture and the teacher has to disseminate the information in a cramped style . the use of various aids like transparencies or powerpoint slides has not really helped in learning as they do not stimulate the processing of information delivered by the student . during contemporary medical education , portable and enduring and might enhance the recall of information leading to improved test performance . note - taking by students is generally seen as an integral and essential part of the process of learning from lectures . few studies have reported that the lecture handouts could improve understanding of the topic and students test performance . to further add on , many lecturers are now routinely publishing lecture handouts on the web for students to access . this method of dissemination of information may provide many advantages over conventional paper - based distribution as it provides links to other online materials . the technological advancement has made all the other methods redundant in the sense that students can learn at their own pace , contact the lecturer anytime , they want to post him questions and get an answer , explanation or clarification from the lecturer . at the same time , the learning process is made easier by providing the students all the information in presentation slides . handouts could be a valuable aid to guide the students to study in an organized way and is easy for them to cope with their exam stress . writing in the blank spaces in handout during a lecture allows the students to be alert and active , and they learn by writing . the present study was undertaken to find out the efficacy of the skeleton handouts against full lecture handout during ophthalmology theory lectures . this was a pre- and post - test based experimental study involving 6 semester medical students ( n = 108 ) . in our syllabus , the ophthalmology theory lectures are covered during the 6 semester of medical training while the clinical posting in ophthalmology takes place during the 7 and 8 semester . the topics of nearly equal difficulty level were identified with lecture gap of around 1-month . during the first intervention , the full lecture handouts were distributed to each member of the class 4 days prior to concerned lecture . on the day of lecture , a pretest in the form of multiple choice questions was conducted for the whole class , and the post - test with the same questions was conducted soon after the lecture . the same type of intervention was done for skeleton handouts as well after a gap of 1-month . one hundred and eight students participated during the intervention with full handout whereas one hundred and four students participated in the skeleton handout intervention . closed question regarding preference of students to either type of handouts was asked after both the interventions and the percentage of preference was noted . data were processed using microsoft excel and analyzed using spss version 12 ibm , malaysia . dependent sample t - test was used to compare the before and after test score in intervention groups , that is , skeleton handouts and full handouts . anova with repeated measures was used to compare the change score from pretest between skeletal handouts and full handouts . all the 108 students of the class participated in the intervention through full lecture handout in comparison to one hundred and four students participating in the skeleton handout intervention . table 1 reveals that the posttest score ( 4.85 0.363 in skeleton handout and 2.61 0.771 in full lecture handout ) were significantly increased in comparison to respective pretest scores ( 1.85 1.275 in skeleton handouts and 1.92 1.09 in full lecture handouts ) [ table 2 ] . paired comparison of pretest and posttest scores in skeletal handout and full handout comparison of change score from pre - test between skeletal handout and full handout ( n=104 ; those who participated in both full handout and skeleton handout interventions ) anova with repeated measures was used for the comparison of change score from pre - test between intervention groups . skeleton handout has a mean change score from pretest 3.0 with sd 1.246 , which is higher than full handout having mean change score from pretest 0.70 with sd 1.238 . there was a significant difference of mean change score between a skeletal handout and full handout ( p < 0.001 ) . in this study , the majority of the students preferring full handouts expressed that it was easy to revise at the last minute , and it could save a lot of work in the preparation and assimilation of the details about the subject taken . however , most of the students among those who preferred skeleton handouts expressed that it encouraged them to stay focused during the class and all of them concurred that they could add points in the skeleton handouts that were being elaborated in the class . they also said that they could get some more points and added on to the skeleton handouts by referring to information beyond textbooks . handouts are a useful supplement that can support further learning in noncontact hours and also give a framework on which the students can learn . they clarify expectations during the lectures and can encourage focused note - making by leaving specific areas for students to add notes in their own words or connect to other areas of learning . provision of full slide presentation has decreased the students attentiveness as they already have the notes on what the lecturer is going to teach in the class with the belief that they can always refer to it at their leisure . however , the skeleton handouts will provide an opportunity for the students to ponder over the subject and increase the understanding of the subject by the scaffold given . the students who were given skeletal handouts out - performed when compared with students who were not given . in our study , the mean post - test value was significantly higher when the students were given skeleton handouts , which were similar to the one was done by russell et al . they have concluded that learning by medical students was improved when they recorded notes in class . however , the majority of the students in our study , preferred to have full handout ; probably for their easy referral during the examination . when students are given full handouts , they often substitute this for class attendance and are less likely to attend the lecture , which sometimes is disheartening to the lecturer . perhaps the compromise that best assists students is the provision of partial or guided notes . skeleton outlines or handouts can provide a scaffold for accurate student note - taking while still requiring their attention , active engagement , and attendance . guided rather than complete notes provided prior to class time can also allow students to prepare and review material for concepts in need of further explanation with the added benefits of increasing student engagement with the content and improving their accuracy of note - taking . it has been stated in one of the literatures that the students who generated their own outlines or study questions learned more from a lecture than did students who used materials provided by the lecturer . the limitation of our study was that we could not conduct a randomized control study , but we plan to conduct a randomized control study with long - term outcome measure in the next batch of students . although the majority of the students wished to have full handouts of the ophthalmology lectures , they could perform better after going through skeleton handouts .

a 16-year - old female patient was referred to our pain clinic from the orthopedic department with persistent pain and swelling of the right forearm . the patient 's history revealed her being admitted to a local hospital with urinary tract infection and receiving an infusion of 5% dextrose with 0.9% saline through an iv cannula placed on the right dorsum of the hand five months previous . later , extravasation of fluid in the surrounding tissue occurred leading to pain and swelling that did not subside but kept increasing . she was treated with analgesics and antibiotics followed by local incision and drainage . despite symptomatic treatment and surgical intervention , the symptoms further worsened leaving surgical scars ( fig . the primary care physician managed conservatively for almost 5 months before she presented to our hospital . clinical examination revealed a tender , shinny , swollen limb with allodynia and stiff joints with no evidence of nerve injury suggestive of crps type-1 . doppler study ruled out any possibility of vascular disease . a diagnostic stellate ganglion block ( sgb ) improved the numerical pain rating score ( 0 - 10 ) from 7 to 2 with enhancement of limb mobility . a multimodal approach of treatment including oral medications ( gabapentin , amitryptyline , tramadol ) , a series of sgb with bupivacaine , and limb physiotherapy were initiated . at 6 months follow - up , pain and swelling subsided drastically with marked functional recovery ( fig . trauma is the most common precipitating event of crps type-1 , the majority following minor insult to an extremity . the inciting event in our case was inadvertent extravasations of a dextrose solution into tissues surrounding the iv cannula . these extravasations to the interstitial space may have led to an inflammatory response resulting in edema , redness and pain . rapid swelling and compression of the surrounding tissue may also cause acute limb compartment syndrome , reducing tissue perfusion . swelling , inflammation , tenderness and skin changes have been reported to confound diagnosis of crps following snake bite also . these mechanisms ' roles can not be ignored in our case as the current understanding of crps pathophysiology involves inflammation and hypoxia along with autonomic and somatic nervous system involvement . however , it is difficult or even impossible to demonstrate a causal relationship between these clinical conditions and crps development . due to its wide range of precipitating factors and pathophysiologic complexity , crps diagnosis is based primarily on established clinical criteria including allodynia , temperature and skin color changes , edema , motor dysfunction and trophic changes . although special investigations , such as autonomic function tests and bone scans , support the diagnosis of crps , these diagnostic modalities add little to the overall accuracy of diagnosing the disease when compared with clinical criteria . hence , distinguishing crps type-1 clinically from other pain conditions , such as undiagnosed deep vein thrombosis , cellulitis and vasculitis , can be challenging even to specialist pain physicians . the clinical course of crps progresses through three stages : acute inflammatory followed by dystrophy , ultimately leading to structural atrophy . all classical signs and symptoms of inflammation are present , similar to that of cellulitis . moreover , in the early course of the disease , all features may not be present making diagnosis difficult . adolescents are less likely to have overt autonomic signs associated with crps , such as hyperhidrosis and trophic changes , compared to adults . this may also lead to improper diagnosis . with a multimodal treatment approach to crps , wide ranges of non - pharmacological therapeutic modalities , including intravenous regional blockade , sympathetic blockade and spinal cord stimulation , use of sgb combined with oral medications and limb physiotherapy not only served as therapy , but also contributed to confirming the diagnosis . it is interesting to note that although no special equipment or testing is required for diagnosis of crps type-1 , an average of 30 months pass and 4.8 different physicians are visited before patients with crps are referred to a pain centre even in well - established centres . these data show lack of disease awareness among general practitioners who often encounter such patients in the early stage , as in our case . this case highlights that crps often remains under - diagnosed in the hands of non - specialist physicians , particularly if the patient presents with various inflammatory features such as cellulitis , arthritis , etc . primary care physicians are often the first group of care providers to whom patients go for consultation . disease awareness among practitioners , its early recognition , appropriate therapy at the beginning and referral to a pain centre shall prevent unnecessary suffering from crps and also may prevent the incidence of its development in the community .

true aneurysms , or diverticulae , of the mitral valve are a rare cause of mitral regurgitation . it occurs most commonly in association with aortic valve infective endocarditis,1 - 5 ) but they may also present with mitral valve infective endocarditis,5 - 7 ) chronic mitral regurgitation8 ) or as a complication of mitral valve prolapse.9 ) the echocardiographic features of mitral valve aneurysms ( mva ) are non - specific,3 - 9 ) and can be misinterpreted as a large vegetation , cystic left atrial myxoma , or degeneration in association with a prolapsing leaflet . delay in making the correct diagnosis increases the risk of infection , embolization , or rupture of the aneurysm ; these complications can be prevented by surgery to repair or replace the valve.3 ) we report a patient in whom a mva of posterior leaflet as initially misdiagnosed as cystic mass , which was confirmed at operation . there was no family history of aortic , collagen , vascular or congenital heart disease . vital signs were blood pressure of 125/59 mmhg , pulse rate of 98 beat / min , respiration rate of 24 breaths / min , and body temperature of 36.5. on the physical examination , there were no peripheral skin lesions and cardiac auscultation revealed a grade 2/6 systolic heart murmur and electrocardiography demonstrated non - specific depression of st segment and t wave changes . the blood chemistries , including coagulation studies , erythrocyte sedimentation rate , c - reactive protein , the complete blood count , and lipid profiles were within normal limits . transthoracic echocardiography showed left ventricular hypertrophy and enlarged left atrium size ( 44.3 mm ) with normal left ventricular ejection fraction ( 68% ) . moderate aortic regurgitation with senile sclerotic aortic valve change , and moderate tricuspid regurgitation with elevated right ventricle systolic pressure ( 49 mmhg ) was shown . interestingly , a large fluttering cystic mass ( 1.3 1.6 cm ) in the left atrium attached to the posterior mitral valve leaflet which bulged into the left atrium and ventricle throughout the cardiac cycle resulted in poor coaptation and opening of mitral valve ( fig . the color doppler flow showed that the cystic mass was filled with flow during systole and also the color flow in the cystic mass was communicated with the left atrium ( fig . continuous wave doppler revealed that the mass caused hemodynamic disturbance and there was moderate increase of mean pressure gradient ( fig . 2c ) . because there was no clinical evidence of infective endocarditis with negative blood culture and no history of previous infective endocarditis or rheumatic fever , the cystic mass was considered as cystic left atrial myxoma , less likely mva . she was taken to the operating room for left atrial cystic mass resection . at operation , histopathologic examination of the resected segments showed myxomatous degenerative change and calcification of mitral valve without evidence of an acute inflammatory process . she made an uneventful postoperative recovery and 2 months later was well with no evidence of mitral regurgitation . mva , which is defined as a localized bulge of the mitral leaflet toward the left atrium with systolic expansion and diastolic collapse,9 ) is uncommon and reported cases are rare and often the consequence of infective endocarditis.1 - 7 ) early and preoperative recognition of its presence is important because it may rupture and produce catastrophic mitral regurgitation in an already seriously ill patient or it may be overlooked at the time of aortic valve replacement . primary or secondary endocarditis from aortic infection , rheumatic disease , and other disorders causing connective tissue degeneration5 ) are the possible cause from which the strength of the mitral leaflet is weakened and the leaflet protrudes into the left atrium under the left ventricular pressure . there are also occasional reports of the mva forming in patients without endocarditis , such as those with connective tissue diseases , including mitral valve prolapse , osteogenesis imperfecta , marfan syndrome , and pseudoxanthoma elasticum.5)10)11 ) in the absence of endocarditis , a regurgitation aortic jet has been speculated to be the most possible cause of mva,12 ) but on the posterior mitral leaflet , any mechanical stress due to aortic regurgitation could be profoundly less compared to that acting on the anterior one . our case seems to be a very rare incident since the aneurysm was detected on the posterior mitral valve leaflet although the aortic regurgitation was not severe whereas endocarditis was never documented . diagnosis of mva by echocardiography has been reported before.3)4 ) echocardiography shows mva as a localized saccular bulge of the leaflet toward the left atrium and communication between the aneurysm and the left ventricle during the cardiac cycle.3)4 ) mitral valve repair or replacement is indicated when aneurysm is ruptured or when the unruptured aneurysm is large or accompanied by significant regurgitation . mva may be confused with cystic degeneration of the valve , a cystic atrial myxoma , or a large vegetation.13 ) our patient shows that transthoracic echocardiography may not be diagnostic in this condition and can be misleading especially when the posterior leaflet is affected.6)8)9 ) until now , we can not find any report of mva of posterior leaflet without evidence of infective endocarditis . our case suggests that mva could happen on posterior leaflet of the mitral valve without apparent involvement of the aortic valve endocarditis . in conclusion , mva should be considered in the differential diagnosis of mass lesions seen on the atrial side of the mitral valve at echocardiography .

the fermentation of fatty low - value renewable carbon sources aiming at the production of various added - value metabolites such as organic acids , single - cell oil , and lipases presents a noticeable interest in the sector of industrial microbiology and biotechnology [ 13 ] . the yeast yarrowia lipolytica degrades very efficiently hydrophobic substrates such as n - alkanes , fatty acids , fats , and oils , which they have specific metabolic pathways , for production of single - cell protein , single - cell oil , organic acids , and lipases [ 47 ] . lipases are used in the production of detergents , cosmetics , pharmaceuticals , flavour enhancers and foods [ 8 , 9 ] . y. lipolytica is one of the most extensively studied lipolytic yeasts , and it secretes lipase . the fact that lipases remain active in various organic solvents has allowed their use in the catalysis of inter- and transesterification reactions of oils and in enantioselective transesterification . the y. lipolytica is able to produce several lipases ( extracellular , membrane - bound , and intracellular activities ) , and its lipase production depends on media composition and environmental conditions [ 1114 ] . cloned and identified two lipase genes ( lip1 and lip3 ) which the deduced amino acid sequences are similar to lipases from candida cylindracea and geotrichum candidum . isolated and characterized the lip2 gene , which encodes the extracellular lipase yllip2 ( 38.5 kda ) . they showed that this gene is responsible for all of the extracellular lipase activity of y. lipolytica . reported that extracellular lipase production was improved by chemical mutation and the properties of mutant lipase were the same of those of the wild type : molecular weight 38 kda , optimum ph 7 and optimum temperature 37c [ 1517 ] . screened a huge number of y. lipolytica strains and reported lipase activities ranging from 1.845.5 u / ml . results for other wild candida species showed a lipase production of the same magnitude with the present investigation ( e.g. , concentrations 215 u / ml for various carbon sources used ) [ 2 , 7 ] . citric acid ( ca ) is the most important organic acid produced in tonnage using fermentation . ca has many industrial applications , for instance , as flavour , acidifying and preservative additive in the food and pharmaceutical industry , as stabiliser for vegetable oils and fats or as complex forming and bleaching component in many washing detergents [ 3 , 18 ] . the y. lipolytica is unique in its ability to produce and excrete into the medium a broad range of organic acids the krebs cycle intermediates , ca , isocitric acid ( ica ) and 2-ketoglutaric acid as well as pyruvic acid . ca production in all developed countries follows a conventional procedure which involves the use of aspergillus niger ( as producer ) and molasses ( as substrate ) . using y. lipolytica would have several advantages compared to the aspergillus , including a wider substrate range , a smaller sensitivity to low dissolved oxygen concentrations and heavy metals , and higher product yield [ 1 , 3 , 18 , 19 ] . furthermore , these strains produce ca under intracellular nitrogen limitation [ 20 , 21 ] . the maximum and minimum ca production values by y. lipolytica strains were reported 217 g / l on petrolatum and 1.4 g / l on glycerol , respectively [ 1 , 22 ] . the aim of the study was to investigate growth of y. lipolytica on media composed of various plant oils utilized as the sole carbon source and effect of the nature of the ( fatty ) carbon source and the nitrogen source used upon the biochemical behavior and metabolites production ( e.g. , organic acids and lipases ) in flask - submerged fermentations . y. lipolytica dsm 3286 strain was obtained from the culture collection of the dsm , germany . the strain was grown at 29c and 200 rpm in 50 ml ypd or production media in 250 ml flasks . the composition of the basal medium for lipase production with some modifications was : olive oil 10 g / l , yeast extract 2 g / l , kh2po4 0.5 g / l , k2hpo4 0.5 g / l , mgso4 7h2o 0.5 initial ph of the media was adjusted to 6 . the soy bean , canola , castor , sesame , wheat bud , sweet almond , bitter almond , walnut and coconut oils ( table 1 ) were added to the basal lipase production medium instead of olive oil at the same volume , when the effects of carbon sources were being studied . for investigation of nitrogen sources effects , similar concentrations of mineral ( nh4cl , nh4no3 , ( nh4)2so4 and nh4h2po4 ) and organic ( casein , urea , peptone , soy bean flour , beef extract and malt extract ) nitrogen compounds were replaced instead of yeast extract . cells were harvested by centrifugation at 4600 g for 6 minutes and washed successively with hexane and centrifuged . biomass was determined from the dry weight at 80c for 24 hours [ 2 , 24 ] . unconsumed substrate fat was measured according to method used by papanikolaou et al . . briefly , 100 l of cultivation medium supernatant was added to a solution of 0.504 mm p - nitrophenyl laurate ( pnpl ) in 50 mm phosphate buffer , ph 7.0 . the production of p - nitrophenol was monitored by spectrophotometer ( uvi light xs , france ) at 410 nm during the linear period of product accumulation . one unit ( u ) of lipase activity is defined as the amount of enzyme that produces 1 mol of product per minute . to confirm that the enzyme detected by the spectrophotometric method was lipase and not merely esterase , in some cases an emulsion created by olive oil at 40% ( v / v ) and polyvinyl alcohol at 2% ( w / v ) was used as substrate for the lipase assay . the solution was emulsified with the aid of a homogeniser for 6 minutes at maximum speed , in order to obtain a satisfactory dispersion of oil particles into the aqueous phase . then the enzyme solution ( 1 ml ) , pure or diluted , depending on the quantity of lipase , was added to 5 ml of substrate emulsion and 4 ml of 100 mm phosphate buffer , ph 8.0 . samples were incubated for 10 minutes on a shaker ( 150 rpm ) at 37c . the reaction was stopped by adding 15 ml ethanol or 4 ml of an acetone - ethanol ( volume ratio = 50 : 50 ) mixture . enzyme activity was determined by titration of the fatty acid released with 50 mm naoh . one activity unit of lipase was defined as the amount of enzyme , which released 1 mol of fatty acid per minute under assay conditions [ 2 , 7 , 15 , 23 ] . the ca and ica production was determined by hplc ( an inertsil ods-3 reversed - phase column at 210 nm ; 20 mm phosphoric acid was used as a mobile phase with the flow rate of 1.0 ml / min ) . y. lipolytica dsm 3286 was cultivated in different media containing various plant oils as the sole carbon source . various mineral and organic nitrogen sources were also tested for their capacity to support the production of metabolites ( lipase , ca and ica ) and biomass by this strain . effect of plant oils and nitrogen sources upon the biochemical behavior and metabolites production of y. lipolytica dsm 3286 was investigated by measuring biomass , lipase activity , ca and ica concentration at various time points within a period of 7 days ( figure 1 ) . for all media , the maximum lipase activity was occurred 48 hours after inoculation and the high level production of organic acids was determined after 72 hours ( figure 1 ) . the highest lipase activity ( 34.6 u / ml ) and ca concentration ( 3.6 g / l ) was detected in wheat bud oil medium ( figure 2 ) . in contrast , marked increase in lipase and ca productivity was observed upon addition of organic nitrogen sources except for malt extract ( figure 3 ) . urea proved to be a suitable nitrogen source for ca production as in olive medium supplemented with urea , ca concentration reached up to 3.9 lipase production by some plant and vegetables oils ( sunflower oil , corn oil , olive oil and so on ) and animal fats has been studied [ 2 , 6 , 26 , 27 ] . ca can be produced from alkanes , vegetables oils , fats , glycerol , ethanol , molasses and starch hydrolysates [ 1 , 4 , 22 , 25 , 2832 ] . nevertheless , only a few studies have examined both lipase and ca productions simultaneously . in this study we investigated y. lipolytica lipase and ca production at the same time with various plant oils and nitrogen sources . plant oils were applied as the sole carbon source , because y. lipolytica produces bioemulsifier . the y. lipolytica degrade and oxidise very efficiently hydrophobic substrates , such as fats , oils , alkanes and fatty acids [ 2 , 4 ] . glucose or other carbon sources which are used as cometabolism for utilization plant oil in other studies [ 8 , 25 ] were not used in this study . the highest lipase activity was detected on medium with olive oil ( figure 2 ) . these results could be justified by the fact that y. lipolytica strains display a lipase activity which acts preferentially on oleyl residues at positions 1 and 3 of the glyceride . the extracellular lipase requires oleic acid as stabilizer / activator , whereas the cell bound lipases dose not , and is different in several other properties from the extracellular enzyme . it has been mentioned that olive oil has 5583% oleic acids [ 34 , 35 ] . considering chemical compound of other plant oils canola oil , for its oleic acid content ( 55% ) , after olive oil is good candidate for lipase production . probably the reason is that an expression system containing the lip2 gene expressed under the control of the oleic acid inducible pox2 promoter . another reason is selective uptake of fatty acids by y. lipolytica , because two hypotheses have been formulated to explain of the transport of hydrophobic substrate such as fatty acids into microorganisms . the hydrophobic substrate can be solubilised ( or pseudosolubilised ) in the presence of surface active compounds ( surfactant - mediated transport ) or they can adhere directly to the cell wall . in y. lipolytica , evidence for both mechanisms has been described . their presence depends on the growth phase and seems to be regulated in an oleate - responsive manner , that is , inducible by alkane and oleic acid and repressed by glucose . concerning fatty - acid uptake , investigations carried out by kohlwein and paltauf in y. ( saccharomycopsis ) lipolytica concluded that below a threshold of 10 m , an energy - free transporter was required , whereas above this concentration fatty acids like lauric or oleic acid diffused freely . moreover , these authors proposed that at least two different chain - length - selective transporters are present : one carrier system is specific for c12 and c14 fatty acids , and the second for c16 and c18 such as oleic acid [ 37 , 38 ] . the production of lipase was low on almond oils media , nevertheless their high level of oleic acid content ( 6075% ) is as well as olive oil ( not showed data ) . the probable reason is that almond oils have antimicrobial and toxic compounds such as benzaldehyde and hydrocyanic ( prussic ) acid . g / l ) , lipase ( yeast extract ; 34.7 u / ml ) and ca ( urea ; 3.9 g / l ) production as compared with mineral nitrogen source ( figure 3 ) . organic nitrogen source provides nutritional needs ( amino acids , vitamins , etc . ) for growth and culture supplements for extracellular lipase production . nitrogen source is limited in media with organic nitrogen source . on the basis of other reports [ 2 , 7 , 8 , 23 ] , the level production of lipase ( 34.6 0.1 u / ml ) by y. lipolytica dsm 3286 on plant oils in flask - submerged fermentation , especially olive oil , is interesting . therefore olive oil , for others analyzed parameters including yx / s ( 0.830 g / g),yl / s ( 3.460 u / g ) , pl ( 0.720 u / h ) and ql ( 0.086 u / g.h ) , is a suitable carbon source for lipase production ( table 1 ) . although in comparison with glucose and other carbon sources , ca production was low on oils and c / n ratio may be not suitable for ca production . in this study lipase production was prior to ca production and then ca as by - product detected on plant oils media . altogether the y. lipolytica dsm 3286 strain is essentially low ca producing yeast , even on glucose and optimized media ( not showed data ) . g / l ) had best results between other organic nitrogen sources and therefore urea is suitable nitrogen source for ca production . on the basis of other reports [ 1 , 22 ] , amount of ca ( 3.6 0.3 g / l ) as by - product in lipase production process by y. lipolytica dsm 3286 on olive oil , with ycit / s ( 0.393 g / g ) , pcit ( 0.054 g / h ) and qcit ( 0.008 g / g.h ) , is acceptable ( table 2 ) . it was interesting that maximum lipase production time ( after 48 hours ) was different from maximum ca production time ( after 72 hours ) on plant oils as sole carbon source . the maximum of the lipase activity was observed for cells of stationary growth phase ( after 48 hours ) , and the transition of yeast cells from the exponential growth phase to the growth retardation caused by the exhaustion of nitrogen in the medium was accompanied by a decrease in the lipase activity ; which is apparently due to a decrease in the rate of synthesis of lipase under conditions of nitrogen limitation . the nitrogen supply was exhausted , which coincided with the maximum biomass accumulation and the beginning of ca and ica production . thus , ca and ica were produced principally by y. lipolytica dsm 3286 on olive oil during the stationary growth phase ; the total ca and ica concentration reached maximum of 8.08 it is suggested that lower yx / s values ( around 0.50.7 g / g ) obtained from high - lipase producing wild y. lipolytica strains are very satisfactory for single - cell protein fermentation from fatty substrates [ 2 , 7 ] . therefore plant oils with high content of oleic acid such as olive oil supplemented with ( nh4)2so4 ( yx / s = 0.710 g / g ) and organic nitrogen , except malt extract ( yx / s = 0.508 g / g ) and urea ( yx / s = 0.666 g / g ) , and also sweet almond oil ( yx / s = 0.8 g / g ) and sesame oil ( yx / s ~ 0.7 g / g ) are very satisfactory for biomass and single - cell protein production ( tables 1 and 2 ) . in previous studies , investigators usually used only one or limited number of substrate and , also they tested either lipase or ca production [ 2 , 18 , 23 ] . one advantage of presents study is testing more than twenty carbon and nitrogen sources and measuring lipase and organic acids production simultaneously . one disadvantage of ca production is same amounts of ca and ica production on plant oils media ( tables 1 and 2 ) . in conclusion , our results show that lipase and ca could be produced by y. lipolytica using renewable low - cost substrates such as plant oils . the maximum organic acids were produced after high level of lipase production that is an interesting relationship between lipase and organic acids ( ca and ica ) production . our study suggests that fair amounts of ca could be produced by this yeast as by - product . furthermore , by mutagenesis and metabolic engineering methods , levels of ca and even lipase production could be increased while ica production , which is an undesirable product , could be decreased . manufacturing of these two important biotechnological products ( lipase and ca ) simultaneously during one simple procedure could be economically very valuable .

papillary cystadenoma of the salivary glands is an uncommon benign neoplasm , which usually occurs in the minor salivary glands , and very rarely in the major salivary glands . extensive oncocytic change may be seen , and these lesions are classified as oncocytic variants . we report three cases of oncocytic papillary cystadenoma ( opc ) arising in the major salivary glands . case 1 was a 38-year - old man who had had a mass on the right neck for 1-month . fine needle aspiration ( fna ) was performed , and the cytologic smear showed irregular sheets and papillary clusters of epithelial cells [ figure 1a ] , with rich granular cytoplasm , bland - appearing nuclei , and conspicuous nucleoli [ figure 1b ] . a warthin tumor was suspected based on these findings . computed tomography ( ct ) revealed a cystic mass with a slight enhancement in the right parotid gland , and the patient underwent right superficial parotidectomy . gross examination showed a well - defined 2.5-cm unilocular cystic mass with multiple papillary projections . microscopically , the epithelial lining comprised cuboidal to low - columnar cells [ figure 1c ] , with abundant granular , eosinophilic cytoplasm , and vesicular nuclei with prominent nucleoli [ figure 1d ] . ( a ) cytologic smear revealing many oncocytic cells in papillary c d clusters ( pap , 100 ) , ( b ) with abundant , granular cytoplasm and prominent nucleoli ( pap , 400 ) , ( c ) histological analysis revealed a multilocular cystic tumor filled with papillary projections ( h and e , 40 ) , ( d ) the papillae were lined by oncocytic cells with eosinophilic , granular cytoplasm ) , ( h and e , 200 ) case 2 was an 84-year - old man who had had a slow - growing mass on the left neck for 1-year . palpation revealed a firm , tender mass in the left parotid gland area , 4 cm in size . fna was performed , and the cytologic smear revealed a few scattered foamy macrophages and some inspissated proteinaceous material without epithelial cells , suggesting a cystic neoplasm [ figure 2a and b ] . a ct scan revealed a 3.1 cm 2.7 cm septated cystic mass in the superficial lobe of the left parotid gland . the patient underwent left superficial parotidectomy , revealing a multilocular cystic tumor with papillary projections . microscopically , the tumor appeared as a well - defined cystic mass , with multiple papillary fronds lined by tall columnar cells [ figure 2c ] . the cells had rich granular , eosinophilic cytoplasm , and bland - appearing nuclei with conspicuous nucleoli [ figure 2d ] . ( a and b ) cytology revealed inspissated proteinaceous material without epithelial cells ( a : pap , 100 ; b : pap , 400 ) , and ( c ) histology revealed a cystic mass with papillary projections in the lumen ( h and e stain , 40 ) ; ( d ) the tumor cells were columnar with rich eosinophilic granular cytoplasm ( h and e stain , 200 ) case 3 was a 57-year - old man who presented with a swelling on the right upper neck , which had slowly increased in size over 1-year . on palpation , a slightly tender 2-cm mass was found below the right submandibular angle . fna was performed , and the cytologic smear showed irregular fragments of epithelial cells [ figure 3a ] . the cells had moderate amounts of cytoplasm and bland - looking nuclei with inconspicuous nucleoli [ figure 3b ] . cytologic examination suggested a benign epithelial tumor , and a ct scan revealed a 3-cm cystic mass containing an inner solid portion . a 2.4-cm well - circumscribed cystic mass filled with papillary projections was found in the resected submandibular gland . microscopy revealed a unilocular cyst with multiple papillary fronds lined by columnar epithelial cells , which had abundant eosinophilic cytoplasm and prominent nucleoli [ figure 3c and d ] . ( a ) cytologic smear revealed irregular fragments of epithelial cells ( pap , 100 ) , ( b ) the cells had moderate amount cytoplasm and inconspicuous nucleoli ( pap , 400 ) , ( c ) histology revealed a cystic mass filled with papillary fronds ( h and e , 40 ) , ( d ) which were lined by columnar , oncocytic cells with prominent nucleoli ( h and e , 200 ) case 1 was a 38-year - old man who had had a mass on the right neck for 1-month . fine needle aspiration ( fna ) was performed , and the cytologic smear showed irregular sheets and papillary clusters of epithelial cells [ figure 1a ] , with rich granular cytoplasm , bland - appearing nuclei , and conspicuous nucleoli [ figure 1b ] . a warthin tumor was suspected based on these findings . computed tomography ( ct ) revealed a cystic mass with a slight enhancement in the right parotid gland , and the patient underwent right superficial parotidectomy . gross examination showed a well - defined 2.5-cm unilocular cystic mass with multiple papillary projections . microscopically , the epithelial lining comprised cuboidal to low - columnar cells [ figure 1c ] , with abundant granular , eosinophilic cytoplasm , and vesicular nuclei with prominent nucleoli [ figure 1d ] . ( a ) cytologic smear revealing many oncocytic cells in papillary c d clusters ( pap , 100 ) , ( b ) with abundant , granular cytoplasm and prominent nucleoli ( pap , 400 ) , ( c ) histological analysis revealed a multilocular cystic tumor filled with papillary projections ( h and e , 40 ) , ( d ) the papillae were lined by oncocytic cells with eosinophilic , granular cytoplasm ) , ( h and e , 200 ) case 2 was an 84-year - old man who had had a slow - growing mass on the left neck for 1-year . palpation revealed a firm , tender mass in the left parotid gland area , 4 cm in size . fna was performed , and the cytologic smear revealed a few scattered foamy macrophages and some inspissated proteinaceous material without epithelial cells , suggesting a cystic neoplasm [ figure 2a and b ] . a ct scan revealed a 3.1 cm 2.7 cm septated cystic mass in the superficial lobe of the left parotid gland . the patient underwent left superficial parotidectomy , revealing a multilocular cystic tumor with papillary projections . microscopically , the tumor appeared as a well - defined cystic mass , with multiple papillary fronds lined by tall columnar cells [ figure 2c ] . the cells had rich granular , eosinophilic cytoplasm , and bland - appearing nuclei with conspicuous nucleoli [ figure 2d ] . ( a and b ) cytology revealed inspissated proteinaceous material without epithelial cells ( a : pap , 100 ; b : pap , 400 ) , and ( c ) histology revealed a cystic mass with papillary projections in the lumen ( h and e stain , 40 ) ; ( d ) the tumor cells were columnar with rich eosinophilic granular cytoplasm ( h and e stain , 200 ) case 3 was a 57-year - old man who presented with a swelling on the right upper neck , which had slowly increased in size over 1-year . on palpation , a slightly tender 2-cm mass was found below the right submandibular angle . fna was performed , and the cytologic smear showed irregular fragments of epithelial cells [ figure 3a ] . the cells had moderate amounts of cytoplasm and bland - looking nuclei with inconspicuous nucleoli [ figure 3b ] . cytologic examination suggested a benign epithelial tumor , and a ct scan revealed a 3-cm cystic mass containing an inner solid portion . a 2.4-cm well - circumscribed cystic mass filled with papillary projections was found in the resected submandibular gland . microscopy revealed a unilocular cyst with multiple papillary fronds lined by columnar epithelial cells , which had abundant eosinophilic cytoplasm and prominent nucleoli [ figure 3c and d ] . ( a ) cytologic smear revealed irregular fragments of epithelial cells ( pap , 100 ) , ( b ) the cells had moderate amount cytoplasm and inconspicuous nucleoli ( pap , 400 ) , ( c ) histology revealed a cystic mass filled with papillary fronds ( h and e , 40 ) , ( d ) which were lined by columnar , oncocytic cells with prominent nucleoli ( h and e , 200 ) cystadenoma of the salivary glands is a rare benign tumor , usually presenting as a slow - growing mass . the tumor usually arises in the minor salivary glands ( 0.9 - 2% of all minor salivary gland neoplasms ) but is extremely rare in the major salivary gland . a review reported only three cases of the tumor arising in major salivary glands , and tsurumi et al . found that all four reported cases of salivary gland cystadenoma , between 1985 and 2003 , had arisen in the minor salivary glands . the latter has well - defined unilocular or multilocular cysts , with intraluminal papillary projections . the lining epithelial cells are cuboidal or columnar , and usually two layers in thickness . oncocytic papillary cystadenoma is often confused with warthin tumor , because of its histologic similarity ; papillary structures , oncocytic cells with rich eosinophilic cytoplasm , and prominent nucleoli . opc may also be misdiagnosed as intraductal papilloma as the latter often has cystically dilated ducts with papillary epithelial projections . however , the papillary cores of intraductal papilloma are more complex and prominent than opc . cystic change may be present in this tumor , but the papillary pattern , typical of opc , is not a feature of oncocytoma . zhang et al . reported diverse cytologic features of two cases of the tumor , one of which showed a cellular smear of oncocytic cells with prominent nucleoli and many histiocytes , and the other showed abundant necrotic debris , and large sheets of polygonal cells with vague papillary features and inconspicuous nucleoli . gupta et al . described somewhat different cytologic findings from two fnas taken from the same opc , with either atypical squamous cells in a necrotic background , consistent with squamous cell carcinoma , or many vacuolated and signet ring cells in a mucoid background . described a case of opc in which the aspirate was insufficient for diagnosis because of paucicellular smear . the first case showed distinct papillary clusters of oncocytic cells and the third case had irregular sheets of epithelial cells without identifiable oncocytic change . these contrast with the second case , in which only proteinous material without epithelial cells was found . it is assumed that the acellular or paucicellular smear of papillary cystadenoma is a result of the dilution of cystic fluid . in summary , opc is extremely rare in major salivary glands and tends to show varied cytologic features . although cytologic diagnosis is challenging , the tumor should be included in the differential diagnosis when oncocytic cells or papillary features are present in cystic tumors of salivary glands .

supplementary material is available in the online version of this article at ( doi : 10.1007/s11671 - 007 - 9043 - 2 ) and is accessible for authorized users .

despite its terrifying impact on health , tobacco use is widespread throughout the world . if no effective interventions are introduced and implemented against it , up to one billion people will die from tobacco use in the 21st century . youth smoking is a concern for industrial , low - income , and middle - income countries , and there is no significant difference between the genders regarding the prevalence of smoking . in 2000 , decree no . 412 ( " on measures for tobacco control and prevention in georgia " ) , issued by the president of georgia , came into force , and the world health organization ( who ) framework convention on tobacco control was signed in 2004 ; however , the smoking rate in georgia remains high and tends to increase , especially among youth . there are approximately 5,000 undergraduate students currently enrolled in tbilisi state medical university ( located in the city of tbilisi , georgia ) and approximately 18,000 currently enrolled in tbilisi state university ( also in tbilisi , georgia ) . the objectives of the present study were to determine the prevalence of smoking in medical and non - medical students at the abovementioned universities ; to determine whether medical education has an impact on smoking ; and to identify the most important risk factors for smoking among undergraduate students . the purpose of the survey was explained to the students , who were subsequently asked to complete anonymous questionnaires . we attempted to survey all available students without using any randomization schemes . because the present study was a cross - sectional study conducted during or immediately after an open , general lecture , it was difficult to predict who would be attending the lecture . therefore , for practical purposes , 200 students ( 100 males and 100 females ) at each university were interviewed during the study period . smoking status was established in accordance with the who criteria . respondents who reported smoking at least 1 cigarette per day ( either every day or occasionally ) were defined as current smokers . those who had been experimenting with smoking at the time of the survey ( having had fewer than 100 cigarettes in their lifetime ) were also defined as smokers . those who had never smoked or who had smoked fewer than 100 cigarettes in their lifetime but who had not smoked within the past 30 days ( including the day of the survey ) were defined as nonsmokers . smokers were asked about the average number of cigarettes smoked daily , age at smoking initiation , and attempts to quit , if any . data collection took place at the two universities during the study semester , between february and march of 2011 . the smoking questionnaire employed in the who multinational monitoring of trends and determinants in cardiovascular disease project was modified to reflect local habits and features . the final version of the structured questionnaire included multiple - choice questions and several open questions addressing smoking status , nicotine dependence , and factors influencing smoking behavior , as well as knowledge and perceptions of smoking . we used the statistical package for the social sciences , version 15.0 for windows ( spss inc . , chicago , il , usa ) , in order to calculate the frequencies of each variable . pearson 's chi - square test was used for comparing proportions , and the student 's t - test was used in order to examine the differences among the means . logistic regression analyses were used in order to determine whether smoking was correlated with demographic and health risk behavior characteristics . the male - to - female ratio in the study population was 0.9:1.1 ( 190 males vs. 210 females ) . the mean age was 20.24 years ( range , 17 - 27 years ) among smokers and 20.26 years ( range , 17 - 25 years ) among nonsmokers . there were no significant differences in the distribution of age , gender , and year of education among the students . on the basis of daily cigarette consumption , smokers were defined as follows : very heavy smokers ( > 20 cigarettes / day ) ; moderate - to - heavy smokers ( 11 - 20 cigarettes / day ) ; or light smokers ( 1 - 10 cigarettes / day ) . of the 195 students who answered that question , 45.8% were light smokers , 40.2% were moderate to heavy smokers , and 14.0% were very heavy smokers ( table 1 ) . smoking was found to have a strong relationship with gender , males accounting for 65% of all smokers and females accounting for 35% . table 1prevalence and frequency of smoking among the students interviewed ( n = 400 ) . of the 195 smokers , 122 ( 62.6% ) stated that they had never attempted to quit . among those who stated that they had attempted to quit smoking , the most common reasons for starting to smoke again were smoking addiction , alcohol consumption , and other external factors ( students being unable to specify such factors ) . of the 195 smokers , 111 ( 56.9% ) stated that they were , whereas 43.1% stated that they were not . we found no statistically significant differences between medical and non - medical students in terms of the prevalence of smoking ( table 2 ) . in addition , the chi - square test showed no statistically significant relationship between the type of education ( medical or non - medical ) and the smoking status . 59.5% expressed willingness to quit smoking , as did 54.2% of the non - medical students ( table 3 ) . the main reasons for being willing to quit were quite similar between medical and non - medical students , with no statistically significant differences between the two groups . in fact , health reasons were slightly more common among non - medical students . smokers were asked whether an increase in the price of tobacco would be an incentive to quit smoking . of the smokers , 69.2% stated that they would not quit smoking if the price of cigarettes increased and 30.8% stated that they would ( p < 0.001 ) . we found no difference between medical and non - medical students in terms of the high price of tobacco being a motivation for quitting smoking . of the sample as a whole , 65% had relatives who were smokers and 35% were living with nonsmokers . however , the chi - square test showed that having smokers in the family had no significant influence on the smoking habits of the students . the present study showed a high prevalence of smoking among undergraduate students in georgia , smokers accounting for 48.75% of the study population . our findings also show that smoking was strongly associated with gender , males accounting for 65% of all smokers and females accounting for 35% . in georgia , male and female smokers account for 56% and 6% of the general adult population , respectively . we found no statistically significant differences between medical and non - medical students in terms of the prevalence of smoking ( 49.5% vs. 48.0% ) . a similar study was conducted in greece , which is the country with the highest per capita consumption of cigarettes in the european union . the prevalence of smoking in that study was 26 - 56% for medical students and 44 - 57% for non - medical students . the results of that study are consistent with ours and suggest that medical education has little impact on student decisions regarding whether or not to smoke . several other authors have reported results that are also in accordance with those of the present study , having reported smoking prevalence rates as high as 47% in medical students and even higher among non - medical students . although attempts at conducting similar studies have focused exclusively on medical students , most such studies have shown high rates of smoking . this can be seen as an indicator of lax enforcement of anti - tobacco measures at universities , as well as being an indicator of lax government policies directed toward reducing tobacco consumption in the general population . a study conducted in brazil showed that the prevalence of smoking among medical students was high and that medical students had insufficient knowledge of smoking . however , several studies have shown declining trends in the prevalence of smoking , especially among medical students . the results of a study conducted in the university of jordan , in the city of amman , jordan , corroborate these findings , having shown a lower prevalence of smoking among health science students . similar findings were reported in a study conducted in 1995 and involving students at the university of cambridge , in the city of cambridge , england . that study showed that the prevalence of smoking was higher among students in the humanities than among medical students ( 17% vs. 7.7% ) . these data reinforce the notion that those studying health - related subjects have a higher level of awareness of the dangers of smoking . however , it is of note that various smoking prevalence studies conducted in other countries have shown characteristics that are different from those found in brazil , jordan , or england . nevertheless , there have been few studies comparing medical and non - medical students in terms of trends in smoking . therefore , it is difficult to determine whether the trend toward a reduction in smoking prevalence is due to anti - smoking education in the medical curriculum . the results of our study show that the prevalence of smoking is alarmingly high , indicating that current medical curricula do not place enough emphasis on the hazards of smoking . in fact , inadequate undergraduate education about tobacco - related health risks has been reported to be one of the main factors contributing to missed opportunities . in addition , most studies involving the general population , as well as most of those involving health professionals , have shown that tobacco consumption is more common in males . although the prevalence of smoking has been decreasing in males and increasing in females , it remains significantly higher in males . our results regarding the proportion of students who had attempted to quit smoking at least once seem to be quite similar to those of the study conducted in greece , according to which 44% of all medical students and 36% of all non - medical students had attempted to quit at least once . these findings show that undergraduate students have poor knowledge of the negative health effects of smoking and indicate the need for educational programs that are more comprehensive , as well as for smoking cessation programs targeting such students . we sought to determine how many students were willing to quit smoking and why they were willing to quit . we found that 56.9% of the smokers were willing to quit , health and financial reasons being the most common reasons for quitting . of the medical students , 59.5% expressed willingness to quit smoking , as did 54.2% of the non - medical students . although the difference is not statistically significant , it suggests that medical students have a better understanding of the harmful effects of tobacco . nevertheless , medical and non - medical students gave similar reasons for quitting smoking . medical education appears to have no preventive effect , or rather , has no influence on the attitudes and behaviors of undergraduate students toward smoking . in any case , it is vital to improve anti - smoking education in the curricula of all medical and non - medical schools . in the case of medical students , it should be borne in mind that they will become physicians . as such , they are expected to play a crucial role in anti - smoking campaigns . this means that they will not only advise patients on smoking issues but will also serve as role models for their patients . therefore , the undergraduate years provide a perfect opportunity for making medical students fully aware of the harmful effects of smoking . the proportion of students who are actually willing to quit smoking can serve as a very good indicator of success in quitting . the results indicate that current medical education is ineffective in making students aware of the hazards of smoking and in advocating smoking cessation among students . although the proportion of male smokers is higher than is that of female smokers , the proportions of males and females expressing willingness to quit smoking are equal . the lack of significant difference between medical and non - medical students regarding willingness to quit smoking suggests a lack of knowledge of the harmful effects of smoking and emphasizes the need to revise existing educational programs . an increase in the price of cigarettes is not enough to reduce the prevalence of smoking . o objetivo deste estudo foi descrever a prevalncia do tabagismo e os fatores que o influenciam em estudantes de medicina e outros universitrios em tbilisi , gergia , bem como determinar se a educao mdica tem impacto no tabagismo . foi realizado um estudo transversal na universidade mdica estadual de tbilisi e na universidade estadual de tbilisi , ambas em tbilisi , gergia . pedimos a 400 alunos de quarto ano ( 200 em cada universidade ) que respondessem a questionrios padronizados . a mdia de idade dos fumantes foi de 20,24 anos e a dos no fumantes foi de 20,26 anos . dos estudantes de medicina , 49,5% eram fumantes , assim como o eram 48,0% dos universitrios que no estudavam medicina . o tabagismo apresentou forte relao com o gnero ; 65% dos fumantes eram do sexo masculino . dos fumantes , 56,9% disseram que gostariam de parar de fumar ( por motivos de sade ou financeiros ) . dos estudantes de medicina , 59,5% disseram que estavam dispostos a parar de fumar , assim como o disseram 54,2% dos universitrios que no estudavam medicina . de educao antitabagismo no currculo das universidades e implantao de campanhas de preveno ao tabagismo em instituies de ensino superior . entretanto , essas medidas s sero eficazes se as polticas de controle do tabaco forem rigorosamente observadas tambm em nvel nacional . embora tenha um impacto terrvel sobre a sade , o tabaco amplamente usado em todo o mundo . se no forem implantadas intervenes eficazes , o tabagismo causar a morte de at um bilho de pessoas no sculo xxi . fumantes comea a fumar na adolescncia ou logo aps o trmino do ensino mdio . o tabagismo entre os jovens uma preocupao para pases industriais , de mdia renda e de baixa renda , e no h diferena significativa entre os gneros quanto prevalncia do tabagismo . em 2000 , entrou em vigor o decreto n 412 ( " das medidas de controle e preveno do tabagismo na gergia " ) , emitido pelo presidente da gergia , e , em 2004 , foi assinada a conveno - quadro para o controle do tabaco , da organizao mundial da sade ( oms ) ; no entanto , a taxa de tabagismo na gergia permanece alta e tende a aumentar , especialmente entre os jovens . h atualmente cerca de 5.000 alunos de graduao matriculados na universidade mdica estadual de tbilisi ( na cidade de tbilisi , na gergia ) e cerca de 18.000 matriculados na universidade estadual de tbilisi ( tambm em tbilisi , gergia ) . os objetivos do presente estudo foram determinar a prevalncia do tabagismo em estudantes de medicina e outros universitrios nas universidades supracitadas ; determinar se a educao mdica tem impacto sobre o tabagismo e identificar os mais importantes fatores de risco de tabagismo em alunos de graduao . o objetivo da pesquisa foi explicado aos estudantes , os quais foram posteriormente convidados a preencher questionrios annimos . como o presente estudo foi um estudo transversal , realizado durante ou imediatamente aps uma palestra geral , aberta a todos , era difcil prever quem estaria assistindo palestra . portanto , para fins prticos , foram entrevistados 200 alunos ( 100 do sexo masculino e 100 do sexo feminino ) de cada universidade durante o perodo de estudo . definiu - se o tabagismo conforme os critrios da oms . os entrevistados que relataram fumar pelo menos 1 cigarro por dia ( todos os dias ou ocasionalmente ) foram considerados fumantes . aqueles que vinham experimentando o tabagismo no momento da pesquisa ( tendo fumado menos de 100 cigarros na vida ) tambm foram considerados fumantes . aqueles que nunca fumaram ou que fumaram menos de 100 cigarros na vida , mas que no haviam fumado nos ltimos 30 dias ( inclusive no dia da pesquisa ) foram considerados no fumantes . os fumantes responderam a perguntas sobre a mdia de cigarros fumados por dia , a idade de incio do tabagismo e as tentativas de parar de fumar , se que haviam feito alguma . a coleta de dados ocorreu nas duas universidades durante o semestre letivo , entre fevereiro e maro de 2011 . o questionrio sobre tabagismo do projeto de monitorao multinacional de tendncias e determinantes em doenas cardiovasculares , da oms , foi modificado para refletir hbitos e caractersticas locais . a verso final do questionrio estruturado incluiu questes de mltipla escolha e vrias questes abertas sobre tabagismo , dependncia de nicotina e fatores que influenciam o comportamento de fumar , bem como sobre conhecimento e percepo do tabagismo . o programa statistical package for the social sciences , verso 15.0 para windows ( spss inc . , chicago , il , eua ) foi usado para calcular as frequncias de cada varivel . o teste do qui - quadrado de pearson foi usado para comparar as propores , e o teste t de student foi usado para examinar as diferenas entre as mdias . anlises de regresso logstica foram usadas para determinar se o tabagismo se correlacionava com caractersticas demogrficas e comportamentos de risco sade . a razo entre os gneros masculino e feminino na populao do estudo foi de 0,9:1,1 ( 190 homens vs. 210 mulheres ) . a mdia de idade dos fumantes foi de 20,24 anos ( variao , 17 - 27 anos ) e a dos no fumantes foi de 20,26 anos ( variao , 17 - 25 anos ) . no houve diferenas significativas entre os estudantes no que tange distribuio de idade , gnero e ano de educao universitria . com base no consumo dirio de cigarros , os fumantes foram definidos da seguinte forma : fumantes muito pesados ( > 20 cigarros / dia ) ; fumantes moderados a pesados ( 11 - 20 cigarros / dia ) ; fumantes leves ( 1 - 10 cigarros / dia ) . dos 195 alunos que responderam a essa pergunta , 45,8% eram fumantes leves , 40,2% eram fumantes moderados a pesados e 14,0 % eram fumantes muito pesados ( tabela 1 ) . o tabagismo apresentou forte relao com o gnero ; 65% dos fumantes eram homens e 35% eram mulheres . tabela 1prevalncia e frequncia de tabagismo entre os estudantes entrevistados ( n = 400 ) . dos 195 fumantes , 122 ( 62,6% ) disseram que nunca haviam tentado parar de fumar . dentre os que afirmaram ter tentado parar de fumar , os motivos mais comuns pelos quais voltaram a fumar foram o vcio de fumar , o consumo de lcool e outros fatores externos ( os quais os estudantes no conseguiram especificar ) . dos 195 fumantes , 111 ( 56,9% ) disseram que sim , ao passo que 43,1% disseram que no . no foram encontradas diferenas estatisticamente significativas entre os estudantes de medicina e os demais universitrios no tocante prevalncia do tabagismo ( tabela 2 ) . alm disso , o teste do qui - quadrado no mostrou relao estatisticamente significativa entre o tipo de educao ( mdica ou no mdica ) e o tabagismo . de parar de fumar , assim como o disseram 54,2% dos universitrios que no estudavam medicina ( tabela 3 ) . a diferena no foi estatisticamente significativa . tabela 3vontade de parar de fumar , de acordo com o tipo de educao . os principais motivos pelos quais os estudantes de medicina e demais universitrios gostariam de parar de fumar eram bastante semelhantes , sem diferenas estatisticamente significativas entre os dois grupos . na verdade , motivos de sade foram ligeiramente mais comuns entre os universitrios que no estudavam medicina . perguntou - se aos fumantes se um aumento do preo do tabaco seria um incentivo para parar de fumar . dos fumantes , 69,2% disseram que no iriam parar de fumar caso o preo dos cigarros aumentasse e 30,8% disseram que iriam ( p < 0,001 ) . no encontramos nenhuma diferena entre os estudantes de medicina e os demais universitrios no tocante ao alto preo do tabaco ser uma motivao para parar de fumar . do total da amostra , 65% tinham parentes que eram fumantes e 35% viviam com no fumantes . no entanto , o teste do qui - quadrado mostrou que ter fumantes na famlia no teve influncia significativa sobre o hbito de fumar dos estudantes . o presente estudo mostrou alta prevalncia de tabagismo em alunos de graduao na gergia : 48,75% da populao do estudo eram fumantes . nossos achados tambm mostram forte associao entre tabagismo e gnero : 65% dos fumantes eram homens e 35% eram mulheres . na gergia , fumantes do sexo masculino e fumantes do sexo feminino representam , respectivamente , 56% e 6% da populao geral adulta . no foram encontradas diferenas estatisticamente significativas entre os estudantes de medicina e os demais universitrios no tocante prevalncia de tabagismo ( 49,5% vs. 48,0% ) . um estudo semelhante foi realizado na grcia , que o pas com o maior consumo de cigarros per capita na unio europeia . a prevalncia de tabagismo naquele estudo foi de 26 - 56% entre os estudantes de medicina e de 44 - 57 % entre os universitrios que no estudavam medicina . os resultados daquele estudo vo ao encontro dos nossos e sugerem que a educao mdica tem pouco impacto na deciso dos estudantes de fumar ou no . vrios outros autores tm relatado resultados que tambm vo ao encontro dos do presente estudo , com taxas de prevalncia de tabagismo de at 47% em estudantes de medicina e ainda maiores em outros universitrios . embora as tentativas de realizao de estudos semelhantes tm sido voltadas exclusivamente a estudantes de medicina , a maioria desses estudos tem mostrado altas taxas de tabagismo . isso pode ser visto como um indicador da falta de rigor em fazer cumprir medidas antitabaco em universidades , alm de ser um indicador da falta de rigor das polticas governamentais voltadas reduo do consumo de tabaco na populao em geral . um estudo realizado no brasil mostrou que a prevalncia do tabagismo em estudantes de medicina era alta e que os estudantes de medicina no tinham conhecimento suficiente a respeito do tabagismo . no entanto , vrios estudos tm mostrado tendncia de queda da prevalncia do tabagismo , especialmente em estudantes de medicina . os resultados de um estudo realizado na universidade da jordnia , na cidade de am , na jordnia , corroboram esses achados , tendo mostrado menor prevalncia de tabagismo em estudantes de cincias da sade . achados semelhantes foram relatados em um estudo realizado em 1995 com alunos da universidade de cambridge , na cidade de cambridge , na inglaterra . o estudo em questo mostrou que a prevalncia de tabagismo era maior nos estudantes de humanidades do que nos estudantes de medicina ( 17% vs. 7,7% ) . esses dados reforam a noo de que aqueles que estudam temas relacionados com a sade esto mais cientes dos perigos do tabagismo . no entanto , importante mencionar que vrios estudos de prevalncia de tabagismo realizados em outros pases apresentam caractersticas que so diferentes das encontradas no brasil , na jordnia ou na inglaterra . no obstante , so poucos os estudos que comparam as tendncias do tabagismo em estudantes de medicina e outros universitrios . portanto , difcil determinar se a tendncia de reduo da prevalncia do tabagismo atribui - se educao antifumo no currculo de medicina . os resultados de nosso estudo mostram que a prevalncia do tabagismo alarmantemente alta e indicam que o atual currculo de medicina no enfatiza como deveria os perigos do tabagismo . com efeito , relatou - se que a educao universitria inadequada a respeito dos riscos sade relacionados com o tabaco um dos principais fatores que contribuem para a perda de oportunidades . alm disso , a maioria dos estudos envolvendo a populao geral , bem como a maioria daqueles envolvendo profissionais de sade mostra que o consumo de tabaco mais comum entre os homens . embora a prevalncia do tabagismo venha diminuindo entre os homens e aumentando entre as mulheres , continua a ser significativamente maior naqueles . nossos resultados a respeito da proporo de estudantes que tentaram parar de fumar pelo menos uma vez parecem ser bastante semelhantes aos do estudo realizado na grcia , segundo o qual 44% dos estudantes de medicina e 36% dos demais universitrios haviam tentado parar esses resultados mostram que os alunos de graduao tm pouco conhecimento dos efeitos negativos do tabagismo sobre a sade e indicam que h necessidade de programas educacionais mais abrangentes , bem como de programas de cessao do tabagismo para esses alunos . procuramos determinar quantos estudantes estavam dispostos a parar de fumar e por que estavam dispostos a parar . constatamos que 56,9% dos fumantes estavam dispostos a parar de fumar , mais comumente por motivos de sade e financeiros . dos estudantes de medicina , 59,5% disseram que estavam dispostos a parar de fumar , assim como o disseram 54,2% dos universitrios que no estudavam medicina . embora no seja estatisticamente significativa , a diferena sugere que os estudantes de medicina tm melhor compreenso dos efeitos nocivos do tabaco . no obstante , os motivos pelos quais os estudantes de medicina e demais universitrios desejavam parar de fumar eram semelhantes . a educao mdica parece no ter efeito preventivo , ou melhor , no tem qualquer influncia sobre as atitudes e comportamentos dos alunos de graduao em relao ao tabagismo . de qualquer forma , fundamental melhorar a educao antifumo no currculo de medicina e demais reas . no caso dos estudantes de medicina , deve - se ter em mente que eles se tornaro mdicos . como mdicos , espera - se que desempenhem um papel fundamental em campanhas antitabagismo . isso significa que no apenas aconselharo seus pacientes a respeito do tabagismo , mas tambm serviro de modelo a eles . portanto , os anos de graduao proporcionam uma oportunidade perfeita para tornar os estudantes de medicina plenamente conscientes dos efeitos nocivos do tabagismo . a proporo de estudantes que esto realmente dispostos a parar de fumar pode ser um bom indicador de sucesso em parar de fumar . os resultados indicam que a educao mdica atual no torna os alunos conscientes dos riscos do tabagismo e no promove a cessao do tabagismo entre os estudantes . embora a proporo de fumantes do sexo masculino seja maior do que a de fumantes do sexo feminino , a proporo de homens e mulheres dispostos a parar de fumar igual . a ausncia de diferena significativa entre os estudantes de medicina e os demais universitrios no que tange vontade de parar de fumar sugere uma falta de conhecimento dos efeitos nocivos do tabagismo e evidencia a necessidade de rever os programas educacionais existentes . um aumento do preo dos cigarros no suficiente para reduzir a prevalncia do tabagismo .

one of the most widely consumed beverages throughout the world is tea produced from the tea plant ( camellia sinensis ) . geographical consumption patterns of the different teas vary greatly with green tea consumed mainly in asia and the middle east and black tea consumed mostly in western countries . this together with evidence from epidemiological studies and high consumption rates worldwide has led to growing interest in tea as a product that may significantly contribute to human health . the polyphenol profile of the different teas is affected by their different methods of production . black tea is produced by wilting , crushing , and partial oxidation and consequently is rich in theaflavins and thearubigins . in green tea production oxidation is minimised resulting in catechins being dominant , particularly epigallocatechin - gallate ( egcg ) , white tea undergoes the least processing and is produced from young leaves and buds resulting in high levels of egcg , though generally lower than the levels found in green tea . reduced risk of coronary heart disease , stroke incidence , chronic inflammation , and cancer incidence is associated with black and green tea consumption . recent studies have focused on the impact of tea and tea polyphenols on blood glucose ( bg ) and insulin sensitivity . black tea has been shown to decrease plasma glucose and enhance insulin concentrations after consumption in comparison to a control and a caffeine drink . aldughpassi and wolever showed that 250 ml of black tea with test meals actually increased overall mean peak bg compared to water though a reduction in the standard error might indicate the ability of tea compounds to improve the precision of the bg response . a study of green tea catechins in insulin resistant induced obese rats suggested that they may impact glucose control through several pathways . the objectives of this study were as follows : to assess the total polyphenol content of different commercial teas;to assess the bioaccessibility of polyphenols from whole and bagged teas after in vitro digestion;to determine the effect of black , white , and green tea infusions on sugar release from bread after an in vitro digestion model . to assess the total polyphenol content of different commercial teas ; to assess the bioaccessibility of polyphenols from whole and bagged teas after in vitro digestion ; to determine the effect of black , white , and green tea infusions on sugar release from bread after an in vitro digestion model . all chemicals and reagents were of analytical grade and purchased from sigma - aldrich ( poole , uk ) . the different teas were sourced directly from a specialist supplier as whole teas or purchased in bag form from a local tesco supermarket . the weight of each whole tea used was approximately 3 g except for the flowering osmanthus and jasmine & lily teas where an entire bulb was used ( the weight of each bulb was recorded ) . for teas in each tea was infused in 200 ml of boiling water ( 90c unless otherwise specified on the manufacturer 's instructions ) for three minutes and then stirred six times before the tea was removed . the resultant sample was then left at room temperature to cool for an additional 17 minutes before testing commenced . all tests were carried out on a minimum of three separate occasions and samples were analysed in triplicate for each test . an aliquot ( 200 l ) of each tea sample was added to 1.5 ml of freshly prepared folin - ciocalteu reagent ( 1 : 10 v / v with water ) . the mixture was allowed to equilibrate for 5 min and then mixed with 1.5 ml of 60 g / l sodium carbonate solution . after incubation at room temperature for 90 min , the absorbance of the mixture was read at 725 nm using the respective solvent as blank . the results were expressed as mg of gallic acid equivalents ( gaes ) per gram of tea . samples from nineteen brands of commercially available tea were analysed using an in vitro digestion model adapted from ryan and others . a total of 4 ml of each 200 ml tea infusion was added to an amber vial and made up to a volume of 15 ml with saline . the samples were acidified to ph 2 by the addition of 1 ml of a porcine pepsin preparation ( 0.04 g pepsin in 1 ml 0.1 m hcl ) and then incubated at 37c in a shaking water bath at 95 rpm for 1 hour . the ph was increased to 5.3 with 0.9 m sodium bicarbonate , followed by the addition of 200 l of the bile salts glycodeoxycholate ( 0.04 g in 1 ml saline ) , taurodeoxycholate ( 0.025 g in 1 ml saline ) and taurocholate ( 0.04 g in 1 ml saline ) , and 100 l of pancreatin ( 0.04 g in 500 l saline ) to each sample . the ph was adjusted to 7.4 using 1 m naoh and overlaid with nitrogen . the samples were then incubated in a shaking water bath for two hours at 37c . duodenal aliquots were taken and samples were frozen until analysis . the relative glycaemic impact ( rgi ) of one black tea , one green tea , and one white tea sample from the clipper brand was measured in order to assess the effect of the tea polyphenols on the inhibition of starch breakdown . this was achieved by subjecting samples of tea combined with bread to an in vitro digestion procedure and measuring the resultant reducing sugars released . white bread dough was made to a recipe of 190 g warm tap water , 1 tbsp virgin olive oil , 1 tsp salt , 1 tbsp sugar , 1 tbsp dried milk powder , 350 g strong white flour , and 1.5 tsp of yeast . the dough was then baked in a russell hobbs bread maker ( model no : 18036 , manchester , uk ) for a total of 3 hours and 20 minutes . samples of the bread were then prepared by weighing 2.5 g samples and placing each into 60 ml specimen pots . the pots were inserted into an aluminium heating block and covered with an insulating sheet in readiness for testing . this consisted of a simulated gastric digestion phase followed by an ileal digestion phase with timed sampling at the end of the gastric phase and during the ileal phase . a volume of 30 ml of each tea infusion ( 1 tea bag / infusion ) was added to its own individual bread sample . a 250 l baseline sample was extracted for each sample at t = 0 min and added to a test tube in a ratio of 1 : 4 in ethanol . this was followed by the addition to each sample of 0.1 ml 10% -amylase , 0.8 ml 1 m hcl , and 1 ml 10% pepsin solution in 0.05 m hcl to each . the resultant mixture was stirred slowly at 130 rpm every 15 s for 30 min at 37c to complete the gastric digestion phase , and then gastric aliquots were taken . the ileal phase was initiated by the addition of 2 ml 1 m nahco3 and 5 ml 0.2 m na maleate buffer ( ph 6 ) to each sample , and the volume was increased to 55 ml with dh2o . in quick succession , 0.1 ml of amyloglucosidase and 1 ml of 2% pancreatin solution ( in maleate buffer , ph 6 ) were added to each sample . samples were then incubated for 120 minutes with constant slow mixing , and aliquots were taken at 20 , 60 , and 120 minutes during ileal digestion . the tubes were centrifuged ( 1000 g , 2 min ) in a biofuge primo centrifuge ( heraeus instruments , kendro laboratory products , germany ) and an aliquot of the supernatant was removed for analysis of reducing sugars . sugar released from the bread during digestion was measured by a colourimetric method adapted from englyst and hudson designed to measure monosaccharides after an amyloglucosidase secondary digestion to complete depolymerisation of starch fragments . a total of 0.05 ml of 10 mg / ml glucose standard or sample from the in vitro digestion was added to 0.25 ml of enzyme solution a ( 1% amyloglucosidase in acetate buffer , ph 5.2 ) . each sample was incubated for 10 minutes at 25c and then 0.75 ml of 3,5-dinitrosalicylic acid ( dns ) mixture ( 0.5 mg / ml glucose : 4 m naoh : dns reagent mixed in ratio 1 : 1 : 5 ) was added . the resultant sample was then heated for 15 minutes at 95c in a water bath . following this , 3 ml of water was added to each sample which was then left to cool for 20 min in a cold water bath . absorbance was read at 530 nm on a shimadzu uv-1201 spectrophotometer ( shimadzu corporation , australia ) and sugar release was measured in mg per g of bread sample . slowly digestible starch ( sds ) was extrapolated by subtracting the rapidly digestible starch ( rds ) measurement at 20 min from the reducing sugars measurement at 120 min during ileal digestion . all experiments were carried out in triplicate and each had a minimum of three replicates for each tea . the data are presented as means ( sem ) and comparisons between samples were carried out by an anova and tukey 's multiple comparison test ( spss , version 17 ; spss inc . , chicago , il , usa ) . a probability of 5% or less was considered statistically significant . table 1 illustrates that all teas were a significant source of polyphenols . of the whole teas , brand had a significantly higher polyphenol content ( p < 0.05 ) compared to the other commercial whole teas analysed . of the bagged varieties , green tea infusion had a significantly higher polyphenol content than both white and black teas , as measured by fcr ( p < 0.05 ) . the in vitro digestion model enables the measurement of polyphenols potentially available for absorption after the gastric and duodenal phases of digestion . bioaccessibility refers to the proportion of polyphenols which are presented to the brush border for absorption after digestion and gives some indication as to their potential bioavailability in vivo . table 2 illustrates that the polyphenol content of all tea infusions was enhanced following the gastric digestion phase . this enhancement continued into the duodenal phase , although some tea polyphenols became less bioaccessible relative to the gastric phase . at 20 minutes into the duodenal phase of digestion , green tea significantly suppressed rds release in white bread to 253.83 mg / g bread sample when compared to white tea and the control bread ( p < 0.05 ; figure 1 ) . black tea showed no significant effect on sugar release at this time point . in all teas the different methods of processing and production impact the polyphenol content of the resultant teas . total black tea polyphenols decrease during fermentation , and the longer tea is subjected to processing , the lower the polyphenol content . turkmen and others found that black tea polyphenol content as measured by fcr reached a maximum of 131.9 mg gae / g tea extract compared to the 87.9 gae / g in the current study indicating that the polyphenol content of the same tea can vary widely . the green tea infusion was shown to have more overall reducing power than both black tea and white tea . theaflavins in black tea are dimers of catechins and contain more hydroxyl groups in their structure . in green tea , catechins remain dominant . this could in part explain the differences in reducing potential between black tea and green tea . there has been very little research to date on the polyphenol content and health effects of white tea . rusak and others found that green tea was a richer source of polyphenols than white tea and the current study supports this . they also found that the concentration of catechins was significantly higher in green tea leaves than white tea leaves . ryan and carolan found that green teas varied in their polyphenol content , ranging from 250 to 750 mg gae / tea bag . they also found that both the structure of the tea bag and the infusion time influenced the polyphenol content of the teas . fcr values in the current study were slightly lower than those found by ryan and carolan , averaging 115.5 mg gae / g tea bag . rusak and others found that extraction of catechins from green tea was affected by the form of tea used , with extraction from loose green tea leaves being more effective than from refined bagged tea leaves . greater tea bag size results in tea solids diffusing into solution faster because of the larger surface area available in which the contents can diffuse . the material of the tea bag can also have an impact on polyphenol diffusion . in the current study , tea bag infusion time and stirring / squeezing of the tea bags tea bags were different weights , the black tea bag weighing more than both white tea and green tea . weights were , however , corrected for upon calculations . of the whole teas analysed , this was the only whole tea that had a higher polyphenol content than any of the bagged teas . whole teas tend to be more compacted and have undergone much less processing than the bagged tea . unpublished data from our laboratory indicate that the grinding of leaves to form the bagged teas has the effect of releasing polyphenols and this may in part explain the higher polyphenol content in the bagged teas . overall , all teas were shown to be good sources of polyphenols . in the current study , polyphenol bioaccessibility increased in all teas from baseline to gastric phases suggesting that the polyphenols in these teas may become more available in humans after consumption . the bioaccessibility increase from baseline to gastric phase was similar for both bagged and whole teas . the compact and relatively unprocessed nature of the whole teas resulted in a lower polyphenol content at baseline . however , the bioaccessibility increases suggest that the digestive enzymes can further release polyphenols from the tea infusion . green and others looked at the effect of in vitro digestion specifically on catechins in green tea and found that catechins had less than 20% recovery after in vitro digestion . however , the current study is the first to report and compare the bioaccessibility of total polyphenols from green , white , and black tea infusions . in the current study , with the exception of white bagged tea , the bioaccessibility decreased slightly from gastric to duodenal phases in bagged teas . however , at the duodenal stage values still remained above baseline for all tea varieties . rusak and others showed that the form of tea , either loose or bagged did not affect white tea catechin stability . therefore , compounds in white tea may be more stable , and thus less susceptible to degradation compared to those in the other teas . green tea was the only tea shown to significantly reduce sugar release from white bread . this is a promising finding in that green tea may reduce the rds of starch rich foods such as bread . wu and others found that increased levels of added purified polyphenols ( 50% egcg ) resulted in decreased retrogradation for resistant starch in rice . from these results , it was predicted that the hydroxyl radical of tea polyphenols combined with rice starch to form hydrogen bonds , preventing the reassociation of the starch chains . in the current study , white tea had no significant effect on sugar release , although there was a slight increase compared to the control bread . certain polyphenols or other compounds in white tea may be responsible for interfering with the natural chemical bonds in the bread , therefore rendering the starch more susceptible to degradation . white tea polyphenols were shown to be the most stable throughout digestion , and therefore they may have a greater impact on starch than those polyphenols which are degraded more readily . different teas contain different polyphenols , and therefore it is plausible that each tea may have a different effect on sugar release . teas were used at low concentrations in this study , that is , one tea bag per infusion . therefore , green tea at low concentrations reduces sugar release from starch samples , whereas white tea at a low dose seems to have the opposite effect . teas were made as infusions and then added to bread samples at the baseline phase of digestion , whereas other studies have baked tea into breads or looked at the effect of purified tea polyphenol rich extracts . therefore , different study designs may account for variability in study results . however , what can be seen is that different types of tea do have an effect on starch breakdown and sugar release from breads . unpublished data from our laboratory found that glycaemic response ( gr ) increased over 180 minutes following the consumption of white bread with added black tea extract in comparison to eating white bread alone . a more pronounced effect was also seen when a tea infusion was consumed alongside the white bread and the time taken for this combination to exert its peak gr was prolonged compared to either the control bread or the bread with tea extract added . however , koh and others found black tea to reduce starch digestion , yet found no effect with green or oolong tea . in the current study , each tea was presented as an infusion alongside bread during digestion and the black tea infusion had no significant effect on sugar release from bread . the form in which the black tea is used , the preparation method , and the concentration used may all be factors to account for the variation in results between studies . deshpande and salunkhe found that in isolation tannic acid and catechins decreased in vitro digestibility of various types of starch sources . also , bjork and nyman found that phytic acid and tannic acid reduced starch hydrolysis in the digestive tract . however , isolated polyphenols may have a different effect on starch digestion compared to phenolics in combination . for example , green tea is one of the richest sources of phenolic compounds and includes catechins such as egcg , procyanidins , and quercetin . therefore , a combination of the phenolics in tea may have synergistic effects , either enhancing or reducing the degree of starch breakdown and sugar release compared to isolated catechins or tannins [ 25 , 26 ] . the reason for the reduction in sugar release seen in the current study may be because of the structural bonding of the green tea polyphenols with starch molecules . both black tea and green tea contain conjugate forms of catechins , and these compounds may interfere with the way in which starch is broken down . therefore , future research could look into accessing the polyphenol profile of the different teas to determine which polyphenols have an effect on reducing starch breakdown . tea polyphenols may have an inhibitory effect on digestive enzymes such as -amylase and -glucosidase . black tea and to a lesser extent green tea were shown to inhibit -amylase in human saliva and removal of tea tannins resulted in loss of the inhibitory activity . hara and honda found that both catechins and theaflavins inhibited salivary -amylase and whilst , various teas differed in the extent of inhibitory activity , black tea showed consistently greater inhibitory activity . kwon and others found that black tea and white tea showed almost a 40% inhibition of -amylase , with green tea showing only a 30% inhibition . they also found black and white tea to have a higher -glucosidase inhibitory activity than green and oolong teas . at higher concentrations of polyphenols , { 50 g gae / ml , 100 g gae / ml and 200 g gae / ml } -glucosidase inhibition was increased in all teas . although enzyme inhibition was not tested in the current study , a future area of study is to investigate the effect of tea polyphenols on digestive enzymes . finally , it should be noted that many studies evaluating the gr to foods contain the addition of tea or coffee in their test meals . based on the current results and on previous studies , the polyphenols in tea may affect bg , thus affecting the results of the test foods studied . it is important that when testing foods for different effects on disease parameters , the combination of ingredients is considered . a single food in isolation may have different effects on gr than that food with an additional tea beverage .

diagnosis and treatment of congenital heart disease have changed dramatically over the last few decades . the relative importance of preoperative morbidity and mortality has been increasing as a result of the great improvements in perioperative and long - term survival . this is especially true for critical congenital heart disease ( cchd ) , a group of morphologically heterogeneous disorders which have in common that early surgical or catheter interventional therapy is mandatory to achieve survival . clinical examination is unable to detect all forms of cchd [ 1 , 32 ] . heart murmurs , one of the hallmarks of non - critical heart disease typically diagnosed later in life may be absent or misleading because of the underlying anatomy , prolonged decline of pulmonary vascular resistance or reduced ventricular function . despite the increasing use of prenatal diagnosis , a significant proportion of affected newborns is still not diagnosed before discharge after birth [ 7 , 31 ] . this problem may be aggravated by recent trends to earlier discharge and other changes in postnatal care . the first manifestation of cchd may be acute with circulatory collapse and the need for cardiopulmonary resuscitation or death , i.e. delayed diagnosis is associated with significant morbidity and mortality [ 4 , 6 , 16 , 18 ] . the current incidence of severe physiologic compromise resulting from previously unrecognised cchd has been estimated to be 1 per 15,000 to 1 per 26,000 live births . hence , there is broad consensus that screening for cchd is warranted [ 17 , 27 , 29 , 30 ] . the use of prenatal ultrasound , close clinical observation during the transitional period and thorough physical examination alone may be sufficient for timely diagnosis of cchd . this may be true in the specific setting of this single centre study , but the prerequisites mentioned are probably not encountered in the majority of hospitals or maternity units . pulse oximetry can detect mild hypoxemia , which is characteristic for many forms of cchd , and may not be recognised by clinical examination . the first abstracts examining pulse oximetry as a screening tool for cchd were published in 1995 [ 5 , 14 ] . since then , there has been an increasing number of single and oligocentric studies on the subject [ 2 , 3 , 13 , 15 , 19 , 23 , 24 , 26 ] . despite differences among the study protocols concerning target lesions , time of screening , cut - off values , probe placement and others , most authors came to the conclusion that pulse oximetry screening ( pos ) is a useful method . recently published meta - analyses tend to share this view but argued that existing evidence is still insufficient to support a general recommendation for implementation of pos in routine care and therefore stated a need for further data [ 17 , 29 , 30 ] . the aim of our study was to test the hypothesis that pos , using currently available pulse oximeters , can effectively reduce the diagnostic gap in cchd in daily clinical routine in centres of primary , secondary and tertiary care . a prospective multicenter field study of 34 neonatal / obstetrical departments in saxony , germany , was performed from july 2006 to june 2008 . all full and post - term neonates ( gestational age 37 weeks ) meeting the following criteria were included in the study : ( 1 ) no prenatal diagnosis / suspicion of congenital heart disease , ( 2 ) normal routine clinical examination ( 3 ) informed parental consent . all forms of cchd were defined as the target of pos , regardless of whether cyanosis , i.e. duct dependency , is an invariable or a possible feature of the particular condition ( table 1 ) . table 1types of critical congenital heart disease ( cchd ) defined as target lesions for pulse oximetry screening ( pos)duct - dependent systemic circulation interrupted aortic arch complex / critical coarctation of the aorta hypoplastic left heart syndrome critical aortic valve stenosisduct - dependent pulmonary circulation pulmonary atresia various forms variants of congenital heart disease with severe pulmonary stenosis / atresia critical pulmonary valve stenosistotal anomalous pulmonary venous drainagetransposition of the great arteriescomplex cyanotic congenital heart disease transposition of the great arteries ventricular septal defect functional univentricular heart various forms types of critical congenital heart disease ( cchd ) defined as target lesions for pulse oximetry screening ( pos ) at the age of 2472 h , pos was performed by midwives , nurses and physicians experienced in the routine use of pulse oximetry . no particular requirements concerning the type of pulse oximeter were made so that a great variety of devices was used . pos was positive when a spo2 of 95% was measured and confirmed after 1 h. pos results were recorded in each newborn s hospital chart as well as in its individual booklet for prevention during childhood and adolescence ( gelbes heft ) . pos was defined as false - negative when a diagnosis of cchd was made after pos in the participating hospitals / at our centre . data acquisition was performed at the study sites , at the central office for quality management in perinatal and neonatal care of the saxonian medical association and at our hospital , which is the referral centre for participants of the study . data analysis and descriptive statistics were performed using commercially available software ( microsoft excel 2008 for mac ) . the study protocol was approved by the ethics committee of the university of leipzig , germany . in 18 of them , clinical symptoms led to the diagnosis of cchd before pos . a total of 6,036 newborns received diagnosis / treatment for other conditions . no parents refused to participate in the study . seven hundred ninety - five did not receive pos , mainly because of early discharge after birth ( n = 727 ; 91% ) . three children were excluded from further analysis because the study protocol had not been correctly followed . pulse oximetry had been performed in these three early on the first day of life ( i.e. at an age of 4 , 6 and 10 h , respectively ) resulting in spo2 ranging from 94% to 95% at initial and control measurement . all were discharged early ( one after exclusion of cchd by echocardiography ) , and none was re - admitted for cchd . this measurement was confirmed , i.e. pos was positive in 54 ( 0.13% ) of 41,445 neonates . pos was false - positive related to the diagnosis of cchd in 40 newborns ( healthy n = 12 , persistent pulmonary hypertension of the newborn n = 15 , sepsis n = 13 ) . pos was true - positive in 14 children ( fig . 1 and table 2 ) . pos was true - negative in 41,384 and false - negative in four children ( table 3 ) . the latter were diagnosed at the age of 38 days before ( n = 1 ) and after ( n = 3 ) discharge from the maternity unit . the patient with transposition of the great arteries and ventricular septal defect presented with dyspnea and cyanosis : 1flow chart of the study populationtable 2types of critical congenital heart disease ( cchd ) detected by pulse oximetry screening ( pos ) ( n = 14)duct - dependent systemic circulation2 persistent truncus arteriosus , interrupted aortic arch 1 hypoplastic left heart syndrome 1duct - dependent pulmonary circulation2 pulmonary atresia , ventricular septal defecttotal anomalous pulmonary venous drainage5transposition of the great arteries2complex cyanotic congenital heart disease3 transposition of the great arteries ventricular septal defect 2 taussig bing syndrome 1table 3types of critical congenital heart disease ( cchd ) missed by pulse oximetry screening ( pos ) ( n = 4)duct - dependent systemic circulation3 critical coarctation of the aorta 2 complex coarctation of the aorta ventricular septal defect 1complex cyanotic congenital heart disease1 transposition of the great arteries ventricular septal defect 1 flow chart of the study population types of critical congenital heart disease ( cchd ) detected by pulse oximetry screening ( pos ) ( n = 14 ) types of critical congenital heart disease ( cchd ) missed by pulse oximetry screening ( pos ) ( n = 4 ) sensitivity , specificity , positive and negative predictive value for pos were 77.78% , 99.90% , 25.93% and 99.99% , respectively . the incidence of cchd ( n = 90 ) in the study population ( n = 48,348 ) was 0.186% . in the perinatal and neonatal registries , maintained at the saxonian medical association , there was no death resulting from cchd outside of our centre , i.e. any being related to delayed diagnosis or non - referral among our study population . 2reduction of the diagnostic gap in critical congenital heart disease ( cchd ) with pulse oximetry screening . all newborns with critical cchd ( n = 90 out of the study population ( n = 48,348 ) ) are shown . colours indicate in percentages whether the diagnosis was made by prenatal ultrasound ( prenatal ) , by physical examination or clinical observation ( clinical ) before pulse oximetry screening , i.e. during the first 24 h of life or by pulse oximetry screening ( pos ) . when pos was used , only 4.4% of newborns had a delayed diagnosis of cchd ( diagnostic gap ) reduction of the diagnostic gap in critical congenital heart disease ( cchd ) with pulse oximetry screening . all newborns with critical cchd ( n = 90 out of the study population ( n = 48,348 ) ) are shown . colours indicate in percentages whether the diagnosis was made by prenatal ultrasound ( prenatal ) , by physical examination or clinical observation ( clinical ) before pulse oximetry screening , i.e. during the first 24 h of life or by pulse oximetry screening ( pos ) . when pos was used , only 4.4% of newborns had a delayed diagnosis of cchd ( diagnostic gap ) the total number of patients , in whom pos has been assessed for earlier detection of cchd exceeds 150,000 [ 9 , 17 ] . our study differs from most of the previously published reports mainly in that we included 42,240 newborns , which is comparable only to recent data from meberg and de - wahl granelli [ 9 , 19 ] . furthermore , a broad spectrum of hospitals providing primary , secondary or tertiary care participated in our study with the aim of examining whether pos is able to reduce the diagnostic gap in cchd not only in controlled single or oligocentric studies but also under real life conditions . in our study , there was a relatively high percentage of cchd detected by prenatal ultrasound ( 60% ) . it would be of interest to know which results would have been obtained by pos in these newborns . however , when a prenatal diagnosis of cchd is made , perinatal management is planned as soon as parents opt for postnatal therapy ( and not for abortion or compassionate care ) . after delivery , these newborns receive immediate medical assessment and treatment if the diagnosis is confirmed . treatment frequently consists of prostaglandin infusion and volume replacement and may include catheter interventions ( balloon atrioseptostomy of a restrictive foramen ovale , balloon angioplasty of critical valvular stenoses and , rarely , stenting of restrictive communications at the atrial or ductal level ) . it is kept within the normal range in non - cyanotic lesions , whereas in many cyanotic lesions , an attempt is made to maintain pulmonary to systemic flow ratio at 1:1 ( corresponding to an spo2 of about 80% ) . thus , spo2 in these newborns rather reflects the underlying hemodynamics and the quality of postnatal care . it does not answer the question whether these children would have been detected by pos ( or by previous clinical observation / physical examination ) . this would only have been possible if the prenatal diagnosis and postnatal care had been withheld , which obviously was not feasible for ethical reasons . after the first physical examination and clinical observation during the first 24 h of life , i.e. before pos was performed , 80% of all cchd had been diagnosed . a second physical examination before discharge from maternity is current practice in our as well in many other regions . however , this does not seem to increase the sensitivity which can be achieved with this method . thus , it seems reasonable to assume that the diagnostic gap without pos would have remained at 20% , which is in agreement with the data reported by wren . using pos to complement prenatal ultrasound and physical examination / clinical observation achieved a diagnostic gap as low as 4.4% ( fig . 2 ) . we believe that this number is more important than the sensitivity of pos alone , which was 77.78% in our study and is comparable to other large series [ 9 , 15 , 19 ] . this relates to the fact that the reduction of the diagnostic gap in cchd reflects the aim of pos at least from a clinician 's perspective . unlike other methods , for example , newborn metabolic or hearing screening pos is not meant as a new test for more or less replacing other existing methods . rather , it can be thought of as a tool which is complementary to prenatal ultrasound , physical examination and clinical observation . it has been well documented that pos sometimes does not detect left heart obstructive lesions which represented 75% of the false - negative cases in our series . this is not surprising , as for example coarctation of the aorta may develop and progress early after birth and is not always duct - dependent . nevertheless , in some cases , there is right to left shunting via a patent arterial duct ; for this reason we did not exclude these lesions from the target group . we would like to underscore the idea that pos is a complementary tool and that thorough physical examination should not only include the search for signs of heart failure but also comparative pulse palpation . complex cyanotic lesions can be also be missed by pos alone , especially in the context of high pulmonary blood flow . however not surprisingly , sensitivity and reliability of pos may also be influenced by human factors , and the training level of the staff performing pos should be adequate . the rate of false - positive results in our study was very low ( 0.10% ) . furthermore , some of these neonates had other diseases , like neonatal sepsis , for which earlier diagnosis does no harm . it has also been shown that earlier screening leads to a higher percentage of false - positive results . therefore , most publications , including one large review , recommend that pos be performed at or after 24 h of age [ 9 , 15 , 30 ] . it has been argued that pos should be done with a new - generation pulse oximeter . on the other hand , existing technology is used to monitor critically ill children and to guide intensive care and theoretical differences may not always be clinically relevant . in our study undertaken with a great variety of pulse oximeters the percentage of cchd diagnosed before discharge from maternity was even higher than in the swedish study , where new - generation pulse oximeters were used ( 95.6 vs. 92% ) . most authors currently use a high cut - off value of 95% or 96% , at which level pos seems to have the best overall performance [ 2 , 9 , 15 , 19 , 23 , 24 , 26 ] . between these two values , differences are probably minimal , and with a higher cut - off , the need for measurement of pre- and postductal difference in spo2 decreases . however , postductal measurement is important , as some forms of cchd associated with predominant right to left shunting at ductal level may be missed when only preductal spo2 is used . our study protocol included repeated spo2 measurements after 1 h if the initial value was < 96% . this reduced the number of newborns undergoing clinical examination and echocardiography from 65 to 54 . after having accumulated more data , further analyses may show that in newborns with an spo2 below a certain threshold , assessment should be initiated directly without repeated measurements . in our study , we did not address the issue of costs which has been extensively examined by griebsch et al . who came to the conclusion that pos is at least cost - neutral in the short term . taking into account the financial burden of death and sequelae of neurologic impairment or disability , which may result from late diagnosis of cchd , pos should be cost effective [ 29 , 30 ] . in reviewing our data and the experience published by others , we agree that there remain some open questions concerning the use of pos . on the other hand , using currently available pulse oximeters pos as an adjunct to prenatal ultrasound , thorough physical examination and clinical observation can substantially reduce the diagnostic gap in cchd . in view of the persisting problem of late diagnosis of cchd and its potential sequelae we would rather suggest that pos be implemented in routine neonatal care while scientific evaluation of the method is continued to allow for modifications . in saxony , after the end of our study , an appropriate recommendation has been put forward by the working groups for perinatology and neonatology of the saxonian medical association . during the study period , there were no reports of suspected death from unrecognised cchd from the participating hospitals . in the saxonian perinatal and neonatal registries , there were no deaths in external hospitals resulting from cchd among our study population . we , therefore , can not fully exclude single missed cases of death from undiagnosed cchd . additionally , after discharge from a maternity unit , there may have been re - admissions for cchd in hospitals in other regions , i.e. not covered by saxonian registries . however , the incidence of cchd in our population was rather high compared to what should be expected from published reports . during the study period , there were no reports of suspected death from unrecognised cchd from the participating hospitals . in the saxonian perinatal and neonatal registries , there were no deaths in external hospitals resulting from cchd among our study population . we , therefore , can not fully exclude single missed cases of death from undiagnosed cchd . additionally , after discharge from a maternity unit , there may have been re - admissions for cchd in hospitals in other regions , i.e. not covered by saxonian registries . however , the incidence of cchd in our population was rather high compared to what should be expected from published reports .

fetal blood can enter the maternal circulation and a diagnosis of clinical fetomaternal hemorrhage ( fmh ) is made when fetal blood loss reaches a significant level leading to clinical symptoms due to fetal anemia , such as an abnormal fetal heart rate ( fhr ) patterns , decreased fetal movement , and/or fetal hydrops . fmh with clinical problems is estimated to occur in 1 of 300010,000 births 14 , and affected infants may die , survive with disability , or exhibit normal development 14 . fmh accounts for approximately 2.0% of japanese infants with cerebral palsy caused by antenatal and/or intrapartum hypoxic conditions 5 . measurement of fetal middle cerebral artery peak systolic velocity ( mca - psv ) is a useful noninvasive test for detection of fetal anemia due to not only rh alloimmunization 6 but also fmh 711 . however , as the mca - psv is measured in women with suspected fetal anemia and/or nonreassuring fetal status ( nrfs ) , neither the time of significant fmh occurrence nor natural history of fmh is well understood . we recently encountered a case of fmh that was diagnosed immediately after birth , but was documented later to have lasted for at least 2 weeks prior to delivery . the hokkaido university hospital institutional review board approved this study and the patient provided signed informed consent . a 43-year - old nulliparous woman with body weight of 63 kg was admitted to our hospital at gestational week ( gw ) 31 due to persistent nausea , proteinuria as evidenced by spot urine protein - to - creatinine ratio ( mg / mg ) of 0.45 , and edema ( weight gain of 2.2 kg in 2 weeks ) in the presence of normal blood pressure of 136/81 mmhg . minimal pericardial effusion without skin edema , pleural effusion , or ascites was present in the fetus . elevation of the aspartate transaminase ( ast)/alanine transaminase ( alt ) from 29/15 iu / l at gw 28 to 58/45 iu / l with mild thrombocytopenia ( 132 10/l ) , and fetal peak systolic velocity of middle cerebral artery ( mca - psv ) of 73.6 cm / sec ( 1.76 multiples of the median [ mom ] according to the report by mari 6 ) on the day of admission ( at gw 31 ) , and nrfs ( fig . a male infant was born by cesarean section , weighing 1746 g with 1- and 5-min apgar scores of 6 and 8 , respectively , and arterial cord blood ph , paco2 , pao2 , and base deficits of 6.97 , 64.5 mmhg , 22.6 mmhg , and 15.2 meq / l , respectively . as the neonate was very pale and had a cord blood hemoglobin ( hb ) concentration of 3.6 g / dl , 50 ml of red blood cells ( rbc ) and 20 ml of fresh frozen plasma ( ffp ) the neonate did not develop respiratory syndrome , did not require tracheal intubation , and exhibited unremarkable findings on brain magnetic resonance imaging ( mri ) 46 days after birth . both the mother and infant had otherwise uneventful clinical courses and left hospital on postpartum day 10 and 53 days after birth , respectively . the medical chart recorded a gradual and consistent increase in mca - psv in this fetus : 31.0 cm / sec ( 1.07 mom ) at gw 23 , 48.7 cm / sec ( 1.46 mom ) at gw 26 , and 59.5 cm / sec ( 1.63 mom ) at gw 28 . nonspecific complaints of the mother were also recorded , including heartburn at gw 26 , and heartburn and generalized itch without skin eruption at gw 28 . investigations were performed to determine the time of fmh occurrence . as the mother had participated in a prospective study unrelated to the present episode , two blood samples obtained at gw 28 and at gw 31 were stocked and available for retrospective analyses . after receiving permission from the patient , flow cytometric assessment of fmh and determination of -fetoprotein ( afp ) were performed using these blood samples as well as blood obtained postpartum . this case already exhibited a high level of hbf fraction , 5.5% at gw 28 ( 16 days prior to delivery ) and 3.2% on the day of admission , and decreased levels of 0.6% on postpartum day 28 ( fig . the afp level was already markedly elevated to 14,704 ng / ml ( 67 mom according to the report of ishijima 12 ) at gw 28 , 23,000 ng / ml ( 82 mom ) on the day of admission , and 23,702 ng / ml ( 85 mom ) on postpartum day 0 . flow cytometric study for assessment of fmh . flow cytometric analysis was performed using a fetal cell count kit ( iq products bv , groningen , the netherlands ) ( a ) and monoclonal antibodies against hb -chain and hb -chain using hemoglobin ( 37 - 8):percp - cy5.5 and hemoglobin (51 - 7):sc-21756 ( santa cruz biotechnology , inc . fetal rbc contains fetal hb ( hbf ) with -chain ( ) and reduced expression of carbonic anhydrase ( ca ) , while f cells composing adult rbcs contain hbf with a high expression of ca . fraction sizes were 5.5% for rectangle area in a and 5.6% for parallelogram area in b on gw 28 , 3.2% and 2.5% for rectangle areas in a and b , respectively , on gw31 , and 0.6% and 0.7% for rectangle areas in a and b , respectively , on ppd 28 . at our hospital , ultrasound ( including doppler ultrasound ) studies were performed after obtaining inclusive informed consent from all pregnant women at the beginning of pregnancy . measurement of fetal mca - psv in otherwise healthy women used to be performed at the attending physician 's discretion in our hospital . however , many physicians measured mca - psv in otherwise healthy women at our hospital ; in a review of medical charts of 256 women who gave birth to singleton infants without clinical fmh at term ( gw 3740 ) in 2014 at our hospital , there were 100 women in whom fetal mca - psv was measured around gw 30 ( between gw 28 and gw 31 ) . the 100 measurements of mca - psv mom performed by seven physicians were distributed normally ( fig . 3 ) , ranging from 0.60 to 1.95 mom . the 25th , 50th , 75th , 90th , 95th , and 98th percentile mom values were 0.85 , 1.02 , 1.12 , 1.28 , 1.44 , and 1.52 , respectively . the 95th percentile mom of 1.44 was consistent with the results obtained in another study population without diagnosis of fmh by kurmanavicius et al . 13 in which 95th percentile mca - psv of 56.1 cm / sec for gw 30 corresponded to 1.40 mom according to mari et al . 6 . thus , the prevalence rate of mca - psv mom 1.5 was estimated as approximately 1 in 50 fetuses without clinical fmh when tested around gw 30 . one hundred women underwent fetal mca - psv measurement between gw 28 and gw 31 and gave birth to term singleton infants without clinical fmh . investigations were performed to determine the time of fmh occurrence . as the mother had participated in a prospective study unrelated to the present episode , two blood samples obtained at gw 28 and at gw 31 were stocked and available for retrospective analyses . after receiving permission from the patient , flow cytometric assessment of fmh and determination of -fetoprotein ( afp ) were performed using these blood samples as well as blood obtained postpartum . this case already exhibited a high level of hbf fraction , 5.5% at gw 28 ( 16 days prior to delivery ) and 3.2% on the day of admission , and decreased levels of 0.6% on postpartum day 28 ( fig . the afp level was already markedly elevated to 14,704 ng / ml ( 67 mom according to the report of ishijima 12 ) at gw 28 , 23,000 ng / ml ( 82 mom ) on the day of admission , and 23,702 ng / ml ( 85 mom ) on postpartum day 0 . flow cytometric study for assessment of fmh . flow cytometric analysis was performed using a fetal cell count kit ( iq products bv , groningen , the netherlands ) ( a ) and monoclonal antibodies against hb -chain and hb -chain using hemoglobin ( 37 - 8):percp - cy5.5 and hemoglobin (51 - 7):sc-21756 ( santa cruz biotechnology , inc . , santa cruz , ca ) ( b ) , respectively . fetal rbc contains fetal hb ( hbf ) with -chain ( ) and reduced expression of carbonic anhydrase ( ca ) , while f cells composing adult rbcs contain hbf with a high expression of ca . fraction sizes were 5.5% for rectangle area in a and 5.6% for parallelogram area in b on gw 28 , 3.2% and 2.5% for rectangle areas in a and b , respectively , on gw31 , and 0.6% and 0.7% for rectangle areas in a and b , respectively , on ppd 28 . at our hospital , ultrasound ( including doppler ultrasound ) studies were performed after obtaining inclusive informed consent from all pregnant women at the beginning of pregnancy . measurement of fetal mca - psv in otherwise healthy women used to be performed at the attending physician 's discretion in our hospital . however , many physicians measured mca - psv in otherwise healthy women at our hospital ; in a review of medical charts of 256 women who gave birth to singleton infants without clinical fmh at term ( gw 3740 ) in 2014 at our hospital , there were 100 women in whom fetal mca - psv was measured around gw 30 ( between gw 28 and gw 31 ) . the 100 measurements of mca - psv mom performed by seven physicians were distributed normally ( fig . the 25th , 50th , 75th , 90th , 95th , and 98th percentile mom values were 0.85 , 1.02 , 1.12 , 1.28 , 1.44 , and 1.52 , respectively . the 95th percentile mom of 1.44 was consistent with the results obtained in another study population without diagnosis of fmh by kurmanavicius et al . 13 in which 95th percentile mca - psv of 56.1 cm / sec for gw 30 corresponded to 1.40 mom according to mari et al . thus , the prevalence rate of mca - psv mom 1.5 was estimated as approximately 1 in 50 fetuses without clinical fmh when tested around gw 30 . one hundred women underwent fetal mca - psv measurement between gw 28 and gw 31 and gave birth to term singleton infants without clinical fmh . retrospective analysis of the maternal blood for hbf fraction and afp concentration demonstrated unequivocally that this patient already suffered from fmh 16 days prior to delivery ( at gw 28 ) . in addition , a consistent increase in fetal mca - psv , beginning with 1.46 mom on gw 26 , suggested that this patient may have suffered from fmh for more than 3 weeks . fetal mca - psv measurement with doppler ultrasound is noninvasive and useful for detection of fetal anemia , having a very high sensitivity of 100% with a cutoff of 1.5 mom 6 . however , this test with a cutoff of 1.5 mom has a low positive predictive value among the general population of pregnant women an approximately 2.0% of women exhibit mca - msv mom 1.5 in the absence of clinical fmh , as reported by kurmanavicius et al . 13 and demonstrated in this study . as clinical fmh occurs in 2 of 10,000 births based on the literature 14 , only 2 of 200 women with a positive mca - psv test result would have clinical fmh , yielding a positive predictive value of 1.0% ( 2/200 ) in the general pregnant population . this may explain why the measurement of mca - psv has not been used as a routine test in japan as well as most other countries . our clinical path inevitably did not include measurement of fetal mca - psv for otherwise healthy women . therefore , we did not have a predefined clinical path for cases that were incidentally found to have mca - psv mom 1.5 . the present review of 100 mca - psv measurements around gw 30 suggested that the prevalence rate of mca - psv mom 1.5 was not high ( approximately 2% ) . therefore , the following treatments were recently introduced into our clinical path for cases with incidental mca - psv mom 1.5 : continuous fhr tracing for 30 min and determination of maternal afp and hbf fraction levels . clinical fmh is rare 14 , with only a few fmh cases with in utero transfusion reported to date 7,8,14 . cases with in utero transfusion were limited to those with hydrops fetalis 7,8,14 , and the effects of in utero transfusion on outcome have not yet been established . however , early suspicion of fmh , early confirmation of cord blood hb concentration in pale neonates , and early blood transfusion may be effective for avoiding neurological sequelae in such anemic neonates 4 . the mother complained of nonspecific symptoms , including heartburn at gw 26 , heartburn and generalized itch without skin eruption at gw 28 , and persistent nausea on the day of admission . rise of ast / alt concomitant with a decreased platelet count on the day of admission suggested impending hellp syndrome . it is unclear at present whether these symptoms were associated with the occurrence of fmh and whether the impending hellp syndrome was associated with fmh , although a case with onset of hellp syndrome soon after massive fmh caused by blunt abdominal trauma was reported previously 15 . in conclusion , this case indicated the existence of persistent fmh . however , a policy of screening with mca - psv for detection of occult fmh targeting the general pregnant population was considered to be too labor intensive and not cost effective .

minor salivary gland tumors include a heterogeneous group of neoplasms with varied histology accounting for 10 - 15% of all salivary gland neoplasms , most of which are located in the palate . it was first described by sheldon et al . in 1943 and was considered a variant of pleomorphic adenoma . in 1991 , the world health organization classified myoepithelioma as a distinct entity . this tumor frequently affects the parotid gland and palate , less commonly it can occur on the skin and soft tissue . no sex predilection is noted and usually it occurs as a slow growing painless mass . various histological types of myoepithelioma described include spindle , plasmacytoid or hyaline , epitheloid , clear and oncocytic ( a variant of spindle type ) . myoepithelioma of parotid occur in older people and are composed more often of spindle and epithelia cells whereas those of minor glands occur in slightly younger individuals and are composed of plasmacytoid cells . recently , hakeem et al . who reviewed 20 reported cases of myoepithelioma came across only two clear cell variants occurring in the palate , making this lesion extremely rare . here we discuss a case of clear cell myoepithelioma of palate with emphasis on the clinical and histological differential diagnosis . a 58-year old male patient reported to the department of oral medicine and radiology of pms college of dental science and research ( kerala , india ) with a slow growing painless swelling on the palate that had been present for almost 4 years . extra oral examination revealed no abnormalities . on intraoral examination , a smooth surfaced sessile swelling was seen at the junction of hard and soft palate , 2 cm from the midline and 1 cm away from the crest of the edentulous alveolar ridge near to the left maxillary tuberosity region . the lesion was slightly yellowish , well circumscribed , and non - tender with a soft consistency . there was no evidence of superficial vascularity as the lesion did not blanch on palpation . the swelling appeared to slip under the mucosal surface on palpation ( figure 1 ) . the patient was largely edentulous except for the three standing teeth ( upper right maxillary central and lateral incisors and maxillary right upper canine ) . the clinical differential diagnosis of a slow growing , soft , non tender , non ulcerated smooth surfaced sessile mass at the junction of hard and soft palate includes a list of lesions comprising of commonly occurring palatal abscess , cysts both odontogenic and non - odontogenic ; soft tissue neoplasms like fibroma , lipoma , neurofibroma , schwannnoma , leiomyoma and also minor salivary gland neoplasms . reactive lesions like pyogenic granuloma was not considered in the differential diagnosis as there was no recognizable irritant like dentures , dental plaque etc . soft and slippery consistency of the lesion helped in eliminating hard tissue tumors ; hard tissue and vascular malformations ; and lesions like fibroma , irritation fibroma and ossifying fibroma . while preparing the diagnostic list , several soft tissue tumors of connective tissue ( muscle , neural , adipocytic ) , and salivary gland origin had to be considered , as they have similar clinical features especially when occurring in the palate . the lesion being a slow growing one , high - grade malignancies were not considered in the differential diagnosis . benign muscle tumor like leiomyoma was considered in the differential diagnosis taking into account the age of the patient and the location , but granular cell myoblastoma was not considered as it was more common in the tongue than in the palate . benign nerve tissue tumors like neurofibroma and neurilemmoma had several features similar to this case . out of the two , neurofibroma was as a more probable option considering its relatively higher frequency on the palate . the color , the size and the feel of the lesion also suggested the possibility of lipoma , in spite of the fact that classical lipomas occur rarely on the palate . some literature describes sialolipoma , a relatively rare lesion with more incidences in the palate than classical lipoma , to be having certain clinical features similar to this case . considering the frequency of occurrence in the palate and the softness of the lesion , benign salivary gland tumors had to be strongly considered probably before any of the muscle or nerve tissue tumors in the panel of diagnostic hypothesis . of the benign salivary gland tumors occurring in the palate , pleomorphic adenoma was the most probable and other tumors like basal cell adenoma , myoepithelioma , and cyst adenoma further down in the differential diagnosis list . the long duration ( 4 years ) , the lesion took to grow up to the present size helped in ruling out the possibility of malignant salivary gland neoplasms , however slow growing polymorphous low grade adenocarcinoma and low grade mucoepidermoid carcinoma could be considered . since palatal involvement of lesions like non - hodgkin lymphoma is usually associated with human immunodeficiency virus infection , it was not considered as the patient was relatively healthy and free from hiv infection . even though rare in the palate , possibility of fibrous histiocytoma was kept in mind as the lesion presented as a painless submucosal nodule in an elderly adult . thus the clinical differential diagnosis list was formulated considering the clinical features of the patient s tumor , prevalence of previously described lesions , and demographic data . the list of lesions suggested had salivary gland tumors like pleomorphic adenoma , basal cell adenoma , myoepithelioma , cyst adenoma , low grade mucoepidermoid carcinoma , polymorphous low grade adenocarcinoma occupying the top slots in the list followed by other lesions like lipoma , neurofibroma , neurilemmoma , leiomyoma and benign fibrous histiocytoma . however , the diagnosis of any soft tissue tumor can only be confirmed by histopathological investigation like fine needle aspiration cytology or incisional / excisional biopsy . the excised tissue was fixed in 10% formaldehyde for 24 h. routine laboratory procedures were followed by paraffin embedding of the tissue . five m thick sections were obtained and stained with hematoxylin and eosin ( h&e ) for histological analysis ( figures 2 - 5 ) . microscopically , it was a thinly encapsulated tumor composed mainly of sheets of clear cells mixed with cells having eosinophilic cytoplasm . the cells showed small round to oval , bland nuclei that was eccentrically placed . in between these cells hyalinized fibrous septae were seen . the differential diagnoses of clear cell tumors that can possibly occur in this region include benign and malignant salivary gland neoplasms and metastatic clear cell lesions especially from kidney and thyroid . malignant primary salivary gland neoplasms include clear cell variant of mucoepidermoid carcinoma , acinic cell carcinoma showing clear cell changes , epithelial myoepithelial carcinoma , clear cell carcinoma , clear cell myoepithelial carcinoma and sebaceous carcinoma . in contrast to carcinomas , myoepitheliomas have a non - infiltrative , well - circumscribed periphery . malignant primary and metastatic lesions were ruled out as there was no infiltrative growth pattern , cytologic pleomorphism , high mitotic rate , coagulative necrosis or lack of encapsulation . the benign clear cell tumors of salivary gland include pleomorphic adenoma , myoepithelioma , oncocytoma , oncocytic hyperplasia and sebaceous adenoma . absence of glanduloductal differentiation and chondromyxoid or chondroid foci made us delete pleomorphic adenoma from the differential diagnosis . oncocytoma and oncocytic hyperplasia show the characteristic appearance of oncocytes . in the clear cell variant of oncocytoma , in which the clear cells are a dominant or partial component sebaceous adenoma is composed of irregularly sized and shaped nests of sebaceous cells without cytologic atypia . when pleomorphic adenoma , oncytoma , oncocytic hyperplasia and sebaceous adenoma were excluded from the histological diagnosis the most consistent was the positive staining for cytokeratin , s100 and sma whereas vimentin and gfap expression vary . among cytokeratins ck7 and ck14 are found usually positive . pan cytokeratin and myoepithial markers like calponin , s-100 , p63 are also generally positive for myoepitheliomas . in our case , immunohistochemical markers pan cytokeratin , s-100 and sma was done and most of the tumor cells showed positivity for all the three markers ( figures 6 - 8 ) . the clinical differential diagnosis of a slow growing , soft , non tender , non ulcerated smooth surfaced sessile mass at the junction of hard and soft palate includes a list of lesions comprising of commonly occurring palatal abscess , cysts both odontogenic and non - odontogenic ; soft tissue neoplasms like fibroma , lipoma , neurofibroma , schwannnoma , leiomyoma and also minor salivary gland neoplasms . reactive lesions like pyogenic granuloma was not considered in the differential diagnosis as there was no recognizable irritant like dentures , dental plaque etc . soft and slippery consistency of the lesion helped in eliminating hard tissue tumors ; hard tissue and vascular malformations ; and lesions like fibroma , irritation fibroma and ossifying fibroma . while preparing the diagnostic list , several soft tissue tumors of connective tissue ( muscle , neural , adipocytic ) , and salivary gland origin had to be considered , as they have similar clinical features especially when occurring in the palate . the lesion being a slow growing one , high - grade malignancies were not considered in the differential diagnosis . benign muscle tumor like leiomyoma was considered in the differential diagnosis taking into account the age of the patient and the location , but granular cell myoblastoma was not considered as it was more common in the tongue than in the palate . benign nerve tissue tumors like neurofibroma and neurilemmoma had several features similar to this case . out of the two , neurofibroma was as a more probable option considering its relatively higher frequency on the palate . the color , the size and the feel of the lesion also suggested the possibility of lipoma , in spite of the fact that classical lipomas occur rarely on the palate . some literature describes sialolipoma , a relatively rare lesion with more incidences in the palate than classical lipoma , to be having certain clinical features similar to this case . considering the frequency of occurrence in the palate and the softness of the lesion , benign salivary gland tumors had to be strongly considered probably before any of the muscle or nerve tissue tumors in the panel of diagnostic hypothesis . of the benign salivary gland tumors occurring in the palate , pleomorphic adenoma was the most probable and other tumors like basal cell adenoma , myoepithelioma , and cyst adenoma further down in the differential diagnosis list . the long duration ( 4 years ) , the lesion took to grow up to the present size helped in ruling out the possibility of malignant salivary gland neoplasms , however slow growing polymorphous low grade adenocarcinoma and low grade mucoepidermoid carcinoma could be considered . since palatal involvement of lesions like non - hodgkin lymphoma is usually associated with human immunodeficiency virus infection , it was not considered as the patient was relatively healthy and free from hiv infection . even though rare in the palate , possibility of fibrous histiocytoma was kept in mind as the lesion presented as a painless submucosal nodule in an elderly adult . thus the clinical differential diagnosis list was formulated considering the clinical features of the patient s tumor , prevalence of previously described lesions , and demographic data . the list of lesions suggested had salivary gland tumors like pleomorphic adenoma , basal cell adenoma , myoepithelioma , cyst adenoma , low grade mucoepidermoid carcinoma , polymorphous low grade adenocarcinoma occupying the top slots in the list followed by other lesions like lipoma , neurofibroma , neurilemmoma , leiomyoma and benign fibrous histiocytoma . however , the diagnosis of any soft tissue tumor can only be confirmed by histopathological investigation like fine needle aspiration cytology or incisional / excisional biopsy . the excised tissue was fixed in 10% formaldehyde for 24 h. routine laboratory procedures were followed by paraffin embedding of the tissue . five m thick sections were obtained and stained with hematoxylin and eosin ( h&e ) for histological analysis ( figures 2 - 5 ) . microscopically , it was a thinly encapsulated tumor composed mainly of sheets of clear cells mixed with cells having eosinophilic cytoplasm . the cells showed small round to oval , bland nuclei that was eccentrically placed . in between these cells hyalinized fibrous septae were seen . the differential diagnoses of clear cell tumors that can possibly occur in this region include benign and malignant salivary gland neoplasms and metastatic clear cell lesions especially from kidney and thyroid . malignant primary salivary gland neoplasms include clear cell variant of mucoepidermoid carcinoma , acinic cell carcinoma showing clear cell changes , epithelial myoepithelial carcinoma , clear cell carcinoma , clear cell myoepithelial carcinoma and sebaceous carcinoma . in contrast to carcinomas , myoepitheliomas have a non - infiltrative , well - circumscribed periphery . malignant primary and metastatic lesions were ruled out as there was no infiltrative growth pattern , cytologic pleomorphism , high mitotic rate , coagulative necrosis or lack of encapsulation . the benign clear cell tumors of salivary gland include pleomorphic adenoma , myoepithelioma , oncocytoma , oncocytic hyperplasia and sebaceous adenoma . absence of glanduloductal differentiation and chondromyxoid or chondroid foci made us delete pleomorphic adenoma from the differential diagnosis . oncocytoma and oncocytic hyperplasia show the characteristic appearance of oncocytes . in the clear cell variant of oncocytoma , in which the clear cells are a dominant or partial component , the sparse granularity of the typical oncocyte will still be evident . sebaceous adenoma is composed of irregularly sized and shaped nests of sebaceous cells without cytologic atypia . when pleomorphic adenoma , oncytoma , oncocytic hyperplasia and sebaceous adenoma were excluded from the histological diagnosis , we came to the conclusion that our case is a clear cell myoepithelioma . the most consistent was the positive staining for cytokeratin , s100 and sma whereas vimentin and gfap expression vary . among cytokeratins ck7 and ck14 are found usually positive . pan cytokeratin and myoepithial markers like calponin , s-100 , p63 are also generally positive for myoepitheliomas . in our case , immunohistochemical markers pan cytokeratin , s-100 and sma was done and most of the tumor cells showed positivity for all the three markers ( figures 6 - 8 ) . they are present in major and minor salivary glands , sweat glands , lacrimal glands , prostate , breast , nasopharynx , lung , retroperitoneal , skin and soft tissue . though myoepitheliomas can develop in any of these regions , its frequency is much less . three - fourths of all cases of myoepitheliomas occur in the parotid gland and palate . our case was also observed in the palate . a common stem cell with a bidirectional differentiation into epithelial or myoepithelial cell the varied histological types ( spindle , plasmacytoid or hyaline , epitheloid , clear and oncocytic ) exhibited by myoepitheliomas can be attributed to the various stages in the differentiation from a cell that has the potential to differentiate into epithelial cells . though different cell types are recognized in myoepitheliomas , spindle and plasmacytoid variants are most common . in a tumor , either a single cell type predominates or there can be a mixture of different cell types . myoepitheliomas can present several architectural patterns which are non - myxoid ( solid ) , myxoid ( pleomorphic adenoma - like ) , reticular ( canalicular - like ) and mixed . it must be because most of the cases showed a solid , non - myxoid arrangement . suggested that clear cell tumors of the salivary glands are almost invariably malignant in nature with rare exceptions in the form of myoepitheliomas and oncocytomas . both benign and malignant variants of myoepithelioma may contain clear cells with latter being less common . the clear cells of myoepithelioma stain positive for glycogen and negative for mucin and fat . accumulation of glycogen in the cytoplasm causing cytoplasmic clearing accounts for the pathogenesis of clear cells demonstrated in the myoepitheliomas . parotid myoepitheliomas are surrounded by a thin fibrous capsule , whereas palatal myoepitheliomas are not . that benign myoepitheliomas present as an asymptomatic mass that slowly enlarges over a period of several months to years . they also added that palatal lesions rarely ulcerate and parotid lesions never produce facial palsy . a malignant counterpart of myoepithelioma with distinct clinical and histological features has also been reported . patients with myoepithelial carcinoma are generally aged over 50 years and a majority presents with a painless mass . the parotid gland is the most common primary site , followed by submandibular gland and minor salivary glands . unusual locations reported earlier include palate , gum , larynx , lateral wall of the nasopharynx , base of the tongue , maxillary sinus and cavernous sinus . it may arise de novo but at least 50% develop in the preexisting pleomorphic adenoma or myoepithelioma . there are also sporadic reports of malignant myoepilethelioma at unusual sites like breast , bone , external auditory canal , skin and vulva . benign myoepitheliomas can undergo malignant transformation especially in long standing tumors or in tumors with multiple recurrences . it is an intermediate to high grade carcinoma with approximately onethird of patients dying , one - third having multiple recurrences and remaining one third being disease free . the recommended management of myoepithelioma is surgical excision with a margin of uninvolved tissue around . recurrence of myoepithelioma of parotid gland presenting as a retroauricular cutaneous nodule has also been reported . myoepitheliomas of the palate are rare with clear cell variant even rarer . only two cases of clear cell myoepithelioma of palate have been reported so far . our case electricon microscopy in these tumors helps in the identification and characterization of the myoepithelial cells . the rarity of this lesion makes this a diagnostic challenge and while screening all benign clear cell lesions of the palate , diagnosis of myoepithelioma should be kept in mind .

a 26-year - old male presented to us following a seizure with left sided weakness ( 4-/5 , medical research council ( mrc ) grade ) . the past medical history was significant for a traumatic brain injury seven years prior that involved a depressed skull fracture after being hit with a baseball bat . a brain computed tomography ( ct ) scan at admission showed a 3.5 2.6 cm hematoma in the right parietal lobe adjacent to the prior craniotomy site . there was also bony separation at the margins of the prior craniotomy with presence of protrusion of the brain parenchyma through the defect ( fig . a magnetic resonance angiogram ( mra ) of the brain suggested a pseudoaneurysm ( fig . 1d ) , which was confirmed on a catheter cerebral angiogram that demonstrated the presence of a pseudoaneurysm arising from the right middle cerebral artery m4 segment measuring 3 3 mm in size with a neck of 1 mm that was successfully coiled ( fig . an excelsior sl-10 micro catheter ( stryker , kalamazoo , michigan , u.s.a . ) was used for navigating the vascular anatomy , followed by deployment of a single platinum coil measuring 2.5 5 to achieve complete occlusion of the aneurysm . a new occlusion of the distal branches of the angular artery , likely thromboembolic , was identif ied on the f inal angiogram , but was regarded as clinically insignificant ( fig . the post - operative course was uneventful with improvement in left - sided hemiparesis to almost normal strength , and the patient was discharged home on day three . he had been doing well at the 12-month follow - up , with no motor or sensory deficits and a modified rankin scale score of 1 . the patient does have occasional partial seizures likely from the prior depressed skull fractures ( engle class ib ) , which are currently well - controlled with the use of medications . intracranial aneurysms can be classified as congenital , atherosclerotic , dissecting , infectious , and post traumatic . traumatic intracranial aneurysms ( ticas ) are rare and account for less than 1% of the intracranial aneurysms and are more common in children and adolescents . they can occur after even mild or seemingly trivial head trauma , and are associated with a morbidity and mortality rate as high as 50% . the mca has the most complex branching pattern of the major intracranial arteries and ticas are frequently found in the cortical branches of middle cerebral artery ( mca ) . ticas may develop beneath an overlying linear or depressed skull fracture and are attributed to direct vessel trauma leading to formation of a pseudoaneurysm . histologically , ticas can be classified as true , false , dissecting or mixed based on the damaged structures of the vascular wall . a pseudoaneurysm ( false aneurysm ) , which is the most common type , occurs when all layers of the arterial wall are lacerated , and is almost always associated with direct injury to cortical arteries , which are often beneath a skull fracture . several mechanisms have been proposed in the formation of traumatic aneurysms , all of which involve either direct injury to the vessel or stretching of the vessel by adjacent forces . the case we encountered is very unusual , as the aneurysm did not develop from the direct trauma of the vessel from the depressed bone fragments ; but rather occurred secondary to the outpouching of the brain parenchyma through the dural defect between the edges of the craniotomy flap and skull made during the initial repair of the depressed skull fracture ( fig . this likely resulted in stretching and tearing of the arterial wall from the bony edges leading to formation of a pseudoaneurysm . while debridement , elevation of the fracture , removal of bone fragments , and delayed cranioplasty ( if needed ) remains the most common way to manage depressed skull fractures , there is an increasing consensus that immediate bone fragment replacement / cranioplasty does not result in any increase in infectious complications and may be preferable as it also obviates the need for a second procedure . also , while prevention of infection and epilepsy remains the main driver of treatment for a compound depressed fracture , the present case highlights an unusual complication of a traumatic pseudoaneurysm formation that can be a cause of delayed morbidity and perhaps mortality . as the pseudoaneurysm likely resulted from the herniation of brain parenchyma through the dural defect , the likely pathogenesis involved presence of an occult dural tear that was sustained at the time of initial injury and was not apparent during the initial surgery . a rare of case of a delayed traumatic pseudoaneurysm of the middle cerebral artery following surgical repair of a compound depressed fracture of the skull is reported . being aware of this complication in providing long - term treatment to patients with depressed skull fractures can be helpful to avoid and treat this unusual complication .

