Datasets:

ewhfef

/

mix_cpt

like 1

the term manual lymph drainage ( mld ) was coined by the vodders during their years of developing many of the techniques in use today . the vodder technique of mld is the most widely practiced method in asia , europe , north america , and countries in other areas of the world1 , 2 . mld is widely employed for medical disorders , such as lymphedema , phlebedema , lipedema , postsurgical edema , arthropathy , and rheumatic disease ; cosmetic disorders ; gynecological disorders ; neurological disorders ; and autonomic retuning and as a health spa treatment3 , 4 . mld affects the treatment of edema and acts on the body in various ways3 , 4 , especially on the psychological and physical changes that result from stress , the environment , and other factors3 . this means that the appropriate application of mld to subjects who are in a state of disharmony with regard to their psychological and physical status restores them to a normal state , with mld having a tonic effect on the smooth muscle of lymph vessels that possess numerous sensory nerve endings with connections to the autonomic nervous system ( ans ) and other human systems4,5,6 . there are many articles have investigated the effects of massage on stress , pain , systems , fatigue , anger , fear , the autonomic nervous system , and brain state7,8,9,10,11 . however , study of the effect of mld on electroencephalography ( eeg ) has not been reported . therefore , the aim of this study was to objectively investigate the eeg performance of subjects with psychological stress under mld of the neck . they were chosen according to the following criteria : 1 ) no history of mental illness , 2 ) not currently taking any medication known to affect eeg signal , and 3 ) no known heart- or muscle - related disease . all responded to the stress responses inventory ( sri)12 and returned it to the authors . they were asked how they respond to stressful situations ( e.g. , emotionally , cognitively , somatically , or behaviorally ) and to write 39 responses to stressful situations . according to the results of the questionnaire , 26 subjects with an sri score > 80 were enrolled in the study . they were randomly allocated to receive a 15-min session of either mld or rest ( control ) . all test protocols were approved by the ethics committee of the physical therapy faculty of kangwon national university . all interventions were completed in a supine position on a massage table with a pillow placed under the knees to relax the low back muscles . data acquisition and mld took place in a quiet temperature - controlled environment ( 2224c ) . talking , phone calls , and noise that could increase the activity of the sympathetic nervous system were minimized13 ) , and the subject s body was covered with a soft , thin sheet so as to avoid discomfort from body exposure . subjects were allowed to rest comfortably for at least 5 min prior to the baseline recording procedure . subjects in the experimental group received mld of the neck for 15 min , while the subjects in the control group relaxed by lying quietly for the same time period . the protocol was standardized in that the stroke category ( type ) and time of each stroke was the same between all participants . a total 18 channels of eeg were recorded , including fp1 , fpz , fp2 , f7 , f3 , fz , f4 , f8 , t3 , c3 , cz , c4 , t4 , t5 , p3 , pz , p4 , and t6 . there were two other electrodes , which were placed on both zygomatic bones as a ground electrode and reference electrode . participants were asked to close their eyes and refrain from talking , falling asleep , or making exaggerated body movements during eeg measurement . analysis was based on a 5-min period of eeg signal acquisition , followed by computerized fourier analysis of the eeg waves using the telescan software package ( laxtha , daejeon , south korea ) . the signal was sampled at a rate of 256 hz and was digitally filtered using a 150 hz band - pass filter . absolute and relative power values were obtained for delta ( 14 hz ) , theta ( 48 hz ) , alpha ( 813 hz ) , and beta ( 1330 hz ) . the spectral power data ( v2 ) were subjected to a log10 transformation . all outcome measures were determined and analyzed by an investigator who was blinded and was not obligated to provide any intervention for subjects . all variables were tested for normality using the one - sample kolmogorov - smirnov test and did have a normal distribution . the paired t - test was used to test for homogeneity and to compare the pre - mld and post - mld results for differences in eeg . the general characteristics did not differ statistically between the two groups at baseline ( p>0.05 , table 1table 1 . homogeneity test for general characteristics the subjects at baselinevariablegroupmld ( n=14)control ( n=12)age ( yrs)21.21.220.91.4sex ( male / female)3/112/10height ( cm)163.74.5165.35.4weight ( kg)58.38.560.16.0body mass index ( kg / m)21.83.021.91.8 ) . the average absolute and relative eeg subband power distribution did not differ statistically between the two groups at baseline ( p>0.05 , tables 2 and 3table 2 . average absolute eeg subband power distribution of all channelstypeprepost ( v)mld1.440.801.610.89control1.610.421.790.59 ( v)mld3.520.563.560.52control3.530.583.610.54 ( v)mld2.520.472.720.43*control2.490.562.520.45 ( v)mld2.620.512.310.64control2.470.322.510.43all variables are means sd . the post - mld average absolute alpha rhythm was significantly higher in the experimental group ( p<0.05 , table 2 ) . there were also significant difference between the pre - mld and post - mld average relative alpha and delta rhythms in the experimental group ( p<0.05 , table 3table 3 . the main purpose of the present study was to investigate the effect on acute brain activity of mld in subjects with psychological stress by using eeg . although the effect of various massages on brain activity has been previously observed15,16,17 , we are not aware of any reports on the effects of mld on brain activity using eeg power . so , to the best of our knowledge , this is the first study to investigate the differences in brain activity resulting from mld . this study demonstrates that mld of the neck area is effective for promoting relaxation , manifested as an increase in post - mld average absolute alpha rhythm , relative delta rhythm and alpha rhythm ( p<0.05 ) . comparison between the two groups in the present study revealed that subjects receiving mld had significantly higher alpha and delta activities than those in the control group . this finding indicates psychological benefits of mld over resting for stress reduction in subjects with stress . the mechanism by which mld promotes relaxation and changes in brain activity may be explained by the findings of kim et al7 . they reported that mld contributed to increased cardiac parasympathetic activity , which led to decreased sympathetic activity in the neuromuscular system ; thus , it can cause relaxation . alpha waves are dominant under relaxed conditions , whereas beta wave becomes dominant under excited conditions . our finding of reduced cortical arousal through mld may also be explained by the findings of other studies14 , 15 . they reported the state induced by massage techniques represent a hypoactive central nervous system state that may by similar to stageisleep . since mld is a kind of light pressure massage , it is possible that the same mechanism was partly responsible for the decrease in psychological stress observed in this study . the clinical implication of the present study is that mld was found to be an effective intervention for stress reduction in subjects with psychological stress . moreover , interpretation of brain activity of subjects in this study demonstrated greater relaxation after mld compared with control group . the beneficial effects found could be used as a guide for good treatment for stress reduction in subjects with stress . regarding limitations of this study , we did not use other secondary outcome measures in order to subjectively quantify subject anxiety and pain intensity and only evaluated the acute effects of mld . further studies should include other outcome measures and specific treatment groups , and longitudinal study of mld effects on brain activity using eeg would be an interesting direction . in conclusion , the present study is the first report of the effect of mld on acute brain responses using eeg . the findings reveal that a single 15-min session of mld at the neck region produces an immediate decrease in brain activity . it is recommended that mld be considered as an alternative treatment for stress in subjects with psychological stress .

conventional hormonal replacement therapy with estrogen and estrogen - progesterone found a place in the management of menopause . clear - cut follow - up schedules are in place for conventional hormone replacement therapy . with passage of time we have more therapies in the basket , for example , a widely acting gonadomimetic hormone ( with combination of all estrogenic , progestrogenic and androgenic actions ) known as tibolone . the women 's health initiative randomized control trial , million women study brought out what was already known to us that conventional hormone replacement therapy slightly increases the risk of cancer breast , cancer endometrium , if estrogen alone therapy is used . therefore careful follow - up is recommended by regular general examination , breast examination , mammography , pelvic examination , pelvic sonography and lipid profile . with the introduction of tissue - specific menopausal therapy by tibolone which was highlighted to reduce risk of breast cancer ( ca ) and ca endometrium , we encouraged its use . but with increasing usage and experience we realized that tibolone is not breast and endometrium - safe . the present study analyzed 50 women who were put on tibolone for management of menopausal symptoms . women received 2.5 mg of tibolone at night with dinner orally for variable period of time depending on their need [ tables 15 ] . the study included the analysis of a range of variants such as : indications for tibolne therapy age distribution of women effectiveness of tibolone therapy analysis of side - effects indication for tibolone therapyage of womanduration of therapyeffectivenessside - effects indication for tibolone therapy the indications for tibolone therapy included prophylactic useurological problemsvulvovaginal symptomsskeletal symptomsreduced libidopsychological symptomsvasomotor symptoms the age groups were as follows - 25 - 30 years31 - 40 years41 - 50 years51 - 60 years61 - 70 yearsabove 70 duration of therapy was recorded.effectiveness of tibolone therapy was categorized as follows : excellentgoodsatisfactorypoorside - effects were recorded . the indications for tibolone therapy included prophylactic useurological problemsvulvovaginal symptomsskeletal symptomsreduced libidopsychological symptomsvasomotor symptoms vulvovaginal symptoms psychological symptoms the age groups were as follows - 25 - 30 years31 - 40 years41 - 50 years51 - 60 years61 - 70 yearsabove 70 duration of therapy was recorded . effectiveness of tibolone therapy was categorized as follows : excellentgoodsatisfactorypoorside - effects were recorded . the total number of women studied was 50 from april 2002 to april 2010 at the potdar hospital . surprisingly nobody reported side - effects , the ones mentioned here have been noted due to high degree of suspicion . surprisingly nobody reported side - effects , the ones mentioned here have been noted due to high degree of suspicion . pharmacology of tibolone - 2.5 mg tibolone when taken orally is quickly metabolized to 3-alpha and 3-beta hydroxy metabolites which bind solely to estrogen receptors , whereas delta-4 isomer has affinity for both progesterone and androgen receptors but not for estrogen receptors . the delta-4 isomer functions as progesterone in endometrial tissue , whereas in brain and liver it is androgenic . in the breast tissue , the main action is strong inhibition of sulfatase activity and weak inhibition of 17-beta hydroxysteroid dehydrogenase activity which prevents conversion of estrone sulfate to estradiol . and thus it was thought to reduce breast pain and density helping better mammographic interpretation as well as protection of breast from cancer . tibolone has an advantage over other forms of hormone replacement therapy as it is easy to use and does not induce withdrawal bleeding in postmenopausal women . the androgenic action of tibolone may help depression and libido more than any other form of hormone replacement therapy . tibolone has been considered and promoted as safe for the endometrium and breast and may provide some cardiovascular protection to these women . tibolone is the therapy of choice for postmenopausal women , more so in elderly women ( 65 years ) with contraindication for estrogen therapy like breast and endometrial cancer , hypertriglyceridemia and endometriosis . the mumbai cancer registry shows a sharp rise in the incidence of cancer with increasing age . in females the cancer of breast is by far the commonest cancer at all ages ( 21.2 / 1 lakh ) but its incidence peaks in the age group 65 - 69 years ( 149.3 / 1 lakh ) . the present study shows that tibolone is very effective in the management of menopausal symptoms , 94% showing excellent response . it is a very well tolerated drug ; only weight gain of 1.5 to 2 kg was seen in most women when the therapy period was of about two years [ table 6 ] . weight gain during tibolone therapy the significant finding of the present study was the development of carcinoma breast in one of the subjects who needed tibolone for more than five years [ table 7 ] . although tapering was repeatedly tried because of recurrence of menopausal symptoms , it had to be restarted . incidence of cancer breast in women who are nonusers of estrogen - progesterone therapy ( eprt ) as against women who use this therapy for 5 to 15 years it appears that this malignancy was as a consequence of prolonged tibolone therapy . it is possible that aromatase in breast tissue which is not suppressed by tibolone might have led to the formation of a significant amount of estradiol in the breast . similar findings were found in the librate study where women treated for cancer breast were put on tibolone therapy for management of menopausal symptoms . the study was completed in april 2010 and showed recurrence of cancer breast with no protection offered by tibolone . the million women study also showed that tibolone had nearly 1.5 times the risk of breast cancer as compared to women who never used hormone replacement therapy . whereas the thebes study showed that the tibolone group had a 3.1fold decrease in risk of invasive breast cancer . the only drawback of this study is that the follow - up is only of two years which is not enough to give reliable results . tibolone has an advantage over other forms of hormone replacement therapy as it is easy to use and does not induce withdrawal bleeding in postmenopausal women . the androgenic action of tibolone may help depression and libido more than any other form of hormone replacement therapy . tibolone has been considered and promoted as safe for the endometrium and breast and may provide some cardiovascular protection to these women . tibolone is the therapy of choice for postmenopausal women , more so in elderly women ( 65 years ) with contraindication for estrogen therapy like breast and endometrial cancer , hypertriglyceridemia and endometriosis . the mumbai cancer registry shows a sharp rise in the incidence of cancer with increasing age . in females the cancer of breast is by far the commonest cancer at all ages ( 21.2 / 1 lakh ) but its incidence peaks in the age group 65 - 69 years ( 149.3 / 1 lakh ) . the present study shows that tibolone is very effective in the management of menopausal symptoms , 94% showing excellent response . it is a very well tolerated drug ; only weight gain of 1.5 to 2 kg was seen in most women when the therapy period was of about two years [ table 6 ] . weight gain during tibolone therapy the significant finding of the present study was the development of carcinoma breast in one of the subjects who needed tibolone for more than five years [ table 7 ] . she was put on tibolone for menopausal symptoms . although tapering was repeatedly tried because of recurrence of menopausal symptoms , it had to be restarted . incidence of cancer breast in women who are nonusers of estrogen - progesterone therapy ( eprt ) as against women who use this therapy for 5 to 15 years it appears that this malignancy was as a consequence of prolonged tibolone therapy . it is possible that aromatase in breast tissue which is not suppressed by tibolone might have led to the formation of a significant amount of estradiol in the breast . similar findings were found in the librate study where women treated for cancer breast were put on tibolone therapy for management of menopausal symptoms . the study was completed in april 2010 and showed recurrence of cancer breast with no protection offered by tibolone . the million women study also showed that tibolone had nearly 1.5 times the risk of breast cancer as compared to women who never used hormone replacement therapy . whereas the thebes study showed that the tibolone group had a 3.1fold decrease in risk of invasive breast cancer . the only drawback of this study is that the follow - up is only of two years which is not enough to give reliable results .

accessory and ectopic liver lobes have a low incidence and are usually identified during imaging studies or autopsy . they are classified as accessory liver lobes when connected to the liver by a stalk and as ectopic liver tissue when there is no connection . reports of ectopic liver are rare although this entity has been reported in the thorax , lung , gallbladder , pancreas , umbilicus , adrenals , pylorus , and inferior vena cava . in the abdomen , patients rarely manifest physical complaints , unless complications occur , such as torsion , bleeding , or obstruction . a 47-year - old male was diagnosed with malignant melanoma of the scalp in may 2015 at our tertiary care hospital . in reviewing treatment options , he underwent imaging surveillance , including an abdominal computed tomography . this showed a 2.6 cm enhancing mass adjacent to the fundus of the stomach and immediately below the diaphragm [ figure 1a and b ] . the radiology differential included a gastrointestinal stromal tumor ( gist ) , splenule , and metastatic melanoma . the patient had opted to receive biochemotherapy for his primary melanoma , and a biopsy of the perigastric mass was needed to exclude a metastasis . ( a and b ) computerized tomography scans of the abdomen showing a subdiaphragmatic mass ( shown with arrow ) . ( d ) clusters of polygonal cells with distinct borders , smooth nuclear contours , and abundant cytoplasm containing pigment ( diff - quik , 40 ) . ( e ) cell block showing hepatic tissue with portal tracts ( h and e , 20 ) . ( f ) hepatic tissue confirmed by arginase-1 immunostain endoscopic ultrasound ( eus ) identified a 2.5 cm oval mass in the perigastric space below the diaphragm , without definite involvement of the stomach . five passes were performed with a 22-gauge shark core needle ( medtronic , dublin , ireland ) to maximize tissue acquisition [ figure 1c ] . rapid on - site evaluation ( rose ) was performed by a cytopathologist . after reviewing the first pass , the cytopathologist communicated with the endoscopist to ask if the needle had passed through the liver . the endoscopist confirmed that the needle was not in or near the liver and that it had been passed through the stomach to obtain the biopsy . small tissue fragments were touched on the slide , and those which did not smear were placed in formalin for the cell block preparation . diff - quik smears of the second fine needle aspiration ( fna ) demonstrated flat sheets , single cells , and small clusters of polygonal cells with distinct cell borders , round to ovoid centrally placed nuclei with some size variation , but smooth nuclear contours , open chromatin , and no pleomorphism . the cell block confirmed this , demonstrating small core fragments of hepatic parenchyma with portal tracts . immunohistochemistry for arginase-1 confirmed that this was hepatic tissue [ figure 1e and f ] and a reticulin stain demonstrated normal hepatic plates ( figure not shown ) . the cytologic and histologic findings , in conjunction with the imaging , were consistent with ectopic liver . the differential diagnosis of an incidentally discovered perigastric mass is broad and includes both benign and neoplastic entities . benign primary lesions and neoplasms include , but are not limited to , leiomyoma , schwannoma , neurofibroma , gist , lymphoma , and carcinoid . the top clinical differential included gist or metastatic melanoma . although it is a rare entity , it is important to realize that ectopic liver is in the differential of abdominal and perigastric lesions . in our case , the fna diff - quik smear demonstrated cytology that appeared consistent with benign hepatocytes during rose , which raised the possibility of ectopic liver . however , confirmation that this was not one of the other entities in the differential was critical , especially since they can have overlapping cytologic features . epithelioid gists , for instance , may be admixed with or entirely lack spindle cells . they usually demonstrate intermediate - sized cells with round to occasionally irregular nuclei , intranuclear inclusions , some binucleations , and scant to moderate clear to granular cytoplasm , which tends to get pulled away from the cell clusters in wispy strands . cells may be spindled , epithelioid , or mixed , and while they may demonstrate frank dysplasia , with large nuclei , prominent nucleoli , and abundant cytoplasm , they can also look bland , and like hepatocytes and epithelioid gists , binucleation , intracytoplasmic vacuoles , and intracytoplasmic pigment are occasional findings . rose , with active intraprocedural communication between the cytologist and the endoscopist , was extremely helpful in this case to rule out the possibility of liver contamination . when cells that appeared to be hepatocytes were noted on the first few passes , there was an immediate discussion with the endoscopist about the placement of the needle . it was confirmed the needle passed through the stomach to obtain the biopsy , and was within the perigastric mass , far from the liver . confirmation that the lesion consisted of benign hepatocytes was ultimately facilitated by use of a new eus needle ( shark core ) designed to give either generous cytology samples by touch prep or core biopsy material for the cell block . in addition to confirming the presence of hepatic tissue , it was also useful to exclude the possibility of hepatocellular carcinoma , which has been reported in ectopic liver . one pilot study on this device currently in press was performed at our institution and showed that the device performs very well when compared to a standard eus - fna needle in terms of yield and reduced number of passes . in summary , the cytology from the material biopsied by fna may have overlapping features with other entities more commonly considered in the differential of perigastric masses . rose is helpful to communicate with the endoscopist about placement of the needle . from our literature review , while fna of presumed ectopic liver within the thorax has been reported , ours is the first report of perigastric ectopic liver diagnosed by fna . da performed the endoscopy , was involved in the clinical management of the patient , and drafted portions of the manuscript . gb and ref performed both the on site evaluation of the case , and arrived at the final diagnosis . as this is a case report without identifiers , our institution does not require approval from the institutional review board ( irb ) . eus - endoscopic ultrasound eus - fna - endoscopic ultrasound - guided fna fna - fine needle aspiration gist - gastrointestinal stromal tumor ivc - inferior vena cava rose - rapid on - site evaluation . to ensure the integrity and the highest quality of cytojournal publications , the review process of this manuscript was conducted under a double blind model ( authors are blinded for reviewers and vice versa ) through automatic online system .

in type i diabetes , the insulin - producing islets of langerhans , scattered throughout the pancreas , are selectively destroyed by an apparent autoimmune process . daily and often multiple injections of exogenous human recombinant insulin are the current standard of care . despite continual advances in insulin therapy and the fact that some patients have now survived for more than 50 years with insulin therapy , the quality of life can be difficult owing to the nonphysiological delivery of an insulin bolus . the transplantation of the entire human pancreas from a deceased organ donor provides another therapeutic option for diabetic patients , especially , for those patients already receiving immunosuppressive therapy for a kidney allograft . alternatively , the insulin - producing islets can be isolated from the majority of the pancreas and transplanted alone as free islets . as noted above , a necessary component of the transplantation of an allogeneic pancreas or the islets alone because complications from such immunosuppressive therapy can include increased susceptibility to infections , malignancy , neurotoxicity , and nephrotoxicity , allotransplantation must be carefully considered and is often not suitable for young patients . further , the availability of human donor pancreas is extremely limited with only about 2,000 pancreas donors per year . worse , only about 16% of procured pancreases meet the criteria for transplantation and less than 3% are used for islet isolation [ 4 , 5 ] . to overcome the major hurdles of organ availability and immunosuppression , we have reported the ability of porcine islets , encapsulated in an agarose - agarose macrobead , to restore normoglycemia in diabetic animal models . the use of pigs as an islet source provides an unlimited number of organs for islet isolation and encapsulation . further , porcine insulin is nearly identical to human insulin , differing in only a single amino acid and it has been used in the clinic since its discovery in 1922 . the double layered agarose encapsulation of the porcine islets also provides at least some protection from recipient immune responses to the xenogeneic islets and is likely to ameliorate the need for immunosuppressive therapy . in fact , we have previously shown the porcine islet macrobeads to enable the discontinuation of exogenous insulin for more than 6 months ( study termination ) in spontaneously diabetic bb rats without immunosuppressive therapy . the current study was initiated to further investigate the effects of multiple implantations of encapsulated porcine islets to pancreatectomized diabetic dogs ( n = 3 dogs that received islet macrobeads and n = 2 dogs that received exogenous insulin only ) . because human diabetic patients implanted with macrobeads would be chronically exposed to porcine islets , we further set out to assess the viral safety of the macrobeads before implantation and also screened various canine tissues for evidence of viral transmission after more than 2 years of porcine islet macrobead exposure . a total of six male devocalized beagle dogs ( ridgelan farms , inc . , mount horeb , wi ) were received at approximately 20 weeks of age . the dogs were individually housed in stainless steel cages , 32 w 42 l 32 h , with rubber - coated mesh flooring and a stainless steel platform , 32 40. science diet growth ( hill 's , 6730 ) was provided twice daily and clean municipal water provided ad libitum . viokase ( henry schein , 9758341 ) powder was added to food at a dose of 1 tsp / meal following pancreatectomy . a 12-hour light cycle was maintained throughout the study with lights on at 0700 hours . all study protocol procedures were approved by the rogosin institute animal care and use committee ( iacuc ) . the rogosin institute - xenia division animal facility holds full accreditation status awarded by the association for assessment and accreditation of laboratory animal care , international ( aaalac , int . ) . donor islets were prepared from newsham sows over two years of age and with multiple parities . after electrical stun and exsanguination ( bob evans farms , xenia , oh ) , pancreata were retrieved and transported to the islet isolation laboratory in cold hank 's balanced salt solution ( hbss ; mediatech , 21 - 020 ) on ice . warm ischemia times averaged 12.26 minutes while cold ischemia times ranged from 30 minutes to one hour . islet isolation was carried out as previously described . briefly , after trimming the gland of fat and connective tissue , the main pancreatic duct was cannulated and injected with hbss containing 2.0 g / l collagenase p ( roche , 11213873001 ) and 0.01 g / l dnase ( sigma aldrich , d5025 ) . twice the gram weight of the pancreas ( in ml ) was perfused through the pancreatic duct at a rate of 50 ml / min at 18c . , 99 - 408)-ficoll ( sigma aldrich , f9378 ) gradients of densities 1.105 g / cm , 1.095 g / cm , and 1.055 g / cm in 50 ml polystyrene conical tubes . the tubes were centrifuged at 2000 rpm , and islet - containing layers were manually collected . islet macrobeads were maintained in rpmi containing 2.5% porcine serum ( mediatech , 35 - 041 ) and 1% antibiotic / antimycotic ( a / a ; gibco life technologies , 15240 ) in an atmosphere of 35% co2 and air at 3638c . macrobeads were examined for uniformity , collected the day prior to implant , and were aliquoted to 175 ml conical tubes . a maximum of 400 macrobeads per tube were stored overnight at room temperature ( 1828c ) in rpmi ( gibco life technologies , 22400 ) containing 1% a / a . prior to islet macrobead implantation , representative samples of macrobeads and culture media were sent to microtest laboratories , inc . ( agawam , ma ) for product bioburden testing ( ansi / aami / iso 11737 - 1 part 1 ) ; see table 1 . macrobeads were aseptically crushed and extracted with fluid d ( emd millipore , 1.46483.0006 ) while the culture media was tested as supplied . using usp 61 membrane filtration , each sample was filtered onto a 0.45 m membrane ( emd millipore , hvlp ) , which was then transferred to tryptic soy agar ( tsa ) plates and cultured at 32.5c 2.5c for 35 days ( aerobic , anaerobic , and aerobic spore formers ) . additional samples were filtered onto 0.45 m membranes , which were transferred to sabouraud dextrose agar ( sda ) plates and incubated at 22.5c 2.5c for 57 days ( yeast and mold ) . a usp 62 microbial limits test for salmonella sp . was also performed by incubation of samples in lactose broth for 48 hours at 3035c , aliquoted 1 ml / tube to sc / tt tubes , followed by an additional 24 hours at 32.5c 2.5c . upon completion of incubation , contents were streaked to brilliant green agar ( bga ) , xylose lysine desoxycholate ( xld ) agar , and bismuth sulfite ( bs ) agar plates for 48 hours at 32.5c 2.5c . all bioburden testing included negative media controls and appropriate positive media controls ( escherichia coli [ atcc # 11229 ] , aerobic ; clostridium sporogenes [ atcc # 11437 ] , anaerobic ; bacillus subtilis [ atcc # 6633 ] , aerobic sporeformer ; candida albicans [ atcc # 10231 ] yeasts and molds ) . colonies were counted using a reichert quebec darkfield colony counter . for each class of organism , the total recoverable bioburden as cfu / sip was calculated ( culture media sip = 5 ml per category ; macrobead sip = 1 bead ) . culture media and islet macrobeads were also sent to iowa state university , veterinary diagnostic laboratory ( vdl ; ames , ia ) for additional microbiological testing including porcine virology prior to macrobead implantation , as summarized in table 1 . porcine virology tests included : porcine reproductive and respiratory syndrome virus ( prrsv ; rt - pcr using qiagen , 282315 ) , swine influenza virus ( siv ; rt - pcr using qiagen , 282615 ) , porcine endogenous retrovirus ( perv ; rt - pcr ) , porcine enterovirus ( pev ; rt - pcr ) , porcine respiratory corona virus ( prcv ; rt - pcr using qiagen , 283615 ) , transmissible gastroenteritis virus ( tgev ; rt - pcr using qiagen , 283615 ) , porcine circovirus ( pcv ; pcr ) , porcine lymphotropic herpes virus type-1 ( plhv-1 ; pcr ) , swine hemagglutinating encephalomyelitis virus ( shev ; rt - pcr ) , porcine parvovirus ( ppv ; pcr ) , porcine cytomegalovirus ( pcmv ; pcr ) , mycoplasma hyopneumoniae ( pcr ) , pseudorabies virus ( prv ; virus isolation ) , encephalomyocarditis virus ( emcv ; virus isolation ) , group a rotavirus ( antigen capture elisa ) , and chlamydia spp . each rt - pcr / pcr assay included a positive control , a no template control , and was performed according to standard operating procedures of vdl . at the time of the surgical procedure for the second macrobead implant ( day 215 post-1st implant ) , or sham surgery for non - implant controls , white blood cells and biopsies of mesenteric lymph node and tonsil samples were shipped on dry - ice to bioreliance ( glasgow , scotland ) for assessment of perv by quantitative real - time pcr ( qrt - pcr ) . the appropriate controls including : positive controls ( negative control nucleic acid spiked with viral target sequence ) , post - extraction spike controls ( to assess target specific pcr inhibition ) , exogenous internal positive controls ( to establish that all negative pcr results were truly negative and not due to failed amplification ) , no - template controls ( to monitor aerosol and reagent contamination ) , negative controls ( to monitor reagent contamination ) , and sentinel extraction controls ( to assess possible airborne sample to sample cross - contamination ) were conducted in triplicate with each assay . at necropsy , samples of retrieved islet macrobeads and swabs of the peritoneal cavity were sent for sterility testing per usp 71 via membrane filtration and product immersion to microtest laboratories , inc . using a direct cultivation and cell culture method , retrieved islet macrobeads were screened for the presence of mycoplasma ( microtest laboratories , inc . , per the fda points - to - consider in the characterization of cell lines used to produce biologicals ) . macrobeads were also screened for the presence of bacterial endotoxin by microtest laboratories , inc . following the procedure depicted in the fda 1987 guidance for bacterial endotoxin testing . samples of retrieved macrobeads , white blood cells , bone marrow , mesenteric lymph node , small intestine , liver , kidney , heart , lung and central nervous system ( brain ) were collected aseptically , snap - frozen , and sent to bioreliance for viral screening . virus selection was based on their ubiquitous nature in swine ( semcv , plhv , shepe , pcv , pcmv , and perv ) , or their specific presence in source animals ( pcv , perv ) , or their trans - species infectivity potential ( semcv , plhv , shepe , pcmv , and perv ) , and clinical significance ( all ) . samples were assessed by qrt - pcr for the presence of specific porcine nucleic acid sequences from both control dogs and islet macrobead implanted dogs . probe sequences used in the viral screening process are proprietary but are available upon specific request . testing was conducted in accordance with the principles of good laboratory practice and under the regulations described in the united states federal register 21 cfr part 58 . animals were sedated with acepromazine at a dose of 0.05 mg / kg i m ( henry schein , 356 - 7290 ) and anesthetized with ketamine at a dose of 5 mg / kg ( fort dodge animal health , 9952949 ) , diazepam at a dose of 1 mg / kg ( butler , gnr04054 ) and isoflurane gas at 13% ( henry schein , 982 - 2413 ) . during surgery all animals received buprenorphine ( butler , gnr02300 ) at doses of 0.0050.020 mg / kg i m during surgery and every 812 hours after surgery , continuing for 2 - 3 days ; and cefotaxime ( claforan antibiotic ; henry schein , 852 - 1633 ) at a dose of 20 mg / kg iv during and 8 hours after surgery . at implant , the abdominal , subcutaneous , and cutaneous incisions were closed with 3 - 0 vicryl ( ethicon , j333 ) , 3 - 0 vicryl , and 3 - 0 nylon ( ethicon , j669 ) sutures and 6.70 mm 3.90 mm precise skin staples ( 3 m health care ) , respectively . at pancreatectomy , the abdominal , subcutaneous , and cutaneous incisions were closed with 2 - 0 vicryl ( ethicon , j332 ) , 4 - 0 polymend ( veterinary products laboratories , v-397 - 1 ) , and 3 - 0 nylon ( ethicon , j669h ) sutures , respectively . fourteen weeks following arrival , all animals underwent pancreatectomy for the induction of insulin - dependent diabetes . the pancreas was exposed by a peritoneal midline incision and removed with blunt dissection . following induction , animals were maintained on humulin 70/30 insulin ( eli lilly , 029363 ) at doses to attempt morning ( 0900 hr ) and evening ( 1800 hr ) normoglycemia . at the time of pancreatectomy , three animals received a 1st implant of porcine islet macrobeads at a dose equivalent to 4 daily insulin requirements . the three dogs that received the first implant underwent a second implant ( dose = 3 ) of islet macrobeads 215 days after the 1st implant . insulin requirements of recipients and the corresponding number of porcine islet macrobeads received are given in table 3 . at the time of the second implant , nonimplanted control animals received a sham surgery . animals receiving porcine islet macrobeads were started on pravastatin ( 80 mg / day ; teva pharmaceuticals ) 48 hr prior to a 1st implant and continued daily throughout the course of study . because the in vivo environment of the dog abdomen would likely impair insulin production from implanted macrobeads as compared to the carefully controlled in vitro culture environment , we opted to implant dogs with enough macrobeads to provide 4 times ( 4 dose for the 1st implant ) or 3 ( 3 dose for the 2nd implant ) the amount of each dog 's daily exogenous insulin requirement . insulin production during a 24 hr period of in vitro culture was determined once a week prior to macrobead implantation for each batch of islet macrobeads ( see below ) so that an average quantity of insulin secretion per macrobead in the four weeks prior to each implant could be calculated ( see table 3 ) . the daily exogenous insulin requirements of each macrobead implanted dog were multiplied by 4 ( first implant ) or 3 ( second implant ) and enough macrobeads implanted to provide either a 4 or 3 dose of insulin . as insulin production varies with every batch of islet macrobeads , each preparation of islet macrobeads was evenly divided amongst the different recipient dogs such that each animal received a proportion of each batch of macrobeads based on exogenous insulin requirements . for the first implant , each dog received 11181380 porcine islet macrobeads with a total macrobead weight of 0.310.36 kg . for the second macrobead implant each dog received 10961904 porcine islet macrobeads weighing 0.290.49 kg . average macrobead age ( time after isolation and encapsulation ) was 13.8 weeks for all dogs at the time of the first implant and 14.1 weeks for the second implant . a peritoneal midline incision was made and the site of pancreatectomy was examined for the presence of any residual pancreatic tissue just prior to placing the macrobeads into the peritoneal cavity . immediately prior to implant , macrobeads were washed three times with rpmi containing 1% a / a and were gently placed into the peritoneal cavity by use of a sterile plastic spoon . as needed , animals were manually fed hill 's prescription diet a / d ( 5670 ) , hill 's canned science diet growth ( 6680 ) , and/or nutri - cal ( evsco pharmaceuticals , 01311 ) to maintain normoglycemia during the first 2448 hours after implant . observations included appetite , bowel movements , body weight , blood glucose ( accu - chek simplicity bg monitor and chemstrips ; maximum value of 600 mg / dl : roche diagnostics ) , urine glucose , and insulin therapy . intraperitoneal fluid was collected during a glucose / arginine challenge procedure and samples tested for the presence of porcine c - peptide as follows . following light anesthesia , an iv catheter was inserted into the peritoneal cavity and 10 ml / kg of a 16.7 mm glucose and 5 mm arginine solution were injected . this infusion procedure was carried out for two of the implanted dogs ( fox-1 and kox-1 ) . hsx-1 received the glucose / arginine challenge , though intraperitoneal fluid was only collected from the initial 3035 minute timepoint following challenge . standard radioimmunoassays from linco research , inc . were used for the detection of porcine insulin ( pi-12 k , sensitivity of 2 u / ml ) and porcine c - peptide ( pcp-22 k , sensitivity of 0.1 ng / ml ) . assays were run according to the manufacturer 's instructions with samples in duplicate and reference , standards , and controls in triplicate . complete necropsies were performed on day 867 after 1st implant of porcine islet macrobeads . following anesthesia , the collection of blood , and exsanguination , macrobeads were randomly retrieved for virology ( n = 60 ; snap frozen , 6 macrobeads / tube ) , histopathology ( n = 50 ; fixed in 10% neutral buffered formalin ( nbf ) , 5 macrobeads / tube ) or microbiology ( n = 12 ; snap frozen , 6 macrobeads / tube ) . tissue samples , including porcine islet macrobeads , were sent to bioreliance for viral screening or sent to pathology associates international ( pai , a charles river company ; west chester , oh ) for histopathology . at the time of pancreatectomy , both pancreas and omentum were fixed in 10% nbf for 24 hrs , washed , and stored in 70% ethanol . at necropsy , the following tissues were collected , fixed as above , and sent to pai : heart , spleen , liver , kidneys , brain , testes , duodenum , jejunum , ileum , mesentery , adrenal glands , stomach , lungs , diaphragm , abdominal musculature , bone ( sternum ) , spinal cord , sciatic nerve , epididymus , eyes , submandibular lymph nodes , bladder , muscle ( thigh ) , thymus , thyroid , and parotid salivary gland . tissues were embedded in paraffin , and 5 m sections were stained with haematoxylin and eosin ( h&e ) . samples were analyzed by a diplomat of the american college of veterinary pathology , and the macroscopic and histopathological findings were documented . culture media and macrobead samples were found to be negative for bacterial growth and salmonella sp . throughout the observation periods . prior to the first macrobead implant , 6 macrobead preparations and samples of the 6 corresponding culture medias were screened for a panel of porcine viruses and microbial agents as described above and shown in table 1 . prior to the second macrobead implant , 2 randomly selected macrobead preparations and samples of the 2 corresponding culture media were screened for the same panel of porcine viruses and microbial agents as the first implant macrobeads . macrobead and media samples were positive for perv and negative for all other screened agents . mean daily insulin requirements between weeks 4 - 5 ranged from 11 to 20 iu per dog ( figure 1 ) . the three implanted dogs remained insulin - free for 1214 days after the first implant of islet macrobeads , at which time the animals were started on 4 units of insulin in the morning and 4 units in the evening in response to rising blood glucose levels ( figure 1(b ) ) . exogenous insulin was gradually increased over the next 1 - 2 weeks until blood glucose levels stabilized . the 2 control dogs did not undergo macrobead implantation or sham surgery at the time of the first implant . following the second islet macrobead implant , the three implanted dogs began exogenous insulin therapy on day 9 ( kox-1 ) or 21 ( fox-1 and hsx-1 ) . presecond implant insulin requirements were reestablished for the dogs after the 2nd implant on day 148 ( fox-1 ) , day 189 ( kox-1 ) , and > day 652 ( hsx-1 ) after 2nd implant . random , nonfasting porcine c - peptide was not detected in the serum of any study animals before pancreatectomy or prior to macrobead implantation nor in the two control dogs throughout the study . serum porcine c - peptide was , however , detected in the three macrobead implanted dogs after transplantation ( figure 2(a ) ) . fox-1 showed only minimal levels of c - peptide following both implant procedures with levels ranging from 0.10.2 ng / ml . c - peptide levels up to 0.31 ng / ml ( hsx-1 ) and 0.29 ng / ml ( kox-1 ) were found 12 - 13 days following the first implant . on days 21 - 22 following 2nd implant , c - peptide levels of 0.34 ng / ml ( hsx-1 ) and 0.45 ng / ml ( kox-1 ) were found . by days 82 - 83 post 2nd implant , only hsx-1 had any evidence of circulating porcine c - peptide ( 0.18 ng / ml ) . porcine c - peptide was detected in the peritoneal fluid of all three implanted dogs approximately 3 months prior to necropsy during an intraperitoneal glucose / arginine challenge procedure ( figure 2(b ) ) . serum c - peptide was not detected at this time in any of the three implanted dogs . control animals did not undergo the peritoneal challenge or fluid collection procedure . at necropsy , porcine c - peptide was detected in the peritoneal fluid of hsx-1 and kox-1 ( 0.41 ng / ml and 0.32 ng / ml , resp . ) but not in the peritoneal fluid from fox-1 . all animals lost body weight immediately after pancreatectomy . over the next several months , study animals regained weight and surpassed preinduction levels . a slight increase in body weight was also observed in the immediate period following both macrobead implantations , as a result of macrobead weight and fluid therapy associated with the surgical procedures . complete necropsies were performed on all study animals on day 867 after macrobead implant . during the exploration of the peritoneal cavity it was noted that the majority of the macrobeads were free - floating and intact . macrobeads were occasionally found to be either entangled in residual omentum tissue or contained within the pelvic omentum - like tissue ( 510% of macrobeads ) . nonetheless , these entrapped macrobeads were free - floating within their tissue cavities . few macrobeads from all three implanted dogs were also found to be lodged between the lobes of the liver . only hsx-1 had any evidence of a broken macrobead(s ) : a few agarose macrobead fragments were observed embedded in a lobe of the liver . no broken macrobeads were observed in the other two implanted dogs . diffuse fibrotic plaques and mesothelial hyperplasia were common findings on the serosal surface of the abdominal wall and abdominal tissues . the proliferative lesions were limited to the serosal surfaces and did not appear to extend into organ parenchyma cells at sectioning . macroscopic views of mesentery from implanted and control dogs were nearly identical ( figure 3 ) demonstrating the biocompatibility of the agarose macrobeads . various lobes of the liver from all 3 implanted dogs presented with occasional indentations due to the presence of macrobeads . during the surgical procedure at the time of second macrobead implant or sham surgery ( control dogs ) , biopsies of tonsil and mesenteric lymph node and white blood cells were collected from all study animals for assessment of perv . at necropsy , nine tissues ( bone marrow , c.n.s ( brain ) , mesenteric lymph node , small intestine , heart , kidney , liver , lung , and pbmcs ) and islet macrobeads were collected and screened by pcr for the presence of 6 different viruses . all tissues were negative for the presence of pcmv , plhv , perv , shev1 and shev2 , and semcv . perv specific sequences were detected in all islet macrobead samples . in samples from both control and implanted dogs , low level amplification signals for pcv were found in a number of different tissues suggestive of a cross - reaction with canine sequences . tissues and macrobeads collected at necropsy were processed and examined by a board - certified pathologist . viable islet cells were not found in the macrobeads examined from the three implanted dogs . some macrobeads were surrounded by a proliferative inflammatory capsule characterized by a minimal to mild degree of fibrous collagen tissue . peritoneal organs and tissues with a serosal surface had minimal to mild proliferative and inflammatory lesions . the lesions were composed of fibrous connective tissue covered with a thin layer of mesothelium or mesothelial cells . the results of this study demonstrate the safety of transplanting agarose - agarose porcine islet macrobeads in a fully discordant large animal model as a treatment for insulin dependent diabetes . importantly , specific immunosuppressive therapy was not administered , although pravastatin was given for its mild anti - inflammatory properties . all three dogs that received porcine islet macrobeads were insulin - free for approximately 2 weeks following the first implant procedure . preimplant insulin requirements were reached over the following week such that by three weeks after implantation all animals were again on full insulin requirements . remarkably , the implanted dogs did not reach pre - insulin requirements following the second implant , until significantly longer periods of time . in fact , one animal still had not reached preimplant insulin requirements , at necropsy , on day 652 following 2nd implant . a significant issue with any xenotransplantation procedure is the possibility of xenozoonosis . in the case of porcine islets , porcine endogenous retrovirus ( perv ) is encoded throughout the porcine genome , and because it is unlikely to be removed , this virus has generated considerable attention of the xenotransplantation community and regulatory authorities ( see review by denner and tonjes ) . although perv was shown to infect human cells in vitro , there is no evidence of transmission to humans including farmers , veterinarians , or abattoir workers , all of whom are exposed daily to numerous porcine secretions , excretions , blood , and tissues . more than 80 years of using porcine insulin in the clinic has also not revealed any transmission of perv . preclinical studies in nonhuman primates have also not detected any evidence of perv transmission even with high dose immunosuppression and high titers of perv . also assuring are the negative human clinical findings of perv transmission to date including burn patients treated with porcine skin , patients with hemophilia treated with porcine factor viii , and patients receiving extracorporeal porcine hepatocyte therapy [ 2224 ] , as well as from two clinical trials of porcine islets following implantation [ 25 , 26 ] . nonetheless , the accidental transmission of animal viruses and perv in particular mandate a carefully thought out strategy to manage such risks [ 27 , 28 ] . the ability of the islet macrobeads to survive extended culture periods up to at least one year provides ample opportunity to not only determine the insulin production from each batch of macrobeads but to also screen the macrobeads for evidence of microbiological and viral pathogens . we have recently demonstrated the proliferation of macrobead encapsulated porcine insulin - producing cells over the first few weeks of culture . during this time , this allows a determination of the precise insulin - producing capacity of encapsulated islets and the subsequent implantation of a patient - tailored dose of macrobeads to meet individual insulin demands . as noted above , the islet macrobeads are also routinely screened for numerous pathogens prior to implantation into study animals . traditional organ allografting techniques do not allow for the extended testing times required to assure the absence of xenorelated infectious diseases , and until now , the promise of islet xenotransplantation has been largely unrealized by this restraint . we consider this an advanced technique to the antemortem screening of donor animals because the tissue to be transplanted can be tested directly . this is a significant technological advance and one with the potential to finally make xenotransplantation a reality . furthermore , additional patient safety is obtained by not requiring the immunosuppression of macrobead recipients . similar to the findings discussed above , we have not found any evidence of viral transmission , including perv , in diabetic rats implanted with porcine islet macrobeads . in the current study , viral transmission was also not found in biopsies of mesenteric lymph node tissue and tonsil tissue and peripheral blood mononuclear cells on day 215 after the 1st implant ( immediately prior to implanting the second dose of macrobeads ) . additionally , numerous tissues were collected at necropsy from all study dogs . at this time , islet macrobead implanted dogs had been exposed to porcine tissue for 2.4 years . all tissues were found to be negative for the screened viral sequences although some low level amplification was observed for pcv in some tissues of all dogs including the nonimplanted control animals . as low level amplification signals for the pcv target were detected in both negative control and macrobead implanted animals , it is likely that this finding is due to a cross - reaction with canine specific sequences . the negative findings from the extensive viral screening of the islet macrobeads prior to implantation also support the notion that porcine islet macrobead implantation , under the conditions of this study , can be performed safely and does not result in trans - species viral transmission . consistent with a previous study , the islet macrobeads were found to be extremely biocompatible . at necropsy , most islet macrobeads were intact and free - floating in the peritoneal cavity . perhaps the most impressive finding at necropsy was the mild degree of inflammatory reaction in the peritoneal cavity which was not thought to impair organ function . furthermore , these data also demonstrate the capacity of the abdomen to accommodate a large number of macrobeads . ongoing improvements in the insulin secretion capacity of each islet macrobead continue to allow for the implantation of fewer macrobeads in order to replace exogenous insulin . finally , current clinical trials of a similarly sized mouse renal adenocarcinoma - containing agarose macrobead for the inhibition of various cancers have shown the human abdomen to easily house several thousand such macrobeads [ 32 , 33 ] . these data support the feasibility of transplanting agarose - agarose porcine islet macrobeads as a treatment strategy for insulin dependent diabetes mellitus . the ability to incubate the porcine islet macrobeads for extended periods in order to prescreen the islets for numerous pathogens further advances the safety profile of this therapy . critically , no evidence of viral transmission was observed after long - term implantation in diabetic study animals .

it is estimated that about 30 to 100 million people in endemic areas of the world are infected , especially in tropical and subtropical countries ( 1 ) . it has variable manifestations from asymptomatic to hyperinfection , or disseminated infection ( 2 ) . most cases are completely asymptomatic ; some patients have mild gastrointestinal , cutaneous , or pulmonary symptoms with or without fever ( 1 ) . gastrointestinal and pulmonary symptoms are common in acute strongyloidiasis ( 3 ) . presence of eosinophilia is the most important laboratory finding in strongyloidiasis patients ( 4 ) . chronic strongyloidiasis causes mild clinical manifestations , but in patients receiving corticosteroid treatment and immunosuppressive therapy or having diseases such as diabetes and hematologic malignancies the disease may change to severe complicated strongyloidiasis ( 5 ) ; and larvae outside the usual migration pattern invade virtually every organ ( 6 , 7 ) . strongyloidiasis is still endemic in some provinces of iran . in 2007 , 4.9% of people in rural areas of mazandaran province were reported infected with this parasite ( 8) . in a recent study in gilan province 42% of a population with eosinophilia were found positive for infectivity with s. stercoralis ( 4 ) . in a mentally retarded institution of southern iran 17.3% of the residents were found infected with this nematode ( 9 ) . s. stercoralis hyperinfection cases have been documented in immunocompromised patients ( 10 - 13 ) , and even leading to the death of the patient ( 11 ) . nevertheless , in spite of recent frequent reference of suspected patients to diagnostic referral centers , analytical description of indigenous patients is lacking . therefore , this paper aims to analyze demographic and clinical characteristics of a large series of strongyloidiasis patients from endemic areas of iran . helminthological laboratories of school of public health ( sph ) , tehran university of medical sciences ( tums ) and school of medicine , gilan university of medical sciences ( gums ) are two referral centers for diagnosis of helminth infections in iran . most admitted patients either coming by themselves or are referred by physicians from private clinics , health care units or hospitals due to the presence of eosinophilia in peripheral blood , clinical symptoms and immunocompromised conditions . in this retrospective descriptive study , the medical records of patients attended these centers during 2009 to 2013 were reviewed . patients who were registered positive for s. stercoralis were identified ; among them anyone whose clinical characteristics and demographic data were completely available was included in this analytical study . there were also some s. stercoralis infected cases with incomplete information ; most of them related to patients who could not refer to the centers by themselves due to long distance , elderliness , hospitalization and so on . identification of s. stercoralis infection in these centers was based on coprological examinations and detection of rhabditiform or filariform larvae by either normal saline direct smear , formalin - ether concentration technique ( fect ) or by nutrient - agar plate culture . for nutrient - agar plate culture , about 3 g of fresh stool sample was placed in the center of the dish , incubating at 26 - 30 c for 48 - 72 h. then , the plates were examined by stereomicroscope and in case of the presence of larvae or their tracks the identification of s. stercoralis and differentiation from other possible nematodes were performed as explained by moghaddassani et al . . parasitic load of s. stercoralis infected patients were categorized in three ranks : -low infection : direct smear and fect negative , 1 - 4 larvae counted on agar plate surface ; -moderate infection : direct smear negative , fect positive , 5 - 10 larvae counted on agar plate surface ; -high infection : direct smear and fect positive , more than 10 larvae counted on agar plate surface . among third group , those cases with enormous number of larvae in their stool examinations ( fig.1 ) , and presence of severe pulmonary symptoms were also recorded as hyperinfection . a checklist was prepared and patients demographic information ( including sex , age , place of birth , current place of residency , travel history , clinical manifestations ( gastrointestinal , skin and pulmonary symptoms ) , relative eosinophil and risk factors were entered . data was analyzed by chi - square test ( x ) or fisher s exact test . t - test was used to compare average of eosinophil counts in asymptomatic and symptomatic patients . mann - whitny u test was used to compare parasitic loads in asymptomatic and symptomatic patients . helminthological laboratories of school of public health ( sph ) , tehran university of medical sciences ( tums ) and school of medicine , gilan university of medical sciences ( gums ) are two referral centers for diagnosis of helminth infections in iran . most admitted patients either coming by themselves or are referred by physicians from private clinics , health care units or hospitals due to the presence of eosinophilia in peripheral blood , clinical symptoms and immunocompromised conditions . in this retrospective descriptive study , the medical records of patients attended these centers during 2009 to 2013 were reviewed . patients who were registered positive for s. stercoralis were identified ; among them anyone whose clinical characteristics and demographic data were completely available was included in this analytical study . there were also some s. stercoralis infected cases with incomplete information ; most of them related to patients who could not refer to the centers by themselves due to long distance , elderliness , hospitalization and so on . identification of s. stercoralis infection in these centers was based on coprological examinations and detection of rhabditiform or filariform larvae by either normal saline direct smear , formalin - ether concentration technique ( fect ) or by nutrient - agar plate culture . for nutrient - agar plate culture , about 3 g of fresh stool sample was placed in the center of the dish , incubating at 26 - 30 c for 48 - 72 h. then , the plates were examined by stereomicroscope and in case of the presence of larvae or their tracks the identification of s. stercoralis and differentiation from other possible nematodes were performed as explained by moghaddassani et al . . parasitic load of s. stercoralis infected patients were categorized in three ranks : -low infection : direct smear and fect negative , 1 - 4 larvae counted on agar plate surface ; -moderate infection : direct smear negative , fect positive , 5 - 10 larvae counted on agar plate surface ; -high infection : direct smear and fect positive , more than 10 larvae counted on agar plate surface . among third group , those cases with enormous number of larvae in their stool examinations ( fig.1 ) , and presence of severe pulmonary symptoms were also recorded as hyperinfection . a checklist was prepared and patients demographic information ( including sex , age , place of birth , current place of residency , travel history , clinical manifestations ( gastrointestinal , skin and pulmonary symptoms ) , relative eosinophil and risk factors were entered . data was analyzed by chi - square test ( x ) or fisher s exact test . t - test was used to compare average of eosinophil counts in asymptomatic and symptomatic patients . mann - whitny u test was used to compare parasitic loads in asymptomatic and symptomatic patients . overall , 70 parasitologically positive indigenous cases of s. stercoralis were subjected in this descriptive analytical study . twenty eight patients were referred to the school of medicine ( gums ) , during 2010 - 2013 ; all of them being both native and resident of gilan province . the other 42 patients were referred to the school of public health ( tums ) , during 2009 - 2013 . the distribution of these people according to their place of birth is illustrated in fig 2 . not all these people were still residents of their native provinces , but rather 16 individuals ( 38.1% ) being immigrants to other provinces , majority ( 14 individuals ) to tehran . table 1 shows distribution of the patients based on the sex , age groups , and clinical manifestations . among all 70 patients described in this study , 43 people ( 61.4% ) the age of the patients varied from 29 years to 83 years of old , with the mean of 60.4 ( sd ) years . a total of 11 patients ( 15.7% ) were asymptomatic ; rest of them ( 59 ; 84.3% ) reported manifestation . the most complain was gastrointestinal symptoms , so that 50 patients ( 71.4% ) reported epigastria pain , abdominal discomfort , borborygmus , intermittent diarrhea and constipation , bloating , and nausea . although 18 patients ( 25.7% ) complained of cutaneous symptoms including itching and rash , but no larva currens eruption was recorded , even in cases of overwhelming infections . pulmonary symptoms including dyspnea , cough and sputum discharge were recorded in 11 patients ( 15.7% ) . determination of relative eosinophil count could be possible for 52 patients ; of them 51 patients ( 98.1% ) showed peripheral eosinophilia , ranging from 6% to 66% , with the mean of 22.7% . the patient without elevated eosinophils was a 63 old man with gastrointestinal disorders , suffering from colitis whose parasite load was high . the mean of relative eosinophilia among asymptomatic and symptomatic patients were 36.28 and 20.63 , respectively ; and this difference was significant ( p = 0.001 ) . there was statistically significant correlation between age and eosinophilia , so that its rate reduced with ageing ( r = -324 , p = 0.019 ) . in 22 patients ( 31.4% ) one or more predisposing conditions were known including diabetes ( 7 , 10% ) , chronic liver diseases ( 7 , 10% ) , chronic renal failure ( 5 , 7.1% ) , cancer ( 3 , 4.3% ) , heart valve replacement ( 2 , 2.8% ) , rheumatoid ( 2 , 2.8% ) , colitis ( 1 , 1.4% ) , bone marrow transplant ( 1 , 1.4% ) , and liver transplant ( 1 , 1.4% ) . five patients were hospitalized , among them a 64 years old lady with pulmonary distress and heart failure was recorded having disseminated infection . she improved after taking ivermectine , and her subsequent examinations for strongyloidiasis , over 13 month after medication were negative . among patients with high infection rate , 8 cases ( 11.4% ) showed hyperinfection syndrome ( 6 male , 2 female ) , as well . this patient was 60 years old male with hematologic malignancy , being under prolonged corticosteroid therapy . his infectivity with s. stercoralis was not noticed till the last day of his life . presence of clinical manifestations had positive association both with parasitic loads ( p = 0.007 ; mann - whitny u test ) , and immunocompromised conditions ( p = 0.015 ; fisher s exact test ) . s. stercoralis is considered as the most neglected among soil - transmitted helminth infections ( 15 ) . in the reports pertaining to previous decades , in temperate and subtropical areas of iran it was coexisted with hookworms , with lower prevalences ( 16 ) . with the sharp decrease in prevalences of most soil - transmitted helminthes during recent two decades in iran ( 17 ) , the actual prevalence of s. stercoralis in the country is unclear but the recent rare records ( 4 , 8 , 9 ) indicate that strongyloidiasis is still prevalent in the same previous known endemic areas , but with higher prevalences than hookworms . this issue is due to the ability of s. stercoralis for the establishment of autoinfection cycle in infected individuals for several decades , even whole life . for this reason s. stercoralis has age cumulative distribution , and is more prevalent in older population . complicated cases of strongyloidiasis , as a consequence of immunocompromised conditions , have also been reported recently ( 10 - 13 ) . additionally , increase of corticosteroid and other immunosuppressive therapy and predisposing risk factors has led to frequent reference of suspected people to referral centers for differential diagnosis . therefore , this descriptive analytical study deals with the largest series of indigenous iranian strongyloidiasis patients . considering the original provinces of the patients ( n = 70 ) in descending order of magnitude , gilan , mazandaran , khuzestan , boushehr , and hormozgan provinces comprised 64.3% , 30% , 2.9% , 1.4% , and 1.4% of patients , correspondingly . the first two provinces are bordering to caspian sea and the others to persian gulf ; therefore , suitable moist environment , especially heavy rainfall and temperate areas of the north , is in favor of s. stercoralis lifecycle . 38.6% ) which is compatible with other studies ( 18 ) is indication of greater exposure of male gender with source of infection as a result of working in farm , rice and tea field , outdoors activities , gardening and so on . the two most initiation reasons to refer the patients for diagnosis of strongyloidiasis to the above mentioned referral centers were peripheral eosinophilia and gastrointestinal symptoms . all the cases in the present study were selective and referred by themselves or by their physicians because of eosinophilia , clinical symptoms or predisposing factors . although 25.7% of patients complained of cutaneous symptoms , but similar to other studies in northern italy ( 20 ) , imported cases to spain ( 3 ) , and hyperinfection cases in iran ( 10 - 12 ) , none of them had larva currens eruption , even cases of overwhelming or disseminated infections . but , in a study on imported strongyloidiasis , it has been found mostly in white patients who acquired their infection in southeast asia ( 21 ) . future comparative molecular studies using isolates from patients with and without larva currens and from different geographical areas of the world will clarify the probable effect of different strains on this cutaneous symptom . based on the parasitic load , patients could be divided in three groups with low , moderate and high infection rate . statistically significant difference was found between presence of clinical manifestations and parasitic load ( p = 0.007 ; mann - whitny u test ) . greater clinical manifestations were coincident with increasing in parasitic load , therefore , leading to easier detection of infections . it could be considered as an important laboratory finding in patients with strongyloidiasis ( 4 , 23 - 25 ) . different studies reported various rates of peripheral eosinophilia for strongyloidiasis patients including 22.5% ( 3 ) , 63.6% ( 19 ) , 82.6% ( 21 ) and 90% ( 25 ) . in this study mean of relative eosinophilia was 22.7% , but the trends changed according to the clinical manifestations and age of the patients . the mean of relative eosinophilia was statistically higher in asymptomatic patients ( 36.3% ) than that of symptomatic ( 20.3% ) ( p = 0.001 ) . moreover , rate of eosinophilia reduced with the increase in age ( r = -324 , p = 0.019 ) . although eosinophilia is a marked indication of s. stercoralis infection but clinicians should bear in mind that in areas with endemicity of other diseases associated with eosinophilia , like fascioliasis , hydatidosis , toxocariasis , trichinellosis , the presence of this clue should be accompanied with the outcomes of other measures in order to discriminate etiological agents . immunosuppressive conditions were recorded for 31.4% of patients ( 22 individuals ) in the current study . these risk factor conditions included of diabetes , chronic liver diseases , chronic renal failure , cancer , heart valve replacement , rheumatoid , colitis , bone marrow transplant , and liver transplant . among these immunosuppressed patients , 5 cases were hospitalized , the fate of one of them , who had hematological malignancy , ended with death ; making the mortality rate of 20% for strongyloidiasis associated hospitalized patients . this rate in a study on immigrant population estimated to be 16.7% ( 26 ) . in another study , half of the cancer patients with strongyloidiasis had solid organ malignancy and the remaining had hematological malignancy ( 27 ) . the previously reported fatal case of strongyloidiasis in iran had chronic lymphocytic leukemia ( 11 ) . therefore , chemotherapy , especially steroid therapy , in such patients with background diseases is life threatening . considering that all the patients analyzed in this study , as well as previous reported strongyloidiasis hyperinfection cases in iran ( 10 , 11 , 13 ) are originated from endemic areas of north and south provinces of the country , the necessity for early diagnosis and proper treatment before initiation of suppressive therapy in peoples with history of residency or travel to these areas is highlighted . establishment of referral diagnostic centers for strongyloidiasis in all endemic provinces of the country is recommended . population based studies to assess true prevalence in each area , finding infected individuals and treatment and following up is necessary . detection of infection in asymptomatic patients requires multiple sampling , utilizing sensitive diagnostic techniques and eosinophil counts .

cartilaginous tumors of larynx are very rare . primary chondroid tumors of larynx are less than 1% of all laryngeal tumors [ 13 ] . 7075% of these tumors detect on endolaryngeal part of cricoid cartilage [ 13 , 6 ] . male : female ratio is 1 : 4 [ 711 ] . although there is no definitive etiologic factor for these tumors , most commonly known is irregular ossification of the laryngeal cartilage [ 912 ] . macroscopic and microscopic appearances of laryngeal chondromas are similar to chondromas of other sites . cartilaginous tumors of larynx are classified in 4 groups : ( 1 ) metaplastic cartilaginous nodules ( chondrometaplasia ) , ( 2 ) chondromas , ( 3 ) chondrosarcomas , and ( 4 ) cartilaginous material is in other neoplasias ( pleomorphic adenoma , chondroblastic osteosarcoma etc . ) . the differential diagnosis of these 4 types is very important . a 31-years - old male patient was referred to the chest medicine department with a history of dysphonia and dyspnea . 2 cm sized , ill - defined , porous , and hard mass on posterior surface of cricoid cartilage in subglottic area was detected in microlaryngoscopic evaluation . macroscopically total volume of 2 cc , 0.20.5 cm sized , fragmented , gray - pink colored , medium - hardness , homogenous materials were seen . microscopically well - defined , lobulated - contoured mass was seen near of the focally calcified cartilaginous tissue ( figure 1 ) . mitosis , necrosis , hemorrhage , and binucleation were not detected ( figure 2 ) . this patient was referred to the department of otolaryngology with stuck feeling in the throat after 2 years from first operation . 1 0.8 cm sized , hard , gray - coloured mass , which is covered by regular mucosa , was detected in same localization ( right subglottic area ) . chondroid tumors of the larynx are uncommon neoplasms and they are comprised up to 1% of all laryngeal neoplasms some studies supported that the incidence of malign chondroid tumors is higher than benign types [ 712 , 15 ] . these tumors are localized in posterior lamina of cricoids cartilage [ 16 , 14 ] . the most common symptoms are dyspnea , hoarseness , and compression to the neighbor tissues . chondroid tumors of larynx can be easily recognized histologically , because of their characteristic features . but morphologic features must be well known for differential diagnosis between subtypes . this lesion is characterized by loose of lobular pattern , mucochondroid changes in soft tissue matriks , and well - defined nodules [ 812 , 15 ] . macroscopically chondromas are smaller than 2 cm ( average 1.5 cm ) but chondrosarcomas are generally larger than 3 cm sized ( average 4.3 cm ) . microscopically chondromas are hypocellular ( 3040 nuclei / hpf ) lesions with homogenous lobular growing pattern . they have no nuclear atypia and mitosis . increased cellularity ( more than 40 nuclei / hpf ) , nuclear atypia , binucleation , mitosis , and pleomorphism are variable in low- and high - grade chondrosarcomas . our lesion was characterized by hypocellularity , no nuclear atypia , mitosis , binucleation , and pleomorphism . our patient referred to the department of otolaryngology with recurrent mass in same localization after 2 years . especially differential diagnosis between chondromas and low - grade chondrosarcomas is important for planning of treatment . we suggested that the differential diagnosis of chondromas must be done carefully and the follows - up of patients must be planned more frequently .