wolff - parkinson - white ( wpw ) syndrome is characterized by ventricular pre - excitation that ventricles are electrically and mechanically pre - excited through an accessory pathway , which connects atria and ventricles . a rare cause of morbidity in infants and children with wpw syndrome is left ventricular ( lv ) dysfunction or dilated cardiomyopathy ( dcm ) , which may attribute to the recurrent or incessant tachycardia.1)2 ) recently however , several small case series and case reports suggested a possible association between accessory pathways and ventricular dysfunction in the absence of sustained tachyarrhythmias.3 - 11 ) the eccentric premature ventricular activation via an accessory pathway results in an asynchronous spread of ventricular depolarization and this asynchronous ventricular activation may provoke ventricular dysfunction.12 ) the occurrence of ventricular dysfunction in asymptomatic wpw syndrome may depend on the location of the accessory pathway , and invariably it was reported the electrophysiological features was the right sided accessory pathway , especially in the septal or paraseptal location.7)10 ) it is thought that this type of accessory pathway induces abnormal interventricular septal wall motion leading to lv dyssynchrony and subsequently ventricular dysfuction or dcm through lv remodeling . this non - tachycardia type of lv dysfunction with wpw syndrome is characterized by complete recovery of the ventricular function after ablation or spontaneous regression of pre - excitation . it is the purpose of this article to review the clinical and electrophysiologic evidence for this association and to discuss the underlying pathophysiological mechanism . the prevalence of lv dysfunction associated with wpw syndrome in the absence of tachyarrhythmias is unknown . age distribution of these patients with pre - excitation related lv dysfunction is wide , ranging from infants to old age . in 1998 yamanaka et al.3 ) suggested the association between lv dysfunction and wpw syndrome for the first time in a 67 year old man with type b wpw syndrome , of whom cardiac function improved after catheter ablation . several years thereafter , in 2004 emmel et al.4 ) described the improvement of ventricular function after the disappearance of pre - excitation in 4 children with dcm associated with wpw syndrome , and suggested a causal relationship between ventricular pre - excitation and dcm . thereafter , only several small case series and case reports have been reported until now and summarized in table 1 . some patients with abnormal wall motion might have undergone an electrophysiological study and catheter ablation before overt ventricular dysfunction developed . it is also possible that some patients have been considered only to have idiopathic dcm coincidentally . patients with manifested antegrade accessory pathway conduction have asynchronous ventricular wall motion because of abnormal impulse propagation by premature eccentric ventricular activation over a bypass tact . it was found in the earlier echocardiographic studies13 - 16 ) that the pattern of abnormal ventricular contraction was dependent on the location of the accessory pathway . left - sided accessory pathways produced premature anterior motion in the posterior lv wall and right - sided accessory pathways showed abnormal interventricular septal motion . left - sided accessory pathways producing typical anterior motion of the lv posterior wall is visible on m - mode echocardiography in early systole as a small , distinct bump and this unopposed early contraction of the posterior wall results in abnormal anterior motion of the interventricular septum . the right - sided accessory pathway related abnormal interventricular septal motion consists of an early systolic posterior and subsequent midsystolic anterior motion , with a delay in the usual late systolic septal movement . these abnormal septal motions in type b wpw syndrome are similar to those described in patients with left bundle branch block ( lbbb ) . grines et al.17 ) observed abnormal interventricular septal motion in patients with lbbb and this abnormal septal motion in lbbb corresponded to periods of asynchrony in contraction , ejection , end systole , and end diastole between the right and left ventricle . they also found that regional ejection fraction of the septum decreased in lbbb compared with that of normal subjects and this loss of septal contribution resulted in a reduction in global ejection fraction in lbbb compared to normal subjects . in the earlier echocardiographic studies , they observed that abnormal septal motion was normalized after loss of pre - excitation in patients with type b wpw syndrome and suggested that abnormal septal motion is related to the altered sequence of ventricular activation during pre - excitation.14)15 ) several recent studies suggested that wpw syndrome may lead to regional and global contractile dysfunction resulting from aberrant impulse propagation.5)9)10)12 ) the right septal and paraseptal accessory pathways have been considered the main location to induce lv dysfunction . tomaske et al.7 ) showed in 56% of patients with right septal or posteroseptal accessory pathways , lv ejection fraction decreased and septal - to - posterior wall motion delay increased . they discussed the importance of septal and posteroseptal location of accessory pathways to cause ventricular dysfunction . the extent of the pre - excited ventricular myocardium depends on the relative timing of normal and eccentric ventricular activation . in left free wall pathways , the amount of pre - excited myocardium is rather small because of long conduction time from the sinus node to the atrial pathway insertion leading to almost normal activation of the left ventricle via atrioventricular nodal conduction . in right free wall pathways , lv activation is almost synchronous over the normal conduction pathway , with a precontraction area being limited to the right ventricular free wall . in contrast to right free wall pathways , septal and posteroseptal pathways may induce earlier interventricular activation , subsequently substantial part of septal pre - excitation and delayed activation of lv free wall . early septal activation may induce paradoxical or hypokinetic interventricular septal motion , similar to septal motion abnormalities seen in patients with right ventricular pacing or lbbb , and this dyskinetic segment may function like an aneurysm and induce adverse remodeling with progressive dilation . the detrimental effect of chronic right ventricular pacing or lbbb on lv function has been well described . chronic asynchronous electric activation in the patients with lbbb or right ventricular pacing disturbs the myocardial regional workload and wall stress , which may result in abnormal wall motion , regional myocardial perfusion defects leading to adverse remodeling with asymmetry of wall thickness and progressive lv dilation.17 - 20 ) in an animal study,21 ) the abnormal impulse propagation by ventricular pacing causes considerable disturbances in regional systolic fiber shortening , mechanical work , blood flow , and oxygen consumption - and in early activated regions , the values are low . an early activation of the basal septum via septal and paraseptal pathways will induce segmental contraction which is unopposed by the activation of the remaining left ventricle . on these early contracting segments , imposed preload will be low , resulting in decreased myocardial work and thinning of the myocardium , which is manifested as extreme systolic bulging of the pre - excited basal septum towards the right ventricle . 1 shows paradoxical systolic bulging of the basal septum in a 9-year - old girl with right anterior accessory pathway . this early basal septal segmental contraction with bulging and delayed free wall activation may lead to significant dyssynchrony in the lv motion and thereby cause ventricular dysfunction . a study using recent echocardiographic technique22 ) showed local ejecting shortening was impaired at the pre - excitation site and at similar electrical and mechanical dyssynchrony , wpw syndrome with septal accessory pathways had more extensive hypokinesia than did wpw syndrome with lv free wall accessory pathways . kwon et al.9 ) reported that lv systolic function was reduced in all patients with septal pathways , compared to patients with left or right free wall pathways . in their study , compared to patients with normal septal motion , patients with septal dyskinesia had a reduced lv ejection fraction and an increased lv end diastolic dimension . tomaske et al.7 ) also suggested that wpw syndrome with right septal or posteroseptal pathways may cause lv dyssynchrony and jeopardize global lv function . they used conventional echocardiographic and recent speckle tracking imaging techniques to see lv dyssynchrony in patients with septal accessory pathways . in these two recent studies , these observations support the importance of dyssynchrony in the pathogenesis of lv dysfunction associated with ventricular pre - excitation . 2 shows a heterogeneous pattern in the radial strain assessment using 2-dimensional speckle tracking echocardiography that changed into a more homogeneous pattern after catheter ablation of the right anterior accessory pathway in a 9-year - old girl with asymptomatic wpw syndrome . however , it is unknown why not all patients with right sided accessory pathways develop ventricular dysfunction , in the absence of incessant tachyarrhythmias . in 1977 , levovitz et al.16 ) suggested that the determinants of abnormal septal wall motions are the location , right ventricular lateral freewall or septal and degree of ventricular pre - excitation . they reported that asynchronous movement was observed only in their patients in whom duration of the qrs complex was greater than 130 msec . in the study by kwon et al.,9 ) all patients with septal dyskinesia and reduced lv ejection fraction showed longer pre - excited qrs duration than those with normal septal motion . another recent study suggested that different locations of paraseptal accessory pathways may induce a variable degree of lv dyssynchrony and dysfunction . tomaske et al.7 ) reported that their patients with midseptal and anteroseptal pathways demonstrated lower lv ejection fraction and a higher degree of intraventricular lv dyssynchrony compared with those with posteroseptal pathways . the degree of pre - excitation and the degree of dyssynchrony induced by the different location of the accessory pathway may be the additional factor for development of ventricular dysfunction in these patients . the prognosis of accessory pathway induced lv dysfunction or dcm is excellent . in the reported cases shown in table 1 , cardiac function improved and normalized acutely or within several months after loss of pre - excitation , either spontaneously or by catheter ablation or appropriate medical therapy . these findings suggest that recovery of ventricular function through lv reverse remodeling occurs after loss of ventricular pre - excitation . in infants and small children , catheter ablation is chosen as a last alternative therapy due to its higher risk of complications and uncertain long - term effect of ablation scars on young myocardium.23)24 ) cadrin - tourigny et al.6 ) reported severe lv dysfunction in two young infants with asymptomatic wpw syndrome and in both infants , successful pharmacologic suppression of posteroseptal accessory pathway conduction with amiodarone resulted in dramatic improvement in lv function . therefore , accessory pathway associated ventricular dysfunction should be suspected in all patients presenting with dcm and ventricular pre - excitation , especially with right sided accessory pathways , when tachyarrhythmias induced ventricular dysfunction is ruled out . however , many asymptomatic patients with wpw and septal and paraseptal accessory pathways may have abnormal ventricular wall motion without overt ventricular dysfunction or dcm and the catheter ablation of accessory pathways in these patients is still controversial . it seems reasonable that catheter ablation should be reserved for those patients in whom progression of adverse lv remodeling and deteriorating cardiac function are demonstrated in the conventional and recently developed techniques of echocardiography during follow - up . the causal relationship between lv dysfunction or dcm and wpw syndrome in the absence of tachyarrhythmias is recently recognized . right - sided accessory pathways may induce lv dyssynchrony , leading to adverse remodeling and ventricular dysfunction . loss of ventricular pre - excitation spontaneously or by catheter ablation results in mechanical resynchronization , reverse remodeling and lv function recovers completely .

insertion of the double j catheter into the ureter , which facilitates drainage of the upper urinary tract , is one of the most common urological procedures this medical procedure is generally thought of as quite easy and safe to perform , with a very low rate of severe complications . nevertheless , as any other medical procedure , it is usually associated with some bothersome symptoms such as pain , dysuria , fever , erythrocyturia , mucose membrane injury , or ureter peristalsis disturbances , all of which have a negative impact on the patient s quality of life . other complications include anxiety , sexual dysfunction , sleep disturbances , and absence at work . sporadically forgotten stents should also be mentioned as they may result in serious complications such as marked incrustation , stent fragmentation , urosepsis , or even renal failure [ 15 ] . it must be , however , underlined that bacterial or fungal colonization of both catheter surface and urine is the most frequent complication that is noted . it is observed especially during long term stent retaining [ 6 , 7 ] . king et al . performed a systematic review , which has disclosed that urinary catheterization was the major risk factor of healthcare associated urinary tract infections . the authors have calculated that 79.3% of this complication could have been prevented if catheter insertion had been avoided . many authors therefore investigate the types of pathogens isolated from catheters and urine as well as the correlation between urine and stent culture . the relationship between colonization and stenting duration , as well as some other aspects , such as age , gender , co morbidities , reason for stenting , and the method of catheter insertion , are also found in their field of interest . clinical trials published recently have shown great inconsistency between microbiological analysis of the stent surface and urine . it concerns both the presence of colonization and the type of pathogen cultured from the double j catheters and the urine sample . available data is controversial , therefore it seems that there is a place for further studies in this field [ 9 , 10 ] . the aim of our study was to estimate the relationship between bacterial colonization of the double j catheter , which was dissected into three separate sections , and the microorganisms isolated from urine . we have also examined the rate of urine and catheter colonization as well as the types of pathogens that were cultured . we decided to use microorganism dna analysis while exploring the catheter colonization as this method is thought to possess the highest diagnostic sensitivity . the study sample comprised 60 patients ( 25 women and 35 men , aged 34 to 67 years ) who had double the participants of the trial were recruited from the 2 department of urology of the medical university of lodz between january 2011 and june 2012 . indications for ureteral catheter insertion were the following : urinary tract lithiasis , hydronephrosis due to ureteropelvical junction stricture , ureter stricture , tumors , and the presence of additional blood vessels . the patients were divided for further analysis into three subgroups , according to stenting duration : 1 ) subjects with their stents kept for 20 to 30 days ( 18 cases ) ; 2 ) participants with the catheters retained longer than 30 but shorter than 90 days ( 30 cases ) ; 3 ) patients with their catheters kept for the period longer than 90 days ( 12 cases ) . there were no statistical differences in age and gender distributions between the three subgroups all the participants had their midstream urine samples taken prior to the stent insertion and removal . a negative urine culture before catheterization was mandatory to participate in the study . j stents were inserted via an open or endoscopic manner ( 9 and 51 cases , respectively ) . in our trial , 500 mg of ciprofloxacin twice daily , was administered orally for five days . no episodes of urinary tract infection symptoms were noted in our subjects during the observation period . significant bacteriuria or funguria was defined as a bacterial or fungal count of more than 10 cfu / ml . double after initial analysis of the whole stent it was further divided , under aseptic conditions , into three sections : pelvic , ureteral , and vesicular , which were analyzed separately . bacterial and fungal dna was identified using electrophoresis in polyacrylamide gel with a denaturing gradient ( pcr dgge ) . the trial protocol was approved by the local ethics committee of the medical university of lodz . all the participants have given written informed consent after a complete description of the study . appropriate tests for statistical analysis were used and p values < 0.05 were considered statistically significant . double j catheter dna analyses were positive in all cases . in 2 cases one type of microorganism was isolated from the stent surface . the remaining 58 catheters were colonized by more than one pathogen , including 12 stents with two species and 46 stents with three types of pathogens . each of three sections of double j catheters was analyzed separately . 32 stents were colonized by the same pathogens on all three sections . in 6 cases , stent analyses have shown two types of microorganisms isolated from the different parts , while the remaining 22 catheters were colonized by three different bacteria or fungi species . bacteria species that were most frequently isolated from the catheter surface were the following : staphylococcus aureus , enterococcus faecalis , pseudomonas aeruginosa and escherichia coli . j catheter sections urine cultures were positive in only 8 patients before the catheter removal . only three bacteria species were isolated from the urine specimens : escherichia coli , pseudomonas aeruginosa , and klebsiella oxytoca . the number of bacteria species isolated from urine specimens is shown in table 2 . microorganisms cultured from urine specimens urine and catheter cultures were consistent in 5 cases . in 3 cases urine culture and stent analyses have shown different bacteria species . of 18 urine specimens taken from the subjects with the shortest period of the stent retaining , only 1 was infected ( 5.5% ) . in case of catheters left in the urinary tract for the period of longer than 30 days but shorter than 90 days , 3 of 30 urine samples urine specimens taken from the patients with catheters retained for longer than 90 days were infected in 6 of 12 cases ( 50% ) . gender , reason for catheter insertion , and the method of stenting were not related to variables such as urine culture and the number of pathogens isolated from the stent surface . they are consistent with our previous observations , where we noted that 64 of 65 stents were colonized . also riedl et al have found a 100% risk for colonization while retaining stents long term . similarly , farsi et al . have observed a 67.9% rate of stent colonization . on the other hand , , point to a nearly 44% risk of catheter colonization [ 12 , 13 ] , and kehinde et al . of nearly 42% . urine culture was positive in 8 of 60 patients in our study , which accounts to about 13% . riedl et al . have noted a 45% risk of urine infection during short term stenting and 100% during long term stenting . . a possible explanation for such differences in the rate of stent colonization and urine infection may be the sample of patients ( number , age , sex , concomitant diseases ) , type of catheter , use of prophylactic antibiotic therapy , or different methodology of colonization estimation ( culture , dna analysis ) . the results of many trials show that the rate of catheter colonization is much higher than the rate of urine infection . farsi et al . noted in their study that urine cultures were positive in 29.9% while catheter colonization was found in 67.9% . point to more than 2.5fold higher risk of catheter colonization ( 42% ) than urine infection ( 17% ) . similarly , riedl et al . have noted that short term catheterization is accompanied with catheter colonization in 69% and urine infection in 45% . our previous study has shown that urine infection was detected in 17 of 65 cases while stent colonization was observed in 64 of 65 cases , so the risk for catheter colonization was 4fold higher . we have noted that the rate of stent colonization was 100% , and was 8fold higher than the incidence of urine infection . available data indicate , similarly to our present observation , that the species most frequently isolated from urine is escherichia coli [ 7 , 14 , 16 ] . among others , enterococcus sp . , proteus sp . , klebsiella sp . , and pseudomonas sp . urine specimens were colonized by only 3 bacteria species , while no fungi were observed . however , other authors point to other species as being the most common . kehinde et al . have noted that staphylococcus was isolated most frequently from urine samples . it must be stressed , however , that no antibiotic prophylaxis was administered in these patients , which could have had a significant impact on obtained results . in our study we have noted one such sample . in case of the double j catheters , the most common species isolated were pseudomonas aeruginosa , staphylococcus aureus , enterococcus fecalis , and escherichia coli . it is worth mentioning that species such as staphylococcus aureus , enterococcus fecalis were not isolated from any urine specimen . bacterial colonization seems to depend on the use of antibiotic prophylaxis . in the previously mentioned study of farsi et al . , in patients not given any antibiotic prophylaxis pseudomonas aeruginosa was the most common pathogen found on the stent surface and cultured from urine . have found that colonization of foley catheters was more frequent in patients not receiving systemic fluoroquinolones . he has also quite often observed polymicrobial colonization , with both gram positive and gram negative species . the above findings remain in agreement with our results , as in almost all of our cases ( 58/60 ) we have isolated more than one pathogen . in our previous study , 22 of 65 stents were colonized by more than one species . this is a very important issue as it points to the need for use of a quite broad spectrum of antibiotics in post procedure prophylaxis . this issue is of great importance , as urinary stenting is the procedure most commonly carried out in urology and also because of accompanying complications , especially high rates of urinary tract infections . the current strategies comprise either stent modifications or additional medical interventions both non pharmacologic and pharmacologic . stents ) , design ( distant coil stents vs. loop stents ) , diameter , or type of material that the stent is made of . modifications of stent surface such as silver coating , diamond like coating , hydrophobicity change , or antimicrobial activity molecules ( heparin , triclosan ) have been created , to inhibit or maybe even in the future to prevent , stent colonization and biofilm formation . use of antibiotic releasing biomaterials has additional advantages , as they deliver the drug to target tissues and have a positive impact on both effectiveness ( influencing directly the biofilm that is often resistant to systemic antibiotic therapy ) and lowering the risk of systemic side effects . additionally , it is suggested that increase in patient s fluid intake , augmentation of urine ph value ( via citrate consumption ) , analgesics , anticholinergics , alpha blockers , and/or prophylactic antibiotic therapy may decrease the rate of complications or minimize the complaints . unfortunately , none of the above described strategies has the power to fully prevent catheter colonization , so there is a need for further investigations and technological innovations in this field . it is well known that there are certain groups of patients who are extremely predisposed to this complication . the risk factors include age , sex , lower urinary tract obstruction , co morbidities such as diabetes , chronic renal failure , or impairment of the immune system . therefore , the indications for stenting in these groups of patients should be thoroughly considered . have achieved a significant reduction of the rate of catheterization thanks to educational intervention that was implemented in a teaching hospital in central italy . stenting duration is thought to be a strong risk factor for urine and/or stent colonization [ 9 , 13 , 17 ] . akay et al . observed the relationship between long term stenting and catheter colonization , but not urine infection . in light of these observations it seems that removal of the catheter should be performed as early as possible . on the contrary , in the group of patients presented in our study , no relationship between the duration of urinary stenting and catheter colonization could be observed , as all stents were colonized regardless of the duration . similarly , in our previous study almost all ( 64 of 65 ) double j catheters were colonized . these results point to a rule that stent insertion practically means it will be colonized . double j catheter retention in the urinary tract is associated with an extremely high risk of bacterial colonization , while the associated risk of urine infection is about 8fold lower . there is a great inconsistency between urine infection and catheter colonization , indicating a low predictive value of urine culture for estimating stent colonization . there is also a great heterogeneity of microorganisms colonizing double j catheters , while there were only three bacteria species isolated from urine specimens .

on rare occasions , pheochromocytoma and ganglioneuroma develop together within a single adrenal medullary tumor1 - 3 ) . it is apparent that composite tumors of that type may display symptoms referable to hormonal hypersecretion by either portion of the tumor1 ) . yet , pheochromocytomas commonly act in the typical manner and presented as cardiomyopathy4 - 6 ) or pancreatitis7 - 11 ) have been rarely documented . the non - specific nature of its manifestations may render prompt recognition elusive12 ) . we recently experienced a case of a composite adrenal medullary tumor of pheochromocytoma and ganglioneuroma that was incidentally discovered and masquerading as acute relapsing pancreatitis . to our knowledge a 48-year - old male was referred to our endocrinologic clinic for the evaluation of an adrenal incidentaloma . two months ago , he had been admitted to our gastroenterologic clinic at which time he complained of acute , severe intense abdominal discomfort . he claimed that the discomfort had a rapid onset reaching a maximum in 10 to 20 minutes . laboratory data revealed elevated serum pancreatic amylase and lipase levels ( reference range shown parenthetically ) of 98.8 u / l ( 8~53 ) and 603.4 u / l ( 0~60 ) , respectively . a diagnosis of acute alcoholic pancreatitis was suggested . seven days after he was discharged , the same symptoms and signs re - developed at which time he also described palpitation , facial flushing and resting hand tremor in the previous five months that were developed slightly , briefly and occasionally . on admission , however , serum pancreatic amylase and lipase levels were elevated to 81.4 u / l ( 8~53 ) and 100.9 u / l ( 0~60 ) , respectively . the serum cpk - mb ( creatinine phosphokinase - mb isoform ) level was 1.8 g / l ( 0.7~3.8 ) and the troponin - i level was 0.3 g / l ( 0~0.5 ) . the findings of a chest roentgenogram , electrocardiogram , and 24-hour holter monitor showed nothing signigficant . echocardiographic findings were normal except for mild concentric left ventricular hypertrophy . an abdominal computed tomographic scan showed a mildly enlarged pancreatic change without abnormal fluid collection and necrosis . in addition , he showed a left adrenal mass , 33 cm in diameter , which was heterogeneously low attenuated and enhanced after intravenous contrast administration ( figure 1a ) . about one month after the second discharge , we performed medical tests for discriminating the functional status of the adrenal incidentaloma . the urinary metanephrine level was elevated to 24.7 mol/24h ( 0.5~8.1 ) and the urinary vma ( vanillylmandelic acid ) level was normal at 27.5 mol/24h ( 7~33 ) . the results of plasma catecholamines , baseline plasma acth and cortisol , urinary - free cortisol , uplight plasma renin activity and aldosterone and serum dhea - s ( dehydroepiandrosterone sulfate ) levels were also within normal ranges . the results of 5-hiaa ( 5-hydroxyindoleacetic acid ) and thyroid function tests were also normal . i - mibg ( metaiodobenzyl guanidine ) scintigraphy was performed for the confirmation of the provisional diagnosis and localization of additional extra - adrenal tumors . i - mibg scintigraphic findings showed a marked uptake in a single location corresponding to the left adrenal mass ( figure 1b ) . ultrasonographic findings of the neck were unremarkable and serum intact pth and calcitonin levels were 14.39 pg / ml ( 15~65 ) and 1.4 ng / ml ( 0~10 ) , respectively . therefore , we could exclude pheochromocytoma from familial association of multiple endocrine neoplasia type ii . an -adrenoceptor blocker ( doxazocin mesylate 4 mg qd for 11 days ) was prescribed preoperatively to prevent an operative adrenal crisis . then , the left adrenal gland was surgically removed trans - abdominally through a subcostal incision . on pathologic examination , a composite adrenal medullary tumor of pheochromocytoma and ganglioneuroma was confirmed . on gross examination , a well - defined solid tumor measuring 2.82.52.5 cm was found within the adrenal gland and cut surface of the tumor was yellowish brown . the microscopic appearance of the tumor showed an abrupt transition of two different patterns of typical pheochromocytoma and a large area of ganglioneuroma ( figure 2a ) . in the pheochromocytoma area , organoid nests , called " zellballen " pattern , of polygonal cells surrounded by fibrovascular stroma were observed . each cell had granular basophilic or amphophilic cytoplasm and intracytoplasmic hyaline globules , positively stained by pas ( periodic acid - schiff 's reagent ) . its round or oval nuclei had a prominent nucleoli which contained inclusion - like structures and showed moderate pleomorphism with hyperchromasia ( figure 2b ) . at the ganglioneuroma area , uncapsulated clusters of fully matured ganglion cells surrounded by fascicles of spindle ( schwann - like ) cells were shown ( figure 2c ) . on immunohistochemical staining , it was weakly positive for nse ( neuron - specific enolase ) , chromogranin and synaptophysin , negative for hmb45 and ck in the pheochromocytoma component , and positive for s-100 protein in the ganglioneuroma component . now , the patient has remained well and free of any symptoms for more than eight months . although pheochromocytoma may occur at multiple sites and in association with a number of other tumors , the presence of both pheochromocytoma and ganglioneuroma within a single tumor is extremely rare . only a few cases have been described previously1 - 3 ) . while pheochromocytoma is a tumor that originated from the adrenal medullary chromaffin cells , ganglioneuroma represents a tumor from autonomic ganglion cells or their precursors . embriologically , both chromaffin and ganglion cells are derived from neural crest cells and migrate to somatic areas13 ) . it is apparent that a composite tumor of pheochromocytoma and ganglioneuroma may display symptoms referable to hormonal hypersecretion by either portion of the tumor1 ) , and moore et al.2 ) described hormonal hypersecretion in approximately three - fourths of the reported cases . clinically active pheochromocytoma may produce the classic symptoms of headache , palpitation , and excessive perspiration in 50% of the cases . in addition , hypertension , either sustained or paroxysmal , is the cardinal feature of pheochromocytoma14 ) . in our case , while the 24-hour urinary metanephrine level was elevated to 24.7 mol/24h ( reference range , 0.5~8.1 ) , no definite manifestations referable to catecholamine hypersecretion he only complained of palpitation , facial flushing and hand tremor which developed only slightly , briefly and occasionally . reviews have placed frequency of hypertension at 72.4% , with that of sustained hypertension at only 47.9% in pheochromocytomas12 ) , and moore et al.2 ) noted that only four of the 13 patients had associated hypertension in composite adrenal medullary tumors . the reason for a lack of endocrine abnormalities and symptoms of pheochromocytoma component in some composite adrenal tumors has not been shown conclusively . one possible hypothesis is the autoregulation of the pheochromocytoma cells by the ganglion cells in the ganglioneuroma component15 ) . pheochromocytomas commonly do not behave in the classic manner , which may render prompt recognition elusive12 ) . the signs and symptoms are often absent and can be unusually presented as catecholamine - induced cardiomyopathy4 - 6 ) or hyperamylasemia7 - 11 ) . in review of the literature , the amylase was almost exclusively of the s - type in the pheochromocytoma- associated hyperamylasemia . moreover , the pancreas itself was morphologically normal on image or surgical locations and lipase was within the normal range . in general , hyperamylasemia was probably caused by tumor production or ischemic injury to amylase containing tissue . in pheochromocytoma , the source of hyperamylasemia was thought to be pulmonary endotherial cells under ischemic damage caused by a potent vasoconstrictive action of circulating catecholamines16 ) . according to the theory of perrier el al.9 ) , functioning pheochromocytoma causes a catecholamine - induced cardiomyopathy , which contributes to the failure of the left ventricle , followed by pulmonary edema and release of amylase from hypoxic lung tissue . however , in our case , there was no clinical evidence of cardiomyopathy or myocardial ischemia based on electrocardiographic , echocardiographic findings and cardiac biochemical marker , and the clinical pictures of the patient suggested acute pancreatitis . the provisional diagnosis seemed reasonable because of the acute onset of abdominal intense discomfort , elevated serum pancreatic enzyme levels and a mildly enlarged pancreas on image . we did not perform an analysis of amylase isoenzyme by the electrophoretic method , but pancreatic amylase , not total amylase , was assayed by a enzymatic photometric test , which is based on the method of monoclonal antibodies to inhibit the salivary enzyme and which distinguish pancreatic and salivary amylase sufficiently17 , 18 ) . therefore , it could be suggested that the pancreatitis was caused by the pheochromocytoma in our case . the mechanism of this was considered to be ischemic damage of the pancreas itself by vasoconstrictive action of circulating catecholamines16 ) . the second suggested mechanism was dysfunction of the sphincter of oddi caused by adrenergic action of circulating catecholamines . this possibility was low because the sphincter of oddi was relaxed by adrenergic stimulation in general19 ) . however , paradoxical contraction by down regulation of adrenoceptors was as probable as circulatory collapse or hypotension seen in pheochromocytoma . in conclusion , pheochromocytoma can be present in a variety ways without hypertension and classic symptoms . therefore , it is vital to recognize the rare presentations of pheochromocytoma to avoid an unsuspected lethal course . we recently experienced a case of 48-year - old male with incidentally discovered a composite adrenal medullary tumor of pheochromocytoma and ganglioneuroma that was manifested as catecholamine - induced relapsing pancreatitis without hypertension .

matrix - assisted laser desorption ionization ( maldi ) is a method of wide analytical interest , representing an effective approach to obtain information on the molecular weight of macromolecules . due to the privileged formation of singly charged molecular species ( [ m + h ] , [ m h ] , alkali cationized molecules ) it leads to information on macromolecules mixtures , even if ion suppression effects can be present to some extent . maldi is widely employed in the protein field and is successfully used to determine the intact protein molecular weight up to 10 da , as well as to obtain information on the protein sequences by a rapid analysis of their digestion products and by ms / ms experiments performed on them . all are in agreement with the presence of two different steps : ( i ) primary ion formation from the matrix ( ma ) ; ( ii ) secondary ion formation of the analyte ( a ) originating from the gas phase interaction of reactive matrix ions and neutral molecules of the analyte . point ( i ) is the most debated one , due to the fact that the photon energy ( for n2 laser h = 3.68 ev , for triplicated nd : yag laser h = 3.55 ev ) is much lower than that required for matrix ionization ( typical ionization energies ( ies ) of the most employed matrices are in the range 710 ev ) . it has been proposed that multiple photon absorption , leading to multiple electronic excited matrix molecules , followed by energy pooling phenomena , would lead to the formation of the matrix odd electron molecular ion ma that , in turn , reacts with neutral matrix molecule leading to [ ma + h ] . the last species must be considered responsible for the analyte ( a ) protonation in gas phase : ( 1)mah++ama+ah+ only if paa > pama , ( pa = proton affinity ) . the possible limitation of this mechanism lies in the low probability of multiphoton absorption and energy pooling , which makes the high ionic yield observed in maldi experiments difficult to explain . alternatively it has been considered that the electronic excitation ( 1 photon required ) leads in general to an increase of the matrix acidity , promoting proton transfer . however , excited - state proton transfer ( espt ) [ 1 , 5 ] does not affect carboxylic acids , and consequently it can not be invoked as effective mechanism for proton transfer for the maldi matrices ; furthermore espt is active for compounds that do not exhibit any activity as maldi matrix . the desorption of preformed ions , already present in the solid state sample ( before laser irradiation ) , has been proposed as maldi ionization mechanism . the laser effect would be , in this case , just the desorption of these ions or matrix clusters containing them . in the latter case ions this mechanism can be invoked in the case of preprotonated compounds , metal complexes , and ionic compounds , and it is well supported by thermal desorption experiments , revealing the presence in gas phase of the same ions generated by laser irradiation . however the presence of mah ions in the matrix before laser irradiation is difficult to prove , and consequently a gas phase protonation mechanism different to that reported in ( 1 ) must be invoked . other ionization mechanisms have been proposed as laser - induced shockwaves ( the laser irradiation produces local high pressure shocks , resulting in the thermal excitation of a microvolume of the matrix - analyte sample ) , thermal ionization ( originating by the photon - phonon transformation in the crystal lattice ) , and ion - electron emission due to piezoelectric effects . all the above described proposals are mainly focused on the physical aspects of maldi ionization mechanism and the chemical nature of the matrix has been only marginally considered . in this paper we propose and discuss another possible mechanism , related to the chemical nature of the matrices effective for maldi experiments and their structural arrangements when crystallized . the solid - state interaction of carboxylic groups present in the matrix molecules could be considered responsible , when irradiated , for some disproportionation reactions , leading to both [ ma + h ] and [ ma h ] ions , that is , the reactants necessary for the [ a + h ] and/or [ a h ] formation . little is known on this aspect but measurements on gas - phase basicity of some matrix neutrals and anions [ 11 , 12 ] indicate that disproportion reaction can be available through a two - photon absorption . this hypothesis has been investigated both from the experimental and from a theoretical point of view . one l of three different matrix solutions ( -cyano-4-hydroxycinnamic acid ( hcca ) , 2,5-dihydroxybenzoic acid ( dhb ) , and sinapinic acid ( sa ) ( 10 mg / ml acetonitrile ) ) was deposited on the maldi plate and left to dry . the three matrix solutions were mixed with a cobalt powder suspension ( 10 mg / ml in h2o ) in order to obtain matrix : cobalt molar ratio : 10 : 1 , 1 : 1 , and 1 : 10 ( w / w ) . one l of the three solutions so obtained was deposited on the maldi plate and left to dry . 0.01 mg of insulin were dissolved in 1 ml of four different solutions ( h2o , h2o containing 0.1% formic acid , h2o containing 0.1% acetic acid , and h2o containing 0.1% trifluoracetic acid ) . 5 l of each insulin solution obtained were mixed with 5 l of a sinapinic solution ( 10 mg / ml in h2o / acn , 1 : 1 , v / v ) . one l of the solutions so obtained was deposited on the maldi plate and left to dry . 0.1 mg of human serum albumin ( hsa ) were dissolved in 1 ml of four different solutions ( h2o , h2o containing 0.1% formic acid , h2o containing 0.1% acetic acid , and h2o containing 0.1% trifluoracetic acid ) . 5 l of each hsa solution obtained were mixed with 5 l of a sinapinic solution ( 10 mg / ml in h2o / acn , 1 : 1 , v / v ) . one l of the solutions so obtained was deposited on the maldi plate and left to dry . maldi / ms measurements were performed using a maldi - tof ultraflextreme ( bruker daltonics , bremen , germany ) , equipped with 1 khz smart beam ii laser ( = 355 nm ) and operating in the positive and negative reflectron ion modes . the instrumental conditions employed to analyze molecular species in the m / z range 503000 in positive ion mode were ion source 1 : 25.00 kv ; ion source 2 : 22.30 kv , lens : 7.70 kv , pulsed ion extraction : 80 ns , reflector : 26.45 kv , reflector 2 : 13.45 kv . the instrumental conditions employed to analyze molecular weight in the m / z range 9807000 were ion source 1 : 25.00 kv ; ion source 2 : 22.40 kv , lens : 8.00 kv , reflector : 26.45 kv , reflector 2 : 13.45 kv , pulsed ion extraction : 120 ns . the instrumental conditions employed to analyze molecular weight in the m / z range 5000080000 were ion source 1 : 25.00 kv ; ion source 2 : 23.30 kv , lens : 6.50 kv , pulsed ion extraction : 450 ns . the instrumental conditions to analyze low molecular weight in the m / z range 502000 in negative ion mode were ion source 1 : 20.00 kv ; ion source 2 : 17.85 kv , lens : 6.15 kv , reflector : 21.15 kv , reflector 2 : 10.75 kv , pulsed ion extraction : 80 ns . external mass calibration was done using the peptide calibration standard , based on the monoisotopic values of [ m + h ] of angiotensin ii , angiotensin i , substance p , bombesin , acth clip ( 117 ) , acth clip ( 1839 ) , somatostatin 28 at m / z 1046.5420 , 1296.6853 , 1347.7361 , 1619.8230 , 2093.0868 , 2465.1990 , and 3147.4714 , respectively , and by using the monoisotopic values of [ m + h ] of insulin at mass / charge the [ ma + h ] and [ ma h ] ion production has been tested by irradiating the matrix sample with the same laser power and with the same number of laser shots ( 2000 ) . the calculation of energy of the photo processes postulated below was performed according to the thermodynamic hess ' law . the formation energy of the products in particular photoreactions was calculated with simultaneous optimization of its geometry . all values of the formation energy were calculated by dft method applying the hybrid functional b3lyp and 6 - 31+g * function base . these supplementary functions make the essential 6 - 31 g base capable to describe correctly the ionic systems . it is interesting to note that the most of effective matrices ( ma ) to perform maldi experiments contain the carboxyl group . at first sight this aspect could suggest that acidity of the matrix directly affects the production of protonated molecules [ a + h ] of the analyte . however it is emphasized that this behaviour can not explain how , with the same acidic matrix , deprotonation reactions can take place , with the formation of [ a h ] ions of the analyte . as discussed in the introduction , the species responsible for the generation of [ a + h ] is considered , in the most of the cases , to be [ ma + h ] , that is , the protonated matrix , and not the ma molecules and/or ma . consequently the [ ma + h ] production should be considered to be the focal point of the maldi mechanism . as above discussed , the most of maldi mechanisms proposed in the literature are mainly focused on physical aspects related to laser irradiation : in the case of pooling mechanism the interaction of the solid sample with the photons leads to an electronic excitation that , only after pooling phenomena , can give account for ma ( and not [ ma + h ] ) ion production . in the case of preformed ion mechanism , the ions are considered to be already present in the solid - state sample , and the only effect of laser irradiation is to make them free in gas phase . here we propose a different mechanism , in which , considering the molecular arrangement in solid state of carboxylic acids , a possible interaction between the carboxylic groups is present , as shown in figure 1 . carboxylic acids are known as compounds which very readily form strong hydrogen bonds , and their carboxylic groups can act as both donors and acceptors [ 1517 ] . in some cases intermolecular hydrogen bonds between such groups are formed . in the cambridge structural database version 5.32 ( conquest version 1.13 ) as much as 4129 ( out of 17615 incorporating carboxyl groups ) structures containing intermolecular carboxyl - carboxyl hydrogen bonds can be found . most of them ( 3431 structures ) form centrosymmetric or quasicentrosymmetric 8-membered ring arrangements described by the graph descriptor r2(8 ) [ 1921 ] . the others form chain structures , including catemer arrangements where the h - bonds are formed between translation symmetry - related molecules ( including screw axes symmetry ) . this feature is characteristic only for chiral compounds which must crystallize in the chiral space groups where the symmetry centre is absent , but also in the case of some simple carboxylic acids , for example , formic [ 22 , 23 ] and acetic [ 22 , 24 ] acids . the preference to form dimeric versus catemer structures is not clear while comparing the structures of acetic , trifluoro- , and trichloro - acetic acid . the laser irradiation of the matrix dimer with the structure shown in figure 1 could be considered responsible for the formation of the ion couple [ ma + h ] and [ ma h ] . indeed , by irradiation of sinapinic acid , both [ ma + h ] and [ ma h ] ions are produced with practically the same abundances . the related spectra , reported in figures 2(e ) and 2(f ) , have been obtained by using the same laser power ( 35% ) and the same delay time ( 80 ns ) . the situation is more complicated in the case of dhb ( figures 2(c ) and 2(d ) ) : [ ma + h ] and [ ma h ] ions with similar abundance ( 80700 ; 96500 , resp . ) are produced , suggesting the occurrence of the disproportionation reaction , but also ma and [ ma 2h ] or [ m h2 ] ions are present with practically identical abundance . the formation of the former can be justified by the low ionization energy of 2,5-dhb ( 8.05 ev ) lower than that of sa ( 8.47 ev ) and hcca ( 8.50 ev ) , while the [ ma 2h ] formation can be due to the high stability of the radical anion produced through h loss from [ ma h ] anion . the formation of [ ma h ] ion is highly favoured ( ion abundances 200000 ) , while [ ma + h ] ion exhibits an abundance of 2000 . this can be justified by its pka value ( around 8) , higher than that of other matrices ( around 4 ) . the main question that arises is as follows : is this dissociation reaction activated by photons or by the local heating due to laser irradiation ? thermal ionization could occur , as described by dreisewerd et al . in the matrix bulk , following the rules of surface ionization . the typical reaction observed by thermal ionization is a disproportion , governed by the saha - langmuir equation ( 2 ) in which the work function is replaced by the matrix electron affinity ( ea ) : ( 2)2mahma+ma+,i = ce(eaip)/kt , where i is the extent of ionization of species i , c is a constant near unity , ea is the matrix electron affinity , ip is the matrix ionization potential , k is the boltzmann constant , and t is the absolute temperature . from this equation it follows that thermal ionization in maldi would require very high temperature , not available by simple irradiation of the matrix . to investigate this point the three matrices under investigation were mixed with powdered cobalt so as to increase the laser power absorption . in these conditions the role of thermal effects would be further enhanced . it can be seen that the presence of cobalt leads to a decrease of [ ma + h ] ion formation , and so the possible increase of temperature related to the higher energy absorption in the presence of cobalt can not be responsible for the [ ma + h ] ion production . in order to evaluate the role of photon irradiation , three calculation models of the primary matrix ionization mechanisms for uv - maldi were proposed that might generate analyte ions . in these model calculations benzoic acid dimer and benzoic acid derivatives dimers 2,5-dihydroxybenzoic acid , 4-methoxybenzoic acid , 4-nitrobenzoic acid , and 4-n , n - dimethylaminobenzoic acid were tested in terms of their properties as potential matrix compounds . benzoic acid derivatives were proposed to allow comparison of the electron - donor or electron - acceptor properties of the substituents on the ability to ionize the analyte molecule . the structures of dimer molecules were taken from cambridge structural data base and used as the starting crystal structures in the quantum chemical calculations of their ionization energies . according to the first model , an absorbed uv photon by the matrix dimer induces its dissociation process into monomers accompanied by its simultaneous heterolysis of one of the hydrogen bonds . this calculation model in the case of benzoic acid ( as an example ) is illustrated by the dissociation reaction reported in scheme 2 . the energies ( per one dimer molecule ) of all studied reactions e were calculated by dft method at b3lyp ( 6-+31 g * ) level and compared with the energies of the photons of two typically used uv lasers emitting pulses of 337 and 355 nm wavelengths e337 and e355 . these photon energies are e337 = 3.68 ev and e355 = 3.50 ev . the values of energy calculated for the studied dimers are given in table 1 . these results unambiguously show that this type of dissociation can not be a one - photon process , and it meets the energetic criterion for the two - photon process , making the probability of such process low . however it is interesting to note that the two - photon process is that invoked by zenobi and knochenmuss as the first step of their theory of ionization by pooling phenomena . the data reported in table 1 indicate that the interaction with two photons is enough to activate the ion pair formation shown in scheme 2 . the experimental data reported in figure 2 and showing a practically identical abundance of [ ma + h ] and [ ma h ] for sinapinic acid and for dhb support this mechanism . the second proposed photoionization mechanism of the matrix dimer molecules corresponds to the reaction reported in scheme 3 . the energies of two processes , a heterodissociation of one of the both o h bonds associated with the proton transfer to carbonyl oxygen atom and a rotation of the benzoic acid moiety about the o h hydrogen bond of 180 , contribute to the energy requirement of this reaction . the assumption of such rotation in modelling of this ion was necessary since the nonrotated ion would be unstable enough tending to return to the starting dimer structure during the optimization . the third photoprocess studied was the homolytic dissociation of the dimers into neutral monomers ( scheme 4 ) . obviously , this is not ionization process itself but it is reasonable to assume that the proton can be transferred through an excited - state proton transfer from the monomer to the analyte molecule and , due to this , may cause the ionization of the analyte . it is also possible , although less probable , that this is two - stage process . according to this supposition , the first absorbed photon induces the reaction of dissociation of the dimer into monomers ( above reaction ) and on the second stage , the next photon could cause the ionization of the monomer . the values of the energies for the dissociation of the studied dimers are collected in table 3 . the comparison of the photon energy ( e355 = 3.50 ev ) with the particular energy of each process studied leads to the conclusion that the one - photon ionization of the matrix dimers can take place according to the second ionization model ( scheme 3 ) and could operate only for 2,5-dihydroxybenzoic acid , 4-methoxybenzoic acid , 4-nitrobenzoic acid , and 4-n , n - dimethylaminobenzoic acid . it seems that the two - stage ionization of an analyte is also probable to some extent since the life - time of the monomers may be long enough that these monomers could be ionized by the next photon absorbed . the analysis of the substituent effect on the calculated energy values ( e ) for the studied compounds leads to the conclusion that there is no correlation between selected properties of the substituents ( e.g. , electron affinity ) and the ability of these compounds to undergo ionization . of course , by having more data it would be possible to search for such a trend . finally we investigate the role of small organic acids ( ac ) usually added to the ( ma + a ) solution before the maldi measurements . usually trifluoroacetic acid ( tfa ) is added at 0.1% , leading to a high enhancement of the analyte ion intensity . to verify this aspect solutions of three matrices ( hcca , dhb , and sa ) containing insulin or albumin were added with 0.1% of formic acid ( hcooh ) , acetic acid ( ch3cooh ) , and trifluoracetic acid ( tfa ) . the results are shown in figures 4 and 5 , respectively , indicating that with tfa a wide increase of signal intensity is obtained . this could be due to a protonation reaction between tfa and the analyte molecules but , alternatively , it could be considered that the different acidity of tfa could lead to a [ ma + tfa ] complex with different electronic distribution : in this case the formation of [ ma + h ] ions would be strongly favoured ( figure 6 ) , due to the higher acidity of tfa . all calculations concerning the above conjugate have been performed according to schemes shown in scheme 2 through scheme 4 . it should be taken into account that in the case of the most probable process shown in scheme 3 , both hydrogen bonds are not equivalent , and therefore the weaker one will be broken ( see scheme 5 ) . also the energy of dissociation of conjugates into substituted benzoic acid cation and tfa anion ( table 5 ) and into separate acid monomers ( table 6 ) was calculated . the values collected in table 5 , similarly to those in table 1 , suggest that such processes are not possible as one - photon processes and the dissociation into monomers are low - energy processes . the obtained data show that considering the matrix molecules in the solid - state sample in a dimeric structure containing intermolecular carboxyl - carboxyl hydrogen bond in centrosymmetric or quasicentrosymmetric 8-membered ring arrangement , the formation of both [ ma + h ] and [ ma h ] ions can be justified by the occurrence of a two - photon - induced disproportion reaction . alternatively one - photon induced process can lead to the opening of the eight - membered ring with the subsequent formation of species containing both cationic and anionic sites . experimental data showed that the processes leading to [ ma + h ] must be considered to be activated by photons , and thermal ionization seems to inhibit this behaviour . it is interesting to observe that the obtained results are somehow well related to the mechanisms already proposed with respect to considering the desorption of clusters containing preformed ions ; our data seem to suggest that the ions are not originally present in the solid - state sample , but they are originated by the laser irradiation . in the case of multiple photon absorption followed by pooling phenomena , necessary for the ma formation , the data discussed above indicate that it is unnecessary to invoke the energy pooling : in fact the absorption of two photons will be enough to activate the disproportion reaction leading to [ ma + h ] and [ ma h ] , that is , the reactants for the analyte ionization .

in the diabetes care in general practice study ( 10 ) , 474 general practitioners agreed to include all subjects on their practice list who fulfilled the following criteria : newly diagnosed diabetes based on hyperglycemia symptoms and/or raised blood glucose values , diagnosed between 1 march 1989 and 28 february 1992 and aged 40 years . the diagnosis was subsequently confirmed with a single fasting whole blood / plasma glucose value of 7.0/8.0 mmol / l , measured in a major laboratory . the protocol - based exclusion criteria were life - threatening somatic disease , severe mental illness , or unwillingness to participate . after exclusion of 162 patients , the original study population consisted of 1,381 newly diagnosed diabetic patients . based on the onset of insulin treatment within 180 days of diagnosis , ~97.5% were considered to have type 2 diabetes ( 10 ) . freshly voided morning urine samples were collected from all patients at the time of diagnosis and again at the sixth annual screening for diabetes complications ( 6-year follow - up ) . in the present investigation , we used data from the 970 patients who attended the screening . the protocol was approved by the ethics committee of copenhagen and frederiksberg , and informed consent was obtained from all patients . the urine samples were assayed between 2009 and 2010 for the oxidatively modified guanine nucleosides 8-oxodg and 8-oxoguo using a validated method of ultraperformance liquid chromatography and tandem mass spectrometry ( 11 ) . for 32 ( 3.3% ) and 34 ( 3.5% ) patients , respectively , no valid assessments of 8-oxodg and 8-oxoguo were obtained . the assessment of the remaining patient characteristics at baseline and at 6-year follow - up has been described previously ( 10 ) . cohabitation status , education level , and height ( used in bmi calculation ) were only assessed at diagnosis . the vital status of all the patients was certified on 1 january 2009 through the danish civil registration system ( 12 ) . the vital status of one patient could not be assessed because this person had emigrated in 1992 . diabetes - related death was defined as at least one of the following entries in the danish national register of causes of death ( 13 ) : sudden death or death from myocardial infarction , stroke , renal disease , hyperglycemia , hypoglycemia , or peripheral vascular disease . the participants were grouped according to the quartiles of their urinary 8-oxodg and 8-oxoguo levels in order to examine the associations between patient characteristics at 6-year follow - up and the corresponding levels of oxidative stress . the medians and interquartile ranges ( for continuous characteristics ) or percentages ( for categorical characteristics ) were reported for each quartile , and associations were assessed by kruskal - wallis or tests , respectively . the associations between oxidative stress and all - cause mortality and diabetes - related mortality , respectively , were analyzed in cox proportional hazards regression models based on time from 6-year follow - up to death or censoring . oxidative stress was represented by the natural logarithm of 8-oxodg and 8-oxoguo and by a four - class ordinal variable corresponding to the quartiles of the distribution . two models were estimated for each of the oxidative stress variables and each of the outcomes : a univariate ( unadjusted ) model and a multivariate model . covariates included in the latter model were age , sex , diabetes duration , smoking status , cohabitation status , physical activity , education , bmi , presence or absence of hypertension and of microalbuminuria , a1c , total cholesterol , triglycerides , serum creatinine , presence or absence of retinopathy and of peripheral neuropathy , history of acute myocardial infarction and stroke , and antidiabetes treatment . for investigation of the influence of changes in oxidative stress from diagnosis to the 6-year follow - up on subsequent mortality , two 16-class variables were created where the classes corresponded to the combination of the quartiles at diagnosis and at 6-year follow - up of 8-oxodg and 8-oxoguo , respectively . these variables were then used in cox proportional hazards models as described above to assess the significance of upward and downward movements between quartiles . only patients with a valid measurement both at diagnosis and at assessment 6 years after diagnosis were included in this analysis . the proportional hazards assumption was tested by adding the interactions between each of the independent variables and the logarithm of time since diagnosis to the model ; a joint test of these interactions tests the assumption . in addition , to determine the possible additional prognostic value of adding urinary 8-oxoguo to established biomarkers and risk factors , we generated receiver operating characteristic ( roc ) curves for all - cause mortality , and the areas under the curves ( aucs ) were calculated . reported p values are two sided . the urine samples were assayed between 2009 and 2010 for the oxidatively modified guanine nucleosides 8-oxodg and 8-oxoguo using a validated method of ultraperformance liquid chromatography and tandem mass spectrometry ( 11 ) . for 32 ( 3.3% ) and 34 ( 3.5% ) patients , respectively , no valid assessments of 8-oxodg and 8-oxoguo were obtained . the assessment of the remaining patient characteristics at baseline and at 6-year follow - up has been described previously ( 10 ) . cohabitation status , education level , and height ( used in bmi calculation ) were only assessed at diagnosis . the vital status of all the patients was certified on 1 january 2009 through the danish civil registration system ( 12 ) . the vital status of one patient could not be assessed because this person had emigrated in 1992 . diabetes - related death was defined as at least one of the following entries in the danish national register of causes of death ( 13 ) : sudden death or death from myocardial infarction , stroke , renal disease , hyperglycemia , hypoglycemia , or peripheral vascular disease . the participants were grouped according to the quartiles of their urinary 8-oxodg and 8-oxoguo levels in order to examine the associations between patient characteristics at 6-year follow - up and the corresponding levels of oxidative stress . the medians and interquartile ranges ( for continuous characteristics ) or percentages ( for categorical characteristics ) were reported for each quartile , and associations were assessed by kruskal - wallis or tests , respectively . the associations between oxidative stress and all - cause mortality and diabetes - related mortality , respectively , were analyzed in cox proportional hazards regression models based on time from 6-year follow - up to death or censoring . oxidative stress was represented by the natural logarithm of 8-oxodg and 8-oxoguo and by a four - class ordinal variable corresponding to the quartiles of the distribution . two models were estimated for each of the oxidative stress variables and each of the outcomes : a univariate ( unadjusted ) model and a multivariate model . covariates included in the latter model were age , sex , diabetes duration , smoking status , cohabitation status , physical activity , education , bmi , presence or absence of hypertension and of microalbuminuria , a1c , total cholesterol , triglycerides , serum creatinine , presence or absence of retinopathy and of peripheral neuropathy , history of acute myocardial infarction and stroke , and antidiabetes treatment . for investigation of the influence of changes in oxidative stress from diagnosis to the 6-year follow - up on subsequent mortality , two 16-class variables were created where the classes corresponded to the combination of the quartiles at diagnosis and at 6-year follow - up of 8-oxodg and 8-oxoguo , respectively . these variables were then used in cox proportional hazards models as described above to assess the significance of upward and downward movements between quartiles . only patients with a valid measurement both at diagnosis and at assessment 6 years after diagnosis the proportional hazards assumption was tested by adding the interactions between each of the independent variables and the logarithm of time since diagnosis to the model ; a joint test of these interactions tests the assumption . in addition , to determine the possible additional prognostic value of adding urinary 8-oxoguo to established biomarkers and risk factors , we generated receiver operating characteristic ( roc ) curves for all - cause mortality , and the areas under the curves ( aucs ) were calculated . tables 1 and 2 show characteristics of the study participants at 6-year follow - up according to quartiles of 8-oxodg and 8-oxoguo . the median age at 6-year follow - up was 69.2 ( interquartile range 59.977.3 ) , with an equal distribution of men and women ( 51.4 and 48.6% , respectively ) . characteristics at 6-year follow - up according to quartiles of 8-oxodg characteristics at 6-year follow - up according to quartiles of 8-oxoguo for both 8-oxodg and 8-oxoguo , participants in the highest quartiles were older , were more often women , and had lower levels of serum creatinine . in addition , participants in the highest quartiles of 8-oxodg had lower total cholesterol , and those in the highest quartile of 8-oxodg had lower bmi than participants in the lower quartiles . the level of a1c differed between the quartiles of 8-oxodg , but no trend across quartiles was observed . participants in the highest quartiles of 8-oxoguo were more often nonsmokers , less physically active , and more often living alone at the time of diagnosis . differences in antidiabetes and antihypertension treatment between the quartiles of 8-oxoguo were observed but with no trend across quartiles , except for the use of diuretics , where those in the highest quartiles more frequently used diuretics ( table 2 ) . the study observation time ranged from 26 september 1995 to 1 january 2009 , and the median duration of follow - up was 9.8 years ( interquartile range 4.213.3 ) . during this period , 654 ( 61.2% ) patients who died between 6-year follow - up and 1 january 2009 had significantly higher urinary 8-oxoguo excretion at 6-year follow - up than survivors ( median 3.89 nmol / mmol creatinine [ interquartile range 3.094.91 ] vs. 3.43 nmol / mmol creatinine [ 2.754.16 ] ; p < 0.0001 ) . in contrast , no significant difference was found for 8-oxodg ( 2.08 nmol / mmol creatinine [ 1.602.73 ] vs. 2.02 nmol / mmol creatinine [ 1.572.63 ] ; p = 0.25 ) . kaplan - meier analysis estimates of survival according to quartiles of urinary 8-oxodg and 8-oxoguo at 6-year follow - up are shown in supplementary fig . the results of the unadjusted and adjusted cox regression analyses are shown in table 3 . relationship of 8-oxodg and 8-oxoguo with all - cause and diabetes - related mortality at 6-year follow - up , only 8-oxoguo was significantly associated with mortality . the unadjusted hazard ratios for all - cause and diabetes - related mortality of patients with 8-oxoguo levels in the highest quartile compared with those in the lowest quartile were 2.09 ( 95% ci 1.622.67 ; p the multivariate - adjusted hazard ratios for all - cause and diabetes - related mortality of patients with 8-oxoguo levels in the highest quartile compared with those in the lowest quartile were 1.86 ( 1.342.58 ; p < 0.001 ) and 1.72 ( 1.112.66 ; p = 0.02 ) , respectively . when log8-oxodg and log8-oxoguo were considered as continuous covariates , the results were similar to those described above ( table 3 ) . the multivariate - adjusted hazard ratios for all - cause and diabetes - related mortality per unit increase in log8-oxoguo were 1.82 ( 95% ci 1.362.42 ; p < 0.001 ) and 1.64 ( 1.132.37 ; p = 0.009 ) , respectively . we analyzed the significance of changes in the nucleic acid oxidation markers from diagnosis to 6-year follow - up . for both 8-oxodg and 8-oxoguo , ~30% of the patients in the second and third quartiles remained in the same quartile as at diagnosis , whereas this figure was ~45% for patients in the highest and lowest quartiles ( table 4 ) . the unadjusted and multivariate - adjusted hazard ratios for all - cause mortality of patients who changed quartile from diagnosis to 6-year follow - up compared with those who remained in the same quartile are shown in table 4 . relationship between changes in 8-oxodg and 8-oxoguo from diagnosis to 6-year follow - up and all - cause mortality for 8-oxoguo , there was a clear and significant trend for increased risk in patients who changed to higher quartiles and decreased risk in patients who changed to lower quartiles . this trend was seen for all quartiles of 8-oxoguo at diagnosis but was most pronounced for patients with 8-oxoguo in the highest quartile : the unadjusted and multivariate - adjusted hazard ratios of patients who changed from the highest to the lowest quartile of 8-oxoguo compared with those who remained in the highest quartile were 0.38 ( 95% ci 0.160.89 ) and 0.31 ( 0.140.68 ) , respectively . for 8-oxodg , this trend was less obvious and was only significant for patients with 8-oxodg in the lowest and highest quartiles at diagnosis . the ability of 8-oxoguo and other biomarkers and risk factors to predict mortality was evaluated by roc curves . the auc was 0.63 ( 95% ci 0.590.67 ) for a model including the traditional biomarkers in diabetes : a1c , fasting triglycerides , total cholesterol , urinary albumin , systolic blood pressure , and bmi . adding 8-oxoguo to this model increased the auc to 0.67 ( 0.640.72 ) ( supplementary fig . 2 ) . aucs for a multivariate model including all the covariates in our multivariate cox proportional hazards regression model with and without 8-oxoguo were 0.88 ( 0.860.91 ) and 0.87 ( 0.860.91 ) , respectively . the study observation time ranged from 26 september 1995 to 1 january 2009 , and the median duration of follow - up was 9.8 years ( interquartile range 4.213.3 ) . during this period , 654 ( 61.2% ) patients who died between 6-year follow - up and 1 january 2009 had significantly higher urinary 8-oxoguo excretion at 6-year follow - up than survivors ( median 3.89 nmol / mmol creatinine [ interquartile range 3.094.91 ] vs. 3.43 nmol / mmol creatinine [ 2.754.16 ] ; p < 0.0001 ) . in contrast , no significant difference was found for 8-oxodg ( 2.08 nmol / mmol creatinine [ 1.602.73 ] vs. 2.02 nmol / mmol creatinine [ 1.572.63 ] ; p = 0.25 ) . kaplan - meier analysis estimates of survival according to quartiles of urinary 8-oxodg and 8-oxoguo at 6-year follow - up are shown in supplementary fig . the results of the unadjusted and adjusted cox regression analyses are shown in table 3 . relationship of 8-oxodg and 8-oxoguo with all - cause and diabetes - related mortality at 6-year follow - up , only 8-oxoguo was significantly associated with mortality . the unadjusted hazard ratios for all - cause and diabetes - related mortality of patients with 8-oxoguo levels in the highest quartile compared with those in the lowest quartile were 2.09 ( 95% ci 1.622.67 ; p < 0.001 ) and 1.94 ( 1.402.69 ; p < 0.001 ) , respectively . the multivariate - adjusted hazard ratios for all - cause and diabetes - related mortality of patients with 8-oxoguo levels in the highest quartile compared with those in the lowest quartile were 1.86 ( 1.342.58 ; p < 0.001 ) and 1.72 ( 1.112.66 ; p = 0.02 ) , respectively . when log8-oxodg and log8-oxoguo were considered as continuous covariates , the results were similar to those described above ( table 3 ) . the multivariate - adjusted hazard ratios for all - cause and diabetes - related mortality per unit increase in log8-oxoguo were 1.82 ( 95% ci 1.362.42 ; p < 0.001 ) and 1.64 ( 1.132.37 ; p = 0.009 ) , respectively . we analyzed the significance of changes in the nucleic acid oxidation markers from diagnosis to 6-year follow - up . for both 8-oxodg and 8-oxoguo , ~30% of the patients in the second and third quartiles remained in the same quartile as at diagnosis , whereas this figure was ~45% for patients in the highest and lowest quartiles ( table 4 ) . the unadjusted and multivariate - adjusted hazard ratios for all - cause mortality of patients who changed quartile from diagnosis to 6-year follow - up compared with those who remained in the same quartile are shown in table 4 . relationship between changes in 8-oxodg and 8-oxoguo from diagnosis to 6-year follow - up and all - cause mortality for 8-oxoguo , there was a clear and significant trend for increased risk in patients who changed to higher quartiles and decreased risk in patients who changed to lower quartiles . this trend was seen for all quartiles of 8-oxoguo at diagnosis but was most pronounced for patients with 8-oxoguo in the highest quartile : the unadjusted and multivariate - adjusted hazard ratios of patients who changed from the highest to the lowest quartile of 8-oxoguo compared with those who remained in the highest quartile were 0.38 ( 95% ci 0.160.89 ) and 0.31 ( 0.140.68 ) , respectively . for 8-oxodg , this trend was less obvious and was only significant for patients with 8-oxodg in the lowest and highest quartiles at diagnosis . the ability of 8-oxoguo and other biomarkers and risk factors to predict mortality was evaluated by roc curves . the auc was 0.63 ( 95% ci 0.590.67 ) for a model including the traditional biomarkers in diabetes : a1c , fasting triglycerides , total cholesterol , urinary albumin , systolic blood pressure , and bmi . adding 8-oxoguo to this model increased the auc to 0.67 ( 0.640.72 ) ( supplementary fig . 2 ) . aucs for a multivariate model including all the covariates in our multivariate cox proportional hazards regression model with and without 8-oxoguo were 0.88 ( 0.860.91 ) and 0.87 ( 0.860.91 ) , respectively . in this study , we found that urinary excretion of the rna oxidation marker 8-oxoguo in a freshly voided morning urine sample 6 years after diagnosis of diabetes independently predicted all - cause and diabetes - related mortality , whereas the dna oxidation marker 8-oxodg did not . the prognostic information of 8-oxoguo was independent of other characteristics of patients with type 2 diabetes that have been linked to mortality : most importantly , age , sex , a1c , lipids , urinary albumin excretion , blood pressure , smoking , and preexisting cardiovascular disease ( 1419 ) . in addition , we found that changes in 8-oxoguo from the time of diagnosis to 6-year follow - up were associated with mortality , with increased risk of death in patients with an increase and decreased risk of death in patients with a decrease in 8-oxoguo . this finding supports the notion that rna oxidation is not only a predictor of risk in diabetes but also a risk factor , i.e. , that modification of rna oxidation also modifies risk . to our knowledge , our population - based study of diabetic patients is the only study of the association between urinary excretion of markers of oxidative stress and mortality in diabetic patients . as in the current study , urinary 8-oxoguo assessed at the time of diagnosis was shown to be an independent predictor of mortality ( 9 ) . the association between 8-oxoguo and mortality seemed stronger at 6-year follow - up than at diagnosis . at diagnosis , the risk of death was ~50% higher among those in the highest quartile than among those in the lowest quartile , whereas this figure was ~90% at 6-year follow - up . roc curve analyses showed that 8-oxoguo added prognostic information to other established biomarkers in diabetes . however , in the multivariate model including all the covariates in our multivariate cox proportional hazards regression model , 8-oxoguo did not significantly improve the predictive accuracy of the model . rna oxidation could be an epiphenomenon rather than a contributing pathophysiological mechanism , but it would still function as a marker of generalized intracellular oxidative stress . however , the fact that only 8-oxoguo was shown to be an independent predictor of mortality suggests that rna oxidation plays a more important role in diabetic patients than dna oxidation and generalized intracellular oxidative stress . it has been suggested that mitochondrial dysfunction , with failure of complete oxidation leading to overproduction of superoxide by the mitochondrial electron - transport chain , plays a key role in the biochemical abnormalities leading to vascular complications in diabetes ( 20 ) . superoxide is readily converted into hydrogen peroxide through the action of superoxide dismutase , and in the presence of iron , hydrogen peroxide is reduced to hydroxyl radicals by a fenton reaction . the highly reactive hydroxyl radicals then oxidize dna and rna among many other macromolecules ( 20 ) . compared with dna , rna is more prone to oxidative damage because of its widespread distribution in close proximity to sites of reactive oxygen species generation , its single - stranded nature , and the lesser association with protecting proteins . surprisingly , the role of rna oxidation in human disease has only recently been investigated , predominantly in degenerative brain diseases ( 2130 ) . on the basis of the limited evidence available , we suggest a possible mechanism by which rna oxidation could be involved in the development of diabetes complications . oxidized bases on mrna may cause ribosome stalling on the mrna with production of truncated proteins or slow the translational process , leading to a reduction in protein expression ( 31 ) . since the noncoding rnas , which include rrna , trna , and mrnas , play critical regulatory roles in mrna translation , oxidation of these could further negatively affect the translational process , leading to smaller quantities of protein overall and/or defective proteins , which may have detrimental effects on a variety of distinct cellular processes that lead to changes in proliferation and apoptosis ( 3133 ) . apoptosis plays an important role in the development of the atherosclerotic plaque ( 34 ) , and through this pathway increased rna oxidation may be involved in the development of the vascular complications in diabetes . it should be emphasized that the described possible mechanism is a suggestion based on available evidence and that no conclusion regarding a causal relationship between rna oxidation , vascular complications , and mortality can be drawn from the present association . important limitations of this study include the reliance on a single measurement of the nucleic acid oxidation markers in a morning spot urine sample and the need to elaborate the potential mechanisms underlying our findings . on the other hand , the fact that a significant association between 8-oxoguo and mortality was shown both at diagnosis ( 9 ) and at follow - up and the relation between changes in rna oxidation and mortality support the assumption that the association is not a chance finding . however , it has been shown that waist circumference and waist - to - hip ratio may be better predictors of mortality than bmi ( 35 ) . in addition , glucose variability , which has been shown to exhibit a more specific triggering effect on oxidative stress than chronic sustained hyperglycemia ( 36,37 ) , was not assessed in the study . finally , the lack of information on diet and consumption of antioxidants , which potentially could affect the level of oxidative stress , should be mentioned . the main strengths of our study include the prospective design , the large sample size , the long follow - up period , and the low attrition rate . the findings of this study may provide new insight into the pathophysiological mechanisms responsible for the development of complications in diabetes but also indicate that measurement of 8-oxoguo could have clinical applications . in the prevention of complications in diabetic patients , our data show that urinary 8-oxoguo predicts mortality in type 2 diabetic patients independent of other conventional risk factors including these currently used biomarkers . measurement of urinary 8-oxoguo could therefore provide additional information about risk that may be useful for identifying patients who would benefit the most from intensified treatment or specific treatment strategies . a multivariate risk factor approach with the combined use of 8-oxoguo and other known risk factors it should be emphasized , though , that the clinical significance of using this marker is unknown , since the clinical utility of 8-oxoguo remains to be determined . although the clinical trials conducted to date have failed to provide adequate support for the use of antioxidants in diabetes , recent studies of bardoxolone methyl ( 39 ) , showing beneficial effect on glomerular filtration rate in type 2 diabetic patients , suggest that targeting the nrf2 ( nuclear factor erythroid 2related factor 2 ) pathway may be a useful strategy to counteract oxidative stress in diabetes . investigating the effect of activators of nrf2 on rna oxidation may provide interesting information that could shed more light on the role of rna oxidation in diabetes . in addition , intervention studies should be initiated to investigate the effect of different treatment modalities of diabetes ( e.g. , glycemic control , exercise , diet , and weight control ) on rna oxidation . further experimental work is needed to establish the exact mechanisms responsible for the association between 8-oxoguo and mortality in diabetes patients , and studies should be initiated to evaluate the potential clinical applications of 8-oxoguo as a biomarker in diabetes that could be used for risk stratification , selection of appropriate therapeutic intervention , or monitoring response to therapy .

coronary heart disease ( chd ) is one of the most common and dangerous human diseases . it is a major cause of disability and the most common cause of death ; especially in industrialized societies . atherosclerosis is a chronic inflammatory disease that many factors are involved in its pathogenesis ( 2 ) . the classic risk factors include aging , obesity , smoking , high blood pressure , diabetes , physical inactivity and stress ( 3 ) . in addition to these factors , the role of inflammatory and infectious agents in pathogenesis of the atherosclerosis has been discussed ( 4 , 5 ) . several studies have been done in correlation between bacterial and viral pathogens such as chlamydia pneumoniae , mycoplasma pneumoniae , helicobacter pylori , herpes simplex virus and human cytomegalovirus with atherosclerosis and coronary artery disease ( 69 ) . to the best of our knowledge , a survey on fungal rdna signatures in human coronary atherosclerotic plaques has been done and recently in another study the correlation of atherogenesis with candida albicans infection has been reported ( 10 , 11 ) . furthermore in an animal study , the role of cryptococcus neoformans was investigated as a potential inducer of atherogenesis ( 12 ) . the aim of the present study was to investigate the possible role of fungi in the atherosclerosis . in the study , to increase the accuracy of the results , we used mycological , pathological and molecular methodes to detect presence of fungal elements in atherosclerotic plaques . elimination of these infectiuos agents could be considered in prevention of coronary heart disease , if fungi have a role in atherogenesis . during 2011 to 2012 , a total of 41 patients with chd hospitalized in imam khomeini and mehr hospitals in tehran , iran , included in our study . artery occlusion was confirmed by angiography in all patients ( more than 50% stenosis in at least three coronary arteries ) then coronary artery bypass graft ( cabg ) as the sole treatment was performed . patient s information such as age , sex , smoking , obesity , hypertension , hyperlipidemia , family history of heart diseases and diabetes were considered . informed consent to participate in this study was signed by all patients and ethics committee of tehran university of medical sciences approved this study . atherosclerotic plaque specimens were obtained during surgery and aseptically divided to three pieces and separately were placed in sterile tubes containing normal saline and formalin used for culture and pathology , respectively . for molecular analysis dna and rna free tubes containing 70% alcohol were used and specimens were immediately transported to the laboratory in a cool box . pieces of the specimens were cultured on chro - magar candida , sabouraud dextrose agar , sabouraud dextrose agar containing chloramphenicol , brain heart infusion agar , blood agar and eosin methylene blue agar . ten percent koh preparation was used for microscopic direct examination of clinical specimens to seek for fungal elements . for pathological examination , tissue sections were stained by gomori methenamine silver ( gms ) , hematoxylin & eosin ( h&e ) , and periodic acid schiff ( pas ) . for molecular analysis , fungal dna was extracted from the specimens using dneasy blood & tissue kit ( qiagen ) according to the manufacturer s instructions and pcr performed for fungal rdna detection using universal primers its1 ( 5-tcc gta ggt gaa cct gcgg-3 ) as forward and its4 ( 5-tcc tcc gct tat tga tat gc-3 ) as reverse primers . dna amplification performed using one initial denaturation at 95c for 5 min , followed by 35 cycles including 30 sec denaturation at 95c , 1 min annealing at 49 c and 90 sec extension at 72 c and 1 cycle of 9 min at 72c for final extension . six positive specimens were randomly selected and the amplicons were subjected to sequencing and data were compared with reliable sequences deposited in genbank using the basic local alignment search tool ( blast ) ( http://www.ncbi.nlm.nih.gov/blast ) . demographic data were analyzed using chi square test by spss ( chicago , il , usa ) version 15 and a p value < 0.05 was considered as statistically significant . direct examination with 10% koh was negative for fungal elements in all specimens , but culture was positive in only one case and it was grown as a mold colony . the microscopic characteristics of this fungus in slide culture revealed hyaline , septate and branched mycelium with unicellular , spherical or cylindrical and hyaline conidia ( fig . microscopic view of monilia sp . isolated from a specimen of coronary atherosclerotic plaques this isolate was microscopically identified as the monilia sp . and confirmed based on pcr and sequencing . the presence of fungi were confirmed in tissue sections stained with gms ( fig . 2 , 3 ) and h&e , in 6 and 2 specimens , respectively . but yeast cells in artery biopsy ( gms 1000 ) yeast cells in artery biopsy ( gms ) 1000 ) by performing pcr with fungal universal primers , 11 out of 41 specimens analyzed in this study , were positive for the fungal organisms . blast analysis of the 6 sequences confirmed the amplicons as c. albicans ( n=3 ) , c. guilliermondii ( n=2 ) and monilia sp . positive ( standard c. albicans ) and negative ( sterile distilled water ) controls are shown . in the recent years , the role of infectious agents in pathogenesis of atherosclerosis has supported by several studies . these studies have focused mainly on viral and bacterial pathogens and few of them have been done on the role of fungi in this process ( 2 , 9 ) . we studied the role of fungi in the pathogenesis of atherosclerosis in patients with angiographically proved coronary heart disease ( more than 50% stenosis in at least three coronary arteries ) using direct examination , culture , histopathology and pcr - sequencing methods . although fungi were found in atherosclerotic plaques in 26.8% of cases , our study did not demonstrate statistically significant association between the presence of fungi and atherosclerosis . also , no significant association was found between age , sex , smoking , obesity , hypertension , hyperlipidemia , family history of heart diseases , and diabetes with the presence of fungal elements in atherosclerotic plaques . in a study conducted by ott and colleagues , the presence of fungal organisms in 92.11% ( 35 of 38 ) of atherosclerotic plaque samples was confirmed . therefore fungi were considered as a non - specific underlying factor in development of atherosclerosis ( 10 ) . difference in geographic area , patient groups and diagnostic method may be effective in different results of two studies . on the other hand results of our study are in agreement with results obtained by maya j nurgeldiyeva et al . that showed the presence of fungi of the genus candida in 31.9% ( 15 out of 47 ) of atherosclerotic plaques ( 11 ) . the oral cavity and gastrointestinal tract could be the major sources of microbial agents present in atherosclerotic plaques ( 13 , 14 ) . fungi , especially candida species are a part of the mouth and gastrointestinal tract flora . yeasts can enter the bloodstream through small wounds during brushing or by therapeutic interventions such as tooth extraction , root canal therapy and surgery and cause transient fungemia ( 2 , 10 , 14 ) . this aggregation causes fungi clinging to the vascular endothelium , which can increase the risk of thrombosis and cardiovascular disease ( 15 ) . taheri and colleagues compared oral candida flora of 90 patients with coronary atherosclerosis and 90 control subjects . although no statistically significant difference was found between candida colonization in patients and controls , the number of colonies and the intensity of candida colonization were significantly lower in controls ( 16 ) . in our study , only patients with angiographically proved coronary heart disease were included . application of different diagnostic methods for detection of fungi in atherosclerotic plaques is an advantage for our study that increased the accuracy of the results . however , the study population size was limited and this could be a disadvantage for the study . although , a significant association between the presence of fungi with atherogenesis and atherosclerotic disease was not found in this study , the presence of fungi in 26.8% of specimens is noteworthy and the results does not exclude the role of fungi in atherosclerosis and coronary artery disease . therefore , more studies with more number of patients is recommended for a better evaluation of fungi role in atherosclerotic disease and chd . ethical issues ( including plagiarism , informed consent , misconduct , data fabrication and/or falsification , double publication and/or submission , redundancy , etc . ) have been completely observed by the authors .

a mobile plaque ( mp ) in the carotid artery is an uncommon condition which can be detected with duplex ultrasound , and it is associated with a high risk of ischemic stroke . carotid endarterectomy ( cea ) is an option for patients who are refractory to medical treatment ; however , the risks of intraoperative embolization and general anesthesia should be considered carefully . here , we report a case of ischemic stroke in evolution due to mp in which carotid artery stenting ( cas ) successfully prevented further infarctions . he had been treated for diabetes mellitus , chronic renal and heart failure . after admission , one month after admission , he suddenly developed right hemiparesis , and an mri disclosed acute multiple infarcts in the left hemisphere ( fig . a carotid ultrasound disclosed an mp located at the bifurcation of the left common carotid artery ( fig . . video ) , and the mp was considered to be the cause of the ischemic lesions . a maximal medical therapy consisting of heparin , statin , and cilostazol had been undertaken ; however , the number of infarcts had increased along with his neurological deteriorations ( fig . f ) . as the antithrombotic therapy was not effective to treat progressive stroke , cea was considered . however , tl scintigraphy suggested a cardiac ischemia involving the anterior and inferior walls , making it difficult to induce general anesthesia . we used a telescoping technique to advance a guide catheter ( 9-f optimo ; tokai medical , kasugai , japan ) . a 0.038-inch wire was parked in the left common carotid artery ( cca ) just proximal to the mp ; however , a 5-fr simmons diagnostic catheter could not track the wire . then , under roadmap guidance , a 0.038-inch wire was navigated carefully to the left external carotid artery ( eca ) while paying attention not to touch the mp . the 5-fr simmons catheter could be advanced over the 0.038-inch wire into the left eca , followed by tracking along the guide catheter into the distal cca with a telescoping technique . we inflated the balloon of the guide catheter to obtain the control of the proximal flow . although eca was not blocked , favorable flow reversal was archived only by blocking the cca . during this proximal flow control , spider fx ( ev3 , plymouth , minn . , usa ) along with a microguidewire was advanced to the internal carotid artery ( ica ) , and once the spider was deployed , the balloon of the guide catheter was deflated . intravascular ultrasound after stenting did not show any mp , and the procedure was completed after the retrieval of spider fx . postoperative carotid ultrasound clearly showed the disappearance of the mp in the stented cca and ica ( fig . there was an mp noted in the left eca which was located outside the stented vessel ( fig . one month later , the mp in the eca could not be observed by ultrasound . a postmortem examination disclosed the patency of the stented carotid artery , and no plaque had prolapsed into the stent ( fig . we used a telescoping technique to advance a guide catheter ( 9-f optimo ; tokai medical , kasugai , japan ) . a 0.038-inch wire was parked in the left common carotid artery ( cca ) just proximal to the mp ; however , a 5-fr simmons diagnostic catheter could not track the wire . then , under roadmap guidance , a 0.038-inch wire was navigated carefully to the left external carotid artery ( eca ) while paying attention not to touch the mp . the 5-fr simmons catheter could be advanced over the 0.038-inch wire into the left eca , followed by tracking along the guide catheter into the distal cca with a telescoping technique . we inflated the balloon of the guide catheter to obtain the control of the proximal flow . although eca was not blocked , favorable flow reversal was archived only by blocking the cca . during this proximal flow control , spider fx ( ev3 , plymouth , minn . , usa ) along with a microguidewire was advanced to the internal carotid artery ( ica ) , and once the spider was deployed , the balloon of the guide catheter was deflated . carotidwall wallstent ( stryker , mass . , usa ) was deployed in the ica down to the cca ( fig . 3 ) . intravascular ultrasound after stenting did not show any mp , and the procedure was completed after the retrieval of spider fx . postoperative carotid ultrasound clearly showed the disappearance of the mp in the stented cca and ica ( fig . there was an mp noted in the left eca which was located outside the stented vessel ( fig . one month later , the mp in the eca could not be observed by ultrasound . a postmortem examination disclosed the patency of the stented carotid artery , and no plaque had prolapsed into the stent ( fig . mp in the carotid artery is a rare condition observed by carotid ultrasound , showing mobile components in a carotid plaque . mobile plaque , floating thrombus , free floating thrombus , or mobile thrombus. the nature of ischemic strokes due to an mp has not been elucidated well because of the small number of reported cases . recently , delgado et al . reviewed 22 reported cases of mp including 3 cases of their own ; only 7 out of the 22 patients were treated solely with antithrombotic therapy , and the remaining 13 patients were treated with surgical interventions . antithrombotic therapy alone seems to be insufficient to treat the ischemic stroke due to mp . studied 21 mps removed by cea , and reported that progressive ischemic strokes were more frequent in patients with mp . according to their histological analysis , the ratio of the necrotic core compared to that of the entire plaque was significantly larger than those in non - mobile plaques . thus , mp is considered to be more embologenic than non - mp . for patients with mp in which maximal medical therapy is not effective , the surgical removal of an mp ( cea ) or coverage of an mp ( cas ) should be considered . cea surgically removes an mp , and it seems to be the ideal treatment ; however , requirements for general anesthesia sometimes make it difficult to perform cea . when cas has to be conducted , a large - sized guide catheter has to be placed in the cca . usually , a 0.035-inch wire is advanced to the ipsilateral eca in order to provide enough support for the tracking of the guide catheter . in patients with mp , toughing of the mp with the wire should be avoided when a guide catheter is placed . in our case , a 0.038-inch wire was placed distal to the cca ; however , the guide catheter could not be advanced along the wire . the advancement of a 0.038-inch wire to the eca was needed for the placement of a guide catheter to the cca . iwata et al . reported 62 cases of successful transbrachial cas using a novel sheath guide ( msk - guide ; medikit , tokyo , japan ) . the system did not require the advancement of wires to the eca , and the use of this system is considered to be effective in patients with mp ; however , a flow reversal can not be achieved with it . proximal protection , which is known as the parodi anti - embolic system , is achieved by establishing a reverse flow in the ica by blocking the flow of the cca and eca . in distal protection , proximal protection can overcome this drawback ; however , the patients should be tolerated to flow reversal , and the injection of contrast medium during the procedure is not allowed , resulting in a difficulty in stent deployment . in our case , however , the free cell area of closed - cell stents is smaller than those of open - cell stents . the use of closed - cell stents with a smaller free cell area is expected to cover a plaque more efficiently than open - cell stents . histological studies suggest that mp is more embologenic than non - mp , and the use of closed - cell stents should be encouraged in terms of plaque coverage . to our knowledge , 2 cases of mp in which cas was undertaken have been reported . both cases were asymptomatic , and this is the first reported case of successful cas in a patient with stroke in evolution due to mp . in conclusion , this present case suggests that cas can prevent ischemic stroke in evolution . however , the accumulation of cases is required to elucidate the nature of cas for mp in terms of prevention of ischemic stroke .

in korea , cancer has ranked highest among all causes of death . during 2009 , the cancer incidence rate for all sites combined showed an annual increase of 3.3% ( 1.6% in males , 5.3% in females ) from 1999 to 2009 . in an effort to reduce the burden of cancer , national cancer control programs were formulated by the korean government in 1996 , and the government established the national cancer screening program ( ncsp ) in 1999 . since then , the target population and types of cancer covered have been expanded . until 2001 , the ncsp provided medical aid recipients with screening for three types of cancer ( stomach , breast , and cervix ) ; this service was provided free of charge . national health insurance ( nhi ) beneficiaries in the lower 20% income stratum were included in the ncsp in 2002 . in 2003 , nhi beneficiaries in the lower 30% income stratum and one additional type of cancer ( liver ) were included . in 2004 , colorectal cancer was included in the ncsp , and , since 2005 , the ncsp has provided medical aid recipients and nhi beneficiaries in the lower half of the income stratum with screenings for five types of cancer ( stomach , liver , colorectal , breast , and cervix ) . nhi beneficiaries in the upper 50% income stratum receive screening services for the same five types of cancer from the nhi corporation ; however , they are required to pay 10% of the cost [ 2 - 6 ] . in addition to these organized cancer screening programs provided by the government , opportunistic cancer screening is widely available in korea . organized cancer screening programs utilize nationally implemented protocols that define a target population , screening interval , and follow - up strategies ( appendix 1 ) . however , opportunistic cancer screening programs also include a variety of options in terms of the items screened , intervals between screening , and target cancer , depending on individual decisions or recommendations from health care providers . all costs of opportunistic cancer screening are paid entirely by users without a governmental subsidy . the current study was conducted in order to report on trends in overall cancer screening rates , including both organized and opportunistic cancer screenings among the korean population . the korean national cancer screening survey ( kncss ) , a nationwide , population - based , cross - sectional survey , has been conducted annually by the national cancer center since 2004 . stratified multistage random sampling based on resident registration population data is conducted according to geographic area , age , and gender . this study was approved by the institutional review board of the national cancer center , korea ( approval no . according to the protocols of the ncsp ( appendix 1 ) , people older than 40 years were eligible to undergo gastric cancer screening ; those older than 50 years were included for colorectal cancer screening ; women older than 40 years were eligible to undergo breast cancer screening ; and women older than 30 years were included for cervical cancer screening . screening for liver cancer was restricted to individuals older than 40 years , including those in high - risk groups , such as those who were positive for hepatitis b virus surface antigen or hepatitis c virus antibody or who had liver cirrhosis . the ncsp has provided screening for all members of the korean population who are of the appropriate age . therefore , the target population of this study was cancer - free men 40 years of age and older and women 30 years of age and older in each year . for stratified multistage random sampling , the number of enumeration districts was designated in proportion to the population size by considering gender , age , and area , and the final study clusters were randomly selected . five to eight households in an urban area and 10 to 12 households in a rural area were chosen randomly . the data were collected by a professional research agency using face - to - face interviews , except in 2004 , when the data were collected using computer - assisted telephone interviews . subjects were recruited through door - to - door contact , and at least three attempts to contact each household were made . one person was selected from each household ; if there was more than one eligible person in the household , the person whose date of birth was closest to the study date was selected . participants were asked about sociodemographic characteristics and their experience with screening for five types of cancer ( stomach , liver , colorectal , breast , and cervix ) . the questions included : " have you ever undergone [ cancer type ] screening ? " and " which screening method have you experienced ? " for the interval between screenings , the question was : " when did you last undergo [ cancer type ] screening with this method ? " to determine the reasons for undergoing screening or not undergoing screening , we asked : " what are your primary reasons for undergoing screening or not undergoing screening ? " distributions of sociodemographic characteristics of the study population for each year are shown in appendix 2 . the " screening rate with recommendation " category was assigned to participants who had undergone screening tests according to the protocols of the ncsp ( appendix 1 ) . however , for colorectal screening , respondents who underwent colonoscopy , double - contrast barium enema ( dcbe ) , or fecal occult blood test ( fobt ) within five , five , or one year , respectively , before 2009 , and within 10 , five , and one year , respectively , in 2009 and afterward were regarded as having undergone screening with recommendation . changes in annual lifetime screening rates and screening rates with recommendation were calculated as the annual percentage change ( apc ) within 95% confidence intervals ( cis ) . monthly household income was subgrouped into three tertiles for each year . due to an inadequate number of individuals within the high - risk group , as well as unstable results that showed a wide 95% ci , lifetime screening rates and screening rates with recommendation showed a continuous increase from 2004 until 2012 . on average , between 2004 and 2012 , the screening rate with recommendation showed an annual increase of 4.3% for gastric cancer , 0.8% for liver cancer , 2.4% for colorectal cancer , 4.5% for breast cancer , and 1.3% for cervical cancer ( table 1 ) . significant increasing trends were observed in the screening rates for gastric , colorectal , and breast cancer , but not in those for liver or cervical cancer . despite a stable pattern , compared with 2010 and 2011 , an increasing trend of screening rates the apc of the screening rate using upper endoscopy was more than twice the screening rate using upper gastrointestinal series ( 4.4% per year vs. 1.8% per year , respectively ) . on average , the colorectal cancer screening rate using fobt showed a more rapid increase , compared with screening using colonoscopy or dcbe ( 3.0% per year vs. 1.9% per year , and 0.1% per year , respectively ) . among men , between 2009 and 2011 , the screening rate with recommendation for stomach cancer showed a significant increase , while that of colorectal cancer showed a plateau ( fig . 1 ) . in women , despite an increase in the rate of screening with recommendation for stomach and breast cancer , the trend for cervical and colorectal cancer uptake according to recommendations plateaued between 2009 and 2011 . screening rates for some types of cancer plateaued between 2009 and 2011 ; on the other hand , all cancer screening rates showed an increase in 2012 , compared with those in 2011 in both men and women . overall screening rates with recommendation showed an increase in all age groups and for all types of cancer , with the exception of cervical cancer among women in their thirties ( fig . the most significant increases were those of gastric cancer screenings and breast cancer screenings among subjects over the age of 70 years , both of which showed sharp increases when compared with other groups . screening rates with recommendation for stomach and breast cancer showed a steady increase at all income levels , and differences in screening rates showed a decrease among all income groups , compared with those in 2004 ( fig . 3 ) . screening rates for colorectal cancer peaked in 2008 , and showed a decreasing pattern between 2008 and 2011 , followed by an increasing pattern in all income groups . the screening rate for cervical cancer was lower in the low - income group than in the middle- and high - income groups , while gaps between groups showed a decrease . lifetime screening rates and screening rates with recommendation for the five herein - described types of cancer have shown a significant increase since 2004 . one of the goals of the second - term 10-year plan for cancer control , 2006 - 2015 was achievement of an increase in cancer screening rates with recommendation to 70% by 2015 . in this study , the screening rates with recommendation for stomach and breast cancer exceeded 70% in 2012 . . in the united kingdom , where nationwide organized cancer screening has been implemented , 73.3% of women aged 45 to 74 years underwent mammography in 2009 - 2010 . differences in screening rates among age groups have shown a continuous decrease , and the screening rates for all age groups were over 70% in 2009 - 2010 . the five - year coverage for cervical cancer screening using the pap smear test has remained at 78.9% for women 25 to 49 years of age since 2008 ; however , in women 50 to 64 years of age , a slight decrease was observed , from 78.9% in 2009 - 2010 to 77.9% 2010 - 2011 . in the united states , the prevalence of having undergone recent screening with either fobt or endoscopy was 46.8% in 2005 and 53.2% in 2008 , indicating a 6.4% increase over the three - year period . the screening rate using colonoscopy was much higher than that using fobt , while in korea , fobt and colonoscopy showed similar total colorectal cancer screening rates . colonoscopy showed an increase from 1987 to 1998 , stabilized until 2000 , and then began to rise again after 2000 and showed a particularly rapid increase ( apc , 7% ) from 2003 to 2010 . in korea , both the lifetime screening rate and screening rate with recommendation for colorectal cancer have shown a steady increase since 2004 . in addition , the screening rate for colorectal cancer using fobt showed a more rapid increase when compared with colonoscopy , which may be due to guidelines of the organized cancer screening program , which designated that only cases showing abnormal results on fobt could undergo inspection by colonoscopy or dcbe . between 2000 and 2005 , the screening rate showed a slight decline of 3.4% in the reported use of mammography in the past two years among women aged 40 years and older . based on the national health interview survey , the annual screening rate for breast cancer was 51% and 53% in 2005 and 2008 , respectively , and declining trends were no longer apparent but instead rose overall and across groups for the recent period of 2005 through 2008 . screening rates for breast cancer of korean women in the same year were lower than those reported ; these rates have also risen steadily in korea since 2004 . in 2008 , 78.3% of american women 18 years of age and older reported having undergone a pap test within the past three years , indicating a small decrease of -1.3 percentage points ( 79.6% in 2005 ) . as with breast cancer screening , no change in absolute differences in the rates of cervical cancer screening according to education was observed . in japan , the screening rate for gastric cancer was 11.8% in 2007 , with a declining trend since the early 1990s . the screening rate for colorectal cancer was 18.8% , trending toward a gradual increase ( apc , 0.5% ) . screening rates for colorectal , breast , and cervical cancer are slightly lower in korea than in western countries , such as the united states [ 14 - 16 ] and the united kingdom [ 10 - 12 ] ; however , they are higher than those in the average of member countries in the organization for economic cooperation and development ( oecd ) for breast cancer screening ( 62.2% in 2009 ) and cervical cancer screening ( 61.1% in 2009 ) . although survey - based results may be affected by recall bias , findings from many studies have demonstrated the reliability of self - reported histories of cancer screening , which have shown good agreement with medical records [ 20 - 22 ] . in addition , stratified multistage random sampling of survey data was performed according to geographic area , age , and gender , but not according to insurance type . in 2012 , the proportion of medical aid recipients was low compared with that in other years . because screening rates for medical aid recipients were lower than those for nhi beneficiaries ( data not shown ) , the proportions of health insurance types may have affected the greater increase in screening rates in 2012 . finally , the response rate in our study ranged from 34.5% to 58.5% ; however , compared with other nationwide studies conducted in korea , in which response rates were less than 50% [ 23 - 25 ] , our response rate can be considered acceptable . despite some limitations , therefore , the data were sufficiently representative for use in investigation of cancer screening rates , including both organized and opportunistic cancer screening , and the cancer screening rate showed a steady increase . screening rates for five types of cancer have shown a steady increase since 2004 , and those of some types of cancer reached the 10-year plan for cancer control target of 70% , excluding liver and colorectal cancer . thus , the national cancer control plan to reduce the economic burden of cancer has been implemented successfully ; however , greater effort is still needed in order to increase the screening rates for liver and colorectal cancer . ugi , upper gastrointestinal series ; afp , alpha - fetoprotein ; fobt , fecal occult blood test . in the case of an abnormality on the ugi , endoscopy is recommended , and a biopsy is performed when an abnormality is found during endoscopy , patients at high risk for liver cancer include those with chronic hepatitis determined from serological evidence of infection with hepatitis b or c virus or liver cirrhosis , in the case of an abnormality on fobt , colonoscopy or a double - contrast barium enema is recommended , and a biopsy is performed when an abnormality is found during colonoscopy . restricted to women aged 30 - 39 years , due to missing data , some row sums are not 100% every year , 1 usd=1,000 krw , others : divorced or separated , the question related to residential area did not distinguish between urban and rural areas in 2004 .

the primary amino acid sequence of a protein contains all the information necessary for protein folding and its biological activity . however , in a normal cellular condition , a nascent polypeptide chain faces a crowded environment and there is a good possibility that protein will be misfolded and will form aggregates that make the protein inactive , and in certain cases it becomes toxic for the cell . both the prokaryotic and eukaryotic cells possess a family of proteins responsible for binding to nascent polypeptide chains and help them fold into biologically functional three - dimensional structures , they are known as molecular chaperones , and they vary in size and complexity [ 26 ] . many of the molecular chaperones are induced in response to stress or heat , and so they got the name hsp ( heat shock protein ) . molecular chaperones like hsp90 , hsp70 , hsp40 , and hsp104 bind to nascent polypeptide chain at hydrophobic regions which are exposed to the crowded environment otherwise buried inside in a completely folded protein [ 710 ] . molecular chaperones have developed multiple and diverse tertiary and quaternary structures to bind nonnative protein substrates . though , there is a lack of sequence similarity among different families of chaperones and only a few of them are represented in all three domains of life ( bacteria , archaea , and eukaryote ) , generally , they use convergent strategies to bind the substrates . crystallographic and other evidence show that many chaperones including prefoldin , trigger factor , hsp40 , and hsp90 have clamp - like structures , possibly responsible for the binding of nonnative substrates . another class of cylindrical - shaped chaperones , known as chaperonins , is found to be conserved in all three domains of life and assist the folding of many cytosolic proteins [ 12 , 13 ] . in some cases , the transient binding of nascent polypeptide chain is sufficient for protecting its hydrophobic regions and promoting its proper folding . however , for the folding of a multidomain protein , more than one class of chaperones might be involved , and they work in a concerted manner to generate a protective passage . for example , nascent polypeptide chain coming out from ribosome will first bind to hsp70/hsp90 which will help attain a quasinative structure and then will be transferred to chaperonin cct for its final folding [ 14 , 15 ] . here we review the current status of understanding of protein folding by the chaperonin cct complex in eukaryotes . the chaperonins are large , multimeric , cylindrical protein complexes consisting of two stacked rings and each ring has 79 subunits [ 2 , 4 , 1618 ] . on the basis of amino acid sequence homology , chaperonins have been categorized into two groups , group i and group ii [ 1924 ] . group i is found in all eubacteria and endosymbiotic organelles like mitochondria , chloroplasts , and related organelles like hydrogenosomes and mitosomes whereas group ii chaperonins are present in archaebacteria and in the cytosol of all eukaryotes [ 2 , 17 , 2527 ] . here we will give brief introduction to group i chaperonin and then will discuss group ii chaperonin , cct . the function of this group has been well studied using groel / groes from escherichia coli . the genes for groel and groes were discovered in a mutagenic screen for genes required for the growth of bacteriophage lambda and later found to be essential for the survival of e. coli itself in all conditions [ 2830 ] . groel / groes has been the subject of extensive structural and functional analysis for understanding protein folding in vitro and in vivo [ 3133 ] . groel is about 800 kda homooligomeric protein complex with atpase activity and is composed of two seven - membered rings of 57 kda subunits . each ring has a central cavity aligned with hydrophobic surfaces for the binding of unfolded or denatured proteins [ 3437 ] . the cochaperonin groes is a heptameric ring complex composed of 10 kda subunits and caps the groel folding chamber [ 3840 ] . first , confinement of substrate protein in the groel / groes complex is necessary for protecting them from aggregation . second , the folding process in the chaperonin would be much faster than that of in a free solution . x - ray studies of groel have revealed three distinct domains : equatorial domain , apical domain , and intermediate domain . the equatorial domain is responsible for most of the intra- and intersubunit interactions as well as for the binding of atp and its hydrolysis . the apical domain encompasses the entrance of central cavity and holds all the hydrophobic residues required for substrate binding . the intermediate domain connects both the domains and acts as a hinge for the movements of apical domain which is induced upon binding of atp and its hydrolysis [ 4345 ] . the substrate binding residues present in the apical domain is also responsible for binding of co - chaperonin , groes , which is essential for groel - mediated protein folding . the mechanism of groel - mediated protein folding has been extensively studied using different approaches [ 16 , 24 , 46 , 47 ] . briefly , protein folding cycle starts with the binding of unfolded or denatured proteins and atp at one end ( the cis end ) of groel , followed by the binding of groes to the same end . the binding of groes caps the entrance and releases the substrate to the central cavity . the binding of groes also promotes atp hydrolysis and the protein substrate gets folded in the anfinsen cage of the groel - groes - adp complex . the binding of protein substrate and atp to trans ring causes the release of groes and substrate . in the group ii chaperonins , both archaeal thermosome and eukaryotic chaperonin containing tcp-1 ( cct ; also known as tcp-1 ring complex , tric ) are being studied using different techniques [ 4952 ] . the subunit tcp-1 of cct complex was first isolated from murine testes and subsequently it was found to be constitutively expressed in other mammalian cells , insects , and yeasts [ 5357 ] . the compelling evidence for cct complex as a chaperonin came from the observations of its involvement in the assembly of actin and tubulin filaments [ 5861 ] . though , initially , it was discovered as a folding machine for actin and tubulin , later , it was found to be involved in the folding of 510% of newly synthesized cytosolic protein substrates [ 12 , 52 , 62 , 63 ] . like groel system , cct also binds atp and hydrolyses it during protein folding cycle , the cct does not have detachable groes - like cochaperonin , rather , a flexible protrusions located in the apical domain in each cct subunit acts as a lid and is responsible for closing the central cavity [ 17 , 64 , 65 ] . the chaperonin cct is a large , cylindrical , multimeric complex having central cavity for binding unfolded or denatured polypeptides . the structure of cct is much more complex compared to groel because each ring of cct is composed of eight different but related subunits ranging between 52 and 65 kda [ 5860 ] . moreover , it has been observed that all the eight subunits of cct are essential in yeast . now the question is when homooligomeric groel is sufficient to fold an array of substrates , why did cct complex evolve eight different subunits ? perhaps , one possible explanation could be that certain specific combinations of subunits interact with specific structural features or motifs of the protein substrates and hence eight different subunits give large number of combinations to accommodate a broad variety of substrates for their folding [ 66 , 67 ] . furthermore , the presence of eight different subunits encoded by eight paralogous genes indicates that every subunit might have some specialized role in the overall functioning of cct . phylogenetic analysis of these eight subunits suggested that functional specialization of the individual subunits took place in the early phase of eukaryotic evolution and associated with its cellular functions and became essential for its survival [ 68 , 69 ] . the electron microscopy and single particle analyses show that the domain structures of cct are similar to those of groel and archaeal thermosome , and three domains ( equatorial , apical , and intermediate domains ) are present in a single subunit . the mammalian subunits of cct are designated as cct , cct , cct , cct , cct , cct , cct , and cct which are corresponding to cct1p - cct8p in yeast . using biochemical and genetic approaches , it has been shown that cct is a single heterooligomer of eight subunits and the arrangement of the subunits has a unique pattern as shown in figure 1 [ 70 , 71 ] . the arrangement of subunits in cct ring also addressed by modeling using existing data , and four possible models have been proposed with clockwise and anticlockwise orientations ; however , the relative positions of the subunits in a ring remain same [ 7174 ] . the phasing between two rings of cct has been addressed by three - dimensional reconstructions generated by electron microscopy and using monoclonal antibodies against cct4p and cct5p . it has been shown that interring communications take place through two different subunits in all the eight positions . moreover , the subunits associated with initiation and completion of the folding cycle cluster together in both inter- and intraring arrangements . however , recent cryo - em analysis of closed cct suggested new positions in a ring for three subunits - cct1p , cct5p , and cct7p , and the subunits in a ring are arranged as cct1pcct7pcct5pcct4pcct8pcct3pcct2pcct6p in a clockwise orientation . it reveals a unique pattern of interring arrangement that generates three heterotypic interring contacts ( cct3p - cct4p , cct2p - cct5p and cct6p - cct7p ) and two homotypic interring contacts ( cct1p - cct1p and cct8p - cct8p ) . as the relative positions of other five subunits remain same in both the models and only three are having deviation in each of these models , fine - tuning of biochemical and em analyses will be required to fix their positions . recently , crystal structure analysis has been done for cct purified from bovine testis along with its natural substrate , tubulin in the open conformation . this structure showed that substrate interacts with cct in the loops of apical and equatorial domains . the organization of atp binding sites indicates that the substrate is stretched inside the cavity . moreover , the fold of each domain ( equatorial , intermediate and apical ) of the eight subunits of cct found to be similar to those of - and -subunit of thermosome . the thermosome equatorial domains can be superimposed on their cct homologues , though small rearrangements in the orientation of helices and loops along the equatorial domain were observed . in contrast to the crystal structures of groel and thermosome , apical domains of cct showed a wide range of conformations , both in the central cavity and in the aperture of the lid domains [ 42 , 78 , 79 ] . this supports the existence of sequential , hierarchical mechanism of conformational changes induced by atp . the binding of atp to the subunits of cct and its hydrolysis is absolutely required for the cct - mediated folding of newly synthesized polypeptide or misfolded protein substrates [ 58 , 60 , 80 ] . in the absence of atp or in presence of adp , cct remains in an open conformation in which substrate binding sites in the apical domains are exposed and bind the substrates . addition of atp induces the conformational changes and subsequently the central cavity is closed by built - in lid structure to confine the protein substrate inside the cavity and provides a secluded environment for its folding . however , just addition of atp to cct does not promote the lid closure as shown using non - hydrolyzable atp analogs [ 65 , 81 ] . using non - hydrolyzable atp analog ( amp - pnp ) , it has been demonstrated that amp - pnp - cct binds to actin and tubulin on one cct ring [ 66 , 82 ] . on the other hand , when cct is blocked in its adp - pi state , this suggests that binding sites in the apical domain of cct will be available for target proteins in atp - cct state but not in adp - pi - cct state . therefore , nucleotide exchange and hydrolysis might be working as a regulatory switch for the binding of target proteins to cct . the bacterial chaperonin groel has a cofactor groes which is used as a lid for the closure of central cavity essential for protein folding . however , the eukaryotic cytosolic chaperonin cct / tric does not have any homologue of groes to cap the central cavity [ 17 , 78 ] . the crystal structure of archaeal chaperonin thermosome has shown the presence of protrusions emerging from the apical domain and arranged in an iris - like sheet which is responsible for closing the central cavity . these apical protrusions are unique to group ii chaperonins , like thermosome and cct , and they have been proposed to have groes - like activity and act as a built - in lid that might open and close in an atp - dependent manner [ 17 , 85 ] . compelling evidence for the requirement of lid responsible for encapsulating the unfolded polypeptides comes from the observations that lidless chaperonins lose the ability to fold stringent substrates [ 65 , 86 , 87 ] . functions for the closing and opening of the central cavity for substrate binding and releasing ? though there are striking similarities between group i and group ii chaperonins , the lid closure mechanism seems to be quite different from each other . the binding of groes to groel occurs upon atp binding to equatorial domain of the groel subunits whereas the lid formation in eukaryotic and archaeal chaperonins is triggered by the transition state of atp hydrolysis , suggesting nucleotide cycle dependent mechanistic difference of lid closure . in one study , using the thermococcus chaperonin , it has been suggested that atp binding / hydrolysis causes independent conformational changes in the subunits . however , complete closure of the lid is induced and stabilized by the interactions of the helical protrusions of different subunits . the high - resolution crystal structure of groel in atp - bound and -unbound formed and that of groes has identified an atp binding domain encompassing n- and c - terminus of groel subunits [ 42 , 90 , 91 ] . this domain contains highly conserved gdgtt ( residues 8690 ) atp binding residues along with loop structural motifs , lgpkg ( residues 3135 ) , itkdg ( residues 4953 ) , and ggg ( residues 414416 ) which are found to be conserved among chaperonins . sequence homology analysis of cct subunits with that of groel identified almost identical atp binding motif containing residues gdgtt and other loop structural motifs suggesting their conserved role in atp binding / hydrolysis in prokaryotes as well in eukaryotes . though the conserved loop elements appear to have certain common functions in atp binding / hydrolysis , they could possess some functions which are specific to certain loop elements in a subunit . this is inferred from the observations that certain alleles of the same gene , affecting residues from different conserved loop structures have different degrees of cytoskeletal dysfunctions [ 92 , 93 ] . besides , there might be functional hierarchy among the paralogous motifs of different subunits . for example , homologous replacement of the fifth conserved glycine residue to glutamic acid in the lgpkg motif causes a lethal phenotype in cct2p whereas the same mutation in cct1p makes it heat sensitive in yeast saccharomyces cerevisiae . however , the same replacement in cct6p does not have much effect ( table 1 and figure 2 ) [ 92 , 94 ] . furthermore , it has been shown that the conserved atp binding / hydrolysis motif gdgttaaaaa replacement in the cct6p in s. cerevisiae does not have much effect whereas same replacement is lethal for cct1p . from the above - mentioned experimental data , it can be suggested that different subunits play different role for atp binding / hydrolysis in the cct . this might be required for folding different substrates and modulating intraring and interring interactions . one of the most well - studied chaperonins is the groel from e. coli , and the recognition of substrate by this chaperonin has been studied to a large extent , and so we will briefly discuss substrate recognition mechanism of groel before we go into the details of substrate recognition by cct complex . a number of techniques have been used to unveil the mechanism of substrate binding by groel . the localization of substrate binding region appears to be at the entrance of central cavity of groel which has been shown by electron microscopy [ 34 , 97 ] . the x - ray crystal structure revealed this substrate binding region in each subunit and has been termed apical domain . a systematic mutational study was used to understand the role of different amino acids in this region , and it revealed that a152 , y199 , s201 , y203 , f204 , l234 , l237 , l259 , and v263 play important roles in binding the substrates as the mutations in these residues in the apical domain affect the substrates binding to groel severely . interestingly , most of these residues have hydrophobic side chains which can generate a hydrophobic surface for the binding of the substrate . on the other hand , mutations of charged amino acids in the apical domain appear to have no effect on the binding of the substrates . this suggests that hydrophobic residues in the apical domain are mainly responsible for creating hydrophobic surfaces in the central cavity for binding of substrates through hydrophobic interactions . however , the single - residue replacements in the intermediate domain ( i150e , s151v , a152e , a383e , a405e , and a406e ) exert global effect on the functioning of groel . all of these mutants showed severe defect in atpase activity though they fall outside the atp - binding domain . also the mutants at positions , 150 , 151 , 383 , and 405 could bind polypeptide but the release of the polypeptide was severely affected . on the other hand , d87k / d87n mutation in the conserved domain , gdgtt , in the equatorial domain , lost the atpase activity completely , though it has the ability to bind atp . it has also reduced the ability to bind polypeptide ; however , there was a complete block of polypeptide release . several techniques have been used to implicate the importance of hydrophobic interactions between groel surface and the substrates [ 98102 ] . however , there are some exceptions to this substrate recognition principle and certain other forces such as electrostatic interactions might play an important role as well for binding the substrate to groel efficiently [ 103105 ] . although group i chaperonin ( groel ) and group ii chaperonin ( cct ) have double ring structure and share sequence similarities , they differ from each other in two major aspects . first , group i chaperonin is composed of identical subunits and has seven subunits per ring whereas group ii chaperonin is composed of 28 paralogous subunits with 3040% homology to one another and each ring has 89 subunits [ 16 , 17 , 26 , 106 , 107 ] . for example , cct is composed of eight paralogous subunits [ 52 , 70 ] . as the sequence divergence in the apical domain is more among the paralogous subunits , it has been hypothesized that different subunit in cct has different substrate specificity [ 107 , 108 ] . second , group ii chaperonins do not have groes - like cofactor ; however , it possesses a helical protrusion that acts as built - in lid . these two major differences might be related to the evolution of these group ii chaperonins for assisting the folding of different archaeal and eukaryotic proteins . the structural and mechanistic differences between two groups might have profound functional impact on the substrate specificity [ 13 , 109 ] . for example , several eukaryotic protein including actin and tubulin can be folded by cct only whereas the bacterial proteins which require the assistance of groel for their folding , are not able to fold in eukaryotic cytosol [ 13 , 58 , 109 , 110 ] . the specificity of groel and cct towards the substrates is thought to be due to chemical nature of their interactions with substrates . it has been well established that groel recognizes the exposed hydrophobic surfaces of unfolded substrates [ 111113 ] . on the other hand , cct subunits possess specific binding sites for unique polar motifs of certain cellular proteins [ 82 , 114116 ] . however , using a biochemical approach , it has been shown that for the binding of actin , von hippel - lindau tumor suppressor and g wd-40 protein to cct , hydrophobic interactions are involved [ 117119 ] . in the absence of well - defined structural surfaces or motifs present in the substrates of cct , three model proteins , actin , tubulin , and von hippel - lindau tumor suppressor have been studied thoroughly to find the recognition sites present in these substrates as well as in the interacting subunits of cct . several studies have pointed out that the nature of actin and tubulin conformations bound to cct are not of any nonspecific structures as in the case of groel substrates rather they must have some kind of defined , quasinative conformations before they are recognized by cct for the final steps of their folding [ 80 , 120122 ] . the quasinative conformation may be achieved themselves or they may be guided by prefoldin kind of cochaperonin to reach that conformation . the atomic structure of actin has shown the presence of two domains , small and large . the three - dimensional reconstruction analysis of alpha actin with cct using electron microscopy has shown that cct interacts with these two domains of actin by two specific and distinct interactions . the small domain of actin interacts with cct4p subunit whereas large domain interacts with either cct2p or cct5p ( both are 1,4 position with respect to cct4p ) . this observation led to the suggestion that cct interacts with actin in subunit - specific and geometry dependent - manner . the three - dimensional reconstruction analysis combined with immunomicroscopy and screening study using actin peptide arrays have identified residues in four regions of actin molecule [ 114117 , 124 ] . two of these regions ( r37-d51 and r62-t66 ) are located at the tip of small domain and interacts with cct4p subunit of cct . the other two regions ( e195-r206 and t229-i250 ) are present at the tip of large domain and interact with cct2p or cct5p subunit . mutational analysis coupled with electron microscopy and biochemical assay have shown that major determinants of actin binding to cct are present at the tip of the large domain [ 116 , 125 ] . a mutation ( g150p ) in the conserved putative hinge region between small and large domains resulted in the accumulation of actin on the chaperonin cct . furthermore , electroncmicroscopic studies have shown that actin interacts with cct2p or cct5p subunits rather than cct4p subunit . it was thought that cct2p and cct5p might have highest substrate affinity , and this possibility has been strengthened by immunoprecipitation experiments of actin - cct complexes . on the other hand , the interaction of tubulin with cct seems to be much more complex and it does not confine to a few regions of tubulin rather it is spread along its entire sequence and interacts with several domains at a time [ 82 , 117 , 122 , 124 , 126 , 127 ] . several studies have shown that cct binding sites in tubulin are present in loops exposed to the surface of native protein [ 67 , 82 , 128 , 129 ] . of the eight binding sites present in tubulin , three are located at n - terminal domain and five are placed in c - terminal domain . immunomicroscopic experiments have shown that residues t33-a57 interact with cct1p or cct4p whereas the residues s126-q133 and e160-r164 interact with cct7p or cct8p . the interaction of c - terminal domain of tubulin with cct is much more complex than that of n - terminal domain . five putative segments present in the c - terminal domain responsible for interaction with cct are t239-k254 , p261-h266 , s277-v288 , v355-p359 , and w407-e417 , and they interact with multiple cct subunits at a time . the segments t239 - 254 and p261-h266 interact with cct6p or cct3p subunits , the segments s277-v288 and v355-p359 interact with cct5p or cct2p whereas the segment w407-e417 interacts with cct2p or cct8p subunits . though it was suggested that tubulin binding sites could have weak interactions with cct , the residues s277-v288 are thought to be hot spot for the binding of tubulin to cct and it might have higher affinity to cct compared to other sites [ 117 , 122 , 126 , 127 , 130 ] . recently , jayasinghe et al . used a computational approach to pinpoint the interactions between gamma subunit of cct and its stringent substrate beta - tubulin . it has been shown that the substrate binding sites in cct are composed of helical region ( hl ) and helical protrusion region ( hp ) . interaction of substrate at helical region involves both hydrophobic and electrostatic contacts while binding to helical protrusion is stabilized by salt bridge network . the tumor suppressor protein von hippel - lindau ( vhl ) has been extensively studied from substrate point of view of cct and has been shown to be an obligate substrate of cct [ 14 , 132 ] . vhl is a subunit of a ubiquitin ligase complex that targets cellular proteins , like hif-1 , for proteolysis [ 133 , 134 ] . loss of function mutations in vhl is responsible for the tumor formation in kidney , adrenal glands , and central nervous system [ 135 , 136 ] . vhl is composed of 213 amino acid residues of which 55-amino acid domain ( 100155 residues ) is necessary and sufficient for binding to cct [ 132 , 137 ] . interestingly , most of the mutations in this domain are responsible for vhl diseases [ 138140 ] . using alanine - scanning mutagenesis procedure , the 55-amino acid segment has been completely analyzed to identify minimal regions responsible for cct binding . this has revealed that two small regions of vhl , amino acid 116119 ( box1 ) and 148155 ( box2 ; figure 3 ) are absolutely required for stable binding with cct . furthermore , contribution of individual amino acid in these two boxes has been evaluated using single alanine substitution mutants . this analysis has shown that alanine replacement of w117 and l118 within box1 or f148 , i151 , l153 , or v155 within box2 ( figure 3 ) substantially reduce the binding of vhl to cct . though these two boxes are distant in the primary structure , they are located in the adjacent strands within the sheet domain of folded vhl and the side chains of two boxes are projected in the same direction to generate a hydrophobic surfaces required for its interaction with cct . allosteric regulation plays an important role for transitions between different functional states among the molecular machines in response to changes in environmental conditions . as it was strongly believed that allosteric regulation of chaperonins is crucial for assisting the protein folding , the chaperonins groel and cct complex were studied from allosteric point of view to understand their functioning [ 47 , 141 ] . the allosteric transitions of groel can be described by a nested allosteric model in which each of its rings is in equilibrium between a t state and an r state . the t state has low affinity for atp and high affinity for unfolded substrate proteins whereas r state possesses high affinity for atp and low affinity for unfolded substrates [ 142144 ] . it has been shown that t and r states interconvert in a concerted manner in accordance with monod - wyman - changeux ( mwc ) model of cooperativity . however , in the presence of high concentration of atp , groel ring switches from tt state to rr state via tr state in a sequential manner in accordance with koshland - nmethy - filmer ( knf ) model of cooperativity . though the overall structure of both groel and cct is similar , cct is different from groel with respect to its subunit composition and so , it was important to unveil whether intra - ring transitions are concerted or sequential for cct . kinetic studies have shown that cct undergoes two atp - dependent transitions and they most likely correspond to each of its two rings . atp - induced conformational changes have been detected by monitoring changes in fluorescence and visualized using cryo - em and single - particle reconstructions [ 66 , 147149 ] . it has been found that the binding of atp to cct generates an asymmetric particle in which one ring will have slight conformational changes whereas the other ring undergoes a substantial movement in the apical and equatorial domains . using the powerful yeast genetics , it has been suggested that intra - ring conformational changes in cct are not concerted rather it occurs in a sequential manner around the ring . this was inferred from the suppression analysis of different mutant alleles of cct1p , cct2p , cct3p , and cct6p . first , a lot of conformational heterogeneity has been observed in the apo state of cct but not in groel . second , atp - induced conformational changes take place in a sequential manner in cct whereas concerted mechanism is observed for groel . biochemical as well as genetic analysis data suggested that atp - induced conformational changes in cct take place in the order cct1pcct3pcct2pcct6p [ 70 , 150 ] . the role of cct has been well established for the folding of a large number of proteins . however , it was not clear in the beginning whether cct alone is sufficient for the folding of nascent chains to their maturity or other components are also required . later , it was found in a genetic screen during the identification of synthetic lethals for gamma - tubulin that gimc , also known as prefoldin ( pfd ) , participates in the maturation of cytoskeletal proteins . using biochemical approach , it has been shown that prefoldin plays an important role for the formation of functional actin and tubulin by transferring unfolded protein substrates to cct . the role of prefoldin ( gimc ) has been established in the folding of actin using chaperone trap and suggested that prefoldin acts along with cct for the maturation of the substrate protein . using in vitro transcription / translation of actin , it has been shown that unfolded actin polypeptide chain remains bound to prefoldin until it is transferred to cct . however , eukaryotic prefoldin is composed of six different subunits whereas archaeal prefoldin has only two different kinds of subunits : and subunits . it is possible that like cct , eukaryotic prefoldin has been developed to more complex structure from simpler archaeal form to heterohexameric structure to participate in the more complex protein folding processes . three - dimensional reconstruction of cct with prefoldin based on electron microscopy analysis has shown that prefoldin interacts with each of cct rings in a unique conformation with two specific subunits that are placed in a 1,4 arrangement . therefore , it is highly desirable that pfd : actin complex will interact with cct through cct4 and cct2 subunits or cct4 and cct5 subunits . a large body of evidence show that heterohexameric complex of prefoldin uses its jellyfish or octopus - like structure to grip nonnative protein substrates and transfer it to cct for proper folding [ 74 , 155 , 156 ] . another set of proteins implicated in the regulation of cct function are phosducin - like proteins ( phlps ) which were originally identified as modulators of heterotrimeric g protein signaling . subsequently , they were found to play an important role in the regulation of cct function [ 158162 ] . phlps are subdivided into three families like phlp1 , phlp2 , and phlp3 and they share structural similarities at n - terminal helical domain , a central thioredoxin - like fold , and a c - terminal extension . phlp1 has been shown to have inhibitory effect on cct and this may be required for regulating the protein folding capacity of cct . electron microscopy reconstruction of mammalian cct : phlp1 has demonstrated that phlp1 binds to apical domains of several chaperonin subunits . furthermore , the interaction of phlp2 with cct was suggested in proteome - wide studies . to substantiate this observation , in vitro study was done using human phlp2a and has been shown that it does inhibit the folding of actin and forms ternary complex with cct and actin in mammalian system . however , recent study has suggested stimulatory role of plp2 in yeast s. cerevisiae . it has been shown that plp2-cct - act1 complexes produce 30-fold more actin than cct - act1 complexes in a single atp driven cycle . plp2 itself can bind to actin through its c - terminal of thioredoxin fold and cct - binding subdomain 4 of actin . the inhibitory effect of human pdcl3 , an orthologue of plp2 , can be relieved by exchanging the acidic c - terminus extension of that of plp2 of yeast . therefore , it seems that higher eukaryotes have developed another level of regulatory control of cct by phosducin - like proteins . the third member of this family , phlp3 , has been shown to bind cct as well . the phlp3 forms ternary complex with cct and actin or tubulin , and does inhibit the folding process . it has been suggested that this negative impact is not due to direct competition for substrates rather by diminishing the atpase activity of cct by phlp3 . moreover , in vivo experiments have shown that yeast phlp3 might coordinate the proper biogenesis of actin and tubulin with prefoldin . so , it is clearly established that phosducin - like proteins are responsible for regulating the function of cct along with normal function of g protein signaling [ 161 , 162 , 164 , 166 ] . therefore , both prefoldin and phosducin - like proteins are working as co - chaperones to modulate the function of cct . in another study , it has been shown that caveolin-1 can interact with cct and modulates its protein folding activity . the caveolin-1-tcp interaction involves the first 32 amino acids of the n - terminal segment of caveolin . phosphorylation at tyrosine residue 14 of caveolin-1 induces the detachment of caveolin-1 from cct and activates actin folding . bag3 protein is known as co - chaperone of hsp70/hsc70 and involved in the regulation of various cell processes , such as apoptosis , autophagy , and cell motility . using rnai , it has been shown that strains lacking bag3 activity slowed down - cell migration and also influenced the availability of correctly folded monomeric actin . altogether , it shows that cct is highly regulated by cochaperones for its folding activity of actin and other proteins and the interaction of different co - chaperones with cct decides the fate of the final folding process . recently , it has been demonstrated that p90 ribosomal s6 kinase ( rsk ) and p70 ribosomal s6 kinase ( s6k ) can phosphorylate cct in response to tumor promoters or growth factors that activate the ras - mitogen activated protein kinase ( mapk ) pathway . furthermore , it has been shown that cct2p plays an important role in regulating cell proliferation and especially the phosphorylation of cct2p at ser-260 contributes substantially to this . though cct2p has been implicated in this process , first , phosphorylated cct2p subunit itself might be interacting with certain factors of ras - mapk and pi3k - mtor - pathways and regulate the cell proliferation in response to multiple agonists in diverse mammalian cells . second , the phosphorylation of cct2p subunit of cct might change the folding rate and reduce the stress - related unfolded proteins in the cell . on the other hand , it has been shown that a fraction of groel is phosphorylated at least one phosphate at each of its subunits in e. coli [ 170 , 171 ] . this phosphorylation of groel subunits enhances 50100 -fold capacity of this chaperonin to bind to denatured proteins . possibly , the phosphorylated form of groel might be responsible for refolding or degradation of certain damaged polypeptides . in another study , it has been shown that groel of thiobacillus ferrooxidans is phosphorylated in response to phosphate starvation suggesting its role in sensing and regulating stress responses in bacteria . it is plausible that cct carrying phosphorylated form of cct2p might be playing similar roles in eukaryotes . many newly translated proteins may interact with several different chaperones before they reach their biologically functional three - dimensional structures . one of the most abundant molecular chaperones is hsp70 which was found to associate with cct in vivo suggesting their cooperation in protein folding [ 59 , 132 ] . in vitro experiments were performed to elucidate the cooperative nature of hsp70 and cct using mammalian cell - free lysates . it has been shown that short chains of actin and firefly luciferase can interact with hsp70 whereas longer ones interact with cct [ 173 , 174 ] . besides , the cooperation between prefoldin ( gimc ) and cct were found for folding of actin and tubulin [ 152154 ] . using three - dimensional reconstruction of cct : pfd based on cryoelectron microscopy , it was shown that prefoldin binds to cct through two specific subunits . moreover , several studies have also suggested the interaction between phosducin - like proteins and cct indicating their cooperative nature for protein folding [ 158 , 159 , 165 ] . furthermore , it has been shown that sequential cooperation between hsp70 and hsp90 plays an important role for the folding of steroid hormone receptors and kinases [ 175 , 176 ] . from the above - mentioned experimental evidence , it appears that cooperation between different chaperones is a central principle to the protein folding process . for example , it has been shown that many chaperones are recruited to the protein synthesis machinery , and as soon as the polypeptide chains are coming out , they are protected by these chaperones including cct . it has been demonstrated that chaperones can bind to ribosome - bound polypeptide chains in both prokaryotes and eukaryotes [ 173 , 174 , 177183 ] . it seems that the cellular proteins follow different chaperone - dependent and chaperone - independent pathways for reaching their biologically functional three - dimensional structures . the proteins in the chaperone - independent pathway will probably be small proteins and they can fold without any help from any chaperones . the chaperone - dependent folding process might follow three different strategies depending upon the nature of the proteins . first , newly synthesized proteins will be bound by hsp70 , hsp90 , or other small chaperones transiently at the exposed hydrophobic regions present on the surface of nonnative proteins and will be protected from aggregation and will fold . second , some proteins will be bound by hsp70 and hsp90 sequentially and will proceed for folding . third , a small fraction of newly synthesized polypeptide chains will bind sequentially to hsp70 , hsp90 , and prefoldin / phosducin - like proteins and then transferred to cct for final folding . the concerted and cooperative action of a large number of molecular chaperones leads to the production of three - dimensional and biologically functional protein molecules . the failure in this process may result in the aggregation and misfolding of many essential proteins and may cause a severe effect on the overall functions in a cell . now it has been well recognized that protein aggregation and misfolding are the root causes for many diseases known as protein misfolding or the toxic effect of the aggregated or milfolded protein could be because of gain - of - function which will have titrating effect on interacting proteins or due to the loss of function of misfolded proteins [ 185 , 186 ] . aggregation process is a multistep process having stable or metastable intermediates that lead to the formation of homotypic fibrillar aggregates that may interact with other proteins and result in the formation of inclusion bodies or plaques that deposit outside or inside the cells [ 187189 ] . a large body of evidence shows that the intermediates of the aggregate formation process are responsible for disease pathogenesis rather than the final products which may be inactive or protective [ 190 , 191 ] . for example , increased level of diffuse polyglutamine- ( polyq- ) expanded huntingtin is thought to be cause of cell death in huntington 's disease and inclusion bodies could enhance the survival . similarly , proteinaceous deposits or inclusion bodies are found to be not associated with toxicity in parkinson 's and alzheimer 's diseases [ 192194 ] . though chaperonins are generally responsible for maintaining the cellular protein homeostasis , they are now implicated in the pathogenesis of misfolding human diseases as well . both the group i and group ii chaperonins are found to be participating as modulators of misfolding diseases . for example , inactivation of mitochondrial hsp60 is responsible for hereditary spastic paraplegia , a late - onset neurodegenerative disease [ 195 , 196 ] . recent studies have clearly shown that polyq - expanded huntingtin is a potential substrate of cct [ 197199 ] . moreover , an rna interference screen in c. elgans has identified six out of eight subunits of cct as suppressors of polyq aggregation , suggesting that cct can bind polyq and inhibit the formation of toxic aggregates . it has been shown that overexpression of cct1p of cct was effective at inhibiting huntingtin aggregation and subsequently increased the viability . on the other hand , the deletion of cct6p of cct increases the huntingtin aggregation and toxicity [ 52 , 199 ] . generally , the cct substrates are large , hydrophobic proteins containing the regions with -strand propensity , and they are highly prone to the formation of toxic aggregates [ 63 , 117 , 118 , 201 ] . aggregation of these proteins may begin with conformational transition from native monomer to mature amyloid fibrils [ 197 , 202 , 203 ] . therefore , it is quite possible that cct binds directly to -sheets and protects the protein from being aggregated which can be otherwise toxic for the cell . it has been shown that overexpression of certain subunits of cct can protect from misfolding diseases . therefore , drug - mediated induction of molecular chaperones can be considered as one of the methods for treating these diseases . otherwise , certain cct subunits can be injected directly on regular basis like insulin in case of diabetic patients and misfolding diseases can be handled . molecular chaperones are crucial for the production of biologically functional three - dimensional protein structures . a large number of molecular chaperones are present in all the three kingdoms of life implying their importance in biological system . chaperonins are cylindrical structures having central cavity for encapsulating unfolded protein substrates and assist in protein folding in an atp - dependent manner . the chaperonin cct is composed of eight different but related subunits of differential functional hierarchy in which catalytic cooperativity of atp binding / hydrolysis takes place in a sequential manner rather than concerted cooperativity as found in groel . moreover , substrate recognition in cct takes place through diverse mechanisms involving hydrophobic and electrostatic interactions . for the fine - tuning of protein process , many cochaperones like prefoldin , phosducin - like proteins act as upstream factors and transfer the substrate to cct . these upstream molecular chaperones and chaperonins might be responsible for generating a protective chaperone cage for the newly synthesized polypeptide chains to minimize the chance of aggregation and misfolding . recently , it has been shown that certain cct subunits are phosphorylated in response to tumor promoters or growth factors suggesting the possible roles of different kinases and possible certain phosphatases in regulating the activity of cct . any abnormal function posed by any chaperone at any stage of protein folding might have severe consequences . many mutations in the molecular chaperones are now linked to parkinson 's and alzheimer 's diseases . better understanding of chaperonin cct and other molecular chaperones will be helpful to develop drugs for the treatment of misfolding or conformational diseases .