the human -herpesviruses , including cytomegalovirus ( cmv ) and human herpesvirus 6 ( hhv-6 ) , are ubiquitous among human populations.1 in brazil , serological prevalence surveys conducted in the north and southeast regions show adult infections rates for hhv-6 and cmv of around 90% among the population studied ( 0 - 40 years of age ) , with the primary infection occurring during the first year of life.2 - 4 both viral agents can cause several human diseases either as a consequence of reinfection or reactivation of latent infection.5 - 7 the reactivation of latent hhv-6 and cmv is common following liver transplantation . the reactivation may possibly be induced and facilitated by allograft rejection and immunosuppressive therapy.8,9 both viruses affect the success of the transplant procedure . viral infection is associated with several observable clinical findings : fever , neutropenia , central nervous system manifestations or other visceral involvements.10 in addition , hhv-6 viremia is an independent significant predictor of invasive fungal infections and is associated with late mortality in liver transplantation recipients.11 in addition , coinfection with the viral agents can lead to a higher frequency of transplant rejection.12,13 the diagnosis of recrudescence or new infection with cmv and hhv-6 is not easy . although serological detection techniques are available , the diagnostic value of a positive result is limited by the high prevalence of infection in adults.2,3 a report of specific anti - cmv or hhv-6 igm in the sera or a four - fold rise in igg antibodies can be used as a diagnostic criterion , but such an assay has limited sensitivity . moreover , the interpretation of serological results is complicated by the fact that both primary and secondary infections with other herpes viruses may be associated with a concurrent antibody response to hhv-6.6,14 amplification techniques are also available for the diagnosis of both viral agents ; however , the results obtained using these techniques can be controversial , as they are dependent on the method of pcr employed.15 - 17 - 17 nested pcr ( n - pcr ) amplifies one dna target - specific sequence and is divided into two stages . in the first stage , a pair of primers amplifies the specific sequence of dna . in the second , a new primer set is used for an internal region of the previously amplified fragment . in this second stage , the dna is maintained at a high concentration to prevent nonspecific annealing , thus making this technique more efficient and specific.18,19 the aim of this study was to simultaneously monitor cmv and hhv-6 active infections using nested pcr and , together with clinical findings gathered over periods of six months to one year , follow the clinical status of patients undergoing liver transplants . patients - thirty patients submitted for liver transplantation at the liver transplant unit , gastro center in the state university of campinas , sp , brazil were studied prospectively for a period of six months to one year using n - pcr for cmv and hhv-6 dna detection . for basic immunosuppressive therapy tacrolimus ( fk 506 ) and mycophenolate mofetyl ( mmf ) were prescribed based on selected patient characteristics and specific protocol studies . all liver transplant recipients received 200 mg of acyclovir every 12 h for 60 days for prophylaxis against herpes simplex infection because of the high seroprevalence of this virus in the brazilian population ( data not reported ) . each patient protocol was revised and checked for clinical findings associated with infection by cmv or hhv-6 . routine prophylaxis for cmv infection was not used unless the donor was positive and the recipient serum negative for cmv . ganciclovir was administered for two weeks for protection against cmv in patients with two or more consecutive positive pcr results.20 no patients received anti - hhv-6 therapy during this study . the study protocol was approved by the ethics committee of the school of medical science at the state university of campinas . all clinical and laboratory records were reviewed for evidence of hhv-6 and cmv - attributable disease symptoms . collection of blood samples - peripheral blood samples were drawn weekly in the first month post - transplant . at thirty to 90 days post - transplantation , blood samples were collected every two weeks . after 90 days , samples were collected monthly up to one year post - transplant . peripheral blood leukocyte dna extraction the procedures for isolating blood leukocytes and dna extraction for cmv have been described previously.14,21 the dna amount and purity were determined by optical density readings obtained using spectrophotometry and by reading the absorbance at a wavelength of 260 nm ( 1 od = 50 ng ) . the final dna concentration present in each sample was determined by the formula od 26050factor of dilution = ng/l . serum dna extraction - dna was extracted from 200 l of serum using a phenol - chloroform protocol after treatment with lysis buffer containing sds and overnight incubation with proteinase k at 65c , followed by dna precipitation with cold ethanol . the resulting dna pellet was eluted in 50 l of te buffer ( tris edta).22 cmv nested pcr - five microliters of dna extracted from pbl , as described above , was used in the nested pcr . the reaction mixture contained primers specific to cmv and was generated following a previously described protocol.15,16 to exclude false - negative results , the dna samples were subjected to pcr with -globin primers.23 hhv-6 nested pcr nested pcr was carried out using 5 l of dna extracted from serum as described above . the primers and protocol used for hhv-6 nested pcr have been previously described.24 all amplifications were carried out on a robocycler 40 ( stratagene , la jolla , ca , usa ) . the nested pcr products were analyzed under uv light after electrophoresis in 2% agarose ( gibco - brl ) and staining of the gel with ethidium bromide . cmv pp65-antigenemia- blood samples were collected in edta - containing tubes and transferred to the laboratory within 6 hours . the cell pellet was suspended in phosphate - buffered saline ( pbs ) , and the polymorphonuclear leukocytes ( pml ) were then centrifuged to prepare cytospin slides ( 310 pml per slide ) . the slides were air - dried and fixed in formaldehyde before being immunostained with monoclonal antibodies ( iq products , netherlands ) and incubated with a peroxidase - labeled anti - mouse secondary antibody ( hrp , biotest , dreieich , germany ) . the test was carried out in duplicate , and the results were expressed as the number of positive cells per 310 pml.25 - 27 hhv-6 specific antigens in peripheral blood mononuclear cells ( pbmcs)- the presence of the hhv-6 specific antigens in the pbmc cytopreparations was demonstrated by indirect immunoperoxidase staining using monoclonal antibodies against both the a and b variants ( mab8533 and mab8535 , chemicon , inc . , temecula , ca ) . a peroxidase - conjugated anti - mouse antibody ( dako , copenhagen , denmark ) and a peroxidase - conjugated goat anti - rabbit antibody ( zymed , san francisco , ca ) were used as the second and third antibodies . a 3-amino-9-ethyl carbazole solution containing hydrogen peroxide mayer 's hematoxylin was used for counterstaining.28,29 definitions- active cmv infection was defined as two or more consecutive n - pcr positive results and/or one positive pp65-antigenemia.5,26,30 - 32 cmv disease infection was determined based on biopsy and symptoms consistent with cmv disease including fever , malaise , myalgia , anorexia and leucopenia accompanied by active cmv infection.5,30 patients were considered to have cmv viral syndrome if they suffered from unexplained fever ( > 37.5c ) for at least 3 days , in combination with at least one of the following features : arthralgia , leukopenia ( < 310/l ) , thrombocytopenia ( < 15010/l ) and liver enzyme elevation ( alt>50 u / l).33 a patient was considered to have asymptomatic cmv infection when active cmv infection occurred without the signs , symptoms , or laboratory abnormalities described above . a patient was considered to have an active hhv-6 infection based on one n - pcr positive result from serum - extracted dna . statistical analysis- categorical variables were compared using a fisher 's exact test or a chi - squared test . the nested pcr for cmv and hhv-6 was standardized with positive and negative dna controls for each virus . figure 1 illustrates the nested pcr products from cmv and hhv-6 positive and negative samples . the median time to initial cmv detection was 29 days following transplantation ( range : 0 - 99 days ) . the median time to initial hhv-6 detection was 23.5 days after transplantation ( range : 0 - 273 days ) . the time to detection of each virus was not statistically different ( p = 0.49 ) . co - infection did not occur frequently ( table 1 ) , and cmv and/or hhv-6 dna detection prior to positive cmv - pp65 antigenemia was observed in most patients . overall , 53.3% ( 16/30 ) of patients presented viral infection - related symptoms and/or laboratory findings such as fever , granulocytopenia , thrombocytopenia , diarrhea , oral or intestinal ulcers and encephalitis ( table 1 ) . twenty percent ( 6/30 ) of patients had detectable cmv dna and/or hhv-6 dna in their serum associated with other laboratory and/or clinical manifestations of infection . several cases demonstrated clinical manifestation , and cmv and/or hhv-6 active infection also were observed , but not concomitantly . the rejection of the transplanted liver was observed in 16.7% ( 5/30 ) of the patients , although the time of the rejection episodes was not correlated to cmv and hhv-6 active infection ( p>0.05 ) ( table 2 ) . the mortality rate at the one - year follow - up was 23% ( 7/30 ) ( table 3 ) , and hhv-6 was detected in several cases . however , the deaths could not be correlated with active infections caused by either virus ( p > 0.05 ) . the results of the present study indicate that hhv-6 and/or cmv infections were frequent in liver transplant recipients in the gastro center hepatic transplant unit at the state university of campinas ( campinas / sao paulo / brazil ) . cmv and/or hhv-6 dnaemia was found in the majority of patients during the one year follow - up period . our results are in agreement with the results of prior studies.9,34 these other studies suggest that the hhv-6 virus does not cause disease in itself but acts as a co - adjuvant to facilitate the emergence of several pathogens such as cmv.6,17,35 in the cohort studied , cmv and hhv-6 active infection data were in agreement with these reports . as there was no predominance of either type of viral infection , it was not possible to observe hhv-6 facilitation activity . the lack of predominance was probably because of the small sample number enrolled in this study ( n = 30 ) . qualitative pcr , such as nested pcr , can not distinguish between low and high viral load , and this may have contributed to the difficulty in determining significant viremia . however , in our center , n - pcr detected cmv dna in two or more consecutive samples . n - pcr carried out with dna extracted from serum is also frequently unable to detect latent infection . previous studies have noted the presence of cmv specific antigens in renal allograft biopsy material together with acute or chronic rejection , suggesting an association between viral infection and rejection.36 in the present study , this association could not be found because only one patient presented active infection by both agents , and hhv-6 dnaemia was not found during follow - up in any of the cases . in addition , the evaluation period of this study was not long enough to exclude the possibility that rejection may occur in patients co - infected after a time greater than one year . active hhv-6 infection was observed in most of the patients who died after the one year follow - up , but this finding was not time - related . the results presented herein indicate that few patients remain free of -herpesviruses following liver transplantation . most patients with an active infection by more than one virus were infected sequentially and not concurrently . most patients with hhv-6 active infections were asymptomatic , although several cases of hhv-6 active infection occurred among patients who died post - transplantation . fac designed the study , contributed to data analysis , performed the immunoassays and wrote the manuscript . scbc contributed to the study design , conducted and coordinated the laboratory studies and wrote the manuscript .

brain drain is defined as the movement of health personnel in search of the better standard of living and life quality , higher salaries , access to advanced technology and more stable political conditions in different places worldwide . brain drain may be within countries ( internal brain drain ) , but in most cases refers to cross - border or international migration and often from the developing countries to the developed ones . international migration of highly skilled professionals first emerged as a major public health issue in the 1940s , when many european health professionals emigrated to the united kingdom and the united states . by the mid 60s , the losses were enough to cause concern . in 1979 , who published a detailed 40-country study on the importance and flow of health professionals , which findings suggested that close to 90% of all migrating physicians were moving to just five countries : australia , canada , the federal republic of germany , the united kingdom , and the united states . the strength of any nation depends to a large extend on its productivity , which in turn depends on the well - being of the population . emigration of health care professionals has both short and long - term consequences on the sustenance of the originating countries . sub - saharan africa in particular faces the greatest challenges , with 11% of the world 's population and 25% of the global disease burden ; yet the region has only 3% of the global health workforce and accounts for less than 1% of health expenditures worldwide . the proportion of effects of brain drain depends on the extent of a country 's development . developed countries , overall , have large numbers of scientists and healthcare and other professionals , the developing countries may have just a handful . a major obligation of any government to its population is to pursue and implement policies that increase numbers of these key professionals to a desirably stable level , or where they are already approaching stability , to maintain them at those levels . the health care system in the developing countries faces many problems , human resource being one of the majors . the system is structurally and systemically fragile and weak to provide effective service where it is most needed . by contrast , north and south america , which together have 14% of the world 's population but only 10% of the global disease burden , employ 37% of the global health workforce and contribute over 50% of the pool of global health expenditure . malawi in southern africa now had about 100 doctors and 2000 nurses to serve a population of 12 million people . staff are unable to keep up with the seemingly endless flow of patients near death . the negative impact of brain drain can be masked in the urban areas of developing countries with higher concentration of public and private health centers , but such effects remain clear in the rural areas . as at 2006 , more than 25% of doctors in the us are foreign trained , and us had an estimated ratio of 25.6 doctors per 10,000 population . in comparison , the small country of lesotho in southern africa has 0.5 doctors per 10,000 population , and an adult hiv prevalence rate of 28.9% , in addition to tuberculosis , malaria , and the host of other lower respiratory and gastrointestinal illnesses that plague that part of the world.v uganda had only one doctor per 24,700 inhabitants , and zambia needs 15,000 physicians for its health care system to work properly , but only around 800 are registered . the physician - to - population ratio is estimated to be 13/100,000 in africa , compared with 280/100,000 in the united states . despite these loop - sided statistics , about 23,000 qualified academics emigrate each year from africa alone . in the year 2001 , a manila based newspaper reported 13,536 filipino -nurses left the country , whereas only 4780 graduated . a survey of ghana 's health - care facilities in 2002 found that 72% of all clinics and hospitals were unable to provide the full range of expected services , due to a lack of personnel . fourty - three percent were unable to provide full child immunizations , 77% were unable to provide 24-hour emergency services and round - the - clock safe deliveries for women in childbirth . ken sagoe , of the ghana health service , these statistics represent a severe deterioration in ghana 's health capacity . sagoe also points out that 604 out of 871 medical officers trained in the country between 1993 and 2002 now practice overseas . zimbabwe , similarly , trained 1200 doctors during the 1990s , but only 360 remain in the country by the year 2000 . in kadoma , eight years ago , there was one nurse for every 700 residents ; currently there is one for every 7,500 . in 1980 , the country was able to fill 90 percent of its nursing positions nationwide ; currently only 30 percent are filled . in addition to scarcity of personnel , the international labor organization estimates that 1841% of the health - care labor force in africa is infected with hiv . over the last five years , the governments in developing countries have lost large segments of health administrators , nurses , and physicians to foreign non - governmental organizations , which can easily outbid the public sectors for the services of local health talents . with continues exodus of the younger age group of medical personnel from the developing countries , the sustenance of medical education depends on the few aging generation of health care personnel , often too weak and exhausted . at the level of research and postgraduate training , there are only a few medical journals published in africa , some of which are published irregularly and are probably of low quality . consequently , researchers do not wish to publish their papers in such journals and therefore a vicious cycle is created ; the small number of submitted papers results in accepting poor quality papers or irregular publication of the journal . irregular publication then also results in the authors not submitting papers as they are not sure whether and when their papers will be published , a vicious circle of inadequacy thus persist that explain why such journals can not make an impact on the scientific world . however , some researchers believe brain drain could impart positively on medical education , as it will lead to beneficial international collaboration in health care research and development . as a rule of thumb , migrates from developing countries are young ( aged between 15 and 45 years ) and have higher levels of education and income than individuals of the same age who remain in the home country . healthcare is one of the knowledge - intensive industries that generate enormous revenues in europe , japan and the united states to the detriment of the countries that invested in training the healthcare personnel . these industries creates and sustain a steep and dynamic gradient of career opportunities that attracts and concentrates talented individuals from around the world , which in most instances came from the developing countries . in addition to laws that confer preferential immigration status on individuals who have advanced degrees in science and engineering , employment opportunities also affect mobility both within countries ( from public to private and academic to productive sectors ) and among countries . in a study conducted in kenya , the cost of tertiary education of a single doctor in kenya is approximately us$ 48,169 . the total cost of secondary education per student is us$ 6865 and that for primary education us$ 10,963 . this figure does not represent the loss incurred by society because of emigration of a single medical doctor . the real loss is the cumulative dollar value of the investment made by the kenyan society in producing a doctor who decides to emigrate for a period of t years . with the assumption that : the average age of emigrating doctors is 30 years ; the average statutory pensionable age for europe and americas is 62 years , an emigrant doctor would work for 32 years before retirement ; and the feasible average interest rate on fixed deposits in kenya is 6.65% , if the amount of us$ 65,997 ( i.e. cost of educating one medical doctor ) were put into a commercial bank for a period of 32 years at a fixed deposit interest rate of 6.65% per annum , the investment will grow to us$ 517,931 . this is obtained by applying the standard compounding formula : [ ( initial investment ) ( 1+r)t ] = [ us$ 65,997 ( 1 + 0.0665)32 ] . therefore , on average , for every doctor that emigrates , a country loses about us$ 517,931 . the economic loss incurred by kenya as a result of the brain drain of 167 medical doctors is us$ 86,494,477 , i.e. 167 doctors us$ 517,931 per doctor . there are often multiple reasons why healthcare workers leave their countries of origin , the so - called push and pull factors . zarmeneh aly identified why medical graduates of pakistan emigrate to the west : the long - standing belief of young doctors and their parents who train outside their home country is superior and a mark of achievement . a reaction against the pakistani government 's poor management of the education system , and the corruption associated with this management , in favor of what are perceived to be the more merit - based medical training systems of the west . other reasons for healthcare workers migration are lack of opportunity , high unemployment in health labour markets , new communication technology such as electronic recruitment of medical workers and deplorable state of health care in most developing countries . curiously , medical schools in developing countries are also seen as role models encouraging brain drain , when they are openly expressing pride having trained students who are practicing in the developed worlds . the answer to this question is as diabolical as brain drain itself . in the past , many attempts aimed at solving brain drain issue were unsuccessful , maybe because even if one adjusts the push factors , it may be outside one 's domain to adjust the pull factors . it therefore takes a concerted , holistic and sincere approach if we are to expect any change . for the developing countries , it goes beyond just increasing salaries ; even if you give medical workers the highest salary today , the next day inflation will take away all that come with the salary hike . some practical steps taken by the british government in recent terms are commendable , in that they tend to have positive result in discouraging medical workers , in commonwealth developing countries from migrating to britain . migration of medical personnel impart more negatively on the health care of the exporting countries than positively . moreover , because of the nature and complexity of this problem , sincere and committed efforts are needed from both the medical workers and the governments of developing and developed countries . there is need to change not just the process , but the entire systems in most of the developing countries . the governments should chose between politics and people , while the medical workers should balance their individual needs with ethics .

hundred married females from an urban area of pune city were taken for this study by random sampling . after due consent , each participant was subjected to psychosocial performa designed for this study ( pretested and validated ) , personal attribute questionnaire ( a 24-paired - item scale , each pair describing contradictory characteristics in which scoring brings out the masculinity and the femininity response of each individual ) , general health questionnaire 12 ( ghq , a self - administered standardized 12-item screening test sensitive for the presence of psychiatric disorders in individuals that is not designed to detect symptoms that occur with specific psychiatric diagnosis ) , beck 's depressive inventory ( bdi , a self - administered standardized 21-item instrument to assess the presence and severity of depression with a reliability of 0.730.92 in nonpsychiatric samples ) , beck 's anxiety inventory ( bai , a self - administered standardized 21-item instrument to assess the presence and severity of anxiety ) , and perceived stress scale ( pss , a most widely used 10-item psychological instrument to measure the degree to which situations in an individual 's life are appraised as stressful , which measures the perceived stress over last 1-month duration ) . the participants were subsequently analyzed on the basis of their presence of masculinity and femininity scores and compared and correlated with various sociodemographic and outcome variables . table 3 shows the mean and median scores of the population on ghq , bdi , bai , and pss . psychopathology was found in 30% , while proportions of anxious , depressed , and stressful were 25 , 23 , and 36% , respectively [ table 4 ] . there was no statistically significant association between age , income , occupation of the spouse , and education of the participants with scores on psychopathology ( ghq ) , depression ( bdi ) , anxiety ( bai ) , and perceived stress ( pss ) . table 5 shows that the number of years since marriage and masculinity and femininity scores were found to have statistically significant association with all these scores . table 6 shows that masculinity scores of the participants were negatively correlated while femininity scores were positively correlated with the scores on ghq , bdi , bai , and pss all these were statistically significant . furthermore , a multivariate analysis ( logistic regression ) was done to assess the impact of independent variables ( masculinity and femininity ) and others such as education of the wives , family structure , and occupation of the spouse that are likely to confound the results [ table 7 ] . the analysis revealed that after controlling for all the variables , masculinity had a protective effect on scores of ghq ( psychopathology ) , bdi ( depression ) , bai ( anxiety ) , and pss ( perceived stress ) whereas raised femininity scores corresponded with increased scores on general psychopathology and depression . sociodemographic profile of the study participants distribution of various sociodemographic and individual characteristics distribution of various dependent variables distribution of participants regarding dependent variables of psychopathology , depression , anxiety , and stress association of masculinity and femininity scores in family and years of marriage with various dependent variables correlation of masculinity and femininity scores in family to various dependent variables multivariate analysis ( logistic regression ) of the dependent and independent variables graphical representation of the relationship between ghq and masculinity scores this study attempts to understand the role of optimal mix of psychological aspect of masculinity and femininity on the psychopathology of the participants , depressive symptoms , anxiety , and stress perceived by the individual . the median masculinity score of 32 ranging from 24 to 42 is more than the median femininity score ranging from 18 to 26 . these suggest , in general , the societal acceptance of psychological androgyny with a stronger emphasis on the masculine pattern in the female population today . the mean score of the participants on the outcome variables was skewed ; however , the median scores on general psychopathology ( ghq ) , depression ( bdi ) , anxiety ( bai ) , and perceived stress ( pss ) were well below the cutoff in all [ table 3 ] . the cutoff - based evaluation of these scales revealed that around one fourth to one third of the sample population was psychologically distressed , which is in accordance with the findings by other researchers in similar population [ table 4 ] . the absence of any significant relationship between age , income , occupation of the spouse , and education of the participants on one side with scores on psychopathology ( ghq ) , depression ( bdi ) , anxiety ( bai ) , and perceived stress ( pss ) on another side lay emphasis on the more stronger role of individual 's intrinsic factor than extrinsic factors . these findings go in favor of the top - down approach in coping as suggested by watson et al . the number of years since marriage was significantly associated with scores of ghq , bdi , bai , and pss , which had statistically significant negative correlation with these . this implied that as the number of years of married life increased , the scores on psychopathology , anxiety , depression , and perceived stress decreased . this reflects increasing pattern of adaptability in people and more specifically female participants over the years in marital life . masculinity scores of the participants were found to be significantly associated with various outcome variables ( i.e. , psychopathology , depression , anxiety , and perceived stress . ) this again emphasizes the role of individual intrinsic factors ( i.e. , top - down model ) in coping with life events . correlation analysis revealed that people with higher masculinity scores tend to have less psychopathology , were less depressed and anxious , and had reduced perception of stress . on the other hand , femininity scores of the participants were found to be significantly affecting the various outcome variables ( i.e. , psychopathology , depression , anxiety , and perceived stress ) reiterating the role of individual intrinsic factors ( i.e. , top - down model ) in coping with life events . correlation analysis revealed that people with higher femininity scores tend to have more psychopathology , get more depressed and anxious , and have increased perception of stress . these findings lead to a construct where masculinity is positively associated with better mental health and coping and wherein increased amount of femininity in the female made them vulnerable manifesting with stress and psychopathology . the finding of similar nature came out in multivariate analysis wherein masculinity was found to have a psychoprotective effect and that of femininity detrimental . similar findings have been shown by various researchers who advocate greater emphasis on intrinsic factors in increasing psychological resilience in people . our findings do find androgyny with more emphasis toward masculinity having psychoprotective attributes in our female participants . to conclude , our study revealed that a strong component of psychological masculinity in feminine individuals increase their resilience and coping and makes them less vulnerable to psychopathology in general , depression , anxiety , or the perception of stress . the role of intrinsic individual factors can not be ignored and fostering the same may lead to better mental health . however , the replication and validation of these findings is a continuous process and should move parallel with simultaneous constructive intervention . our findings establish the point that targeting the individual 's own intrinsic factor during psychotherapeutic sessions might improve therapeutic outcome and empowerment - oriented approach would be more valuable in effective preventive and remediation strategies rather than mere stress reduction or environmental manipulation . this study does not attempt to underplay the role of stress reduction or environmental manipulation but wants to make a conscious effort to understand individual vulnerabilities and empowering them , such as with more psychological masculine attributes in a feminine individual . the study aimed to target only the female gender and to study the protective value of masculine traits in them . whether the prominence of masculine trait is effective or the optimal androgynic trend could have been generalized can not be commented upon , as it did not involve both the genders . there could be a varied number of extrinsic and intrinsic factors that may affect the outcome variable ; the exclusion of the same in a general population is impractical . this study has just attempted a cross - sectional analysis in a normative sample of the female population for the role of gender orientation in psychological distress . this study generates an insight into this direction , and more research , especially longitudinal studies , in this dimension would be further conclusive . our findings establish the point that targeting the individual 's own intrinsic factor during psychotherapeutic sessions might improve therapeutic outcome and empowerment - oriented approach would be more valuable in effective preventive and remediation strategies rather than mere stress reduction or environmental manipulation . this study does not attempt to underplay the role of stress reduction or environmental manipulation but wants to make a conscious effort to understand individual vulnerabilities and empowering them , such as with more psychological masculine attributes in a feminine individual . the study aimed to target only the female gender and to study the protective value of masculine traits in them . the male population was not included . whether the prominence of masculine trait is effective or the optimal androgynic trend could have been generalized can not be commented upon , as it did not involve both the genders . there could be a varied number of extrinsic and intrinsic factors that may affect the outcome variable ; the exclusion of the same in a general population is impractical . this study has just attempted a cross - sectional analysis in a normative sample of the female population for the role of gender orientation in psychological distress . this study generates an insight into this direction , and more research , especially longitudinal studies , in this dimension would be further conclusive .

the discrimination of tissue types , densities , and volumes from 2d and 3d images is of particular interest in the medical imaging research community . the post - processing of 3d ct images via colorization of these morphologies holds particular utility with regards to the segmentation of skeletal muscle . specific attenuation values are assigned to individual volumetric elements ( voxels ) , based upon the degree to which ct x - rays of given energies transmit through the volume element . the degree of attenuation depends on the energy spectrum of the incident x - rays , as well as the average atomic number of the tissues of the patients . since most computers utilize hardware that requires integer values , linear attenuation coefficients are rescaled to an integer range that encompasses 4096 values , between -1000 and + 3095 . this scale is known as the ct number , which may likewise be readily converted to hounsfield units ( hu ) . comparatively dense tissues , such as bone , have large attenuation coefficients and thereby large , positive ct numbers ; conversely , large , negative ct numbers are typical for low - density tissues , such as lung and adipose tissue . muscles are normally displayed with ct number between 50 and 100 hounsfield units , although within a normal muscle belly , other tissue elements are typically present , such as loose connective tissue and fat , which have much lower ct numbers . if a singular tissue type occupies a particular voxel , that element will possess a readily - identifiable ct number corresponding singularly to that tissue type ; however , typical voxels simultaneously contain several tissue types and thereby express an average ct number whose value is proportional to the ratio of the tissue types . this phenomenon explains the wide range of values present within a particular dataset and suggests the necessity for increased voxel resolution and the development of novel intervoxel segmentation methods to optimize the study of muscle structural changes , in particular for 3d color muscle ct .. muscular degeneration is typically characterized by the progressive loss of muscle function and mass , and has been readily identified as an independent risk factor for high mortality in aging populations and patients suffering from neuromuscular pathology or injury . in aging , this phenomenon is typically defined as sarcopenia , and while its prevalence has been consistently detrimental to an individual s physical vitality , defining its diagnosis and etiology remains debated . despite the absence of a universally accepted definition , extant clinical literature commonly correlates sarcopenia with the loss of both skeletal muscle function and structure , and a variety of mechanisms have been implicated to dictate these phenomena . aging skeletal muscle has significantly diminished myofibril contractility dues to a progressive reduction of the proportion of glycolytic type ii muscle fibers . additionally , these myofibers elicit a significant reduction in triglyceride processing capability , resulting in excessive lipid droplet storage along myocyte cell membranes . in general , the concordant loss of muscle mass and infiltration of non - contractile tissue confers an increased risk for frailty , disability , and eventual mortality . in an increasingly aging world , it is clear that optimizing extant assessment methodologies and identifying a normative clinical definition for sarcopenia is of growing importance . the mechanisms that may govern muscular degeneration in sarcopenia have likewise been indicated within the context of neuromuscular pathology and sequela of trauma . indeed , the dramatic deleterious changes in muscle exhibited in these patients have been implicated as analogous to the changes evidenced in sarcopenia . aging of muscle can occur as the result of many chronic diseases or traumatic events , and as such , there exist many clinical definitions that may be potentially obfuscated by their coincidence . for example , as in sarcopenia , skeletal muscle degeneration due to illness , known as cachexia , has been analogously associated with increases in relative muscle adiposity , which has likewise been correlated to increased rates of cachectic patient morbidity and mortality . however , these phenomena are particularly evident in patients with spinal cord injury ( sci ) , as paralysis from lower motor neuron denervation drastically reduces skeletal muscle mass and increases local muscle fibrosis and adiposity . one extreme case of a sci sequela is irreversible conus and cauda equina syndrome a condition in which leg muscles may be completely and permanently disconnected from the nervous system . when these innervated connections between the spinal cord and the leg muscles are severed , volitional leg muscle contractility disappears downstream from the sci lesion . without this contractility , muscle fibers are gradually replaced by loose and fibrous connective tissues a phenomenon that typically occurs within three to five years post - sci . to emphasize the utility of the advanced muscle imaging analysis techniques described herein , this review focuses on reported literature regarding the assessment of muscle degeneration and regeneration , via home - based functional electrical stimulation ( h - b fes ) of sci patients . many of the tissue analyses employed to study structural changes occurring in lower motor neuron ( lmn ) denervated muscle are usually performed with biopsies i.e. , the analysis of only a few milligrams of muscle . complementary imaging techniques , such as ct scans , are also employed to assess and validate histological information and to study macroscopic changes . the combinatorial value of ct imaging methods , post - processing techniques , and segmentation can be demonstrated by studying the effect of h - bfes training entire muscle volumes . an excellent example of such a case study involved the assessment of a patient with irreversible conus and cauda equina syndrome ( patient a ) who underwent five years of this stimulation treatment , followed by five years of non - compliance to h - bfes . the main novelty introduced in this work was the morphological analysis of the whole quadriceps in different pathological conditions and the quantification of the tissue compositions within the muscle volumes . figure 2 presents the results for patient a. the 3d model and voxel assessment of the rectus femoris shows a clear increase in muscle volume and density during the h - bfes compliant period , and likewise shows a clear decline in these muscle characteristics after five years of non - compliance . in the second and third of these reported case studies , analogous soft tissue analyses and segmentation methods were used on ct images of two patients with different degrees of partial denervation . the first of these patients ( patient b ) , was a female , 53 years . during the last 5 years progressive difficulties of walking with muscle fatigue especially of those involved in ankle flexion - extension ( tibialis anterior , peroneals and tricep surae ) . during recent 3-month hospitalization the patient underwent electric muscle stimulation sessions with schuhfried stimulette den2x , by current for denervated muscles at the lodge side of the leg , bilaterally . the gait is neuropathic , with foot drop especially on the right side , for deficit of peroneal muscles and dorsiflexors . she has complete autonomy in adl , but presents difficulties in supine - sitting and sitting - standing position changes , that are possible only with the aid of the upper limbs . the patient walks with two sticks for long distances and without aids for short distances . waddling gait due to remain also other muscular deficits detected in pre - treatment evaluation . improved resistance to fatigue and balance , both static and dynamic . improved awareness of the present deficit and strategies for dealing with them . improved the strength of the upper limbs and abdominal muscles ( transversus abdominis , internal oblique , external oblique ) . the second study reports the results of patient c a 53 year old male patient 15 years post sci before and after h - bfes . figure 3 depicts the comparison between patient b s soft tissues before and after six weeks of h - bfes rehabilitation using the soft tissue segmentation method detailed herein . the subject was treated each day for 30 minutes with electrical stimulation for denervated calf muscles bilaterally on the anterior and lateral side of the leg . additionally , the histograms containing hu distributions show that fat is the dominant tissue type within the volume , especially on the left side where muscle content is almost absent . however , the distribution after the rehabilitation period ( red ) shows an increase in muscle density and a reduction in fat within the right leg . furthermore , it is possible to analyze the specific soft tissue compositional changes within these muscles . the pie charts in figure 3 depict these particular changes ; indeed , it is evident that there was an increase of dense connective or fibrous tissue in both legs , to the detriment of loose connective tissue . figure 4 contains the results from patient c s analyses , detailing hu histograms that show , once again , the differences between the left and right sides , both pre and post h - bfes . analogous segmentation analyses were performed on the middle of the rectus femoris thigh muscles to compare whether there were significant changes in the muscular composition outside of the h - bfes rehabilitation volume . however , intriguingly , there was a slight shift towards higher density hu values on both legs . indeed , patient c s residual asymmetric innervation of the leg muscles and the effects of six weeks of h - bfes rehabilitation regime allowed the patient to perform short walks without crutches a monumental improvement in function and quality of life after years of only heavily - supported walking and many daily hours of bed rest . the gait is neuropathic , with foot drop especially on the right side , for deficit of peroneal muscles and dorsiflexors . she has complete autonomy in adl , but presents difficulties in supine - sitting and sitting - standing position changes , that are possible only with the aid of the upper limbs . the patient walks with two sticks for long distances and without aids for short distances . waddling gait due to remain also other muscular deficits detected in pre - treatment evaluation . improved resistance to fatigue and balance , both static and dynamic . improved awareness of the present deficit and strategies for dealing with them . improved the strength of the upper limbs and abdominal muscles ( transversus abdominis , internal oblique , external oblique ) . the second study reports the results of patient c a 53 year old male patient 15 years post sci before and after h - bfes . figure 3 depicts the comparison between patient b s soft tissues before and after six weeks of h - bfes rehabilitation using the soft tissue segmentation method detailed herein . the subject was treated each day for 30 minutes with electrical stimulation for denervated calf muscles bilaterally on the anterior and lateral side of the leg . additionally , the histograms containing hu distributions show that fat is the dominant tissue type within the volume , especially on the left side where muscle content is almost absent . however , the distribution after the rehabilitation period ( red ) shows an increase in muscle density and a reduction in fat within the right leg . furthermore , it is possible to analyze the specific soft tissue compositional changes within these muscles . the pie charts in figure 3 depict these particular changes ; indeed , it is evident that there was an increase of dense connective or fibrous tissue in both legs , to the detriment of loose connective tissue . figure 4 contains the results from patient c s analyses , detailing hu histograms that show , once again , the differences between the left and right sides , both pre and post h - bfes . analogous segmentation analyses were performed on the middle of the rectus femoris thigh muscles to compare whether there were significant changes in the muscular composition outside of the h - bfes rehabilitation volume . however , intriguingly , there was a slight shift towards higher density hu values on both legs . indeed , patient c s residual asymmetric innervation of the leg muscles and the effects of six weeks of h - bfes rehabilitation regime allowed the patient to perform short walks without crutches a monumental improvement in function and quality of life after years of only heavily - supported walking and many daily hours of bed rest . the results from these analyses are presented as the average hu values within the muscle reconstruction , as well as the percentage of these tissues with respect to the total muscle volume . the methods outlined for these assessments demonstrate the reliability of these tools to monitor , quantify , and characterize muscle degeneration . 3d ct image segmentation captures the minimal , but clinically significant extent to which muscle may change in size and/or quality in such studies , which was utilized to highlight the importance of rehabilitation trainings for sci - induced and disuse atrophy . in general , these studies altogether suggest the criticality of continuing to identify and exploit the strengths of imaging modalities the context of muscle research . further optimization of these methods will be pivotal in discerning their respective utility in serving the complex and challenging field of translational myology .

the southern community cohort study ( sccs ) is a prospective cohort study designed to identify causes of health disparities between african americans and whites in the incidence and mortality rates of several cancers and other chronic diseases ( 13 ) . the study was approved by the institutional review boards at vanderbilt university and meharry medical college . participants were enrolled in person at community health centers ( chcs ) in the states of alabama , arkansas , florida , georgia , kentucky , louisiana , mississippi , north carolina , south carolina , tennessee , virginia , and west virginia . participants were enrolled by approaching people entering the chcs ( patients and individuals accompanying patients ) and screening them for eligibility and interest in study participation . participants were required to be between the ages of 40 and 79 , to speak english , and to not have been treated for cancer ( excluding nonmelanoma skin cancer ) within the past year . after providing informed consent , the baseline interview contained questions on demographic information , medical history , family history , environmental and occupational exposures , diet , physical activity , and psychosocial variables . depressive symptoms were assessed using a 10-item version of the centers for epidemiological studies depression ( ces - d ) scale , a valid and reliable measure of the presence and severity of depressive symptoms ( 14 ) . cutoffs of 10 , 15 , and 20 define mild , moderate , and severe levels of depressive symptoms ( 14 ) . the ces - d has demonstrated measurement equivalency in samples with differential characteristics including race and sex ( 15 ) . participants were asked if they were taking any antidepressant medication or antianxiety medication and then asked for the name(s ) of all medications of this type being taken . individuals with missing values for bmi ( n = 1,575 ) , education ( n = 45 ) , income ( n = 736 ) , smoking status ( n = 100 ) , marital status ( n = 8) , insurance ( n = 336 ) , and alcohol consumption ( n = 310 ) and those who were aged < 40 years ( n = 2 ) , had a depressive symptoms score ( n = 99 ) , did not answer the question about having diabetes or hypertension ( n = 53 ) , or did not answer the question about using antidepressants ( n = 37 ) were excluded , resulting in 65,767 individuals selected for analysis . participants with diabetes were identified using the question , has a doctor ever told you that you have diabetes ? participants who answered yes to this question were selected for analysis , consisting of 21.7% of the original sample or 14,279 individuals . in two validation studies , one involving a review of medical records and the other assessing a1c percentages , the self - reported diagnoses were confirmed for 98% of those reporting diabetes ( 16 ) . data were analyzed using means , frequencies , and cross - tabulations to calculate descriptive statistics . antidepressant use was coded as absent = 0 and present = 1 and served as the dependent variable . the model was built in three steps . in step 1 , sex , age , insurance status , income , education , level of depressive symptoms ( no depressive symptoms , mild depressive symptoms , moderate depressive symptoms , or severe depressive symptoms ) was entered . in the final step , race was coded into three categories ( whites , african americans , and others / mixed race ) and was entered into the regression model , with whites used as the reference group for calculating odds ratios ( ors ) . in the same model , this analytic strategy tested whether rates of antidepressant use differed for african americans and others / mixed race relative to whites after controlling for sex , age , insurance status , income , education , bmi , smoking status , alcohol consumption , and level of depressive symptoms . all analyses were done using spss ( version 17.0 ; spss , chicago , il ) . depressive symptoms were assessed using a 10-item version of the centers for epidemiological studies depression ( ces - d ) scale , a valid and reliable measure of the presence and severity of depressive symptoms ( 14 ) . cutoffs of 10 , 15 , and 20 define mild , moderate , and severe levels of depressive symptoms ( 14 ) . the ces - d has demonstrated measurement equivalency in samples with differential characteristics including race and sex ( 15 ) . participants were asked if they were taking any antidepressant medication or antianxiety medication and then asked for the name(s ) of all medications of this type being taken . depressive symptoms were assessed using a 10-item version of the centers for epidemiological studies depression ( ces - d ) scale , a valid and reliable measure of the presence and severity of depressive symptoms ( 14 ) . cutoffs of 10 , 15 , and 20 define mild , moderate , and severe levels of depressive symptoms ( 14 ) . the ces - d has demonstrated measurement equivalency in samples with differential characteristics including race and sex ( 15 ) . participants were asked if they were taking any antidepressant medication or antianxiety medication and then asked for the name(s ) of all medications of this type being taken . individuals with missing values for bmi ( n = 1,575 ) , education ( n = 45 ) , income ( n = 736 ) , smoking status ( n = 100 ) , marital status ( n = 8) , insurance ( n = 336 ) , and alcohol consumption ( n = 310 ) and those who were aged < 40 years ( n = 2 ) , had a depressive symptoms score ( n = 99 ) , did not answer the question about having diabetes or hypertension ( n = 53 ) , or did not answer the question about using antidepressants ( n = 37 ) were excluded , resulting in 65,767 individuals selected for analysis . participants with diabetes were identified using the question , has a doctor ever told you that you have diabetes ? participants who answered yes to this question were selected for analysis , consisting of 21.7% of the original sample or 14,279 individuals . in two validation studies , one involving a review of medical records and the other assessing a1c percentages , the self - reported diagnoses were confirmed for 98% of those reporting diabetes ( 16 ) . data were analyzed using means , frequencies , and cross - tabulations to calculate descriptive statistics . antidepressant use was coded as absent = 0 and present = 1 and served as the dependent variable . the model was built in three steps . in step 1 , sex , age , insurance status , income , education , level of depressive symptoms ( no depressive symptoms , mild depressive symptoms , moderate depressive symptoms , or severe depressive symptoms ) was entered . in the final step , race was coded into three categories ( whites , african americans , and others / mixed race ) and was entered into the regression model , with whites used as the reference group for calculating odds ratios ( ors ) . in the same model , two - way interactions between race and the other predictor variables were also examined . this analytic strategy tested whether rates of antidepressant use differed for african americans and others / mixed race relative to whites after controlling for sex , age , insurance status , income , education , bmi , smoking status , alcohol consumption , and level of depressive symptoms . all analyses were done using spss ( version 17.0 ; spss , chicago , il ) . the mean sd age of the sample with diabetes was 54.5 9.0 , with african americans being slightly but significantly younger ( p < 0.0001 ) than whites ( 54.2 vs. 55.4 years ) . table 1 provides a description of the 14,279 participants who reported a diabetes diagnosis stratified by race . level of income reported in this sample was very low , with 61% of each race / ethnicity subgroup reporting < $ 15,000 per year in family income . there were significant racial differences in the crude ( i.e. , unadjusted ) demographic variables . specifically , insurance status , income , education , bmi , smoking status , alcohol consumption , depressive symptoms , and antidepressant use differed by race . african americans were more likely to report having health insurance , incomes of < $ 15,000 , dropping out of high school , lower bmi levels , never having smoked , and consuming more alcohol on a daily basis than whites or others / mixed race . african americans were more likely to have no depressive symptoms , whereas whites and others / mixed race were more likely to have moderate and severe depressive symptoms . finally , among participants reporting diabetes , 17.4% of african americans reported using antidepressant medications compared with 39.0% of whites and 31.3% of individuals of other / mixed race . characteristics of 14,279 participants in the sccs reporting a diagnosis of diabetes test for categorical variables . figure 1 presents a bar graph of the rates of antidepressant use stratified by race . whites and others / mixed race were more likely to report using antidepressants than african americans , regardless of the severity of depressive symptoms . table 2 presents the results ( ors and 95% confidence limits ) of the logistic regression analysis predicting antidepressant use . sex and health insurance status were strong predictors of antidepressant use , with women and insured individuals being approximately twice as likely to be taking antidepressants as men and uninsured individuals . usage tended to decrease with advancing age and increasing income but rose with education and ( slightly ) with increasing bmi . use of antidepressant medications was also positively associated with smoking , with former smokers being 1.21 times and current smokers being 1.46 times more likely to report antidepressant use than participants who had never smoked , but inversely associated with alcohol consumption . as expected , participants with severe symptoms were 6.55 times more likely to be taking antidepressants than those with the lowest level of depressive symptoms . logistic regression predicting antidepressant use from sex , age , insurance status , income , education , bmi , smoking status , alcohol consumption , severity of depressive symptoms , and race data are or ( 95% ci ) . african americans were 0.32 times as likely and others / mixed race were 0.63 times as likely to be taking antidepressants than whites . there were significant interactions for race and income ( p = 0.004 ) , race and smoking status ( p = 0.001 ) , and race and level of depressive symptoms ( p = 0.009 ) . an examination of these interactions showed that the direction of the effect was always consistent , with only a modest change in the magnitude of the effect across the various strata . the mean sd age of the sample with diabetes was 54.5 9.0 , with african americans being slightly but significantly younger ( p < 0.0001 ) than whites ( 54.2 vs. 55.4 years ) . table 1 provides a description of the 14,279 participants who reported a diabetes diagnosis stratified by race . level of income reported in this sample was very low , with 61% of each race / ethnicity subgroup reporting < $ 15,000 per year in family income . there were significant racial differences in the crude ( i.e. , unadjusted ) demographic variables . specifically , insurance status , income , education , bmi , smoking status , alcohol consumption , depressive symptoms , and antidepressant use differed by race . african americans were more likely to report having health insurance , incomes of < $ 15,000 , dropping out of high school , lower bmi levels , never having smoked , and consuming more alcohol on a daily basis than whites or others / mixed race . african americans were more likely to have no depressive symptoms , whereas whites and others / mixed race were more likely to have moderate and severe depressive symptoms . finally , among participants reporting diabetes , 17.4% of african americans reported using antidepressant medications compared with 39.0% of whites and 31.3% of individuals of other / mixed race . characteristics of 14,279 participants in the sccs reporting a diagnosis of diabetes test for categorical variables . figure 1 presents a bar graph of the rates of antidepressant use stratified by race . there were disparities in antidepressant use at all levels of depressive symptoms . whites and others / mixed race were more likely to report using antidepressants than african americans , regardless of the severity of depressive symptoms . table 2 presents the results ( ors and 95% confidence limits ) of the logistic regression analysis predicting antidepressant use . sex and health insurance status were strong predictors of antidepressant use , with women and insured individuals being approximately twice as likely to be taking antidepressants as men and uninsured individuals . usage tended to decrease with advancing age and increasing income but rose with education and ( slightly ) with increasing bmi . use of antidepressant medications was also positively associated with smoking , with former smokers being 1.21 times and current smokers being 1.46 times more likely to report antidepressant use than participants who had never smoked , but inversely associated with alcohol consumption . as expected , participants with severe symptoms were 6.55 times more likely to be taking antidepressants than those with the lowest level of depressive symptoms . logistic regression predicting antidepressant use from sex , age , insurance status , income , education , bmi , smoking status , alcohol consumption , severity of depressive symptoms , and race data are or ( 95% ci ) . african americans were 0.32 times as likely and others / mixed race were 0.63 times as likely to be taking antidepressants than whites . there were significant interactions for race and income ( p = 0.004 ) , race and smoking status ( p = 0.001 ) , and race and level of depressive symptoms ( p = 0.009 ) . an examination of these interactions showed that the direction of the effect was always consistent , with only a modest change in the magnitude of the effect across the various strata . we investigated the relationship between racial status and antidepressant use in a large low - income cohort of individuals with diabetes . although rates of antidepressant use increased as levels of depressive symptoms increased , a large number of individuals with moderate and severe depressive symptoms were not being treated with antidepressant medication . there was a racial disparity in the use of antidepressants at all levels of depressive symptoms . after adjustment for confounders , for every 10 white participants only 3 african american participants were likely to receive antidepressant medications . the prevalence of antidepressant use was consistently lowest for african americans , even after controlling for factors that interacted with racial status ( e.g. , income , smoking , and severity of depressive symptoms ) . the present study was conducted among a diverse sample of adults receiving care from chcs in the southeastern u.s . two other studies performed in other regions of the u.s . also reported racial disparities in antidepressant medication use among persons with diabetes ( 17,18 ) . however , to our knowledge , our study is the first to show this disparity in a large low - income cohort of older adults with diabetes and depressive symptoms . although socioeconomic status has been an important determinant of many health disparities ( 19 ) , socioeconomic status measured by self - reported household income and education was associated with antidepressant use in this study , but the patterns were conflicting , with lower income and higher education being associated with greater antidepressant use . the disparity between african americans and whites in this sample is unlikely to be explained by differences in income or education , as the difference persisted after adjustment for these factors . the reasons for a disparity in antidepressant use are not easily apparent and probably represent a combination of factors including the recognition and diagnosis of depression in african americans , as well as access and attitudes toward antidepressant medication in this population . in some primary care settings , providers recognize depression and recommend treatment for african americans at rates equal to those for whites ( 20 ) . however , even when depression is recognized , african americans were less likely to take antidepressant medications than whites . african americans are less likely than whites to accept antidepressant treatment ( 20 ) and have been shown to prefer counseling over antidepressant medication ( 21 ) . african americans may also use spirituality and religion as a coping mechanism more frequently than whites . first , because this is a cross - sectional study , inferences regarding causal relationships can not be made . second , diagnosis of diabetes was self - reported and not based on chart review or biological testing . in a separate validation study , 124 medical records of sccs participants reporting a prior diagnosis of diabetes were reviewed , and 98% of the self - reports were validated based on criteria including icd-9 codes , physician notes or prescriptions , and/or a1c or blood glucose measurements from the medical records ( 16 ) . third , antidepressant use may have been differentially reported because of differences in beliefs , attitudes , and preferences for treatment ( 20,21 ) . fourth , unmeasured differences in presenting depression - related complaints were unaccounted for , as were physicians ' treatment priorities . providers may be less likely to discuss depression treatment with african americans who are often sicker when seeking care and present with more somatic attributions ( 22 ) . finally , although the ces - d has been validated in various populations , including multiethnic groups with diabetes ( 23 ) , it may not be psychometrically equivalent across race - ethnicity or immigrant generations ( 24 ) and has demonstrated false - positive rates in low - income chc settings . in one study , the standard cutoff for major depressive disorder yielded a sensitivity of 0.95 and specificity of 0.70 , but more than two - thirds of those who screened positive did not actually meet the criteria for major depressive disorder ( 25 ) . thus , it is possible that depression would not be diagnosed in some study participants who reported mild and moderate depressive symptoms or they would not be considered good candidates for treatment after a clinician conducted a careful diagnostic interview . despite these limitations , our findings suggest a sizable racial disparity in antidepressant use in a large , low - income cohort of individuals with diabetes and depressive symptoms . given this is a population with large numbers of people with diabetes and with limited access to health care services , our findings provide important evidence of the need for appropriate management of depression as part of regular diabetes care . treatment for depression in individuals with diabetes may improve glycemic control , overall functioning , and quality of life ( 2 ) , thereby reducing one 's risk of adverse health outcomes associated with untreated depression . conversely , untreated depression might contribute to racial disparities in diabetes outcomes , complications , and premature death . the mechanisms underlying the association between racial status and antidepressant use is likely to be multifactorial . prospective studies are needed to more closely examine the association between racial status and depression treatment in low - income , ethnic minority , adults with diabetes .

in addition to reducing the bone height , it alters the morphologic features of bone leading to an array of osseous defects or deformities . such periodontal bony defects need to be treated since the pattern of behavior of gingiva is conditioned by the architecture of the underlying supporting alveolar bone . these deformities of the alveolar bone unless corrected may interfere with the eradication of the pockets and make the maintenance difficult , thereby leading to recurrence of the disease . the ideal goal of periodontal therapy is the reconstruction of the bone and ligamentous attachment that have been destroyed by the disease . several studies have shown that conventional periodontal therapeutic procedures do not result in regeneration of the supporting tissues , to a predictable degree.[25 ] this has led to various attempts to reconstruct lost periodontium by using different regenerative methods , one of which is bone grafting procedure . the use of bone grafts to promote periodontal regeneration has been the subject of extensive interest . from the time of hegedus in 1923 and through the studies of schallhorn in 1970 's showed the usefulness of osseous grafts in infrabony defects in order to improve bone regeneration and achieve a greater amount of new connective tissue attachment . currently , a bone substitute based upon the inorganic component of bone , a natural derived porous bone mineral of bovine origin called bio - oss manufactured in wolhusen and marketed by geistlich biomaterials , a swiss company , has been used in periodontics and oral and maxillofacial surgery . its morphology , porosity , internal surface , crystalline structure , and chemical composition are reported to be similar to that of human bone . in experimental studies , bio - oss was found to resorb slowly and integrate in natural remodeling process . considering all the above factors the present study has been taken up with the following aims and objectives : to evaluate bio - oss a bovine - derived xenograft in the repair of human infrabony vertical defects resulting from moderate to advanced periodontal disease and compare with conventional open flap debridement.to determine the amount of reduction from baseline osseous defect depth parameters and amount of bone fill subsequent to grafting.to determine whether the use of this graft would improve the periodontal status of the involved teeth , such as reduction in probing pocket depths , gain in clinical attachment after six months and compare the results obtained with conventional open flap operation without using any graft . to evaluate bio - oss a bovine - derived xenograft in the repair of human infrabony vertical defects resulting from moderate to advanced periodontal disease and compare with conventional open flap debridement . to determine the amount of reduction from baseline osseous defect depth parameters and amount of bone fill subsequent to grafting . to determine whether the use of this graft would improve the periodontal status of the involved teeth , such as reduction in probing pocket depths , gain in clinical attachment after six months and compare the results obtained with conventional open flap operation without using any graft . bio - oss spongiosa granules size 0.25 - 1 mm was taken as the implant material . this commercially available xenograft material is a unique , natural , nonantigenic , osteoconductive bone matrix obtained from cattle . twelve patients comprising 5 males and 7 females diagnosed to have adult periodontitis being routinely treated in the department of periodontology were selected to participate in this study . the age of the subjects ranged from 30 to 50 years . to be included in the study the subjects had to fulfil the following criteria . all subjects had to be free of any systemic disease and must not be on any medicationpresence of two or more vertical osseous defects as verified by radiographs with associated probing pocket depths of 5.0 mm following initial nonsurgical therapy . the defects selected were either one - walled , two - walled , wide three - walled , or combination defects . final selection included 24 defectsteeth with furcation involvement and those that were endodontically involved were not included in this studyteeth subjected to occlusal disharmonies , severe attrition , and facets and excessive mobility were also excluded from the studypatients must not have had antibiotic therapy in the past 3 months . all subjects had to be free of any systemic disease and must not be on any medication presence of two or more vertical osseous defects as verified by radiographs with associated probing pocket depths of 5.0 mm following initial nonsurgical therapy . the defects selected were either one - walled , two - walled , wide three - walled , or combination defects . final selection included 24 defects teeth with furcation involvement and those that were endodontically involved were not included in this study teeth subjected to occlusal disharmonies , severe attrition , and facets and excessive mobility were also excluded from the study patients must not have had antibiotic therapy in the past 3 months . approximately 4 - 6 weeks following the hygiene phase of therapy , patients underwent a re - evaluation examination during which time all the base line scores were recorded . plaque index ( silness and loe ) , gingival index ( loe and silness ) were recorded at baseline and 6 months for the group of teeth selected for the study . a groove was made on the stent by using a fissure bur in an occlusoapical direction at a point where the graft material had to be placed . the base of the stent served as a reference point to record the soft tissue measurements . evaluation of the periodontal soft tissue changes namely probing pocket depth , probing attachment level , and gingival recession were determined by recording the following measurements using williams periodontal probe . reference point ( rp ) to gingival margin ( gm)reference point ( rp ) to cemento - enamel junction ( cej)reference point ( rp ) to base of the pocket ( bop ) reference point ( rp ) to gingival margin ( gm ) reference point ( rp ) to cemento - enamel junction ( cej ) reference point ( rp ) to base of the pocket ( bop ) probing pocket depth was recorded by noting the difference between measurements from the reference point to gingival margin and reference point to base of the pocket . probing attachment level was calculated by subtracting the distance between reference point to the cemento - enamel junction from the distance between the reference point to base of the pocket . rp to free gingival margin was measured to determine any change in gingival recession before and after surgery . prior to surgery , an intraoral periapical radiograph was taken using an extension cone paralleling device [ figures 1a and 2a ] . to standardize radiographic assessment , radiographs were obtained in a constant and reproducible plane using film holders with a template containing impression compound , which was placed in a constant position on a group of teeth and an extension arm that can be precisely attached to both film holder and the x - ray tube . to facilitate recording of precise measurements from the radiographs , a profile projector was used . it consists of a micrometer with which measurements upto 0.01 mm can be made accurately . ( a ) infrabony defect ( mesial to lateral incisor ) in the experimental group after debridement ; ( b ) baseline radiograph showing infrabony defect mesial to lateral incisor ; ( c ) bio - oss packed into the defect ; ( d ) 6-month post - operative radiograph showing bone fill ( a ) baseline radiograph showing infrabony defect mesial to first premolar ; ( b ) 6-month post - operative radiograph showing bone fill the defects chosen for the study were randomly assigned either open flap debridement ( ofd ) or the bovine - derived xenograft ( bdx ) bio - oss through the flip of a coin . upon such determination , the site was either filled with bdx particles or left empty after curettage as a control . the surgical procedure was performed utilizing local anaesthetics , 2% xylocaine with epinephrine ( 1:80,000 ) . intrasulcular incisions with reflection of full thickness flaps were utilized to retain as much soft tissue as possible in order to obtain primary closure . the surgical site was repeatedly irrigated with sterile normal saline in order to remove all the debris from the defect . a non - absorbable black silk suture ( 4 - 0 ethicon mersilk ) was placed loosely , left untied in a vertical mattress fashion prior to the filling of the defect . the graft material bio - oss spongiosa was mixed with a drop of sterile saline as per manufacturer 's instructions . the defect was filled to the existing alveolar crest with care being taken not to overfill the defect [ figure 1c ] . pressure was applied with a moist gauze for a minimum of 2 minutes post - operatively followed by placement of a periodontal dressing ( coe - pak ) . the patients were kept under systemic tetracycline therapy of 250 mg qid for 5 days post operatively along with anti - inflammatory analgesics and chlorhexidine ( 0.2 % ) mouth wash . the periodontal dressing and sutures all the patients were recalled at every one - month interval during which the surgical area was irrigated and further oral hygiene instructions were given . all the soft and hard tissue measurements taken preoperatively were repeated post - operatively at 6 months to calculate the attachment gain , reduction in probing pocket depth , gingival recession , and amount of bone fill [ figures 1d and 2b ] . the following method was employed to determine the radiographic changes before and after the study . all the data obtained in this study were evaluated statistically by using student 's t test . to be included in the study the subjects had to fulfil the following criteria . all subjects had to be free of any systemic disease and must not be on any medicationpresence of two or more vertical osseous defects as verified by radiographs with associated probing pocket depths of 5.0 mm following initial nonsurgical therapy . the defects selected were either one - walled , two - walled , wide three - walled , or combination defects . final selection included 24 defectsteeth with furcation involvement and those that were endodontically involved were not included in this studyteeth subjected to occlusal disharmonies , severe attrition , and facets and excessive mobility were also excluded from the studypatients must not have had antibiotic therapy in the past 3 months . all subjects had to be free of any systemic disease and must not be on any medication presence of two or more vertical osseous defects as verified by radiographs with associated probing pocket depths of 5.0 mm following initial nonsurgical therapy . the defects selected were either one - walled , two - walled , wide three - walled , or combination defects . final selection included 24 defects teeth with furcation involvement and those that were endodontically involved were not included in this study teeth subjected to occlusal disharmonies , severe attrition , and facets and excessive mobility were also excluded from the study patients must not have had antibiotic therapy in the past 3 months . approximately 4 - 6 weeks following the hygiene phase of therapy , patients underwent a re - evaluation examination during which time all the base line scores were recorded . plaque index ( silness and loe ) , gingival index ( loe and silness ) were recorded at baseline and 6 months for the group of teeth selected for the study . a groove was made on the stent by using a fissure bur in an occlusoapical direction at a point where the graft material had to be placed . the base of the stent served as a reference point to record the soft tissue measurements . evaluation of the periodontal soft tissue changes namely probing pocket depth , probing attachment level , and gingival recession were determined by recording the following measurements using williams periodontal probe . reference point ( rp ) to gingival margin ( gm)reference point ( rp ) to cemento - enamel junction ( cej)reference point ( rp ) to base of the pocket ( bop ) reference point ( rp ) to gingival margin ( gm ) reference point ( rp ) to cemento - enamel junction ( cej ) reference point ( rp ) to base of the pocket ( bop ) probing pocket depth was recorded by noting the difference between measurements from the reference point to gingival margin and reference point to base of the pocket . probing attachment level was calculated by subtracting the distance between reference point to the cemento - enamel junction from the distance between the reference point to base of the pocket . rp to free gingival margin was measured to determine any change in gingival recession before and after surgery . prior to surgery , an intraoral periapical radiograph was taken using an extension cone paralleling device [ figures 1a and 2a ] . to standardize radiographic assessment , radiographs were obtained in a constant and reproducible plane using film holders with a template containing impression compound , which was placed in a constant position on a group of teeth and an extension arm that can be precisely attached to both film holder and the x - ray tube . to facilitate recording of precise measurements from the radiographs , a profile projector was used . it consists of a micrometer with which measurements upto 0.01 mm can be made accurately . ( a ) infrabony defect ( mesial to lateral incisor ) in the experimental group after debridement ; ( b ) baseline radiograph showing infrabony defect mesial to lateral incisor ; ( c ) bio - oss packed into the defect ; ( d ) 6-month post - operative radiograph showing bone fill ( a ) baseline radiograph showing infrabony defect mesial to first premolar ; ( b ) 6-month post - operative radiograph showing bone fill the defects chosen for the study were randomly assigned either open flap debridement ( ofd ) or the bovine - derived xenograft ( bdx ) bio - oss through the flip of a coin . upon such determination , the site was either filled with bdx particles or left empty after curettage as a control . the surgical procedure was performed utilizing local anaesthetics , 2% xylocaine with epinephrine ( 1:80,000 ) . intrasulcular incisions with reflection of full thickness flaps were utilized to retain as much soft tissue as possible in order to obtain primary closure . the surgical site was repeatedly irrigated with sterile normal saline in order to remove all the debris from the defect . a non - absorbable black silk suture ( 4 - 0 ethicon mersilk ) was placed loosely , left untied in a vertical mattress fashion prior to the filling of the defect . the graft material bio - oss spongiosa was mixed with a drop of sterile saline as per manufacturer 's instructions . the defect was filled to the existing alveolar crest with care being taken not to overfill the defect [ figure 1c ] . pressure was applied with a moist gauze for a minimum of 2 minutes post - operatively followed by placement of a periodontal dressing ( coe - pak ) . the patients were kept under systemic tetracycline therapy of 250 mg qid for 5 days post operatively along with anti - inflammatory analgesics and chlorhexidine ( 0.2 % ) mouth wash . all the patients were recalled at every one - month interval during which the surgical area was irrigated and further oral hygiene instructions were given . all the soft and hard tissue measurements taken preoperatively were repeated post - operatively at 6 months to calculate the attachment gain , reduction in probing pocket depth , gingival recession , and amount of bone fill [ figures 1d and 2b ] . the following method was employed to determine the radiographic changes before and after the study . all the data obtained in this study were evaluated statistically by using student 's t test . all surgical sites showed uneventful healing . no post - operative complications like swelling , hemorrhage or excessive pain there were no instances of any reaction of the tissues to the graft material or rejection of the graft in any patient . no graft material could be detected outside the defects either at the time of suture removal or subsequent check - up visits . while there was no distinct variability in the clinical parameters between the experimental and control sites pre - surgically [ table 1 and figures 3 and 4 ] , a marked improvement was observed in all the parameters in experimental sites at the end of the study period [ table 2 and figures 58 ] . comparison of variables between study groups before experiment comparison of variables among study groups before experiment comparison of variables among study groups before experiment comparison of mean changes in variables between control and experimental groups after treatment comparison of reduction in plaque and gingival index at the end of study period comparison of mean changes in soft tissue variables between study groups comparison of mean changes in hard tissue variables ( in millimeters ) between study groups comparison of mean changes in hard tissue variables ( in percentage ) between study groups there has been a complete shift from resective pocket eradicative procedures to techniques and methods aimed at regeneration and conservation of the periodontium . a number of bone grafts have been used in different clinical trials with different degree of success . upon analysis of the various clinical reports it is seen that most of the bone graft materials such as dfdba , fdba , hydroxyapatite , ceramics etc . , have resulted in significant improvement in the clinical parameters . however , the amount of bone fill , the type of bone tissue that has formed , the exact nature of healing have not been consistent . many of these bone grafts have been found to be no more than fillers of bone defects without demonstrating any osteogenic or osteoconductive potential . in the present study , bio - oss the natural porous bone mineral resulted in significant improvement of all clinical parameters with a mean bone fill of 54.065% . this compares well with the results obtained in other studies using other types of bone grafts.[1417 ] this is also comparable to the study of richardson et al . , where the bone fill was reported to be 55.8% using bio - oss. another significant aspect of this study is the negligible amount of bone fill in sites of open flap debridement without the use of graft . this feature was seen despite considerable improvement in other clinical parameters like reduction in probing pocket depth and gain in attachment level . it should be noted here that all the other factors remained the same between experimental and control sites . hence , this study proves yet again that conventional periodontal therapy does not result in significant bone fill . most of the sites treated with simple surgical debridement demonstrated some degree of crestal bone loss ( mean - 0.20 mm ) . four sites showed no difference in the crestal height before and after the study period . seven sites demonstrated some degree of increase in the height of the alveolar crest ( mean 0.10 mm ) . this compares with the studies of meffert et al . , where hydroxyapatite was used and schallhorn rg and hiatt wh where cross - matched iliac allograft of cancellous bone and marrow was used . the reason for crestal bone loss in the solitary case could have been due to the presence of a very wide osseous defect where it would not have been possible to fill up the entire defect with the graft material . one of the advantages of using any of the popular bone graft material in vogue the minimum recession of the gingiva that is seen post - operatively . in the present study also sites treated with like in earlier studies bio - oss exhibited favorable handling properties such as : ease of delivery to the site , ease of packing the material into the defect , ability of the material to demonstrate an adhesion once placed in the defect , even with significant hemorrhage of the wound site , providing a stable graft . ease of delivery to the site , ease of packing the material into the defect , ability of the material to demonstrate an adhesion once placed in the defect , even with significant hemorrhage of the wound site , providing a stable graft . the type of bone defects taken up in this study varied from three - walled defects to one - walled defects , often combined ones . whereas , in strictly three - walled so called intrabony defects the potential for bone fill is quite predictable , this is not so with combined or bizarre defects . hence , from the results of this study , it appears that morphologic variations in the osseous defects did not interfere with the efficacy of bio - oss. the methods employed for evaluating the results of this study were simple . many other studies advocated reentry procedure at the end of the study period to assess the results . however , re - entry was not considered in this study as the procedure although enables changes in the bone levels to be measured visually , still does not show the actual type of healing that has occurred . since none of the teeth included in the study required extraction histological study was obviously not taken up . the results of this study suggested that bio - oss as a bone graft material is quite predictable when used in infrabony defects in bringing about bone fill and improvement in clinical parameters . however , further investigation with a larger sample on a prolonged post - operative follow - up is required to conclusively establish the efficacy of this material . further , histological evaluation to determine other changes such as the type of new bone that is formed , any evidence of cementogenesis , regeneration of the attachment apparatus should be taken up in the future studies . this study envisaged to evaluate the efficacy of a bovine derived xenograft bio - oss and to compare with open flap debridement in human infrabony periodontal defects . twelve patients who were diagnosed to have adult periodontitis having at least two angular defects were taken up for the study . twelve sites were filled with bio - oss constituting the experimental group and the other 12 sites were surgically debrided without graft placement thereby serving as controls . the results showed that significant improvement in all clinical parameters were observed in sites treated with bio - oss than compared to control sites . this included reduction in probing pocket depth , gain in attachment level and bone fill . in addition sites treated with bio - oss demonstrated minimum post - surgical gingival recession compared to control sites . the control sites although exhibited considerable reduction in probing pocket depth and gain of attachment did not however show any appreciable bone fill . bio - oss was well tolerated by the tissues with no cases showing any exfoliation of the graft , incompatibility or any other complications . it is concluded that bio - oss is a bone graft material of considerable promise .

neurological sufferings associated with hiv infection , especially those involving the central nervous system , are well known to the medical staff . peripheral neuropathies are the most common neurological manifestations occurring in hiv - infected patients , sometime rising diagnostic or therapeutic problems to the clinicians in romania , for the last 20 years knowledge about hiv / aids infections have become largely available to doctors , however so far no consistent data are available on the peripheral nervous system sufferings associated to hiv infections among romanian patients . the present study aims of detecting risk factors for symptomatic peripheral neuropathy in a group of hiv infected romanian patients ( px ) . this is a retrospective study based on data colected from the hiv infected px admitted in the infectious diseases department ( hospital no all hiv infected individuals diagnosed with symptomatic peripheral neuropaties ( n=26 , 7,44% from all the hiv infected px known with neurological , mental or behavioral sufferings ) have been selected ; the control group ( c ) consist of 40 px ( 7,31% from the remaining hiv infected persons ) ( fig.1 ) . selection of the px ( group n and c ) all cases of peripheral neuropathy have been confirmed by the neurologist . for both groups the following data have been compared : age , height , hiv mode of transmission , number of px who developed aids , average and nadir of the cd4 count during monitoring , mean of hiv viral load , median duration of art , use and duration of d - drugs ( ddi - didanosine , ddc - dideoxicitidine , d4 t - stavudine ) , presence of certain coinfections and conditions ( hepatitis b , cmv , toxoplasmosis , tuberculosis , diabetes , concomitant ethanol abuse ) . statistical analysis : differences between groups are based on chi test ( two tailed , with yates correction calculated using epi6 program ) who estimated the odd ratio ( or ) and relative risk ( rr) , respectively unpaired t test ( online graphpad quickcalcs ) for comparing averages and standard deviations between the two groups , statistical significance being considered for p - value less than 0.05 . to detect the optimal level of differentiation ( sensitivity vs specificity ) between the two groups roc analysis has been used ( receiver operator curve , calculated with medcalc 10.1.3.0 statistical program ) to calculate the area under the curve ( auc , required level > 0 5 ) and the p coefficient ( significant if less than 0.05 ) . this is a retrospective study based on data colected from the hiv infected px admitted in the infectious diseases department ( hospital no all hiv infected individuals diagnosed with symptomatic peripheral neuropaties ( n=26 , 7,44% from all the hiv infected px known with neurological , mental or behavioral sufferings ) have been selected ; the control group ( c ) consist of 40 px ( 7,31% from the remaining hiv infected persons ) ( fig.1 ) . selection of the px ( group n and c ) all cases of peripheral neuropathy have been confirmed by the neurologist . for both groups the following data have been compared : age , height , hiv mode of transmission , number of px who developed aids , average and nadir of the cd4 count during monitoring , mean of hiv viral load , median duration of art , use and duration of d - drugs ( ddi - didanosine , ddc - dideoxicitidine , d4 t - stavudine ) , presence of certain coinfections and conditions ( hepatitis b , cmv , toxoplasmosis , tuberculosis , diabetes , concomitant ethanol abuse ) . statistical analysis : differences between groups are based on chi test ( two tailed , with yates correction calculated using epi6 program ) who estimated the odd ratio ( or ) and relative risk ( rr) , respectively unpaired t test ( online graphpad quickcalcs ) for comparing averages and standard deviations between the two groups , statistical significance being considered for p - value less than 0.05 . to detect the optimal level of differentiation ( sensitivity vs specificity ) between the two groups roc analysis has been used ( receiver operator curve , calculated with medcalc 10.1.3.0 statistical program ) to calculate the area under the curve ( auc , required level > 0 5 ) and the p coefficient ( significant if less than 0.05 ) . the demographic characteristics of the patients are : age 31.54 14.64 years ( group n ) vs 23.9 12.03 years ( group c ) , p=0.024 ( t test ) ; gender ratio ( male / female ) 11/15 ( group n ) vs 23/17 ( group c ) , p = ns ( chi ) ; provenance ( urban / rural ) 10/16 ( group n ) vs 24/16 ( group c ) , p = ns ( chi ) . the height of the px was 1.69 0.085 m. ( group n ) , respectively 1.66 0.11 m. ( group c ) , with no statistical difference between the two groups . the average time of clinical monitoring was 5.31 3.07 years ( group n ) vs 7.75 5.4 years ( group c ) , p=0.043 ( t test ) . regarding the acquisition of the virus , for the group n we have counted 13 px with parenterally hiv transmission and 13 px with viral transfer through sexual route , while for the group c we have recorded 28 parenterally transmision and only 8 sexual acquisition of hiv ( 4 cases had unknown mode of infection ) . considering the parenteral vs sexual mode of infections , there was a statistical significant difference between the two groups ( p=0.044 , chi2 ) . aids has been diagnosed in 21 px from group n vs 29 px from group c ( p = ns , chi2 ) . other elements of comparison ( regarding hiv infections and art ) and the significance of the differences between the two groups are shown in table 1 elements of comparison regarding hiv infections and art table 2 highlights the differences between groups regarding the presence of certain coinfections and associated medical conditions . comparison between groups regarding the presence of certain coinfections and associated conditions differences regarding the chance of detection ( or ) and the relative risks ( rr ) for symptomatic peripheral neuropathy based on the age of the subjects are shown in table 3 ; the level of comparison is represented by the first row of the table ( patients aged 60 ) . or and rr for developing symptomatic peripheral neuropathy depending on the age gap in fig.2 is shown the results of the roc analysis for the differentiation between px with or without peripheral neuropathy depending on their age ( auc=0.683 , p - value=0.0081 ) ; 29 years of age represents the optimum point of differentiation between those subjects ( sensitivity = 50% , specificity = 80% ) . roc analysis for the differentiation between px with or without peripheral neuropathy depending on their age according to expert studies the prevalence of hiv - associated peripheral neuropathy varies widely between 1.2 and 69.4 % [ 1 , 2 ] . the incidence of this sufferings dropped down from 27 cases / 100 px / year prior to highly active antiretroviral therapy ( haart ) to 6 cases / 100 px / year currently ; however , when aids is diagnosed , the incidence reaches 30% ( based on clinical observations ) , while post - mortem studies reveals an incidence of almost 100% [ 3 , 4 ] . our data shows that only 26 hiv infected px from a total of 896 subjects have been diagnosed with symptomatic peripheral neuropathy - 2,6% or 0.16 cases / 100 px / year much under the level revealed by the international data . the medical literature describes several risk factors for developping peripheral neuropathy in hiv - positive patients : advanced age , chronic alcoholism , presence of other concomitant severe sufferings or aids stage , anemia , low cd4 count ( or low cd4 nadir ) , high viral load , antiretroviral therapy used in combination ( especially the nucleoside reverse transcriptase inhibitors ) , or concomitant use of anti - tuberculosis drugs [ 1 , 5 , 6 , 7 ] . they are produced near the root of the nerve and then distributed along the nerve with its elongation [ 8 ] . longer nerve means longer distances between mitochondria , so tall persons are more prone to developing hiv associated peripheral neuropathy [ 5 ] . mitochondria dna damage due to aging or abnormal oxidative metabolism is associated with nerve sufferings [ 9 ] . this study revealed no differences in the height of the patients ( seen as a surrogate marker for nerve leg length ) of both n and c groups . it is worth mentioning that in parenterally hiv - infected px ( for which the hiv infection has been diagnosed in childhood ) they have recently completed the period of body growth , which means that with the increase in length new ( fully functional ) mitochondria attached to the axons were produced , balancing the old one . there was a statistically significant difference regarding the age of the subjects and half of the cases have been recorded after 2006 , in older px infected through sexual route . they are also responsible for the differences seen in the duration of monitoring and art . given the fact that , both in romanian subjects infected during childhood or adulthood , the f1 subtype of hiv prevails ( i.e. there are no differences in the neurovirulence of the hiv strains ) [ 10 ] directs us to consider the higher age of patients in group n is the most important risk factor for symptomatic peripheral neuropathy in our group of px . our data shows that the greater the age gap the higher the odds of developing hiv associated symptomatic peripheral neuropathy . the cd4 nadir below 200 cells / mmc , lower in group n , close to the point of statistical significance , suggests that hiv itself and/or opportunistic infections ( characteristic of advanced stage of immunosuppression ) may contribute to the developing of hiv - associated peripheral neuropathy . in the analyzed group no differences were found regarding the use of d - drug regimens used for the treatment of hiv , which is explained by the history of art in romania ( most px were exposed to d - drugs especially during the bi - therapy period ) ; however the lenght of the d - drugs use is higher among patients diagnosed with peripheral neuropathy , and the difference is close to the point of statistical significance , so if we take into account the small size of the groups , maybe this factor can be retained for further analysis on larger cohots . the present study reveals that higher age and advanced immunosupression are the most important risk factors for developing symptomatic peripheral neuropathy in romanian hiv infected patients ; taking into account the small number of cases studied , although not statistically significant , it should be noted the cd4 nadir and the length of d - drug use . further studies ( with a larger number of subjects ) are needed to clarify these issues .

fifty - seven newly hatched male chickens ( samvirke kylling norway , ross 308 ) from the same hatching farm were used in this experiment conducted at the norwegian university of life sciences . animal treatment and animal facilities all complied with the guide for the care and use of laboratory animals ( 15 ) . all animals were cared for according to norwegian laws and regulations for experiments with live animals ( the animal protection act of december 20th , 1974 , and the animal protection ordinance concerning experiments with animals of january 15th , 1996 ) . the chickens were randomly assigned into one of four dietary 22 day treatments . during the first 10 days each group of 14 to 15 animals were kept in deep littered pens ; 75 cm150 cm . at day 10 they were weighed and individually placed in wire - floored metabolism cages up until day 22 . the room temperature was maintained at 32c from days 03 , then gradually reduced by 0.5c per day until day 22 . they were exposed to 23 h light and 1 h dark photoperiod days 07 , the next 2 weeks they were exposed to 24 h dark ; the dark periods were between 1721 h and 0004 h. the chickens had free access to feed and water . water troughs and wire - floors were cleaned daily , with small possibility of coprophagy . none of the birds were sick or died during the feeding period . at day 22 the chickens were weighed , killed by carbon dioxide narcosis and dissected immediately after death . organs and limbs were packed in aluminum foil , frozen in liquid nitrogen and stored at 20c before being analyzed . rapeseed oil was purchased from afb , askim , norway and contained 60% oleic acid , 20% linoleic acid and 10% alpha linolenic acid . rendered animal fat ( raf ) was obtained locally and contained fat from ruminants ( about 70% ) and pigs ( about 30% ) . the composition of raf was ( by weight ) : 26% palmitic acid , 18% stearic acid , 36% oleic acid and 8% linoleic acid . fully hydrogenated soybean oil ( hso ) , refined to remove k1 and ineffective dihydrophylloquinone together with trans - fatty acids , were purchased from aak ( aarhus - karlshamn ) sweden ab . the level of k1 in hso was less than 10% of the original amount in untreated soybean oil . all four feeds were wheat based and composed according to guidelines adjusted to the requirements of chickens ( table 1 ) . the feeds were manufactured at fortek , aas , norway , with a matrix type 2.5 mm46 mm at a rate of 600 they had the same basic composition with regard to protein , carbohydrates , minerals and vitamins except vitamin k. all diets contained lipid in the form of oils and solid fats . the control ( ctr ) feed group contained vitamin k1 from rapeseed oil and soy bean oil ; mk-4 from raf and added k3 . the experimental feeds contained natural levels of k1 and mk-4 from raf , fully hso or a mixture of animal rendered fat and fully hso ( mix ) . the experimental diets contained 2% natural soybean oil to provide a small supplement of k1 ( table 1 ) . the experimental feeds had mk-4 levels from 20 to 50% of the k1 level . composition of the experimental feeds including levels of vitamin k notes : all feeds contained monocalcium phosphate 2% ; ground limestone 1.85% ; nacl 0.25% ; dl - methionine 0.4% , l - lysine 0.3% ; l - threonine 0.1% ; choline chloride 0.13% and mgo 6.5710% . vitamin and mineral mixture provided the following per kg feed : retinyl acetate , 2.7 mg ; cholecalsiferol , 0.07 mg ; dl - alpha tocopheryl acetate , 38 mg ; pyridoxine , 3.4 mg ; riboflavin , 9 mg ; ca - pantothenate , 12.5 mg ; biotin , 0.19 mg ; thiamine , 1.9 mg ; niacin , 37.5 mg ; cobalamine , 0.02 mg ; folic acid , 1.5 mg ; choline chloride , 500 mg ; manganese , 75 mg ; zinc , 75 mg ; iron , 95 mg ; cupper , 10 mg ; iodine , 0.6 mg ; selenium , 0.43 mg . vitamin k in liver , pancreas and feeds were analyzed by hplc using a c-18 reversed phase column and fluorometric detection after postcolumn electrochemical reduction according to thijssen et al . bending resistance was tested in a ta - xt2 texture analyzer from stable micro systems , godalming , surrey , uk ( www.stablemicrosystems.com ) . probe velocity was at 0.1 mm / s , and the distance between the supports was 32.5 mm . load - versus - time curves were generated and analyzed by means of texture expert exceed v2.64 software . bending resistance was calculated from the initial linear segment of the graphs and expressed as n / mm . statistical calculations ( linear regression ) on the effect of the four dietary treatments were performed by spss 15.0 ibm spss , statistical analyses software ( http://www.spss.com/ ) , using comparative means by one - way anova and bonferroni test for assumption of equal variances . fifty - seven newly hatched male chickens ( samvirke kylling norway , ross 308 ) from the same hatching farm were used in this experiment conducted at the norwegian university of life sciences . animal treatment and animal facilities all complied with the guide for the care and use of laboratory animals ( 15 ) . all animals were cared for according to norwegian laws and regulations for experiments with live animals ( the animal protection act of december 20th , 1974 , and the animal protection ordinance concerning experiments with animals of january 15th , 1996 ) . the chickens were randomly assigned into one of four dietary 22 day treatments . during the first 10 days each group of 14 to 15 animals were kept in deep littered pens ; 75 cm150 cm . at day 10 they were weighed and individually placed in wire - floored metabolism cages up until day 22 . the room temperature was maintained at 32c from days 03 , then gradually reduced by 0.5c per day until day 22 . they were exposed to 23 h light and 1 h dark photoperiod days 07 , the next 2 weeks they were exposed to 24 h dark ; the dark periods were between 1721 h and 0004 h. the chickens had free access to feed and water . water troughs and wire - floors were cleaned daily , with small possibility of coprophagy . at day 22 the chickens were weighed , killed by carbon dioxide narcosis and dissected immediately after death . organs and limbs were packed in aluminum foil , frozen in liquid nitrogen and stored at 20c before being analyzed . rapeseed oil was purchased from afb , askim , norway and contained 60% oleic acid , 20% linoleic acid and 10% alpha linolenic acid . rendered animal fat ( raf ) was obtained locally and contained fat from ruminants ( about 70% ) and pigs ( about 30% ) . the composition of raf was ( by weight ) : 26% palmitic acid , 18% stearic acid , 36% oleic acid and 8% linoleic acid . fully hydrogenated soybean oil ( hso ) , refined to remove k1 and ineffective dihydrophylloquinone together with trans - fatty acids , were purchased from aak ( aarhus - karlshamn ) sweden ab . the level of k1 in hso was less than 10% of the original amount in untreated soybean oil . all four feeds were wheat based and composed according to guidelines adjusted to the requirements of chickens ( table 1 ) . the feeds were manufactured at fortek , aas , norway , with a matrix type 2.5 mm46 mm at a rate of 600 kg / h . they had the same basic composition with regard to protein , carbohydrates , minerals and vitamins except vitamin k. all diets contained lipid in the form of oils and solid fats . the control ( ctr ) feed group contained vitamin k1 from rapeseed oil and soy bean oil ; mk-4 from raf and added k3 . the experimental feeds contained natural levels of k1 and mk-4 from raf , fully hso or a mixture of animal rendered fat and fully hso ( mix ) . the experimental diets contained 2% natural soybean oil to provide a small supplement of k1 ( table 1 ) . the experimental feeds had mk-4 levels from 20 to 50% of the k1 level . composition of the experimental feeds including levels of vitamin k notes : all feeds contained monocalcium phosphate 2% ; ground limestone 1.85% ; nacl 0.25% ; dl - methionine 0.4% , l - lysine 0.3% ; l - threonine 0.1% ; choline chloride 0.13% and mgo 6.5710% . vitamin and mineral mixture provided the following per kg feed : retinyl acetate , 2.7 mg ; cholecalsiferol , 0.07 mg ; dl - alpha tocopheryl acetate , 38 mg ; pyridoxine , 3.4 mg ; riboflavin , 9 mg ; ca - pantothenate , 12.5 mg ; biotin , 0.19 mg ; thiamine , 1.9 mg ; niacin , 37.5 mg ; cobalamine , 0.02 mg ; folic acid , 1.5 mg ; choline chloride , 500 mg ; manganese , 75 mg ; zinc , 75 mg ; iron , 95 mg ; cupper , 10 mg ; iodine , 0.6 mg ; selenium , 0.43 mg . vitamin k in liver , pancreas and feeds were analyzed by hplc using a c-18 reversed phase column and fluorometric detection after postcolumn electrochemical reduction according to thijssen et al . was tested in a ta - xt2 texture analyzer from stable micro systems , godalming , surrey , uk ( www.stablemicrosystems.com ) . probe velocity was at 0.1 mm / s , and the distance between the supports was 32.5 mm . load - versus - time curves were generated and analyzed by means of texture expert exceed v2.64 software . bending resistance was calculated from the initial linear segment of the graphs and expressed as n / mm . statistical calculations ( linear regression ) on the effect of the four dietary treatments were performed by spss 15.0 ibm spss , statistical analyses software ( http://www.spss.com/ ) , using comparative means by one - way anova and bonferroni test for assumption of equal variances . weight at day 11 and 22 showed that the chickens in the experimental groups ate slightly more and the raf and hso group grew significantly faster than the mix and ctr groups indicating a satisfactory nutritional status ( table 2 ) . information on feed and weight of the chicken from day 11 to day 22 notes : values are means and sd . means with different letters within the same row are significantly different at the 5% level . total amount of mk-4 eaten by the chicken was significantly different , being highest in the ctr group and lowest in the hso group , while for k1 no difference between experimental group but significantly higher in the control group ( table 2 ) . pancreas had higher vitamin k concentration than liver ( table 3 ) . in liver , mk-4 was the most abundant vitamer , while in pancreas mk-4o was the main vitamer . the association between the amounts of mk-4 consumed and mk-4 in liver was statistically significant . levels of vitamin k in liver and pancreas and femur bending resistance notes : values are means and sd . means with different letters within the same row are significantly different at the 5% level . the concentration of total mk-4 and mk-4o in pancreas as well as in liver is associated with mk-4 consumed . the bending resistance of the femur was slightly higher in the experimental groups than in the control group , with no difference between the experimental groups ( table 3 ) . weight at day 11 and 22 showed that the chickens in the experimental groups ate slightly more and the raf and hso group grew significantly faster than the mix and ctr groups indicating a satisfactory nutritional status ( table 2 ) . information on feed and weight of the chicken from day 11 to day 22 notes : values are means and sd . means with different letters within the same row are significantly different at the 5% level . total amount of mk-4 eaten by the chicken was significantly different , being highest in the ctr group and lowest in the hso group , while for k1 no difference between experimental group but significantly higher in the control group ( table 2 ) . pancreas had higher vitamin k concentration than liver ( table 3 ) . in liver , mk-4 was the most abundant vitamer , while in pancreas mk-4o was the main vitamer . the association between the amounts of mk-4 consumed and mk-4 in liver was statistically significant . levels of vitamin k in liver and pancreas and femur bending resistance notes : values are means and sd . means with different letters within the same row are significantly different at the 5% level . the concentration of total mk-4 and mk-4o in pancreas as well as in liver is associated with mk-4 consumed . the bending resistance of the femur was slightly higher in the experimental groups than in the control group , with no difference between the experimental groups ( table 3 ) . our experimental model seems to suggest that conversion of k1 to mk-4 did not take place in the presence of adequate levels of mk-4 in the feed , not even in the feed group with the lowest level of mk-4 ( hso ) . in the test groups recently , a specific enzyme that converts k1 to mk-4 has been discovered and described ( 10 ) . the production capacity of this enzyme has not been compared with or distinguished from the menadione prenylation system ( 5 ) , so published evidence is as yet insufficient for a full quantitative picture of the various sources and pathways of synthesis in normally fed animals . liver and pancreas appears to use mk-4 in different ways ; in liver 1/3 of mk-4 is converted to mk-4o while in pancreas almost all is as mk-4o . the chickens were in good health with good bone resistance without supplements of k3 in the feed but at least a natural content of 23 ng mk-4/g feed .

acalvaria is an extremely rare congenital malformation characterized by an absence of flat bones of skull , dura mater , and associated muscles in the presence of normal cranial contents and facial bones.[13 ] although its pathogenesis is unclear it has been explained by the theory of post - neurulation defect . it has been considered a fatal congenital anomaly and only a handful of cases have been reported in english literature till date.[13 ] thus , we herein present the present case which to the best of our knowledge is third living case of acalvaria.[13 ] the pertinent literature is reviewed with the aim to make physicians aware regarding the clinical features of this uncommon fatal clinical anomaly so that undue associated morbidity could be avoided by prompt antenatal diagnosis and subsequent management . a 1-month - old female infant presented with soft , lax skull without any scalp abnormality . the baby was born at term by cesarean section and was second issue of consanguineous marriage , the first one being a male . the available antenatal record suggested a normal ultrasound examination and the other complications of pregnancy including any history of drug intake , radiation exposure , etc . the post - natal history suggested normal milestones and development for age without any other clinical symptoms . on examination , the baby appeared active weighing 3,910 g with head circumference of 35 cm . on palpation , the frontal , temporal , and occipital bones were absent in upper part , whereas parietal bones could not be palpated . the facial bones and rest of the physical examination were normal without any associated anomaly . the roentgenogram of skull suggested complete absence of parietal bones , and the frontal , occipital , and temporal bones were not visualized in upper aspect [ figure 1 ] . the computed tomography scan of brain confirmed the bony defect with presence of normal cranial contents and ventricular system [ figure 2 ] . plain roentgenogram skull showing absence of skull bones computed tomography scan head ( slice image ) showing absence of skull bones with normal cranial contents the baby was managed conservatively with a vigilant examination on close follow - up along with parental counseling and reassurance . the patient is presently having normal development without any apparent neurological abnormality on regular follow - up for the last 2 months . acalvaria is an extremely rare congenital malformation in pediatric age group.[13 ] it is characterized by the absence of bones of cranial vault , dura mater , and associated muscles.[13 ] although central nervous system and cranial contents are usually unaffected , but still neuropathological defect of some extent has been reported in few cases . similarly , acalvaria has been reported in association with other anomalies such as holoprosencephaly , micropolygyria , facial clefts , cardiac anomalies , club foot , etc . , thereby emphasizing need for a thorough clinical evaluation so as to assess the prognosis and the final outcome in such rare cases.[14 ] the etiopathogenesis of acalvaria is still unknown . it is considered a heterogenous disorder without any genetic predilection and correlation to folic acid intake . a review of literature suggests that different investigators have explained the embryological basis of this rare malformation by theory of post - neurulation defect . normally , around 4 week of gestation and after closure of anterior neural pore , there is migration of mesenchymal tissue under ectoderm which forms skin and scalp , but the mesenchymal tissue develops into skull bones and associated muscles . thus , faulty migration of mesenchymal tissue in presence of normal development of ectoderm results in an absence of flat bones of skull and associated muscles . although not widely accepted , but still some investigators consider acalvaria to be a part of spectrum of anencephaly , whereas others explain its pathogenesis on the basis of failure of primary closure of neural tube . the clinical presentation as experienced in present and reported cases is heralded by the presence of soft , lax skin covered skull due to absence of flat bones of cranial vault and associated muscles.[13 ] the facial bones and cranial contents are usually normal , although some neuropathological abnormality has also been reported.[13 ] since acalvaria remains a fatal congenital malformation , an insight into its radiological features demands special emphasis because a timely antenatal diagnosis followed by an appropriate intervention could prevent the undue morbidity , mortality , and psychological trauma resulting at term . although different craniofacial anomalies including anencephaly , hydrocephalous , osteogenesis imperfecta , hypophosphatasia , etc . , remain important radiological differential diagnosis , but still as experienced by different investigators , a vigilant transvaginal ultrasound scan examination around 12 weeks of gestation can accurately diagnose acalvaria in most of the cases . the presence of normal cerebral hemispheres remains main sonographic feature differentiating acalvaria from anencephaly which remains most common antenatal differential diagnosis.[68 ] among laboratory investigations , alpha - fetoprotein level is usually raised , whereas unconjugated estradiol levels are usually undetectable . the initial treatment of acalvaria is mainly conservative aimed at supportive care and management of any associated anomalies if present . being a rare anomaly with only two reported living cases , the surgical treatment for correcting the skull defect has not been discussed in literature till date . the presence of spontaneous bone growth as observed in few cases of scalp defect , such as cutis aplasia , highlights the importance of initial conservative management and supportive care in patients with acalvaria . they can subsequently undergo skull reconstruction by bone grafting and cranioplasty at school going age . since the only living case of this fatal anomaly with long - term follow - up is severely mentally retarded and disabled requiring specialized medical and social care , so we conclude that antenatal diagnosis of this fatal anomaly by a vigilant radiological evaluation is extremely important so that the morbidity , mortality , and the associated psychological trauma resulting at the time of birth and on subsequent management could be prevented .

intra - articular administration of hyaluronic acid ( ha ; hyaluronan ) is a widely applied treatment for osteoarthritis ( oa ) ( reviewed by simon and strand).1 although ha injection provides relief from pain , albeit transiently , its mechanism of action has not been fully clarified . in a recent recommendation on the management of hip and knee oa by the osteoarthritis research society international ( oarsi),2 the strength of recommendation ( sor ) of intra - articular ha was calculated to be relatively low ( 64% ) , whereas that of nonsteroidal anti - inflammatory drugs ( nsaids ) for oral administration as analgesics was 92%93% . the effect of ha in oa has often been attributed to its potential use in viscosupplementation , ie , supplementation of viscosity ; however , the benefits of ha from some other action and not by its biomechanical properties is also suggested ( reviewed by neustadt and colleagues).3 more specifically , a direct effect of ha against inflammation or cartilage degradation in oa has also been suggested . in addition to its use in oa , intra - articular injection of ha has been used in patients with inflammatory arthritis such as rheumatoid arthritis ( ra ) . in some reports , it is indicated that ha has beneficial effects on inflammatory arthritis.46 this study briefly reviews the recent findings related to the function of ha , focusing on its reported effects in chondrocytes , not only in supplementing viscosity , but also as a potential anti - inflammatory anti - arthritic agent . since it is well - known that oligosaccharide hyaluronan is inversely involved in inflammation,7 whenever ha is mentioned in this paper , it should be understood that the reference is to high molecular weight hyaluronan . hyaluronic acid is a polysaccharide composed of repeating disaccharide units , ie , 1,4-glucuronic acid ( glcua ) and 1,3-n - acetylglucosamine ( glcnac ) . ha belongs to the group of glycosaminoglycans , but unlike chondroitin sulfate or keratan sulfate , ha is not sulfated . ha is synthesized by bioactivity of hyaluronan synthase ( has ) , which has been reported to have three isoforms ( has1 , has2 , and has3 ) in humans.8 ha is widely distributed but mainly localized to the extracellular matrix and body fluid . it contributes to the viscoelasticity of the fluid and elasticity in connective tissues , which absorb mechanical stress , for example , between cartilage and cartilage surfaces.3 in addition to this property of providing viscoelasticity , recent investigations have continued to reveal the variety of actions of ha on cartilage . for example , in normal diarthrodial joints , ha not only works as a mechanical / physical barrier , but also has anti - inflammatory and analgesic functions.3 as for pain suppression , it has been shown that ha attenuates prostaglandin- or bradykinin - induced pain in experimental oa animals.9,10 ha binds to the signal - transducing receptor molecules cd44 and receptor for ha - mediated motility ( rhamm ; reviewed by taylor and colleagues).11 in this regard , cd44 is the primary receptor of ha , and has also been found to bind with other matrix components , including collagen , chondroitin sulphate , and osteopontin.1113 binding of the components to cd44 leads to modulation of cellular functions;1416 for example , ha is reported to inhibit proteoglycan degradation and anti - fas - induced apoptosis of chondrocytes through cd44 expressed on the cells.17,18 in inflammatory or degenerative joints , cartilage matrix is degraded by the action of an array of proteinases . in particular , the families of matrix metalloproteinases ( mmp ) and a disintegrin and metalloproteinase with thrombospondin motifs ( adamts ) are mainly responsible for cartilage degradation . more specifically , the collagenases ( mmp-1 and mmp-13 ) and stromelysin-1 ( mmp-3 ) play a pivotal role in collagen degradation , whereas the aggrecanases ( adamts ) induce aggrecan degradation . these proteinases are often induced by inflammatory stimuli , such as interleukin-1 ( il-1 ) or tumor necrosis factor- ( tnf- ) , which are activated by inflammation or mechanical or physical stress in joints.19,20 subsequently , in an inflamed joint , cartilage exposed to the induced adamts-4 and adamts-5 would release ha , indicating deterioration of matrix integrity;21 this loss of ha from the cartilage matrix is thought to result in the progression of cartilage degradation . on the other hand , it has been demonstrated that ha can enhance synthesis of chondroitin sulfate and proteoglycans,22,23 and reduce the production and activity of mmps and adamts.5,24,25 il-1 is considered to be the dominant catabolic cytokine present in the inflamed articular joints . in this regard , ha is reported to counteract the catabolic process induced by il-1 . for example , fioravanti and colleagues stimulated human chondrocytes under hydrostatic pressure and showed that ha ( mw 500730 kda ) abrogates a decrease in proteoglycan and an increase in nitric oxide ( no ) levels , both induced by il-1 . in a study by karna and colleagues , ha ( 500730 kda ) was found to counteract the effect of il-1 , as ha inhibited il-1-induced downregulation of type ii collagen mrna expression.26 julovi and colleagues27 used 800-kda ha to assess the inhibitory effect of ha on il-1-induced mmps in cartilage and found that the 800-kda ha penetrated cartilage tissue , localized in the pericellular matrix around chondrocytes , and bound with cd44 on chondrocytes , leading to the suppression of il-1-stimulated mmp production by chondrocytes . in this context , we found that ha ( 800 kda and 1900 kda ) suppressed il-1-induced mmp-1 production both at protein and mrna levels in cultured human chondrocytes , and this effect was cancelled by treatment with anti - cd44 antibody.28 as for synovial fibroblasts , shimizu and colleagues29 reported that ha inhibited tnf-- and il-1-induced mmp-1 production from rheumatoid synovial fibroblasts . for this effect , ha was suggested to bind with cd44 expressed in synovial fibroblasts . more recently , the interaction between synovial fibroblasts and ha via intracellular adhesion molecule-1 ( icam-1 ) was reported by hiramitsu and colleagues.29 the mechanism of this inhibition was speculated to occur through ha binding to icam-1 , leading to down - regulation of nuclear factor-b ( nf-b ) and p38 mitogen - activated protein kinase ( mapk ) phosphorylation . in addition to the effects shown against mmps , a recent report by yatabe and colleagues indicated the inhibition of adamts4 ( aggrecanase-1 ) expression in human chondrocytes by ha ( 2700 kda ) through the cd44 and icam-1 signaling pathways.25 these findings collectively suggest a therapeutic potential of ha against matrix breakdown in arthritic cartilage . fibrinolysis is one of the major pathological changes occurring in arthritic joints , in which the plasminogen system is known to play a key role . the balance between plasminogen activator ( pa ) and plasminogen activator inhibitor ( pai ) determines the direction in which plasminogen is activated into plasmin , which in turn further promotes fibrinolysis by activating proteinases , including gelatinases and other mmps . pa occurs in two forms : urokinase - type pa ( upa ) and tissue - type pa ( tpa ) . for example , nonaka and colleagues30 reported that ha suppressed the secretion of u - pa and pai-1 from oa synovial fibroblasts . further , it was found that the receptor for u - pa ( u - par ) was expressed in the synovium in both oa and ra and that the expression was attenuated by treatment with ha.30 these results suggested that ha could suppress fibrinolytic activity mediated by the upa and upar systems . more recently , hsieh et al measured the levels of mmp-2 , mmp-9 , u - pa , and pai-1 in a series of chondral , meniscal , and synovial cultures of early oa after treatment with or without three ha products with different molecular weights ( mw ) , and demonstrated that all of the ha products could decrease the secretion of mmp-2 and mmp-9 . they suggested that ha with high mw might be more effective in inhibiting mmp-2 , mmp-9 , u - pa , and pai-1 expression.31 further investigation on the crosstalk between pa / pai and ha would provide important clues to the protection of cartilage degradation by these proteinases . acidic nsaids are a major subset of anti - inflammatory drugs that inhibit the activity of cyclooxygenase ( cox ) , leading to suppression of prostaglandins and leukotrienes in inflamed tissue . in this regard , interaction has been demonstrated between ha or its fragment and cox-2 expression in a variety of cells.3235 several studies showed upregulation of cox-2 expression by ha . for example , ha ( 200-kda ) was reported to increase the expression of glomerular cox-2 in rat renal glomerular cells34 and in human vascular endothelial cells,36 respectively . the effect of ha has been reported to be mimicked by an engagement of cd44 ( an ha receptor).35,36 further , stuhlmeier reported prostaglandin e2(pge2 ) as a potent activator of has1 in il-1-treated type - b - synoviocytes.37 in that study , the author tested a series of eicosanoids and found that pge2 , and to a lesser extent , pgi2 and thromboxane a2 analogues activated has1 expression in synoviocytes after il-1 treatment , whereas the lipoxygenase product leukotriene b4 had no effect on has1 induction . therefore , ha may upregulate cox-2 , causing the activated cox-2 to induce pge2 , which would then activate has1 through a positive feedback mechanism ( figure 1 ) . conversely , mitsui and colleagues demonstrated an inhibitory effect of ha on the expression of cox-2 mrna in il-1-stimulated subacromial synovial fibroblasts.33 in that study , however , ha with a molecular weight of 90 10 was used ; therefore , the different effect of ha might have been due to the difference in the molecular weight . in addition , kinugasa and colleagues reported that inhibition of cox-2 using a cox inhibitor or cox-2 antisense oligonucleotide in a tumor cell line resulted in downregulation of cd44 expression.38 collectively , the has / ha system and cox / pg system may have a regulatory network via cd44 , which would be regulated differently by each ha component present in each cell type , and further modulated by the presence of inflammatory cytokines and mediators ( figure 1 ) . still , inconsistency among studies concerning the interaction between ha and cox / pg may also , at least in part , be due to the expression or activation of has isoforms ( has1 - 3 ) , the expression pattern of prostanoid receptors , or the amount of concomitant stimuli by other inflammatory mediators such as il-1 . for example , isoforms of has or ep receptors are expressed and function differentially among tissues and cells.8,39 elucidation of the interaction between ha and cox systems in articular chondrocytes would give important information about the clinical application of ha with simultaneous clinical use of nsaids , and may clarify the possible beneficial effects of ha with larger molecular weight in this regard . in inflammatory or degenerative joints , cartilage matrix is degraded by the action of an array of proteinases . in particular , the families of matrix metalloproteinases ( mmp ) and a disintegrin and metalloproteinase with thrombospondin motifs ( adamts ) are mainly responsible for cartilage degradation . more specifically , the collagenases ( mmp-1 and mmp-13 ) and stromelysin-1 ( mmp-3 ) play a pivotal role in collagen degradation , whereas the aggrecanases ( adamts ) induce aggrecan degradation . these proteinases are often induced by inflammatory stimuli , such as interleukin-1 ( il-1 ) or tumor necrosis factor- ( tnf- ) , which are activated by inflammation or mechanical or physical stress in joints.19,20 subsequently , in an inflamed joint , cartilage exposed to the induced adamts-4 and adamts-5 would release ha , indicating deterioration of matrix integrity;21 this loss of ha from the cartilage matrix is thought to result in the progression of cartilage degradation . on the other hand , it has been demonstrated that ha can enhance synthesis of chondroitin sulfate and proteoglycans,22,23 and reduce the production and activity of mmps and adamts.5,24,25 il-1 is considered to be the dominant catabolic cytokine present in the inflamed articular joints . in this regard , ha is reported to counteract the catabolic process induced by il-1 . for example , fioravanti and colleagues stimulated human chondrocytes under hydrostatic pressure and showed that ha ( mw 500730 kda ) abrogates a decrease in proteoglycan and an increase in nitric oxide ( no ) levels , both induced by il-1 . in a study by karna and colleagues , ha ( 500730 kda ) was found to counteract the effect of il-1 , as ha inhibited il-1-induced downregulation of type ii collagen mrna expression.26 julovi and colleagues27 used 800-kda ha to assess the inhibitory effect of ha on il-1-induced mmps in cartilage and found that the 800-kda ha penetrated cartilage tissue , localized in the pericellular matrix around chondrocytes , and bound with cd44 on chondrocytes , leading to the suppression of il-1-stimulated mmp production by chondrocytes . in this context , we found that ha ( 800 kda and 1900 kda ) suppressed il-1-induced mmp-1 production both at protein and mrna levels in cultured human chondrocytes , and this effect was cancelled by treatment with anti - cd44 antibody.28 as for synovial fibroblasts , shimizu and colleagues29 reported that ha inhibited tnf-- and il-1-induced mmp-1 production from rheumatoid synovial fibroblasts . for this effect , ha was suggested to bind with cd44 expressed in synovial fibroblasts . more recently , the interaction between synovial fibroblasts and ha via intracellular adhesion molecule-1 ( icam-1 ) was reported by hiramitsu and colleagues.29 the mechanism of this inhibition was speculated to occur through ha binding to icam-1 , leading to down - regulation of nuclear factor-b ( nf-b ) and p38 mitogen - activated protein kinase ( mapk ) phosphorylation . in addition to the effects shown against mmps , a recent report by yatabe and colleagues indicated the inhibition of adamts4 ( aggrecanase-1 ) expression in human chondrocytes by ha ( 2700 kda ) through the cd44 and icam-1 signaling pathways.25 these findings collectively suggest a therapeutic potential of ha against matrix breakdown in arthritic cartilage . fibrinolysis is one of the major pathological changes occurring in arthritic joints , in which the plasminogen system is known to play a key role . the balance between plasminogen activator ( pa ) and plasminogen activator inhibitor ( pai ) determines the direction in which plasminogen is activated into plasmin , which in turn further promotes fibrinolysis by activating proteinases , including gelatinases and other mmps . pa occurs in two forms : urokinase - type pa ( upa ) and tissue - type pa ( tpa ) . for example , nonaka and colleagues30 reported that ha suppressed the secretion of u - pa and pai-1 from oa synovial fibroblasts . further , it was found that the receptor for u - pa ( u - par ) was expressed in the synovium in both oa and ra and that the expression was attenuated by treatment with ha.30 these results suggested that ha could suppress fibrinolytic activity mediated by the upa and upar systems . more recently , hsieh et al measured the levels of mmp-2 , mmp-9 , u - pa , and pai-1 in a series of chondral , meniscal , and synovial cultures of early oa after treatment with or without three ha products with different molecular weights ( mw ) , and demonstrated that all of the ha products could decrease the secretion of mmp-2 and mmp-9 . they suggested that ha with high mw might be more effective in inhibiting mmp-2 , mmp-9 , u - pa , and pai-1 expression.31 further investigation on the crosstalk between pa / pai and ha would provide important clues to the protection of cartilage degradation by these proteinases . acidic nsaids are a major subset of anti - inflammatory drugs that inhibit the activity of cyclooxygenase ( cox ) , leading to suppression of prostaglandins and leukotrienes in inflamed tissue . in this regard , interaction has been demonstrated between ha or its fragment and cox-2 expression in a variety of cells.3235 several studies showed upregulation of cox-2 expression by ha . for example , ha ( 200-kda ) was reported to increase the expression of glomerular cox-2 in rat renal glomerular cells34 and in human vascular endothelial cells,36 respectively . the effect of ha has been reported to be mimicked by an engagement of cd44 ( an ha receptor).35,36 further , stuhlmeier reported prostaglandin e2(pge2 ) as a potent activator of has1 in il-1-treated type - b - synoviocytes.37 in that study , the author tested a series of eicosanoids and found that pge2 , and to a lesser extent , pgi2 and thromboxane a2 analogues activated has1 expression in synoviocytes after il-1 treatment , whereas the lipoxygenase product leukotriene b4 had no effect on has1 induction . therefore , ha may upregulate cox-2 , causing the activated cox-2 to induce pge2 , which would then activate has1 through a positive feedback mechanism ( figure 1 ) . conversely , mitsui and colleagues demonstrated an inhibitory effect of ha on the expression of cox-2 mrna in il-1-stimulated subacromial synovial fibroblasts.33 in that study , however , ha with a molecular weight of 90 10 was used ; therefore , the different effect of ha might have been due to the difference in the molecular weight . in addition , kinugasa and colleagues reported that inhibition of cox-2 using a cox inhibitor or cox-2 antisense oligonucleotide in a tumor cell line resulted in downregulation of cd44 expression.38 collectively , the has / ha system and cox / pg system may have a regulatory network via cd44 , which would be regulated differently by each ha component present in each cell type , and further modulated by the presence of inflammatory cytokines and mediators ( figure 1 ) . still , inconsistency among studies concerning the interaction between ha and cox / pg may also , at least in part , be due to the expression or activation of has isoforms ( has1 - 3 ) , the expression pattern of prostanoid receptors , or the amount of concomitant stimuli by other inflammatory mediators such as il-1 . for example , isoforms of has or ep receptors are expressed and function differentially among tissues and cells.8,39 elucidation of the interaction between ha and cox systems in articular chondrocytes would give important information about the clinical application of ha with simultaneous clinical use of nsaids , and may clarify the possible beneficial effects of ha with larger molecular weight in this regard . although articular injection of ha is frequently used in the treatment of arthritic conditions , the precise mechanism by which ha suppresses degradation of cartilage or intra - articular inflammation is still under vigorous investigation . a disease - modifying effect of ha has been suggested by some in vitro studies ; however , there has been little clinical evidence thus far . understanding the wide array of signaling pathways of anti - inflammatory effects by ha will encourage novel anti - degradation strategies for both inflammatory and degenerative arthritides , using ha in combination with other disease - modifying anti - rheumatic drugs and/or nsaids .

the executives at children 's hospital colorado are well aware of the affects that caring for sick children and the pressure associated with it have on our entire staff . understanding what compassion fatigue looks like as well as the importance of stress management and its role in overall wellness for each of our employees led to the interest and support of heartmath / caritas workshops . heartmath / caritas training transformed into a program to help staff connect with why they got into this profession and provides the tools to help staff members function in the immense stress they are faced with every day . six - hour workshops are offered to every employee of children 's hospital colorado , supported and paid for by the wellness program in the human resource department . these trainings intentionally brought caritas and heartmath together with an understanding that the two programs match passion with science , trust with hope , and conviction with confidence . both qualitative data , in the form of written feedback from participants , and quantitative results ( table ) support the continued need for these workshops with more exposure to ensure all employees can attend . quantitative results of heartmath / caritas workshops at the children 's hospital , aurora , colorado heartmath / caritas workshops are one way children 's hospital colorado supports its staff in dealing with compassion fatigue and burnout . the passion for sustaining this work comes from understanding how these programs have personally affected those employees who have attended and the compelling mission to pay it forward to all staff at children 's hospital colorado .

low temperature is a critical environmental factor that affects growth and survival of many plant species and limits their geographical distribution . overwintering plants are able to increase their freezing tolerance when exposed to low but non - freezing temperatures , a process known as cold acclimation . during cold acclimation , cold - regulated ( cor ) genes are highly induced during cold acclimation and are involved in the acquisition of freezing tolerance ( thomashow , 1998 ) . expression of cor genes is activated by c - repeat binding factors ( cbfs ) , the best - characterized transcription factors related to acquiring freezing tolerance ( jaglo - ottosen et al . , 1998 ; liu et al . , 1998 ) . since cbfs are plant specific ( riechmann and meyerowitz , 1998 ; riechmann et al . , 2000 ; 2002 ) , it is thought that the cbf signal transduction pathway is conserved only among plants . therefore , it is reasonable to speculate that there are mechanisms for adaptation to low temperature that are conserved throughout evolution , as is known for heat - shock responses . to date , it is not well understood whether there are conserved responses to low temperature within prokaryotes and eukaryotes . cold acclimation is also observed in bacteria and has been extensively studied in escherichia coli . the major cold shock protein ( csp ) in e. coli , cspa , is predominantly induced after exposure to low temperature and accumulates to up to 10% of the total protein in the cell ( goldstein et al . , e. coli contains nine cspa family genes ( cspa to cspi ) and four of them ( cspa , cspb , cspg , and cspi ) are induced by cold shock ( yamanaka et al . , 1998 ; wang et al . , 1999 the cspc and cspe genes are expressed constitutively and involved in the chromosome partitioning ( yamanaka et al . , 1994 ) . overexpression of cspd results in a lethal phenotype in e. coli ( yamanaka and inouye , 1997 ; xia et al . , 2001 ) . the e. coli cspa , cspb , cspg , and cspe quadruple deletion mutant shows a cold - sensitive phenotype , which can be complemented by overexpression of any one of the csps except cspd ( xia et al . , 2001 ) . rna molecules tend to form stable secondary structures at low temperatures , which may impede rna function such as in translation and transcription . therefore , it has been suggested that csps act as rna chaperones to destabilize rna secondary structures , enabling efficient translation at low temperature ( jiang et al . , 1997 ; phadtare et al . , in addition , cspa , cspc , and cspe are transcription antiterminators , which regulate expression of a set of cold - inducible genes ( bae et al . , 2000 ) . bacterial csps are composed of a single nucleic acid binding domain ( of about 70 amino acid residues ) called the cold shock domain ( csd ) . the csd consists of a five - stranded -barrel containing two consensus rna binding motifs ( rnp-1 and rnp-2 ) , which contribute to single - stranded dna / rna binding ( schroder et al . , 1995 ; in animals , the homologous genes to bacterial csps have been identified , and have been shown to contain an n - terminal csd and c - terminal auxiliary domains . the vertebrate y - box binding ( yb ) proteins have a highly conserved csd , which shares about 40% amino acid sequence identity with bacterial csps ( wistow , 1990 ; wolffe et al . , 1992 ) . yb proteins contain an n - terminal ala and pro rich domain ( a / p domain ) followed by a csd and c - terminal alternating clusters of basic and acidic amino acid residues ( b / a repeat ; wolffe , 1994 ; graumann and marahiel , 1998 ) . the c - terminal b / a repeat was proposed to have dna and rna binding activity and the ability to bind other proteins ( evdokimova and ovchinnikov , 1999 ) . yb-1 , the best - characterized yb protein , is a multifunctional protein that is involved in the regulation of transcription and translation , drug resistance , cell proliferation , and stress adaptation ( kohno et al . , 2003 ) . after a temperature downshift , no increase in cell numbers was observed for yb-1-depleted chicken cells , indicating an essential function for yb-1 in cell proliferation under cold conditions ( matsumoto et al . , yb-1 binds to dna and rna and shows concentration - dependent melting and annealing activities ( matsumoto and wolffe , 1998 ; skabkin et al . , 2001 ) . another example of a eukaryotic csd protein is lin-28 , which was originally identified as an essential regulator of larval development in c. elegans ( moss et al . , 1997 ) . lin-28 comprises a single csd and two cchc retroviral - like zinc fingers at the c - terminus . mammalian lin-28 is a translational enhancer of igf-2 , which is essential for the growth and differentiation of muscle tissue ( polesskaya et al . , 2007 ) . in addition , it has been demonstrated that lin-28 plays an important role in reprogramming human somatic cells into pluripotent stem cells ( yu et al . lin-28 post - transcriptionally inhibits the biogenesis of let-7 mirna , which is a regulator of cell growth and differentiation in embryonic cells ( roush and slack , 2008 ; viswanathan et al . , 2008 ) . collectively , these studies suggest that animal csd proteins play important roles in a variety of biological processes , not only stress adaptation . cold shock domain proteins are widespread among lower and higher plants including monocots and dicots ( karlson and imai , 2003 ) . plant csd proteins so far characterized typically contain a large glycine - rich region interspersed with cchc zinc fingers at the c - terminus ( karlson et al . , 2002 ) , a domain structure , and arrangement resembling that of lin-28 . we thus conducted a database search using plant gdb ( http://www.plantgdb.org/ ) to identify csd proteins in plant species whose genome sequences are available ( figure 1 ) . all known genome sequences contain at least two csd proteins that are composed of a csd , glycine - rich regions and cchc zinc fingers . the sequence information also indicates that genomic sequences for csd proteins commonly contain no introns . the number of gene family members in each species ranges from two ( oryza sativa , zea mays , sorghum bicolor , and vitis vinifera ) to seven ( glycine max ) . in addition , individual species have both long and short versions of csd proteins . in monocots , csd proteins contain two or four cchc zinc fingers , whereas the number of cchc zinc fingers is one , two , four , five , or seven in dicots . the glycine - rich and cchc zinc finger regions are probably involved in binding of nucleic acids and other proteins ( karlson et al . , 2002 ; nakaminami et al . , 2006 ; since all plant csd proteins contain a highly conserved csd , it is speculated that the diverse combinations of cchc zinc fingers are necessary for binding to specific nucleic acids and proteins . schematic representation of the domain organization of cold shock domain proteins in plant species whose genome sequences are available . only very short est sequences , or none at all , are available for ljcsp1 ( lotus japonicas ) and ptcsps ( populus trichocarpa ) . whereas a considerable amount of research has been carried out to characterize csd proteins in bacteria and animals , little is known about their functions in plants . the first functionally characterized plant csd protein was the wheat csp ( wcsp1 ; karlson et al . wcsp1 contains a glycine - rich region interspersed with three c - terminal cchc zinc fingers . wcsp1 mrna is up - regulated in response to cold and the corresponding protein is substantially accumulated in crown tissue during prolonged cold acclimation . transcript levels of wcsp1 are not modulated by other environmental stresses such as salt , drought and heat , or treatment with abscisic acid ( karlson et al . , 2002 ) , suggesting that the function of wcsp1 is specific to cold adaptation . wcsp1 binds to dna and rna and melts double - stranded nucleic acids in vitro and in vivo ( karlson et al . , 2002 ; nakaminami et al . , in addition , wcsp1 complements a cold - sensitive phenotype of the e. coli csp mutant ( nakaminami et al . , 2006 ) . these data suggest that wcsp1 shares a conserved function with e. coli csps and is involved in the regulation during cold acclimation . up indicates up - regulation of the gene expression by each abiotic stress , while rice has two csd proteins [ oscsp1 ( os02g0121100 ) and oscsp2 ( os08g0129200 ) ] , which exhibit nucleic acid binding activity and complement the cold sensitivity of the e. coli csp mutant ( chaikam and karlson , 2008 ) . expression of oscsps was slightly increased in shoot and root tissues by short term low temperature treatment ( chaikam and karlson , 2008 ; table 1 ) . however , oscsp protein levels were not increased in crown tissue during 10 days of low temperature treatment ( chaikam and karlson , 2008 ) . tissue specific expression patterns of oscsps revealed that oscsp proteins are highly accumulated in the developing panicle , flower , and seed ( chaikam and karlson , 2008 ) . thus , the functions of oscsps may be more associated with developmental processes than with cold tolerance . in arabidopsis , four csd proteins ( atcsp1atcsp4 ) were identified and functional analyses of atcsps have been performed with overexpression lines and mutants ( table 1 ) . an atcsp3 ( at2g17870 ) knock - out mutant ( atcsp3 - 2 ) was more sensitive to freezing than was wild - type under both non - acclimated and cold - acclimated conditions ( kim et al . , 2009 ) . overexpression of atcsp3 confers increased freezing tolerance in arabidopsis without obvious developmental defects ( kim et al . , 2009 ) . atcsp3 does not affect the expression of cbfs and cor genes , but it regulates the expression of stress - related genes whose roles in freezing tolerance are unknown ( kim et al . , 2009 ) . interestingly , several genes down - regulated in atcsp3 - 2 are known to be up - regulated in the ada2b-1 mutant , which is more freezing tolerant than wild - type without up - regulation of cor gene expression ( vlachonasios et al . , 2003 ) . since ada2b is a component of histone acetyltransferase complexes , it has been demonstrated that atcsp2 ( atgrp2/csdp2 ; at4g38680 ) transcript is highly expressed in meristematic and developing tissues ( fusaro et al . , 2007 ; consistent with its expression patterns , functional analyses using rnai knock - down transgenic plants indicated that atcsp2 negatively regulates flowering time , and positively regulates seed / embryo development ( fusaro et al . , 2007 ) . recently , it was demonstrated that atcsp4 ( atgrp2b ; at2g21060 ) , the closest paralog of atcsp2 , also plays an important role in development . overexpression of atcsp4 resulted in reduced silique length and embryo lethality ( yang and karlson , 2011 ) . expression of several mads - box and endosperm development genes is altered in the atcsp4-overexpressing line during floral and silique development . park et al . ( 2009 ) reported that overexpression of atcsp1 ( csdp1 ; at4g36020 ) delays seed germination under dehydration or salt stress conditions , whereas overexpression of atcsp2 accelerated seed germination under salt stress conditions . although overexpression of atcsp1 or atcsp2 did not enhance freezing tolerance in arabidopsis , they each complement the freezing - sensitive phenotype of grp7 , which is a mutant of glycine - rich rna binding protein 7 ( grp7 ) with rna chaperone activity ( kim et al . these functional studies of plant csd proteins reveal that the expressions of multiple csd proteins are differentially regulated by developmental and stress cues ( table 1 ) . furthermore , plant csd proteins commonly exhibit rna chaperone activity and function as regulatory proteins . the evolutionarily conserved structures and biochemical activities of csd proteins suggest that these proteins are indispensable for cold adaptation in both prokaryotes and eukaryotes . in addition , regulatory functions of csd proteins extend to developmental processes in both animals and plants . whereas information regarding the biological functions of plant csd proteins is accumulating whether or not csd proteins have specific target mrnas in plant cells needs to be addressed . in chlamydomonas reinhardtii , the nab1 csd protein stabilizes the mrna of lhcbm ( major light - harvesting complex of photosynthesis ii ) and represses its translation at the pre - initiation stage ( mussgnug et al . , 2005 ) . however , rna stabilization and translational repression have been described for animal csd proteins such as yb-1 and frgy2 ( matsumoto et al . , 1996 ; evdokimova et al . , 2001 ) . the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest .

it has great tensile strength , and is the main component of ligaments and tendons . it is responsible for skin elasticity , and its degradation leads to wrinkles that accompany aging . the native tropocollagen molecule ( molecular mass 300 kda ) consists of three polypeptide chains of about 1000 amino acid residues each , wound round one another to form a triple helix . the atoms in the individual chains are held together with covalent bonds , while the three chains are held in the triple - helical structure by weaker bonds . when the protein is heat - denatured , these weak bonds are broken but the covalent bonds stay intact and the three chains separate from one another and collapse into random coils [ 13 ] . collagen fibers in vivo must be stable enough to withstand the disruptive influence of thermal agitation , but capable of assembly and disassembly of the component molecules . in solution , the unfolding temperatures of a wide range of fibrous collagens are within only a few degrees of the animal 's body temperature , but when the molecules are aggregated to form fibers , there is an increase in the transition temperature of ~27c [ 4 , 5 ] . thermal denaturation of collagen depends on water content , ph of environmental medium , and degree of cross - linking [ 610 ] . . a number of environmental and anthropometric risk factors may contribute to it , and recent reports have suggested the importance of genetic factors as well . studies of the effect of disc disease on the properties of the collagen molecule are rather limited [ 1113 ] . differential scanning calorimetry dsc has been used to prove the differences between the stages of disc degeneration in calorimetric measures . it has been presented that the annulus fibrosus ( af ) and nucleus pulposus ( np ) show thermodynamically distinct behavior . the thermal denaturation of normal af and np is almost identical regarding the main transition temperatures , but completely different in the total calorimetric enthalpy changes . the comparison of calorimetric curves of the thermal denaturation of degenerated and healthy specimens shows significant differences . it was demonstrated in the literature that dsc is an applicable method for the demonstration of thermal consequences of local as well as global conformational changes in the structure of the human intervertebral discs . it comes from the inherent nature of this method that we can not assign any thermal event to any molecular process directly , but the results suggest that definitive differences exist between the stages of disc degeneration in calorimetric measurements going on either on a local or global level . the diseases of the intervertebral disc such as degenerative disc disease , and scoliosis are both characterized by changes in the extracellular matrix components that will affect the mechanical function of the tissue . the stability of the collagenous components and hence the mechanical integrity of connective tissues such as the disc are dependent on the degree and type of cross - links between the collagen molecules . this paper records an experiment that investigates calorimetrically the effect of disc disease on the collagen helix - coil transition for tissue extracted from patients during surgical operation . forty discs were obtained from patients with degenerative disc disease undergoing surgery for low back pain . the data allow the comparison between thermal stability of collagen tissue from healthy patients and from patients suffering from disc disease . forty discs were obtained from patients with degenerative disc disease undergoing surgery for low back pain in department of neurosurgery , military institute of the health services , central clinical hospital of the department of national defence , warsaw , poland . the routine was approved by ethical commission and all participating patients gave their informed consent . immediately after operation , the specimens were frozen at 70 degrees c. in the frozen state , the specimens were transported to the laboratory . before dsc experiments , the specimens were kept in room temperature for a while to unfroze them . tissues were scanned in a computer - controlled perkin - elmer dsc-7 , fitted with an intracooler , and running software supplied by the manufacturer was used for the calorimetric measurements . weighed samples ( 0.01 mg ) were heated at 5c per minute and at 0.5c per minute from 20c to an appropriate specified temperature using an empty pan as a reference . dsc curves for collagen in healthy disc and collagen in disc degenerated by discopathy are presented in figures 1 and 2 . the curves in figure 1 have been obtained with heating rate of 0.5c / min . the curves in figure 2 have been obtained with heating rate of 5c / min . as can be seen the heating rate has an influence on the position of the peak of denaturation temperatures of collagen in disc tissues . the helix - coil transition for collagen from healthy disc appears near 94.5c ( when dsc curves were recorded with heating rate of 5c / min ) and near 50c ( when dsc curves were recorded with heating rate of 0.5c / min ) . for collagen in degenerated disc , dsc peak appears in the range of 84104c ( when dsc curves were recorded with heating rate of 5c / min ) and in the range of 51 - 52c ( when dsc curves were recorded with heating rate of 0.5c / min ) . dsc profiles show that disc disease induced changes in molecular structure of collagen in disc tissue that alters position in dsc peak of collagen ( highly energetic and sharp denaturation endotherm that is a characteristic of the triple helix ) . higher helix - coil transition temperature in degenerated disc collagen suggests that additional intermolecular cross - linking of collagen fibers occurs . as a healthy disc came from female disc , we compare dsc curves for healthy and degenerated only for female disc ( we did not have male healthy disc ) . we observed that the maximum of peak responsible for thermal denaturation of collagen in male disc is in the range of 80.9597.87c , whereas for female it is in the range of 86.25104.47c ( figure 3 ) . it may suggest that collagen in female disc possesses more cross - linking linkages than collagen in male discs . the high enthalpy of unfolding of collagen is thought to derive mainly from the breaking of the hydrogen bonds between triple helixes and hydrogen bonds forming the hydration network around the collagen molecule . the enthalpy values ( h ) for the endothermic transition of collagen in disc are shown in tables 1 and 2 . the results show that h decreases with the age of patients and degree of degeneration . the decrease of the enthalpy of this degenerated structure is attributed to the loss of bound water and thermal cooperation of the components . it was also suggested by the widening of the thermal transition period and the asymmetry of the curves themselves . the drop in the main transient temperature in the degenerated disc is mostly due to the loss of the immensely hydrated proteoglycans . the fragmentation of this structure results in the decrease of bound water clusters , and so consequently the decrease of the thermal capacity ( ability to store heat energy ) . in calorimetry , the significantly lower thermal capacity is an important sign of the loss of water clusters , resulting in a greater baseline shift when compared to the native stage . the consequence is the significantly smaller changes in the enthalpy and less thermical cooperation in the degenerated disc in comparison with the control ( healthy ) specimens . we employed the x - ray diffraction ( xrd ) analysis to find out whether the crystal domains are present in disc tissue . however , we could not find a dependence between the amount of crystal and amorphous phase in collagen and the age of patients nor the gender . disc disease seems to be induced by a process leading to changes in fully hydrated collagen . these changes caused both stabilization and destabilization of the triple helix in fibers . for collagen fibers from patients suffering from disc disease , the heating rate has an influence on the position on denaturation temperatures of collagen in disc tissues . higher helix - coil transition temperature in degenerated disc collagen suggests that additional intermolecular cross - linking of collagen fibers occurs . denaturation temperatures of collagen in degenerated male disc possess smaller values than in female ones . disc disease induces changes in collagen structure and leads to formation of additional crosslinks between collagen fibers .

open pyeloplasty remains the gold standard for correcting ureteropelvic junction obstruction with a success rate between 90% to 98% . although endopyelotomy and retrograde dilation are alternative methods of managing ureteropelvic junction obstruction in children , the success of these 2 procedures is inferior to that reported for conventional dismembered pyeloplasty . laparoscopic pyeloplasty is rapidly becoming an acceptable procedure for ureteropelvic junction obstruction ( upjo ) in the pediatric population . the advent of smaller instrumentation and more experience with intracorporeal suturing allows this well - accepted procedure in adults to be implemented in the pediatric population . four children between 7 months to 8 years of age ( mean , 3.14 years ) underwent transperitoneal laparoscopic pyeloplasty for a upjo in a pelvic kidney . all patients except the 7-month - old patient presented with antenatal diagnosed hydronephrosis , which worsened and was associated with parenchymal thinning of the pelvic kidney . preoperatively , all patients had ultrasounds and diuretic radionuclide imaging ( dri ) evaluations consistent with upjo . all patients underwent cystoscopic evaluation with retrograde ureteropyelogram ( rgp ) and stent placement before laparoscopic positioning ( figure 1 ) . the transperitoneal laparoscopic approach previously described in the literature was implemented but slightly modified for the pelvic kidney . one was placed lateral to the contralateral rectus in the midclavicular line at the level of the umbilicus ; another at the umbilicus to hold a 30-degree 3-mm laparoscope ; and the remaining one near the midaxillary line lateral to the ipsilateral rectus 3 cm medial and cephalad from the anterior superior iliac spine ( figure 2 ) . three patients had a lower pole - crossing vessel , and 1 patient had a stenotic proximal segment . all reap - proximations were performed with 6 0 polyglactic acid sutures in an interrupted fashion . twelve sutures were placed at the upj with or without renal pelvis tapering , which was performed with a running 6 0 polyglactic acid suture . all pyeloplasties were performed with a double pigtailed ureteral stent placed during cystoscopic evaluation at the onset of the procedure . outcome measures included operative time , length of hospital stay , postoperative symptomatology , and resolution of obstruction by symptoms and ultrasonography , or dri , or both . all the patients were followed with ultrasonography 2 months and then every 6 months after the procedure . average operative time was 2.1 hours ( range , 1.5 to 2.8 ) . mean hospital stay was 2.15 days ( range , 1 to 7 ) . mean time to return to normal activity was 2 weeks ( range , 0.5 to 6 ) . after surgical repair , all 3 patients ( 100% ) were completely pain free at a mean follow - up of 12 months . all patients were evaluated 2 months postoperatively for an ultrasonographic evaluation revealing mild to moderate hydronephrosis . at a mean ultrasonographic follow - up of 11 months ( range , 6 to 20 ) , all 4 patients had resolution of the hydronephrosis . current published data support at this time that it is not recommended in children younger than 6 months . generally , the indications for laparoscopic pyeloplasty are identical to those of the open surgical approach , including failed previous retrograde or antegrade endopyelotomy . we were able to mobilize the ureter sufficiently that a culp - deweerd spiral , scardino - prince vertical flap , or a dismembered tubularized renal pelvic wall flap described by gill et al were not necessary . however , though the cohort is small , we feel that the same principles used in orthotopic laparoscopic pyeloplasty are easily recapitulated for the pelvic kidney . even with a small number of patients , our results seem to show that laparoscopic pyeloplasty is an option for treating upjo in pelvic kidneys . we feel that the transperitoneal laparoscopic approach is feasible in this specific , complex , pediatric population .