febrile infection - related epilepsy syndrome ( fires ) , also known as devastating epileptic encephalopathy in school - aged children or acute encephalitis with refractory repetitive partial seizures , is an acute and explosive - onset epileptic encephalopathy with a fatal outcome , which occurs in previously healthy children following a nonspecific febrile illness , . the precise etiology of this syndrome and the effective treatment options remain unclear . herein , we present our experience in treating a 12-year - old girl with fires , who dramatically recovered from this devastating encephalitis after treatment with a combination of lidocaine and mgso4 . a 12-year - old girl was admitted to our emergency department with complaints of severe headache , dizziness , and fever ( 38 c ) . prior to her admission , she was healthy without any epileptic disorders and had normal development . the patient did not have a familial history of any epileptic or genetic metabolic disease . a week prior to admission , she experienced fever , sore throat , and fatigue and was treated for acute pharyngitis . on admission , she had a glasgow coma scale ( gcs ) score of 12 . on day 2 of admission , her condition worsened rapidly , and she fell into a state of coma ( gcs score of 3 ) . clonic seizures , accompanied by tachycardia , respiratory compromise , and hypotension alternating with myoclonic jerks of both lower limbs . although se was temporarily stabilized by administration of bzd agents , the focal myoclonic seizures , involving both lower limbs alternatively , did not stop . initially , central nervous system infection was highly suspected , and so we initiated antibiotic and antiviral treatments ; however , these were ineffective . simultaneously , multiplex polymerase chain reaction , microarray , high - throughput sequencing , and molecular biological detection to detect encephalitis with unknown pathogens based on taiwan pathogenic microorganism genome database were performed , but all test results were negative . unrevealing laboratory investigations included arterial blood gas analysis ; complete blood count ; blood glucose level ; electrolyte panel ; serum concentrations of blood urea nitrogen , creatinine , calcium , magnesium , phosphate , and liver enzymes ( aspartate aminotransferase , alanine transaminase , coagulation studies , and serum concentrations of bilirubin and ammonia ) ; and cultures of blood and cerebrospinal fluid . specialized metabolic tests were also conducted including quantitative plasma amino acids , evaluation of plasma lactate and pyruvate levels , acylcarnitine profile , and qualitative urine organic acids . drug testing using appropriate panels was performed on samples of blood and urine to determine the presence of drugs that can cause altered levels of consciousness , such as sedatives , salicylates , hallucinogenic agents , anticholinergic agents , opioids , monoamine oxidase inhibitors , acetaminophen , selective serotonin reuptake inhibitors , and tricyclic antidepressants and was negative . in addition , we examined for the presence of environmental toxins , such as organophosphates , lead or other heavy metals , and hydrocarbons in the aforementioned biological samples all were unremarkable . screening for autoimmune and paraneoplastic panel in blood or cerebrospinal fluid [ i.e. , antibodies of anti - hu , anti - yo , anti - ri ( anna2 ) , anti - ma2 ( ma2/ta ) , anti - cv2/crmp5 , antiamphiphysin , anti - nmdar , anti - caspr2 , anti - ampar , anti - lgi1 ] revealed negative results . status epilepticus ( se ) was treated using various drugs at different time points , specifically using phenobarbital ( phb ; 15 mg / kg for loading , 3 mg / kg / day for maintenance ) , phenytoin ( 15 mg / kg for loading , 5 mg / kg / day for maintenance ) , valproic acid ( 15 mg / kg / day gradually titrated to 60 mg / kg / day ) , levetiracetam ( lev , 1500 mg q12hr ) , intravenous immunoglobulin ( 1 g / kg ; up to 3 courses ) , dexamethasone ( 7 days ) , midazolam ( 20 mg / kg / min continuous intravenous infusion ) , pyridoxine , and a ketogenic diet ( stopped by gastrointestinal bleeding ) . however , se occurred up to dozens of times a day ( > 30 times overall ; fig . drug - induced comas were induced four times ( two times with pentobarbital , one time with propofol , and one time with thiopental ) . throughout the course of hospital admission , frequent eeg monitoring was performed . electroencephalography showed burst - suppression features with suppression periods lasting 45 s and without electroclinical seizures . however , every time the dose of anesthetics was tapered , se relapsed ( fig . brain magnetic resonance imaging was performed two times , which demonstrated abnormal hyperintensities bilaterally over the external capsules , medial temporal lobes , hippocampi , and posteromedial thalami ( fig . 3 ) . on day 27 of admission , we started lidocaine therapy at a dose of 1.25 mg / kg / h . over the next 2 days , the severity and frequency of her generalized - type se , we added mgso4 at a dose of 20 mg / kg / h , which markedly improved the se and eeg readings from day 1 of administration ( fig . 2 ) ; her blood magnesium level was 3.3 mg / dl the next day . therefore , we discontinued the barbiturate - induced coma and gradually lowered the dose of midazolam infusion over several days before stopping it completely . she was discharged from the intensive care unit ( icu ) with intravenous lidocaine , mgso4 , and lev . at this time , no clinical seizure had been noted . an overview of the patient 's seizure evolution is as follows : the ictal discharge in the right frontal temporal region along with left - sided tonic seizure started approximately 810 s before the generalized seizure occurred , and when this was stopped by administering an anesthetic or bzd , the seizure transformed into clinical myoclonic jerks of both lower limbs ( l > r ) with eeg slow wave with / without visible epileptiform discharges . she then received rehabilitation therapy to counteract the prolonged period during which she had been bedridden in the icu . although seizures did not occur when all intravenous - type antiepileptic drugs ( aeds ) were stopped , the discharge medication was a prophylactic polytherapy with pb , lev , topiramate , lorazepam , and pyridoxine . neither seizures nor significant neurological complications were observed during this period ; however , her short - term memory was slightly impaired " by amnesia " . neuropsychological evaluation with the wechsler intelligence scale for children iv revealed no difference in the four factor indices compared with expected values for her age . to most neurologists , treating fires is extremely problematic because its etiology and treatment remain unclear . although the severe seizures can be controlled , the patient is usually left with a grave psychomotor impairment . in this case , we not only found that a combined regimen of lidocaine and mgso4 achieved a favorable control of se but also found that it resulted in minimal psychomotor impairment . with the exceptions of comas induced by anesthetics or barbiturates , most aeds fail to stop the se that results from fires . however , the significant danger is that the longer the burst - suppression coma is induced , the worse the cognitive outcome . a review of the literature showed that few regimens have been proposed for this condition . used lidocaine treatment and eventually controlled the refractory se after 9 weeks of numerous ineffective aed treatments . lin et al . reported that moderate therapeutic hypothermia at 33 c resulted in a sustained control of refractory se . in another study on 12 patients with fires , immunoglobulin and a ketogenic diet three courses of intravenous immunoglobulin ( 1 g / kg ) were administered but were ineffective . the presence of voluminous amounts of coffee grounds and gastric juices in the nasogastric tube made it difficult to implement the ketogenic diet . after administering lidocaine for 2 days , the frequency of generalized seizures reduced to one - third , but when mgso4 was added , the number of seizures dropped markedly to no more than three per day within 3 days . the mechanism that caused lidocaine and mgso4 to apparently work in our case is unclear . capizzi et al . assumed that lidocaine , being a local anesthetic drug as well as an antiarrhythmic drug ( class ib ) , has membrane - stabilizing effects and exhibits a central local anesthetic action on the inhibitory pathway fibers involved in direct cortical stimulation . magnesium sulfate ( mgso4 ) , a drug commonly used in eclampsia but rarely in fires , is a seizure prophylactic ; its mechanism of action , however , remains unclear . zeiler et al . reviewed 19 original articles and suggested a trend toward improved seizure control with the use of intravenous mgso4 for noneclamptic refractory se . one hypothesis is that severe preeclampsia is a state of increased seizure susceptibility resulting from blood brain barrier disruption and neuroinflammation , which lowers the seizure threshold . although the pathophysiology of fires is undetermined , the mechanism that caused the combination of lidocaine and mgso4 to apparently work successfully in our case provides a clue for fires research and understanding . this is the first report describing the combined use of lidocaine and mgso4 with successful treatment outcomes . this experience provided a different treatment option for control of status epilepticus due to fires when all conventional aeds failed . however , more studies are needed to explore the mechanisms and effects of lidocaine and mgso4 in fires .

in rodent brains images may become distorted due to instrument imperfections or , in the case of histology , tissue processing . for example , magnetic field inhomogeneity causes geometric distortions in echo planar imaging ( epi ) mri ; mechanical forces acting on a harvested brain during slicing may cause tissue tearing . and chemical preparation for histological analysis may cause deformation of tissue , evident in histological micrographs . correcting ( or rectifying , warping ) the distorted images is required to adequately represent rodent brains ( in the case of epi distortion in mri ) , or to compare different acquisition modalities ( e.g. , comparison of in vivo images and histological micrographs ) . normally , affine ( linear + translation ) transformation can not reconcile severe distortions making nonlinear transformation necessary . among the nonlinear transformation techniques , point - based interpolating transformation techniques are widely employed because they are easy to implement and flexible for different applications [ 13 ] . a typical point - based interpolating approach is comprised of three steps : ( 1 ) placing homologous point landmarks on the source image ( image to be transformed ) and the target image ( image used as the reference ) , respectively , ( 2 ) computing the interpolating transformation ( e.g. , polynomial splines , b splines and thin - plate splines [ 4 , 5 ] ) between the source and target images , and ( 3 ) aligning the landmarks exactly and mapping other parts of image using the computed transformation . manual identification of landmark points is time consuming and prone to intra- and interobserver variations . a number of investigators have attempted to automate the landmark definition process by exploiting the geometry of anatomical or biological structures . typical geometrical features include line intersections [ 6 , 7 ] , local curvature maxima [ 810 ] , and centroid of closed boundary region . employing the anatomical and biological features however , some biomedical images do not contain well - distributed features to generate reliable landmarks for accurate registration . more importantly , noise , artifacts , and other factors can cause errors when identifying the features using either automated or manual selection . developed a method to relax the exact landmark matching ( i.e. , allowing the algorithm to relocate the landmarks ) using thin - plate splines by minimizing the bending energy functional [ 13 , 14 ] . bookstein [ 15 , 16 ] used a linear regression model and a technique called curve dcolletage to relax the interpolation condition . image properties such as edges of objects have been used to relax the exact landmark matching . however , the effect of the landmark relaxation on the registration accuracy was unknown in prior studies . in the current investigation , we developed a technique to automate and optimize the landmark generation using the local curvature on anatomical contours and validated the technique . the technique presented here is for two dimensional ( 2-d ) brain image nonlinear registration . although the rodent brain is a three dimensional ( 3-d ) object , some brain imaging studies are essentially carried out on 2-d planes such as in histological microscopy , and in 2-d mri studies . more importantly , nonlinear distortion mostly occurs on 2-d as well in these imaging studies . for example , tissue tearing , shearing , shrinkage , and enlargement , during sectioning and section handling , and eddy current in 2-d epi mri acquisition , mostly cause 2-d in - plane nonlinear distortions . to guarantee the correspondence of the two brain images to be registered , some prior process 3-d registration may be necessary . the strategy of this method is first to generate contours on corresponding anatomical features on the source and target images and then generate landmarks on homologous anatomical contours . the landmarks are then relaxed from the original locations and allowed to slide along the contours to achieve optimal matching . the relocation of the landmarks is governed by a cost function constituted by the local curvatures of landmarks and their displacements . the homologous contours on which the landmarks are generated can be manually drawn on the images or identified as the borders of objects using border detection methods , or first identified automatically using border detection methods and then manually modified to correct errors resulting from noise and artifacts . depending on the image properties , appropriate border detection methods such as dynamic programming or active contour models can be used . the homologous contours are manually split into several corresponding source and target curves that are relatively regular in shape ( c.f . figures 2(a ) , 2(b ) , 3(a ) , and 3(b ) . the splitting usually improves the computational stability and efficiency of the nonlinear transformation in our experience ( we used the thin - plate splines for the transformation calculations ) . the first two landmarks and their homologues are fixed at the two ends of the corresponding source and target curves ( figure 1(a ) ) . the remaining landmarks ( from the 3rd to the nth ; n is the user - preset number of landmarks on the curve ) are generated and optimized in an iterative fashion as shown in table 1 . the landmarks split the source and target curves into homologous curve segments ( e.g. , i 1 landmarks split the curve into i 2 segments ) . the ith landmark is placed in the middle of the longest segment of the source curve ( can be on the target curve depending on the user ; the source curve was used in this study ) , and its homologue is placed on the corresponding target curve segment ( figure 1(b ) ) . before the ( i + 1)th landmark is added , the landmarks are relaxed from their initial locations and slid on the curve segments to match each other ( this procedure is called landmark optimization , figures 1(b ) and 1(c ) ) by minimizing the cost function ( 1)m=|ismax ( s)itmax ( t)|+(i1sls+i1tlt ) , where s and t indicate the source and target , respectively , i1 is the displacement of landmarks on the ( i 1)th curve segment , l is the length of the curve segment , and is the local curvature defined by ( 2)=||||||||3 , where is a curve , || || represents the euclidean distance , and and are the first and second derivatives , respectively . the first term of the cost function is the difference between the local curvatures ( normalized by the maximum curvatures of the curve segments ) of a source landmark and the homologous target landmark . the second term is the displacements of the landmarks from their initial locations normalized by segment length . the cost function is a combination of the curvature similarity of the source and target landmarks and the displacements of the landmarks . the landmarks are relaxed and moved to match their curvatures ( the first term in ( 1 ) ) to minimize the cost function , and their displacements are weighed by the second term . the weighting parameter > 0 regulates the sliding distances of the landmarks along the contours . by increasing , equation ( 1 ) is minimized using the nelder - mead algorithm . when the landmarks have been optimized , any point - based registration method can be used to warp the source image and register the source and target images . we utilized the thin - plate splines to generate the warping field for registering the target images to the source in this work . the landmark generation and optimization technique was evaluated using two types of mouse brain imaging studies . in the first study , the second study examined the ability of the algorithm to correct geometric distortion of epi a fast mri acquisition technique . the convergence criterion of the cost function minimization was set as either landmark displacement of less than 10 pixels in subsequent iterations or a maximum of 500 iterations , and the weighting factor was set to between 0.2 and 0.3 in both of these studies . the thin - plate splines , which have been extensively used in medical image registration [ 7 , 1930 ] , were used for the nonlinear transformations . the accuracy of registration between the transformed source image ( mri in the first study , and epi in the second study ) and the reference image was measured by two methods using the normalized mutual information ( nmi ) and target registration error ( tre ) , respectively . nmi is defined as ( 3)nmi=(h(st)+h(t))h(st , t ) , where h(st ) and h(t ) are the marginal entropies , respectively , of the transformed source image st and t , and h(st , t ) denotes their joint entropy . tre is the distance between a set of target points ( pt ) and warped corresponding source points ( ps ) . in general , ( 4)tre = t(ps)pt , where t is the transformation , which is the thin - plate splines in this study . the size of the point set was dependent on the nature of the images , and at least four points were identified on each pair of images . mouse brains were harvested after 3d t2 * -weighted mri , fixed and embedded in paraffin for histological sectioning . brain slices were stained with prussian blue and hematoxylin . the mri volume was linearly registered to the blockface volume , and then computationally resliced in the coronal plane to match the corresponding histological sections [ 31 , 32 ] . the mri slices and histological sections were then nonlinearly registered by three experienced technicians using the thin - plate splines with the landmarks generated and optimized by the presented technique . the technicians were instructed to draw the source and target contours and generate curves according to their knowledge of anatomy . the automatic landmark optimization technique was evaluated by comparing two registration results : the first was from landmarks generated on the user defined contours but adjusted manually by the technicians , and the other registration was conducted using discrete landmarks manually selected by the technicians . five mice were scanned with t2-weighted ( t2-wt ) spin - echo imaging and diffusion tensor imaging ( dti ) with epi acquisition . the mri slices were prescribed at the same anatomical locations in these scans . in the dti epi scan , a baseline image without diffusion weighting ( b0 ) was also acquired . the b0 images were used as the source images , and the t2-wt images were used as the target images for registration . since the b0 image and all diffusion - weighted images undergo same geometric distortion , the transformation procured from the t2-wt / b0 registration is applied to correct the diffusion - weighted images . the b0 images were registered to the t2-wt images by the same three technicians in the mri / histology registration using manually selected landmarks , landmarks manually adjusted on anatomical contours , and automatically generated and optimized on contours , respectively . figures 2(a ) and 2(b ) demonstrate a pair of mri and histological slices before coregistration . the curves drawn by a technician and landmarks generated and optimized on the curves are also shown on these figures . the transformed mri and its overlay on the histological section are shown on figures 2(e ) and 2(f ) . figures 2(c ) and 2(d ) show the discrete manually selected landmarks by a technician . registration results with landmarks generated using different methods in five pairs of mri and histological slices ( from pair i to v ) were compared . the mean and standard error of the nmi and tre of each pair of images by the three technicians is presented in figures 2(g ) and 2(h ) , respectively . nmi and tre are shown from the manually selected discrete landmarks ( discrete landmarks ( lms ) in figures 2(g ) and 2(h ) ) , landmarks generated on anatomical curves and optimized using ( 1 ) ( optimized lms on curves in figures 2(g ) and 2(h ) ) . the nmi and tre of landmarks on curves but not optimized ( nonoptimized lms on curves ) and of landmarks manually adjusted on curves ( manually adjusted lms ) are also presented to investigate the improvement in registration accuracy resulting from landmark optimization . the number of optimized , manually adjusted and nonoptimized landmarks , was the same on each curve . the nonoptimized landmarks were obtained by simply generating the landmarks on the curves without performing optimization using ( 1 ) , keeping each landmark at its initial position . as demonstrated in figure 2(g ) ( nmi results ) and figure 2(h ) ( tre results ) , generating landmarks on anatomical curves ( no matter the landmarks were optimized or not ) either significantly improved registration accuracy ( p < 0.05 , paired t - test ) or showed a trend towards improvement ( 0.05 p < 0.10 ) in all image pairs compared to manually selecting discreet landmarks . the automatic landmark optimization using ( 1 ) resulted in a trend towards registration accuracy improvement ( 0.05 p < 0.10 ) in one image pair by nmi calculations ( figure 2(g ) ) compared to nonoptimized landmarks , and a trend towards improvement ( 0.05 p < 0.10 ) in two image pairs by tre calculations ( figure 2(h ) ) . compared to nonoptimized landmarks , manually adjusted landmarks showed a trend toward improvement in registration accuracy ( 0.05 p < 0.10 ) on two image pairs by nmi ( figure 2(g ) ) . the tre results showed a significant accuracy improvement ( p < 0.05 ) or a trend towards improvement ( 0.05 p < 0.10 ) on three image pairs by manually adjusting the landmarks ( figure 2(h ) ) . a typical pair of b0 and t2-wt images is shown in figures 3(a ) and 3(b ) . the geometrical distortion is evident on the b0 by visual comparison to the t2-wt images . figures 3(a ) and 3(b ) show the anatomical curves and optimized landmarks on a pair of t2-wt / b0 images , and the discrete landmarks manually selected by the same technician are shown in figures 3(c ) and 3(d ) . the result of distortion correction using the optimized landmarks is shown in figure 3(e ) . for an improved visualization , the corrected b0 ( in grayscale ) is overlaid on the t2-wt image plotted in pseudocolor in figure 3(f ) . the same statistical analysis was performed in the epi distortion correction as for the mri / histology registration , allowing a direct comparison of the results ( figures 3(g ) and 3(h ) ) . generating landmarks on anatomical curves ( irrespective of whether or not the landmarks were optimized ) either significantly improved registration accuracy ( p < 0.05 ) or showed a trend towards improvement ( 0.05 p < 0.10 ) in all image pairs compared to manually selecting discreet landmarks except in one image pair ( in figure 3(g ) , by nmi calculations ) . the automatic landmark optimization using ( 1 ) resulted in significant accuracy improvement ( p < 0.05 ) in one image pair and a trend towards registration accuracy improvement ( 0.05 p < 0.10 ) in two image pairs by nmi calculations ( figure 3(g ) ) compared to nonoptimized landmarks , and a trend towards improvement ( 0.05 p < 0.10 ) in one image pair by tre calculations ( figure 3(h ) ) . compared to nonoptimized landmarks , manually adjusted landmarks showed a significant improvement ( p < 0.05 ) in registration accuracy on two image pairs by nmi , and a trend toward improvement ( 0.05 p < 0.10 ) on one image pair ( figure 3(g ) ) . the tre results showed a trend towards improvement ( 0.05 p < 0.10 ) on one image pair by manually adjusting the landmarks ( figure 3(h ) ) . no difference was found between automatically optimized and manually adjusted landmarks . in this study , we developed a landmark generation and optimization technique for point - based nonlinear image registration methods . this technique extracts landmarks from the anatomical contours and optimizes the landmark positions by minimizing the cost function constituted by the displacements and the local curvatures of the landmarks . the technique was evaluated in two distinct applications : the registration of mri and histological slices and distortion correction of epi mri images . statistical analyses have shown that the automation of landmark selection resulted in significant accuracy improvement in image registration compared to manually selected landmarks . although in most experiments the improvement in nmi and tre resulting from the landmark optimization was not statistically significant compared to the results using nonoptimized landmarks , the trends towards improvement in registration accuracy was demonstrated in several experiments . manually adjusting the landmarks on curves could improve registration accuracy on several experiments and show a trend towards improvement in some other experiments . however , automated landmark selection provides increased efficiency by minimizing the required user intervention . it is not a monotonic function of the image similarity and , thus , may be trapped at local minima when used as a driving force for image registration . in this study , the image pairs to compare have all been already registered ; thus , nmi was only calculated on a small interval on which it is reasonable to think that nmi is monotonic . tre is a measure of the registration accuracy of a set of points on the images . the points for tre calculation are usually identified on anatomical features ; thereby , the evaluation may be more meaningful than nmi with regard to the anatomical accuracy of registration . it is reasonable to think that automatically generating contours can minimize inter- and intrauser variance . but on the other hand , some automatic border detection techniques are more susceptible to noise and artifacts compared to manual delineation . not only can this method be used in imaging studies similar to those presented here , but also can be used for multiple brain registration , or similarly , for registration to an atlas with minimum modification . before using this method , a 3-d affine transformation is likely necessary to first align the brain volumes together , or to the atlas , and then each brain volume needs to be resliced to match individual brain slices , or to the atlas slices . however , this technique still requires user intervention and thus suffers inter- and intra - investigator inconsistencies . we are currently improving this technique by including the image intensity and more geometrical information in addition to displacement and curvature to fully automate the landmark generation process . in this study , the number of landmarks either manually selected or automatically generated on curves was determined by the technicians according to their experience . it is desirable to precalculate the necessary landmark number for different landmark generation methods to improve registration accuracy .

multiple primary malignancies ( mpms ) were initially described by billroth in 1889 and later defined by warren and gates as the presence of at least 2 unrelated primary malignancies in a single patient . occurrences of mpms are rare and are described mostly as case reports , although there are reports from single institutions and from the registries of certain countries . the mechanism of development of mpms is unclear and likely multifactorial ; identified risk factors include previous cancer treatment , smoking , diet , and genetic mutations . as advances have been made in tumor detection and treatment , cancer survivorship has improved , which may contribute to the increasing likelihood of the development of additional cancers . here , we present the case of a 57-year - old woman presenting with melanoma , breast cancer , diffuse large b - cell lymphoma ( dlbcl ) , nodular lymphocyte - predominant hodgkin lymphoma ( nlphl ) and a giant cell tumor , with all 5 diagnoses made within 4 months . to our knowledge a 57-year - old woman was evaluated for a skin lesion in the right popliteal space . her family history is significant for melanoma ( paternal grandmother , age unknown ) and colon cancer ( mother , age 74 ) . she has never smoked . her skin lesion was diagnosed via excisional biopsy in april 2014 as superficial spreading melanoma . pathology showed a breslow thickness of 3.4 mm , no ulceration , and clark level iv , with no satellite lesions ( fig . surgical margins were widely clear ( > 2 cm ) and inguinal sentinel lymph node biopsy was negative . the patient had a screening mammogram the same month , which revealed an 8-mm irregular asymmetry in the left breast . the diagnostic mammogram revealed a lymph node in the left axillary tail that appeared highly suggestive of malignancy . biopsy of the suspicious axillary node was significant for dlbcl as well as epstein - barr virus ( ebv)-positive nlphl ( fig . pet / ct obtained for lymphoma staging in june 2014 showed an area of intense fdg avidity in a left axillary lymph node that was suspicious for lymphoma . the scan also revealed an intense discrete focus of hypermetabolic activity in the proximal left tibia , also suspicious for malignancy . in july 2014 , she underwent bilateral mastectomy with right sentinel lymph node biopsy and left axillary lymph node dissection . pathological analysis revealed stage iia , t1cn1am0 grade 1 left breast invasive lobular carcinoma ( ilc ) with 3 synchronous lesions , 0.5 - 1.5 cm in diameter ( fig . staining was positive for estrogen receptor ( er ) and progesterone receptor but negative for her2 . left axillary lymph node dissection revealed 2 out of 23 lymph nodes positive for metastatic ilc and 1 lymph node with isolated tumor cells , focal lymphovascular space invasion , and negative margins . ct - guided core biopsy of the left tibial soft tissue mass revealed cd68/cd45-positive cells , most consistent with giant cell tumor of tendon sheath / pigmented villonodular synovitis . it was the consensus of the tumor board that , for her excised melanoma , no further therapy was indicated . for a pt3an0 melanoma with negative margins by at least 2 cm and a negative sentinel lymph node biopsy , adjuvant radiation to the primary site similarly , in the absence of positive nodes with high - risk features such as 3 lymph nodes positive in the inguinal region , bulky lymph node involvement ( 4 cm in the axillary or inguinal region , 3 cm for cervical region ) , or extracapsular extension , nodal irradiation is also not indicated . for her 2 synchronous lymphomas , the consensus was to treat her dlbcl with systemic therapy followed by involved site radiation therapy to the left axilla . the standard of care for her nlphl would be observation or radiation therapy after surgical excision . in her case , the affected area was planned for radiation due to the proximity of her other tumors . for her stage iia breast ilc , the decision was to treat her with chemotherapy followed by postmastectomy radiation therapy due to multiple risk factors , including focal lymphovascular space invasion and 2 positive lymph nodes . given the overlap in chemotherapy drugs for locally advanced breast cancer and dlbcl , r - chop therapy was recommended , as it includes both adriamycin and cyclophosphamide and covers her lymphoma as well as her breast cancer . in terms of radiotherapy , we planned to treat the chest wall , the supraclavicular fossa , and her undissected axillary lymph nodes for her breast cancer . she did not warrant radiation to the dissected axilla ( level i ) for the management of her breast cancer as she was without gross extracapsular extension . however , for her dlbcl , she did warrant radiation to the site of initial disease , which was the dissected axilla ( level i ) . the patient completed 6 cycles of r - chop and her planned course of radiation therapy , which she tolerated well . a wide local resection with curettage was planned for the giant cell tumor of the left proximal tibia . more patients are living longer after a cancer diagnosis and unfortunately become susceptible to the development of further malignancies , especially if there is an underlying oncogenic predisposition . indeed , elderly patients are more likely to develop mpms , underscoring the importance of time to the process . diagnostic criteria require that each cancer must be definitively malignant by histopathology , each must be histopathologically distinct , and the possibility of metastasis among each tumor must be excluded . moertel defined synchronous tumors as occurring within 6 months of one another . it is conceivable that our patient 's melanoma is metastatic from her lesion in 1988 , but each of her tumors is histopathologically distinct and came to attention within 4 months . the data are limited , but spratt and hoag found that the reported prevalence of mpms varies from 0.7 to 11.7% . performed a retrospective review of 52,398 patients and found that 3.8% had at least 2 primary malignancies , and that 2.8% of patients with a second malignancy had at least 3 tumors . interestingly , patients with multiple malignancies demonstrated improved 5-year survival compared to patients with corresponding solitary malignancies . it is unclear whether these patients have some manner of increased tumor resistance or if these survival rates were simply necessary for the development of multiple cancers in the first place . there are many potential causes of mpms , including genetic factors , prior cancer therapy , smoking , alcohol consumption , and diet . however , it may be important to consider detection bias when identifying certain types of malignancy and attributing causality to prior cancer treatment or other factors . patients with an initial cancer diagnosis may be followed more closely and may have other malignancies detected that otherwise may not have come to attention . regardless , random chance clearly also seems to be a factor , especially given our patient 's lack of established risk factors . her family history is significant for melanoma and colon cancer , but there is no evidence suggestive of a hereditary cancer syndrome and she is a never - smoker . it appears that the incidence of mpms is increasing , which implies that clinicians should be vigilant to the possibility that patients are at continued risk for new and separate malignancies even after an initial diagnosis . more studies are needed to further elucidate the contemporary incidence of mpms and to investigate risk factors . each case must be considered individually , ideally by a multidisciplinary team , accounting for the type and stage of each tumor , response to treatment , and the patient 's overall health status . radical therapy is indicated for curable cancers , and more conservative approaches are appropriate if radical therapy is not feasible for one or more malignancies .

oxygen is vital for all aerobic biological processes . however , about 5% of it is converted into reactive oxygen species ( ros ) . methionine residues in proteins are susceptible to oxidation by reactive oxygen and nitrogen species leading to formation of methionine sulfoxide ( metso ) . organisms developed a complex antioxidant defence system that includes enzymatic and nonenzymatic antioxidants [ 35 ] . methionine sulfoxide reductases ( msrs ) are redox repairing enzymes which reduce metso back to methionine ( met ) . a number of published reports describe the role of methionine sulfoxide reductases in antioxidant defence and the regulation of protein function [ 610 ] . methionine sulfoxide reductases reduce both free and protein - bound metso , back to met in the presence of thioredoxin ( trx ) . two distinct families constitute msrs : msra , which specifically reduces s - epimer , and msrb , which is stereospecific for r - epimer of methionine sulfoxide [ 1114 ] . mammalian msrb enzymes constitute three different subclasses : msrb1 , msrb2 , and msrb3 [ 1520 ] . all of them contain zn ion , coordinated by two motifs cxxc ( two cysteines separated by two residues ) , which stabilize their structure . whereas mammalian msrb1 contains a resolving cysteine ( cys4 ) and catalytic selenocysteine ( sec95 ) in its active site , the other two subclasses , msrb2 and msrb3 , in analogy with bacterial msrbs , contain only catalytic cysteine [ 2124 ] . nmr solution and x - ray crystal ( pdb entry 3mao ) structures of mammalian msrb1 have recently been determined . reduction of methionine sulfoxide by selenocysteine containing msrb1 is a multistep process [ 24 , 25 ] . first , a catalytic sec95 attacks a sulfoxide moiety of the substrate , metso , resulting in the formation of selenenic acid intermediate and concomitant release of methionine . second , a resolving cys4 attacks the selenenic acid intermediate to form an intramolecular selenide - sulfide bond , and finally , a fully reduced enzyme is regenerated by reduction of the latter bond with thioredoxin , a natural electron donor ( scheme 1 ) . it has been established that thioredoxin is the natural reducing agent for the members of msr family , though dtt can be used as the reductant in vitro [ 14 , 24 , 26 ] . reduction of oxidized msrb2 and msrb3 as well as of the most bacterial msrbs occurs through a similar mechanism without formation of the intramolecular disulfide bridge . however , it was reported that bacterial msrb from neisseria meningitides upon oxidation form intramolecular disulfide bridge . it was shown that its reduction by trx is overall rate - limiting step of msrb cycle and it is described as an apparent irreversible process . thioredoxin is a ubiquitous protein that plays an important role in maintaining redox balance in cells . in most of its reactions , the proposed mechanism of trx - catalysed substrate disulfide reduction [ 2931 ] includes nucleophilic attack by thiolate of cys32 supported by hydrophobic interactions resulting in transient mixed disulfide formation followed by nucleophilic attack of the resolving cys35 generating trx - s2 and reduced protein . summarising , reduction of msrbs ( mammalian and bacterial ) disulfide bond by trx , can be described by the following steps . the just - formed intermolecular bond gets resolved by the thioredoxin resolving cysteine 35 , followed by the formation of reduced msrb and oxidized trx . s bridge of oxidized thioredoxin is further reduced by nadph and the specific fad - containing enzyme thioredoxin reductase . some studies suggest that specific structural recognitions exist between oxidized substrate disulfide bond and reduced trx [ 14 , 32 ] . the knowledge of the structure aspects of the msrb / trx complex formation would help to understand full catalytic mechanism of methionine sulfoxide reductases . here , we have investigated the structural aspects of the interaction between mammalian oxidized msrb1 and reduced thioredoxin . mutants of msrb1 and trx were produced in order to get an intermolecular complex suitable for its investigation using nmr spectroscopy . firstly , since sec - containing msrb1 is not possible to express in bacterial cells , sec95 of msrb1 was replaced by cys giving sec95cys mutant ( hereafter msrb1 ) . further , thioredoxin containing a cys35ser mutation ( hereafter trx ) was obtained as this modification removes the second resolving cys35 leading to the formation of a detectable intermediate msrb1-trx ( scheme 2 ) . the resultant msrb1-trx complex is stable and could , therefore , be structurally characterized by nmr spectroscopy and with the aid of computational approach . the genes of c - terminal his - tagged mouse msrb1 ( sec95cys ) and human trx ( cys35ser ) mutants , cloned into pet21 expression vectors were kindly provided by professor v. gladyshev 's group . escherichia coli strains er2566 ( new england biolabs ) and bl21 ( de3 ) ( novagen ) cells were respectively transformed with the constructs using the standard protocol . the transformed cells were spread on several lb agar plates containing 100 mg / l of ampicillin and were further stored at 4c . e. coli bl21(de3 ) cells carrying plasmid pet21-trx and e. coli er2566 cells containing pet21-msrb1 were grown in lb - media with 100 mg / l ampicillin . to uniformly label proteins either with n or with n - c , cells were grown in m9 minimal media containing 1 g / l 99%-enriched nh4cl , 4 g / l c6-glucose and 100 mg / msrb1 and trx were expressed , by growing host cells at 37c until an od600 reached 0.8 , followed by induction of protein synthesis with 1 mm iptg and subsequent incubation for 3 h at 37c . cells were harvested by centrifugation and disrupted by sonication in 50 mm phosphate buffer , ph 8.0 , containing 400 mm nacl , 0.01% tween 20 ( sigma aldrich ) ; 5 mm -mercaptoethanol as well as complete protease inhibitor cocktail ( roche ) ( 1 tab for 50 ml ) . clear supernatant was loaded onto the preequilibrated in binding buffer ( 50 mm na phosphate buffer , ph 8.0 , 400 mm nacl , 5 mm -mercaptoethanol , and 5 mm imidazole ) ni - nta column ( novagen ) . the column was step - washed with the increasing concentration of imidazole ( 5 , 10 , and 20 mm ) in binding buffer followed by elution of protein with 250 mm imidazole in binding buffer . the purity of the samples was examined with sds - page gel ( invitrogen ) . the yield of the proteins per liter of growth medium was 20 mg and 10 mg for msrb1 and trx , respectively . the obtained reduced msrb1 was further subjected to oxidation by excess of dabsyl - met - r - so ( kindly provided by professor h - y . kim ) for 3 h at 25c in 20 mm phosphate buffer , 20 mm nacl , and ph 7.5 . the final nmr msrb1 and trx samples contained 1,52 mm of protein in 10 mm nacl , 10 mm phosphate , ph 5.5 , 90%h2o , 10% d2o buffer . the sample for the backbone assignment of reduced double enriched trx ( n- and c ) also contained 5 mm dtt . avance 600 mhz spectrometer , equipped with 5 mm z - gradients txi ( h / c / n ) cryoprobe at the nmr centre of nt faculty , ntnu or varian directdrive nmr system 700 mhz spectrometer , equipped with 5 mm z - gradients salt tolerant h / c / n probe at the spbspu . proton chemical shifts were referenced to external 3-(trimethylsilyl)-propane - sulfonic acid sodium salt ( dss ) , while n and c chemical shifts were referenced indirectly to a liquid ammonia and dss , respectively , based on the absolute frequency ratios . the comparison between reduced and oxidized forms of msrb1 was performed by analyzing 2d n - h hsqc spectra of both reduced and oxidized msrb1 . n - h coupling constant was set to 90 ms , the relaxation delay in hsqc experiments was of 1 s. 2048 complex points were collected in f2 dimension , while 256 were collected in f1 dimension . h , c , and n backbone resonance assignments for trx protein were achieved using n hsqc , hnca , cbca(co)nh , cbcanh , hbhanh , hbha(co)nh , and n noesy nmr spectra from the standard pulse sequence library . the nmr data were processed with the bruker xwinnmr version 3.5 and varian vnmrj version 2.2c software . spectral analysis was performed using cara version 1.8.4.2 . in order to map the interacting sites of both trx and msrb1 proteins , that is , to determine which aminoacids are involved in the formation of the interprotein complex , nmr titration of each protein was performed . n - h hsqc of n labeled oxidized msrb1 was recorded followed by acquisition of the series of hsqc spectra of oxidized msrb1 in the presence of increasing amounts of n - unlabeled trx . likewise , the n - h hsqc spectrum of n - enriched pure trx was recorded first , followed by the set of hsqc spectra of trx containing an increasing amount of unlabeled oxidized msrb1 . all titration experiments were carried out three times at 298 k at two different ph values 5.5 and 6.5 in order to have statistically significant results . structural calculations for msrb1 and trx and their complex were performed using molecular modelling techniques , including molecular mechanics and protein docking ( molsoft icm pro 3.6 program package , ecepp/3 force field ) as well as molecular dynamics simulations ( gromacs program package version 4.0.7 , g53a6 force field ) . vmd and icm pro 3.6 program packages were used to visualize and analyze molecular dynamics ( md ) trajectories and the resulting averaged spatial structures of the proteins and their complexes . the initial sets of atomic coordinates of the proteins ( reduced forms ) were taken from pdb ( 2kv1 and 3trx for msrb1 and trx , resp . ) . md simulations were carried out using a standard protocol including the following steps : energy minimization , water box equilibration , and productive run . both structures of msrb1 protein and four complexes of its oxidized form with reduced trx were placed in dodecahedral water box . the box dimensions were chosen in such a way that water shell around the protein structures was no less than 12 . the intrinsic msrb1 positive charge was neutralized with two cl ions , and the negative charge of the protein complex was neutralized by three na ions . the energy minimization procedure was executed with steepest descent method . during 300 ps equilibration of the water molecules surrounding protein , spatial position of its atoms the equilibration procedure was followed by two productive runs with 5 ns and 20 ns in order to separate proteins and their complexes , respectively . distances between zn ion and s atoms of the coordinating cysteines of msrb1 ( cys23 , cys26 , cys71 , and cys74 ) were restricted with help of harmonic potential having a minimum at a value of 2.35 . the averaged molecular structures were obtained by the cluster analysis after each productive run of md . lincs algorithm was used to constrain the covalent bond distances and the valence angle values . the temperature of the system was held at 300 k with velocity - rescale algorithm . to hold system pressure at 1 atm value , berendsen barostate with the time constant for coupling , = 0.5 ps was used . the cut - off distance of nonbonded interactions was set to 10 for van der waals interactions and to 14 for electrostatic interactions . to obtain the disulfide bond between cys95 and cys4 residues in oxidized msrb1 , md simulations in explicit water box with the described above protocol were used , except that the distance between s atoms of two cysteines was restrained at 2.8 with the force exceeding van der waals repulsion of these atoms . the disulfide bond itself was formed and its geometry was optimized by molecular mechanic procedures in ecepp/3 force field implemented in icm pro 3.6 program package . to construct the mutant form cys35ser of trx , the side chain of the mutated aminoacid together with its neighbours situated within 5 radius , were subjected to the energy minimization procedure in ecepp/3 potential . the average structures of oxidized msrb1 and reduced trx have been subsequently subjected to the protein - protein docking in icm pro 3.6 . this algorithm uses optimal docking area parameter representing the protein surface regions with the maximum dehydration energy arising upon formation of the tight protein - protein contact . to reduce conformational sampling calculations , only the hot points that were situated near msrb1 and trx active sites were used within the study . interaction energies were calculated using potential grids with 0.5 cells , and the truncated van der waals potential with maximum 1.0 kcal / mol was used . the conformations of msrb1-trx complex , obtained from docking , having a representative distance between s cys95 of msrb1 and s c32 of trx less than 10 , were taken for further analysis and sorted into four groups depending on the mutual orientations of the two proteins . then , the representative conformations from each of four groups were refined by the above - described molecular dynamics simulation methods . upon oxidation , msrb1 undergoes structural changes caused by the intramolecular cys95/cys4 disulfide bond formation . in order to assign h and n nmr spectra of the oxidized msrb1 , the hsqc spectra of msrb1 protein in both oxidized and reduced states were compared [ 25 , 47 ] . this analysis revealed close similarity between the hsqc spectra of both redoxed forms : the majority of the cross - peaks assigned in the case of reduced msrb1 protein remained either at the same position in h - n hsqc spectra or their positions were slightly altered . this overall spectral correspondence between reduced and oxidized forms of msrb1 allowed us to unambiguously identify and assign the cross - peaks related to the most of the aminoacids of the oxidized msrb1 . nevertheless , the signal broadening and the shift of some of the cross - peaks in the oxidized protein corresponding to the residues either belonging to the protein active site or situated in its vicinity were observed ( cys4 , phe82 , cys95 , and ile96 ) . the performed comparison provided us with the assignment of the h and n chemical shifts of oxidized msrb1 protein and indicated that no major structural changes occur in msrb1 protein upon oxidation . the availability of the assignment of native human trx ( 313 k , ph = 5.5 ) assisted in the obtainment of the present assignment . as a result , the backbone assignments of trx 's h , c , and n nuclei were deposited in biomagresbank under accession number bmrb-16850 . the obtained within the study assignments for both oxidized msrb1 and reduced nmr spectroscopy is a commonly used technique for mapping the interacting site of a protein upon complex formation with its ligands . within the study , it was performed the nmr titration of oxidized msrb1 with reduced trx and vice versa . to determine whether the interaction of these proteins can be observed on nmr time scale and , in the positive case , to determine which aminoacids from those proteins are involved in the interaction , the following approach was used . n - enriched protein ( either msrb1 or trx ) was titrated with nonenriched partner , and their interaction was monitored by comparative analysis of h - n hsqc spectra of a single protein and the protein in the presence of its partner . since only one protein was enriched with n nmr active isotope in each single experiment , the presence in solution of another protein did not hamper the observation of h - n spectrum under analysis , and it was possible to clearly identify aminoacids changing their nmr parameters ( either chemical shifts ( shifting of the cross - peak ) or relaxation rates ( broadening of the cross - peak ) ) , which indicated that these residues are involved in interaction with another protein . this analysis allowed us to determine which aminoacids from each protein are directly involved in the complex formation . the integral chemical shift differences calculated for all msrb1 residues using the relationship h + n/7 ( h + n/5 for glycine residues ) and plotted as a function of the aminoacid number , are shown in figure 1(a ) . these data show that the residues belonging to the msrb1 active site as well as their neighbouring residues directly participate in interaction with reduced trx ( c4-f7 , e65 , and c95-f97 ) . it was also observed that the msrb1 hsqc cross peaks corresponding to the residues revealing the maximal shifts upon interaction with trx became broader ( increase of transverse relaxation rates ) , thus supporting our finding in pinpointing of the interaction relevant aminoacids . altogether , our studies indicate that these msrb1 residues are involved in protein complex formation with trx . the integral h - n hsqc chemical shift differences for all trx residues plotted as a function of the aminoacid number were obtained as described above and are shown in figure 2(a ) . the titration of trx has revealed three aminoacid areas which are involved in interaction with msrb1 : d26-s35 , v57-e68 , and k72-m74 . our results indicate that : ( i ) trx active site ( as expected ) is involved in the interaction ; ( ii ) the monitored segments are situated mostly on the trx external loops ; ( iii ) these segments are rather close in space , thus supporting and validating our findings . the average structures of oxidized msrb1 and reduced trx were obtained from the molecular dynamics simulations starting from coordinates of the reduced proteins taken from pdb ( 2kv1 and 3trx for msrb1 and trx , resp . ) . the calculated structures are shown in figures 1(b ) and 2(b ) . upon formation of an intermolecular protein complex , catalytic cys32 ( active residue ) and cys35 ( resolving residue ) of trx should become close in space to the disulfide bond , connecting cys4 and cys95 residues of oxidized msrb1 . a protein docking procedure was carried out in order to identify possible options for mutual arrangement of these proteins . hot points of docking , were found ( evidenced for both proteins in figures 1(b ) and 2(b ) in red ) , and more than 1000 spatial structure orientations were generated . our experimental data on residues involved in complex formation and application of 10 distance restrains between the s atoms of cys32 of trx and cys4-cys95 disulfide bond of msrb1 were used to sort these structures into four groups . the neighbouring contacts between residues and the distances between sulphur atom of cys32 ( trx ) and the nearest sulphur atom of oxidized msrb1 disulfide bond , differentiating each group , are shown in table 1 . among the four represented complexes , the fourth ( d ) is the most plausible since the distance in this structure between cys32 of trx , and two sulphur atoms of disulfide bond in msrb1 were found to be minimal ( 5.3 ) . further , each of the four representative structures has been subjected to 5 ns molecular dynamics simulations . the aim of the molecular dynamics simulation was to refine the possible structure of the trx - msrb1 complex taking into account the obtained nmr data , the effects of water environment , and flexibility of proteins backbone and side chains . the analysis of the averaged structures of the complexes obtained after 5 ns of md simulations reveal that only for complex d the distance scys32 trx therefore , the md trajectory for this complex was extended to 20 ns ( figures 3(a ) and 3(c ) ) . it is necessary to emphasize that even if these data suggest that the scys32 trx is closer to scys4 of oxidized msrb1 , the analogous distance to cys95 is rather comparable . this observation precludes us to determine which cys residue from intramolecular msrb1 disulfide bridge will be subsequently targeted to form intermolecular disulfide bond . as it was calculated by our md , the n - terminal tail of msrb1 upon oxidation gets closer to the -strand of the protein formed by arg93-ile96 ( figures 3(a ) and 3(a ) ) . further , elongation of the mentioned msrb1 's -strand occurs additionally involving residues i96-s98 accompanied by formation of three additional hydrogen bonds with residues s5-f7 ( figures 3(b ) and 3(b ) ) . this newly constituted strand belongs to the n - terminus of msrb1 , which now is stabilized by the intramolecular disulfide bond formed upon oxidation . at the final stage of the interaction , trx adjusts its unstructured loop to the newly formed n - terminal -strand of msrb1 , leading to formation of interprotein -sheet , composed of six -strands , where five of them belong to msrb1 and the sixth to trx ( residues k72-m74 ) ( figures 3(c ) and 3(c ) ) . thus , in developing of two new -strands ( one intramolecular msrb1 and one intermolecular ) , nine aminoacids participate . this observation is in good agreement with our nmr titration results ( figures 1 and 2 ) , thus further validating our calculations . in addition , -strand of msrb1 formed by g77-f82 extends periodically to residues g75-g77 allowing residues g75-l78 to form hydrogen bonds with the second antiparallel -strand of msrb1 constituted by a66-g72 . the distance between scys32 of thioredoxin and scys95 of msrb1 , a reference distance , oscillates during the interaction from the smallest value of about 3.2 ( first 100 ps of the trajectory ) to 9.5 and then down to 4.8 . such approaching may cause a catalytic act , upon which the disulfide bond becomes resolved followed by reduction of msrb1 . upon the formation of the protein intermolecular complex , six new hydrogen bonds evolve ( three of them are interproteins ) , thus stabilizing the intermolecular complex . analysis of md trajectories for msrb1-trx complex ( prior formation of the intermolecular disulfide bond ) indicates an existence of two conformations of c4-c95 msrb1 bond . the degree of strain could be estimated by comparison of the energies for the van der waals ( vw ) and torsion ( to ) terms of disulfide bond for oxidized msrb1 in the step preceding the complex formation and in the free state . in accordance with ecepp/3 force field , the following energies have been obtained ( kcal / mol ) : 3.1 and 3.2 ( vw ) , 0.5 and 1.7 ( to ) , 2.6 and 1.5 ( total ) for strained and relaxed s s bond , respectively . it is worth to mention that during the md simulations this msrb1 intramolecular s s bond prefers its strained conformation . this bond thus becomes totally destabilized by 2.6 kcal / mol for strained or by 1.5 kcal / mol for relaxed conformation . however , the presence of six hydrogen bonds inside the developed interprotein -sheet obviously overcompensates the straining of cys4-cys95 bond in msrb1 , as energy profit from one hydrogen bond formation in -sheet can be estimated by 0.78 kcal / mol . in this study , both the experimental and the computational approaches were combined to investigate msrb1-trx protein complex formation . through the analysis of the nmr data , the aminoacids involved in protein complex formation have been determined . the average 3d structures of oxidized msrb1 and reduced trx were generated and subsequently subjected to protein docking and molecular dynamics . complementary use of the mentioned approaches indicates that the formation of the interprotein -sheet , stabilized by new hydrogen bonds , distorting the msrb1 intramolecular disulfide bond .

left atrial myxoma is a benign primary cardiac tumor and a cause of embolic stroke . nearly half of the benign heart tumors are myxomas , making it the most common heart tumor . although classified as benign tumors , myxomas seem to grow and spread , but there is still debate as to whether they metastase or fragments simply embolize . there is a 2:1 female preponderance and the age at onset is usually between 3060 years . although atrial myxoma is largely sporadic , at least 7% of cases are familial . stroke appears to be responsible for 80% of the neurological presentations of myxomas , and only 40% of these have a typical pattern of cardioembolism involving several vascular territories . myxoma usually causes ischemic stroke by embolism of tumor or thrombus , but aneurismal dilations at sites of earlier embolic vascular occlusion can cause intracerebral or subarachnoid hemorrhage . cerebral imaging often demonstrates multiple infracts suggestive of an embolic cause , but in some cases it may show only small subcortical ischemic lesions , as seen in lacunar infracts . transthoracic echocardiography has approximately 90% sensitivity in the detection of left atrial myxoma , while the sensitivity of transesophageal examination is 100% . transesophageal examination is also preferred because of its ability to detect other cardioemoblic sources , such as intracardiac thrombus , vegetations , or aortic arch plaque . general or constitutional manifestations , such as fatigue , fever , erythematous rash , arthralgia , myalgia , and weight loss , as well as laboratory abnormalities , such as anemia and elevated esr and serum c reactive protein and globulin levels , have been observed in many patients irrespective of the site and size of the myxoma . the risk of recurrent myxoma is 13% for sporadic cases , and it is usually attributed to multifocal myxoma embolization or incomplete resection . it has not yet been established as to whether transcranial doppler ( tcd ) can be used as a means to examine the emboliogenic potential of cardiac myxoma in patients with acute stroke . a 43-year old woman from the philippines was brought to the emergency department of rambam medical center in haifa , israel because of acute onset of generalized tonic - clonic seizures and left hemiplegia . her vital signs were normal , and cardiac exam revealed sinus rhythm without heart murmurs . neurological examination showed left central facial palsy , eye deviation to the right , left hemiplegia , brisk deep tendon reflexes , and extensor plantar reflex on the left . a ct scan of the brain on admission revealed a small old right parietal lobe infraction . another ct was performed 24 hours later and revealed a large acute infraction in the territory of the right middle cerebral artery ( mca ) and another smaller one in the territory of the posterior cerebral artery on the same side . carotid duplex , including common , internal and external carotid arteries and vertebral arteries , was normal . tcd ( pioneer , tc 8080 , viasys , nicolet ) microemboli monitoring was performed for 30 minutes , with both mcas monitored . cardiac holter showed a sinus rhythm average of 74 ( 62119 ) without tachy or brady - arrhythmia or conduction disturbances . transesophagial echocardiography ( tee ) identified a 5 cm left atrial mass , which was highly suspected to be an atrial myxoma ( figure 1 ) attached to the interatrial septum and prolapsed through the mitral valve ( without significant lv inflow obstruction in the supine position ) . after the tee results were obtained , another tcd monitoring was performed and continued this time for one hour . transthoracic echocardiography was performed three days after surgery and showed no evidence of residual myxoma or atrial septal defect . figure 1multiple trans - esophageal images of a large left atrial myxoma ( marked by * ) . long - axis mid - esophageal views of the heart ( a systole , b early diastole , c mid - diastole ) demonstrating prolapse of the myxoma from the left atrium into the left ventricle during diastole . multiple , mobile , finger - like projections of the tumor were evident ( thin arrows ) . four - chamber mid - esophageal view ( d ) showing the connection of the tumor to the interatrial septum ( thick arrow ) . la = left atrium , lv = left ventricle , ao = ascending aorta , aov = aortic valve , rv = right ventricle . multiple trans - esophageal images of a large left atrial myxoma ( marked by * ) . long - axis mid - esophageal views of the heart ( a systole , b early diastole , c mid - diastole ) demonstrating prolapse of the myxoma from the left atrium into the left ventricle during diastole . multiple , mobile , finger - like projections of the tumor were evident ( thin arrows ) . four - chamber mid - esophageal view ( d ) showing the connection of the tumor to the interatrial septum ( thick arrow ) . la = left atrium , lv = left ventricle , ao = ascending aorta , aov = aortic valve , rv = right ventricle . this technique provides useful information about possible sources of embolism , as well as their activity . acute ischemic stroke , carotid stenosis , and cardiac diseases are the most studied areas in the field of mes monitoring . the significance of mes in patients with cerebrovascular diseases has been the subject of many studies . the short pubmed literature search by combination of mes and stroke retrieved 122 related items . the first line of research is devoted to revealing the prevalence and timing of mes appearance in patients with acute ischemic stroke . it is now well established that mes is a frequent phenomenon in patients with ischemic stroke and that the amount of mes is related to stroke onset , with the peak occurring in the first hours and days after stroke . many other important studies have been devoted to the key issue of using the presence and amount of mes as predictors of the short- and long - term clinical outcomes during the index event , as well as possible predictors of recurrent ischemic stroke . studies exploring the correlation between prevalence and amount of mes and the etiology of ischemic stroke can also be found in the literature . another line of research estimates the impact of mes in both symptomatic and asymptomatic carotid disease . in patients suffering from carotid stenosis , the presence of mes can be indicative of an unstable structure of carotid plaque that is prone to be a source of emboli . the existence of mes in patients with carotid disease is also an important indicator of the substantial danger of the first and recurrent cerebrovascular event . another area in which the presence of mes determines the increased risk of peri- and post - procedural complications is that of carotid surgery and stenting . in both cases , the appearance of a significant amount of mes reflects damage of the plaque , resulting in multiple emboli with neurological and cognitive impairment . in cardiology , tcd is a reliable technique for the detection of mes from cardiac embolic sources , such as prosthetic cardiac valves , aortic plaques , and atrial fibrillation . it is also used as an especially sensitive technique for detecting right - to - left cardiac or pulmonary shunts . the microembolic signals obtained from patients with artificial heart valves and during examinations for pfo detection have also been used as a model , alongside other methods , in an attempt to differentiate between gaseous and solid microemboli . the results of mes monitoring may also be found in some other clinical situations , where their presence or absence helps us to better understand the role of embolism in the development of the disease . our case emphasizes that mes monitoring may be useful not only in detecting the emboliogenic nature of the disease , but also in exploring the precise timing of embolization ( i.e. , either permanent , as in the many clinical situations mentioned above , or sporadic , as in our case ) . although cardiac myxoma is a rare disease , it should be considered in young patients with stroke and elevated esr , even without systemic symptoms . there are no data in the literature about tcd monitoring in patients with atrial myxomas . our findings showed the absence of mes on two consecutive tcd examinations , suggesting a spontaneous occurrence , rather than the permanent presence , of embolization , even in the most acute phase of stroke . these data are compatible with literature sources pointing to the simultaneous appearance of multiple emboli at different target sites in patients presenting with cardiac myxomas . another line of evidence supporting our data is the large number of silent lesions commonly found in patients with stroke and myxomas , as in the case of the patient presented here . thus , it seems that in many cases , despite silent embolic events , the patient may remain clinically intact for years until a symptomatic event occurs . the tendency of myxomas to spontaneously produce severe and multiple emboli emphasizes the need for the surgical excision of myxomas when possible anticoagulation seems to be doubtful .

in western countries , more than half of pregnant women take prescription medication , and nearly all pregnant women use over - the - counter medications , vitamins or other dietary supplements.14 drugs that are safe for adults may be teratogenic for the developing fetus . the majority of drugs or their metabolites cross the placental barrier,5 and metabolites may be more fetotoxic than their source substances , as was noted in the case of thalidomide - induced phocomelias.6 because pregnant women rarely participate in randomized studies of medicines , evidence from observational studies is central in establishing safety of prenatal drug exposure.7 since birth defects are rare , assembling cohorts to observe their occurrence is expensive in terms of time , money , and resources , leading to widespread use of the case - control design . case - control studies , often based on interviews or questionnaires , are susceptible to selection and recall bias , and they do not allow estimation of absolute risks ( prevalences ) of birth defects . existing medical databases are increasingly being used to conduct pharmacoepidemiologic cohort studies , including studies of drug teratogenicity.8 medical databases , some of which have been in existence for decades,9,10 prospectively and routinely record health data , enabling relatively quick and inexpensive analyses of data from large populations.8 such analyses allow direct estimation of birth defects prevalences , while estimates of association are less susceptible to selection or recall bias obtained in studies with primary data collection.11 medical databases have their own limitations , however , which must be considered when interpreting findings of studies based on their data.8 in this paper , we first provide examples of established north american and european medical databases suitable for studying teratogenicity of drugs , and then describe common epidemiologic biases as manifested in studies based on such databases . medical databases are data repositories that contain heath - related data , including electronic medical records , administrative databases such as claims records , and registries of diseases and rendered health services.8,12 medical databases are typically maintained for surveillance or reimbursement , meaning that the influx of data into them is decoupled from research purposes . therefore linkage of records from different databases covering the same population may be required in order to combine in the same dataset information on prenatal drug exposure , occurrence of birth defects , and relevant covariates . independence of data collection from research hypotheses reduces risk of self - selection bias , but limits the variables available for analysis to those routinely collected by the databases . in the united states , medicaid , a health care plan for low - income persons , maintains claims databases of its enrollees . low - income pregnant women , and children under the age of 6 years , are eligible for medicaid coverage , which includes access to prescribed drugs.16 each state administers its own medicaid program , and patient eligibility and available services vary from state to state.17 private insurers in the us also maintain claims databases that compile data on maternal use of prescribed drugs and on birth defects as a part of an overall diagnostic record . for example , the health management organization ( hmo ) research network combines data maintained by several managed - care health plans ( such as kaiser permanente or harvard pilgrim health care).18 the network is part of the national initiative under the aegis of the department of health and human services to increase awareness of the benefits and risks of therapeutics.18 each participating health plan maintains computerized databases of member enrollment , filled prescriptions , and diagnoses made during outpatient visits and hospitalizations.18 in canada , the saskatchewan health services databases cover 99% of the population of the saskatchewan province ( about 1 million persons or 3.2% of the canadian population ) . . drug teratogenicity may be studied by linkage of databases on vital statistics ( live and still births ) , outpatient prescriptions , and hospitalization services . the linked data are available for research use , provided they remain unidentifiable to ensure data protection.19 the united kingdom s medicines and healthcare products regulatory agency ( mhra ) hosts the general practice research database ( gprd ) , which is one of the largest and the most complete databases , containing medical records on more than 4 million patients , including prescribed medications , referrals and diagnoses made during hospitalizations and visits to general practitioners ( gps).1315 the database was established in 1987 , and its data are linkable to the uk s other medical databases.13 in the course of the past two decades , all nordic countries ( denmark , finland , iceland norway , and sweden ) have established prescription databases,2123 tracking prescription medications dispensed in outpatient pharmacies . the earliest database ( in denmark ) was established in 1989 , while the newest ( in sweden ) was launched in 2005 ( for a recent review of nordic prescription databases , see review by furu and colleagues21 ) . in sweden , maternal use of medication in pregnancy is also available , since 1995 , as measured by self - reporting during the first antenatal visit.24 drawbacks of north american databases maintained by health insurers or based on residence include nonuniform eligibility ; selective coverage ( eg , the poor or the employed ) ; and potential loss to follow - up if patients cease to be eligible for coverage after changes in income , employment , or residence . by contrast , medical databases in nordic countries are derivatives of universal and uniform health coverage of welfare states.20 thus , in contrast to the north american databases , membership in a nordic medical database is independent of income , employment , or residence.21 sources of data on birth defects and other reproductive outcomes in nordic countries include birth registries,9,24,25 hospital discharge registries,26 and registries of congenital malformations.24,26,27 birth registries , with records dating back to the 1960s or 1970s,9,25 typically record birth defects discovered immediately after birth , and therefore must be supplemented with data from hospital registries and registries of congenital malformations.24,27,28 hospital registries , congenital malformation registries , or induced abortion registries26 can also be used to ascertain reproductive outcomes other than live or still births . miscarriages , and elective or therapeutic pregnancy terminations , including those done after prenatal diagnosis , and some data on malformations also may be available.26,28 data from medical databases can be linked to other registries containing demographic , social , and labor - market data . this is especially true for denmark , whose network of population databases has been described as the most complete and interwoven collection of statistics touching on almost every aspect of life.10,26 a crucial advantage of nordic databases is the possibility of across - the - board data linkage via unique identification number , assigned at birth and encoding date of birth and sex , which follows each citizen from cradle to grave.10,21,29 birth registry records contain the maternal identification number , which is a necessary link for unambiguous ascertainment from prescription databases of maternal drug intake during pregnancy.26 teratology information services ( tis ) counsel newly pregnant women , or women who are trying to conceive , regarding safety of medication use . the european network of teratology information services lists about 25 european and south american tis,30 and a similar number is listed by the us - based organization of teratology information specialists of the united states and canada.31 tis record the women s demographic , obstetrical , medical , and drug - exposure history.31 during the year after the expected delivery , the tis conducts a follow - up interview , collecting data on malformations . reporting to tis is initiated by women and is thus not systematic . therefore , despite availability of large numbers of computerized records , tis - based studies on teratogenicity of drugs are similar to epidemiologic studies with primary data collection in their susceptibility to self - referral bias , and nonrandom losses to follow - up . furthermore , tis may cover diverse geographic areas , making it difficult to establish a reference for an expected number of malformations in the source population . medical databases ( summarized in table 1 ) are widely used for addressing teratogenicity of drugs . examples include use of the gprd study on anticonvulsants;32 the tennessee medicaid study on angiotensin - converting enzyme inhibitors,33 a tis - based study of prenatal loratadine exposure;34 studies of antidepressants from us claims databases,35 the saskatchewan healthcare databases,36 the population databases of sweden,37 denmark,38 and on tis.39 large sample sizes , obtainable from medical databases , may reduce random error around the resulting estimates , but systematic error remains a problem.40 all three main types of epidemiologic bias selection bias , information bias , and confounding occur in studies of medical databases . in an ideal cohort study of drug teratogenicity , an investigator would recruit cohorts of women exposed and unexposed to a given agent before conception and examine birth defects detected among the fetuses throughout gestation , at birth , and several years postnatally . in such an ideal setting , the incidence rate of a birth defect is the number of all fetuses or neonates with that defect , detected at any time during the follow - up , divided by the total person - time contributed by all fetuses at risk . in reality , neither reproductive outcomes nor total person - time contributed by the initial conceptuses is fully observable ( respectively , the numerator and the denominator of the incidence rate ) . two major sources of selection bias are relevant to the study of drug teratogenicity : spontaneous fetal loss ( extrauterine pregnancy , miscarriage , stillbirth ) , and induced abortion . malformations associated with first - trimester miscarriages are not always recorded or observable ( table 2).28 elective pregnancy terminations during the first trimester are usually unrelated to medication use or suspected birth defects.41,42 second - trimester induced abortions are commonly carried out after diagnosis of malformation by prenatal diagnosis . the proportion of malformations diagnosed prenatally varies geographically ( eg , 25% in croatia vs 88% in paris)43 and according to local availability of relevant procedures.44 furthermore , rates of second - trimester pregnancy terminations depend on local laws , severity of birth defect , and long - term prognosis.43 up to 94% of fetuses with prenatally diagnosed fatal malformations ( eg , anencephaly ) are aborted , compared with 30% to 40% of fetuses with treatable malformations ( eg , diaphragmatic hernia or transposition of great arteries).43 in sweden , 60% of spina bifida cases were diagnosed at elective termination of pregnancy between 18 and 22 weeks gestation , and the level of ascertainment of spina bifida was inversely related to achieved gestational age at pregnancy end ( birth or pregnancy termination).28 in contrast , diagnosis of cleft palate rarely led to pregnancy termination.28 in the us , between the 1970s and the 1990s , use of ultrasonography or amniocentesis for prenatal diagnosis has increased from 7% to nearly 90% , while the rate of elective abortions for any malformation increased from 0.8% to 18% , with a larger absolute increase among terminations for nonfatal malformations.45 finally , access to and utilization of prenatal diagnosis may depend on a pregnant woman s socioeconomic status , race / ethnicity , or age.46 thus , severity of selection bias resulting from second - trimester pregnancy terminations may vary according to geography , type of malformation , timing of termination , calendar time , and maternal characteristics . in summary , in cohort studies of birth defects , inability to observe birth defects at all reproductive outcomes represents loss to follow - up of a potentially nonrandom subgroup of embryos and fetuses . selection bias ensues if either the medication or the malformation affects an embryo s survival until malformation can be observed . such bias can cause a spurious apparent association between drug exposure and medicinal agent or , alternatively , lead to erroneous conclusions about the lack of an association.47 to reduce selection bias , whenever possible , all observable reproductive outcomes should be ascertained as well as malformations detected both prenatally and at birth.48 in database studies of drug teratogenicity , relying on dispensed prescription information to ascertain drug use in pregnancy may lead the investigator to erroneous assumptions regarding the fact , the timing , and the dosage of medication intake.49 such misclassification is an important limitation given the short duration of gestation and even shorter duration of developmental critical periods , during which birth defects can plausibly occur as a result of drug exposure . a major drawback of prescription registries is lack of data on adherence once medication is dispensed . furthermore , medication dispensed during hospitalization or in outpatient clinics are not recorded in prescription registries potentially leading to under - ascertainment of medication use.21 nondifferential misclassification of maternal drug exposure , if severe , may nullify the observed estimate of effect , if an effect exists . the proportion of true cases of birth defects captured by electronic sources ( completeness , analogous to sensitivity)50 may vary widely by type of anomaly and type of data source.51,52 in the saskatchewan health databases , for example , data in the hospitalization records may be suitable for studying only major birth defects.19 imperfect sensitivity of birth defect measure leads to underestimation of true prevalence of birth defects , but imperfect sensitivity alone does not bias a relative estimate of effect . if no other bias is at work , relative estimate of effect will be unbiased53 in the absence of false - positive records of birth defects ( 100% specificity ) , which is usually the case for electronic records of birth defects.54 in summary , data on medication use and occurrence of birth defects in medical databases are of varying quality , depending on method of data collection and on the type of medication and birth defect under study . a researcher embarking on a study of teratogenicity should obtain information about validity of data on the variables of interest in a selected data source . predictors of medication use by a pregnant woman that are independent risk factors for a given birth defect can confound the estimate of association between the medication and the birth defect under study . examples of potential confounding factors include geography , maternal age , race , socioeconomic status , and the disease for which the medication is prescribed.55 unmeasured or unknown confounding can not be controlled in an analysis , except indirectly , if unmeasured traits happen to correlate with measured and controlled characteristics . residual confounding , which can be viewed as a special case of unmeasured confounding , occurs when controlling for a variable used to measure a confounding factor does not completely remove confounding by that factor . this may occur when the variable is misclassified owing to poor measurement or inadequate categorization . the estimate of effect adjusted for a misclassified version of a confounder is biased in the direction of confounding . if adjusting for a misclassified confounder variable attenuates the crude estimate , adjustment for a perfectly measured confounder is expected to result in further attenuation , while amplification of effect estimate by a misclassified confounder variable indicates that the true effect may be larger than the apparent one . confounding by indication is common in studies of unintended effects of drugs , because of the difficulty in separating the effect of a given drug from the effect of the disease for which the drug is given ( the indication ) . thus , a maternal diseases itself rather than medication used to treat it may increase risk of malformation in offspring . to counter confounding by indication , one may examine risks of birth defects among offspring of mothers taking the same medication prescribed for different indications and among offspring of women with similar indications taking different drugs . these methods may only partially address confounding by indication since use of different medications for the same indication may vary according to severity or etiology of disease , both of which may affect fetal risks . one way to address confounding by indication is by taking advantage of the time - sensitive nature of the relation between drug exposure and the possible birth defect . for example , causation between cardiac malformations and drug exposure can not be inferred if the drug exposure occurred only during second and third trimester , ie , after the heart had been formed.12 selection bias , information bias , and confounding are all at work simultaneously in a given epidemiologic study , and may bias estimates in the opposing directions . theory and software have been developed to quantify the impact of study estimates by unmeasured confounding,41,56 and misclassification of study variables.5762 the methods are based on subjecting study results to an array of informed assumptions62 about the source and the magnitude of systematic error . many available methods tend to apply to simple situations , such as those characterized by dichotomous study variables . however , even rough quantification of bias is an improvement over sometimes insufficiently justified assertions and beliefs regarding its direction and impact.57 in an ideal cohort study of drug teratogenicity , an investigator would recruit cohorts of women exposed and unexposed to a given agent before conception and examine birth defects detected among the fetuses throughout gestation , at birth , and several years postnatally . in such an ideal setting , the incidence rate of a birth defect is the number of all fetuses or neonates with that defect , detected at any time during the follow - up , divided by the total person - time contributed by all fetuses at risk . in reality , neither reproductive outcomes nor total person - time contributed by the initial conceptuses is fully observable ( respectively , the numerator and the denominator of the incidence rate ) . two major sources of selection bias are relevant to the study of drug teratogenicity : spontaneous fetal loss ( extrauterine pregnancy , miscarriage , stillbirth ) , and induced abortion . malformations associated with first - trimester miscarriages are not always recorded or observable ( table 2).28 elective pregnancy terminations during the first trimester are usually unrelated to medication use or suspected birth defects.41,42 second - trimester induced abortions are commonly carried out after diagnosis of malformation by prenatal diagnosis . the proportion of malformations diagnosed prenatally varies geographically ( eg , 25% in croatia vs 88% in paris)43 and according to local availability of relevant procedures.44 furthermore , rates of second - trimester pregnancy terminations depend on local laws , severity of birth defect , and long - term prognosis.43 up to 94% of fetuses with prenatally diagnosed fatal malformations ( eg , anencephaly ) are aborted , compared with 30% to 40% of fetuses with treatable malformations ( eg , diaphragmatic hernia or transposition of great arteries).43 in sweden , 60% of spina bifida cases were diagnosed at elective termination of pregnancy between 18 and 22 weeks gestation , and the level of ascertainment of spina bifida was inversely related to achieved gestational age at pregnancy end ( birth or pregnancy termination).28 in contrast , diagnosis of cleft palate rarely led to pregnancy termination.28 in the us , between the 1970s and the 1990s , use of ultrasonography or amniocentesis for prenatal diagnosis has increased from 7% to nearly 90% , while the rate of elective abortions for any malformation increased from 0.8% to 18% , with a larger absolute increase among terminations for nonfatal malformations.45 finally , access to and utilization of prenatal diagnosis may depend on a pregnant woman s socioeconomic status , race / ethnicity , or age.46 thus , severity of selection bias resulting from second - trimester pregnancy terminations may vary according to geography , type of malformation , timing of termination , calendar time , and maternal characteristics . in summary , in cohort studies of birth defects , inability to observe birth defects at all reproductive outcomes represents loss to follow - up of a potentially nonrandom subgroup of embryos and fetuses . selection bias ensues if either the medication or the malformation affects an embryo s survival until malformation can be observed . such bias can cause a spurious apparent association between drug exposure and medicinal agent or , alternatively , lead to erroneous conclusions about the lack of an association.47 to reduce selection bias , whenever possible , all observable reproductive outcomes should be ascertained as well as malformations detected both prenatally and at birth.48 in database studies of drug teratogenicity , relying on dispensed prescription information to ascertain drug use in pregnancy may lead the investigator to erroneous assumptions regarding the fact , the timing , and the dosage of medication intake.49 such misclassification is an important limitation given the short duration of gestation and even shorter duration of developmental critical periods , during which birth defects can plausibly occur as a result of drug exposure . a major drawback of prescription registries is lack of data on adherence once medication is dispensed . furthermore , medication dispensed during hospitalization or in outpatient clinics are not recorded in prescription registries potentially leading to under - ascertainment of medication use.21 nondifferential misclassification of maternal drug exposure , if severe , may nullify the observed estimate of effect , if an effect exists . the proportion of true cases of birth defects captured by electronic sources ( completeness , analogous to sensitivity)50 may vary widely by type of anomaly and type of data source.51,52 in the saskatchewan health databases , for example , data in the hospitalization records may be suitable for studying only major birth defects.19 imperfect sensitivity of birth defect measure leads to underestimation of true prevalence of birth defects , but imperfect sensitivity alone does not bias a relative estimate of effect . if no other bias is at work , relative estimate of effect will be unbiased53 in the absence of false - positive records of birth defects ( 100% specificity ) , which is usually the case for electronic records of birth defects.54 in summary , data on medication use and occurrence of birth defects in medical databases are of varying quality , depending on method of data collection and on the type of medication and birth defect under study . a researcher embarking on a study of teratogenicity should obtain information about validity of data on the variables of interest in a selected data source . predictors of medication use by a pregnant woman that are independent risk factors for a given birth defect can confound the estimate of association between the medication and the birth defect under study . examples of potential confounding factors include geography , maternal age , race , socioeconomic status , and the disease for which the medication is prescribed.55 unmeasured or unknown confounding can not be controlled in an analysis , except indirectly , if unmeasured traits happen to correlate with measured and controlled characteristics . residual confounding , which can be viewed as a special case of unmeasured confounding , occurs when controlling for a variable used to measure a confounding factor does not completely remove confounding by that factor . this may occur when the variable is misclassified owing to poor measurement or inadequate categorization . the estimate of effect adjusted for a misclassified version of a confounder is biased in the direction of confounding . if adjusting for a misclassified confounder variable attenuates the crude estimate , adjustment for a perfectly measured confounder is expected to result in further attenuation , while amplification of effect estimate by a misclassified confounder variable indicates that the true effect may be larger than the apparent one . confounding by indication is common in studies of unintended effects of drugs , because of the difficulty in separating the effect of a given drug from the effect of the disease for which the drug is given ( the indication ) . thus , a maternal diseases itself rather than medication used to treat it may increase risk of malformation in offspring . to counter confounding by indication , one may examine risks of birth defects among offspring of mothers taking the same medication prescribed for different indications and among offspring of women with similar indications taking different drugs . these methods may only partially address confounding by indication since use of different medications for the same indication may vary according to severity or etiology of disease , both of which may affect fetal risks . one way to address confounding by indication is by taking advantage of the time - sensitive nature of the relation between drug exposure and the possible birth defect . for example , causation between cardiac malformations and drug exposure can not be inferred if the drug exposure occurred only during second and third trimester , ie , after the heart had been formed.12 selection bias , information bias , and confounding are all at work simultaneously in a given epidemiologic study , and may bias estimates in the opposing directions . theory and software have been developed to quantify the impact of study estimates by unmeasured confounding,41,56 and misclassification of study variables.5762 the methods are based on subjecting study results to an array of informed assumptions62 about the source and the magnitude of systematic error . many available methods tend to apply to simple situations , such as those characterized by dichotomous study variables . however , even rough quantification of bias is an improvement over sometimes insufficiently justified assertions and beliefs regarding its direction and impact.57 harmful , neutral , or protective has important implications for pregnant women and their offspring . bias masking a true teratogenic drug effect would result in continued use of a harmful agent , while bias creating false appearance of teratogenicity may limit treatment options available to pregnant women . these could include treatments for chronic conditions that may themselves detrimentally affect pregnancy outcome if left untreated . provided that methodological problems are understood and effectively handled , computerized health care databases are a valuable source of data for cohort studies of teratogenicity of drugs .

radiographs are an important method of diagnosing and monitoring oral diseases , dentofacial development , and the progress or prognosis of therapy.1 panoramic radiography is recommended for patients in several age ranges : children with transitional dentition ( after eruption of the first permanent tooth ) ; adolescents with permanent dentition ( prior to the eruption of the third molars ) ; and adults who are dentate or partially edentulous . panoramic radiographs are also recommended in new patients being evaluated for oral diseases and in patients undergoing follow - up , based on clinical signs , symptoms , and treatment plans.1 intraoral radiographs are more accurate than panoramic radiographs ; however , panoramic examinations may be advantageous because they involve a reduced radiation dosage , cost less , and allow a larger area to be imaged . thus , although these issues have been addressed in prior studies , it is important that dentists understand the mechanisms associated with the formation of real and ghost images in panoramic radiographs . the panoramic radiograph technique is based on the principle of computed tomography , since both techniques involve synchronized rotation between an x - ray source and an image receptor around the patient 's head that remains stationary.2 this is a radiological technique that is relatively simple and is commonly used in all dental specialties , 34 since patients have shown a good tolerance to this technique , it is fast and comfortable , and , most importantly , it includes the entire maxillomandibular structure ( dental arches , temporomandibular joint , and associated structures ) in a single film.456 it is used in a broad range of clinical settings , including the evaluation of deciduous and permanent teeth ; orthodontic evaluations ; the evaluation of bone lesions , erupted teeth , periodontal bone supports , third molars , lesions in the maxillary sinus , and fractures of the mandible ; and evaluations of foreign bodies and the loss of bone mineral density ( osteopenia and osteoporosis ) ; among others.4678 however , this technique also presents certain disadvantages , such as the overlap of the images of certain anatomical structures , the amplification and reduction of images ( geometric distortion ) , and the production of ghost images.356910 ghost images , or artifacts , are created by anatomical structures or objects that are located outside of the zone of focus or image layer but have a sufficient density to attenuate the x - ray beams , in turn producing distorted radiographic images.2 although panoramic radiographs play a crucial role in diagnosis , these ghost images complicate the use of panoramic radiographs , since they may closely resemble the lesions themselves , be confounded with dental structures in inappropriate locations , or mask anomalies by obstructing the view of subjacent anatomical structures.4 panoramic radiographs of a patient with earrings inserted in his or her earlobe are one of the most common examples of how ghost images of non - anatomic origin may be formed.2 in such cases , one can observe images of the earrings in the molar region , the coronoid process , the mandibular notch , and/or the mandibular condyle , resembling an impacted tooth or a cyst , among other abnormalities . it is therefore important to understand the appearance of anatomical structures and their variations in panoramic radiographs,4 with the additional challenge of identifying structures that fall outside of the standards of normality in order to make correct and coherent diagnoses . since many patients have piercings or other metal objects in their facial region , and as these objects can impair the quality of the panoramic radiograph image and its interpretation , it is important to understand the errors and artifacts that accompany this radiological technique . therefore , the aim of the present study was to present a study of the formation of ghost images in panoramic radiographs caused by metal objects in different regions of the skull , as well as to report a clinical case , in an attempt to aid dentists in understanding the mechanisms associated with the formation of real and ghost images in panoramic radiographs . after having received approval for the present study from our institution 's ethics committee ( cae : 45441815.4.0000.5137 ) , we conducted this study using a macerated adult skull placed in a kodak 9000c three - dimensional panoramic device ( eastman kodak company , rochester , new york , usa ) . the skull was positioned in the x - ray device according to standard procedures , with its sagittal plane placed in the center of the zone of focus of the x - ray unit and perpendicular to the horizontal plane and the frankfurt plane parallel to the horizontal plane . according to the bitewing of the device , the top teeth were arranged to place the dental arches in the zone of focus or image layer . a round metal object was attached in various areas of the skull to assess the formation of real and ghost images ( the region around tooth 46 , the upper region of the ear , the anterior nasal spine , the nasal wing , the lateral pole of the temporomandibular joint , the region around tooth 18 , the mandibular angle , the earlobe , the hyoid bone , and the dorsum of the tongue ) . one image was taken with no metal object , which was defined as the control radiograph . the radiographs were taken using the following exposure factors : 60 kv , 2.0 ma , and 14.3 s of exposure . the present study mapped the maxillomandibular areas according to the diagram proposed by reuter et al.,11 who demonstrated the formation of one , two , or three similar images after the strategic positioning of a metal object in different anatomic structures of the face . reuter et al.11 referred to the zones where the test object was portrayed once as zone 1a and zone 1b , the zone where the test object was portrayed twice as zone 2 , and the zone where the test object was portrayed three times as zone 3 ( fig . 1 ) . in the areas corresponding to zone 1a , which comprises the anterior regions of the mandible and the maxilla , the formation of one single real image occurs . in zone 1b , which includes the most posterior portion of the zone of focus or image layer ( i.e. , the regions of the occipital bone and the foramen magnum ) , the production of a single ghost image occurs . the mandibular ramus and angle , the mandibular condyles , the hard palate , and the earlobes are located in the areas corresponding to zone 2 , where two images are produced : one ghost image and one real image . in zone 3 , comprising the spine and hyoid bone , the formation of a triple image can be observed , including two real images lateral to the film and one ghost image in the center of the film . in this study , the images obtained from the skull were examined directly in the kodak dental imaging software ( kodak dental systems , rochester , ny , usa ) by two trained oral radiologists , one with ten years of experience and the other with eight years of experience . they used a computer with a geforce 9500 gt graphics card ( nvidia corporation santa clara , ca , usa ) , and an led lg flatron e2241 monitor ( lg electronics , seoul , korea ) , with a resolution of 19201080 pixels and the brightness and contrast levels of the monitor set to the default configuration . the analysis of the images was performed following the schematic design of zones 1a , 1b , 2 , and 3 . disagreements were resolved by discussion , and a final consensus was reached . in order to exemplify the importance of the anatomic and technical knowledge of the formation of panoramic radiographic images , we also report the clinical case of a 24-year - old male patient referred to the radiology clinic to undergo dental exams necessary for orthodontic treatment . panoramic and lateral cephalometric radiographs were taken using the orthopantomograph op 100 ( instrumentarium imaging , tuusula , finland ) . for the panoramic radiograph , the x - ray tube was operated with settings of 57 kvp , 3.2 ma , and an exposure time of 17.6 s. for the lateral cephalometric radiograph , the x - ray tube was operated with settings of 80 kvp , 10 ma , and an exposure time of 0.7 s. after radiographic exposure , all films were processed in a macrotec mx-2 automatic processor ( macrotec , so paulo , brazil ) with picker rp type - s processing chemistry ( eastman kodak company , rochester , ny , usa ) . first , a panoramic radiograph was taken of the macerated skull with a metal sphere attached in the region where tooth 46 was missing . this region was located within the focal plane , corresponding to the formation of images in zone 1a . 2 ) . figure 3 presents a panoramic radiograph taken with a metal sphere that was attached to the crown of tooth 18 . this region is located in the focal plane but corresponds to image formation in zone 2 . the result was the production of a real image in the region of the crown of tooth 18 ( ipsilateral ) and a ghost image on the opposite side ( contralateral ) . figure 4 shows another example of a panoramic radiograph taken with a metal sphere located in zone 2 . in this case , the metal sphere was attached in the region of the right earlobe , producing a real ipsilateral image and a distorted contralateral ghost image . in the last panoramic radiograph , the metal sphere was attached in the region of the hyoid bone ( fig . thus , three images were formed , with two real and bilateral images projected in the region of the right and left mandibular angle and one ghost image in the center of the film , overlapping the region of the inferior incisors . in this case report , the panoramic radiograph presented a radiopaque image overlapping the roots of the molars , premolars , and canines , located in the upper right hemiarch ( fig . the lateral cephalometric radiograph was analyzed to evaluate the existence of a metal object that could explain the formation of the radiopaque band observed in the panoramic radiograph ( fig . this study demonstrated the formation of real and ghost images in panoramic radiographs through the placement of metallic objects in different maxillomandibular regions of a macerated adult skull . the images were analyzed using the schematic design of zones where the test object was portrayed once ( zone 1a and 1b ) , twice ( zone 2 ) , and three times ( zone 3 ) . panoramic radiographs may contain single or double real images , as well as ghost images . a real image is formed when the object is located between the rotation center of the x - ray beam and the image receptor , and the image is taken with high definition and sharpness when the object is near the central layer of the zone of focus ( fig . 7).1213 the central ray of the x - ray beam must also be perpendicular to the object and image detector.14 the shape of the panoramic radiograph zone of focus or image layer is similar to that of the dental arch . figure 2 shows the formation of a single real image in a panoramic radiograph taken with one metal sphere attached in the region where tooth 46 was missing ( zone 1a ) . a ghost image occurs when the object or anatomic structure is located between the x - ray source and the center of rotation , and has a density that is sufficient to attenuate the x - ray beams.212 this image subsequently presents the same morphology as the object , but with distortion , and appears on the opposite side and at a higher point than the corresponding real object . but may or may not be expanded horizontally.10 the ghost image also contains a corresponding real image , which may or may not be visible in the panoramic radiograph.12 figures 3 and 4 , as well as the case report , illustrate the formation of a real ipsilateral image and a distorted contralateral ghost image in panoramic radiographs with a metal object located in zone 2 . the double image is formed when an object or anatomic structure is located between the center of rotation of the x - ray beams and the image receptor , but is intercepted twice during the exposure cycle.212 the distance of the object to the image receptor is the same in both instances in which the object is exposed.2 the double images are as sharp as the real images.211 double images are caused by objects or anatomic structures that are located in the midline . figure 5 demonstrates the formation of a double image caused by a metal sphere placed in the region of the hyoid bone ( zone 3 ) . another clinical example is the cervical spine , from which a real image is portrayed bilaterally at the extreme sides of the radiograph.12 triple images can consist of two lateral images and a central ghost , as in case of the cervical spine and hyoid bone.11 in this situation , a real image is portrayed bilaterally at the extreme sides of the radiograph , and the central image is a ghost.1112 figure 5 exemplifies the formation of a triple image caused by the placement of a metal sphere in the region of the hyoid bone ( zone 3 ) . in this case report , the panoramic radiograph presented a radiopaque image located in the upper right hemiarch . also observed was a voluminous metal restoration in the occlusal region of tooth 28 . therefore , it was confirmed that the radiopaque image formed in the region of the maxilla on the right side of the panoramic radiograph was in fact a ghost image caused by the restoration of the amalgam present in the maxillary left third molar . the metal restoration was located in zone 2 and produced an ipsilateral real image ( restoration in tooth 28 ) and a distorted contralateral ghost image ( radiopaque band ) . in conclusion , knowledge of the principles of the formation of ghost images in panoramic radiographs , as well as of normal anatomy , prevents incorrect diagnoses that could result in inappropriate prognoses and treatment plans . it is recommended that , whenever possible , all metal objects should be removed from the head and neck region in order to prevent the formation of ghost images created by these objects .

the bromeliaceae is one of the morphologically and ecologically most diverse flowering plant families native to the tropics and subtropics of the new world ( givnish et al . , 2011 ) . its geographical distribution ranges from the states of virginia , texas , and california in the usa ( latitude 37 n ) to northern patagonia in argentina ( latitude 44 s ) . bromeliads have different habits , varying from terrestrial to epiphytical , and are found from sea level to altitudes above 4,000 m , in both desert and humid regions , as well as in soils subject to regular floods and in places with very low or high luminosity . they can thrive on scalding sands and rocks , and withstand temperatures near 0 c ( benzing , 2000 ) . traditionally , the family has been divided into three subfamilies , bromelioideae ( 650 spp . ) , pitcairnioideae ( 890 spp . ) , and tillandsioideae ( 1000 spp . ) , based on smith and downs ( 1979 ) ; this classification is adopted in the present study . however , in a recent phylogeny based on eight plastid regions , with representatives from 46 of 58 genera , givnish et al . the new classification splits the paraphyletic pitcarnioideae into six subfamilies and proposes that they are related to each other as follow : ( brocchinioideae , ( lindmanioideae , ( tillandsioideae , ( hechtiooideae , ( navioideae , ( pitcarnioideae , ( puyoideae , bromelioideae ) ) ) ) ) ) ) . bromeliads are especially appreciated for their ornamental value , but some species have proven medicinal properties ( e.g. , bromelia antiacantha ) or are cultivated as tropical fruits ( e.g. , pineapple : ananas comosus ) . here , we review the main genetic and evolutionary topics concerning bromeliaceae , from a conservation standpoint . among the plant families , bromeliaceae is the one with the highest diversity of pollination modes ( ornithophily , chiropterophily , entomophily , mixed / unspecific , and autogamy ) throughout its geographic distribution ( kessler and krmer , 2000 ; canela and sazima , 2005 ; wendt et al . , 2008 ; schmid et al . , bromeliads have evolved floral displays with a great diversity of colors , shapes , and scents , which are related to pollinator attraction , with nectar being the usual reward ( benzing , 2000 ) . the presence of bromeliaceae in the new world has provided an important resource base , largely absent in the old world , for small , hovering vertebrate pollinators ( fleming and muchhala , 2008 ) . a recent study ( krmer et al . , 2008 ) strongly supports the hypothesis that the composition of nectar sugars in bromeliaceae is correlated with the pollinator syndrome ( lepidopterophilous , trochilophilous , or chiropterophilous ) . although the majority of bromeliads are pollinated by vertebrates , mainly hummingbirds and bats , bees are among the most frequent visitors to some short - corolla species with ornithophilous features . nevertheless , few studies have identified insects as effective pollinators of these bromeliads ( kamke et al . , 2011 ) . simultaneously with the divergence of bromeliad subfamilies ( see evolution below ) , the first split of modern hummingbird lineages appears to have occurred in the andes about 13 mya , with several other andean lineages diverging during the pliocene and pleistocene ( givnish et al . , 2011 ) . this might have contributed to the rapid expansion of the range of bromeliads and pollinators throughout the neotropics . however , plant - pollinator interactions , seed dispersal , and the mechanisms promoting or constraining species diversification via these interactions are complex and poorly studied in the neotropics ( antonelli and sanmartn , 2011 ) . bromeliads possess specialized floral features such as herkogamy and dichogamy , which prevent spontaneous self - fertilization and facilitate animal - mediated outcrossing ( benzing , 2000 ; martinelli g , 1994 , phd thesis , university of st . floral morphology , hand - pollination experiments , and population genetics studies have shown that selfing and mixed are the most common mating systems in a large part of the family ( bush and guilbeau , 2009 ; matallana et al . , 2010 ; table 1 ) , although self - incompatibility systems can be found in all of the subfamilies ( pitcairnioideae : vosgueritchian and buzato , 2006 ; bromelioideae : canela and sazima , 2003 , 2005 ; schmid et al . , 2010 ; kamke et al . , 2011 ; tillandsioideae : hietz et al . , the tillandsioideae subfamily has a particularly high frequency of selfing and mixed systems in various genera , including alcantarea , guzmania , racinea , tillandsia , vriesea , and werauhia ( benzing , 2000 ; lasso and ackerman , 2004 ; paggi et al . , 2007 , 2012 ; clonality is another reproductive strategy present in the family ( murawski and hamrick , 1990 ; izquierdo and pinero , 2000 ; sarthou et al . , 2001 ; sampaio et al . , 2002 ; sgorbati et al . , 2004 ; cascante - marn et al . , 2006 ; barbar et al . , 2009 ) , with important ecological and evolutionary consequences ( gonzales et al . , 2008 ) such as recruitment and population maintenance ( villegas , 2001 ) . vriesea gigantea presented a high natural production of flowers , fruits , and seeds , with high rates of viable seeds , with an average germination rate of 94% ( paggi et al . , 2007 , 2010 ) . furthermore , the species showed regular chromosome segregation and high pollen viability ( 8498% , palma - silva et al . , 2008 ) , which indicated that the populations analyzed were fertile . manual hand - pollination indicated that v. gigantea is self - compatible ( paggi et al . , 2007 ) and showed low to moderate levels of inbreeding depression ( = 0.02 to 0.39 ; sampaio et al . , 2012 ) . in a study with vriesea friburgensis we highlighted that it is pollinated by hummingbirds and produces high flower , fruit , and seeds together with high seed and pollen viability . self - sterility was observed from spontaneous selfing and manual self - pollination treatments , which may be a consequence of late - acting self - incompatibility . we proposed that this self - sterile species depends on pollinator services to maintain its population fitness and viability through cross - pollination ( paggi et al . , 2012 ) . we compiled data from all diversity and genetic structure studies published before june 2011 ( table 1 ) . of the 58 genera and about 3,140 bromeliad species ( givnish et al . , 2011 ) , only 20 species of the following nine genera have been previously evaluated : aechmea , alcantarea , bromelia , dyckia , encholirium , pitcairnia , puya , tillandsia , and vriesea . the use of co - dominant markers has been the preferred method for studying bromeliad population genetics , with nuclear microsatellite markers being the most frequently used molecular markers ( nine species ) , followed by allozymes ( eight species ) . dominant markers such as amplified fragment length polymorphisms have been used in only one study of one species , and random amplified polymorphic dna was applied in another study of three species ( table 1 ) . a comparison of genetic diversity parameters among such studies is difficult , as the highly polymorphic ssrs usually show higher observed and expected heterozygosity ( ho and he , respectively ) compared with other markers . for example , populations of pitcairnia geyskesii have been evaluated using allozymes ( sarthou et al . , 2001 ) and ssrs ( boisselier - dubayle et al . , 2010 ) . with allozymes , ho and he were 0.188 and 0.246 , respectively ; with ssrs , ho and he were 0.293 and 0.324 , respectively . we found low inbreeding coefficient indices ( fis ) in salmost all species with outcrossing mating systems . the exceptions were b. antiacantha ( fis = 0.431 ) , possibly due to the wahlund effect and/or null alleles , and alcantarea glaziouana ( fis = 0.156 ) , owing to biparental inbreeding . pitcairnia staminea , which is autogamous , had a high inbreeding coefficient ( fis = 0.240 ; table 1 ) . v. gigantea and dyckia ibiramensis , which have a mixed mating system , also showed high inbreeding coefficients ( fis = 0.273 and 0.436 , respectively ; table 1 ) . the degree of differentiation among populations ( fst ) of species evaluated ranged from 0.043 to 0.961 . these differences in plant population structure can be influenced by pollen and seed dispersal effects , clonal growth ( gliddon et al . , 1987 ) , gene flow rates , and connectivity among populations . compared with species from continuous forest habitats , species restricted to inselberg habitats ( barbar et al . , 2007 , 2009 ; palma - silva et al . , 2011 ; table 1 ) showed more highly structured populations , with extremely high population differentiation and isolation based on the distance among inselbergs . thus , rock outcrops could be highly useful venues for studies regarding the molecular ecology and genetics of continental radiations . chromosome numbers have been determined for nearly 12% of the known species ( cotias - de - oliveira et al . , 2004 ) , owing to the scarcity of cytogenetic data , the chromosomal evolution of the family has not been completely elucidated . the major hindrances to cytogenetic studies are probably the very small size and poor staining ability of the chromosomes , together with a marked cytoplasmic content ( sharma and ghosh , 1971 ; brown and gilmartin , 1986 ) . billings ( 1904 ) was the first to determine the chromosome number of a bromeliad , using tillandsia usneoides , after which several studies were carried out . the first reports revealed a great variety of diploid numbers ( 2n = 16 , 34 , 36 , 46 , 48 , 50 , 52 , 54 , 56 , 64 , 96 , and 100 ) and basic numbers ( x = 5 , 8 , 9 , 16 , 17 , and 25 ; brown and gilmartin , 1986 ; bellintani et al , 2005 ) . in contrast , most of the 72 bromeliad species studied by marchant ( 1967 ) showed a basic number of x = 25 ( except cryptanthus : x = 17 ) . since then , studies in several different species have generally found the basic chromosome number to be a multiple of x = 25 , corroborating marchant s finding ( brown and gilmartin , 1989 ; cotias - de - oliveira et al . , 2000 , 2004 ; palma - silva et al . , 2004 ; gita et al . , 2005 ; ceita et al . , 2008 ; louzada et al . , polyploidy of this base number ( 2n = 4x = 100 and 2n = 6x = 150 ) has been observed in all subfamilies , but with low frequency ( brown and gilmartin , 1989 ; gita et al . brown and gilmartin ( 1989 ) have proposed a model to explain the evolution of the chromosome base number . in their model , two paleodiploids ( x = 8 and x = 9 ) hybridized , resulting in a paleotetraploid lineage ( x = 17 ) , which in turn hybridized with the x = 8 paleodiploid , and the poliploidization stabilized at the hexaploid level of x = 25 . . , 1987 ) suggest that a diploidization of the dibasic paleohexaploid occurred . the dibasic model could explain the origin of the distinctive chromosome number in cryptanthus , which may represent a paleotetraploid with 2n = 34 . one alternative hypothesis is that cryptanthus evolved from x = 25 via aneuploidy ( brown and gilmartin , 1989 ) . flow cytometric results obtained by ramrez - morillo and brown ( 2001 ) indicated that the cryptanthus chromosome number originated by descending aneuploidy . bromeliaceae chromosomes are usually exceedingly small ( 0.212.72 m ) , although the size varies widely among species . ( 2005 ) , larger chromosomes are usually found at lower ploidy levels , with diploids exhibiting a higher contrast between maximal and minimal chromosome sizes compared with polyploids . chromosome banding and triple staining with cma3/actinomycin / dapi has revealed that bromeliads have relatively little heterochromatin , with only one or two cma / dapi terminal bands corresponding to nucleolus organizing regions . b chromosomes have been reported in three bromelioideae species ( cotias - de - oliveira et al . , 2000 , 2004 ; bellintani et al . , recently , givnish et al . ( 2011 ) reinforced the i.e. of smith ( 1934 ) that bromeliads arose in the guayana shield roughly 100 mya during the cretaceous period , with the extant subfamilies beginning to diverge only about 19 mya . ( 2011 ) also suggested that about 15.4 mya , bromeliads began to spread from that hyper - humid , extremely infertile center to other parts of tropical and subtropical america , and probably arrived in tropical africa about 9.3 mya , in a recent long - distance dispersal event . during the evolution of this family , events such as climatic oscillations throughout the pleistocene have resulted in the dispersion of some clades , including bromelioidae ( givnish et al . , , v. gigantea has survived glaciation periods in two fragmented refugia in southeastern brazil ( palma - silva et al . , 2009 ) . the bromeliad revolution probably occurred after the uplift of the northern andes and shift of the amazon to its present course ( givnish et al . , 2007 ) . some morphological and physiological adaptations , including crassulacean acid metabolism ( cam ) photosynthesis and the formation of rosettes and leaf absorptive scales , might have been crucial to the adaptive radiation of bromeliads ( benzing , 2000 ; crayn et al . , an ecological peculiarity of bromeliaceae , compared with other families of the order poales , is their epiphytic habit ( linder and rudall , 2005 ) . based on plastid loci , crayn et al . ( 2004 ) proposed that the epiphytic habit of bromeliads evolved a minimum of three times , most likely in response to geological and climatic changes in the late tertiary . the more than 3,000 bromeliad species that currently occupy the neotropical region have evolved to fill numerous niches , with an incredible diversity of adaptations . some aspects of the complex evolutionary history of this family are still unclear , indicating the need for further molecular studies , in combination with paleontological data , to explain the evolutionary gaps in the wide diversity of bromeliad forms and adaptations . bromeliads are widely distributed in the neotropics , with three centers of diversity : the brazilian atlantic rainforest ; the andean slopes of peru , colombia , and ecuador ; and mexico and adjacent central america ( zizka et al . , many species are presently distributed in endangered biomes , are endemic , or have a relict distribution , threatening the survival of many members of this family . for example , the brazilian atlantic rainforest is a diverse biome with multiple extremely endangered vegetation types occupying only 7.91% of the extent of their original distribution ( fundao sos mata atlntica and instituto nacional de pesquisas espaciais , 2009 ; carnaval and moritz , 2008 ) . as the atlantic rainforest contains at least 803 bromeliad species , 653 of which are endemic and 40% of which are endangered , the preservation of the atlantic rainforest is vital for the conservation of bromeliaceae ( martinelli et al . , 2008 ) . all of the works cited in the above section diversity and genetic structure contain data that could be used in making conservation decisions . considerations of the clonal and sexual reproduction , demography , genetic structure within and among populations , gene flow , and mating systems of bromeliaceae are of primary importance in developing successful conservation strategies ( bizoux and mahy , 2007 ) . our group has studied mainly brazilian bromeliads , and our field records show a significant reduction in the current distribution of species , compared with the first records in the literature . we believe that genetic , cytogenetic , and reproductive data will be essential for diagnosing species status and for assisting conservation programs and will help to elucidate aspects of evolution and environmental and climate change for bromeliaceae and the brazilian atlantic rainforest .

management options for patients with low or intermediate risk prostate cancer and a life expectancy of less than 10 years include active surveillance ( 1 ) , radical prostatectomy ( 2 ) , external beam radiation therapy ( ebrt ) , low - dose rate ( ldr ) brachytherapy monotherapy ( 3 , 4 ) , or high - dose - rate ( hdr ) brachytherapy ( 5 , 6 ) . since cure rates are similar among these treatment options ( 7 ) , health - related quality of life ( hrqol ) is an important factor in a patient s decision - making process ( 8) . in prostate cancer patients , physician - assessed hrqol changes do not correlate with patient - assessed changes . physicians under - estimate hrqol changes and over - estimate improvement in symptoms relative to patients ( 9 ) . discrepancies are particularly large for symptoms like pain and fatigue ( 9 ) . as a result the expanded prostate cancer index composite ( epic ) is a validated questionnaire used to assess hrqol in prostate cancer patients . epic includes 4 domains : urinary , bowel , sexual , and hormonal ( 10 ) . there are summary ( i.e. , overall ) scores and function and bother subscale scores for each of the 4 domains . domains and subscales are scored using a 0 - 100 grading system , with a higher score indicating a higher quality of life . hrqol changes in prostate cancer patients undergoing radical prostatectomy , ldr brachytherapy monotherapy or ebrt vary significantly between treatment modalities ( 11 ) . there has been only one prior report on hrqol changes due to hdr brachytherapy monotherapy for prostate cancer ( 12 ) . as a result recurrence risk was defined according to the national comprehensive cancer network ( nccn ) guidelines ( 13 ) . low recurrence risk was defined as patients with clinical t1-t2a disease , prostate - specific antigen ( psa ) < 10 ng / ml , and a gleason score 6 . intermediate recurrence risk patients were those with clinical t2b - t2c disease , psa=10 - 20 ng / ml , or a gleason score = 7 . favorable intermediate risk patients were defined as those with a gleason score of 3 + 4=7 , ct2b disease , and 50% positive core biopsies ( 5 ) . low risk and favorable intermediate risk patients may be treated with hdr brachytherapy monotherapy ( 5 , 6 , 12 , 14 ) . unfavorable intermediate risk patients had a gleason score of 4 + 3=7 , ct2c disease , or > 50% positive core biopsies ( 15 , 16 ) . patients with unfavorable intermediate risk prostate cancer and patients who received intensity modulated radiation therapy ( imrt ) or androgen deprivation therapy were excluded from this study . after obtaining institutional review board approval , we treated 84 low risk and favorable intermediate risk prostate cancer patients with hdr brachytherapy monotherapy at the h. lee moffitt cancer center & research institute between february 2011 and april 2013 . after providing informed consent , patients underwent hdr brachytherapy monotherapy to the prostate to 2,700 - 2,800 cgy in two 1,350 - 1,400 cgy fractions separated by 2 - 3 weeks . over a one - year period following hdr brachytherapy , approximately half of the patients were placed on phosphodiesterase-5 inhibitors such as one sildenafil 50 mg tablet by mouth three times per week for erectile dysfunction . sixty - four of 84 ( 76% ) patients completed the 50-question form prior to hdr brachytherapy monotherapy , i.e. , at baseline , and 6 and/or 12 months after treatment patients who failed to complete the 50-question epic questionnaire commonly stated that it was too long . table 1patient characteristics.number of patients64mean follow - up9 monthsage at diagnosis , mean ( range)65 years ( 48 - 83)bmi , kg / m , mean ( range)29.5 ( 22.0 - 43.0)psa , ng / ml , median ( range)5.3 ( 1.0 16.1)prostate size , cc , median ( range)54 ( 24 - 108 ) ajcc clinical t stage t1c58 t2a5 t2b1 gleason score 3 + 3=643 3 + 4=721 nccn recurrence risk group low39 intermediate25 ajcc= american joint committee on cancer ; cc : cubic centimeters ; nccn= national comprehensive cancer network ; psa= prostate - specific antigen . ajcc= american joint committee on cancer ; cc : cubic centimeters ; nccn= national comprehensive cancer network ; psa= prostate - specific antigen . in accordance with prior reports ( 12 , 17 , 18 ) , we calculated mean epic scores for each time point . pre - treatment epic scores were compared to scores obtained 6 months and 12 months after treatment using a student s t - test . linear regression was used to analyze the relationship between patient characteristics ( body mass index ( bmi ) , age , prostate volume , psa , gleason score , and recurrence risk group ) and epic scores . pre - treatment urinary overall , function , bother , incontinence , and irritative / obstructive scores were 87 , 91 , 85 , 87 , and 85 , respectively ( figure 1 ) . six months after treatment , urinary overall , function , bother , incontinence , and irritative scores decreased to 76 , 79 , 75 , 76 , and 75 respectively ( p<0.01 ) . twelve months after treatment , all urinary scores had increased and were not significantly different from baseline values . figure 1epic urinary overall , function , bother , incontinence , and irritative / obstructive scores before hdr brachytherapy monotherapy and 6 and 12 months after treatment . pre - treatment bowel overall , function , and bother scores were 95 , 90 , and 95 , respectively ( figure 2 ) . six months after treatment , there was a significant decrease in bowel overall , function , and bother scores to 86 , 84 , and 86 respectively ( p<0.001 ) . twelve months after treatment , bowel overall and bother scores increased to 88 and the bowel function score had increased to 86 . figure 2epic bowel overall , function , and bother scores before hdr brachytherapy monotherapy and 6 and 12 months after treatment . pre - treatment sexual overall , function , and bother scores were 46 , 43 , and 53 , respectively ( figure 3 ) . six months after treatment , there was a significant decrease in sexual overall and bother scores to 34 and 42 , respectively . twelve months after treatment , sexual overall and bother scores increased and were not statistically different from baseline values . figure 3epic sexual overall , function , and pre - treatment hormonal overall , function , and bother scores were 91 , 88 , and 92 , respectively ( figure 4 ) . six months after treatment , there was a non - significant decrease in sexual hormonal scores . figure 4epic hormonal overall , function , and bother scores before hdr brachytherapy monotherapy and 6 and 12 months after treatment . morton et al . ( 19 ) reported hrqol changes in intermediate risk prostate cancer patients who received ebrt and an hdr brachytherapy boost without androgen deprivation therapy . patients experienced clinically significant decreases in epic urinary , bowel , and sexual overall scores 12 months and 24 months after treatment . in contrast , the epic hormonal overall score did not change significantly due to radiotherapy . similarly , in this study , the epic bowel overall score remained significantly below baseline 12 months after radiotherapy ( figure-2 ) ; however , the decrease in the epic hormonal overall score was not statistically significant ( figure-4 ) . to date , only one study has reported patient - assessed hrqol changes in prostate cancer patients treated with hdr brachytherapy monotherapy . ( 12 ) treated 79 low and intermediate risk prostate cancer patients with hdr brachytherapy monotherapy . they observed a decline in epic scores across all 4 domains as early as one month after treatment . this compares favorably with our findings , where epic urinary and sexual scores did not improve until 12 months after hdr brachytherapy ( figures 1 and 3 ) . epic scores may have taken longer to improve after hdr brachytherapy in this report because we delivered a higher biologically effective dose of radiotherapy ( 14 ) . observed that urinary , bowel , and hormonal scores remained stable 3 - 48 months after treatment . also , they reported a decline in sexual overall scores as early as one month after treatment with no recovery thereafter . as in the report by barkati et al . , baseline sexual scores in this study were considerably lower than urinary , bowel , and hormonal scores ( figures 1 - 4 ) . like barkati et al . , we observed a significant decrease in sexual overall and bother scores at 6 months ( figure-3 ) . however , in this report , there was improvement in sexual scores at 12 months . this was probably due to early use of a phosphodiesterase-5 inhibitor after brachytherapy in approximately half of our patients ( 20 ) . ( 21 ) used the common terminology criteria for adverse events v4 grading system to determine incidence rates of erectile dysfunction 3 years after hdr brachytherapy monotherapy vs. imrt . rates of erectile dysfunction requiring medical intervention for both hdr brachytherapy monotherapy and imrt were low and equivalent . in this study , 64/84 ( 76% ) prostate cancer patients treated with hdr brachytherapy monotherapy completed 50 questions comprising the most recent version of the epic questionnaire . similarly , others have reported 36 - 78% compliance rates ( 12 , 22 ) . since men who did not complete the form commonly stated that it was too long , we have switched to a 26-item , short - form version of epic in an effort to improve patient compliance ( 23 , 24 ) . hdr brachytherapy monotherapy is well - tolerated in patients with low and favorable intermediate risk prostate cancer . epic urinary and sexual domain hrqol scores returned to close to baseline 12 months after treatment .

it is an irony of science that the discovery of fundamental principles is often delayed . the nucleus is arguably the functionally most prominent cellular organelle as it houses the vast majority of the genetic information and is the exclusive site of essential processes including transcription , rna processing , replication and dna repair . yet , our understanding of how the nucleus is organized and how nuclear architecture contributes to cellular function is only now emerging and still lags behind that of most other cellular compartments . fortunately , pioneering studies over the past decade have uncovered several of the fundamental principles which determine nuclear architecture and function ( lanctot et al . these studies have revealed the presence of distinct structural elements , particularly lamin proteins and possibly short actin filaments , within the nucleus and they have led to the identification of a large number of distinct nuclear subcompartments in which specific nuclear functions , such as the synthesis and processing of ribosomal rna in the nucleolus , occur ( burke and stewart 2006 ; gruenbaum et al . 2005 ; handwerger and gall 2006 ; hernandez - verdun 2006 ; lamond and sleeman 2003 ) ( fig . in addition , it has become clear that the genome is non - randomly organized within the spatial confines of the nucleus with specific chromosomes and genes preferentially localizing to specific sites ( lanctot et al . for example , despite the requirement to accommodate 2 m of dna within the cell nucleus of a diameter of typically 10 m , chromatin is estimated to only occupy about 15% of the nuclear volume . fig . 1nuclear architecture and function . the mammalian cell nucleus contains a large number of non - random and structural features . a nuclear lamins ( green ) form a network of architectural elements at the periphery . this nuclear lamina is implicated in mechanical support of the nucleus , organization of chromatin ( red / blue ) in the nuclear interior , as a platform for signaling events and in the sequestration of nuclear proteins . b each chromosome occupies a distinct chromosome territory ( red chromosome 14 ; green chromosome 17 in human breast epithelial cells ) . image courtesy : a gianluca pegoraro , b karen meaburn , c manjari mazumdar , all national cancer institute , nih nuclear architecture and function . the mammalian cell nucleus contains a large number of non - random and structural features . a nuclear lamins ( green ) form a network of architectural elements at the periphery . this nuclear lamina is implicated in mechanical support of the nucleus , organization of chromatin ( red / blue ) in the nuclear interior , as a platform for signaling events and in the sequestration of nuclear proteins . b each chromosome occupies a distinct chromosome territory ( red chromosome 14 ; green chromosome 17 in human breast epithelial cells ) . image courtesy : a gianluca pegoraro , b karen meaburn , c manjari mazumdar , all national cancer institute , nih a key question in understanding nuclear architecture is how protein and rnas move within the nucleus . this is a key issue if we are to gain a full appreciation of what the nuclear environment is like . more importantly , the mode of nuclear motion of molecules has physiological implications as it affects how proteins find their target sequences in the genome , how rnas are exported from the nucleus upon transcription and how proteins are sequestered within the nucleus for regulatory purposes . we have long known that proteins and rnas must be able to move within the cell nucleus . messenger rnas traverse the nucleus and are promptly exported from the nucleus after their synthesis and complete processing . newly synthesized proteins on the other hand are imported into the nucleus and rapidly disperse throughout its volume , implying effective intranuclear trafficking . furthermore , proteins rapidly relocalize within the nucleus upon experimental or physiological changes in conditions . for example , inhibition of protein synthesis leads to rapid and dramatic relocalization of a large number of nucleolar proteins ( andersen et al . 2005 ) , and pre - mrna splicing factors swiftly respond to transcriptional activation and inhibition ( jimnez - garca and spector 1993 ; misteli et al . how proteins and rnas move within the nucleus and how they find their targets , however , was unknown for long . it was not clear whether movements are energy dependent , whether they occur by directed transport or what the speed of intranuclear trafficking is . the investigation of intranuclear protein and rna dynamics was made possible by the development of techniques to visualize and measure the motion of these molecules by time - lapse microscopy . tracking of proteins by photobleaching methods revealed a surprising degree and speed of intranuclear trafficking . the analysis of tracers such as gfp or dextran which do not undergo specific interactions with dna or other proteins indicated that the nuclear environment is permissive for rapid diffusional motion ( grisch et al . the diffusion of molecules within the nucleus is only limited by the steric constraints imposed by chromatin and nuclear bodies ( grisch et al . 2005 ; misteli 2001 ; seksek et al . 1997 ) . the typically measured diffusion coefficients of 10100 m s are similar to those observed in the cytoplasm and are only 5 times less than that in solution . as expected for diffusion , this rapid motion is energy - independent and non - directional . several methodological approaches involving fluorescently labeled , microinjected , engineered or endogenous rnas demonstrate that ribosomal rnas as well as polya - rnas move freely in a non - directional manner with a diffusion coefficient of 0.030.1 m s within the nucleus ( ritland - politz et al . a diffusion coefficient of this magnitude is sufficient to ensure transport of an rna particle from deep within the nucleus to the cytoplasm within a few minutes , consistent with biochemical observations on kinetics of rna maturation and transport . thus energy - independent , diffusion - based movement of rna particles alone can account for the observed kinetics of rna export , and no active mechanisms are required to ensure rapid export . an impressive demonstration of the non - directional motion of mrna comes from studies in which a nascent rna is visualized at its site of transcription and its export to the cytoplasm measured in living cells ( ritland - politz et al . 2006 ; shav - tal et al . these studies show that rnas synthesized from genes positioned in proximity to the nuclear envelope diffuse away from their site of synthesis in all directions rather than follow a direct path to the nearest nuclear pore . this observation powerfully demonstrates the non - directed , diffusion - based motion of rnas in the nucleus . one of the conceptually most challenging problems in cell biology is the question of how molecules find their specific targets within a cell or within an organelle . this problem is particularly complex in the cell nucleus where transcriptional regulators need to find their specific target genes amongst the myriad of potential binding sites within the genome . somewhat counter - intuitively , the non - directional , but rapid , motion of proteins within the cell nucleus provides a means to ensure targeting of proteins to specific genome locations ( misteli 2001 ) . the power of targeting by random diffusion within the nucleus is best illustrated when considering how a transcription factor finds its target genes but it also applies to targeting of proteins to nuclear compartments or any other nuclear site . proteins a priori do not know where their targets are and we do know of any directed transport systems , such as a molecular motor - based mechanism , that would bring a factor to a specific gene or a specific location within the nucleus . thus , the only way for a transcription factor to find its target is to scan the genome . the intrinsic ability of proteins to rapidly move within the nucleus by diffusion - based mechanisms permits such genome scanning ( misteli 2001 ) . this occurs by a transcription factor freely diffusing within the nucleoplasm until it interacts by chance with chromatin . the molecule will now probe whether the sequence it has encountered is a specific binding site such as in the promoter of one of its target genes . if it is , the transcription factor will be captured and stably associated with its specific target site . if the sequence is not a binding site , the molecule , after a short interaction , will dissociate from chromatin and continue its diffusional journey through the nucleus . it might appear at first glance that such a stop - and - go model for genome scanning would be insufficiently effective . however , the observed dynamic properties of transcription factors are entirely consistent with this model . we know that the residence time of most transcription factors even on specific dna binding sites is in the order of a few seconds and that their interaction with non - specific sites is even faster , most likely in the order of tens of milliseconds ( gorski et al . 2006 ; houtsmuller et al . 1999 ; sprague et al . 2006 ) . assuming these time scales for binding , one can calculate that it takes a single transcription factor molecule only a few minutes to search the entire genome space . considering that most transcription factors exist in several thousand copies and have multiple target genes , their random diffusional motion is entirely sufficient to ensure a steady supply at their target genes . this assumption is further supported by direct measurement of transcription factor flux on an endogenous rrna promoter demonstrating the collision of several hundred molecules per second ( dundr et al . since proteins similarly move by diffusional motion within the cytoplasm , it stands to reason that the random scanning is also a key mechanism of protein targeting in the cytoplasm and represents a universal mechanism for how proteins find their targets . a key feature , and a requirement , in a genome - scanning model of targeting is that the interactions of proteins with chromatin are transient . this has been confirmed by photobleaching methods on a large number of dna binding proteins ( gorski et al . first , it allows proteins to maintain a high rate of motion and thus allows faster scanning . chromatin interactions static , they would get stuck at non - specific or incorrect binding sites which would slow down their overall motion . second , the short - life of protein chromatin interactions continuously makes available binding sites which can then be scanned by diffusing transcription factors . if proteins interacted for extended periods of time on chromatin , non - specific or improper binding would block access of the correct factors were protein complexes permanently bound to their target sites , changes in transcriptional activity such as in response to physiological stimuli could only occur after active removal of the bound complex , presumably by dedicated and specialized molecular machinery . in contrast , in a dynamic binding model the natural flux of proteins provides a window of opportunity for association of a distinct regulator each time a bound protein or complex dissociates as part of its normal binding cycle . a hallmark of the mammalian cell nucleus is the presence of distinct subnuclear compartments and domains in which particular functions occur ( handwerger and gall 2006 ; hernandez - verdun 2006 ) ( fig . 2 ) . the prototypical nuclear compartment is the nucleolus , a distinct intranuclear compartment in which ribosomal rnas are synthesized and partially processed . other prominent nuclear domains include splicing factor compartments which serve as storage and assembly sites for spliceosomal components , and the cajal bodies which are possibly involved in maturation of small nuclear rnps . the structure of intranuclear bodies is not determined by a membrane , and the principles underlying their biogenesis are extremely poorly understood . the recently revealed dynamic properties of proteins in nuclear compartments give a hint as to the principles involved in subnuclear compartment assembly . the nucleolus is the site of ribosomal rna synthesis and is a prototypical nuclear body . the complex organization of the nucleolus is revealed by multi - color staining of distinct nucleolar components . rpa43 rna polymerase i transcription factor , fibrillarin snrnp component , b23 rrna processing factor . image courtesy of miroslav dundr , rosalind franklin university of medicine and science , chicago intranuclear compartments . the nucleolus is the site of ribosomal rna synthesis and is a prototypical nuclear body . the complex organization of the nucleolus is revealed by multi - color staining of distinct nucleolar components . . image courtesy of miroslav dundr , rosalind franklin university of medicine and science , chicago the key property in understanding the formation of nuclear compartment is the remarkable fact that the association of proteins with their compartments is highly dynamic ( dundr et al . typical residence times of proteins within a compartment are in the seconds range . in practical terms the dynamic behavior of nuclear body proteins means that they undergo repeated rapid cycles of association and dissociation between the nuclear body and the nucleoplasm . as a consequence , a nuclear body is in perpetual flux and its structure is determined by the ratio of on - rate versus off - rate of its proteins . given these observations , the simplest scenario to explain the formation of nuclear subdomains is a self - organization model in which the collective sum of all interactions amongst proteins in a body establishes and determines its structure ( carrero et al . nuclear bodies are essentially dynamic protein aggregates which form as a consequence of multiple , transient interactions amongst a large number of proteins . this is consistent with the lack of defining intranuclear membranes and with the absence of any known dedicated structural elements within intranuclear bodies . while dynamic observations on nucleoli , splicing factor compartments and cajal bodies support this view , this model has not been rigorously tested experimentally . a key prediction , and a way to experimentally address this fundamental question , is that it should be possible to create nuclear bodies de novo and that any nuclear body component should be able to nucleate the formation of a body . experimental systems to test these predictions are now available , and we are eagerly awaiting the results of these key experiments . the observation of dynamic properties of proteins in the cell nucleus of living cells has suggested that dynamic trafficking is an intrinsic property of proteins and rnas . the dynamic behavior of proteins and rnas clearly contributes to their proper function . however , is dynamic trafficking important for physiological regulation ? several observations demonstrate that dynamic trafficking of both proteins and rna indeed can have regulatory function by several means . of particular importance as a regulatory mechanism is intranuclear sequestration ( fig . 3 ) . accumulation of a protein at a particular nuclear site or within a nuclear compartment can serve to either increase the local concentration of a factor at that site or to reduce its abundance in the rest of the nucleus . several proteins are now known to accumulate , in particular nuclear locations in response to physiological cues and their sequestration is thought to contribute to their cellular function . a retention of proteins within intranuclear subcompartments regulates their nuclear concentration and their rate of nuclear export . b sequestration of proteins at the nuclear periphery , particularly via interaction with the lamina ( blue ) controls their nucleoplasmic concentration and their availability at target genes . sequestration of an activator leads to repression , sequestration of a repression leads to activation . c retention of partially or fully processed rna within intranuclear compartments modulates rna export rate intranuclear retention and sequestration as a regulatory means . a retention of proteins within intranuclear subcompartments regulates their nuclear concentration and their rate of nuclear export . b sequestration of proteins at the nuclear periphery , particularly via interaction with the lamina ( blue ) controls their nucleoplasmic concentration and their availability at target genes . sequestration of an activator leads to repression , sequestration of a repression leads to activation . c retention of partially or fully processed rna within intranuclear compartments modulates rna export rate prototypical example of sequestration as a regulatory mechanism is the accumulation of the ubiquitin ligases mdm2 and the von hippel - lindau tumor suppressor protein vhl in the nucleolus ( mekhail et al . 2005 , 2007 ) . these two proteins are responsible for the ubiquitination of the p53 tumor suppressor and the hypoxia - induced factor hif , respectively . modification of these two targets leads to their nuclear export and degradation in the cytoplasm . since ubiquitination is a major regulatory mechanism in their function , the concentration of the ubiquitin ligase in the nucleus significantly determines their fate . however , in response to physiological cues , both mdm2 and vhl can become sequestered in the nucleolus , thus reducing their nucleoplasmic concentration and the extent of ubiquitination of their targets ( mekhail et al . 2005 , 2007 ) ( fig . sequestration of these proteins appears to occur by increased retention of diffusing molecules within the nucleolus . the possibility that retention is a highly controlled mechanism comes from observations on nucleostemin , a nucleolar protein implicated in stem cell maintenance and cancer proliferation ( meng et al . the retention of nucleostemin in the nucleolus is controlled by a gtp gdp binding cycle which might be directly or indirectly linked to signaling pathways . in addition to nucleostemin the localization of several additional gtp - binding domain containing nucleolar proteins seems to be controlled in a similar fashion ( meng et al . intranuclear sequestration can also be a means of negative regulation . the basic helix loop helix transcription factor hand1 is expressed in trophoblast stem cells and is required for their differentiation along several lineages ( martindill et al . hand1 is sequestered in the nucleolus and upon stimulation during differentiation is rapidly released and moves into the nucleoplasm where it presumably acts on target genes . hand1 is retained in the nucleolus by its interaction with i - mfa , and this interaction is sensitive to phosphorylation of hand1 . upon phosphorylation hand1 dissociates from i - mfa and interestingly , both the sequestration of hand1 in the nucleolus and its release are functionally important . premature release of hand1 triggers differentiation , whereas the presence of hand1 is also required to maintain the stem - cell potential of trophoblast stem cells . thus nucleolar sequestration of this key cell fate regulator serves a dual regulatory role ( martindill et al . however , the nuclear lamina has recently also emerged as a major site for transcription factor sequestration ( heessen and fornerod 2007 ) ( fig . prominent transcription factors which associate with the nuclear periphery either via interaction with the lamins or with the inner nuclear membrane proteins include c - fos , oct-1 and rb ( imai et al . it is tempting to speculate that the association of these factors with the periphery reduces the intranuclear concentration and thus their availability at target genes ( heessen and fornerod 2007 ) . a physiological role for such peripheral sequestration is most strongly suggested by observations on the pro - proliferation factor c - fos which associates with the lamina in quiescent cells but is released upon entry of cells into the cell cycle correlating with activation of some of its target genes ( ivorra et al . release of oct-1 from the lamina correlates with activation of collagenase , one of its prime target genes ( imai et al . the sequestration of transcription factors at the nuclear periphery is a potentially powerful and simple mechanism of transcriptional regulation . it will be important to determine how widespread this mechanism is and how precisely the association of transcription factors with the lamina is controlled . dynamic trafficking of rna is similarly used as a regulatory mechanism ( prasanth and spector 2007 ) . in particular , it appears that rna retention can act as a quality control mechanism for rna export . for one , incompletely spliced or processed rnas are retained at their sites of transcription and not released into the nucleoplasm for export . in addition , pools of stable polyadenylated rnas are enriched in the nuclear splicing factor compartments , although their function remains unclear ( huang et al . but retention is also emerging as a mechanism for regulation of specific rnas . a non - coding rna transcribed from the mouse cat2 gene encoding a cationic amino acid transporter is retained in the nucleus via a dedicated 3 end containing extensive repeat sequences ( prasanth et al . 2005 ) . these sequences are responsible for accumulation of the rna in a nuclear compartment identified as paraspeckles ( fig . this retention serves a physiological function because upon stress the cat2 rna is cleaved , released from paraspeckles and rapidly exported into the cytoplasm for translation , thus ensuring a rapid physiological stress response . although the cat2 rna is to - date the best characterized example of such retention , a similar mechanism is likely at work for the migration - stimulating factor ( msf ) mrna whose 3 end resembles that of cat2 and is retained within the nucleus ( kay et al . the rna appears to be released from the nucleus and rapidly translated . a further candidate for control via nuclear retention interestingly , gomafu does not seem to encode for any protein and might thus be a non - coding regulatory rna which is retained in the nucleus ( sone et al . the past few years have seen a dramatic change in how we view the cell nucleus . we have come to appreciate the presence of distinct structural elements within the nucleus , the presence of a multitude of intranuclear bodies and the fact that genomes are non - randomly organized within the nuclear space . one of the most consequential findings has been the realization that just about every aspect of nuclear organization is highly dynamic . both proteins and rnas move rapidly within the nucleus , and they only transiently interact with chromatin and nuclear bodies . it is now clear that the dynamic nature of nuclear components is a fundamental property and has implications for how molecules are targeted to their final destinations and for how intranuclear compartments form . most importantly , it is becoming clear that the dynamic properties of nuclear proteins are critical for various mechanisms of physiological regulation , particularly via sequestration and retention of proteins and rna . it is likely that these recently discovered examples of regulation by modulation of dynamic interactions within the cell nucleus are only the proverbial tip of the iceberg , and it stands to reason that we should consider the contribution of dynamic interactions and trafficking in any nuclear event we investigate in the future .

primary therapy for locally invasive disease consists of external and intracavitary or interstitial irradiation , except in selected early cases that may be treated surgically with local excision or with brachytherapy alone . radical surgery , which may involve partial or total pelvic exenteration , is generally reserved for pelvic recurrence after irradiation and for patients with fistulas at diagnosis . we report a case of stage iii primary vaginal papillary serous adenocarcinoma with metastasis to the left inguinal lymph nodes treated definitively with external beam radiation concurrent with cisplatin followed by high - dose - rate ( hdr ) brachytherapy . a 56-year - old woman with unconfirmed in utero diethylstilbestrol exposure presented in early 2007 with intermittent spotting of a 4-month duration . a vaginal cystic structure was present for 30 years , requiring intermittent aspiration . on vaginal examination , a smooth , non - tender , globular cystic structure was visualized on the right wall of the vagina measuring about 3 to 4 cm in diameter in the middle third of the vagina . metastatic workup included normal endocervical and endometrial biopsies , colonoscopy , mammography , and chest . 2-[f ] fluoro-2-deoxy - d - glucose positron emission tomography ( fdg - pet ) scan showed a large , slightly irregular , lobulated hypermetabolic mass near the midline centered slightly to the right 3 cm in the area of the middle third of the vagina and metastasis to the left inguinal lymph nodes . computed tomography ( ct ) showed a 6-cm vaginal mass and 2-cm mass in the left inguinal area . mri revealed a 6-cm tumor between the bladder and vagina with no invasion of the bladder mucosa and a left inguinal lymph node measuring 3.3 cm in largest dimension ( fig . the vaginal incisional biopsy measured 0.7 0.3 0.2 cm and was read at an academic medical center as grade 2/3 clear cell and focal papillary serous adenocarcinoma with angiolymphatic involvement. immunohistochemistry staining showed positive ck7 , progesterone receptor , and carcinoembryonic antigen with negative ck20 and estrogen receptor . clinically , her tumor was stage iiic by the ajcc staging system , t2 n1 m0 and stage ii by the figo staging system . discussions were held with the patient regarding all treatment options , including possible acute and long - term serious sequelae . three - dimensional simulation was conducted using a computed tomography simulator ( pet - ct discovery -st ; ge medical systems , waukesha , wisc . target volumes were contoured on a pinnacle system ( phillips healthcare , andover , mass . the patient was treated using 6-mv photons delivered with a 9-field intensity modulated radiotherapy technique to a total dose of 50 gy in 25 fractions to the tumor and to 45 gy in 25 fractions to the inguinal and pelvic lymph nodes ( fig . she then received an electron boost using 12-mev photons using a direct field technique to total dose of 30 gy in 15 fractions to the 90% isodose line to the left inguinal lymph nodes . six cycles of weekly cisplatin at 100 mg / m were administered during external beam radiation . 2b ) . a multiple channel vaginal catheter cylinder ( intracavitary mould applicator ; nucletron inc . , the balloon was inflated with 30 ml of saline with 5% contrast to position the rectum away from the high dose rate sources and to attenuate the radiation to further protect the posterior wall of the vagina and rectum . four catheters and 80 dwelling source positions were employed to deliver 5 gy per treatment session using a hdr unit . the patient experienced a limited amount of side effects during treatment with only grade 2 dermatitis from external beam radiation . complete response was confirmed by fdg - pet and ct scan 6 months after treatment . at 3 , 6 and 18 months , two cases of adenocarcinoma were included in a series of 28 primary vaginal cancers treated with external beam radiotherapy and brachytherapy . frank et al . reported 26 patients with primary vaginal adenocarcinoma treated with external beam radiotherapy and brachytherapy with mostly grade 1 - 2 toxicities . the combination of external beam radiotherapy and cis - platinum was described by samant et al . , concurrent cisplatin appeared to sensitize the tumor to radiation since the tumor responded at a lower dose and at a faster rate than expected . concurrent chemotherapy with irradiation appears to significantly enhance radiation effects on cancer and cycling epithelial cells , but it does not appear to proportionately increase late effects in normal tissues . the present case is unique in that we utilized both chemoradiation and brachytherapy to treat vaginal adenocarcinoma . hdr brachytherapy is a technique that delivers radiation to the tumor bed while minimizing dose inaccuracies from internal organ and patient motion . vaginal hdr brachytherapy has a very low incidence of side effects [ 6 , 7 ] and considering the short treatment time , it appears cost beneficial . side effects from vaginal brachytherapy for cervical cancer include acute mucositis , proctitis , and cystitis , as well as late proctitis and cystitis , telangiectasia , atrophy , fibrosis and stenosis . in this case , the patient experienced only vaginal discharge during and after radiation therapy and grade 2 dermatitis during external - beam treatment . despite the tumor 's proximity to the bladder , the patient did not develop a fistula between the bladder and vagina . in summary , chemoradiation followed by brachytherapy provided adequate dose to the tumor and lymph nodes for complete clinical response while distributing the dose to prevent severe toxicity .

the annotations , sequences , and orthologous relationships of mouse and rat genes were retrieved from ensembl version 53 ( www.ensembl.org ) , whereas those of yeast genes were obtained from the saccharomyces genome database ( www.yeastgenome.org ) . based on go , immune - related proteins have the annotation of go:0002376 ( immune system process ) . essentiality mouse genes were defined based on mouse genome informatics 4.21 ( www.informatics.jax.org ) , following liao and zhang ( 2007 ) . essentialities and expression levels of yeast genes were obtained from zhang and he ( 2005 ) . a zero fitness upon gene deletion is used to define essential genes in both the yeast and mouse . properties of mouse gene expression were defined based on the microarray data of 61 mouse tissues ( su et al . expression level was calculated by averaging expression signals in the 61 tissues , whereas tissue - specificity ( ) , which ranges from 0 to 1 ( higher values indicate stronger tissue - specificity ) , was calculated according to liao et al . ( 2007 ) , and those for human were compiled from six sources : human protein reference database ( www.hprd.org ) , munich information center for protein sequences ( mips.helmholtz-muenchen.de ) , molecular interaction database ( mint.bio.uniroma2.it ) , reactome ( www.reactome.org ) , intact ( www.ebi.ac.uk ) , and database of interacting proteins ( dip.doe-mbi.ucla.edu ) . the human ortholog 's number of interacting proteins ( kppi ) was used as a proxy for a mouse protein 's kppi . to calculate mammalian or yeast protein evolutionary rate , signal peptides , annotated by spdb ( proline.bic.nus.edu.sg/spdb/ ) , orthologous coding sequences without signal peptides were aligned following the protein alignment by clustalw ( www.ebi.ac.uk/clustalw/ ) . when a gene has multiple isoforms , the longest isoform was used . values of dn and ds between mouse and rat and between s. cerevisiae and s. paradoxus were computed using paml 4 ( yang 2007 ) . supplementary figures s1s4 and supplementary tables 1 and 2 are available at genome biology and evolution online ( http://www.oxfordjournals.org/our_journals/gbe/ ) .

stroke is a leading cause of long - term disability , and the absolute number of patients with stroke is increasing . of the neurological sequelae that cause functional disability , the incidence of gait disturbance due to hemiparesis is reportedly relatively high among chronic stroke patients1 , 2 . the occurrence of this disability leads to marked impairment of quality of life and the sense of well - being3 , 4 . in addition , the burden of caregivers is anticipated to increase when patients are in need of assistance for walking5 . improved walking ability is one of the most common goals for post - stroke hemiparetic patients6 , 7 . a slant board has been used as a therapeutic device for patients with spastic lower hemiparesis . standing on the toe - up inclination surface stretches the calf muscles , which reduces the pathologically increased lower limb muscle tone in post - stroke hemiparetic patients . standing without a back rest on the slant board can produce a forward shift in the center of pressure in healthy adults and hemiparetic patients , as reported in our previous study . kluzak reported that standing on an inclined surface resulted in an after - effect of learning in healthy , blindfolded subjects when they returned to standing on a horizontal surface8 . recent reports described other benefits of using the board in healthy adults and hemiparetic patients , such as movement of center of pressure9 , and possible increase in the maximum range of center of gravity in the antero - posterior direction10 . neurophysiological studies have also demonstrated increased contraction of the anterior tibialis muscle in healthy subjects standing on the slant board11 . daily home - based rehabilitation using the slant board was hypothesized to improve gait in post - stroke hemiparetic patients . to test the hypothesis , the present study investigated the effects of a 30-day home - based daily rehabilitation program using the slant board on walking function in post - stroke hemiparetic patients . the intervention and study protocol were approved by the ethics committee of jikei university school of medicine ( 24 - 274 7040 ) , and informed consent was obtained from all patients before beginning the study . the following were the inclusion criteria : 1 ) clinical confirmation of gait disturbance ( unable to walk as before the onset of stroke ) and brunnstrom recovery stage for lower limb classification of 35 ; 2 ) ability to walk without the use of canes or orthoses ; 3 ) more than six month after stroke onset ; 4 ) history of a single symptomatic stroke only ( no bilateral cerebrovascular lesions ) . 5 ) independency on daily living and no physical or mental illness requiring medical management ; 6 ) no previous local treatment for lower limb spasticity with motor point block or botulinum toxin injections . table 1table 1.clinical characteristics of the studied patientspatientno.age at intervention ( yrs)gendertime after stroke ( m)stroke subtypehandednessside of hemiparesisbrs for lower limbsbarthel indexrom of the ankle joint ( )mas for plantar flexorsnrs for lower limbs150male9cirightright31003027252male10cirightright31003028366male12ichrightright5100351 + 7465male18ichleftright4100351 + 5559male16ichrightright5100251 + 10660male11ichrightright4100251 + 8ci : cerebral infarction , ich : intracerebral hemorrhage , brs : brunnstrom recovery stage , bi : barthel index , mas : modified ashworth scale , nrs : numeral rating scale , rom : range of motion summarizes the clinical characteristics of the study patients . the time between stroke onset and the intervention ranged from 9 to 18 months . stroke was classified into cerebral infarction in 2 patients and intracerebral hemorrhage in 4 patients . ci : cerebral infarction , ich : intracerebral hemorrhage , brs : brunnstrom recovery stage , bi : barthel index , mas : modified ashworth scale , nrs : numeral rating scale , rom : range of motion after the initial evaluation of walking function , the patient was instructed to perform the home - based training using a slant board . follow - up evaluation was performed 30 days later after the initial evaluation to assess the effect of the training program on walking function . the slant board ( asahi corporation , tokyo , japan ) has a plastic construction and the angle of the slant was set at 20 degrees for all patients ( fig . the home - based rehabilitation program using the slant board was explained in detail to the patients at the beginning of the program . the standing position was maintained with both ankle joints dorsiflexed for 3 minutes . during each session , the patient was instructed to perform two sets of 3-min training using the slant board with > 3 min of rest between the tasks , three times a day ( in the morning , daytime , evening ) and to perform this protocol daily for 30 days . the patients were required to immediately inform the hospital by telephone in case of adverse events . at the end of the 30-day protocol , self - reported questionnaires were used to evaluate whether the patients performed and completed the daily protocol as instructed . for all patients , the brunnstrom recovery stage ( brs ) , barthel index ( bi ) , range of motion ( rom ) in the ankle joint , modified ashworth scale ( mas ) of calf muscle , sensory impairments with numeral rating scale ( nrs ) , maximum walking speed ( mws ) , number of steps and timed up and go test ( tug ) were serially evaluated at the beginning and end of the 30-day protocol . walking velocity is a reliable objective measure of walking function and has been reported to correlate with more sophisticated methods used to assess walking function12 . first , patients walked 10 m at a self - selected comfortable pace . second , they were instructed to walk on the same walkway as fast and as safely as possible . the walking velocity in these trials was recorded as comfortable walking velocity and maximum walking velocity , using a series of motor tasks , the test evaluates balance control , muscle strength , and coordination . the reported reliability of the test is excellent , with high intra - class correlation coefficients . in this test , patients are instructed to stand up from a chair with armrests , walk 3 meters , turn around , return to the chair , and sit down . the time taken to complete this task represented the tug performance time . the time was measured when turning towards the paralytic side as well as towards the non - paralytic side . for all evaluations , two consecutive measurements were taken at each time point and the average value was calculated and recorded . the slant angle was set at 20 degrees data are expressed as mean sd . differences in the applied measures before and after the 30-day protocol were examined for statistical significance using the signed wilcoxon rank sum test for paired samples . all statistical analyses were performed using spss package 17.0 ( spss , chicago , il , usa ) . at least 89% of the scheduled training time was completed by all patients over the 30 days . none of the patients experienced pathological symptoms or any deterioration of walking function during the study period . all patients reported that home - based training was not particularly stressful and the training did not interfere with their activities of daily living . comfortable walking velocity increased significantly from 0.72 0.14 m/ at baseline to 0.83 0.26 m/ after the 30-day program ( p<0.05 ) . similarly , the maximum walking velocity increased significantly from 0.98 0.32 m/ at baseline to 1.25 0.31 m/ after the training ( p<0.05 , table 2table 2.comparison of performance pre- and post - interventionpre - interventionpost - interventioncomfortable waking velocity(m / s)0.72 0.140.83 0.26*(steps)22.3 3.320.4 3.6*maximum waking velocity(m / s)0.98 0.321.25 0.31*(steps)19.5 4.217.1 3.3*tug ( turn of paralysis side)(s)14.0 5.310.2 2.3*tug ( turn of non - paralysis side)(s)14.0 5.610.1 2.2*teps : number of steps during the 10-m walking . the number of steps increased significantly when measured at comfortable walking velocity ( baseline : 22.3 3.3 steps , end of program : 20.4 3.6 steps , p<0.05 ) as well as at maximum walking velocity ( baseline : 19.5 4.2 steps , end of program : 17.1 3.3 steps , p<0.05 , table 2 ) . furthermore , tug performance time decreased significantly in tests involving turning towards the paralytic side ( baseline : 14.0 5.3 seconds , end of program : 10.2 2.3 , p<0.05 ) . in addition , the tug also decreased significantly in tests involving turning towards the non - paralytic side ( baseline : 14.0 5.6 seconds , end of program : 10.1 2.2 , p<0.05 , table 2 ) . this study showed that home - based training using a slant board is safe and significantly improves walking function in post - stroke hemiparetic patients . to our knowledge , this is the first study that reports the beneficial effects of home - based slant board - rehabilitation program for gait improvement for post - stroke patients . in this study , all patients showed improvement in walking velocity , with a decrease in the number of steps to complete the pre - selected distance . previous studies indicated that using the slant board the stretches the spastic calf muscles such as gastrocnemius and soleus muscles11 . however , this mechanism does not seem to be the only factor responsible for the increased walking velocity observed in our study . using electromyographic recording , we previously demonstrated that maintenance of standing position with dorsiflexion of the ankle joint in healthy subjects is associated with increased muscle activity of the tibialis anterior ( ta ) muscle and other anteriorly located leg muscles . the increase in ta muscle activity can produce an anterior shift in the center of gravity . this shift is considered beneficial since it promotes body motion in the anterior direction . furthermore , the increase in ta muscle activity is expected to strengthen hip joint muscles , such as the rectus femoris muscle , gluteus maximus muscle and rectus abdominis muscle11 . considered together , it is likely that these mechanisms contributed to the observed improvement in gait function in this study . so far , the ankle strategy and hip strategy suggested by horak et al . have been accepted widely as important concepts for rehabilitative management of patients with gait disturbances14 . suggest that the hip strategy is used to restore equilibrium when the support surface is smaller than that of the feet14 , 15 . therefore , therapeutic approaches that strengthen the ta muscle seem to be meaningful in clinical settings for stroke rehabilitation , in addition to the above two strategies . this study also confirmed that the use of the slant board improved tug performance time . the tug evaluates balance control ability as well as mobility of the lower limbs13 . the observed improvement in tug performance time suggests that this rehabilitation program improves balance control . although the mechanism underlying this beneficial effect on balance control remains unknown , strengthening of hip joint muscles is speculated to be responsible . furthermore , maintaining standing position on the slant board can be considered static activity , in contrast with the dynamic training in stand - up exercise and treadmill exercise . first , during static training , the therapist can easily assist the patients so that they maintain the standing position . the risk of falling during such training is markedly low , which can encourage the patient to practice daily . these characteristics of static training could have also been involved in the gait function improvement observed in the present study . further studies using kinematic and/or dynamic gait analyses are warranted to further investigate various aspects of the protocol . we confirmed that the board is safe to use for both patients and health professionals . therefore , we recommend the application of this program as a home - based rehabilitation program for post - stroke hemiparetic patients with gait disturbances of different age groups . first , the study involved only a small number of patients and lacked a control group . the finding of this study should be confirmed in another randomized controlled study with a larger sample size . second , the long - term effects of the 30-day training program on walking function remain to be investigated . third , the duration of board use in one session of the rehabilitation program ( 3 minute ) was arbitrarily selected in this study . in addition , whether the improvement in walking speed and tug was unrelated to the measure of the other evaluations needs to be clarified . the optimal duration of the rehabilitation program should be investigated , although the duration in the current protocol seems to have produced satisfactory improvement . the 30-day home - based daily training using the slant board is safe and improved walking function in post - stroke patients with gait disturbances .

the 14 human ascariasis cases were reported by human and animal health care providers to the maine department of health and human services ( dhhs ) or the maine department of agriculture , conservation and forestry . patients with confirmed infection had excreted in stool 1 worm that was subsequently laboratory - identified as ascaris sp . patients with probable infection reported excreting 1 worm in stool and were epidemiologically associated with a confirmed patient . patients with suspected infection were persons with symptoms consistent with larval migration who had been on the same implicated farm as a patient with confirmed infection or persons who excreted 1 worm in stool without laboratory confirmation or association with a patient with confirmed illness . the figure displays geographic information regarding the 14 human cases ( 8 confirmed , 4 probable , and 2 suspected ) from 7 unrelated small - scale farms in 6 counties in maine . workers or residents did not rotate among farms , but all 14 case - patients reported contact with pigs . of the 4 patients with a history of international travel , 2 reported receiving previous treatment for parasites . the table shows the location of farms where infections were found , the laboratory results , and case classifications made during april 2010march 2013 . patients were 153 years of age ( median 25 years ) ; 93% were female . * boldface indicates samples from farm x. cdc , centers for disease control and prevention ; cornell , cornell university animal health diagnostic center ; na , not available . maine dhhs conducted a site visit of farm x. a pooled fecal specimen was collected from 10 pigs . a worm specimen from a person was sent to the centers for disease control and prevention ( cdc ) for identification . pooled pig feces and pig and human worm specimens were sent to private , university , and cdc laboratories for identification . farm x grew and sold organic vegetables and raised conventional and organic livestock , including dairy cows , laying hens , sheep , and pigs . among 12 persons who worked at farm x during the fall of 2012 , a total of 3 ascariasis cases occurred ; all patients had gastrointestinal illness onsets after may 2012 and were treated with albendazole . infection was confirmed , had 3 stool samples reportedly test negative for parasites in february 2012 before returning to united states . infection was reportedly treated for unspecified parasites in december 2011 while abroad . the third patient , who also had probable ascaris spp . infection , reported travel in asia within the previous 2 years but was not previously screened or treated for parasitic infections . in october 2012 , worms were recovered from 5 of 10 pigs from farm x at slaughter . farm x had purchased these pigs as piglets from a local supplier in may 2012 . during july 2012 , these pigs were penned where pigs had been raised 1.5 years previously , in an area 15 feet from active vegetable plots . a mixture of hay used as bedding material for pigs , and pig manure was used as fertilizer for growing vegetables . three hand - washing stations were observed at farm x. at farm x , we detected ascaris eggs in pooled feces by zinc sulfate and sugar flotation testing methods . human and pig worm specimens collected at farm x were confirmed as ascaris spp . at cdc . we were unable to determine if pigs from any of the 7 affected farms had a source in common ( e.g. , common swine stock or breeder ) . we determined that direct or indirect exposure to pigs was the single common factor in all 14 cases of human ascariasis we investigated . at farm x , where detailed information was available regarding pig husbandry , the timing of illness among the farm workers was consistent with acquisition of ascaris infection from pigs . cross - transmission of ascaris infections between humans and pigs likely occurred on these farms . ascariasis occurred at multiple locations in maine where farm workers had no history of travel to parts of the world where transmission occurs , strengthening our position that pigs introduced infection . we believe that ascaris eggs persisted in farm soil for extended periods , which led to ongoing transmission ( 11 ) . laboratory rrna analyses of the pig and human isolates contribute to a growing body of evidence that a. lumbricoides and a. suum are genetically very closely related and might , in fact , be a single species ( 1214 ) . certain farm practices might have contributed to human exposure to ascaris eggs , including , as noted at farm x , use of pig manure as fertilizer , use of pig bedding for compost , and location of pig pens near where produce is grown . none of the 7 farms we investigated managed pigs according to organic farming standards , and pigs were not regularly dewormed . the human infections occurred on maine farms with limited numbers ( < 30 ) of pigs , where farmers might be unaware of their risk for acquiring ascariasis . maine does not have a wild pig population ; therefore , wild pigs were not a potential source of infection or environmental contamination . nematodes include keeping pig pens separate from vegetable fields and avoiding use of pig manure for fertilizer , especially on produce . ideally , farms should have dedicated equipment for handling animal waste and stall cleaning . farm workers should wash hands before and after contact with pigs , pig waste , or soil contaminated with pig waste . because ascaris eggs can remain viable for extended periods in soil , raw produce should be washed thoroughly before consumption . for optimal animal health , pigs should be dewormed before introduction to the farm and should be regularly dewormed , and humans and pigs should be treated concurrently when human cases occur . preventing ascaris infections requires an integrated , onehealth approach that addresses on - farm practices , animal husbandry , and health education efforts .

a 66-year - old woman presented to the emergency department with chest pain , dyspnea and sweating for the previous 4 hours after making a speech in front of a large group of people . the initial electrocardiogram on admission showed t - wave inversion in leads v3 through v6 , and q - wave in leads v4 through v6 ( fig . 1a ) , and this was all newly developed compared to the ecg taken one year previously . the troponine - t , ck - mb and ck levels were slightly elevated to 0.565 ng / ml ( normal range : 0 - 0.1 ng / ml ) , 10.98 ng / ml ( normal range : 0 - 5 ng / ml ) , and 164 iu / l ( normal range : 32 - 135 iu / l ) , respectively . an emergency echocardiography showed a 49% ejection fraction and extensive akinesis from the mid to apical left ventricle , except for the base . an aortogram showed the same findings as echocardiography , that is , akinesis from the mid to apical left ventricle with sparing of the basal segments ( fig . however , a coronary angiography showed no significant stenosis of the epicardial coronary arteries ( fig . cine imaging , the contrast - enhanced first - pass and delayed imaging , and the respiratory compensated coronary angiography with a navigator echo were performed . there was severe hypokinesis from the mid to apical left ventricle with sparing of the basal segments ( fig . the dysfunction of the left ventricle was slightly improved compared to the initial left ventriculogram . coronary mr angiography showed no significant stenosis of the major epicardial coronary arteries ( fig . she completely recovered her ventricular function and from her symptoms after two weeks , the former as determined on follow - up with performing echocardiography , with only supportive treatment . a report from japan demonstrated that incidence of acute cardiovascular events after an earthquake was triple the incidence before the earthquake ( 6 ) . this phenomenon has recently come to be recognized as a reality in clinical medicine and it is reported on as a novel cardiac syndrome . first described this unique syndrome in 1991 in five patients who presented with acute chest pain and electrocardiographic abnormalities that matched the manifestation of acute myocardial infarction , but they had no coronary artery stenosis ( 7 ) . since then , this syndrome has been reported , mostly from japan , by the name of ' tako - tsubo cardiomyopathy ' or ' ampulla cardiomyopathy ' , which reflected the characteristic shape of the left ventriculogram at end - systole that resembled a japanese pot ( tako - tsubo ) with a narrow neck and wide base . the tako - tsubo - like shape results from apical and mid - ventricular akinesis with the basal hyperkinesis , which is not easily explained by any distribution of coronary artery disease ; this has been considered as one of the evidences that this syndrome is a distinct entity that differs from acute myocardial infarction . since this characteristic left ventricular dysfunction resolved in days or weeks for most reported cases , ' transient left ventricular apical ballooning ' has recently been used to name this syndrome and to emphasize the reversibility of the left ventricular dysfunction . the reversible ventricular dysfunction seen in this syndrome can be attributed to myocardial stunning , which is defined as prolonged postischemic left ventricular dysfunction after a short duration of myocardial ischemia . markedly elevated and sustained levels of plasma catecholamines and stress - related neuropeptides that are related to intense stress in most patients with the syndrome have been reported , and they are considered to play a central role between the severe stress and the myocardial stunning in this syndrome ( 3 , 4 ) . however , the mechanism of how catecholamine and stress - related neuroepeptides can induce myocardial stunning is not fully understood . the possible mechanisms that have been suggested to account for the stress - related myocardial stunning include ischemia resulting from vasoconstriction of the coronary arteries that 's due to increased sympathetic tone , sympathetically - mediated microcirculatory dysfunction and catecholamine - induced direct myocyte injury . contraction - band necrosis , which is compatible with catecholamine - induced myocyte damage , was found on myocardial biopsy in some patients with this syndrome ( 3 ) . the universally accepted diagnostic criteria for transient left ventricular apical ballooning are not yet available . a report from the mayo clinic suggested the diagnostic criteria for the syndrome , which should meet all of the following ; 1 ) transient akinesis or dyskinesis of the left ventricular apical and mid - ventricular segments , 2 ) absence of obstructive coronary artery disease or acute plaque rupture , 3 ) new electrocardiographic abnormalities , and 4 ) exclusion of any apparent etiology causing acute myocardial stunning , including head trauma , intracranial or subarachnoid bleeding , pheochromocytoma , obstructive epicardial coronary artery disease , myocarditis and hypertrophic cardiomyopathy ( 8) . even though coronary arterial imaging can reveal no obstructive epicardial coronary artery disease , syndrome x or idiopathic dilated cardiomyopathy should be excluded from the differential diagnosis ; however , transient dysfunction of the left ventricular apical and midventricular segments is not a typical presentation for these entities . a prior stressful event as a precipitating factor or increased catecholamines levels are not a diagnostic requirement , although both are helpful to diagnose the syndrome . in our case , the characteristic tako - tsubo - like left ventricular geometry was demonstrated by cine mri , like was demonstrated by left ventriculography and echocardiography . the absence of obstructive epicardial coronary artery disease was confirmed by respiratory compensated coronary mr angiography , the same was done by x - ray coronary angiography . in addition , contrast - enhanced delayed mri revealed an absence of hyperenhanced myocardial segments , which implicated the absence of acute or chronic myocardial infarction , regardless of coronary artery disease ( 9 ) . although we did not performed stress perfusion mri in the patient , the lack of hypoperfusion on the first - pass imaging at rest suggested there was neither profound compromise of the epicardial flow nor any microvascular obstruction . therefore , mri may not only substitute for x - ray coronary angiography and left ventriculography , but it can also confirm that acute or chronic myocardial infarction is absent and the left ventricular dysfunction is reversible ( 9 ) . the morbidity and mortality related to invasive x - ray coronary angiography can be avoided by using mri for the diagnosis of this syndrome . although the mechanism of transient left ventricular apical ballooning syndrome has still not been fully elucidated , a typical presentation of tako - tsubo - like apical ballooning , the absence of obstructive epicardial coronary artery disease and the reversibility of myocardial dysfunction are very characteristic and these factors can be determined by performing mri .

chchd2 contains a chch domain , which is characterized by a twin cx9c motif ( 2 pairs of cysteines spaced by 9 residues ) . similar to other chch domain - containing proteins , the cx9c motif is responsible for the import of chchd2 into the ims through the mia40-erv-1 system . as it contains an amino - terminal mitochondrial targeting sequence and a transmembrane domain , chchd2 may be embedded in the i m , where the core electron transport chain ( etc ) machinery assembles and functions . like its yeast homolog mic17 and its mammalian paralog chch domain containing 10 ( chchd10 ) , chchd2 promotes mitochondrial oxygen consumption . recent studies have showed that chchd2 is involved in electron transport through 2 mechanisms : ( 1 ) chchd2 translocates to the nucleus and transcriptionally upregulates isoform 2 of cytochrome c oxidase subunit 4 ( cox4i2 ) , and ( 2 ) chchd2 binds to cytochrome c oxidase ( cox ) thereby maintaining cox enzymatic activity . intriguingly , bcl - xl has also been identified in the mitochondrial i m cristae , where it binds to the subunit of atp synthase to prevent wasteful ion flux and thus maintain the membrane potential for optimal atp production . given that chchd2 and bcl - xl interact with one another , co - localize to the i m , and play similar functions in metabolism and apoptosis , one could speculate that the chchd2bcl - xl complex functions as a sensor to probe the status of mitochondrial metabolism and coordinate metabolism with apoptosis . to understand how chchd2 coordinates mitochondrial metabolism with apoptosis , it is important to identify the metabolic cues monitored by chchd2 . as the major by - product of mitochondrial oxidative phosphorylation ( oxphos ) , ros serve as signaling molecules for normal biological processes but also represent a potent damage - inducing agent for cellular proteins and nucleic acids . as a protective strategy these defensive functions are generally considered to be carried out by cellular detoxification enzymes ( catalase , superoxide dismutase , and glutathione peroxidase ) and by the low molecular weight thiol glutathione ( gsh ) or cysteine residues in the active sites of proteins such as thioredoxin and peroxiredoxin ( trx / prx ) . in addition to gsh and the trx / prx proteins , recent evidence suggests that some proteins with thiols exposed on their surface could function to neutralize cellular ros . given that the characteristic feature shared by all chch domain - containing proteins is the twin cx3c or cx9c motif , which provides free thiols under reducing conditions , the many mitochondria - localized chch domain - containing proteins may function as an additional thiol pool to detect and scavenge cellular ros in the ims . as shown in our study , downregulation of chchd2 sensitizes cells to apoptosis induced by a variety of stimuli , including uv irradiation , cisplatin , staurosporine , etoposide , and doxorubicin . although these apoptotic stimuli induce cell death via different mechanisms , they all induce ros , suggesting that chchd2 could function as a ros scavenger . . additionally , oxidation of chchd2 by ros could lead to a conformational change ( our unpublished data ) , which correlates with the ros - dependent translocation of chchd2 from the mitochondria to the nucleus , where chchd2 can transactivate the expression of genes involved in mitochondrial respiration . these data suggest that chchd2 is not only a ros scavenger but also a prosurvival transcription factor that promotes oxidative phosphorylation and compensates for the energy crisis that results from ros - induced dna damage and mitochondrial dysfunction . based on our data showing that the mitochondrial protein chchd2 interacts with bcl - xl to facilitate cell survival , and on previous observations suggesting that chchd2 participates in the metabolic regulation of ros , we propose a model whereby under unstressed conditions chchd2 promotes mitochondrial respiration whereas under oxidative stress chchd2 helps neutralize ros through its chch domain ( fig . 1 ) . therefore , based on our data showing that inhibition of chchd2 can sensitize cancer cells to apoptosis , chchd2 could represent a novel therapeutic target for the treatment of cancers . figure 1.chchd2 promotes cell survival by counteracting ros generation and facilitating oxidative phosphorylation . under normal conditions , chchd2 localizes to the mitochondria and helps maintain normal oxphos flux by binding to cox or bcl - xl . in the presence of oxidative stress , chchd2 becomes oxidized and translocates from the mitochondria to the nucleus where it binds to dna and promotes the expression of cox4i2 , which in turn facilitates oxphos , thereby counteracting ros - induced inhibition of atp production and facilitating survival . abbreviations : bcl - xl , bcl2-like 1 isoform 1 ; chchd2 , coiled - coil - helix - coiled - coil - helix domain containing 2 ; cox , cytochrome c oxidase ; cox4i2 , cytochrome c oxidase subunit 4 , isoform 2 ; oxphos , oxidative phosphorylation ; ros , reactive oxygen species . chchd2 promotes cell survival by counteracting ros generation and facilitating oxidative phosphorylation . under normal conditions , chchd2 localizes to the mitochondria and helps maintain normal oxphos flux by binding to cox or bcl - xl . in the presence of oxidative stress , chchd2 becomes oxidized and translocates from the mitochondria to the nucleus where it binds to dna and promotes the expression of cox4i2 , which in turn facilitates oxphos , thereby counteracting ros - induced inhibition of atp production and facilitating survival . abbreviations : bcl - xl , bcl2-like 1 isoform 1 ; chchd2 , coiled - coil - helix - coiled - coil - helix domain containing 2 ; cox , cytochrome c oxidase ; cox4i2 , cytochrome c oxidase subunit 4 , isoform 2 ; oxphos , oxidative phosphorylation ; ros , reactive oxygen species .

body balance is defined as the organism s ability to maintain the body s position without the aid of another person , excluding uncontrolled falls . in the evaluation of body balance various methods and testing instruments are used , including the observation of oculovestibular reflexes , vestibulospinal reflexes , and tests using posturography . in academic research in general the sensitivity in the behavior of the analyser of balance in various conditions of an external and internal environment has been touched on . far less often is there an evaluation of exercise programs that aim to stimulate the forming of the abilities to maintain the balance carried out . it is known that better body balance allows one to obtain better results in sport . this is linked to the necessity to find methods of stimulation and training programs that would increase this ability . at present we know standard training programs for body balance but we also know that the effectiveness of these programs is not always satisfactory , hence also the global search for new forms of body balance stimulation . recent years have seen new solutions , in which a vibration platform has been utilized allowing for a stimulation of the system of balance . academic reports allow one to speculate on an appropriate program of vibration exercises influencing the shaping of the ability to maintain body balance . the vibration platform creates muscle - nerve stimulation through mechanical vibration at an appropriate frequency ( hz ) and amplitude ( mm ) . the vibration brought about by this device causes a natural contraction of the muscles in all the parts of the body involved in the training program . the use of mechanical vibrations in training leads to an increase in the gravitational burdening for the muscles . in addition , this leads to the occurrence of reflex tension of the muscle in reaction to vibration . the vibrations that are the consequence of vibration training influence , by means of proprioceptors , the stimulation of the circumferential and central nerve system . at the same time it leads to a weakening in the mechanism of braking caused by the antagonistic muscles . the alternating mechanism of arousal and braking leads , as a consequence , to the generation of greater values of muscle strength . testing the effectiveness of the action of the vibration platform amongst competitive professionals and amateurs in a vertical jump test showed an improvement in the value of the maximum strength of the skeletal muscles . issurin has observed that as a result of standard training the increase in the value of maximum strength of the skeletal muscles was 16% , while as a result of applying a vibration platform the increase in the strength value was 50% . in addition , he observed an improvement in the mobility of joints as well as a change in the composition and mass of the body . there were also noted metabolic changes in the form of a reduction in the uptake of oxygen by 50% in the test of maximum physical exertion ( the so - called test until refusal to continue the effort ) . in addition , there was an increase in the volume of blood within the working muscles , and even changes in the concentration of testosterone and the growth hormone . the aim of the research was an evaluation of 2 training programs covering the same standard physical activity in the initial stage ( warm - up ) and the main ( motor exercises ) as well as a separate end part in program a of stretching and in program b of vibration training designed to improve the level of body balance . the tests were administered to 40 women ( randomly chosen female students of the academy of physical education and sport in gdansk ) . they were divided into 2 equal 20-person groups : control group ( c ) and experimental group ( e ) . this takes into consideration the age of the participants , the body mass , the bmi indicator [ kg / m ] , and the rohrer indicator [ g / m ] . the students in each group were of a similar average age and body height : for the students from group c the average value of this feature was 167.205.4 cm , while for students from group e it was 168.894.91 cm . the body mass of the students from group c formed on the mean level 57.149.14 kg , while in the group e it was 55.79.16 kg . we found a similar dependence in the bmi indicator , where its average value in students from the c group did not exceed 21.682.36 kg / m2 , while for students from group e it was 20.612.11 kg / m2 . a statistical analysis of the results obtained for age , height , and body mass show that there is an absence of significant differences between group c and e , which means that the tested groups are homogeneous in this respect . the impact of the programed motor exercises on the level of the female student body balance was evaluated in a carefully programed experiment involving an 8-week training program , 3 times a week , for 60 minutes according to the 2 programmes a and b. these programs had a joint introductory and main part though with a different finish . the initial stage lasted 15 minutes and entailed a warm - up preparing the participant for the increasing main part , which lasted 30 minutes and covered balance exercises , exercises shaping the muscle strength of the lower limbs , the abductors and adductors of the lower limbs , and exercises shaping the stomach muscles and those of the extensors and flexors of the shoulders in supporting exercises . the end part lasting 15 minutes involved a standard program : a ) stretching and in the experimental programme b ) exercises on a vibration platform . the frequency of the vibrations was increased every 2 weeks , starting from 5 hz ( for the first 2 weeks ) to 20 hz ( for the final 2 weeks ) . such a regulation was dictated by the safety and effectiveness of the exercises carried out with limited scopes of the parameters above . the intensity of the motor exercises was directed by means of music utilizing a beat per minute ( bpm ) device . in the initial stage 130140 bpm was used , while in the main part 130150 bpm was used . in the end students from group c exercised according to program a , while those from group e according to program b. evaluation of the ability to maintain body balance was carried out by means of tests : a stabilographic tests with posture - graphical method in its basic form with eyes open in which the field of developed surface is defined ( mm ) ; after completing 7 turns in the longitudinal axis of the body in conditions of having the eyes open . the examined person had to stand on only the right leg and maintain balance for the longest possible period of time . the tests were conducted 3 times : the first test was carried out prior the experiment , the second after a month , and the third test after 2 months of the experiment s duration . to verify the significance of the differences between the groups c and e , in which various forms of programmed exercises were employed , the assumptions of a model on the uniformity of variants and configurations were verified by means of the tests of cochran et al . . the effect of repeated measurements was given the name training * and related to their change in state prior to the commencement of the experiment , to the states after 1 and 2 months of its duration . in table 2 are presented the mean values and standard deviation of the developed surface [ mm ] in the test with open eyes in the test group categories with regard to various methods of motor training . analysis of the variants of the anova system of repeated measurements for the field of changed developed surface [ mm ] showed the existence of a statistically significant effect of training in relation to both groups ( table 3 ) . the interesting effect from the point of view of the aim of this work is that the interaction of the type of training applied in relation to the various research groups ( figure 1 ) did not turn out to be statistically significant ( p=0.5154 ) . in table 4 are presented the mean values of the variable field of developed surface [ mm ] in the successive tests in the categories of affiliation to the research test groups . in the column ( 6 ) are given the values of the testing function ( f ) and the tested probability ( p ) for the planned comparisons of the field of developed surface [ mm ] between the group c and the group e in the successive tests that occurred . it was found that the group c and the group e do not significantly differ in terms of the values of the field of developed surface in test 1 preceding the commencement of the training cycle . the comparison of the group after a month of training ( test 2 ) equally did not show significant differences in the values of the field of developed surface between group c and the group e. requiring analysis is the fact of a statistically significant regression ( p=0.0030 ) in the results between the first and second stage of the testing both in the control group as in the experimental group . the testing in the third test again did not show significant differences between the group c and the group e for the end of the training cycle ; but the fall in the value of the field of developed surface in the experimental group between tests 2 and 3 ( p=0.0055 ) was significant . the mean values and standard deviation of the field of developed surface [ mm ] in a test following 7 turns in the categories of the tested groups in relation to the various methods of motor training employed are presented in table 5 . analysis of the variance of the anova system of repeated measurements for the varied developed field surface [ mm ] in a test following 7 turns showed the existence of a statistically significant effect of the training concerning both of the test groups are presented in table 6 . the interaction of the applied form of training with regard to the various test groups ( figure 2 ) did not turn out to be statistically significant ( p=0.1163 ) . table 7 presents the mean values of the variable field of developed surface [ mm ] in the test after turns in the successive tests in the categories of affiliation to test groups . column ( 6 ) gives the values of the testing function ( f ) and the test probability ( p ) for the planned comparisons of the field of developed surface [ mm ] in the test after turns between the control and experimental groups in the successively conducted tests . the results of the analysis showed that the group c and the group e did not differ significantly in relation to the value of the field of developed surface after turns in the first test that preceding the commencement of the training cycle . test 2 equally did not display significant differences in the values of the field of developed surface between the group c and the group e. we can not therefore talk of the appearance of an experiment effect , although the actual increase in the value of the field of developed surface in the control group between tests 1 and 2 turned out to be statistically significant ( p=0.0076 ) . test 3 again did not show significant differences between the group c and the group e at the end of the training cycle . however , the fall in the value of the field of developed surface in the control group noted between tests 2 and 3 ( p=0.0005 ) was significant . in table 8 are presented the mean values and the standard deviation for the time balance was maintained on the right lower limb in the closed eyes test in the test group categories relating to the application of various methods of motor training . analysis of the anova system variance of repeated measurements for the variable time maintained on the lower right extremity in the eyes closed test showed the existence of a statistically significant effect of the training for both test groups ( table 8) . of interest from the view point of the aim of this work is that the effect of interaction of the applied form of training in relation to the various test groups ( figure 3 ) did not turn out to be statistically significant ( p=0.4817 ) . in table 9 are presented the mean values of the variable time maintained on the lower right extremity [ s ] in the closed eyes test in the successive tests in the categories of affiliation to the test groups . column ( 6 ) gives the values of the testing function ( f ) and the test probability ( p ) for the planned comparisons between the group c and the group e in the successive tests . the results of the analysis showed that the group c and the group e did not differ significantly in relation to the time maintained on the lower right extremity in test 1 , which preceded the commencement of the training cycle . test 2 equally did not show significant differences in the values of time maintained on the lower right extremity between the group c and the group e. we can not therefore speak of the appearance of an experiment effect , although there was noted a significant increase in the time value maintained on the lower right extremity between tests 1 and 2 both in the control group ( p=0.0002 ) , as equally in the experimental group ( p=0.0034 ) . the analysis in test 3 again did not show any significant differences between the group c and the group e at the end of the training cycle ; however , there was found a significant increase in the values of time maintained on the lower right extremity but only in the experimental group and this being between tests 2 and 3 ( p=0.0213 ) . the impact of the programed motor exercises on the level of the female student body balance was evaluated in a carefully programed experiment involving an 8-week training program , 3 times a week , for 60 minutes according to the 2 programmes a and b. these programs had a joint introductory and main part though with a different finish . the initial stage lasted 15 minutes and entailed a warm - up preparing the participant for the increasing main part , which lasted 30 minutes and covered balance exercises , exercises shaping the muscle strength of the lower limbs , the abductors and adductors of the lower limbs , and exercises shaping the stomach muscles and those of the extensors and flexors of the shoulders in supporting exercises . the end part lasting 15 minutes involved a standard program : a ) stretching and in the experimental programme b ) exercises on a vibration platform . the frequency of the vibrations was increased every 2 weeks , starting from 5 hz ( for the first 2 weeks ) to 20 hz ( for the final 2 weeks ) . such a regulation was dictated by the safety and effectiveness of the exercises carried out with limited scopes of the parameters above . the intensity of the motor exercises was directed by means of music utilizing a beat per minute ( bpm ) device . in the initial stage 130140 bpm was used , while in the main part 130150 bpm was used . in the end students from group c exercised according to program a , while those from group e according to program b. evaluation of the ability to maintain body balance was carried out by means of tests : a stabilographic tests with posture - graphical method in its basic form with eyes open in which the field of developed surface is defined ( mm ) ; after completing 7 turns in the longitudinal axis of the body in conditions of having the eyes open . the examined person had to stand on only the right leg and maintain balance for the longest possible period of time . the tests were conducted 3 times : the first test was carried out prior the experiment , the second after a month , and the third test after 2 months of the experiment s duration . to verify the significance of the differences between the groups c and e , in which various forms of programmed exercises were employed , the assumptions of a model on the uniformity of variants and configurations were verified by means of the tests of cochran et al . . the effect of repeated measurements was given the name training * and related to their change in state prior to the commencement of the experiment , to the states after 1 and 2 months of its duration . in table 2 are presented the mean values and standard deviation of the developed surface [ mm ] in the test with open eyes in the test group categories with regard to various methods of motor training . analysis of the variants of the anova system of repeated measurements for the field of changed developed surface [ mm ] showed the existence of a statistically significant effect of training in relation to both groups ( table 3 ) . the interesting effect from the point of view of the aim of this work is that the interaction of the type of training applied in relation to the various research groups ( figure 1 ) did not turn out to be statistically significant ( p=0.5154 ) . in table 4 are presented the mean values of the variable field of developed surface [ mm ] in the successive tests in the categories of affiliation to the research test groups . in the column ( 6 ) are given the values of the testing function ( f ) and the tested probability ( p ) for the planned comparisons of the field of developed surface [ mm ] between the group c and the group e in the successive tests that occurred . it was found that the group c and the group e do not significantly differ in terms of the values of the field of developed surface in test 1 preceding the commencement of the training cycle . the comparison of the group after a month of training ( test 2 ) equally did not show significant differences in the values of the field of developed surface between group c and the group e. requiring analysis is the fact of a statistically significant regression ( p=0.0030 ) in the results between the first and second stage of the testing both in the control group as in the experimental group . the testing in the third test again did not show significant differences between the group c and the group e for the end of the training cycle ; but the fall in the value of the field of developed surface in the experimental group between tests 2 and 3 ( p=0.0055 ) was significant . the mean values and standard deviation of the field of developed surface [ mm ] in a test following 7 turns in the categories of the tested groups in relation to the various methods of motor training employed are presented in table 5 . analysis of the variance of the anova system of repeated measurements for the varied developed field surface [ mm ] in a test following 7 turns showed the existence of a statistically significant effect of the training concerning both of the test groups are presented in table 6 . the interaction of the applied form of training with regard to the various test groups ( figure 2 ) did not turn out to be statistically significant ( p=0.1163 ) . table 7 presents the mean values of the variable field of developed surface [ mm ] in the test after turns in the successive tests in the categories of affiliation to test groups . column ( 6 ) gives the values of the testing function ( f ) and the test probability ( p ) for the planned comparisons of the field of developed surface [ mm ] in the test after turns between the control and experimental groups in the successively conducted tests . the results of the analysis showed that the group c and the group e did not differ significantly in relation to the value of the field of developed surface after turns in the first test that preceding the commencement of the training cycle . test 2 equally did not display significant differences in the values of the field of developed surface between the group c and the group e. we can not therefore talk of the appearance of an experiment effect , although the actual increase in the value of the field of developed surface in the control group between tests 1 and 2 turned out to be statistically significant ( p=0.0076 ) . test 3 again did not show significant differences between the group c and the group e at the end of the training cycle . however , the fall in the value of the field of developed surface in the control group noted between tests 2 and 3 ( p=0.0005 ) was significant . in table 8 are presented the mean values and the standard deviation for the time balance was maintained on the right lower limb in the closed eyes test in the test group categories relating to the application of various methods of motor training . analysis of the anova system variance of repeated measurements for the variable time maintained on the lower right extremity in the eyes closed test showed the existence of a statistically significant effect of the training for both test groups ( table 8) . of interest from the view point of the aim of this work is that the effect of interaction of the applied form of training in relation to the various test groups ( figure 3 ) did not turn out to be statistically significant ( p=0.4817 ) . in table 9 are presented the mean values of the variable time maintained on the lower right extremity [ s ] in the closed eyes test in the successive tests in the categories of affiliation to the test groups . column ( 6 ) gives the values of the testing function ( f ) and the test probability ( p ) for the planned comparisons between the group c and the group e in the successive tests . the results of the analysis showed that the group c and the group e did not differ significantly in relation to the time maintained on the lower right extremity in test 1 , which preceded the commencement of the training cycle . test 2 equally did not show significant differences in the values of time maintained on the lower right extremity between the group c and the group e. we can not therefore speak of the appearance of an experiment effect , although there was noted a significant increase in the time value maintained on the lower right extremity between tests 1 and 2 both in the control group ( p=0.0002 ) , as equally in the experimental group ( p=0.0034 ) . the analysis in test 3 again did not show any significant differences between the group c and the group e at the end of the training cycle ; however , there was found a significant increase in the values of time maintained on the lower right extremity but only in the experimental group and this being between tests 2 and 3 ( p=0.0213 ) . in table 2 are presented the mean values and standard deviation of the developed surface [ mm ] in the test with open eyes in the test group categories with regard to various methods of motor training . analysis of the variants of the anova system of repeated measurements for the field of changed developed surface [ mm ] showed the existence of a statistically significant effect of training in relation to both groups ( table 3 ) . the interesting effect from the point of view of the aim of this work is that the interaction of the type of training applied in relation to the various research groups ( figure 1 ) did not turn out to be statistically significant ( p=0.5154 ) . in table 4 are presented the mean values of the variable field of developed surface [ mm ] in the successive tests in the categories of affiliation to the research test groups . in the column ( 6 ) are given the values of the testing function ( f ) and the tested probability ( p ) for the planned comparisons of the field of developed surface [ mm ] between the group c and the group e in the successive tests that occurred . it was found that the group c and the group e do not significantly differ in terms of the values of the field of developed surface in test 1 preceding the commencement of the training cycle . the comparison of the group after a month of training ( test 2 ) equally did not show significant differences in the values of the field of developed surface between group c and the group e. requiring analysis is the fact of a statistically significant regression ( p=0.0030 ) in the results between the first and second stage of the testing both in the control group as in the experimental group . the testing in the third test again did not show significant differences between the group c and the group e for the end of the training cycle ; but the fall in the value of the field of developed surface in the experimental group between tests 2 and 3 ( p=0.0055 ) was significant . the mean values and standard deviation of the field of developed surface [ mm ] in a test following 7 turns in the categories of the tested groups in relation to the various methods of motor training employed are presented in table 5 . analysis of the variance of the anova system of repeated measurements for the varied developed field surface [ mm ] in a test following 7 turns showed the existence of a statistically significant effect of the training concerning both of the test groups are presented in table 6 . the interaction of the applied form of training with regard to the various test groups ( figure 2 ) did not turn out to be statistically significant ( p=0.1163 ) . table 7 presents the mean values of the variable field of developed surface [ mm ] in the test after turns in the successive tests in the categories of affiliation to test groups . column ( 6 ) gives the values of the testing function ( f ) and the test probability ( p ) for the planned comparisons of the field of developed surface [ mm ] in the test after turns between the control and experimental groups in the successively conducted tests . the results of the analysis showed that the group c and the group e did not differ significantly in relation to the value of the field of developed surface after turns in the first test that preceding the commencement of the training cycle . test 2 equally did not display significant differences in the values of the field of developed surface between the group c and the group e. we can not therefore talk of the appearance of an experiment effect , although the actual increase in the value of the field of developed surface in the control group between tests 1 and 2 turned out to be statistically significant ( p=0.0076 ) . test 3 again did not show significant differences between the group c and the group e at the end of the training cycle . however , the fall in the value of the field of developed surface in the control group noted between tests 2 and 3 ( p=0.0005 ) was significant . in table 8 are presented the mean values and the standard deviation for the time balance was maintained on the right lower limb in the closed eyes test in the test group categories relating to the application of various methods of motor training . analysis of the anova system variance of repeated measurements for the variable time maintained on the lower right extremity in the eyes closed test showed the existence of a statistically significant effect of the training for both test groups ( table 8) . of interest from the view point of the aim of this work is that the effect of interaction of the applied form of training in relation to the various test groups ( figure 3 ) did not turn out to be statistically significant ( p=0.4817 ) . in table 9 are presented the mean values of the variable time maintained on the lower right extremity [ s ] in the closed eyes test in the successive tests in the categories of affiliation to the test groups . column ( 6 ) gives the values of the testing function ( f ) and the test probability ( p ) for the planned comparisons between the group c and the group e in the successive tests . the results of the analysis showed that the group c and the group e did not differ significantly in relation to the time maintained on the lower right extremity in test 1 , which preceded the commencement of the training cycle . test 2 equally did not show significant differences in the values of time maintained on the lower right extremity between the group c and the group e. we can not therefore speak of the appearance of an experiment effect , although there was noted a significant increase in the time value maintained on the lower right extremity between tests 1 and 2 both in the control group ( p=0.0002 ) , as equally in the experimental group ( p=0.0034 ) . the analysis in test 3 again did not show any significant differences between the group c and the group e at the end of the training cycle ; however , there was found a significant increase in the values of time maintained on the lower right extremity but only in the experimental group and this being between tests 2 and 3 ( p=0.0213 ) . body balance , as has been shown by many academics worldwide , is a genetically conditioned ability , which may be developed particularly during the early stages of biological development . during the later stage the development of this ability is inherently hampered by the fact that the brain already is in possession of a completely formed and appropriately however , the level of these abilities , including that of balance , may be unsatisfactory for those who engage in sports . as a result there exists the need to develop a set of programed exercises to aid development or maintain these abilities along with the possibility for a further verification of the effectiveness of training . we have evaluated in our experiment the effectiveness of a sports training program in which there has been utilized a vibration platform emitting mechanical vibrations to the whole body or parts of it . in the subject literature there exist reports and studies that indicate the time a stimulus acts as well as the frequency of these vibrations may bring about various effects . a lot of discussion has also been devoted to the question of the impact of vibrations on the development of motor abilities . some authors emphasize the absence of an influence on the part of vibrations on the shaping of muscle strength and also force directed into a vertical jump . certain authors did not observe significant changes following the application of vibration exercises in relation to body balance . the phenomenon of fatigue impact through mechanical vibrations is emphasized and even a connecting of the effectiveness of vibration exercises with the duration of their application . this is also maintained by the results obtained in our experiment , where the application of a short vibration training time duration did not result in statistically significant changes in the level of body balance amongst the female students . there equally exist works where the effectiveness of vibration exercises in effective body balance is linked to the frequency of these vibrations . damjan has claimed that under the influence of applied vibrations of a low frequency there is observed a significant change in the nerve - muscle coordination and that of static balance . the presented effects are often identified with changes occurring in the muscle structure , as well as the bone , but first and foremost these concern the broad spectrum of nerve - muscle conductivity and the higher structures of the nervous system [ 2729 ] . an evaluation of the training effects measured by the manifested level of ability to maintain balance is also not unequivocal in meaning , the symptoms of which may be discerned in the present work ( figure 4 ) . the changes noted after the elapse of the first 4-week period in the experiment and control group were deterioration in the results characterizing an increase in the mean field of the centre of body mass deflections . the fact of the negative impact of a programme of physical activity in the standard model a realized in the control group and in particular in the experimental model b realized in the experimental group on the system of the body balance of exercising female students is undoubtedly something in need of further observation . after a subsequent 4 weeks of the experiment the evaluation of effectiveness for the body balance system , in the test under static conditions , brought a different characterization of the effects than the evaluation in the test following kinetic arousal . the mean measurement value for the experimental group gained the initial level , while for the control group there was noted a subsequent lowering in the maintenance of the body balance level . a different direction in relation to training effects may result from positive stimulation by means of exercises on a vibration platform , accelerating the eradication of fatigue processes . however , the test after kinetic arousal allowed one to see in both groups a heightened level of ability for the balance system . it is also important to note in this test an almost constant measurement value for 75% of those tested in the experimental group ( i test q3q1=1171 mm ; ii test q3q1=1122 mm ; iii test q3q1=1001 mm ) and significant changes in the control group , which again may point to the manifestation of fatigue effects ( i test q3q1=885 mm ; ii test q3q1=2288 mm ; iii test q3q1=1122 mm ) . the absence of unequivocal tendencies for the changes to end at a level of balance ability after 8 weeks of the experiment raise the question as to whether it should not be continued until the training effects have been established . simultaneously , it seems necessary to conduct the evaluation of the impact of mechanical vibrations on the ability to maintain body balance with consideration of the time used for stimulation in the training process , the time duration of a single stimulus , and the frequency of the vibrations . a sizeable part of the experiments conducted are based on the testing of the effects of regaining the ability to maintain body balance , following multi - dimensional vibration stimulation to the whole body , for individuals with illness syndromes handicapping the ability to maintain stability and locomotion [ 4,3032 ] . the results obtained by us in the testing of healthy female students of the academy of physical education and sport may be used as a model for the testing and training of the above - mentioned individuals suffering from these disease syndromes . a sizeable part of the experiments conducted are based on the testing of the effects of regaining the ability to maintain body balance , following multi - dimensional vibration stimulation to the whole body , for individuals with illness syndromes handicapping the ability to maintain stability and locomotion [ 4,3032 ] . the results obtained by us in the testing of healthy female students of the academy of physical education and sport may be used as a model for the testing and training of the above - mentioned individuals suffering from these disease syndromes . it has been shown that the obtained results in relation to body balance by students from the group c in which program a was administered together with stretching displayed only a slightly worse set of results than those obtained by the examined students from group e in which program b was implemented together with exercises on a vibration platform . therefore , we were unable to confirm the hypothesis of the effectiveness of a vibration platform with regard to the ability to maintain body balance in female students studying physical education . however , there exists a significant difference in the testing of maintaining balance on a single lower limb in the group of female students training under the program b when compared to those following program a.

the term metabolic syndrome ( mets ) has been used to describe the combination and co - occurrence of abdominal obesity , insulin resistance , impaired glucose tolerance , hypertension , and dyslipidemia ( increased low - density lipoprotein / high - density lipoprotein [ ldl / hdl ] and triglycerides ) . these cardiovascular risk factors interact with each other and accelerate the progression of atherosclerosis and arterial thrombosis.13 although mets is considered to represent heterogeneous traits , insulin resistance and glucose intolerance associated with obesity are considered to be central to the pathophysiology . mets depends on genetic predisposition , as well as environmental conditions that allow its progression . an abundant high - calorie , high - fat diet , and sedentary lifestyle are considered central to the development of mets in individuals at risk . estimates suggest that approximately 24% of americans suffer from mets as defined by adult treatment panel iii ( atp iii ) guidelines.4 the onset of mets and further progression to type 2 diabetes is associated with findings of endothelial dysfunction , increased platelet activation , and platelet hyperreactivity.1,59 the ossabaw miniature swine has recently been identified as a large animal model for the study of the pathogenesis of mets.10,11 ossabaw swine , from ossabaw island off the coast of georgia , exhibit a thrifty genotype , which allows them to store large amounts of fat for survival during seasonal famine accentuated by the isolated location of the island . when fed an excess calorie atherogenic diet over several months , ossabaw swine develop all the pathological aspects of mets , including hypertension , dyslipidemia , glucose intolerance , endothelial dysfunction , steatohepatitis , as well as atherosclerosis in the coronary vasculature.1020 so far no study has examined the effects of mets in ossabaw swine on platelet aggregation . we hypothesized that ossabaw swine with mets exhibit a pro - aggregatory phenotype evidenced by increased reactivity of platelets to agonists compared with lean pigs without mets . all experimental procedures involving animals were approved by the indiana university animal care and use committee and complied fully with the guide for the care and use of laboratory animals and the american veterinary medical association panel on euthanasia.21,22 for all surgical procedures and euthanasia , anesthesia was induced by tiletamine - zolazepam ( 5 mg / kg ) and xylazine ( 2.2 mg / kg ) given intramuscularly ; anesthesia was maintained with isoflurane gas ( < 4% ) . ossabaw miniature swine ( sus scrofa ) were provided from the colony in the comparative medicine program of indiana university school of medicine and purdue university . swine aged 6 to 7 months at the beginning of the study were fed for 44 weeks either a lean standard chow of 2400 kcal / day containing 8% fat ( lean control group , n = 10 ) or an excess calorie atherogenic diet of 6000 kcal / day containing 45% fat ( mets group , n = 10).17 the details of this pig model , including the composition of the atherogenic diet ( 5b4l ; purina testdiet , richmond , in ) and lean standard chows ( 5l80 ; purina testdiet ) have been published previously.17 in this model , total cholesterol increased about 5-fold and triglycerides about 3-fold . blood pressure was measured with a tail cuff sphygmomanometer in conscious swine in a low - stress restraint sling.10,11 swine were acclimatized to low - stress restraint in a specialized sling for 5 to 7 days before the intravenous glucose tolerance test ( ivgtt).10,11 swine were then fasted overnight and after inducing anesthesia , the right jugular vein was catheterized percutaneously.10,11 as previously described and after collecting fasting blood samples , pigs were administered an intravenous bolus of 1 g glucose / kg body weight , and blood samples were obtained at 5 , 10 , 20 , 30 , 40 , 50 , and 60 minutes after injection.10,11 blood glucose was measured using a ysi 2300 stat plus analyzer ( ysi , yellowsprings , oh ) . plasma insulin levels were determined as previously described.11 the area under the curve ( auc ) was calculated with the baseline at 0 . fasting plasma samples obtained the day of the ivgtt from the jugular catheter were analyzed for triglycerides , total cholesterol and hdl fraction by using a standard enzymatic kit ( thermo trace , melbourne , australia ) as previously described.11 ldl was calculated using the friedewald equation ( ldl = total cholesterol hdl intravascular ultrasound ( ivus ) study of both left anterior descending and circumflex coronary arteries using a 30 or 40 mhz ivus imaging catheter ( ultracross 3.2 or discovery ; boston scientific , natick , ma ) was performed as previously described.1013,15,16,18 atheroma was defined as the presence of soft or fibrous plaque adjacent to the lumen and separated from the adventitia by an echolucent medial layer as previously described.1013,15,16,18 all segments were analyzed at 1-mm intervals along the length of the arteries . percent degrees atheroma ( % circumferential wall coverage ) was calculated and compared between groups . whole blood ( 6070 ml ) was collected from large bore central catheters into vacutainers containing 3.2% citrate prior to administration of heparin or contrast material and was used immediately for platelet experiments . platelet - rich plasma ( prp ) was obtained from citrate blood after centrifugation at 120 g for 5 minutes . after recovering the prp , the blood samples were subjected to further centrifugation at 850 g for 10 minutes to recover platelet - poor plasma ( ppp ) . the resulting prp and ppp were kept at room temperature for use within 1 hour . platelets were stimulated at 37c with adenosine diphosphate ( adp ) at concentrations of 0.5 , 1 , 2.5 , 5 , 10 , and 20 m , collagen at 1 g / ml and arachidonic acid ( aa ) at 0.5 mm ( chrono - log corporation , havertown , pa ) as previously described.23 thrombin was chosen over thrombin receptor agonist peptide ( trap ) as agonist due to inability of trap to induce consistent aggregation of porcine platelets by activation of platelet protease activated receptor-1 ( par1).24 platelets for thrombin experiments were washed by further centrifugation of prp at 850 g for 10 minutes to remove plasma and reduce fibrin formation induced by thrombin during platelet aggregation . the supernatant was discarded and the remaining pellet resuspended in hepes buffer ( sigma - aldrich , st . aggregation of washed platelet suspension was measured after stimulation with thrombin ( chrono - log corporation ) at concentrations of 0.01 , 0.05 , 0.1 , and 0.5 u / ml . aggregation measurements were performed with a chronolog lumi - aggregometer ( model-700 ; chrono - log corporation ) with the aggrolink software package as described previously.23 platelet aggregation was expressed as the maximal percent change in light transmittance from baseline ( mpa ) or as auc within 8 minutes , using animal specific ppp or hepes buffer as a reference . dose response curves and half maximal effective concentrations ( ec50 ) were calculated for each animal using single ligand regression analysis for the different agonist concentrations of thrombin and adp . all statistical analyses were performed using sigma plot ( version 11 ; systat software , san jose , ca ) and spss statistics ( version 18 ; spss inc . , somers , ny ) . as appropriate , t - tests were used to compare platelet aggregation between groups and means of ec50 , with p < 0.05 considered significant . unless specified otherwise , values are represented as means standard deviation ( sd ) . ossabaw miniature swine ( sus scrofa ) were provided from the colony in the comparative medicine program of indiana university school of medicine and purdue university . swine aged 6 to 7 months at the beginning of the study were fed for 44 weeks either a lean standard chow of 2400 kcal / day containing 8% fat ( lean control group , n = 10 ) or an excess calorie atherogenic diet of 6000 kcal / day containing 45% fat ( mets group , n = 10).17 the details of this pig model , including the composition of the atherogenic diet ( 5b4l ; purina testdiet , richmond , in ) and lean standard chows ( 5l80 ; purina testdiet ) have been published previously.17 in this model , total cholesterol increased about 5-fold and triglycerides about 3-fold . blood pressure was measured with a tail cuff sphygmomanometer in conscious swine in a low - stress restraint sling.10,11 swine were acclimatized to low - stress restraint in a specialized sling for 5 to 7 days before the intravenous glucose tolerance test ( ivgtt).10,11 swine were then fasted overnight and after inducing anesthesia , the right jugular vein was catheterized percutaneously.10,11 as previously described and after collecting fasting blood samples , pigs were administered an intravenous bolus of 1 g glucose / kg body weight , and blood samples were obtained at 5 , 10 , 20 , 30 , 40 , 50 , and 60 minutes after injection.10,11 blood glucose was measured using a ysi 2300 stat plus analyzer ( ysi , yellowsprings , oh ) . plasma insulin levels were determined as previously described.11 the area under the curve ( auc ) was calculated with the baseline at 0 . fasting plasma samples obtained the day of the ivgtt from the jugular catheter were analyzed for triglycerides , total cholesterol and hdl fraction by using a standard enzymatic kit ( thermo trace , melbourne , australia ) as previously described.11 ldl was calculated using the friedewald equation ( ldl = total cholesterol hdl ( triglycerides/5 ) ) . intravascular ultrasound ( ivus ) study of both left anterior descending and circumflex coronary arteries using a 30 or 40 mhz ivus imaging catheter ( ultracross 3.2 or discovery ; boston scientific , natick , ma ) was performed as previously described.1013,15,16,18 atheroma was defined as the presence of soft or fibrous plaque adjacent to the lumen and separated from the adventitia by an echolucent medial layer as previously described.1013,15,16,18 all segments were analyzed at 1-mm intervals along the length of the arteries . percent degrees atheroma ( % circumferential wall coverage ) was calculated and compared between groups . whole blood ( 6070 ml ) was collected from large bore central catheters into vacutainers containing 3.2% citrate prior to administration of heparin or contrast material and was used immediately for platelet experiments . platelet - rich plasma ( prp ) was obtained from citrate blood after centrifugation at 120 g for 5 minutes . after recovering the prp , the blood samples were subjected to further centrifugation at 850 g for 10 minutes to recover platelet - poor plasma ( ppp ) . the resulting prp and ppp were kept at room temperature for use within 1 hour . platelets were stimulated at 37c with adenosine diphosphate ( adp ) at concentrations of 0.5 , 1 , 2.5 , 5 , 10 , and 20 m , collagen at 1 g / ml and arachidonic acid ( aa ) at 0.5 mm ( chrono - log corporation , havertown , pa ) as previously described.23 thrombin was chosen over thrombin receptor agonist peptide ( trap ) as agonist due to inability of trap to induce consistent aggregation of porcine platelets by activation of platelet protease activated receptor-1 ( par1).24 platelets for thrombin experiments were washed by further centrifugation of prp at 850 g for 10 minutes to remove plasma and reduce fibrin formation induced by thrombin during platelet aggregation . the supernatant was discarded and the remaining pellet resuspended in hepes buffer ( sigma - aldrich , st . aggregation of washed platelet suspension was measured after stimulation with thrombin ( chrono - log corporation ) at concentrations of 0.01 , 0.05 , 0.1 , and 0.5 u / ml . aggregation measurements were performed with a chronolog lumi - aggregometer ( model-700 ; chrono - log corporation ) with the aggrolink software package as described previously.23 platelet aggregation was expressed as the maximal percent change in light transmittance from baseline ( mpa ) or as auc within 8 minutes , using animal specific ppp or hepes buffer as a reference . dose response curves and half maximal effective concentrations ( ec50 ) were calculated for each animal using single ligand regression analysis for the different agonist concentrations of thrombin and adp . all statistical analyses were performed using sigma plot ( version 11 ; systat software , san jose , ca ) and spss statistics ( version 18 ; spss inc . , somers , ny ) . as appropriate , t - tests were used to compare platelet aggregation between groups and means of ec50 , with p < 0.05 considered significant . unless specified otherwise , values are represented as means standard deviation ( sd ) . swine fed high calorie , high fat / cholesterol / fructose atherogenic diet were considerably more obese than those fed standard chow ( 107.5 8.7 vs 65.8 6.7 kg , p < 0.01 ) and exhibited all the features of mets compared with lean controls ( figure 1 , table 1 ) . both systolic and diastolic hypertension were present in mets pigs compared with lean controls ( 148/95 mmhg vs 126/80 mmhg , p < 0.01 ) . total cholesterol was almost 4-fold higher and triglycerides were roughly 2-fold increased in obese pigs compared with lean controls . obese pigs with mets showed an almost 10-fold increase in ldl levels compared with lean controls with similar concentrations of hdl ( table 1 ) . mets pigs demonstrated insulin resistance as evidenced by higher insulin concentrations and higher glucose concentrations following ivgtt ( table 1 , figure 2 ) . it is noteworthy that animals with mets exhibited significantly higher levels of insulin than leans even before administration of intravenous glucose bolus . intravascular ultrasound showed larger coronary atheroma volume as defined by percent circumferential wall coverage in obese pigs with mets compared with lean controls ( 33 2% vs 25 2% , p = 0.013 ) . an example of a coronary atheroma from a mets animal is depicted in figure 1 . platelet aggregation results for increasing doses of adp ( figure 3 ) and for thrombin as agonists ( figure 4 ) are shown . mets animals showed a trend towards higher maximal platelet aggregation at submaximal concentrations of adp , compared with leans with similar aggregation at maximal concentrations of adp . for both adp and thrombin ec50 overall , platelets from pigs with mets were more sensitive to aggregation by adp than platelets from lean controls with lower ec50 concentration ( ec50 [ mpa ] : 1.83 1.3 m vs 3.64 2.2 m ; p = 0.02 ) ( figure 5 ) . higher velocity of platelet aggregation as defined by the slope of initial aggregation was measured for submaximal concentrations of adp in animals with mets vs lean controls with similar velocity for maximal concentrations of adp ( ec50 [ slope ] : 1.06 0.9 m vs 2.42 1.6 m ; p = 0.02 ) ( figures 3 , 5 ) . there was a nonsignificant trend towards lower ec50 : ( 0.27 0.3 vs 0.36 0.6 u / l ; p = 0.36 ) ( figure 5 ) for aggregation induced by thrombin in platelets from mets pigs compared with lean controls . no significant difference was found for slope of thrombin - induced platelet aggregation between mets and lean controls ( data not shown ) . obese pigs with mets demonstrated higher platelet aggregation in response to collagen than lean pigs ( auc : 286 74 vs 198 123 ; p = 0.037 ) and a trend towards higher platelet aggregation in response to aa ( auc : 260 151 vs 178 145 ; p = 0.13 ) ( figure 4 ) . swine fed high calorie , high fat / cholesterol / fructose atherogenic diet were considerably more obese than those fed standard chow ( 107.5 8.7 vs 65.8 6.7 kg , p < 0.01 ) and exhibited all the features of mets compared with lean controls ( figure 1 , table 1 ) . both systolic and diastolic hypertension were present in mets pigs compared with lean controls ( 148/95 mmhg vs 126/80 mmhg , p < 0.01 ) . total cholesterol was almost 4-fold higher and triglycerides were roughly 2-fold increased in obese pigs compared with lean controls . obese pigs with mets showed an almost 10-fold increase in ldl levels compared with lean controls with similar concentrations of hdl ( table 1 ) . mets pigs demonstrated insulin resistance as evidenced by higher insulin concentrations and higher glucose concentrations following ivgtt ( table 1 , figure 2 ) . it is noteworthy that animals with mets exhibited significantly higher levels of insulin than leans even before administration of intravenous glucose bolus . intravascular ultrasound showed larger coronary atheroma volume as defined by percent circumferential wall coverage in obese pigs with mets compared with lean controls ( 33 2% vs 25 2% , p = 0.013 ) . an example of a coronary atheroma from a mets animal is depicted in figure 1 . platelet aggregation results for increasing doses of adp ( figure 3 ) and for thrombin as agonists ( figure 4 ) are shown . mets animals showed a trend towards higher maximal platelet aggregation at submaximal concentrations of adp , compared with leans with similar aggregation at maximal concentrations of adp . for both adp and thrombin overall , platelets from pigs with mets were more sensitive to aggregation by adp than platelets from lean controls with lower ec50 concentration ( ec50 [ mpa ] : 1.83 1.3 m vs 3.64 2.2 m ; p = 0.02 ) ( figure 5 ) . higher velocity of platelet aggregation as defined by the slope of initial aggregation was measured for submaximal concentrations of adp in animals with mets vs lean controls with similar velocity for maximal concentrations of adp ( ec50 [ slope ] : 1.06 0.9 m vs 2.42 1.6 m ; p = 0.02 ) ( figures 3 , 5 ) . there was a nonsignificant trend towards lower ec50 : ( 0.27 0.3 vs 0.36 0.6 u / l ; p = 0.36 ) ( figure 5 ) for aggregation induced by thrombin in platelets from mets pigs compared with lean controls . no significant difference was found for slope of thrombin - induced platelet aggregation between mets and lean controls ( data not shown ) . obese pigs with mets demonstrated higher platelet aggregation in response to collagen than lean pigs ( auc : 286 74 vs 198 123 ; p = 0.037 ) and a trend towards higher platelet aggregation in response to aa ( auc : 260 151 vs 178 145 ; p = 0.13 ) ( figure 4 ) . obesity and mets are important cardiovascular risk factors involved in the development of atherosclerotic disease . it is well established that insulin resistance and the emergence of diabetes mellitus are associated not only with atherosclerotic plaque formation and progression , but also influence the thrombotic phenotype of affected individuals.1,59 numerous studies have documented that patients with diabetes mellitus exhibit higher levels of activated platelets and are more likely to show inadequate inhibition of platelet aggregation by aspirin or thienopyridines.2527 as platelets become activated they form aggregates with leukocytes through binding by p - selectin and experience cross talk with leukocytes that leads to secretion of tissue factor by leukocytes and generation of thrombin.28,29 platelet leukocyte aggregates that are increased in patients with diabetes mellitus promote rolling and delivery of leukocytes to the endothelium and promote transmigration of leukocytes , and may thus promote vascular inflammation and atherosclerosis formation.1,8,28,29 consistent with these ex vivo findings , patients with coronary disease and diabetes mellitus are at highest risk for recurrent myocardial infarction and stent thrombosis after percutaneous coronary interventions and more likely to be clopidogrel nonresponders than nondiabetic patients.30 several mechanisms have been postulated to play a role in increasing platelet reactivity associated with mets , including direct effects of free fatty acids , adiponectin , and leptin on platelets.1,31,32 a recent study examining the effect of weight loss suggested altered platelet inhibitory effects of nitric oxide and prostacyclin in obese individuals.33 also , previous studies have suggested that insulin resistance may lead to a loss of insulin - mediated inhibition of p2y12 receptor signaling , thereby increasing adp - mediated platelet aggregation.34 platelets from obese ossabaw swine with mets were more sensitive to lower doses of adp as defined by a lower ec50 concentration and showed evidence of faster initial platelet aggregation in response to submaximal concentrations of adp . similarly , higher platelet aggregation was shown in platelets from mets pigs compared with platelets from lean controls when using a fixed concentration of collagen and aa as agonist . it can be deduced , however , that platelets from animals with mets may have a higher tendency for aggregation under shear conditions than platelets from animals without mets . although numerous rodent and small animal models are available for atherosclerosis research , we still lack large animal models that allow study of the progression of atherosclerosis and use of experimental devices in human - sized animals.35 other miniature swine models of mets are limited by lack of hypertension and also coronary atherosclerosis has not been documented.36 the ossabaw swine model has appeared as a viable option for the study of coronary interventional devices and coronary disease in an animal that is close to human size and exhibits the same metabolic changes that are associated with vascular disease in the human population.1018 within our study ossabaw swine who were fed excess calorie atherogenic diet for 44 weeks developed all the features of mets compared with a lean control group , including central abdominal obesity , systolic and diastolic hypertension , hypertriglyceridemia , hypercholesteremia , insulin resistance , impaired glucose tolerance , and coronary atherosclerosis . the effect of type 1 diabetes mellitus on platelet aggregation has been studied in swine models.37 this is the first study to examine the platelet aggregatory phenotype of obese ossabaw swine with mets and insulin resistance compared with non - mets pigs . shukla et al included an atherogenic diet fed group of sinclair miniature swine , who failed to show increased platelet reactivity , although that group was dyslipidemic , similar to the mets ossabaw swine in the current study.37 thus , the increased sensitivity of mets ossabaw platelets to adp and collagen may potentially be due to other factors in mets , such as insulin resistance . clearly , this is a highly distinguishing feature of ossabaw miniature swine compared with sinclair or yucatan swine , which do not show primary insulin resistance when fed excess calorie diet.20,35 this study adds additional information to the phenotypic characterization of the obese ossabaw swine and suggests the presence of a pro - aggregatory environment in ossabaw swine with mets that may be comparable to humans with mets . combined with the finding of spontaneous coronary atherosclerosis in obese ossabaw swine , these data support the use of this model to study intravascular devices in a metabolic environment comparable to human patients with mets . the obese ossabaw swine may be an ideal large animal model for the study of the effects of high caloric - high fat diet on the genesis of mets and may be useful to further examine the mechanisms associated with increased platelet reactivity in humans with mets .

percutaneous nephrolithotomy ( pcnl ) is the favored endourologic procedure for large ( > 20 mm ) renal calculi , offering patients a low - morbidity procedure with high efficacy in terms of stone clearance . pcnl has proved its efficacy and has stood the test of time compared with open surgical techniques and extracorporeal shock - wave lithotripsy . over time , developments in radiologic imaging , urologic instruments , and techniques have advanced the frontiers for the success and safety of pcnl . the procedure is now recognized to have a low failure rate and to be a superior alternative to shock - wave lithotripsy for treating large renal calculi . the kidney puncture is critical , and it is difficult to obtain an appropriate renal tract . although fluoroscopy and ultrasound guidance provide well - established methods of percutaneous renal access , these interventions are technically demanding and risky , especially in unfavorable settings such as complex anatomy . both ultrasound- and fluoroscopy - guided puncture techniques provide only a 2-dimensional view of the anatomy , and reaching the exact site of puncture can be more difficult . there have been reports of the use of computed tomography and magnetic resonance imaging , but these tools are time consuming , are not ergonomic , and do not take the movements of the kidney into account . overall , c - arm fluoroscopy remains the most commonly used method to obtain initial renal puncture for pcnl , but it involves patient and operator irradiation . it has been shown that when a fornix is targeted , there is decreased risk for injury to the large vessels in the kidney . repeated attempts at gaining the accurate puncture increases the risk for renal injury and bleeding , which can lead to abandoning the surgery or rarely requiring intervention . in the past , several technical modifications were attempted to simplify the procedure for initial kidney puncture , such as laser guidance , robot assistance , and retrograde endoscopy - guided approaches . however , until now , none of these ideas has become routine , mainly because of poor cost - effectiveness . thus , it has been felt that there is a need for a device that would ( 1 ) help in increasing the accuracy and safety of renal puncture , ( 2 ) decease the time needed for renal procedure , ( 3 ) reduce the radiation dose to the patient and surgeon , ( 4 ) avoid the direct exposure of surgeons ' hands to radiation when handling the puncture needle under fluoroscopy , and ( 5 ) make it easier to transfer the puncture technique to the next generation . given these needs , there have been a few recent studies reported in the literature that have attempted special techniques or used devices to help in renal puncture . it is well understood that in vivo , muscle and fascial deformation as well as kidney movement may diminish calyceal puncture success . we aimed to design a mechanical device that would aid in renal puncture and thus improve the safety and efficacy of pcnl . also , to overcome the drawbacks of other reported instruments , we planned to do the trial in vivo using a live porcine model so as to closely replicate the operative process in humans and thus undertake a better evaluation of its clinical reliability . the purpose of the kidney access device ( kad ) is to stabilize the needle and align it for pcnl needle puncture , thus allowing precise needle placement . the base of the kad is an electromagnet , which when it is switched on attaches firmly to a landing platform that is attached to the operating table . the platform can be fixed to any presently used operating table along the side rails . our device has 2 hydraulic arms with a translucent tube attached distally , which can rotate and allow a large degree of manipulation to allow access to upper , middle , and lower pole calyces . the hydraulic arms are fixed to the operating table with the help of an anchoring magnetic base . the puncture needle passes through a radiolucent tube that is secured to the hydraulic arm , allowing accurate alignment ( figure 1 ) . the needle - holding tube can move in the horizontal direction and rotate in any axis , and being radiolucent , only the needle is visible under fluoroscopy . fine needle compensation for renal movement because of breathing can be done by a special adjusting screw . for full description of the device 's parts , ( a ) top marker : base magnet , which can be switched on to anchor the kad to the platform attached to the operating table . golden - colored hydraulic arms of the kad for gross alignment of the puncture needle . these arms allow targeting of upper , middle , and lower pole calyces and can be fixed in positon by tightening of the central golden screw . ( c ) third marker from top marker : screw for finer horizontal movements of the puncture needle . this allows finer movements of the needle holding tube and the needle to compensate for the kidney movements with respiration . ( d ) 4th marker from top : the horizontal slider allows the medial and lateral movements of the needle , required for step 3 of the renal puncture . ( e ) 5th marker from top : shows the small hole in the distal radiolucent tube , through which the puncture needle passes . the hole is made such that it is snug , such that it holds the needle in place and allows it to be pushed through when in position . ( f ) bottom marker : shows the radiolucent distal tube , which holds the puncture tube . the protocol for the research was approved by the institutional review board as well as the institutional animal care and use committee . this was based on the fact that pigs have comparable renal size and anatomy with that of humans . , we targeted 3 calyces of each kidney in the 4 pigs for a total of 24 punctures . the pig was intubated , and under general anesthesia , the pig was positioned in a supine position . the urinary bladder was identified in the pelvis , and the bilateral ureters were identified and dissected free close to the urinary bladder . ureteric catheters were then inserted into the ureters to reach the respective renal pelvis and were secured in place . the animal was then positioned in a prone position on the operating table and secured on the operating room table . the kad was then placed on the landing platform attached to side of the operating table and anchored in place by switching on the base magnet ( figure 2a ) . radiopaque dye was instilled into the renal pelvis through the previously placed ureteric catheters to opacify the renal pelvicalyceal system . for the first puncture , the upper pole posterior calyx was chosen ; this was subsequently followed by the midposterior calyx , and the lower pole posterior calyx was targeted last . ( a ) step 1 of using the kad for renal access . the kad is mounted on the operating table attached platform using the base magnet . using the golden hydraulic kad arms , ( b ) step 2 : the puncture needle is aligned in the vertical axis of the targeted calyx , under fluoroscopy , such that it is parallel to the calyx . ( c ) step 3 : using the distal slider , the puncture needle is moved forward , such that the needle superimposes the targeted calyx , and confirmed under fluoroscopy . the needle can then be pushed in to puncture the kidney until a sensation of giveaway is felt and confirmed by the return of urine . the first step was to align the needle in the horizontal direction of the targeted calyx under fluoroscopic guidance ; this was done after positioning the image intensifier ( fluoroscopy ) vertically , such that it was at a right angle to the animal and the puncture needle . the second step was to rotate the needle - holding tube ( of the kad ) in the same vertical axis as the targeted calyx . this was done after rotating the fluoroscope to approximately 30 such that the targeted calyx appeared as a black dot on fluoroscopy as the image intensifier was in the line of the targeted calyx , and the puncture needle looked the same , which confirmed that both the targeted calyx and the needle were in a parallel vertical plane ( figure 2b ) . in the final step , the needle was superimposed over the targeted calyx by moving the kad holding tube forward . appearance of a single dot on fluoroscopy confirmed that the needle was overlying the targeted calyx ( figure 2c ) . the respiration of the kidney was withheld at this time for approximately 30 seconds so that renal movements with respiration were avoided . for the final step , the puncture needle was pushed through the skin and subcutaneous structures until a feeling of giveaway was felt . a successful puncture was confirmed under fluoroscopy and also by the return of clear urine through the puncture needle ( figures 3a and 3b ) . a guidewire was then inserted through the needle and manipulated down the ureter and confirmed in place with fluoroscopy ( figures 3c and 3d ) . after the guidewire was secured in place , the subsequent midpole and lower pole calyceal punctures were done following the same puncture steps . this was followed by similar renal punctures done on the contralateral kidney with insertion of guidewires that were secured in place . ( b ) accurate placement of needle in the renal calyx , confirmed on fluoroscopy . ( c ) guidewire placed into the renal collecting system through the kad - aided renal puncture . ( d ) guidewire placement in the renal pelvicalyceal and down the ureter through the renal puncture needle confirmed under fluoroscopy . during the whole procedure the pig was then kept under anesthesia for 1 hours while being monitored for any change in hemodynamic or ventilator settings . vertical incisions were made on bilateral flanks along the lateral borders of the psoas muscles . any injury to surrounding organs , such as the pleura , liver , or bowel , was evaluated , along with any retroperitoneal hematoma or active bleeding from the kidney . the kidneys were then dissected , and the positions of the guidewires in relation to the targeted calyces were noted . the purpose of the kidney access device ( kad ) is to stabilize the needle and align it for pcnl needle puncture , thus allowing precise needle placement . the base of the kad is an electromagnet , which when it is switched on attaches firmly to a landing platform that is attached to the operating table . the platform can be fixed to any presently used operating table along the side rails . our device has 2 hydraulic arms with a translucent tube attached distally , which can rotate and allow a large degree of manipulation to allow access to upper , middle , and lower pole calyces . the hydraulic arms are fixed to the operating table with the help of an anchoring magnetic base . the puncture needle passes through a radiolucent tube that is secured to the hydraulic arm , allowing accurate alignment ( figure 1 ) . the needle - holding tube can move in the horizontal direction and rotate in any axis , and being radiolucent , only the needle is visible under fluoroscopy . fine needle compensation for renal movement because of breathing can be done by a special adjusting screw . for full description of the device 's parts , ( a ) top marker : base magnet , which can be switched on to anchor the kad to the platform attached to the operating table . golden - colored hydraulic arms of the kad for gross alignment of the puncture needle . these arms allow targeting of upper , middle , and lower pole calyces and can be fixed in positon by tightening of the central golden screw . ( c ) third marker from top marker : screw for finer horizontal movements of the puncture needle . this allows finer movements of the needle holding tube and the needle to compensate for the kidney movements with respiration . ( d ) 4th marker from top : the horizontal slider allows the medial and lateral movements of the needle , required for step 3 of the renal puncture . ( e ) 5th marker from top : shows the small hole in the distal radiolucent tube , through which the puncture needle passes . the hole is made such that it is snug , such that it holds the needle in place and allows it to be pushed through when in position . ( f ) bottom marker : shows the radiolucent distal tube , which holds the puncture tube . the protocol for the research was approved by the institutional review board as well as the institutional animal care and use committee . this was based on the fact that pigs have comparable renal size and anatomy with that of humans . , we targeted 3 calyces of each kidney in the 4 pigs for a total of 24 punctures . the pig was intubated , and under general anesthesia , the pig was positioned in a supine position . a 2-inch lower abdominal midline incision was made , and the peritoneum was entered . the urinary bladder was identified in the pelvis , and the bilateral ureters were identified and dissected free close to the urinary bladder . ureteric catheters were then inserted into the ureters to reach the respective renal pelvis and were secured in place . the animal was then positioned in a prone position on the operating table and secured on the operating room table . the kad was then placed on the landing platform attached to side of the operating table and anchored in place by switching on the base magnet ( figure 2a ) . radiopaque dye was instilled into the renal pelvis through the previously placed ureteric catheters to opacify the renal pelvicalyceal system . for the first puncture , the upper pole posterior calyx was chosen ; this was subsequently followed by the midposterior calyx , and the lower pole posterior calyx was targeted last . the kad is mounted on the operating table attached platform using the base magnet . using the golden hydraulic kad arms , ( b ) step 2 : the puncture needle is aligned in the vertical axis of the targeted calyx , under fluoroscopy , such that it is parallel to the calyx . ( c ) step 3 : using the distal slider , the puncture needle is moved forward , such that the needle superimposes the targeted calyx , and confirmed under fluoroscopy . the needle can then be pushed in to puncture the kidney until a sensation of giveaway is felt and confirmed by the return of urine . the first step was to align the needle in the horizontal direction of the targeted calyx under fluoroscopic guidance ; this was done after positioning the image intensifier ( fluoroscopy ) vertically , such that it was at a right angle to the animal and the puncture needle . the second step was to rotate the needle - holding tube ( of the kad ) in the same vertical axis as the targeted calyx . this was done after rotating the fluoroscope to approximately 30 such that the targeted calyx appeared as a black dot on fluoroscopy as the image intensifier was in the line of the targeted calyx , and the puncture needle looked the same , which confirmed that both the targeted calyx and the needle were in a parallel vertical plane ( figure 2b ) . in the final step , the needle was superimposed over the targeted calyx by moving the kad holding tube forward . appearance of a single dot on fluoroscopy confirmed that the needle was overlying the targeted calyx ( figure 2c ) . the respiration of the kidney was withheld at this time for approximately 30 seconds so that renal movements with respiration were avoided . for the final step , the puncture needle was pushed through the skin and subcutaneous structures until a feeling of giveaway was felt . a successful puncture was confirmed under fluoroscopy and also by the return of clear urine through the puncture needle ( figures 3a and 3b ) . a guidewire was then inserted through the needle and manipulated down the ureter and confirmed in place with fluoroscopy ( figures 3c and 3d ) . after the guidewire was secured in place , the subsequent midpole and lower pole calyceal punctures were done following the same puncture steps . this was followed by similar renal punctures done on the contralateral kidney with insertion of guidewires that were secured in place . ( b ) accurate placement of needle in the renal calyx , confirmed on fluoroscopy . ( c ) guidewire placed into the renal collecting system through the kad - aided renal puncture . ( d ) guidewire placement in the renal pelvicalyceal and down the ureter through the renal puncture needle confirmed under fluoroscopy . during the whole procedure , the animal was carefully monitored for hemodynamic stability and stable ventilator settings . the pig was then kept under anesthesia for 1 hours while being monitored for any change in hemodynamic or ventilator settings . vertical incisions were made on bilateral flanks along the lateral borders of the psoas muscles . any injury to surrounding organs , such as the pleura , liver , or bowel , was evaluated , along with any retroperitoneal hematoma or active bleeding from the kidney . the kidneys were then dissected , and the positions of the guidewires in relation to the targeted calyces were noted . intraoperatively , all calyces in the upper , middle , and lower poles of the kidney could be easily accessed with the kad . postprocedural dissection of the kidneys and surrounding organs revealed no retroperitoneal hematoma , visceral organ injury , or active bleeding from kidneys in any of the pigs . after the dissection of kidneys , analysis of the position of guidewires in relation to the targeted calyxes was done . the precise intrarenal placements of guidewires in relation to targeted calyces were noted at all 24 sites . pcnl is a specialized , minimally invasive urologic procedure that remains the surgical procedure of choice for treating large renal calculi and calculi resistant to extracorporeal shock - wave lithotripsy treatment . despite its popularity , it is an invasive procedure that can result in significant morbidity and even mortality . pcnl has a steep learning curve and is a difficult procedure to master , taking about 60 pcnl procedures for surgical competence and 115 procedures for excellence . with technological advances , there have been endeavors to invent a device that would make this procedure safe while decreasing the learning curve associated with it . even though many innovative devices have been reported , each has its drawbacks , and most have not been evaluated in an animal trial . the main concern when performing pcnl is the location of the stone and an accurate initial renal puncture to target it . also if the renal puncture is not done at end on calyx , it can result in torrential bleeding from the vessels which run on the sides of the calyces . an inaccurate renal puncture could also result in injury to visceral organs , substantial bleeding , or loss of kidney . among the many techniques and instruments that have been reported to perform safe pcnl , a micro optical light lead is inserted through the puncture needle and then connected to an endoscopic camera system , so that the calyces can be identified and the stone can then be located . however , this technique has several limitations , chiefly that the scopes used for the procedure cost thousands of dollars , making the device not a cost - effective alternative . pcnl renal puncture in itself is a difficult procedure to master , and along with the endoscopic camera system , the amount of expertise required increases dramatically . a method that uses computer software along with a gantry also exists . in this method , images of the renal calyceal system are taken and then sent to a laptop computer containing the software . with these images , the surgeon can mark the area to puncture by clicking the computer mouse on a specific image . once the surgeon clicks on the image , the software determines the needle trajectory and provides the settings for 3 axes on the gantry . after this is done , the needle can be placed on the needle holder , and by using the calculations the software provided , the targeted calyx can be punctured . this software - based method involves a lot of equipment and requires knowledge on how to use the software , and these limitations make it difficult to use on a routine basis . these elaborate methods are reserved for those with extensive training and resources and not meant for routine use in hospitals , and with rising health care costs , they are not practical to be used on a regular basis . the kad is cost effective , costing < $ 700 to make . other reported devices require complicated and advanced software and other parts , resulting in higher costs . we made the kad using 2 hydraulic arms , a radiolucent needle holder , and some clamps ; our device provides a precise means by which to enter the renal calyces . in each of our punctures , it can easily be used by a novice and accurately applied with a 3-step technique as we describe . in addition to increasing the safety of renal puncture , the kad avoids the direct exposure of radiation to the surgeon 's hands while performing the procedure . also , the kad reduces the radiation exposure to the patient , as the amount of fluoroscopy used is much less , because the time required for the renal puncture is reduced . although our device has many advantages , we do feel that it needs to be critically evaluated in a human trial , as minor problems could be encountered when using the kad in humans . some of the possible anticipated problems of using the kad in humans are as follows : minor adjustments with the finer movements of the needle - holding tube may be required to compensate for the differences in renal anatomy between humans and pigs.depending on the body wall thickness ( which can be variable in humans depending on body mass index ) the lengths of the hydraulic arms of the kad may need to be increased when using the device on obese patients.human anatomy can be variable , and more careful planning would be needed when accessing kidneys with congenital or developmental variations.we did not measure the actual radiation used during this initial trial of the kad . minor adjustments with the finer movements of the needle - holding tube may be required to compensate for the differences in renal anatomy between humans and pigs . depending on the body wall thickness ( which can be variable in humans depending on body mass index ) the lengths of the hydraulic arms of the kad may need to be increased when using the device on obese patients . human anatomy can be variable , and more careful planning would be needed when accessing kidneys with congenital or developmental variations . we did not measure the actual radiation used during this initial trial of the kad . other reported devices require complicated and advanced software and other parts , resulting in higher costs . we made the kad using 2 hydraulic arms , a radiolucent needle holder , and some clamps ; our device provides a precise means by which to enter the renal calyces . in each of our punctures , it can easily be used by a novice and accurately applied with a 3-step technique as we describe . in addition to increasing the safety of renal puncture , the kad avoids the direct exposure of radiation to the surgeon 's hands while performing the procedure . also , the kad reduces the radiation exposure to the patient , as the amount of fluoroscopy used is much less , because the time required for the renal puncture is reduced . although our device has many advantages , we do feel that it needs to be critically evaluated in a human trial , as minor problems could be encountered when using the kad in humans . some of the possible anticipated problems of using the kad in humans are as follows : minor adjustments with the finer movements of the needle - holding tube may be required to compensate for the differences in renal anatomy between humans and pigs.depending on the body wall thickness ( which can be variable in humans depending on body mass index ) the lengths of the hydraulic arms of the kad may need to be increased when using the device on obese patients.human anatomy can be variable , and more careful planning would be needed when accessing kidneys with congenital or developmental variations.we did not measure the actual radiation used during this initial trial of the kad . minor adjustments with the finer movements of the needle - holding tube may be required to compensate for the differences in renal anatomy between humans and pigs . depending on the body wall thickness ( which can be variable in humans depending on body mass index ) the lengths of the hydraulic arms of the kad may need to be increased when using the device on obese patients . human anatomy can be variable , and more careful planning would be needed when accessing kidneys with congenital or developmental variations . we did not measure the actual radiation used during this initial trial of the kad . the kad , along with the 3-step technique , aids in the safe and accurate placement of the puncture needle , even in the hands of a novice . the device also allows less operative time and subsequently less exposure to fluoroscopy for both the surgeon and the patient . the hydraulic arms and rotating axis of the radiolucent tube allow precise placement of the puncture needle . in our trial this is the first report of a kidney puncture device with a successful animal trial . this device can also be used in many other surgical procedures and allows a minimally invasive approach .

vaginal hysterectomy with repair ( vhr ) of the anterior and posterior vaginal wall is often required to provide functional and lasting relief to women who present with prolapse of the vagina , cervix , and uterus out of introitus , especially near or after menopause . according to studies by wu et al1 and luber et al,2 it is predicted that over the next 40 years , the demand for services for such pelvic floor disorders will double from the present rate , in view of longevity of women . significant pelvic structure weakening occurs after menopause due to deficient estrogen , which maintains pelvic tonicity . olsen et al3 have also reported that the chance of a woman having genital prolapse increases with age ; hence , the incidence of prolapse will rise as life expectancy increases . ranee et al4 reported that the incidence of genital prolapse is difficult to determine , as many women do not seek medical advice . beck et al5 reported that half of parous women lose pelvic floor support , resulting in some degree of prolapse , and of these women , only 10% to 20% seek medical care . genital prolapse accounts for 20% of women on the waiting list for major gynaecological surgery . in the beginning , women may not have symptoms ; however , as relaxation progresses , there is feeling of heaviness , pressure in the vagina , something ( vagina , cervix , uterus ) coming out of the introitus , and leakage of urine , which can worsen during heavy weight lifting , coughing , laughing , or sneezing . in addition , there may be symptoms related to urinary tract infection or sexual dysfunction . the present study was conducted to investigate trends in vaginal hysterectomy with anterior and posterior vaginal repair for genital prolapse and associated morbidity by analysis of case records of women who had surgery for this condition . the present study was conducted to investigate trends in vaginal hysterectomy with anterior and posterior vaginal repair for genital prolapse and associated morbidity by analysis of case records of women who had surgery for this condition . the present study was an analysis of case records of women who underwent vaginal hysterectomy for genital prolapse in the last 20 years . the cases were divided into four five - year blocks , a ( 19891993 ) , b ( 19941998 ) , c ( 19992003 ) , and d ( 20042008 ) . the study began at the end of 1998 ; hence , for retrospective cases , information from records from previous years was collected and analyzed , and for the remainder , similar information was collected prospectively and analyzed . the study does not include all cases of genital prolapse that presented to the outpatient department or that were admitted . since relaxation of the supports of the uterus with or without obvious genital prolapse continues to be a common problem , many women present with a feeling of something coming out of the vagina and do not return after active exercises have been recommended . the present study also does not include cases of vault prolapse , which occurs after vaginal hysterectomy for genital prolapse or nondescent vaginal hysterectomy for other benign disorders . we do not see vault prolapse after obstetric hysterectomy , which is not a common procedure because alternative modalities are used for postpartum haemorrhage and rupture of the uterus is uncommon now . the present study is analysis of case records of vaginal hysterectomy with repair of genital prolapse . the records of 1818 of 2424 ( 75% ) hysterectomies performed during the retrospective period could be obtained , and 100% of the 2407 records in the prospective period were obtained . therefore , a total of 4225 case records were available for analysis , and , of these , 911 ( 21.6% ) were of vaginal hysterectomy with repair of the anterior and posterior vaginal wall for genital prolapse ( the study subjects ) . these cases represented 2.7% of 34,080 gynecological admissions during the period of analysis . in each time period , the following proportion of cases were vaginal hysterectomy with anterior and posterior vaginal wall repair for genital prolapse : 100 of 522 ( 19.1% ) in block a , 701 of 167 ( 23.8% ) in block b , 388 of 1211 ( 21.1% ) in block c , and 388 of 1789 ( 21.68% ) in block d. over the study years , therefore , there was no change in the proportion of hysterectomies that were vaginal hysterectomies with vaginal wall repair for genital prolapse ( fig . a , 73/100 ( 73% ) women were over 40 years of age , and 324/388 ( 83.5% ) of women in block d were in this age group , that is , in the most recent group , a higher percentage of women were above 40 years of age ( p = 0.03 ) . analysis of parity revealed fluctuations . in block a , x / x ( 10% ) women had had two to four births , and in block d , x / x ( 38% ) women had done so . the proportion of women who had had five or more births was 43/100 ( 43% ) in block a , 68/167 ( 40.7% ) in block b , 92/256 ( 36% ) in block c , and 180/388 ( 46.4% ) in block d. ( table 1 ) . overall , only 4/911 ( 0.4% ) women had not given birth , 492/911 ( 54% ) had two to four births , and 383/911 ( 42% ) had five or more births . of the 911 women , 803 ( 88.1% ) had third degree cervico - uterine prolapse ( cervix outside the introitus ) , 84 ( 9.2% ) had second degree ( cervix up to introitus ) , and 24 ( 2.6% ) had first - degree cervico - uterine prolapse ( cervix inside the introitus ) with varying degrees of vaginal prolapse , that is , vagina either remaining just inside the introitus or coming up into the introitus or outside of it . of all 911 women , 94.2% had presented with something coming out of the vagina , but 5.8% presented with other symptoms as the chief complaint ( 4.7% abnormal uterine bleeding and 1.1% lower abdominal pain ) . of the 911 women , 92 ( 10.1% ) had other gynecological disorders in addition to genital prolapse . the percentage of women presenting with no other disorders was 86/100 ( 86% ) in block a,152/167 ( 91.10% ) in block b , 222/256 ( 86.72% ) in block c , and 358/388 ( 92.27% ) in block d , again with no change over the years ( fig . 2 ) . of the 911 study subjects , 818/911 ( 89.8% ) had no intra - operative complications . the problem of intra - operative shock decreased from 1/100 ( 1% ) in block a to nil in block d , and cardiac arrest decreased from 1/100 ( 1% ) in block a to nil in block d. a decreasing trend was observed in all the postoperative complications : postoperative vaginal discharge decreased from 6/100 ( 6% ) in block a to 9/388 ( 2.3% ) in block d ; urinary tract infection , from 5/100 ( 5% ) to 2/388 ( 0.5% ) in block d ; and fever , from 1/100 ( 1% ) in block a to 2/388 ( 0.5% ) in block d. however , heavy vaginal bleeding occurred in 1/100 ( 1% ) cases in block a and in 3/388 ( 0.8% ) of cases in block d ( fig . uterovaginal prolapse is a common gynecological disorder , predominantly seen in middle and old age . genital prolapse was found to be a common indication for surgery with no change in trends over the years . tinelli et al7 reported that even if the etiology of pelvic prolapse is poorly defined and multifactorial , risk factors associated with aging , such as biomechanical abnormalities in connective tissue composition , hormonal deficiency , and irregular tissue metabolism are nonmodifiable and , therefore , often seen in clinical practice . according to a study by olsen et al,3 pelvic floor dysfunction is a major health issue for older women , indicated by the 11.1% lifetime risk of undergoing a single operation for pelvic organ prolapse and urinary incontinence , as well as by the large proportion of repeat operations . the initial damage to pelvic floor integrity generally occurs during childbirth and later with the climacteric , a second insult occurs . so if there are many births , there is more damage , and the climacteric effect adds to it , which often leads to prolapse of the vagina , cervix , and uterus . as is evident from the study by romanzi et al8 lower urinary tract symptoms are common in women with genital prolapse . voiding difficulty , bladder outlet obstruction , and occult stress incontinence may coexist , and they are associated with prolapse . in a study by mehboob et al,9 of the 98 women who had vaginal hysterectomy with vaginal wall repair for genital prolapse , the majority had developed prolapse between 41 and 60 years of age . jones et al10 also reported that weakening of the pelvic floor muscles and a decrease in pelvic connective tissue resilience due to menopause facilitate progression to symptomatic pelvic visceral prolapse . in the present study , although 80% women were more than 40 years of age at diagnosis , many had had the disorder for years . our institution is rural with 60% to 65% of patients living in a rural area . these rural women complete childbearing at a young age , and the disorder actually starts at a young age and is aggravated near menopause , but the women continue to live with it . in the present analysis , no trends were found . in the study by mehboob et al,9 there were no woman who had not given birth , 25.5% had had 3 to 5 births , and 75% had had 5 or more births , a major factor responsible for prolapse . however , in the present study , 4 ( 0.4% ) women had not given birth , and 383 ( 42% ) had had 5 or more births . overall 874 ( 96% ) women had had two or more births . the decrease in multiple births is a recent phenomenon ; therefore multiple births as a cause of prolapse is likely to further decrease in the future . in the study by ugboma et al,11 genital prolapse ( 118 cases ) accounted for 11.8% of gynecological admissions ( a very high proportion of genital prolapse ) , 70% study subjects were above 45 years of age , and 73% had had 5 or more births . authors also report that the most common symptom was something coming down the vagina ( 95% of study subjects ) . in the present study , out of 34,080 gynecological admissions , 4831 ( 14.2% ) had hysterectomy , 911 had hysterectomy for genital prolapse or 2.7% of all admissions ( a high number ) , and 21.6% of women who had hysterectomies and 94.2% women who underwent vaginal hysterectomy with vaginal wall repair had presented with something coming out of the introitus ; however , approximately 5% women had had genital prolapse for years but had presented with abnormal menstruation or pain . in a study of surgical management of 200 cases of genital prolapse , dutta et al12 reported that 40% of their cases had third degree uterine prolapse , 42% had second - degree uterine prolapse , and 18% had first- degree uterine prolapse . in the present study , of the 911 women , 803 ( 88.1% ) had third - degree uterine prolapse with varying degrees of vaginal prolapse , and only 2.6% had surgery for first - degree cervico uterine prolapse with varying degrees of anterior / posterior vaginal wall prolapse . in this area , rural women usually wait until their day - to - day life is affected . furthermore , practice decisions regarding surgery are taken more often in cases of third - degree uterocervical prolapse , and the present analysis is only of cases where surgery was performed . sometimes the cervix might have only first - degree descent , but vaginal wall prolapse is more than cervical descent and may be responsible for the symptomatology , and , in such cases , vaginal hysterectomy is the preferred mode of therapy . pakbaz et al,13 also reported that vaginal hysterectomy is an efficient treatment for uterovaginal prolapse with swift recovery and a low rate of complication . many women have urinary stress incontinence ; therefore , efforts to disclose latent stress incontinence should be undertaken preoperatively . overall , complications occurred in 14.7% of women : 15% in block a , 21.0% in block b , 10.6% in block c , and 4% in block d , a highly significant decrease in complications over the years , especially in block d ( the rates of complications in all block are compared with rate of complication in block d ) ( p < 0.001 ) . this is probably because of preoperative evaluation and therapy of lower genital tract infection and possibly better antibiotics and overall better care . the issues related to prevention need to be looked into . surgical morbidity can be reduced by proper preoperative , intraoperative and postoperative care . preoperative meticulous evaluation and care are imperative . treatment of urinary and genital infection and evaluation of biochemical and hematological parameters to prevent intraoperative and postoperative morbidity are essential . these could be the reasons for the decreasing morbidity trends observed in the present study . pakbaz et al14 reported lack of information on pelvic organ prolapse in the public domain . with an aging population , however , attempts need to be made to have safe birth and healthy lifestyles so as to prevent genital prolapse and , in case it occurs , to provide advocacy and therapy that prevents progression so that hysterectomy is averted , and if hysterectomy is needed , the aim should be to have the least morbidity .