studies in human volunteers have demonstrated clonidine like analgesic , sedative , sympatholytic and cardiovascular effects.[810 ] in recent studies , dexmedetomidine has been shown to have clinically significant effects on anaesthetic requirements , haemodynamic responses induced by anaesthesia and surgery in patients . it has also been observed that an intraoperative infusion of dexmedetomidine combined with inhalation anaesthetics provided satisfactory intraoperative conditions without adverse haemodynamic effects and decreases emergence agitation in children . dexmedetomidine is increasingly being used as a sedative for monitored anaesthesia care ( mac ) because of its analgesic properties , cooperative sedation , and lack of respiratory depression . the study was undertaken to assess the efficacy and safety of dexmedetomidine in attenuating sympathoadrenal response to tracheal intubation and to analyse reduction in intraoperative anaesthetic requirement . after obtaining approval from the institutional ethical committee , a randomised controlled study was formulated . the study population comprised 60 patients with asa physical status i and ii , aged 1865 years , scheduled for elective surgery of duration 3 hours or more . written informed consent pregnant and nursing women , patients with morbid obesity , heart block , and hypertensive patients on blockers were excluded from the study . the patients were randomly assigned to one of the two groups , each containing 30 patients , using a slips of paper in a box technique . the grouping is as follows : group c : control group : isoflurane opioid - saline anaesthesia group d : dexmedetomidine group : isoflurane opioid - dexmedetomidine anaesthesia all the patients were premedicated with inj . glycopyrrolate 0.2 mg intramuscularly , 30 min prior to induction of anaesthesia . on arrival in the operating room , the patients baseline heart rate , blood pressure , oxygen saturation ( spo2 ) , and respiratory rate were recorded after 5 min settling in the operative room . dexmedetomidine in a dose of 1 g / kg over a period of 10 min prior to induction of anaesthesia through an infusion pump . during the infusion , heart rate , systolic blood pressure , diastolic blood pressure , respiratory rate , oxygen saturation and sedation score were recorded at 5 min intervals and at 10 min ( end of infusion ) . laryngoscopy was performed with a macintosh laryngoscope and trachea was intubated with appropriate size endotracheal tube . the isoflurane was used in lowest possible concentration necessary to keep the blood pressure and heart rate within 20% limits of patient 's preoperative baseline values . the inspiratory concentration of isoflurane was adjusted in steps of 0.2% when needed to keep the haemodynamic parameters to acceptable values . fentanyl in increments of 0.4 g / kg was given when inspiratory isoflurane concentration exceeded by 1% . the dexmedetomidine infusion was continued after intubation in a dosage of 0.20.7 g / kg / hour in group d , till the start of skin closure . all the patients in group d received isoflurane in minimum concentration of 0.4% , which was further increased when requirement of inj . dexmedetomidine exceeded 0.7 g / kg / hr to keep the haemodynamic parameters within acceptable range . similarly , isoflurane was terminated at the start of skin closure and n2o was discontinued after skin closure . at the end of anaesthesia , patients were extubated when respiration was deemed sufficient and patients were able to obey simple commands . all the parameters and the results from the two groups ( group c and group d ) were entered in the predesigned study proforma sheet . ( 1)sedation score at 5 min and 10 min after administration of loading dose of dexmedetomidine in group d according to ramsay sedation score.(2)heart rate , systolic and diastolic blood pressure , spo2 at 5 min and 10 min after dexmedetomidine administration , preinduction , induction , 0 min , 1 min , 5 min after intubation.(3)the dose of the inj . thiopentone for induction of anaesthesia.(4)total fentanyl requirement throughout the operative procedure.(5)the average inspiratory isoflurane concentrations was calculated as the sum of the products of inspiratory concentrations and times divided by total anaesthesia time.(6)the intraoperative need for adjuvants such as inj . sedation score at 5 min and 10 min after administration of loading dose of dexmedetomidine in group d according to ramsay sedation score . heart rate , systolic and diastolic blood pressure , spo2 at 5 min and 10 min after dexmedetomidine administration , preinduction , induction , 0 min , 1 min , 5 min after intubation . the average inspiratory isoflurane concentrations was calculated as the sum of the products of inspiratory concentrations and times divided by total anaesthesia time . the intraoperative need for adjuvants such as inj . statistical analysis was conducted with spss ( version 10 , 2010 ) for windows statistical package using paired and unpaired student 's t test . p<0.05 was regarded as statistically significant , p<0.001 was taken as highly significant , and p>0.05 was regarded as nonsignificant . a sample size of 20 patients per group was needed to detect an intergroup difference of at least 20% with two - sample t - test . all the parameters and the results from the two groups ( group c and group d ) were entered in the predesigned study proforma sheet . ( 1)sedation score at 5 min and 10 min after administration of loading dose of dexmedetomidine in group d according to ramsay sedation score.(2)heart rate , systolic and diastolic blood pressure , spo2 at 5 min and 10 min after dexmedetomidine administration , preinduction , induction , 0 min , 1 min , 5 min after intubation.(3)the dose of the inj . thiopentone for induction of anaesthesia.(4)total fentanyl requirement throughout the operative procedure.(5)the average inspiratory isoflurane concentrations was calculated as the sum of the products of inspiratory concentrations and times divided by total anaesthesia time.(6)the intraoperative need for adjuvants such as inj . sedation score at 5 min and 10 min after administration of loading dose of dexmedetomidine in group d according to ramsay sedation score . heart rate , systolic and diastolic blood pressure , spo2 at 5 min and 10 min after dexmedetomidine administration , preinduction , induction , 0 min , 1 min , 5 min after intubation . the average inspiratory isoflurane concentrations was calculated as the sum of the products of inspiratory concentrations and times divided by total anaesthesia time . the intraoperative need for adjuvants such as inj . statistical analysis was conducted with spss ( version 10 , 2010 ) for windows statistical package using paired and unpaired student 's t test . p<0.05 was regarded as statistically significant , p<0.001 was taken as highly significant , and p>0.05 was regarded as nonsignificant . a sample size of 20 patients per group was needed to detect an intergroup difference of at least 20% with two - sample t - test . dexmedetomidine was well tolerated and no drug - related adverse events were observed . about 10 min after receiving dexmedetomidine , patients were drowsy but arousable ( sedation score 2 ) . patient characteristics [ mean ( sd ) ] and type of surgeries the mean sleep dose of inj . thiopentone required in group c was 6 mg / kg , while it was 4.4 mg / kg in group d [ table 2 ] . the decrease in the dose requirement was by 30% in dexmedetomidine group as compared to control group ( p=0.00 ) . anaesthesia characteristics [ mean ( sd ) ] the average inspiratory concentration of isoflurane required during anaesthetic maintenance was 0.8% in group c and 0.54% in group d. a decrease of 32% was observed in group d compared to group c ( p=0.00 ) . also , the requirement of inj . fentanyl was 1.8 g / kg in group c as opposed to 1.1 g / kg in group d. group c patients required 33% more fentanyl as compared to group d patients ( p=0.00 ) [ table 2 ] . before administration of the study drugs in the operating room , heart rate and blood pressure values between the two groups did not differ . in group d patients receiving dexmedetomidine loading infusion , a fall in the heart rate and blood pressure was observed , which was not more than 5% of the baseline . patients were sedated but arousable with sedation score of 2 . in both the groups , the maximal increase in heart rate and blood pressure occurred immediately after tracheal intubation ( 0 min ) when compared to the baseline arterial blood pressure . the increase in heart rate after intubation was 21% in group c as compared to 7% in group d ( p=0.00 ) . similarly , significant increase in systolic pressure was observed in group c which was 40% as compared to 8% in group d ( p=0.00 ) , while increase in diastolic pressure was 25% and 11% in group c and group d , respectively ( p=0.001 ) [ figure 1 ] . cardiovascular response during tracheal intubation in group c ( ) and group d ( ) . hr = heart rate ; sap = systolic arterial pressure ; dap = diastolic arterial pressure . time points : 1 = baseline ; 2 = preinduction ; 3 = induction ; 4 = 0 min after intubation ; 5 = 1 min after intubation ; 6 = 5 min after intubation in order to avoid analgesics that have sedative effect like inj . diclofenac sodium 75 mg intravenously , while 7 out of 30 patients in group d required inj . atropine was repeated in the postoperative period after extubation in one patient out of the two . all the patients were immediately able to obey commands upon arrival into recovery room . in the recovery room , none of the patients had explicit recall of awareness or complained of any discomfort when interviewed after operation . we conducted this prospective randomised study in an attempt to examine whether administration of dexmedetomidine to a commonly administered balanced anaesthetic regimen improves perioperative haemodynamic stability in patients undergoing major surgical procedure . dexmedetomidine is a highly selective 2 agonist that has been shown to have sedative , analgesic and anaesthetic sparing effects . it causes a dose - dependent decrease in arterial blood pressure and heart rate , associated with decrease in serum norepinephrine concentration . dexmedetomidine was well tolerated , and no serious side effects or adverse reactions occurred in the present study . the dose of thiopentone needed for induction was reduced significantly ( 30% ) in the patients receiving dexmedetomidine , as also found by aantaa and coworkers , demonstrating the anaesthesia potentiating effects of the drug . it has also been shown that dexmedetomidine potentiates analgesia caused by fentanyl and reduces its dose requirements in humans during surgery . the fentanyl dose in the control group was almost twice that given in the dexmedetomidine group . the requirement of fentanyl was reduced by 33% in the dexmedetomidine group in our study . analgesic property has been demonstrated earlier in a study with dexmedetomidine in experimental ischaemic pain in healthy volunteers and as the sole analgesic after surgery . dexmedetomidine has been widely studied as an anaesthetic adjuvant and its anaesthetic sparing effects are well known . in studies by aho and colleagues and aantaa and coworkers , it has been shown to reduce the isoflurane requirement dose dependently up to 90% . in our study , isoflurane was used as the main anaesthetic agent , the requirement of which was decreased by 32% in group d , in accordance with earlier studies . tracheal intubation is associated with increases in arterial pressure , heart rate and plasma catecholamine concentrations . increases in arterial pressure and heart rate observed in the control group in the present study were similar to those reported in earlier studies . in the present study , pretreatment with dexmedetomidine 1 g / kg attenuated , but not totally obtunded , the cardiovascular response to tracheal intubation after induction of anaesthesia . in patients undergoing general or gynaecological surgery , numerous studies have shown that dexmedetomidine blunts cardiovascular response to intubation and our findings are in accordance with them . in addition to this beneficial property of -2 agonists , they have also been reported to increase the risk of hypotension and bradycardia . these effects have most often been seen in young healthy volunteers on rapid bolus administration . in our study , bradycardia was observed in two patients receiving dexmedetomidine , with no fall in blood pressure , which responded promptly to iv atropine . in our study , three patients were of craniotomies for supratentorial tumours . increase or decrease in blood pressure may cause bleeding or edema or predispose the patient to cerebral ischaemia . the haemodynamic responses to emergence from anaesthesia and extubation are blunted with dexmedetomidine and the centrally mediated sympatholytic effect is continued well in postoperative period , which was advantageous in these patients . tanskanen and coworkers , in their study using dexmedetomidine as an anaesthetic adjuvant for intracranial tumour , concluded that there was an increased perioperative haemodynamic stability in patients undergoing brain tumour surgery without postoperative respiratory depression . also , dexmedetomidine has been studied as a supplement to isoflurane for vitreoretinal surgeries , without causing undue haemodynamic fluctuation , and has been shown to decrease the excitatory response during extubation with acceptable reduction in intraocular pressure . a possible limitation of our study may have been the use of subjective criteria to determine dose of thiopentone , isoflurane and fentanyl for each patient . estimating anaesthesia depth by changes mediated by autonomic nervous system is difficult during dexmedetomidine infusion as it increases the haemodynamic stability . intraoperative bispectral index ( bis ) monitoring would have been definitely more objective in deciding the depth of anaesthesia and the requirement of anaesthetic agent . also , measurement of qt interval and plasma catecholamine levels , more objective means of haemodynamic response , was not done because of practical difficulty . measurement of the end - tidal isoflurane concentration would have been ideal to indicate the depth and for quantifying decrease in utilisation between groups than the inspired dial concentration . postoperative requirement of analgesics was not taken into consideration as it was not part of our study . measurement of time to recovery following extubation would have given an idea of recovery in both the groups . bradycardia , a possible consequence of administration of 2 agonist , was counteracted by the use of atropine . dexmedetomidine , as a pre - anaesthetic medication and intraoperative infusion , decreases intraoperative anaesthetic requirement .

in the last decade the necessity for multicenter studies has arisen in order to increase sample sizes and therefore reliability of results . this multicenter approach is particularly important for rare disorders , such as huntington 's disease ( hd ) , because it permits collecting data from a sufficiently large number of participants in a reasonable time frame . cerebral white matter ( wm ) changes in hd have been reported in a number of studies ( for reviews see bohanna et al . ( 2009 ) ) , mainly using mr - based diffusion weighted imaging ( dwi ) and diffusion tensor imaging ( dti ) ( douaud et al . , 2009 ; rosas et al . , 2006 ) . these techniques use diffusion directions of water molecules to calculate a tensor , which can be converted into metrics such as fractional anisotropy ( fa ) ( basser and jones , 2002 ; pierpaoli et al . , 1996 ) , a dimensionless scalar ranging from a value of zero ( isotropy ) to a value of one ( high directionality , anisotropy ) . there is , however , a downside to multicenter imaging studies focusing on dti , in that scanner and environmental noise , as well as systematic inter - subject variables may contribute to the variability of dti results . some effort has already been made to investigate the influence of these variables on scanning results . pagani and colleagues demonstrated between - center differences in dti repeatedly scanning a number of subjects ( between 7 and 13 ) at 8 different scanner sites ( pagani et al . , by contrast , other authors reported only small differences in dti metrics between sites when scanning a physical phantom ( teipel et al . other studies focused on comparability of dti metrics from different scanners by region of interest ( roi ) analyses of coefficients of variation ( cv ) . they found that fa is the most comparable measure across different scanners and that nonlinear co - registration of fa maps reduced average inter - site cv ( fox et al . 2010 ) . a common way to reduce variability is to calibrate all scanners involved in a multicenter study , with the optimal procedure being to scan a number of participants as a reference group at each study center . however , this is a time consuming and expensive procedure , and not viable for many important but underfunded studies . therefore , other equally valid ways of ensuring comparability of dti results from different scanners should be explored . as a proof of concept , we used dti data from early hd and control participants acquired from four different scanners . for comparison , we defined five rois based on previous findings in dti studies with hd patients , i.e. basal ganglia , external and internal capsules , thalamic region and corpus callosum ( douaud et al . these five structures have been identified as being particularly affected in people with manifest hd with a specific pattern of decreases and increases in fa values . the regions most affected were the basal ganglia with an increase in fa and the internal and external capsules and the thalamic regions which showed fa decreases ( rosas et al . , 2006 ) . it should be noted that these studies were not multicenter studies and that all participants were scanned with the same mri scanner . the aim of our study was to provide an evaluating framework for combining dti data acquired from different study centers without time- and cost - intensive repeated scanning of the same reference group at each study site . as the primary outcome variable the present study focused on fa data as the relevant dti metric since fa differences between hd subjects and controls have already been reported ( douaud et al . , 2009 ; fox et al . , 2012 ; rosas et al , we suggest a three - step procedure as outlined in fig . 1 . in the first step we tested whether the cv of fa values in predefined rois differed between centers . in this step we used data from control participants to estimate potential influences independent of disease related factors . in the second step we tested whether group differences between patients and controls differed between centers . in the third step patient and control data from different study sites were pooled and a voxel - wise whole - brain analysis was performed to assess whether putative group differences were present in the selected rois . data in this study were collected as part of the european paddington project at four study sites in europe ( leiden , the netherlands ; london , uk ; paris , france ; ulm , germany ) . the paddington project , which involves five partners ( siena biotech spa , university of ulm , university college london , the london school of hygiene and tropical medicine and kcr poland ) , is an international initiative that aims to provide pharmacodynamic approaches for disease - modifying clinical trials . the work package 2 of this project entails the collection of volumetric 3 tesla mri and dti scans acquired with standardized acquisition protocols from patients with hd in an early disease stage and from healthy control participants , with the objective of identifying biomarkers of disease progression . dti data from work package 2 were used to evaluate our three - step procedure . hd patients had a genetically confirmed diagnosis with a trinucleotide ( cag ) repeat length of 36 or higher , and had clinical features of mild hd at least of stage i based on the unified huntington 's disease rating scale ( uhdrs ) with a total functional capacity ( tfc ) score of 1113 . subjects with claustrophobia and conditions not permitting mr scanning were excluded from the study . in total 61 hd and 40 control subjects ( co ) the acquisition parameters for the different sites are listed in table 1 , and sample sizes are listed in table 2 . the in - house developed dti analysis software tensor imaging and fibre tracking ( tift ; mller et al . , 2007a , 2007b ) with additional routines for eddy current correction was used for post - processing and statistical analysis . prior to fa - calculation motion artifacts were eliminated in each volume and each subject separately by a recently described procedure ( mller et al . in order to perform spatial normalization to a common space ( brett et al . , 2002 ) a study - specific b0-template and an fa - template were created ( mller et al . normalization to an fa - template was further included into the process of normalization because a non - affine registration to an fa - template has the advantage of providing more contrast in comparison to b0-images ( smith et al . , 2006 ) . in a next step a non - linear normalization of the dti data sets was performed by minimizing the squared differences of regional intensities between the normalized fa - maps and the fa - template ( following the basic ideas of ashburner and friston ( 1999 ) ) . scanner- and sequence specific b0- and fa - templates were created by arithmetically averaging data sets of all participants for each site after linear transformation according to manually set landmarks . after initial normalization , templates were created during an iterative step to further optimize the normalization matrices . this process was repeated until the correlation was > 0.7 between the individual fa - maps and the fa - template , which was achieved by one iteration . according to standard methods ( basser and jones , 2002 ) , the second - rank diffusion tensor d , the eigenvalues ( 1 , 2 , 3 ) , the eigenvectors ( 1,2,3 ) , and the fa for quantification of the diffusion anisotropy were calculated after normalization of individual dti data sets . since filter size may influence the results of the dti data analysis ( henley et al . , 2010 ; jones et al . , 2005 ) , the ideal adjustment of the smoothing kernel requires application of the matched filter theorem which states that the filter size should be tailored to the size of the expected difference ( rosenfeld and kak , 1982 ) . based on results from previous studies that had already tested several filter sizes between 6 and 10 mm full width at half maximum ( fwhm ) in dti analysis of fa and mean diffusivity values ( mller et al . , 2012 ; unrath et al . , 2010 ) and informed by previous voxel - based morphometry studies of t1-weighted data ( henley et al . , 2010 ; 2004 ) we used an estimated gaussian filter size of 8 mm fwhm for smoothing to balance the trade - off between sensitivity and specificity . the cv is defined as the ratio of the standard deviation of the measurements divided by the mean multiplied by 100 . it is an intuitive estimate of measurement variance expressed as relative percentage regardless of the absolute measurement value and reflects the inverse of the signal - to - noise ratio . in previous studies on dti test retest reliability the cv is a frequently reported statistical measure ( vollmar et al . , 2010 ) . since testing whether or not differences between centers are beyond chance imposes a comparable reliability problem cv values of fa data were computed in different rois . using previous reference studies ( douaud et al . a priori defined anatomical rois were located in the basal ganglia , the external capsule , the thalamus , the internal capsule , and the corpus callosum ( fig . 2 ) . cvs were averaged per roi and subject and were then tested on significant center effects using a one - way analysis of variance with main factor center ( 4 levels ) . for each roi used in the between - center comparisons of the cvs , averages of fa values were computed for each individual and entered into an analysis of variance with main factors group ( 2 levels : controls , patients ) and center ( 4 levels ) . in this analysis , the group - by - center interaction is particularly informative because it permits inference whether or not group differences of the mean fa values would significantly differ between centers . voxel - wise statistical comparison between hd and control subjects was performed by student 's t - tests implemented in the tift software ( mller et al . fa values below 0.2 were not considered for calculation since cortical gray matter may show fa values of up to 0.2 ( kunimatsu et al . , 2004 ) . correction for multiple comparisons used the false - discovery - rate ( fdr ) algorithm at a nominal level of p < 0.05 ( genovese et al . , 2002 ) . to further reduce alpha error a spatial correlation algorithm data in this study were collected as part of the european paddington project at four study sites in europe ( leiden , the netherlands ; london , uk ; paris , france ; ulm , germany ) . the paddington project , which involves five partners ( siena biotech spa , university of ulm , university college london , the london school of hygiene and tropical medicine and kcr poland ) , is an international initiative that aims to provide pharmacodynamic approaches for disease - modifying clinical trials . the work package 2 of this project entails the collection of volumetric 3 tesla mri and dti scans acquired with standardized acquisition protocols from patients with hd in an early disease stage and from healthy control participants , with the objective of identifying biomarkers of disease progression . dti data from work package 2 were used to evaluate our three - step procedure . hd patients had a genetically confirmed diagnosis with a trinucleotide ( cag ) repeat length of 36 or higher , and had clinical features of mild hd at least of stage i based on the unified huntington 's disease rating scale ( uhdrs ) with a total functional capacity ( tfc ) score of 1113 . subjects with claustrophobia and conditions not permitting mr scanning were excluded from the study . in total 61 hd and 40 control subjects ( co ) the acquisition parameters for the different sites are listed in table 1 , and sample sizes are listed in table 2 . the in - house developed dti analysis software tensor imaging and fibre tracking ( tift ; mller et al . , 2007a , 2007b ) with additional routines for eddy current correction was used for post - processing and statistical analysis . prior to fa - calculation motion artifacts were eliminated in each volume and each subject separately by a recently described procedure ( mller et al . , 2007a , 2007b ) . in order to perform spatial normalization to a common space ( brett et al . , 2002 ) a study - specific b0-template and an fa - template were created ( mller et al . normalization to an fa - template was further included into the process of normalization because a non - affine registration to an fa - template has the advantage of providing more contrast in comparison to b0-images ( smith et al . , 2006 ) . in a next step a non - linear normalization of the dti data sets was performed by minimizing the squared differences of regional intensities between the normalized fa - maps and the fa - template ( following the basic ideas of ashburner and friston ( 1999 ) ) . scanner- and sequence specific b0- and fa - templates were created by arithmetically averaging data sets of all participants for each site after linear transformation according to manually set landmarks . after initial normalization , templates were created during an iterative step to further optimize the normalization matrices . this process was repeated until the correlation was > 0.7 between the individual fa - maps and the fa - template , which was achieved by one iteration . according to standard methods ( basser and jones , 2002 ) , the second - rank diffusion tensor d , the eigenvalues ( 1 , 2 , 3 ) , the eigenvectors ( 1,2,3 ) , and the fa for quantification of the diffusion anisotropy were calculated after normalization of individual dti data sets . since filter size may influence the results of the dti data analysis ( henley et al . , 2010 ; jones et al . , 2005 ) , the ideal adjustment of the smoothing kernel requires application of the matched filter theorem which states that the filter size should be tailored to the size of the expected difference ( rosenfeld and kak , 1982 ) . based on results from previous studies that had already tested several filter sizes between 6 and 10 mm full width at half maximum ( fwhm ) in dti analysis of fa and mean diffusivity values ( mller et al . 2010 ) and informed by previous voxel - based morphometry studies of t1-weighted data ( henley et al . 2004 ) we used an estimated gaussian filter size of 8 mm fwhm for smoothing to balance the trade - off between sensitivity and specificity . the cv is defined as the ratio of the standard deviation of the measurements divided by the mean multiplied by 100 . it is an intuitive estimate of measurement variance expressed as relative percentage regardless of the absolute measurement value and reflects the inverse of the signal - to - noise ratio . in previous studies on dti test retest reliability the cv is a frequently reported statistical measure ( vollmar et al . , 2010 ) . since testing whether or not differences between centers are beyond chance imposes a comparable reliability problem , 2009 ; rosas et al . , 2006 ) these a priori defined anatomical rois were located in the basal ganglia , the external capsule , the thalamus , the internal capsule , and the corpus callosum ( fig . 2 ) . cvs were averaged per roi and subject and were then tested on significant center effects using a one - way analysis of variance with main factor center ( 4 levels ) . for each roi used in the between - center comparisons of the cvs , averages of fa values were computed for each individual and entered into an analysis of variance with main factors group ( 2 levels : controls , patients ) and center ( 4 levels ) . in this analysis , the group - by - center interaction is particularly informative because it permits inference whether or not group differences of the mean fa values would significantly differ between centers . voxel - wise statistical comparison between hd and control subjects was performed by student 's t - tests implemented in the tift software ( mller et al . , 2007a ) to infer significant differences of fa maps between both groups . fa values below 0.2 were not considered for calculation since cortical gray matter may show fa values of up to 0.2 ( kunimatsu et al . , 2004 ) . correction for multiple comparisons used the false - discovery - rate ( fdr ) algorithm at a nominal level of p < 0.05 ( genovese et al . , 2002 ) . to further reduce alpha error a spatial correlation algorithm the results section follows the outline of the suggested three - step procedure by first analyzing cv in control participants and then analyzing differences of fa values between hd and control participants . these two steps were based on pre - defined regions of interest . in the third step , a voxel - wise analysis of fa maps is performed , to see whether the overall pattern of impairments matches the expected one ( fig . 1 ) . 1 ) as a potential source of variability in control participants we first examined the distribution of demographic variables of age and sex between sites . an analysis of variance ( anova ) on age showed a significant center effect ( f(3,36 ) = 3.99 , p = 0.02 ) . post - hoc newman keuls tests ( nominal alpha level of p < 0.05 ) revealed that control participants at center c were significantly older than control participants at center d ( p = 0.01 ) . distribution of sex ( chi = 2.02 ; degrees of freedom ( d.f . ) = 3 ; p = 0.57 ) did not differ between study centers ( center - specific demographic details are summarized in table 2 ) . due to the results above , age was added as a covariate to the anova when testing for center effects on cv . one - way anovas with age as a covariate showed no significant differences of cv between centers for the five predefined rois ( highest f - value : f(3,35 ) 1.73 , p = 0.18 ; external capsule ) . 2 ( f - values and associated p - values are shown above the bars ; d.f . was 3.35 for each analysis ) . one - way anovas without age as covariate did not change the pattern of results ( highest f - value : f(3,36 ) = 2.47 ; p = 0.08 ; external capsule ) . at one specific site it had become necessary to scan subjects with shorter echo times ( te ) than at the three other sites ( see table 1 ) . since diffusion quantification may change with te ( e.g. qin et al . , 2009 ) we used te as a second covariate . with this , the overall pattern of insignificant center effects remained stable for each roi ( highest f - value : f(3,34 ) = 2.03 ; p = 0.13 ; external capsule ) . finally , the same analysis was repeated but now on fa values . center effects were again not significant for all rois ( basal ganglia : f(3,34 ) = 0.35 ; p = 0.79 ; external capsule : f(3,34 ) = 1.94 ; p = 0.14 ; thalamus : f(3,34 ) = 1.45 ; p = 0.25 ; internal capsule : f(3,34 ) = 1.74 ; p = 0.18 ; corpus callosum : f(3,34 ) = 1.54 ; p = 0.22 ) . as for the cv statistics , the demographic variables age and sex distribution were tested for significant group - by - center interactions by two separate analyses . an anova with age as the dependent variable did not show a significant effect for group ( f(1,93 ) = 2.34 ; p = 0.13 ) or center ( f(3,93 ) = 1.48 ; p = 0.23 ) , and no significant interaction thereof ( f(3,93 ) = 1.89 ; p = 0.14 ) . sex distribution , however , differed across centers ( chi = 16.06 ; d.f . = 7 ; p = 0.03 ) , in that centers a , b and d included unequal numbers of male and female hd participants ( table 2 ) . statistical tests for significant group - by - center interactions were computed with fa as the dependent variable and sex as covariate . for each of the five rois we found a significant effect of group ( table 3 ) indicating differences in mean fa values between patients and controls for each region when averaging across centers . the group - by - center interactions ( table 3 ) were far from significance for all rois , supporting that group differences between patients and controls were not systematically influenced by being collected at a specific center ( see fig . did not change the pattern of results ( see table 3 and fig . 3 for details ) . whole brain - based spatial statistics for pooled fa data showed patterns of significant group differences encompassing all five rois ( fig . 4 ) . basal ganglia fa was significantly increased in hd patients compared to controls , whereas decreases of fa were found in the thalamic region , the internal and external capsules and the corpus callosum in hd patients . 1 ) as a potential source of variability in control participants we first examined the distribution of demographic variables of age and sex between sites . an analysis of variance ( anova ) on age showed a significant center effect ( f(3,36 ) = 3.99 , p = 0.02 ) . post - hoc newman keuls tests ( nominal alpha level of p < 0.05 ) revealed that control participants at center c were significantly older than control participants at center d ( p = 0.01 ) . distribution of sex ( chi = 2.02 ; degrees of freedom ( d.f . ) = 3 ; p = 0.57 ) did not differ between study centers ( center - specific demographic details are summarized in table 2 ) . due to the results above , age was added as a covariate to the anova when testing for center effects on cv . one - way anovas with age as a covariate showed no significant differences of cv between centers for the five predefined rois ( highest f - value : f(3,35 ) 1.73 , p = 0.18 ; external capsule ) . results are shown in fig . 2 ( f - values and associated p - values are shown above the bars ; d.f . was 3.35 for each analysis ) . one - way anovas without age as covariate did not change the pattern of results ( highest f - value : f(3,36 ) = 2.47 ; p = 0.08 ; external capsule ) . at one specific site it had become necessary to scan subjects with shorter echo times ( te ) than at the three other sites ( see table 1 ) . since diffusion quantification may change with te ( e.g. qin et al . , 2009 ) we used te as a second covariate . with this , the overall pattern of insignificant center effects remained stable for each roi ( highest f - value : f(3,34 ) = 2.03 ; p = 0.13 ; external capsule ) . center effects were again not significant for all rois ( basal ganglia : f(3,34 ) = 0.35 ; p = 0.79 ; external capsule : f(3,34 ) = 1.94 ; p = 0.14 ; thalamus : f(3,34 ) = 1.45 ; p = 0.25 ; internal capsule : f(3,34 ) = 1.74 ; p = 0.18 ; corpus callosum : f(3,34 ) = 1.54 ; p = 0.22 ) . as for the cv statistics , the demographic variables age and sex distribution were tested for significant group - by - center interactions by two separate analyses . an anova with age as the dependent variable did not show a significant effect for group ( f(1,93 ) = 2.34 ; p = 0.13 ) or center ( f(3,93 ) = 1.48 ; p = 0.23 ) , and no significant interaction thereof ( f(3,93 ) = 1.89 ; p = 0.14 ) . sex distribution , however , differed across centers ( chi = 16.06 ; d.f . = 7 ; p = 0.03 ) , in that centers a , b and d included unequal numbers of male and female hd participants ( table 2 ) . statistical tests for significant group - by - center interactions were computed with fa as the dependent variable and sex as covariate . for each of the five rois we found a significant effect of group ( table 3 ) indicating differences in mean fa values between patients and controls for each region when averaging across centers . the group - by - center interactions ( table 3 ) were far from significance for all rois , supporting that group differences between patients and controls were not systematically influenced by being collected at a specific center ( see fig . 3 ) . removing the covariate sex from the analysis did not change the pattern of results ( see table 3 and fig . 3 for details ) . whole brain - based spatial statistics for pooled fa data showed patterns of significant group differences encompassing all five rois ( fig . 4 ) . basal ganglia fa was significantly increased in hd patients compared to controls , whereas decreases of fa were found in the thalamic region , the internal and external capsules and the corpus callosum in hd patients . in order to comply with the requirements for increased statistical power and consistency of results in clinical imaging studies , it is necessary to scan increasingly larger sample sizes . this demands data to be collected across multiple study sites , since one single center is unlikely to be able to acquire such sample sizes in a reasonable timeframe . ideally , measured diffusion metrics should be identical across different mr scanners after their calibration . in reality , however , differences between scanning parameters at different sites and with various scanner types can influence the homogeneity of diffusion - derived dependent variables . further sources of variation include environmental noise and inter - site and inter - subject variability . also , since one single voxel covers multiple axon bundles , slight differences in positioning may result in averaging different fa values of the same voxel because even a slight shift in the voxel position could lead to a different coverage of axon bundles . the variability of diffusion data has already been extensively studied in previous research ( pagani et al . , 2010 ; teipel et al . , 2011 ; nevertheless , a specific multicenter study may not have the resources to repeatedly scan a large number of participants at each site and will have to develop a procedure to overcome potential problems associated with between - site variability within practical and financial constraints unique to that project . therefore , the aim of the present study was to provide an alternative approach to evaluate the feasibility of pooling multicenter data in a valid and robust fashion . within the framework of a three - step testing procedure we observed that in control participants cvs calculated in different a priori selected rois did not significantly differ between study sites . although the specific contribution of any individual source of variation can not be detected by this approach , the consistency of this measure across centers suggests that a systematic bias due to one or more centers is unlikely . especially when controlling for influences of control participants ' age all f - values in the present analyses of center effects on cv data were clearly below the critical threshold of f(3,35 ) = 2.25 which would have indicated effects at a level of p < 0.10 , hereby also ruling out the idea that keeping the null - hypothesis may have been an issue of insufficient power . in a next step we tested whether roi based group differences between patients and controls would differ between centers . again , all f - values of the group - by - center interactions of interest were clearly below the critical value of f(3,91 ) = 2.15 ( associated p = 0.10 ) when controlling for additional effects of unbalanced sex distributions across groups and centers . in the absence of this covariate only one ( basal ganglia ; table 3 ) of the five analyses of variances showed an f - value above the critical threshold of f(3,93 ) = 2.14 ( p = 0.10 ) which however was still beyond significance , thus supporting the conclusion that none of the study centers had contributed systematically biased differences . the results of our analysis demonstrate that multicenter pooling of dti data can be performed in a valid manner . the resulting voxelwise pattern of fa group differences between patients and controls showed a high consistency with previous reports performed at single centers ( douaud et al . 2009 ) and also encompassed all rois from which previous test statistics had been derived . rosas et al . ( 2006 ) compared fa maps of hd subjects against controls on a single center level , and they found decreases in the internal and external capsules , the corpus callosum and the thalamic region , whereas an increase was found in the basal ganglia . this increase in the basal ganglia appears to be a main feature of hd pathology probably reflecting a decline of medium spiny neurons . in healthy participants , the complex neural connectivity of the basal ganglia results in a net diffusion with low fa values however , the disease related atrophy of those medium spiny neurons which have a distinct orientation results in a net increase of fa values for the basal ganglia because other neurons with a different orientation remain unaffected . therefore , an increase of directionality of water molecules ' diffusion within the basal ganglia most likely indicates a disturbance in this region ( douaud et al . anatomical structures affected by the disease process in hd were also identified by structural mri studies ( for review see bohanna et al . ( 2009 ) ) as well as by functional mri studies ( for review see albin et al . the identified key structures were again the basal ganglia , the thalamus , the external and internal capsules as well as the corpus callosum . accordingly , the clinical symptoms in hd most likely result from an abnormal functioning of the basal - ganglia - thalamo - cortical circuitry that may originate from striatal degeneration ( albin et al . patterns of changes in fractional anisotropy associated with early hd need to be reproduced at larger scales in order to improve reliability and to meet the requirements of clinical trials , which imposes the necessity of multicenter studies . in the present study we present a new approach for assessing feasibility and validity of pooling dti data from different centers if time or cost limitations do not permit scanning of a reference sample at each study site . this study may serve as a reference for future multicenter trials employing mri to measure disease - specific progression independent of site specific conditions . another technical application of this methodological framework may be the interim quality control of dti data during an ongoing multicenter imaging trial .

it is defined as a transient loss of consciousness attributable to global cerebral hypo - perfusion , further characterized by rapid onset , brevity , and spontaneous recovery . neurally - mediated syncope ( nms ) is by far the most common cause of transient loss of consciousness but diagnosis can be challenging in some patients . in patients with recurrent syncope of unexplained origin , implantable loop recorders ( ilr ) are now well established as a diagnostic tool as outlined in the guidelines of the european society of cardiology . however , these implantable cardiac electronic devices currently do not monitor hemodynamic parameters beyond heart rate and rhythm . importantly , it is now evident that the circulatory adjustments resulting in hypotension often occur just prior to a sudden loss of consciousness . nms prediction by early and noninvasive measurement of the underlying circulatory adjustments would protect patients from syncope - related falls and accidents . advanced signal processing in combination with the integration of these systems in wearable sensors have paved the way to develop novel approaches for early detection of impending syncopes . the avoidance of even a small routine surgical procedure is another advantage of wearable multi - sensor systems . in contrast to the relative benign nms , in arrhythmia - related syncopes , especially in structural heart disease , immediate therapeutic intervention by electrical defibrillation is often mandatory . this is important because these syncopes often have a rapid onset with a high risk of sudden cardiac death . in these patients therefore , the implantable cardioverter - defibrillator has become the therapy of first choice for a large number of patients with heart failure who have an impaired left ventricular ejection fraction . however , in patients with a transient risk of arrhythmia - related syncope , icd and accompanying risks , including device infections , lead defects , and traumatizing inappropriate shocks , might outweigh their potential therapeutic benefits . for these patients the wearable cardioverter - defibrillator ( wcd ) has become a second therapeutic option besides implants , translating wearable sensors into clinical practice . herein , we review recent evidence and future perspectives indicating that wearable sensor technologies open up new avenues beyond implantable cardiac electronic devices in the management of patients with syncope . the high incidence and prevalence of syncope and related morbidity underpin the necessity of an early detection of an approaching syncope . in nms since pharmacological and interventional therapies are limited , acute syncope prediction would prevent falls in these patients . the prediction of potentially life - threatening arrhythmia - related syncopes is especially important in patients with structural heart disease to prevent sudden cardiac death . the hemodynamic interaction of various systems , including vascular- and heart rate / rhythm - related parameters , underscores the need for multimodality of wearable sensor systems , which are addressed here . the triggering mechanisms of nms are complex and incompletely understood . during the development of nms , the resulting neurally - mediated hemodynamic adaptations typically result in a decline in blood pressure ( bp ) and a change ( decrease or increase ) in heart rate ( hr ) . hemodynamic studies indicate that vasodilatation is the most common reproducible phenomenon occurring just prior to the onset of dizziness or a sudden loss of consciousness . several approaches , predominantly based on hr and/or bp , have been proposed to acutely predict syncope , with mixed results as discussed elsewhere in detail [ 1520 ] . this approach , however , is not yet technically feasible in an ambulatory setting . in recent years we and others [ 2426 ] have been working on algorithms , taking into account photoplethysmogram ( ppg ) morphology features , towards a better characterization of patient status in different settings . in line with previous studies , we found that pulse wave characteristics are useful to characterize bp changes . in this context , the pulse arrival time ( pat ) is a simple and reliable parameter to characterize blood pressure changes ( figure 1 ) . pat is defined as the time between the r peak in the electrocardiogram and the onset of the pulse wave in the periphery . in particular , it has been demonstrated that a pertinent relation of pat measures with systolic bp can be observed shortly before syncope , when pat starts to steadily increase . this might explain the predictive power of pat to detect critical events related to bp regulation failures based on this simple and easy measure . after having investigated a wide range of male and female patients ( age : 1880 years ) with and without various cardiovascular diseases , we found that pat changes preceding syncope appear to be relatively homogenous . however , larger patient numbers are needed to clarify the impact of age - dependent and pathophysiological changes in cardiac and vascular function . because pat includes ( 1 ) the pre - ejection period and ( 2 ) the microvascular circulation ( ppg from finger / ear / forehead ) , it appears to be useful in reflecing acute changes in bp , but might be limited in characterizing arterial stiffness in the context of acute syncope prediction . importantly , pat has , in general , a complex relation to systolic bp and hr that is rather difficult to interpret unless detailed context information is available . however , since electrocardiogram ( ecg ) and ppg can be easily and comfortably acquired synchronously , these measures suggest a range of interesting applications in nms diagnostic as well as training procedures ( figure 2 ) . in patients undergoing head - up tilt table testing , we developed a syncope warning system and demonstrated the feasibility of pat - based syncope prediction in patients without any evidence of heart rhythm disorders , including bundle branch block , atrioventricular block , or tachyarrhythmias . a prospective study testing our previously developed algorithms for nms prediction in patients undergoing head - up tilt table testing and orthostatic self training , addressed the predictive value of this syncope warning system in a larger patient population ( nct01262508 ) . in contrast to the almost always relatively benign nms , arrhythmia - related syncopes often occur very fast without any prodromal symptoms . affected patients have an increased risk of sudden cardiac death due to the sudden loss of cardiac output within seconds after the initiation of arrhythmias . icd have been shown to protect patients at high risk for ventricular tachycardias from sudden cardiac death . however , defining a high - risk patient is still challenging . while the risk of death from early post - myocardial infarction is large , clinical trials have failed to demonstrate a mortality benefit from the early use of implantable defibrillators . moreover , many patients are only transiently at high risk , limiting the value of an icd in this population . this especially holds true for patients with various causes of transient heart failure . for high - risk patients with arrhythmia - related syncope who are not candidates for an icd whether wearable defibrillators reduce mortality in ( 1 ) heart failure patients who are not candidates for an icd ( clinicaltrials.gov : nct01326624 ) , or ( 2 ) after a myocardial infarction ( nct01446965 ) is currently under investigation . the triggering mechanisms of nms are complex and incompletely understood . during the development of nms , the resulting neurally - mediated hemodynamic adaptations typically result in a decline in blood pressure ( bp ) and a change ( decrease or increase ) in heart rate ( hr ) . hemodynamic studies indicate that vasodilatation is the most common reproducible phenomenon occurring just prior to the onset of dizziness or a sudden loss of consciousness . several approaches , predominantly based on hr and/or bp , have been proposed to acutely predict syncope , with mixed results as discussed elsewhere in detail [ 1520 ] . this approach , however , is not yet technically feasible in an ambulatory setting . in recent years we and others [ 2426 ] have been working on algorithms , taking into account photoplethysmogram ( ppg ) morphology features , towards a better characterization of patient status in different settings . in line with previous studies , we found that pulse wave characteristics are useful to characterize bp changes . in this context , the pulse arrival time ( pat ) is a simple and reliable parameter to characterize blood pressure changes ( figure 1 ) . pat is defined as the time between the r peak in the electrocardiogram and the onset of the pulse wave in the periphery . in particular , it has been demonstrated that a pertinent relation of pat measures with systolic bp can be observed shortly before syncope , when pat starts to steadily increase . this might explain the predictive power of pat to detect critical events related to bp regulation failures based on this simple and easy measure . after having investigated a wide range of male and female patients ( age : 1880 years ) with and without various cardiovascular diseases , we found that pat changes preceding syncope appear to be relatively homogenous . however , larger patient numbers are needed to clarify the impact of age - dependent and pathophysiological changes in cardiac and vascular function . because pat includes ( 1 ) the pre - ejection period and ( 2 ) the microvascular circulation ( ppg from finger / ear / forehead ) , it appears to be useful in reflecing acute changes in bp , but might be limited in characterizing arterial stiffness in the context of acute syncope prediction . importantly , pat has , in general , a complex relation to systolic bp and hr that is rather difficult to interpret unless detailed context information is available . however , since electrocardiogram ( ecg ) and ppg can be easily and comfortably acquired synchronously , these measures suggest a range of interesting applications in nms diagnostic as well as training procedures ( figure 2 ) . in patients undergoing head - up tilt table testing , we developed a syncope warning system and demonstrated the feasibility of pat - based syncope prediction in patients without any evidence of heart rhythm disorders , including bundle branch block , atrioventricular block , or tachyarrhythmias . a prospective study testing our previously developed algorithms for nms prediction in patients undergoing head - up tilt table testing and orthostatic self training , addressed the predictive value of this syncope warning system in a larger patient population ( nct01262508 ) . in contrast to the almost always relatively benign nms , arrhythmia - related syncopes often occur very fast without any prodromal symptoms . affected patients have an increased risk of sudden cardiac death due to the sudden loss of cardiac output within seconds after the initiation of arrhythmias . icd have been shown to protect patients at high risk for ventricular tachycardias from sudden cardiac death . however , defining a high - risk patient is still challenging . while the risk of death from early post - myocardial infarction is large , clinical trials have failed to demonstrate a mortality benefit from the early use of implantable defibrillators . moreover , many patients are only transiently at high risk , limiting the value of an icd in this population . this especially holds true for patients with various causes of transient heart failure . for high - risk patients with arrhythmia - related syncope who are not candidates for an icd , whether wearable defibrillators reduce mortality in ( 1 ) heart failure patients who are not candidates for an icd ( clinicaltrials.gov : nct01326624 ) , or ( 2 ) after a myocardial infarction ( nct01446965 ) is currently under investigation . the feasibility of integrating a syncope warning system into a body - worn network was the first step in conducting hemodynamic studies in patients with syncope . based on a standard electrocardiogram , use of a ppg and an accelerometer enable hemodynamic changes to be characterized in syncope patients in standardized settings . current research focuses on more robust feature extraction approaches , since standard ppg signals are prone to artifacts . during standardized diagnostic procedures ( e.g. , head - up tilt table testing ) , ppg - derived signals have been found to provide representative parameters for left ventricular and peripheral vascular performance . characterization of hemodynamics using decomposition of the ppg signal into its forward and reflection waves has been investigated ( figure 3 ) . in this context , a novel algorithm for assessment of left ventricular ejection time from the ppg waveform has been introduced . we propose the use of gaussian functions to model both systolic and diastolic phases of the ppg beat and consequently determine the onset and offset of the systolic ejection from the analysis of the systolic phase 3 derivative . the results achieved by the proposed methodology revealed better estimation of left ventricular ejection time , and similar correlation with the echocardiographic reference , as compared with previously proposed algorithms . the pat methodology is based on hemodynamic surrogate measures , which are also sensitive to patient activities such as posture changes that are not necessarily related to blood pressure variations . the impact of posture on the pat measure and related hemodynamic parameters such as the pre - ejection period in well - defined procedures has been characterized . additionally , the pat of a monitored subject has been investigated in an unsupervised scenario illustrating the complexity of such a measurement . our results show the failure of blood pressure inference based on simple calibration strategies using the pat measure only . however , there are opportunities to compensate for the observed effects towards the realization of wearable cuff - less blood pressure monitoring devices . these findings emphasize the importance of accessing context information in personal healthcare applications , where vital sign monitoring is typically unsupervised . the presence of motion artifacts in ppg signals is one of the major obstacles in the extraction of reliable cardiovascular parameters in real time and continuous monitoring applications . recently , a novel algorithm for motion artifact detection , which is based on the analysis of the variations in the time and period domain characteristics of the ppg signal , has been introduced . the extracted features are ranked using a feature selection algorithm ( nmifs ) and the best features are used in a support vector machine classification model to distinguish between clean and corrupted sections of the ppg signal . the results achieved show that period domain features play an especially important role in the discrimination of motion artifacts from clean ppg pulses . in this connection , the sensatron is a multi - sensor device that records an ecg , an impedance cardiogram ( icg ) , near - infrared ppg , infrared ppg , and a thoracic inductive plethysmogram , as well as recording sound signals from 2 thorax locations . up to three 3-axis acceleration sensors at the thorax , arms , or legs provide information on posture and movements . the design already takes into account an easy and planned adaptation to home monitoring scenarios for integration into functional textiles . a first textile prototype was created within the heartcycle project by clothingplus in oy , finland . in addition , a novel visualization concept for ppg signals has been developed ( figure 4 ) . this approach enables quick evaluation of ppg amplitude and amplitude oscillation , ppg slopes , and the existence of a local minima and maxima during the diastolic phase . this might be useful because morphological changes in the ppg during blood pressure break - down have been found . our approach is based on a phase - space representation of the raw or pre - processed ppg signals , where basically the ppg signal is plotted versus the derivative of the same ppg sequence . the ppg signal is displayed based on this concept of a ppg pulse ( i ) until the next pulse ( i+1 ) . the beginning of the systolic phase ( point ai ) appears at the lowest y - value at the zero - crossing of the x - axis , whereas the diastolic phase starts at the highest y value again at the zero - crossing of x ( point ci ) . the difference of the highest and lowest y - value ( at point ci and point ai ) represents the amplitude of the ppg . a zero - crossing during the diastolic phase indicates a fully developed dicrotic notch within the signal the maximum slope during the systolic segment ( point bi ) can be easily extracted and coincidences in this example with the zero - crossing of y , which is in general not the case . pre - processing steps include filtering , artefact detection and removal , optional normalization , and the noise - reduced calculation of the derivative . a storage unit is used to compare ppg signals acquired at different time segments ( e.g. , during a procedure ) . in addition , the rate of changes of heart rate , stiffness and reflection indexes , pre - ejection period , left ventricular ejection time , pat , and pulse transit time parameters change preceding syncope . preliminary evidence indicates that integration of these hemodynamic surrogate measures into existing syncope prediction algorithms reduces the number of false - positive event detections . the wcd is available for selected patients at increased risk for tachyarrhythmia - related syncope ( figure 5 ) . the wcd consists of 2 main components : the garment and the monitor . in recent years the system uses 3 defibrillation electrodes , as well as 4 capacitive electrodes for long - term ecg monitoring ( figure 6 ) . if a ventricular tachyarrhythmia is detected and therefore the risk for syncope is high , a vibration plate generates tactile notification as the first of a series of alarms . simultaneous activation of the 2 patient response buttons offers the possibility to withhold defibrillator discharge as long as the user is conscious . ecg recording of events and daily use are transmitted and stored by an internet - based information system . the efficacy of the algorithm has been reviewed through a retrospective analysis of appropriate shocks , inappropriate shocks , and arrhythmia detections during a 1-year period . the authors found that by incorporating a patient responsiveness test , as well as features that eliminate or reduce signal interference common to external ecg electrodes , the wcd detection algorithm has a low risk of inappropriate shocks . in a retrospective analysis of about 100 patients at the university of duesseldorf ( unpublished ) , we found an even lower rate of inappropriate defibrillation in patients using the wcd , resulting in high patient satisfaction . the feasibility of integrating a syncope warning system into a body - worn network was the first step in conducting hemodynamic studies in patients with syncope . based on a standard electrocardiogram , use of a ppg and an accelerometer enable hemodynamic changes to be characterized in syncope patients in standardized settings . current research focuses on more robust feature extraction approaches , since standard ppg signals are prone to artifacts . during standardized diagnostic procedures ( e.g. , head - up tilt table testing ) , ppg - derived signals have been found to provide representative parameters for left ventricular and peripheral vascular performance . characterization of hemodynamics using decomposition of the ppg signal into its forward and reflection waves has been investigated ( figure 3 ) . in this context , a novel algorithm for assessment of left ventricular ejection time from the ppg waveform has been introduced . we propose the use of gaussian functions to model both systolic and diastolic phases of the ppg beat and consequently determine the onset and offset of the systolic ejection from the analysis of the systolic phase 3 derivative . the results achieved by the proposed methodology revealed better estimation of left ventricular ejection time , and similar correlation with the echocardiographic reference , as compared with previously proposed algorithms . the pat methodology is based on hemodynamic surrogate measures , which are also sensitive to patient activities such as posture changes that are not necessarily related to blood pressure variations . the impact of posture on the pat measure and related hemodynamic parameters such as the pre - ejection period in well - defined procedures has been characterized . additionally , the pat of a monitored subject has been investigated in an unsupervised scenario illustrating the complexity of such a measurement . our results show the failure of blood pressure inference based on simple calibration strategies using the pat measure only . however , there are opportunities to compensate for the observed effects towards the realization of wearable cuff - less blood pressure monitoring devices . these findings emphasize the importance of accessing context information in personal healthcare applications , where vital sign monitoring is typically unsupervised . the presence of motion artifacts in ppg signals is one of the major obstacles in the extraction of reliable cardiovascular parameters in real time and continuous monitoring applications . recently , a novel algorithm for motion artifact detection , which is based on the analysis of the variations in the time and period domain characteristics of the ppg signal , has been introduced . the extracted features are ranked using a feature selection algorithm ( nmifs ) and the best features are used in a support vector machine classification model to distinguish between clean and corrupted sections of the ppg signal . the results achieved show that period domain features play an especially important role in the discrimination of motion artifacts from clean ppg pulses . in this connection , the sensatron is a multi - sensor device that records an ecg , an impedance cardiogram ( icg ) , near - infrared ppg , infrared ppg , and a thoracic inductive plethysmogram , as well as recording sound signals from 2 thorax locations . up to three 3-axis acceleration sensors at the thorax , arms , or legs provide information on posture and movements . the design already takes into account an easy and planned adaptation to home monitoring scenarios for integration into functional textiles . a first textile prototype was created within the heartcycle project by clothingplus in oy , finland . in addition , a novel visualization concept for ppg signals has been developed ( figure 4 ) . this approach enables quick evaluation of ppg amplitude and amplitude oscillation , ppg slopes , and the existence of a local minima and maxima during the diastolic phase . this might be useful because morphological changes in the ppg during blood pressure break - down have been found . our approach is based on a phase - space representation of the raw or pre - processed ppg signals , where basically the ppg signal is plotted versus the derivative of the same ppg sequence . the ppg signal is displayed based on this concept of a ppg pulse ( i ) until the next pulse ( i+1 ) . the beginning of the systolic phase ( point ai ) appears at the lowest y - value at the zero - crossing of the x - axis , whereas the diastolic phase starts at the highest y value again at the zero - crossing of x ( point ci ) . the difference of the highest and lowest y - value ( at point ci and point ai ) represents the amplitude of the ppg . a zero - crossing during the diastolic phase indicates a fully developed dicrotic notch within the signal the maximum slope during the systolic segment ( point bi ) can be easily extracted and coincidences in this example with the zero - crossing of y , which is in general not the case . pre - processing steps include filtering , artefact detection and removal , optional normalization , and the noise - reduced calculation of the derivative . a storage unit is used to compare ppg signals acquired at different time segments ( e.g. , during a procedure ) . in addition , the rate of changes of heart rate , stiffness and reflection indexes , pre - ejection period , left ventricular ejection time , pat , and pulse transit time parameters change preceding syncope . preliminary evidence indicates that integration of these hemodynamic surrogate measures into existing syncope prediction algorithms reduces the number of false - positive event detections . the wcd is available for selected patients at increased risk for tachyarrhythmia - related syncope ( figure 5 ) . the wcd consists of 2 main components : the garment and the monitor . in recent years the system uses 3 defibrillation electrodes , as well as 4 capacitive electrodes for long - term ecg monitoring ( figure 6 ) . if a ventricular tachyarrhythmia is detected and therefore the risk for syncope is high , a vibration plate generates tactile notification as the first of a series of alarms . simultaneous activation of the 2 patient response buttons offers the possibility to withhold defibrillator discharge as long as the user is conscious . ecg recording of events and daily use are transmitted and stored by an internet - based information system . the efficacy of the algorithm has been reviewed through a retrospective analysis of appropriate shocks , inappropriate shocks , and arrhythmia detections during a 1-year period . the authors found that by incorporating a patient responsiveness test , as well as features that eliminate or reduce signal interference common to external ecg electrodes , the wcd detection algorithm has a low risk of inappropriate shocks . in a retrospective analysis of about 100 patients at the university of duesseldorf ( unpublished ) , we found an even lower rate of inappropriate defibrillation in patients using the wcd , resulting in high patient satisfaction . predicting the most common cause of transient loss of consciousness neurally - mediated syncope is feasible by ecg- and ppg - based wearable multi - sensor systems . miniaturized devices for ambulatory pulse arrival time measurement are on the horizon and might improve syncope warning systems in the future . syncope prediction by pat may also be realized in ambulatory blood pressure devices , which are extremely prevalent today in clinical practice . the option to use pat for assessment of arterial stiffness is already available in some models of such devices . how the concept from hutt can be translated to ambulatory settings integration of recently presented algorithms ( including parameters for left ventricular function ) and peripheral vascular performance ( including pre - ejection period , pulse transit time , and reflection indexes ) in such wearable devices might additionally improve syncope management . for patients with structural heart disease who are at risk for potentially life - threatening tachyarrhythmias and related syncopes , the wearable cardioverter defibrillator has been found to be a relatively novel therapeutic option to prevent sudden cardiac death , and it is currently under investigation in randomized controlled trials . since syncope is a highly complex entity representing the interplay of various systems , including vascular and heart rate / rhythm - related hemodynamics , the multimodality of wearable sensor systems may prove to be useful in various settings in and out of hospital . these include syncope management units , dedicated units for the treatment of complex ventricular arrhythmias , and home monitoring of patients with heart failure . in conclusion , wearable sensor technologies open up new diagnostic and therapeutical possibilities in the management of patients with syncope .

basosquamous carcinoma ( bsc ) is a rare epithelial neoplasm with features of both basal cell carcinoma ( bcc ) and squamous cell carcinoma ( scc ) and is linked by a transition area . like scc , bsc is more locally invasive , aggressive , and more likely to metastasize than forms of bcc . clinically , most bscs are located in the head and neck , mainly on the nose and in the auricular and periocular regions . bsc is usually a slow - growing tumor and the low rate of correct diagnosis of bsc is probably due to small biopsy specimens . moreover , bsc has a nonspecific clinical presentation and diagnosis is made only after biopsy . excision is probably the best treatment , and surgical margins should be wider than those for low - risk bcc due to the infiltrative growth pattern of this tumor . nevertheless , high recurrence rates are reported despite wide local incision , making complete excision essential . a 63-year - old caucasian male had a sessile lesion removed from his left ear . seven years later , a local recurrence was widely excised and histological analysis confirmed bsc . histological analysis with hematoxylin and eosin ( h and e ) stain revealed multiple islands of malignant cells throughout the dermis , exhibiting both basaloid and squamous differentiation . the areas had bcc , with large and rounded basaloid cells [ figure 1 ] , and scc , with polygonal squamoid cells with abundant eosinophilic cytoplasm , large nuclei , and prominent nucleoli . the scc component exhibited frequent atypical mitosis , cell and nuclear pleomorphism and hyperchromatism of the nuclei [ figure 2 ] . the transition zone , which appeared abruptly , had intermediate cells and was between the bcc and scc areas [ figure 1 ] . the squamoid component exhibited dyskeratotic cells and metastatic cells invading blood vessels [ figure 3 ] . the patient has been followed up , and after 6 months , with no sign of further disease . bsc with a transition zone appearing abruptly ( intermediate cells ) between scc and bcc areas ( h and e , 200 ) squamoid component with cellular and nuclear pleomorphism and nuclear hyperchromatism ( h and e , 400 ) squamoid component with dyskeratotic cells ( star ) and invasion of the tumor cell in blood vessel ( arrow ) ( h and e , 400 ) many malignant skin tumors are easily classified as either classic bcc or scc , with the correct prediction of their biological behavior . some tumors , however , are intermediate in their histology , while others are somewhat variable from one microscopic field to another . the diagnosis of the present case was bsc , with a transition zone between bcc and scc . the bsc areas have small , uniform , hyperchromatic cells , with peripheral palisading mitoses and stromal collagen deposition with proliferative fibroblasts . in contrast to pure bcc , some basaloid cells in bsc have eosinophilic cytoplasm , often lack the characteristic peripheral palisading and retraction artifact and exhibit variable cytoplasmic keratinization.[810 ] basaloid cells are mitotically more active than indolent growth bcc , with greater numbers of apoptotic nuclei . scc areas are characterized by large polygonal squamoid cells with eosinophilic cytoplasm reflecting cytoplasmic keratinization , larger open nuclei with prominent nucleoli , frequent mitosis , dyskeratotic cells , and intercellular bridges . the transition zone between bcc and scc may or may not exist and has a fibroblast - rich and collagenized stroma . however , according to martin et al . , bsc is not a collision carcinoma of bcc and scc and the transition zone between the basal cell and squamous cell types is not necessary for the diagnosis of bsc . clinically , bsc is most commonly seen on the head and neck , mainly involving the central face , and has a significant predominance in male caucasians . in a case series involving 178 patients , leibovitch found that 95% of the tumors were located in the head and neck , with the nose ( 33.1% ) , auricular region ( 18.5% ) , and periocular region the most commonly affected sites . the patient in the present case had a tumor located in the auricular region , had a very pale skin and worked in the sun due to his occupation as a farmer . the predilection of bsc for the head and neck region may be explained by the greater exposure to sunlight . there is a significant risk of local recurrence and metastasis with bsc , which is one the most aggressive types of bcc , considering its infiltrative growth and stromal reaction patterns . long - term follow - up for the detection of local recurrence and distant metastatic spread is recommended . , some metastatic cells were found invading blood vessels , indicating the aggressive potential of the tumor cells . excision of the lesion with a surgical margin was performed and the patient has been followed up for 6 months . according to martin , the more significant indicators of recurrence were positive surgical margins , lymphatic invasion , and perineural invasion , whereas the degree of differentiation and the size of the initial lesion were not significant . there is a high incidence rate of lymph node involvement and patients with perineural invasion have significantly higher rates of metastatic disease and local recurrence when compared to those without perineural invasion . the importance of distinguishing bsc from scc and bcc lies in its significant propensity to metastasize .