antimalarial compounds consisting of two 4-amino-7-chloroquinoline ( 4,7-acq ) components linked by flexible tethers were identified as some of the first small molecule botulinum neurotoxin serotype a light chain ( bont / a lc ) inhibitors . shortly thereafter , we discovered that antimalarial agents composed of a 4,7-acq component coupled with either an adamantane or a cholic acid derived component provided effective dual inhibitors of the bont / a lc and plasmodium falciparum strains d6 and w2 strains . this discovery led to a model in which 4,7-acq - based antimalarials are / have been modified with the intent of also generating novel bont / a lc inhibitors and vice versa . activity relationships for such chemotypes and to generate more potent bont / a lc inhibitors that , because they incorporate a 4,7-acq moiety , would be expected to be prospective antimalarial agents . botulinum neurotoxins ( bonts ) , exotoxins secreted by anaerobic , spore - forming bacterium clostridium botulinum , are the most potent of biological toxins . bonts are responsible for the potentially fatal disease botulism , which is most commonly associated with food contamination , wound infection , and colonizing infection in infants . however , because of ease of production , dissemination , lethality , and amenability for use as biological weapons , bonts are classified as category a biothreat agents by the centers for disease control and prevention ( cdc ) . each cleaves a specific peptide bond in one or more of three proteins that form the soluble n - ethylmaleimide - sensitive factor attachment protein receptor ( snare complex ) . the interaction of these three proteins facilitates the transport of acetylcholine into neuromuscular junctions . botulinum neurotoxins are secreted as single polypeptide chains and , following post - translational processing , are composed of a heavy chain ( hc ) subunit and light chain ( lc ) subunit ; the two subunits are covalently linked by a disulfide bridge . the hc , which comprises two domains of 50 kda , is responsible for mitigating toxin internalization and release into the neuronal cytosol via an acidic endosome . the c - terminal domain ( i.e. , the ganglioside and protein receptor - binding domain ) binds to the neuronal cell membrane and mediates the internalization of toxin containing endosomes ; the n - terminal domain facilitates the release of the lc from the endosome into the cell cytosol . this proteolysis eliminates neurotransmitter release into the synaptic cleft , thereby causing the flaccid paralysis that is medically diagnosed as botulism . bont serotypes a and e cleave synaptosomal - associated protein of 25 kda ( snap-25 ) . serotypes b , d , f , and g cleave vesicle - associated membrane protein , and serotype c1 cleaves both snap-25 and syntaxin . the bont / a lc is the most potent and longest acting of the bont lc serotypes in the neuronal cytosol . for example , the bont / a lc is 10-fold more potent than cobra toxin and 10-fold more deadly than cyanide . the lethal dose of bont / a holotoxin is estimated to be between 1 and 5 ng kg for humans . while antibody - based vaccines are available , this avenue of treatment is limited by a short window of therapeutic opportunity , since such biomolecules can not penetrate into the cell cytosol . hence , developing small molecules that will effectively inhibit bont lc proteolytic activity postneuronal intoxication is of particular interest . a variety of bont / a lc inhibitors have been reported and range from compounds bidentate coordinating the enzyme catalytic zn to allosteric inhibitors . in general , the most potent bont / a lc inhibitors reported to date possess ki values ranging from 0.1 to 10 m and include hydroxamic acid based compounds 1 and 3 , 2,5-diphenylthiophene derivative 2 , betulin derivative 4 , naphthopyrone 5 , and chrysene 6(24 ) ( chart 1 ) . malaria is a devastating global health threat , with nearly half of the world population at risk of being infected . malaria is caused by five plasmodium species : p. falciparum , p. ovale , p. vivax , p. malarie , and p. knowlesi , of which p. falciparum , which causes cerebral malaria , is the major cause of mortality . malaria parasites contain an acidic food vacuole ( fv ) that digests hemoglobin ( hb ) , and it is generally accepted that the fv is the site of action for a number of aminoquinoline ( acq ) based drugs . the heme obtained from hb degradation is toxic to the parasite and is therefore transformed into insoluble hemozoin pigment , while the globin is hydrolyzed into individual amino acids . antimalarial drugs that are active within the fv appear to kill the parasite by either producing toxic free radicals or blocking hemozoin formation , as in the case of acq - based drugs . the development of widespread drug resistance to chloroquine ( 9 , cq , chart 2 ) , one of the most successful antimalarial drugs , has resulted in severe health issues for countries in malaria endemic regions . although collected data indicate that mutations in the p. falciparum cq - resistant transporter ( pfcrt ) , multidrug resistance protein 1 ( pfmdr1 ) , and multidrug resistance - associated protein ( pfmrp ) are responsible for malaria parasite resistance to cq and its analogues , additional analyses appear necessary to fully corroborate such data . therefore , significant focus has been placed on the syntheses of peroxide antimalarials , as well as on the development of other chemotypes that prevent heme polymerization . nevertheless , various acq - based derivatives are being investigated for their antimalarial activity , either since they appear highly active and nontoxic , such as pyrrolizidine - acq compounds and aminoquinoline aq-13 , or because of their contribution to the development of sar , for example , compounds such as 4-n- , 4-s- , or 4-o - alkylaminoquinoline derivatives . hybrid adducts of various biologically active compounds covalently bonded to the acq moiety , such as reversal agents , complexes with au and ru , antibiotic azithromycin , 2-imidazolidine and ferrocenyl derivatives , and tetraoxanes , are of special interest . very recently , a breakthrough in the antimalarial field has been achieved : quinolone antimalarials that target the parasite s liver and blood stages , as well as forms that are crucial to disease transmission , have been reported . as indicated above , we previously described the synthesis and inhibitory activity of bont / a lc inhibitors possessing a 4,7-acq moiety covalently tethered to either a cholic acid derived or an adamantane component . the compounds were database mined using a pharmacophore for bont / a lc inhibition and were originally synthesized as antimalarial agents . testing indicated that the compounds inhibit the bont / a lc with ic50 values in the low micromolar range . furthermore , it is important to note that several of the derivatives provide potent antimalarial activity against cqr strain w2 and multidrug resistant strain ( mdr ) tm91c235 . for example , 7 was very active in vitro against p. falciparum cqr and cq susceptible ( cqs ) strains , with a resistance index ( ri ( w2/d6 ) ) of 0.65 . this compound also cured mice infected with p. berghei in a thompson test . herein , we present second generation 4,7-acq - cholate based inhibitors of the bont / a lc that are significantly more potent than initially discovered leads . in particular , we expanded our understanding of the sar for this inhibitor chemotype via the generation of compounds with a variety of substitutions on the c(3 ) position of the cholic acid component . in addition , it was anticipated that such an approach would facilitate the synthesis of bis(4,7-acq)-cholic acid derivatives , which became desired target molecules ( based on the promising activities of compounds 7 , 8 , and 12 ( scheme 1 ) . finally , because initially discovered derivatives of the 4,7-acq - cholic acid chemotype were originally developed as antimalarial agents , the compounds prepared during this study were also examined for potency against three p. falciparum strains . overall , the unique capacity of this general chemotype to provide both inhibition of the bont / a lc and antimalarial activity provides a paradigm that facilitates the repositioning of derivatives based on target potency . the ability of derivative 12 ( scheme 1 ) both to inhibit the bont / a lc and to act as an antimalarial agent prompted the development of a new generation of 4,7-acq - cholic acid based derivatives . the design of the compounds focused on investigating effects on potency resulting from ( 1 ) an additional basic group at position c(3 ) of the cholic acid component , ( 2 ) the incorporation of a second acq component , and ( 3 ) the length of the ,-diaminoalkylidene spacer between the steroid and 4,7-acq moiety(ies ) . furthermore , functionalization of the c(3 ) basic group ( acylation , mesylation , and alkylation ) , as well as changes in the basic character of the substituent ( i.e. , h - bond donor / acceptor strength and voluminosity ) , was examined in the context of inhibitory activity and metabolic stability . to aid in rationalizing differences in the in vitro potencies of the derivatives , detailed docking simulations were performed in the bont / a lc substrate cleft . in addition , assessment of the in vitro antimalarial activities of the new derivatives against the cqs and cqr strains of p. falciparum was also conducted , and the most active antimalarial of the series was examined in a p. berghei rodent model . hydrolysis of the acetate at c(3 ) afforded corresponding alcohols 1619 , which were further oxidized with ibx in dmso in the presence of tfa to ketones 2225 ( scheme 1 ) . derivatives 12 and 13 were selectively n - methylated using 37% formaldehyde and nabh(oac)3 . key intermediates for the synthesis of n - alkylated congeners 3657 , i.e. , compounds 3335 , were obtained from precursor 26(45 ) ( scheme 2 ) . in the first step , the free amine was protected as an acetate , mesylate , or boc derivative ( 27 , 28 , or 29 , respectively ) . subsequently , selective hydrolysis and reduction of the intermediate mixed anhydrides ( reaction steps b and c , respectively ) ( scheme 2 ) afforded compounds 3335 in 5480% overall yield . the three intermediate compounds were subsequently transformed ( via the above indicated procedure ) using n-(7-chloroquinolin-4-yl)ethane-1,2-diamine ( acq2 ) or n-(7-chloroquinolin-4-yl)propan-1,3-diamine ( acq3 ) into respective derivatives 3641 ( scheme 3 ) . n - mesyl and n - boc derivatives 3841 were methylated using 37% formaldehyde to yield 4245 , respectively . after removal of the n - boc protecting group , permethylated products 56 and 57 were obtained in two steps from 44 and 45 , respectively . on the other hand , 40 and 41 were transformed into 3-(n , n - dimethyl ) derivatives 50 and 51 by protective group manipulation using fmoc and boc protection groups ( scheme 4 ) . finally , derivatives 52 and 53 , which contain primary amino groups , were obtained from 40 and 41 via the removal of the n - boc group with 50% tfa in dcm ( scheme 3 ) . in the above reductive amination of the aldehyde derived from 35 with acq2 or acq3 , bis - steroidal 4,7-acq derivatives 58 and 59 were also isolated as double alkylation byproducts ( scheme 5 ) . bis - steroidal 4-aminoquinoline derivatives 60 and 61 , which possess free amino groups at position c(3 ) , were obtained after the deprotection of 58 and 59 , respectively . additional targets of the present work , bis-4,7-acq derivatives 6268 ( scheme 6 ) , were obtained as diastereomers at c(3 ) from 24-acq - ketones 2225 ( scheme 1 ) via reaction with respective acq - amines . however , the products were obtained in rather low yield , and derivatives with shorter linkers , i.e. , 62 and 63 , having two and three methylene groups , respectively , were not separated into single isomers and were therefore tested in the biological assays as mixtures . the structures of all synthesized compounds were confirmed by spectral and analytical techniques . the purity of all tested compounds was determined with waters and agilent hplc instruments and was 95% for all . the inhibitory efficacies of derivatives 1268 at 20 m , employing a well - established hplc - based assay for bont / a lc inhibition , are shown in table 1 . the assay uses a synthetic substrate that contains the snap-25 scissile bond ( snap-25 residues 187203 of snap-25 ) , and proteolytic activity is determined via comparison of the peak areas of the products versus the intact substrate . in the assay , cq exhibited only marginal inhibition of the bont / a lc ( 6.51% at 20 m ) , which clearly emphasizes the importance of the cholic acid component ; the cholic acid component not only contributes to the overall amphiphilic character of the inhibitors but also results in additional interactions with amino acid residues in bont / a lc binding cleft ( vide infra ) . percent inhibition measurements were performed in duplicate , and standard deviations were less than ( 25% ) for all . ic50 calculations were determined by measuring enzyme activity at nine different smnpi concentrations and in the absence of the small molecule . the small molecule concentrations used in the measurements were determined by estimating the ic50 value and moving in 1 log increments in either direction of the estimated value . nsc240898 , used as the control for comparison , displayed dose dependent inhibition of bont / a induced snap-25 cleavage in neurons with no toxicity at concentrations as high as 40 m ( ref ( 57 ) ) . average value from more than 20 measurements . tested as diphosphate salt . for ki determination , reaction velocity versus substrate concentration was plotted for multiple small molecule concentrations . subsequently , the data were fit to a model of competitive inhibition and analyzed by nonlinear regression analysis . the first set of inhibitors ( 1215 ) was prepared to evaluate the effect on activity of increasing the length of the methylene bridge between the steroid core and the 4,7-acq component ; the results indicated that increasing the length of the methylene bridge did not significantly affect inhibitory potency . moreover , the results indicated that within the acq2 ( compounds 12 , 16 , 22 ) , acq3 ( compounds 13 , 17 , and 23 ) , and n-(7-chloroquinolin-4-yl)hexane-1,3-diamine ( acq6 , compounds 15 , 19 , and 25 ) series , transformations at c(3 ) from ac to oh to c = o generally diminished the inhibitory activities of the respective compounds . n-(7-chloroquinolin-4-yl)butane-1,3-diamine ( acq4 ) derivatives ( compounds 14 , 18 , and 24 ) were the exception . among derivatives with nonbasic substituents at c(3 ) ( scheme 1 ) , the most active were acetate 13 ( ic50 = 2.28 m ) and keto derivative 23 ( ic50 = 2.32 m ) . the introduction of a basic amino substituent at the c(3 ) position of the steroid component markedly increased inhibitory potency . among this set of compounds , the most active was acq2 derivative 52 , which contains an nh2 group at the c(3 ) position and a secondary amino group at the c(24 ) position ( ic50 = 0.81 m ) . the activity of this compound was followed by acq2 derivative 50 ( ic50 = 1.02 m ) , which possesses a tertiary amine at the c(3 ) position . the observed results suggest that for potent bont / a lc inhibitory activity the presence of an ionizable amino group at the c(3 ) position is critically important . the least active compounds are cholic acid derivatives with acidic nh groups ( i.e. , n - ms and n - boc ) at c(3 ) ( see 42 ( ic50 = 24.97 m ) and 44 ( ic50 = 6.78 m ) ) ; both are acq2 derivatives . for derivatives in which amino groups at c(24 ) and/or c(3 ) were methylated , bont / a lc inhibitory activity was not significantly affected ( for comparisons see 52 54 , 53 55 , 54 56 , and 55 57 ) . it has been shown that the catalytic cleft of the bont / a lc is very plastic because of the mobility of flexible loop regions . hence , we hypothesized that the cleft can conformationally adapt to accept large molecules , consequently , we examined two additional subclasses : bis - steroidal 4,7-acq derivatives ( 5861 ) and bis-4,7-acq cholic acid derivatives ( 6268 ) . a significant increase in inhibitory activity was observed after removal of the n - boc protecting group , 59 ( 6% ) vs 61 ( 93% , ic50 = 0.63 m ) , which confirmed the importance of the presence of a basic amino group at c(3 ) for good activity . the bis-4,7-acq derivatives all inhibited the enzyme by 95% at 20 m and retained comparable levels of potency at 10 m ( see table 1s in supporting information ) . the ki values of these derivatives were found to be lower than 0.4 m , with compound 67 being the most potent derivative ( ki = 0.103 m ) . as an important step in further evaluating the inhibitors , three derivatives that were found to enter cells were examined for the ability to protect snap-25 during bont / a challenge in primary neurons ( table 2 ) . western blot analysis was used to determine the relative concentrations of intact and cleaved snap-25 in treated and control cells . the experiments were performed using 1.5 l of a 10 mm concentration of ligand in the presence of 10 l of a 50 nm solution of bont / a . the results indicate that 3-amino derivative 52 significantly protects snap-25 versus corresponding 3-(n - acetyl ) derivative 36 ( 77 % vs 16 % , respectively ) , further confirming the importance of a basic amino substituent at cholic acid position c(3 ) . somewhat better results were obtained with bis - steroid derivative 60 . in the presence of 5 l of a 100 nm solution of antibiotic bafilomycin ( baf ) snap-25 protection is slightly higher ( 77% with 52 only and 88% upon baf addition vs 60 ( 89% alone and 96% upon 5 l of baf ( 100 nm ) addition ) ) . using 25 l of a 100 nm solution of baf the results suggest that 4,7-acq - cholate - based inhibitors can successfully protect snap-25 in the presence of the bont / a holotoxin , with a slight additive effect when given in combination with baf ( a universal antagonist of all bonts in cell culture that acts by inhibiting the release of the lc into the neuronal cytosol ) . a : 488.5 l of medium + 1.5 l ( 10 mm ) of compound + 10 l of bont - a ( 50 nm ) . b : 483.5 l of medium + 1.5 l ( 10 mm ) of compound + 5 l of baf ( 100 nm ) + 10 l of bont - a ( 50 nm ) . c : 463.5 l of medium + 1.5 l ( 10 mm ) of compound + 25 l of baf ( 100 nm ) + 10 l of bont - a ( 50 nm ) . blank : 490 l of medium + 10 l if bont - a ( 50 nm ) . average value from four measurements . to rationalize the inhibitory potencies of the new derivatives , structure - based docking simulations were performed using schrdinger suite 2011 and the modules therein . the structure of the bont / a lc used in this study was obtained from x - ray crystal structure pdb code 3ds9 . for compounds 1257 , which are composed of a single steroid and acq component , all docking studies indicated that a carbonyl oxygen from the acetoxy ( aco- ) group at c(7 ) ( steroid numbering ) coordinates the enzyme s zn ( figure 1 ) while concomitantly expelling the catalytic water . this effectively disables the enzyme s proteolytic activity ( see ligand interactions diagrams , figures s1s3 ) . importantly , this is a main difference versus previously described models in which no coordinate interaction with the zn was observed . strong salt bridges and h - bonds between the n - c(24 ) ammonium cations of the ligands ( i.e. , from secondary or tertiary nitrogens ) and glu164 and/or glu55 ( figure 1 ; see also figure s1 ) were observed . specifically , derivatives possessing secondary ammonium cations engaged in interactions with both glu164 and glu55 , while derivatives with tertiary ammonium cations , because of steric hindrance , interacted with only with glu55 . these results appear to rationalize the improved inhibitory potencies of compounds possessing secondary ammonium cations versus derivatives with tertiary ammonium cations but which possess the same substituent at the c(3 ) position : 12 ( 74.00% ) vs 20 ( 33.28% ) ; 53 ( 84.80% , ic50 = 1.04 m ) vs 55 ( 81.38% , ic50 = 1.13 m ) ; 52 ( 89.34% , ic50 = 0.81 compounds 52 ( yellow carbons ) and 54 ( turquoise carbons ) docked in the catalytic cleft of the bont / a lc with key amino acid residues emphasized in stick . the carbonyl oxygen from the acetoxy group at the cholate c(7 ) ( steroid numbering ) coordinates the enzyme s zn ( not explicitly indicated ) . for full ligand interaction diagrams the presence of a methyl group ( cyan ) on the c(24 ) nitrogen , 54 , limits hydrogen bonding to only glu55 . another key interaction observed for inhibitors 1257 was a strong cation interaction between the ligand s 4,7-acq moiety and arg231 , with surrounding residues forming a pocket in which the aromatic component engages in hydrophobic collapse ( figures s1s6 ) . favorable interactions were also observed between substituents on the c(3 ) positions of the inhibitors cholic acid components and proximal residues . importantly , these interactions appear to be heavily weighted with respect to rationalizing the inhibitory efficacies of ligands having neutral and basic c(3 ) substituents . ligands with acetoxy and hydroxy groups can engage in an h - bond with tyr366 ( figure s1 and figure s2 ) . moreover , the methyl group from the acetoxy substituent may additionally stabilize the ligand enzyme binding event through hydrophobic interactions with phe369 and tyr366 . hence , these interactions explain the more efficacious inhibition provided by the 3-acetoxy vs 3-hydroxy derivative ( cf . 12 vs 17 ) . ligands possessing a c(3 ) amino group engage in favorable salt bridges and h - bonds with glu252 and/or asp370 ( figure 1 ; see also figures s1 and s3 ) . ammonium cations derived from primary amino groups engage in interactions with both amino acid residues ( 52 , 53 , 54 , and 55 ) , while derivatives with tertiary nitrogens achieve interactions with only asp370 ( 50 , 51 , 57 , and 56 ) . these strong electrostatic interactions could explain the increase in activity for the ligands with basic c(3 ) substituents when compared to noncharged substituents . furthermore , ligands with primary ammonium cations can form two stabilizing interactions , while those with tertiary ammonium cations can engage in only one salt bridge . this is in accord with their activities and explains why compounds with the same substitution at c(24 ) follow the same relative activity patterns : 52 ( ic50 = 0.81 m ) vs 50 ( ic50 = 1.02 m ) ; 54 ( ic50 = 1.14 m ) vs 56 ( ic50 = 2.27 m ) . the same relationship was also observed for compounds from the acq3 series , e.g. , 53 ( ic50 = 1.04 m ) vs 51 ( ic50 = 1.34 m ) . the above observations can also explain the change in inhibitory activity in the sequence 52 50 56 . in this series of ligands , the introduction of methyl groups on positions c(24 ) and c(3 ) leads to a decrease in interaction strength with acidic residues and consequently to a decrease in the activities of the ligands . the experimental results seem to further support the models based on observed decreases in the activities of ligands possessing bocnh or meso2nh groups ( 42 , 44 vs 54 , and 43 , 45 vs 55 ) . next we examined derivatives possessesing 4,7-acq substituents linked to both positions c(3 ) and c(24 ) of the steroidal core . one series possesses an -bonded acq substituent at c(3 ) ( 66 and 68 , figure s4 ) , and the other possesses a -bonded acq at c(3 ) ( 64 , 65 , and 67 , figure 2 ; see also figure s6 ) . the simulation results indicate that compounds of the -c(3 ) series , i.e. , 68 and 66 , oriented similarly to the members of the mono - acq series but with some emerging differences . most distinctive was a lack of any interactions by the amino group at c(3 ) . for these ligands , three points of binding are most interesting : ( 1 ) the carbonyl oxygen of the oriented acetoxy group at c(7 ) coordinates the enzyme s zn ( figure s4 ) ; ( 2 ) a salt bridge interaction between the protonated amino group at c(24 ) and glu164 ; ( 3 ) a subset involving favorable hydrophobic contacts between the 4,7-acq component tethered to the c(3 ) position of the cholic acid component and a subpocket formed by amino acids tyr366 , tyr251 , val245 , phe243 , leu263 , phe273 , and leu367 . both 68 and 66 achieve further binding stabilization through stacking with tyr366 , and for 68 , an additional h - bond with the hydroxyl substituent of tyr366 is predicted ( figure s4 ) . in addition , the second 4,7-acq ring is stabilized via h - bonding with thr176 . furthermore , model analysis indicates proximity between the side chain of pro239 and the quinoline core of the c(24 ) 4,7-acq component . the role of proline interactions with aromatic moieties in the stabilization of small molecule binding has been reported ( figure s4 ) . compounds 64 ( orange carbons ) , 65 ( purple carbons ) , and 67 ( turquoise carbons ) docked in the catalytic cleft of bont / a lc with key amino acid residues emphasized in stick . the carbonyl oxygen from the acetoxy group at c(12 ) ( steroid numbering ) coordinates the enzyme s zn ( not explicitly indicated ) . for full ligand interaction diagrams -c(3 ) derivatives are predicted to dock in the substrate binding cleft in the opposite direction versus -c(3 ) derivatives . however , the -c(3 ) derivatives retain the three major binding points indicated above : i.e. , coordination of the enzyme s catalytic zn , a salt bridge with glu164 , and hydrophobic stabilization of the 4,7-acq aromatic core are all observed . in the -c(3 ) series the coordination to zn is achieved via the carbonyl oxygen of aco - c(12 ) and the salt bridge with glu164 is formed via the protonated amino group at c(3 ) ( figure 2 ; see also figure s6 ) . favorable hydrophobic interactions are observed between the 4,7-acq core linked to c(24 ) and the pocket composed of the same amino acid residues observed for -c(3 ) derivatives . additional interactions in this area are stacking and h - bonding with tyr366 . new , stabilizing interactions include h - bonds or salt bridges between c(24 ) ammonium groups and glu252 and asp370 . for 67 and 65 , the second 4,7-acq component engages in h - bonding with asn174 ; this interaction is possible because of a longer alkyl chain spacer compared to 64 . the carbonyl oxygen from the acetate at c(7 ) of compounds 67 , 64 , and 65 is positioned within interaction distance with the backbone nitrogen atom of ser259 ( 3.14 for 67 , 2.90 for 64 , and 3.87 for 65 ) , and because of the relative flexibility of the binding site , it is feasible to assume that a hydrogen bond can be formed ( figure 2 ) . the docking scores for these three derivatives followed the same trends as their ki values ( table 4 ) . examination of an overlap of the docked structures of the -c(3 ) series ( 66 and 68 ) and -c(3 ) series ( 64 , 65 , and 67 ; figure s5 , 3-derivatives in pink shades , 3-derivatives in green shades ) indicated that the latter is rotated 180 in the binding site around the midpoint of the c(8)c(9 ) bond . however , the key functional groups of compounds in both series superimposed well . for example , superimpositions of the compounds aliphatic aminos , aromatic aminos , quinolone cores , and acetate groups that coordinate the enzyme s zn ion are all uniform . hence , it appears that differences caused by alterations in the stereochemistry at c(3 ) are compensated for by the length of the alkyl spacers connecting the 4,7-acq fragments and the central cholate components . finally , the described overlap of the - and -series of bis-4,7-acq derivatives rationalizes the good activities of diastereomer mixtures 62 and 63 . falciparum strains : d6 ( cq susceptible ( cqs ) strain ) , w2 ( cq resistant ( cqr ) strain ) , and tm91c235 ( thailand , a multidrug - resistant ( mdr ) strain ) , following well - established protocols . in brief , the malaria sybr green i based fluorescence ( msf ) assay is a microtiter plate drug sensitivity assay that uses the presence of malarial dna as a measure of parasitic proliferation in the presence of antimalarial drugs or experimental compounds . the intercalation of sybr green i dye , and its resulting fluorescence , is relative to parasite growth , and a compound that inhibits the growth of the parasite will result in lower fluorescence . all of the compounds exhibited better ic90 activity against both cqr strains in comparison to cq , and nine are more potent ( ic90 ) against cqs strain d6 than cq . when compared to mfq , seven compounds are more active against cqr strain w2 , and 19 are more active against mdr strain tm91c235 ( ic90 ) . in addition , three inhibitors are more active than artemisinin ( art ) against the w2 and tm91c235 strains : 56 , 54 , 16 , and 56 , 54 , 42 and 16 , respectively ( ic90 ) . resistance index ( ri ) is defined as the ratio of the ic90 for the resistant versus sensitive strain , tm91c235/d6 and w2/d6 , respectively . selectivity index ( si ) is defined as ratio of the ic50 for hepg2/w2 , hepg2/d6 , and hepg2/tm91c235 , respectively . stability of compounds during incubation with human ( hlm ) and mouse ( mlm ) liver microsomes . for this compound index is defined as the ratio of the ic50 for the resistant versus sensitive strain , tm91c235/d6 and w2/d6 , respectively ; average of greater than eight replicates . a lower score indicates better binding of a given ligand in the substrate cleft . in general , acq2 derivatives are more active than acq3 derivatives , with an increase in the length of the alkyl chain resulting in a further decrease in antimalarial activity ( e.g. , 14 , 15 vs 12 , 13 , and the compounds may further lose activity via the following set of transformations aco in contrast to bont / a lc inhibitory activity , further analysis of the compounds antimalarial activities did not establish a unique pattern of substituent influence on potency . highly active ( ic90 < 5 nm ) and appreciably active ( ic90 < 10 nm ) compounds possess c(3)-amino , c(3)-oh , and c(3)-n - boc substituents on the steroidal core ; the same holds for the least active compounds ( ic90 > 100 nm ) . however , alkylation of the secondary aliphatic amino group at c(24 ) ( yielding a tertiary amine ) favors antimalarial activity , as 56 and 54 are the most active derivatives . in general , the antimalarial behavior of the new derivatives suggests that the contribution of substituents to antimalarial activity is more complex than appears at first glance . in this regard , the compounds will be submitted for thorough analysis of their respective physicochemical parameters . the resistance index ( ri , table 3 ) clearly indicates that most of the compounds are more active against the cqs d6 strain than against the cq resistant strains . the above results clearly indicate that the mode of action of the steroid - derived aminoquinolines is similar to that of 9 ( i.e. , cq ) , although the compounds are much more active than the parent compound , as shown in table 3 . specifically , derivative 23 was selected for the evaluation of its ability to inhibit -hematin formation using a -hematin inhibitory activity assay ( bhia ) . subsequently , it was found that 23 has an ic50 that is 3 times lower than that of 9 ( cq ) ( i.e. , 23 ( ic50 = 0.399 ) vs 9 ( ic50 = 1.09 ) ) , thereby demonstrating a significantly better ability to interact with hematin than 9 . hence , these results indicate that our present compounds , like other acq - based antimalarials , most likely interfere with the heme detoxification mechanism of malaria . in general , the examined derivatives showed low metabolic stability ( table 3 ) . out of the complete set of compounds , 57 , 50 , and 54 showed desirable in vitro stability , with t1/2 > 60 min in both human liver microsomes ( hlms ) and mouse liver microsomes ( mlms ) . somewhat less stable were derivatives 57 , 43 , and 17 , which are stable against mlm but are moderately stable ( 56 ) or insufficiently stable ( 43 and 17 ) against hlm . the toxicity of the compounds was estimated using human liver carcinoma cell line ( hepg2 ) and peripheral blood mononuclear cells ( pbmc ) . in general , the hepg2 assay for cytotoxicity estimation revealed that almost all of the compounds are well tolerated by this cell line , possessing ic50 1000 nm . in addition , high selectivity indices ( sis ) were calculated for the most promising antimalarials 54 and 56 ( si > 2300 ) . in addition , 50 , 42 , 40 , 22 , and 16 also displayed low toxicity ( table 3 ) . of the tested compounds , the results indicate that 54 and 56 are the most prominent antimalarial agents . the only difference between the two compounds is substitution at the c(3 ) amino group : nh2 ( 54 ) and ( ch3)2n ( 56 ) . derivative 56 showed somewhat better in vitro activity against all examined malarial strains ( ic50 , ic90 ) ; however , lower toxicity against hepg2 ( 54 , ic50 = 14 380 nm , si = 50002326 ; 56 , ic50 = 9212 nm , si = 68233177 ) and better metabolic stability against hlm led to the selection of 54 for in vivo testing . compound 54 was administrated po at 40 and 160 mg kg day on day 3 , day 4 , and day 5 after infection using the thompson test in icr female mice with 10 1 parasites infected with rodent p. berghei strain . under both treatments mice died on day 13 postinfection . all mice died of malaria , and no signs of tissue damage caused by compound toxicity were observed upon necropsy . unfortunately , this is one of many situations when compound activity in a living organism does not match excellent in vitro activity . second generation 4,7-acq - cholate - based inhibitors of the bont / a lc that are significantly more potent than initially discovered leads were described . the introduction of a basic amino group at the c(3 ) position of the cholic acid component markedly increased inhibitor potencies ( ic50 = 0.812.27 m ) . the observation that the substrate binding cleft of the bont / a lc is very flexible indicates that the enzyme can conformationally adapt to accept large molecules . this led us to evaluate two additional subclasses : bis(steroidal)-4,7acq derivatives and bis(4,7-acq)cholic acid derivatives . the bis(4,7-acq)cholic acid derivatives were found to possess ki values of < 0.4 m , and compound 67 was the most potent derivative ( inhibition ( 20 m ) of 95% , ki = 0.103 m ) . compounds tested during bont / a challenge in primary neurons were found to protect snap-25 by up to 89% . in the presence of 5 l of 100 nm solution of baf , snap-25 protection was slightly higher ; however , upon the addition of 25 l of 100 nm solution of baf , no further increase in protection was observed . these results suggest that our inhibitors can successfully protect snap-25 in the presence of the bont / a holotoxin , with only slight additive effects when given in combination with baf . docking simulations rationalized the bont / a lc inhibitory activities of the compounds in two main terms : ( 1 ) coordination of a carbonyl acetate oxygen to zn and the expulsion of the catalytic water and ( 2 ) formation of strong interactions between ligands and catalytically important amino acid residues , including salt bridges with asp370 and glu164 and stacking / h - bonding with tyr366 . with respect to antimalarial activity , all of the compounds exhibited better ic90 potencies against cqr strains versus cq , and nine compounds are more potent ( ic90 ) against cqs strain d6 than cq . when compared to mfq , seven compounds are more active against cqr strain w2 , and 19 are more active against mdr strain tm91c235 ( ic90 ) . the two most active compounds exhibit very high in vitro activity against cqr strain w2 ( 1.35 nm and 3.74 nm , respectively ) ; at the same time both compounds are very active against the bont / a lc ( 2.27 m , 1.14 m ) . however , it is disappointing that compound 54 , which possesses excellent in vitro antimalarial activity , failed to protect / cure mice when administered at 40 and 160 mg kg day in a thompson test . melting points were determined on a boetius pmhk or a mel - temp apparatus and were not corrected . optical rotations were measured on a rudolph research analytical automatic polarimeter , autopol iv , in dichloromethane ( dcm ) or methanol ( meoh ) as solvent . ir spectra were recorded on a perkin - elmer spectrophotometer ft - ir 1725x . h and c nmr spectra were recorded on a varian gemini-200 spectrometer ( at 200 and 50 mhz , respectively ) and on a bruker ultrashield advance iii spectrometer ( at 500 and 125 mhz , respectively ) employing indicated solvents ( vide infra ) using tms as the internal standard . chemical shifts are expressed in ppm ( ) values and coupling constants ( j ) in hz . esi mass spectra were recorded on an agilent technologies 6210 time - of - flight lc ms instrument in positive ion mode with ch3cn / h2o 1/1 with 0.2% hcooh as the carrying solvent solution . the selected values were as follows : capillary voltage = 4 kv , gas temperature = 350 c , drying gas = 12.l min , nebulizer pressure = 45 psig , fragmentator voltage = 70 v. the elemental analysis was performed on the vario el iii c , h , n , s / o elemental analyzer ( elementar analysensysteme gmbh , hanau , germany ) . thin - layer chromatography ( tlc ) was performed on precoated merck silica gel 60 f254 and rp-18 f254 plates . column chromatography was performed on lobar lichroprep si 60 ( 4063 m ) and rp-18 ( 4063 m ) columns coupled to a waters ri 401 detector and on biotage sp1system with uv detector and flash 12 + , flash 25 + , or flash 40 + columns charged with kp - sil ( 4063 m , pore diameter 60 ) , kp - c18-hs ( 4063 m , pore diameter 90 ) , or kp - nh ( 4063 m , pore diameter 100 ) as adsorbent . compounds were analyzed for purity ( hplc ) using a waters 1525 hplc dual pump system equipped with an alltech select degasser system , and dual 2487 uv vis detector . for data c and d involved use of agylent technologies 1260 liquid chromatograph equipped with quaternary pump ( g1311b ) , injector ( g1329b ) , 1260 als , tcc 1260 ( g1316a ) , and detector 1260 dad vl+ ( g1315c ) . for data processing , n1-(7-chloroquinolin-4-yl)ethane-1,2-diamine ( acq2 ) , n1-(7-chloroquinolin-4-yl)propane-1,3-diamine ( acq3 ) , n1-(7-chloroquinolin-4-yl)butane-1,4-diamine ( acq4 ) , and n1-(7-chloroquinolin-4-yl)ethane-1,6-hexane ( acq6 ) were prepared according to known procedures . alcohol 11 ( 1.08 g , 2.07 mmol ) was dissolved in dcm ( 100 ml ) . pcc ( 670 mg , 3.11 mmol ) was added , and the mixture was stirred at room temperature for 3.5 h. reaction mixture was filtered through a short column of sio2 ( eluent dcm / ea = 7/3 ) . acq2 ( 1.06 g , 4.78 mmol ) was added , and mixture was stirred 15 min , followed by addition of nabh(oac)3 ( 1.01 g , 4.76 mmol ) , and stirring was continued at room temperature overnight . water was added , and following the intensive shaking , the water layer was adjusted to ph 12 with 0.5 m naoh solution . solvent was removed under reduced pressure and crude mixture was prepared for column purification ( dry flash , sio2 , eluent ea , ea / meoh gradient 95/5 9/1 , ea / meoh / nh3 gradient 18/0.1/0.1 18/0.7/0.7 and flash chromatography ( biotage sp1 rp column , eluent meoh / h2o gradient 8/2 9/1 ) ] . mixture of 12 ( 501 mg , 0.69 mmol ) and anhydrous k2co3 ( 169 mg , 1.22 mmol ) in dry meoh ( 14 ml ) was stirred at room temperature for 3 h and poured onto ice / water mixture . [ ]d20 + 0.040 ( c 1.4 10 g / ml , dcm ) . ir ( kbr ) : 3309w , 2930w , 2863w , 1715 m , 1612w , 1580s , 1535w , 1449w , 1373 m , 1332w , 1241s , 1138w , 1074 m , 1019 m , 967w , 937w , 897w , 878w , 850w , 805w , 767w cm . h nmr ( 500 mhz , cdcl3 , ) : 8.51 ( d , j = 5.1 , h - c(2 ) ) , 7.94 ( d , j = 1.8 , h - c(8 ) ) , 7.74 ( d , j = 9.1 , h - c(5 ) ) , 7.36 ( dd , j1 = 9.1 , j2 = 2.1 , h - c(6 ) ) , 6.38 ( d , j = 5.4 , h - c(3 ) ) , 5.95 ( bs , h - n ) , 5.115.07 ( m , h - c(12 ) ) , 4.924.88 ( m , h - c(7 ) ) , 3.553.47 ( m , h - c(3 ) ) , 3.363,30 ( m , 2h - c(9 ) ) , 3.02 ( t , j = 5.6 , 2h - c(10 ) ) , 2.672.57 ( m , 2h - c(24 ) ) , 2.09 ( s , ch3coo ) , 2.07 ( s , ch3coo ) , 0.90 ( s , ch3-c(10 ) ) , 0.82 ( d , j = 6.5 , ch3-c(20 ) ) , 0.71 ( s , ch3-c(13 ) ) . c nmr ( 125 mhz , cdcl3 , ) : 170.61 , 170.58 , 151.97 , 149.91 , 149.03 , 134.80 , 128.58 , 125.18 , 121.34 , 117.34 , 99.12 , 75.43 , 71.56 , 70.82 , 49.75 , 47.62 , 47.48 , 45.02 , 43.39 , 41.97 , 41.01 , 38.65 , 37.74 , 34.94 , 34.84 , 34.25 , 33.28 , 31.33 , 30.49 , 28.94 , 27.29 , 26.69 , 25.55 , 22.77 , 22.52 , 21.60 , 21.40 , 17.88 , 12.20 . ( + ) esi - hrms ( m / z ) : [ m + 2h ] 341.7034 ( error , 2.20 ppm ) , [ m + h ] 682.3980 ( error , 0.22 ppm ) . combustion analysis ( c39h56cln3o5h2o ) : calculated c 66.88 , h 8.35 , n 6.00 ; found c 66.51 , h 7.82 , n 6.10 . mixture of 12 ( 200 mg , 0.28 mmol ) , 37% formaldehyde ( 68.3 l , 0.84 mmol ) , nabh(oac)3 ( 237 mg , 1.12 mmol ) in dcm ( 9 ml ) was stirred at room temperature until disappearance of starting compound ( tlc , sio2 , ea / meoh / nh3 = 9:1:1 ) . ph of water layer was adjusted to ph 12 using 0.5 m naoh , and the mixture was worked up in the usual manner . product was isolated after column chromatography ( dry flash , sio2 , gradient hexane / ea 4:6 ea ) . [ ]d20 + 0.089 ( c 1.3 10 g / ml , dcm ) . ir ( kbr ) : 3276w , 2947 m , 2872w , 1732s , 1678s , 1614 m , 1456w , 1379 m , 1333w , 1251s , 1201s , 1134 m , 1025w , 966w , 889w , 832w , 801w cm . h nmr ( 500 mhz , cdcl3 , ) : 8.53 ( d , j = 5.0 , h - c(2 ) ) , 7.96 ( d , j = 2.0 , h - c(8 ) ) , 7.68 ( d , j = 9.0 , h - c(5 ) ) , 7.37 ( dd , j1 = 2.0 , j2 = 9.0 , h - c(6 ) ) , 6.37 ( d , j = 5.5 , h - c(3 ) ) , 6.08 ( bs , h - n , exchangeable with d2o ) , 5.10 ( s , h - c(12 ) ) , 4.954.90 ( m , h - c(7 ) ) , 4.614.54 ( m , h - c(3 ) ) , 3.313.27 ( m , 2h - c(9 ) ) , 2.772.30 , ( m , 2h - c(10 ) ) , 2.472.34 ( m , 2h - c(24 ) ) , 2.28 ( s , ch3-n ) , 2.08 ( s , ch3coo ) , 2.07 ( s , ch3coo ) , 2.05 ( s , ch3coo ) , 0.92 ( s , ch3-c(10 ) ) , 0.83 ( d , j = 6.6 , ch3-c(20 ) ) , 0.72 ( s , ch3-c(13 ) ) . c nmr ( 125 mhz , cdcl3 , ) : 170.52 , 170.47 , 170.35 , 152.05 , 149.85 , 149.01 , 134.79 , 125.13 , 121.24 , 117.35 , 99.25 , 75.41 , 74.04 , 70.67 , 57.85 , 55.15 , 47.93 , 45.08 , 43.52 , 41.26 , 40.91 , 39.60 , 37.73 , 35.24 , 34.64 , 34.59 , 34.28 , 33.59 , 31.89 , 31.21 , 29.66 , 28.95 , 27.42 , 26.85 , 25.60 , 24.15 , 22.80 , 22.54 , 21.58 , 21.47 , 21.34 , 17.98 , 12.23 . ( + ) esi - hrms ( m / z ) : [ m + h ] 738.42320 ( error , 1.55 ppm ) . combustion analysis for ( c42h60cln3o61.5h2o ) : calculated c 65.91 , h 8.30 , n 5.49 ; found c 65.86 , h 8.33 , n 5.40 . solution of 16 ( 460 mg , 0.64 mmol ) and ibx ( 896 mg , 3.2 mmol ) was dissolved in dmso . cf3co2h ( 104.4 l ) was added , and the mixture was stirred at room temperature . after 6 h mixture was poured onto water and ph was adjusted to ph 11 using naoh . the obtained solid was filtered off , washed with h2o , and dried in vacuo . crude product was purified by column chromatography ( dry flash , sio2 , eluent ea / meoh gradient 95/5 9/1 , ea / meoh / nh3 gradient 18/1/1 9/1/1 ) and flash chromatography ( biotage sp1 rp column , eluent meoh / h2o gradient 8/2 9/1 ) . [ ]d20 + 0.069 ( c 1.3 10 g / ml , dcm ) . ir ( atr ) : 2933w , 2861w , 2610w , 1714 m , 1609w , 1578 m , 1532w , 1443w , 1374w , 1330w , 1239s , 1169w , 1137w , 1073w , 1023w , 964w , 877w , 847w , 813w , 767w cm . h nmr ( 500 mhz , cdcl3 , ) : 8.52 ( d , j = 5.3 , h - c(2 ) ) , 7.95 ( d , j = 2.1 , h - c(8 ) ) , 7.71 ( d , j = 8.9 , h - c(5 ) ) , 7.35 ( dd , j1 = 8.9 , j2 = 2.1 , h - c(6 ) ) , 6.38 ( d , j = 5.5 , h - c(3 ) ) , 5.91 ( bs , h - n ) , 5.14 ( bs , h - c(12 ) ) , 5.034.96 ( m , h - c(7 ) ) , 3.373.29 ( m , 2h - c(9 ) ) , 3.062.94 ( m , h-c(4 ) and 2h - c(10 ) , 2.682.57 ( m , 2h - c(24 ) ) , 2.09 ( s , ch3coo ) , 2.07 ( s , ch3coo ) , 1.02 ( s , ch3-c(10 ) ) , 0.84 ( d , j = 6.6 , ch3-c(20 ) ) , 0.75 ( s , ch3-c(13 ) ) . c nmr ( 125 mhz , cdcl3 , ) : 212.05 , 170.37 , 170.09 , 152.02 , 149.84 , 149.07 , 134.74 , 128.66 , 125.13 , 121.24 , 117.33 , 99.15 , 75.24 , 70.55 , 49.69 , 47.67 , 47.45 , 45.05 , 44.52 , 43.22 , 42.22 , 42.10 , 41.91 , 37.71 , 36.57 , 36.08 , 34.94 , 34.35 , 33.27 , 30.87 , 29.77 , 27.24 , 26.74 , 25.79 , 22.77 , 21.59 , 21.42 , 21.28 , 17.90 , 12.22 . ( + ) esi - hrms ( m / z ) : [ m + 2h ] 340.6947 ( error , 0.49 ppm ) , [ m + h ] 680.3812 ( error , 1.86 ppm ) . combustion analysis ( c39h54cln3o5 ) : calculated c 68.85 , h 8.00 , n 6.18 ; found c 68.35 , h 8.33 , n 6.32 . hplc purity , method a : tr = 8.484 , area 95.76% . mixture of alcohol 33 ( 200 mg , 0.38 mmol ) and pcc ( 123 mg , 0.57 mmol ) in dry dcm ( 40 ml ) was stirred at room temperature for 3 h. reaction mixture was filtered through a short chromatography column . fractions that contained desired aldehyde were collected ( dry flash , sio2 , eluent dcm / ea = 7/3 ) . to the stirred mixture ( 10 min ) of crude aldehyde obtained above , acq2 ( 193 mg , 0.874 mmol ) in dcm ( 20 ml ) at room temperature nabh(oac)3 ( 185 mg , 0.874 mmol ) was added , and stirring was continued at room temperature for 6 h , followed by additional amount of nabh(oac)3 ( 2.75 mg , 13 mmol ) . after the reaction was completed et3n was added , solvent was removed under reduced pressure , and product was isolated after column chromatography ( dry flash , sio2 , ea , ea / meoh gradient 95:5 9:1 , ea / meoh / nh3 = 18:0.1:0.1 18:0.7:0.7 , lobar lichroprep b rp meoh / h2o = 9:1 ) . yield 112.7 mg ( 40% ) . [ ]d20 + 0.036 ( c 1.0 10 g / ml , dcm ) . ir ( kbr ) : 3273 m , 2950 m , 2869 m , 1716 m , 1634 m , 1614 m , 1582s , 1539 m , 1449 m , 1377 m , 1333w , 1250 m , 1142w , 1024w , 969w , 817w , 729w , cm . h nmr ( 200 mhz , cdcl3 , ) : 8.31 ( d , j = 5.7 , h - c(2 ) ) , 8.11 ( d , j = 9.0 , h - c(5 ) ) , 7.77 ( d , j = 1.7 , h - c(8 ) ) , 7.28 ( dd , j1 = 1.6 , j2 = 8.8 , h - c(6 ) ) , 6.29 ( d , j = 5.7 , h - c(3 ) ) , 5.58 ( d , j = 7.7 , h - n ) , 5.06 ( bs , h - c(12 ) ) , 4.89 ( bs , h - c(7 ) ) , 3.60 ( bs , h - c(3 ) and 2h - c(9 ) ) , 3.23 ( bs , 2h - c(10 ) ) , 2.83 ( bs , 2h - c(24 ) ) , 2.08 ( s , ch3coo ) , 2.07 ( s , ch3coo ) , 1.96 ( s , ch3conh ) , 0.90 ( bs , ch3-c(10 ) ) , 0.80 ( d , j = 6.2 , ch3-c(20 ) ) , 0.69 ( bs , ch3-c(13 ) ) . c nmr ( 50 mhz , cdcl3 , ) : 170.38 , 170.24 , 169.33 , 151.12 , 149.39 , 146.17 , 136.03 , 125.81 , 123.30 , 116.80 , 98.63 , 75.43 , 70.77 , 49.65 , 49.42 , 47.52 , 46.69 , 45.01 , 43.32 , 41.39 , 40.84 , 37.62 , 36.07 , 35.36 , 34.76 , 34.21 , 32.90 , 31.25 , 28.86 , 28.04 , 27.26 , 25.51 , 24.56 , 23.53 , 22.67 , 21.63 , 21.36 , 17.72 , 12.15 . h nmr ( 500 mhz , cdcl3 , ) : 8.27 ( d , j = 5.5 , h - c(2 ) ) , 8.24 ( d , j = 8.9 , h - c(5 ) ) , 7.81(s , h - c(8 ) ) , 7.357.30 ( m , h - c(6 ) ) , 6.27 ( d , j = 6.0 , h - c(3 ) ) , 5.37 ( d , j = 7.8 , h - n ) , 5.07 ( bs , h - c(12 ) ) , 4.90 ( d , j = 2.3 , h - c(7 ) ) , 3.903.40 ( m , h - n , exchangeable with d2o , h - c(3 ) and 2h - c(9 ) ) , 3.333.23 ( m , 2h - c(10 ) ) , 2.932.80 ( m , 2h - c(24 ) ) , 2.10 ( s , ch3coo ) , 2.08 ( s , ch3coo ) , 1.97 ( s , ch3conh ) , 0.91 ( bs , ch3-c(10 ) ) , 0.82 ( d , j = 6.4 , ch3-c(20 ) ) , 0.70 ( bs , ch3-c(13 ) ) . c nmr ( 125 mhz , cdcl3 , ) : 170.38 , 170.20 , 169.24 , 151.49 , 148.68 , 145.44 , 136.43 , 126.05 , 125.19 , 123.61 , 116.66 , 98.55 , 75.43 , 70.79 , 49.66 , 49.45 , 47.54 , 46.55 , 45.05 , 43.37 , 41.43 , 40.82 , 37.67 , 36.14 , 35.42 , 34.81 , 34.26 , 32.92 , 31.28 , 29.64 , 28.90 , 28.09 , 27.30 , 25.56 , 24.32 , 23.59 , 22.73 , 21.68 , 21.41 , 17.76 , 12.21 . ( + ) esi - hrms ( m / z ) : [ m + h ] 745.406 89 ( error , 0.26 ppm ) . combustion analysis ( c41h59n4o5 ) : calculated c 68.07 , h 8.22 , n 7.75 ; found c 68.17 , h 8.19 , n 7.51 . hplc purity , method a : tr = 2.088 , area 98.43% . compounds 40 and 58 were obtained according to procedure described for 36 , using alcohol 35 as starting material ( 271 mg , 0.468 mmol ) . column chromatography ( dry flash , sio2 , ea , ea / meoh gradient 9:1 6:4 , ea / meoh / nh3 = 18:0.5:0.5 18:1.5:1.5 , lobar lichroprep rp column meoh / h2o = 9:1 ) . yield 123 mg ( 34% ) of 40 and 168 mg ( 26% ) of 58 . [ ]d20 + 0.057 ( c 1.0 10 g / ml , dcm ) . ir ( kbr ) : 3343w , 2932 m , 2866w , 1712s , 1610w , 1579s , 1527 m , 1448 m 1367s , 1330w , 1235s , 1167 m , 1137 m , 1062w , 1021 m , 998 m , 964w , 937w , 879w , 850w , 804w , 766w , 720w cm . h nmr ( 500 mhz , cdcl3 , ) : 8.51 ( d , j = 5.1 , h - c(2 ) ) , 7.94 ( d , j = 2.2 , h - c(8 ) ) , 7.72 ( d , j = 8.8 , h - c(5 ) ) , 7.35 ( dd , j1 = 9.0 , j2 = 2.0 , h - c(6 ) ) , 6.38 ( d , j = 5.5 , h - c(3 ) ) , 5.95 ( bs , n - h ) , 5.105.07 ( m , h - c(12 ) ) , 4.914.89 ( m , h - c(7 ) ) , 3.353.32 ( m , 2h - c(9 ) ) , 3.28 ( bs , h - c(3 ) ) , 3.03 ( t , j = 5.7 , 2h - c(10 ) ) , 2.702.55 ( m , 2h - c(24 ) ) , 2.10 ( s , ch3coo ) , 2.06 ( s , ch3coo ) , 1.44 ( s , boc - n ) , 0.91 ( s , ch3-c(10 ) ) , 0.82 ( d , j = 6.2 , ch3-c(20 ) ) , 0.71 ( bs , ch3-c(13 ) ) . c nmr ( 125 mhz , cdcl3 , ) : 170.37 , 170.25 , 151.99 , 149.88 , 149.04 , 134.80 , 128.63 , 125.18 , 121.29 , 117.34 , 99.15 , 75.45 , 70.86 , 65.81 , 49.71 , 47.67 , 47.43 , 45.03 , 43.36 , 41.89 , 41.53 , 37.71 , 36.40 , 35.48 , 34.96 , 34.24 , 33.30 , 31.29 , 28.85 , 28.40 , 27.31 , 26.67 , 25.50 , 22.80 , 22.69 , 21.61 , 21.37 , 17.91 , 15.24 , 12.20 . ( + ) esi - hrms ( m / z ) : [ m + h ] 781.464 78 ( error , 2.25 ppm ) . combustion analysis for ( c44h65cln4o6h2o ) : calculated c 66.10 , h 8.45 , n 7.01 ; found c 65.93 , h 8.60 , n 6.12 . [ ]d20 + 0.106 ( c 1.1 10 g / ml , dcm ) . ir ( kbr ) : 3365w , 2934 m , 2867w , 1712s , 1610w , 1580 m , 1520mw , 1448w , 1366s , 1234s , 1166 m , 1061w , 1021 m , 998w , 965w , 938w , 880w , 851w , 804w , 721w cm . h nmr ( 500 mhz , cdcl3 , ) : 8.54 ( d , j = 5.1 , h - c(2 ) ) , 7.97 ( d , j = 1.9 , h - c(8 ) ) , 7.67 ( d , j = 8.6 , h - c(5 ) ) , 7.35 ( dd , j1 = 8.8 , j2 = 1.8 , h - c(6 ) ) , 6.37 ( d , j = 5.4 , h - c(3 ) ) , 6.11 ( bs , n - h ) , 5.08 ( bs , 2 h - c(12 ) ) , 4.924.87 ( m , 2 h - c(7 ) ) , 3.27 ( bs , 2h - c(9 ) and 2 h - c(3 ) ) , 2.882.76 ( m , 2h - c(10 ) ) , 2.532.40 ( m , 2 2h - c(24 ) ) , 2.06 ( s , 2 ch3coo ) , 2.05 ( s , 2 ch3coo ) , 1.45 ( s , 2 boc - n ) , 0.91 ( s , 2 ch3-c(10 ) ) , 0.80 ( d , j = 6.7 , ch3-c(20 ) ) , 0.69 ( bs , 2 ch3-c(13 ) ) . c nmr ( 125 mhz , cdcl3 , ) : 170.34 , 170.25 , 155.10 , 151.98 , 149.82 , 148.93 , 134.83 , 128.65 , 125.16 , 121.18 , 117.35 , 99.27 , 79.20 , 75.39 , 70.83 , 53.89 , 51.76 , 50.79 , 47.94 , 45.06 , 43.37 , 41.53 , 39.70 , 37.69 , 36.39 , 35.48 , 35.18 , 34.24 , 33.56 , 31.28 , 28.85 , 28.40 , 27.34 , 25.51 , 23.90 , 22.78 , 22.69 , 21.61 , 21.30 , 17.97 , 12.22 . ( + ) esi - hrms ( m / z ) : [ m + 2h ] 670.929 92 ( error , 0.94 ppm ) . combustion analysis for ( c77h118cln5o12h2o ) : calculated c 68.04 , h 8.90 , n 5.15 ; found c 67.84 , h 9.05 , n 5.02 . mixture of alcohol 35 ( 3.96 g , 6.84 mmol ) and pcc ( 2.12 g , 9.86 mmol ) in dry dcm ( 349 ml ) was stirred at room temperature for 3 h. mixture was purified through a short chromatography column . fractions containing aldehyde were collected ( dry flash , sio2 , eluent dcm / ea = 7/3 ; tlc , sio2 , ea / hex = 1/1 , visualization by cam ) . acq3 ( 1.48 g 6.3 mmol ) was added , and resulting mixture was stirred at room temperature for 12 h. nabh4 ( 477 mg , 12.6 mmol ) was added in one portion , and stirring was continued for 1 h. the solvent was removed under reduced pressure , and product was isolated upon column chromatography [ dry flash , sio2 , eluent hex / ea gradient 1/1 2/8 , ea , ea / meoh gradient 95/5 6/4 , ea / meoh / nh3 gradient 18/0.5/0.5 18/2.1/2.1 and flash chromatography ( biotage sp1 rp column , eluent meoh / h2o gradient 7/3 meoh ) ] . [ ]d20 + 0.125 ( c 1.4 10 g / ml , dcm ) . ir ( kbr ) : 3313 m , 2937 m , 2868 m , 1717s , 1613 m , 1582s , 1536 m , 1452 m , 1372 m , 1248s , 1211 m , 1173 m , 1137 m , 1064w , 1024 m , 985w , 848w , 852w , 803w , 768w , 608w cm . h nmr ( 200 mhz , cdcl3 , ) : 8.50 ( d , j = 5.6 , h - c(2 ) ) , 7.93 ( d , j = 2.2 , h - c(8 ) , 7.87 ( s , h - n - boc , exchangeable with d2o ) , 7.74 ( d , j = 9.0 , h - c(5 ) , 7.34 ( dd , j1 = 9.0 , j2 = 2.2 h - c(6 ) ) , 6.31 ( d , j = 5.6 , h - c(3 ) ) , 5.11 ( bs , h - c(12 ) ) , 4.94/4.85 ( m , h - c(7 ) ) , 4.39 ( bs , h - n ) , 3.453.20 ( m , 2h - c(9 ) and h - c(3 ) ) , 3.002.85 ( m , 2h - c(11 ) ) , 2.73/2.56 ( m , 2h - c(24 ) ) , 2.11 ( s , ch3coo ) , 2.03 ( s , ch3coo ) , 1.45 ( s , ( ch3)3c - n(boc ) ) , 0.91 ( s , ch3-c(10 ) ) , 0.86 ( d , j = 6.2 , ch3-c(20 ) ) , 0.74 ( s , ch3-c(13 ) ) . c nmr ( 50 mhz , cdcl3 , ) : 170.49 , 170.40 , 155.20 , 152.18 , 150.57 , 149.14 , 134.53 , 128.53 , 124.65 , 122.17 , 117.60 , 98.27 , 79.22 , 75.49 , 70.79 , 50.56 , 49.65 , 47.47 , 45.01 , 44.14 , 43.50 , 41.50 , 37.65 , 36.31 , 35.45 , 34.92 , 34.20 , 33.32 , 31.23 , 28.92 , 28.39 , 27.28 , 27.19 , 26.53 , 25.53 , 22.69 , 21.54 , 21.38 , 17.90 , 12.20 . h nmr ( 500 mhz , cdcl3 , ) : 8.49 ( d , j = 5.3 , h - c(2 ) ) , 7.93 ( d , j = 2.1 , h - c(8 ) , 7.83 ( s , h - n - boc , exchangeable with d2o ) , 7.75 ( d , j = 8.9 , h - c(5 ) , 7.34 ( dd , j1 = 8.8 , j2 = 1.9 h - c(6 ) ) , 6.31 ( d , j = 5.5 , h - c(3 ) ) , 5.10 ( bs , h - c(12 ) ) , 4.934.86 ( m , h - c(7 ) ) , 4.39 ( bs , h - n ) , 3.40 ( t , j = 5.7 , 2h - c(9 ) ) , 3.28 ( bs , h - c(3 ) ) , 2.972.89 ( m , 2h - c(11 ) ) , 2.732.56 ( m , 2h - c(24 ) ) , 2.10 ( s , ch3coo ) , 2.02 ( s , ch3coo ) , 1.45 ( s , ( ch3)3c - n(boc ) ) , 0.91 ( s , ch3-c(10 ) ) , 0.85 ( d , j = 6.4 , ch3-c(20 ) ) , 0.73 ( s , ch3-c(13 ) ) . c nmr ( 125 mhz , cdcl3 , ) : 170.42 , 170.32 , 151.94 , 150.62 , 148.93 , 134.65 , 128.35 , 124.73 , 122.20 , 117.56 , 98.26 , 75.49 , 70.82 , 50.49 , 49.46 , 47.54 , 45.06 , 43.97 , 43.50,41.55 , 37.71 , 36.36 , 35.49 , 34.97 , 34.24 , 33.35 , 31.28 , 28.94 , 28.41 , 27.32 , 27.10 , 26.44 , 25.57 , 22.78 , 22.71 , 21.56 , 21.38 , 17.93 , 12.24 . ( + ) esi - hrms ( m / z ) : [ m + h ] 795.4822 ( error , + 0.06 ppm ) . combustion analysis for ( c45h67cln4o60.5h2o ) : calculated c 67.18 , h 8.52 , n 6.96 ; found c 67.01 , h 8.42 , n 6.74 . method c : tr = 15.915 , area 98.61% . into stirred mixture of crude aldehyde ( 3.80 g , 6,6 mmol ) and acq3 ( 3.11 g , 13 mmol ) in dcm ( 126 ml ) for 10 min at room temperature , nabh(oac)3 ( 2.8 mg , 13 mmol ) was added . stirring was continued for 12 h , followed by additional amount of nabh(oac)3 ( 2.8 mg ) and stirring for 3 h. then the solvent was removed under reduced pressure and products were isolated upon column chromatography [ dry flash ( sio2 , eluent ea , ea / meoh gradient of 95/5 9/1 , ea / meoh / nh3 gradient of 18/0.5/0.5 9/1/1 , and flash chromatography ( biotage sp1 , rp column , eluent meoh / h2o gradient 75/25 95/5 , n - h column , eluent ea / hex gradient 6/3 ea ) . yield 1.44 g of 41 ( 20% ) and 800 mg of bis - steroidal amine 59 ( 13% ) . [ ]d20 + 0.131 ( c 1.6 10 g / ml , dcm ) . ir ( kbr ) : 3392 m , 2950s , 2869 m , 1735s , 1612 m , 1581s , 1529 m , 1450 m , 1372s , 1244s , 1171s , 1065 m , 1023 m , 965 m , 939w , 882w , 852w cm . h nmr ( 500 mhz , cdcl3 , ) : 8.50 ( d , j = 5.4 , h - c(2 ) ) , 7.93 ( d , j = 1.9 , h - c(8 ) ) , 7.757.65 ( m , h - c(5 ) and h - n - boc , exchangeable with d2o ) , 7.33 ( dd , j1 = 8.8 , j2 = 1.9 , h - c(6 ) ) , 6.32 ( d , j = 5.4 , h - c(3 ) ) , 5.05 ( bs , 2 h - c(12 ) ) , 4.88 ( d , i = 2.2 , 2 h - c(7 ) ) , 4.45 ( bs , h - n ) , 3.453.20 ( m , 2h - c(9 ) and 2 h - c(3 ) ) , 2.722.58 ( m , 2h - c(11 ) ) , 2.542.40 ( m , 2 2h - c(24 ) ) , 2.04 ( s , 4 ch3coo ) , 1.44 ( s , 2 ( ch3)3c - n(boc ) ) , 0.90 ( s , 2 ch3-c(10 ) ) , 0.75 ( d , j = 6.4 , 2 ch3-c(20 ) ) , 0.67 ( s , 2 ch3-c(13 ) ) . c nmr ( 125 mhz , cdcl3 , ) : 170.30 , 170.24 , 155.11 , 152.06 , 150.48 , 149.08 , 134.53 , 128.47 , 124.59 , 121.98 , 117.49 , 98.41 , 79.17 , 75.36 , 70.77 , 54.98 , 54.23 , 50.71 , 47.81 , 44.98 , 44.41 , 43.31 , 41.49 , 37.63 , 36.31 , 35.44 , 35.02 , 35.18 , 33.68 , 31.23 , 28.83 , 28.36 , 27.16 , 25.47 , 24.53 , 23.59 , 22.70 , 22.64 , 21.55 , 21.26 , 17.84 , 12.15 . ( + ) esi - hrms ( m / z ) : [ m + h ] 1354.8695 ( error , 0.02 ppm ) . combustion analysis for c78h120cln5o12 : calculated c 69.13 , h 8.92 , n 5.17 ; found c 69.05 , h 8.84 , n 5.37 . hplc purity , method c : tr = 11,964 , area 97.93% . method d : tr = 5.892 , area 96.83% . into stirred solution of 38 ( 121 mg , 0.16 mmol ) and formaldehyde ( 37% , 54 l , 0.72 mmol ) in 5 ml of dcm when starting 38 was completely consumed ( tlc , sio2 , ea / meoh / nh3 = 9:1:1 ) , the reaction mixture was suspended in dcm / h2o mixture , ph was adjusted to 12 with 0.5 m naoh , and transferred to separatory funnel and further worked - up in usual manner . product was isolated after column chromatography ( flash , biotage sp1 nh column , eluent hexane / ea 4:6 ea ) . [ ]d20 + 0.048 ( c 1.0 10 g / ml , dcm ) . ir ( kbr ) : 3406 m , 2949 m , 2869 m , 1731s , 1611w , 1582s , 1538w , 1452 m , 1377 m , 1320 m , 1247s , 1150 m , 1081w , 1023 m , 969w , 880w , 849w , 812w , 763w , 733w cm . h nmr ( 500 mhz , cdcl3 , ) : 8.54 ( d , j = 5.5 , h - c(2 ) ) , 7.99 ( d , j = 2.0 , h - c(8 ) , 7.73 ( d , j = 9.1 , h - c(5 ) , 7.36 ( dd , j1 = 8.9 , j2 = 2.0 h - c(6 ) ) , 6.37 ( d , j = 5.5 , h - c(3 ) ) , 6.29 ( bs , h - n , exchangeable with d2o ) , 5.09 ( bs , h - c(12 ) ) , 4.954.90 ( m , h - c(7 ) ) , 4.604.37 ( m , h - n , exchangeable with d2o ) , 3.34 ( bs , 2h - c(9 ) ) , 3.203.10 ( m , h - c(3 ) ) , 2.96 ( s , ch3-n ) , 2.80 ( t , j = 5.8 , 2h - c(10 ) ) , 2.522.40 ( m , 2h - c(24 ) ) , 2.32 ( s , ch3-so2n ) , 2.07 ( s , 2 ch3coo ) , 0.92 ( s , ch3-c(10 ) ) , 0.82 ( d , j = 6.6 , ch3-c(20 ) ) , 0.71 ( s , ch3-c(13 ) ) . c nmr ( 125 mhz , cdcl3 , ) : 175.77 , 170.41 , 170.24 , 151.41 , 150.17 , 148.33 , 135.13 , 127.96 , 125.31 , 121.49 , 117.23 , 99.02 , 75.36 , 70.66 , 57.70 , 54.89 , 54.02 , 47.84 , 45.06 , 43.38 , 41.92 , 41.61 , 41.22 , 39.53 , 37.69 , 37.36 , 35.43 , 35.16 , 34.07 , 33.44 , 31.19 , 29.40 , 28.86 , 27.37 , 25.49 , 23.64 , 22.78 , 22.61 , 22.14 , 21.57 , 21.30 , 17.94 , 12.21 . ( + ) esi - hrms ( m / z ) : [ m + h ] 773.407 72 ( error , + 0.53 ppm ) . combustion analysis for ( c41h61cln4o6s2h2o ) : calculated c 60.83 , h 8.09 , n 6.92 , s 3.96 ; found c 60.55 , h 7.92 , n 6.81 , s 4.00 . hplc purity , method a : tr = 1.868 , area 99.31% . method b : tr = 1.859 , area 98.31% . into a stirred solution of 40 ( 1.00 g , 1.28 mmol ) and formaldehyde ( 37% , 190 l , 2.55 mmol ) in dry meoh ( 30 ml ) was added a suspension of nabh3cn ( 81 mg , 1.28 mmol ) and anhydrous zncl2 ( 88 mg , 0.64 mmol ) in meoh ( 10 ml ) . after 2 h of stirring at room temperature the additional amount of nabh3cn ( 41 mg , 0.64 mmol ) and anhydrous zncl2 ( 44 mg , 0.32 mmol ) were added as meoh ( 10 ml ) suspension and stirring was continued for 1 h. solvent was evaporated under reduced pressure , and remaining residue was transferred into a separatory funnel as dcm suspension . water was added and ph was adjusted to ph 12 with 0.5 m naoh , and the organic layer was worked up in the usual manner . product was obtained after flash column chromatography ( biotage sp nh column , eluent hexane / ea 4:6 2:6 ) . [ ]d20 + 0.163 ( c 2.0 10 g / ml , dcm ) . ir ( atr ) : 3376w , 2949 m , 2868w , 2802w , 1731s , 1611w , 1581s , 1528 m , 1452 m , 1374s , 1331w , 1246s , 1171 m , 1063w , 1023 m , 965w , 940w , 881w , 850w , 807w , cm . h nmr ( 500 mhz , cdcl3 , ) : 8.46 ( d , j = 5.4 , h - c(2 ) ) , 7.89 ( s , j = 2.0 , h - c(8 ) ) , 7.71 ( d , j = 8.9 , h - c(5 ) ) , 7.30 ( dd , j1 = 8.8 , j2 = 2.1 , h - c(6 ) ) , 6.33 ( d , j = 5.4 , h - c(3 ) ) , 6.07 ( s , h - n - boc , exchangeable with d2o ) , 5.06 ( s , h - c(12 ) ) , 4.924.88 ( m , h - c(7 ) ) , 4.42 ( bs , h - c(3 ) ) , 3.45 ( s , ch3-n ) , 3.33 ( bs , 2h - c(9 ) ) , 3.052.95 ( m , 2h - c(11 ) ) , 2.702.43 ( m , 2h - c(24 ) and h - n , exchangeable with d2o ) , 2.07 ( s , ch3coo ) , 2.04 ( s , ch3coo ) , 1.42 ( s , ( ch3)3c - n(boc ) ) , 0.88 ( s , ch3-c(10 ) ) , 0.79 ( d , j = 6.6 , ch3-c(20 ) ) , 0.68 ( s , ch3-c(13 ) ) . c nmr ( 125 mhz , cdcl3 , ) : 170.34 , 170.27 , 151.80 , 149.92 , 148.87 , 134.77 , 128.36 , 125.11 , 121.43 , 117.29 , 99.98 , 75.41 , 70.82 , 50.43 , 49.66 , 47.60 , 47.33 , 44.97 , 43.30 , 41.83 , 41.48 , 37.65 , 36.35 , 35.44 , 34.89 , 34.19 , 33.23 , 31.24 , 28.79 , 28.35 , 27.25 , 26.45 , 25.45 , 22.74 , 22.63 , 21.57 , 21.31 , 17.85 , 12.15 . ( + ) esi - hrms ( m / z ) : [ m + h ] 795.481 85 ( error , 0.43 ppm ) . combustion analysis for c45h67cln4o6 : calculated c 67.94 , h 8.49 , n 7.04 ; found c 67.72 , h 8.63 , n 6.75 . method c : tr = 9.936 , area 98.20% . into a stirred mixture of crude 48 ( 817 mg , 0.90 mmol ) and formaldehyde ( 37% , 0.2 ml , 2.8 mmol ) in dry methanol ( 8 ml ) at room temperature was added a suspension of anhydrous zncl2 ( 61 mg , 0.45 mmol ) and nabh3cn ( 56.8 mg , 0.90 mmol ) in dry methanol ( 8 ml ) . the reaction mixture was stirred for 24 h , and the solvent was removed under reduced pressure . the residue was transferred into a separatory funnel as dcm solution , and water was added . water layer was extracted with dcm ( 2 15 ml ) , and combined organic layers were washed with saturated nahco3 and brine and dried over anhydrous na2so4 . solution was filtered off . the solvent was removed under reduced pressure , and crude product ( 625 mg ) was dissolved in solution of piperidine in dcm ( 20% , 27 ml ) . reaction mixture was stirred for a further 24 h at room temperature , and the solvent was removed under reduced pressure , transferred into a separatory funnel as dcm solution , and worked up in the usual manner . the product was isolated after column chromatography purification [ dry flash , sio2 , eluent meoh , meoh / nh3 gradient 95/5 9/1 and flash chromatography ( biotage sp1 , eluent ea / hex , gradient 6/4 ea , gradient ea / meoh 95/5 9/1 ) ] . [ ]d20 + 0.056 ( c 1.8 10 g / ml , dcm ) . ir ( kbr ) : 331w , 2935s , 2866 m , 2772w , 1730s , 1610w , 1580s , 1534w , 1449 m , 1375 m , 1330w , 1242s , 1140w , 1069w , 1024w , 966w , 877w , 808w cm . h nmr ( 200 mhz , cdcl3 , ) : 8.52 ( d , j = 5.1 , h c(2 ) ) , 7.94 ( d , j = 2.2 , h - c(8 ) ) , 7.73 ( d , j = 9.0 , h - c(5 ) ) , 7.35 ( dd , j1 = 9.0 , j2 = 2.2 , h - c(6 ) ) , 6.39 ( d , j = 5.6 , h - c(3 ) ) , 5.94 ( bs , h - n , exchangeable with d2o ) , 5.08 ( bs , h - c(12 ) ) , 4.904.70 ( m , h - c(7 ) ) , 3.403.26 ( m , 2h - c(9 ) ) , 3.082.96 ( m , 2h - c(11 ) ) , 2.702.54 ( m , 2h - c(24 ) ) , 2.27 ( s , ( ch3)2n ) , 2.10 ( s , ch3coo ) , 2.07 ( s , ch3coo ) , 0,90 ( s , ch3-c(10 ) ) , 0.82 ( d , j = 6.2 , ch3-c(20 ) ) , 0.71 ( s , ch3-c(13 ) ) . c nmr ( 50 mhz , cdcl3 , ) : 170.68 , 170.58 , 152.05 , 149.86 , 134.74 , 128.63 , 125.12 , 121.28 , 117.31 , 99.12 , 75.49 , 70.94 , 64.98 , 49.71 , 47.56 , 47.43 , 44.96 , 43.30 , 42.19 , 41.90 , 41.68 , 37.64 , 35.67 , 34.89 , 34.32 , 33.23 , 32.74 , 31.50 , 28.86 , 27.24 , 26.66 , 25.40 , 24.05 , 22.67 , 21.60 , 21.40 , 17.81 , 12.13 . ( + ) esi - hrms ( m / z ) : [ m + h ] 709.446 54 ( error , + 1.59 ppm ) . combustion analysis for c41h61cln4o42h2o : calculated c 66.06 , h 8.79 , n 7.52 ; found c 66.22 , h 8.98 , n 7.38 . method b : tr = 7.942 , area 96.45% . compound 40 ( 100 mg , 0.128 mmol ) was stirred at room temperature in dcm / tfa mixture ( 2 ml , 1:1 , v / v ) for 3 h. solvent was removed under reduced pressure , and the residue was transferred as dcm solution to separatory funnel and worked up in the usual manner . [ ]d20 + 0.046 ( c 1.1 10 g / ml , dcm ) . ir ( atr ) : 3286w , 2932 m , 2860 m , 1724s , 1610w , 1578s , 1533w , 1448 m , 1374 m , 1330w , 1238s , 1135w , 1077w , 1021 m , 964w , 936w , 878w , 847w , 806w , 766w cm . h nmr ( 500 mhz , cdcl3 , ) : 8.51 ( d , j = 5.4 , h - c(2 ) ) , 7.94 ( d , j = 2.0 , h - c(8 ) ) , 7.72 ( d , j = 8.7 , h - c(5 ) ) , 7.35 ( dd , j1 = 8.9 , j2 = 2.2 , h - c(6 ) ) , 6.37 ( d , j = 5.4 , h - c(3 ) ) , 5.95 ( bs , h - n , ) , 5.08 ( bs , h - c(12 ) ) , 4.924.86 ( m , h - c(7 ) ) , 3.373.30 ( m , 2h - c(9 ) ) , 3.03 ( t , j = 5.7 , 2h - c(11 ) ) , 2.682.56 ( m , 2h - c(24 ) and h - c(3 ) ) , 2.10 ( s , ch3coo ) , 2.07 ( s , ch3coo ) , 0.90 ( s , ch3-c(10 ) ) , 0.82 ( d , j = 6.4 , ch3-c(20 ) ) , 0.71 ( s , ch3-c(13 ) ) . c nmr ( 125 mhz , cdcl3 , ) : 170.64 , 170.60 , 151.98 , 149.88 , 149.01 , 134.80 , 128.59 , 125.17 , 121.31 , 117.33 , 99.13 , 75.49 , 70.89 , 51.64 , 49.72 , 47.62 , 47.43 , 45.03 , 43.39 , 41.89 , 41.49 , 39.41 , 37.76 , 35.50 , 34.94 , 34.36 , 33.28 , 31.42 , 31.16 , 28.98 , 27.29 , 26.64 , 25.58 , 22.78 , 21.64 , 21.46 , 17.87 , 12.19 . ( + ) esi - hrms ( m / z ) : [ m + 2h ] 341.210 68 ( error , 0.04 ppm ) . combustion analysis for c39h57cln4o4h2o : calculated c 66.98 , h 8.50 , n 8.01 ; found c 66.84 , h 8.32 , n 7.92 . method b : tr = 7.973 , area 95.08% . compound 44 ( 800 mg , 1.0 mmol ) was stirred at room temperature in dcm / tfa mixture ( 16 ml , 1:1 , v / v ) for 1.5 h. then the solvent was removed under reduced pressure , and the residue was transferred as dcm solution into a separatory funnel and worked up in the usual manner . product was isolated after flash column chromatography ( biotage sp , sio2 , eluent ea / meoh , gradient 9:1 55/45 ) . [ ]d20 + 0.080 ( c 2.0 10 g / ml , dcm ) . ir ( atr ) : 3409 m , 2947s , 2865 m , 1729 m , 1668w , 1612w , 1582s , 1536w , 1451 m , 1378 m , 1331w , 1244s , 1157w , 1136w , 1077w , 1024 m , 964w , 879w , 848w , 807w cm . h nmr ( 500 mhz , cdcl3 , ) : 8.49 ( d , j = 5.3 , h - c(2 ) ) , 7.92 ( d , j = 2.0 , h - c(8 ) ) , 7.65 ( d , j = 8.9 , h - c(5 ) ) , 7.31 ( dd , j1 = 8.9 , j2 = 2.1 , h - c(6 ) ) , 6.33 ( d , j = 5.3 , h - c(3 ) ) , 6.026.96 ( m , h - n , exchangeable with d2o ) , 5.085.03 ( m , h - c(12 ) ) , 4.86 ( d , j = 2.7 , h - c(7 ) ) , 3.24 ( q , j = 5.5 , 2h - c(9 ) ) , 2.70 ( d , j = 5.9 , 2h - c(11 ) ) , 2.602.52 ( m , h - c(3 ) ) , 2.432.30 ( m , 2h - c(24 ) ) , 2.23 ( s , ch3-n ) , 2.05 ( s , 2 ch3coo ) , 0.87 ( s , ch3-c(10 ) ) , 0.79 ( d , j = 6.6 , ch3-c(20 ) ) , 0.68 ( s , ch3-c(13 ) ) . c nmr ( 125 mhz , cdcl3 , ) : 170.56 , 152.05 , 149.73 , 149.04 , 134.64 , 128.64 , 125.00 , 121.19 , 117.29 , 99.18 , 75.43 , 70.84 , 57.80 , 55.11 , 51.59 , 47.78 , 45.02 , 43.38 , 41.46 , 41.25 , 39.57 , 39.49 , 37.71 , 35.49 , 35.13 , 34.29 , 33.46 , 31.36 , 31.23 , 28.94 , 27.33 , 25.54 , 24.04 , 22.73 , 21.59 , 21.37 , 17.90 , 12.15 . ( + ) esi - hrms ( m / z ) : [ m + h ] 695.429 22 ( error , 0.78 ppm ) . combustion analysis for c40h59cln4o4h2o : calculated c 67.34 , h 8.62 , n 7.85 ; found c 66.98 , h 8.73 , n 7.62 . method c : tr = 11.413 , area 96.83% . into a stirred solution of 54 ( 190 mg , 0.27 mmol ) and formaldehyde ( 37% , 50 l , 0.67 mmol ) in dry meoh ( 2 ml ) was added a suspension of nabh3cn ( 13 mg , 0.21 mmol ) and anhydrous zncl2 ( 29 mg , 0.21 mmol ) in dry meoh ( 2 ml ) . after the reaction was completed ( tlc , sio2 , ea / nh3/meoh = 9:1:1 ) the solvent was evaporated under reduced pressure . the ph was adjusted to ph 12 with 0.5 m naoh , and the reaction was further worked up in the usual manner . product was obtained after column chromatography ( dry flash , sio2 , eluent ea , ea / meoh gradient 9:1 8/2 , ea / nh3/meoh = 18:0.5:0.5 18:1.5:1.5 ) . [ ]d20 + 0.023 ( c 2.0 10 g / ml , dcm ) . ir ( atr ) : 3424s , 2929s , 2867 m , 1731s , 1612w , 1582s , 1531w , 1452 m , 1377 m , 1330w , 1244s , 1163w , 1070w , 1026 m , 966w , 879w , 807w cm . h nmr ( 500 mhz , cdcl3 , ) : 8.53 ( d , j = 5.4 , h - c(2 ) ) , 7.95 ( d , j = 2.0 , h - c(8 ) ) , 7.68 ( d , j = 9.0 , h - c(5 ) ) , 7.35 ( dd , j1 = 8.9 , j2 = 2.1 , h - c(6 ) ) , 6.37 ( d , j = 5.4 , h - c(3 ) ) , 6.00 ( bs , h - n , exchangeable with d2o ) , 5.115.07 ( m , h - c(12 ) ) , 4.924.87 ( m , h - c(7 ) ) , 3.313.26 ( m , 2h - c(9 ) ) , 2.74 ( t , j = 5.8 , 2h - c(11 ) ) , 2.462.35 ( m , 2h - c(24 ) ) , 2.27 ( s , ( ch3)2n - c(3 ) ) , 2.26 ( s , ch3-n ) , 2.08 ( s , ch3coo ) , 2.07 ( s , ch3coo ) , 0.91 ( s , ch3-c(10 ) ) , 0.83 ( d , j = 6.6 , ch3-c(20 ) ) , 0.72 ( s , ch3-c(13 ) ) . c nmr ( 125 mhz , cdcl3 , ) : 170.64 , 170.59 , 152.15 , 149.81 , 149.14 , 134.75 , 128.78 , 125.10 , 121.22 , 117.38 , 99.28 , 75.54 , 70.99 , 65.02 , 57.86 , 55.20 , 47.80 , 45.10 , 43.43 , 42.10 , 41.81 , 41.30 , 39.62 , 37.77 , 35.77 , 35.27 , 34.44 , 33.56 , 32.61 , 31.59 , 28.96 , 27.43 , 25.50 , 24.15 , 23.93 , 22.82 , 22.76 , 21.65 , 21.40 , 17.98 , 12.23 . ( + ) esi - hrms ( m / z ) : [ m + h ] 723.460 48 ( error , 0.80 ppm ) . combustion analysis for c42h63cln4o4 : calculated c 69.73 , h 8.78 , n 7.74 ; found c 69.52 , h 8.60 , n 7.63 . compound 58 ( 140 mg , 0.104 mmol ) was stirred at room temperature in dcm / tfa mixture ( 2 ml , 1:1 , v / v ) for 3 h. solvent was removed under reduced pressure , and the residue was dissolved in dcm containing 5% meoh and transferred into a separatory funnel . the organic layer was washed twice with saturated na2co3 , once with brine , and dried over anhydrous na2so4 . [ ]d20 + 0.032 ( c 1.2 10 g / ml , dcm ) . ir ( kbr ) : 2936 m , 2864 m , 1726s , 1611w , 1580 m , 1526w , 1446w , 1375s , 1331w , 1238s , 1199.6 m , 1132 m , 1076w , 1021s , 964w , 937w , 878w , 832w , 801w , 765w , 720w cm . h nmr ( 500 mhz , cdcl3 , ) : 8.53 ( d , j = 5.3 , h - c(2 ) ) , 7.96 ( d , j = 2.1 , h - c(8 ) ) , 7.64 ( d , j = 9.2 , h - c(5 ) 7.35 ( dd , j1 = 8.9 , j2 = 2.1 , h - c(6 ) ) , 6.37 ( d , j = 5.3 , h - c(3 ) ) , 6.035.99 ( m , h - n , exchangeable with d2o ) , 5.07 ( bs , 2 h - c(12 ) ) , 4.914.86 ( m , 2 h - c(7 ) ) , 3.293.21 ( m , 2h - c(9 ) and 2 h2n ) , 2.842.76 ( m , 2h - c(11 ) ) , 2.642.55 ( m , 2 h - c(3 ) ) , 2.512.37 ( m , 2 2h - c(24 ) ) , 2.07 ( s , 2 ch3coo ) , 2.06 ( s , 2 ch3coo ) , 0.90 ( s , 2 ch3-c(10 ) ) , 0.80 ( d , j = 6.6 , 2 ch3-c(20 ) ) , 0.69 ( s , 2 ch3-c(13 ) ) . c nmr ( 125 mhz , cdcl3 , ) : 170.60 , 152.16 , 149.74 , 149.13 , 134.72 , 128.81 , 125.09 , 121.06 , 117.37 , 99.33 , 75.45 , 70.90 , 53.85 , 51.74 , 51.65 , 47.93 , 45.07 , 43.39 , 41.51 , 39.73 , 39.53 , 35.17 , 34.36 , 33.58 , 31.42 , 31.28 , 28.99 , 27.33 , 25.59 , 23.97 , 22.79 , 21.65 , 21.41 , 17.95 , 12.21 . ( + ) esi - hrms ( m / z ) : [ m + 2h ] 570.878 12 ( error , 1.85 ppm ) [ m + h ] 1140.748 97 ( error , 2.51 ppm ) . combustion analysis for c67h102cln5o8h2o : calculated c 69.43 , h 9.04 , n 6.04 ; found c 69.39 , h 9.27 , n 5.91 . mixture of 22 ( 385 mg , 0.57 mmol ) and acq2 ( 251 mg , 1.13 mmol ) in dry meoh ( 9 ml ) was stirred at room temperature for 12 h. then nabh3cn ( 45 mg , 0.72 mmol ) was added in one portion , and after 1 h of stirring at room temperature glacial acoh ( 3 drops ) was added . solvent was removed under reduced pressure , and product was isolated by column chromatography ( dry flash , sio2 , eluent ea , ea / meoh gradient 95/5 1/9 , ea / meoh / nh3 gradient 18/1/1 9/1/1 , flash , biotage sp1 , rp column , eluent gradient meoh / h2o 8/2 9/1 ) . [ ]d20 + 0.070 ( c 1.4 10 g / ml , dcm ) . ir ( atr ) : 3379w , 2929 m , 2863w , 1722 m , 1611w , 1581s , 1535w , 1450w , 1375 m , 1330w , 1248 m , 1170w , 1137w , 1078w , 1024w , 965w , 879w , 849w , 807w cm . h nmr ( 200 mhz , cdcl3 , ) : 8.50 ( d , j = 5.6 , 2 h - c(2 ) ) , 7.94 ( d , j = 1.7 , 2 h - c(8 ) ) , 7.787.68 ( m , 2 h - c(5 ) ) , 7.377.26 ( m , 2 h - c(6 ) ) , 6.426.32 ( m , 2 h - c(3 ) ) , 6.115.89 ( m , 2 h - n exchangeable with d2o ) , 5.11(bs , h - c(12 ) ) , 4.90 ( bs , h - c(7 ) ) , 3.33 ( bs , 2 2h - c(9 ) ) , 3.082.92 ( m , 2 2h - c(10 ) and h - c(3 ) ) , 2.702.55 ( m , 2h - c(24 ) ) , 2.10 ( s , ch3coo ) , 2.08 ( s , ch3coo ) , 0.96 ( s , ch3-c(10 ) ) , 0.83 ( d , j = 6.2 , ch3-c(20 ) ) , 0.72 ( s , ch3-c(13 ) ) . c nmr ( 50 mhz , cdcl3 , ) : 170.60 , 170.51 , 170.44 , 151.96 , 149.92 , 148.97 , 134.77 , 128.56 , 128.49 , 125.18 , 125.14 , 121.39 , 121.22 , 121.13 , 117.31 , 99.16 , 99.09 , 75.51 , 71.01 , 70.90 , 57.83 , 52.04 , 49.71 , 47.60 , 47.43 , 44.96 , 44.79 , 43.30 , 42.72 , 42.32 , 41.92 , 41.44 , 37.65 , 36.96 , 36.13 , 35.34 , 34.87 , 34.71 , 33.47 , 33.25 , 31.37 , 31.10 , 29.99 , 28.93 , 28.44 , 28.26 , 27.26 , 26.62 , 25.62 , 24.87 , 23.22 , 22.74 , 21.56 , 21.29 , 17.83 , 12.13 . h nmr ( 500 mhz , cdcl3 , ) : 8.50 ( d , j = 5.3 , 2 h - c(2 ) ) , 7.977.91 ( m , 2 h - c(8 ) ) , 7.767.64 ( m , 2 h - c(5 ) ) , 7.377.30 ( m , 2 h - c(6 ) ) , 6.416.34 ( m , 2 h - c(3 ) ) , 6.075.84 ( m , 2 h - n exchangeable with d2o ) , 5.145.04 ( m , h - c(12 ) ) , 4.934.86 ( m , h - c(7 ) ) , 3.393.25 ( m , 2 2h - c(9 ) ) , 3.072.9 ( m , 2 2h - c(10 ) ) 2.682.56 ( m , h - c(3 ) ) , 2.262.13 ( m , 2h - c(24 ) ) , 2.09 ( s , ch3coo ) , 2.07 ( s , ch3coo ) , 2.01 ( s , ch3coo ) , 0.96 ( s , ch3-c(10 ) ) , 0.92 ( s , ch3-c(10 ) ) , 0.850.79 ( m , ch3-c(20 ) ) , 0.72 ( s , ch3-c(13 ) ) , 0.71 ( s , ch3-c(13 ) ) . c nmr ( 125 mhz , cdcl3 , ) : 170.44 , 151.96 , 149.91 , 149.01 , 134.79 , 128.61 , 128.55 , 125.19 , 125.16 , 121.34 , 121.09 , 117.34 , 99.18 , 99.11 , 75.54 , 71.05 , 57.87 , 52.11 , 49.71 , 47.67 , 47.47 , 45.02 , 44.87 , 43.35 , 42.75 , 42.39 , 41.96 , 41.52 , 37.73 , 36.19 , 34.93 , 34.76 , 33.54 , 33.31 , 31.16 , 30.06 , 28.99 , 28.33 , 27.31 , 26.70 , 25.67 , 25.53 , 24.95 , 23.25 , 22.79 , 21.58 , 21.31 , 17.91 , 12.19 . ( + ) esi - hrms ( m / z ) : [ m + 2h ] 443.2334 ( error , + 0.03 ppm ) [ m + h ] 885.4587 ( error , + 0.96 ppm ) . combustion analysis ( c50h66cl2n6o41.5 h2o ) : calculated c 65.77 , h 7.62 , n 9.20 ; found c 65.30 , h 7.24 , n 9.07 . hplc purity , method b : tr = 1.788 , area 98.83% . method c : tr1 = 10.371 , tr2 = 10 566 , area 95.15% . the structure of bont / a lc and binding site ( bs ) used in this study was obtained from previously published crystal structure of protein ( pdb code 3ds9 ) . all ligand structures were built in maestro ( maestro , version 9.3 ; schrdinger , llc : new york , ny , 2012 ) , and their structures were optimized in semiempirical nndo module of schrdinger suite 2012 ( schrdinger suite 2012 semiempirical nddo module ; schrdinger llc : new york , ny , 2012 ) , using the rm1 method . ionization states for all ligands were examined using sequential pka estimation in the epik module ( epik , version 2.3 ; schrdinger , llc : new york , ny , 2012 ) . ligand log p were calculated using qikprop module ( qikprop , version 3.5 ; schrdinger , llc : new york , ny , 2012 ) . grid docking of the ligands were performed using glide ligand docking ( glide , version 5.8 ; schrdinger , llc : new york , ny , 2012 ) , using standard precision and flexible ligand sampling , without any additional constraints . generated docking structures were refined and used for the next step . all structures were docked in ionization state proposed by results of epik pka prediction for ph 7.0 2.0 , with protonated aliphatic nitrogen atoms in structure . for the next step , induced fit docking was used ( schrdinger suite 2012 induced fit docking protocol ; glide , version 5.8 ; schrdinger , llc : new york , ny , 2012 ; prime , version 3.1 ; schrdinger , llc : new york , ny , 2012 ) . all modeling was performed within a ph range of 7.0 1.0 , which encapsulates the ph at which the in vitro experiments were conducted ( i.e. , ph 7.3 ) . for the derivatives presented herein , computational predictions showed that the pka values of the aliphatic nitrogen atoms range between 9.50 and 10.80 1.0 , while the pka values of nitrogen atoms that are part of the acq moiety range between 7.40 and 8.00 1.20 . accordingly , it is plausible to assume that all aliphatic nitrogen atoms in the ligand structures are protonated and that the aromatic acq nitrogens are only partially protonated ( around 50% ) . thus , simulations of ligand binding were conducted with protonated aliphatic nitrogen atoms and neutral aromatic nitrogens . refine only option . considering the relative inflexibility of the steroidal core and different conformational space of the - and -substituents at c(3 ) , differences in binding modes could be expected as well . taking into account the overall flexibility of the bont / a lc substrate cleft , it is clear that residues in the enzyme s substrate cleft would undergo some additional conformational changes compared to smaller ligands . for these reasons , the substrate cleft was slightly relaxed via induced fit dockings , which was followed by glide grid docking . the highest scoring models were further optimized using the opls2005 force field with water as a solvent ( the solvent effects were simulated using the analytical generalized born / surface area mode ) , using polak ribiere conjugate gradient with a maximum of 5000 iterations and gradient convergence threshold of 0.05 kj / mol . during minimization , the backbone of the protein was constrained and the catalytic engine zn and his223 , his227 , and glu262 were frozen in space . in previous binding simulations with smaller inhibitors , it was oriented into the binding cleft , thereby adding to the hydrophobic nature of the pocket . now , met253 reorients away from the binding cleft , thus providing enough space for the docking of a larger steroidal bis - acq system . the hplc - based assay was used to evaluate small molecule mediated inhibition of the bont / a lc in vitro . in brief , this assay utilizes an n - terminal acetylated , c - terminal aminated synthetic peptide identical in sequence to residues 187203 of snap-25 . compounds with intrinsic fluorescence quenching capability do not interfere with the activity measurements of this assay because substrate hydrolysis is determined by hplc separation of the products from the substrate , followed by measurement of the peak areas . the assay mixture contained 40 mm hepes0.05% tween ( ph 7.3 ) , bont / a recombinant lc , peptide substrate , 0.5 mg / ml bovine serum albumin , and various concentrations of small molecule . the assay was run at 37 c , quenched by the addition of tfa , and analyzed using reverse - phase hplc . zinc ( 50 m excess ) was added to the assays to remove compounds that are zinc chelating agents . percent inhibition measurements were performed in duplicate , and standard deviations were less than 25% for all . ic50 calculations were determined by measuring enzyme activity at nine different smnpi concentrations and in the absence of the small molecule . the small molecule concentrations used in the measurements were determined by estimating the ic50 value and moving in 1 log increments in either direction of the estimated value . the resulting kinetic data were subsequently fit to the langmuir isotherm , vi / vo = 1/{1 + ( [ i]/ic50 ) } , using nonlinear regression analysis . for ki determination , reaction velocity versus substrate concentration was plotted for multiple small molecule concentrations . these plots were analyzed using global kinetic analysis . subsequently , the data were fit to a model of competitive inhibition and analyzed by nonlinear regression analysis . ventral spinal cords were isolated from day 6 embryonic chicken embryos and cultured by the method of kuhn as previously described . after trypsinization for 25 min , the cells were dissociated by trituration and preplated in dulbecco s modified eagle medium for 1 h to enrich the neuronal population by adherence of non - neuronal cells to the culture dish . cells remaining in suspension were centrifuged for 6 min at 1250 rpm , resuspended in liebowitz s l15 medium with 10% fetal bovine serum and n3 supplements , and plated onto six - well culture plates that were first coated with poly - l - lysine ( 1 mg / ml overnight ) and then with laminin ( 710 g / ml for 34 h ) . , the cells were incubated with 10 nm bont / a and inhibitor for 3.5 h. the cells were then rinsed with fresh culture medium , scraped , and collected into tubes . the tubes were centrifuged , and supernatant was removed and replaced with 30 l of lysis buffer . after overnight storage at 80 c , the tubes were thawed , vortexed briefly , and incubated on ice for 10 min . lysates were centrifuged at high speed at 4 c for 30 min to remove debris . 12% gels were used for sds page , and proteins were blotted onto pvdf membranes . western blots were probed with a 1:2500 dilution of smi-81 anti - snap-25 antibody ( covance research products inc . , berkeley , ca ) and 1:20000 horseradish peroxidase ( hrp ) conjugated goat anti - mouse ( pierce , rockford , western blots were developed using a chemiluminescent substrate and imaged with a biorad versadoc system . protein bands were quantitated by densitometry with quantity one software ( biorad , hercules , ca ) . the in vitro antimalarial drug susceptibility screen is a modification of the procedures first published by desjardins et al . , with modifications developed by milhous et al . , and the details are given in ref ( 47 ) . all synthesized aminoquinolines were screened in vitro against p. falciparum strains : cq and mfq susceptible strain d6 ( clone of sierra i / unc isolate ) , cq resistant but mfq susceptible strain w2 ( clone of indochina i isolate ) , and cq and mfq resistant strain tm91c235 ( clone of southeast asian isolate ) . the compounds were assayed for hepg2 toxicity according to protocol given in ref ( 47 ) . peripheral blood mononuclear cells ( pbmc ) were separated from whole heparinized blood of healthy volunteers . blood was diluted with phosphate buffered saline ( pbs ) ( 1:1 ) and layered on histopaque solution . after centrifugation , interface cells were collected and washed three times with pbs . after counting , cells were resuspended in nutrient medium . nutrient medium was rpmi-1640 medium , supplemented with 10% fetal calf serum ( fcs ) , 1% glutamine ( 200 mm ) , 1% -mercaptoethanol ( 5 m ) , 1% penicillin ( 10000 iu ml ) , and 1% streptomycin ( 10 mg ml ) . cells were seeded ( 200 000 cells per well ) in 96-well plates in nutrient medium with 0.5% phytohemagglutinin ( pha ) ( 1 mg ml ) and different concentrations of organic compounds in dmso . cells were incubated for 72 h at 37 c in a humidified atmosphere with 6% co2 . absorbances were recorded at = 540 and 670 nm on a recorder ( lkb 5060 - 006 , lkb , vienna , austria ) . percent of cell growth inhibition were calculated from formula ( asample aneg.control)/(apos.control aneg.control ) = x and ( 1 x ) 100 = % inhibition . ic50 concentrations were defined as the concentration of a drug needed to inhibit cell survival by 50% , compared with vehicle - treated control .

spine disorders affect various sections of the spine and have a variety of causes1 , 2 . most back pain occurs in the lumbo - sacral and cervical regions of the spine . low back pain is defined as pain localized between the ribs and the gluteal folds , with or without symptoms in a lower limb . pain in the upper part of the spine usually means pain in the cervical spine and upper thorax , with or without syndrome in an upper limb3 . back pain is divided into two groups that differ in pathogenesis and in therapeutic procedure . it is caused by a specific disease of the motor system or injury due to external causes . the second group consists of non - specific low back pain which does not have a specific cause and may be caused by musculoskeletal disorders or injury due to other reasons . non - specific low back pain occurs in 90% of people who suffer from back pain4 . according to the duration of the pain , back pain is divided into acute up to six weeks , subacute from 6 to 12 weeks , and chronic lasting more than 12 weeks5 . the international association for the study of pain states that chronic pain is a condition lasting more than three months6 . the occurrence of pain in the cervical and lumbar spine in children and adolescents is influenced by : anthropometric , lifestyle , mechanical strain on the back , psychological , social and behavioral factors7 , 8 . pain in the lumbar spine is most frequently associated with sports activities such as judo , golf , rugby , basketball , baseball , soccer , athletics and volleyball9 . pain causes a change in the muscle tension of particular muscle groups , contributes to changes in body shape , disturbs motor control and causes abnormal motion patterns10 , 11 . taking into consideration the universality of the back pain problem in children and adolescents we decided to check if symptoms occur in children and young people studying in primary , junior high and high schools of ballet . the predetermined objectives of this research were to assess in which segments of spine pain arises most frequently before and now and to investigate whether there was a statistically significant difference in the range of pain sensation between before and now . our hypothesis was that the feeling of back pain is weaker now than before at particular levels of education ( primary school level , junior high school and high school ) . the subjects were 237 children and young people learning in ballet schools in gdask and d in poland . the average age of the respondents at this school level was 11.240.77 years ( mean standard deviation ) , and their time of study in ballet school was 2.14 0.74 years . their average age was 14.01 0.84 years , and their mean time of study in ballet schools was 4.641.24 years . their average age was 17.010.77 years , and their mean time of study in ballet school was 7.471.54 years . a numeric rating scale ( nrs ) was used to determine the sense of pain in the cervical , thoracic and lumbar spine12 . students were asked to point to the level of pain on the day of research now using a numerical scale for cervical , thoracic and lumbar spine pain , ( mark in red ) , and then to mark the feeling of pain for each section of the spine at any time during school attendance , before the assessment of pain using the nrs was performed once . for analyzing the result , the reported of pain feelings was only taken into account in terms of before and now for the various sections of the spine . the children and young people did not report the occurrence of musculoskeletal injuries and did not report pain as meningeal or radicular . the feelings of back pain were analyzed using , percentage assessment , point statistics and statistical tests to compare the groups . this research was carried out with the permission of the local bioethics committee of poznan university of medical sciences , and after receiving the consent of the subjects parents or guardians . among the 80 children at primary level , 14 students reported pain in the cervical spine , 6 in the thoracic spine and 15 in the lumbar part of the spine . regarding pain now , 12 children declared feelings of back pain in the cervical spine , and 10 declared pain in the thoracic and lumbar regions of the spine ( table 1table 1.number of students reporting pain before and now in different sections of the spine at the primary , junior high , and high school levelscbeforethbeforelbeforecnowthnowlnownumer of pupilsprimary school level146151221080junior high school level19931751593high school level201041753064number of people532587261255237c : cervical spine , th : thoracic spine , pain in the cervical spine , 9 in the thoracic spine and 31 in the lumbar section of spine . regarding pain now , 7 students reported pain in the cervical spine , 5 in thoracic spine and 15 in the lumbar part of the spine . at the high school level , among the students 64 , 20 reported pain before in the cervical spine , 10 in thoracic spine , and 41 in the lumbar spine . regarding pain now 7 students reported pain in the cervical spine , five in the thoracic segment , and 30 in the lumbar part of the spine . c : cervical spine , th : thoracic spine , l : lumbar spine the assessment of pain at the primary , junior high and high school levels showed that reported pain occurred in a smaller number of respondents now rather than before in each part of the spine . considering the occurrence of pain in each section of spine for the whole group of students , it should be noted that 53 pupils reported pain before in the cervical spine but only 26 respondents now . in the thoracic spine 25 students felt pain in the lumbar spine before but only 12 students reported pain now . eighty - seven pupils reported pain in the lumbar spine before , but only 55 students reported pain now however , it should be pointed that the most reported location of feelings of pain was the lumbar spine both before and now . it can be seen that for learners at the primary level , pain sensations were reduced in different sections of the spine . in the cervical spine pain , it was reduced by 20% , in the thoracic spine by 66.67% , and in the lumbar section of the spine by 33.33% ( table 2table 2.the percentages of students at the primary , junior high , and high school levels reporting pain both before and nowprimary school leveljunior high school levelhigh school levelcthlcthlcthla80.00%33.33%66.67%40.00%55.56%59.38%35.00%45.45%75.61%b20.00%66.67%33.33%60.00%44.44%40.63%65.00%54.55%24.39%a : % of people declaring pain now to declaring pain before , b : percentage % of people reporting fewer pain sensations now than before , c : cervical spine , th : thoracic spine , l : lumbar spine ) . a : % of people declaring pain now to declaring pain before , b : percentage % of people reporting fewer pain sensations now than before , c : cervical spine , th : thoracic spine , l : lumbar spine reduced pain sensations were also reported among junior high school students . for the low - cervical spine segment , pain decreased by 60.00% , in the thoracic spine by 44.44% , and in the lumbar spine by 40.63% ( table 2 ) . respondents at the high school level , students also reported a reduction in pain : in the cervical part by 65.00% , in the thoracic spine by 54.55% , and in the lumbar spine by 24.39% ( table 2 ) . the basic characteristics of the primary , junior high and high school students are shown in table 3table 3.basic results of the primary , junior high , and high school level studentsprimarymiddlehighbeforepainnowpainbeforepainnowpainbeforepainnowpainmin.0.000.000.000.000.000.001st qu0.000.000.000.001.000.00median0.000.001.000.001.001.00mean0.450.300.660.341.130.673rd qu1.001.001.001.002.001.00max.2.002.003.002.003.002.00 . at the primary level all the results are the same for pain feeling now , and before with the exception of the mean . the mean value for feeling pain before is 0.45 and that for now is 0.30 ( table 3 ) . for the junior high school level , the minimum , 1st and 3rd quartile values of pain are the same for before and now . however , the median value for the feelings of pain before is 1.00 and that of now is 0.00 . the values for the mean of before and now are different : the value of 0.66 for before is bigger than the mean of now , 0.34 ( table 3 ) . also the maximum value for before=3.00 , is bigger than the maximum value of all values for before are bigger than now , except for the minimum ( 0.00 ) and median ( 1.00 ) . the 1st quartile value of before is 1.00 , and that of now is 0.00 . the mean value of now is 1.13 , and that of before is 0.67 . the 3rd quartile value of before is 2.00 , and that of now is 1.00 . the maximum value of before is 3.00 and that of now is 2.00 ( table 3 ) . subsequently whether there were significant differences between pains before and now among the tested groups at the three education levels was determined . since the p values of the paired t - test for the primary , junior high and high school levels are less than 0.05 , there are a statistically significant differences in pain sensation between before and now . moreover , the p value of the t - test between the primary and junior high school ballet students is 0.5926 , the p value of the t - test between the primary and high school ballet students is 0.0004 , and the p value of t - test between the junior high ballet school and high ballet school is equal 0.0015 . now are weaker than before at the various levels of education ( table 1 ) . perhaps the reason for was the time of the survey ( it was conducted at the beginning of the school year the assessments of pain expressed as percentages for each of the levels of education and the various sections of the spinal column show a reduction of pain between before and now the values of the basic characteristics are different at primary level for the mean values ( table 3 ) , at the secondary level for the median , mean and maximum values ( table 3 ) , and at the high school level for the values for the mean , 3rd quartile and maximum ( table 3 ) . in the comparison of the results for all levels of teaching only the mean the p value of the t - test between the primary and junior high school ballet is nt significant different ( 0.5926 ) , the p value of the t - test between the primary and high school ballet is significant different ( 0.0004 ) , and the p value of the t - test between the junior high and high school ballet is also significantly different ( 0.0015 ) . sports activities and , a high level of physical activity affects low back pain occurrence7 , 9 , 14 . wedderkopp and co - authors did not find a relationship between the level of physical activity and back pain presence15 . however , in ballet low back pain is more often seen in boys and men . it is related to their requirement to lift and hold female dancers of the ground16 . previously conducted studies indicate that ballet , as a form of physical activity , causes low back pain9 . poor core stability is , perhaps , a predisposing factor for injuries to the lower limbs and lumbar spine in dancers , due to poor stabilization of the lumbo - pelvic complex17 . studies show that segmental muscle stabilization training as part of a core stability program decreased low back pain18 . yang and co - authors point out that the main cause of pain in the lumbar spine is abnormal function of the core muscles in this segment19 . headache , stomach pain and sleeping trouble can have a strong influence on the occurrence of back pain in children20,21,22,23 . the occurrence of low back pain may also be affected by the short height of a child . shorter children report more psychosocial problems in group relationships , which may predispose them to pain occurrence14 . sensory receptors are associated with fast - conductive fibers , a , which are stimulated by harmless stimuli . according to the results of research and observation , daily stimuli which stimulate sensitized nociceptive nerve paths nociceptors exhibit , like nerve cells , the phenomenon of adaptability , which is associated with peripheral sensitization in the formation of muscle - skeletal pain conditions . however , it has been shown in the assessment of pain formation that psychological and social factors are the most important elements that help to predict the presence of pain and the degree of its severity24 . pain in muscles caused by exercise is short and provoked by impaired blood flow to working muscles25 . it is possible that pain felt in the cervical , thoracic and lumbar spine reported by the respondents was associated with a weakening of the stabilizing muscles26,27,28 . davarian and colleagues have shown that there is a correlation between the intensity of pain and disability29 .

hepatitis c virus ( hcv ) is a small , hepatotropic , positive - strand rna virus with a genome of approximately 9.6 kb in length.1,2 the hcv genome consists of a single open reading frame that encodes viral proteins and a 5 and 3 noncoding region ( ncr ) . the 5 ncr contains the internal ribosome entry site that initiates the translation of the hcv genome into a single polyprotein;2 the 3 ncr is required for efficient hcv rna replication,3 as it has a specific tripartite structure : a variable region that is important for efficient rna replication ; a poly(u / uc ) tract of variable length ; and a highly conserved x tail.46 studies have demonstrated that the conserved elements in the 3 ncr , including a minimal poly(u ) tract of about 25 bases , are essential for hcv replication in cell culture and in vivo.710 although the detailed mechanisms by which the 3 ncr elements act on rna replication are not clear , it is likely that binding of one or more viral or host proteins to this rna structure is necessary to establish the replication complex.11 the open reading frame encodes a polyprotein precursor of about 3000 amino acids that is cleaved by host and viral proteases into three structural proteins ( core , e1 , and e2 ) and seven nonstructural proteins ( p7 , ns2 , ns3 , ns4a , ns4b , ns5a , and ns5b ) ( fig . 1).3,12 hcv has six major genotypes and each genotype contains numerous variants.13 each genotype has its own epidemiologic characteristics . genotype 1 is predominant in america14,15 and europe,16,17 followed by genotypes 2 and 3;18,19 and genotype 4 is the most common genotype in africa and the middle east,2022 and is often seen among immigrants or indigenous injection drug users in north america and europe.23,24 genotype 5 is mainly distributed in south africa25 and genotype 6 has been found primarily in asia.2628 hcv infection is a major cause of liver disease , with a high possibility of chronic infection . if left untreated , chronic hcv infection frequently results in progressive fibrosis , cirrhosis , and an increased risk of hepatocellular carcinoma.2 the most recent report from the world health organization estimates that about 150 million people worldwide are chronically infected with hepatitis c virus ( www.who.int ) . unfortunately , there is still no effective vaccine for hcv.29 to date , the standard of care for chronic hcv infection in most countries is combination therapy with pegylated interferon ( ifn ) and ribavirin . however , the sustained virologic response ( svr ) rate is just 4050% in patients infected with hcv genotype 1 and 80% in patients infected with hcv genotypes 2 or 3.2,30,31 since 2011 , treatment for hcv infection has been improved by adding one of the hcv nonstructural protein ns3/4a serine protease inhibitors , telaprevir or boceprevir , to pegylated ifn and ribavirin.32 this regimen improves the svr to 75% in patients infected with hcv genotype 1.33 ns3/4a serine protease plays at least two roles in the hcv life cycle . first , it is responsible for cleaving the hcv polypeptide into individual viral proteins ; it is essential for viral replication and virion assembly.32,34,35 second , ns3/4a protease inhibits the innate immune response to facilitate hcv persistence . therefore , ns3/4a protease is a good target for inhibitors that inhibit hcv replication and restore the host 's innate immunity . following the successful determination of the crystal structure of ns3/4a protease , small molecules specifically binding to the catalytic site of the ns3/4a protease were developed34 and two protease inhibitors , telaprevir and boceprevir , are now used to treat hcv infection in some countries . although protease inhibitor - based therapy significantly improves the svr,32,34 the high cost , severe adverse events , and rapid emergence of resistance mutations associated with these drugs limit their use in the developing countries where treatment is needed most.34 therefore , there is a pressing need to identify new treatment options that are cost- and clinically effective for all hcv genotypes . micrornas ( mirnas ) belong to a noncoding gene family involved in eukaryotic posttranscriptional gene regulation.3639 transcription of mirna genes forms primary transcripts ( pri - mirnas ) that usually contain a hairpin structure . the stem - loop structure is then cleaved by an rnaseiii - like enzyme called drosha , together with its binding partner dgcr8 ( digeorge syndrome critical region 8) , to yield precursor mirnas ( pre - mirnas ) that are subsequently transferred from the nucleus to the cytoplasm with the help of exportin-5 . in the cytoplasm , pre - mirnas are processed by dicer rna polymerase iii into a duplex structure , from which one strand is separated and functions as the guide strand ( functional strand ) to be loaded together with argonaute proteins into an rna - induced silencing complex that recognizes and binds to the target mrna . the other strand is degraded and nonfunctional ( fig . 2).36,4044 mirna exerts its function by degrading the target mrna ( if the mirna has perfect base pairing with its target mrna ) or inhibiting mrna translation ( if there is only partial pairing).4547 the human genome encodes more than 1500 mirnas ( www.mirbase.org , released january 2012 ) . each mirna can regulate numerous target genes and each mrna is likely to be regulated by several mirnas.48,49 the interaction of mirnas and their target mrnas results in a complex network that is involved in almost every cell process , including development , differentiation , proliferation , death , disease pathology , and antiviral defence.5053 most recently , increasing evidence suggests that cellular mirnas and other components of the mirna pathway can interact with viruses at multiple levels to influence viral replication.54,55 in this review , we summarize the mirnas involved in hcv infection and their proposed connection with antiviral innate immunity . type i ifn is a major player in the innate immune response , and is induced through pathways mediated by two distinct pathogen - associated molecular - pattern receptors : toll - like receptors ( tlrs ) and retinoic acid - inducible gene i ( rig - i)/melanoma differentiation - associated gene 5 . the mirnas mir-155 and mir-146a / b have been shown to be involved in these pathways.56 the potential target genes of mir-146a , such as interleukin-1 receptor - associated kinase 1 and 2 , tnf receptor - associated factor 6 , rig - i , and ifn - regulatory factor 5 , play a critical role in these type i induction pathways.57 inducible expression of mir-155 has been observed in both bacterial and viral infections , and may act as a negative feedback regulator of the tlr pathway to dampen the innate immune response.5860 in addition , mir-155 has been shown to be involved in carcinogenesis.6164 two oncogenic viruses , kaposi 's sarcoma - associated herpesvirus and the avian -herpesvirus marek 's disease virus type 1 , can encode mir-155 mimics mir - k12 - 11 and mir - m4 , respectively.6567 both of the virus - encoded mirnas share high sequence similarity with mir-155 and therefore function as an ortholog of cellular mir-155.6568 although numerous lines of evidence point to the fact that more and more mirnas are involved in host innate immunity either directly or indirectly,53,55,69,70 we are just at the very start of understanding the diverse roles of mirna in regulating host immunity . more studies are needed to understand how cellular mirnas interact with the antiviral innate immune response . type i ifn plays an irreplaceable role in anti - hcv defense . quite interestingly , as the key player in hcv therapy , ifn treatment can rapidly modulate the expression of numerous cellular mirnas.71 using microarray technology , pedersen et al . analyzed the expression of cellular mirnas in ifn - stimulated cells and found that the expression levels of about 30 mirnas were altered ( increased or decreased).71 furthermore , they analyzed the sequence of these mirnas and hcv genomic rna and identified eight ifn -induced mirnas ( mir-1 , -30 , -128 , -196 , -296 , -351 , -431 , and -448 ) that matched with hcv genome perfectly . functional assays showed that overexpression of these mirnas by transfection of the mirna mimics reproduced the antiviral effect of ifn in huh7 cells , while their neutralization with anti - mirnas dampened the antiviral effects of ifn against hcv.71 similarly , another independent study reported that mir-196 inhibited hcv rna replication in hcv replicon cells ( genotypes 1b and 2a).72 both studies illustrated that one of the mechanisms for ifn inhibition of hcv rna replication is probably the induction of cellular mirnas that can directly degrade hcv rna . in addition to the ifn - inducible mirnas described above , there are many other mirnas that can also inhibit hcv replication . for example , the overexpression of mir-199a has been reported to suppress hcv rna replication , while inhibition by a specific antisense oligonucleotide upregulated viral replication in two cell lines bearing the replicons hcv-1b or -2a.73 in addition to the direct inhibition , some mirnas inhibit hcv rna replication indirectly by activating the ifn pathway . for example , mir-130a has been shown to be able to inhibit hcv replication in both replicon ( genotype 1b ) and jfh1 infectious models . transfected mir-130a mimic into cultured huh7.5.1 cells stably expressing an hcv genome and found that replication of hcv rna was significantly inhibited.74 further studies showed that the expression levels of type i ifns ( ifn and ifn ) were significantly increased . as huh7.5.1 cells are deficient in both tlr3 and rig - i , two important mediators of type i ifn induction , these cells can not recognize hcv infection and , as a result , almost no type i ifns are produced following virus infection . these results imply that mir-130a may inhibit hcv replication indirectly , probably by restoring the host innate immune response in tlr3- and rig - i - deficient cells.74 however , other investigators have demonstrated that hcv infection upregulates mir-130a , and that interferon - induced transmembrane protein 1 ( ifitm1 ) is a direct target for this mirna : knockdown of mir-130a enhanced ifitm1 expression and reduced hcv replication.75 this study demonstrated that hcv evades innate immune attack by decreasing antiviral ifitm1 expression through mir-130a . since li et al found mir-130a up - regulated the ifn expression , the expression of the ifn - stimulated gene ifitm1 should increase ( although not tested in their study ) . these two studies reached seemingly contradictory results , which may indicate that mir-130a has more than one target , and the interaction of this mirna with the host innate immune system is complex . in any case , the interaction between mir-130a and the host innate immune system deserves further study . mir-122 is the most abundant liver - specific mirna , accounting for around 70% of the total mirna content in mammalian liver tissue.76 it is undetectable in other tissues in mice.76 studies have demonstrated that mir-122 acts as a regulator of fatty - acid metabolism in mouse liver,77 and reduced mir-122 levels are associated with hepatocellular carcinoma.7881 in addition , of all the mirnas , mir-122 is unique in its stimulatory , not inhibitory , role in hcv replication.82 the discovery that mir-122 is required for hcv replication linked a host mirna to a human infectious disease for the first time.83 the role of mir-122 in hcv replication was first reported by jopling et al . in 2005.82 they sequestrated mir-122 with antisense oligonucleotides and found that hcv rna accumulation decreased.82 in addition , they also identified two target sites in the hcv 5 untranslated region that are necessary for hcv replication . since then , a large number of elegant studies have been performed to show that the interaction between mir-122 and viral 5ncr is essential to promote hcv replication.8488 the role of mir-122 as an important , possibly essential , host factor for hcv production makes it an attractive candidate for antiviral therapy . modified antisense agents and small - molecule inhibitors have been developed as potential new lead compounds for drug discovery.89 most recently , treatment with miravirsen ( spc3649 ) efficiently suppressed hcv genotype 1a and 1b infections in chimpanzees , with no evidence of viral resistance or side effects.90 miravirsen , a locked nucleic acid modified dna phosphorothioate antisense oligonucleotide of mir-122 , has also been trialed in patients with chronic hcv genotype 1 , with the results showing a dose - dependent reduction in hcv rna levels without the occurrence of viral resistance.91 in addition to targeting hcv rna directly , yoshikawa et al . found that overexpression of mir-122 suppresses the activity of ifn - stimulated response element ( isre ) while significantly silencing mir-122-enhanced ifn - induced isre activity.92 isres are specific nucleotide sequences located in the promoters of ifn - stimulated genes that encode antiviral proteins and can induce transcription of these genes by binding with ifn - stimulated gene factor 3.93 furthermore , yoshikawa et al . found that silencing mir-122 decreased the expression of suppressor of cytokine signaling , a negative regulator of ifn signaling , in mouse liver , leading to increased ifn anti - hcv activity.92 this study indicates that the anti - hcv effect of mir-122 might also be mediated by decreasing the expression of antiviral proteins or increasing negative regulators of cytokine production . most recently , it has been reported that mir-141 , which can be induced by hcv infection , may also be necessary for efficient hcv replication.94 mir-141 belongs to the mir-200 family , which is believed to play an essential role in tumor suppression by inhibiting epithelial transfected an mir-141 mimic or antagonist into hcv1a - infected hepatocytes to increase or deplete intracellular mir-141 expression , respectively.94 the results showed that overexpression of mir-141 enhanced hcv replication , while depletion of mir-141 inhibited virus replication . meanwhile , they identified dlc-1 ( deleted in liver cancer 1 ) as one of the target genes.94 increasing mir-141 decreased dlc-1 protein levels without a parallel decrease in dlc-1 mrna levels , suggesting that mir-141 primarily targets translational inhibition of dlc-1.94 dlc-1 encodes a member of the rho - gtpase activating protein ( gap ) family of proteins . the rho - gap proteins can specifically catalyze the conversion of the active gtp - bound rhoa protein into the inactive gdp - bound protein.97,98 active rhoa protein is required for ras - mediated tumorigenic transformation,99 and rho - gaps may therefore act as important negative regulators in human carcinogenesis . in hepatocellular carcinoma , homozygous deletion or loss of dlc-1 mrna expression usually occurs in vivo and in vitro.98,100 in addition , restoration of dlc-1 in hepatoma cell lines lacking dlc-1 results in reduced cell proliferation as well as reduced metastatic activity.100 since hcv - infected cells express mir-141 , and its overexpression significantly suppresses dlc-1 expression,94 the results indicate the presence of a novel mechanism of hcv infection - associated mirna - mediated regulation of a tumor suppressor protein , which is worth further exploration . mirnas involved in hcv infection and their interactions with the innate immune system are summarized in table 1 . hcv , hepatitis c virus ; ifn , interferon ; isre , interferon - stimulated response element ; mirna , microrna type i ifn plays an irreplaceable role in anti - hcv defense . quite interestingly , as the key player in hcv therapy , ifn treatment can rapidly modulate the expression of numerous cellular mirnas.71 using microarray technology , pedersen et al . analyzed the expression of cellular mirnas in ifn - stimulated cells and found that the expression levels of about 30 mirnas were altered ( increased or decreased).71 furthermore , they analyzed the sequence of these mirnas and hcv genomic rna and identified eight ifn -induced mirnas ( mir-1 , -30 , -128 , -196 , -296 , -351 , -431 , and -448 ) that matched with hcv genome perfectly . functional assays showed that overexpression of these mirnas by transfection of the mirna mimics reproduced the antiviral effect of ifn in huh7 cells , while their neutralization with anti - mirnas dampened the antiviral effects of ifn against hcv.71 similarly , another independent study reported that mir-196 inhibited hcv rna replication in hcv replicon cells ( genotypes 1b and 2a).72 both studies illustrated that one of the mechanisms for ifn inhibition of hcv rna replication is probably the induction of cellular mirnas that can directly degrade hcv rna . in addition to the ifn - inducible mirnas described above , there are many other mirnas that can also inhibit hcv replication . for example , the overexpression of mir-199a has been reported to suppress hcv rna replication , while inhibition by a specific antisense oligonucleotide upregulated viral replication in two cell lines bearing the replicons hcv-1b or -2a.73 in addition to the direct inhibition , some mirnas inhibit hcv rna replication indirectly by activating the ifn pathway . for example , mir-130a has been shown to be able to inhibit hcv replication in both replicon ( genotype 1b ) and jfh1 infectious models . transfected mir-130a mimic into cultured huh7.5.1 cells stably expressing an hcv genome and found that replication of hcv rna was significantly inhibited.74 further studies showed that the expression levels of type i ifns ( ifn and ifn ) were significantly increased . as huh7.5.1 cells are deficient in both tlr3 and rig - i , two important mediators of type i ifn induction , these cells can not recognize hcv infection and , as a result , almost no type i ifns are produced following virus infection . these results imply that mir-130a may inhibit hcv replication indirectly , probably by restoring the host innate immune response in tlr3- and rig - i - deficient cells.74 however , other investigators have demonstrated that hcv infection upregulates mir-130a , and that interferon - induced transmembrane protein 1 ( ifitm1 ) is a direct target for this mirna : knockdown of mir-130a enhanced ifitm1 expression and reduced hcv replication.75 this study demonstrated that hcv evades innate immune attack by decreasing antiviral ifitm1 expression through mir-130a . since li et al found mir-130a up - regulated the ifn expression , the expression of the ifn - stimulated gene ifitm1 should increase ( although not tested in their study ) . these two studies reached seemingly contradictory results , which may indicate that mir-130a has more than one target , and the interaction of this mirna with the host innate immune system is complex . in any case , the interaction between mir-130a and the host innate immune system deserves further study . mir-122 is the most abundant liver - specific mirna , accounting for around 70% of the total mirna content in mammalian liver tissue.76 it is undetectable in other tissues in mice.76 studies have demonstrated that mir-122 acts as a regulator of fatty - acid metabolism in mouse liver,77 and reduced mir-122 levels are associated with hepatocellular carcinoma.7881 in addition , of all the mirnas , mir-122 is unique in its stimulatory , not inhibitory , role in hcv replication.82 the discovery that mir-122 is required for hcv replication linked a host mirna to a human infectious disease for the first time.83 the role of mir-122 in hcv replication was first reported by jopling et al . in 2005.82 they sequestrated mir-122 with antisense oligonucleotides and found that hcv rna accumulation decreased.82 in addition , they also identified two target sites in the hcv 5 untranslated region that are necessary for hcv replication . since then , a large number of elegant studies have been performed to show that the interaction between mir-122 and viral 5ncr is essential to promote hcv replication.8488 the role of mir-122 as an important , possibly essential , host factor for hcv production makes it an attractive candidate for antiviral therapy . modified antisense agents and small - molecule inhibitors have been developed as potential new lead compounds for drug discovery.89 most recently , treatment with miravirsen ( spc3649 ) efficiently suppressed hcv genotype 1a and 1b infections in chimpanzees , with no evidence of viral resistance or side effects.90 miravirsen , a locked nucleic acid modified dna phosphorothioate antisense oligonucleotide of mir-122 , has also been trialed in patients with chronic hcv genotype 1 , with the results showing a dose - dependent reduction in hcv rna levels without the occurrence of viral resistance.91 in addition to targeting hcv rna directly , yoshikawa et al . found that overexpression of mir-122 suppresses the activity of ifn - stimulated response element ( isre ) while significantly silencing mir-122-enhanced ifn - induced isre activity.92 isres are specific nucleotide sequences located in the promoters of ifn - stimulated genes that encode antiviral proteins and can induce transcription of these genes by binding with ifn - stimulated gene factor 3.93 furthermore , yoshikawa et al . found that silencing mir-122 decreased the expression of suppressor of cytokine signaling , a negative regulator of ifn signaling , in mouse liver , leading to increased ifn anti - hcv activity.92 this study indicates that the anti - hcv effect of mir-122 might also be mediated by decreasing the expression of antiviral proteins or increasing negative regulators of cytokine production . most recently , it has been reported that mir-141 , which can be induced by hcv infection , may also be necessary for efficient hcv replication.94 mir-141 belongs to the mir-200 family , which is believed to play an essential role in tumor suppression by inhibiting epithelial transfected an mir-141 mimic or antagonist into hcv1a - infected hepatocytes to increase or deplete intracellular mir-141 expression , respectively.94 the results showed that overexpression of mir-141 enhanced hcv replication , while depletion of mir-141 inhibited virus replication . meanwhile , they identified dlc-1 ( deleted in liver cancer 1 ) as one of the target genes.94 increasing mir-141 decreased dlc-1 protein levels without a parallel decrease in dlc-1 mrna levels , suggesting that mir-141 primarily targets translational inhibition of dlc-1.94 dlc-1 encodes a member of the rho - gtpase activating protein ( gap ) family of proteins . the rho - gap proteins can specifically catalyze the conversion of the active gtp - bound rhoa protein into the inactive gdp - bound protein.97,98 active rhoa protein is required for ras - mediated tumorigenic transformation,99 and rho - gaps may therefore act as important negative regulators in human carcinogenesis . in hepatocellular carcinoma , homozygous deletion or loss of dlc-1 mrna expression usually occurs in vivo and in vitro.98,100 in addition , restoration of dlc-1 in hepatoma cell lines lacking dlc-1 results in reduced cell proliferation as well as reduced metastatic activity.100 since hcv - infected cells express mir-141 , and its overexpression significantly suppresses dlc-1 expression,94 the results indicate the presence of a novel mechanism of hcv infection - associated mirna - mediated regulation of a tumor suppressor protein , which is worth further exploration . mirnas involved in hcv infection and their interactions with the innate immune system are summarized in table 1 . hcv , hepatitis c virus ; ifn , interferon ; isre , interferon - stimulated response element ; mirna , microrna mirnas are endogenous , short , noncoding rnas that function at the posttranscriptional ( mrna ) level through mrna degradation or inhibition of translation . host mirnas play various important roles in many cellular processes , including host innate immunity and cell defense . increasing lines of evidence suggest that many mirnas are involved in the viral life cycle . for hcv , mirnas can either stimulate or inhibit viral rna replication through distinct mechanisms . in the past few years , many studies have focused on identifying differentially expressed mirnas before and after hcv infection . identifying their target mrnas remains a great challenge , as a complex network of interaction exists between mirnas and mrnas . although high - throughput screening methods such as genome - wide association studies and microarrays may reveal the complicated network of regulation and eventually identify targets for intervention , functional studies will have to be performed to validate these targets experimentally . remarkably , the advent of anti - mir-122 drugs is opening a new era for hcv therapy . although many mirnas have been identified in vitro , and the function of many of these has not been verified in vivo , mirnas have great potential as therapeutic targets for viral infection , in addition to many other diseases .

any pink color within a melanocytic lesion can be regarded as a marker for inflammation and has long been recognized as a clinical clue to melanoma [ 1 , 2 ] . in melanomas without significant pigmentation , either amelanotic or hypomelanotic , a subset of these dermoscopically visible areas has been called milky - red areas listed more than one shade of pink being a statistically significant positive indicator of melanoma ( odds ratio = 2.3 , sensitivity = 32.4% , and specificity = 82.9% ) versus all nonmelanomas . our goal in this study was to see if the presence of pink can be detected , even by those unskilled in dermoscopy , with the goal of earlier identification of melanoma . in this research , the term pink is not defined other than the color pink as understood by dermoscopically nave observers . preliminary analysis determined that pink located in the periphery of dermoscopic lesions could assist in early detection of melanoma . the image set used in this study consists of 1290 digital , contact nonpolarized dermoscopic images of melanocytic lesions , acquired during the national institutes of health - funded study sbir r44 ca-101639 - 02a2 . this image set included 296 invasive malignant melanomas and melanomas in situ and 994 benign melanocytic lesions including 461 dysplastic nevi with mild atypia , 70 dysplastic nevi with moderate atypia , 271 nevocellular nevi without atypia , and 192 congenital nevi . images were acquired at four clinics : skin and cancer associates ( plantation , fl ) , the dermatology center ( rolla , mo ) , columbia dermatology ( columbia , mo ) , and sheard and drugge pc ( stamford , ct ) . the phelps county regional medical center institutional review board ( rolla , missouri ) approved this research and each subject or subject 's parent or guardian signed a consent form for this research . all melanomas were biopsied and examined by a dermatopathologist and all benign lesions were either biopsied or followed and determined to have no change . the high in situ to invasive melanoma ratio ( 1.35 ) reflects the detection of melanomas at an early stage in four dermatology private practices in the usa . lesions were included if they were biopsied or if either patient or physician suspected the possibility of melanoma . the nih study lesions comprised a convenience sample of all lesions seen in these four clinics . all melanocytic lesions in this nih study were included in the current study except the following classes : spitz - reed nevi , nevi with known previous treatment ( nevus recurrens ) , lesions with multiple diagnoses / collision tumors , and lesions with high - grade atypia , blue nevi , and metastatic melanomas . each image was analyzed without knowledge of diagnosis by three untrained observers : two premedical students with eight months ' clinical research experience finding borders for lesions in dermoscopic images ( rkr , ksp ) and an engineering graduate student ( ug ) with no clinical experience and six months of image processing experience . the three observers independently determined if any peripherally located or centrally located pink areas were present . the study was supervised by a dermatologist with 18 years of experience in dermoscopy ( wvs ) . we offer several examples of pink , including light pink ( figures 1 and 2 ) , dark pink ( figure 4(a ) ) , and pink - orange ( figure 5(a ) ) . we did not demand any particular shade or any medical diagnosis , such as erythema , but rather sought to include all lesions for which the untrained observers agreed that there was some shade of pink present . for people with fair skin , with an identifiably pink background , pink must be increased from the background , as in figures 1(a ) and 1(b ) . the lesion was not considered pink if there was no change in pink from the background skin color . for peripheral pink , the pink color was present at the lesion rim in a large enough area to be easily detected ( area diameter > 1 mm on 10 mm wide dermoscopic images ) but need not be present circumferentially . similarly , central pink was considered present if pink was present somewhere inside the lesion rim , occupying the same minimal area . any discrepancies between the two observers were resolved by consensus on presence or absence of pink area . figures 16 show dermoscopic views of malignant and benign lesions that had central pink , peripheral pink , or pink throughout the lesion . histopathological imaging for figures 3(a ) , 4(a ) , and 5(a ) are seen in figures 7 , 8 , and 9 , respectively . the p values were computed from the chi - square distribution using the wald chi - square value ( pearson uncorrected ) . standard sensitivity and specificity were calculated as sensitivity : ( true positive/(true positive + false negative ) ) and specificity : ( true negative/(false positive + true negative ) ) . odds ratios were calculated as the odds of melanoma with peripheral pink present divided by the odds of melanoma without the feature : ( ( true positive true negative)/(false negative false positive ) ) . of the entire pigmented lesion set , 1154/1290 ( 89% ) had some pink in the lesion . of the 296 malignant melanomas overall , 93% had pink color present in the lesion . the majority 899/1290 ( 70% ) of lesions had pink color present somewhere in the lesion periphery . the invasive melanomas had a slightly higher percentage of peripheral - only pink ( 21/126 = 16.7% ) compared to the in situ melanomas ( 22/170 = 12.9% ) . of the 994 benign nevi , 64 ( 6.4% ) had peripheral - only pink . the odds ratio for the presence of peripheral - only pink for all malignant melanomas is 2.51 ( 95% ci : 1.73.8 , p < 0.0001 ) while the odds ratio for central - only pink is 0.462 ( 95% ci : 0.320.67 , p = 0.204 ) . pink color was present confined to the center of the lesion in 38/296 ( 12.8% ) of melanomas and in 217/994 ( 21.8% ) of benign lesions . a rim of pink at the outside of a pigmented lesion , in some cases even resembling the sun visible around the darker lunar object in a lunar eclipse or a rim of sun at sunset , can be termed the pink rim sign . the pink rim sign is present in the melanoma in situ in figure 1(a ) and the two invasive melanomas in figures 4(a ) and 5(a ) . the differentiation in the clinic of early malignant melanoma from benign pigmented lesions , even with dermoscopy , remains a difficult challenge . at the earliest stages , especially in melanomas lacking significant pigmentation , few dermoscopic clues irregular pigment network , negative network , atypical vascular patterns , and blue - gray granular peppering are helpful features if they are present [ 3 , 711 ] . yet these structures have specific patterns which can be challenging for those still learning dermoscopy . pink color observed without magnification is a clinical clue for presence of malignancy , including primarily nonmelanocytic lesions , such as basal cell and squamous cell carcinoma . data presented here supports the hypothesis that any pink coloration observed dermoscopically confined to the periphery of a lesion increases the likelihood of melanoma ( odds ratio 2.5 ) , whereas pink observed only in the central , nonperipheral portion of the lesion decreases the likelihood of melanoma ( odds ratio 0.46 ) . for example , pink - orange , light pink , and dark pink were all counted as pink . menzies et al . noted that milky - red pink areas conferred an odds ratio of 2.5 for the diagnosis of melanoma among a group of amelanotic lesions and an odds ratio of 2.3 for the finding of more than one shade of pink thus , our findings extend the findings of the menzies group to most nevocellular lesions , with a similar odds ratio for peripheral pink . as our study demonstrates with dermoscopically naive observers , training in dermoscopy where other structures are absent , such as for hypomelanotic melanoma ( figure 3(a ) ) or symmetric lesions with few other features of melanoma ( figure 1(a ) ) , the finding of pink color in the periphery can help lead to earlier excision . the type of dermoscopy can affect results . dermoscopic features related to vascularity , including pink color , can be altered by excess pressure caused by direct skin contact with the dermatoscope . however , our image set shows pink coloration preserved in the majority of contact dermoscopy images , demonstrating that contact dermoscopy can be a reliable detection method for pink coloration . in addition , benvenuto - andrade et al . , in a study comparing dermoscopy techniques , showed greater preservation of pink veil by polarized dermoscopy . however , our image set shows that with proper minimal - pressure technique using a gel interface , pink coloration is preserved in the majority of these contact dermoscopy images . images in the benvenuto - andrade study show that pink coloration becomes redder in some non contact polarized dermoscopy images and appears in some benign images , creating a false - positive pink rim sign , such as what was seen in a dysplastic nevus and a blue nevus in that study . we conclude that contact non polarized dermoscopy using proper technique , with gel and minimal pressure , gives the best color balance to allow reliable detection of the pink rim sign . we may apply this clinical observation to analytic techniques to increase the accuracy of both computer - based diagnostic systems and human detection of melanoma . in computer - based systems , it is anticipated that combined color and location analysis can provide diagnostic assistance to practitioners . the pink rim sign might serve as an adjunct feature for such systems by applying the ratio of pink in the periphery to pink in the center of the lesion . for practitioners , in cases where it is difficult to determine whether structural features of melanoma are present , the finding of a pink rim sign can increase the index of suspicion for melanoma and may lead to earlier excision . additionally , the presence of pink may be determined on clinical examination , without dermoscopy . therefore pink serves as not only a dermoscopy warning sign but also an aid to localization of significant lesions . . pink veil may be seen in benign lesions , including dermatofibroma , as well as basal cell carcinoma and melanoma . for milky - red areas , who found for a group of benign and malignant amelanotic and hypomelanotic lesions that milky - red areas are present more frequently in melanomas ( 93% in thick melanomas ; 31% in thin melanomas ) versus 17% in benign lesions . the three observers were not given any specific structural definition that they needed to learn but rather were instructed to just decide whether pink color was present at the lesion rim or center . our definition of pink is more inclusive than milky - red areas , and more lesions will be described as pink . in our experience , nevi in people with fair skin ( fitzpatrick 1 and 2 skin types ) generally contain pink color . the frequency of pink color observed in lesions will vary among populations . thus the high frequency of pink color observed in our series can not be generalized to all populations . we found that , in our thicker melanomas , for example , the 0.8 mm melanoma in figure 4 , pink is confined to the rim of the lesion because of more advanced central pigmentation , whereas in earlier melanomas pink was found throughout the lesion , as in the melanoma in situ in figure 1 and the 0.55 mm melanomas in figures 2 and 3 . there is experimental support for the hypothesis that pink color in the periphery can indicate malignancy . . showed that the blood volume visible by transillumination techniques exceeds the area of pigmented lesions seen on dermoscopy . they also showed a correlation between the degree of atypia and the blood volume . on the spectrum from mild to moderate to severe atypia to malignant melanoma , blood volume the finding of increased blood volume could help explain the reason that pink in the periphery is correlated with malignancy in pigmented lesions . we also found an increase in the presence of peripheral pink with more advanced diagnosis . peripheral pink was present in 6.6% of nevocellular nevi , 12.9% of in situ melanomas , and 16.7% of invasive melanomas . this correlation did not extend to degree of atypia in benign lesions , however , as more nevi with mild atypia ( 5.64% had peripheral pink than those with moderate atypia ( 2.86% ) ) . otherwise , the progressive increase in blood volume with increasing atypia observed in the terushkin study correlates overall with our observed increase in peripheral pink percentages as well as the progression of milky - red areas in the pizzichetta study ( 93% in thick melanomas ; 31% in thin melanomas ) versus 17% in benign lesions . the findings of the terushkin transillumination study provide a physiologic reason why pink in the periphery may have more discriminatory power for any skin cancer than pink in other areas . although pink is correlated with erythema , vascularity , and blood volume , there is no 1 : 1 correlation . the advantage to pink as a sign for melanoma is that it can be detected by naive observers . our study is limited by the nonconsecutive acquisition of a convenience sample of lesions in the four clinics above , with clinical and dermoscopic digital photographs obtained between 2007 and 2011 . these lesions comprise the majority of melanomas found in these clinics during this interval . for this study , we eliminated two of the most difficult diagnostic categories of lesions collision tumors and melanocytic lesions with high - grade dysplasia . the first category , collision tumors , those difficult lesions that can show features of both benign and malignant lesions , for the second category , high - grade dysplastic lesions , the analysis can be complicated by disagreement among dermatopathologists . another limitation present in our study is that we employed only student observers , neither of whom was formally trained in dermoscopy . these nonphysician observers may nonetheless model the skills of physicians beginning training in dermoscopy , and , as dermoscopy is more widely employed by nonspecialists , the pink rim sign could be used by these physicians . another limitation is the possibility of unconscious introduction of bias , using a different technique such as applying less central pressure when acquiring melanoma images , resulting in more apparent erythema in the periphery . nevi have less erythema and when capturing the image the photographer would not likely have paid attention to the amount of pressure being applied resulting in the vasculature being blanched . as this is a pilot study , future studies are needed to confirm this observation . future studies could be done with noncontact dermoscopy , and lesions could be evaluated by those with experience in dermoscopy to further explore these findings . a pink rim in a melanocytic lesion is a clue to the diagnosis of melanoma . an odds ratio of 2.5 was calculated for any shade of pink confined to the periphery of dermoscopic images of melanocytic lesions . when pink color is observed in the periphery of a dermoscopic image of a melanocytic lesion , this feature suggests consideration of melanoma within the differential diagnosis .

this can cause alveolar ridges with a reduced width and height leading to a great challenge in prosthetic reconstruction as well as during endosseous dental implant placement . a lot of techniques have been developed to achieve alveolar ridge augmentation before dental implant placement . autogenous bone graft harvested from the patient s skeleton is considered the gold standard , and it seems to be the most predictable technique used in alveolar defects , although there are some limitations with it , such as the second surgical site involvement , inadequate donor site and the morbidity and discomfort along with it [ 13 ] . another method for reconstruction of bone defects is guided bone regeneration , which has been successful based on animal and clinical studies [ 46 ] . clot contraction under the membrane ; however , probably reduces its efficacy [ 710 ] . graft materials such as allografts and xenografts are also proper alternatives for ridge augmentation , in order to overcome the autogenous bone restrictions . demineralized freeze - dried bone allograft ( dfdba ) is a type of bone graft that has been extensively used , although lack of osteoinductivity , getting washed out in bleeding sites and difficulty in handling have questioned the effectiveness of the material . to overcome this problem , dbm graft materials have been improved by using a carrier to maintain the particles together . it has some characteristics such as being hemostatic , expanding to fill the bony defect and having good stability . materials such as glycerol , polymers , polymeric gels , and collagen have been utilized as a carrier to keep up the integrity of the particles . irvine , ca ) is a trade name for a type of dbm combined with a carrier that exists in different forms of gel , matrix and putty depending on the type of vehicle used . poloxamer 407 is the carrier for the putty form of dynagraft . at low temperatures , based on histologic analysis , this material inhibits epithelial cell migration , playing the role of a barrier . in addition , it has been demonstrated to be guided tissue regenerative inherently , in human and animal studies [ 11 , 12 ] . bio - oss ( geistlich biomaterials , wolhusen , switzerland ) is an inorganic osseous matrix that is produced after elimination of the organic components of medular bovine bone using a thermal treatment . after removal of the organic components , bio - oss preserves its trabecular artichecture and porosity and it acts as an osteoconductive material . the physical properties permit clot stabilization and revascularization to let osteoblast migration , leading to osteogenesis . the aim of this study was to evaluate the quality and quantity of bone healing after application of dynagraft and bio - oss in experimentally induced bone defects in rabbit calvaria compared to natural healing . the animal selection , management , and experimental protocol were approved by the animal care and use committee of tehran university of medical sciences . the study was performed strictly according to the advices of helsinki consensus for the use and care of animals . twelve new zealand white male rabbits with 2.5 kg mean weight were included in the study . the animals were kept on standard laboratory chow with free access to water for 2 weeks preceding the day of surgery . an intra - muscular injection of 2% ( 5mg / kg ) xylazine and 10% ( 40mg / kg ) ketamin ( alafason , woeden , holland ) was used to anesthetize the rabbits . subsequent to shaving the surgical sites on the calvaria , 7% betadine was used to scrub the area for 5 minutes . a 10 cm anteroposterior incision 15 surgical blade , and then the skin ( dermal and sub dermal tissues ) and the periosteum were reflected using a fine periosteal elevator . after that , a round bur was used to create three identical holes with an external diameter of 5 mm in the calvaria , under abundant irrigation with physiologic serum . anatomic landmarks such as the occipital process and craniocaudal sutures were used to standardize the location of the defects . in the center of the calvaria , these two landmarks meet each other and make a plus sign ( + ) . depending on the type of material , the defects were categorized into three groups : the control group , in which the defect was left without any treatmentthe bio - oss group , in which the defect was filled with bio - oss ( particles sized 0/25 mm1mm)the dynagraft group , in which dynagraft was applied to fill the defect the control group , in which the defect was left without any treatment the bio - oss group , in which the defect was filled with bio - oss ( particles sized 0/25 mm1 mm ) the dynagraft group , in which dynagraft was applied to fill the defect the periosteum was closed using an absorbable 4/0 suture ( vicryl johnson & johnson somerville . ni ) , and the skin was sutured with a non - resorbable 4/0 suture ( monofil polyamid , surgi pro . the animals were transferred to a warm place , and they underwent medication with 3 days of narcotics ( ketoprofen 0/1 mg / day ) and 7 days of antibiotic therapy ( enrofloxacin 0/6 mg / day ) subcutaneously . the animals were sacrificed via pentobarbital overdose ( 100mg / kg ) , injected intravenously after one month of healing . then , the calvaria was removed and placed in 10% buffered formalin solution for 2 weeks and then it was de - calcified in 10% formic acid for 14 days . subsequently , they were dried out in graded alcohols and surrounded in paraffin . before final preparation for sectioning , all samples were placed in formalin for 48 hours . ten histologic sections with a thickness of 6 m were prepared from each defect containing an intact border of the bone , and then the samples were routinely stained with hematoxylin and eosin . the amount of inflammation , foreign body reaction , bone vitality , type of bone formation ( sequence of collagen fibers ) were evaluated by a light microscope ( bx41 , olympus co. , tokyo , japan ) at a magnification of 40 . a five - tiered grading system was utilized to scale the inflammation as follows : 0 , without any inflammatory cells ; i , slight inflammation ; ii , focal inflammation containing 5 to 10 inflammatory cells ; iii , focal inflammation with 10 to 50 inflammatory cells ; and iv , focal inflammation with more than 50 inflammatory cells . in case of observing multinucleated giant cells in granulomatous response , the type of bone formation was characterized as woven bone alone ( type i ) , both woven and lamellar bone ( type ii ) , and lamellar bone alone ( type iii ) . collagen bundles in concentric form in the bony trabecules were determined as lamellar bone ; whereas , irregularly oriented collagen fibers in the trabeculae were considered as woven bone . the amount of bone formation ( percent ) and remaining biomaterial were evaluated by means of graphic software ( photoshop 8.0 cs , adobe photoshop cs ) . statistical analysis for the amount of inflammation and type of bone formation wilcoxon sign rank test was used to evaluate the intergroup differences ( two by two ) about the type of bone formation . repeated measure anova and paired lsd test were applied to analyze the amount of bone formation . in order to compare the remaining bone material between groups , paired t test was performed accordingly . the animals were sacrificed via pentobarbital overdose ( 100mg / kg ) , injected intravenously after one month of healing . then , the calvaria was removed and placed in 10% buffered formalin solution for 2 weeks and then it was de - calcified in 10% formic acid for 14 days . subsequently , they were dried out in graded alcohols and surrounded in paraffin . before final preparation for sectioning , all samples were placed in formalin for 48 hours . ten histologic sections with a thickness of 6 m were prepared from each defect containing an intact border of the bone , and then the samples were routinely stained with hematoxylin and eosin . the amount of inflammation , foreign body reaction , bone vitality , type of bone formation ( sequence of collagen fibers ) were evaluated by a light microscope ( bx41 , olympus co. , tokyo , japan ) at a magnification of 40 . a five - tiered grading system was utilized to scale the inflammation as follows : 0 , without any inflammatory cells ; i , slight inflammation ; ii , focal inflammation containing 5 to 10 inflammatory cells ; iii , focal inflammation with 10 to 50 inflammatory cells ; and iv , focal inflammation with more than 50 inflammatory cells . in case of observing multinucleated giant cells in granulomatous response , the type of bone formation was characterized as woven bone alone ( type i ) , both woven and lamellar bone ( type ii ) , and lamellar bone alone ( type iii ) . collagen bundles in concentric form in the bony trabecules were determined as lamellar bone ; whereas , irregularly oriented collagen fibers in the trabeculae were considered as woven bone . the amount of bone formation ( percent ) and remaining biomaterial were evaluated by means of graphic software ( photoshop 8.0 cs , adobe photoshop cs ) . statistical analysis for the amount of inflammation and type of bone formation wilcoxon sign rank test was used to evaluate the intergroup differences ( two by two ) about the type of bone formation . repeated measure anova and paired lsd test were applied to analyze the amount of bone formation . in order to compare the remaining bone material between groups , the results are shown in table 1 . evaluating the amount of inflammation using friedman test , all specimens showed slight infiltration ( grade i and ii ) and there was no significant difference between groups ( p=0/478 ) . seventy five percent of the control group , 66/66% of bio - oss group and 58/33% of dynagraft group showed grade i inflammation ; whereas , other specimens presented with grade ii inflammation . comparison of histologic characteristics in groups all specimens consisted of both lamellar and woven bone . considering the type of bone formation , in the control group , 16/67 % and 83/33 % of the specimens displayed type i and ii bone formation , respectively . in the dynagraft group , 41/67% of the specimens showed type ii bone formation , and bone type iii was detected in 58/33% of the defects in this group . in the bio - oss group , type i , ii and iii bone formation was observed in 16/66% , 58/33% , and 25/01% of the specimens , respectively . according to friedman test , the difference between groups was statistically significant ( p value < 0/05 ) . intergroup differences ( two by two ) were evaluated using wilcoxon sign rank test , and a significant difference was detected comparing dynagraft group with the control and bio - oss groups ( p value < 0/017 ) ; whereas , there was no significant difference between bio - oss and the control group ( p value > 0/017 ) . the mean amount of new bone formation was 28/6% 11/2 in the control group , and the dynagraft and bio - oss groups demonstrated 56/1% 15/1 and 53/55% 13/5 mean amount of new bone formation , respectively . according to repeated measure anova , the difference between groups was statistically significant ( p value < 0/001 ) . for evaluation of the two by two differences , lsd test showed a significant difference comparing the control group with dynagraft and bio - oss groups ( p value < 0/05 ) . considering the remaining material , using paired t test , no significant difference was observed between bio - oss and dynagraft groups ( p value < 0/1 ) . foreign body reaction was seen in two defects in the dynagraft group , and four specimens in the bio - oss group , and the difference was not statistically significant ( p value < 0/342 ) . many experimental studies have been carried out to evaluate different techniques and materials in bone regeneration procedures [ 1417 ] . since the amount of bone formation and its quality are of significant importance , a large number of studies have compared the characteristics and treatment outcomes of a variety of materials and techniques [ 18 , 19 ] . in the present study , we compared the effect of two types of bone grafts on the quality and quantity of bone healing in experimental defects in rabbit calvarium . the bone formation process of calvarium is intra membranous , similar to that of the alveolar bone . thus , we investigated the calvarium of the rabbit in order to make an appropriate comparison . this could be related to its good stability and its ability to act as a barrier membrane . moreover , these characteristics interpret the osteoinductive and osteoconductive effect of dynagraft in comparison to bio - oss with its osteoconductive properties . this is in accordance with the study of callan et al . in their case series study , four to six months prior to implant placement , they carried out a bone augmentation procedure . subsequent to material placement , a membrane barrier material was used in order to facilitate site closure . they established similar results about the new allograft material and they claimed it can be used in bone defects within a short time healing . they used the material for osseous reconstruction associated with dental implants . in their case series , they performed fresh socket implantation in the maxillary anterior segment . following extraction and fixture insertion , the residual defect was filled with the material . after approximately 6 months , at the second stage of implant surgery , a bone core was harvested from the distal aspect of the implant . . evaluated bone regeneration of critical sized calvarial defects in rabbits utilizing demineralized bone matrix putty . they established a complete bone fill and closure of the defects with vital bone at 12 weeks . since a true critical - sized cranial defect in the rabbit model is 15 mm , in the present study , due to the small size of the cranium , three critical - sized defects could not be prepared in the rabbit calvarium . moreover , many studies have shown that bio - oss is effective in bone regeneration procedures [ 2326 ] . the amount of new bone was comparable with bio - oss in conjunction with membrane . in the present study , we found similar results regarding bio - oss , but dynagraft group was better in amount of bone formation , although not statistically significant . investigated the healing of alveolar ridge defects augmented with cancellous bovine bone mineral and they found bio - oss an appropriate material for ridge reconstruction in humans . on the contrary , pinholt et al . implanted bio - oss subperiosteally for ridge construction purposes and heterotopically in the abdominal muscles of rats . in the microscopic evaluation , they found no osteoinduction or osteoconduction and a foreign body reaction was observed around the material . in their previous investigation , the authors of this study concluded that using dbbm alone in the bone defects of rabbit calvaria did not increase bone regeneration compared to the control group . they also showed that osteogenesis in rabbit calvarium may improve by adding prgf to dbbm . it seems that prgf accelerates the rate of degradation of the biomaterial . in the current study , regarding the negligible inflammatory and foreign body reaction in histological analysis these characteristics were previously confirmed by hammerle et al . , slotte and lundgren , and khoshkhoonejad et al . displayed similar characteristics about dynagraft [ 1 , 21 , 22 ] . in the present study , we did not use barrier membranes to cover the defects in order to eliminate its confounding effect ; therefore , we could evaluate the role of dynagraft as a barrier membrane in addition to its other properties . in the systematic review performed by khojasteh et al . moreover , khoshkhoonejad et al . demonstrated that using a membrane did not increase the amount of bone regeneration in their study . it is worth mentioning that membranes lack enough mechanical strength and they collapse into the defect , they separate the periosteum from the bone , and their early degradation and displacement may also contribute to their inefficacy [ 30 , 31 ] . considering the amount of remaining biomaterial the authors of this study , in their previous experiment showed a considerable amount of residual material in defects with bio - oss that reduced significantly when prgf was added . bio - oss is a hydroxyapatite compound and it is expected to have a slower rate of absorption . since our histological sections were prepared 4 weeks after the grafting procedure , this result could be different between groups if the samples were prepared after a longer period . according to the type of bone formation , the majority of new bone consisted of type ii in bio - oss and control groups ; whereas , dynagraft group mostly contained bone type iii . the difference between groups was statistically significant . as we mentioned earlier in the present study , dynagraft benefits from its osteoinductive and osteoconductive properties ; it can accelerate bone regeneration while playing the role of a membrane . our results were consistent with another study performed by khoshkhoonejad et al . , in which bio - oss group showed type ii bone formation in all specimens after one month . in the current study , considerable new bone formation was observed in both dbbm and dfdba groups in comparison with the control group . the dynagraft group showed slightly more bone formation compared to the bio - oss group .

otogenic infections rarely cause pneumocephalus . in 1884 , chiari reported the first case of pneumocephalus on an autopsy done in patient of ethmoiditis . in 1926 review of literature by andrew and canalis . revealed head trauma as the leading cause of otogenic pneumocephalus ( 36% ) . other causes were otogenic infections ( 30% ) , mastoid surgeries ( 30% ) and idiopathic ( 3% ) . the middle ear diseases lead to intra - cranial complications in 0.54% of cases . of these intra - cranial complications , pneumocephalus secondary to chronic otitis media in a child is very rarely reported and ours is probably the first case in children below 12 years . a 10-year - old child was admitted in our hospital with chief complaints of left ear discharge and earache since 8 days ; fever and headache since 5 days . he had history of left ear discharge 2 years back . on examination , he was febrile , neck rigidity was present . otological examination revealed pus discharge from 2 to 6 oclock position of left tympanic membrane and a reddish polypoid mass at 610 oclock position . contrast enhanced computed tomography ( cect ) head [ figures 1 and 2 ] revealed pneumocehalus in left cerebellopontine angle with mild compression at brainstem . interestingly the air pockets showed there path of entry from middle ear through trautman 's triangle / sinodural angle . ( a - f ) computed tomography head plain axial cuts showing pneumocephalus in left cerebellopontine angle with mild effacement of fourth ventricle . small air pockets showing the site of entry from sinodural angle ( a - f ) contrast enhanced computed tomography head axial cuts to rule out any abscess child was immediately started on emperical intravenous ( i.v . ) a 10-year - old child was admitted in our hospital with chief complaints of left ear discharge and earache since 8 days ; fever and headache since 5 days . he had history of left ear discharge 2 years back . on examination , he was febrile , neck rigidity was present . otological examination revealed pus discharge from 2 to 6 oclock position of left tympanic membrane and a reddish polypoid mass at 610 oclock position . contrast enhanced computed tomography ( cect ) head [ figures 1 and 2 ] revealed pneumocehalus in left cerebellopontine angle with mild compression at brainstem . interestingly the air pockets showed there path of entry from middle ear through trautman 's triangle / sinodural angle . ( a - f ) computed tomography head plain axial cuts showing pneumocephalus in left cerebellopontine angle with mild effacement of fourth ventricle . small air pockets showing the site of entry from sinodural angle ( a - f ) contrast enhanced computed tomography head axial cuts to rule out any abscess pneumocephalus implies air inside the cranial vault , which usually results from cranio - facial trauma . it may be as a result of erosion of the skull due to neoplasm or infection , neurosurgical procedures or fistulous tract formation . the reported rate of intra - cranial complications related to middle ear diseases ranges from 0.5% to 4% . review of literature of last 35 years yielded only 21 cases of pneumocephalus secondary to chronic otitis media . and this is probably the first case in children below 12 years of age . the physio - pathologic mechanism that can explain the genesis of pneumocephalus is a break between the middle ear and either middle or posterior cranial fossa , which allows air to enter . in addition , an increase in middle ear pressure over the intra - cranial pressure must be present to permit air to enter the cranium . this may be explained by two mechanisms : ball valve effect : the intra - cranial contents may act as a ball valve allowing air to enter through a fistula especially during sneezing or coughinginverted bottle effect : when a csf leak is present ; a negative intra - cranial pressure is built up , leading to the replacement of uid with air . ball valve effect : the intra - cranial contents may act as a ball valve allowing air to enter through a fistula especially during sneezing or coughing inverted bottle effect : when a csf leak is present ; a negative intra - cranial pressure is built up , leading to the replacement of uid with air . in our patient , the imaging suggests leak of air between the middle ear and posterior cranial fossa . in the clinical picture of otogenic pneumocephalus and meningitis , rigor nucalis is another early sign , as well . as lethargy and poor oral intake . otologic symptoms are frequently present , and may include otorrhea , postauricular edema and otalgia . when associated with trauma or surgery , bed rest and close monitoring may suffice , although needle aspiration or re - exploration may be needed . when secondary to otitis media or a congenital defect , control of any infection and repair of the defect are mandatory . once the defect is identified and has been located , cartilage , free fascia , or temporalis muscle - fascia flap are best used to plug the site , depending on its size . recommendations for the management of otogenic meningitis with pneumocephalus include a third - generation cephalosporin , either i.v . cefotaxime ( 75 mg / kg every 6 h ) or ceftriaxone ( 50 mg / kg every 6 h ) , and vancomycin ( 15 mg / kg every 6 h ) . the need to add vancomycin to the therapeutic regimen is a consequence of the escalating prevalence of strains of germs resistant to penicillin and cephalosporins . adding rifampin ( 20 mg / kg as a single daily dose , i.v . ) or meropenem ( 40 mg / kg every 8 h ) may be taken into consideration . the usefulness of adding dexamethasone to the therapeutic regimen has been controversial ; patients receiving steroids have been shown to have a 50% reduction of neurological sequelae , without improvement regarding the audiological outcome . we were not able to do complete management in this case as a child left the hospital . we are still reporting the case because otogenic pneumocephalus is very rare especially in children , it needs immediate therapy to prevent complications and this case has interesting imaging findings . pneumocephalus is a very rare complication of chronic otitis media , especially in children . in spite of its rarity , the diagnosis must be considered when a patient has neurologic complaints and chronic otitis media . immediate therapy is required as it carries a potential risk of increased intra - cranial pressure or meningitis . although neurosurgical complications of chronic otitis media need priority management , we stresses the need of treatment of ear disease in same admission period of the patient to prevent further complications .

after independence days , health care coverage has evolved a great deal in african countries . in the 1970s in senegal country located in west africa , however , the state s disengagement from social sectors , in particular , healthcare , due to the economic downturn has dealt a blow to the free health care coverage inherited from the settlers system . facing a soaring of prices in the provision of care and drug prescriptions along with the population s financial restrictions to ensure direct and immediate payment of health care , authorities have developed strategies implementing a number of social protection institutions such as the disease prevention institute ( dpi ) of business or interprofessional and mutual for care coverage . the implemented policies have hugely improved the health care system though without eradicating the problem due to a still growing cost of most provided care services such as oral care ; a burden to those institutions budget . it is then imperative for them to contain the moral risk through a well - managed oral care coverage . moral risk indicates an insured person s behavior to use health care services more reasonably than if non - assured , due to the elimination or reduction of financial barrier towards health services . the study was conducted in dakar , where 90% of social protection institutions are located . requirements to be included in the analysis were to be active since at least 2005 , date of the first established national health accounts in senegal . data about the institutions was gathered via correspondences to the leadership of the senegalese disease prevention institute federation and the united health mutual . upon receipt , self - administered questionnaires were sent to the managers for a lock - up period of 1 month . the study targeted the offered services , the method of coverage with co - payments ( i.e. part of the responsibility of the recipient ) , the availability or not of a coverage ceiling or of a dentist council to prevent any potential abuse of care services from both providers and patients . cspro ( us census bureau , washington , dc ) and spss ( ibm corp . of the 220 self - administered questionnaires , 127 were received with only 40 with usable data on 25 dpi , specified as 13 businesses , 12 interprofessional , and 15 health mutual . the amount of information received , despite the forged relationship with sps s managers , denotes the non - reliability of autoadministrated questionnaires . thus , we think a meeting - based survey would maximize the collect of information . however , because answers to some questions require access to the archives , it was not a proper method . nevertheless , the survey would provide with better results if the targeted institutions managers were made aware of their potential impact on the improvement of people s oral care . we think that the results from the surveyed institutions ( business , interprofessional and community - based ) show the barriers confronted by the involved businesses , interprofessional and community - based institutions . all institutions but the community - based ones ( 6.6% ) provide with conservative and extractions coverage ( table 1 ) . for the prosthetic care it is covered with a relatively low percentage of 27.6% by businesses based institutions and 33.7% by the interprofessional . such coverage is non - existent ( 0% ) with community - based institutions despite the fact that lo s survey in dakar indicates 4 out of 28 of mutual ( 14.3% ) provide with prosthesis coverage . the method used to conduct the survey might explain that fact ( table 1 ) . the orthodontic coverage is very high among interprofessional - based institutions ( 72.3% ) , relatively low in those of businesses ( 27.6% ) and nonexistent in community - based institutions ( table 1 ) . such disproportion might be due to the fact that dentures coverage is more about aesthetic , which requires big resources , than functionality both prosthetic and orthodontic care are less important among institutions ( 22 and 42.4% ) compare to another type of care . in fact , larger is an insurance structure , more shared is the risk and less costly it becomes for everyone . the inequalities in dental care coverage along with the renouncement of the less - favored to health care due to financial restrictions are blocking factors to prosthetic and orthodontic coverage by institutions . the ability of the insurance system s leadership to use techniques to contain the impact of risks they are facing , determines their viability . mainly co - payment and coverage ceiling were the methods of controlling used by institutions for risk containment . the implemented co - payment seems to be an adequate method of control and does not incur additional costs for the institution . thus , 97% of surveyed institutions have used it including 100% of community - based and interprofessional institutions ( figure 1 ) . according to a review by bit ( 2007 ) , the patient co - payment is a double standard as it is an efficient means to contain moral risk but might contribute , when high , to limit the accessibility of treatment as well ; contrary to social protection policy . the medical coverage ceiling appears to be efficient in containing potential frauds and abuses and does not incur additional costs . thus , it is mainly used by mutual due to their financial restrictions ( 49% ) ( figure 2 ) and according to a survey conducted by lo and colleagues ( 2011 ) , 75% have used it . the dentist council enables the control of compliance with therapeutic schemes to prevent any abuse in care services by both providers and consumers . however , such method entails additional cost to the institutions , which makes it not accessible by community - based institutions due to their financial restrictions . having said that , as a point of order , a protocol of treatment by compelling services provider to prescribe functional treatment and generic drugs were adopted . nevertheless , the strategy became a burden among mutual when some of them were refused a reimbursement of their due . the results show that most institutions ( 85% ) lack means to evaluate the quality of care provided and application management of current pricing ( figure 3 ) . that s why we think it is necessary to regulate the system of the convention of care providers . such system should incite them to accept to conform to the norms of quality , the protocols of evaluation , and to the current pricing defined by the ministry of health in collaboration with providers . it should be noted that even if these mechanisms reduce the moral risk , they could be a discouraging factor to insured patients who postpone their treatments . such action can contribute to a high care cost due to the aggravation of disease . the amount of information received , despite the forged relationship with sps s managers , denotes the non - reliability of autoadministrated questionnaires . thus , we think a meeting - based survey would maximize the collect of information . however , because answers to some questions require access to the archives , it was not a proper method . nevertheless , the survey would provide with better results if the targeted institutions managers were made aware of their potential impact on the improvement of people s oral care . we think that the results from the surveyed institutions ( business , interprofessional and community - based ) show the barriers confronted by the involved businesses , interprofessional and community - based institutions . all institutions but the community - based ones ( 6.6% ) provide with conservative and extractions coverage ( table 1 ) . for the prosthetic care it is covered with a relatively low percentage of 27.6% by businesses based institutions and 33.7% by the interprofessional . such coverage is non - existent ( 0% ) with community - based institutions despite the fact that lo s survey in dakar indicates 4 out of 28 of mutual ( 14.3% ) provide with prosthesis coverage . the method used to conduct the survey might explain that fact ( table 1 ) . the orthodontic coverage is very high among interprofessional - based institutions ( 72.3% ) , relatively low in those of businesses ( 27.6% ) and nonexistent in community - based institutions ( table 1 ) . such disproportion might be due to the fact that dentures coverage is more about aesthetic , which requires big resources , than functionality both prosthetic and orthodontic care are less important among institutions ( 22 and 42.4% ) compare to another type of care . in fact , larger is an insurance structure , more shared is the risk and less costly it becomes for everyone . the inequalities in dental care coverage along with the renouncement of the less - favored to health care due to financial restrictions are blocking factors to prosthetic and orthodontic coverage by institutions . the ability of the insurance system s leadership to use techniques to contain the impact of risks they are facing , determines their viability . mainly co - payment and coverage ceiling were the methods of controlling used by institutions for risk containment . the implemented co - payment seems to be an adequate method of control and does not incur additional costs for the institution . thus , 97% of surveyed institutions have used it including 100% of community - based and interprofessional institutions ( figure 1 ) . according to a review by bit ( 2007 ) , the patient co - payment is a double standard as it is an efficient means to contain moral risk but might contribute , when high , to limit the accessibility of treatment as well ; contrary to social protection policy . the medical coverage ceiling appears to be efficient in containing potential frauds and abuses and does not incur additional costs . thus , it is mainly used by mutual due to their financial restrictions ( 49% ) ( figure 2 ) and according to a survey conducted by lo and colleagues ( 2011 ) , 75% have used it . the dentist council enables the control of compliance with therapeutic schemes to prevent any abuse in care services by both providers and consumers . however , such method entails additional cost to the institutions , which makes it not accessible by community - based institutions due to their financial restrictions . having said that , as a point of order , a protocol of treatment by compelling services provider to prescribe functional treatment and generic drugs were adopted . nevertheless , the strategy became a burden among mutual when some of them were refused a reimbursement of their due . the results show that most institutions ( 85% ) lack means to evaluate the quality of care provided and application management of current pricing ( figure 3 ) . that s why we think it is necessary to regulate the system of the convention of care providers . such system should incite them to accept to conform to the norms of quality , the protocols of evaluation , and to the current pricing defined by the ministry of health in collaboration with providers . it should be noted that even if these mechanisms reduce the moral risk , they could be a discouraging factor to insured patients who postpone their treatments . such action can contribute to a high care cost due to the aggravation of disease . implemented methods such as the co - payment , coverage ceiling and the dentist council by social protection institutions in senegal might allow containing the hike of dental care expenditure . thus , besides those strategies , communication and information of health professionals , and patients about their rights and duties is essential . however , we must make efforts to end abuses and deviance noticed on both sides .

a central but as yet unanswered question in cell biology concerns the amount of phospholipids or tag that is required in a living cell : how is lipid homeostasis regulated in coordination with nutritional and environmental conditions ? the cellular tag content in yeast varies dramatically between different stages of growth and development ( 1 , 2 ) , which underscores its important metabolic role and the active involvement of its associated organelle , the ld , in cellular physiology . the key anabolic and catabolic enzymes involved in tag metabolism are conserved between yeast and mammals ( fig . 1 and supplemental table 1 ) and therefore , this minireview will focus on the regulatory and physiological aspects of tag metabolism in yeast and what we can potentially learn from studies in this organism about tag homeostasis in mammalian cells . gene names ( italicized ) and their functions are provided in supplemental table 1 and in text . the area in light blue indicates the er ; the area in red indicates ld . the inset in gray indicates alternative pathways for the synthesis of potential tag precursors ; their specific contribution to tag formation is unclear . ffa , free fa ; lpa , lysophosphatidic acid ; dag - pp , dag pyrophosphate ; mag , monoacylglycerol ; gro , glycerol ; dhap , dihydroxyacetone phosphate ; pl , phospholipid ; pi , phosphatidylinositol ; pg , phosphatidylglycerol ; gpi , glycosylphosphatidylinositol . note the dual functionality of enzymes encoded by tgl3 and tgl5 genes as tag lipases and tag : phospholipid acyltransferases ( 18 ) . the specific requirements for storage and membrane lipids oscillate as cells grow and divide or enter the non - replicative stationary ( quiescent ) phase . during these periods , it appears to be crucial to control the metabolic flux of fa either into phospholipids ( proliferative state ) or into or out of tag ( nutrient limitation conditions ) . because yeast cells typically do not feed on exogenous fa , all net cellular requirements for membrane , signaling , and storage lipids need to be satisfied by de novo fa synthesis , carried out by acetyl - coa carboxylase and the fa synthase complex , encoded by the acc1 and the fas1 and fas2 genes , respectively . acetyl - coa carboxylase is the first and rate - limiting enzyme and is controlled at the transcriptional level in coordination with phospholipid synthesis ( 7 ) and by phosphorylation by snf1p , the yeast amp - activated protein kinase catalytic subunit and ortholog of mammalian ampk ( 8) . although acetyl - coa carboxylase activity is controlled by the snf1p kinase , it remains to be determined whether tag homeostasis is also directly regulated by this major energy - sensing kinase . besides the potential impact of fa de novo synthesis on tag content , its synthesis is largely controlled by the activity of the pah1-encoded pa phosphatase , a heavily phosphorylated and strictly regulated protein ( 912 ) that is the functional ortholog of mammalian lipin ( see below ) ( 13 ) . yeast mutants lacking pa phosphatase are characterized by a lipodystrophy phenotype in analogy to their mouse counterparts . the regulation of dgats , encoded by dga1 ( functional homolog of mammalian dgat2 ) and lro1 genes ( homolog of mammalian lecithin : cholesterol acyltransferase and phosphatidylcholine : cholesterol acyltransferase and functionally characterized as a pdat ) , is less clear : during logarithmic growth , pdat appears to be more active , whereas dgat activity may contribute more significantly to tag formation in early stationary phase in yeast ( 14 ) . yeast cells entering vegetative growth display a highly stimulated turnover of tag , which is thought to release lipid precursors for rapid initiation of ( membrane ) growth ( 1 ) . quantitative simulation of this initial phase of growth by dynamic flux balance analysis indeed demonstrates the requirement for tag degradation for membrane lipid production and cell - surface expansion ( 2 ) . during this adaptation to the presence of glucose , de novo fa synthesis appears not to be sufficient to satisfy the cellular requirements , and the tag lipases encoded by tgl3 and tgl4 genes are most active , leading to almost complete tag degradation ( see below ) ( 1 , 6 ) . the tgl4-encoded lipase is the functional ortholog of mammalian adipose triglyceride lipase ( desnutrin , pnpla2 ( patatin - like phospholipase domain - containing protein 2 ) ) ( 1 ) . because formation and function of peroxisomes , the only site of -oxidation in yeast , are repressed by glucose , it is evident that lipolysis serves another purpose in addition to releasing fa for energy production during that phase of growth . both quantitative modeling and microscopic evidence suggest that replenishment of tag pools already takes place while lipolysis is still active in cells entering vegetative growth . at the cellular level , tag synthesis and degradation indeed oscillate in coordination with the cell division cycle : tgl4p lipase - dependent tag breakdown is activated by cdk1 ( cyclin - dependent protein kinase 1)/cdc28p at the g1/s transition , which is characterized by the morphogenesis checkpoint and bud emergence . lipolysis contributes to bud formation , presumably by providing precursors for membrane or signaling lipids ( 15 ) ; in the absence of lipolysis , cell cycle progression is significantly delayed and becomes solely dependent on de novo fa synthesis . conversely , tag synthesis is down - regulated by cdk1/cdc28p - dependent phosphorylation of pa phosphatase ( 16 , 17 ) . notably , phosphorylation / inhibition of this enzyme occurs at g2/m of the cell cycle , suggesting that tag formation and lipolysis operate in parallel from g1/s until g2/m , albeit at different subcellular locations . the reason for this apparent discrepancy may reside in the essential cellular requirement to tightly control the metabolic flux of fa into tag or phospholipids during cellular growth . in that respect , it deserves mentioning that tag lipases encoded by tgl3 and tgl5 genes may also act as transacylases , independent of their pure lipolytic activity , supporting their important function also to control phospholipid acyl composition ( 18 ) . complete tag breakdown generates glycerol and free fa that may be reactivated to acyl - coa by acsls , of which six activities are known in yeast ( 19 ) and which can be functionally complemented , at least in part , by the mammalian orthologs ( 19 , 20 ) . in contrast to mammalian cells , fa synthase in yeast releases acyl - coa rather than free fa , which makes the yeast acsl activities dispensable for utilizing de novo synthesized fa . notably , the two faa4- and fat1-encoded acsl enzymes are localized to ld , i.e. in close proximity to the site of tag storage and lipolytic action ( 21 , 22 ) . it is tempting to speculate that these acsl activities are therefore metabolically coupled to lipolysis , i.e. for channeling free fa released from tag ( or steryl ester ) breakdown toward activation and further metabolic utilization . the apparent futile cycle of anabolic and catabolic processes of tag homeostasis acting in parallel during the cell division cycle ( 15 , 17 ) is perhaps better understood in view of the spatial separation of the biosynthetic and lipolytic processes . most of the tag biosynthetic steps occur in the er , including the sct1- , gpt2- , slc1- , and ale1-encoded acyltransferases ( fig . 1 ) . however , more subtle localization patterns of the glycerol-3-phosphate acyltransferases sct1p and gpt2p to distinct er subfractions may also exist ( 23 ) . pa phosphatase , which generates dag , is associated with membranes ( presumably the er ) in a salt - extractable form and may also be present in the cytosol and in the nucleus ( 11 ) , where it may act as a repressor of phospholipid biosynthetic genes . pdat , encoded by lro1 , localizes to the er , consistent with its preferred utilization of pc as the acyl donor ( 24 ) . on the other hand , dgat , encoded by dga1 , localizes to both the er and ld in yeast ( 21 , 22 , 25 ) . thus , it appears that this enzyme moves along with the nascent ld that is believed to emerge from the er membrane ( 26 , 27 ) . the dual localization to the er and ld of ergosterol biosynthetic enzymes encoded by erg1 , erg6 , and erg7 , the fa - activating enzymes encoded by faa4 and fat1 , and the lyso - pa acyltransferase encoded by slc1 further underscores the close functional relationship between the er and ld , in particular with respect to lipid fluxes ( 5 , 6 , 2830 ) . the specific mechanisms that direct and sequester these proteins to the ( nascent ) ld are unknown but may involve hydrophobic interactions with multiple protein domains rather than specific ld targeting sequences ( 31 ) . notably , all the enzymes identified so far localizing to both the er and ld catalyze anabolic enzymatic steps , whereas the catabolic enzymes , such as the tag lipases and steryl ester hydrolases encoded by tgl3 , tgl4 , tgl5 , and tgl1 and by yeh1 , respectively , are restricted to the ld ( 1 , 5 , 6 , 30 ) . this differential localization for anabolic and catabolic enzymes suggests different routes for their ld association either through sequestration from the cytosol or via targeting to the er . it should also be noted that yeast ( or bacterial ) ld do not appear to contain homologs of mammalian perilipins ( pat proteins ( 32 ) : perilipin , adrp ( adipose differentiation - related protein ) , tip47 ( tail - interacting protein of 47 kda ) , s312 , and oxpat ) , which regulate cellular lipid stores , e.g. by mediating phosphorylation - dependent access of lipases to the ld surface . yeast tag lipases encoded by tgl3 , tgl4 , and tgl5 are constitutively present on ld and do not alter their localization during periods of lipolysis or lipogenesis ( 15 ) . thus , the lipolytic process in yeast is largely regulated by direct activation or inactivation of these proteins on ld . tag were in the past considered mainly as an efficient storage form of fa that may serve as energy substrates in the absence of other carbon sources . this view has considerably changed during the last few years , with emerging evidence that tag serve specific metabolic functions . in mammalian cells , tag formation was found to be crucial for the detoxification of lipotoxic fa ( 33 , 34 ) . similarly , yeast mutants that are lacking all four acyltransferases involved in tag formation are highly sensitive to unsaturated fa supplementation ( 35 , 36 ) . in the absence of tag synthesis , oleic acid supplementation leads to a rapid block of the secretory pathway at the level of the er , up - regulation of the unfolded protein response , and ultimately cell death ( 36 ) . multiple genes involved in iron and phospholipid metabolism are repressed in the absence of tag synthesis ( 35 ) . notably , saturated fa are not toxic to wild - type yeast or to tag - deficient mutants and may indeed suppress unsaturated fa - induced lipotoxicity in such mutant strains by generating a more balanced fa composition in cellular phospholipids ( 36 ) . unsaturated fa - induced lipotoxicity in yeast mutants lacking tag is also suppressed by expression of the human diacylglycerol acyltransferase dgat2 , providing an intriguing model system for functional studies of the heterologous enzyme in yeast ( 35 ) . in addition to providing a buffer for excess fa detoxification , tag also provide important metabolites for sporulation or cell cycle progression ( 15 , 18 ) . the specific nature of the lipid - dependent cell cycle checkpoint is currently unclear , but the temporal coincidence of lipolysis requirements with bud emergence in the cell division cycle indicates the involvement of the major morphogenesis checkpoint regulator , the swe1p protein kinase , in this process . the tag - derived lipid species required to grease the cell cycle are unknown ( 15 ) . multiple homozygous diploid lipase - deficient mutants are unable to sporulate , demonstrating the importance of tag degradation for providing energy substrate for peroxisomal -oxidation or for phospholipid remodeling in the context of spore membrane formation ( 18 ) . because tag and phospholipids share common precursors , it is evident that shifting the balanced synthesis either way will affect the steady - state concentration of the other components . for instance , defective pc synthesis in mutants lacking the cho2- or opi3-encoded phosphatidylethanolamine and phospholipid methyltransferases , respectively , results in increased cellular tag content ( 37 ) . the potential cross - talk between the kennedy ( cdp - choline ) pathway and tag metabolism is less clear : in the presence of choline or ethanolamine , dag resulting from lipolysis may potentially be directly utilized for phospholipid synthesis , which would involve a dag translocation step from the ld to the er for its incorporation into phospholipid . however , the position and stereoselectivity of the yeast lipases have not been determined yet , and it is unclear at present if indeed the phospholipid precursor sn-1,2-dag or rather sn-1,3- or sn-2,3-dag species or a mixture thereof is generated by tag lipolysis . alternatively , utilization of dag for phospholipid synthesis in the presence of ethanolamine or choline may compete with tag formation ; however , no evidence for either aspect is currently available . even less evident is the connection between tag accumulation in mutants lacking the yeast tafazzin ortholog encoded by the taz1 gene , which harbors monolysocardiolipin acyltransferase activity required for mitochondrial cardiolipin remodeling ( 38 , 39 ) . it was speculated that lack of this activity may increase pc and dag levels , giving rise to increased tag synthesis ( 38 ) . for instance , a block of the secretory pathway that is induced by inactivating the copii component sec13p involved in er - to - golgi trafficking leads to dysregulation of phospholipid synthesis and concomitant tag accumulation ; thus , tag synthesis may function as an alternative exit for excess lipid intermediates that are not disposed off from the er through the secretory pathway ( 40 ) . the storage compartment for tag , the ld , interacts with numerous other cellular organelles and processes ( 41 ) , and multiple experimental approaches are undertaken to identify and characterize novel regulators of ld / tag homeostasis . microscopic analyses are particularly powerful to identify mutants with altered ld and tag content . fig . 2 illustrates the ld phenotypes of wild - type yeast and various defective mutants : snf1 mutants lack the yeast ampk and harbor hyperactive acetyl - coa carboxylase ( 8) , leading to fa overproduction and subsequent tag accumulation ; fld1 ( few lipid droplets ) mutants lack the yeast seipin ortholog ( 42 , 43 ) and are characterized by abnormally shaped ld ; tgl3 tgl4 mutants lack the major tag lipases encoded by the genes tgl3 and tgl4 , which results in obese cells ( 1 ) ; and a quadruple mutant lacking dga1 , lro1 , are1 , and are2 genes encoding dag acyltransferase activities is devoid of tag and steryl esters and of ld altogether ( 35 , 36 ) . even subtle changes in cellular tag content or ld morphology may become easily microscopically visible or result in changes of biophysical properties of the cells . in an attempt to isolate mutants with increased tag content based on flotation , kamisaka et al . ( 44 ) identified , among other factors , the protein snf2p , which is the catalytic subunit of the swi / snf chromatin - remodeling complex , as a potential regulator of lipid metabolism . a physiological link between lipid homeostasis and chromatin structure is intriguing and deserves further attention . imaging - based screens of the entire yeast deletion collection comprising some 4700 viable mutants and making use of the lipophilic dyes nile red ( 42 ) and bodipy 493/503 ( 43 ) yielded a large number of factors regulating ld content and morphology . the most prominent hit in both screens identified mutants that lacked the gene fld1 , which is a functional ortholog of human bscl2 , implicated in a severe inherited disease termed berardinelli - seip congenital lipodystrophy type 2 ( fig . indeed , heterologous expression of the wild - type bscl2 gene , but not of mutant variants , restored wild - type ld morphology to a yeast fld1 mutant , demonstrating the functional conservation of the protein ( 42 ) . further factors with a potential role in neutral lipid metabolism resulting from these imaging - based screens include endosomal / vacuolar and mitochondrial proteins but also kinases and transcription factors ( 4143 ) . it should be noted that there was surprisingly little overlap ( 10% ) among the mutants identified in both mutant screens , which suggests that ( a ) these screens are far from being saturated and ( b ) ld morphology is very dynamic and strongly dependent on the respective growth and analysis conditions . thus , the chapter of identifying all the relevant components involved in tag metabolism and ld biogenesis is far from being complete and may hold many more surprises . analyzing ld morphology as an indicator of tag homeostasis using different staining and microscopic techniques . e , left panels , fluorescence ; right panels , transmission ( differential interference contrast ) . a , ld visualization in wild - type cells using a green fluorescent protein - tagged reporter construct ( 1 ) . b , accumulation of ld in an snf1 deletion strain that displays hyperactive acc1p ( 8) and tag accumulation . note that the size of ld does not increase in this mutant but rather their number ( bodipy 493/503 staining ) . c , morphologically altered ld in an fld1 mutant ( 42 , 43 ) lacking the yeast ortholog of mammalian bscl2 , implicated in berardinelli - seip congenital lipodystrophy type 2 ( bodipy 493/503 staining ) . d , ld accumulation in an obese yeast mutant lacking the two major tag lipases , tgl3p and tgl4p ( nile red staining ) ( 1 ) . e , a quadruple mutant lacking the four acyltransferases involved in tag synthesis ( 35 , 36 ) that also lacks ld and any detectable nile red staining in the 550570-nm emission range ( see ref . f , coherent anti - stokes raman scattering microscopy of a wild - type strain ( left panel ) and the quadruple mutant ( right panel ) . coherent anti - stokes raman scattering enables label - free detection of ld based on the spectroscopic properties of lipid molecules . c and e are courtesy of heimo wolinski ( university of graz ) ; f is courtesy of lu fake and huang zhiwei ( national university of singapore ) . the commonly used term neutral lipid to describe the major ld components steryl esters and tag solely reflects their biophysical property as being uncharged but by no means does justice to their biological functions . the conservation of key metabolic steps makes yeast an intriguing model for functional analyses of heterologously expressed mammalian genes involved in tag metabolism . studies in yeast also contribute to understanding complex metabolic networks , such as those involved in nutrient and lipid signaling , lipotoxicity , and the metabolic syndrome ( 45 , 46 ) . notably , some key enzymatic functions were first identified and characterized in yeast at the molecular level , such as the pa phosphatase activity of lipin ( 12 ) , a mammalian lipodystrophy factor for many years in search of a biochemical function ( 13 ) . replacement of the yeast orthologs by their mammalian counterparts , either wild - type or mutant forms derived from patients , has proven to be a viable strategy to assess biological activity in an in vivo setting that provides an impressive arsenal of genetic , genomic , and cell biological tools for functional studies ( 35 , 42 ) . now that the basic enzymatic steps involved in tag homeostasis have been worked out , what are the next challenges ? why does tag homeostasis require so many redundant activities , such as acyltransferases and lipases ? obviously , these enzymes display distinct substrate specificities that may be relevant in the context of maintaining cellular fa and phospholipid homeostasis and regulation under different environmental , nutritional , and developmental conditions . the most intriguing problems concern the biogenesis of the organelle that accommodates tag and steryl esters , the ld , and the functional interplay between the er and ld with respect to controlling lipid fluxes . these great balls of fat were only recently recognized as a highly dynamic cellular compartment ( 41 , 4749 ) , which is now subject to extensive studies in microorganisms , invertebrates , and mammalian and plant cells . imaging - based large - scale functional genomic screens performed in various cell types , including yeast , are likely to uncover the critical factors required for ld formation , morphology , catabolism , and inheritance ( 27 , 42 , 50 ) . the combination of such refined imaging - based screens in different cell types with proteomic and lipidomic analyses of isolated ld is expected to uncover and converge at a critical and conserved set of proteins and lipids relevant for ld biogenesis and metabolism in eukaryotes . these studies will also contribute to solving the puzzles as to the highly redundant activities involved in tag formation and breakdown and their relevance for cellular physiology and disease .

a retrospective cohort study was conducted on 31 type 1 diabetic recipients of itx ( itx alone , n = 25 ; islet after kidney , n = 6 ) between 2001 and 2007 . procedures were performed as described ( 1 ) under protocols approved by the university of miami health research ethics board , and informed consent was obtained from each subject . hypoglycemia unawareness was assessed using the clarke hypoglycemic score ( minimum = 0 ; maximum = 7 ; no hypoglycemia = 0 ; hypoglycemia unawareness 4 ) ( 4 ) twice , including pre - itx and at the most recent follow - up ( 47.2 21.3 months after first itx ) . subjects were grouped based on graft function : off - insulin ( n = 8) , graft dysfunction ( restarted insulin and stimulated c - peptide 0.3 ng / ml , n = 13 ) , and graft failure ( stimulated c - peptide < 0.3 ng / ml ; n = 10 , evaluated 11.5 14.5 months after graft failure ) . plasma glucose ( hexokinase method ) and c - peptide ( double antibody radioimmunoassay ) were measured during a mixed - meal test . a1c ( high - performance liquid chromatography [ hplc ] ; bio - rad , richmond , ca ; normal values : 4.26.1% ) was determined before itx and at the most recent follow - up . the paired t test was used to compare variables pre- and post - itx , and one - way anova was used to compare the post - itx hypoglycemic score among groups . p values ( two - tailed ) < 0.05 and < 0.016 ( bonferroni 's correction for differences among subgroups ) were considered significant . patient age at baseline was 43.8 8.7 years , and diabetes duration was 29.3 11.8 years . a total of 13 subjects ( 42% ) had hypertension , and 11 ( 35% ) had dyslipidemia . diabetes complications at baseline included retinopathy ( 71% ; n = 22 ; 14 proliferative ) , neuropathy ( 45% ; n = 14 ; 4 autonomic , 7 peripheral , and 3 both ) , and nephropathy ( 29% ; n = 9 ; 3 microalbuminuria and 6 with a kidney transplant 9.1 6.3 years old ) . mean hypoglycemic score pre - itx was 5.29 1.51 and was inversely correlated with pre - itx glycemic control , as measured by a1c ( r = 0.370 , p = 0.040 ) . a decrease in hypoglycemic score was observed post - itx ( 1.35 1.92 , p < 0.001 ) . similarly , there was a reduction in the proportion of patients with hypoglycemia unawareness ( pre- vs. post - itx : 87 vs. 13% , p < 0.001 ) and an increase in glycemic threshold that resulted in symptoms ( pre- vs. post - itx : 41.4 17.6 vs. 58.4 10.3 mg / dl , p = 0.001 ) . results were sustained even after the patient 's stratification based on islet function ( pre- vs. post - itx off insulin : 5.63 2.00 vs. no hypoglycemia reported ; graft dysfunction : 5.31 1.49 vs. 1.15 1.63 , p < 0.001 ; and graft failure : 5.00 1.16 vs. 2.70 2.26 , p = 0.014 ) ( fig . 1 ) ; however , an increase in post - itx hypoglycemic score was observed as patients lost graft function ( p = 0.007 ) . a1c improved in off - insulin ( 6.8 0.8 vs. 6.0 0.6 , p = 0.038 ) and graft dysfunction ( 7.5 1.2 vs. 6.7 0.5 , p = 0.022 ) subjects , and no further deterioration was observed after graft failure ( 7.0 0.9 vs. 7.4 0.9 , p = 0.318 ) , suggesting that recovery of awareness was a consequence of glycemic stability rather than metabolic deterioration associated with a less strict therapy . additionally , a lower hypoglycemic score correlated with better -cell function ( r = 0.440 , p = 0.013 , and r = 0.496 , p < 0.001 , post - itx fasting and stimulated c - peptide , respectively ) . follow - up was similar for all groups ( 49.3 20.3 vs. 42.7 22.2 vs. 51.5 21.9 months , p = 0.602 ) , and no clinical or laboratory factors were associated with hypoglycemia unawareness post - itx , including diabetes duration and presence of neuropathy ( data not shown ) . in this sample of unstable type 1 diabetic patients , glucose stabilization after itx was associated with restoration of hypoglycemia awareness . medical treatment is driven by hypoglycemia avoidance but can lead to metabolic deterioration if it is necessary to lower the insulin dose ( 5 ) . pancreatic transplantation reestablishes normal glucose homeostasis ( 6 ) , but the surgical risks are a limitation . no hypoglycemia has been reported after itx in patients achieving insulin independence ( 1 ) . after developing graft dysfunction and reintroducing exogenous insulin , hypoglycemic episodes may resume , although with a much lower frequency than pre - itx . based on these observations , it is conceivable that itx recipients regain adrenergic symptoms . indeed , an improvement in hypoglycemia awareness after itx was suggested by a small - sample short - term clamp study ( 2 ) , although the literature is not consistent ( 3 ) . to the best of our knowledge , this is the first report of restoration of hypoglycemia awareness after itx , which includes more than a few patients and has a longer follow - up , and we have demonstrated statistically significant benefits even after graft failure no hypoglycemia was reported by off - insulin subjects ; the recovery of hypoglycemia awareness found in patients with graft dysfunction was predictable and was probably related to tight glycemic control due to itx and prevention of hypoglycemia . in graft failure patients , avoidance of hypoglycemia during the early post - itx period possibly led to increased awareness , which was maintained after loss of graft function . hypoglycemia unawareness relapse was observed in this group , even though the score remained in the normal range . the retrospective design of this study did not allow for more frequent evaluations and better description of patient symptoms that would have enhanced the study . despite this limitation we were able to identify the restoration of hypoglycemia awareness . in conclusion , the stable metabolic control achieved by itx restores hypoglycemia awareness in type 1 diabetic subjects . these benefits were accomplished without metabolic deterioration , independent of diabetes duration or presence of autonomic neuropathy , and persisted beyond the duration of islet graft function .

patients were recruited between february 2005 and june 2007 from the university hospitals of st . olav , akershus , stavanger , haukeland , and north norway as well as levanger hospital . inclusion criteria were age 1840 years , insulin - dependent diabetes with positive test for gada and/or ia-2a ( antibodies against gad65 and ia-2 ) , fasting c - peptide level 0.2 nmol / l , and diabetes duration 12 weeks . further , we did not include women who were pregnant or who did not use contraception . the study protocol was approved by the regional ethics committee and the norwegian drug agency . details of study design can be found in the online appendix ( supplemental table 1 in the online appendix [ available at http://care.diabetesjournals.org/cgi/content/full/dc09-1436/dc1 ] ) . before inclusion , all patients underwent a clinical examination and blood sampling for assessment of blood count , lipid profile , and renal and liver function . all patients received a standard multi - injection regimen consisting of mealtime monomeric short - acting insulin and long - acting nph insulin twice daily ( except for two subjects in the diazoxide group and three in the placebo group who received nph insulin only at night ) . after obtaining fasting blood glucose consistently between 4 and 6 mmol / l and postprandial levels between 5 and 7 mmol / l , the patients were double - blindly randomized to either 100 mg diazoxide at night or placebo . the primary end point was -cell function , assessed by fasting and glucagon - stimulated c - peptide . secondary end points were glycemic control ( a1c and blood glucose ) , insulin dosage , and markers of autoimmunity ( gada , ia-2a , and tregs ) . each visit included a clinical investigation and measurements of fasting blood glucose , blood pressure , and body weight . patients were also examined for presence of edema and asked about the occurrence of hypoglycemia and adverse events . we registered current insulin dosage ( fast- and long - acting ) at each visit . c - peptide glucagon stimulation tests were performed in duplicate at baseline and every 3 months thereafter . in total blood was sampled in the overnight - fasted state and 6 min after the intravenous injection of 1 mg glucagon . patients were to perform seven - point home glucose monitoring ( 7phgm ) during 3 consecutive days at baseline , during intervention , and between 3 and 6 months after intervention . blood glucose was measured fasting , 2 h after breakfast , before lunch , 2 h after lunch , before dinner , 2 h after dinner , and at bedtime using the patients ' own blood glucose measuring device . manufacture of drug and placebo capsules was performed by the krager tablet production unit ( krager , norway ) . at baseline and at the end of intervention , we isolated peripheral blood mononuclear cells ( pbmcs ) from 11 subjects in the diazoxide - treated and 9 subjects in the placebo - treated group . cells were isolated by separation on lymphoprep ( axis - shield , oslo , norway ) and then stored below 140c . we also isolated pbmcs from 20 nondiabetic , age- and sex - matched blood donors . after thawing , the cells were processed through a facscanato flow cytometer ( bd biosciences , san jose , ca ) . a1c was assayed by a dca 2000 ( bayer as diagnostics , oslo , norway ) . c - peptide , insulin , glucagon , and proinsulin were assayed by radioimmunoassay ( linco research , st . gadas and ia-2as were determined by enzyme - linked immunosorbent assay ( medizym ; medipan diagnostica , selchow , germany ) . titers of gada > 5 units and of ia-2a > 10 units were regarded as positive . the coefficient of variation ( cv ) for fasting c - peptide is 13% ( 16 ) . to detect a 20% difference with 80% certainty , one would only need eight patients in each group . considering a spontaneous variation in c - peptide decline , we planned to include 50% more patients . assuming a 10% dropout , 36 patients for c - peptide glucagon tests , we used the mean values of duplicates for analyses . for 7phgm , we used the median because of high spread in some of the values . dichotomous variables were analyzed using the pearsons test . to assess insulin sensitivity , we used the homeostasis model assessment ( homa ) calculator from oxford university ( http://www.dtu.ox.ac.uk ) . a1c was assayed by a dca 2000 ( bayer as diagnostics , oslo , norway ) . c - peptide , insulin , glucagon , and proinsulin were assayed by radioimmunoassay ( linco research , st . gadas and ia-2as were determined by enzyme - linked immunosorbent assay ( medizym ; medipan diagnostica , selchow , germany ) . titers of gada > 5 units and of ia-2a > 10 units were regarded as positive . the coefficient of variation ( cv ) for fasting c - peptide is 13% ( 16 ) . to detect a 20% difference with 80% certainty , one would only need eight patients in each group . considering a spontaneous variation in c - peptide decline , we planned to include 50% more patients . assuming a 10% dropout , 36 patients for c - peptide glucagon tests , we used the mean values of duplicates for analyses . for 7phgm , we used the median because of high spread in some of the values . , we used the homeostasis model assessment ( homa ) calculator from oxford university ( http://www.dtu.ox.ac.uk ) . forty - one patients were randomized to either diazoxide ( no . 22 ) or placebo ( no . 19 ) . six patients were excluded during intervention , all of these during the first 3 months of intervention . three patients dropped out for personal reasons ( one from the diazoxide group and two from the placebo group ) . three patients were excluded due to assumed side effects ( from the diazoxide group , one due to a rash and another due to dizziness ) . in the placebo group , one patient experienced sleep disturbances , which disappeared after drug cessation . compared with completers , the dropouts were slightly older , more overweight , had higher blood pressure , and had higher fasting c - peptide at baseline but comparable glycemic control ( table 1 ) . there was a significant decrease in c - peptide levels of 0.17 0.04 ( p = 0.003 ) in subjects as a whole from the time of diagnosis to inclusion . clinical examination and routine blood testing ( see research design and methods ) were normal ( data not shown ) . blood pressure , bmi , glycemic control , and fasting c - peptide levels did not differ between the groups . the use of nicotine was equally distributed . in the diazoxide group , two patients used oral contraceptives , one used a statin , one used a proton pump inhibitor , and one used gabapentin . in the placebo group , three patients used oral contraceptives and one was on asthma inhalation therapy containing glucocorticoids ( table 1 ) . the placebo group gained 1.8 1.0 kg during the first 3 months and had a total weight gain of 2.0 1.5 kg . there was no significant difference in insulin dosage ( total and long acting ) between groups ( supplemental table 2 ) . a1c improved in the diazoxide - treated group compared with the placebo group both during intervention and during follow - up ( fig . neither fasting blood glucose at the study visits nor the 7phgm registration showed significant changes during intervention . however , 3 months after intervention , the fasting blood glucose decreased significantly , by 1.4 mmol / l in the placebo group ( p = 0.017 for difference between groups ) . the same tendency was seen 6 months after intervention . only 51% of the patients completed the 7phgm registrations after the intervention . by these data , there was a tendency ( p = 0.158 ) for a reduction in blood glucose before and after breakfast in the diazoxide - treated versus placebo group . changes in a1c from baseline , recorded after 3 , 6 , 9 , and 12 months of the study ( means se ) . the self - reported frequency of hypoglycemic events before inclusion was comparable between the two groups . during intervention , five patients in the diazoxide group reported frequent minor hypoglycemic events ( > 10 events during 6 months ) . five patients in the placebo group reported frequently minor hypoglycemia and two reported one major event each . fasting and glucagon - stimulated c - peptide levels decreased similarly in both groups throughout the study . from comparable baseline values , the fasting c - peptide in the diazoxide group decreased by 0.05 0.03 nmol / l after intervention and by 0.13 0.02 the decrement in the placebo group was 0.02 0.05 and 0.08 0.04 , respectively ( supplemental table 3 ) . stimulated c - peptide levels showed the same pattern ; from a baseline value of 0.53 0.07 nmol / l , the decrement was 0.1 0.04 and 0.18 0.04 in the diazoxide group . in the placebo group , the baseline value was 0.6 0.1 nmol / l and decreased by 0.04 0.07 and 0.09 0.07 , respectively . to adjust for different glucose concentrations , we calculated the ratio of c - peptide to glucose ( fig . no significant difference between groups was observed . also fasting levels of insulin , proinsulin , and glucagon fasting and stimulated values of c - peptide to glucose ratio at 0 , 3 , 6 , 9 , and 12 months . , diazoxide - treated subjects ; , placebo - treated subjects . insulin sensitivity as assessed by homa - s% was comparable between the groups at baseline . after 3 , 9 , and 12 months , the homa - s% increased by , respectively , 16 , 14 , and 32% in the diazoxide group . sensitivity remained stable in the placebo group ( 3 , 0 , and 4% for the same time periods , p = 0.020 , 0.147 , and 0.072 , respectively , for differences between the groups ) . 74% in the diazoxide and 63% of subjects in the placebo group were positive for both antibodies . participants taken together had a mean gada titer at the start of 182 15 and 168 15 units after 12 months . the decline in gada was nonsignificantly smaller in the diazoxide - treated group ( p = 0.133 ) . the mean ia-2a ( all participants ) was 188 30 units at baseline and 168 31 units after 12 months . subjects who were positive for both gada and ia-2a had a tendency for a more pronounced decline in fasting c - peptide after 6 ( p = 0.092 ) and 12 months ( p = 0.128 ) compared with subjects who were only gada positive , with a similar aggravating effect on stimulated c - peptide after 12 months ( p = 0.083 ) . the proportion of cd4 lymphocytes to the total number of lymphocytes did not differ between the diabetic groups and the healthy control group ( supplemental fig . the proportion of rtregs and atregs , expressed , respectively , as the proportion of cd4/cd45ra / cd25/foxp3 cells to the total number of cd4/cd45ra cells and the proportion of cd4/cd45ra / cd25/foxp3 to the total number of cd4/cd45ra cells , did not differ between the diazoxide- and placebo - treated groups ( supplemental figs . 2 and 3 ) . the results were the same when the groups were split into c - peptide decline over or below the median ( supplemental fig . 4 ) . likewise , when tregs were compared in high versus low titers of gada and ia-2a or numbers of antibodies ( i.e. , one or two antibodies ) , there was no detectable correlation of the treg ratio to disease activity ( data not shown ) . however , we found evidence for a small overall downregulation of tregs in the diabetic group versus the nondiabetic group . this significant difference was found both for the rtregs and the atregs ( supplemental figs . forty - one patients were randomized to either diazoxide ( no . 22 ) or placebo ( no . 19 ) . six patients were excluded during intervention , all of these during the first 3 months of intervention . three patients dropped out for personal reasons ( one from the diazoxide group and two from the placebo group ) . three patients were excluded due to assumed side effects ( from the diazoxide group , one due to a rash and another due to dizziness ) . in the placebo group , one patient experienced sleep disturbances , which disappeared after drug cessation . compared with completers , the dropouts were slightly older , more overweight , had higher blood pressure , and had higher fasting c - peptide at baseline but comparable glycemic control ( table 1 ) . there was a significant decrease in c - peptide levels of 0.17 0.04 ( p = 0.003 ) in subjects as a whole from the time of diagnosis to inclusion . clinical examination and routine blood testing ( see research design and methods ) were normal ( data not shown ) . blood pressure , bmi , glycemic control , and fasting c - peptide levels did not differ between the groups . the use of nicotine was equally distributed . in the diazoxide group , two patients used oral contraceptives , one used a statin , one used a proton pump inhibitor , and one used gabapentin . in the placebo group , three patients used oral contraceptives and one was on asthma inhalation therapy containing glucocorticoids ( table 1 ) . the placebo group gained 1.8 1.0 kg during the first 3 months and had a total weight gain of 2.0 1.5 kg . there was no significant difference in insulin dosage ( total and long acting ) between groups ( supplemental table 2 ) . a1c improved in the diazoxide - treated group compared with the placebo group both during intervention and during follow - up ( fig . neither fasting blood glucose at the study visits nor the 7phgm registration showed significant changes during intervention . however , 3 months after intervention , the fasting blood glucose decreased significantly , by 1.4 mmol / l in the placebo group ( p = 0.017 for difference between groups ) . the same tendency was seen 6 months after intervention . only 51% of the patients completed the 7phgm registrations after the intervention . by these data , there was a tendency ( p = 0.158 ) for a reduction in blood glucose before and after breakfast in the diazoxide - treated versus placebo group . changes in a1c from baseline , recorded after 3 , 6 , 9 , and 12 months of the study ( means se ) . the self - reported frequency of hypoglycemic events before inclusion was comparable between the two groups . during intervention , five patients in the diazoxide group reported frequent minor hypoglycemic events ( > 10 events during 6 months ) . five patients in the placebo group reported frequently minor hypoglycemia and two reported one major event each . fasting and glucagon - stimulated c - peptide levels decreased similarly in both groups throughout the study . from comparable baseline values , the fasting c - peptide in the diazoxide group decreased by 0.05 0.03 nmol / l after intervention and by 0.13 0.02 the decrement in the placebo group was 0.02 0.05 and 0.08 0.04 , respectively ( supplemental table 3 ) . stimulated c - peptide levels showed the same pattern ; from a baseline value of 0.53 0.07 nmol / l , the decrement was 0.1 0.04 and 0.18 0.04 in the diazoxide group . in the placebo group , the baseline value was 0.6 0.1 nmol / l and decreased by 0.04 0.07 and 0.09 0.07 , respectively . to adjust for different glucose concentrations , we calculated the ratio of c - peptide to glucose ( fig . no significant difference between groups was observed . also fasting levels of insulin , proinsulin , and glucagon fasting and stimulated values of c - peptide to glucose ratio at 0 , 3 , 6 , 9 , and 12 months . insulin sensitivity as assessed by homa - s% was comparable between the groups at baseline . after 3 , 9 , and 12 months , the homa - s% increased by , respectively , 16 , 14 , and 32% in the diazoxide group . sensitivity remained stable in the placebo group ( 3 , 0 , and 4% for the same time periods , p = 0.020 , 0.147 , and 0.072 , respectively , for differences between the groups ) . 74% in the diazoxide and 63% of subjects in the placebo group were positive for both antibodies . participants taken together had a mean gada titer at the start of 182 15 and 168 15 units after 12 months . the decline in gada was nonsignificantly smaller in the diazoxide - treated group ( p = 0.133 ) . the mean ia-2a ( all participants ) was 188 30 units at baseline and 168 31 units after 12 months . subjects who were positive for both gada and ia-2a had a tendency for a more pronounced decline in fasting c - peptide after 6 ( p = 0.092 ) and 12 months ( p = 0.128 ) compared with subjects who were only gada positive , with a similar aggravating effect on stimulated c - peptide after 12 months ( p = 0.083 ) . the proportion of cd4 lymphocytes to the total number of lymphocytes did not differ between the diabetic groups and the healthy control group ( supplemental fig . 1 ) . the proportion of rtregs and atregs , expressed , respectively , as the proportion of cd4/cd45ra / cd25/foxp3 cells to the total number of cd4/cd45ra cells and the proportion of cd4/cd45ra / cd25/foxp3 to the total number of cd4/cd45ra cells , did not differ between the diazoxide- and placebo - treated groups ( supplemental figs . 2 and 3 ) . the results were the same when the groups were split into c - peptide decline over or below the median ( supplemental fig . when tregs were compared in high versus low titers of gada and ia-2a or numbers of antibodies ( i.e. , one or two antibodies ) , there was no detectable correlation of the treg ratio to disease activity ( data not shown ) . however , we found evidence for a small overall downregulation of tregs in the diabetic group versus the nondiabetic group . this significant difference was found both for the rtregs and the atregs ( supplemental figs . this study is the longest intervention trial with diazoxide so far performed in subjects with diabetes . the main findings are that 6 months of low - dose diazoxide treatment was accompanied with few side effects and did not measurably affect residual insulin production but was associated with improved metabolic control . the absence ( or near absence ) of well - known side effects ( 7,8 ) is in line with our previous studies in type 2 diabetic patients ( 9,10 ) . this study extends these observations to a younger age - group and a longer treatment period . one may conclude that further long - term studies with low - dosage diazoxide , if they are indeed indicated , is not hampered by concerns about safety or compliance . in contrast to the previous studies ( 7,8 ) , we did not observe any postintervention effect on fasting or glucagon - stimulated levels of c - peptide . ( 7 ) , which reported beneficial effects of diazoxide treatment , is the study most similar to ours ; a discussion of differences of potential importance is thus in order . age , c - peptide levels at baseline , and method of testing insulin production were similar . study were included within a week after diagnosis , while intervention in our subjects started up to 12 weeks after initiation of insulin treatment ( in line with current recommendations ) . it seems possible that our subjects were in partial remission at baseline , whereas the participants in the bjrk et al . the implications of such a difference are however not clear . a simple explanation for the lack of measurable effects on insulin production might be the lower dosage of diazoxide . we used a dose of diazoxide that was about one - third of the dose used in the bjrk et al . we reasoned that a longer intervention period ( i.e. , 6 rather than 3 months in the previous studies ) could compensate for a lower dose . such a putative effect may , however , have been nullified by a blunting of the effects of diazoxide with time . thus , there was at least a tendency for inhibition of fasting and stimulated c - peptide after 3 months of intervention but no such effect at the end of the 6-month intervention period . the notion of time - dependent blunting of effects is compatible with the bjrk et al . study , in which the suppression of c - peptide during intervention was blunted already at the end of the 3-month intervention period . assumed blunting to be due to a recovery of the secretory capacity of the -cell . this explanation can not be ruled out but is less likely in the light of the postintervention data in our study . one possibility is improved insulin production that was not picked up by the present methods of testing ( i.e. , fasting and glucagon - stimulated c - peptide ) . the fact that the beneficial effect lingered postintervention would be compatible with a hidden postintervention effect of diazoxide on -cell function . one can not rule out that other tests of -cell function , such as meal - stimulated secretion , would have detected a difference . an alternative or additional possibility is that diazoxide favorably affects metabolic control by improving insulin sensitivity . such a notion is compatible with the insulin requirement not being enhanced in the diazoxide - treated group despite the expected inhibitory effect of diazoxide on insulin secretion . similar observations were made in the two previous clinical studies ( 7,8 ) , and data from animal studies have indicated improved sensitivity ( 18 ) . further , when we calculated homa - s% ( rarely done in type 1 diabetes but deemed admissible ) , there was significant improvement in the diazoxide - treated group during the intervention . the molecular mechanisms for an insulin - sensitizing effect of diazoxide remain to be clarified . of possible significance is the fact that body weight increased more in the placebo group during the first 3 months . in animal studies , diazoxide decreases appetite ( 20 ) , and a similar , albeit unproven , effect could be operative in humans ( 21 ) . to our knowledge , ours is the first study to prospectively analyze the relative treg proportion in pbmcs during the early phase of type 1 diabetes . the foxp3 marker was unaltered through the 6- month intervention period both in the diazoxide and the placebo group . on the other hand , the treg ratio differed significantly between age - matched nondiabetic subjects and the type 1 diabetic subjects . these observations are in contrast to a previous report ( 22 ) , which , however , did not focus specifically on differences between newly diagnosed type 1 diabetes versus healthy subjects . in our study , the samples from the healthy control subjects were assembled in one center ( trondheim ) , thus avoiding the transportation necessary for many of the diabetes samples . however , we did not find any differences between samples from trondheim and those from other centers ( results not shown ) . nevertheless , the observed differences between nondiabetic and diabetic subjects need to be confirmed by an independent study , ideally performed in freshly isolated cells . as to humoral autoimmunity , we , as others ( 7,8 ) , did not detect any significant influence by diazoxide on gada levels . are further studies with diazoxide in type 1 diabetes warranted ? at first glance , the lack of effects on endogenous insulin production would speak against such endeavors . however , since diazoxide at higher dosage does exert beneficial effects , a beneficial effect on insulin production at the present dose in a combination therapy , for instance together with an immunosuppressant , does not seem unreasonable . in any case , the beneficial effects of diazoxide on metabolic control that we observe could be welcome in a combination therapy .

asthma ( mim 600807 ) is a common chronic respiratory disease resulting from the complex interaction of genetics and environmental factors . short - acting b2-adrenergic receptor agonists are commonly used bronchodilators for symptom relief in asthmatics . activated b2-adrenergic receptor ( adrb2 ) stimulates adenylyl cyclase leading to increased cyclic adenosine monophosphate ( camp ) level . a primary function of camp is to activate protein kinase a ( pka ) , which in turn activates myosin light chain phosphatase , reducing phosphorylation and generating relaxation . camp - activated pka also activates and phosphorylates phosphodiesterase 4 ( pde4 ) isoforms which then degrade camp providing thus a pivotal acute feedback mechanism [ 24 ] . pde enzymes , ubiquitously distributed in mammalian tissues , are essential to control numerous physiological processes . pdes are encoded by 11 gene families , each encompassing 1 to 4 subfamilies of distinct genes [ 5 , 6 ] . the pde4 family has gained interest in view of their involvement in the regulation of processes such as inflammation and cognition [ 3 , 7 ] . four genes ( 4a d ) encode a large number of pde4 isoforms through activation of different promoters or alternative splicing [ 3 , 7 ] . the dominant pde4 type expressed in airway smooth muscle cells is pde4d [ 810 ] . pde4d encode a variety of long and short isoforms which are summarized in figure 1 . long pde4d5 isoform was shown to be a key regulator of b2-adrenoceptor - induced camp turnover in human airway smooth muscle cells . chronic elevation of camp could therefore lead to desensitization of the receptor to b2-agonists following long - term exposure . this differs from the acute transient desensitization of the receptor caused by pka phosphorylation and activation or by agonist - triggered b - arrestin - mediated recruitment to the b2-adrenoreceptor [ 12 , 13 ] . experiments using knockout mice for pde4d gene further supported pde4d role in a regulation of airway smooth muscles contractility [ 9 , 14 ] . it is thus not surprising that recent genome - wide association study ( gwas ) for asthma identified pde4d as a highly plausible candidate gene . pde4d was also associated with mineral bone density , chronic obstructive pulmonary disease ( copd ) in japanese population , and stroke [ 15 , 19 ] . pde4d associations with diverse phenotypes are most likely due to its multiple splicing forms with exons scattered over 1.5 mb , each regulated in a tissue - and - cell specific manner and involved in multiple signalling processes [ 3 , 7 , 20 ] . as the role of pde4 enzymes was discovered , the interest in controlling their activity was apparent , as summarized in several reviews [ 2 , 2023 ] . pde4 inhibitors have been also viewed as a promising therapy for the treatment of inflammatory airway disorders . the potentially beneficial action of pde4 inhibitors has been successfully translated in clinical trials for copd treatment [ 22 , 25 , 26 ] , and their efficacy for asthma control is presently under investigation . no data are yet available on the involvement of this gene in the response to drugs used in asthma treatment . the aim of this study was to test the genetic variants in pde4d gene as potential predictive factor of the response to a single dose of short - acting b2-agonist . patients ( n = 133 ) were recruited from the asthma and outpatient pulmonary clinics at sainte - justine hospital between march 2007 and september 2010 . subjects were eligible if they were caucasians , aged 5 to 18 years , able to perform an appropriate spirometric maneuver under the supervision of technicians specifically trained in pulmonary function testing of children , and meeting standard criteria for a diagnosis of asthma . subjects were eligible for the recruitment irrespectively of the type of maintenance therapy ( inhaled corticosteroids , ics , or leukotriene modifiers ) and irrespectively of disease control and severity . atopy was defined as presence of physician - diagnosed eczema and/or serum food - specific ige and/or positive skin test for 1 or more aeroallergens or food antigens . exclusion criteria were as follows : use of long - acting bronchodilators in the last month , prematurity , a history of significant pulmonary disease other than asthma , and known immunodeficiencies . the study protocol was reviewed and approved by the sainte - justine hospital ethics committee , and informed consent was signed by each patient 's parent . to assess bronchodilator responsiveness , four inhalations of albuterol were administered via metered dose inhaler ( 100 g / inhalation ) with valved holding chamber and mouthpiece ( aerochamber ; invacare corp , elyria , ohio , usa ) . spirometry was performed before ( pre ) and 15 min after ( post ) receiving albuterol sulfate in accordance with the american thoracic society guidelines . responsiveness to albuterol was reported as the percent change in forced expiratory volume in one second ( fev1 ) after albuterol administration : 100 ( ( fev1(post ) fev1(pre))/fev1(pre ) ) . the equations proposed by polgar and promadath were selected for the calculation of predicted and reference values . dna was extracted from saliva using oragen kit according to manufacturer ( dna genotek , http://www.dnagenotek.com ) . two polymorphisms , located at positions 1345 ( rs1504982 ) and 984 ( rs10940648 ) relative to the first nucleotide of pde4d5 mrna isoform ( af12073 ) , were the only snps available at ncbi data base that reside in the 2 kb region preceding pde4d5 transcription start site . two other pde4d polymorphisms were selected based on published data : rs1544791 , located in the intron 2 of the longest pde4d7 isoform ( nm_001165899.1 ) , was a top - ranking snp in the gwas of asthma and the other , rs829259 , located in 3utr of all pde4d isoforms , was reported in association with the copd in adults . all dna segments containing the polymorphic sites were amplified by pcr in a total volume of 20 ul using 15 ng of genomic dna , 0.4 m of each primer , 140 m dntps , 10 mm tris - hcl ( ph 8.3 ) , 2.3 mm mgcl2 , 50 mm kcl , and 0.4 u of platinum taq polymerase ( invitrogen ) . amplification was performed for 37 cycles composed of 30 s at 94c , 30 s at 57c , and 30 s at 72c , following initial denaturation of 3 min at 95c . genotyping was performed by allele - specific oligonucleotide ( aso ) hybridization as described previously . genotype was included in the model as fixed factor , and the baseline fev1% predicted value was included as continuous covariate . the analyses were restricted to patients having airway obstruction , for example , baseline fev1/fvc ( fvc forced vital capacity ) ratio lower than 90% [ 33 , 34 ] ( see table 2 for patients characteristics ) . hierarchical multivariate linear regression was used to assess the association between genotypes of rs1544791 and rs1504982 with the inclusion of additional covariates ( age , sex , atopy , and use or not of ics and leukotriene modifiers ) . asthma severity of patients recruited in our study is comparable to that observed in the childhood asthma management program ( camp ) and the leukotriene modifier or corticosteroid or corticosteroid salmeterol trial ( loccs ) populations used in a number of pharmacogenetic studies . significant differences were obtained for pde4d5 promoter 1345 ( rs1504982 , p = 0.03 ) and for rs1544791 polymorphisms ( p = 0.006 ) . the mean % fev1 change se for rs1504982 c carriers was 7.1 0.7 versus 10.3 1.2 in noncarriers , and for rs1544791 g carriers , the mean % fev1 change se was 7.4 0.7 as compared to 13.7 2.1 in individuals without this allele . the association remained significant for both snps with inclusion of age , sex , atopy , and controller medication into the multivariate model ( p = 0.02 and p = 0.004 , resp . ) . the recent association of pde4d to asthma phenotype , as well as its known function , makes pde4d an excellent candidate gene for the association with the outcome of asthma treatment . pde4d is a well - established regulator of airway smooth muscles contractility [ 9 , 14 ] and plays a key role in adrb2-induced camp regulation . study conducted in vitro demonstrated that a single nucleotide substitution in one of camp - responsive elements in pde4d5 promoter created by site - directed mutagenesis , causes camp - driven upregulation of pde4d5 expression and functional activity in human airway smooth muscle cells . based on the publicly available data , it does not seem that there is a naturally occurring genetic variant in pde4d5 camp - responsive elements . among snps analyzed here , 1345 variation of the pde4d5 promoter was associated with the change in fev1 following a single dose of short - acting b2-agonist . the change in fev1 was also significantly associated with rs1544791 , the top ranking snp in gwas study of asthma susceptibility . the same g asthma predisposing allele was associated in our study with the poor treatment outcome . schematic representation of pde4d gene structure and relation to pde4d isoforms is given in figure 1 . snps tested within this study are depicted as well as those associated with variety of phenotypes reported by others [ 1517 ] . each of the phenotypes associated with pde4d gene ( stroke , asthma , bmd , and copd ) maps to different linkage disequilibrium ( ld ) blocks . the same is true for the snps significantly associated with b2-agonist response in this study . the snp rs1544791 is located within 181 kb intron 2 of the longest isoform ( pde4d7 , nm_001165899.1 ) . this snp resides within 75 kb long ld block and is separated by 374 kb from pde4d5 transcription start . despite its distant location , it could act as a remote cis - acting element . indeed , functional - snp database ( f - snp ) integrating information from 16 bioinformatics tools predicts that it might be a transcription regulator . we further assessed rs1544791 in allelic expression dataset generated in hapmap lymphoblastoid ( ceu ) cell lines of european ancestry where allelic differences within individual were renormalized as recently described . population differences for allelic expression of uc010iwj.1 isoform ( also corresponding to the first few exons unique to the longest pde4d7 isoform nm_001165899.1 ) were significantly associated ( p = 0.005 ) with rs1544791 genotype . snp 1345 is also recognized as transcription regulator in f - snp data base and in ucsc browser ( http://genome.ucsc.edu ) . in the analysis , we used % fev1 change as a continuous variable with the correction for baseline fev1% predicted values rather than categorical phenotype of responders and nonresponders based on a % fev1 change above or below a given predefined value . the reason for this was a strong positive correlation between poor response to the administered albuterol and high baseline fev1% predicted values . since about 50% of our patients were on regular ics and 20% were on leukotriene modifiers , it is reasonable to assume that prior treatment with these drugs had improved lung function in a proportion of our study subjects , therefore reducing the magnitude of the response to albuterol . because of ethical considerations , we did not withdraw anti - inflammatory treatment prior to assessment , but we therefore restricted our analysis to the patients with airway obstruction . since airway obstruction is defined by a reduced fev1/fvc ratio which normally reaches a value of 0.9 in children , we limited our analysis to patients with fev1/fvc ratio lower than that threshold . an alternative strategy would have been to recruit new asthma patients prior to initiation of any asthma controller therapy . however , onset of childhood asthma usually occurs early during the preschool age and valuable spirometric data can not be obtained in preschoolers . a multicenter study would have been necessary in order to recruit the appropriate numbers of patients . this study demonstrates the first attempt to explore the association of pde4d genetic variations and response to asthma b2-agonists treatment in caucasian children . results obtained are not necessarily applicable to other populations as frequency of tested polymorphisms and ld structure differ . if confirmed in a larger study , this finding will build upon existing knowledge in asthma pharmacogenetics ultimately assisting personalized patient treatment . the work was supported by a grant from the canadian institutes of health research ( mop-81154 ) ; m. krajinvoic is a scholar of the fonds de la recherche en sant du qubec .

an estimated 157,500 surgical site infections ( ssis ) associated with inpatient surgeries occurred in the united states in 2011 according to a prevalence survey by the u.s . centers for disease control and prevention ( cdc ) 1 . in spite of improvements in operating room practices , sterilization methods , physical barriers , surgical techniques , and routine systemic antimicrobial prophylaxis 2 - 5 , ssis remain the most common healthcare - associated infection 6 , with an incidence of up to 2.5% after primary hip and knee arthroplasty and 10% following revision surgery 7 and an increased relative risk of infection in high - risk patients 8 - 11 . these figures could be underestimated , however , owing to the increasing occurrence of multidrug - resistant pathogens 12 , 13 . periprosthetic infection often necessitates implant removal and is responsible for a disproportionate burden of morbidity and mortality 14 and elevated economic and social costs 15 , 16 . recognizing these problems coatings of devices would be most useful in the prevention and treatment of implant - associated infection 17 and a similar recommendation was delivered at a consensus meeting on periprosthetic joint infections 18 . however , developing new antibacterial implant coatings appears extremely challenging 19 , 20 . biocompatible hydrogels are able to deliver pharmacological agents locally and may be designed to meet the desired elution pattern 21 . recently , a fast - resorbable hydrogel coating that works as a physical barrier to bacterial adhesion and that can be intra - operatively loaded with various antibacterials has been developed 22 . based on the observation that bacterial colonization , from microbial adhesion to an established mature biofilm layer , only takes few hours 23 and given the known efficacy of short - term systemic antibiotic prophylaxis 24 , a fast - resorbable antibacterial coating may provide short - term local protection , while limiting possible long - term unwanted side effects 25 . this novel hydrogel coating ( defensive antibacterial coating [ dac ] , novagenit srl , mezzolombardo , italy ) , composed of covalently linked hyaluronan and poly - d , l - lactide , is designed to undergo complete hydrolytic degradation within 72 hours , during which it completely releases a variety of antibacterials at concentrations ranging from 2% to 10% . the hydrogel showed synergistic activity with various antibiotics and antibiofilm agents in vitro 26 . also , it has been proven safe and effective in vivo in a rabbit model of highly contaminated implant both with 27 and without systemic antibiotic prophylaxis 28 . following a brief previous report 29 , we here present the results of a multicenter european trial on this novel technology applied to total hip and knee joint prosthesis . from november 2013 to june 2015 , 380 patients were included in this prospective , observational multicenter study ( fig . the study protocol was approved by the local ethical committees of the six participating centers . the study was performed within the 7th european framework programme ( project # 277988 ) and funded by the european commission and the participating partners ( clinical institutions and the following private companies : novagenit srl , mezzolombardo , italy , acting as project leader ; adlerortho srl , bologna , italy ; arcos sarl , brignoles , france ; belgafix sprl , drogenbos , belgium ) . treatment assignment at each study center was generated by an electronic system ; patients were assigned to receive an implant either with the antibiotic - loaded dac ( dac ) coating ( treatment group ) or without the coating ( control group ) . inclusion criteria were : the need for a cementless or hybrid ( partially cemented ) total hip or knee prosthesis and age > 18 years . exclusion criteria were : pregnancy , breast - feeding or planning to become pregnant during the study period , the presence of an active infection at the site of surgery , severe malignancies with a life expectancy of less than 3 months , previous diagnosis of immune depression or immunosuppressive treatment for organ transplantation , known allergy to the antibiotics or the dac hydrogel constituents , unwillingness or inability to present for follow - up examinations or refusal to sign the informed consent documents or inability to do so . after routine pre - operative workup , all patients underwent total joint replacement according to the current practice of each participating center . the surgical approach and type of implant was left to the surgeon 's discretion ; similarly , systemic antibiotic prophylaxis was given according to each center 's routine protocol 30 . all patients received low - molecular - weight heparin for deep vein thrombosis prophylaxis or oral anticoagulants for 4 to 6 weeks postoperatively . patients undergoing hip arthroplasty received prophylaxis with indomethacin or celecoxib for 2 weeks after surgery to prevent heterotopic bone formation . 2 ) , was filled at surgery with a solution of 5 ml sterile water for injection and the desired antibiotic to obtain in approximately 3 to 5 minutes the antibiotic - loaded hydrogel at a dac concentration of 6% ( w / v ) and an antibiotic concentration ranging from 20 mg / ml to 50 mg / ml , depending on the surgeon 's choice . the surgeons could choose the antibiotic from among a list of antibacterials previously tested as being compatible with the hydrogel ( novagenit srl , data on file ) . a few minutes after reconstitution , the hydrogel was directly spread onto the implant , which was then inserted into the body in the usual way ( figs . 3 and 4 ) . the main outcome was the absence of adverse events and side effects related to the hydrogel coating , as assessed by clinical , laboratory and radiographic examinations . another targeted main outcome was a reduction in ssis , as defined below , in the treatment group as compared with the controls . secondary outcomes were a reduction in delayed wound healing in the treatment versus the control group . wound healing was assessed at postoperative days 7 and 14 using the asepsis scoring method 32 ; serum laboratory tests , x - ray and clinical evaluations were scheduled at 6 4 weeks , 3 months 4 weeks , 6 , 12 , 18 , and 24 months 8 weeks postoperatively . clinical evaluation was performed using the sf-12 health survey score and the harris hip or the knee society scores as appropriate ; any signs of early ssi , including pain , redness , warmth , swelling , draining wound , fistulas , etc . , according to the cdc procedure - associated module ssi ( http://www.cdc.gov/nhsn/pdfs/pscmanual/9pscssicurrent.pdf ) were also recorded . ssis were defined as the presence of positive local clinical signs of acute inflammation and/or a draining sinus requiring further surgery , including early debridement or implant removal and/or unplanned antibiotic treatment with or without a positive cultural examination . delayed wound healing was defined as an incomplete healing of the wound at 4 weeks after surgery , including the presence of wound dehiscence , necrosis or serum leakage , that may need further medication , but that did not require any additional surgical treatment . serious adverse events and complications or side effects were also recorded whenever present at follow - up . radiographic examination , including evaluation of osteolysis or progressive ( > 2 mm ) radiolucent lines around the implant or signs of implant loosening or sinking , was performed by an independent radiologist unaware of assignment to dac treatment . categorical data were analyzed using fisher`s exact test ; continuous data were compared using student 's t test ( http://graphpad.com/ ) . sample size was calculated assuming an overall ssi incidence of 1% in the treatment group and of 6% in the control group and a 10% dropout rate ( alpha error : 0.05 , power 0.80 ; cf . after routine pre - operative workup , all patients underwent total joint replacement according to the current practice of each participating center . the surgical approach and type of implant was left to the surgeon 's discretion ; similarly , systemic antibiotic prophylaxis was given according to each center 's routine protocol 30 . all patients received low - molecular - weight heparin for deep vein thrombosis prophylaxis or oral anticoagulants for 4 to 6 weeks postoperatively . patients undergoing hip arthroplasty received prophylaxis with indomethacin or celecoxib for 2 weeks after surgery to prevent heterotopic bone formation . 2 ) , was filled at surgery with a solution of 5 ml sterile water for injection and the desired antibiotic to obtain in approximately 3 to 5 minutes the antibiotic - loaded hydrogel at a dac concentration of 6% ( w / v ) and an antibiotic concentration ranging from 20 mg / ml to 50 mg / ml , depending on the surgeon 's choice . the surgeons could choose the antibiotic from among a list of antibacterials previously tested as being compatible with the hydrogel ( novagenit srl , data on file ) . a few minutes after reconstitution , the hydrogel was directly spread onto the implant , which was then inserted into the body in the usual way ( figs . 3 and 4 ) . the main outcome was the absence of adverse events and side effects related to the hydrogel coating , as assessed by clinical , laboratory and radiographic examinations . another targeted main outcome was a reduction in ssis , as defined below , in the treatment group as compared with the controls . secondary outcomes were a reduction in delayed wound healing in the treatment versus the control group . wound healing was assessed at postoperative days 7 and 14 using the asepsis scoring method 32 ; serum laboratory tests , x - ray and clinical evaluations were scheduled at 6 4 weeks , 3 months 4 weeks , 6 , 12 , 18 , and 24 months 8 weeks postoperatively . clinical evaluation was performed using the sf-12 health survey score and the harris hip or the knee society scores as appropriate ; any signs of early ssi , including pain , redness , warmth , swelling , draining wound , fistulas , etc . , according to the cdc procedure - associated module ssi ( http://www.cdc.gov/nhsn/pdfs/pscmanual/9pscssicurrent.pdf ) were also recorded . ssis were defined as the presence of positive local clinical signs of acute inflammation and/or a draining sinus requiring further surgery , including early debridement or implant removal and/or unplanned antibiotic treatment with or without a positive cultural examination . delayed wound healing was defined as an incomplete healing of the wound at 4 weeks after surgery , including the presence of wound dehiscence , necrosis or serum leakage , that may need further medication , but that did not require any additional surgical treatment . serious adverse events and complications or side effects were also recorded whenever present at follow - up . radiographic examination , including evaluation of osteolysis or progressive ( > 2 mm ) radiolucent lines around the implant or signs of implant loosening or sinking , was performed by an independent radiologist unaware of assignment to dac treatment . categorical data were analyzed using fisher`s exact test ; continuous data were compared using student 's t test ( http://graphpad.com/ ) . sample size was calculated assuming an overall ssi incidence of 1% in the treatment group and of 6% in the control group and a 10% dropout rate ( alpha error : 0.05 , power 0.80 ; cf . 1 ) ; a total of 373 patients ( 189 treated and 184 controls ) were available at an average follow - up of 14.5 5.5 months ( range 6 to 24 ) ( table 1 ) . there were no significant differences in age , sex , site of surgery , and host type between the two groups . peri - operative data ( table 2 ) showed that the majority of the patients required a primary hip joint replacement and approximately 30% in both groups underwent revision surgery . revision surgery was performed as a part of a two - stage procedure for infection in 48 ( 92.3% ) patients in the control group and in 51 ( 94.4% ) patients in the treatment group . primary hip and knee replacement was performed at five centers ; revision procedures were performed at three centers . cefazolin was the most often used antibiotic for short - term prophylaxis in both groups ; combined systemic short - term prophylaxis was administered in approximately half of the patients ( table 2 ) . on average , the most often used antibiotics were vancomycin and gentamicin at a concentration of 5% and 3.2% , respectively . fifteen patients received an implant with a combined vancomycin and meropenem antibiotic coating ; 4 patients received an implant coated with teicoplanin 5% or ceftazidime 5% or amphotericin b 5% , all in a second - stage procedure for previous infection ( microorganism - targeted local antibiotic coating ) . there was no difference in time to early wound healing between the two groups : the mean asepsis score at 7 and 14 days was 2.6 4.9 and 2.1 6.1 in the control group and 1.9 4.2 and 1.9 5.3 in the treatment group , respectively . unplanned antibiotic treatment during hospital stay , for reasons other than ssi ( mainly urinary or respiratory tract infections ) , was recorded in 8 ( 4.3% ) patients in the control group and in 9 ( 4.7% ) in the treatment group . there were no significant differences in serum laboratory values and average clinical scores between the two groups at the first follow - up visit ( tables 3 and 4 ) . at the 6-month control visit , / l in 12 ( 6.5% ) patients in the control group and in 5 ( 2.6% ) patients in the treatment group ( p=0.08 ) . delayed wound healing was recorded in 7 ( 3.8% ) patients in the control group and in 2 ( 1.2% ) in the treatment group ( p=0.1 ) . four ssis were reported in the control group of patients undergoing primary joint arthroplasty ( n=132 ; 3% ) and one ssi in the treatment group ( n=135 ; 0.7% ) ; the mean infection rate after primary joint arthroplasty in the control group was 0% in two centers and 2.5% , 3.7% , and 4.5% in the three other centers , respectively . seven post - surgical infections were observed in the controls undergoing a revision procedure ( n=52 ; 13.4% ) and none in the treatment group ( n=54 ) . the mean infection rate after revision joint arthroplasty in the control group was 10.3% , 16.7% , and 17.6% in three centers . overall , 11 ssis were reported in the control group and only one in the treatment group ( 6.0% vs. 0.6% ; p=0.003 ) . when only patients with a minimum follow - up of 12 months were included in the analysis , the overall incidence of post - surgical infection was 6.4% in the controls ( 8/125 ) and 0% ( 0/126 ) in the treatment arm ( p=0.003 ) . the complications included : hip prosthesis dislocation ( n=1 ) treated with modular parts exchange , knee stiffness ( n=2 ) , and deep vein thrombosis after total knee replacement ( n=2 ) in the control group ; hip revision prosthesis dislocation ( n=1 ) treated conservatively , deep vein thrombosis after hip revision surgery ( n=2 ) , and stiff knee after revision ( n=1 ) in the treatment group . no allergic or skin reactions or adverse events related to the hydrogel coating were reported in either group . radiographic examination revealed no signs of focal osteolysis around the implant in either group ; progressive ( > 2 mm ) radiolucent lines around the implant were observed in 3 patients in the treatment group and in 7 patients in the control group ; no signs of implant loosening or sinking were reported in either group and no brooker 3 or 4 heterotopic ossifications were observed . this is the first clinical trial to report on the safety and efficacy of a fast - resorbable antibiotic - loaded hydrogel coating for total joint replacement . consistent with previous in vivo studies 26 , 28 , our data show that use of the hydrogel coating is clinically safe and devoid of any detectable local or systemic side effects in the short - term follow - up . the biocompatibility of the hydrogel basic components and the short time to resorption 19 , 22 make the possible occurrence of delayed side effects quite unlikely . also , our results showed a ten - fold reduction in the occurrence of early ssis in the treated patients as compared with the controls . this confirms prior in vivo studies 26 , 28 and is the first demonstration of clinical efficacy that short - term local prophylaxis may reduce post - surgical septic complications after joint replacement . although still observed after primary joint replacement , the protective effect was much more evident in the patients undergoing revision surgery . it should be noted that approximately 75% of the patients in both groups were type b or c hosts and that the majority of revision surgeries were performed in previous infection . were the first to popularize the incorporation of antibiotics into polymethylmethacrylate ( pmma ) bone cement for local antibiotic prophylaxis in cemented total joint arthroplasty 33 . clinical studies have shown that antibiotic - loaded bone cement can decrease deep infection rates of cemented total hip arthroplasties and revision rates due to suspected aseptic this solution has proven both effective and economically sound , especially in high - risk patients 35 , 36 . however , because pmma was not designed as a local delivery carrier of antibiotics it may have some limitations . antibiotic - loaded pmma may not prevent biofilm formation and may be associated with the development of antibiotic - resistant small - colony variants 37 , 38 . furthermore , the increasing use of cementless implants worldwide , especially at the hip , make this option possible for only a limited number of patients . silver coating is probably the most extensively studied 40 , though concerns have been raised about the possible toxicity of silver ions 41 . to overcome this problem , research efforts have recently focused on new silver coating technologies 42 , 43 . however , despite the demonstrated clinical efficacy in two comparative studies on a small series of patients 44 , 45 , the routine use of silver - coated implants remains rather limited . excellent clinical efficacy was recently demonstrated for iodine coating of titanium alloys in a continuous , non - comparative series of patients undergoing joint replacement 46 . however , the unknown long - term , questionable large- scale applicability , and the complex regulatory pathway still limit this technology . biodegradable polymers and sol - gel coatings have been shown to provide controlled antibiotic release on titanium 47 , 48 or hydroxyapatite implants 49 , offering ease of use , versatility , and possible large - scale application . in the present application , the anti - adhesive ability of the dac hydrogel has been coupled to local antibiotic administration , to reduce the chance for planktonic bacteria to colonize the implant . although the minimum of 6 months monitoring appears adequate to detect early post - surgical septic complications , as defined by infectious diseases society of america ( idsa ) guidelines 50 , longer follow - up periods are necessary to further investigate delayed or late periprosthetic infections . in this regard , it should also be noted that the analysis of the patient subgroups observed for a minimum follow - up of 12 months did confirm the difference between the treatment and the control groups . second , the study design deliberately left the choice of systemic antibiotic used for prophylaxis and the one added to the hydrogel locally at the discretion of each study center . indeed , there is no scientific evidence showing the superiority of one antibiotic prophylaxis over another after joint prosthesis 51 . other limitations are the exclusion of infected joint prostheses and other potentially challenging clinical situations in which an antibacterial coating could be useful .