herein , we present an adult retinoblastoma case who was misdiagnosed as melanoma in fine - needle aspiration biopsy with intravitreal hemorrhage and later neovascular glaucoma after biopsy . the diagnosis of retinoblastoma was confirmed after enucleation . retinoblastoma should be considered in the differential diagnosis of an intraocular mass in adults , especially when media haziness precludes adequate ophthalmoscopic visualization . the frequency of retinoblastoma ranges from 1 in 14,000 to 1 in 20,000 live births and about 90% of patients are diagnosed under 3 years of age . in the majority of cases , the diagnosis is made before 5 years of age . the literature pertaining to retinoblastoma affecting adults is scarce , however the vast majority of cases have been isolated reports . despite clinical improvements in the field of ocular oncology for establishing a preoperative diagnosis , a small number of atypical cases still defy accurate clinical diagnosis . herein we report a isolated of late presentation of retinoblastoma in a 29-year - old woman that was not diagnosed by cytology in a vitrectomy specimen but proved to be retinoblastoma tumor later in the enucleation specimen . a 29-year - old woman with a complaint of diminished vision in her left eye since 7 months ago was referred to our clinic . she had a history of left eye redness which did not respond to topical medications . in past medical history , she had a seizure attack 4 years before and had received appropriate treatment . examination in the right eye ( od ) was normal . in the left eye ( os ) , visual acuity was one meter finger count , anterior segment examination was normal , intraocular pressure was 14 mmhg , and relative afferent pupillary defect was 2 + . in the posterior segment examination , there was a dome - shaped partially pigmented mass with vitreous hemorrhage obscuring tumor features . orbital magnetic resonance imaging ( mri ) revealed an enhancing nodular mass lesion in the posterior aspect of the left globe protruding into the vitreous and associated with exudative or nearby hemorrhagic retinal detachment [ figures 1a , 1b and 1c ] . b scan of left eye revealed a dome - shaped solid mass with high internal reflectivity 16.5 12.5 11 mm in size with point calcifications [ figures 1d and 1e ] . ( a ) magnetic resonance imaging ( mri ) t1 image shows a hyperintense lesion in the posterior part of the left globe that is hypointense in t2 mri image ( b ) . ( d and e ) b - scan echography reveals a dome - shaped lesion with a calcification area associated with retinal detachment . ( f ) gross pathology of the enucleated eye shows partly a pigmented lesion in the posterior and inferior part of the globe occupying > 50% of the globe . ( g and h ) the microscopic appearances of well - differentiated retinoblastoma in our case . intraocular tumor , possibly retinoblastoma or aggressive astrocytoma , was considered as a provisional diagnosis and biopsy was suggested . fine - needle aspiration biopsy ( fnab ) was performed but the specimen lacked enough cells for definitive diagnosis . the sample was obtained from the vitreous after removing the vitreous hemorrhage and also from the tumor by high aspiration and low cut level . with a histologic diagnosis of choroidal melanoma , after one month , the patient presented with severe left eye pain , visual acuity was no light perception , anterior segment showed circumcilliary congestion , neovascularisation of the iris and angle with intraocular pressure of 60 mmhg . due to severe pain and loss of vision , at the time , the left eye was enucleated . in the pathology report , macroscopically , a blood filled globe cavity and a tumor with hypo and hyperpigmented areas and foci of hemorrhage 20 mm in diameter and 5 mm in thickness was shown [ figure 1f ] . the microscopic section was composed of atypical proliferation of cellular elements with small round hyperchromatic nuclei and scant cytoplasm , arranged as closely packed cells forming a nodule occupying half of the posterior chamber . the neoplastic cells were differentiated in some areas accompanied by flexner - wintersteiner rosette formation [ figure 1 g and 1h ] . ihc stains were positive for neuron - specific enolase ( nse ) , retinoblastoma protein and synaptophysin and negative for human melanoma black 45 ( hmb45 ) and leukocyte common antigen in the tumor cells and hence the final diagnosis was retinoblastoma . retinoblastoma , a tumor originating from the sensory retina , is the most common primary intraocular tumor in childhood . according to shields et al only 8.5% of patients with retinoblastoma are older than 5 years at the time of diagnosis and all reported cases were sporadic . the presence of vitreous hemorrhage , inflammation , and cataract further complicate the diagnosis as in our patient in whom vitreous hemorrhage was the presenting sign precluding careful funduscopy and b scan revealed the point calcified mass . although late presenting retinoblastoma is uncommon , there are a number of case reports of these cases ( 30 cases ) . ultrasonography and computed tomography scan may not reveal calcification , which is characteristic of retinoblastoma in children . until date , in the reported cases the presentation age of retinoblastoma in adults ranged from 16 to 74 years . the third decade of life was the most common presenting age for adult retinoblastoma cases ( 13 patients ) . flexner - wintersteiner rosettes were reported in 10 patients , homer - wright 's rosettes in 6 patients , non - specified rosettes in 6 patients , no rosettes in 7 patients , and in 2 patients the reports did not state the existence of rosettes . the use of fnab and ihc ( with nse ) may be helpful and confirmatory . in our case in general , preoperative fnab is not routinely performed in retinoblastoma to minimize the theoretical risk of tumor seeding , however occasionally , fnab could be a useful diagnostic procedure , when the diagnosis is clinically uncertain . despite the rare use of fnab in the diagnosis of intraocular tumors , the risk of tumor recurrence in enucleated eyes was not increased after enucleation according to the literature . shields et al stated some risks of retinoblastoma dissemination after vitrectomy in eyes with unsuspected retinoblastoma , in older pediatric patients . the tumor may also arise from previously undiagnosed , spontaneously regressed / arrested retinoblastomas which have been reactivated . gallie et al suggested a modification in the original mutation model of oncogenesis proposed by knudson to explain the presence of retinoma and low penetrance retinoblastoma 1 ( rb1 ) mutations in some families . it is well - accepted that inherited retinoblastoma with germinal mutation will arise if a second event ( mutation ) occurs in susceptible immature ( primitive ) retinoblasts , whereas retinoma will be likely if a second event occurs in almost mature retinoblasts . this proposal states that rb1 mutations are necessary , but not sufficient to induce retinoblastoma and some mutations in rb1 do not inactivate the rb1 protein completely , leading to partial penetrance . normal fundi were not previously documented in the reported patients and hence perhaps they did harbor a retinoma in which additional oncogenic mutations eventually occurred , leading to retinoblastoma tumor . retinomas are clinically well - characterized quiescent retinal masses , clearly linked to rb1 gene mutation , but they do not generally progress to malignant growth . if retinoblastoma could be diagnosed in its early stage , less aggressive treatment modalities such as chemotherapy , photocoagulation , cryotherapy , brachytherapy , external beam radiation , and diathermy would be extremely useful . management of retinoblastoma should be guided by the objectives save life , retain anatomical integrity of the eye , preserve vision , and obtain good cosmetic results . enucleation is indicated for unilateral retinoblastomas which fill most of the globe and when there is little hope of salvaging vision . enucleation was the primary treatment modality in the majority of reported adult cases of retinoblastoma , as the lesions were detected at a fairly advanced stage and each patient had one normal unaffected eye . risk factors for orbital and/or metastatic disease from retinoblastoma include massive choroidal invasion , tumor invasion into extrascleral tissue , the retrolaminar optic nerve and the optic nerve resection line , which in turn correlate with poorer survival . the question regarding the long - term survival of adults with retinoblastoma compared with usual retinoblastoma in children remains to be answered . in the case reported herein , the difficulty in establishing the diagnosis of retinoblastoma in adults was illustrated . in conclusion , retinoblastoma should be considered in the differential diagnosis of an intraocular mass in adults , especially when media haziness precludes adequate ophthalmoscopic visualization and any amelanotic or partially melanocytic lesion in the fundus of a patient at any age should raise the suspicion of retinoblastoma .

uas - dark 14 , uas - dronc , uas - dronc 16 , uas - hid and uas - p35 20 lines were as described previously . to co - express dronc and apaf-1 , we recombined uas - dronc and uas - apaf-1 onto the 3 chromosome . these transgenes were misexpressed through en - gal4 , gmr - gal4 , arm - gal4 or tubulin > frt > y+ , gfp > frt > gal4 ( abbreviated in the text as tub > gfp > gal4 ) drivers . mosaic loss - of - function clones and flip out clones were induced through the standard flp / frt system 31,32 . the loss of function mutant strains , apaf-1 ( originally described as dark ) , dronc , dronc and diap1 had been described previously 9,12,26 . to analyze gfp - apaf-1 , we fused egfp to the n - terminal side of either the full length apaf-1 coding sequence , or a truncated apaf-1 that has a stop codon engineered in place of a.a . 1292 ( referred to as apaf-1 ) , subcloned into puast - attb and targeted to the 51d locus for insertion through the phic31-integrase system 17,18 . all fluorescent images were obtained with a zeiss lsm510 confocal microscope , using 20x or 40x objective lenses . following are the antibodies used for this study : guinea pig anti - dronc raised against the full length recombinant protein 33 , monoclonal 9e10 ( dshb , univ . iowa ) and rabbit anti - myc ( santa cruz ) for myc - tag detection , rabbit anti - cleaved caspase ( cell signaling technologies ) , guinea pig anti - hsc3 34 and rabbit anti - gfp ( molecular probes ) . for fluorescent in situ hybridization - dronc western blots in figure 1 , schneider cells ( figure 1c ) or whole larvae ( figure 1d ) were ground in 5% sds - loading buffer before analysis . for anti - myc western blots in figure 3 and 4 , we loaded into each lanes extracts prepared from forty eye - antennal imaginal discs isolated from late 3-instar larvae . for quantification of western bands and whole - mount immuno - labelings , we used image j software ( http://rsbweb.nih.gov/ij ) . the intensity of the band of interest was normalized with either an anti - hsc3 band ( figure 3 ) or a nonspecific 120kda band present in the same lane ( figure 4b ) . the statistical significance was assessed through t - test ( two - tailed ) . to assess gfp - apaf-1 levels in schneider cell cultures , we transfected uas - gfp - apaf-1 constructs together with an actin - gal4 plasmid . standard procedures were followed for sample preparation , including fixation in 2% glutaraldehyde , dehydration and drying in hmds solvent . gold - paladium was used to coat adult flies and images were taken at a 180x magnification . the mutant diap1 proteins were derived from previously reported alleles 24 . the dronc protein used in the in vitro binding assay was near full length , catalytically dead and has been described previously 33 . the recombinant apaf-1 protein was from its natural initiation met to amino acid 729 , and thus lacked the c - terminal wd repeats . uas - dark 14 , uas - dronc , uas - dronc 16 , uas - hid and uas - p35 20 lines were as described previously . to co - express dronc and apaf-1 , we recombined uas - dronc and uas - apaf-1 onto the 3 chromosome . these transgenes were misexpressed through en - gal4 , gmr - gal4 , arm - gal4 or tubulin > frt > y+ , gfp > frt > gal4 ( abbreviated in the text as tub > gfp > gal4 ) drivers . mosaic loss - of - function clones and flip out clones were induced through the standard flp / frt system 31,32 . the loss of function mutant strains , apaf-1 ( originally described as dark ) , dronc , dronc and diap1 had been described previously 9,12,26 . to analyze gfp - apaf-1 , we fused egfp to the n - terminal side of either the full length apaf-1 coding sequence , or a truncated apaf-1 that has a stop codon engineered in place of a.a . 1292 ( referred to as apaf-1 ) , subcloned into puast - attb and targeted to the 51d locus for insertion through the phic31-integrase system 17,18 . all fluorescent images were obtained with a zeiss lsm510 confocal microscope , using 20x or 40x objective lenses . following are the antibodies used for this study : guinea pig anti - dronc raised against the full length recombinant protein 33 , monoclonal 9e10 ( dshb , univ . iowa ) and rabbit anti - myc ( santa cruz ) for myc - tag detection , rabbit anti - cleaved caspase ( cell signaling technologies ) , guinea pig anti - hsc3 34 and rabbit anti - gfp ( molecular probes ) . for fluorescent in situ hybridization - dronc western blots in figure 1 , schneider cells ( figure 1c ) or whole larvae ( figure 1d ) were ground in 5% sds - loading buffer before analysis . for anti - myc western blots in figure 3 and 4 , we loaded into each lanes extracts prepared from forty eye - antennal imaginal discs isolated from late 3-instar larvae . for quantification of western bands and whole - mount immuno - labelings , we used image j software ( http://rsbweb.nih.gov/ij ) . the intensity of the band of interest was normalized with either an anti - hsc3 band ( figure 3 ) or a nonspecific 120kda band present in the same lane ( figure 4b ) . the statistical significance was assessed through t - test ( two - tailed ) . to assess gfp - apaf-1 levels in schneider cell cultures , we transfected uas - gfp - apaf-1 constructs together with an actin - gal4 plasmid . standard procedures were followed for sample preparation , including fixation in 2% glutaraldehyde , dehydration and drying in hmds solvent . gold - paladium was used to coat adult flies and images were taken at a 180x magnification . the mutant diap1 proteins were derived from previously reported alleles 24 . the dronc protein used in the in vitro binding assay was near full length , catalytically dead and has been described previously 33 . the recombinant apaf-1 protein was from its natural initiation met to amino acid 729 , and thus lacked the c - terminal wd repeats .

the trigeminal neuralgia is defined as sudden , usually unilateral , severe , brief , stabbing , recurrent episodes of pain in the distribution of one or more branches of the trigeminal nerve ( fifth cranial nerve ) . in 80 - 90% of cases , it is thought to be caused by compression of the trigeminal nerve by a loop of artery or vein . the incidence increases with age and is rare in people younger than 40 years of age . most common adverse effects observed with carbamazepine are drowsiness , unsteadiness , constipation , nausea , and vomiting . it is also reported to cause hypertension ; however , the frequency of occurrence is rare ( 1/10,000 to < 1/1,000 ) . a 74-year - old female presented to a private practitioner with complaint of facial pain on the left side . on examination her blood pressure ( b.p ) was 117/80 mmhg . her dental examination was normal and based on the symptoms a provisional diagnosis of trigeminal neuralgia was made . after 2 days , she complained of headache , restlessness and on examination b.p was found to be 290/110 mmhg . as the pain was persisting , she presented to the neurology opd of the institute with complaint of left side facial pain and the diagnosis was confirmed as trigeminal neuralgia . the patient was started with carbamazepine 50 mg tablet bd and was gradually titrated to 200 mg bd . the mri findings confirmed the diagnosis of trigeminal neuralgia . on examination , her b.p . she was advised to get thyroid profile , random blood sugar and lipid profile done , all of which were within normal range . she presented after 4 days with severe nausea , vomiting and dizziness with persisting constipation . the dose of carbamazepine was reduced to 100 mg bd and rest all the medicines were continuing . the ecg and fundus examination were normal , biochemistry reports revealed low uric acid 2.3 ( 2.5 - 6.2 mg / dl ) ; low serum sodium 119 mg / dl ( 135 - 155 ) ; low chloride 96 mmol / l ( 98 - 107 ) ; high total protein 9.3 g / dl ( 6.3 - 8.2 ) ; and high serum globulin 4.3 g / dl ( 2.5 - 3.5 ) . next day the patient was admitted to a private hospital with complaints of severe headache , epigastric pain and vomiting . on examination b.p . records were normal and the patient is off all the antihypertensive treatment . the patient is stable now [ table 1 ] . the trigeminal neuralgia resolved after the carbamazepine was stopped . no other treatment modality was used . sequence of events summary meanwhile , based on literature search and detailed review of the patient 's medical and family history , carbamazepine was suspected to be the causal agent for this adverse reaction as there was no underlying cardiovascular disease . after oral carbamazepine high dose ; and non - resolution of hypertension despite anti - hypertensive therapy confirmed that hypertension was induced by oral carbamazepine therapy as it resolved only after carbamazepine discontinuation . a causal association between hypertension and carbamazepine was assessed by world health organization ( who ) probability method and naranjo 's adverse drug reaction probability scale . the who probability method and naranjo 's adverse drug reaction probability scale showed certain and definite ( naranjo 's score 10 ) association , respectively . as per modified hartwig and siegel scale , the severity of the reaction was moderate : level 4b . literature search was conducted which showed six reports on carabamazepine - induced hypertension from various countries . all the cases had patients who developed hypertension during carbamazepine treatment which resolved on discontinuation . to the best of our knowledge , discussed the possible role of anti - diuretic hormone in the production of this infrequent side effect . the possible mechanism as to why the antihypertensive therapy is unresponsive as explained by downey et al . is that antiepileptic agents could induce drug - metabolizing system and thus reduce the effects of antihypertensive medications . another author stated that carbamazepine induces the cytochrome p450 , which catalyze the metabolism of most of the antihypertensive used . the time sequence of start of the suspected drug and onset of hypertension are consistent with the diagnosis . the rechallenge by another physician led to raised blood pressure , not successfully managed by the appropriate treatment . the symptoms recovered after withdrawal of the suspected drug and the patient did not develop hypertension further owing to cessation of carbamazepine which is suggestive of possible association between carbamazepine and hypertension . carbamazepine - induced hypertension is rare and this report may act as alerting mechanism to the health care professionals . neurologists , doctors and experts in various fields , pharmacist and patients need to be aware of the potential of carbamazepine - induced hypertension .

the incidence of unstable intertrochanteric fractures has grown substantially in the elderly along with an increasing life expectancy . intertrochanteric fractures are more common in elderly patients and result in a high morbidity and a more difficult rehabilitation resulting from a deterioration of muscle strength and proprioceptive function12 ) . for these reasons , stable bone healing is the most critical element after a fracture occurs . when selecting hip arthroplasty for the treatment of fractures , stable fixation of the greater trochanteric fragment is essential for complete bone union and functional recovery of the hip joint . a variety of internal fixation devices and fixation methods have been developed to achieve bone healing with stable fixation of fracture fragments , but the rate of sequelae , such as nonunion after early failure of fixation , has reportedly increased up to 50%3456 ) . in particular , elderly patients with unstable intertrochanteric fractures of the femur associated with osteoporosis have difficulty performing early weight bearing , an outcome that can potentially prolong treatment time and result in an increased rate of systemic complications and poor functional recovery and clinical outcomes . because of unfavorable prognosis , instead of osteosynthesis , hip arthroplasty has become increasingly more popular as a treatment option . stern and goldstein7 ) have recommended prosthetic replacement to allow for early weight bearing in elderly patients with an unstable intertrochanteric fracture , and cho et al.8 ) have demonstrated favorable results at short - term follow - up after performing cementless total hip arthroplasty for unstable intertrochanteric fractures . after arthroplasty for unstable intertrochanteric fractures with the greater trochanteric fragment , nonunion of the greater trochanteric fragment may cause pain in the trochanteric region , functional gait abnormality and dislocation due to reduced hip abductor strength . therefore , anatomical reduction and rigid fixation of the greater trochanter are important . previously introduced fixation techniques or devices include : i ) tension band wiring , ii ) modified tension band wiring , iii ) greater trochanteric reattachment device ( gtrd ) , iv ) claw plate , and v ) others . nam et al.9 ) have reported satisfactory results with cementless total hip arthroplasty and double tension band wiring in fixation of unstable intertrochanteric fractures with the greater trochanteric fragment . moreover , cho et al.10 ) and kho et al.11 ) documented satisfactory results with hip hemiarthroplasty and cerclage wiring in fixation of unstable intertrochanteric fractures . the authors of this study aimed to classify fracture patterns based on fracture type of the greater trochanter and report clinical and radiological results by applying fixation techniques that vary depending on the fracture pattern . for the purposes of this study , unstable intertrochanteric fractures were those classified as type 4 and type 5 ( using evan 's criteria ) and associated with a fracture of the medial cortex including the lesser trochanter . of the 137 patients who underwent hip arthroplasty for an intertrochanteric fracture with osteoporosis ( bone mineral density , < 2.5 ) between march 2014 and october 2015 , 63 met our eligibility criteria and were included in this study . patients were divided into three groups according to fracture pattern of the greater trochanter ( as determined using three - dimensional computed tomography [ 3d ct ] images ) and different fixation methods were then applied by a single orthopaedic surgeon . the mean age at time of surgery was 81.2 years ( range , 71 - 95 years ) ; 49 patients were female and 14 were male . the average follow - up period after the surgery was 11.6 months ( range , 5 - 24 months ) . all operations were done in a lateral position . using a modified gibson approach , about a 15-cm skin incision was made and the fractured head and neck of the femur was removed . after temporary fixation using a 1.0-mm wire with double strands , the appropriate femoral component was inserted . to examine the potential benefits of differential fixation techniques , fracture patterns were divided into three groups ( a , b , and c ) based on 3d ct images . patients in group a ( n=18 ) presented with an oblique or spiral fracture with relatively large fragment ( fig . group b ( n=4 ) included the rarer cases of a transverse fracture of the greater trochanter ( fig . group c ( n=41 ) included those with comminuted fracture of the greater trochanter ( fig . a , fixation was accomplished using a 1.0-mm roll wire and a spiral band in a figure of 8 wiring the intertrochanteric area between the greater and lesser trochanter ( fig . this conventional technique can prevent escape of the greater trochanteric fragment with resultant hip abductor weakness . for group b , we used tension band wiring using a 1.0 double - stranded wire after inserting two 1.8 k - wires from the tip of the greater trochanter to the distal part ( fig . this method can help prevent bone loss of the greater trochanteric fragment by increasing compression across the transverse fracture of the greater trochanter . for group c , solid fixation of comminuted fractures was carried out using two 2.0 cables with the gtrd ( fig . comminuted fragments were reattached in place using cables by connecting a metal claw to the bone fragment12 ) . to evaluate postoperative function in patients , we measured : i ) recovery of ambulation ability , ii ) thigh and groin pain , and iii ) the harris hip score ( hhs ) . for radiographic assessment , bone union was evaluated based on anteroposterior views on plain x - rays taken pre- and postoperatively and at final follow - up . in the present study , the mean operative time , defined as the time from skin incision to the end of the skin closure , was 75 minutes and the mean intra - operative blood loss was 800 ml . partial weight loading and standing were allowed in an average of 7 days after surgery ( range , 4 - 10 days ) . clinically , 50 out of 63 patients returned to normal daily activities without a decrease in walking distance , and we observed improvements in hhs from 74.8 to 85.7 points at the latest follow - up ( considered relatively favorable scores ; e.g. , fair to good ) . upon radiological assessment , bone union was observed in 62 ( 98.4% ) cases , with the lone exception being a patient who experienced osteolysis , and the average time to radiographic union was 9 months ( range , 8 - 12 months ) . the most common postoperative complications were irritation or impingement caused by wire or cable breakage ( n=3 ) , and in each case the metallic fragments were removed . a single case of traumatic dislocation was observed and managed with closed reduction without additional surgical intervention . there were no cases of deep vein thrombosis , pressure ulcer , pulmonary embolism or other medical complications . intertrochanteric fractures of the femur commonly occur in the elderly and anatomical reduction of this fracture type is challenging ; the frequency of total hip arthroplasty has been increasing in recent years . even though the fracture is reduced , stable fixation and maintenance of anatomical reduction are difficult . the incidence of complications ( e.g. , pressure ulcer , pneumonia , pulmonary embolism , atelectasis , urinary tract infection , and others ) is relatively high as a result of patients being bedridden for prolonged periods of time13 ) , and is considered a critical factor impacting mortality in the elderly population . the importance of early ambulation after surgery has been highlighted as it may reduce morbidity and increase the likelihood of favorable clinical results14 ) . previous studies have demonstrated that the rates of sequelae range between 18% and 50% after open reduction and internal fixation for unstable intertrochanteric fractures345715 ) . laros and moore5 ) have stated that the failure rate after internal fixation of unstable intertrochanteric fractures were 25% , and of these , 29% underwent secondary revision . chan and gill16 ) and haentjens et al.1718 ) compared the clinical results between bipolar arthroplasty and internal fixation for treatment of unstable intertrochanteric and subtrochanteric fractures . rehabilitation was easier and faster and the incidence of pressure ulcers , pneumonia and atelectasis was dramatically lower in the bipolar arthroplasty group , an outcome that was found to be attributable to early ambulation . furthermore , stern and angerman6 ) have suggested that hip arthroplasty can achieve more favorable clinical results ( i.e. , recovery of functional activities ) than internal fixation for unstable intertrochanteric fracture of the femur in elderly patients . kim et al.19 ) have recommended cemented bipolar hemiarthroplasty as a useful option for the treatment of unstable intertrochanteric fractures in elderly patients aged 65 years and have obtained good results in 88% of patients . since a comminuted fracture of the greater and lesser trochanters is commonly associated with an intertrochanteric femur fracture in arthroplasty , surgeons may face technical challenges during surgery in cases of non - rigid wire fixation or bone deformity in unwanted directions despite slight wire tension . since the greater trochanter serves as the main attachment site for abductor muscles , anatomical reduction and maintenance of reduction depending on leg positions are uneasy in cases of comminution of the greater trochanter . hamadouche et al.20 ) have achieved satisfactory outcomes with the use of the gtrd for greater trochanteric nonunion following revision total hip arthroplasty . in this study , we thought more deeply about acquiring solid fixation in various fracture types and fragment patterns . we aimed to reduce the time and effort of fixation by applying different fixation methods to three types of fracture patterns ( classified using 3d ct images ) . recovery of preoperative ambulation status was obtained in 79% of patients , and we observed substantial improvements in hhs , both considered to be relatively favorable outcomes . the mean operative time was 75 minutes , confirming that fixation was completed in a relatively short period of time . this retrospective study was limited by the relatively short follow - up period and small sample size . choosing an appropriate fixation method ( e.g. , cerclage wire fixation , tension band wiring and greater trochanteric reattachment ) for distinct fracture patterns will help us to easily acquire proper reduction and fixation of greater trochanteric fractures and shorten bone union time when performing hip arthroplasty for unstable intertrochanteric fractures . it will also assist with early ambulation and rehabilitation , allowing early return to activities of daily living .

although the phenomenology of coexisting obsessive compulsive disorder ( ocd ) and bipolar disorder ( bd ) has been identified for more than 100 years,1 the etiological and nosological aspects of this medical issue remain largely unknown . symptoms of ocd may occur before , during , or after the first mood episode and , in most cases , fluctuate with mood swings.2 in this regard , the majority of bd ocd comorbidities are considered to be a subtype of bd , rather than two separate diseases.3 to date , no standard pharmacotherapy for cooccurring bd antidepressants , especially for selective serotonin reuptake inhibitors ( ssris ) , which are first - line options for ocd treatment , are strictly restricted for bd ocd individuals because of the potential to elicit manic / hypomanic switching.4 in majority of the cases , a combination of multiple mood stabilizers or augmentative antipsychotics to mood stabilizers constitute the routine medication regimens.5 however , due to the lack of well - designed clinical trials , case reports of successful treatment in patients with comorbid bd we herein present a case of a young female patient who suffered from recurrent mood episodes and symptoms of ocd . a combination of quetiapine and valproate stabilized her mood ; however , it did not lead to apparent improvement of her ocd symptoms . therefore , a small dose of aripiprazole was added to the mood stabilizers , which consequently achieved significant remission in her obsessive compulsive symptoms . this case report favors the use of augmentative aripiprazole in patients with comorbid bd ocd who respond poorly to mood stabilizers . mrs a , a 28-year - old woman , was admitted to the department of psychiatry in our hospital because of recurrent depressive episodes and aggravating compulsive behaviors . two years prior to admission , this patient developed her first depressive episode after breaking up with her ex - boyfriend . thereafter , this patient became depressed and had insomnia , loss of appetite , and loss of interest . meanwhile , she began to repetitively tidy up her belongings and check whether the windows and doors were closed . there was no suicidal ideation reported at that moment and she could still adhere to the work as a bank staff . her obsessive behaviors gradually aggravated , with repetition rate remarkably increasing from several times per day to dozens of times per day . about half a year prior to admission , this patient reported the first suicidal attempt after fighting with her parents . she went into emotional outburst , stood at the window , and claimed to end her own life . she would rush to the balcony or windows and would attempt to commit suicide by jumping down from the building , while she was also reported with dangerous driving behaviors . consequently , her daily life and job were seriously disrupted . a primary diagnosis of ocd was made , and paroxetine was prescribed at a daily dose of 40 mg . about a week later , the patient became extremely excited , talkative , and vigorous . although paroxetine was discontinued soon afterward and quetiapine was initiated , her manic state lasted for nearly 3 weeks . therefore , hospitalization was suggested by her doctor . after admission , a comprehensive physical and laboratory examinations were performed to exclude unknown underlying physical diseases . besides , cranial magnetic resonance imaging scan was also normal . according to the diagnostic and statistical manual of mental disorders , fifth edition , this patient was dually diagnosed with bipolar i disorder and ocd . quetiapine was gradually titrated up to 700 mg per day with concomitant valproate at 1,000 mg per day . although her emotion became stable with these mood stabilizers , the patient reported an inadequate response in symptoms of ocd . fascinatingly , an add - on therapy of aripiprazole 10 mg per day promoted accelerating alleviation of her obsessive behaviors . meanwhile , no severe adverse events were observed . her remission in emotional and ocd symptoms this work was approved by the institute ethical committee of the first affiliated hospital , zhejiang university school of medicine , and written informed consent was obtained from the patient and her guardians . mrs a , a 28-year - old woman , was admitted to the department of psychiatry in our hospital because of recurrent depressive episodes and aggravating compulsive behaviors . two years prior to admission , this patient developed her first depressive episode after breaking up with her ex - boyfriend . thereafter , this patient became depressed and had insomnia , loss of appetite , and loss of interest . meanwhile , she began to repetitively tidy up her belongings and check whether the windows and doors were closed . there was no suicidal ideation reported at that moment and she could still adhere to the work as a bank staff . her obsessive behaviors gradually aggravated , with repetition rate remarkably increasing from several times per day to dozens of times per day . about half a year prior to admission , this patient reported the first suicidal attempt after fighting with her parents . she went into emotional outburst , stood at the window , and claimed to end her own life . she would rush to the balcony or windows and would attempt to commit suicide by jumping down from the building , while she was also reported with dangerous driving behaviors . a primary diagnosis of ocd was made , and paroxetine was prescribed at a daily dose of 40 mg . about a week later , the patient became extremely excited , talkative , and vigorous . although paroxetine was discontinued soon afterward and quetiapine was initiated , her manic state lasted for nearly 3 weeks . a comprehensive physical and laboratory examinations were performed to exclude unknown underlying physical diseases . besides , cranial magnetic resonance imaging scan was also normal . according to the diagnostic and statistical manual of mental disorders , fifth edition , this patient was dually diagnosed with bipolar i disorder and ocd . quetiapine was gradually titrated up to 700 mg per day with concomitant valproate at 1,000 mg per day . although her emotion became stable with these mood stabilizers , the patient reported an inadequate response in symptoms of ocd . fascinatingly , an add - on therapy of aripiprazole 10 mg per day promoted accelerating alleviation of her obsessive behaviors . her remission in emotional and ocd symptoms was well maintained in the follow - up visits . this work was approved by the institute ethical committee of the first affiliated hospital , zhejiang university school of medicine , and written informed consent was obtained from the patient and her guardians . the condition of bd ocd comorbidity has always been common in clinical practice . however , spurious ocd comorbidity in bd should be carefully differentiated from the true comorbidity . in most cases of comorbid subjects , the ocd symptoms appear to be closely linked to affective symptoms , manifesting dominantly an episodic and mood - dependent course.6 in other words , the ocd symptoms in these individuals were part of bd rather than a separate disease . to date , the optimal treatment protocols for bd our study indicates that low - dose augmentation with aripiprazole may help to relieve the accompanying symptoms of ocd in bd patients . although a combination of mood stabilizers effectively alleviated the emotional symptoms , additional aripiprazole may be necessary to address the remaining symptoms of ocd . to our knowledge , the dose of aripiprazole ( 10 mg per day ) in our patient was lower than other documented cases ( 1525 mg per day).7,8 moreover , no published literature has ever discussed the impact of augmentative medications on suicidal risk in bd ocd patients . aripiprazole , an atypical antipsychotic , acts as a partial agonist at the d2 and 5-ht1a receptors , as well as an antagonist at 5-ht2a receptor.9 aripiprazole monotherapy , or as an adjuvant to mood stabilizers , is efficacious in managing acute mania and stabilization phases of bd , but not bipolar depression.10 besides , recent evidence favors the use of augmentative aripiprazole for ssris - refractory ocd.11,12 however , long - term use of aripiprazole is associated with increased risk of extrapyramidal syndromes ( epss).10 therefore , defining the minimum effective dose of aripiprazole is essential for preventing epss . in our patient , of note , the add - on therapy to mood stabilizers was initiated after partial remission in emotional symptoms . the aforementioned information implies a promising two - step strategy for bd ocd treatment . that is , first to attenuate the mood problem with mood stabilizers and then , if necessary , deal with the obsessive behaviors with an adjuvant , such as the employment of aripiprazole indicated in our study . this two - step strategy not only helps avoid the unnecessary add - on medications if adequate response was observed with mood stabilizers but also facilitates the determination of minimum effective dose of the adjuvant . the suicidal risk in bd ocd patients has not been fully elucidated , as well as drug - induced suicidal ideations . ocd comorbidity reported more suicidal attempts than those without morbidity.13 several antipsychotics , including risperidone,14 olanzapine,15 quetiapine,16 and aripiprazole,7,8 were suggested due to the benefits in relieving the ocd symptoms in bd patients . however , in these studies , little attention has been paid to the emergent suicidal ideations following the antipsychotic augmentation . a few case reports documented the possible link of using aripiprazole with suicidal ideation.17 however , epidemiological data in large sample revealed that consuming aripiprazole was not associated with more suicide events compared to other atypical antipsychotics.18 moreover , aripiprazole users had reported significantly lower risk of all - cause mortality among various antipsychotics.19 given the recurrent onset of suicidal attempts in our patient , special attention should be paid to the suicidal profiles of antipsychotic augmentation in bd to conclude , the present case study provides supporting evidence that aripiprazole augmentation to mood stabilizers was efficacious in patients with bd besides , paying special attention to suicidal risk surround antipsychotic augmentation was also suggested . in the future ,

the left atrial ( la ) calcification is a relatively uncommon complication of long - standing rheumatic valvular heart disease.1 ) completed calcification of la has been described as a ' coconut atrium'2)3 ) or ' porcelain atrium'.4)5 ) porcelain atrium involves the la appendage , the free wall of the la and the mitral valve apparatus , except interatrial septum , whereas coconut atrium involves all areas of the la.2 - 5 ) porcelain atrium or coconut atrium is rare disease entity , furthermore the massive calcification of la after mitral valve replacement has been reported very few the cases.6)7 ) authors experienced a 67-year - old woman who had massive la calcification after mitral valve replacement . a 67-year - old woman was admitted to our hospital presenting with congestive heart failure ( new york heart association functional class iv ) and peripheral pitting edema . the patient had a history of mitral valve replacement for chronic rheumatic mitral valvular disease ( severe mitral stenosis ) in 1987 without evidence of la calcification at the operation finding . 1 ) . a chest x - ray revealed diffuse calcified outline of the left atrial wall and enlarged cardiac silhouette with cardiothoracic ratio of 82% ( fig . 2 ) . transthoracic echocardiogram showed a good motion of prosthetic mitral valve with increased mean diastolic pressure gradient about 6 mmhg and a mild mitral regurgitation . three chamber enlargement except left ventricle with 87 mm - sized antero - posterior diameter of the la ( estimated volume index was 180 mm / m ) and a severe tricuspid regurgitation were also shown . transesophageal echocardiogram showed decreased la contraction and increased spontaneous echo contrast in the la chamber ( fig . the estimated peak systolic pulmonary artery pressure was 70 mmhg and demonstrated severe pulmonary hypertension . we performed tricuspid valve annuloplasty with annular ring ( 33 mm - sized tailor flexible ring , st . paul , mn , usa ) and removal of subvalvular structure with myotomy and endoatriectomy of right atrium under cardiac arrest with a cardiopulmonary bypass . he first described the post - mortem findings on a 44-year - old male who died of congestive heart failure due to mitral stenosis . massive calcification of the la was predominant in woman as frequent as 74% and was usually associated with rheumatic mitral stenosis . it took an average duration of 19.7 years to diagnose massive la calcification after rheumatic mitral stenosis.9 ) however , massive calcification throughout the la wall is very rare . the pathophysiology of the la calcification associated with mitral valvular disease were 1 ) a previous ulceration of the atrial wall as the origin of the calcification and 2 ) a response to the chronic strain forces in the setting of mitral disease.1 ) it is also rarely reported in chronic renal failure.10 - 12 ) massive calcification of the la wall can cause recurrent thrombosis despite the therapeutic anticoagulant level of the patient following a surgical thrombectomy.13 ) roberts et al.14 ) reported that calcific deposits prevented the la from dilating , decreased its compliance , and caused the elevated la pressure to be transmitted to the pulmonary vessels and the right ventricle , leading to tricuspid insufficiency . in our case , la calcification was progressed after mitral valve replacement despite there was no definite la wall calcification at the time of surgery . we thought it was due to previous formed rheumatic ulceration of the atrial wall as the origin of the calcification and its progression . radiography of the left side of the chest is recommended to assess long - standing rheumatic mitral valve disease . the image of computed tomography can display massive atrial calcification rather than the plane chest x - ray . in general , transesophageal echocardiography permits admirable visualization of the la compared with transthoracic echocardiography because of the anatomic accessibility of the esophagus . however transesophageal imaging is significantly impaired in the presence of calcified walls of la.15 ) adequacy of transesophageal and transthoracic echocardiographic imaging possibly depends on the extent and the density of calcification.16 ) magnetic resonance imaging may demonstrate the presence of thrombus , but its ability to depict the atrial calcification is suboptimal.3 ) surgery should be considered as one option of the treatment for atrial calcification.1 ) but no postoperative investigation has been done to evaluate the long - term mortality and the progression of the atrial compliance after the operation . in conclusion , in patient with long lasting rheumatic valvular heart disease it is possible that progression of la calcification after mitral valve replacement despite there was no la wall calcification at the time of surgery . thus , if patient who had rheumatic heart disease and underwent mitral valve replacement , suffer right heart failure , extensive la calcification should be considered .

a lot of natural herbs distributed around the mediterranean sea have been traditionally used by local people . most of the studies on plants to grow for mediterranean climate have been investigated mainly in tolerance for the drying and relationship with environmental stress . however , the physiological function of natural herb is still poorly understood though many species grow allover the place . in an area of north africa , the several species of natural herb were employed in traditional medicine as active against helicobacter pylori , oxidative stress , hypertension , and hypoglycaemic [ 24 ] . cistus monspeliensis is a perennial plant which in widely distributed from south europe to north africa . the genus cistus is popular on tea and spice but not utilized for the antiaging effects . c. monspeliensis has been reported to have an antioxidation , antibacterial , and anti - inflammatory effects . in the small intestine , the ingested capsaicin was carried into the intestinal epithelium , which is in contact with a high concentration of food ingredients [ 7 , 8 ] . considering the relevance of the food components and the effects of the oral route for human exposure , we have investigated the effect of capsaicin on the energy metabolism of intestine , by using caco-2 cell line , a well - known in vitro model of intestinal epithelium . the intracellular atp accumulation is important for optimal integrity of the mucosa and has been suggested to play a specific role in the regulation of absorption and barrier functions [ 9 , 10 ] . from these reports , it was supposed that the intracellular atp accumulation contributes to the homeostasis of caco-2 cells , because the regulation of absorption and barrier functions are necessary for caco-2 cells differentiation . in this study , the energy metabolism underlying the effect of c. monspeliensis extract on intestinal epithelium was clarified by performing the capillary electrophoresis time - of - flight mass spectrometry ( ce - tof / ms ) analysis . moreover , we performed real - time pcr to quantitate mrna using the primers , related with atp production , and the luciferase assay was performed to measure intracellular atp production in the intestinal epithelium . this is the first report that the extract of c. monspeliensis was induced by the activation of energy metabolism in human intestinal epithelial cells . human intestinal epithelial caco-2 cells were obtained from american type culture collection ( manassas , va , usa ) and were used between passages 10 to 30 . the cells were maintained in dulbecco 's modified eagle 's medium ( dmem , sigma , st . louis , mo ) supplemented with 10% ( v / v ) fetal bovine serum ( hyclone co. , ltd . ) , 1% ( v / v ) penicillin - streptomycin ( lonza , walkersville , md usa ) , and 1% ( v / v ) nonessential amino acids ( cosmo bio co. , ltd . , tokyo , japan ) and incubated in an atmosphere of 5% co2 at 37c . 23 plants were collected between june and july in 2008 from jendouba , kairouan , and kasserine areas in tunisia and air - dried in the shade at room temperature and ground to powder with an electrical blender and stored at room temperature . each plant sample ( 1 g ) was extracted with 10 ml of distilled water at 105c for 15 minutes or with 10 ml of 70% ( w / v ) ethanol at room temperature for 1 week . the extract water was filtered and sterilized using 0.22 m membrane filter and stored at 80c . dpph ( 2 mg ) was dissolved in ethanol ( 12.76 ml ) . the ethanol solution ( 6 ml ) , 400 mm 2-morpholinoethanesulfonic acid monohydrate ( mes ) solution ( 1.5 ml ) , and milliq water ( 4.5 ml ) were mixed in one tube . furthermore , plants extract ( 10 l ) and the mixing solution ( 190 l ) were added in 96-well plates and incubated for 10 min at room temperature . the absorbance was spectrophotometrically determined at 520 nm using a multidetection microplate reader ( powerscan ht , dainippon pharmaceutical , usa ) . after incubating seeded plates for 24 h , total rna was purified using the isogen kit ( nippon gene co. , ltd . , total rna was quantified by measurement at 260 nm with a uv spectrophotometer and was also measured at 280 nm to assess purity . only rna with a 260/280 ratio higher than 1.8 was used for the real - time pcr . template cdna was obtained from total rna using the superscript reverse transcriptase system ( invitrogen ) . briefly , rna was denatured at 65c for 5 min and incubated with 1 ml oligo ( dt ) 1215 primers and chilled at 4c . after adding superscript ii reverse transcriptase ( 200 units ) , the reaction mix was incubated at 42c for 60 min , then 10 min at 70c . for the quantification of mrna quantitative pcr reactions were performed in a miniopticon instrument ( bio - rad , usa ) . briefly , the rt mix ( 2 ml ) was used as template for the real - time pcr mix containing 0.5 mm forward and reverse nested primers ( 2 l each ) and 2 sybr green supermix ( 10 l ) . the primers used were checked using the blastn program of the genebank ; their sequence ( tpi : forward : 5-cttggctgagagatggaagg-3 , reverse : 5-cagtgaaggcagacaaacca-3 ; pgm : forward : 5-gcggagaacttcatccagag-3 , reverse : 5-tgtcagaatgatcccaccaa-3 ; atp synthase : forward : 5-ctggaggacctgttgatgct-3 , reverse : 5-tggggtttttcgatgacttc-3 ) was based on the known sequences in the cording region of the human genes . the amplification conditions were 3 min at 95c , 10 sec at 95c , 30 sec at 62c , and 30 sec 72c for 41 cycles . at the end of the reaction , a melting curve analysis was carried out to check for the presence of primer dimers . after incubating seeded plates for 24 h , the extract was added at 0.1% ( w / v ) concentration and the cells were incubated for 12 h. after treatment , 5% ( w / w ) mannitol solution was added at 10 ml and was removed . once again , 5% ( w / w ) mannitol solution was added at 2.0 ml and was removed . cells were scraped in 1.3 ml of methanol which includes 10 m each of 2 internal standards ( isc1 and isa1 ) , and methanol including scraped cells was transferred at 1.0 ml to centrifuge tube . 1 ml of chloroform and 400 l of milliq water were added to the solution and then thoroughly mixed . followed by centrifugation at 2,300 g for 5 min at 4c , the 250 l of water layer was removed and centrifugally filtered through a 5-kda - cutoff filter ( millipore , usa ) to remove proteins . the filtrate was desiccated and dissolved in 20 l of milliq water prior to injection . the capillary electrophoresis time - of - flight mass spectrometry ( ce - tof / ms ) experiments were performed using an agilent ce - tof / ms system ( agilent technologies co. ) unit 5 . separations were carried out on a fused silica capillary ( 50 m i.d . 80 cm total length ) . run buffer : anion buffer solution ( p / n : h3302 - 1021 ) , rinse buffer : anion buffer solution ( p / n : h3302 - 1022 ) , sample injection : pressure injection 50 mbar , 25 sec , ce voltage : positive , 30 kv , ms ionization : esi negative , ms capillary voltage : 3,500 v , ms scan range : m / z 501,000 , sheath liquid ( p / n : h3312 - 1020 ) . atp was assessed by firefly bioluminescence using the luminescence luciferase assay kit ( toyo ink , tokyo , japan ) . caco-2 cells were plated in 96-well plates at 1.0 10 cells / ml in 100 l . after 24 h incubation , the extract was added at 0.1 , 0.01 , and 0.001% ( w / v ) concentration , and the cells were incubated for 3 , 6 , and 12 h. after treatment , 100 l of luciferin - luciferase solution was added and stirred for 3 min using a microplate genie , 100 v ( scientific industries , inc . the luminescence was determined using a multidetection microplate reader ( powerscan ht , dainippon pharmaceutical , usa ) . the viability of cells was determined by mtt ( 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide ) assay . briefly , caco-2 cells were plated in 96-well plates at 1.0 106 cells / ml in 100 l . after 24 h incubation , extract diluents with medium were added to obtain final concentrations from 0.1% to 0.001% ( w / v ) , and the cells were cultured for 24 h , followed by the addition of 10 l of 5.0 mg / ml of mtt . after 12 h incubation , 150 l of 10% sodium dodecyl sulfate ( wako ) was added and incubated for 48 h. the absorbance was spectrophotometrically determined at 570 nm using a multidetection microplate reader ( powerscan ht , dainippon pharmaceutical , usa ) . dpph radical is well known as a stable organic free radical which has been used for estimation of the antioxidant capacity . we selected c. monspeliensis in twenty three plants that had high antioxidation to assess atp production because this plant was used to traditional foods . to investigate the effect of c. monspeliensis extract on the activation of energy metabolism in human intestinal epithelium ( figure 1 ) , real - time pcr was employed to evaluate the mrna expression of genes , related with glycolysis and tca cycle . in this experiment , we used the primers of triosephosphate isomerase ( tpi ) , phosphoglycerate mutase ( pgm ) , and atp synthase that have a deep relationship with intracellular atp production and relatively higher expression in caco-2 cells . especially , to treat 0.1% ( w / v ) c. monspeliensis for 6 h , mrna expression levels of tpi , pgm , and atp synthase were upregulated by 170% , 161% , and 310% , respectively . pgm is an enzyme that catalyzes the internal transfer of a phosphate group from c-3 to c-2 which results in the conversion of 3-phosphoglycerate to 2-phosphoglycerate through a 2 , 3-bisphosphoglycerate intermediate . tpi is an enzyme that catalyzes the reversible interconversion of the triose phosphate isomers , dihydroxyacetone phosphate , and d - glyceraldehyde 3-phosphate . atp synthase is an important enzyme that creates energy for the cell to use through the synthesis of atp in mitochondria . in figure 1 , mrna expression of tpi and pgm , glycolytic enzymes , also , atp synthase , electron transport chain enzyme , was increased . as a result of the metabolomics analysis , c. monspeliensis extraction increased production of each metabolite in glycolysis and tca cycle ( table 2 ) . three metabolites on rate - limiting step in glycolysis ( glucose 6-phosphate , fructose 1,6-diphosphate , and pyruvic acid ) were increased by c. monspeliensis extraction . the production of pyruvic acid is related to promote function of atpase that involved atp synthesis . atp is not produced in tca cycle ; however , a lot of atp is produced in electron transport chain . antiaging on promoting energy metabolism is expected to promote function of glycolytic and electron transport chain enzymes that are pathways of atp production . in the results of real - time pcr and metabolomics , c. monspeliensis was upregulated metabolite and mrna expression involved atp production . we determined atp production of c. monspeliensis extract using the luminescence luciferase assay kit ( figure 2 ) . 0.1 , 0.01 , and 0.001% ( w / v ) of c. monspeliensis extraction increased atp production on caco-2 compared with nontreated cells . especially , to treat 0.01% ( w / v ) of c. monspeliensis for 3 h , atp production was upregulated by 172% . as a result , we considered that c. monspeliensis had effect of atp production and to promote energy metabolism on caco-2 . we determined cell proliferation on caco-2 using mtt assay . from the result of mtt assay , 0.01 and 0.001% ( w / v ) of c. monspeliensis extraction were not changed cell proliferation on caco-2 cells , whereas 0.1% ( w / v ) of c. monspeliensis extraction induced the increase of cell proliferation on caco-2 cells compared with nontreated cells . as a result , we considered that 0.1 to 0.001% ( w / v ) c. monspeliensis extract had not toxic effect for caco-2 cells . atp is a multifunctional nucleotide that is the most important as a molecular currency of intracellular energy transfer . in this role , atp transports chemical energy within cells for metabolism . the glycolytic and tca cyclic enzymes played an important role in the intracellular atp production [ 14 , 15 ] . tpi enzyme is essential for energy production , allowing two molecules of glyceraldehyde 3-phosphate to be produced for every glucose molecule , thereby doubling the energy yield . the activity of tpi and pgm was not influenced by age or caloric restriction , while the other glycolytic enzymes were influenced . furthermore , the activity and expression of these two enzymes , however , are decreased by oxidation and disorders ( alzheimer 's disease , hemolytic anemia , erythrocyte destruction , etc . ) these observations indicate that the expression and activity of these glycolytic enzymes were upregulated in response to specific signals , such as c. monspeliensis extraction . atp synthase uses the proton motive force to make atp from adp and inorganic phosphate ( pi ) in mitochondria . however , these enzymes have contribution toward not only atp production but also regulation of cellular function . its deficiency has been known to cause a severe multisystemic disease with autosomal recessive inheritance and neurodegeneration . decreased tpi activity induces the accumulation of dhap , which produces methylglyoxal instead of gap . the methylglyoxal induces oxidative damage to proteins and dna and accumulation of advanced glycation end products ( ages ) , which leads to structural degeneration and functional decline of brain cells . also pgm is related with the apoptosis of human prostate cancer cells , lncap , du145 , and pc-3 . monoubiquitination of phosphoglycerate mutase , as well as formation of a noncovalent complex containing ubiquitin and phosphoglycerate mutase , increased in colorectal cancer , which may suggest a potential pathophysiological event . a decreased level of phosphoglycerate mutase isoenzymes was reported in breast carcinoma indicating its differential expression . active regulation of the mitochondrial atp - synthase ( complex v ) in response to the cellular energy demand has been demonstrated in several species like rat , dog , and humans and different types of tissue like heart muscle , skeletal muscle , fibroblasts , and brain . regulation of the atp - synthase seems to be a central physiological phenomenon which is presumably present in many other species and other organs . as energy supply via the mitochondrial atp - synthase plays such a vital role in almost every cell of the body , more diseases will probably be identified where ( primary or secondary ) abnormalities of this enzyme occur . in addition , one of the causes of aging , free radical , is focused on oxidative cytotoxic . mitochondrion that is important as metabolism organ of atp production caused impairment accompanied effect of aging and ros . the effect of enhancement component on atp production can make use of antiaging effector as well as functional foods for antiaging . our research showed that c. monspeliensis extract induced the expression of enzymes , related with intracellular atp production , in human intestinal epithelial cells . and it is known that species of cistus contain quercetin , kaempferol , aesculin , myricetin , and flavan-3-ols . for example , quercetin has antioxidation and has a function to improve memory impairment that accompany alzheimer 's disease . moreover , it is known that an aging is caused for decline of intestinal absorption [ 30 , 31 ] . we expect that c. monspeliensis extract can contribute to prevent several diseases like senescence , alzheimer 's disease , cardiosclerosis , stenocardia , cancer , and so forth and recover the function of impairment intestinal absorption . 0.001 , 0.01% ( w / v ) of c. monspeliensis increased atp production ( figure 2 ) and , however , did not changed cell viability as compared with control ( figure 3 ) . on the other hand , 0.1% ( w / v ) of c. monspeliensis decreased atp production as compared with low - concentration sample and , however , up - regulated cell viability in mtt assay . from these results , we considered that atp was consumed for cell proliferation in 0.1% ( w / v ) of c. monspeliensis , and atp accumulation a little decreased as compared with low concentration ( 0.01 and 0.001% ( w / v ) ) . it was reported that the atp accumulation was contributed to the proliferation and homeostasis on caco-2 cells . we expect that c. monspeliensis extract can induce the atp accumulation and then the activation of proliferation and homeostasis on caco-2 cells . we considered that isolation of active component from c. monspeliensis extract should be fulfilled and further in vivo studies should focus on the confirmation of activation and safety of c. monspeliensis extract . our findings indicate that c. monspeliensis extract had high antioxidant ability . in addition , we show that c. monspeliensis extract has the function of promoting energy metabolism pathways , including glycolysis , tca cycle , and electron transport chain , in human intestinal epithelial cells . also , c. monspeliensis extract enhanced the production of intracellular atp on intestinal epithelium . from these results , c. monspeliensis extract has an ability to enhance the energy metabolism .

until recently , most general obstetrician - gynecologists in the united states ( us ) had little opportunity to provide care to hiv - infected women . screening guidelines published in 2006 and 2008 [ 1 , 2 ] and advances in treatment have changed the landscape of treatment and care of hiv - infected women . as more women test positive for hiv and as hiv - infected women live longer , healthier lives , increasing numbers of women of reproductive age will be living with an hiv diagnosis , and general obstetrician - gynecologists will increasingly encounter women who have been diagnosed with hiv infection . recognizing the increased need for information and guidance for providing optimal gynecologic care for hiv - infected women , the american college of obstetricians and gynecologists recently published a practice bulletin on the topic . the literature on the risk of postoperative complications among hiv - infected patients is mixed . for example , a large retrospective study of surgical outcomes that used data from the kaiser permanente medical care program revealed greater 12-month mortality and greater incidence of post - operative pneumonia ; however , no other post - operative complications were elevated among hiv - infected patients . notably , despite hiv - infected women having an elevated risk for some conditions leading to gynecologic surgery [ 610 ] , little systematic evidence is available on the risk of complications of gynecologic surgeries among hiv - infected women . the few reports that exist are inconsistent in their findings with one study observing a significantly higher rate of complications among hiv - infected women and two other studies yielding null results [ 12 , 13 ] . the objective of this analysis is to add to the knowledge base by describing complications among hiv - positive women undergoing certain common gynecological surgeries . we used national data from the us to compare frequencies of complications among hiv - infected and - uninfected women undergoing common gynecological surgical procedures in inpatient settings . we included several years of data in order to have a sufficient number of discharge records among hiv - infected women to create reliable estimates for selected surgical procedures . the healthcare cost and utilization project ( hcup ) includes databases and software tools developed through a partnership among private industry , states , and the federal government . the nis , the largest all - payer database of inpatient stays in the united states , is a key component of hcup . the nis incorporates data from approximately 8 million hospital stays per year , and it approximates a 20% stratified sample of community hospitals in the us ( ahrq , 2010 ) . sampling is stratified on location ( rural or urban ) , hospital size , region of the country , teaching status , and type of ownership ( public or private ) . as of 2007 , 40 states contributed data to the nis , and hospitals in the sampling frame comprised approximately 90% of us hospital discharges . we analyzed discharge records from women aged 15 and older , excluding hospitalizations that included delivery ( international classification of diseases , 9th revision , clinical modification ( icd-9-cm ) codes 650 ; v27 ) . we further limited the study population to include only hospitalizations during which one of the following procedures had been performed : oophorectomy ( procedure codes 65.3 , 65.31 , 65.39 , 65.4 , 65.41 , 65.49 , 65.5 , 65.51 , 65.52 , 65.53 , 65.54 , 65.6 , 65.61 , 65.62 , 65.63 , and 65.64 ) , salpingectomy for ectopic pregnancy ( procedure code 66.62 ) , bilateral tubal sterilization ( procedure codes 66.2 , 66.21 , 66.22 , 66.29 , 66.3 , 66.31 , 66.32 , and 66.39 ) , dilation and curettage ( procedure codes 69.0 , 69.01 , 69.02 , and 69.09 ) or hysterectomy . hysterectomy was defined as abdominal hysterectomy ( 68.3 , 68.39 , 68.4 , and 68.49 ) , vaginal hysterectomy ( 68.5 ; 68.59 ) , or total laparoscopic hysterectomy / laparoscopic - assisted hysterectomy ( 68.31 , 68.41 , and 68.51 ) . any type of hysterectomy with the code 54.21 was also coded as laparoscopic - assisted . we focused on these gynecologic surgical procedures because they were the most common ( at least 150 surgeries performed ) among hospitalizations of hiv - infected women in our dataset . with the exception of hysterectomy with concomitant oophorectomy , we excluded hospitalizations during which multiple gynecologic surgeries were performed . the nis does not include patient identifiers , and the unit of analysis is the hospital discharge record . although some patients may have been admitted multiple times during the study period for procedures we examined , we expect this to be rare . our primary outcome , experiencing at least one complication of surgical procedures , was defined as experiencing extended length of stay ; transfusion ; anemia due to acute blood loss ; accidental puncture or laceration during a procedure ; hemorrhage , hematoma , or seroma complicating a procedure ; urinary tract infection ; fever ; other postoperative infection ; urinary tract complications including urinary retention and ureteral obstruction ; paralytic ileus ; any of several less common complications ( e.g. , thromboembolism and postoperative shock ) . extended length of stay was defined as being at or above the 90th percentile for that specific surgical procedure . this was equivalent to 5 days for hysterectomy with oophorectomy , 4 for hysterectomy alone , 9 for oophorectomy alone , 4 for salpingectomy for ectopic pregnancy , 5 for bilateral tubal sterilization , and 6 for dilation and curettage . our primary independent variable was hiv status ( icd-9-cm codes 042 , 043 , 044 , 079.53 , 279.10 , 279.19 795.71 , 795.8 , and v08 ) . we defined comorbidity as presence of 1 of the following conditions / behaviors that could put women at increased risk for complications of the gynecologic surgeries we examined : obesity , diabetes , cardiac condition or hypertension , anemia , gastrointestinal ulcers , smoking , and alcohol or substance abuse . based on review of the literature , we selected relevant icd-9 codes for these conditions / behaviors , and we defined them accordingly . we compared discharge records of hiv - infected and -uninfected women undergoing the gynecologic surgeries we examined on various descriptive characteristics of patients and hospitals , including age , primary payer , hospital teaching status / location , hospital region , and presence of any comorbidity . race was not examined because some states do not report race / ethnicity data , and , among states that do report this , there are often inconsistencies and missing values in the data . comparisons were evaluated with chi - squared tests ( alpha = 0.05 ) . for each surgery , we used multivariable logistic regression to estimate the association between hiv infection status and experiencing 1 complication of surgery , adjusting for patient age , primary payer , year of hospitalization , and presence of any comorbidity . because of the possibility that associations between hiv infection status and the occurrence of complications might differ depending on whether comorbidity was present , we tested for interaction between hiv infection status and presence of any comorbidity . associations for which statistically significant interaction was detected ( alpha = 0.05 ) are presented separately for women with and without comorbidity . in addition , we conducted multivariable logistic regression to estimate the association between hiv infection and the 4 most common complications in our sample . these included extended length of stay , transfusion , anemia due to blood loss , and all infectious complications combined ( i.e. , experience of urinary tract infection ; fever ; other postoperative infection ; or contaminated or infected blood , other fluid , drug , or biological substance ) . again , we adjusted models for patient age , primary payer , year of hospitalization , and presence of comorbidity , and we tested for interaction between hiv infection and any comorbidity . finally , for each surgery we examined , we tested for major shifts over time by using multivariable logistic regression ( with adjustment for the same variables ) , to compare , for hospitalizations among hiv infected women , the odds of extended length of stay , infectious complications , and all other complications combined during the time periods preceding ( 19942000 ) and during ( 20012007 ) widespread implementation of highly active antiretroviral treatment ( haart ) in the us . we used sas - callable sudaan 9.0 software ( rti international , research triangle , durham , nc , usa ) to account for the multistage probability sampling design . all results are based on weighted estimates of hospitalizations in the us during the period of study . in 1998 , the nis sample design changed to better reflect the population of hospitals in the sample . specifically , short - term rehabilitation hospitals were excluded , stratification variables were redefined , the discharge definition was changed , and previous - year nis hospitals were no longer given sampling precedence . to account for the change in sample design , we applied an alternate set of nis discharge and hospital weights ( based on the 1998 design ) to 19941997 data . all programming was independently duplicated by a second data analyst . because the study utilized deidentified data from a publicly available data set , the centers for disease control and prevention determined that human - subject research review was not required . during the years 19942007 , there were an estimated 14,922,397 surgeries of interest ( hysterectomy , oophorectomy , salpingectomy for ectopic pregnancy , bilateral tubal sterilization , or dilation and curettage ) among women aged 15 and older . excluding delivery hospitalizations ( n = 4,303,344 ) and hospitalizations during which more than one of the gynecologic surgeries we examined were performed ( n = 1,682,573 ) resulted in an analytic sample of an estimated 8,939,780 surgeries , 18,177 of which were among hiv - infected women , and 8,921,603 of which were among hiv - uninfected women . the distribution of type of gynecological surgery differed among hospitalizations of hiv - infected and - uninfected women , with bilateral tubal sterilization ( 12.6% versus 3.4% ) and dilation and curettage ( 12.7% versus 6.8% ) being more common among hiv - infected women in our sample ( table 1 ) . for hospitalizations that included hysterectomy , the distribution of surgical approach also varied according to the woman 's hiv infection status . regardless of whether an oophorectomy was performed concomitantly , hiv - infected women in our sample more often underwent abdominal hysterectomies ( 87.0% versus 82.6% for hysterectomy with oophorectomy and 61.8% versus 55.9% for hysterectomy alone ) . laparoscopic hysterectomy was performed less often among hiv - infected women ( 9.3% versus 11.1% for hysterectomy with oophorectomy and 8.3% versus 12.7% for hysterectomy alone ) . compared to hiv - uninfected women , hiv - infected women hospitalized for gynecological surgeries were more often under 35 years of age ( 34.6% versus 20.6% ) , less often relied on private insurance as the primary payment source ( 29.1% versus 69.6% ) , more often received care in urban teaching hospitals ( 67 . 7% versus 42.5% ) and in the southern or northeastern us ( 83.7% versus 57.6% ) . hiv - infected women more often presented with comorbidity ( 44.7% versus 34.1% ) . for hysterectomy with or without oophorectomy , oophorectomy without hysterectomy , salpingectomy , and dilation and curettage , hiv infection was positively associated with experiencing 1 complication of surgery in models adjusted for age , primary payer , year , and presence of comorbidity ( table 2 ) . however , for bilateral tubal sterilization , the association between hiv infection status and experiencing 1 complication differed according to whether comorbidity was present ( interaction p value < 0.001 ) , and hiv infection was positively associated with experiencing complications only among women without comorbidity . the magnitude of the association between hiv infection and experiencing 1 complication ranged from two - fold for hysterectomy with oophorectomy ( adjusted odds ratio ( aor ) : 2.0 ; 95% confidence interval ( ci ) : 1.7 , 2.2 ) to more than three - fold for bilateral tubal sterilization in the absence of comorbidity ( aor : 3.1 ; 95% ci : 2.4 , 4.0 ) . when we examined the effect of hiv infection on the occurrence of common specific complications , we found that hiv infected women were more likely than uninfected women to experience infectious complications of all the gynecologic surgeries we examined ( table 3 ) . however , for bilateral tubal sterilization , the association between hiv infection and extended length of stay differed by the presence of comorbidity ( interaction p value < 0.001 ) ; hiv - infected women more often experienced extended length of stay only when comorbidity was absent . for the following types of surgery , transfusion was more often performed on hiv - infected women than hiv - uninfected women : hysterectomy without oophorectomy , bilateral tubal sterilization , and dilation and curettage . for dilation and curettage , but not for the other types of gynecologic surgeries we examined , hiv - infected women more often experienced anemia due to acute blood loss . among hospitalizations of hiv - infected women , there were no statistically significant changes from 19942000 to 20012007 in the frequency of any of the three groups of complications we examined for changes over time ( extended length of stay , infectious complications , and all others combined ) . hiv infection was associated with elevated frequencies of surgical complications of the gynecologic surgeries we examined . not only was positive hiv status associated with experiencing any of several surgical complications , but it also showed higher frequencies of specific complications such as needing to undergo transfusion . in most cases , the presence of comorbidity did not alter the association between hiv and surgical complications . overall , we observed an increased occurrence of complications among hiv - infected women undergoing gynecologic surgeries . comorbidity altered the association between hiv status and surgical complications of bilateral tubal sterilization . among women who had this procedure , the frequency of experiencing at least one complication was essentially equal among hiv - infected and - uninfected women with any of the comorbidities we examined ; however , when comorbidity was absent , hiv - infected women more often experienced at least one complication . we suspect that , for tubal sterilization , which is a relatively minor procedure , comorbidity may be more important than hiv status in putting women at risk of surgical complications . nevertheless , hiv status appeared to play an important role among women without comorbidity ; among these women , hiv infection was associated with increased occurrence of complications . this is consistent with the increased occurrence of complications that we observed for other surgical procedures . notably , abdominal approaches were more often used ( and laparoscopic approaches less often used ) among hiv - infected women in our sample who underwent hysterectomy , as compared to uninfected women who underwent hysterectomy . this likely contributed to the increased frequency of surgical complications that we observed among hiv - infected women undergoing hysterectomy . little has been published on complications of gynecologic surgical procedures among hiv - infected women . a study by grupert and colleagues included 235 gynecologic and obstetric surgeries among hiv - infected women . they reported a higher complication rate among hiv - infected women , as compared to hiv - negative controls . another study by franz and colleagues reviewed the hospital course of 24 hiv - infected patients who underwent hysterectomy and compared them to uninfected controls , finding that hiv - infected women and controls did not differ on complication rates . they found that hiv - infected women had approximately twice the odds of experiencing complications ( 17% of hiv - infected women versus 9% of controls ) ; however , these results did not achieve statistical significance , perhaps because of small sample size . in contrast , we examined associations between infection with hiv and frequency of complications of selected gynecologic surgeries in a large nationally representative database , and we observed statistically significant associations between hiv infection status and experiencing complications . although frequencies of complications were higher among hiv - infected women , we could not determine the extent to which this was due to immunosuppression . our findings are , therefore , only applicable to inpatient settings ; results in other settings may differ . finally , the quality of our data is dependent on the accuracy of diagnoses and procedures listed in discharge records . some of the conditions and behaviors that we classified as comorbidity may have been characterized by differential accuracy or completeness , depending on a woman 's hiv status . for example , a provider may have been more or less likely to ascertain substance abuse and note it in the medical record , depending on a woman 's hiv status . despite these limitations , it is based on a large , nationwide data set , making our findings more generalizable to inpatient gynecologic surgeries across the us . the large sample size allowed for the ability to detect associations that may have been missed in other studies . finally , unlike the earlier studies we cited , we sought to account for the relationship between hiv and comorbidity in putting women at risk for complications of gynecologic surgery . in conclusion , the issue of hiv infection in gynecological treatment and care will become more prominent as more women in the us screen positive for hiv , and treatment adds years to women 's lives . our study adds to the evidence that hiv infection is an important consideration in gynecologic treatment and care . adequately powered prospective studies that examine factors that put hiv - infected women at higher risk of surgical complications , and that identify potentially modifiable risk factors , are needed . results of such studies can inform development of prevention strategies to protect the health of hiv - infected women , and help reduce disparities between hiv - infected and -uninfected women in complications of gynecologic surgeries . our finding that the frequency of infectious and other surgical complications among hospitalizations of hiv - infected women did not decrease following widespread implementation of haart suggests that women 's access to and adherence to treatment for hiv disease could be improved . strategies for improving access and adherence to haart should be developed , evaluated , and prioritized . additionally , the high proportion of hiv - infected women in our sample with comorbidities underscores the importance of health providers being well prepared to address a wide range of medical comorbidities as well as behaviors that may negatively impact the health of hiv - infected women . treatment by multidisciplinary teams of providers may be another approach for ensuring that the health needs of hiv - infected women are addressed .