this is based on visual recognition of particular patterns of distinct cells indicative of specific or general cellular processes . however , it can be difficult or impossible to disambiguate cells in fixed sections using standard illumination methods . in the context of solid tumors , multiple molecular markers particularly at the protein level using immunohistochemistry can be imaged using multispectral microscopy . this is an imaging technique in which a section is imaged multiple times , each time illuminated using a distinct and narrow range of wavelengths . the result is a cube of optical density data or , equivalently , a spectrum of optical densities at each pixel location . this data can be processed to identify regions or cells that are positive for particular markers ( e.g. , via thresholding of optical densities and other techniques ) . however , even with sophisticated analysis and visualization tools , it is difficult for expert clinicians to interpret images acquired using more than a few markers . furthermore , while it is believed that cells with particular immunohistochemical ( ihc ) profiles may interact to varying degrees of patient detriment , there are few quantitative methods available for investigating these hypotheses . the present paper presents such a method and demonstrates the application of it to a study of follicular lymphoma ( fl ) . given a cube of data from a multiple - stained section imaged using multispectral microscopy , we assumed that it is possible to determine whether each cell in a section is positive ( 1 ) or negative ( 0 ) for each marker and to obtain cells spatial ( x , y ) co - ordinates . this can be achieved , for example , using commercially available software ( e.g. , nuance ; perkinelmer inc . , let denote a set of n markers and the positivity of a cell for stain s as s . we adopt the term combinatorial molecular phenotype ( cmp ) from a previous study that describes the positivity of a cell for all stains in as the binary vector = ( s1 , , sn ) , where si denotes the i stain . for example , a possible cmp for an image stained with three markers may be = ( 1 , 0 , 0 ) . no ( x , y ) co - ordinates will be recorded for cells that are negative for all markers in . therefore , any of 2 - 1 cmps may be observed . each possible cmp can be assigned a unique number according to a suitable binary counting scheme . if the right - most element of a cmp represents the least significant binary digit and the next right - most element represents the next least significant digit and so on , then the vectors ( 0 , 1 , 0 ) , ( 1 , 0 , 0 ) , and ( 1 , 1 , 1 ) can be numbered 2 , 4 , and 7 , respectively . because this numbering system begins at 1 , it is useful for indexing the matrices we introduce later in this paper however , less sophisticated software is limited to segmenting regions from particular sub - bands , giving rise to separate sets of region centroid co - ordinates for each stain , some of which may encompass multiple cells ( i.e. , inaccurate segmentation ) . however , it is possible to use the region centroid co - ordinates obtained for each marker independently to identify regions that are so sufficiently close that they are likely to correspond to the same cell , and , therefore , to approximate cmps . first , regions likely to encompass multiple cells are rejected by discarding those that are larger than the maximum area expected for typical cells . then , co - ordinates from different markers that are within a pairing distance , dp , are combined into a single co - ordinate to which the appropriate cell cmp is associated . later in the paper , we have shown how distance dp may be chosen statistically . we hypothesized that two cells sufficiently close together and with identical or different cmps may interact to varying degrees of patient benefit or detriment . we call this a hypothesized interaction . by extension , we hypothesize that if a particular hypothesized interaction does indeed act in this way , then many such interactions between pairs of cells will have a larger effect on patient outcome . we formalize the notion of hypothesized interactions and quantify their occurrence by introducing a commutative arrow operator . a particular hypothesized interaction y consider all possible pairings of cells within a given section , such that no two paired cells are located further than an interaction distance , di , from one another . the arrow notation specifies that a proportion , p , of all pairings , are between a cell with cmp number i and another with j. this notation is convenient because it can be used to form graphs , which may aid visualization . later in this paper , we explain how an optimal value of di can be chosen statistically . the proportion p can also be interpreted as a probability . by calculating p for each possible let d be a ( 2 1 ) ( 2 1 ) symmetric matrix , whose ( i , j ) element is the probability of observing a cell with cmp number i within a distance di of a cell with cmp number j. if y is a random variable that represents a hypothesized interaction , then the statistical distribution of y can be written as y ~ d , and we call d a hypothesized interaction distribution ( hid ) . example hypothesized interaction distributions and their corresponding graphs we are interested in testing the hypothesis that cells of particular types may interact to varying degrees of patient benefit or detriment . specifically , we are interested in investigating whether hids facilitate the assignment of patients into groups with good versus poor survival . taking inspiration from grey - level co - occurrence matrix analysis methodology in the texture analysis literature , table 1 defines three scalar summary statistics that can be used to characterize a hid . note that the indexing used in the summations accounts for the fact that the upper and lower triangles are symmetric and that only one of these triangles is required to specify the hid ( i.e. , the elements in one of the triangles sum to unity ) . definitions and descriptions of scalar summary statistics distribution peakiness is characterized in different ways by de and h(d ) ; dmax characterizes the degree to which the most common hypothesized interaction occurs . shannon entropy , h(d ) , measures the expected information ( in bits ) required to transmit a randomly chosen hypothesized interaction , drawn from the distribution d , over an idealized channel . hypothesized interactions associated with uniform distributions require more information than those from non - uniform distributions . given a vector of values of a particular summary statistic ( e.g. , dmax ) for n patients , = ( 1 , , n ) , we can choose an optimal threshold , t , on that statistic that best assigns patients to groups on the basis of survival via the optimization here , the i element of the vector s is the survival time for the i patient and the function gt returns a vector whose i element is the group assignment of the i patient resulting from the application of threshold t to the i element of the vector . the objective function computes the value of the statistic associated with the null hypothesis that the survival curves arising from the group assignments are equal ; high values of have corresponding low p values . the distances di and dp may in principle be selected by the investigator , for example , by drawing upon expert clinical knowledge . however , this approach is subjective and we therefore proposed an objective method . optimal values ( di , dp ) can be chosen statistically via the optimization this is a modification of equation 1 , where (di , dp ) returns a vector of summary statistics for hids computed using distances di and dp . the aims of this experiment were to demonstrate the application of hid analysis to data from multispectral microscopy of multiple - stained tissue sections from a sample of fl patients and to evaluate the method 's ability to identify patient groups that differ in survival . twenty six archived human lymph nodes from patients ( 16 male , 10 female , aged 36 - 77 years , mean 55 years ) with a diagnosis of fl were collected at initial diagnosis , prior to the treatment . written informed consent and local research ethics committee approval were obtained . clinical data and up to 14 years of follow - up and survival data were available for each sample ( 19 dead , 7 alive ; median time to death 32 months ; range 2 - 170 months ; 171 months follow - up in all patients alive at the end of the study period ) . duplex quantum dot - based immunofluorescence was used to co - localize yy1 with cd20 using a duplex qd - if protocol . briefly , tissue sections were subjected to antigen retrieval by microwave heating in tris - edta buffer and incubated with yy1 monoclonal primary antibody ( santa cruz biotechnology , santa cruz , ca , usa ) diluted 1:50 ( v / v ) in 1:10 ( v / v ) goat serum overnight at 4c . secondary detection was performed using a goat anti - mouse secondary antibody diluted 1:250 ( v / v ) in 1:10 ( v / v ) goat serum , followed by 655 nm quantum dot solution ( invitrogen , carlsbad , ca , usa ) ( 1:50 ( v / v ) in 1:10 ( v / v ) goat serum ) . sections were washed and incubated with avidin solution followed by incubation with biotin solution ( vector laboratories , burlingame , ca , usa ) . an anti - cd20 monoclonal primary antibody ( dako , glostrup , denmark ) diluted 1:50 ( v / v ) in 1:10 ( v / v ) goat serum was then applied for 1 hour at room temperature . secondary detection was performed using the same protocol as for yy1 with the exception of use of a 605 nm quantum dot solution ( invitrogen ) diluted 1:50 ( v / v ) in 1:10 ( v / v ) goat serum . finally , sections were rinsed and mounted in polyvinyl mounting medium with dabco ( sigma - aldrich , st . areas positive for yy1 or cd20 expression were identified by spectral imaging using a leitzdiaplan fluorescence microscope and a nuance spectral analyzer . image cubes were collected and subjected to spectral unmixing using nuance software version 2.4.2 to generate 2d intensity distributions of yy1 and cd20 positivity ; up to five images from different portions of each specimen were taken to help ensure that inferred characteristics were representative . composite images taken of sections double stained for yy1 and cd20 were unmixed using spectra for 605 nm and 655 nm quantum dots and the resultant 2d intensity maps for each of yy1 and cd20 thresholded to generate biologically relevant positive regions of interest ; regions of interest were at least 100 pixels in area . region centroid co - ordinates , region area , maximum intensity , and average intensity were exported to excel ( microsoft corporation , redmond , wa , usa ) . all images were captured at a total magnification of 400 with standardized and uniform image illumination and magnification such that all images captured were directly comparable in terms of intensity and scale . very large regions representing incorrect discrimination between distinct cells were rejected by discarding regions with area greater than 200 pixels . the topology of the function ( equation 2 ) for each summary statistic was estimated via thin plate spline interpolation of values of function computed on a regular 14 24 grid of ( di , dp ) values . we therefore selected optimal values of di and dp for each summary statistic by inspecting contour plots , favoring maxima near ( di , dp ) = ( 0 , 0 ) . each patient was represented by the median of each summary statistic , taken over all images acquired for the patient . finally , kaplan meier survival curves were estimated using leave - one - out validation : at the i iteration , the i patient was left out of the analysis and an optimal threshold for each summary statistic calculated for the left - in patients ( equation 1 ) ; this threshold was used to allocate the left - out patient to one of two groups . given a cube of data from a multiple - stained section imaged using multispectral microscopy , we assumed that it is possible to determine whether each cell in a section is positive ( 1 ) or negative ( 0 ) for each marker and to obtain cells spatial ( x , y ) co - ordinates . this can be achieved , for example , using commercially available software ( e.g. , nuance ; perkinelmer inc . , let denote a set of n markers and the positivity of a cell for stain s as s . we adopt the term combinatorial molecular phenotype ( cmp ) from a previous study that describes the positivity of a cell for all stains in as the binary vector = ( s1 , , sn ) , where si denotes the i stain . for example , a possible cmp for an image stained with three markers may be = ( 1 , 0 , 0 ) . no ( x , y ) co - ordinates will be recorded for cells that are negative for all markers in . therefore , any of 2 - 1 cmps may be observed . each possible cmp can be assigned a unique number according to a suitable binary counting scheme . if the right - most element of a cmp represents the least significant binary digit and the next right - most element represents the next least significant digit and so on , then the vectors ( 0 , 1 , 0 ) , ( 1 , 0 , 0 ) , and ( 1 , 1 , 1 ) can be numbered 2 , 4 , and 7 , respectively . because this numbering system begins at 1 , some software can automatically and accurately segment individual cells using full multispectral data . however , less sophisticated software is limited to segmenting regions from particular sub - bands , giving rise to separate sets of region centroid co - ordinates for each stain however , it is possible to use the region centroid co - ordinates obtained for each marker independently to identify regions that are so sufficiently close that they are likely to correspond to the same cell , and , therefore , to approximate cmps . first , regions likely to encompass multiple cells are rejected by discarding those that are larger than the maximum area expected for typical cells . then , co - ordinates from different markers that are within a pairing distance , dp , are combined into a single co - ordinate to which the appropriate cell cmp is associated . later in the paper , we have shown how distance dp may be chosen statistically . we hypothesized that two cells sufficiently close together and with identical or different cmps may interact to varying degrees of patient benefit or detriment . we call this a hypothesized interaction . by extension , we hypothesize that if a particular hypothesized interaction does indeed act in this way , then many such interactions between pairs of cells will have a larger effect on patient outcome . we formalize the notion of hypothesized interactions and quantify their occurrence by introducing a commutative arrow operator . consider all possible pairings of cells within a given section , such that no two paired cells are located further than an interaction distance , di , from one another . the arrow notation specifies that a proportion , p , of all pairings , are between a cell with cmp number i and another with j. this notation is convenient because it can be used to form graphs , which may aid visualization . later in this paper , we explain how an optimal value of di can be chosen statistically . the proportion p can also be interpreted as a probability . by calculating p for each possible hypothesized interaction , we can define a statistical distribution over them . let d be a ( 2 1 ) ( 2 1 ) symmetric matrix , whose ( i , j ) element is the probability of observing a cell with cmp number i within a distance di of a cell with cmp number j. if y is a random variable that represents a hypothesized interaction , then the statistical distribution of y can be written as y ~ d , and we call d a hypothesized interaction distribution ( hid ) . we are interested in testing the hypothesis that cells of particular types may interact to varying degrees of patient benefit or detriment . specifically , we are interested in investigating whether hids facilitate the assignment of patients into groups with good versus poor survival . taking inspiration from grey - level co - occurrence matrix analysis methodology in the texture analysis literature , table 1 defines three scalar summary statistics that can be used to characterize a hid . note that the indexing used in the summations accounts for the fact that the upper and lower triangles are symmetric and that only one of these triangles is required to specify the hid ( i.e. , the elements in one of the triangles sum to unity ) . definitions and descriptions of scalar summary statistics distribution peakiness is characterized in different ways by de and h(d ) ; dmax characterizes the degree to which the most common hypothesized interaction occurs . shannon entropy , h(d ) , measures the expected information ( in bits ) required to transmit a randomly chosen hypothesized interaction , drawn from the distribution d , over an idealized channel . hypothesized interactions associated with uniform distributions require more information than those from non - uniform distributions . given a vector of values of a particular summary statistic ( e.g. , dmax ) for n patients , = ( 1 , , n ) , we can choose an optimal threshold , t , on that statistic that best assigns patients to groups on the basis of survival via the optimization here , the i element of the vector s is the survival time for the i patient and the function gt returns a vector whose i element is the group assignment of the i patient resulting from the application of threshold t to the i element of the vector . the objective function computes the value of the statistic associated with the null hypothesis that the survival curves arising from the group assignments are equal ; high values of have corresponding low p values . the distances di and dp may in principle be selected by the investigator , for example , by drawing upon expert clinical knowledge . however , this approach is subjective and we therefore proposed an objective method . optimal values ( di , dp ) can be chosen statistically via the optimization this is a modification of equation 1 , where (di , dp ) returns a vector of summary statistics for hids computed using distances di and dp . the aims of this experiment were to demonstrate the application of hid analysis to data from multispectral microscopy of multiple - stained tissue sections from a sample of fl patients and to evaluate the method 's ability to identify patient groups that differ in survival . twenty six archived human lymph nodes from patients ( 16 male , 10 female , aged 36 - 77 years , mean 55 years ) with a diagnosis of fl were collected at initial diagnosis , prior to the treatment . written informed consent and local research ethics committee approval were obtained . clinical data and up to 14 years of follow - up and survival data were available for each sample ( 19 dead , 7 alive ; median time to death 32 months ; range 2 - 170 months ; 171 months follow - up in all patients alive at the end of the study period ) . duplex quantum dot - based immunofluorescence was used to co - localize yy1 with cd20 using a duplex qd - if protocol . briefly , tissue sections were subjected to antigen retrieval by microwave heating in tris - edta buffer and incubated with yy1 monoclonal primary antibody ( santa cruz biotechnology , santa cruz , ca , usa ) diluted 1:50 ( v / v ) in 1:10 ( v / v ) goat serum overnight at 4c . secondary detection was performed using a goat anti - mouse secondary antibody diluted 1:250 ( v / v ) in 1:10 ( v / v ) goat serum , followed by 655 nm quantum dot solution ( invitrogen , carlsbad , ca , usa ) ( 1:50 ( v / v ) in 1:10 ( v / v ) goat serum ) . sections were washed and incubated with avidin solution followed by incubation with biotin solution ( vector laboratories , burlingame , ca , usa ) . an anti - cd20 monoclonal primary antibody ( dako , glostrup , denmark ) diluted 1:50 ( v / v ) in 1:10 ( v / v ) goat serum was then applied for 1 hour at room temperature . secondary detection was performed using the same protocol as for yy1 with the exception of use of a 605 nm quantum dot solution ( invitrogen ) diluted 1:50 ( v / v ) in 1:10 ( v / v ) goat serum . finally , sections were rinsed and mounted in polyvinyl mounting medium with dabco ( sigma - aldrich , st . areas positive for yy1 or cd20 expression were identified by spectral imaging using a leitzdiaplan fluorescence microscope and a nuance spectral analyzer . image cubes were collected and subjected to spectral unmixing using nuance software version 2.4.2 to generate 2d intensity distributions of yy1 and cd20 positivity ; up to five images from different portions of each specimen were taken to help ensure that inferred characteristics were representative . composite images taken of sections double stained for yy1 and cd20 were unmixed using spectra for 605 nm and 655 nm quantum dots and the resultant 2d intensity maps for each of yy1 and cd20 thresholded to generate biologically relevant positive regions of interest ; regions of interest were at least 100 pixels in area . region centroid co - ordinates , region area , maximum intensity , and average intensity were exported to excel ( microsoft corporation , redmond , wa , usa ) . all images were captured at a total magnification of 400 with standardized and uniform image illumination and magnification such that all images captured were directly comparable in terms of intensity and scale . very large regions representing incorrect discrimination between distinct cells were rejected by discarding regions with area greater than 200 pixels . the topology of the function ( equation 2 ) for each summary statistic was estimated via thin plate spline interpolation of values of function computed on a regular 14 24 grid of ( di , dp ) values . we therefore selected optimal values of di and dp for each summary statistic by inspecting contour plots , favoring maxima near ( di , dp ) = ( 0 , 0 ) . each patient was represented by the median of each summary statistic , taken over all images acquired for the patient . finally , kaplan meier survival curves were estimated using leave - one - out validation : at the i iteration , the i patient was left out of the analysis and an optimal threshold for each summary statistic calculated for the left - in patients ( equation 1 ) ; this threshold was used to allocate the left - out patient to one of two groups . figure 2 shows the topology of the objective function ( equation 2 ) for h(d ) under the constraint that dp < di ( as we must pair regions before considering that paired regions may interact ) . table 2 shows the results of the leave - one - out survival analyses and the ( di , dp ) used . there was a statistically significant difference between the survival curves defined by the summary statistic h(d ) , but not for dmax and de ; the p value for h(d ) would survive bonferroni correction for multiple comparisons at the = 0.05 significance level . kaplan meier survival curves for h(d)estimated via leave - one - out validation are shown in figure 3 . low h(d ) ( peaky distributions ) were associated with poor survival and these peaks were often observed for hypothesized interactions between cells that were positive for both yy1 and cd20 . figure 1 shows example hids and their graphs for patients with poor ( d1 ) versus good ( d2 ) survival . contour plot showing the value of the test statistic as a function of pairing distance dp and interaction distance di for the summary statistic dmax leave - one - out analysis results . the statistically significant ( p < 0.05 ) result for h ( d ) would survive bonferroni correction survival curves estimated using leave - one - out validation for groups defined by thresholding h(d ) . the method defines a statistical distribution over hypothesized interactionsa pairing of cells with particular combinations of ihc characteristics , located within a given distance of one another . relatively simple scalar summaries of these distributions can be used to assign patients to groups that may differ in terms of survival , and the distributions themselves can be inspected to identify possible cellular interactions that are associated with short versus long survival . hid analysis of retrospective data from a study of fl showed that shannon entropy , h(d ) , was able to assign patients to groups with significantly different survival curves . our results suggest that cells that are positive for both yy1 and cd20 may interact to patient detriment . this result is consistent with our understanding of the biology of these cells , but existing analysis tools do not allow testing of this hypothesis . one limitation of this work is the visual ( albeit quantitative and objective ) selection of distances dp and di prior to , and outside of , the leave - one - out analysis . unfortunately , the high computational cost of evaluating the function and the fact that the function tends to have multiple maxima made automated selection infeasible within the scope of this work . future research should address this problem . in conclusion , hid analysis provides a largely automated quantitative method for making inferences about possible interactions between proximal cells in terms of their ihc characteristics as measured using multispectral microscopy and multiple ihc markers with potential value for diagnosis and prognosis .

chronic mitral regurgitation ( mr ) results in a state of chronic volume overload for the left ventricle ( lv ) , resulting in compensatory dilatation . this process , known as remodeling , is similar to changes occurring after myocardial infarction or with dilated cardiomyopathy , ultimately resulting in lv failure [ 15 ] . mitral valve surgery for regurgitation acutely removes the condition of lv volume overload , but also increases lv afterload . relatively few data are available documenting the changes in lv size and function over time following mitral surgery for severe regurgitation in unselected populations . we therefore examined for echocardiographic changes in lv size and function following mitral surgery in consecutive patients referred for mitral surgery . we also investigated the effect of clinical and surgical factors on changes in lv size and function over time following surgery . the cardiology database at our institution ( apollo advance ; lumedx , oakland , ca ) was queried for subjects having mitral valve surgery performed between 1/1/01 and 12/31/06 . operative reports were reviewed to determine if concomitant coronary artery bypass graft surgery ( cabgs ) was performed . subjects were included if the preoperative echocardiogram demonstrated severe mr and if they had at least one pre- and one post - operative echocardiogram . subjects were excluded if a preoperative echocardiogram showed aortic valve stenosis ( more than mild ) , aortic insufficiency ( more than mild ) , mitral stenosis ( any ) , or any prior valve prosthesis or repair . subjects were also excluded if repair or replacement was performed on any valve other than the mitral valve . postoperative echocardiograms demonstrating mr ( more than mild ) were not included since we were interested in the effect of removing mr on changes in lv size and function . 454 echocardiograms were available ( 277 postoperative and 177 preoperative ) . during the first postoperative month , 108 echoes were performed . between one month and one year , 78 echoes were performed . after the first year , 91 more echoes were performed ( range , 372 to 1,962 days ) . normal data are presented as mean 1 sd and compared with t - test or paired t - test as appropriate . non - normal data are presented as median ( inter - quartile range ) and are compared using the mann - whitney test . linear regression was used to assess the change in cardiac chamber sizes and lv fractional shortening over time , both before and after cardiac surgery . variables of interest were age , sex , race , cabgs at the time of mitral surgery , presence of mitral leaflet disease ( opposed to functional mr ) , mitral valve replacement ( opposed to repair ) , and low ejection fraction ( < 50% ) . the clinical characteristics and demographics of the study sample at the time of surgery are shown in table 1 . compared to those without cabgs , subjects with and without cabgs were similar with respect to age , sex , prevalence of mitral replacement ( as opposed to repair ) , lv ejection fraction ( eyeball method ) , lv fractional shortening ( measured ) , lv systolic and diastolic diameters , left atrial ( la ) diameter , body mass index ( bmi ) , and race . figure 1 displays the lv diastolic diameter ( lvdd ) of all subjects relative to the number of days before and days after mitral valve surgery . lvdd was smaller on postoperative echoes compared to preoperative exams ( 539 mm vs. 579 mm , p<0.001 ) , largely reflecting a change in the lvdd occurring at the time of surgery . although there was a slight downward trend , the regression line reflecting the change in lvdd over time following mitral valve surgery ( beta=0.5 mm / year [ 95% ci : 1.5 , 0.5 ] , p=0.4 ) did not show a significant secular trend . when patients were stratified by cabgs performance , the regression line reflecting the change in lvdd over time did not demonstrate a significant secular trend in either group [ cabg ( = 0.02 mm / year [ 95% ci : 1.2 , 1.2 ] , p=0.98 ) ; no cabg ( = 0.9 mm / year [ 95% ci : 2.5 , 0.8 ] , p=0.3 ) ] . using multiple linear regression , the interaction term for cabg and change in lv diameter was not significant ( p=0.42 ) , indicating that the change in lv diameter over time was not significantly different between those with and without cabg . despite the lack of statistical significance , the downward trend over time in the group without cabg is noteworthy , especially since no such trend is seen in the group without cabg ( figure 2 ) . because the sample sizes are fairly small in the subgroups , it is plausible that there is a true difference between the groups that was not detected . no significant secular trends in postoperative lvdd were seen when stratified by mr etiology ( functional vs. degenerative ) , by surgery type ( repair vs. replacement ) , by the presence or absence of mitral leaflet disease , or by ejection fraction . using multivariate linear regression to adjust for potential confounders , the coefficient for lvdd still did not show a significant secular change ( table 2 ) . lv fractional shortening ( fs ) was unchanged on postoperative echoes compared to preoperative exams ( 2212% vs. 2211% , p=0.71 ) . overall , lv fractional shortening increased by 1.6% per year ( 95% ci : 0.3 , 2.9 , p=0.02 ) after surgery ( figure 3 ) . postoperative secular trends in lv fs , stratified by cabgs performance are displayed in figure 4 . the regression line reflecting the change in lv fs over time following surgery was significant for an increase in lv fs in those without cabgs ( beta=2.3% per year [ 95% ci : 0.4 , 4.2 ] , p=0.02 ) . lv fs did not demonstrate a significant change over time in those with cabg ( =1.1% per year , 95% ci : 0.5 , 2.7 , p=0.20 ) . using multiple linear regression , the interaction term for cabgs and change in lv fs over time was not significant ( p=0.42 ) , indicating that the change in lv fs over time was not significantly different between those with and without cabgs . this is intuitive from the regression lines , which appear to have a similar slope in those with and without cabgs ( figure 4 ) . increase in lv fs over time was confined almost entirely to those who had decreased lvef at baseline [ low lvef ( =7.7% per year [ 95% ci : 2.1 , 13.3 ] , p=0.07 ) ; normal lvef ( =0.4% per year [ 95% ci : 2.7 , 3.6 ] , p=0.78 ) ] . no significant secular trends in postoperative lv fs were seen when stratified by mr etiology ( functional vs. degenerative ) , by surgery type ( repair vs. replacement ) , or by the presence or absence of mitral leaflet disease . using multivariate linear regression to adjust for potential confounders , the coefficient for lv fs remained significant for an increase over time after mitral valve surgery ( table 2 ) . the same analysis performed above for lvdd was repeated to examine for changes in la diameter over time , both before and after mv surgery . regression of la diameter with time after surgery ( beta=0.3 mm / year [ 95% ci : 0.7 , 1.3 ] , p=0.4 ) did not show significant secular trends . mean la diameter was unchanged on postoperative echoes ( 499 mm ) compared to preoperative exams ( 517 mm ) ( p<0.07 ) . we found that lv diastolic size decreased at the time of mitral valve surgery , but no subsequent change was observed during follow - up postoperatively . postoperative lv function remained unchanged from the preoperative exams ; however , function did steadily improve over time following surgery . increase in lf function over time after surgery was mainly seen in those with reduced lv function before surgery . . also found that lvdd on echocardiography decreased at the time of surgery for patients with functional regurgitation undergoing cabgs with mv repair . lvdd remained stable on follow - up exams at 1 , 3 and 5 years after surgery . similar to patients in our study , their cohort did not experience change in lv systolic function immediately following surgery . however , they did report that ejection fraction remained unchanged at 1 , 3 and 5 years postoperatively . although their study sample was large , with 251 patients , they did not include subjects with degenerative disease nor did they include subjects who did not require cabgs . westenberg et al . performed cardiac magnetic resonance imaging in 20 subjects with dilated cardiomyopathy before and after mitral valve repair without cabgs . they similarly found that lv diastolic size decreased significantly early after surgery but did not have subsequent change on repeat imaging at one year . they also found that lv ejection fraction had increased at 2 months postoperatively , with sustained improvement at one year . studied 121 patients who underwent mitral repair for severe functional mr with or without cabgs . using echocardiography , they also found that subjects had reduction in lv diastolic size immediately following surgery and that lv size did not change postoperatively by 2.5 years . lv fs was similar to preoperative values immediately after surgery , with subsequent improvement noted at 2.5 years . in contrast to our results , however , they found that the improvement in lv function that occurred during the follow - up period was limited to those that received concomitant cabgs at the time of mv surgery . the literature is therefore consistent that lv diastolic size generally reduces at the time of mitral surgery for mr and then tends to remain stable subsequently . similarly , all of the aforementioned studies agree that lv function generally remains similar to preoperative function in the early postoperative period . there remains some discrepancy , however , regarding subsequent improvement in lv function in the years following mitral valve surgery . factors such as baseline lv function , type of surgery ( repair vs. replacement ) , need for cabg and primary mr ( compared to functional mr ) likely account for differences between study populations . gelsomino et al noted that the effect of mitral valve surgery on lv size and function can be variable . accordingly , they described a subset of patients that experienced progressive reduction in lv size , with improved lv function over time following the early postoperative period . this group was thought to undergo a process called reverse remodeling and were found to have increased survival in their study . due to the retrospective nature of the study , echocardiograms were not acquired at pre - specified intervals . therefore , recurrent imaging may have been preferentially acquired in those with more symptoms or in those who survived long enough to have follow - up echoes months and years after surgery . data regarding change in cardiac chamber size beyond the peri - operative period are scarce . understanding the degree to which mv surgery interrupts the natural history of mr is extremely important . changes in chamber size are increasingly being used as surrogate markers of prognosis in many studies of congestive heart failure [ 1015 ] . also , improved understanding of how the lv adapts to changing loading conditions is important when considering adoption of new therapies . for this reason , we excluded patients who had documented mr on postoperative exams , since loading conditions were incompletely modified in this group . following mv surgery for mr , lv size becomes reduced at the time of surgery , but then remains stable over time thereafter . improvement in lv function over time postoperatively was only seen in those without concomitant cabgs , possibly related to less baseline myocardial scarring in this group .

traumatic spinal cord injury ( sci ) affects over 200,000 people in the usa , with nearly 10,000 new injuries reported annually [ 13 ] . people affected by sci are usually young ( average age 32 years ) and life expectancy is marginally lower when compared with a non - sci population . hence , the ramifications of the injury itself as well as the medical decisions made can result in enormous economic burden and social cost . long - term outcome after sci comprises a combination of the initial extent of the injury , natural recovery from injury , medical interventions , rehabilitation , and social / community reintegration . initially , acute management includes medical agents administered with the goal of reducing secondary injury cascade , and an initial surgical treatment that usually includes either all or a combination of reduction , decompression , and stabilization . physical therapy and rehabilitation , which can lead to a significant impact on overall recovery is an additional component . finally , late onset sci problems , such as bowel and bladder dysfunction , pain , spasticity , and problems with automatic breathing , play a role . later interventions for other sequelae of sci , for example , spasticity management , and chronic pain management are not discussed . research using animal models has provided evidence that early decompressive surgery can lead to improved neurological recovery after sci [ 5 , 6 ] , which highlights the importance of early intervention . unfortunately , the number of human spinal surgical studies using prospective , randomized , or controlled methodologies is limited . a review of the current literature reveals varying results , some indicate the potential neurological benefit associated with early spinal decompression [ 7 , 8 ] while others do not [ 6 , 911 ] . choice of surgical procedure for individual types and severity of sci is also controversial . in this paper , we place emphasis on currently available evidence for indications and outcomes of the most commonly available surgical options including presurgical reduction as well the choice of the surgical option for any given particular injury pattern . traumatic cervical spine fractures or dislocations are often associated with cervical sci as a result of several mechanisms , including narrowing of the cervical spinal canal or by direct displacement of one cervical spine component with direct focal compression on the spinal cord . reduction of the cervical spine displacement can restore the spinal cord diameter or relieve the focal compression on the spinal cord . as reported in a few case reports or small case series [ 9 , 1214 ] the argument is that focal traumatic disc herniation , which is often associated with traumatic spine injuries , can lead to worsening neurological condition if reduction is attempted . we review the controversy surrounding whether reduction is safe , or not , and whether prereduction magnetic resonance ( mr ) imaging to rule out traumatic disc herniation is essential in the next few paragraphs . several authors [ 9 , 10 , 1214 ] believe that traumatic disc herniation associated with fracture - dislocation or facet dislocation increases the risk of spinal cord herniation after reduction . however , the association of a high percentage of disc herniation with dislocated facets ( 42% ) and the absence of a clear relation between outcome and pre- or postreduction mr imaging results has raised questions about the real value of obtaining mr images before reduction . cumulative evidence suggests that early reduction influences outcome [ 10 , 1518 ] , hence delaying reduction by obtaining mr images , which requires transport of a patient with an unstable spine fracture to the mr imaging suite , may influence outcome and requires adequate justification . initial closed reduction is generally part of the early intervention paradigm in treating acute sci and is generally undertaken on an urgent basis . the finer details of the reduction technique are not covered in this paper . however , in summary , the technique is generally performed using gardner - wells tongs , crutchfield , or halo rings . gardener wells and halo rings models are available that are now compatible with mr imaging . craniocervical traction typically begins with a small weight that is gradually increased until anatomic alignment or near - alignment is achieved . no specific recommendations regarding ideal or maximum weight are advised . however , approximately 5 pounds per level is generally recommended after 10 pounds for the head are added . caution and clinical judgment are required during traction with careful consideration paid to whether the patient is awake or not , paralyzed or not , and x - ray control to avoid overdistraction . in awake patients , the reduction attempt end point is determined when reduction and realignment , or worsening pain or neurological function is achieved . in comatose patients , clinical judgment or if reduction is deemed impossible , further imaging is required to determine the cause of irreducibility , which is usually anatomical and usually requires an open surgical reduction [ 19 , 20 ] . in an attempt to develop sci management guidelines , the cervical spine trauma group developed jointly by the american association of neurological surgeons ( aans ) and congress of neurological surgeons ( cns ) , has reviewed all pertinent literature regarding c spine reduction through the year 2001 . their resultant cumulative meta - analysis found 1200 cases of closed reduction with class iii evidence . overall permanent neurological injury rate was < 1% ( the equivalent of 11 patients ) . in those 11 patients , two had root injuries , two had ascending spinal cord injuries at the time of reduction , and seven had a worse american spinal injury association ( asia score ) , but neither the nature nor the cause of this change in asia score was identified . twenty other patients had temporary neurological deterioration , the causes of which were : over distraction , disc herniation , unrecognized rostral injury , epidural hematoma , or spinal cord edema . the review group 's final conclusion was that closed reduction of fracture - dislocation injuries of the cervical spine by traction - reduction was safe and effective for the reduction of spinal deformity in awake patients . the group concluded that with closed reduction approximately 80% of patients will experience injury reduction . overall permanent neurological complication rate for closed reduction was approximately 1% and the associated risk of a transient injury was 2% to 4% . these include inadequate immobilization , unrecognized rostral injuries , overdistraction , loss of reduction , cardiac , respiratory , and hemodynamic instability . although prereduction mr imaging demonstrates disc herniation in up to half of patients with facet subluxation injuries , the clinical importance of these findings is questionable . the use of prereduction mr imaging has not been shown to improve the safety or efficacy of closed traction reduction in awake patients . mr imaging before fracture - dislocation reduction may result in unnecessary delays in accomplishing fracture realignment and decompression of the spinal cord . class iii evidence exists in support of early closed reduction of cervical fracture - dislocation injuries with respect to neurological function , therefore prereduction mr imaging in this setting is not necessary . the ideal timing of reduction is unknown , but many investigators favor performing reduction as soon as is possible after injury to maximize the potential for neurological recovery . in those patients where attempted closed reduction of a cervical fracture injury fails a higher incidence of anatomic obstacles to reduction , including facet fractures and disc herniation is observed . in patients where closed reduction fails further detailed radiographic studies should be undertaken before attempts at open reduction . patients with cervical fracture - dislocation , who can not be examined , because of head injury or intoxication , can not be assessed for neurological deterioration during attempted closed traction - reduction . for this reason , an mr imaging study before an attempted reduction is recommended as a treatment option . in patients with bilateral locked facets resulting in closed reduction failure , surgical reduction in incomplete tetraplegic patients the role that timing of surgical intervention plays in sci management remains one of the most controversial topics . despite immense research efforts related to sci treatment any treatment components that can contribute to improved outcomes are welcomed , and early surgical intervention still offers hope . secondary injury in cervical spine trauma have sparked interest in the concept of time - window . secondary injury is defined a cascade of events initiated by trauma and involves vascular changes , electrolytes shifts , excitotoxic neurotransmitters accumulation , inflammation , and loss of energy metabolism [ 25 , 26 ] . the first clinical human trial to address and highlight the concept of secondary injury was the national acute spinal cord injury study ( nascis ) trial . in the nascis-2 trial , methyl - prednisolone was used to treat acute sci within a specific window of time , and although results were statistically marginally significant , some clinical evidence to support the secondary injury concept was indicated . the fundamental question was whether early surgery would lead to improved outcome or not , if not performed within what was a poorly defined time - window . before discussing surgical timing any further , we would like to highlight the heterogeneity of human sci , for example , population demographics , mechanism of injury , injury pathology , injury severity and the level of injury . this heterogeneity makes it difficult to conclude whether early surgery for a sci is helpful or not . it is possible that certain sci patient subsets may benefit more than others while some may not benefit at all . many investigators have examined different injury models in different animals for varying periods of time , the vast majority of which have shown some beneficial effect from early decompression [ 5 , 2838 ] . however , this experimental evidence of benefit was not translated to clinical evidence with the same degree of certainty . this was most likely due to patient heterogeneity and the inherent difficulty in study design . in an attempt to determine whether early surgery improves outcome or not , some investigators attempted a meta - analysis of related and current sci literature to form an evidence based answer to the question [ 39 , 40 ] . a toronto group of investigators reviewed data that included all human sci trials conducted between 1966 and 1998 and concluded that despite strong experimental evidence there was no clear census as to the appropriate timing of surgical intervention and that there was no compelling evidence that early surgical decompression influences patient neurological outcome [ 22 , 23 , 39 ] . on the other hand , an italian group of investigators reviewed data that included human sci surgical trials undertaken between 1996 and 2000 . they concluded that despite the presence of some statistically significant neurological advantage with early surgery in an incomplete injury subset of patients , they were unable to determine with any confidence a neurological advantage to surgery within 24 hours of injury in any of the groups they studied . the toronto group recently updated their meta - analysis data and included studies between 2000 and 2005 . interestingly , in a multicenter retrospective study that included 36 north american centers information regarding the timing of intervention in acute sci varied widely . surgery was performed < 24 hours after injury in 23.5% of patients , between 25 and 48 hours after injury in 15.8% of patients , between 48 and 96 hours in 19% of patients , and more than 5 days post injury in 41.7% of patients . the results of this study clearly revealed a lack of consensus on optimum timing of surgical treatment . a recent retrospective study , that looked at the outcome of early surgical management versus late or no surgical management after acute sci in all acute nonpenetrating , traumatic sci from 1995 to 2000 found no difference in neurological or functional improvements between the early versus the late surgical group . the only class i evidence study addressing this issue is by vaccaro et al . . they randomized cervical sci ( c3-t1 ) levels of different grades of injury to early < 72 hours surgery and late > 5 days surgery . the study failed to show any difference in outcome between the two groups ( mean follow up 305 days ) . another aspect of the controversy surrounding surgical treatment timing is whether surgery , especially early surgery has any influence on complication rate or length of stay after sci . it is obvious that surgery might be risky in more critically ill patients who sustain sci ; especially if the cervical injury is high , and is associated with other significant injuries . . showed that there was no significant difference in the length of stay between operative and nonoperative groups or between the early versus late surgery groups . there is clear evidence that injury severity is crucial in determining the ultimate outcome after traumatic sci , but the potential impact of injury mechanism or sci type remains elusive . pollard and apple were unable to identify an association between mechanism of injury and neurological recovery or outcome . however , they were able to observe that patients with brown - sequard syndrome or a central cord syndrome had relatively improved outcomes . the relatively improved outcome in central cord syndrome patients when compared to other mechanisms of sci is a notable observation and affects decision making regarding timing of surgery in this particular sci model . contrary to all other sci types , central cord syndrome is one type where early surgery is not classically recommended in the acute setting ; typically these patients are managed conservatively with a collar and rest . recent consensus is that there is no standard recommendation regarding the role and timing of surgery in acute sci . there are guideline recommendations supporting the safety of early surgery ( < 72 hours ) in hemodynamically stable patients , and data supports recommendations for urgent reduction of bilateral locked facets in patients with incomplete tetraplegia and urgent decompression in patients with neurological deterioration . there are also recommendations supporting decompression as reasonable for acute cervical sci ; when possible , excluding patients with life - threatening multisystem trauma , it is recommended that urgent decompression be performed within 24 hours of a sci . there is class iii evidence that early ( < 24 hours ) surgery reduces length of stay in patients with acute sci and may reduce postinjury medical complications [ 22 , 23 ] . there is an obvious need for well - designed , executed , and randomized controlled trials comparing early versus late surgery in sci . questions regarding the relation between a particular sci type , level or severity and surgery outcome would be logical . it has yet to be determined with certainty which type of sci would benefit most , if at all , from early surgery . finally , it is worth mentioning that currently the toronto group , in collaboration with thomas jefferson university and the spine trauma study group , has started a multicenter , prospective trial to evaluate the effect of early ( < 24 hours after injury ) versus late ( > 24 hours ) decompressive surgery for cervical sci . the study ( surgical timing in acute spinal cord injury study , i.e. , stascis ) is not randomized because of the ethical concerns related to allocating a neurologically declining patient to a delayed decompression group . the study is currently open and when complete will have enrolled up to 450 patients . spinal cord injury patients are a complex and heterogeneous group , as previously mentioned , therefore , a unified approach to the spine is impossible . in acute sci patients , the choice of the surgical procedure is a function of the objective of the surgery ; fixation of instability and decompression of the spinal cord . the choice of one procedure or another is a function of the severity of injury , the level of injury , the mechanism of injury , and the location and the extent of compression . goal setting before the procedure is critical to procedure choice ; hence a surgical plan should be individualized to each patient . in patients with complete spinal cord injuries , overall outcome is generally poor , and usually the primary goal of surgery is spinal stabilization . this allows the patient better postural management , reduces pain , improves pulmonary functions and allows the initiation of physical therapy and rehabilitation . although spontaneous fusion is eventually the fate of many cases even without surgery , surgical stabilization allows the early benefit of fusion and helps prevent possible future kyphotic deformity . in incomplete sci patients , the choice of the procedure is dependent on the mechanism of injury and the other factors mentioned above . approaches to the spine can be classified as anterior or ventral and posterior or dorsal . either objective , that is , stabilization or decompression can be achieved from either route , but in severe cases combined anterior and posterior approaches ( staged or in a single setting ) may be required . in acute traumatic sci , the classic controversy of short acute sci trauma usually involves one or more specific segments and short segment instrumented fusion is therefore almost always the preferred choice . posterior approaches are usually used in flexion type injuries and in most thoracolumbar injuries provided there is no severe ventral compression that is thought to be related to a partial sci . instrumentation can be lateral mass , halifax clamp or wires in the cervical spine , transpedicular screws , and sublaminar hooks or wires in the thoracolumbar spine . a posterior approach allows easy and quick spinal decompression via laminectomy , if needed , and also allows ligamentotaxis in thoracolumbar injuries . the ventral or anterior approach is used most commonly in cervical spine traumatic sci , much less so in thoracolumbar injuries , because of the anatomical barrier of the chest and abdomen and the related difficulty in surgical approach . anterior approaches are used in extension injuries and allow for corpectomy and ventral decompression if needed . an anterior approach can often be combined with a posterior approach ; the sequence of this combination is an individual surgeon 's choice . several authors have attempted to devise an algorithm for decision making that employs the factors mentioned above , however approach choice remains individually tailored , by the surgeon , to meet the specific goals of the individual patient [ 49 , 50 ] . from the stand point of evidence - based medicine , it is unlikely that evidence supporting one specific approach or another to treat a particular injury pattern , injury type , or level , is possible . the severity of injuries is too complex and heterogeneous to be broken down to essentially a handful types and there are multiple surgical options available and used by many surgeons . traumatic sci is a major source of morbidity , mortality , and imposed cost on society as a whole . surgical treatment remains an essential tool in management and the controversy regarding the timing of surgery needs to be resolved . collection of outcome data that would suffice as evidence - based is a huge undertaking , and hopefully this will be accomplished in the future . presurgical reduction is considered safe and essential in the management of sci with loss of alignment , at least as an initial step in the overall care of a sci patient . the authors report no conflict of interests concerning the materials or methods used in this study or the findings specified in this paper .

the carotid body ( cb ) , a neural - crest - derived paired organ located in the carotid bifurcation , is the main arterial chemoreceptor in mammals and facilitates reflex hyperventilation during hypoxia ( lpez - barneo et al . , 2001 ) . in addition , the cb is able to undergo neurogenesis to contribute to acclimatization of the organism during a persistent hypoxia ( edwards et al . , 1971 , arias - stella and valcarcel , 1976 , heath et al . , 1982 , we have previously shown that glia - like sustentacular , or type ii , cells surrounding chemosensory neuron - like glomus cells are neural crest stem cells ( ncscs ) and contribute to cb growth in hypoxia by differentiating into new glomus cells ( pardal et al . in addition to this neurogenic activity , a profound angiogenic process takes place in the hypoxic cb ( wang and bisgard , 2002 , chen et al . , 2007a ) . whether neural - crest - derived adult cb stem cells ( cbscs ) are able to differentiate into vascular cells , contributing to angiogenesis in vivo , in addition to neurogenesis , is largely unknown we have recently described the presence of mesectoderm - restricted progenitors within cb parenchyma and have shown that these cells are able to convert into endothelial cells in vitro ( navarro - guerrero et al . , 2016 ) . therefore , the cb constitutes an ideal model where to study the potential plasticity of an adult population of ncscs . utilizing both in vitro assays and in vivo cell - fate mapping experiments , we show that adult cbscs do indeed retain multipotent differentiation capacity , being able to convert into endothelial cells ( ecs ) contributing to hypoxia - induced angiogenesis during acclimatization of the organ . moreover , we provide mechanistic insight into this process by showing that cbscs differentiate into ecs via a hypoxia inducible factor ( hif)-dependent mechanism and in response to hypoxia - released molecules such as erythropoietin . thus , cbscs use plasticity to facilitate physiological adaptation of the cb , and hence the organism , to hypoxia . in order to test the ability of cbscs to contribute to vascular cell types , we decided to perform in vivo cell - fate mapping using gfap - cre / floxed lacz or yfp transgenic mice . these animals express the -galactosidase enzyme ( -gal ) , or the yellow fluorescent protein ( yfp ) , in gfap cbscs and their derivatives ( pardal et al . , 2007 ) . however , the human gfap promoter in this type of construct is not expressed in all gfap mouse cells , as previously revealed in brain astrocytes ( malatesta et al . , 2003 ) . in the case of cb , a first exposure to hypoxia ( 7 days ; figure s1 ) needs to be performed in order to activate the construct ( pardal et al . , 2007 ) , and only around 50% of gfap cbscs get labeled ( figure s1f ) . after this first exposure to hypoxia , only gfap cb stem cells and their derivatives in resting conditions , i.e. , some nestin progenitor cells , appear labeled ( figures s1 g and s1h ) , confirming the specificity of the construct . after construct activation , we exposed gfap - cre / floxed lacz mice to hypoxia for 7 days ( for hypoxic stimulus confirmation , see figures 4k and s5c ) and interrogated vascular cells for the expression of lacz . by using an antibody against -gal , and gsa i , an endothelium - specific lectin , we found that 16.3% 2.7% of ecs were positive for -gal ( 39 out of 229 gsa i cells analyzed ; figures 1a and s1j ) . no single -gal gsa i cell was found in the cbs of normoxic gfap - cre / floxed lacz littermate mice ( 99 gsa i cells analyzed ) , a result that was confirmed with yfp transgenic mice ( figure s1d ) . differentiation of gfap cbscs into ecs was confirmed in cb dispersed cells from hypoxic gfap - cre / floxed lacz mice , by using classical x - gal staining to reveal the activity of -gal , and fluorescent cytochemistry to detect gsa i ( figure 1b ) , or the endothelial markers cd31 ( figure 1i ) , or von willebrand factor ( vwf ; figures s1k and s1l ) . moreover , by using this cb ex vivo preparation , we confirmed neuronal differentiation capacity of gfap cbscs in the same mice ( figure 1c ) . interestingly , by using a smooth muscle cell marker ( sma ; figure 1d ) and a pericyte marker ( ng2 ; figure 1e ) , we also uncovered the ability of cb neural progenitors to convert into other mesenchymal cell types forming the vascular compartment , suggesting that cb progenitors can give rise to complete blood vessels . the use of a second type of reporter mouse strain ( gfap - cre / floxed yfp mice ) corroborated the cell - fate mapping results , both at the immunochemical ( figures 1f1h ) and flow cytometry ( fluorescence - activated cell sorting [ facs ] ; figure s1i ) levels . the percentage of yfp gsa i cells , obtained from gfap - cre / floxed yfp mouse cbs , was smaller ( 9.31% 0.59% ) than from the lacz mouse model , probably due to differences in cre - mediated excision efficiencies between the two reporter genes used . the use of a different ec marker , such as cd31 , for cell - fate mapping ( figures 1h , 1i , and s1i ) yielded qualitatively very similar results . no single ec , labeled with the reporter gene , was found when analyzing the cb of th - cre / floxed lacz mice ( figure s1 m ) , ruling out an unspecific expression of cre recombinase by ecs , and confirming the differentiation of gfap cbscs into vascular cells . we corroborated the multipotent differentiation capacity of cbscs in vivo , by performing bluo - gal staining and electron microscopy ( em ) on the cb of hypoxic gfap - cre / floxed lacz mice ( see figure s2 and its legend ) . altogether , in vivo cell - fate mapping experiments identify cbscs as multipotent neural - crest - derived adult stem cells contributing to both neurogenesis and angiogenesis during hypoxic acclimatization of the organ . to directly prove the multipotential capacity of cbscs to differentiate into both neuronal and vascular cell types , we performed single - cell deposition experiments in vitro ( figure 2 ) . out of 1,536 dispersed primary neurosphere ( ns ) cells plated , 200 of them gave rise to secondary ns . upon plating onto adherent conditions , 15 of those ns ( 7.5% ) contained both neuronal and mesectodermal cells within the colony ( figures 2a2d and s3a s3f ) , confirming differentiation into both cell lineages from a single progenitor cell . the percentage of multipotent progenitor cells is likely being underestimated due to the lack of appropriate neuronal differentiation conditions in the single - cell plating assay . in addition to tuj1 and gsa i markers ( figure 2b ) , we have corroborated the presence of neuronal and mesectodermal cells with different widely used markers : tyrosine hydroxylase ( th ) for dopaminergic neuronal cells and vwf , ng2 , and cd34 for vascular cells ( figures 2d and s3 ) . we also performed single - cell plating analysis with facs - sorted rat cb cells previously transfected with a pgfap - egfp construct , where 100% of gfp - sorted cells were gfap cbscs ( figures 2e and 2f ) . although the frequencies of ns formation and neuronal differentiation were quite low , likely due to transfection- and facs - induced damage , and lack of optimal neuronal differentiation conditions , we were able to find some examples of multipotent differentiation from single cbscs ( figures 2 g and 2h ) . consistently , we also found multipotent differentiation when plating single gfp cells from cbs of gfap - egfp transgenic mice ( figure 2i ) . we next decided to explore the mechanisms by which cbscs differentiate into ecs by using in vitro assays and rat cb cells , since the organ in rats is more manageable than in mice and the cells grow better in culture . rat cbsc - derived ns can be plated onto adherent conditions where , upon mitogen withdrawal , cells are induced to differentiate . using this assay , we found that cb progenitors differentiate into ecs ( gsa i ) when exposed to adherent substrate ( control in figures 3a and 3b ) . however , this differentiation was dramatically enhanced when the cells were exposed to hypoxia ( see figures 3c and 3d for hypoxia confirmation in vitro ) or vascular factors ( figures 3a and 3b ) , such as erythropoietin ( epo ) , endothelin-1 ( et-1 ) , platelet - derived growth factor ( pdgf ) , vascular endothelial growth factor ( vegf ) , or adenosine ( ado ) . this differentiation effect appeared to be specific for vascular factors since the addition of typical cb neuronal factors , such as dopamine ( da ) or methionine - enkephalin ( met - enk ) ( gonzlez - guerrero et al . , 1993 , gauda and gerfen , 1996 ) , did not increase endothelial differentiation ( figures 3a and 3b ) . interestingly , most vascular factors studied are consistently expressed in cb ecs , as assessed by rt - pcr on facs - sorted cb cells ( figure s3h ) . we next evaluated the expression of mature ec markers in gsa i ns cells , using gsa i cb dispersed cells and liver cells as positive controls ( figure 3e ) . we confirmed the expression of vwf , endothelial no synthase ( enos ) , and cadherin v , all of them specific for mature endothelium . we also confirmed the expression of the receptors for the different vascular factors that activate endothelial differentiation in cbscs ( figure s3i ) . both cb- and ns - derived gsa i cells lack expression of the receptor for da ( drd2 ; figure s3j ) , a neurotransmitter that did not increase endothelial differentiation ( figures 3a and 3b ) . as a control , we tested the expression of a cb neuronal marker ( th ; figure 3f ) , corroborating the lack of neuronal cell contamination in gsa i populations . moreover , we studied the functionality of cbsc - derived ecs obtained in vitro , by performing a low - density lipoprotein ( ldl ) uptake assay ( voyta et al . , 1984 ) . out of 216 gsa i cells analyzed from three independent cultures , 95% 1.5% of them were also positive for dii - conjugated ac - ldl ( figure 3 g ) , confirming the uptake and intracellular storage of lipoproteins , and hence the functionality of ecs differentiated from cbscs in vitro . finally , we decided to confirm the vasculogenic capacity of cb progenitor cells by using matrigel and a subcutaneous vasculogenesis assay ( see figure s4 ) . taken together , in vitro and in vivo functional assays and endothelial marker expression analysis indicate that neural - crest - derived cbscs are able to differentiate into mature and functional ecs , supporting their capacity to participate in cb angiogenesis . interestingly , in vitro differentiation assays indicate that cbsc conversion into ecs is directly favored by low oxygen ( see figure 3a ) . therefore , we decided to check whether cbscs were intrinsically sensitive to hypoxia , and whether prolyl hydroxylase ( phd)/hif pathway had a role in the vascular differentiation process . our data ( see figure s5 ) clearly establish an important , although partial , role for hif2 in the endothelial differentiation process . in addition , we decided to further study the role of a relevant hypoxia - induced vascular cytokine , epo . epo is a hematopoietic factor , finely regulated by oxygen tension ( franke et al . , 2013 ) , that has been described to participate in angiogenesis ( heeschen et al . , 2003 ) . in vitro results described above show that epo increases the number of ecs obtained from cbscs ( see figures 3a and 3b ) , and this is achieved without affecting neuronal differentiation ( figures 4a and 4b ) . moreover , epo receptor ( epor ) is expressed by both ns- and cb - derived nestin - activated progenitors ( figure 4c ) , suggesting a direct role of this cytokine on cbsc fate determination . therefore , we decided to further examine the role of epo on cb angiogenesis both in vitro and in vivo . the addition to adherent ns cultures of an antibody against epo , that sequesters the hematopoietic cytokine , completely abrogated the increase in endothelial differentiation ( figures 4d and 4e ) . ag490 is an inhibitor of epor - associated janus kinase ( jak ) 2 that has been described to block epo intracellular signaling ( lester et al . , 2005 ) . addition of ag490 to epo - treated adherent ns provoked a significant decrease in the number of ecs obtained ( figures 4d and 4e ) . thus , our results suggest a specific role for epo in the induction of endothelial differentiation from cbscs in vitro . we then decided to corroborate that epo was being endogenously produced in the cb ( lam et al . , 2009 ) . as assessed by rt - pcr , cb cells produce epo especially in hypoxic conditions ( figure 4f ) . in order to test whether cell - released epo was able to direct vascular differentiation from cb progenitors , we performed hypoxic co - cultures of ns progenitor cells and ecs ( figure 4 g ) . for these co - culture experiments , we used a well - characterized human ec line ( huvec ) , which produces epo ( figure 4h ) . the presence of huvecs in the surrounding wells increased the amount of functional ecs in cb ns , an effect that was abrogated after the addition of a blocking antibody against epo ( figures 4i and 4j ) , further corroborating the role of epo on cb progenitor endothelial differentiation in vitro . finally , we wanted to test whether epo was having a relevant role in hypoxia - induced cb angiogenesis in vivo . to this end , we used gfap - cre / floxed lacz transgenic mice exposed to hypoxia . systemic injection of epo signaling blocker ag490 in these mice significantly attenuated the typical hypoxia - induced epo - mediated hematocrit increase ( figure 4k ) , corroborating the systemic action of the inhibitor . cbsc - derived newly formed ecs are labeled by -galactosidase expression and hence appear with blue precipitate after x - gal staining ( figure 4l ) . quantification of x - gal cells in dispersed cb of these ag490-treated hypoxic mice revealed a decrease in the number of gsa i ecs derived from cbscs ( figure 4 m ) , confirming the importance of epo release and epo signaling in cbsc - dependent angiogenesis . in the present study , we show that adult cbscs display multipotent differentiation capacity , giving rise to both neural and non - neural derivatives during physiological acclimatization of cb to hypoxia . our results show that around 10%15% of total ecs , after a single exposure of the organ to chronic hypoxia , are derived from cbscs , and this value is likely being underestimated due to inefficient expression of transgenic constructs ( figure s1 ) . considering that ecs are described to double their number in the organ in response to hypoxia ( chen et al . , 2007a ) , we therefore estimate that around one every three or four newly formed ecs is cbsc derived . the ability of nscs to differentiate into ecs has been previously described only in some in vitro studies ( wurmser et al . , 2004 , ii et al . , 2009 ) or in the context of nervous system oncological disease ( ricci - vitiani et al . , 2010 , wang et al . , 2010 , however , to the best of our knowledge , no other example has been reported about adult neural progenitors participating in a physiological process of angiogenesis in vivo . in addition to the direct effect of hypoxia ( see the role of hif2 in the supplemental information ) , conversion of cbscs into ecs is also clearly enhanced by the exposition of cells to vascular factors , which are particularly released during hypoxia ( fandrey , 1995 ) . among these factors , epo has been described to regulate cns progenitor behavior ( shingo et al . , 2001 , chen et al . , 2007b ) and has a clear role enhancing vasculogenesis ( bahlmann et al . , 2004 , wang et al . , 2004 ) . our results show that cb cells produce this vascular factor mainly during hypoxia , indicating that locally produced epo might play a role in angiogenesis in the cb . this result is consistent with the described production of epo by neuronal glomus cells in the organ ( lam et al . moreover , we have recently described a role for cb neuronal cells on the proliferative activation of cbscs through a different cytokine , et-1 ( platero - luengo et al . , 2014 ) . the additional release of epo by cb glomus cells might represent another example of neuronal activity - dependent regulation of stem cell behavior , in this case promoting progenitors to participate in angiogenesis . nevertheless , we can not formally discard an additional role for circulating epo in cb angiogenesis . altogether , our results suggest that cbscs retain mesectodermal differentiation potential , resembling neural progenitors in the peripheral nervous system during development ( le douarin et al . , 2008 ) . this ability allows them to participate in physiological angiogenesis , in addition to neurogenesis , during adaptation of the organ to hypoxia . this finding might be relevant to understand the pathophysiology of this chemoreceptor organ and to improve the therapeutic use of these stem cells . male and female transgenic mice ( 46 weeks old or 89 months old for dimethyloxalylglycine [ dmog ] in vivo experiments ) and wistar rats ( 6 weeks old ) were housed and treated according to the animal care guidelines of the european community council ( 86/609/eec ) . all experimental procedures were approved by the committee on animal research at the university of seville . rat and transgenic mouse cbs were dissociated by enzymatic treatment and cultured to form ns , following protocols previously described ( pardal et al . , 2007 ) . immunostainings were performed following standard procedures described elsewhere ( pardal et al . , 2007 ) . dispersed cb cells from gfap - cre / floxed lacz and th - cre / floxed lacz mice were plated in ultralow binding petri dishes and allowed to recover superficial epitopes , lost upon enzymatic dispersion , in an incubator overnight . after that , standard protocols for x - gal staining were applied , as previously described ( pardal et al . , 2007 ) . once x - gal staining was performed , we processed the cells for antibody staining as indicated previously . carotid bifurcations were embedded in gelatin ( 80100 bloom ; panreac ) supplemented with 2% glutaraldehyde ( electron microscopy sciences ) , and 50-m - thick slices were cut on a vibratome ( vt1000s ; leica microsystems ) . primary ns were collected in 15-ml conical tubes and centrifuged at 100 g for 1 min . following aspiration of supernatant , ns were dispersed by accutase ( sigma ) , gently pipetting up and down during 10 min . . dissociated ns cells were centrifuged for 5 min at 300 g at 4c . afterward , cells were resuspended and cultured at clonal density on an ultralow binding 96-well plate . secondary ns were re - plated onto adherent conditions using fibronectin pre - treated plates , as described before . after 3 days in culture , adherent ns colonies were fixed in 4% paraformaldehyde ( pfa ) and processed for immunolabeling and gsa i staining ( see above ) . ns obtained from dispersed rat cbs were re - plated onto adherent conditions using 5 g / ml fibronectin ( biomedical technologies ) pre - treated 24-well plates , at a density of 4 or 5 ns / well . drug treatments are described in the supplemental information . after growing for 3 days in adherent conditions , flat ns colonies were fixed and stained with rodhamine or fluorescein - conjugated gsa i ( see immunocytochemistry section ) . total rna was extracted from gsa i positive and negative cell fractions from dispersed adherent ns or dispersed rat cbs , using a commercial kit ( rneasy micro kit ; qiagen ) , following manufacturer s instructions . for retrotranscription , cdna was synthesized with quantitec reverse transcription kit ( qiagen ) , as indicated by manufacturer s instructions . all sorts and analysis were performed in a moflo three - laser flow cytometer ( dako cytomation ) and a lsr fortessa ( bd biosciences ) , respectively . whole - cell protein extracts were obtained from huvec cell line , cultured during 3 days in hypoxic conditions ( 3% o2 ) in ec basal medium-2 ( ebm-2 , lonza ) for epo protein detection . analyses of significant differences between means were carried out using the unpaired or paired two - tailed student s t test . newman - keuls post hoc or tukey s multiple comparison tests were used whenever a significant difference between three or more sample means was revealed by a one - way anova . tukey or newman - keuls test was used depending upon whether or not additional power is required to detect significant differences between means .

educator highlights for cbe lse show how professors at different kinds of institutions educate students in life sciences with inspiration and panache . if you have a particularly creative teaching portfolio yourself , or if you wish to nominate an inspiring colleague to be profiled , please e - mail laura hoopes at [email protected] .

3d printing, has been used in the clinical sciences for planning complex surgeries and designing custom implants . the introduction of low - cost consumer - grade 3d printers enables this technology to be used for more routine procedures , although its application in rt has not been widely published . these 3d printers deposit 0.1 mm- to 0.3 mm - thick layers of hot acrylonitrile butadiene styrene or polylactic acid plastic . the resulting model ( 3d printout ) in addition , there are several software packages that import medical magnetic resonance or computed tomography ( ct ) images and enable the creation of models suitable for printing . the use of medical imaging and 3d printing had previously been used for radiation therapy applications at cancercare manitoba . additionally , the institution s medical devices department regularly modifies tracheostomy tubes and , thus , has developed a strong collaborative relationship with the respiratory therapists of the long term ventilator service ( ltvs ) , winnipeg regional health authority ( manitoba ) . it was natural then that this unique collaboration may lead to an innovative approach to client care by applying 3d printing technology to rt practice . the ltvs provides rt services to long - term clients in the community who require support for medically complex needs . these needs frequently include tracheostomy airway management in clients who may or may not also require mechanical ventilation . there are several well - known long - term effects of tracheostomy , including dysphagia , inability to phonate , tracheal granuloma and tracheomalacia ( 9,10 ) . respiratory therapists at the ltvs use various methods of customizing tracheostomy tubes to mitigate anatomical or other therapeutic challenges , or to manage complications resulting from long - term use of the airway itself . routine diagnostic techniques , such as bronchoscopy , broncheolar lavage , radiography and ct , have been used to diagnose and monitor these long - term effects . although a cornerstone of management , these diagnostic techniques have occasionally proven insufficient for identifying the etiology of symptoms related to long - term tracheostomy use that have appeared clinically . this has limited the ability of the respiratory therapists to determine appropriate clinical interventions such as tracheostomy type , position and need for customization . for example , occasionally complicated tracheal anatomy may make airway visualization highly challenging and , thus , limit the ability to determine a differential diagnosis in the presence of multiple long - term complications . one approach that has been used to give a detailed and informative understanding of the etiology of client symptoms has been the use of 3d printing technology to reproduce the results of high - resolution ct scanning . the genesis of the idea to use 3d printing technology in cases such as these came as a result of collaborative review and a shared desire to design a solution for these clinical concerns . the existing relationship between the respiratory therapists of the ltvs and personnel in the medical devices department at cancercare manitoba served as an important environmental factor , which facilitated the connection of ideas . encouraging professionals to connect ideas and expertise with those of colleagues and others in this manner brought individuals together and supported a sense of collective cognitive responsibility ( 11 ) . through sharing in the responsibility for knowledge creation , both the individual goals of the respiratory therapist and the patient , as well as the collective goals of the organization were achieved in an innovative way . as a result , 3d printing was used as a means of assessing airway abnormalities and symptomology not explained by traditional diagnostic and assessment techniques . the resulting images were then imported into 3d modelling software ( 3d doctor v5 , able software corp , usa ) in which the trachea was outlined . although parts of this task were automated , several hours of manual work by the medical devices personnel was necessary . a significant challenge is that because a trachea tube was in place during the ct scan , it was subsequently necessary to remove it from each image manually ( figure 1 ) . the resulting outline of the trachea was then exported as a stereolithography ( stl ) file from which tracheal models were printed using a consumer - grade fused - deposition printer ( solidoodle 3 , solidoodle llc , usa and makergear m2 , makergear llc , usa ) ( figures 2 and 3 ) . printing the tracheal models was relatively straightforward as either a positive that was a copy of the patient anatomy , or as a negative that revealed the lumen of the trachea . furthermore , the ability to physically fit the tracheostomy tube into the positive model enabled the team to better determine the most appropriate airway specification relative to the anatomical limitations and other complications . figures 4 , 5 and 6 demonstrate three different models of the trachea that were printed using the 3d printer . these models provided a profound visual aid in understanding the unexpectedly extensive anatomical abnormality of this symptomatic client . an additional strength of 3d printing is that the models may be sliced in any plane before printing , and producing additional copies is simple . although 3d printing technology holds much potential for use in rt and in tracheostomy management , one notable limitation of this approach is the lengthy process of outlining the trachea on the ct images . although this step is partially automated , the presence of the trachea tube in the images complicates the process considerably . the use of 3d printing technology to enhance clinical evaluation of clients with long - term tracheostomy provides an example of how innovative ideas lead to practical solutions using new technologies . the adoption of 3d printing in rt practice has not previously been reported in the literature . as is the case in many innovation - generating organizations , the roots of a new practice concept rest in sharing and synthesizing multiple perspectives , problem solving and collective knowledge construction ( 12 ) . through dissemination of this idea , it is hoped that others will be prompted to share in further exploration and development of this particular idea , thus fostering new insights . an interesting array of issues worthy of further inquiry have emerged from this innovation . to evaluate the possible scope of applicability of 3d printing in tracheostomy management , it would be valuable to quantify the prevalence and morphology of tracheal abnormalities in clients with long - term tracheostomy . additionally , it would also be of great interest to determine whether routine assessment using 3d printing technology could impact client outcomes . finally , we need to consider its application as a tool for professional and client education . beyond spurring interest and inquiry in this new assessment technique , it is also hoped that this discussion will promote wider awareness and use of the structural mechanisms available to respiratory therapists for sharing knowledge . supporting an environment of information exchange and community knowledge generation will lead to enhancements in the support we provide to those with respiratory challenges .

the mammalian cns is inferior to that of invertebrates in the sense that regeneration is extremely limited ( tanaka and ferretti , 2009 ) . in that sense the optic nerve has the same organization and three types of glial cells as the cns white matter . breakthrough in the field of optic nerve regeneration not only has enabled vision restoration for millions of patients with glaucoma or other secondary optic neuropathies , but also provides insight into the cure of neurodegenerative diseases like alzheimer 's disease and motor neuron diseases . glial scars form in response to trauma and inhibit axonal extension across the injured site both physically and chemically . thus , diffusion of growth factors is inhibited due to the presence of a physical barrier and inhibitory molecules . injured cns axons are exposed to inhibitory proteins of myelin , such as nogo and myelin - associated glycoprotein ( mag ) . unlike fish and amphibians , adult mammals lose their neuronal growth and plasticity with age ( tanaka and ferretti , 2009 ) . compared to the peripheral nervous system , there is lower expression of growth factors and regeneration associated genes , such as growth associated protein-43 , an important growth associated protein in the nervous system ( linda et al . , 1992 ; huebner and strittmatter , 2009 ) . the extracellular matrix also lacks laminin , which is necessary for cell differentiation and migration ( luebke et al . , 1995 ) . the onset and progression of apoptosis is dependent on the distance of injured site to the eye : the closer the injury site is to the eye , the quicker the onset and progression of apoptosis is . the current hypothesis is that rgc death is protracted due to retrograde transport of neurotrophic factors , which temporarily support rgc survival ( almasieh et al . , 2012 ) . the closer the injury site is to the eye , the quicker the reservoir of neurotrophic factors will be depleted . an alternate theory is that there is active uptake of extracellular neurotoxic signals such as calpains , which undergoes retrograde transportation , triggering apoptosis . the triggered apoptosis in turn leads to further rgc death by activation of downstream factors through p53 , activating microglia , further neuronal degeneration and cell death ( fischer and leibinger , 2012 ) . the autonomous endoplasmic reticulum stress pathway in neurons has also been implicated in axotomy - induced rgc death ( hu et al . , 2012 ) . understanding the obstacles to optic nerve regeneration provides us with a rough framework to understand the essential steps to bring about optic nerve regeneration : enhancing neuronal survival , axon regeneration , remyelination and the establishment of functional synapses to the original neuronal targets . current potential treatments for optic nerve regeneration include neurotrophic factor provision , inflammatory stimulation , growth inhibition suppression , intracellular signalling modification and modelling of bridging substrates . neurotrophic factors are a family of diffusible proteins that mediate neuronal cell growth , differentiation and survival during development . the classical neurotrophic factors can be broadly classified into the neurotrophin family , the neurokines and other growth factors . the neurotrophin family comprises four peptides , namely brain - derived neurotrophic factor ( bdnf ) , nerve growth factor ( ngf ) , neurotrophin 3 ( nt3 ) and neurotrophin 4/5 ( nt4/5 ) . these polypeptides are structurally and functionally related , and mediate their effects through specifically binding to tropomyosin kinase receptors a - c ( henderson , 1996 ; fischer and leibinger , 2012 ) . ciliary neurotrophic factor ( cntf ) and leukaemia inhibitory factor ( lif ) make up the neurokine family . both act through glycoprotein 130 ( gp130)-containing signal transduction receptor ( henderson , 1996 ) . other growth factors include hepatocyte growth factor ( hgf ) , fibroblast growth factor ( fgf ) and glial cell line derived neurotrophic factor . rgcs express receptors for these growth factors , and intravitreal injection has been shown to enhance rgc survival upon optic nerve injury . the biological actions of these neurotrophic factors are broad , and include enhancing cell differentiation , cell survival , and synapse stabilization ( mey and thanos , 1993 ) . bdnf is one of the most effective agents in enhancing survival of axotomized rgcs ( mansour - robaey et al . , 1994 ) . however , trophic factors slow but do not prevent rgc death ( benowitz and yin , 2010 ) . hgf has been shown to increase neuronal survival and axonal regeneration both in vitro and in vivo ( tonges et al . , 2011 ) . fgf and bdnf have been reported to stimulate axonal sprouting , however the effects are mild . recent evidence showed that administration of osteopontin significantly potentiates the regenerative response of alpha retinal ganglion cells upon insulin - like growth factor 1 ( igf-1 ) or bdnf stimulation ( duan et al . , 2015 ) . studies are needed to further investigate whether osteopontin has similar enhancing effects on other neurotrophic factors . efforts have been made to activate growth - promoting pathways in injured cns neurons by exogenously providing trophic factors . intravitreal application of bdnf promotes survival of axotomized rgcs in optic nerve injury models , while bdnf injection into the superior colliculus also reduces developmental rgc death ( ma et al . , 1998 ; cntf is more potent in stimulating axonal regeneration in addition to promoting axonal survival than bdnf ( muller et al . intravitreal bdnf injection failed to convey any protective effect while cntf showed a significant protective effect ( pease et al . , 2009 ) . the action of cntf is closely linked with camp level , inflammatory stimulation and activation of the stat3 pathway ( muller et al . inflammatory stimulation has been shown to transform rgcs into an active regenerative state ( leibinger et al . , 2013a ) . following axotomy , intravitreal transplantation of a peripheral nerve segment has been found to enhance the regenerative response of axotomized rgc ( lau et al . , 1994 ) . initially interpreted as the result of trophic factors from the peripheral graft , the stimulated axon regeneration has been primarily attributed to inflammatory stimulation . infiltrated inflammatory cells such as macrophages and neutrophils both have been found to play an important role and as sources of oncomodulin ( singh and plemel , 2014 ) . oncomodulin is a calcium - binding protein from the parvalbumin family that is secreted by activated macrophages and neutrophils present in the vitreous and retina . lens injury and injection of zymosan and other inflammatory conditions give rise to an influx of inflammatory cells , producing high levels of oncomodulin ( yin et al . oncomodulin has been proposed to play an important role in axonal regeneration after lens injury ( yin et al . , 2009 ) . its effect is dependent on the presence of elevated camp and mannose ( benowitz and yin , 2010 ) . oncomodulin has been found to bind with rgcs only when camp is elevated or when the rgc membrane is permeabilized via other methods . thus it is hypothesized that camp is responsible for oncomodulin receptor translocation ( meyer - franke et al . , 1998 ) . thus , while the effect of oncomodulin has been shown to be dependent on the presence of both camp and mannose , mannose in turn exerts its effect only in the presence of camp . however , genetic evidence also supports the role of injury - induced neurokines . in cntf mice or cntf lif double knockout mice , the growth - promoting effect after lens injury is either reduced or largely blocked , suggesting injury - induced neurokines are necessary in inflammation - induced activation of axonal regeneration ( leibinger et al . , 2009 ) . key mediators of the beneficial effects of inflammatory stimulation have been identified as cntf , lif and interleukin-6 ( il-6 ) ( leibinger et al . , 2013b ) . signalling pathways involved include jak / stat3 and pi3k / atk / mtor ( leibinger et al . , 2013a ) . growth inhibitory signals highly expressed in cns myelin are nogo - a , mag , oligodendrocyte - myelin gp ( omgp ) and chondroitin sulphate proteoglycans ( cspgs ) . the first three are myelin proteins and the latter is generated by reactive scar tissues . contrary to common misconception , both the cns and the peripheral nervous system ( pns ) express these myelin proteins . the difference between their permissiveness for axonal regeneration lies in their difference in response to injury . in the pns , macrophages and schwann cells rapidly clear and downregulate myelin proteins following injury . in the cns , oligodendrocytes continue to express myelin proteins with no downregulation of expression ( filbin , 2003 ) . nogo - a , an isoform of nogo , is found in the endoplasmic reticulum , oligodendrocyte surface and inner surface of the myelin membrane . thus when an axon is injured and exposed , it comes into contact with nogo - a . mag is located in the periaxonal membrane while omgp is expressed by oligodendrocytes and neurons . intriguingly , while nogo , mag & omgp have been shown to induce growth cone collapse and inhibit neuronal outgrowth , the neuronal response to mag varies from inhibition to promotion , depending on the stage of neuronal development ( filbin , 2003 ) . a combination of neurotrophic factors and neutralized myelin - associated growth inhibitors have been shown to have a complementary effect on axonal regeneration ( weibel et al . , 1994 ) . glial scars inhibit axonal growth by acting as a physical barrier and through generating growth inhibiting factors , such as cspg . cspgs inhibit axonal growth through multiple mechanisms . due to their large size and dense negative charges , they non - specifically hinder the binding of matrix molecules to their cell surface receptors . evidence shows that cspg also interacts with nogo and mag ( sharma et al . , at the molecular level , rgc axonal regeneration can be enhanced through modifying intracellular signalling pathways . mammalian sterile 20-like kinase 3b ( mst3b ) is a purine - sensitive protein kinase that regulates axonal outgrowth in both the embryonal and mature nervous system ( lorber et al . , 2009 ) . suppression of mst3b blocks axon - promoting effects of oncomodulin and other trophic factors such as bdnf and ngf , whereas expression of active mst3b still enables axonal regeneration despite the absence of these trophic factors ( lorber et al . consistent with mst3b as an erk downstream - signalling molecule , in a conditional b - raf gain - of - function mouse model , activation of raf mek signalling promotes axon regeneration after optic crush ( odonovan et al . , 2014 ) . signalling cascades associated with neurotrophic factors include cam kinase , map kinase , jak / stat and pi 3-kinase / mtor . effects of oncomodulin can be blocked by inhibiting cam kinase while effects of other trophic factors can be blocked by inhibiting map kinase , jak / stat and pi 3-kinase / mtor . alternatively , deleting genes that encode suppressors of these pathways have been shown to result in sustained axonal regeneration . pten gene has been identified as a tumor suppressor and is a negative regulator of pi 3-kinase / mtor . deletion of either gene has been shown to promote optic nerve regeneration ( park et al . double knockout of both genes was found to result in a synergistic increase in axonal regeneration , allowing regenerating axons from the injured optic nerve to reach the optic chiasm ( sun et al . , 2011 ) . rho - associated protein kinase ( rock ) is a kinase that mediates various cellular functions such as cell motilitiy , secretion , proliferation and gene expression . there are mainly two isoforms , with rock1 mainly expressed in the lung , liver and kidney , and rock2 distributed mostly in the brain and heart . rock inhibitors are a novel potential class of glaucoma therapeutics , exerting its effect through its effects on the cytoskeleton and many other mechanisms ( wang and chang , 2014 ) . the rock signalling cascade is a known negative regulator of neurite extension , with pten being one of its downstream targets . in turn , the rock pathway can be activated by several cns growth inhibitors expressed in myelin , including mag , nogo and omgp ( van de velde et al . , 2015 ) . the kruppel - like family ( klf ) of transcription factors regulates the development and regeneration of the nervous system , showing different levels of expression pre- and post - natally ( moore et al . , 2009 ) . the regeneration induced by klf-4 deletion can be greatly enhanced by exogenous cntf , or removal of socs3 , suggesting a crosstalk mechanism between klf4 and stat3 ( qin et al . , 2013 ) . camp is a second messenger that augments axon regeneration through multiple mechanisms , but it has little effect on its own ( kurimoto et al . , 2010 ) . camp renders growth cones unresponsive to growth inhibitory signals , increases the expression of growth - promoting proteins and alters gene expression . elevation of the intracellular camp level also enhances the response of the injured rgcs to trophic factors . camp also appears to enable oncomodulin binding to rgc surface receptors , possibly by stimulating receptor translocation . it is well established that optic axons regenerate along a peripheral nerve graft , which provides a bridge to guide axons across barriers , a growth permissive environment and also a bed of neurotrophic factors ( so and aguayo , 1985 ) . the peripheral nerve graft thus enhances neuronal survival and studies show that regenerating rgcs are able to establish connections into the superior colliculus and also form functional synaptic connections ( bray et al . , 1987 ; sauve et al . , 1995 ) . a peptide nanofiber scaffold has been shown to stimulate axonal regeneration with return of functional vision in hamsters ( ellis - behnke et al . the scaffold prevents scar formation , provides a permissive environment for axonal regrowth and breaks down into peptides that can be potentially used by the surrounding tissue . there have been breakthroughs at multiple levels in the search of a solution to optic nerve regeneration since the pioneer work by so and aguayo ( 1985 ) . although these advancements are encouraging , there is a long way to secure functional restoration of the visual pathway . anamniotes ( lower vertebrates ) such as fish and salamanders retain the capacity not only for axonal regrowth , but also for neurogenesis following cns injury . birds and mammals , however , lose this capacity during embryonic development ( tanaka and ferretti , 2009 ) . one major reason for this is attributed to glial cells , astrocytes and their derivatives like myelin and the astrocytic scar ( johnson , 1993 ; garcia and koke , 2009 ) . the differences and similarities of cns regeneration between lower vertebrates and higher vertebrates are being intensely studied . since many molecular pathways in the cns are shared , the adult zebrafish is a powerful model to study cns regeneration ( becker and becker , 2008 ) . retinal projections are topographically arranged and orderly reconnection of axons with their central target is required . whether mature axons can regenerate into the brain at their full length , navigate to appropriate target areas and restore visual function should be further investigated . ( 2012 ) showed that in a mouse model subjected to a combination of zymosan injection , camp injection and deletion of pten gene , the powerful synergistic effect allowed rgc to regenerate into the lateral geniculate nucleus , superior colliculus and other visual centers . more importantly , this resulted in partial restoration of visual functions like the optomotor response and depth perception ( so and aguayo , 1985 ) . optic nerve regeneration to the optic tectum was possible but was shown to be dysfunctional ( beazley et al . , 1997 ) . a randomized trial comparing monocular visual stimulation with prey to the injured eye and the non - injured eye showed that consistent visual stimulation to the injured eye resulted in return of pupil responses and ability to capture prey at 1 year ( beazley et al . , 2003 ) . other methods to facilitate neural function reorganization include pulsed magnetic field stimulation ( rodger et al . , 2012 ; makowiecki et al . , analysis of visual recovery in zebrafish showed two phases of recovery ( kato et al . , 2004 ) . in the first phase , retinal projections are limited to the outer layer of the optic tectum , and the fish show a gross optomotor response ( bilotta , 2000 ) . the slow phase allows for recovery of high resolution vision , and involves complete restoration of visual circuits and refinement of synaptic terminals ( mcdowell et al . , 2004 ) .

the annual incidence of aneurysmal subarachnoid hemorrhage ( asah ) is about nine people per 100,000 population.2 ) the population - based mortality of subarachnoid hemorrhage ( sah ) is 38%,8 ) 1015% of patients with asah die before they arrive at the hospital , and a further 5% of patients die within the first 24 hours after hemorrhage.17 ) obliteration of the ruptured aneurysm is the only effective treatment for sah . the platinum coils are inserted in the lumen of ruptured aneurysm , and then the local thrombus , formed around the coils , finally obliterates the sac . after the international subarachnoid aneurysm trial ( isat ) , evt with coil is a first - line treatment for managing a ruptured aneurysm.7)14 ) the coil embolization can be performed under local anesthesia ( la ) or general anesthesia ( ga ) , and the ga in more common in general.3 ) evt may lead to several complications . the two most common evt procedure - related complications are thromboembolic complication ( tec ) and intraoperative rupture ( ior).13)15 ) both of these complications are somewhat more common in the setting of sah than for unruptured aneurysm.16 ) as proven , these two procedure - related complications ( tec , ior ) have a significant effect on the mortality and morbidity of asah patients,5)21 ) studies of the various risk factors associated with tec and ior have continued.6)12)14 ) however , most research studies were performed under ga . there are currently few studies about the relationship of procedure - related complications with la . in our hospital , we performed evt on patients with asah under la . there are currently few studies about complications and risk factors under la , so we analyzed the results of patients who underwent evt under la in our hospital from 2009 to 2013 and compared these results with those of patients who underwent the same procedure under ga . this study enrolled 189 patients who underwent evt for ruptured aneurysm with la in one institute between january 2009 and december 2013 . the study subjects include all patients who had obvious asah identified by computed tomography ( ct ) , magnetic resonance imaging ( mri ) , or lumbar puncture , and who had suspected rupturing intracranial aneurysm in digital subtraction angiography ( dsa ) or ct angiography and were judged able to be treated by evt . we performed surgical clipping under ga in 3 patients since we were unable to proceed with coil embolization under la due to their uncooperativeness even after administration of intravenous sedatives such as ativan ( lorazepam ) or midacum ( midazolam ) , but there was no case of coil embolization under ga . the patients ' heart rate , electrocardiographic data , blood pressure , respiratory rate , and oxygen saturation were continuously monitored . intravenous sedatives was considered whilst monitoring patient 's vital signs when the patient complains of pain or discomfort , increased heart rate or high blood pressure was noted , or the patient was unable to maintain immobility . patients with stupor or worse conscious level were likely to show unstable respiratory function and , therefore , underwent the procedure aided by endotracheal intubation . for all 186 patients , we checked whether there were complications ( tec , ior ) . then , complication rate and risk factors were analyzed with respect to the following factors : patient related factors ( age , sex , hypertension , diabetes mellitus [ dm ] , chronic kidney disease [ ckd ] , hyperlipidemia , smoking , previous antiplatelet use , and sah grade ) ; aneurysm - related factors ( aneurysm neck size , aneurysm greatest diameter , and aneurysm height - to - neck ratio ) ; physician- and technique - related factors ( whether the single catheter technique or additional techniques such as stent assist embolization or double catheter technique was used ) . the aneurysm neck size was defined as wide if the aneurysm neck size was 4 mm or larger , and narrow if it was less than 4 mm . aneurysm greatest diameter was defined as large if the greatest diameter was 7 mm or larger , and small if it was less than 7 mm . the aneurysm height - to - neck ratio was defined as favorable when the ratio was 1.5 or greater and unfavorable when it was less than 1.5 . good grade ' and ' poor grade ' were defined by the grade 1 to 3 and 4 to 5 , respectively . tec were identified during procedure with angiography regardless of type ( thrombus formation near the neck of the aneurysm , thromboembolism in the distal branches , and parent vessel occlusion ) . postoperative thromboembolic events were diagnosed with mri and/or dsa , which were performed in the case of sudden neurologic compromise . ior was diagnosed if the tip of the coil or microcatheter was seen outside the limit of the aneurysmal sac and/or there was extravasation of the contrast material . statistical analysis was carried out using spss version 20.0 software ( ibm spss inc . , armonk , ny , usa ) , and significance was determined at p < 0.05 . the study sample was composed of 64 men ( 34.4% ) and 122 women ( 65.6% ) . patients ' mean age was 55.5 13.74 years old , and 133 patients ( 71.5% ) were under 65 years . sixty - one patients ( 32.8% ) had hypertension and 21 patients ( 11.3% ) had dm . in addition , 42 patients ( 22.6% ) were smokers and 156 patients ( 83.9% ) had good grade . the aneurysms were located in the internal carotid artery ( ica ) in 57 ( 30.6% ) , anterior cerebral artery ( aca)/anterior communicating artery ( acoma ) in 84 ( 45.2% ) , middle cerebral artery ( mca ) in 22 ( 11.8% ) , vertebrobasilar system ( vb ) in 20 ( 10.8% ) , and dorsal wall of the ica in three patients ( 1.6% ) . the aneurysm neck size was wide in 51 ( 27.4% ) and narrow in 135 patients ( 72.6% ) . the aneurysm size was small in 131 ( 70.4% ) and large in 55 patients ( 29.6% ) . the height - to - neck ratio was favorable ( 1.5 ) in 101 ( 54.3% ) and unfavorable ( < 1.5 ) in 85 aneurysms ( 45.7% ) ( table 1 ) . procedure - related complications ( tec or ior ) occurred in 47 cases ( 25.3% ) and among these , tec occurred in 23 cases ( 12.4% ) and ior occurred in 24 cases ( 12.9% ) ( table 2 ) . in the analysis of patient - related factors that affected the outcome , age , sex , hypertension and dm in addition , ckds , hyperlipidemia , smoking , previous antiplatelet medication and sah grade also did not show statistically significant correlation ( table 3 ) . in the correlation analysis of outcome factors with aneurysm - related factors such as aneurysm neck size , greatest diameter , and height - to - neck ratio , neck size and height - to - neck ratio however , aneurysm greatest diameter showed a significant correlation with tec occurrence ; the tec occurrence rate was 21.8% for those with a large aneurysm and 8.4% for those with a small aneurysm ( p = 0.048 ) . also , the location of the aneurysm did not show a statistically significant correlation with procedure - related complications ( table 4 ) . three surgeons performed 55 ( 29.6% ) , 94 ( 50.5% ) , and 37 ( 19.9% ) evt cases , respectively . regarding technique factor , there was no significant difference between the groups who underwent single catheter coil embolization and those who used additional techniques such as stentassisted coil embolization or double catheter coil embolization ( table 5 ) . prior to the introduction of the endovascular coil in the 1990s , craniotomy and surgical clipping was a standard treatment for cerebral aneurysm.7 ) recently , endovascular coiling has become the gold standard in aneurysm treatment , as endovascular coiling has shown greater stability and better outcomes in isat and other studies.7)10)11 ) however , evt can cause some complications during the procedure ; the endovascular procedure - related complications of sah include device failure , coil migration , dissection , etc . , and the most frequent complications are tec and ior.7)12)21 ) the incidence of tec and ior have been reported in 1112.5%12)14 ) and in 2.55%5)6)19 ) of patients who underwent evt , respectively . the major causes of tec are distal vascular thrombosis formation caused by the original thrombus of vessel wall tearing by microcatheter or microwire , local thrombosis formation , and emboli shedding after procedure.21 ) the incidence of ior is related to the microcatheter , or guide - wire , as well as oversizing the coils , overpacking the aneurysm , or using stiff three - dimensional coils . in general coil embolization of asah was carried under ga . the advantage of evt under ga is that we can perform interventions in optimal conditions . this results in fewer technical failures and complications , therefore obtaining better clinical outcomes.1 ) but endovascular coil embolization could be carried without ga because it does not involve extensive surgical excision . coiling under la allows us to evaluate a patient 's neurological status intra - procedurally . in addition , it can lower the additional risks associated with ga and mechanical ventilation , such as cardiopulmonary morbidity rates , hospital stay length , and hospital costs.4 ) and emergency coil embolization with ga is not always possible in real clinical field . as mentioned earlier , several studies reported the complication rate of evt , but they are confined to that performed under ga only . however , few studies have examined complication rates for procedures carried out under la . in our hospital , from the time of the first evt , we performed evt under la for all cases . therefore , we studied how procedure - related complications and factors affecting complications differed under la compared to ga . the incidence of tec in our study was 12.4% , showing a similar incidence to previous studies.12)14 ) however , the incidence of ior was 12.9% , which was more than double compared with other studies ( 2.55%).5)6)19 ) this was a visible part of the notable differences with other studies performed under ga , and could be viewed as the effects of the type of anesthesia . the most likely cause of ior is perforation by a microcatheter and microwire.20 ) discomfort at the femoral cannulation site , severe headache , hot sensation upon injection of contrast into the cerebral artery and the electrolysis of the detachable coils in the procedure can cause pain.9 ) long procedure time could be stressful for patients9 ) and poor patient cooperation due to hemorrhage could cause movement . these factors could cause the movement of the microcathter or microwire , by moving the patient , these may lead to the ior during the coil embolization under la . these reasons mean that there is always the possibility of movement of the patients and changes in vital signs . as changes and movement could be unpredictable under la , operators might be less able to concentrate on the procedure itself . and these unstable factors may cause impatience to the operators , and they also enable you to make the wrong decision during procedure . for most patients in this study , there was unexpected movement of the head . this is thought to be the reason why ior showed a higher incidence under la . on the other hand , reducing patients ' movements with ga but in this study , tec rate was similar to that of the others studies.12)14 ) this is probably because the rate of tec was so high as over 10% that the rates under ga and la did not differ . patient - related factors that are known to influence the incidence of tec or ior during coil embolization are as follows : smoking , absence of hypertension , and age under 65 years.14 ) however , none of the aforementioned factors along with other patients factors , such as patients ' gender , dm , ckd , previous antiplatelet treatment and sah grade , had a significant correlation with the complication in this study . the incidence of tec was higher when the aneurysm was 10 mm or larger at its greatest diameter , and when the neck was 4 mm or wider.6)14)18 ) in addition , the incidence of ior was higher when the aneurysm was located in a mca bifurcation , and the aneurysm 's greatest diameter was less than 7 mm.12)14 ) in this study , the only factor related to tec occurrence was the greatest diameter of the aneurysm , and the other factors did not show a significant relationship with tec . the ior was not significantly correlated with any patient - related or aneurysm - related factors . a larger aneurysm was a risk factor for tec in this study , the same as the results of previous studies.6)14)18 ) for large aneurysms , a lot of intra - aneurysmal clotting prior to evt and a larger clot volume induced by coil occlusion in the aneurysm may elevate the risk of tec.14 ) unlike other studies,14 ) a wide neck aneurysm was not a significant risk factor for tec . a previous study explained the reason for the higher tec rate in wide neck aneurysms as it required a large coil surface , which has a higher potential for thrombus formation , higher possibility of intra - aneurysmal clot migration , and a higher risk of coil protrusion into the parent vessel.14 ) however , this does not show statistical significance in our study ; it is difficult to describe the clear reason . it is difficult to generalize the results due to the insufficient number of patients available for analyzing how patients ' factors affected procedure complications . second , a comparison between a ga group and a la group was not performed at the one institute ; differences between institutes may have affected outcomes . third , differences in the time before performing evt and time taken for evt for each patient might have affected the results . fourth , there was a potential selection bias , because patients were selected on a case - by - case basis . lastly , the subjects in our study were not randomized , with adverse events being self - reported . nevertheless , there was an obvious difference in the complication rate for evt in accordance with the type of anesthesia used . compared to ga , the tec rate was similar for patients treated under la , but the ior rate was significantly higher . it is necessary another local anesthetic technique that reduce patients movement by reducing the pain and anxiety of the patients . if not , switching from la to ga may be necessary to reduce complications and to improve outcomes .

increasing awareness of the important role of vitamin d for bone and other diseases has led to increased 25-hydroxyvitamin d [ 25(oh)d ] testing ( d represents d3 and d2 forms ) . however , variability within and between methods and laboratories has often compromised correct diagnosis and the ability to compare results from different studies and national surveys [ 15 ] . automated antibody - based , radioimmunoassays , high performance liquid chromatography ( hplc ) , and mass spectrometry methods for 25(oh)d testing are subject to variability issues that can arise from a variety of sources , such as differential detection of the d3 and d2 forms , interference by detection polyclonal antibodies , and nonspecific detection of other vitamin d metabolites such as the 3-epimer form of 25(oh)d [ 3-epi-25(oh)d ] and 24,25(oh)2d3 . in addition , incomplete release of 25(oh)d from the vitamin d binding protein ( dbp ) has been identified as a potential source of variability for both manual and automated immunoassays . establishing an immunoassay for 25(oh)d is challenging because the majority of the highly hydrophobic 25(oh)d is tightly bound ( dissociation constant , kd , 5 10 m ) to a vast excess of dbp from which it must be separated ; almost no 25(oh)d is found free ( non - protein bound ) in the circulation , and less than 5% of the available dbp binding sites are occupied by vitamin d compounds . in addition , dbp binds vitamin d3 along with other metabolites and vitamin d2 , whose similar structures may be easier to release from dbp and difficult to differentiate ; dbp has a higher affinity for vitamin d3 than other metabolites and vitamin d2 ; and generating specific antibodies against small antigenic molecules , such as 25(oh)d , is difficult , but it is mandatory because the vitamin d standardization program ( vdsp ) states that 25(oh)d assays should measure equimolar amounts of 25(oh)d3 and 25(oh)d2 ( total vitamin d ) . measuring total vitamin d is required because some supplements contain the d2 form , and not measuring d2 would lead to lower 25(oh)d values . in methods such as radioimmunoassay , hplc , and mass spectrometry , an initial extraction step with organic solvents releases all bound 25(oh)d from dbp [ 1013 ] . however , organic solvents are not compatible with most automated immunoassays , and alternative releasing agents , which are proprietary , are used instead . recent studies performed in populations with different levels of dbp have questioned the effectiveness of these proprietary releasing agents to completely free 25(oh)d from dbp . the goal of this study was to examine the ability of the advia centaur vitamin d total assay by comparison with an lc - ms / ms method to accurately measure 25(oh)d levels in serum samples from healthy adults ( endogenous ) and healthy adults with exogenous dbp ( endogenous + spiked ) and from pregnant women and chronic kidney disease ( ckd ) patients receiving dialysis , who have higher and lower than normal serum levels of dbp , respectively [ 7 , 14 , 15 ] . the advia centaur vitamin d total assay is traceable to the nist - ghent reference measurement procedure ( rmp ) for vitamin d testing . ( this version of the advia centaur vitamin d total assay is not currently available commercially in all regions , including the usa . ) in order to determine the influence of dbp on a vitamin d immunoassay , a study examining dbp as an endogenous interference , similar to how hemoglobin , cholesterol , or total protein would be measured , following clinical and laboratory standards institute ( clsi ) document ep7-a2 was performed at the siemens r&d facility in tarrytown , ny , usa . human native dbp ( > 95% pure ) was purchased from athens research & technology , inc . the lc - ms / ms method used in this study is traceable to the esoterix id - lc - ms / ms method , which is traceable to nist . the lc - ms / ms method performed at siemens used the waters acquity h - class ultrahigh performance liquid chromatography ( uplc ) and triple quadrupole ( tqd ) tandem mass spectroscopy ( ms ) with masslynx and quanlynx software ( waters acquity tqd system , waters corporation , manchester , uk ) . this method is able to separate , identify , and separately quantify the concentrations of 25(oh)d2 , 25(oh)d3 , and 3-epi-25(oh)d3 in a serum sample . as reported by the manufacturer , the lc - ms / ms method demonstrated a dynamic assay range of 2.5220 ng / ml ( 6.25550 three levels of 25(oh)d2 and 25(oh)d3 concentrations tested over five consecutive days yielded intra - assay coefficients of variation ( cvs ) of 7.7% and interassay precision cvs of < 12% for 25(oh)d2 and 25(oh)d3 . the advia centaur vitamin d total assay used in this study is traceable to the ghent university id - lc - ms / ms 25(oh)d rmp . ( this version of the assay is not currently available commercially in all regions , including the usa . ) the ghent university is a reference laboratory for the vitamin d standardization program ( vdsp ) . the sample reference material ( srm ) used for the ghent university method is traceable to the nist srm 2972 [ 4 , 9 , 17 ] . recently , siemens received confirmation from the centers for disease control and prevention ( cdc ) that the advia centaur vitamin d total assay is now a certified procedure of the vitamin d standardization - certification program ( vdscp ) . certification was achieved by demonstrating that the total vitamin d [ 25(oh)d ] results for the 40 vdscp samples ( 10 quarterly challenges ) agreed with the results from the id - lc - ms / ms rmp method . the advia centaur vitamin d total assay achieved a mean bias of 0.3% ( acceptance criterion was 5.0% ) and a mean imprecision of 5.5% ( acceptance criterion was < 10.0% ) . the advia centaur vitamin d total assay is a one - pass , 18-minute antibody competitive chemiluminescent immunoassay . release of the 25(oh)d metabolites from the dbp is accomplished by denaturing and blocking agents . 25(oh)d in serum competes with a 25(oh)d analog ( labeled with fluorescein ) for an anti-25(oh)d monoclonal mouse antibody ( labeled with acridinium ester ) . detection occurs after the remaining anti-25(oh)d monoclonal antibody ( labeled with acridinium ester ) complexes with vitamin d analog ( labeled with fluorescein ) and anti - fluorescein monoclonal antibody covalently bound to paramagnetic particles . the standardized assay demonstrates equimolar cross - reactivity with 25(oh)d2 ( 104.5% ) and 25(oh)d3 ( 100.7% ) , minimal cross - reactivity with 3-epimer-25(oh)d3 ( 1.1% ) , and a broad dynamic assay range of 4.2150 ng / ml precision was determined by assaying six samples twice a day in replicates of 4 , over 20 days ( n = 160 replicates per sample ) according to the clinical and laboratory standards institute ( clsi ) protocol ep5-a2 ; the run - to - run cvs were in the range of 4.2% and 11.9% . all samples were run in singlicate on both the lc - ms / ms and a single advia centaur system . clinical serum samples from 18 healthy adults were purchased from a commercial vendor ( promeddx , norton , ma , usa ) . serum samples from 36 pregnant women in their third trimester and 40 ckd hemodialysis patients were purchased from another commercial vendor ( research sample bank , delray beach , fl , usa ) . peripheral venous blood samples were collected , placed at 4c , and centrifuged ; serum aliquots were prepared and stored for less than four months at 20c until analysis . generally , no difference was found in the serum concentrations of dbp for men and women [ 11 , 15 ] . serum samples were sent to siemens healthcare diagnostics ( tarrytown , ny , usa ) for dbp and 25(oh)d measurements . the serum samples from the 18 healthy adults were divided into five serum pools ; each of the four pools contained 4 individual serum samples and one pool contained 2 individual serum samples . the 25(oh)d concentrations in these five serum pools were measured by using a lc - ms / ms method at siemens healthcare diagnostics , ( tarrytown , ny , usa ) according to a protocol that allowed resolution of 25(oh)d2 and 25(oh)d3 from 3-epi-25(oh)d3 . the lc - ms / ms values for the five individual pools ( pools 15 ) resulted in mean 25(oh)d concentrations of 24 , 32 , 51 , 41 , and 75 ng / ml , respectively . the endogenous levels of dbp were measured in each of the five serum pools using the quantikine elisa vitamin d binding protein bp kit , dvdbp0 ( r&d systems , inc . ) . subsequently , each of the five serum pools was divided into six aliquots , and dbp ( ranging from 50 to 250 ug / ml in 50 ug / ml increments ) was spiked into 5 of the 6 aliquots from each pool ( table 3 ) . the dbp content in the resulting thirty samples was then reanalyzed to confirm higher dbp concentrations in spiked samples , and 25(oh)d measurements were performed according to routine procedures using the advia centaur vitamin d total assay traceable to the ghent university id - lc - ms / ms 25(oh)d rmp . ( this version of the assay is not currently available commercially in all regions , including the usa . ) bias of 25(oh)d values to the original lc - ms / ms values was determined . in addition , the 36 clinical serum samples from third - trimester pregnancy patients and the 40 clinical serum samples from ckd patients were evaluated for endogenous dbp and 25(oh)d using the advia centaur vitamin d total assay ; and bias of 25(oh)d values to the original lc - ms / ms values was determined . only four samples from pregnancy subjects had detectable 25(oh)d2 ( 3.2 , 5.2 , 8.0 , and 10.7 g / ml ) . nineteen samples from dialysis patients had detectable 25(oh)d2 ( range 1.6 to 35 g / ml ) , eight of which had levels above 10 g / ml . the 3-epi-25(oh)d3 was present at levels greater than 1.5 ng / ml in samples from 23 dialysis and 32 pregnancy subjects . difference plots and bias values were obtained using microsoft excel ( 2010 ) ; analyze - it add - in program in excel was used to compare the different sets of data in order to obtain the 95% confidence interval ( ci ) and standard deviations ( sd ) for 95% limits of agreement . correlation plots and correlation and deming regression analyses were generated using graphpad prism , version 6 . the mean serum concentrations of dbp in healthy subjects ( endogenous and endogenous + spiked ) , pregnant women , and dialysis patients are presented in table 1 . for the five serum pools , the average endogenous serum dbp concentration ( sd ) was 348 106 g / ml ( range 260.7 to 519.0 g / ml ) , which is consistent with the results of other studies [ 6 , 20 , 21 ] . for healthy serum samples spiked with dbp , the average dbp concentration was higher ( 545 185 g / ml , range 261.2 to 980.6 g / ml ) than endogenous levels . for pregnancy samples , the average dbp concentration was also greater ( 415 245 g / ml , range 82.2 to 874.5 g / ml ) than that for healthy serum samples . in contrast , for ckd patients receiving dialysis , the average dbp concentration was lower ( 198 173 g / ml , range 63.4 to 1115.7 g / ml ; median 142.1 g / ml ) than levels in healthy serum and pregnancy samples . the mean total serum 25(oh)d concentrations and range as measured by the lc - ms / ms method and the advia centaur vitamin d total assay are presented in table 2 . the mean 25(oh)d levels ( sd ) were 44.6 19.8 and 44.8 20.1 ng / ml for healthy serum samples ( endogenous ) , 44.6 18.0 and 43.5 16.7 ng / ml for healthy ( endogenous + spiked ) serum samples , and 44.6 18.0 and 43.7 17.0 ng / ml for both endogenous and endogenous + spiked healthy serum samples , and they were lower for pregnancy serum samples , 27.3 9.6 and 25.3 8.7 ng / ml , and dialysis serum samples , 28.1 14.8 and 29 15.3 ng / ml . consistent with previous reports , no correlation was found between the dbp and 25(oh)d concentrations for serum from dialysis patients ( pearson 's correlation coefficient r = 0.1 ) [ 11 , 14 , 15 ] . pregnancy samples demonstrated a positive correlation ( r = 0.35 ; p = 0.013 ) between serum concentrations of dbp and 25(oh)d for lc - ms / ms , but no correlation was found for advia centaur ( r = 0.15 ; p = 0.37 ) . there were too few non - spiked healthy samples for valid 25(oh)d and dbp correlation assessment . the overall average bias of all samples from healthy individuals ( endogenous and endogenous + spiked ) for the advia centaur vitamin total assay to the lc - ms / ms method was 1.4% ; for all third - trimester pregnancy samples , the average bias was 6.1% ; and for all renal dialysis samples , the average bias was 4.4% . the results for bias , percent bias , 95% ci , and sd ( 95% limits of agreement = 1.96 sd ) as a function of dbp concentration for each population separate and combined are presented in figure 1 . when all populations were combined , positive bias ( versus lc - ms / ms ) was observed at very low serum dbp concentrations and negative bias was observed at very high serum dbp concentrations ( figure 1 ) . with respect to dialysis samples with generally lower dbp concentrations , we do not know if uremic serum properties contributed to bias , and we question the validity of analyzing combined populations . nevertheless , we examined how well the methods in subjects with serum dbp concentrations at extremes of the serum dbp concentration range ( very low and very high)for combined and separate populations ( figures 2 , 3 , and 4 ) . very low and very high serum dbp concentrations were defined as two sd below and above the mean for healthy subjects which is 348 106 g / ml ; hence , the very low dbp group comprised samples having concentrations of 136 g / ml , and high dbp group comprised samples having concentrations of 560 g / ml . the middle range group had samples with dbp concentrations ranging from 137 to 559 g / ml . the following populations were analyzed : ( 1 ) healthy , spiked , pregnancy , and dialysis ( figure 2 ) ; ( 2 ) healthy ( which had no low or high groups ) [ figure 3(a ) ] ; ( 3 ) healthy and spiked ( which had no low group ) [ figure 3(b ) ] ; ( 4 ) spiked ( which had no low group ) [ figure 3(c ) ] ; ( 5 ) pregnancy [ figure 4(a ) ] ; ( 6 ) dialysis ( which had no high group ) [ figure 4(b ) ] . analysis of 25(oh)d values for advia centaur and lc - ms / ms as a function of low , medium , and high serum dbp concentrations demonstrated that correlations between the two methods were acceptable at low and high serum dbp levels for all populations analyzed ( combined and separate ) ( figures 2 , 3 , and 4 ) , with pregnancy samples demonstrating the lowest correlation at very high serum dbp concentrations ( r = 0.87 , p < 0.0002 ) . healthy samples ( endogenous and endogenous + spiked ) showed very good correlations and agreement between methods . the mean bias obtained for combined populations and each population separately for their low , medium , and high range dbp groups represented acceptable assay performance ( table 4 ) . only four samples from pregnant subjects had detectable 25(oh)d2 ( 3.2 , 5.2 , 8.0 , and 10.7 g / ml ) . nineteen samples from dialysis patients had detectable 25(oh)d2 ( range 1.6 to 35 ng / ml ) , eight of which had levels above 10 ng / ml . the mean percent bias of the eight 25(oh)d2 samples which had greater than 10 ng / ml was 9.0 0.12% ( mean sd ) , whereas the mean percent bias of the remaining samples was 3.0 0.12% . of the 40 dialysis patients , 17 had less than 1.5 ng / ml 3-epi-25(oh)d3 and 23 ( 58% ) had 3-epi-25(oh)d3 concentrations ranging from 1.7 to 3.6 ( mean sd , 2.5 0.57 ng / ml ) . of the 36 pregnancy subjects , four had less than 1.5 ng / ml 3-epi-25(oh)d3 and 32 ( 89% ) had 3-epi-25(oh)d3 concentrations ranging from 1.6 to 6.3 ng / ml ( mean sd , 3.3 1.3 ) . this study addressed the influence of dbp on the accuracy of the advia centaur vitamin d total assay by comparison with an lc - ms / ms method . the advia centaur vitamin d total assay results in this study are traceable to id - lc - ms / ms 25(oh)d reference method procedure and the standard reference materials established by nist and the university of ghent [ 4 , 9 , 17 , 22 ] . in healthy individuals , endogenous serum dbp concentration ( 347.6 g / ml ) was found to be within the range reported by others ( 300600 g / ml ) and increased after dbp spiking ( 545 g / ml ) . despite the increase in dbp concentrations after spiking , 25(oh)d measurements in individual samples were equivalent between the two methods . there were no healthy ( endogenous or endogenous + spiked ) samples in the low dbp range , as defined by two sd values below the mean of healthy samples ( i.e. , 136 g / ml ) ( figure 3 ) . the high dbp range , as defined by two sd values above the mean of healthy samples ( i.e. , 560 g / ml ) , comprised twelve spiked samples ( figure 3 ) , and 25(oh)d agreement was good between the methods for 25(oh)d values ( r = 0.9927 , p < 0.0001 ; bias 3.0 thus , there was not a significant bias observed for the advia centaur for healthy samples ( endogenous + spiked ) . this demonstrates that dbp concentrations as high as 980 g / ml did not appear to interfere with the assay for this population . by comparison , endogenous serum dbp concentrations peaked at 519 g / ml in healthy subjects . because the use of dbp - spiked samples may be suspect , these results will be confirmed in future studies which evaluate a greater number of samples from healthy subjects containing endogenous serum dbp concentrations in the higher range ( although it is unlikely that normal healthy subjects exist with dbp concentrations that can be achieved at the high spiking concentrations ) . depending on hormonal status or disease state serum matrix components may be different , and the levels of dbp may be higher or lower than those of healthy individuals [ 14 , 15 ] . in women who are receiving estrogen therapy and those who are pregnant , higher serum estrogen levels correlate with increases in circulating dbp and total 1,25(oh)2d . during pregnancy , increased 1,25(oh)2d3 occurs in response to the growing calcium demands of the fetus [ 14 , 15 ] . consistent with these reports , the mean dbp concentration was greater ( 415 g / ml ) for samples from pregnant women than for those from healthy subjects ( 347.6 g / ml ) and dialysis patients ( 198 g / ml ) . despite the overall higher dbp concentrations in pregnancy serum , 25(oh)d results for those samples with low , medium , and high dbp concentrations demonstrated acceptable agreement between the advia centaur vitamin d total assay and the lc - ms / ms method ( r = 0.96 , p < 0.0018 , bias 2.0 10.9% ; r = 0.96 , p < 0.0001 , bias 3.0 12.6% ; r = 0.87 , p < 0.0002 , bias 14.0 9.9% , resp . ) . although samples in the low and medium dbp range showed less bias than those with very high dbp concentrations , the assay performance was acceptable for all groups . four samples out of 36 contained measureable 25(oh)d2 ; it is unlikely that 25(oh)d2 influenced the assay bias because several samples lacking 25(oh)d2 demonstrated similar levels of bias . in contrast to a previous study that found higher 25(oh)d levels in pregnant women compared to nonpregnant healthy women , this study found overall lower levels in pregnant women ; this difference may relate to differences in vitamin d supplementation . nephrotic syndrome and ckd predialysis and dialysis patients demonstrate diminished serum levels of the bioactive 1,25(oh)2d , likely due , in part , to impaired renal synthesis , nutritional deficit , and lower 25(oh)d substrate levels [ 2326 ] . although some studies report no change in serum dbp levels in renal failure patients compared with healthy individuals , other studies demonstrate lower serum levels and increased dbp urinary excretion ; lower serum concentrations of dbp likely reflect increased urinary loss due to proteinuria , which is a common finding in ckd patients [ 15 , 27 , 28 ] . in this study , the overall mean 25(oh)d level was equivalent between the advia centaur vitamin d total assay and the lc - ms / ms method and for the low and medium range dbp groups ( r = 0.97 overall ; r = 0.98 low range dbp group ; r = 0.97 middle range dbp group , p < 0.0001 ; bias was 4.35 12.4% overall , 10.0 10.6% for the low range dbp group and 0.0 12.4% for the middle range dbp group , resp . ) , indicating acceptable performance of the advia centaur vitamin d total assay in the presence of dbp and uremic serum . it is not known whether unique components of uremic serum contributed to the bias observed . nineteen samples from dialysis patients had detectable 25(oh)d2 ( range 1.6 to 35 ng / ml ) , eight of which had levels above 10 ng / ml . the 25(oh)d2 containing samples appeared to contribute to the positive bias in this patient population . this result is consistent with the performance of the advia centaur vitamin d total assay which demonstrates a slight difference in recovery for 25(oh)d2 and 25(oh)d3 ( 104.5% versus 100.7% ) as stated in the instructions for use manual . only one uremic sample was found in the higher range [ 1115.7 g / ml dbp ; 19.1 ng / ml 25(oh)d3 by lc - ms / ms and 21.4 ng / ml 25(oh)d by advia centaur ; 12% bias of advia centaur to lc - ms / ms ] . whether an error in dbp measurement was the cause for the unusually high dbp concentration is not known . although the serum 25(oh)d concentrations in dialysis patients were lower than those found in healthy individuals , the values were approximately normal ( according to the endocrine society guidelines ) . this is likely due to patient adherence to vitamin d supplementation which is indicated for end - stage renal disease patients on dialysis . it is worth noting that lower levels of serum 25(oh)d concentrations in predialysis patients correlate with a greater risk of mortality . this underscores the need to accurately evaluate and monitor serum 25(oh)d levels in the ckd patient population . a recent study implicated ineffective 25(oh)d - dbp extraction procedures as the cause of variability in an evaluation of five automated assays compared to an rmp lc - ms / ms method . the study , which included samples from healthy individuals , pregnant women , dialysis patients , and intensive care patients , found that the bias was , at least in part , dependent on dbp concentration . the advia centaur vitamin d total assay in the present study differed from the assay in the previous study in that it has a different standardization ; this version is standardized with internal standards traceable to the nist - ghent vdsp rmp . this may have had some impact on why the results of this study differ from those previously reported . the small positive bias found in renal dialysis patients with dbp concentrations below those found in normal healthy subjects and small negative bias found in pregnant subjects with dbp levels above those found in normal healthy subjects were within the acceptable range for the assay . thus , for populations with different levels of dbp , the 25(oh)d results obtained by the advia centaur vitamin d total immunoassay were equivalent to the sum of 25(oh)d2 and 25(oh)d3 using the lc - ms / ms method especially for individuals with serum dbp concentrations within the range for the healthy population ( 137 to 559 g / ml ) .