streptococci in man are responsible for a variety of infections , ranging from relatively mild illnesses , such as pharyngitis and impetigo , to clinically severe pathologies such as pneumonia , septicaemia , necrotizing fasciitis or myositis . classically , arthritis secondary to group a streptococcal ( gas ) pharyngitis is attributed to acute rheumatic fever ( arf ) , but nowadays post - streptococcal reactive arthritis ( psra ) must be considered as well [ 25 ] . dissimilarities between arf and psra may exist genetically , on the level of humoral hyperresponsiveness [ 79 ] , and on the level of clinical characteristics [ 1016 ] . in individual cases diagnostic criteria may be helpful for diagnosing probably gas - induced psra in particular . in 1997 , ayoub and ahmed have proposed a set of clinical and serological criteria for the diagnosis of psra : ( a ) acute - onset arthritis , symmetric or asymmetric , usually nonmigratory , ( b ) a protracted course or a recurrent type of arthritis , ( c ) poor responsiveness to salicylates / nonsteroidals , ( d ) evidence of antecedent streptococcal infection , ( e ) absence of any jones major manifestation . though these criteria were meant for classification and not meant for diagnostic use , they can be applied as a starter for diagnostic use for clinicians : particularly criterion ( b ) is unclear from the onset . clinically , rheumatologists need to homogenize groups of psra patients as so far literature describes a heterogeneous spectrum of psra without proven prognostic implications . this study reflects all new patients presenting in a five - year period with early arthritis and at presentation elevated streptococcal antibody titre suggestive of a recent streptococcal infection , and not known with a previous rheumatic condition . a cohort of patients was studied comprising consecutive patients with early and acute onset of arthritis and at presentation a serological suggestion of a recent infection with -haemolytic streptococci , presenting to our outpatient clinics of rheumatology from april 1998 until january 2003 : a period of 57 months . before inclusion , other causes of arthritis were excluded : septic arthritis , rheumatoid arthritis , connective tissue disease , crystal deposition disease , reactive arthritis attributable to parvovirus b19/salmonella / shigella / campylobacter / chlamydia / gonococ - ci / spirochetes , and arthritis secondary to inflammatory bowel disease . an elevated antistreptolysin - o and/or antideoxy - ribonuclease - b titre with / without tonsillopharyngitis was a prerequisite for inclusion into the present study . all patients were according to good clinical practice regularly seen on outpatient basis for followup . there was no strict study protocol for psra patients , but the following tests were obtained on routine basis in these patients : full blood count , erythrocyte sedimentation rate , serum creatinine , alkaline phosphatase , gamma - glutamyltranspeptidase , aspartate amino or alanine aminotranspeptidase , uric acid , igm rheumatoid factor ( igm rf ) , antinuclear antibodies ( ana ) , c reactive protein , and urine microscopy . an echocardiogram was obtained in cases with a cardiac murmur or with a conduction block on the electrocardiogram . clinical evaluation was performed by a rheumatologist , that is , the number of joints involved was counted according the 36 swollen joint index ( sji ) . during follow - up visits ( t = 0 , 6 weeks , 12 weeks , and every 3 month afterwards ) , the diagnosis was reevaluated by patient 's own rheumatologist , and when after one year no alternative explanation for the arthritis had been presented , the diagnosis of psra became definite . this procedure was checked by an experienced clinician before entrance into this study . in all patients antistreptolys - in - o ( aso ) and antideoxyribonuclease - b ( antidnaseb ) titres were measured simultaneously and sequentially , at least at three time points : at presentation , at 410 weeks after pharyngitis , and once afterwards . when a sore throat was not recollected , the titres of aso and antidnaseb were measured every 4 weeks during a period of 3 months . the suggestion of a recent streptococcal infection was accepted when an increased titre of aso and/or antidnaseb and subsequently decrease / normalization of titres were found : ( 1 ) aso > 200 300 u / l in adolescents ; antidnaseb > 200 u / l irrespective of age ; ( 2 ) one or both titres must primarily show a significant rise and subsequent decrease : critical difference between consecutive aso values 26% and between consecutive antidnaseb values 14% . psra was diagnosed by the patient 's own rheumatologist when after at least one year no alternative diagnosis could be made . definite psra patients were subdivided into two separate groups , based on the aso / antidnaseb ratio taken at 410 weeks after pharyngitis / tonsillitis : ratio < 1.40 a plea for gas - induced reactive arthritis , whereas ratio > 1.5 , a plea for ngas - induced psra . patients diagnosed by their own rheumatologist or the experienced clinician as non - psra are grouped together into a third separate group . ayoub and ahmed have proposed criteria for the classification of psra which may be quite applicable for clinical use : a triad regarding characteristics of arthritis , one regarding evidence of antecedent streptococcal infection , and a duplet regarding the exclusion of acute rheumatic fever : ( a ) acute onset of , symmetric or asymmetric , nonmigratory arthritis , ( b ) protracted course of arthritis or recurrent arthritis , ( c ) poor response of arthritis to salicylates / nonsteroidals , ( d ) serological evidence of antecedent streptococcal infection , ( e ) not fulfilling any modified jones major criterion . the combination of this quintet of criteria is tested in our study groups . criterion ( b ) is however difficult to score prospectively in a diagnostic phase from the onset in an individual patient . a previous study from the region of arnhem , in the central region of the netherlands , revealed that the higher aso / antidnaseb ratios plea for psra types not due to gas , that is , ratios > 1.5 plea for nongroup a streptococcal ( ngas ) infection which may be helpful particularly in cases with a negative throat culture ; ratios < 1.4 plea for gas - induced reactive arthritis . therefore a serological criterion which is clear from the beginning at the diagnostic phase , is formulated for presumably gas - induced psra : ( f ) aso / antidnb ratio at 410 weeks < 1.4 . a further serological criterion is formulated as ( g ) antidnaseb > 400 u / l which is twice above the upper limit of normal range ( normal range : < 200 the presented patient population is fitted into five sets of several combinations of criteria : starting with the five ( ( a ) , ( b ) , ( c ) , ( d ) , plus ( e ) ) criteria adapted from ayoub and ahmed , versus modification 1 , that is , a sextet consisting of the aforementioned quintet plus ( f ) , versus modification 2 , that is , a sextet consisting of criteria by ayoub and ahmed plus ( g ) and modifications 3 and 4 consisting of the sextets minus ( b ) : ( a ) , ( c ) , ( d ) , ( e ) , plus ( f ) , and ( a ) , ( c ) , ( d ) , ( e ) , plus ( g ) , respectively . using the aforementioned tests , the following test characteristics are calculated : sensitivity ( se ) , specificity ( sp ) , positive predictive value ( ppv ) , likelihood ratio of a positive test ( lr+ ) measured as the ratio between probability on a positive test result in gas - induced psra patients versus nonindex patients ideally reaching to infinite , and likelihood ratio of a negative test ( lr ) which is measured as the ratio between probability on a negative test result in gas - induced psra versus nonindex patients ideally reaching to nil : using a 2 2 table with gas - induced psra diagnosed by a rheumatologist at classification time 1 year after presentation as the golden standard , and using several combinations of criteria as index test , and in which a = true test positive , b = false test positive , c = false test negative , and d = true test negative : ( legend : v[x ] = square root of x ; exp[x ] = 2.7182818284 ) se = a/(a + c ) with 95% confidence interval : se + / ( 95%ci ) 1.96*v[ac/(a + c ) ] ; sp = d/(b + d ) with 95%ci : sp + /1.96*v[bd/(b + d)];ppv = a/(a + b ) with 95%ci : ppv + /1.96*v[ab/(a + b)];npv = d/(c + d ) with 95%ci : npv + /1.96*v[cd/(c + d ) ] ; lr+ = se/(1 sp ) with 95%ci : exp[ln(lr+ ) + /1.96*v[1/a 1/(a + c ) + 1/b 1/(b + d)]];lr = ( 1 se)/sp with 95%ci : exp[ln(lr ) + /1.96*v[1/c 1/(a + c ) + 1/d 1/(b + d ) ] ] . se = a/(a + c ) with 95% confidence interval : se + / ( 95%ci ) 1.96*v[ac/(a + c ) ] ; sp = d/(b + d ) with 95%ci : sp + /1.96*v[bd/(b + d ) ] ; ppv = a/(a + b ) with 95%ci : ppv + /1.96*v[ab/(a + b ) ] ; npv = d/(c + d ) with 95%ci : npv + /1.96*v[cd/(c + d ) ] ; lr+ = se/(1 sp ) with 95%ci : exp[ln(lr+ ) + /1.96*v[1/a 1/(a + c ) + 1/b 1/(b + d ) ] ] ; lr = ( 1 se)/sp with 95%ci : exp[ln(lr ) + /1.96*v[1/c 1/(a + c ) + 1/d 1/(b + d ) ] ] . intergroup comparison between probable gas and ngas patients was done by mann - whitney 's two - sample test . nonparametric spearman 's rho correlations are calculated between esr , crp , aso / adb ratio , number of arthritic joints , and number of weeks arthritis persists . during a period of nearly five years 45 patients with a sterile acute arthritis with at presentation a positive streptococcal serology presented to our outpatient department . all cases were sporadic , and clustering of cases was not seen . in 36 patients a significant change in streptococcal serology was observed , and in 16 a probability of psra due to group a streptococci could not be confirmed as they had group c or g streptococci in throat culture ( n = 2 ) and a compatible high serological ratio suggestive of nongroup a streptococci ( ngas ) , that is , aso / antidnaseb > 1.5 . suspicion rose in a group of 20 patients during followup that indeed group a streptococci ( gas ) were the causative organisms . this indicates an estimated annual incidence rate for psra of 36 in our dutch population , that is , 1.26/100 000 : gas - related psra ( n = 20 ) indicates an annual incidence rate of 0.70/100 000 , with ngas - related psra ( n = 16 ) 0.56/100 000 . a group consisting of 9 patients presented with arthritis plus elevated aso but did not have a significant increase nor decrease in streptococcal antibody titres ; and during followup an alternative diagnosis had to be be made . therefore these patients are categorized into a third group of non - psra . during followup they were diagnosed as coincidental streptococcal carriers in rheumatoid arthritis ( n = 3 ) , psoriatic arthritis ( n = 3 ) , reactive arthritis due to chlamydia ( n = 1 ) , lfgren syndrome ( n = 1 ) , and lyme arthritis ( n = 1 ) . in gas - induced psra , 4 out of 20 patients had positive antinuclear antibodies , without any specificity . by definition all psra patients have an increased aso titre at 410 weeks after a sore throat / pharyngitis , since seronegative cases were excluded . in the 20 presumed gas - induced psra we obtained 7 positive throat cultures ( 35% ) : all indeed consisted gas ; whereas in the 16 presumed ngas cases 2 positive throat cultures ( 12% ) were found : once group c and once group g streptococci . the number of arthritic joints according to the 36 swollen joint count ( sjc36 ) is higher in the presumed gas- than in the presumed ngas - induced cases : means ( sd ) 5.2 ( 1.3 ) versus 1.9 ( 0.4 ) . in gas - related psra the following systemic involvement was encountered : pericarditis ( 1 out of 20 patients ) , and pneumonitis ( 1 out of 20 patients ) , whereas in ngas - related psra similar extraarticular manifestations were not seen . gas - related psra patients all presented with a nonmigratory type of arthritis ; 45% had arthritis which persisted for more than 3 months ( by our definition protracted course ) , and in 10% arthritis recurred . good response to salicilates / nonsteroidals was seen in 1 out of 20 patients . all patients had proof of recent streptococcal infection in serology , either in aso and/or in antidnaseb . none of the patients presented with a major jones ' criterion other than arthritis , nor fulfilled jones ' criteria on arf . by definition all gas - related cases presented with a low aso / antidnaseb ratio . altogether 95% of gas - induced psra patients fulfilled the criteria a + c + d + e + f , by including criterion b this percentage dropped to 50% . when criterion b is excluded , 75% of ngas - induced patients meet criteria set a + c + d + e. in the 9 non - psra cases criteria set a + c + d + e + f is met in 33% still . in 45 patients with acute arthritis consisting of gas - induced psra in 20 , ngas - induced psra in 16 , and other cases in 9 , the following test characteristics for finding gas - induced psra are calculated : sensitivity ( se ) , specificity ( sp ) , predictive value of a positive test result , that is , positive predictive value ( ppv ) , predictive value of a negative test result , that is , negative predictive value ( npv ) with 95% confidence intervals ( 95%ci ) . also likelihood ratio of positive test ( lr+ ) and likelihood ratio of negative test ( lr ) are calculated . prevalence of index cases , that is , gas - induced psra in this population is 44% . only 50% of patients meet a + b + c + d + e , which represents se , 76% sp , with predictive values : 62.5% ppv and 65.5% npv , and likelihood ratio for a positive test 2.1 lr+ . by adding additional criterion f specificity increases to 88% and lr+ increases to 4.2 . by eliminating criterion b a significant improvement is obtained of sensitivity and likelihood ratios : lr+ 7.9 and lr0.06 . the additional serological criteria ( f ) or ( g ) give similar results regarding their additive diagnostic value . for further data spearman 's rho in the 36 psra patients between period arthritis persists , mean ( sd ) 17 ( 35 ) weeks and swollen joint count ( sjc ) 3.5 ( 4.5 ) joints , is 0.55 ( p < .001 ) . in the 20 gas - induced cases spearman 's correlation between period of persisting arthritis and sjc is 0.66 ( p < .005 ) , versus 0.26 ( p > .05 ) in ngas - induced cases . other correlations are insignificant between the period arthritis persists on the one hand and erythrocyte sedimentation rate(esr)/c - reactive protein(crp ) on the other . lancefield group a streptococci ( gas ) [ 25 ] , but also nongroup a streptococci , group c and group g , are supposed to be capable to evoke reactive arthritis , though proof is lacking in literature . post - streptococcal reactive arthritis ( psra ) due to gas may well be considered part of the spectrum or possibly a forme fruste of acute rheumatic fever ( arf ) which currently seems to have vanished from welfaring communities . the clinical significance of a more benign psra secondary to ngas remains unclear : it is not known whether carditis and/or chorea risks in ngas - induced psra are similar to the risks in patients who developed full - blown rheumatic fever . it is a challenge for clinicians to separate these entities in a prospective manner . previously arf and psra have been differentiated and correctly classified by paediaticians applying simple clinical and laboratory variables . in this paper similar variables are used to define clusters of criteria to differentiate subtypes of psra , which enables homogeneity of studied cohorts in future . of course some circularity in reasoning can not be ruled out completely as most patients referred have already been diagnosed using serological criteria which are generally applied in the netherlands due to previous publications [ 4 , 5 , 10 ] . a resurgence over the last decades of other post - streptococcal syndromes has been related to the spread of different possibly more virulent clones of streptococci , higher number of patients with conditions interfering with immunity , and alterations in patterns of child care . in our region of the netherlands we find a low estimated annual incidence for psra , only 0.70 per 100 000 inhabitants for gas - induced psra . despite the resurgence of post - streptococcal syndromes as claimed , these low incidence rates on gas - induced psra stress the importance of multicentre cooperation in order to increase insight into risk profiles for developing severe sequelae such as carditis , and may increase our insight into long term prognosis . next to pyogenic sequelae , gas infections are known for their molecular mimicry resulting in non - pyogenic , sterile but potentially devastating sequelae such as in acute rheumatic fever ( arf ) . patients may also develop a more benign manifestation of post - streptococcal reactive arthritis ( psra ) , particularly when caused by nongroup a streptococci ( ngas ) a predictor for benign courses . in children probably more than in adults , gas - induced psra should be considered a potential predecessor of rheumatic heart disease , which is reflected in penicillin prophylaxis regimens . currently , clinicians should be capable to separate gas - induced psra from ngas - induced psra , and this may be relevant as presently more specific risk factors for developing a subsequent carditis are lacking . a recent analysis of psra cases from literature revealed a significant clinical heterogeneity of the term psra . prior to an evidence - based advice of therapy clarity is needed of the clinical entity of the psra diagnosis . only a weak genetic difference between patients with arf and psra has been demonstrated : arf being possibly associated with hla drb1 * 16 , and psra with hla drb1 * 01 . it is therefore doubtful whether genetics are responsible for an individual 's genetic susceptibility for developing the type of post - streptococcal syndrome . humoral hyperresponsiveness then might be hypothesized to explain the specificity of response following streptococcal infection . a high frequency of in vitro elevated d8/17 binding to b lymphocytes has been found as a susceptibility marker for developing arf more than psra [ 7 , 8 , 24 ] . another explanation could be in an individual 's defense , for example , dependent on toll - like receptors . next to host factors , microbacterial factors may be important : in gas - induced psra so far , the m - serotypes 9 , 11 , and 28 have been found , and these are m - serotypes not known from acute rheumatic fever . there is a need for criteria considering the clinical heterogeneity in the psra diagnosis in current literature . more homogeneity may result form an additional serological criterion of high aso titres with lower aso / antidnaseb ratios , to exclude nongroup a streptococci . already in 1997 ayoub and ahmed proposed a set of criteria for psra . however , some of these can not be applied at the patient 's presentation , which thwarts diagnostic applicability . in the present study , a retrospective evaluation of the potential clinical applicability of these criteria half of the presented gas - induced psra patients , however , do not fulfil the previously proposed criteria by ayoub and ahmed on psra . particularly the criterion of a protracted course of arthritis is met in only half of presumably gas - induced psra patients . it is unclear yet whether this gas - related psra patient group without protracted arthritis bears a higher risk than the other psra subgroups to develop , for example , carditis . on the other hand , one can not apply this feature for diagnosing purposes at presentation as clearly a certain delay in time is needed to meet this criterion . interestingly , a protracted course was not present in any of our ngas - induced patients , diagnosed by serological data . epidemiologically , in adulthood psra due to gas is estimated to occur in 0.7 per 100 000 inhabitants . there may be some underdiagnosis as clearly not all suspected patients will have been referred to our department . in childhood these epidemiologic data may be quite different , probably higher . epidemiological reasoning to prove that psra really exists can not be done by using the presented data . immunologically , the classic paradigm for molecular mimicry in gas - induced psra is similar as in acute rheumatic fever after infection with gas , underscoring the existence of psra . however , the presence of antibodies to an antigen in the membrane of the organism , the type 5 streptococcal m protein cross - reacting with tissue , has not been convincingly demonstrated in psra . the assumed causal role of streptococcal infection therefore remains to be demonstrated pathogenetically . ayoub and ahmed have originally proposed criteria that are applicable only in full - blown clinical situations , not specifically for diagnostic purposes from the onset ; as these appear to exclude half of our presumably gas - induced psra patients . an additional serological criterion , that is , either the elevated antideoxyribonuclease - b or the lower antistreptolysin - o / antideoxyribonuclease - b ratio ( < 1.4 ) , as described previously , may be applied elegantly and leads to an improved criteria set in our presented population . also the criterion on a protracted course , resulting in excluding all ngas - induced cases , may become abundant . in order to homogenize the psra patient group one might study : ( a ) acute onset and nonmigratory type of arthritis , ( c ) poor responsiveness of arthritis to salicylates / nonsteroidals , ( d ) evidence of antecedent streptococcal infection , ( e ) not fulfilling modified jones criteria , plus ( f ) antistreptolysin - o / antidnaseb ratio < 1.4 , or ( g ) antidnase - b > 400 u / l . prospective studies in other gas - induced psra patient groups are needed for validation and proving their prognostic value . for purposes of homogeneity in studies the aforementioned criteria may have some merits . for practical purposes in an individual during the diagnostic phase a serological criterion plus ( a ) and ( c ) seem to be quite applicable .

with rising life expectancy , the number of elderly persons in developed communities increases . according to the world factbook , life expectancy for those born in 2013 is 84.19 years for the japanese and 80.32 years for the german population . diseases and injuries of the musculoskeletal system specific to the aging population will become even more frequent [ 2 , 3 ] . particularly in elderly females , osteoporosis represents a very common medical condition and a significant health care problem with a growing incidence of osteoporotic fractures most typically occurring in the hip and spine . clinical pathways characterized by multidisciplinarity have been recently re - worked to cope with problems specific to these patients and to secure their best possible outcome . their clinical picture , radiological morphology and intrinsic instability range widely from nearly stable to completely unstable conditions . although the number of case studies and publications on this topic in the current literature is incremental , there is no consensus about the optimal treatment strategy for these lesions . in this paper , we review literature data on the management of fragility pelvic fractures and compare them with our recommendations that are based on a previously published classification . we reviewed 245 patients with fragility fractures of the pelvic ring who received in - patient treatment in our department between 2007 and 2012 . included were only patients older than 65 years of age without any history of cancer . patients with acetabular fractures were also excluded . in a few immobilized patients , even patient transfer from a bed to a chair was sufficient to cause a fragility fracture . at admission , the patients suffered from severe , immobilizing pain in the groin or at the pubic region . some of these patients also suffered from deep pain in the lower back or in the sacral region . 1a c ) : an anteroposterior ( ap ) ; the pelvic inlet view ; and the pelvic outlet view . on the ap view of the pelvis , fractures of the superior and inferior pubic rami or the pubic bone near the symphysis are often visible . in case of lateral impact , the fracture line at the superior pubic ramus runs horizontally and there is a slight overriding of the fracture fragments ; the lateral fracture fragment is displaced medially . in case of an ap or posteroanterior impact , the fracture lines run vertically through the pubic bone or through the obturator foramen . the pelvic inlet view gives a good impression of the amount and direction of the fracture fragment displacement in the anterior pelvic ring . in this projection , the inner curve of the innominate bone and the anterior cortex of the sacrum are also well visible . the pelvic outlet view , especially , gives information about the dorsal pelvis , the shape and symmetry of the sacrum , the neuroforamina and the sacroiliac joints . assessment of the dorsal pelvic ring on conventional pelvic overviews is difficult in these patients because of disturbing bowel and bladder content . further , interruptions within the bony structures of the dorsal pelvis are less clearly visible due to fragile bones with rarefaction of spongious and cortical structures . in case of an anterior pelvic fracture , we therefore recommend to always perform a computed tomography ( ct)-scan of the whole pelvis . this precludes lesions of the dorsal pelvic ring being overlooked and the degree of instability consequently underestimated . multiplanar reconstructions of the ct data help us to fully appreciate the fracture configuration of the dorsal pelvis . in the coronal reconstructions , a complete fracture of the lateral mass of the sacrum is sometimes better visible than in transverse sections . a horizontal component of sacral fractures , if present , even with a slight angulation , can be recognized best in sagittal reconstructions.fig . 1 a pelvic ap overview of type ia ffp in an 82-year old woman . there is slight overriding of the fracture fragments at the right superior pubic ramus ; b pelvic inlet overview . a slight internal rotation of the right hemipelvis at the fracture of the right anterior pelvic ring is visible ; c pelvic outlet overview . there is no lesion of the posterior pelvic ring a pelvic ap overview of type ia ffp in an 82-year old woman . there is slight overriding of the fracture fragments at the right superior pubic ramus ; b pelvic inlet overview . a slight internal rotation of the right hemipelvis at the fracture of the right anterior pelvic ring is visible ; c pelvic outlet overview . there is no lesion of the posterior pelvic ring when analyzing the conventional radiographs and the ct - data of 245 patients , we detected a spectrum of different fracture morphologies . the fractures may involve the anterior pelvic ring only , the posterior pelvic ring only or a combination of both . anteriorly , the fractures run through the superior and inferior pubic rami , the pubic bone in the proximity of the symphysis or more seldom there is a disruption of the symphysis itself . in the dorsal pelvis , the instability may run through the ilium , through the lateral mass of the sacrum or through the sacroiliac joint . a new , comprehensive classification system was established , distinguishing the fracture morphology and degree of instability ( fig . the absolute number of these lesions in our series is presented in brackets ( n =) after each fracture type.fig . e ffp type iic : non - displaced sacral , sacroiliac or iliac fracture with anterior disruption . i ffp type iva : bilateral iliac fractures or bilateral sacroiliac disruptions together with anterior disruption . k ffp type ivc : combination of different posterior instabilities together with anterior disruption classification of fragility fractures of the pelvis . e ffp type iic : non - displaced sacral , sacroiliac or iliac fracture with anterior disruption . i ffp type iva : bilateral iliac fractures or bilateral sacroiliac disruptions together with anterior disruption . k ffp type ivc : combination of different posterior instabilities together with anterior disruption ffp type i lesions include anterior pelvic ring fractures only ; ffp type ia is a unilateral anterior lesion and ffp type ib is a bilateral anterior lesion . bilateral isolated anterior lesions were very rare in our series ( n = 1 ) ; unilateral lesions are much more common ( n = 43 ) , but they were far from comprising the majority of all ffp ( 43/245 = 17.5% ) . this data supports the need for ct - evaluation of all low - energy pelvic ring lesions with pubic rami fractures ; whenever an anterior pelvic ring lesion is present , there is a high risk of a concomitant posterior ring lesion often missed on conventional radiographs . ffp type ii lesions are non - displaced posterior lesions ; ffp type iia is a non - displaced posterior lesion only ( n = 3 ) ; ffp type iib is a sacral crush with anterior disruption ( n = 59 ) ; and ffp type iic is a non - displaced sacral , sacroiliac or iliac fracture with anterior disruption ( n = 65 ) . together , ffp type iib and ffp type iic lesions formed half of all ffp lesions in our series ( 124/245 = 50.6% ) . ffp type iii lesions are characterized by a displaced unilateral posterior injury combined with an anterior pelvic ring lesion . ffp type iiia involves a displaced unilateral ilium fracture ( n = 20 ) , ffp type iiib is a displaced unilateral sacroiliac disruption ( n = 4 ) and ffp type iiic is a displaced unilateral sacral fracture ( n = 3 ) . non - displaced unilateral posterior lesions ( all ffp types ii , n = 127 ) were much more common in our series than displaced unilateral ones ( all ffp types iii , n = 27 ) , the ffp type ii versus ffp type iii ratio being 4.7:1 . ffp type iva have bilateral iliac fractures or bilateral sacroiliac disruptions ( n = 2 ) . ffp type ivb is characterized by a spinopelvic dissociation containing a bilateral vertical fracture through the lateral mass of the sacrum with a horizontal component connecting them ( n = 37 ) . ffp type ivc is a combination of different posterior instabilities ( n = 8) . the frequency of spinopelvic dissociations in this series was striking , although not always visible on the conventional radiographs ( 37/245 = 15.1 % ) . this underlines the importance of two - dimensional ct - reconstructions ; only in the sagittal reconstruction of the sacrum can the horizontal component of an h - type sacrum fracture be identified . . described typical anatomical patterns in insufficiency sacrum fractures and empasised that the h - type fracture pattern is not uncommon , with 61% of isolated sacral insufficiency fractures . this comprehensive classification describes all different fracture morphologies and also categorizes the lesions into different degrees of instability . isolated anterior lesions ( ffp type i ) are more stable than non - displaced posterior lesions ( ffp type ii ) . displaced posterior lesions are less stable than non - displaced ones , and bilateral posterior lesions ( ffp type iv ) are less stable than unilateral ones ( ffp type iii ) . depending on the clinical presentation of the patient at initial presentation and his or her fracture type , different treatment strategies will have to be chosen . depending on the healthcare system , diagnosis and treatment of anti - osteoporosis medication is started or continued . studies have shown low rates of follow - up for osteoporosis diagnostics after fragility fractures . the orthopedic trauma surgeon treating the pelvic fragility fracture plays an important role in initiating anti - osteoporotic management . diagnostic work - up on the state of bone metabolism should follow after fracture treatment . management consists of life style changes , fall prevention , vitamin d , calcium supplementation and antiresorptive drugs . promising results of enhanced fracture healing in ffp via administering the anabolic agent parathyroid hormone were published recently [ 13 , 14 ] . the latter will not contribute to fracture healing in the acute setting , but they may avoid recurrent fragility fractures in the same or other skeletal regions . after a few days or a week of bed rest and with significant pain relief , careful mobilization is started . we hypothesize that inadequate , premature and aggressive mobilization may lead to additional trauma of the weak bony structures with more complex and more unstable fracture types as a consequence . mobilization is done in the presence of physiotherapists . in the meantime , a high index of suspicion for the appearance of additional , secondary lesions must be present . in case pain intensity and pain frequency do not decrease , or even increase , after days or weeks , we recommended repeating the ct - scan evaluation in order to rule out fracture types of higher instability , or performing magnetic resonance imaging ( mri ) to rule out occult sacral fractures . the patient should be seen regularly on an outpatient basis until ( radiographic ) evidence of fracture healing and relief from complaints . the fracture is situated in the lateral mass , lateral to the neuroforamina and medial to the sacroiliac joint . in an osteoporotic anatomical specimen , alar void was shown in the lateral mass of the sacrum ( fig . patients with ffp type ii lesions present with pain in the dorsal pelvis and also experience pain in the groin in cases of pubic rami fractures . due to acute and intense pain , the patients can hardly be mobilized within the first days after admission . if no pain relief is observed within days with adequate pain medication and mobilization remains impossible , surgical stabilization of the dorsal fracture should be considered . in ffp type alternatives for invasive treatment are sacroiliac screw osteosynthesis , sacroplasty , bridging plate osteosynthesis or insertion of a transsacral positioning bar.fig . zones of low bone density in both sacral alae and a fracture of the left sacral ala are visible transverse ct cut through dorsal pelvis of 83-year old female . zones of low bone density in both sacral alae and a fracture of the left sacral ala are visible sacroiliac screw osteosynthesis is a well - known procedure in dorsal sacral instabilities after high - velocity trauma . two large fragment screws are inserted into the body of s1 or one screw into the body of s1 and s2 each ( fig . consequently , there is a higher risk of screw loosening because of a lower pull - out force of these screws . however , there is little experience using these techniques and information regarding clinical outcome is limited . adequate instruments and implants are scarcely available .fig . 4 a pelvic ap overview of 72-year old female with anterior and posterior intervertebral fusion between l4 and s1 . the patient had a history of more than three months of severe pain after a fall at home . a bilateral fracture of the pubic rami is visible with sclerotic margins , demonstrating a non - union . a fracture of the dorsal pelvic ring is not clear ; b axial ct reconstruction shows a bilateral fracture of the sacral ala at the s1 level ; c coronal reconstruction gives another view of the bilateral sacral ala lesions . this is an unstable lesion of the pelvic ring classified as ffp type ivb ; d pelvic ap overview five months after operative reconstruction . insertion of a transsacral bar was not possible due to the pedicular screws in s1 . the bilateral anterior instability was bridged with a long plate and screws construct ; e pelvic inlet overview ; f pelvic outlet overview . note the long screws into the posterior column providing stability in this osteoporotic bone . the patient is free of complaints in the pelvic region and able to walk without crutches a pelvic ap overview of 72-year old female with anterior and posterior intervertebral fusion between l4 and s1 . the patient had a history of more than three months of severe pain after a fall at home . a bilateral fracture of the pubic rami is visible with sclerotic margins , demonstrating a non - union . a fracture of the dorsal pelvic ring is not clear ; b axial ct reconstruction shows a bilateral fracture of the sacral ala at the s1 level ; c coronal reconstruction gives another view of the bilateral sacral ala lesions . this is an unstable lesion of the pelvic ring classified as ffp type ivb ; d pelvic ap overview five months after operative reconstruction . insertion of a transsacral bar was not possible due to the pedicular screws in s1 . the bilateral anterior instability was bridged with a long plate and screws construct ; e pelvic inlet overview ; f pelvic outlet overview . note the long screws into the posterior column providing stability in this osteoporotic bone . the patient is free of complaints in the pelvic region and able to walk without crutches sacroplasty has been recommended as an alternative treatment option for sacral insufficiency fractures in osteoporotic bone [ 2123 ] . here , a small portion of bone cement is injected into the fracture area through a long needle . similar to kyphoplasty , this technique provides quick pain relief and early mobilization is possible . biomechanically , there are important differences between vertebral kyphoplasty and sacroplasty . in osteoporotic vertebral fractures , the plane of the fracture is horizontal , whereas in sacral fragility fractures , it is vertical . in standing position , axial loading is perpendicular to the fracture plane of a vertebral fracture , but parallel to the plane of the sacral fracture . thus , on the longer term , these patients will be prone to treatment failure due to the high likelihood of fracture displacement . in cases of recurrent fractures and consecutive revision surgery , internal fixation of the sacrum therefore , the authors can not recommend sacroplasty for the treatment of ffp . for bridging plate osteosynthesis , a pre - contoured long plate connects both dorsal iliac crests and is curved around the posterior inferior iliac spines . several angular stable screws are inserted into the dorsal ilium through the plate holes on each side . the plate construct does not give absolute stability ; it only bridges the fracture area , but does not realize any compression in the fracture side . we consider this technique of osteosynthesis less suitable for the stabilization of insufficiency fractures of the sacrum . , a solid bar with a diameter of 6 mm is placed horizontally in a coronal plane through the body of s1 , alternatively through s2 , from one ilium to the opposite . the procedure bears the same risks as sacroiliac screw placement , such as perforation of the anterior cortex of the sacrum and damage to the cauda equine , nerve roots and vessels . the morphology of the sacrum is highly variable and , in some patients , there is no safe transsacral corridor through s1 . in contrast , although smaller , the transsacral corridor in s2 seems to be constantly available . the correct entry portal for the insertion of the bar is determined in a perfect lateral view of the sacrum at the level of s1 and s2 . by tightening the nuts , a compressive force , which is perpendicular to the plane of the sacral fracture , the amount of compression is not dependent on the strength of the spongious bone in the body of s1 , as is the case in sacroiliac screw osteosynthesis , but on the strength of the cortical bone at the dorsal ilium . when the nut and washer threatens to perforate through the very weak dorsal ilium cortex while tightening , we replace the usual washer by a larger one ( e.g. , a small conventional plate ) . by using a larger washer , we distribute the contact pressure between the washer and the cortex over a larger surface . an additional s1 screw can be inserted at the unstable side to minimize rotational instability in the dorsal pelvis ( fig . 5a h ) . a small series of 11 patients treated with this technique has been published with excellent to good results by mehling et al . . loosening , pull - out or cut - out of the transsacral positioning bar was not observed in this case series.fig . a clear lesion of the dorsal pelvis is not visible ; b pelvic inlet view ; c pelvic outlet view . instability of the symphysis pubis is visible as a step - off ; d coronal ct reconstruction of the dorsal pelvis . there is an undisplaced , yet complete , fracture of the left massa lateralis of the sacrum ; e the fracture of the left lateral mass of the sacrum runs down through the neuroforamina s1 and s2 ; f postoperative pelvic ap view . the instability of the symphysis pubis has been fixed with a double plate osteosynthesis and long screws into the posterior column , the anterior plate being angular stable ; g postoperative pelvic inlet view ; h postoperative pelvic outlet view . three months after surgery , the patient was walking independently without complaints at the pelvis seventy - two - year old female with an ffp iic lesion . a pelvic ap overview . a clear lesion of the dorsal pelvis is not visible ; b pelvic inlet view ; c pelvic outlet view . instability of the symphysis pubis is visible as a step - off ; d coronal ct reconstruction of the dorsal pelvis . there is an undisplaced , yet complete , fracture of the left massa lateralis of the sacrum ; e the fracture of the left lateral mass of the sacrum runs down through the neuroforamina s1 and s2 ; f postoperative pelvic ap view . a sacral bar has been placed through the body of s1 . on the left side , an additional sacroiliac screw has been placed . the instability of the symphysis pubis has been fixed with a double plate osteosynthesis and long screws into the posterior column , the anterior plate being angular stable ; g postoperative pelvic inlet view ; h postoperative pelvic outlet view . three months after surgery , the patient was walking independently without complaints at the pelvis non - displaced superior pubic rami fractures are splinted in a retrograde manner with a large fragment screw inserted through a small skin incision from the pubic bone near to the symphysis ( fig . , reduction can be achieved through a small suprapubic midline incision prior to screw insertion . in case of symphysis pubis instability or a fracture very near to the joint , a bridging angular stable plate osteosynthesis has to be considered ( fig . 4a h ) . in most patients with long history of pain in the pubic region , a very unstable condition with osteoporotic bone was found intraoperatively in our case series . therefore , a long bridging plate with long screws placed through the infra - acetabular corridor using the modified stoppa - approach on both sides is preferred to achieve high stability of the construct ( figs . 4d f , 5f h , 7b d).fig . 6 a seventy - four - year old female with an ffp type iiia lesion . a complete right - sided crescent fracture of the ilium and superior and inferior pubic ramus fractures with vertical displacement are visible in the pelvic a.p . view ; b pelvic inlet view ; c pelvic outlet view ; d postoperative pelvic a.p . view . the ilium fracture has been fixed with a large fragment angular stable plate and two long lag screws . the pubic ramus fracture has been splinted with a minimally invasive retrograde transpubic screw ; e postoperative pelvic inlet view ; f postoperative pelvic outlet viewfig . 7 a surface rendering of the pelvic bone in an 87-year old female after a fall at home . an incomplete fracture of the dorsal ilium , a displaced fracture of the superior pubis ramus and an undisplaced inferior pubic ramus fracture are visible . the very low bone density in both sacral ala and in the center of the iliac wing are clearly visible . the lesion is classified as an ffp type iic ; b postoperative pelvic a.p . overview . the incomplete ilium fracture has been fixed with a single screw , the anterior instability with a bridging plate and screw osteosynthesis . to avoid loosening of the screws , an we chose the longest possible screw trajectory in the pubic bone and ischium ; c postoperative pelvic inlet overview ; d postoperative pelvic outlet overview . six months after surgery , the patient walked independently and without complaints a seventy - four - year old female with an ffp type iiia lesion . a complete right - sided crescent fracture of the ilium and superior and inferior pubic ramus fractures with vertical displacement are visible in the pelvic a.p . view ; b pelvic inlet view ; c pelvic outlet view ; d postoperative pelvic a.p . view . the ilium fracture has been fixed with a large fragment angular stable plate and two long lag screws . the pubic ramus fracture has been splinted with a minimally invasive retrograde transpubic screw ; e postoperative pelvic inlet view ; f postoperative pelvic outlet view a surface rendering of the pelvic bone in an 87-year old female after a fall at home . an incomplete fracture of the dorsal ilium , a displaced fracture of the superior pubis ramus and an undisplaced inferior pubic ramus fracture are visible . the very low bone density in both sacral ala and in the center of the iliac wing are clearly visible . the lesion is classified as an ffp type iic ; b postoperative pelvic a.p . overview . the incomplete ilium fracture has been fixed with a single screw , the anterior instability with a bridging plate and screw osteosynthesis . to avoid loosening of the screws , an we chose the longest possible screw trajectory in the pubic bone and ischium ; c postoperative pelvic inlet overview ; d postoperative pelvic outlet overview . six months after surgery , the patient walked independently and without complaints in contrast to other authors , pelvic external fixation for anterior stabilization is not recommended in fragility fractures . there is a high risk of loosening and pin track infection and , always critical , a lack of patient comfort . internal fixators placed between both anterior superior or inferior iliac spines have been presented in recent literature . although they can be inserted in a minimally invasive manner , there is a high risk of damage to the lateral cutaneous femoral nerve and occurrence of heterotopic ossification . the implantation is in the proximity of the femoral vessels and nerve ; furthermore , there is a little distance between the skin and screw head . additionally , the implant must be removed in most cases , which involves a second operative procedure . displaced fragility fractures of the pelvic ring ( ffp types iii and iv ) necessitate a more aggressive surgical approach . as closed reduction is not possible , open reduction and internal fixation will be mandatory . after open reduction of displaced sacral fractures or sacroiliac instabilities , similar techniques for stabilization can be used , as previously described , for the stabilization of non - displaced sacral fractures . in chronic cases , i.e. , after failure of conservative treatment , gross instability is often combined with joint widening or bone loss . in those cases , we prefer performing a sacroiliac arthrodesis , as we estimate excessive risk of non - union in simple osteosynthesis . an anterior approach to the sacroiliac joint through the first window of the ilioinguinal approach is chosen . the joint is debrided and filled with cortico - spongious bone grafts of the ipsilateral iliac crest . two short plates are inserted over the sacroiliac joint at an angle of 6090 to each other . one large fragment cancellous bone screw is inserted in the sacrum parallel to the joint and one or two screws are placed in the ilium . sacroiliac screws or a transsacral positioning bar can be placed additionally to enhance stability . in case of transiliac instabilities , the fracture runs from the inner curve of the ilium proximally and laterally . for stabilization , we prefer insertion of an angular stable plate placed parallel to the sacroiliac joint and the pelvic brim , bridging the medial edge of the fracture . as in other skeletal regions , angular stable plates have higher pull - out forces ; therefore , there is a lower risk of loosening . the angular stable plate has to be pre - shaped and twisted to fit on the innominate bone as best as possible . the proximal screws should be directed parallel to the sacroiliac joint , the distal screws in the sagittal plane or slightly towards lateral ( fig . the ilium fracture running proximally and laterally ends at the iliac crest and is stabilized there with one long screw , which is inserted perpendicular to the fracture plane and between the inner and outer cortex of the crest ( fig . , a small fragment plate is placed over the iliac crest . in ffp type iv fractures , there is bilateral dorsal instability with displacement . in bilateral fractures of the ilium , bilateral angular stable plate osteosynthesis is done in the same technique as in unilateral transiliac instabilities . in minimally - displaced bilateral fractures of the lateral mass of the sacrum the bilateral sacral fractures are compressed when the washers and nuts on both ends of the bar are pressed against the lateral cortex of the dorsal ilium . for absolute rotational stability this can only be done in cases where a transsacral corridor large enough for additional screw placement is available ( fig . bilateral sacroiliac screws as the only measure of stabilization are not reliable , as there is a high risk of loosening due to poor bone stock . iliolumbar fixation , which involves a much more prolonged surgery with bridging of the lumbosacral junction , is preserved for cases with gross instability or for patients in which a safe transsacral corridor is not available . transpedicular screws are placed bilaterally in the l4 and l5 pedicles and in the dorsal ilium . the screw in the ilium is inserted between the two cortices in the direction of the greater sciatic notch . the pedicle screws are connected with a bar on each side and the two bars are connected with a transverse connector . iliolumbar fixation prevents further intrusion of the lumbosacral segment into the pelvic ring . in very unstable situations , the lumbosacral segment seems displaced distally when related to the pelvic ring ; d axial ct - reconstruction of the dorsal pelvis showing a severe rarefication of the bone substance and a complete bilateral fragility fracture of the lateral mass of the sacrum ; e coronal ct - reconstruction confirms the complete bilateral fragility fractures of the lateral mass of the sacrum ; f postoperative pelvic a.p . overview . small plates were used as washers under the nuts of the sacral bar to hinder cut - through of the washers ; g postoperative pelvic inlet view ; h postoperative pelvic outlet view ; i a representative axial ct - cut one year after surgery , showing complete healing of the sacral fragility fractures ; j a representative coronal ct - cut showing the position of the transpedicular screws and the transsacral positioning bar . the lumbosacral segment seems displaced distally when related to the pelvic ring ; d axial ct - reconstruction of the dorsal pelvis showing a severe rarefication of the bone substance and a complete bilateral fragility fracture of the lateral mass of the sacrum ; e coronal ct - reconstruction confirms the complete bilateral fragility fractures of the lateral mass of the sacrum ; f postoperative pelvic a.p . overview . small plates were used as washers under the nuts of the sacral bar to hinder cut - through of the washers ; g postoperative pelvic inlet view ; h postoperative pelvic outlet view ; i a representative axial ct - cut one year after surgery , showing complete healing of the sacral fragility fractures ; j a representative coronal ct - cut showing the position of the transpedicular screws and the transsacral positioning bar . the patient is able to stand with auxiliary means patients receiving conservative treatment should be mobilized and bear weight , as tolerated . physiotherapy should be adapted to the functional status of the patient before the fragility fracture occurred and certainly not enforced . too aggressive mobilization may lead to additional fragility fractures in the pelvic ring with enhanced instability . short transfers and sitting in a wheelchair can be allowed , but long standing and walking with full weight bearing is not recommended . due to low bone strength , there is a risk of loosening of the implants with delayed healing , non - union and revision surgery . usually , after six weeks , conventional radiographs will confirm ongoing bone healing without implant migration . 1a c ) : an anteroposterior ( ap ) ; the pelvic inlet view ; and the pelvic outlet view . on the ap view of the pelvis , fractures of the superior and inferior pubic rami or the pubic bone near the symphysis are often visible . in case of lateral impact , the fracture line at the superior pubic ramus runs horizontally and there is a slight overriding of the fracture fragments ; the lateral fracture fragment is displaced medially . in case of an ap or posteroanterior impact , the fracture lines run vertically through the pubic bone or through the obturator foramen . there is no overriding , sometimes even a slight diastasis . the pelvic inlet view gives a good impression of the amount and direction of the fracture fragment displacement in the anterior pelvic ring . in this projection , the inner curve of the innominate bone and the anterior cortex of the sacrum are also well visible . the pelvic outlet view , especially , gives information about the dorsal pelvis , the shape and symmetry of the sacrum , the neuroforamina and the sacroiliac joints . assessment of the dorsal pelvic ring on conventional pelvic overviews is difficult in these patients because of disturbing bowel and bladder content . further , interruptions within the bony structures of the dorsal pelvis are less clearly visible due to fragile bones with rarefaction of spongious and cortical structures . in case of an anterior pelvic fracture , we therefore recommend to always perform a computed tomography ( ct)-scan of the whole pelvis . this precludes lesions of the dorsal pelvic ring being overlooked and the degree of instability consequently underestimated . multiplanar reconstructions of the ct data help us to fully appreciate the fracture configuration of the dorsal pelvis . in the coronal reconstructions , a complete fracture of the lateral mass of the sacrum is sometimes better visible than in transverse sections . a horizontal component of sacral fractures , if present , even with a slight angulation , can be recognized best in sagittal reconstructions.fig . 1 a pelvic ap overview of type ia ffp in an 82-year old woman . there is slight overriding of the fracture fragments at the right superior pubic ramus ; b pelvic inlet overview . a slight internal rotation of the right hemipelvis at the fracture of the right anterior pelvic ring is visible ; c pelvic outlet overview . there is no lesion of the posterior pelvic ring a pelvic ap overview of type ia ffp in an 82-year old woman . there is slight overriding of the fracture fragments at the right superior pubic ramus ; b pelvic inlet overview . a slight internal rotation of the right hemipelvis at the fracture of the right anterior pelvic ring is visible ; c pelvic outlet overview . when analyzing the conventional radiographs and the ct - data of 245 patients , we detected a spectrum of different fracture morphologies . the fractures may involve the anterior pelvic ring only , the posterior pelvic ring only or a combination of both . anteriorly , the fractures run through the superior and inferior pubic rami , the pubic bone in the proximity of the symphysis or more seldom there is a disruption of the symphysis itself . in the dorsal pelvis , the instability may run through the ilium , through the lateral mass of the sacrum or through the sacroiliac joint . a new , comprehensive classification system was established , distinguishing the fracture morphology and degree of instability ( fig . the absolute number of these lesions in our series is presented in brackets ( n =) after each fracture type.fig . 2classification of fragility fractures of the pelvis . a ffp type ia : unilateral anterior pelvic ring disruption . c ffp type iia : dorsal non - displaced posterior injury only . d ffp type iib : sacral crush with anterior disruption . e ffp type iic : non - displaced sacral , sacroiliac or iliac fracture with anterior disruption . i ffp type iva : bilateral iliac fractures or bilateral sacroiliac disruptions together with anterior disruption . k ffp type ivc : combination of different posterior instabilities together with anterior disruption classification of fragility fractures of the pelvis . e ffp type iic : non - displaced sacral , sacroiliac or iliac fracture with anterior disruption . i ffp type iva : bilateral iliac fractures or bilateral sacroiliac disruptions together with anterior disruption . k ffp type ivc : combination of different posterior instabilities together with anterior disruption ffp type i lesions include anterior pelvic ring fractures only ; ffp type ia is a unilateral anterior lesion and ffp type ib is a bilateral anterior lesion . bilateral isolated anterior lesions were very rare in our series ( n = 1 ) ; unilateral lesions are much more common ( n = 43 ) , but they were far from comprising the majority of all ffp ( 43/245 = 17.5% ) . this data supports the need for ct - evaluation of all low - energy pelvic ring lesions with pubic rami fractures ; whenever an anterior pelvic ring lesion is present , there is a high risk of a concomitant posterior ring lesion often missed on conventional radiographs . ffp type ii lesions are non - displaced posterior lesions ; ffp type iia is a non - displaced posterior lesion only ( n = 3 ) ; ffp type iib is a sacral crush with anterior disruption ( n = 59 ) ; and ffp type iic is a non - displaced sacral , sacroiliac or iliac fracture with anterior disruption ( n = 65 ) . together , ffp type iib and ffp type iic lesions formed half of all ffp lesions in our series ( 124/245 = 50.6% ) . ffp type iii lesions are characterized by a displaced unilateral posterior injury combined with an anterior pelvic ring lesion . ffp type iiia involves a displaced unilateral ilium fracture ( n = 20 ) , ffp type iiib is a displaced unilateral sacroiliac disruption ( n = 4 ) and ffp type iiic is a displaced unilateral sacral fracture ( n = 3 ) . non - displaced unilateral posterior lesions ( all ffp types ii , n = 127 ) were much more common in our series than displaced unilateral ones ( all ffp types iii , n = 27 ) , the ffp type ii versus ffp type iii ratio being 4.7:1 . ffp type iva have bilateral iliac fractures or bilateral sacroiliac disruptions ( n = 2 ) . ffp type ivb is characterized by a spinopelvic dissociation containing a bilateral vertical fracture through the lateral mass of the sacrum with a horizontal component connecting them ( n = 37 ) . ffp type ivc is a combination of different posterior instabilities ( n = 8) . the frequency of spinopelvic dissociations in this series was striking , although not always visible on the conventional radiographs ( 37/245 = 15.1 % ) . this underlines the importance of two - dimensional ct - reconstructions ; only in the sagittal reconstruction of the sacrum can the horizontal component of an h - type sacrum fracture be identified . . described typical anatomical patterns in insufficiency sacrum fractures and empasised that the h - type fracture pattern is not uncommon , with 61% of isolated sacral insufficiency fractures . this comprehensive classification describes all different fracture morphologies and also categorizes the lesions into different degrees of instability . isolated anterior lesions ( ffp type i ) are more stable than non - displaced posterior lesions ( ffp type ii ) . displaced posterior lesions are less stable than non - displaced ones , and bilateral posterior lesions ( ffp type iv ) are less stable than unilateral ones ( ffp type iii ) . depending on the clinical presentation of the patient at initial presentation and his or her fracture type , different treatment strategies will have to be chosen . all patients immediately require bed rest and painkillers . depending on the healthcare system , diagnosis and treatment of anti - osteoporosis medication is started or continued . studies have shown low rates of follow - up for osteoporosis diagnostics after fragility fractures . the orthopedic trauma surgeon treating the pelvic fragility fracture plays an important role in initiating anti - osteoporotic management . diagnostic work - up on the state of bone metabolism should follow after fracture treatment . management consists of life style changes , fall prevention , vitamin d , calcium supplementation and antiresorptive drugs . promising results of enhanced fracture healing in ffp via administering the anabolic agent parathyroid hormone were published recently [ 13 , 14 ] . the latter will not contribute to fracture healing in the acute setting , but they may avoid recurrent fragility fractures in the same or other skeletal regions . in ffp type i lesions , after a few days or a week of bed rest and with significant pain relief , careful mobilization is started . full - weight bearing as tolerated by the patient is allowed . however , forced mobilization must be avoided until radiographic evidence of fracture healing . we hypothesize that inadequate , premature and aggressive mobilization may lead to additional trauma of the weak bony structures with more complex and more unstable fracture types as a consequence . mobilization is done in the presence of physiotherapists . in the meantime , a high index of suspicion for the appearance of additional , secondary lesions must be present . in case pain intensity and pain frequency do not decrease , or even increase , after days or weeks , we recommended repeating the ct - scan evaluation in order to rule out fracture types of higher instability , or performing magnetic resonance imaging ( mri ) to rule out occult sacral fractures . the patient should be seen regularly on an outpatient basis until ( radiographic ) evidence of fracture healing and relief from complaints . the fracture is situated in the lateral mass , lateral to the neuroforamina and medial to the sacroiliac joint . in an osteoporotic anatomical specimen , an alar void was shown in the lateral mass of the sacrum ( fig . patients with ffp type ii lesions present with pain in the dorsal pelvis and also experience pain in the groin in cases of pubic rami fractures . due to acute and intense pain , if no pain relief is observed within days with adequate pain medication and mobilization remains impossible , surgical stabilization of the dorsal fracture should be considered . in ffp type ii lesions , alternatives for invasive treatment are sacroiliac screw osteosynthesis , sacroplasty , bridging plate osteosynthesis or insertion of a transsacral positioning bar.fig . zones of low bone density in both sacral alae and a fracture of the left sacral ala are visible transverse ct cut through dorsal pelvis of 83-year old female . zones of low bone density in both sacral alae and a fracture of the left sacral ala are visible sacroiliac screw osteosynthesis is a well - known procedure in dorsal sacral instabilities after high - velocity trauma . two large fragment screws are inserted into the body of s1 or one screw into the body of s1 and s2 each ( fig . consequently , there is a higher risk of screw loosening because of a lower pull - out force of these screws . however , there is little experience using these techniques and information regarding clinical outcome is limited . adequate instruments and implants are scarcely available .fig . 4 a pelvic ap overview of 72-year old female with anterior and posterior intervertebral fusion between l4 and s1 . the patient had a history of more than three months of severe pain after a fall at home . a bilateral fracture of the pubic rami is visible with sclerotic margins , demonstrating a non - union . a fracture of the dorsal pelvic ring is not clear ; b axial ct reconstruction shows a bilateral fracture of the sacral ala at the s1 level ; c coronal reconstruction gives another view of the bilateral sacral ala lesions . this is an unstable lesion of the pelvic ring classified as ffp type ivb ; d pelvic ap overview five months after operative reconstruction . insertion of a transsacral bar was not possible due to the pedicular screws in s1 . the bilateral anterior instability was bridged with a long plate and screws construct ; e pelvic inlet overview ; f pelvic outlet overview . note the long screws into the posterior column providing stability in this osteoporotic bone . the patient is free of complaints in the pelvic region and able to walk without crutches a pelvic ap overview of 72-year old female with anterior and posterior intervertebral fusion between l4 and s1 . the patient had a history of more than three months of severe pain after a fall at home . a bilateral fracture of the pubic rami is visible with sclerotic margins , demonstrating a non - union . a fracture of the dorsal pelvic ring is not clear ; b axial ct reconstruction shows a bilateral fracture of the sacral ala at the s1 level ; c coronal reconstruction gives another view of the bilateral sacral ala lesions . this is an unstable lesion of the pelvic ring classified as ffp type ivb ; d pelvic ap overview five months after operative reconstruction . insertion of a transsacral bar was not possible due to the pedicular screws in s1 . the bilateral anterior instability was bridged with a long plate and screws construct ; e pelvic inlet overview ; f pelvic outlet overview . note the long screws into the posterior column providing stability in this osteoporotic bone . the patient is free of complaints in the pelvic region and able to walk without crutches sacroplasty has been recommended as an alternative treatment option for sacral insufficiency fractures in osteoporotic bone [ 2123 ] . here , a small portion of bone cement is injected into the fracture area through a long needle . similar to kyphoplasty , this technique provides quick pain relief and early mobilization is possible . biomechanically , there are important differences between vertebral kyphoplasty and sacroplasty . in osteoporotic vertebral fractures , the plane of the fracture is horizontal , whereas in sacral fragility fractures , it is vertical . in standing position , axial loading is perpendicular to the fracture plane of a vertebral fracture , but parallel to the plane of the sacral fracture . thus , on the longer term , these patients will be prone to treatment failure due to the high likelihood of fracture displacement . in cases of recurrent fractures and consecutive revision surgery , internal fixation of the sacrum therefore , the authors can not recommend sacroplasty for the treatment of ffp . for bridging plate osteosynthesis a pre - contoured long plate connects both dorsal iliac crests and is curved around the posterior inferior iliac spines . several angular stable screws are inserted into the dorsal ilium through the plate holes on each side . the plate construct does not give absolute stability ; it only bridges the fracture area , but does not realize any compression in the fracture side . we consider this technique of osteosynthesis less suitable for the stabilization of insufficiency fractures of the sacrum . , a solid bar with a diameter of 6 mm is placed horizontally in a coronal plane through the body of s1 , alternatively through s2 , from one ilium to the opposite . the procedure bears the same risks as sacroiliac screw placement , such as perforation of the anterior cortex of the sacrum and damage to the cauda equine , nerve roots and vessels . the morphology of the sacrum is highly variable and , in some patients , there is no safe transsacral corridor through s1 . in contrast , although smaller , the transsacral corridor in s2 seems to be constantly available . the correct entry portal for the insertion of the bar is determined in a perfect lateral view of the sacrum at the level of s1 and s2 . by tightening the nuts , a compressive force , which is perpendicular to the plane of the sacral fracture , the amount of compression is not dependent on the strength of the spongious bone in the body of s1 , as is the case in sacroiliac screw osteosynthesis , but on the strength of the cortical bone at the dorsal ilium . when the nut and washer threatens to perforate through the very weak dorsal ilium cortex while tightening , we replace the usual washer by a larger one ( e.g. , a small conventional plate ) . by using a larger washer , we distribute the contact pressure between the washer and the cortex over a larger surface . an additional s1 screw can be inserted at the unstable side to minimize rotational instability in the dorsal pelvis ( fig . 5a h ) . a small series of 11 patients treated with this technique has been published with excellent to good results by mehling et al . . loosening , pull - out or cut - out of the transsacral positioning bar was not observed in this case series.fig . a clear lesion of the dorsal pelvis is not visible ; b pelvic inlet view ; c pelvic outlet view . instability of the symphysis pubis is visible as a step - off ; d coronal ct reconstruction of the dorsal pelvis . there is an undisplaced , yet complete , fracture of the left massa lateralis of the sacrum ; e the fracture of the left lateral mass of the sacrum runs down through the neuroforamina s1 and s2 ; f postoperative pelvic ap view . a sacral bar has been placed through the body of s1 . on the left side , the instability of the symphysis pubis has been fixed with a double plate osteosynthesis and long screws into the posterior column , the anterior plate being angular stable ; g postoperative pelvic inlet view ; h postoperative pelvic outlet view . three months after surgery , the patient was walking independently without complaints at the pelvis seventy - two - year old female with an ffp iic lesion . a pelvic ap overview a clear lesion of the dorsal pelvis is not visible ; b pelvic inlet view ; c pelvic outlet view . instability of the symphysis pubis is visible as a step - off ; d coronal ct reconstruction of the dorsal pelvis . there is an undisplaced , yet complete , fracture of the left massa lateralis of the sacrum ; e the fracture of the left lateral mass of the sacrum runs down through the neuroforamina s1 and s2 ; f postoperative pelvic ap view . a sacral bar has been placed through the body of s1 . on the left side , the instability of the symphysis pubis has been fixed with a double plate osteosynthesis and long screws into the posterior column , the anterior plate being angular stable ; g postoperative pelvic inlet view ; h postoperative pelvic outlet view . three months after surgery , the patient was walking independently without complaints at the pelvis non - displaced superior pubic rami fractures are splinted in a retrograde manner with a large fragment screw inserted through a small skin incision from the pubic bone near to the symphysis ( fig . , reduction can be achieved through a small suprapubic midline incision prior to screw insertion . in case of symphysis pubis instability or a fracture very near to the joint , a bridging angular stable plate osteosynthesis has to be considered ( fig . 4a h ) . in most patients with long history of pain in the pubic region , a very unstable condition with osteoporotic bone was found intraoperatively in our case series . therefore , a long bridging plate with long screws placed through the infra - acetabular corridor using the modified stoppa - approach on both sides is preferred to achieve high stability of the construct ( figs . 4d f , 5f h , 7b d).fig . 6 a seventy - four - year old female with an ffp type iiia lesion . a complete right - sided crescent fracture of the ilium and superior and inferior pubic ramus fractures with vertical displacement are visible in the pelvic a.p . view ; b pelvic inlet view ; c pelvic outlet view ; d postoperative pelvic a.p . view . the ilium fracture has been fixed with a large fragment angular stable plate and two long lag screws . the pubic ramus fracture has been splinted with a minimally invasive retrograde transpubic screw ; e postoperative pelvic inlet view ; f postoperative pelvic outlet viewfig . 7 a surface rendering of the pelvic bone in an 87-year old female after a fall at home . an incomplete fracture of the dorsal ilium , a displaced fracture of the superior pubis ramus and an undisplaced inferior pubic ramus fracture are visible . the very low bone density in both sacral ala and in the center of the iliac wing are clearly visible . the lesion is classified as an ffp type iic ; b postoperative pelvic a.p . overview . the incomplete ilium fracture has been fixed with a single screw , the anterior instability with a bridging plate and screw osteosynthesis . to avoid loosening of the screws , an we chose the longest possible screw trajectory in the pubic bone and ischium ; c postoperative pelvic inlet overview ; d postoperative pelvic outlet overview . six months after surgery , the patient walked independently and without complaints a seventy - four - year old female with an ffp type iiia lesion . a complete right - sided crescent fracture of the ilium and superior and inferior pubic ramus fractures with vertical displacement are visible in the pelvic a.p . view ; b pelvic inlet view ; c pelvic outlet view ; d postoperative pelvic a.p . view . the ilium fracture has been fixed with a large fragment angular stable plate and two long lag screws . the pubic ramus fracture has been splinted with a minimally invasive retrograde transpubic screw ; e postoperative pelvic inlet view ; f postoperative pelvic outlet view a surface rendering of the pelvic bone in an 87-year old female after a fall at home . an incomplete fracture of the dorsal ilium , a displaced fracture of the superior pubis ramus and an undisplaced inferior pubic ramus fracture are visible . the very low bone density in both sacral ala and in the center of the iliac wing are clearly visible . the lesion is classified as an ffp type iic ; b postoperative pelvic a.p . overview . the incomplete ilium fracture has been fixed with a single screw , the anterior instability with a bridging plate and screw osteosynthesis . to avoid loosening of the screws , an we chose the longest possible screw trajectory in the pubic bone and ischium ; c postoperative pelvic inlet overview ; d postoperative pelvic outlet overview . six months after surgery , the patient walked independently and without complaints in contrast to other authors , pelvic external fixation for anterior stabilization is not recommended in fragility fractures . there is a high risk of loosening and pin track infection and , always critical , a lack of patient comfort . internal fixators placed between both anterior superior or inferior iliac spines have been presented in recent literature . although they can be inserted in a minimally invasive manner , there is a high risk of damage to the lateral cutaneous femoral nerve and occurrence of heterotopic ossification . the implantation is in the proximity of the femoral vessels and nerve ; furthermore , there is a little distance between the skin and screw head . additionally , the implant must be removed in most cases , which involves a second operative procedure . displaced fragility fractures of the pelvic ring ( ffp types iii and iv ) necessitate a more aggressive surgical approach . as closed reduction is not possible , open reduction and internal fixation will be mandatory . after open reduction of displaced sacral fractures or sacroiliac instabilities , similar techniques for stabilization can be used , as previously described , for the stabilization of non - displaced sacral fractures . in chronic cases , i.e. , after failure of conservative treatment , gross instability is often combined with joint widening or bone loss . in those cases , we prefer performing a sacroiliac arthrodesis , as we estimate excessive risk of non - union in simple osteosynthesis . an anterior approach to the sacroiliac joint through the first window of the ilioinguinal approach is chosen . the joint is debrided and filled with cortico - spongious bone grafts of the ipsilateral iliac crest . two short plates are inserted over the sacroiliac joint at an angle of 6090 to each other . one large fragment cancellous bone screw is inserted in the sacrum parallel to the joint and one or two screws are placed in the ilium . sacroiliac screws or a transsacral positioning bar can be placed additionally to enhance stability . in case of transiliac instabilities , the fracture runs from the inner curve of the ilium proximally and laterally . for stabilization , we prefer insertion of an angular stable plate placed parallel to the sacroiliac joint and the pelvic brim , bridging the medial edge of the fracture . as in other skeletal regions , angular stable plates have higher pull - out forces ; therefore , there is a lower risk of loosening . the angular stable plate has to be pre - shaped and twisted to fit on the innominate bone as best as possible . the proximal screws should be directed parallel to the sacroiliac joint , the distal screws in the sagittal plane or slightly towards lateral ( fig . the ilium fracture running proximally and laterally ends at the iliac crest and is stabilized there with one long screw , which is inserted perpendicular to the fracture plane and between the inner and outer cortex of the crest ( fig . , a small fragment plate is placed over the iliac crest . in ffp type iv fractures , , bilateral angular stable plate osteosynthesis is done in the same technique as in unilateral transiliac instabilities . in minimally - displaced bilateral fractures of the lateral mass of the sacrum the bilateral sacral fractures are compressed when the washers and nuts on both ends of the bar are pressed against the lateral cortex of the dorsal ilium . for absolute rotational stability this can only be done in cases where a transsacral corridor large enough for additional screw placement is available ( fig . bilateral sacroiliac screws as the only measure of stabilization are not reliable , as there is a high risk of loosening due to poor bone stock . iliolumbar fixation , which involves a much more prolonged surgery with bridging of the lumbosacral junction , is preserved for cases with gross instability or for patients in which a safe transsacral corridor is not available . transpedicular screws are placed bilaterally in the l4 and l5 pedicles and in the dorsal ilium . the screw in the ilium is inserted between the two cortices in the direction of the greater sciatic notch . the pedicle screws are connected with a bar on each side and the two bars are connected with a transverse connector . iliolumbar fixation prevents further intrusion of the lumbosacral segment into the pelvic ring . in very unstable situations , the lumbosacral segment seems displaced distally when related to the pelvic ring ; d axial ct - reconstruction of the dorsal pelvis showing a severe rarefication of the bone substance and a complete bilateral fragility fracture of the lateral mass of the sacrum ; e coronal ct - reconstruction confirms the complete bilateral fragility fractures of the lateral mass of the sacrum ; f postoperative pelvic a.p . small plates were used as washers under the nuts of the sacral bar to hinder cut - through of the washers ; g postoperative pelvic inlet view ; h postoperative pelvic outlet view ; i a representative axial ct - cut one year after surgery , showing complete healing of the sacral fragility fractures ; j a representative coronal ct - cut showing the position of the transpedicular screws and the transsacral positioning bar . the lumbosacral segment seems displaced distally when related to the pelvic ring ; d axial ct - reconstruction of the dorsal pelvis showing a severe rarefication of the bone substance and a complete bilateral fragility fracture of the lateral mass of the sacrum ; e coronal ct - reconstruction confirms the complete bilateral fragility fractures of the lateral mass of the sacrum ; f postoperative pelvic a.p . small plates were used as washers under the nuts of the sacral bar to hinder cut - through of the washers ; g postoperative pelvic inlet view ; h postoperative pelvic outlet view ; i a representative axial ct - cut one year after surgery , showing complete healing of the sacral fragility fractures ; j a representative coronal ct - cut showing the position of the transpedicular screws and the transsacral positioning bar . physiotherapy should be adapted to the functional status of the patient before the fragility fracture occurred and certainly not enforced . too aggressive mobilization may lead to additional fragility fractures in the pelvic ring with enhanced instability . short transfers and sitting in a wheelchair can be allowed , but long standing and walking with full weight bearing is not recommended . due to low bone strength , there is a risk of loosening of the implants with delayed healing , non - union and revision surgery . usually , after six weeks , conventional radiographs will confirm ongoing bone healing without implant migration . fragility fractures of the pelvic ring have become a new entity within the spectrum of insufficiency fractures resulting from osteoporosis . we experience a rapidly growing ratio of these lesions in our total population of patients , with the need of in - patient treatment for pelvic injuries . there must be a high index of suspicion for dorsal lesions when patients present with a fracture of the anterior pelvic ring and further diagnostics with a ct scan should be carried out to appreciate the full dimension of the instability . a literature review of management alternatives has been given and compared with our own experience at treating 245 patients . the type of treatment is based on the localization and severity of instability , which is reflected in a new classification system . ffp type ii is often stabilized with a percutaneous procedure , whereas ffps types iii and iv are usually stabilized with an open procedure . sacroliliac screw osteosynthesis , bridging plate osteosynthesis , transsacral positioning bar and angle stable plating are valid alternatives . in ffp type iv lesions , bilateral stabilization or iliolumbar fixation are recommended . further clinical and biomechanical work is needed to shed light on the optimal treatment of these pelvic lesions .

antibody - secreting cells were at one point divided into two categories : the plasmablast and the more mature pcs ( 7 ) . the pc compartment has been further divided into short- and long - lived cells ( 4 , 5 , 8) , with the latter being considered more mature . within such a simple scheme , however , precursor product relationships have been difficult to define . for example , the plasmablasts that appear within the foci in the extrafollicular areas of lymphoid tissues shortly after immunization ( 9 ) have a limited lifespan and are considered to die in situ ( 10 ) . it is unknown whether plasmablasts give rise to short- or long - lived pcs ( 11 ) . antibody production continues well after the loss of the extrafollicular foci due to pcs located in the splenic red pulp , for example , and in bone marrow ( 12 , 13 ) . despite knowing for some time that plasma cells with varying life - span exist in these locations ( 8) , it remains unclear whether one ( long ) is derived from the other ( short ) . in addition to longer lifespan , pc maturation is considered to involve changes in phenotype . certain pcs do not express mhc class ii , cd19 , and cd45 ( 10 , 14 ) , and in general loss of b cell differentiation markers correlates with increased lifespan and is considered a sign of maturity . however , it is not clear if the phenotypic changes represent distinct stages in a linear progression or are to some extent stochastic ( 15 ) . in mice , pcs from all defined subsets have been shown to express cd138 ( 16 ) ( although not all pcs are necessarily cd138 nor are all cd138 cells pc ) making it something of a beacon in pc biology as it is almost the only marker that allows the prospective identification of pcs rather than retrospectively measuring immunoglobulin secretion . in humans , cd138 expression is restricted to pc of the bone marrow ( 18 ) , and cd38 expression provides a somewhat less robust alternative marker ( 19 ) . collectively , ample heterogeneity has been identified among pcs to satisfy the most ardent immunological subsetter , but it has yet to allow the construction of a developmental scheme whereby a particular phenotype can be associated with a particular function , lifespan , or fate . added to this complexity is the fact that in autoimmune diseases immune reactions occurring outside lymphoid tissues ( 20 ) and pc accumulating in inflamed tissues ( 21 ) will contribute to antibody production . are particular pc phenotypes or the pcs found in particular locations associated with distinct cellular origins ? such relationships may determine the pathogenicity of the antibodies being produced . for example , pcs derived from germinal centers ( gcs ) may secrete antibody of a mature ( igg ) isotype and of high affinity for antigen , both factors that may exacerbate tissue damage . thus , knowing the etiology of a pc may identify points and means of clinical intervention . there are several developmental schemes for pcs that collectively encompass virtually every possible connection ( fig . 1 ) . although plasmablasts are potential pc precursors of long standing ( 7 ) , several other populations have joined them recently . the gc is broadly accepted to produce pcs that are high affinity , long - lived , and that reside primarily in the bone marrow ( 13 , 22 ) . defining the developmental processes that drive a gc b cell toward becoming a long - lived bone marrow pc may identify points at which the development of pathogenic autoantibodies can be interrupted , assuming that the long - lived pc - secreting pathogenic autoantibodies in the bone marrow are gc derived , which may not always be the case ( 23 ) . if , for example , the process of pc differentiation depends on antigen - induced signaling ( 24 ) , the creation of environmental niches ( 25 ) , and chemokine gradients ( 26 ) , then modulating any of these may be sufficient to modulate disease severity . one must include also the traditional memory b cell population as containing pc precursors since such cells rapidly differentiate into short - lived pc upon reexposure to antigen ( 27 , 28 ) . in mouse , two additional precursor populations have been identified : both b220 and both concentrated in the bone marrow . one of these is cycling ( 29 ) and therefore presumably susceptible to the treatment applied by hoyer et al . the role of this population in the maintenance of long - lived autoreactive pcs in the bone marrow therefore remains open . the other b220 population is derived from the gcs , lacks expression of cd138 , is developmentally dependent on a transcriptional regulator of pc development , blimp1 ( 30 , 31 ) , and is responsive to antigen , which long - lived pcs are not ( 14 ) . again , the role of this population in the maintenance of long - lived pcs is unresolved . in humans , pc precursor diversity is less well defined , although stages of maturation have been identified in blood ranging from a plasma cell precursor ( 32 ) to more discrete stages within committed pc ( 33 , 34 ) . interestingly , one of these studies noted a significant increase in plasma cell precursor frequency in the blood of children with sle ( 32 ) , which complements other studies reporting a correlation between disease activity and circulating pcs ( 35 ) . these data raise the possibility that aspects of the immune response are dysfunctional in these patients , and if the developmental origin of the pc were known , it may be possible to intervene in a relatively precise manner . the definition of relationships between precursors and pcs is going to be crucial if selective therapies are to be developed that target only the relevant pc subset . the tangled web of pcs and their precursors . schematic representation of pc subsets and their postulated or proven precursors and products . sl , short - lived ; ll , long - lived ; ag , antigen . what is required to elucidate pc development is relatively simple to write but more complicated to effect . first is the efficient identification of the earliest b cell committed to becoming a pc , thereby allowing its isolation and manipulation . currently pc identification relies on the enrichment provided by various fortuitously identified markers such as cd138 . the identification of early stages of pc development would allow the developmental potential and differentiation requirements of such cells to be defined in vitro and in vivo . this would allow also for the rapid accumulation of additional markers in a developmentally ordered manner , creating the schemes that have been so useful in the analysis of white blood cell development . second is the exploitation of in vitro systems to define the impact of various factors that affect pc production . small changes in responsiveness to differentiation stimuli such as the cytokines il-5 and il-10 can , over the course of an immune response , culminate in huge differences in the size and composition of the effector populations generated ( 15 ) . appropriate and targeted therapy of antibody - mediated autoimmune diseases , as highlighted by hoyer et al . ( 6 ) , is going to depend on identifying the pcs involved in a disease process and targeting the critical component(s ) of its development , be that the cycling precursor , the stimuli driving its proliferation , the factors promoting its maturation , or elements of its survival niche , both stromal and soluble . potential approaches include : the cytotoxic and antiinflammatory drugs already in use ; inhibiting b t cell interactions ( 36 ) ; blocking factors that promote pc maturation , such as baff / blys ( 37 ) ; interfering with chemotaxis ( 38 ) ; and blocking the interaction between pc and stromal elements , such as adhesion molecules and cytokines ( 39 ) .

pemphigus vulgaris ( pv ) is an uncommon ( 0.53.2 cases per 100,000 ) , potentially life - threatening ( 5%15% ) , autoimmune bullous mucocutaneous disease,1 which can rarely affect the eyes . autoantibodies are formed against desmoglein , the glue that attaches adjacent epidermal cells . when autoantibodies attack desmogleins , the cells become separated from each other , a phenomenon called acantholysis . this causes blisters and erosions.2,3 we present a case of a patient with ocular manifestations of pv resistant to systemic and topical medication , which was successfully treated with subconjunctival injections of triamcinolone acetonide ( ta ) . a 76-year - old woman , suffering from pv , was referred to our department due to acute onset of intense burning and foreign body sensation in her right eye , despite her treatment with systemic methylprednisolone 15 mg / day and azathioprine 100 mg / day due to pv . pv was diagnosed clinically and by performing ( a ) histopathology from the edge of a blister , ( b ) direct immunofluorescence ( dif ) on normal - appearing perilesional skin , and ( c ) indirect immunofluorescence ( idif ) . acantholytic cells were found in a tzanck smear , taken from the base of an erosion , which helped to differentiate pv from pemphigus foliaceous . slitlamp examination revealed pronounced conjunctival hyperaemia , mucus secretions , ulcerative erosions involving the palpebral conjunctiva at the right lower eyelid margin ( figure 1 ) and punctate corneal epithelial erosions involving the inferior part of the cornea of the same eye . schirmer s test without anesthetic was markedly reduced ( 3 mm ) as well as the tear break up time ( but ) ( 3 seconds ) . therefore the dose of methylprednisolone was increased to 40 mg / day , and the azathioprine was replaced by cyclophosphamide 100 mg daily . in addition , the patient was treated with topical dexamethasone 0.1% daily and frequent instillation of unpreserved carbomer 0.3% . hence , a subconjunctival injection of ta 20 mg in 0.5 ml ( kenacort 40 mg / ml ; bristol - myers squibb ) was given after informed consent was obtained , but without stopping the systemic treatment . two weeks following the ta injection the patient s symptoms were reduced significantly the eye was less inflamed and the conjunctival ulcers were reduced in size . due to the initial positive response to the ta injection three months later the symptoms had improved significantly and the ulcers had disappeared ( figure 2 ) , enabling us to taper further the systemic treatment . the schirmer s test was also increased to 6 mm as well as the but was prolonged to 6 seconds . one year after the second injection the patient is still asymptomatic without signs of recurrence . ocular pemphigus is probably underdiagnosed and its frequency appears underestimated.3 pv is recognized by a dermatologist from the appearance and distribution of the skin lesions . definitive diagnosis requires examination of a skin biopsy from the edge of a blister by a pathologist . definitive diagnosis also requires the demonstration of anti - desmoglein autoantibodies by direct immunofluorescence on the skin biopsy . these antibodies appear as igg deposits along the desmosomes between epidermal cells , a pattern reminiscent of chicken wire . anti - desmoglein antibodies can also be detected in a blood sample using the elisa technique . a high titer of canca is claimed to be an important feature of the disease.3,4 ocular involvement with pv is very rare.2 the most characteristic f indings are conjunctival hyperemia , conjunctivitis , eye irritation and mucus secretions.5 catarrhal conjunctivitis is very common in pv , while bullae and conjunctival erosions are seen rarely.5,6 conjunctival fibrosis is very rare2 as is corneal involvement.7 the therapeutic approach for the treatment of pv requires a systemic therapy with steroids and immunosuppressive drugs . in our case , neither the increase of the initial systemic doses , nor the replacement of azathioprine by cyclophosphamide , had any effect on the ocular signs of the disease . even topical steroids ( drops ) and lubricants were used , but with no symptomatic effect , probably due to persistent erosion of the lid margins , affecting the quality of the tear film and further causing severe dryness of the eye . a considerable improvement was seen following 2 subconjunctival injections of ta 20 mg with an interval of 2 weeks , probably due to normalization of the lid margin anatomy , which improved ocular surface dryness . the subconjunctival ta injections provided sustained release of steroids at the side of greatest need , ie , the palpebral conjunctiva , with no systemic adverse effects . this was of particular importance in our patient who started to present signs of cushing s syndrome from the long - term use of systemic steroids . to the best of our knowledge , this is the first reported case of subconjunctival injections of ta for the treatment of ocular manifestations of pv . daouds et al7 reported 11 cases of ocular manifestations of pv treated mainly with systemic steroids , and in 1 case they used ta . the authors concluded that the disease could recur ( mean follow up 48.9 months ) , but that finally all patients eventually recovered fully without sequelae . although no long case series are available , ta could be regarded as an effective alternative for patients with serious ophthalmic manifestations of pv unresponsive to systemic therapy or those with serious adverse effects to systemic treatment .

dermoid cysts are the most frequently occurring tumors among ovarian germ cell tumors , accounting for more than 20% of all ovarian neoplasms.1 they present most commonly in women younger than 20 years of age , but sometimes occur in menopausal women at a rate of about 10% to 20% . fibrothecomas are ovarian tumors of gonadal stromal origin , mesenchymal cell tumors composed of theca - like elements and fibrous tissue , and account for about 0.4% to 8% of all ovarian tumors.2 a collision tumor is defined as a tumor in which the different neoplastic components remain histologically distinct and are separated from each other by narrow stroma or their respective basal lamina . we report the case of a 77 year old woman who presented with postmenopausal bleeding ( pnb ) due to the torsion of a collision tumor comprising a dermoid cyst and fibrothecoma . a 77-year - old postmenopausal woman presented with vaginal bleeding with a week - long history and a large pelvic mass associated with lower abdominal discomfort for 3 months . the patient complained of pain , but had no fever , chills and significant gastrointestinal symptoms . she had gone through menopause at the age of 52 and was not taking any hormone replacement . she had no significant past medical history , familial history or operation history . on pelvic examination , a relatively hard , movable , non - tender mass as large as a double man 's fist was palpated on the right lower abdomen . the hemogram revealed anemia with a hemoglobin level of 9.8 g / dl and a hematocrit of 29.8% . abdominal magnetic resonance imaging ( mri ) showed a 12 12 11.5 cm sized , well - marginated , bilobulated cystic mass with some solid areas in the right pelvic cavity . with enhancement 1 ) . there was no other abnormal finding on images and no pelvic lymph node enlargement was observed . endometrial aspiration was done , confirming historically normal proliferative endometrium . under general anesthesia , surgical exploration the uterus and left adnexa appeared normal , and a large right ovarian tumor of approximately 12 cm diameter was rotated counterclockwise with a 720 degree arc . right salpingo - oophorectomy was performed for frozen biopsy . after confirming the frozen pathologic results as fibrothecoma and benign dermoid cyst , total hysterectomy and left salpingo - oophorectomy were performed . macroscopically , the resected tumors in both cases showed a unilocular cystic tumor adjacent to a solid tumor . microscopically , the cystic tumors were composed of cutaneous tissues and the solid tumors consisted of spindle cells with lipid - rich cytoplasm , arranged in interlacing bundles . the cystic tumor and the solid tumor were completely separate and no transitional features were recognized histologically ( fig . dermoid cyst is the most frequently occurring ovarian germ cell tumor , accounting for 20% of all ovarian tumors , usually in patients of child bearing age . unlike all other germ cell tumors , malignant changes in benign dermoid cysts have been recorded as occurring in 1.0% to 1.8% of cases , usually in patients older than 40 years of age or menopausal women . fibrothecomas are ovarian tumors of gonadal stromal origin , composed of theca - like elements and fibrous tissue . a collision tumor represents the coexistence of two adjacent but histologically different neoplasms occurring in the same organ with completely different basal layers or stroma . collision tumors occur in various organs such as the esophagus , stomach and thyroid , but they are extremely rare in the ovaries . the most common histologic combination of collision tumors of the ovary consists of teratoma and mucinous tumors.3 during clinical evaluation , differentiation of the characters of these ovarian tumors is important for appropriate treatment strategies and prognosis . ultrasound is the most common tool used to evaluate ovarian masses , but it is impossible to determine the exact size and direction if the mass is huge . computed tomography and mri are more useful diagnostic tools for huge ovarian masses . in this case , the mass was very large and was located just beneath the skin so the mri was helpful . pmb is vaginal bleeding that occurs at least 12 months after your periods have stopped . pmb is a common problem representing 5% of all gynecology outpatient attendances.4 the most common causes of bleeding after menopause include thinning of the reproductive tract tissues and hormone therapy.5 in some cases , bleeding can signal cancer of the uterine lining or cervical cancer . other possible causes include fibroids , small growths in the uterus or cervix , known as polyps , and ovarian cancer , especially estrogen - secreting ovarian tumors . although most women with pmb will not have significant pathology , the priority is to exclude malignancy.6 torsions of ovary , tube , paratubal cysts or adnexa are not common , but they are major causes of acute abdominal pain , local infarction of abdominal organs , and sometimes gynecologic emergencies , accounting for approximately 2.7% of all gynecologic emergencies . torsion may occur with no specific cause , but there are risk factors like bowel movement , bladder changes , and changes in uterine size during pregnancy , intra - abdominal pressure during vomiting , coughing or abdominal trauma.7 the incidence of ovarian torsion is about 1.5 times more frequent on the right side than on the left because of the narrow space on the left side due to the sigmoid colon as well as the increased bowel movement of the terminal ileum and cecum on the right side . it is accompanied by nausea , vomiting and anxiety due to a sensitized autonomic nervous system . the period of symptoms can be variable from a few hours to a few months , but sometimes , there may be no symptoms at all . the diagnosis of ovarian torsion is nonspecific , and it is suspected on the basis of peritoneal signs , sonographic findings such as cystic , mixed pelvic mass and fluid collection in the douglas pouch . recent studies have reported that color doppler flow can be used as a diagnostic tool by proofing ovarian arterial and venous flow , but partial torsion of the ovary can not be observed by color doppler due to the dual blood supply of the ovary.8 menopausal women with acute abdominal pain and a palpable mass in the adnexa usually have the probability of an ovarian tumor with a chance of torsion . however , it is not easy to diagnose before surgical treatment due to nonspecific symptoms , clinical and physical findings and radiographic findings . the treatment of ovarian tumor in menopausal women is generally total hysterectomy and bilateral salpingo - oophorectomy . with the suspicion of malignancy , we present a successfully treated case of a 77 year old woman with collision tumor composed of dermoid cyst and fibrothecoma in the right ovary with a review of literature . this study shows that the hormonal changes caused by sex cord stromal tumor can cause postmenopausal uterine bleeding and can have significant influence on collision tumors involving dermoid cysts and fibrothecoma . the torsion of this kind of unique combination of tumors has never been reported , thus this study has its remarkability .

since the discovery of helicobacter pylori ( h. pylori ) by warren and marshall in 1983 , more than 30 non - pylori - helicobacter species have been described [ 2 , 3 ] . to date , h. bizzozeronnii , h. canadensis , h. canis , h. cinaedi , h. fennelliae , h. felis , h. heilmannii , h. pullorum , h. rappini , h. salomonis , h. winghamensis , and h. westmeadii have been found in humans with gastritis , enteritis , and septicaemia [ 38 ] . h. fennelliae was first described in 1985 as a new campylobacter species isolated from asymptomatic homosexual men with enteritis and proctitis . this organism was subsequently reclassified as a helicobacter species based on 23s rrna hybridisation studies . h. fennelliae is a fastidious organism and difficult to culture ; thus , there are very few reports of the clinical relevance of the organism . in 2000 , tee et al . described a novel species of helicobacter isolated from the blood of a young aboriginal child with diarrhoea and vomiting which was most closely related to h. fennelliae . the authors proposed , this may be a new species of helicobacter . from 1977 to 1990 , the routine microbiological laboratories at red cross war memorial children 's and groote schuur hospitals in cape town , south africa used a variety of antibiotic - containing media plates and standard microaerophilic atmospheric growth conditions for the isolation of campylobacter and other epsilonproteobacteria . h. fennelliae and other fastidious h2-requiring and antibiotic - sensitive epsilonproteobacteria were never isolated under these conditions . with the introduction of the cape town protocol , an isolation method that uses membrane filtration onto antibiotic - free plates and subsequent incubation in an h2-enriched microaerophilic atmosphere , h. fennelliae and other fastidious epsilonproteobacteria were , and still are , routinely isolated from paediatric stool and blood cultures in cape town . over an 18-year period , since the introduction of this protocol in october 1990 , h. fennelliae has been isolated from 5.6% ( 347/6249 ) stool samples from children with diarrhoea . in addition , h. fennelliae was isolated from 15/174 ( 8.6% ) paediatric blood culture samples negative for campylobacter or other helicobacter species . this is highly suggestive of the fact that h. fennelliae may be a significant pathogen and is probably considerably underreported due to inadequate isolation methods . phenotypic and biochemical tests are usually used to identify bacterial isolates in the clinical setting . however , there are limitations to these assays , and thus sequencing and the phylogenetic analysis of the 16s rrna are often utilised to identify new isolates . the access to a large number of h. fennelliae isolates from cape town provided the opportunity to look at the genetic diversity of these isolates and compare the data to that available in the genbank database . the 16s rrna and a portion of the rna polymerase subunit b ( rpob ) gene were analysed . forty previously characterised strains of h. fennelliae collected from paediatric stool and blood samples over an 18-year period , from 19902008 , were analysed in this study . briefly , a couple of microbeads maintained at 80c were removed and added to 100 l of distilled water . the beads were boiled for 5 minutes and the supernatant used in the pcr reaction . initially a portion of the rpob gene was amplified with primers designed by lim et al . . to improve phylogenetic resolution , an additional antisense primer ( 5 ttgcatcatcatgctcc ) amplifying a larger region ( 704 bp ) was designed , based on the sequence data of kuhnert and burnens . two microlitres of extracted dna or boilate was added to a 50 l pcr mix consisting of 2 u super - therm polymerase , 1x pcr reaction buffer , 1.5 mm mgcl2 ( jmr holdings , kent , uk ) , and 200 m of each dntp ( roche biochemicals , mannheim , germany ) . the cycling conditions were 1 cycle 95c 2 minutes , 40 cycles of 95c 15 sec , 52c 25 sec , and 72c 35 sec followed by a final 7-minute extension cycle at 72c . the pcr products were analysed by 2% agarose gel electrophoresis and visualised with uv irradiation after staining with ethidium bromide . pcr products of the 16s rrna and rpob genes were purified ( qiagen , hilden , germany ) and sequenced directly using the bigdye terminator ver1.1 commercial kit ( applied biosystems , ca , usa ) . the nucleotide sequences were aligned with known sequences from genbank using the clustal - x software . a neighbour - joining phylogenetic tree was constructed using the treecon software program ( version 1,3b ) with 500 bootstrap resamplings . the secondary structure of the 16s rrna intervening sequence ( ivs ) of h. fennelliae with the lowest free energy was predicted using mfold version 3.2 [ 19 , 20 ] . an ivs has also been found in the 23s rrna of h. fennelliae isolate ccug 18820 ( accession number ay596237 ) , and a comparison between the ivs of the 23s rrna and 16s rrna was made . the accession numbers of the 16s rrna sequences of h. fennelliae from this study are gq867137-gq867176 . the 16s rrna of all 40 isolates of h. fennelliae were successfully amplified , yielding a 1340-bp product . this is larger than the other helicobacter species due to the presence of ivs in h. fennelliae . a representative sample of 16s rrna pcr products amplified from different helicobacter clinical isolates is shown ( figure 1(a ) ) . the rpob gene was amplified from 32/40 ( 80% ) isolates , with some examples shown ( figure 1(b ) ) . the mean age of the 40 children from whom h. fennelliae was isolated was 14 months ( range : 148 months ) . the male : female ratio was similar , 1.0 : 1.1 . the majority of the children presented with symptoms of diarrhoea ( 31/40 , 77.5% ) . h. fennelliae was isolated from blood cultures in 5 cases ; 2 children had pneumonia , 2 had diarrhoea , and there was a single case of meningococcaemia . dual infection with other enteric organisms was found in 21 samples ( 52.5% ) of which the majority ( 19/21 , 90% ) were campylobacter species , and in 2 samples shigella and giardia species were found . in the remaining 19 samples , the isolates from south africa all branched with an australian helicobacter strain ( accession number af237612 ) , while those from europe and usa formed a separate branch with a bootstrap value of 100% ( figure 2(a ) ) . this branching pattern was not present using the smaller rpob pcr fragment ( data not shown ) , but on a limited number of h. fennelliae samples ( n = 13 ) , where the larger fragment was successfully amplified , the separation of the south african isolates from the other strains is observed ( figure 2(b ) ) . a single nucleotide change ( c - t ) at position 295 of the 16s rrna differentiated the types of strains nctc 11612 , lmg 7546 , and ccug 18820 from the south african isolates . the 16s rrna sequence of isolate 283.94 , although clustered with the south african h. fennelliae isolates , had a region of 57 bp ( nt 10391096 ) with 10 nucleotide changes . blast analysis showed the sequence to have closest homology ( 97% ) with the 16s rrna of campylobacter hyointestinalis subsp . h. fennelliae isolated from the blood did not cluster separately to those from stool samples ( figure 2(a ) ) . the intraspecies variation of south african strains of h. fennelliae was 0.10.6% while between south african and the 3 type strains was 0.70.9% . the dna sequence was inserted at nucleotide position 175 ( based on h. pylori type strain nctc 11637 , accession number z25741 ) . the ivs sequences were identical to the type strains nctc 11612 , lmg 7546 , and ccug 18820 with the exception of isolates 327 - 92 , 249 - 92 , 355 - 93 , 274 - 94 , and 334 - 94 . blast analysis showed that 122 bp of the ivs had a high similarity of 79% and 77% to the 23s rrna of h. canis nctc 12743 and helicobacter sp . in addition , there was a region of 70 bp which showed significant homology ( 90% ) to h. mesocricetorum atcc 700931 . no open reading frames were noted . the secondary structure of the 16s rrna ivs predicted a conformation as seen in figure 3(a ) with free energies of 166.3 kcal.mol at 37c . the 5 and 3 ends of the 16s rrna ivs were complementary resulting in a 24 bp stem structure . the 271 bp 23s rrna ivs of h. fennelliae ccug 18820 had a similar 21 bp stem structure ( figure 3(b ) ) . a region of homology ( 26 bp ) between the two ivs sequences was noted ( boxed area figure 3 ) . this study identified 2 distinct genotypes of h. fennelliae based on the phylogenetic analysis of the 16s rrna and rpob genes . all isolates from south africa clustered with the helicobacter strain af237612 isolated by tee et al . , while the 3 isolates from the northern hemisphere , nctc 11612 , lmg 7546 , and ccug 18820 , formed a separate branch with bootstrap value of 100% . proposed that their novel helicobacter strain may be a new species based on the 16s rrna sequence analysis and a sequence similarity of 97% with h. fennelliae ccug 18820 ( accession number m88154 ) . the discrepancy between these observations can possibly be explained by the fact that the original m88154 sequence , deposited in genbank in 1993 by dewhirst and paster , was subsequently replaced in 2004 by the same authors . sequencing of the housekeeping gene , rpob , is increasingly being used to confirm 16s rrna - generated phylogenetic trees and identify bacteria and closely related species in the clinical setting . the taxonomic resolution of this gene is more than 3 times greater than that of 16s rrna for a number of different bacteria , including vibrio , bacillis , and pseudomonas [ 2527 ] . in this study phylogenetic analysis of the larger rpob fragment confirmed the separation of h. fennelliae isolates into 2 genotypes . intervening sequences of variable lengths and sequences can be found in both the small ( 16s rrna ) and large subunit ribosomal rna ( 23s rrna ) of many bacteria . ivss have been described in a number of campylobacter ( c. coli , c. curvus , c. fetus , c. helveticus , c. hyointestinalis , c. jejuni , c. sputorum , c. rectus , and c. upsaliensis , ) and helicobacter ( h. bilis , h. canis , h. fennelliae , h. mustelae , and h. muridarum ) species [ 2937 ] . the ivs found in the 16s rrna of h. fennelliae isolates examined in this study is highly conserved . the c - t transition noted in 5 isolates did not alter the secondary structure of the ivs ( data not shown ) . although the ivs of the 23s rrna is shorter than that of the 16s rrna , there is a region of homology indicating a possible common ancestral element . the predicted secondary structures of both the 23s rrna and 16s rrna contained a stem of 21 bp and 24 bp formed , respectively , by the 5 and 3 inverted repeats . these may act as recognition sites for excision by rnase iii during rrna maturation [ 28 , 34 ] . h. fennelliae is infrequently reported as causing bacteraemia , and this may in part be due to the fastidious nature of the organism [ 11 , 36 , 3840 ] . in this study 5 of the 15 h. fennelliae strains isolated from the blood were examined . there were no sequence differences between h. fennelliae strains isolated from blood or stool samples . in conclusion , this molecular and phylogenetic study is the largest undertaken of h. fennelliae with results indicating the presence of 2 genotypes . the south african isolates are more closely related to the australian helicobacter strain , a probable h. fennelliae species , isolated by tee et al . than to the 3 type strains .

giant inguinal hernias ( gih ) are rarely encountered in high income countries ; however , in low resource setting such conditions present more frequently often do to a lack of patient knowledge , trust in the health system , or funds to pay for treatment . additionally , in low income countries , resources may preclude management that is the hallmark of more resourced environments where a pneumoperitoneum is used to increase the capacity of the abdominal cavity and a mesh is used for definitive repair ( 1,2 ) . in sierra leone , a small west - african country with a population of 6 million and only 10 surgeons ( 3 ) , the presentation of a gih is less of a surprise . a noted aphorism is that in africa there are only two types of hernias : above the knee and below the knee . the reality in clinical practice , however , where there are many cases of incarcerated and sliding hernias , is that true gih , defined as a sac with a capacity greater than 2000 ml , are fortunately rare . however , when such cases appear , surgeons in these locations are faced with the problems of how to safely treat a patient with their limited resources . a 55 year old male with a right gih was referred to a tertiary care hospital in sierra leone from a smaller district hospital . the patient was known to have had the hernia since childhood but it had grown large over the years with excoriation of the skin at the apex of the hernia resulting in a painful , foul smelling ulcer measuring 4 x 6 cm . the hernia reached to the patient s knee while in the upright position and his penis was invaginated into the enlarged scrotal area ( figure 1 ) . a smaller left inguinal hernia was also noted . pre - operative counseling was given regarding the planned three stage repair that would be accomplished over a number of months . stage i , a 2 hour operation under spinal anesthesia , resulted in the excision of the ulcerated skin and partial replacement of the hernia contents into the abdomen as much as possible without compromising the patients venous return and breathing ( figure 2 ) . two months later , after the wound had healed , stage ii , a second 2 hours operation under spinal anesthesia , reduced the hernia by an additional 10 cm , again careful not to cause any respiratory compromise or abdominal compartment syndrome and the excessive scrotal skin was removed to maintain the hitched up level of the hernia ( figure 3 ) . the patient was advised to rest as much as possible and not to undertake any weight - bearing activities , to allow the abdominal muscles to relax and accommodate well the additional abdominal contents . giant inguinal hernia after stage i : resection of ulcer and surrounding scrotal skin , partial reduction of hernia sac contents . giant inguinal hernia after stage ii : partial reduction of hernia sac contents and resection of scrotal skin five months later , stage iii lasted 7.5 hours and commenced under spinal anesthesia and ended with general anesthesia to fully relax the abdominal musculature . on exploration , bowel loops were adhered together and could not be easily separated from the sac and scrotal skin . this final procedure included a lysis of adhesions to separate the small bowel from the ascending colon followed by an en bloc resection of the colon and attached scrotal skin . the specimen included 15 cm of distal ileum and ascending and transverse colon to the splenic flexure . a primary end - to - end anastomosis of the ileum to the descending colon was carried out and a bassini repair was performed . a scrotal skin reduction allowed ex - vagination of the penis ( figure 4 ) giant inguinal hernia after stage iii : bowel resection , ileo - colic anastomosis , hernia repair and resection of scrotal skin ) after discharge from the hospital the patient was followed up four years later in 2009 . the operational site looked good , the hernia repair was intact and the patient did not have any complaint . the patient returned to his village and was able to ultimately gain employment as a hospital porter . repair of a gih needs careful planning as the hernia sac can contain urinary structures , omentum and large and small bowel ( 4 ) . if the contents of the sac remain unreduced for an extended period of time attempts at sudden reduction can lead to raised intra - abdominal pressure ( 5 ) . an abdominal compartment syndrome with its consequences of respiratory and circulatory collapse and multi - organ failure can be the results of such an attempt . furthermore the diaphragm will be displaced into the thoracic cavity , resulting in respiratory difficulty and compromised venous return . management options can be grouped into either enlarging the abdominal cavity or debulking the abdominal contents . the abdominal cavity can be enlarged by insufflation with co2 over time causing a pneumoperitoneum ( 1,2 ) , a phrenectomy , or by use of a large prostetic mesh to repair the defect ( 5,6 ) . splenectomy , omentectomy , small or large bowel resections can be optional if debulking of the abdominal contents are necessary . these procedures are often combined and contribute to long operative times and usually necessitate an icu admission and post - operative ventilation ( 7 ) . furthermore neither phrenectomy nor splenectomy is advisable for a patient who must earn a living performing difficult manual labor or where infectious diseases , such as malaria , are common . long intensive care unit stays and ventilators are also rarely available in a low - resource setting context . therefore this case in sierra leone was best managed in a multi - stage operation where the abdomen was gradually accustomed to more organs being returned and finally a debulking procedure for organs that could not be safely reduced . for this case a 3-stage procedure was needed , however , earlier experiences ( n=21 ) by the senior author ( ssd ) show that 2 stage procedures can be undertaken for less complicated gih . for surgeons working or visiting such a setting , it is important to be aware of the pitfalls of trying to repair such a hernia in a single stage , but also to understand that such cases are not infrequent and that safe techniques are available . time must also be taken to explain to the patient and family that this is a multi - stage procedure that will be undertaken over a protracted period of time .

pneumocystis species are eukaryotic organisms detected world - wide and infect mammalian hosts including animals and humans . pneumocystis jiroveci causes lethal pneumocystis pneumonia ( pcp ) in immune - suppressed hosts such as patients with aids and those receiving immune suppressive treatment for malignancy treatment or transplantation . currently , pcp most commonly occurs as aids - associated illness in patients who are not aware that they are hiv - positive or those who are not receiving combined anti - retroviral therapy ( cart ) [ 13 ] . although the most common infection site is the lungs , extra - pulmonary infection also occurs [ 413 ] . definitive diagnosis of pneumocystosis is usually achieved by the demonstration of pneumocystis spp . using staining methods , pcr and immunofluorescence techniques . the diagnosis of pcp has been achieved by non - invasive techniques such as induced sputum , oropharyngeal washes and nasopharyngeal aspirates . consequently , more invasive techniques such as bronchoalveolar lavage ( bal ) , transbronchoscopic or surgical lung biopsy used to increase the sensitivity . molecular diagnostic techniques are more sensitive than staining methods , but may have difficulty in distinction of infection versus colonization . demonstration of p. jiroveci in any specimen collected from the respiratory tract of an individual without signs and symptoms of pcp is defined as colonization . due to colonization current research has focused on developing novel quantitative pcr techniques to distinguish between colonization and infection . during oropharyngeal or nasopharyngeal sampling , colonization of p. jiroveci in the pharynx may be transported to the oral or nasal cavity . pneumocystis carinii organisms have been detected in oral swabs of immune - competent rats using pcr . nasal swab sampling has been used in comparison with samples obtained by oropharyngeal washing to determine colonization in elderly individuals , using pcr . therefore , non - invasive oral or nasal swab sampling emerges as a solution to determine the colonization of p. jiroveci in the uppermost parts of the respiratory tract . dexamethasone is commonly used as an immune suppressive agent to induce protozoan parasitic diseases in animal models [ 2629 ] . in the present study , oral and nasal swabs , blood samples and first - time eye swabs were collected from rats prior to administration of dexamethasone and at fixed time points after immune suppression . at the end of immune suppression , samples were collected from lung , heart , liver , kidney , diaphragm , brain , spleen , tongue , muscle , eye , intestine , and feces . microscopic examination and pcr were performed on all samples to assess the presence of p. carinii infection and colonization . two- to three - month - old male outbred rattus norvegicus rats weighing 6080 g were obtained from the ege university experimental animal production facility and used during the experiments . the experimental plan was performed under the instructions and approval of the institutional animal care and use committee of ege university for animal ethical norms . two groups of rats ( each containing 4 rats ) were administrated oral ( 2 mg / l in drinking water ) and subcutaneous injection ( 3 mg / week ) of dexamethasone . prior to immune suppression and 2 , 6 , and 12 weeks after administration of dexamethasone , blood , nasal , oral and eye swab samples ( collected by rubbing the cotton swab ) were collected from anesthetized rats . during the collection of nasal swabs , 50 l sterile 0.9% nacl was pipetted into each nostril of the rats positioned face down and absorbed immediately by sterile dry cotton - tipped swabs . all oral swabs were collected by rubbing the dry cotton - tipped swab in the oral cavity and around the tongue . each swab was broken in a sterile dnase / rnase - free 1.5 ml tube containing 200 l serum physiologic . all swabs were vortexed vigorously , centrifuged at 14,000 g for 5 minutes . after discarding the swabs , lung , heart , liver , kidney , brain , spleen , eye , diaphragm , tongue , muscle , intestine samples and fecal material were collected from each rat . tissues were homogenized using a sterile mortar and pestle in 200 l sterile serum physiologic . morbidity was confirmed by observation of dyspnea , loss of appetite , weight loss , and change in the color of the fur . smears of tissues , fecal material , blood , nasal , oral and eye swabs were stained by giemsa and gram weigert as described [ 3032 ] to examine the presence of p. carinii cysts and trophozoites , respectively . during giemsa staining , slides were covered with methanol and air dried , then slides were covered with giemsa solution [ 10% giemsa ( v / v ) ( merck ) ] and incubated for 30 minutes at room temperature . then , slides were washed with distilled water to remove excess dye and examined under light microscopy with immersion oil . in gram weigert staining , air dried slides were stained with 1% eosin - y ( merck , germany ) solution for 5 minutes . then , slides were rinsed with distilled water for 2 minutes to remove excess dye and stained with crystal violet solution [ 5% crystal violet ( w / v ) ; 10% ethanol ( v / v ) ( applichem ) ; 2% aniline oil ( v / v ) ( merck ) ] for 5 minutes . excess crystal violet is rinsed off with gram s iodine solution [ 3.61 mm potassium iodide ; 1.18 mm iodine ( merck ) ] . rinsed and blot dried slides were washed with aniline oil - xylene solution [ 50% aniline ( v / v ) ; 50% xylene ( v / v ) ( merck ) ] for decolorization . isolation of dna from rat tissues , blood , nasal , oral and eye swabs was performed with the qiaamp dna mini kit according to the manufacturer s protocol ( qiagen ) . during the procedure , 200 l blood , 100 l nasal , oral and eye swab samples , 10 mg spleen and 25 mg from the remaining tissues were used . dna extraction from fecal material was performed with zr fecal dna kit according to the manufacturer s protocol ( zymo research ) . during the procedure , conventional pcr targeting the major surface glycoprotein ( msg ) gene of p. carinii ( genbank no . briefly , the primers to amplify 338 base pair ( bp ) gene fragment in pcr reaction were 5-atggcacggccggttaagag-3 ( 20 nt , aug forward primer ) and 5-atacatttttcttcatgtttt-3 ( 21 nt , c2 reverse primer ) . the 25 l amplification reactions included 3 l template dna , the primers ( 0.8 m each ) , 1.25 u platinum taq dna polymerase ( invitrogen ) , 200 m dntps , and 1 platinum taq reaction buffer . the pcr amplification reaction was performed using the following calculated - control protocol : 5 minutes initial denaturation step at 95c , followed by 30 cycles of 1 minute at 94c , 2 minute at 50c , and 1 minute at 72c , and a final extension of 10 minute at 72c . each pcr included dna from the lung of an infected rat as a positive control and negative control , prepared by the replacement of template dna with distilled water . briefly , the number of p. carinii organisms was counted in stained slides prepared from 20 l lung homogenates of infected rats . dna was isolated from 100 l lung homogenate and pcr was performed as described above with 10-fold dilutions of dna from 1010 organisms . data obtained during the study were processed using prism 3.03 ( graphpad , san diego , ca ) . a two - tailed unpaired t test with 95% confidence interval was used to determine the significance between the results of assays . two- to three - month - old male outbred rattus norvegicus rats weighing 6080 g were obtained from the ege university experimental animal production facility and used during the experiments . the experimental plan was performed under the instructions and approval of the institutional animal care and use committee of ege university for animal ethical norms . two groups of rats ( each containing 4 rats ) were administrated oral ( 2 mg / l in drinking water ) and subcutaneous injection ( 3 mg / week ) of dexamethasone . prior to immune suppression and 2 , 6 , and 12 weeks after administration of dexamethasone , blood , nasal , oral and eye swab samples ( collected by rubbing the cotton swab ) were collected from anesthetized rats . during the collection of nasal swabs , 50 l sterile 0.9% nacl was pipetted into each nostril of the rats positioned face down and absorbed immediately by sterile dry cotton - tipped swabs . all oral swabs were collected by rubbing the dry cotton - tipped swab in the oral cavity and around the tongue . each swab was broken in a sterile dnase / rnase - free 1.5 ml tube containing 200 l serum physiologic . all swabs were vortexed vigorously , centrifuged at 14,000 g for 5 minutes . after discarding the swabs , lung , heart , liver , kidney , brain , spleen , eye , diaphragm , tongue , muscle , intestine samples and fecal material were collected from each rat . tissues were homogenized using a sterile mortar and pestle in 200 l sterile serum physiologic . morbidity was confirmed by observation of dyspnea , loss of appetite , weight loss , and change in the color of the fur . smears of tissues , fecal material , blood , nasal , oral and eye swabs were stained by giemsa and gram weigert as described [ 3032 ] to examine the presence of p. carinii cysts and trophozoites , respectively . during giemsa staining , slides were covered with methanol and air dried , then slides were covered with giemsa solution [ 10% giemsa ( v / v ) ( merck ) ] and incubated for 30 minutes at room temperature . then , slides were washed with distilled water to remove excess dye and examined under light microscopy with immersion oil . in gram weigert staining , air dried slides were stained with 1% eosin - y ( merck , germany ) solution for 5 minutes . then , slides were rinsed with distilled water for 2 minutes to remove excess dye and stained with crystal violet solution [ 5% crystal violet ( w / v ) ; 10% ethanol ( v / v ) ( applichem ) ; 2% aniline oil ( v / v ) ( merck ) ] for 5 minutes . excess crystal violet is rinsed off with gram s iodine solution [ 3.61 mm potassium iodide ; 1.18 mm iodine ( merck ) ] . rinsed and blot dried slides were washed with aniline oil - xylene solution [ 50% aniline ( v / v ) ; 50% xylene ( v / v ) ( merck ) ] for decolorization . isolation of dna from rat tissues , blood , nasal , oral and eye swabs was performed with the qiaamp dna mini kit according to the manufacturer s protocol ( qiagen ) . during the procedure , 200 l blood , 100 l nasal , oral and eye swab samples , 10 mg spleen and 25 mg from the remaining tissues were used . dna extraction from fecal material was performed with zr fecal dna kit according to the manufacturer s protocol ( zymo research ) . during the procedure , conventional pcr targeting the major surface glycoprotein ( msg ) gene of p. carinii ( genbank no . briefly , the primers to amplify 338 base pair ( bp ) gene fragment in pcr reaction were 5-atggcacggccggttaagag-3 ( 20 nt , aug forward primer ) and 5-atacatttttcttcatgtttt-3 ( 21 nt , c2 reverse primer ) . the 25 l amplification reactions included 3 l template dna , the primers ( 0.8 m each ) , 1.25 u platinum taq dna polymerase ( invitrogen ) , 200 m dntps , and 1 platinum taq reaction buffer . the pcr amplification reaction was performed using the following calculated - control protocol : 5 minutes initial denaturation step at 95c , followed by 30 cycles of 1 minute at 94c , 2 minute at 50c , and 1 minute at 72c , and a final extension of 10 minute at 72c . each pcr included dna from the lung of an infected rat as a positive control and negative control , prepared by the replacement of template dna with distilled water . briefly , the number of p. carinii organisms was counted in stained slides prepared from 20 l lung homogenates of infected rats . dna was isolated from 100 l lung homogenate and pcr was performed as described above with 10-fold dilutions of dna from 1010 organisms . data obtained during the study were processed using prism 3.03 ( graphpad , san diego , ca ) . a two - tailed unpaired t test with 95% confidence interval was used to determine the significance between the results of assays . loss of appetite , dyspnea , and change of fur color from white to yellowish were observed at 3 and 5 weeks in orally and subcutaneously immune suppressed rats , respectively . after 12 week of immune suppression , the mean weight of the rat group administered oral dexamethasone significantly decreased from 7910 g to 656 g ( p<0.05 ) ; rats administered subcutaneous dexamethasone increased from 715 g to 772 g ( p=0.06 ) ; and the control group increased from 867 g to 17323 g ( p=0.0004 ) . the mean weights of oral ( p<0.0001 ) and subcutaneous ( p=0.0002 ) dexamethasone - administered rats were significantly lower than in control groups ( figure 1 ) . the percent weight losses in rats administered oral and subcutaneous dexamethasone were 62.4% and 55.5% , respectively . in gram weigert and giemsa stained slides , p. carinii was observed only in lungs of immune - suppressed rats . numerous cysts and trophozoites were detected in lung smears of rats administered oral dexamethasone , compared to few cysts observed in rats administered subcutaneous dexamethasone . trophozoites or cysts of p. carinii were not observed in the stained smears of remaining rat samples . the p. carinii msg gene was detected in immune suppressed and control group rats oral swab samples prior to dexamethasone administration , 2 , 6 and 12 week after immune suppression . in addition , pcr was positive in lungs of all rats , including the control group , after 12 week of immune suppression ( table 1 ) . pcr was positive in nasal swabs of rats administered oral dexamethasone at 2 , 6 and 12 weeks of immune suppression . the p. carinii msg gene was detected in nasal swabs of rats administered subcutaneous dexamethasone after 6 and 12 week of immune suppression . at 12 weeks of immune suppression , pcr detected msg gene in kidney and spleen tissues of immune - suppressed rats , in addition to lung tissues ( table 1 ) . each pcr reaction amplifying 338 bp fragment of msg gene has a detection limit of 10 organisms ( data not shown ) . loss of appetite , dyspnea , and change of fur color from white to yellowish were observed at 3 and 5 weeks in orally and subcutaneously immune suppressed rats , respectively . after 12 week of immune suppression , the mean weight of the rat group administered oral dexamethasone significantly decreased from 7910 g to 656 g ( p<0.05 ) ; rats administered subcutaneous dexamethasone increased from 715 g to 772 g ( p=0.06 ) ; and the control group increased from 867 g to 17323 g ( p=0.0004 ) . the mean weights of oral ( p<0.0001 ) and subcutaneous ( p=0.0002 ) dexamethasone - administered rats were significantly lower than in control groups ( figure 1 ) . the percent weight losses in rats administered oral and subcutaneous dexamethasone were 62.4% and 55.5% , respectively . in gram weigert and giemsa stained slides , p. carinii was observed only in lungs of immune - suppressed rats . numerous cysts and trophozoites were detected in lung smears of rats administered oral dexamethasone , compared to few cysts observed in rats administered subcutaneous dexamethasone . trophozoites or cysts of p. carinii were not observed in the stained smears of remaining rat samples . the p. carinii msg gene was detected in immune suppressed and control group rats oral swab samples prior to dexamethasone administration , 2 , 6 and 12 week after immune suppression . in addition , pcr was positive in lungs of all rats , including the control group , after 12 week of immune suppression ( table 1 ) . pcr was positive in nasal swabs of rats administered oral dexamethasone at 2 , 6 and 12 weeks of immune suppression . the p. carinii msg gene was detected in nasal swabs of rats administered subcutaneous dexamethasone after 6 and 12 week of immune suppression . at 12 weeks of immune suppression , pcr detected msg gene in kidney and spleen tissues of immune - suppressed rats , in addition to lung tissues ( table 1 ) . each pcr reaction amplifying 338 bp fragment of msg gene has a detection limit of 10 organisms ( data not shown ) . colonization is defined as detection of p. jiroveci in respiratory tract specimens without signs and symptoms of pcp . positive pcr results for p. jiroveci in patients without any symptom of pcp may cause undue treatment . to assist clinicians in the management of patients , current research has focused on developing novel quantitative pcr or sampling techniques to distinguish colonization from infection [ 1,2,14,15,1820 ] . colonization of p. jiroveci is demonstrated in nasopharyngeal aspirates and oropharyngeal washes of immune - competent individuals as well as immune - suppressed patients . due to its presence in the pharynx and lungs , detection of p. jiroveci by pcr in induced sputum , oropharyngeal or nasopharyngeal sampling does not definitively indicate that the oral or nasal cavity contains p. jiroveci , and therefore does not determine if the patient has active infection or colonization . to clarify the patient s clinical status , nasal and eye swabs , in addition to oral swabs , were first - time collected from a rat model in the present study . the immune - suppressed rat model has been frequently used in p. carinii studies , and administration of corticosteroids initiates deadly pcp in rats . in the present study , 2 groups of rats were administrated with oral and subcutaneous dexamethasone to initiate pneumocystosis . after 12 week of immune suppression , the mean weights of rat groups significantly decreased in oral ( p<0.0001 ) and subcutaneous ( p=0.0002 ) dexamethasone - administered rats compared to control rats ( figure 1 ) . the percent weight losses in rats administered oral and subcutaneous dexamethasone were 62.4% and 55.5% , respectively . the percent weight loss of various type of rats ( sprague - dawley , wistar , long - evans , brown norway ) was between 6070% after 12 week of subcutaneous methylprednisolone acetate administration ( 22 ) . the percent weight loss of sprague - dawley rats administered oral dexamethasone for 12 weeks was 47.4% , comparable with results of the present study . comparison of the weight losses of immune - suppressed rat groups in the present study showed that weight loss was significantly higher in rats administered oral dexamethasone ( p<0.05 ) . to determine the presence of p. carinii , stained slides containing smears of tissues , fecal material , blood , nasal , oral and eye swabs were examined with microscopy ; p. carinii was only detected in lung smears of immune suppressed rats . examination of the slides showed that numerous cysts and trophozoites were detected in lung smears of rats administered oral dexamethasone compared to few cysts observed in rats administered subcutaneous dexamethasone . however , p. carinii was detected by pcr in oral swabs of all rats prior to immune suppression and 2 , 6 and 12 week after immune suppression . in addition , pcr was positive in lungs of all rats , including the control group , after 12 week of immune suppression . p. carinii dna was investigated in rat fetuses , pups and their placentas just after birth . pcr was positive in all oral swabs of pups by 48 hours and was negative in fetuses and their placentas , indicating that p. carinii is acquired just after birth , but is not transplacental . in the present study , p. carinii dna was detected by pcr in nasal swabs of all immune - suppressed rats , but not in control rats . similarly , oz et al investigated nasal and pharyngeal aspirates and lung samples of rats using pcr . in immune - competent rats , p. carinii was not detected in any respiratory sample . in immune - suppressed rats , p. carinii dna was detected in nasal and pharyngeal aspirates , as well as in lung samples . in a study conducted in elderly individuals without any symptoms of pcp , p. jiroveci was detected in 12.8% of oropharyngeal washes and 10.6% in nasal swabs collected with a modified technique to avoid the nasopharynx . however , in another study , conducted in humans without pcp , p. jiroveci dna was scarce or absent in the oropharyngeal secretions . these results demonstrate that nasal sampling technique is adequate in rat models , but it has to be improved in humans . in the present study , in addition to lungs , p. carinii was also detected by pcr in kidney and spleen tissues of all immune - suppressed rats ( table 1 ) . p. carinii dissemination was investigated in rats ( n : 12 ) administered oral dexamethasone for 12 weeks and a control group ( n : 2 ) using pcr and microscopic examination . p. carinii dna was detected in immune - suppressed rats lungs ( 100% ) , spleen ( 58.3% ) , liver ( 50% ) , and kidney ( 50% ) and in control group rats lungs ( 100% ) , spleen ( 50% ) , and kidney ( 100% ) . p. carinii was detected by microscopic examination in immune - suppressed rats lungs ( 83.3% ) , spleen ( 16.6% ) , liver ( 8.3% ) , and kidney ( 8.3% ) . in another study , rats were fed an 8% protein diet and were administered methylprednisolone acetate for 8 weeks to exacerbate pcp . p. carinii was detected by pcr in rats adrenal gland ( 60% ) , bone marrow ( 60% ) , blood ( 40% ) , heart ( 70% ) , kidney ( 80% ) , liver ( 90% ) , lymph node ( 100% ) , lungs ( 100% ) , spleen ( 60% ) , and thyroid tissue ( 20% ) . the intense involvement of tissues detected in this study was associated with low - protein ( 8% ) diet , compared to the present study using a 14% protein diet . in a similar study , rats ( n : 6 ) were administered intra - muscular injection of hydrocortisone for 6 weeks ; pcr was only positive in lungs and was negative in blood , liver , spleen , and kidney . in the present study , weight losses was significantly higher in rats administered oral dexamethasone ( p<0.05 ) , and nasal swab of rats administered oral and subcutaneous dexamethasone were pcr positive after 2 and 6 week of immune suppression , respectively . the results of the present study show that oral administration of dexamethasone induces immune suppression slightly better than does subcutaneous administration . pcr was positive in lungs and oral swabs of rats prior to the administration of dexamethasone . after the administration of dexamethasone , the p. carinii msg gene was detected in oral swabs , lungs , spleen , kidney and first - time in nasal swabs . nasal swab pcr was negative before immune suppression in healthy rats , showing that colonization has not occurred . pcr was positive in nasal swabs during the second and sixth weeks of oral and subcutaneous administration of dexamethasone , respectively . in a patient without any symptoms of pcp the presence of p. carinii in nasopharyngeal aspirate , oropharyngeal wash , oral swab , induced sputum or bal , and absence in nasal swab , may support the clinician s decision that colonization has or has not occurred . overall , detection of p. carinii first - time in nasal swabs of rats by pcr demonstrated that nasal sampling can be used for the diagnosis of p. carinii pneumonia . therefore , non - invasive nasal swab sampling emerges as a better solution for determining the colonization of p. carinii in the upper respiratory tract .

protein transduction domains ( ptds ) such as hiv-1 tat , pantp43 - 58 , and polyarginine ( r9 ) are small peptides that are able to transduce a variety of peptides and proteins into several kinds of cells [ 13 ] . however , protein transduction technology using ptds has the disadvantage of entrapping the ptd - fused protein within the endosomal vesicles . it has been reported that the main mechanism of protein transduction is the penetration into cells by macropinocytosis ; therefore , much of the material becomes entrapped in the macropinosome [ 47 ] . in fact , pan et al . published a report on their attempt at reprogramming human fibroblast cells using tat fusion recombinant proteins , which was unsuccessful even with the help of an endosomal acidification inhibitor , chloroquine and an endosome - disruptive peptide , and hemagglutinin-2 subunit ( ha2 ) . also , it is reported that methanol fixation causes permeabilization of cell membranes and results in the artificial import of ptd - fused proteins . we focused on developing the transduction technology of proteins using the 30-amino acid peptide / transporter , wr - t , which has an enlarged hydrophobic pocket fused with nine d - enantiomer polyarginines via a gly - pro - gly spacer . to permit the efficient escape of proteins from the endosome , we used cationic lipids to enhance the proton sponge or endosome buffering effect , which is thought to induce osmotic swelling and the consequential rupture of the endosome . in this study , we developed a protein transduction method that can be cultured continuously for adherent living cells using both a functionally reinforced peptide transporter and commercially available cationic lipid reagents . wr - t peptide was synthesized at operon biotechnologies by fmoc solid - phase peptide synthesis . crude peptide was purified by reverse - phase high - performance liquid chromatography ( purity : 82.6% ) . protein expression plasmids were constructed using pew - destination vectors and a gateway entry clone by the gateway lr recombination reaction ( invitrogen life technologies ) . the cationic lipid reagents used for protein transduction included fugene6 ( roche diagnostics ) , lipofectamine ltx ( invitrogen life technologies ) and multifectam ( promega ) in dna transfection reagent , and prodeliverin ( oz biosciences ) and bioporter ( genlantis ) in protein delivery reagent . automated protein in vitro synthesizer protemist dt ( cell free science ) synthesized proteins using a wheat germ cell - free system and bilayer reaction . column affinity purification is also conducted available for purifying synthesized gst- or his - tagged fusion proteins by the use of the protemist dt . placing glutathione 4b ( ge healthcare ) or ni - sepharose high performance ( ge healthcare ) resin in each column , phosphate buffered saline , his ; 20 mm na - phosphate ph7.5 , 0.3 m nacl , 20 mm imidazole ) pass through the column , purified proteins were eluted by elution buffer ( gst ; 50 mm tris - hcl , 10 mm reduced glutathione , ph8.0 , his ; 20 mm na - phosphate ph7.5 , 0.3 m nacl , 500 mm imidazole ) . hela and mrc-5 cells were cultured in dmem containing 10% fetal bovine serum ( fbs ) and antibiotics ( 100 u / ml penicillin , 100 mg / ml streptomycin ) at 37c in an incubator with 5% co2 . to determine the intracellular localization of fusion proteins , hela cells then , wr - t peptide ( 3 m ) and the cargo protein ( 1 - 2 g ) were mixed in 100 l of pbs at room temperature for 15 min , and then cationic lipid reagents were added as follows : fugene6 , 1.5 l ; lipofectamine ltx , 1 l ; multifectam , 10 l ; prodeliverin , 1.5 l ; bioporter , 2.5 l . the solution was incubated at room temperature for 15 min and overlaid onto the cells , which had been aspirated and washed once with pbs before use . serum - free medium ( 100 l ) was added , and cells were incubated at 37c in 5% co2 for 3 h. intracellular localization of transduction proteins was visualized in living cells by an inverted fluorescence microscope ( olympus ix51 ) . cell viability was evaluated with celltiter - glo luminescent cell viability assay ( promega ) to determine the number of metabolically active cells in the culture based on quantitation of the atp present . a previous study demonstrated that wr - t induced the intracellular incorporation of cargo protein in fixed cells . to confirm that alexa-488-labeled bsa protein indeed diffused throughout the cytoplasm , the localization of bsa was compared to that of lysotracker red lysosomal probe ( lonza ) for endocytotic vesicles in living cells . no bsa was detected in the cytoplasm , which indicated that it was entrapped within the endosomal vesicles . therefore , we hypothesized that bsa could escape from endocytotic vesicles if the proton sponge effects of wr - t / bsa complexes were increased by the addition of cationic lipids . to characterize the functions of commercially available cationic lipid reagents for protein transduction , we investigated the localization of bsa in living hela and mrc-5 cells ( figures 1(b ) and s1a of supplementary material available at doi : 10.1155/2011/872065 ) . wr - t / fugene6 , wr - t / prodeliverin , and wr - t / bioporter mixtures of bsa appeared in punctuate vesicular structures , and it was suggested that these cationic lipid reagents did not induce the escape of bsa from endosomes . in contrast , we observed dramatic uptake and distribution of bsa throughout the cytoplasm of each hela cell when wr - t / multifectam and wr - t / lipofectamine ltx mixtures were used . thus , a mixture of wr - t and cationic lipid reagents ( multifectam or lipofectamine ltx ) was required to effectively enhance cell permeability and endosome escape of bsa . similar results were observed when alexa-555-labeled ovalbumin protein mixed with wr - t and cationic lipid reagents were transduced into living cells . in many cases , thus , we compared the degree of toxicity of wr - t and cationic lipid reagents mixtures in living cells . the cationic lipid reagents had low toxicity , while cell viability was increased by the wr - t and cationic lipid reagents mixture ( figure 1(c ) ) . we determined whether venus proteins ( variants of yellow fluorescent protein ) were delivered into hela cells . venus expression vectors were used to produce venus protein with a polyhistidine tag at the c - terminus ( 3h - venus ) . the soluble fractions of the recombinant venus proteins were purified on a ni sepharose column ( figure 2(a ) ) . even with a mixture of wr - t and cationic lipid reagents , 3h - venus was poorly delivered into the cells . furthermore , we detected aggregate fluorescence , which might have occurred because 3h - venus , wr - t and cationic lipid reagents partly form unstable complexes such as large aggregates ( figure 2(b ) ) . it is therefore reasonable to propose that wr - t and cationic lipid reagents mixtures would not be able to deliver all proteins into the cells . therefore , we devised a cargo protein fused with glutathione s - transferase ( gst ) and flag tag to promote stable association and not agglutination with wr - t peptide and cationic lipid reagents . we had previously confirmed that gst and flag tag used together increased the solubility and stability of expressed proteins , compared with gst tag alone . we produced venus protein with gst and flag tag at the c terminus ( 3gf - venus ) using a wheat germ cell - free protein synthesis system . the soluble fractions of the recombinant venus proteins were purified on a glutathione - sepharose affinity column ( figure 2(a ) ) . 3gf - venus migrated into hela cells and diffused throughout the cytoplasm with a mixture of wr - t and multifectam or lipofectamine ltx ( figure 2(c ) ) . in addition , the mixture of wr - t and bioporter markedly enhanced the cytoplasmic localization of 3gf - venus , unlike the case of bsa . we questioned whether these results were truly dependent on gst and flag tag , or whether they were due to variation in molecular weight . thus , we evaluated the delivery of two different tag - fused venus proteins , mcherry tag , and mkg tag , which have molecular weights approximately equal to those of gst and flag tag ( figure 2(d ) ) . virtually none of the venus - mcherry and venus - mkg proteins with wr - t and bioporter were able to enter the cells ( figure 2(e ) ) , and little fluorescence was observed throughout the cytoplasm . taken together , these data indicated that when venus protein is fused with gst and flag tag , they associate through noncovalent interactions with wr - t and bioporter and form stable complexes . to test whether a venus protein with nuclear transport signal ( nls ) can be selectively delivered to the nucleus , we produced venus protein fused with nls at the n terminus and gst and flag tag at the c terminus ( 3gf - nls venus ) in the same way as the 3gs - venus proteins . the 3gf - nls venus / wr - t / bioporter complexes were delivered to the nucleus in > 90% of hela cells ( figures 3(a ) and 3(b ) ) . on the other hand , < 30% of the cells exhibited localization to the nucleus with wr - t / multifectam or lipofectamine ltx complexes . similar results were shown in the case of mrc-5 cells ( figure s1b of supplementary material available at doi : 10.1155/2011/872065 ) . our findings showed that gst and flag tag - fused cargo proteins can be selectively delivered to the nucleus by using nls peptides with wr - t and cationic lipid reagents . furthermore , our results imply that by fusing high stability proteins such as gst and flag tag , cargo proteins agglutinated or not associated with wr - t and cationic lipid reagents could form stable complexes with them and be delivered into the target organelles . in conclusion , we described a strategy for the delivery of full - length proteins into living cells , based on association with wr - t and cationic lipid reagents . this allowed the delivery of distinct proteins into the living cells and escape from the endosomal vesicles , without the need for crosslinking or denaturation steps . this strategy is expected to be a useful tool for studying the role of proteins and protein - protein interactions in living cells , as well as cell reprogramming using recombinant proteins . this strategy may also be a potential clinical tool for tissue engineering and regenerative medicine .