particle - stabilized emulsions , otherwise known as pickering emulsions , have been recognized for more than a century . many classes of particles including silica , polymer latexes , and clays can be used to stabilize such emulsions , with surface wettability usually dictating the emulsion type . thus relatively hydrophilic particles tend to favor the formation of oil - in - water ( o / w ) emulsions , whereas relatively hydrophobic particles usually produce water - in - oil ( w / o ) emulsions . over the past decade or so , increasing attention has been paid to the use of highly anisotropic particles . for example , noble et al . reported the use of polymeric microrods to prepare water - in - oil emulsions and ultimately colloidosomes . more recently , kalashnikova et al . evaluated various types of cellulose - based pickering emulsifiers of ribbon - like shape . similarly , wege et al . utilized hydrophobic anisotropic cellulose microparticles to stabilize water - in - oil emulsions . vermant and co - workers employed a multiple backscattering technique to demonstrate that more stable pickering emulsions are obtained when employing ellipsoidal polystyrene latexes ( mean aspect ratio 9 ) compared to conventional spherical latex particles . similar results were also reported for ellipsoidal hematite particles ( mean aspect ratio 6 ) . over the past decade , we and others have utilized polymerization - induced self - assembly ( pisa ) to prepare a wide range of diblock copolymer nano - objects of tunable size , shape , and surface chemistry in the form of concentrated colloidal dispersions . of particular relevance to the present work , pisa provides an extremely attractive route to highly anisotropic block copolymer worms , enabling their synthesis on a multigram scale in either polar solvents ( e.g. , water or ethanol ) or non - polar solvents ( e.g. , n - alkanes ) . reproducible pisa syntheses of such worms usually require the construction of phase diagrams , although ad hoc syntheses can sometimes also be effective . recently , we compared the performance of hydrophilic linear and cross - linked poly(glycerol monomethacrylate)-poly(2-hydroxypropyl methacrylate ) [ pgma phpma ] diblock copolymer worms prepared via pisa in aqueous solution as pickering emulsifiers for the production of o / w emulsions . the linear worms did not survive the high - energy homogenization conditions required to generate the oil droplets : instead , worm dissociation occurs to generate individual copolymer chains , which then act as a polymeric surfactant to stabilize the emulsion . however , the corresponding cross - linked worms ( which were obtained via addition of a small amount of ethylene glycol dimethacrylate ) survived homogenization , leading to the formation of genuine pickering emulsions . in related work , hydrophobic linear poly(lauryl methacrylate)poly(benzyl methacrylate ) [ plma pbzma ] worms prepared via pisa in n - dodecane survived homogenization to produce w / o pickering emulsions . in this case the worms exhibited thermoresponsive behavior : heating to 150 c led to a worm - to - sphere transition that was essentially irreversible if it is conducted in sufficiently dilute solution ( 1.0% w / w ) . thus this system provided a unique opportunity to compare the effect of particle morphology on pickering emulsifier performance for chemically identical spheres and worms . it was found that the worms were more effective stabilizers because they produced finer , more stable oil droplets than the spheres when directly compared under the same conditions . this is understandable because worms are 12 orders of magnitude more strongly adsorbed at the oil water interface than spheres , yet have a comparable surface area per unit mass , as ( the as for highly anisotropic worms is estimated to be only approximately 33% less than the as for the corresponding spheres ) . given these intrinsic advantages , and the relative ease with which block copolymer worms can now be accessed via pisa syntheses , further exploration of the use of such anisotropic particles as pickering emulsifiers is clearly warranted . in this work , pbzma triblock copolymers can form sufficiently robust worms to act as pickering emulsifiers for o / w emulsions . more specifically , we examine the scope and limitations of the pisa synthesis of such worms , explain why the copolymer morphology does not evolve further to produce vesicles , characterize the worm dimensions using transmission electron microscopy ( tem ) and small - angle x - ray scattering ( saxs ) , and assess the performance of such worms as hydrophilic pickering emulsifiers for the production of millimeter - sized oil droplets . for the sake of brevity , a shorthand notation is utilized throughout this article to describe the various block copolymers . thus g , h , and b are used to represent glycerol monomethacrylate ( gma ) , 2-hydroxypropyl methacrylate ( hpma ) , and benzyl methacrylate ( bzma ) , respectively . hence gxhybz denotes poly(glycerol monomethacrylate - block-2-hydroxypropyl methacrylate - block - benzyl methacrylate ) , where x , y , and z indicate the mean degrees of polymerization ( dp ) of the three respective blocks . the initial raft solution polymerization of gma was conducted in ethanol at 70 c to generate a near - monodisperse g37 macromolecular chain transfer agent ( macro - cta ) ( mw / mn = 1.19 ; see figure s1 and table 1 ) . after purification , this water - soluble macro - cta was utilized for the in situ raft aqueous solution polymerization of hpma at 15% w / w solids , yielding a 100 g batch of g37h60 diblock copolymer precursor ( see figure 1a ) . h nmr studies indicated that more than 99% hpma conversion was achieved within 2 h at 70 c ( see figure s2 ) , as expected from previous studies . gel permeation chromatography ( gpc ) studies indicated that a near - monodisperse diblock copolymer was obtained with high blocking efficiencies and minimal macro - cta contamination ( mw / mn = 1.14 ; see figure s1 and table 1 ) . the gpc trace was unimodal but a high molecular weight shoulder was discernible , which has been attributed to low levels of dimethacrylate impurity in the hpma monomer ( approximately 0.07 mol % as judged by hplc analysis ) ; this results in light branching of the phpma chains . dynamic light scattering ( dls ) studies of this g37h60 diblock copolymer reveal a relatively low count rate of 50 kcps , and h nmr studies confirm that the phpma block is fully soluble in water ( see figure s3 ) , suggesting that self - assembly does not occur for this relatively short phpma block . dmf gpc data recorded using a refractive index detector and calibrated using a series of poly(methyl methacrylate ) standards . synthesis of ( a ) g37h60b10550 triblock copolymer via raft aqueous solution polymerization of hpma followed by raft seeded emulsion polymerization of bzma and ( b ) g92b28 diblock copolymer prepared via raft aqueous emulsion polymerization of bzma . furthermore , no nanoparticles can be observed by tem ( see image in figure 2 ) , which again indicates that the phpma block is not sufficiently long to induce micellar nucleation . this is consistent with observations made by blanazs and co - workers , who found that a minimum phpma dp of around 90 was required to induce nucleation when using a pgma47 macro - cta . however , it should be noted that this minimum critical dp is expected to be rather sensitive to the precise pisa formulation . tem images obtained for dried dilute aqueous dispersions of the g37h60 diblock copolymer precursor , a series of seven g37h60bz triblock copolymers ( where z ranges from 10 to 550 ) , the g37h90 diblock copolymer worms , and the g92b28 diblock copolymer spheres . this g37h60 diblock copolymer precursor was then utilized as a macro - cta for the raft seeded emulsion polymerization of bzma at 70 c to produce a series of seven g37h60bz triblock copolymers , where z ranged from 10 to 550 ( see figure 1a ) . h nmr studies confirmed that bzma conversions greater than 92% were obtained in each case ( see figure s2 and table 1 ) . dimethylformamide ( dmf ) gpc studies indicated that near - monodisperse triblock copolymers were obtained ( mw / mn < 1.20 , see table 1 ) with high blocking efficiencies ( see figure s1 ) . tem images shown in figure 2 and dls studies ( see table 1 ) indicated that spheres with a hydrodynamic diameter ( dh ) of 41 nm were formed when z = 10 ; thus chain extension with just 10 units of bzma is sufficient to induce micellar nucleation . when targeting a pbzma dp of 30 ( and achieving a dp of 28 ) , tem studies indicated the formation of highly anisotropic worms ( figure 2 ) , similar to those reported recently . the worm model provided an excellent fit to the saxs pattern over six orders of magnitude of x - ray scattering intensity ( see figure s4a ) . the mean worm contour length ( lw ) was determined to be 653 nm , which is consistent with tem observations . assuming a circular worm cross - section , the mean worm width ( ww ) , was calculated to be 25.6 1.7 nm , which is also consistent with that estimated from tem images ( for which ww = 24.2 3.2 nm ) , where ww = 2rsw + 4rg , with rsw representing the radius of the worm core cross section and rg representing the radius of gyration of the corona chains . the rg of the g37 corona block of these worms was determined to be 1.7 nm from the data fit to the saxs pattern ( see figure 4a ) . this experimental value is comparable to a theoretical estimate : the projected contour length of a single gma monomer is 0.255 nm ( two carbon bonds in an all - trans conformation ) , the total contour length of a g37 block , lpgma = 37 0.255 nm = 9.44 nm , and the literature value of the kuhn length for poly(methyl methacrylate ) is 1.53 nm , resulting in an rg of ( 9.44 1.53/6 ) , or 1.55 nm . a worm model fit to the saxs data pattern of g37h60b28 ( figure s4a ) indicated that the solvent volume fraction in the core ( xsol ) is 0.03 , which suggests that the hydrophobic worm cores are essentially non - solvated . for g55hy diblock copolymer vesicles , which ranged from 0.38 to 0.66 as y was increased from 200 to 1000 . it is evident that extension with approximately 28 units of bzma not only changes the nanoparticle morphology from spheres to worms but also drastically changes the extent of hydration of the nanoparticle cores . based on the pisa literature , it was anticipated that vesicular morphologies should be obtained for these g37h60bz triblock copolymers as the target dp of the pbzma block was gradually increased . however , only branched worms and spheres were obtained when z = 47 ( see tem images in figure 2 ) . furthermore , both tem and dls studies indicated that only spheres were obtained when z 92 ( see figure 2 and table 1 , respectively ) . the spheres progressively increase in mean diameter from 45 to 120 nm as z was systematically varied from 92 to 550 , but vesicular morphologies were never obtained . hypothetically , the spherical morphology observed by tem might actually correspond to small vesicles . however , the saxs pattern recorded for the g37h60b186 triblock copolymer has a gradient that tends to zero at low q ( see figure s4b ) , indicating typical spherical particles rather than hollow spheres ( or vesicles ) . analysis of the g37h60b186 saxs pattern using a spherical micelle model gave an excellent data fit over six orders of magnitude of x - ray scattering intensity ( figure s4b ) . the saxs - derived mean sphere diameter ( ds ) was calculated to be 56.2 5.4 nm , which is similar to that reported by dls ( 63 nm , see table 1 ) . the structure factor peak observed in the saxs pattern at q 0.05 nm ( figure s4b ) suggests that the spheres are weakly aggregated . the percus yevick correlation radius of packed spheres ( rpy ) was obtained to be 50.5 nm . the tem images obtained for dispersions when z 92 also show that the spheres may be partially fused / weakly aggregated . however , the number - average diameter estimated from tem images recorded for g37h60b92550 triblock copolymer spheres corresponds quite closely to the hydrodynamic diameter obtained from dls studies ( see entries 69 in table 1 ) . although these results are somewhat counterintuitive when compared to most of the recent pisa literature , it is perhaps not too surprising that only spheres are obtained when targeting higher dps for the pbzma block . for example , cunningham et al . prepared a series of g51by diblock copolymer spheres via raft aqueous emulsion polymerization of bzma , with y ranging from 50 to 1000 . only spherical nanoparticles were obtained in all cases , regardless of the total solids content . in the present study , a weakly hydrophobic phpma block lies between the hydrophilic pgma and highly hydrophobic pbzma blocks , which allows triblock copolymer worms to be prepared for compositions containing just 31 mol % pbzma . however , targeting higher pbzma contents only leads to the formation of triblock copolymer spheres . the most likely explanation for these unexpected observations is that the pbzma block is enthalpically highly incompatible with the phpma block , whereas the phpma block is only rather weakly incompatible with the pgma block . thus , when the g37h60 diblock copolymer is chain - extended with bzma , at least some fraction of the partially hydrated phpma block is gradually driven out of the increasingly hydrophobic core to become co - located with the pgma stabilizer chains in the hydrophilic corona ( see the schematic cartoon shown in figure 3 ) . if this is the case , it would lead to an effectively longer stabilizer block , with a theoretical maximum dp of 97 ( i.e. , the sum of g37 and h60 ) . schematic cartoon to illustrate the conformational behavior of g37h60bz triblock copolymer chains as z is systematically increased . the packing parameter , p , is given by p = /aolc where v is the volume of the hydrophobic chains , ao is the optimal area of the head - group , and lc is the length of the hydrophobic tail . initially , the g37h60 diblock copolymer precursor chains are molecularly dissolved in the aqueous phase . for z 10 , the relatively short pbzma block induces nucleation , producing spherical micelles with mixed cores comprising the phpma60 and the pbzma10 blocks . for z 30 , the growing pbzma block leads to an increase in p , which drives a sphere - to - worm transition during pisa . when z 47 , the phpma block becomes at least partly co - located within the stabilizer corona layer , rather than the core . this is because the weakly hydrophobic phpma block is actually less enthalpically incompatible with the hydrophilic pgma block than with the highly hydrophobic pbzma block . this inevitably causes a reduction in p , which leads to a worm - to - sphere transition . a saxs pattern was collected for a 10% w / w aqueous solution of the g37 macro - cta ( i.e. , for molecularly dissolved chains below their overlap concentration ) . a satisfactory data fit was obtained for this pattern using a gaussian coil model , which indicated a rg of 1.7 nm ( see figure 4a ) . this is very close to the rg value for the stabilizer chains obtained from fitting the g37h60b28 saxs pattern using the worm model ( see table s1 ) . this suggests that all of the weakly hydrophobic phpma60 blocks are located within the core of the worms , while the hydrophilic pgma37 blocks occupy the worm corona . to test this hypothesis , the worm model was slightly modified ( see saxs models given in the supporting information ) by incorporating an additional fitting parameter ( ) corresponding to the volume fraction of the phpma block within the core domain . this parameter enables the volume of the core and corona to be determined , rather than fixing these values based on the known block compositions . by definition , if the whole phpma block is located within the core , should be equal to unity . in contrast , should be zero if the phpma block is solely located in the corona . a good data fit was obtained for the g37h60b28 saxs pattern using the modified worm model ( see figure 4b ) . the fitting parameters were similar to those obtained when using the unmodified , original worm model ( see table s1 ) . the rg for the g37 corona block of this triblock copolymer was determined to be 1.7 nm , which is identical to that obtained for the g37 macro - cta alone ( see figure 4a ) . moreover , tends toward unity , indicating that all of the phpma block is located in the worm core ( see figure 3 ) . saxs data ( open black circles ) and fits ( red lines ) for ( a ) a g37 macro - cta and dilute aqueous dispersions of ( b ) g37h60b28 triblock copolymer worms , ( c ) g37h60b186 triblock copolymer spheres , and ( d ) g92b28 diblock copolymer spheres . insets : schematic cartoons of the corresponding morphologies , where lw = the contour length of the worm , ww = width of the worm , rg = radius of gyration , ds = diameter of the sphere , and rpy = percus yevick correlation radius of densely packed spheres ( see table s1 ) . note : structural morphologies are not drawn to scale . saxs data were collected at ( a ) university of sheffield and ( b d ) diamond light source ( didcot , uk ) . a spherical micelle model was similarly modified by incorporating as an additional fitting parameter ( see saxs models and table s1 ) . analysis of the g37h60b186 spheres using this more sophisticated model gave a reasonably good data fit to the saxs pattern over six orders of magnitude of x - ray scattering intensity ( figure 4c ) . again , the fitting parameters were similar to those obtained when using the original unmodified sphere model ( see table s1 ) . however , the rg of the g37 corona block for this g37h60b186 triblock copolymer was determined to be 3.3 nm from this analysis , which is significantly larger than that obtained for the g37h60b28 worms . notwithstanding the imperfect data fit at high q , this indicates that the stabilizer corona is somewhat thicker in the former case , even though the same g37h60 diblock precursor was used for the pisa synthesis of the g37h60b28 and g37h60b186 triblocks . moreover , was found to be 0.62 , which suggests that a significant fraction of the phpma block is now located in the corona , rather than in the core ( see figure 3 ) . this provides direct experimental evidence for a higher effective dp for the corona block when targeting a longer pbzma core - forming block . for the g37h60b186 triblock copolymer spheres , saxs analysis indicates that around 23 hpma repeat units [ ( 1 0.62 ) 60 23 ] in each phpma block are located within the corona , while the remaining 37 repeat units occupy the core along with the pbzma chains . this increase in the effective stabilizer block dp leads to a reduction in the packing parameter , p , which in turn causes the observed worm - to - sphere transition ( see figure 3 ) . the driving force for relocating approximately one - third of the phpma block within the corona is the greater incompatibility within the phpma and pbzma blocks as the dp of the pbzma block is increased . in this context , mable et al . recently reported that systematically varying the pbzma block dp ( or z ) from 25 to 400 led to an evolution in framboidal morphology for a series of g63h350bz triblock copolymer vesicles . thus it is not really surprising that enthalpic demixing between the phpma and pbzma blocks leads to a dramatic change in morphology in the present work . in summary , saxs provides useful insight into the unusual ( and at first sight counterintuitive ) evolution in copolymer morphology for this particular pisa formulation , which can be rationalized by considering subtle changes in the relative enthalpic incompatibilities between the three blocks during the growth of the pbzma core - forming block . in order to examine whether the intermediate phpma block is really essential for worm formation , a g92b28 diblock copolymer was synthesized via raft aqueous emulsion polymerization of bzma using a g92 macro - cta ( see figure 1b ) . the g92 block was designed to have a comparable dp to that of the combined dp of the g37 and h60 blocks , while a pbzma dp of 30 was targeted because this produced worms for the abc triblock formulation . h nmr studies indicated that 94% bzma conversion was achieved after 4 h at 70 c ( see figure s5 ) . gpc studies indicated that a low - polydispersity diblock copolymer was obtained with a high blocking efficiency and minimal macro - cta contamination ( mw / mn = 1.14 ; see figure s6 and table 1 ) . tem images confirmed the formation of very small spheres of around 11.3 2.5 nm diameter ( based on analyzing 100 nanoparticles ) with no evidence for the presence of any worms ( see figure 2 ) . saxs analysis confirmed that spheres are indeed formed because the gradient of the saxs pattern tends to zero in the low q region , which is characteristic of spheres . analysis of this saxs pattern using a star - like micelle model provided a satisfactory data fit over five orders of magnitude of x - ray scattering intensity ( see figure 4d ) . the mean sphere diameter , ds , was calculated to be 21.0 1.4 nm , which is comparable to that suggested by dls , while the rg of the g92 corona block for this g92b28 diblock copolymer was determined to be 3.0 nm . this experimental value is larger than the theoretical estimate ( where rg was calculated to be 2.45 nm ) due to the star - like nature of the spheres . the spherical core diameter was determined to be 9.0 1.4 nm , which is comparable to that estimated from tem images . the correlation radius for densely packed spheres , rpy , was determined to be 19.3 nm . this is simply a consequence of the star - like nature of the former copolymer , which has a much higher effective volume fraction and hence a significantly lower critical overlap concentration . there is a pronounced upturn in the x - ray scattering intensity at low q ( below 0.017 nm ; see figure 4d ) . this could indicate the formation of aggregates ( or mass fractals ) most likely due to the extensive overlap between stabilizer chains of the micelles . the formation of spherical star - like micelles by this g92b28 diblock copolymer suggests that an intermediate phpma block is an essential prerequisite for obtaining a worm morphology . a reasonable explanation for this unexpected observation is outlined in figure 3 . recently , thompson et al . reported that g45h200 diblock copolymer vesicles were unstable with respect to dissociation when used as a pickering emulsifier . however , chemical cross - linking of such vesicles using ethylene glycol dimethacrylate as a third block dramatically improved their stability toward high - shear homogenization : tem studies of dried emulsion droplets confirmed that such covalently stabilized vesicles were adsorbed intact at the oil water interface . more recently , thompson et al . reported that g45h140 diblock copolymer worms similarly could not withstand high - shear homogenization , whereas g37h60b30 triblock copolymer worms proved to be highly efficient pickering emulsifiers . moreover , dls studies showed that the former worms were thermoresponsive , as expected based on previous work by verber et al . in contrast , the g37h60b30 triblock copolymer worms were not thermoresponsive ; this indicates that introducing the more hydrophobic pbzma block stabilizes the worm morphology . in the present study , we have used raft aqueous dispersion polymerization ( see figure 1a ) to prepare g37h90 diblock copolymer worms , which were designed to be analogous to the g37h60b28 triblock copolymer worms . h nmr studies confirmed that more than 99% hpma conversion was achieved after 2 h at 70 c ( see figure s7 ) . gpc studies indicated that a near - monodisperse diblock copolymer was obtained with a high blocking efficiency and minimal macro - cta contamination ( mw / mn = 1.11 ; see figure s8 and table 1 ) . dls studies ( see table 1 ) and tem images ( see figure 2 ) were consistent with the targeted pure worm morphology . rheology experiments for the g37h60b28 triblock copolymer worm gel were performed at 1.0% strain using an angular frequency of 1.0 rad s ( see figure s9 ) . figure 5 shows the minimal change in gel moduli for this dispersion during a 25 c to 2 c to 25 c thermal cycle . these g37h60b28 worms proved to be non - thermoresponsive , with a g of approximately 400 pa being maintained over the entire temperature range . variation of storage moduli ( g , red ) and loss moduli ( g , blue ) for a g37h60b28 triblock copolymer worm gel at 13% w / w during temperature cycling at 1 c min with 5 min equilibration at each temperature : ( i ) cooling from 25 to 2 c ( g = open red squares , g = open blue squares ) and ( ii ) subsequent warming from 2 to 25 c ( g = red crosses , g = blue crosses ) . inset : digital image of the worm gel at 20 c during the tube inversion test . measurements conducted using oscillatory mode at 1 rad s angular frequency and 1% strain amplitude . incorporating the highly hydrophobic pbzma block enables the g37h60b28 worms to act as an effective pickering emulsifier . previously , we reported that g37h60b28 worms can survive the high - shear homogenization conditions required for emulsification , whereas g45h140 worms undergo dissociation to form individual copolymer chains under these conditions . in the present study , we examined homogenization under much lower shear conditions , i.e. , hand - shaking . more specifically , both g37h60b28 and g37h90 worms were evaluated as putative pickering emulsifiers for the stabilization of n - dodecane emulsion droplets in water . aqueous worm dispersions ( 1.88 10 to 1.00% w / w ) were hand - shaken with 20 vol % n - dodecane for 2 min at 20 c to produce emulsions . in order to examine whether the worms were adsorbed intact at the oil water interface , optical microscopy ( om ) and laser diffraction were used to determine the mean oil droplet diameters ( see figure 6 ) . according to om studies , the oil droplets became larger as the g37h60b28 worm concentration was lowered , as shown in figure 6a . these observations were corroborated by laser diffraction studies : the mean oil droplet diameter increased from 115 to 483 m as the worm dispersion concentration was reduced from 1.00 to 0.0075% w / w ( see figure 6c ) . this concentration - dependent behavior is consistent with the formation of genuine pickering emulsions ( see figure 7 ) . recently reported that such triblock copolymer worms can withstand high - shear homogenization , so they should also survive low - shear homogenization . it is worth emphasizing that the mean oil droplet diameters are much larger when using hand - shaking for emulsification ( approximately 115 m at 1.00% w / w ) compared to those obtained using high - shear homogenization ( approximately 45 m at 1.00% w / w ) . when the worm dispersion concentration was lowered to 1.88 10 % w / w , the oil droplets proved to be too unstable to be assessed by laser diffraction . however , the droplet diameter was estimated ( from digital photographs recorded immediately after emulsification ) to be 1.11 0.42 mm ( based on measuring 120 droplets ) . droplet coalescence occurs within a few hours , but reconstitution of the original emulsions could be achieved via further hand - shaking . this differs from the highly stable millimeter - sized emulsions prepared using partially hydrophobized silica particles reported by arditty et al . the instability observed in the present work suggests that worm desorption occurs ; similar observations have been recently reported by rizelli and co - workers for worm - stabilized pickering non - aqueous emulsions . in contrast , it is emphasized that the finer o / w emulsions prepared at higher worm concentrations ( 0.03% w / w ) remain stable indefinitely . optical microscopy images obtained for n - dodecane - in - water emulsion droplets prepared using either ( a ) g37h60b28 or ( b ) g37h90 worms under low - shear conditions ( i.e. hand - shaking ) . ( c ) plots of mean droplet diameter ( obtained by laser diffraction ) vs worm concentration for emulsions prepared by hand - shaking dilute aqueous dispersions of g37h60b28 worms ( red , ) and g37h90 worms ( blue , ) with 20 vol % n - dodecane . schematic illustration of the attempted formation of pickering emulsions using either g37h90 or g37h60b28 worms under low - shear conditions ( i.e. , hand - shaking ) . remarkably , both om and laser diffraction studies indicated that the mean oil droplet diameter remained relatively constant on lowering the concentration of the g37h90 worms ( figures 6b and 6c ) . this indicates that these linear worms are so delicate that they can not survive even low - shear hand - shaking . instead , dissociation to form individual g37h90 copolymer chains occurs , which then adsorb at the oil water interface to stabilize the oil droplets ( see figure 7 ) . again , mean oil droplet diameters were significantly larger ( 136 m ) than those reported previously when using high - shear homogenization ( 45 m ) . for emulsions stabilized using either g37h90 or g37h60b28 worms , creaming of the low - density oil droplet phase occurred on standing for 24 h at 20 c . the lower aqueous phase , which contained excess non - adsorbed copolymer , was carefully removed and analyzed by dls to examine whether the worms remained intact after hand - shaking . dls studies of the g37h90 aqueous phase indicated a hydrodynamic diameter of 41 nm ( polydispersity = 0.18 ) and a much lower count rate ( 2500 kcps ) than that observed for the original worms ( 37 000 kcps ) . this 93% reduction in count rate is fully consistent with substantial worm dissociation occurring during hand - shaking . in contrast , dls studies of the aqueous phase removed from the g37h60b28-stabilized emulsion indicated an apparent hydrodynamic diameter of 153 nm , a polydispersity of 0.23 , and count rate of 21 000 kcps , which are comparable to the dls data obtained before emulsification . these observations confirm that these g37h60b28 worms remain intact after emulsification via hand - shaking . finally , closely related emulsions were prepared using n - hexane instead of n - dodecane to enable more convenient removal of the oil phase via evaporation at ambient temperature . figure 8 shows tem images obtained from emulsions prepared using the g37h60b28 and g37h90 worms . in the latter case , the surface of the dried emulsion droplet is smooth and featureless , with no evidence for any adsorbed nanoparticles ( see figure 8a ) . similar tem observations were reported for both g45h150 diblock copolymer worms and g45h200 diblock copolymer vesicles in previous studies of shear - induced dissociation of diblock copolymer nano - objects . in contrast , the dried emulsions prepared using the g37h60b28 worms clearly comprise intact worms adsorbed at the oil water interface ( see figure 8b ) . thus all the experimental evidence suggests that , regardless of their morphology , gxhy nanoparticles are not sufficiently robust to survive emulsification under any conditions , even low - shear hand - shaking . however , incorporating highly hydrophobic pbzma as a third block produces much more robust linear worms that can withstand high - shear homogenization and allow the formation of millimeter - sized emulsion droplets . tem images obtained for n - hexane - in - water emulsion droplets dried at 20 c using ( a ) 0.25% w / w g37h90 diblock copolymer worms and ( b ) 0.25% w / w g37h60b28 triblock copolymer worms . the edge ( blue ) and top surface ( red ) of the dried emulsion droplets are shown at higher magnification on the right - hand side . no worms are visible when using the g37h90 diblock copolymer since this undergoes dissociation even during low - shear homogenization ( hand - shaking ) . in contrast , worms are clearly discernible when using the g37h60b28 triblock copolymer , indicating that a genuine pickering emulsion had been obtained . a series of pgma phpma pbzma triblock copolymer nano - objects have been prepared in concentrated aqueous solution via polymerization - induced self - assembly ( pisa ) . for certain triblock compositions , highly anisotropic worm - like nanoparticles can be obtained with a mean contour length of 653 nm as determined by saxs studies . surprisingly , chain extension of the hydrophobic core - forming block of these worm - like nanoparticles does not lead to vesicle formation , with spherical micelles being formed instead . saxs studies shed some light on these unexpected observations , which are best explained by considering changes in the relative enthalpic incompatibilities between the pgma , phpma and pbzma blocks during the in situ growth of the latter block . in particular , saxs data fits suggest that the effective rg of the corona block actually increases as the pgma phpma diblock copolymer is chain - extended with bzma , even though the same diblock precursor was used for all copolymer syntheses . thus , at least some fraction of the partially hydrated phpma blocks must be gradually driven out of the increasingly hydrophobic core to become co - located with the hydrophilic pgma stabilizer chains within the corona . saxs analysis suggests that approximately one - third of the hpma repeat units are displaced from the particle cores via this mechanism . this counterintuitive finding highlights the subtle switch from weak to strong segregation between incompatible blocks and its hitherto unappreciated effect on the evolution in copolymer morphology during pisa . finally , the pgma phpma pbzma triblock copolymer worms were evaluated as pickering emulsifiers for n - dodecane oil droplets in water . unlike the pgma phpma diblock copolymer worms reported previously , these triblock worms do not exhibit thermoresponsive behavior . however , they are much more robust when subjected to high - shear , which makes them much more effective pickering emulsifiers . low - shear emulsification ( hand - shaking ) enables the formation of metastable millimeter - sized oil droplets . remarkably , the linear pgma - phpma worms do not survive such mild shear conditions . benzyl methacrylate ( bzma ) , n - dodecane , 2-cyano-2-propyl benzodithioate ( cpdb ) , and 4,4-azobis-4-cyanopentanoic acid ( acva ) were purchased from sigma - aldrich ( uk ) . bzma inhibitor was removed by passing this monomer through an inhibitor removal column . ethanol , dichloromethane , dmso , and dmf were purchased from fisher scientific ( uk ) . glycerol monomethacrylate ( gma ) was kindly donated by geo specialty chemicals ( hythe ) and used without further purification . 2-hydroxypropyl methacrylate ( hpma ) was purchased from alfa aesar ( uk ) and contained 0.07 mol % dimethacrylate impurity , as judged by high performance liquid chromatography ( hplc ) . deuterated methanol ( d4-cd3od ) , dimethyl sulfoxide ( d6-dmso ) , and dimethylformamide ( d7-dmf ) nmr solvents were purchased from goss scientific ( uk ) . deionized water was obtained using an elga elgastat option 3a water purifier ; its ph was approximately 6.2 , and its surface tension was 72.0 mn m at 20 c . the g37 macro - cta was synthesized by dr vincent ladmiral and the g92 macro - cta was synthesized by rheanna perry , following previously reported protocols . g37 macro - cta ( 5.00 g , 0.813 mmol ) , hpma monomer ( 7.04 g , 48.8 mmol ) , deionized water ( 68.6 g ) , and acva ( 76.0 mg , 0.271 mmol , cta / acva molar ratio = 3.0 ) were weighed into a 100 ml round - bottomed flask and purged with n2 for 30 min prior to immersion in an oil bath set at 70 c for 2 h. finally , the polymerization was quenched by cooling to room temperature with subsequent exposure to air . protocol for g37h60b30 triblock copolymer worms : g37h60 diblock copolymer precursor ( 8.00 g of a 10% w / w copolymer solution , 1.00 g of copolymer , 0.0541 mmol ) , acva ( 3.03 mg , 0.0108 mmol , cta / acva molar ratio = 5.0 ) , and bzma monomer ( 0 . 286 g , 1.62 mmol , target dp = 30 ) were weighed into a 25 ml sample vial and purged with n2 for 20 min prior to immersion in an oil bath set at 70 c for 4 h. the polymerization was quenched by cooling to room temperature and subsequent exposure to air . a series of similar copolymer syntheses were performed for which the pbzma target dp ranged from 10 to 550 using bzma masses varying from 0.0953 to 5.23 g ( 0.541 to 29.7 mmol ) , respectively , with the copolymer solids concentration increasing from 11 to 46% w / w . g37 macro - cta ( 1.00 g , 0.163 mmol ) , hpma monomer ( 2.11 g , 14.6 mmol ) , deionized water ( 20.9 g ) , and acva ( 15.1 mg , 0.0542 mmol , cta / acva molar ratio = 3.0 ) were weighed into a 50 ml round - bottomed flask and purged with n2 for 30 min prior to immersion in an oil bath set at 70 c for 2 h. finally , the polymerization was quenched by cooling to room temperature with subsequent exposure to air . g92 macro - cta ( 0.5 g , 0.0334 mmol ) , bzma monomer ( 0.177 g , 1.00 mmol ) , deionized water ( 4.55 g ) , and acva ( 3.12 mg , 0.011 mmol , cta / acva molar ratio = 3.0 ) were weighed into a 25 ml vial and purged with n2 for 30 min prior to immersion in an oil bath set at 70 c for 4 h. finally , the polymerization was quenched by cooling to room temperature with subsequent exposure to air . either n - dodecane or n - hexane ( 20 vol % ) was shaken by hand with 2.0 ml of a 0.001881.0% w / w aqueous worm dispersion for 2 min at 20 c . the droplets were imaged by om , and the mean droplet diameter was determined by laser diffraction . all nmr spectra were recorded using a 400 mhz bruker avance-400 spectrometer , and 64 scans were averaged per spectrum . copolymer molecular weights and polydispersities were determined using a dmf gpc setup operating at 60 c and comprising two polymer laboratories pl gel 5 m mixed c columns connected in series to a varian 390 lc multidetector suite ( only the refractive index detector was utilized ) and a varian 290 lc pump injection module . the gpc eluent was hplc grade dmf containing 10 mm libr at a flow rate of 1.0 ml min . calibration was conducted using a series of ten near - monodisperse poly(methyl methacrylate ) standards ( mn = 625618 000 g mol ) . the chromatograms were analyzed using varian cirrus gpc software ( version 3.3 ) provided by the instrument manufacturer ( polymer laboratories ) . intensity - average hydrodynamic diameters of the copolymer dispersions were determined using a malvern zetasizer nanozs instrument . dilute aqueous dispersions ( 0.10% w / w ) were analyzed using disposable cuvettes , and all data were averaged over three consecutive runs to give the hydrodynamic diameter ( dh ) . as - synthesized copolymer dispersions were diluted at 20 c to generate 0.10% w / w dispersions . copper / palladium tem grids ( agar scientific ) were surface - coated in - house to yield a thin film of amorphous carbon . the grids were then plasma glow - discharged for 30 s to create a hydrophilic surface . individual samples of aqueous copolymer dispersions ( 0.1% w / w , 12 l ) were adsorbed onto the freshly glow - discharged grids for 20 s and then blotted with filter paper to remove excess solution . to stain the copolymer dispersions , uranyl formate ( 0.75% w / v ) solution ( 9 l ) was soaked on the sample - loaded grid for 20 s and then carefully blotted to remove excess stain . imaging was performed on a phillips cm100 instrument at 100 kv , equipped with a gatan 1 k ccd camera . the emulsion was shaken and a sample ( 12 l ) was adsorbed onto the freshly glow discharged grid . the grids were not blotted with filter paper to remove excess dispersion instead , the hexane oil droplet evaporated after several minutes at ambient temperature . saxs patterns were recorded at diamond light source ( station i22 , didcot , uk ) . a monochromatic x - ray radiation ( of wavelength = 0.1239 nm ) and 2d saxs detector ( pilatus 2 m ) were used for the experiment . the saxs camera length setups covered the q range from 0.02 to 1.9 nm , where q = 4 sin / is the modulus of the scattering vector and is half of the scattering angle . a glass capillary cell of 1 mm thickness was used as the sample holder . x - ray scattering data were reduced by dawn software and were further analyzed using irena sas macros for igor pro . saxs measurements were conducted on various aqueous dispersions , for which the copolymer concentration was diluted to 1.0% w / w for data collection . a scattering pattern of the homopolymer solution was collected using a laboratory saxs instrument ( a modified bruker axs nanostar equipped with a microfocus genix 3d cu k radiation x - ray source and a collimator composed of two sets of motorized scatterless slits by xenocs , a camera length of 1.46 m , and a 2d histar multiwire gas detector ) ; glass capillaries of 2 mm diameter were used as a sample holder . an ar - g2 rheometer ( ta instruments ) equipped with a variable temperature peltier plate and a 40 mm 2 aluminum cone was used for all experiments . percentage strain sweeps and angular frequency sweeps were conducted at 25 c using a constant percentage strain of 1% and a constant angular frequency of 1 rad s , respectively . the storage modulus and loss modulus were measured as a function of temperature at a fixed percentage strain ( 1% ) and angular frequency ( 1 rad s ) . optical microscopy images were recorded using a motic dmba300 digital biological microscope with a built - in camera and equipped with motic images plus 2.0 ml software . a malvern mastersizer 2000 instrument equipped with a small volume hydro 2000sm sample dispersion unit ( ca . 50 ml ) , a hene laser operating at 633 nm , and a solid - state blue laser operating at 466 nm was used to size each emulsion . the stirring rate was adjusted to 1000 rpm in order to avoid creaming of the emulsion during analysis . after each measurement , the cell was rinsed once with ethanol , followed by three rinses with doubly distilled water ; the glass walls of the cell were carefully wiped with lens cleaning tissue to avoid cross - contamination , and the laser was aligned centrally to the detector prior to data acquisition . the volume - average diameter was measured and repeated four times for each emulsion .

it has long been established that t cells are mediators of the pathology of multiple sclerosis ( ms ) in both murine models and patient studies [ 16 ] . although , the impact of b cells and their antibody products in mediating the pathology of ms has long been considered [ 710 ] , their contributions have been more recently highlighted by the demonstration that rituximab , an anti - cd20 monoclonal antibody that specifically depletes b cells , was a potent immunomodulatory therapy for the treatment of ms [ 11 , 12 ] . more importantly , however , the efficacy of rituximab in the treatment of ms patients is independent of secreted antibody since rituximab does not affect plasma cell frequencies or serum and cerebrospinal fluid ( csf ) antibody levels . thus , scientists in the field have refocused their attention on the possible roles of b cells in ms that are independent of their antibody secreting function . this paper summarizes the possible antibody secretion - independent roles of b cells on t cell activation and regulation , the relative impact of the b cell subpopulations on t cell activation and regulation , evidence that these mechanisms are altered in ms , and how current and emerging immunotherapies may impact b - t cell interactions in ms . it has long been assumed that b cells are unlikely to play a significant role as antigen - presenting cells ( apcs ) in the induction of effector t cells since human b cells are less potent apcs in vitro on a per - cell basis compared to dendritic cells . however , in 1982 , investigators published for the first time that human b cells could present antigens . in fact , b cells are potent apcs in humans in vitro in the context of both alloantigen [ 16 , 17 ] and exogenous - foreign - antigen responses . studies in mouse models in which the b cells can not secrete antibodies have further highlighted the importance of antibody independent b cell responses [ 19 , 20 ] . these results demonstrated that b cells are required for generating optimal primary and secondary t cell responses and are implicated as apc in a number of disease models in the mouse including rheumatoid arthritis and type 1 diabetes [ 2123 ] . more recently , it has been demonstrated that activated b cells are more effective in activating t cells than their resting or nave counterparts in the mouse [ 2426 ] . this finding has been confirmed with human b cells as well , since human nave b cell alloantigen presentation can be increased with cpg - odn stimulation . antigen - specific b cell apc function can also be increased with cd40l stimulation [ 2731 ] . the most well - studied consequence of b - t cell interactions , however , is the induction of t cell tolerance or expansion of regulatory t cells [ 3234 ] . for example , in mice , antigen specific nave b cells induce nave t cells to proliferate and differentiate into regulatory t cells . hel - specific cd43 ( nave ) b cells do not elicit t cell proliferation or il-2 and interferon - gamma ( ifn ) secretion , suggesting that they induce t cell tolerance . in mouse models , nave b cells did not participate in t cell priming in vivo , implicating nave b cells as a source of regulatory b cells [ 25 , 37 , 38 ] . however , few have examined the role of memory b cell antigen presentation in humans , which is a crucial consideration since memory b cells are antigen experienced . our group demonstrated that memory b cells from healthy donors express sufficient levels of cd80 and hla - dr to operate as antigen presenting cells to induce antigen specific t cell proliferation and ifn secretion . our group has also found that memory b cells from these same healthy donors also produce high concentrations of lymphotoxin- alpha ( lt ) in comparison to their nave counterparts . another group demonstrated that memory b cells from healthy donors induce t cell proliferation in response to glatiramer acetate ( ga ) , a peptide mimic of the neuro - antigen , myelin basic protein ( mbp ) , with some indication that cd80 expression is important for this function . in a separate study , it was demonstrated that resting memory b cells from healthy donors elicit robust allogeneic t cell proliferation that was significantly inhibited by anti - cd80 and anti - cd86 antibodies in contrast to nave b cells , which were unable to stimulate allogeneic t cell proliferation . with the exception of these three studies , memory b cell antigen presentation in healthy donors has not been explicitly examined . memory b cells from treatment nave rrms patients , however , have the unique ability to present neuroantigens to autologous t cells and generate a proliferative response and ifn production . memory b cells from healthy donors are unable to support t cell proliferation and ifn production in response to neuroantigens . taken together it appears that memory b cells might directly contribute to t cell activation by presenting neuro - antigens and secreting cytokines that enhance the th1/ifn producing t cell subset . we are currently testing whether memory b cells from treatment - exposed rrms patients maintain their capacity to incite t cell activation in a neuroantigen specific manner . these findings have generated considerable interest in dissecting the mechanism of b - t cell interactions , especially as they relate to the antigen experience of b cells . the two primary antibody secretion independent ways that b cells potentially impact t cell activation or regulation are by ( 1 ) providing costimulatory signals through direct b - t cell interactions and ( 2 ) cytokine secretion . the following sections detail the primary surface molecules and secreted factors that are thought to contribute to t cell activation or regulation by b cells . figure 1 depicts aspects of the currently accepted model of important b - t cell interactions [ 40 , 41 ] . interactions between b7.1/b7.2 ( cd80/cd86 ) expressed on b cells and their ligands and cd28 and ctla4 expressed on t cells have long been studied in ms [ 42 , 43 ] , and much is understood regarding the influence of these interactions on t cell responses [ 4446 ] . in fact , circulating cd80 + b cells are increased during active relapse phases of ms , compared to patients in remission or controls , suggesting that this population may be involved during the active phase of the disease . importantly , others have demonstrated that a subset of memory b cells , but not nave b cells , express cd80 [ 16 , 18 ] cd86 , and cd25 , although this expression is not necessarily concurrent [ 18 , 48 ] . the impact of cd80 expression on b cells and their ability to activate t cells is certainly of interest since the stimulation of memory t cells is cd28 independent . interactions between mhcii , peptide , and the tcr are certainly central to effective b - t cell costimulation , especially in the context of ms , in which hla - dr haplotypes have a strong association [ 5052 ] . t cell proliferation in the presence of cd19 + b cells ( containing both nave and memory b cells ) activated with cd40l and antigen is reduced by ~60% in the presence of anti - hla - dr antibodies . interactions between cd40 expressed on b cells and cd40 ligand ( cd40l ) expressed on t cells are also critical to mount an effective t cell response . this consistent observation led to the development of anti - cd40l biologics to dampen t cell responses but became problematic due to the increase in thromboembolic events associated with these biologics , and the discovery that activated platelets express cd40l [ 54 , 55 ] . anti - cd40 agonists , however , are being developed for applications in tumor immunotherapy to mobilize t cells in patients with cancer . yet it is unclear what types of t cells are mobilized by cd40-activated b cells . this is a critical point to consider since purified b cells stimulated with one of these new cd40 agonists in combination with cpg ( a tlr9 agonist ) secreted high levels of both il-6 and il-10 [ 57 , 58 ] . interactions between ox40l expressed on apcs and ox40 expressed on t cells have profound effects on t cell responses . have reported that co - culturing ifn-activated microglia from mouse brain tissue with murine cd4 + t cells significantly increased t cell proliferation , which was readily blocked by the addition of anti - ox40l in the cultures . in humans , exposure of human t cells to ox40l prevents the development of il-10-producing t cells and il-17 producing t cells . thus , biologics that block or enhance these interactions may be useful therapeutics for autoimmunity and infection , respectively , and are currently being considered for development in these arenas . however , the impact of ox40-ox40l ligation on b - t cell interactions has not been formally investigated . the influence of t cell derived cytokines in health and disease has dominated scientific literature for at least 2 decades . however , it has become apparent that activated b cells have a high capacity to produce both inflammatory and regulatory cytokines ( table 1 ) . for example , recent investigations have demonstrated that il-10-producing b cells have an immunomodulatory role in experimental allergic encephalomyelitis ( eae ) , which is the mouse model of ms [ 93 , 94 ] . however , cd19 + b cell pools from ms patients have a diminished capacity to produce il-10 [ 95 , 96 ] , suggesting that the inflammatory responses in ms patients may be partially attributable to a defect in il-10 production by b cells . in vitro activation of purified nave and memory b cells from the rrms patient cohort used in our studies using cd40l ( as was done by ) , demonstrates that nave b cells produced similar amounts of il-10 reported by [ 66 , 95 , 97 ] , but memory b cells did not produce appreciable amounts of il-10 . interestingly , when we cultured nave b cells with t cells in our b - t cell culture system , we did not observe appreciable t cell proliferation in response to neuro - antigens , suggesting that the amount of il-10 produced by nave b cells from rrms patients may regulate t cell responses . in support of this concept , il-10 treatment of cd4 t cells from patients with rheumatoid arthritis significantly decreased the numbers of th17 cells in vitro . so what pro - inflammatory cytokines do b cells generate that have a high likelihood of effecting t cell function in response to neuro - antigens ? lt and tumor necrosis factor alpha ( tnf- ) are classic candidates of the tnf superfamily with similar structure [ 99 , 100 ] . their expression by b cells is required for germinal center formation and the migration of antigen - loaded myeloid dendritic cells ( mdcs ) to follicles . lt is also required for the generation of memory b cells and isotype switch . lt and tnf- are readily produced by activated memory b cells from healthy donors , and t cells express receptors for these cytokines . furthermore , tnf and lt levels are increased in ms lesions and mediate oligodendrocyte toxicity in vitro . b cells from rrms patients showed increased lt and tnf and decreased il-10 production in response to polyclonal stimuli ( cd40l and bcr crosslinking ) in the presence of a tlr9 agonist or ifn . in fact , when cd19 + cells are removed in vitro from peripheral blood mononuclear ( pbmc ) cultures , cd4 + and cd8 + t cell proliferation is decreased and may be due to a lack of b cells secreting lt and tnf , which would support t cell proliferation . transforming growth factor - beta ( tgf- ) and il-6 are readily secreted by activated b cells as well , and there is certainly much excitement regarding the ability of tgf and il-6 in combination to mediate th17 cell activity since th17 cells are central to the inflammation associated with eae [ 1 , 109114 ] . however , the classic role of tgf has been to induce tolerance in the immune system , and indeed , anti - tgf treatment of mice led to worsening of eae disease and more severe clinical pathology in these mice . tgf has been attempted ineffectively as a treatment for progressive ms . in vitro recombinant tgf decreased neuroantigen - specific t cell frequency and ifn and il-4 producing t cells from ms patients . more recently , others have demonstrated that murine th17 cells can be generated by either il-6 alone or il-6 in combination with tgf , but only those th17 cells that had been generated with il-6 alone are encephalitogenic . il-6 is increased in the csf of other inflammatory neurological disorders including transverse myelitis ( tm ) and neuromyelitis optica ( nmo ) [ 118 , 119 ] , in the lesions of ms patients , and in some cases , correlates with relapse or general neurodegeneration in the progressive phase . however , il-6 is not increased in the csf of ms patients and il-6 levels do not correlate with other csf parameters including oligoclonal bands , pleocytosis or igg index [ 118 , 122 , 123 ] . whether il-6 is central to the generation of human th17 cells remains under investigation [ 124 , 125 ] . even with confounding and often negative results of other studies in ms , it has been suggested to use the il-6 receptor blocking antibody , tocilizumab , as a therapeutic agent . interestingly , il-6 is increased after treatment of ms patients with ifn , suggesting that this pleiotropic cytokine might not play an inflammatory pathogenic role in ms as il-6 can have both systemic and local effects . because this cytokine differs in its necessity for eae , it might represent an insurmountable task to go to trial due to the uncertain / unpredictable nature of the outcome without predictive animal models . as discussed earlier in this paper , t cells from rrms patients treated with rituximab had a poor response to antigen stimulation , suggesting that the lack of b cells in the periphery of these patients has a profound impact on t cell activation . interestingly , circulating memory b cells are also reduced in rrms patients during mitoxantrone therapy and ifn therapy . ifn treatment decreases mbp - elicited ifn and tnf , but also decreases il-10 and increases il-6 . another study demonstrated that ifn increases the expression of a phosphatase ( shp-1 ) that negatively regulates inflammatory t cell signaling from tnf , ifn , il-4 , and il-13 . from these results , one may speculate that the effects of ifn on t cells modulate their responsiveness to cytokine signals , some provided by b cells . on the other hand , in vitro studies show that th17 cells and associated cytokines ( il-23 , tgf and il-1 ) were reduced , while anti - inflammatory cytokines produced by b cells and dendritic cells were increased . reemerging b cells from rrms patients treated with mitoxantrone therapy recovered the ability to produce il-10 , but again , the impact of these il-10-secreting b cells on neuroantigen - specific t cell responses was not tested . table 2 lists other current and emerging therapies and their possible impact on b cell function and emphasizes the urgent need to investigate how these drugs may affect b - t cell interactions in the patients receiving them . in summary , the influence of b cells on t cell function is only beginning to be realized . further investigations are required in order to fully comprehend the impact of current and emerging therapeutics on b cell responses , which in turn , may have profound impact on t cell function in autoimmune diseases such as ms .

pain is a significant problem in cancer patients and is often the most feared aspect of the disease ; thus , effective analgesia is an essential component of pain management . cancer patients require a comprehensive pain treatment plan that not only addresses the moderate to severe background cancer pain ( bcp ) but also provides adequate management of breakthrough cancer pain ( btcp ) that has a separate and characteristically different presentation . btcp was initially defined by portenoy and hagen in 1989 as a transitory flare of pain in the setting of chronic pain managed with opioid therapy . according to more recent and comprehensive definitions , btcp is a transient exacerbation of pain that occurs either spontaneously or in relation to a specific predictable or unpredictable trigger , despite relatively stable and adequately controlled bcp . as there is a lack of consistency in use of the term btcp , most studies did not report the diagnostic criteria used in screening patients for btcp or included patients with inadequately controlled bcp . indeed , btcp is widely used to describe any exacerbation of pain in patients with bcp or intermittent episodes of pain in patients without bcp . btcp differs from bcp due to its high intensity ( numerical rating score [ nrs ] , 7 ) , the short time interval between onset and peak intensity ( median interval , 3 minutes to peak pain ) , short duration ( median , 30 to 60 minutes ) , potential recurrence over 24 hours ( 3 - 4 daily episodes ) , and non - responsiveness to treatments for bcp [ 3,6 - 9 ] . because patients with btcp are often less satisfied with their analgesic therapy , their functioning decreases due to pain , and they may also experience social and psychosocial consequences , such as increased anxiety and depression . according to a large european observation study and a canadian study , btcp impacts daily living including normal work , mood , sleep , and walking activities . despite the increasing awareness of the need for evaluation and management of btcp , only a few studies have investigated btcp in korea . we conducted this study to characterize the prevalence and treatments for btcp in korea , identify factors associated with the prevalence of btcp , and to show the impact of btcp on qol . this multicenter nationwide study was conducted from 11 september 2010 , to 14 october 2010 in 78 hospitals using a patient questionnaire and retrospective review of medical records . the survey instrument was developed by the palliative care committee of the korean cancer study group . inclusion criteria were hospitalized patients whose bcp is controlled to a mild degree ( nrs 3 or less ) , were receiving analgesic treatments , with a history of hospitalization for > 4 days . patients who could not voluntarily participate in the survey due to clouded consciousness or who did not want to participate were ineligible . patients completed a questionnaire including ( 1 ) average pain intensity for the last 24 hours ; ( 2 ) prevalence , frequency , and treatments for btcp ; ( 3 ) time interval between onset and treatment of breakthrough pain ; ( 4 ) sleep disorder ; and ( 5 ) satisfaction with pain treatment for the last week . patients medical charts were reviewed with respect to pain management until the date the patient entered the study . the following information was obtained from medical records : patient characteristics ( age , sex , diagnosis , stage , current treatment , and type of facility ) , eastern cooperative oncology group performance status ( ecog ps ) , and treatments for background pain and btcp . the patient s demographic and pain characteristics were summarized by a number of subjects ( percentage ) . association between the patient characteristics and presence of btcp was examined using the chi - square test . degrees of difference in patient s qol ( in terms of sleep disorder and satisfaction with pain control ) between patients with and without btcp were examined using crude odds ratios and 95% confidence intervals . cary , nc ) . a p - value of < 0.05 was considered significant . of the 1,841 patients with bcp , 496 ( 26.9% ) complained of high nrs pain , and 736 patients ( 40% ) complained of moderate nrs pain . the remaining 609 patients ( 33.1% ) who complained of pain 3 nrs were analyzed . most patients ( 86% ) were admitted to the general ward , and most were diagnosed with a solid cancer ( 87% ) . of all patients , approximately 80% of patients had stage iv cancer , and 84% were ecog ps < 3 ( table 1 ) . patients with btcp were more frequently admitted to the hospice ward and were less frequently treated with chemotherapy compared to those without btcp ( p=0.0397 and p=0.0642 , respectively ) ( table 1 ) . however , the results of multivariate analysis showed no significant predictors of btcp frequency ( table 2 ) . the frequency of btcp per day was 73 patients complained once , 62 complained twice , 25 patients complained three times , 11 patients complained four times , and six patients complained more than five times . patients with btcp 4.42 times more frequently complained sleep disorder , which caused patients to wake up two or more times during sleep , compared to those without btcp ( p < 0.0001 ) ( table 3 ) . and they were 1.74 times more unsatisfied with their pain control than patients who did not have btcp ( p=0.0498 ) ( table 3 ) . according to the patient questionnaire results , 77% of patients with btcp answered that they were treated with a short - acting analgesic during an attack . a total of 110 patients answered that they were treated with a short - acting analgesic within 10 minutes , 29 patients within 11 to 20 minutes , five patients within 11 to 20 minutes , three patients within 31 to 60 minutes , and six patients after 60 minutes . according to the medical records , 77 of 177 patients ( 44% ) with btcp were prescribed short - acting analgesics on the day of their enrollment in this study . thirty - six patients were treated with oxycodone - immediate release ( ir ) , 30 patients with morphine ( 29 intravenous morphine and one s - morphine ) , six patients with tramadol , three with hydromorphone , one with actiq , and one with acetaminophen . there was no clinical predictor for the reason why patients did not take and short - acting analgesics . a cognate short - acting analgesic , such as oxycodone - ir , was more frequently prescribed for patients treated with oxycodone as a long - acting analgesic ( p=0.0038 ) ( table 4 ) . of the 1,841 patients with bcp , 496 ( 26.9% ) complained of high nrs pain , and 736 patients ( 40% ) complained of moderate nrs pain . the remaining 609 patients ( 33.1% ) who complained of pain 3 nrs were analyzed . most patients ( 86% ) were admitted to the general ward , and most were diagnosed with a solid cancer ( 87% ) . of all patients , approximately 80% of patients had stage iv cancer , and 84% were ecog ps < 3 ( table 1 ) . patients with btcp were more frequently admitted to the hospice ward and were less frequently treated with chemotherapy compared to those without btcp ( p=0.0397 and p=0.0642 , respectively ) ( table 1 ) . however , the results of multivariate analysis showed no significant predictors of btcp frequency ( table 2 ) . the frequency of btcp per day was 73 patients complained once , 62 complained twice , 25 patients complained three times , 11 patients complained four times , and six patients complained more than five times . patients with btcp 4.42 times more frequently complained sleep disorder , which caused patients to wake up two or more times during sleep , compared to those without btcp ( p < 0.0001 ) ( table 3 ) . and they were 1.74 times more unsatisfied with their pain control than patients who did not have btcp ( p=0.0498 ) ( table 3 ) . according to the patient questionnaire results , 77% of patients with btcp answered that they were treated with a short - acting analgesic during an attack . a total of 110 patients answered that they were treated with a short - acting analgesic within 10 minutes , 29 patients within 11 to 20 minutes , five patients within 11 to 20 minutes , three patients within 31 to 60 minutes , and six patients after 60 minutes . according to the medical records , 77 of 177 patients ( 44% ) with btcp were prescribed short - acting analgesics on the day of their enrollment in this study . thirty - six patients were treated with oxycodone - immediate release ( ir ) , 30 patients with morphine ( 29 intravenous morphine and one s - morphine ) , six patients with tramadol , three with hydromorphone , one with actiq , and one with acetaminophen . there was no clinical predictor for the reason why patients did not take and short - acting analgesics . a cognate short - acting analgesic , such as oxycodone - ir , was more frequently prescribed for patients treated with oxycodone as a long - acting analgesic ( p=0.0038 ) ( table 4 ) . recent surveys have reported btcp prevalence of 28%-71% for in - patients with cancer . here , we reported prevalence of 29% among hospitalized cancer pain patients . this is a lower percentage than previously reported [ 12 - 14 ] , although hagen et al . two surveys conducted in 2001 and 2006 enrolled cancer pain patients who visited the outpatient clinic or were admitted in hospital . these also included patients with uncontrolled bcp , and patients with advanced stage cancer and poor ps . in comparison with previous studies , the current survey , conducted in 2010 , only enrolled hospitalized patients with controlled bcp , as the exact definition of btcp . the lowest prevalence rates were detected in studies conducted in out - patient clinics and the highest were reported in studies conducted in the hospice ward . hospitalized patients who maintained generally good ps and received chemotherapy another possible explanation for the lower prevalence of btcp in the current study is that patients might have recall bias . as a matter of fact , 77% of our patients answered that they have received short - acting analgesics for btcp but the medical records reported that only 44% of the patients were treated with short - acting analgesics . thus , patients with btcp may not have reported their pain because of difference between patients memory of pain and the medical record of the study day . in addition , as no validated btcp assessment tool was available at the time of this study , we could not reliably determine the prevalence of btcp only by asking patients to describe the frequency , intensity , and duration . reported that btcp impacts daily living activities of european and canadian patients and raj et al . reported that patients with btcp have more mood and sleep disturbances and are less able to keep up with normal work we also found that btcp was related to dissatisfaction with pain control and sleep disorders . however , there was no available clinical predictor to distinguish patients with and without btcp . thus , successful management of btcp depends on a combination of adequate assessment and appropriate treatment . a systematic review of the literature in 2010 identified 10 tools for assessment of btcp , seven of which were discussed but have not been made available and have only been used in one study . portenoy and hagen developed the breakthrough pain questionnaire ( bpq ) specifically for assessment of btcp . this tool assesses the severity , location , pathophysiology , cause , and precipitating and palliative factors for btcp , as well as its relationship to scheduled analgesic use through patient s self - report . although the bpq has been used in epidemiological and pharmaceutical studies , it has not been validated . hagen et al . developed the alberta breakthrough pain assessment tool ( abpat ) , using the delphi process , specifically for assessment of btcp . this tool was developed for research purposes and was formally validated for clinical use in 2014 . the episodic pain documentation sheet and a modified version of the original potenoy and hagen questionnaire were also developed for assessment of btcp . their advantages include easy administration , relatively rapid onset , and extensive experience in use . concordance was observed between oxycodone as a long - acting analgesic and oxycodone ir as a short - acting analgesic because physicians had more experience in prescribing oxycodone as an oral preparation and it could be easily titrated . many recent studies have suggested that buccal , sublingual , or oral / nasal transmucosal formulations of fentanyl are effective for btcp . fentanyl is a highly lipophilic synthetic opioid , which shows rapid diffusion across the blood brain barrier to elicit a rapid pain response . a recent study demonstrated that physicians are more aware of the benefits of fentanyl , as a higher proportion of patients are prescribed this agent for control of btcp . actiq was first available for use in korea in 2008 and fentora and abstral became available in 2014 . therefore , nowadays , with an established definition of btcp , different assessment tools , and many treatment options for btcp , this result did not coincide exactly with treatment of btcp . however , this study might be useful in showing the state of epidemiology and clinical significance of btcp and help support further studies in korea . this study had some limitations related to the methods used for classification of patients with btcp . the study was designed to describe the epidemiology and pain management strategy for patients with cancer in korea with special emphasis on the prevalence and treatment patterns for cancer pain in 2010 . therefore , this study did not report subtypes of btcp such as spontaneous or incidental type or the impact of btcp on daily living . in addition , we rely solely on patient self - reporting techniques , which can be inaccurate , as patients are not always specific when reporting their pain , and we did not use a btcp - specified assessment tool , as no validated assessment tool for btcp was available at the time of the study . neverthless , this study presents the first report about btcp in korea based on a valid definition of btcp . in summary , approximately 30% of hospitalized patients with adequately controlled bcp complained of btcp . unfortunately , there were no clinical factors predicting the presence of btcp , and btcp impacted qol of cancer patients with controlled bcp . therefore , physicians should perform more appropriate evaluation , and provide adequate management of btcp in cancer patients with controlled bcp . this is the first study to show the prevalence and clinical characteristics of btcp in korea based on a valid definition of btcp . further advances in btcp diagnosis and treatment , as well as knowledge of predictors will continue to inform us about the evolving , complex nature of cancer pain classification and management .

a rpbphp2 gene was kindly provided by eric giraud ( institute for research and development , france ) . ifp1.4 gene was de novo synthesized by genscript company , based on the available protein sequence . the dna sequence was optimized with proprietary optimumgene algorithm ( gensript ) , taking into account the codon usage bias ( human cells ) , gc content , cpg dinucleotides content , mrna secondary structure , and other parameters . the genes encoding a bacteriophytochrome rpbphp2 and an ifp1.4 protein were amplified using polymerase chain reaction ( pcr ) as the bglii - ecori or psti - hindiii fragments and inserted into the bglii - ecori sites of a pbad / his - b vector or the psti and hindiii sites of a pbad / his - c vector , respectively ( invitrogen ) . biliverdin synthesis in bacteria was facilitated by co - transformation with a ppl - bv plasmid , which was obtained after digestion with bamhi and subsequent ligation of the ppl - pcb plasmid . site specific mutagenesis was performed using a quickchange mutagensis kit ( stratagene ) . for simultaneous mutagenesis at several positions , including a site - specific saturated mutagenesis ( i.e. , all 20 amino acids were encoded using the mixture of primers ) , an overlap - extension method was applied . random mutagenesis was performed with a genemorph ii random mutagenesis kit ( stratagene ) , using conditions that resulted in the mutation frequency of up to 16 mutations per 1,000 base pairs . after the mutagenesis , a mixture of mutants was electroporated into the lmg194 host cells ( invitrogen ) containing the ppl - bv plasmid . lmg194 bacteria were grown overnight in lb medium with an addition of 2% glucose to repress synthesis of both proteins . next morning , the bacterial cells were centrifuged and re - suspended in rm medium with 0.002% arabinose , 0.001 mm iptg , 100 m 5-aminolevulinic acid ( ala ) , and 50 m fecl3 . the cells were then grown up to 24 hours either at 37c or 37c followed by 25c , depending on the experiment . for facs screening , protein mutants were expressed in bacteria at 37c followed by an overnight incubation at 25c . the next morning , bacteria were washed with phosphate buffered saline ( pbs ) and then diluted with pbs to an optical density of 0.02 at 600 nm . a moflo xdp cell sorter ( beckman coulter ) , equipped with the standard ar , kr , ar - kr mixed - gas lasers ( coherent ) and a 592 nm solid - state laser ( mpb communications ) , was used . typically , about 10 sizes of each library were facs - sorted , using a 676 nm kr laser line for excitation and a 700lp nm emission filter for positive selection . the brightest collected nir bacterial cells were rescued in a soc medium at 37c for one hour , and then plated on petri dishes made of rm / agar supplemented with ampicillin and kanamycin . the next day , the expression of proteins in bacterial colonies on the dishes were induced with 0.002% of arabinose and 0.001 mm of iptg . the dishes were left for 4 - 6 hours at 37c and then incubated at room temperature overnight . fluorescence of the colonies was analyzed the next day using a leica mz16f fluorescence stereomicroscope equipped with the 650/45 nm excitation and 690lp nm emission filters ( chroma ) , and a ccd camera . a mixture of several selected mutants was then utilized as a template for the next round of mutagenesis . rpbphp2 mutants and ifp1.4 protein with polyhistidine tags were expressed in lmg194 bacterial cells , as described above , and then purified using ni - nta agarose ( qiagen ) . the excitation and emission spectra were measured using a fluoromax-3 spectrofluorometer ( jobin yvon ) . for absorbance measurements , the first approach was based on a direct measurement of the protein concentrations with a bca protein essay kit ( pierce ) , followed by the calculation of extinction coefficients using a beer lambert bouguer equation . the second approach , which has been introduced in the original ifp1.4 paper , was based on a comparison of absorbance values for the protein at the main peak ( 692 nm or 694 nm ) with the absorbance value at the 391 nm peak assuming the latter to have the extinction coefficient of the free bv which is 39,900 mcm . for determination of quantum yield , fluorescence signal of irfp was compared to that of the equally absorbing ifp1.4 and nile blue dye ( quantum yield is 0.27 in an acidic ethanol ) . ph titrations were performed using a series of buffers ( 100 mm naoac , 300 mm nacl for ph 2.55.0 , and 100 mm nah2po4 , 300 mm nacl for ph 4.59.0 ) . to study protein maturation , lmg194 bacterial cells were grown at 37c overnight in a lb medium supplemented with ampicillin , kanamycin , and 2% of glucose . the next morning , the cells were centrifuged , resuspended , and cultured in rm medium with 0.002% arabinose , 0.001 mm iptg , 100 m ala , and 50 m fecl3 for 1 hour . then , the cells were washed and cultured in rm medium supplemented with 0.001 mm iptg , 100 m ala , and 50 m fecl3 ( no arabinose was added this time ) at 37c . fluorescence intensity of the cell suspension was measured every hour initially and after longer time periods later . a pshuttle - cmv vector ( stratagene ) was utilized to construct plasmids for mammalian expression of both irfp and ifp1.4 proteins . the fp genes were pcr - amplified and inserted into the bglii and sali sites of the vector , thus generating pshuttle - cmv - irfp and pshuttle - cmv - ifp1.4 plasmids . hek293 and hela cell lines were grown in dulbecco s modified eagle media ( dmem ) containing 10% fetal bovine serum ( fbs ) , penicillin - streptomycin , and 2 mm glutamine ( invitrogen ) . plasmid transfections were performed using an effectene reagent ( qiagen ) according to the manufacturer s protocol . for adenoviral infection , hela cells were grown in 60 mm petri dishes until ~80% confluence and then infected with 310 pfu per dish . for facs analysis of hela cells co - transfected with egfp and nir fps , 488 nm ar and 676 nm kr laser lines and the respective 530/40 nm and 700lp nm emission filters were used . to quantify cells fluorescence , a mean fluorescent intensity of the double - positive population in the nir channel was divided by a mean fluorescent intensity of the same population in the green channel , thus normalizing the nir signal to the transfection efficiency . cells were imaged using an olympus ix81 inverted epifluorescence microscope equipped with 200w metal halide arc lamp ( prior ) , a 100 1.4 na oil immersion objective lens ( uplansapo , olympus ) , and standard cy5.5 filter set ( 665/45 nm exciter and 725/50 nm emitter ) ( chroma ) . photobleaching measurements of cytoplasmically expressed nir fps in live hela cells were performed similarly to the purified proteins with the 100x objective substituted with a 60 1.35 na oil immersion objective lens ( uplansapo , olympus ) . normalization of the curves was made the same way as for the purified proteins . for protein degradation assay , hek293 cells transfected with nir fps were treated with 1 mm of puromycin 48 hours post - transfection . to achieve brighter fluorescence signal , cells were kept with 25 m of bv during the whole experimental course , starting 2 hours before the addition of the puromycin . to study bv binding and bv influence on protein stability , hela cells were harvested 2 days post adenoviral infection and lysed by 3 freezing - thawing cycles . the lysates were clarified by centrifugation , and the total fluorescence was measured using the fluoromax-3 spectrofluorometer ( jobin yvon ) . the adenovirus serotype v based particles containing the irfp or ifp1.4 genes were created using an adeasy xl adenoviral vector system ( stratagene ) according to the manufacture s recommendations and then purified using a cesium chloride gradient centrifugation , as described . the viral titers were assessed by an adeno - x rapid titer kit ( clontech ) . albino c57bl/6 mice ( female 6 - 8 weeks old , b6(cg)-tyr / j , jackson laboratory ) were intravenously injected with 210 infectious units of an adenovirus . belly fur was removed using a depilatory cream , and the mice imaging was performed with an ivis spectrum instrument ( caliper lifesciences ) in epifluorescence mode equipped with 675/30 nm and 720/20 nm filters for excitation and emission , respectively , starting the second day after an infection . 250 nmol of bv were iv injected on the day 5 post - infection , and the mice imaging started 1 hour later . quantitative measurements of fluorescence signal were made with a living image software 4.0 ( caliper lifesciences ) . for an ex vivo liver imaging , livers were dissected 1 hour after mice were injected with 250 nmol of bv . all animal experiments were performed in an aaalac approved facility using protocols approved by the albert einstein college of medicine animal usage committee . bacterial plasmids encoding several far - red gfp - like fps were kindly provided by benjamin glick ( university of chicago , usa ) and dmitry chudakov and konstantin lukyanov ( both from institute of bioorganic chemistry , russia ) . the recombinant gfp - like fps were expressed in the lmg194 bacterial cells and then purified using the ni - nta agarose ( qiagen ) . the purified fps were diluted to the equal concentrations of 16 m , calculated based on the extinction coefficients at the chromophore absorbance maxima . the 5 l volume of each fp was placed 15 mm deep inside into one of two available bores in a xfm-2 phantom mouse ( caliper lifesciences ) . the bores were located at 7.0 mm and 18.1 mm distance from the imaging surface . images were taken in 23 different combinations of the far - red and nir excitation and emission channels using an epifluorescence mode of the ivis spectrum instrument . a signal - to - background ratio was calculated for each wavelength combination for each fp , using the phantom mouse without protein sample inside as a background reference .

every year thousands of infants , children , adolescents and young adults die suddenly and unexpectedly.1 of these a significant proportion are autopsy negative and are classified as autopsy negative sudden , unexplained death.1 sudden unexpected death in infancy ( sudi ) is defined as clinically unexpected death in infants from one week to one year of age.2 sudi affects around 1 in 1000 infants , with a peak prevalence at three months of age.2 detailed autopsy examinations , including various ancillary examinations reveal a specific cause of death in only about one third of cases.2,3 these ancillary examinations include radiological skeletal surveys to identify rib fractures,4 bacteriological examination,5,6 post - mortem genetic testing to identify the subset of channelopathic sudi , known as the cardiac channel molecular autopsy,1 as well as dna and rna extraction from a variety of tissues to detect serotonin transporter gene polymorphisms.7 various cardiovascular abnormalities , isolated or associated with central nervous system alterations have been observed in cases of sudi.5 these include : accessory atrioventricular pathways ( mostly mahaim fibres ) , hypoplasia of the cardiac conduction system or the central fibrous body , splitting of the atrioventricular node or the his bundle and a zahn node.5 in this proof of concept study we aimed to explore the feasibility of post - mortem echocardiography in order to alert the pathologist to any possible underlying cardiovascular anomalies before the actual dissection of the heart , in order to avoid any possible damage to underlying delicate structures . this proof of concept study was undertaken with approval of the ethical committee of the faculty of health sciences , university of pretoria . the heart of a three month old male infant , whose death fulfilled the criteria for sudi was used . at autopsy , the major vessels were tied of with string , in order to maintain the filling of the left ventricle with formalin to facilitate the subsequent echocardiogram . the left ventricle was then dissected by cutting it open from the lateral aspect in order to prevent any damage to the observed muscular band . the presence of a thick muscular band was confirmed macroscopically ( see figure 2 ) . autopsy has a very low yield for a specific diagnosis in cases of sudi.2,3 weber et al performed the largest single - institution autopsy study of sudi.3 they analyzed 1516 paediatric post - mortem examinations and found 546 sudi cases . death could be explained in 37% of these cases by the autopsy findings implying that 63% of sudi cases remained unexplained . the authors suggest that alternative and/or additional diagnostic techniques are needed to improve the detection rate of an identifiable cause of death at autopsy in an attempt to lower the high number of unexplained sudi cases . an intriguing new concept is the so - called molecular autopsy where post - mortem genetic analysis in cases of sudi explores the prevalence of channelopathies as the pathogenic basis for sudden unexplained death in infants.8 we propose that post - mortem echocardiography is an additional concept worth exploring . this single case report merits a larger study to determine whether post - mortem echocardiography can ultimately guide the cardiac dissection method in order to preserve delicate underlying cardiac anomalous structures which may ultimately turn out to play a causal role in cases of sudi .

many lifestyle interventions use calorie restriction ( cr ) , defined as a reduction in caloric intake without malnutrition , as a key tool to improve health . indeed , cr appears to beneficially affect physiology and biomarkers related to cardiometabolic health , even in non - obese populations . a prime example of the cardiometabolic benefits of cr is the comprehensive assessment of long - term effects of reducing intake of energy ( calerie ) study trials , involving 25% calorie reduction over 6 months to 2 years [ 15 ] . cr is also well - known to extend lifespan in multiple non - human species [ 6 , 7 ] . despite the fact that delayed immune system aging is thought to be a major pathway through which this happens , the effects of cr on the immune system are less studied than the cardiometabolic system in non - obese humans . our goal was to assess the immunosenescence profile of a sample of long - term calorie restrictors to test whether cr is associated with decreased immunosenescence and longer telomeres in participants without excess adiposity . existing evidence for delayed immune system aging are predominantly in model organisms with conflicting results . in the mouse model , one study observed that cr enhances adaptive immunity during a viral challenge , whereas other studies have found impaired immunity [ 11 , 12 ] . in non - human primates , studies have found no enhancement of peripheral blood mononuclear cell ( pbmc ) response to stimulation in cr versus normally - fed controls [ 13 , 14 ] , whereas another study observed an overall markedly delayed t cell senescence . as adherence to long - term cr is one of its main challenges , we recruited participants through two international cr organizations ( cr society international and the cr way ) who all demonstrated successful long - term cr . much like the larger human cr literature , others using a similar approach to ours have predominantly focused on cardiometabolic health rather than immune aging [ 1719 ] . one study did examine whether long - term cr had lower levels of insulin - like growth factor 1 ( igf-1 ) , a factor implicated in cell - mediated anti - tumor immunity and many types of cancer risk , but found no differences compared to western - diet controls . in the context of a 2-year randomized controlled trial of 25% cr , the calerie-2 study examined multiple immune - related outcomes [ 3 , 23 , 24 ] . they observed lower levels of crp and tnf and slightly fewer white blood cells , lymphocytes , and monocytes in the cr group relative to ad - libitum eating controls , but no differences in vaccine response . as some have called into question whether a 25% cr for 2 years is sufficient to elicit processes activated in the rodent models , our first goal was to examine a sample averaging a longer period of restriction 10 years . our second goal was to augment the few prior studies of immune phenotype in non - obese human cr by expanding the scope of the immune measures [ 24 , 26 , 27 ] . we conducted immunophenotyping to examine percentages of nave and senescent t cells , hypothesizing that cr would have significantly greater percentages of cd28cd57 and cd28cd57pd1 t cells compared to normal weight and overweight / obese groups . we also examined telomere length , considered to serve as a marker of cumulative biological aging [ 2832 ] , in pbmcs as well as granulocytes , and the reverse transcriptase enzyme telomerase in pbmcs . to our knowledge , the study presented here is the first to examine telomere length in humans with cr , and further , to examine telomere length specifically within these two main immunologic compartments . we hypothesized that the cr group would have longer pbmc ( primary outcome ) and granulocyte telomere length and greater expression of telomerase . the committee on human research of the university of california , san francisco ( ucsf ) approved all procedures and all participants provided written informed consent . cr participants were recruited from the cr society international , followers of the cr way , and by referral from participants . inclusion criteria and indicators for cr were reporting calorie restriction without malnutrition for a minimum of two years , bmi < 24.99 , and fasting glucose < 80 mg / dl . when possible , the president of the cr way longevity center and vice president for research of the cr society international or the chairman of the board of the cr society international and treasurer and vice president of the cr way longevity center confirmed each participant s self - reported duration of and adherence to calorie restriction . calorie restriction was verified via four weekly random fasting glucose tests ( < 80 mg / dl ) with bayer glucometers . because individuals who have the ability to restrict their eating behavior for long periods may be phenotypically different from the average population , we matched the groups on self - reported age , race / ethnicity , gender , and educational attainment . matched participants were recruited from the local area once the cr group was completed and their information on these matching variables obtained . additionally , we recruited siblings of cr participants to attempt to control for genetic and environmental factors . sample size was dictated by the maximum number of participants we were able to recruit in a predetermined data collection period of one year . all participants went through a four - week washout period leading up to the on - site visit . during the washout period , we asked participants to refrain from vigorous exercise and from taking any supplements not prescribed by a physician . participants traveled to the ucsf clinical and translational science institute clinical research center ( ccrc ) . to minimize confounds due to sleeping in an unfamiliar setting , participants spent two acclimation nights in the ccrc before the blood draw . to minimize confounds due to jetlag and diurnal activity patterns , participants woke up , ate meals , and slept according to their usual schedules in their home time zone . a subset ( n = 26 ) of local participants ( n = 38 ) completed the study on an outpatient basis . the ccrc metabolic kitchen served tailored calorie - restricted meals to the calorie restriction group . venous blood was collected immediately upon waking , after participants had fasted for a minimum of 8 hours . pbmcs were isolated by gradient density centrifugation by use of ficoll - paque ( ge healthcare , piscataway , nj , usa ) . granulocytes were isolated from the red blood cell pellets after ficoll separation by ammonium - chloride - potassium solution . genomic dna was isolated from whole blood , pbmc and granulocytes using qiaamp dna mini kit ( qiagen ) . the cells were plated in a 96-well v - bottom plate and stained with the following fluorescently - conjugated monoclonal antibodies from bd biosciences unless otherwise noted : brilliant violet 605-conjugated anti - cd8 ( sk1 ) and brilliant violet 711-conjugated anti - cd3 ( ucht1 ) , allophycocyanin ( apc)-efluor780-conjugated anti - cd28 ( cd28.2 ) ( ebioscience ) , alexa fluor 647-conjugated anti - cd57 ( hcd57 ) , pe - conjugated anti - cd38 ( hb7 ) , brilliant violet 421-conjugated anti - pd-1 ( eh12.1 ) and fitc - conjugated anti - hla - dr ( l243 ) . live / dead fixable aqua dead cell stain kit ( invitrogen ) was added into all stains to exclude non - viable cells . staining was performed at 4c for 30 minutes , then washed once with facs buffer ( phosphate - buffered saline containing 0.5% bovine serum albumin and 1 mm ethylenediaminetetraacetic acid ) . cells were then fixed in 0.5% formaldehyde and data was acquired on a bd lsr ii flow cytometer ( bd biosciences ) , with 200,000 lymphocytes collected for each sample . cpt beads ( bd bioscience ) were used for instrument set up for each run and rainbow beads ( spherotec ) standardized instrument settings between runs . aliquots of a control specimen were thawed with every run and assessed in triplicate to confirm run - to - run reproducibility . fluorescence minus one ( fmo ) controls were also prepared on control samples for each run , to check that gates were set consistently between runs . cd3cd8 t cells were defined after standard lymphocyte , singlet and dead cell exclusion gates were applied to the data . fmo controls were used to define positive gates for expression of cd28 , cd38 , cd57 , hla - dr and pd-1 on cd3cd8 t cell populations . the telomere length measurement assay was adapted from the published original method by cawthon and colleagues [ 27 , 34 ] . tubes containing 26 , 8.75 , 2.9 , 0.97 , 0.324 and 0.108 ng of a reference dna ( from hela cancer cells ) were included in each pcr run so that the quantity of targeted templates in each research sample could be determined relative to the reference dna sample by the standard curve method . telomerase activity was measured in pbmcs by the telomerase repeat amplification protocol ( trap ) with a commercial assay ( trapeze , telomerase detection kit , upstate / chemicon , temecula , ca ) using procedures described previously . two concentrations corresponding to 5,000 and 10,000 cells were assayed for each sample in order to ensure that the assay was in linear range . the assay reaction was performed in accordance with the trapeze kit manufacturer s instructions , and radioactive products fractionated by 8% polyacrylamide8 m urea sequencing gel electrophoresis . telomerase activity is expressed as the equivalent of number of 293 t cells per 10,000 pbmcs , and activity was quantified using the imagequant 5.2 software ( ge healthcare ) as described previously . fasting serum samples were assayed by elisa for qualitative detection of cytomegalovirus ( cmv ) igg antibodies by the university of california , los angeles , clinical immunology research laboratories . statistical analyses consisted of ancova models comparing the three groups ( cr vs. normal weight vs. overweight / obese ) all controlling for chronological age , and additionally covarying potential confounding variables when they were statistically significantly related to each respective outcome measure . these potential covariates consisted of self - reported gender and race / ethnicity , as well as cmv serological status due to its known effects on immunosenescence . descriptive statistics of key study variables are summarized in table 1 . for the overall sample of 71 participants , the mean age was 55.01 years ( sd = 14.53 , range = 2184 ) , 54 were male , race / ethnicity was 88.6% white and 11.4% east asian / asian - american , and highest level of educational attainment was as follows : 6.7% high school , 6.7% some college , 20% bachelor s degree , 20% master s degree , and 46.7% doctoral degree . the cr group ( n = 30 ) had been restricting their caloric intake without malnutrition for an average of 10.03 years ( sd = 6.84 , range = 333 ) . by design , the cr group had lower bmi than both the normal weight ( n = 16 ; 5 siblings of cr participants ) and overweight / obese ( n = 25 ; 8 obese ; 1 sibling of cr participant ) groups , f(2 , 68 ) = 106.62 , p < 0.001 . as expected , the cr group had lower average fasting glucose levels f(2 , 61 ) = 11.58 , p < 0.001 . forty - seven percent of the sample was cmv seropositive with no significant differences between groups , f(2 , 66 ) = 0.08,p = 0.92 . descriptive statistics of study variables note : mean ( standard deviation ) [ minimum - maximum ] except for sex , ethnicity / race , education , and cmv status , where n ( % ) . differing superscripts refer to statistically significant differences between groups with p < 0.05 , two - tailed . telomerase was non - normally distributed and therefore values reflect median ( interquartile range)[minimum - maximum ] . telomere and telomerase analyses control for chronological age . for all measures of immune activation and immunosenescence in cd4 t cells , no statistically significant differences were observed between the three groups ( data not shown ) . table 2 displays results for cd8 t cells , where a statistically significant group difference emerged in activated cd8 t cells defined as cd38hla - dr cells , f(2 , 64 ) = 3.28 , p = 0.045 , wherein the overweight / obese group had a lower percentage of activated cd8 t cells than both the cr and normal weight groups . additionally , the normal weight group had a higher percentage of cd8cd28 ( i.e. memory ) t cells than the overweight / obese group , f(2 , 64 ) = 3.16 , p = 0.045 . group differences in frequencies of cd8 t cell activation and maturation phenotypes note : mean ( standard deviation ) [ minimum - maximum ] . differing superscripts refer to statistically significant differences between groups with p < 0.05 , two - tailed . all covariates other than age are significantly related to the dependent variable at p < 0.05 . * we hypothesized greater percentages of these phenotypes in the cr group . the cr group had shorter pbmc telomere length ( f(2,62 ) = 4.75 , p = 0.012 ) than the overweight / obese group ( mean difference : 0.14 , p = 0.009 , or 233.32 base pairs ; fig . 1 ) . furthermore , no significant difference in granulocyte telomere length ( f(2,54 ) = 0.89 , p = 0.42 ) was observed between the cr and the normal weight or overweight / obese groups ( fig . no statistically significant differences in telomerase activity , f(2,65 ) = 1.48 , p = 0.236 were observed within pbmc or granulocytes between cr and normal weight and overweight / obese persons(fig . 2 ) . ancova analyses indicate the calorie restriction group has significantly shorter telomere length compared to the other groups ; p = 0.012 . in the largest study to date of long - term human calorie restrictors averaging over a decade of calorie restriction , we set out to examine whether cr is associated with enhanced immunosenescent and cellular aging profiles of t cells . although research has observed beneficial effects of cr on the cardiometabolic system in non - obese populations [ 4 , 5 ] , or beneficial effects of cr on cancer risk in populations with excessive weight , we did not observe these benefits in this context . as delayed immunologic aging is purported to be a main mechanism of cr s lifespan - extending effects , we expected to see a strong and consistent pattern of lower senescent and higher nave cell percentages as well as longer telomere length in the cr group compared to the normal weight and overweight / obese groups . in contrast , we observed very few differences between the three groups in markers of immunosenescence , and the cr group had shorter pbmc telomere length than the comparison groups . in fact , the telomere length in the cr group was significantly shorter when compared to the obese group . it is unclear why we observed differences in pbmcs and not granulocytes , but it is important to examine them separately as pbmc and granulocyte populations have different roles in immune function , and reflect different immune responses . pbmc telomere length is affected by both history of infections ( which cause more replicative turnover ) as well as the biochemical environment of the blood . due to their rapid turnover , granulocyte telomere length is thought to more directly represent the common myeloid progenitor stem cell compartment,[38 , 39 ] . thus , it is possible that the short lived granulocytes may have less variance in turnover , between people , and may be less influenced by environmental exposures , like dietary factors , in the blood . we do not know why cr was associated with shorter pbmc telomere length , but we can offer speculation . given the pattern of findings here , one possibility is that the cr diet as practiced left those individuals more vulnerable to infection , despite that cmv seropositive status was no different between the groups . cmv exposure typically occurs during early childhood , such as during daycare , and therefore was likely acquired before cr started . however , there could be differences in how well people are suppressing reactivation of the cmv virus , which we did not measure and would require cmv viral load analyses as well as specific immune responses such as igg concentrations or cmv - specific t cell responses . there may be differences in the immune cell populations that require greater subset measurement to observe than we undertook here . t cells make up 60% of the pbmcs , and so differences in t cell telomere length could have a large influence on pbmc telomere length . the difference in pbmc telomere length could potentially be due to a skewing in major memory t cell subsets ( i.e. effector vs central memory ) in the cr group due either to increased subclinical infections in this group or how their immune system maintains latent viral infections such as herpes viruses ( e.g. cmv , epstein barr virus ) . these overall null findings add to a sparse , but already mixed , literature in animal and non - human primate species . in mice , cr led to fewer senescent intestinal crypt enterocytes and liver hepatocytes and better telomere maintenance in these cells . however , in primates , cr did not lead to changes in telomere length in leukocytes or other tissues . in the context of mixed evidence of the relationship between cr and immune aging in model organisms such as mice and macaques , it perhaps is not surprising that such a relationship would be difficult to observe in human cr . this means human cr can be practiced in infinitely variable ways with similarly low caloric intake but different macro and micronutrient content , eating patterns , and consistency . most humans find it difficult to adhere to intense long - term cr [ 6 , 7 , 16 ] , and indeed , our research has demonstrated that this group of long - term cr practitioners have a distinct psychosocial profile that suggests a predisposition to the ability to sustain long - term cr . moreover , cr can put individuals at risk for malnutrition [ 6 , 16 ] , calling into question whether cr should be pursued as an intervention for immune system aging for non - obese humans . however , our results should be interpreted with caution in light of conflicting findings from meydani and colleagues , who found beneficial effects on circulating inflammatory markers in the context of a randomized trial of cr . this study had the following limitations : although this is the largest study of long - term cr practitioners , and they had been calorically restricting on average over a decade , it is possible that the effect of cr on immune system aging is too small to be detected by this sample size . our primary analysis had an observed power value of 0.77 , but nonetheless our study is not ideally powered . furthermore , only t cell maturation and activation profiles were assessed , so it remains unknown whether cr practitioners exhibit differences in other immune cell subsets ( e.g. b cells , monocytes or nk cells ) or in the ability of innate and adaptive immune cells to respond to appropriate stimuli by cytokine production or proliferation . however , one strength of this study is that we examined pbmc and granulocyte telomere length separately , which allays concerns that our telomere length variable was an artifact of cell distribution . we also examined cmv status , to rule out the confounding effect of cmv infection on immunosenescence and telomere length . to our knowledge , this is the first study to examine human cr in relation to pbmc and granulocyte telomere length . instead of a strong pattern indicating beneficial effects of human cr on t cell immune activation and immunosenescence , we observed no clear evidence that cr may delay immune aging compared to normal bmi and overweight / obese matched individuals . in fact , the few results emerging from our study suggested potentially negative effects of cr , particularly in the context of pbmc telomere length . delayed immune aging , therefore , must be further studied before it is put forth as a major mechanism of cr s lifespan - extending effects , at least as it is most faithfully practiced in non - obese humans .

ninety six well plates ( flat bottom with lid ) ( corning inc . , one riverfront plaza , ny , usa ) , muller hinton broth ( mhb ) and muller hinton agar ( mha ) ( becton dickinson , franklin lakes , nj , usa ) and chloramphenicol ( sigma - aldrich , buchs sg , switzerland ) were used . sodium chloride and dimethysulphoxide ( dmso ) were purchased from p. c. drug center co. , ltd . , table 1 shows , 11 bacterial strains obtained from 8 different species that were tested for antimicrobial activity . the three mdr bacteria , isolated by tangjitcharoenkun et al . , used in this study were the esbl - producing escherichia coli pmk200400209 and p. aeruginosa ( mdr ) pmk503010109 strain . bacterial strains / species used in this study to screen for antimicrobial activity the a. baumannii pmk200170209 strain reported in this study was isolated as a pan - drug resistant ( pdr ) strain from a thai patient who was treated for lower urinary tract infection at phamongutklao hospital in thailand on february 2009 . the strain showed resistance to all beta - lactam antibiotics , aminoglycosides , quinolones / fluroquinolones and trimethoprim - sulphamethoxazole , whereas it was sensitive to colistin and tigecycline ( data not shown ) . the stamen , calyx , meat of the fruit , persistent calyx of the fruit , seeds , leaves and pneumatophores of the crabapple mangrove tree were collected from samuthsongkram , thailand , in october 2008 . voucher specimens were deposited at department of pharmacognosy , silpakorn university in nakhon pathom , thailand , with voucher reference numbers sc 01 - 07 . the plant parts were dried with hot air oven at 60 and then ground by cutting mill using sieve # 30 . the high performance liquid chromatography ultraviolet spectrophotometric ( hplc - uv ) method was used by our research group for the simultaneous determination of the three active compounds ( gallic acid , luteolin and luteolin-7-o - glucoside ) to evaluate the quality of the extracts as described . the hplc separation was performed on a rp-18 semi - preparative column ( 2504.6 mm i.d . ; 10 m ) with a gradient of acetonitrile and 0.1% ( v / v ) aqueous phosphoric acid , at a flow rate of 5.0 ml / min , detected at 280 nm . the ground powder of the different parts of the crabapple mangrove tree , selected as the leaves ( l ) , pneumatophore ( e ) , flower [ stamen ( t ) and calyx ( p ) ] and fruit [ meat of fruit ( f ) , persistent calyx of fruit ( c ) and seeds ( s ) ] were individually weighed and subsequently macerated either with methanol alone or in four organic solvents , in sequential order at 1:4 ( w / v ) ratio of dried sample ( g ) and solvent ( ml ) . when methanol was used , the extraction was performed at room temperature ( 28 ) for 72 h. when organic solvents were used , sequential extraction was performed , using soxhlet 's apparatus , wherein the dried plant samples were extracted , with each solvent in the following order : ( 1 ) hexane , ( 2 ) dichloromethane , ( 3 ) ethyl acetate and ( 4 ) methanol , for 24 h. in all cases , the percolate was filtered through whatman no . 1 filter paper until clear and the extracts were then subjected to evaporation using a rotary evaporator ( buchi r205 , buchi ( thailand ) ltd . , the dried extract was then dissolved in dmso and subsequently diluted into mhb for screening in antibacterial activity assays . mha plates and mhb were used as the solid and liquid culture media , respectively , for the assay of the number of viable bacteria by total plate counting , and the growth of the bacteria , respectively . screening for antibacterial activity of each extract was assessed using the agar well diffusion assay , modified slightly from that described in hood et al . the indicator strains from mha plates were individually inoculated into mhb and were then incubated at 37 for 24 h. once an actively growing broth culture or suspension of microbes was obtained , the turbidity was adjusted to match that of the standard 0.5 mcfarland solution , which correlated to a cell density of approximately 10 cells / ml . then 30 l of suspension , containing 2.5 , 10 or 20 mg / ml of the test extract , was separately added to each well ( 6 mm in diameter ) of a mha agar plate freshly spread with 0.1 ml of the test bacterial cell suspension . the plates were incubated at 37 for 24 h and then observed visually for the presence of any inhibition zone around the well . agar wells loaded with the same amount ( 30 l ) of dmso / mhb solvent at the same concentrations and were used as negative controls . the minimum inhibition concentration ( mic ) and minimal bactericidal concentration ( mbc ) were measured using the broth microdilution method according to the clinical and laboratory standards institute ( clsi ) protocol . the assay was performed in a 96-well microtitre plate with a final volume of 100 l per well . each well contained 20 l of the serially diluted plant extract under test , 20 l of the test bacteria suspension in mhb at 10 cfu / ml and 60 l of fresh mhb culture broth . chloramphenicol was employed as a positive control whereas the mhb - diluted dmso solvent ( same concentrations as the test plant extracts ) without the test plant extracts was used as the negative control . the assay plate was incubated at 37 for 24 h , whereupon the growth of the test bacteria was examined by visual observation in terms of the turbidity of the culture . the mics were determined as the lowest concentration of the test compound at which no turbidity from microbial growth could be observed . the mbc values were obtained by re - inoculating 10 l of each supernatant fluid from the mic tested wells with no visible turbidity onto fresh mha plates . the mbc is defined as the lowest concentration of the test compound at which no viable cells were detected on replating . the stamen , calyx , meat of the fruit , persistent calyx of the fruit , seeds , leaves and pneumatophores of the crabapple mangrove tree were collected from samuthsongkram , thailand , in october 2008 . voucher specimens were deposited at department of pharmacognosy , silpakorn university in nakhon pathom , thailand , with voucher reference numbers sc 01 - 07 . the plant parts were dried with hot air oven at 60 and then ground by cutting mill using sieve # 30 . the high performance liquid chromatography ultraviolet spectrophotometric ( hplc - uv ) method was used by our research group for the simultaneous determination of the three active compounds ( gallic acid , luteolin and luteolin-7-o - glucoside ) to evaluate the quality of the extracts as described . the hplc separation was performed on a rp-18 semi - preparative column ( 2504.6 mm i.d . ; 10 m ) with a gradient of acetonitrile and 0.1% ( v / v ) aqueous phosphoric acid , at a flow rate of 5.0 ml / min , detected at 280 nm . the ground powder of the different parts of the crabapple mangrove tree , selected as the leaves ( l ) , pneumatophore ( e ) , flower [ stamen ( t ) and calyx ( p ) ] and fruit [ meat of fruit ( f ) , persistent calyx of fruit ( c ) and seeds ( s ) ] were individually weighed and subsequently macerated either with methanol alone or in four organic solvents , in sequential order at 1:4 ( w / v ) ratio of dried sample ( g ) and solvent ( ml ) . when methanol was used , the extraction was performed at room temperature ( 28 ) for 72 h. when organic solvents were used , sequential extraction was performed , using soxhlet 's apparatus , wherein the dried plant samples were extracted , with each solvent in the following order : ( 1 ) hexane , ( 2 ) dichloromethane , ( 3 ) ethyl acetate and ( 4 ) methanol , for 24 h. in all cases , the percolate was filtered through whatman no . 1 filter paper until clear and the extracts were then subjected to evaporation using a rotary evaporator ( buchi r205 , buchi ( thailand ) ltd . , the dried extract was then dissolved in dmso and subsequently diluted into mhb for screening in antibacterial activity assays . mha plates and mhb were used as the solid and liquid culture media , respectively , for the assay of the number of viable bacteria by total plate counting , and the growth of the bacteria , respectively . screening for antibacterial activity of each extract was assessed using the agar well diffusion assay , modified slightly from that described in hood et al . the indicator strains from mha plates were individually inoculated into mhb and were then incubated at 37 for 24 h. once an actively growing broth culture or suspension of microbes was obtained , the turbidity was adjusted to match that of the standard 0.5 mcfarland solution , which correlated to a cell density of approximately 10 cells / ml . then 30 l of suspension , containing 2.5 , 10 or 20 mg / ml of the test extract , was separately added to each well ( 6 mm in diameter ) of a mha agar plate freshly spread with 0.1 ml of the test bacterial cell suspension . the plates were incubated at 37 for 24 h and then observed visually for the presence of any inhibition zone around the well . agar wells loaded with the same amount ( 30 l ) of dmso / mhb solvent at the same concentrations and were used as negative controls . the minimum inhibition concentration ( mic ) and minimal bactericidal concentration ( mbc ) were measured using the broth microdilution method according to the clinical and laboratory standards institute ( clsi ) protocol . the assay was performed in a 96-well microtitre plate with a final volume of 100 l per well . each well contained 20 l of the serially diluted plant extract under test , 20 l of the test bacteria suspension in mhb at 10 cfu / ml and 60 l of fresh mhb culture broth . chloramphenicol was employed as a positive control whereas the mhb - diluted dmso solvent ( same concentrations as the test plant extracts ) without the test plant extracts was used as the negative control . the assay plate was incubated at 37 for 24 h , whereupon the growth of the test bacteria was examined by visual observation in terms of the turbidity of the culture . the mics were determined as the lowest concentration of the test compound at which no turbidity from microbial growth could be observed . the mbc values were obtained by re - inoculating 10 l of each supernatant fluid from the mic tested wells with no visible turbidity onto fresh mha plates . the mbc is defined as the lowest concentration of the test compound at which no viable cells were detected on replating . the agar well diffusion assay was employed for the screening of antibacterial activity in different extracts ( different solvents and different plant parts ) of the crabapple mangrove tree . the methanol extracts of all the plant parts examined , revealed antibacterial activity against all 11 test strains ( 8 different species ) of bacteria , in terms of the appearance of a zone of growth inhibition , around the application well ( data not shown ) . the broth microdilution method was then used to determine the mic and mbc of each of these extracts , and the results are summarised in table 2 . by comparing the mic of the standard control group and the standard from clsi ( 2010 ) , the mic value against s. aureus atcc 29213 and e. coli atcc 25922 were found to be within the acceptable range of clsi ( table 2 ) although an antibacterial activity ( mic ) was detected in the methanol extracts of all 7 crabapple mangrove tree parts against all 11 test bacterial strains , the mbc values ( bactericidal activity ) showed a different pattern of activity from that of mic ( table 2 ) . although all of the methanol samples showed bactericidal effect on all tested gram - negative bacteria , including both the drug - sensitive e. coli and p. aeruginosa , and mdr esbl - producing e. coli , p. aeruginosa and pdr - a . baumannii strains , the only bactericidal activity against gram - positive bacteria was observed with the leaf extract sample against mrsa and the leaf , seeds and pneumatophore extracts against bacillus megaterium and s. aureus , but not bacillus subtilis , for the drug - resistant and drug - sensitive bacteria , respectively . the rest of the samples only showed a bacteriostatic effect against the gram - positive bacteria . based on the differences in the pattern of the mbc and mic values against the test bacterial strains , the samples from the seven different parts of the crabapple mangrove tree could be divided into three groups . the first group comprising of leaves showed the strongest antibactericidal activity against two drug - sensitive strains ( b. megaterium and s. aureus ) and one drug - resistant gram - positive bacteria ( mrsa ) . the second group comprising pneumatophore and seed samples could eradicate only one gram - positive bacteria ( b. megaterium ) and displayed a bacteriostatic effect against the rest of the test gram - positive bacteria . the third group containing the samples from the stamen , calyx and the meat and calyx of the fruit only displayed a bacteriostatic activity against all test gram - positive bacteria ( table 2 ) . antibacterial activity of the methanol extracts of different plant parts from the crabapple mangrove tree further , we selected one sample ( plant part ) from each of the above three groups as representative samples for sequential fractional purification and determination of the antibacterial activity , again based upon the mic and mbc values ( table 3 ) . it was found that fractions obtained from the sequential extraction with hexane ( l1 , s1 and p1 ) , dichloromethane ( l2 , s2 and p2 ) and ethyl acetate ( l3 , s3 and p3 ) showed no detectable antibacterial activity against any of the eleven test strains . rather , the antibacterial activity was only detected in the methano fractions ( samples l4 , s4 and p4 ) . as noted before with the direct methanol extracts ( table 2 ) , the antibacterial effect against the gram - negative bacteria was mostly bactericidal whereas that against the gram - positive bacteria was bacteriostatic except for b. megaterium that displayed a bactericidal action ( table 3 ) . indeed , the mic and mbc activity for the methanol extract following hexane , dichloromethane and ethyl acetate extraction ( table 3 ) were the same as that of the direct methanol extraction ( table 2 ) in most cases of the seed and calyx samples , suggesting that the hexane , dichloromethane and ethyl acetate extractions did not remove the active component ( s ) . however , the bactericidal activity of the leaf extract against the gram - negative bacteria was slightly reduced from 0.2 to 0.4 mg / ml in 4/5 cases . antimicrobial activity of the sequential extracts of various part of the crabapple mangrove tree it was noted that the crabapple mangrove tree extracts exhibited antimicrobial activity against both gram - positive and gram - negative bacteria , including against the mdr strains . the extract exhibited a higher bactericidal action against gram - negative pathogens than gram - positive ones . the active fractions were only found in the methanol extractions and were not affected by the prior sequential extraction of hexane , dichloromethane and ethyl acetate ( except for perhaps a slight reduction in the bactericidal activity in the leaf extract ) , suggesting that the antibacterial compound ( s ) is / are rather hydrophilic . although all 7 evaluated parts of the crabapple mangrove tree showed antibacterial activity , they differed in their activity levels with the most pronounced being found in the methanol extract of leaves ( table 2 ) . this is of interest since leaves are the easiest source to harvest and the most abundant source . indeed , the crabapple mangrove tree can recover rapidly after branch removal , regenerating from buds below the bark surface , and is reasonably productive ( fast growing ) . the antimicrobial action could be due to the flavonoids in this plant but awaits further enrichment and identification of the bioactive component(s ) for confirmation . nevertheless , this result is very interesting since most reports on the effect of plant extracts have , in contrast , shown a greater susceptibility to gram - positive bacteria than to gram - negative bacteria[2629 ] . such observations could be explained by the fact that most of the antimicrobial active components identified are less polar compounds that are not water - soluble and so the organic solvent extracts showed a more potent activity . the outer phospholipidic membrane of gram - negative bacteria with the structural lipopolysaccharide component makes their cell walls largely impermeable to lipophilic solutes , and the porin - based pores form a selective barrier to the hydrophilic solutes with an exclusive limit of about 600 da . gram - positive bacteria are , however , more susceptible to non - polar antibacterial agents since their outer peptidoglycan layer is not an effective permeability barrier . in this case reported here , the effective antimicrobial activity was found in the methanol extract ( table 3 ) , suggesting a rather polar structure and so is consistent with the observed higher antibacterial activity against the gram - negative bacteria than the gram - positive bacteria tested . it may be possible that multiple bioactive compounds are contained in the extracts and that they differentially contribute to the bactericidal effect in gram - negative and bacteriostatic effect in gram - positive bacteria . further isolation and purification of the methanol extracts , especially those of the leaves , are needed to address this and is a currently ongoing study . the overall results provide a promising basis for the evaluation of the potential use of crabapple mangrove tree leaves as a source of antibiotics for the treatment of specific microbial infections associated with the sensitive pathogens as well as the mdr bacteria studied . purification of the effective fractions for further elucidation on its pharmacological and cytotoxicity studies is being conducted to confirm the extracts as potential therapeutic agents . methanol extracts of all tested parts of the crabapple mangrove tree , s. caseolaris linn . , exhibited broad - spectrum antibacterial activity against both gram - positive and gram - negative pathogens , and revealed a significantly higher bactericidal activity against gram - negative bacteria than gram - positive bacteria . the methanol extract from leaves contained the highest antibacterial activity and could even kill the mdr gram - positive ( mrsa ) and gram - negative pathogens ( esbl - producing e. coli , mdr p. aeruginosa and the pdr - a . such a high efficacy of the leaf methanol extracts of the crabapple mangrove tree against mdr and pdr strains suggests its potential to further develop as an antibacterial agent .

endothelial polarity is assumed to be mechanistically similar to that of epithelial cells , where it is well studied . undoubtedly , the morphological and molecular organization of the vascular endothelial cells ( ecs ) at any of the diverse blood - tissue interphases must reflect the highly polarized environment in which they operate . for instance , vascular lumen formation is entirely dependent on segregation of apical and basal membrane compartments and subsequent ec polarization ( lizama and zovein , 2013 ) . a huge body of work has also demonstrated that at vascular blood - brain or blood - retinal barriers , neuronal homeostasis is maintained by highly polarized localization of transporters and channels , which regulate the directional movement of ions , drugs , metabolites , and toxins ( abbott et al . , 2010 ) . thus , it is assumed that , in their naturally highly polarized environment , ecs have adopted differential apicobasal signaling . however , experimental proof of such polarized signaling processes is still missing . vascular endothelial growth factor ( vegf ) family members , and in particular its most studied representative vegf - a , are central to the creation of new blood vessels during normal development and growth but also in pathological situations such as tumorigenesis and ocular neovascular disease ( koch et al . , 2011 ; takahashi and shibuya , 2005 ) . besides its angiogenic role , vegf - a has additional effects on the vascular endothelium . it was originally discovered for its ability to trigger vascular permeability , and this permeability - enhancing property of vegf - a is linked to interstitial fluid accumulation in tumors and psoriatic lesions , as well as tissue edema and concomitant vision loss in neovascular eye disease ( ferrara et al . , 2007 ) . vegf - a also has beneficial roles in the systemic vasculature , regulating normal vascular tone and acting as a trophic factor for the vascular endothelium ( maharaj and damore , 2007 ) . vegf - a mediates its diverse functions through the use of multiple receptors , mainly the receptor tyrosine kinases vegfr1 ( flt-1 ) and vegfr2 ( flk-1/kdr ) ( koch et al . , 2011 ) . the most common splice isoform vegf - a(165 ) , subject of the present study , additionally interacts with neuropilin 1 and various cell - surface heparan sulfate proteoglycans to modulate the intracellular response . vegf - a belongs to a family of growth factors with five genes encoding vegf - a , -b , -c , and -d and placental growth factors ( plgfs ) , each binding vegfrs with different affinities . for instance , plgf-1 and the viral gene product vegf - e specifically bind and activate vegfr1 and vegfr2 , respectively ( takahashi and shibuya , 2005 ) . vegf - a triggers a plethora of intracellular signaling steps in ecs ( koch et al . , 2011 ) . standout mediators associated with the vegf - a - induced endothelial permeability response are the mitogen - activated protein kinases erk and p38 , phospholipases , protein kinase c , phosphatidylinositol 3-kinase ( pi3k)-activated akt , and endothelial nitric oxide synthase ( enos ) ( weis and cheresh , 2005 ) . however , given the complexity of vegf - a signaling and the permeability response , as well as the morphological and cytological differences of various endothelia , it is not surprising that these signaling components remain under intense scrutiny , in particular with respect to their specific role in particular experimental models and their interaction with each other in controlling various effector mechanisms . because of vegf - a s central role in the regulation of endothelial functions in health and disease , anti - vegfs have become irreplaceable tools in treating pathological angiogenesis and permeability ( ferrara et al . , 2007 ) . however , systemic anti - vegf - a therapies are associated with endothelial dysfunction leading to bleeding , inflammation , hypertension , proteinuria , and even lethality ( chen and cleck , 2009 ) . consistent with these observations in patients , animal models demonstrate that vegf - a is constitutively required to maintain vascular tone and vascular ec survival and that reductions in plasma vegf - a levels cause vascular attrition and functional abnormalities ( sugimoto et al . , 2003 ) . we therefore hypothesized that circulating and tissue - produced vegf - a induce distinct responses in ecs . in light of the central role of vegf - a as a permeability - inducing factor in brain ( merrill and oldfield , 2005 ; argaw et al . , 2012 ) and eye pathologies ( miller et al . , 2013 ) , our study focused on vascular ecs at blood - neural barriers and on acute endothelial permeability ( as opposed to chronic interstitial fluid accumulation during inflammation , cancer , and wound healing , which may involve cells other than ecs ) ( nagy et al . , our study is also restricted to the in vitro and in vivo analysis of the microvasculature as opposed to the macrovasculature , because this is where physiological and pathological vascular permeability occurs . here we report that vegf - a induces unequal responses depending on whether it acts on the luminal or abluminal side of neural microvascular ecs ( mvecs ) . mechanistically , this differential ec response is predicated on polarized vegfr expression and distinct downstream signaling . using contrast - enhanced magnetic resonance imaging ( mri ) in the mouse , we determined tissue extravasation of intravenous ( i.v . ) injected gadolinium ( gadolinium diethylenetriamine pentaacetic acid [ gd - dtpa ] , 742 da ) . basal leakage of gd - dtpa was significantly higher in peripheral tissues such as the lung than the brain or the eye , reflecting the significant difference in baseline permeability between neural and nonneural vascular beds . intravenous injection of vegf - a ( 3 g / mouse ) significantly enhanced leakage of gd - dtpa into lungs within 10 min ( figure 1a ) , in agreement with previous reports of vegf - a - induced permeability in nonneural vasculature , such as that of the trachea or the mesentery ( sun et al . , 2012 ; bates and curry , 1996 ) . significantly , no leakage was observed in the brain or the eye over a 30 min observation period . however , when vegf - a was directly injected into the brain cortex or the vitreous of the eye , rapid and significant accumulation of gd - dtpa in the vicinity of the injection site was observed ( figures 1b and 1c ) , indicating that only tissue - borne but not circulating vegf - a induced vascular hyperpermeability in the neural tissues . this was further studied in single pial microvessels , which exhibit barrier properties closely resembling those found at the intact blood - brain barrier ( bbb ) and allow permeability measurements that are tightly controlled with respect to kinetics and dose ( butt et al . vegf - a levels up to 2 g / ml within the lumen of tight pial microvessels did not affect baseline permeability ( figures 1d and 1f ) , even when bradykinin triggered strong vascular permeability ( figures 1e and 1f ) . in contrast , exposure to vegf - a from the tissue side led to a rapid increase of permeability ( figures 1 g and 1h ) , which was maximal at a concentration of ca . 50 ng / ml . taken together , these data demonstrated that cerebral microvascular permeability is strongly induced by vegf - a , albeit only when presented to the abluminal face of microvessels . the microvasculature in the brain and retina constitutes a network of ecs tightly interacting with mural cells such as pericytes and glial cells including astrocytes and mller cells ( abbott et al . , 2010 ) . because microvascular permeability operates primarily on the level of endothelium , we tested whether the ecs themselves responded to vegf in a polarized fashion . for this , we used highly purified nonpassaged , primary mvecs isolated from brain or retina , which were characterized by preservation of interendothelial junctions , apicobasal polarity , and very good in vitro barrier properties ( figure s1 available online ) . macromolecular apical - to - basal flux was enhanced nearly 2-fold when 50 ng / ml vegf - a was added to the basal side of rat brain mvec monolayers , whereas doses of up to 1 g / ml applied to the apical side did not change flux ( figures 2a2d ) . a similar , polarized response to vegf - a was also observed in cultures of murine brain ( data not shown ) and rat or porcine retinal mvecs ( figures 2e and 2f ) , suggesting that functional polarity operates at all vascular blood - brain and -retinal barriers and across species . importantly , both barrier properties and the sided response to vegf - a were lost following repeated subculturing of mvecs ( figures s1d s1f ) , offering a plausible explanation as to why this phenomenon has not yet been universally observed . vegf - a also induced changes in transendothelial electrical resistance ( teer ) and significant reduction in junctional claudin 5 ( cldn5 ) ( figures 2 g and 2h ) , a reliable molecular indicator of mvec barrier and bbb integrity ( nitta et al . , 2003 ) , indicating that paracellular rather than transcellular permeability was operational ( steed et al . , 2010 ) . vegf - a provoked flux and teer changes within minutes ( figures 2b and 2i ) , thus resembling the acute response observed in vivo . a sided permeability response similar to that with vegf - a was also observed with histamine and insulin - like growth factor - binding protein 3 ( igfbp3 ) ( figure 2j ) , whereas opposite responsiveness was observed with lysophosphatidic acid ( lpa ) . thrombin and bradykinin induced significant flux in brain mvecs but without predilection for either endothelial face . significantly , the sided response to histamine and lpa was consistent with previous results from pial microvessels in vivo ( sarker et al . one explanation for the polarized responses to vegf - a is that distinct signaling pathways , and in particular different vegf - a receptors , operate on the apical and basal sides of brain or retinal mvecs . to test this hypothesis , we studied the distribution of vegfr1 ( flt-1 ) and vegfr2 ( kdr / flk-1 ) ( koch et al . , 2011 ) . 70% of vegfr2 was found at basal membranes ( figure 3a ; table s1 ) . analysis by domain - specific biotinylation ( figure 3c ) or analysis by confocal microscopy ( figures s2a and s2b ) confirmed this highly polarized distribution pattern . importantly , distribution of vegfr1 and vegfr2 was also predominantly luminal and abluminal , respectively , in the microvascular endothelium of the mouse hippocampus ( figure 3b ; table s1 ) . vegf receptor distribution was also interrogated in vivo : the luminal side of the mouse vasculature was exposed for 5 min to anti - vegfr1 or -r2 antibodies by cardiac injection . following perfusion , vegfr antibody binding was studied by fluorescent secondary antibody labeling of retinae and lungs ( figure 3d ; figure s2c ) . vegfr1 antibodies bound to retinal and lung microvessels , whereas vegfr2 antibodies strongly labeled lung but not retinal microvessels . in contrast , when the same antibodies were applied abluminally for 5 min to unfixed and nonpermeabilized retinae , vegfr2 but not -r1 was detectable in retinal vessels . in addition , both antibodies strongly reacted with other cells in the retinal ganglion cell layer , consistent with the reported expression of vegf receptors in ganglion and mller cells ( saint - geniez et al . , 2008 ; foxton et al . , , the majority of vegfr1 and -r2 was found to be inversely located to the luminal and abluminal sides , respectively , of mvecs in the brain and the retina but not the lung . these data strongly suggested that different downstream signals operate in response to apical or basal vegf . indeed , we found that the time - dependent activation of p38 and akt was significantly different following apical or basal exposure of brain mvecs to vegf - a . phosphorylation of p38 , increasing over time , was observed in response to basal but not apical vegf - a ( figure 4a ) . in contrast , akt activation was only seen following apical exposure to vegf - a . apical but not basal exposure to plgf-1 , which selectively binds to vegfr1 ( takahashi and shibuya , 2005 ) , also induced akt phosphorylation , with a peak at ca . in contrast , stimulation with the vegfr2-selective ligand vegf - e ( takahashi and shibuya , 2005 ) did not lead to akt activation from any side of the ecs . instead , strong phosphorylation of p38 was observed within 5 min of basal but not apical exposure . the hallmarks of this differential p38 and akt activation pattern could be recapitulated by vegf treatment in vivo . in rats , intravenous injection of neither vegf - a , plgf-1 , nor vegf - e ( 120 g / kg ) led to any activation of p38 in pial microvessels ( figure 4c ) nevertheless , in alveolar lung vessels of the same animals , p38 was strongly and rapidly activated in response to i.v . vegf - a or vegf - e but not plgf-1 ( figure s3 ) . in pial microvessels , vegf - a and vegf - e led to rapid p38 activation , but only when growth factors were administered from the tissue side through a cranial window ( figure 4c ) . however , none of six different commercial anti - phospho - akt antibodies produced significantly altered staining patterns in brain , lung , or retinal tissues of rats treated with time courses of luminal or abluminal vegfs or indeed insulin , suggesting that reliable phospho - akt detection was not possible by immunohistochemistry ( data not shown ) . we therefore opted to study akt activation in whole - cell lysates of retinal tissue . intravenous injection of vegf - a or plgf-1 but not vegf - e led to strong akt activation after 25 min ( figure 4d ) . direct injection of vegfs into the retina ( i.e. , abluminal application ) did not lead to any significant akt phosphorylation . taken together , these data demonstrated that in cerebral or retinal mvecs , rapid p38 activation occurred downstream of abluminal vegfr2 . this polarized signaling pattern was not only in full agreement with the vegfr1 and -r2 localization described in figure 3 but also with the endothelial permeability response . enhanced macromolecular flux across brain mvecs was seen following basal ( but not apical ) vegf - e stimulation , whereas plgf-1 had no effect on barrier function , neither in vitro nor in vivo ( figures 5a and 5b ) . furthermore , we found that sb202190 , but not wortmannin or ly294002 , which inhibited vegf - induced p38 or akt activation , respectively ( figures s4a and s4b ) , abrogated vegf - induced hyperpermeability in vitro and in vivo ( figures 5c and 5d ; figure s4c ) . taken together , this clearly indicated that the permeability response to vegf at blood - neural barriers is mediated by abluminal vegfr2 . in agreement , the vegfr2 inhibitor su1498 completely abolished vegf - a - induced flux in brain mvecs ( figure s4c ) . abundant data suggest that systemic ( hence , luminal ) vegf - a is a trophic factor for the vasculature ( maharaj and damore , 2007 ; gerber et al . , 1998 ) . in light of the specific activation of akt following luminal vegfr1 stimulation , we investigated a potential role in endothelial cytoprotection . rat brain mvecs were stimulated with the microbial alkaloid staurosporine , widely used to induce apoptosis in most cell types , including ecs ( kabir et al . , 2002 ) . apoptosis , measured as an increase in caspase 3/7 activity , was significantly reduced by apical vegf - a or plgf-1 but not vegf - e ( figure 6a ) . this vegf - mediated cytoprotection was sensitive to wortmannin but not to sb202190 ( figure 6b ) . to this end , a potential role of vegf in cytoprotection of retinal ecs could not be interrogated in vivo , because staurosporine was ineffective in inducing ec death . staurosporine was applied to mouse or rat retinae both intravitreally and through the carotid artery at up to 40 m for up to 12 hr . this treatment induced severe retinal ganglion cell death but did not affect ecs at all ( data not shown ) . even when additionally circulating vegf - a was reduced by coadministration of anti - vegf antibodies , we could not measure any annexin v binding or activated caspase 3 staining in retinal ecs ( data not shown ) . thus , vegf mediates a cytoprotective response in rat brain mvecs in culture , with signaling via vegfr1 and akt but not vegfr2 and p38 . in the neuronal vasculature in vivo , vegf - a is likely to be only part of a complex network of environmental cytoprotective factors ( see below ) . the vascular endothelium operates in a highly polarized environment , but to date there has been little exploration of polarized endothelial signaling . here we demonstrate that luminal and abluminal ec surfaces of blood - neural barriers display different functionality during acute responses to vegf - a as well as histamine , igfbp3 , and lpa . in particular , we found that cerebral and retinal vascular permeability was completely refractory to circulating vegf - a . in contrast , the lung vasculature responded to circulating vegf - a and vegf - e with enhanced permeability and p38 activation . because mesenteric and tracheal microvessels also induce permeability in response to luminal vegf - a ( sun et al . , 2012 ; bates and curry , 1996 ) , our findings appear to have revealed a clear specialization of the blood - neural barrier endothelium . importantly , our data are consistent with vegf - a s well - established role as a paracrine factor produced by hypoxic / ischemic tissue ( see also figure 7 ) , for instance by astrocytes during central nervous system inflammatory disease ( argaw et al . , 2012 ) and pathological retinal angiogenesis ( weidemann et al . , 2010 ) or by mller cells during diabetes - induced retinal ischemia ( wang et al . , 2010 ) the sided responsiveness of these mvecs to vegf - a was attributable to highly polarized expression of the receptor tyrosine kinases vegfr1 and -r2 and distinct downstream activation of key signaling pathways . nevertheless , the permeability response and the signaling response of the microvascular endothelium was completely polarized . general computational modeling of vegf receptor surface distribution ( mac gabhann and popel , 2007 ) suggests that the distribution pattern we observed in cerebral and retinal ecs would result in luminal vegfr1 homodimers and some vegfr1-r2 heterodimers but no vegfr2 homodimers . in contrast , basal membranes would be rich in vegfr2 homodimers ( and heterodimers ) but devoid of vegfr1 homodimers ( see also figure 7 ) . we speculate that this absence of vegfr2 and vegfr1 homodimers on the luminal and abluminal surface of neural ecs produces exclusivity with regard to location and receptor specificity of ligands . in particular , in this model , p38 and akt activation would be exclusively associated with vegfr2 and vegfr1 homodimer signaling , respectively . indeed , have recently shown that vegfr2 yields different cellular responses depending on whether it is engaged in homo- or heterodimers ( cudmore et al . , 2012 ) . in addition , coreceptors such as neuropilin 1 may also play a role ( becker et al . future experiments using heterodimeric ligands such as plgf-1-vegf - e ( cudmore et al . , 2012 ) could establish the role of vegfr1-r2 heterodimers in the differential signaling response of brain and retinal ecs . in line with generally accepted views ( takahashi and shibuya , 2005 ; issbrcker et al . , 2003 ) , vegfr2 and p38 activities were associated with the permeability - enhancing response of vegf - a . in contrast , and judging by activation profiles and pi3k inhibition , we did not detect any involvement of akt in vegf - induced permeability in the brain . indeed , conflicting results have been reported regarding a direct role of akt in vascular permeability ( six et al . , 2002 ; chen et al . , 2005 ) , and its involvement has frequently been inferred by the need for activation of the akt substrate enos during vegf - mediated permeability ( takahashi and shibuya , 2005 ) . however , in cerebral mvecs , enos activation can also occur in an akt - independent manner , in particular during inflammation ( martinelli et al . this receptor has been proposed to mediate permeability in a more chronic and auxiliary way ( takahashi and shibuya , 2005 ; koch et al . , 2011 ) , which would not likely be captured in our model systems . in addition , a direct role of vegfr1 in permeability may be restricted to nonneural vascular beds ( odorisio et al . , 2002 ) . instead , and in line with it activating akt , a kinase regarded as synonymous to the cell - survival response ( datta et al . , 1999 ) , we found a clear role of vegfr1 in cytoprotection . a cytoprotective role of vegf - a for ecs is well documented ( gerber et al . , 1999 ) , with most mechanistic evidence derived from in vitro culture models . for instance , in umbilical vein ecs , the main vegf receptor mediating survival appears to be vegfr2 ( gerber et al . , 1998 ; dela paz et al . , 2012 ) , whereas in microvascular retinal or dermal ecs there is evidence that vegfr1 is involved ( cai et al . , 2003 ; zhang et al . , 2010 ) , suggesting that receptor usage may differ between vascular beds . whereas we were able to find clear experimental support for vegfr2-mediated permeability in vitro and in vivo , the cytoprotective function of vegfr1 has so far only been observed in cultured cerebral mvecs . our failure to induce measurable apoptosis in retinal ecs in vivo through staurosporine and vegf - a withdrawal suggests that additional growth ( walshe et al . , 2009 ) or environmental factors such as flow ( dela paz et al . , 2012 ; dimmeler and zeiher , 2000 ) contribute to ec survival , in particular in the brain and the eye ( ueno et al . , 2008 ) . numerous experiments also show that the apoptotic response of ecs in vivo is relatively slow and only measurable after several days ( meeson et al . , 1999 ; sugimoto et al . , 2003 ) , and thus not within the time frame of our current experimental setup . overall , our findings are compatible with previous reports but clearly identify a high degree of specialization of vegf signal transduction at blood - neural barriers . specification of vegf - a action occurs through many mechanisms , including the creation of functionally distinct protein isoforms , the presence of multiple surface , soluble , and coreceptors , and the sequestration of the ligand in the extracellular matrix ( koch et al . , in addition , our data suggest that ecs are able to control the response to vegf - a through compartmentalizing cellular signaling , and provide an intriguing explanation of how blood - neural barriers can be maintained despite constant exposure to circulating vegf - a . finally , the ability to map the vascular permeability response to a distinct topological signaling domain within the ec provides a new opportunity to specifically target pathological aspects of vegf - a while preserving essential cardiovascular functions . all procedures were performed in accordance with uk and ireland animal welfare acts and with the association for research in vision and ophthalmology statement for the use of animals in ophthalmic and vision research and the animal welfare and the ethical review bodies of the ucl institute of ophthalmology , king s college london , and trinity college dublin . recombinant rat vegf - a(165 ) was purchased from r&d systems , vegf - e was from cell sciences ( or a kind gift from kurt ballmer - hofer ) , and human plgf-1 was from peprotech . vascular permeability in lungs , brain , and eyes was assessed in vivo via mri ( campbell et al . , 2012 ) using a dedicated small rodent 7 t mri system ( http://www.tcd.ie/neuroscience/infrastructure/neuroimaging/index.php#7tesla ) . the pial microvasculature of wistar rats ( age 2530 days ) was exposed and the permeability was measured as described previously ( easton and fraser , 1994 ; easton et al . , 1997 ) . microvessels were isolated from rat or mouse cortical gray matter or rat or porcine retinae by collagenase dispase digestion and bsa and percoll density gradient centrifugation ( abbott et al . , 1992 ) . purified vessels were seeded onto collagen iv / fibronectin - coated tissue - culture ware or costar transwells ( 3460 ) at high density ( vessels from 6 rat brains or 12 retinae per 40 cm or 3 cm , respectively ) . cells were grown in egm2-mv ( lonza ) ( with 5 g / ml puromycin during the first 5 days ; perrire et al . , 2007 ) for 23 weeks until their teer plateaued at values above 200 /cm . mvecs were grown on collagen iv / fibronectin - coated tissue - culture ware or 12 mm costar transwell filters . alternatively , they were fixed and permeabilized simultaneously in 80% meoh , 3.2% formaldehyde , 50 mm hepes ( ph 7.4 ) ( martins et al . , 2013 ) . 2004 ) using antibodies against von willebrand factor ( dako ) , ve - cadherin ( martins et al . , 2013 ) , occludin and cldn5 ( invitrogen ) , vegfr1 ( sc-31173 ; santa cruz biotechnology ) , vegfr2 ( ab11939 ; abcam ) , and p - glycoprotein ( clone c219 ) . paraformaldehyde ( pfa)-fixed brains from 10-month - old mice were isolated and 50 m hippocampal slices were dissected out and postfixed for 2 hr in 4% pfa . fixed samples were processed as previously described ( eden et al . , 2010 ) . briefly , samples were embedded in 12% gelatin in 0.1 m phosphate buffer ( ph 7.4 ) . after infusion with 2.3 m sucrose at 4c overnight , 80 nm sections were cut at 120c using a cryoultramicrotome and collected in a 1:1 mixture of 2.3 m sucrose and 2% methylcellulose . , 1991 ) using antibodies against vegfr1 ( sc-31173 ) ( af471 ; r&d systems ) and vegfr2 ( ab11939 ) ( dc101 ) . samples were viewed with a jeol 1010 transmission electron microscope , and images were gathered using a gatan orius sc100b charge - coupled device camera . gold particles were considered luminal ( apical ) or abluminal ( basal ) when found within 20 nm of the respective plasma membrane ( ottersen , 1989 ) . fluorescein isothiocyanate ( fitc ) dextran ( 4 kda ) flux across mvecs was measured as previously described ( martins et al . , 2013 ) . alternatively , teer was assessed by impedance spectroscopy using cells grown either on 12 mm transwells and a cellzscope ( nanoanalytics ) or on gold electrodes ( eight - well 8w1e ) and an ecis ( 4,000 hz ) ( applied biophysics ) . caspase activity of mvecs grown in 96-well plates was measured using the apo - one homogeneous caspase-3/7 assay kit ( promega ) . apical and basal biotinylation was performed using a method adapted from gottardi et al . basal biotinylation was very weak , presumably because of reduced access through the filter and the basement membrane ( gottardi et al . , 1995 ) . therefore , apical and basal domains were labeled simultaneously in the presence of edta . biotinylated proteins were isolated on streptavidin beads and , following immunoblotting for vegf receptors , apical and basal signals were quantified by densitometry and normalized against input signals . basal signal was calculated from the combined apical and basal labeling minus the apical signal . for immunoblot analyses , samples were lysed in 50 mm tris / cl ( ph 6.8 ) , 2% sds , 10% glycerol , 100 mm dithiothreitol ( dtt ) , 100 nm calyculin a ( 50 l / cm of cells ) , separated by sds - page , electrotransferred to nitrocellulose or polyvinylidene fluoride , and immunodecorated with phospho - specific and total antibodies as previously described ( martinelli et al . , 2009 ) . akt ( phospho - s473 and total ) , p38 ( phospho - t180/y182 and total ) , and anti - vegfr2 antibodies were from cell signaling ( 55b11 ) , and anti - vegfr1 was from abcam ( y103 ) . anti - vegfr1 ( sc-31173 ) and -vegfr2 ( dc101 ) antibodies ( each at 6 mg / kg ) were injected into the left ventricle of anesthetized p20 mice ( c57 bl/6j ) . after 5 min , the animals were perfused with pbs followed by 4% pfa . retinae were dissected out and processed for whole - mount staining ( west et al . , 2005 ) . for abluminal detection , dissected unfixed retinae of pbs - perfused mice were overlaid with anti - vegfr1 and -vegfr2 antibodies ( 20 g / ml ) for 5 min , washed three times briefly with pbs , and then fixed in 4% pfa for 1 hr . all retinae were stained using anti - f4/80 ( mca497el ; abd serotec ) and/or biotinylated isolectin b4 ( b1205 ; vector labs ) and secondary antibodies to detect bound vegfr1 and -r2 antibodies . transversal ( 1 mm ) sections of lungs from animals having undergone cardiac injections were analyzed in a similar manner . retinal flat mounts were imaged with a pinhole aperture allowing capture of the entire primary plexus depth in a single scan , whereas sections of lung were analyzed from projections of sequential image stacks . for luminal stimulation , 120 g / kg of vegf - a , vegf - e , or plgf-1 or an equal volume ( 100 l ) of saline was injected into the tail vein of anesthetized p23 wistar rats . for abluminal stimulation , cranial windows were surgically introduced and vegfs were added at 100 ng / ml ( final concentration ) to the liquid pool superfusing the pial microvasculature . five minutes after injection / vegf application , animals were perfused and fixed and pial or lung sections were processed for immunohistochemistry ( powner et al . , 2010 ) . for akt analysis , animals were injected with vegfs in the tail vein as above or in the vitreous ( using 100 ng of vegf - a , vegf - e , or plgf-1 or an equal volume [ 5 l ] of saline ) . after ca . 25 min , retinae were isolated and lysed in 50 mm tris / cl ( ph 6.8 ) , 2% sds , 10% glycerol , 100 mm dtt , 100 nm calyculin a and subjected to immunoblot analysis .