a pleural loose body , which is also known as a ' pleural mouse ' , ' pleural fibrin body ' , or ' thoracolithiasis ' , is a rare benign condition . there have been several case reports of this benign condition , in which most of the loose bodies were calcified and up to 1.5 cm in size ( 123 ) . we report a pleural loose body found incidentally that was unusually large and non - calcified , mimicking a pedunculated pleural tumor on imaging studies . this case report was approved by our institutional review board and the patient 's informed consent was waived . a 58-year - old woman presented with a mass found incidentally on a chest radiograph . the posteroanterior chest radiograph showed a well - defined mass with a smooth margin in the right paratracheal area ( fig . precontrast computed tomography ( ct ) showed a 4 cm mass in the subcarinal area ( fig . 1b , c ) , but on the postcontrast ct obtained 2 minutes later , the mass was observed in the right paravertebral area ( fig . the attenuation value of the mass was 42 - 44 hounsfield units on precontrast ct , and the mass did not show contrast enhancement . in a follow - up posteroanterior chest radiograph , the mass had migrated to the subcarinal area ( fig . the impression based on the imaging studies was a pedunculated solitary fibrous tumor of the pleura , and the differential diagnosis included a benign neurogenic tumor . cystic mass types , such as the bronchogenic cyst , were not included in the differential diagnosis due to the mass ' mobile nature . a 3 4 cm " soap - like " white hard mass was found floating freely in the pleural cavity . the mass did not have a stalk connecting it to the parietal pleura ( fig . histopathologically , the mass consisted of hypocellular hyalinized collagen and the surface of the tumor showed some scattered chronic inflammatory cells and the characteristic " basket - weave " configuration of laminated hypocellular mature collagen ( fig . there have been a few case reports and case series , mainly from japan , which variably describe this condition as an ' intrapleural loose body ' , ' pleural fibrin body ' , or ' thoracolithiasis ' ( 123456 ) . the pleural loose body is rarely symptomatic and there are no age or sex predilections . most of the cases were found incidentally on imaging studies or at autopsy , although there have been a few case reports in which the mass developed after a thoracotomy or traumatic pneumothorax ( 56 ) . the size usually ranges from 5 - 15 mm ( 123 ) and it may or may not be calcified . the largest reported example was a 5 cm calcified mass ( 7 ) . in our case , the loose body was a 4 cm non - calcified fibrin body , which is the largest reported non - calcified loose body . although this mass was found around the carina , they tend to be located in the dependent portion of the thorax around the diaphragm . the mobile nature on sequential imaging studies is the most characteristic finding of a pleural loose body . ( 3 ) demonstrated the continuous migration of 11 calcified loose bodies on multiple sequential cts . our case also showed spontaneous shifting in every sequential imaging study , even in the 2 minute interval between the pre- and postcontrast cts . in a retrospective review of our case , another important diagnostic clue differentiating it from soft tissue tumors was the absence of contrast enhancement , although some benign tumors , such as benign neurogenic tumors , can show little contrast enhancement . they consist of an outer wall of fibrous tissue and a variable central core which is usually fatty tissue with or without necrosis ( 8) . calcification may or may not be present . in our case , the entire mass consisted of hyalinized collagen without a fatty core . in cases with a fatty core , magnetic resonance imaging can help with the differential diagnosis , since the fatty core shows high signal intensity on both t1- and t2-weighted images ( 9 ) . ( 3 ) proposed epipericardial fat necrosis as a possible mechanism for loose bodies with a fatty core , in which twisted , torn epipericardial fat undergoes aseptic fat necrosis and then may drop into the intrapleural space , resulting in a mobile loose body . for loose bodies not containing a fatty core , such as in our case , loose bodies have occurred after a thoracotomy ( 5 ) or traumatic pneumothorax ( 6 ) in which an exudative pleural effusion or hemothorax occurs , we postulated that a fibrin clot might form during resolution of the effusion or hemothorax . although our patient did not have a history of chest - related problems , she might have once suffered from self - limited pleurisy because tuberculous pleurisy is endemic in our country . a small calcified nodule migrating freely in the pleural cavity is virtually pathognomonic for thoracolithiasis , although a loose body with no or little calcification might be misdiagnosed as a benign neoplasm and resected with unnecessary surgery . some loose bodies have been reported to grow at follow - up ( 710 ) . our case was also misdiagnosed pre - surgically as a benign pedunculated solitary fibrous tumor of the pleura because of its non - calcified nature , mobility , and location between the lung and pleura , although the absence of contrast enhancement on ct did not favor a neoplastic condition . pre- and postcontrast ct may play an important role in the differential diagnosis and prevention of unnecessary surgery . in a mass abutting the pleura or mediastinum , the combination of mobility and no contrast enhancement is an important clue to diagnosing a pleural loose body . in patients with these imaging findings

bone scaffolds , which provide the benefit of avoiding unwanted immunological responses and eliminate the risk of acquiring infectious diseases from autografts and allografts , are widely used by orthopedic surgeons when repairing different types of bone defects . the traditional methods of manufacturing scaffolds mainly focus on reshaping the structure of specific types of materials and give the scaffold some biomedical properties via processes such as leaching or soaking . these methods can make scaffolds with higher porosity , but their size , shape , and interconnectivity are not easy to control , which may limit the prognosis in many aspects ; thus , getting satisfactory outcomes when treating bone defects using bone scaffolds is still very challenging . the development of three - dimensional ( 3d ) printing technology has dramatically changed scaffold designs in regenerative medicine . scaffold microstructures are able to regulate cell behaviors such as proliferation , differentiation , and apoptosis . with the recent rapid development of 3d printing technology , not only can we print 3d scaffolds with controllable inner microstructures but also we can have scaffolds composed with components in the extracellular matrix to deliver biomaterials . platelets represent a type of specific source of growth factors and cytokines that are involved in wound healing and tissue repair . many platelet - derived factors play important roles in cell proliferation and differentiation including platelet - derived growth factor ( pdgf ) , transforming growth factor ( tgf)-1 , and insulin - like growth factors-1 . in this study , we designed a 3d printed scaffold with gelatin and platelets , examined the proliferation of preosteoblasts in the scaffolds using a cell counting kit-8 ( cck-8 ) assay and the growth factor release at various time points . some of the pdgfs that were measured include tgf-1 and vascular endothelial growth factor ( vegf ) , which play significant roles in wound healing and tissue regeneration . blood from healthy donors was collected into 3.8% ( mass fraction ) sodium citrate - containing tubes ( lankang company , jinan , china ) . blood samples were centrifuged at 1500 g for 6 min , after which the samples were divided into three layers : a bottom layer composed of red blood cells , an intermediate layer composed of white blood cells , and a top player composed of plasma . all of the plasma layer and 3 mm of the intermediate layer were then centrifuged for another 6 min at 1000 g to obtain a two - part plasma : the upper three - quarters consisted of platelet - poor plasma and the lower quarter consisted of platelet - rich plasma ( prp ) . the scaffolds possess an internal porous structure designed in a computer aided design environment using the software rhino 5.0 ( robert mcneel & associates , usa ) . the internal pores were orderly arranged regular dodecahedrons within the scaffolds with = 1500 m . the titanium skeletons that formed the internal porous structure and the external appearance of scaffolds were 100 m in diameter . the 3d porous titanium was printed based on laser sintering technology ( concept laser mlab , germany ) in two sizes : 5 mm diameter and 3 mm thickness for cultivating cells in 96-well plate and 8 mm diameter and 3 mm thickness for implanting in vivo , respectively . three groups of scaffolds were prepared including clear scaffolds ( blank group ) , scaffolds with gelatin ( control group ) , and scaffolds with gelatin and platelets ( treated group ) . to prepare the scaffold with gelatin , 1 g nacl was added to 5 ml of gelatin solution ( 5% ) , and then the solution was perfused into a 3d printed scaffold and lyophilized well . the nacl was absterged using distilled water , and then freeze - dried for the second time . the sterilization process was treatment with ethylene oxide , and the scaffolds were prepared with gelatin . the treated groups were prepared by adding 20 l of prp to the scaffolds with gelatin then freeze - dried . the structure of the clear scaffold and scaffolds with gelatin and platelets was imaged as shown in figure 1 via scanning electron microscopy . all scaffolds were molded at 3 mm height and 5 mm diameter , which fit the wells of the 96-well microplate ( 3559 , 96wl , corning , usa ) . ( a ) the internal pores of the blank group ( scaffolds with gelatin ) , diameters ranging from 150 to 220 m . bar = 20 m . ( b ) the internal pores of the treated group ( scaffolds with gelatin and platelet ) , diameters ranging from 70 to 200 m . bar = 20 m . preosteoblasts ( mc3t3-e1 , osteoblast cell line ) were bought from the chinese academy of medical sciences . the cells were cultured in an alpha - minimum essential medium ( -mem ) supplemented with 5% fetal bovine serum , 2 mmol / l glutamine , and 100 g / ml penicillin streptomycin and incubated at 37c in a humidified atmosphere with 5% co2 . first , 5000 cells were seeded on a 75 cm culture flask with 8 ml medium . after the cells adhered to the flasks , the medium was removed and replaced by a new 8 ml medium . the medium was replaced every 3 days until the cells were filled in the flasks . then , 1 ml 0.25% trypsin was added to the flask , which was lightly joggled . the flask was cultivated in an incubator for 23 min and 6 ml of a new medium was added to stop digestion . the whole mixture medium was removed to a 15 ml centrifuge tube so that it could be centrifuged for 5 min at 1000 g . the supernatant was dumped , and 5 ml of a new medium was added followed by mechanical isolation . we set four groups including a control group and three types of scaffold groups in a 96-well microplate . three types of scaffolds with 200 l -mem were placed into the wells of the treated groups . each well in the 96-well microplate then received 100 l of cell suspension ( cell concentration : 2 10 cells / ml ) . the 96-well microplate was then incubated in a co2 incubator until we measured the cell viability and cytotoxicity . the gelatin scaffolds group and the gelatin and platelet scaffolds group were compared for release levels of vegf and tgf- after 3 , 6 , 9 , 12 , 15 , 18 , and 21 days via enzyme - linked immunosorbent assay ( elisa ) analysis . the levels of vegf and tgf- release were also measured via elisa after the scaffolds were stored at 25c for 3 months . samples were treated at room temperature , and the concentrations of vegf and tgf- were measured using company kits ( r&d systems , minneapolis , usa ) . two wells in each group were measured simultaneously and averaged , and then 10 l of cck-8 was added to each well of the 96-well microplate . the absorbance was measured at 450 nm with a microplate reader ( thermo scientific , multiskan go , usa ) . the time points chosen to evaluate the cytotoxicity were 1 , 3 , 5 , and 7 days . two wells in each group were measured simultaneously and averaged , then 10 l of cck-8 was added to each well of the 96-well microplate . the absorbance at 450 nm was measured with a microplate reader ( thermo scientific ) . chicago , il , usa ) by student 's single - sample t - test . blood from healthy donors was collected into 3.8% ( mass fraction ) sodium citrate - containing tubes ( lankang company , jinan , china ) . blood samples were centrifuged at 1500 g for 6 min , after which the samples were divided into three layers : a bottom layer composed of red blood cells , an intermediate layer composed of white blood cells , and a top player composed of plasma . all of the plasma layer and 3 mm of the intermediate layer were then centrifuged for another 6 min at 1000 g to obtain a two - part plasma : the upper three - quarters consisted of platelet - poor plasma and the lower quarter consisted of platelet - rich plasma ( prp ) . the scaffolds possess an internal porous structure designed in a computer aided design environment using the software rhino 5.0 ( robert mcneel & associates , usa ) . the internal pores were orderly arranged regular dodecahedrons within the scaffolds with = 1500 m . the titanium skeletons that formed the internal porous structure and the external appearance of scaffolds were 100 m in diameter . the 3d porous titanium was printed based on laser sintering technology ( concept laser mlab , germany ) in two sizes : 5 mm diameter and 3 mm thickness for cultivating cells in 96-well plate and 8 mm diameter and 3 mm thickness for implanting in vivo , respectively . three groups of scaffolds were prepared including clear scaffolds ( blank group ) , scaffolds with gelatin ( control group ) , and scaffolds with gelatin and platelets ( treated group ) . to prepare the scaffold with gelatin , 1 g nacl was added to 5 ml of gelatin solution ( 5% ) , and then the solution was perfused into a 3d printed scaffold and lyophilized well . the nacl was absterged using distilled water , and then freeze - dried for the second time . the sterilization process was treatment with ethylene oxide , and the scaffolds were prepared with gelatin . the treated groups were prepared by adding 20 l of prp to the scaffolds with gelatin then freeze - dried . the structure of the clear scaffold and scaffolds with gelatin and platelets was imaged as shown in figure 1 via scanning electron microscopy . all scaffolds were molded at 3 mm height and 5 mm diameter , which fit the wells of the 96-well microplate ( 3559 , 96wl , corning , usa ) . ( a ) the internal pores of the blank group ( scaffolds with gelatin ) , diameters ranging from 150 to 220 m . bar = 20 m . ( b ) the internal pores of the treated group ( scaffolds with gelatin and platelet ) , diameters ranging from 70 to 200 m . preosteoblasts ( mc3t3-e1 , osteoblast cell line ) were bought from the chinese academy of medical sciences . the cells were cultured in an alpha - minimum essential medium ( -mem ) supplemented with 5% fetal bovine serum , 2 mmol / l glutamine , and 100 g / ml penicillin streptomycin and incubated at 37c in a humidified atmosphere with 5% co2 . first , 5000 cells were seeded on a 75 cm culture flask with 8 ml medium . after the cells adhered to the flasks , the medium was removed and replaced by a new 8 ml medium . the medium was replaced every 3 days until the cells were filled in the flasks . then , 1 ml 0.25% trypsin was added to the flask , which was lightly joggled . the flask was cultivated in an incubator for 23 min and 6 ml of a new medium was added to stop digestion . the whole mixture medium was removed to a 15 ml centrifuge tube so that it could be centrifuged for 5 min at 1000 g . the supernatant was dumped , and 5 ml of a new medium was added followed by mechanical isolation . we set four groups including a control group and three types of scaffold groups in a 96-well microplate . three types of scaffolds with 200 l -mem were placed into the wells of the treated groups . each well in the 96-well microplate then received 100 l of cell suspension ( cell concentration : 2 10 cells / ml ) . the 96-well microplate was then incubated in a co2 incubator until we measured the cell viability and cytotoxicity . the gelatin scaffolds group and the gelatin and platelet scaffolds group were compared for release levels of vegf and tgf- after 3 , 6 , 9 , 12 , 15 , 18 , and 21 days via enzyme - linked immunosorbent assay ( elisa ) analysis . the levels of vegf and tgf- release were also measured via elisa after the scaffolds were stored at 25c for 3 months . samples were treated at room temperature , and the concentrations of vegf and tgf- were measured using company kits ( r&d systems , minneapolis , usa ) . two wells in each group were measured simultaneously and averaged , and then 10 l of cck-8 was added to each well of the 96-well microplate . the absorbance was measured at 450 nm with a microplate reader ( thermo scientific , multiskan go , usa ) . the time points chosen to evaluate the cytotoxicity were 1 , 3 , 5 , and 7 days . two wells in each group were measured simultaneously and averaged , then 10 l of cck-8 was added to each well of the 96-well microplate . the absorbance at 450 nm was measured with a microplate reader ( thermo scientific ) . chicago , il , usa ) by student 's single - sample t - test . as shown in figure 1 , the gelatin microscaffolds were successfully created in pores of the 3d printed porous titanium . we calculated that 100 gelatin pores existed in microscaffolds and found that their diameter was in the range of 100300 m . after freeze - drying the prp , the microscaffold could also stay in the titanium pores because of the expansibility of gelatin when absorbing water . the scaffold with platelets became smaller than before because the platelets adhered to the gelatin . elisa was used to measure the vegf and tgf-1 released over 21 days , with results as shown in figure 2 . it could be determined that there was a triggered release after the first 3 days and the growth factors released would be low and plain at later days , which presented a better sustained release function . all the treated groups showed significantly higher release of vegf and tgf-1 than that in the control group ( p < 0.05 ) . transforming growth factor-1 and vascular endothelial growth factor releasing in the supernatant of mc3t3-e1 cultured with scaffolds of treated group ( a ) tgf-1 releasing in the supernatant of mc3t3-e1 cultured with scaffolds of treated group after the scaffolds were made . ( b ) vegf releasing in the supernatant of mc3t3-e1 cultured with scaffolds of treated group after the scaffolds were made . data are shown as mean standard deviation , n = 3 . * p < 0.05 versus control . tgf-1 : transforming growth factor-1 ; vegf : vascular endothelial growth factor . after the scaffolds were stored at 25c for 3 months , vegf and tgf-1 were measured for another 21 days , with results as shown in figure 3 . the releasing curve not only seemed irregular compared to fresh scaffolds but also proved that the release function was sustained after 3 months of storage at room temperature . at 3 , 9 , 12 , 15 , 18 , and 21 days time points , treated group exhibited significantly higher than that in the control group ( p < 0.05 ) . transforming growth factor-1 and vascular endothelial growth factor releasing in the supernatant of mc3t3-e1 cultured with scaffolds of treated group ( a ) tgf-1 releasing in the supernatant of mc3t3-e1 cultured with scaffolds of treated group after the scaffolds were stored at 25c for 3 months . ( b ) vegf releasing in the supernatant of mc3t3-e1 cultured with scaffolds of treated group after the scaffolds were stored at 25c for 3 months . * p < 0.05 versus control group . tgf-1 : transforming growth factor-1 ; vegf : vascular endothelial growth factor . we chose three wells in each group randomly three times and measured the optical density at 450 nm to determine the number of viable cells [ figure 4 ] . after 21 days of culture , the cck-8 cell count of the control and treated groups was significantly higher than that of the blank group ( p = 0.019 and 0.024 , respectively ) . however , there was no significant difference ( p = 0.364 ) between the control group and the treated group after 21 days of culture . cell proliferation assay of the mc3t3-e1 using cell counting kit-8 assay after a 21-day culture . * p < 0.05 versus the blank group . the number of the living cells was determined by the cck-8 assay to detect cytotoxicity [ figure 5 ] . at the 3 days time point of culturing , the value of the cck-8 cell count of the control and treated groups was obviously higher than that of the blank group ( p < 0.05 ) . at 5 day of culture , the value of the cck-8 cell count of the control group remained above the level of the blank group ( p < 0.05 ) , with the treated group almost equal to the blank group . however , at 7 d of culture , cck-8 cell count of the control and treated groups was lower than that of the blank group ( p < 0.05 ) . as shown in figure 1 , the gelatin microscaffolds were successfully created in pores of the 3d printed porous titanium . we calculated that 100 gelatin pores existed in microscaffolds and found that their diameter was in the range of 100300 m . after freeze - drying the prp , the microscaffold could also stay in the titanium pores because of the expansibility of gelatin when absorbing water . the scaffold with platelets became smaller than before because the platelets adhered to the gelatin . elisa was used to measure the vegf and tgf-1 released over 21 days , with results as shown in figure 2 . it could be determined that there was a triggered release after the first 3 days and the growth factors released would be low and plain at later days , which presented a better sustained release function . all the treated groups showed significantly higher release of vegf and tgf-1 than that in the control group ( p < 0.05 ) . transforming growth factor-1 and vascular endothelial growth factor releasing in the supernatant of mc3t3-e1 cultured with scaffolds of treated group ( a ) tgf-1 releasing in the supernatant of mc3t3-e1 cultured with scaffolds of treated group after the scaffolds were made . ( b ) vegf releasing in the supernatant of mc3t3-e1 cultured with scaffolds of treated group after the scaffolds were made . data are shown as mean standard deviation , n = 3 . * p < 0.05 versus control . tgf-1 : transforming growth factor-1 ; vegf : vascular endothelial growth factor . after the scaffolds were stored at 25c for 3 months , vegf and tgf-1 were measured for another 21 days , with results as shown in figure 3 . the releasing curve not only seemed irregular compared to fresh scaffolds but also proved that the release function was sustained after 3 months of storage at room temperature . at 3 , 9 , 12 , 15 , 18 , and 21 days time points , treated group exhibited significantly higher than that in the control group ( p < 0.05 ) . transforming growth factor-1 and vascular endothelial growth factor releasing in the supernatant of mc3t3-e1 cultured with scaffolds of treated group ( a ) tgf-1 releasing in the supernatant of mc3t3-e1 cultured with scaffolds of treated group after the scaffolds were stored at 25c for 3 months . ( b ) vegf releasing in the supernatant of mc3t3-e1 cultured with scaffolds of treated group after the scaffolds were stored at 25c for 3 months . * p < 0.05 versus control group . tgf-1 : transforming growth factor-1 ; vegf : vascular endothelial growth factor . we chose three wells in each group randomly three times and measured the optical density at 450 nm to determine the number of viable cells [ figure 4 ] . after 21 days of culture , the cck-8 cell count of the control and treated groups was significantly higher than that of the blank group ( p = 0.019 and 0.024 , respectively ) . however , there was no significant difference ( p = 0.364 ) between the control group and the treated group after 21 days of culture . cell proliferation assay of the mc3t3-e1 using cell counting kit-8 assay after a 21-day culture . * p < 0.05 versus the blank group . the number of the living cells was determined by the cck-8 assay to detect cytotoxicity [ figure 5 ] . at the 3 days time point of culturing , the value of the cck-8 cell count of the control and treated groups was obviously higher than that of the blank group ( p < 0.05 ) . at 5 day of culture , the value of the cck-8 cell count of the control group remained above the level of the blank group ( p < 0.05 ) , with the treated group almost equal to the blank group . however , at 7 d of culture , cck-8 cell count of the control and treated groups was lower than that of the blank group ( p < 0.05 ) . as modern medicine advances , many varied methodologies are being explored to treat severe bone diseases . however , curing bone defects it remains very challenging , not only because of the complex stratified architecture of bone itself but also due to changes in the microenvironment including cytokines and blood supplies . our work is a combination of 3d printing technology that gives the scaffold an accurate structure , and biological treatments with platelets to enhance the osteogenesis effect , thus helping patients with bone defects obtain a better prognosis . the 3d printed scaffold appears gray , bright , and clean . using a scanning electron microscope the pores are equally distributed and connected , with a diameter of 1500 m , and are visible within the scaffold . the micropore structure is clearly visible at 200 magnification , with diameters in the range of 100300 m . it has been reported that when the pore diameter of a porous structure is 1540 m , fibrous tissue would grow into the interior of the material . when the diameter of pores is 40100 m when the diameter of the pores exceeds 150 m , the bone tissue can infiltrate completely into the pore structure of the scaffold , potentially improving the osteogenesis effect . the 3d geometric structure and interconnected pores of our scaffold offer a quite large internal surface area and a 3d structure , in which preosteoblasts are able to absorb more large molecules , promote cell adhesion , proliferation , and osteogenic capabilities . during the design process , we treated the scaffold with platelets for the sake of utilizing its cytokines secretion to enhance the regeneration of the defected area . platelets attract considerable attention for their bioactivation of scaffolds due to their simplicity , safety , and cost - effectiveness of autologous blood preparation . we tested the vegf and tgf-1 release for 21 days for different times at which the platelets were made immediately and stored for 3 months at 25c [ figures 2 and 3 ] to address whether the control group and treated group had different effects on cytokines control - release . from these results , it was indicated that the scaffolds with platelets adhered on the microscaffold could release growth factors effectively and be restored for a long time via this special preparation method . in the study the cells proliferated further and numerous cells were measured at day 21 via cck-8 assay , the cell count of the control group and treated groups was significantly higher than that of the blank group . however , there was no significant difference between the control group and the treated group . this result demonstrated that the gelatin and platelets in the 3d porous structure of the scaffold could also promote cytokine secretion and the infiltration of nutrients and metabolites in the medium , which enhanced the 3d printed scaffolds biocompatibility . therefore , this kind of scaffold may be used as a bone graft substitute material . the method we used to evaluate the viability was cck-8 assay , which calculated the relative cell proliferation rate based on 450 nm light absorption indirectly , to assess the degree of cytotoxicity . a higher cell proliferation rate was associated with the lower toxicity of different types of scaffolds . in this study , detection based on cytotoxicity test standards in vitro showed that preosteoblast proliferation increased rapidly in the early stage ( days 15 ) and then the velocity decreased ; we can see the results in figure 5 . in three groups with scaffolds and one group without a scaffold , preosteoblasts maintained remarkably good proliferation in the experimental group . however , the preosteoblasts proliferation seems poor in the treated group compared to the other two groups with scaffolds at day 7 because the cells became confluent on the well surface . this finding is in agreement with previous studies that prp at low concentration in a culture medium has positive effects on cell growth , while high concentrations of prp suppress viability and proliferation . therefore , the results indicate that the scaffolds we designed are not toxic to preosteoblast cells and may be safe to implant in animals . in conclusion , the results of this study confirm that our 3d printed scaffolds provided good tissue compatibility and improved the adhesion and proliferation of preosteoblasts . furthermore , the platelets we treated also showed a terrific sustained release effect for vegf and tgf-1 , even after being stored for 3 months . therefore , a 3d printed scaffold with gelatin and platelets is a suitable implant for cell proliferation and may be a good bone graft substitute material for bone defects . in the present study , 3d scaffolds were only observed in vitro for 3 months ; therefore , a longer observation period and in vivo experiments will be performed in future studies . this research was supported by a grant from the youth foundation of chinese academy of medical sciences ( no . this research was supported by a grant from the youth foundation of chinese academy of medical sciences ( no .

middle ear surgery under general anaesthesia is revolutionised with the introduction of hypotensive anaesthesia that provides a relatively bloodless field while using an operating microscope . the primary methods to minimise blood loss during middle ear surgery included mild head elevation of 15 , and infiltration or topical application of epinephrine ( 1 : 50,000 or 1 : 200,000 ) . currently , many inhalational or intravenous anaesthesia techniques were evaluated to offer ideal intra - operative conditions for middle ear surgery with their advantages and disadvantages . sodium nitroprusside and nitroglycerine precisely control the blood pressure due to their rapid onset and short duration of action , but intra - arterial blood pressure monitoring and electrocardiogram ( ecg ) with s - t segment analysis are mandatory . an infusion of 10 - 20 g / kg / h remifentanil is also useful but is associated with side effect of hyperalgesia . dexmedetomidine , a potent and selective 2-adrenoceptor agonist , is used as adjuvant to general anaesthesia during surgery at pre - operative state ( sedation ) , intra - operative state ( analgesia and hemodynamic stability ) and during post - operative period ( no respiratory depression ) . it is valuable because of its anaesthetic and analgesic - sparing effects with predictable and dose - dependent haemodynamic effects . the present prospective double blind placebo control randomised study was aimed to evaluate the effects of dexmedetomidine infusion on the requirement of isoflurane concentration to lower systolic blood pressure below 30% of baseline values , quality of oligaemic surgical field , and awakening time in patients undergoing middle ear surgery . after approval from institutional ethical committee and written informed consent , 64 adult patients of american society of anaesthesiologists ( asa ) physical status i and ii of both genders , aged 18 - 58 years , weighing 45 - 65 kg , scheduled for elective middle ear surgery , were enrolled for this prospective double - blind placebo controlled randomised study . exclusion criteria were presence of cardiac or respiratory disease , hypertension , obesity ( body mass index > 26 kg / m ) , hepatic or renal dysfunction , bleeding or coagulation disorders . patients with a history of anticipated difficult airway , those on sedatives , hypnotics or antihypertensive medication or allergy to any anaesthetic medications were also excluded from the study . patients were randomly divided into two groups of 32 patients each by computer generated random table number . patients of group i received infusion of dexmedetomidine 0.5 g / kg / h and patients of group ii received placebo infusion of normal saline during middle ear surgery after induction of anaesthesia till 20 min before completion of surgery . the study drug solution was prepared by an anaesthesiologist who was blinded to study protocol and was not involved for intra - operative data collection . all patients were admitted prior to the day of the surgery , and fasting of 6 h was ensured . on arrival to the operation theatre , the baseline systemic blood pressure , heart rate , peripheral oxygen saturation ( spo2 ) and ecg were recorded . after establishing the intravenous line , lactate ringer solution was started and they were pre - medicated with palonosetron ( 75 g ) , glycopyrrolate ( 0.2 mg ) , midazolam ( 2 mg ) and fentanyl ( 2 g / kg ) , 15 min before induction of anaesthesia . after pre - oxygenation for 3 min , anaesthesia was induced with propofol ( 2 mg / kg ) till loss of verbal command and tracheal intubation was facilitated with vecuronium 0.1 mg / kg . anaesthesia was maintained with 60% nitrous oxide in oxygen and isoflurane dial concentration was titrated to achieve a systolic blood pressure 30% below the baseline values . patients were mechanically ventilated to maintain the end - tidal concentration ( etco2 ) between 30 and 35 mm hg . intra - operatively , the heart rate , arterial blood pressure , ecg , etco2 and peripheral pulse oximetry ( spo2 ) were monitored and recorded at 5 min intervals till end of surgery . hypotension was treated by decreasing the dial concentration of isoflurane or rate of infusion and bradycardia was treated with intravenous atropine . during procedure the bleeding at surgical site was assessed by the surgeon as grade 0-no bleeding - excellent surgical conditions ; grade i - minimum bleeding , sporadic suction needed ; grade ii - diffuse bleeding , repeated suction needed ; and grade iii - considerable , troublesome bleeding , and continuous suction was needed . after surgery , the residual neuromuscular blockade was antagonized with neostigmine ( 0.05 mg / kg ) and glycopyrrolate ( 0.008 mg / kg ) . this duration of awakening time comprised from administration of reversal of neuromuscular blockade till sustained eye opening on command . patients were transferred to post - anaesthesia care unit for observation of any respiratory depression , haemodynamic changes , nausea / vomiting or any other drug - induced side - effects or complications . the sample size was based on previous studies in which 28 patients were required to detect 20% decrease in bleeding in the dexmedetomidine group with type 1 error of 0.01 and power of 90% . assuming 5% drop out rate , the final sample size was set at 64 patients . the demographic data for categorical variables were compared using chi - square test and statistical significance in time related variables were analysed using student 's t - test . the present study evaluated the clinical effects of dexmedetomidine infusion during middle ear surgery using operating microscope under general anaesthesia . it was successfully completed on 64 adult consenting patients , and all patients were included in the data analysis . the demographic data of age , sex , weight , asa physical status and duration of surgery were comparable between the groups [ table 1 ] . patient demographic characteristics the baseline values of mean heart rate and systolic blood pressure were comparable between the groups with no statistical significance . though mean heart rate values were comparable during intra - operative period between the groups but bradycardia ( heart rate < 54 beats / min ) was observed in two patients of group i which promptly responded to intravenous atropine . the mean heart rate was found to be higher in patients of group ii after the extubation while patients of group i did not show much variation in their mean heart rate values [ table 2 ] . changes in heart rate during anaesthesia the required percentage of isoflurane concentration was significantly less ( p < 0.05 ) to maintain the mean systolic blood pressure 30% below baseline values in patients of group i who received intra - operative dexmedetomidine infusion [ table 3 ] . comparison of mean percentage of isoflurane requirement to reduce systolic blood pressure 30% below control value the operating microscope was used throughout the middle ear surgery and surgeons observed grade i bleeding ( minimum bleeding with sporadic suction ) at surgical site in majority of patients of group i and none of the patients had bleeding of grade iii [ table 4 ] . none of the patients of group ii had significant reduction in bleeding at surgical site , thus it is evident that patients receiving dexmedetomidine infusion had a better surgical field as compared to patient of group ii ( p < 0.05 ) . assessment of intra - operative bleeding by surgeon ( n : 64 patients ) all patients were able to obey the commands , and the duration of awakening time and recovery were comparable between the groups . post - operative respiratory rate and peripheral spo2 were comparable with no episode of desaturation at any time . middle ear surgeries require good surgical field visibility with no post - operative nausea and vomiting . in the present study , the dexmedetomidine infusion was used to produce oligaemic surgical field during middle ear surgery using operating microscope . it is evident from the study that the patient receiving dexmedetomidine infusion has oligaemic surgical field and better visibility when compared to patient receiving placebo . these findings can be attributed to the fact that dexmedetomidine reduces sympathetic activity , resulting in lower blood pressure and reduced heart rate thereby decreasing blood loss at the surgical site to improve the quality of the surgical field . dexmedetomidine is a highly selective 2 adrenergic agonist and used as adjuvant in anaesthesia to reduce the intra - operative anaesthetic and analgesic requirement . it regulates the autonomic and cardiovascular systems by acting on blood vessels and inhibiting norepinephrine release at sympathetic terminals , thereby attenuating the heart rate and blood pressure responses to intra - operative stressful events of anaesthesia . currently , many inhalational or intravenous anaesthesia techniques were evaluated to offer ideal intra - operative conditions for middle ear surgery . judged that the intravenous anaesthesia technique provided better haemodynamic control , less movement , and faster emergence during middle ear surgery . short acting inhalational anaesthetics such as desflurane and isoflurane produced excellent operating conditions for otological surgery . even small amount of blood can obscure the microscopic operating field and decreasing the extravasation of blood may improve the results of surgical procedures . different techniques , to minimise intra - operative blood loss during middle ear surgery are used . the conventional techniques of electively lowering the blood pressure are positive pressure ventilation and administration of hypotensive drugs . in the study of bekker , patients received an initial loading dose of 1 g / kg of dexmedetomidine over 10 min , followed by a continuous infusion of 0.5 g / kg / h and they determined that intra - operative dexmedetomidine infusion was effective for blunting the perioperative haemodynamic responses with no incidence of hypotension or bradycardia . our present study was in accordance with their study as all patients were haemodynamically stable , and none of them required vasopressor support or bolus administration of fluid to maintain haemodynamic status . a meta - analysis of previous studies showed that the incidence of bradycardia requiring intervention was increased when maintenance dosages of dexmedetomidine were used in excess of 0.7 g / kg / h . in our study , no patients suffered from bradycardia as dexmedetomidine infusion was given in dose of 0.5 g / kg / h and loading dose of dexmedetomidine was not given . the result of the present study indicates that the use of dexmedetomidine infusion reduced the percentage of isoflurane concentration to maintain a systolic blood pressure 30% below baseline values . these findings confirm with a previous study of khan et al . which also showed that use of dexmedetomidine reduces the requirement of inhalational anesthetic . aho et al . and aantaa et al . also reported a reduction of isoflurane requirement in their study , thus confirm the synergism between isoflurane and dexmedetomidine . dexmedetomidine was well tolerated , and none of the patients developed any drug - related side - effects or complications in the perioperative period . the dexmedetomidine infusion did not affect the awakening time or delay the recovery from anaesthesia . coughing on the tracheal tube during awakening will increase venous pressure and may cause post - operative bleeding , so deep extubation with sooth recovery is preferable . guler et al . found that the increase in blood pressure and heart rate during extubation is decreased , and the quality of extubation is improved by dexmedetomidine . ebert et al . did not observe any apnoea , airway obstruction and hypoxemia with bolus doses of dexmedetomidine in their study , and they reported that the depression of respiration may be seen due to deep sedation . in our study , none of the patients suffered from respiratory depression as we did not use dexmedetomidine in high doses . ear surgery may cause post - operative dizziness ( vertigo ) , nausea and vomiting as inner ear is intimately involved with a sense of balance . induction with propofol decreases the post - operative nausea and vomiting in patients undergoing middle ear surgery . prophylaxis with palonosetron , a 5-hydroxytryptamine 3 receptor blocker in premedication was considered for the present study and none of the patients suffered from post - operative nausea and vomiting . during surgery , middle ear is open to the atmosphere , and there is no pressure build up . once the tympanic membrane graft is placed , the middle ear becomes a closed space . if nitrous oxide is allowed to diffuse into this space , middle ear pressure will rise and may interfere with tympanic membrane reconstruction . withdrawing nitrous oxide 1020 min before placement of graft is the usual technique used in our institution . dexmedetomidine infusion was safe to provide oligaemic surgical field for better visualisation under operating microscope for middle ear surgery keeping the haemodynamic variations within the physiological range . it also reduced the requirement of isoflurane and recovery from anaesthesia was complete and smooth .

the interposition of sural nerve grafts for the correction of an iatrogenic sectioning of the obturator nerve during oncologic pelvic surgeries has been rarely described in the literature ( zhang et al . , 2013 , benes , 1999 ) . the obturator nerve is an important landmark during pelvic lymph node dissection and its identification is mandatory . this injury ( indirect thermal or direct mechanical injury as a result of a sharp dissection ) can occur in association with pelvic lymphadenectomy for uterine or cervical cancer and its incidence does not appear to differ between open surgical and laparoscopic approaches . also gynecologic oncology patients undergoing exenterating or extensive retroperitoneal surgery are at increased risk of this injury ( harma et al . , 2014 ) . clinical consequences of this type of lesions vary and may include pain down the medial thigh , variable gait disturbance , weakness of adduction of the thigh and inconstant sensory loss over the medial thigh ( rothmund et al . , 2011 ) . nerve grafting is a reconstructive procedure extensively described in the literature for the correction of different instances of nerve injury . some variation can be observed in the technique , concerning materials utilized , choice of donor site , number of grafts used and specific characteristics of the injury to be treated . this range of technical possibilities depicts the ongoing pursuit of the ideal surgical method and may explain the variability in the currently observed outcomes ( ricciardi et al . , 2012 , spiliopoulos and williams , 2011 ) . other nerves are also used less frequently : the superficial branch of the radial nerve ( upper limb ) , the medial cutaneous nerve of the forearm ( upper limb ) , the greater auricular nerve ( cervical ) , and the saphenous nerve ( lower limb ) . what they have in common is that all are sensitive nerves and its removal causes only sensory deficits without motor alteration . the number of grafts has to be sufficient to occupy the whole area of the transected nerve and to allow the bulk of regenerating axons reaching the distal stump of the nerve . thus , it is common to utilize the maximum possible grafts ( martins et al . , was referred to the sao paulo state cancer institute with a histologically confirmed poorly differentiated endometrioid endometrial carcinoma . a preoperative ct - scan showed no evidence of secondary lesions or enlarged retroperitoneal lymph nodes , therefore a video laparoscopy was indicated for total hysterectomy , bilateral salpingo - oophorectomy , pelvic and paraaortic lymphadenectomy . during the right iliac lymphadenectomy , because nerves consist mostly of connective tissue and not neural tissue , at the time of any section , the collagen and elastin present in the nerve cause a retraction of the divided ends , hindering primary anastomosis . at this moment the laparoscopy was interrupted and converted to a laparotomy because the neurosurgeon who performed the correction of the lesion was more used to laparotomy , and reconstruction of the obturator nerve requires the use of delicate instruments and magnification by a microscope , which are difficult to achieve with laparoscopy . after careful analysis of the characteristics of the lesion ( mechanical total section plus thermal injury and presence of a considerable gap between the extremities ) , the neurosurgeon proceeded to a microsurgical repair of the nerve injury through the interposition of three sural nerve grafts ( fig . 1 , fig . 2 , fig . 3 ) , because the epineural end to end tension free anastomosis was not possible . to obtain a sample of the sural nerve , a continuous longitudinal incision was employed . it has started at the most distal portion of the incision , the ankle , where the sural nerve was located in the subcutaneous tissue between the lateral malleolus and the achilles tendon . once identified , the nerve was dissected proximally . when the dissection approached the proximal third of the leg , it was necessary to open the fascia of the leg on the gastrocnemius and soleus muscles , to expose the nerve . at the end of the dissection , , under magnification , the excess soft tissue ( fascia and fat ) adhering to the graft was removed while maintaining it in a saline solution ( martins et al . , 2013 ) . after intense physiotherapy , the patient showed complete recovery of the adduction of the right leg , and electroneuromiography performed six months later showed clear signs of reinnervation of the adductor longus muscle . a 58 year - old woman was referred to the sao paulo state cancer institute with a histologically confirmed poorly differentiated endometrioid endometrial carcinoma . a preoperative ct - scan showed no evidence of secondary lesions or enlarged retroperitoneal lymph nodes , therefore a video laparoscopy was indicated for total hysterectomy , bilateral salpingo - oophorectomy , pelvic and paraaortic lymphadenectomy . during the right iliac lymphadenectomy , the nerve was transected by the vessel sealing system . because nerves consist mostly of connective tissue and not neural tissue , at the time of any section , the collagen and elastin present in the nerve cause a retraction of the divided ends , hindering primary anastomosis . at this moment , a neurosurgeon experienced in peripheral nerves was involved . the laparoscopy was interrupted and converted to a laparotomy because the neurosurgeon who performed the correction of the lesion was more used to laparotomy , and reconstruction of the obturator nerve requires the use of delicate instruments and magnification by a microscope , which are difficult to achieve with laparoscopy . after careful analysis of the characteristics of the lesion ( mechanical total section plus thermal injury and presence of a considerable gap between the extremities ) , the neurosurgeon proceeded to a microsurgical repair of the nerve injury through the interposition of three sural nerve grafts ( fig . 1 , fig . 2 , fig . 3 ) , because the epineural end to end tension free anastomosis was not possible . to obtain a sample of the sural nerve , a continuous longitudinal incision was employed . it has started at the most distal portion of the incision , the ankle , where the sural nerve was located in the subcutaneous tissue between the lateral malleolus and the achilles tendon . when the dissection approached the proximal third of the leg , it was necessary to open the fascia of the leg on the gastrocnemius and soleus muscles , to expose the nerve . at the end of the dissection , , under magnification , the excess soft tissue ( fascia and fat ) adhering to the graft was removed while maintaining it in a saline solution ( martins et al . , 2013 ) . after intense physiotherapy , the patient showed complete recovery of the adduction of the right leg , and electroneuromiography performed six months later showed clear signs of reinnervation of the adductor longus muscle . the anatomy and function of the obturator nerve have been well studied , and are known to be occasionally damaged during extensive pelvic surgery , which most noticeably causes impairment of ipsilateral leg adduction . it traverses along the medial border of the psoas muscle , passes lateral to the hypogastric vessels and ureter , and follows along the lateral wall of the lesser pelvis until reaching the obturator foramen with the medially converging obturator vessels . before emerging from the obturator canal these branches provide motor function to leg adductor muscles and sensory fiber to the hip joint , knee joint and the medial aspect of the middle and lower thirds of the thigh ( fishman et al . ideally , a complete section of the obturator nerve should be immediately repaired , preferably using microsurgical techniques ( grbz et al . , 2012 ) . although epineural end - to - end tension - free coaptation is currently considered the most efficient method to restore anatomy and function of the structure , in the present case the loss of neural substance led to the impossibility of a tension - free reattachment of the ends ( kavoussi et al . , 1993 ) . prognosis of obturator nerve transection depends on the nature and the severity of the injury . however , patients with a repaired nerve transection usually develop complete resolution of the symptoms within one year after the incident ( hallgren et al . , 2013 ) . if intraoperative evaluation by a neurosurgeon is not possible , the correction should be performed in a second operation . the postoperative fibrosis associated with a tissue retraction , makes the identification of the retracted stumps difficult . at the time of the injury and with unsuitable conditions to perform the repair , a way to try to avoid excessive shrinkage and to facilitate the subsequent identification of the stumps , can be obtained by passing a prolene suture of 5 - 0 or 6 between the epineurium ( the tissue that surrounds the nerve ) and a fixed structure of the region as a fascia . the repair should be performed as soon as possible ( 13 days after the injury ) ( ghaemmaghami et al . , 2009 ) . we performed the correction of the obturator nerve injury using the technique of sural nerve graft interposition . the sural nerve receives sensory afferents from the lateral part of the foot , i.e. it is related to the sensitivity of this region . their withdrawal is generally well tolerated by patients , with complications being rare ( such as infection of the incision or dehiscence ) . the sensory loss is well tolerated and , over time , there is a progressive reduction of this loss ( ghaemmaghami et al . , 2009 ) .

purple urine bag syndrome ( pubs ) is an uncommon condition where the urine bag and tubing of long - term catheterized patients turn purple . first reported by barlow and dickson in 1978 it is more commonly seen in chronically catheterized women and associated with chronic constipation . the process of purple discoloration has been described extensively beginning with deamination of tryptophan to tryptophase by the intestinal anaerobic bacteria which form indole , pyruvic acid , and ammonia . indole is transported via portal circulation and converted to indoxyl sulfate ( indicant ) in the liver . indican excreted through urine is converted to indoxyl by indoxylsulfatase and indoxyl phosphatise produced by urinary bacteria . indoxyl on oxidation in alkaline urine produces pigments indigo ( blue ) and indirubin ( red ) . indirubin reacts with the urine bag and along with indigo to produce a purple color . dehydration is also an important factor as the serum concentration of indoxyl sulfate was found to have a linear correlation with the severity of the azotemia . we encountered an 83-year old male with the history of benign enlargement of prostate and chronic renal failure on long - term catheterization who presented with purple discoloration of his urine bag and catheter [ figure 1 ] . there was no associated fever , altered sensorium , abdominal pain , or constipation with no findings suggestive of systemic infection . urine analysis yielded alkaline urine [ ph-7 ] with few white blood cells and microscopic hematuria , serum creatinine was 3.06 mg% while blood sugars and electrolytes were within normal range . the urine culture yielded insignificant growth of klebsiella pneumonia , morganellamorganii , enterococcus , citrobaterdiversus , and pseudomonas aeruginosa . this the urine color changed normal but purple discoloration reappeared after 2 weeks and persisted . the prevalence of pubs has been reported globally ranging from 9.8% to 8.3% among hospitalized patients and 16.7% to as high as 42.1% among patients with prolonged catheter use . it was found to be associated with alkaline urine , urinary tract infections , renal failure , polyvinyl chloride urine bag , and dementia . though most cases of pubs turn out to be clinically harmless underlying uti can lead to serious complications as typical symptoms of dysuria are absent . the awareness of this syndrome and need to initiate treatment for urinary tract infection are necessary as the outcome may be fatal if progresses to generalized septicemia . considering the changing demography of the indian population with increasing life expectancy , improved geriatric care and geriatric specialty centers , it would serve better if practicing clinicians in primary and secondary health care setting be aware of this condition . it would serve to avoid expensive investigations , unjustified antibiotic use , and unwarranted anxiety among patients , care takers and treating physicians while promoting early diagnosis and management of pubs . creating awareness among patients and care - providers would benefit them in reducing anxiety and unwarranted cost of repeated health visits .

primary -cells and ins1 cells were transfected with matrix - targeted photoactivatable green fluorescent protein ( pagfpmt ) expressed under the insulin promoter and allowed to accumulate in the mitochondrial matrix for 4872 h. mitochondria were labeled with 7 nmol / l tmre ( tetramethylrhodamine - ethyl - ester - perchlorate ; invitrogen , eugene , or ) for 45 min prior to imaging to allow visualization . the transition to active green fluorescent protein ( gfp ) was achieved by photoisomerization using 2-photon laser ( 750 nm ) excitation to give a 375 nm photon equivalence at the focal plane . this allowed for selective activation of regions < 0.5 m ( zeiss lsm510 ) or > 4 m ( leica tcs sp2 ) . in the absence of photoactivation , the presence of preactivated pagfpmt was detected using high - intensity excitation at 488 nm in combination with a fully opened collection pinhole . we used the dilution of pagfpmt into the mitochondrial web as an index for mitochondrial fusion ( 16 ) . a region of interest ( roi ) occupying 1015% of the cell in one confocal section was activated using a 2-photon laser ( 350 nm equivalence at the focal plane ) . the dilution of activated pagfpmt within the mitochondrial web was assessed by repeatedly scanning the entire cell volume ( six confocal z - axis section projection ) at 3- to 10-min time intervals for 50 min . quantification of fusion was performed using metamorph ( molecular devices , sunnyvale , ca ) by measuring the average fluorescence intensity ( fi ) of the mitochondria that became pagfpmt positive based on a fixed threshold . the gfp fi values of pagfpmt dilution were normalized to the gfp fi value immediately after photoactivation and then fitted to a hyperbolic function : f(t ) = 1 fplateau t/(t + t50 ) . f and t50 denotes the time interval to a 50% decrease in normalized gfp fi ( [ 1 fplateau]/2 ) . all fitting procedures and statistical tests were conducted using kaleida - graph software ( synergy software , reading , pa ) . paired student 's t tests were performed to calculate statistical significance . prior to measuring fi , we used an integrated morphometry analysis function designed for these experiments to extract pagfpmt - positive structures that were larger than 10 pixels . this procedure enabled the selection of mitochondrial structures using very low threshold levels in the green channel ( 10% of the image average intensity ) , assuring that > 90% of the mitochondrial pixels were included for analysis . to set the threshold level , a test - threshold function first measured the average green fi of the mitochondria . the lower ( inclusive ) threshold was set at two - thirds of this average . prior to analysis , all images were scanned to verify that all intensity measurements were below saturation ; therefore , an upper threshold was not necessary . primary -cells and ins1 cells were transfected with matrix - targeted photoactivatable green fluorescent protein ( pagfpmt ) expressed under the insulin promoter and allowed to accumulate in the mitochondrial matrix for 4872 h. mitochondria were labeled with 7 nmol / l tmre ( tetramethylrhodamine - ethyl - ester - perchlorate ; invitrogen , eugene , or ) for 45 min prior to imaging to allow visualization . the transition to active green fluorescent protein ( gfp ) was achieved by photoisomerization using 2-photon laser ( 750 nm ) excitation to give a 375 nm photon equivalence at the focal plane . this allowed for selective activation of regions < 0.5 m ( zeiss lsm510 ) or > 4 m ( leica tcs sp2 ) . in the absence of photoactivation , the presence of preactivated pagfpmt was detected using high - intensity excitation at 488 nm in combination with a fully opened collection pinhole . we used the dilution of pagfpmt into the mitochondrial web as an index for mitochondrial fusion ( 16 ) . a region of interest ( roi ) occupying 1015% of the cell in one confocal section was activated using a 2-photon laser ( 350 nm equivalence at the focal plane ) . the dilution of activated pagfpmt within the mitochondrial web was assessed by repeatedly scanning the entire cell volume ( six confocal z - axis section projection ) at 3- to 10-min time intervals for 50 min . quantification of fusion was performed using metamorph ( molecular devices , sunnyvale , ca ) by measuring the average fluorescence intensity ( fi ) of the mitochondria that became pagfpmt positive based on a fixed threshold . the gfp fi values of pagfpmt dilution were normalized to the gfp fi value immediately after photoactivation and then fitted to a hyperbolic function : f(t ) = 1 fplateau t/(t + t50 ) . f and t50 denotes the time interval to a 50% decrease in normalized gfp fi ( [ 1 fplateau]/2 ) . all fitting procedures and statistical tests were conducted using kaleida - graph software ( synergy software , reading , pa ) . paired student 's t tests were performed to calculate statistical significance . prior to measuring fi , we used an integrated morphometry analysis function designed for these experiments to extract pagfpmt - positive structures that were larger than 10 pixels . this procedure enabled the selection of mitochondrial structures using very low threshold levels in the green channel ( 10% of the image average intensity ) , assuring that > 90% of the mitochondrial pixels were included for analysis . to set the threshold level , a test - threshold function first measured the average green fi of the mitochondria . the lower ( inclusive ) threshold was set at two - thirds of this average . prior to analysis , all images were scanned to verify that all intensity measurements were below saturation ; therefore , an upper threshold was not necessary . primary -cells and ins1 cells were transfected with matrix - targeted photoactivatable green fluorescent protein ( pagfpmt ) expressed under the insulin promoter and allowed to accumulate in the mitochondrial matrix for 4872 h. mitochondria were labeled with 7 nmol / l tmre ( tetramethylrhodamine - ethyl - ester - perchlorate ; invitrogen , eugene , or ) for 45 min prior to imaging to allow visualization . the transition to active green fluorescent protein ( gfp ) was achieved by photoisomerization using 2-photon laser ( 750 nm ) excitation to give a 375 nm photon equivalence at the focal plane . this allowed for selective activation of regions < 0.5 m ( zeiss lsm510 ) or > 4 m ( leica tcs sp2 ) . in the absence of photoactivation , the presence of preactivated pagfpmt was detected using high - intensity excitation at 488 nm in combination with a fully opened collection pinhole . we used the dilution of pagfpmt into the mitochondrial web as an index for mitochondrial fusion ( 16 ) . a region of interest ( roi ) occupying 1015% of the cell in one confocal section was activated using a 2-photon laser ( 350 nm equivalence at the focal plane ) . the dilution of activated pagfpmt within the mitochondrial web was assessed by repeatedly scanning the entire cell volume ( six confocal z - axis section projection ) at 3- to 10-min time intervals for 50 min . quantification of fusion was performed using metamorph ( molecular devices , sunnyvale , ca ) by measuring the average fluorescence intensity ( fi ) of the mitochondria that became pagfpmt positive based on a fixed threshold . the gfp fi values of pagfpmt dilution were normalized to the gfp fi value immediately after photoactivation and then fitted to a hyperbolic function : f(t ) = 1 fplateau t/(t + t50 ) . f and t50 denotes the time interval to a 50% decrease in normalized gfp fi ( [ 1 fplateau]/2 ) . all fitting procedures and statistical tests were conducted using kaleida - graph software ( synergy software , reading , pa ) . paired student 's t tests were performed to calculate statistical significance . prior to measuring fi , we used an integrated morphometry analysis function designed for these experiments to extract pagfpmt - positive structures that were larger than 10 pixels . this procedure enabled the selection of mitochondrial structures using very low threshold levels in the green channel ( 10% of the image average intensity ) , assuring that > 90% of the mitochondrial pixels were included for analysis . to set the threshold level , a test - threshold function first measured the average green fi of the mitochondria . the lower ( inclusive ) threshold was set at two - thirds of this average . prior to analysis , all images were scanned to verify that all intensity measurements were below saturation ; therefore , an upper threshold was not necessary . a three - dimensional reconstruction of mitochondria within an entire -cell has been projected onto a single plain to illustrate the density of these organelles ( fig . this morphology suggests the presence of either large continuous networks throughout the cell or , alternatively , small discrete networks juxtaposed with one another . to decipher this complex architecture to its individual components , because individual mitochondria are often in apposition with one another , tagging individual mitochondria with pagfpmt targeted to the matrix allows morphological and biophysical studies of mitochondria within a complex web . in addition , this methodology takes into account organellar movement and mitochondrial dynamics that continuously change the mitochondrion 's size , shape , and location ( 6 ) . with adenoviral delivery , pagfpmt is expressed in all -cell mitochondria but remains in its inactive form ( nonfluorescent ) . c : summary of mitochondrial size distribution performed by 90 photoactivation steps in 9 cells . d : tagging and tracking individual mitochondria in primary -cells reveals fusion events ( transfer of activated pagfpmt to juxtaposed unlabeled mitochondria ) that are then followed by fission events ( separation of previously connected mitochondria ) . ( a high - quality digital representation of this figure is available in the online issue . ) precise ( 0.5 m ) photoactivation of pagfpmt molecules was achieved using a 2-photon laser . equilibration of activated pagfpmt was immediate and revealed the boundaries of matrix continuity of individual mitochondria . figure 1b shows five sequential photoactivations revealing the boundaries of multiple individual mitochondrial networks in a mouse -cell . whereas tmre staining may show continuous mitochondrial structures , the spread of matrix pagfpmt is limited to short segments , indicating that these segments do not form a continuous lumen . in 90 photoactivation events performed in nine different cells , we found that most ( > 95% ) individual networks are rod - like in shape with 76% exhibiting a length < 2 m ( fig . a series of control studies confirmed that laser intensity was kept at levels that did not affect mitochondrial function or morphology . these included time - lapse studies of mt in cells exposed to sequential laser scanning and dose - response studies with the 2-photon laser ( experiments are described in an online appendix at http://diabetes.diabetesjournals.org/cgi/content/full/db07-1781/dc1 and in ref . the small network size of -cell mitochondria raises the possibility that mitochondrial fusion is absent in these cells or , alternatively , that fusion events are transient and brief . to address this issue , fusion events were identified by the transfer of pagfpmt from a labeled mitochondria to a previously unlabeled one when the two came in contact as shown in fig . fusion events were commonly followed by fission ( 11 of 12 events ) in a manner that reverted to prefusion size and morphology of the involved mitochondria . we undertook a series of experiments designed to manipulate the balance between fusion and fission to assess the consequence on mitochondrial architecture in primary and clonal -cells . of particular interest is the fusion protein opa1 because transcriptom databases suggest its expression levels in the -cell is uniquely high ( www.t1dbase.org ) . to test the significance of opa1 in modulating -cell mitochondrial architecture , we overexpressed the protein by adenoviral transduction . overexpression of opa1 in primary -cells caused mitochondria to fragment into even smaller units ( fig . in ins1 cells , transduction with the same number of opa1 adenoviral particles caused mild overexpression and increased density in mitochondrial architecture . increasing the amount of virus further increased the expression level and led to fragmentation of the mitochondrial network ( fig . these findings suggest that the increased degree of fusion may promote elongation ; however , further increases in opa1 levels can lead to fragmentation . gfp infections were performed to control for potential effects of viral transduction and normalized so that cells were exposed to the same number of viral particles . we found that expression of gfp did not affect mitochondrial morphology in any of the experiments performed . a : in primary -cells , overexpression of the fusion protein opa1 leads to mitochondrial fragmentation . overexpression was achieved by adenoviral transduction of gfp or opa1 at equal concentrations of viral particles . b : level of opa1 overexpression in ins1 cells was controlled by the dose of viral particles used . mild overexpression causes mitochondria to take on a dense and elaborate morphology compared with controls . c : in primary -cells , overexpression of dn drp1 to reduce mitochondrial fission leads to mitochondrial swelling . d : in ins1 cells , overexpression of dn drp1 leads to network superfusion and a limited amount of mitochondrial swelling . the number and concentration of viral particles used in a , c , and d was identical . ( a high - quality digital representation of this figure is available in the online issue . ) an alternative approach to examining the consequences of a pro - fusion state is to prevent fission . we found that manipulating the pro - fission protein drp1 also caused dramatic changes in mitochondrial architecture . disruption of fission in primary -cells by overexpressing dominant negative ( dn ) drp1 resulted in swelling of the mitochondria ( fig . 2c ) . in ins cells , similar treatment with . the overall rate of mitochondrial fusion activity within an isolated -cell was assessed by photoactivating a subpopulation of mitochondria and monitoring the spread as well as the dilution of active pagfpmt throughout the rest of the cell 's mitochondria ( fig . the gradual spread of pagfpmt signal to other parts of the -cell , which were not photoactivated , is accompanied by dilution of pagfpmt ( fig . 3a and b ) . as a result , the average pagfpmt fluorescence intensity in mitochondria that contain photoactivated pagfpmt gradually decreases as long as the process of spreading is in progress . a : fusion of the photoactivated fraction ( 10% ) with the rest of the mitochondrial web dilutes activated pagfpmt and leads to a reduction in fluorescence intensity . b : dilution of gfp fi is shown for individual cells ( n = 6 ; gray lines ) and their average se ( ) . the average dilution values were fitted ( r = 0.99 ) to a hyperbolic function yielding t50 of 12.1 min . c and d : -cells within an intact islet also exhibit mitochondrial dynamics as revealed by the diffusion of the activated pagfpmt signal . ( see research design and methods and supplemental material for imaging and image analysis methodology ) . e : fusion activity in primary -cells is reduced after 24 h in hfg media . images taken during the assay show that the number of pagfpmt - positive mitochondria and the fi of pagfpmt remained unchanged . the hfg - treated cells possess significantly brighter gfp intensity ( p = 0.04 ) than controls , indicating reduced mitochondrial fusion activity . ( a high - quality digital representation of this figure is available in the online issue . ) to rule out the possibility that decay in pagfpmt fi was the result of either nonspecific leak of activated pagfpmt from tagged mitochondria or bleaching of pagfp by the laser , two experiments were conducted : gfp fi was measured over time 1 ) in individual mitochondria that did not engage in fusion events and 2 ) in the presence of fccp , a mitochondrial inner membrane uncoupler that collapses m and arrests mitochondrial dynamics . in both experiments gfp fi remained unchanged and stable over a period of 20 min , indicating a negligible component of nonspecific pagfpmt leak or laser - induced bleach ( see mitochondrial fusion assay in supplemental material ) . individual -cells within an islet were tested for mitochondrial fusion activity using pagfpmt that is under the insulin promoter ( fig . 3c ) . we found that fusion activity is functional and comparable with that in isolated -cells . interestingly , there is greater heterogeneity in the overall fusion activity among individual cells within intact islets exhibiting varied rates of gfp dilution ( fig . exposure to increased levels of glucose and fatty acids has been shown to compromise -cell function and viability ( 10 ) . incubation in 20 mmol / l glucose along with the fatty acid palmitate ( 0.4 mmol / l complexed with 0.5% bsa ) for 48 h resulted in fragmentation of mitochondrial morphology and inhibition of mitochondrial dynamics in -cells within mouse islets ( fig . the mitochondrial fusion capability of -cells decreased significantly under hfg ( 20 mmol / l glucose and 0.4 mmol / l palmitate ) which was revealed by the decreased ability of cells to dilute the gfp signal 2 h after photoactivation ( fig . ( 10 ) showed that in ins1 cells , exposure to 20 mmol / l glucose and > 0.2 mmol / l palmitate for 24 h leads to apoptosis and that caspase inhibitors do not rescue the cells from death ( 10 ) . we used confocal imaging of mitochondria to examine alterations in morphology caused by alterations in nutrient level ( fig . cells were assessed visually , and those possessing > 50% punctate mitochondria were considered fragmented . exposure to 0.4 mmol / l palmitate in combination with 5 , 11 , or 20 mmol / l glucose ( hfg ) caused fragmentation in 40 , 75 , and 88% of cells after 4 h and in 51 , 61 , and 79% of cells after 24 h , respectively ( fig . 4a ) . only 16 and 28% of cells exposed to 20 mmol / l glucose without palmitate for 4 and 24 h , respectively , exhibited fragmented mitochondrial architecture . these data indicate that increasing levels of glucose in the presence of palmitate is deleterious to mitochondrial architecture . however , increased glucose alone has a minor effect on mitochondrial fragmentation . culturing ins1 cells in media with high levels of fatty acids and glucose impairs mitochondrial morphology and dynamics . a : confocal images of ins1 cells stained with tmre after 24 h in control ( 11 mmol / l glucose ) , hg ( 20 mmol / l glucose ) , hflg ( 5 mmol / l glucose and 0.4 mmol / l palmitate ) , hf ( 11 mmol / l glucose and 0.4 mmol / l palmitate ) , and hfg ( 20 mmol / l glucose and 0.4 mmol / l palmitate ) media . hf and hfg induce mitochondrial fragmentation within 4 h as opposed to incubation with control , hflg , and hg media , which induce little or no effect on morphology . a group of mitochondria were labeled using a 2-photon laser ( , inset ) . through fusion events , photoactivated gfp distributed itself throughout the mitochondrial network within 50 min in cells treated with control media . redistribution is accompanied by dilution of the photoactivated form of pagfpmt , revealed by the decreased pagfpmt fi . in cells pretreated with hfg for 24 h ( right panel ) , the activated pagfpmt remained segregated in the mitochondria where it was initially photoactivated ; dilution does not occur and pagfp fi remains high . c : quantitative summary of pagfpmt dilution within the mitochondrial population after 24 h of hfg . , cells exposed to hfg for 24 h ; , cells incubated in normal growth media for the same amount of time . average gfp dilution values were fitted ( r = 0.99 ) to a hyperbolic function yielding t50 of 10.1 min only for the normal media group . d : mitochondrial fusion activity , measured by the ability to dilute pagfpmt after 50 min . histogram shows steady - state values of gfp fi obtained 50 min after photoactivation , when the pagfpmt dilution reached equilibrium . hfg- and hf - treated cells show reduced fusion activity compared with control and hflg- and hg - treated cells ( p < 0.05 ) . e : a 4-h hfg treatment is sufficient to reduce the fusion activity of ins1 cell mitochondria to levels similar to those found with 24-h hfg treatment . mitochondrial fusion activity is not affected by 30-min challenge with 8 and 15 mmol / l glucose . glucose media was changed from 2 to 8 or 15 mmol / l 10 min prior to photoactivation . the plateau gfp fi level after 50 min is similar to that of the ins1 cells that remained in 2 mmol / l glucose . ( a high - quality digital representation of this figure is available in the online issue . ) to determine the effect of hfg on mitochondrial fusion , we measured the spread of pagfpmt across the mitochondrial population by quantifying its dilution . 4b and c , 24-h hfg treated cells with fragmented mitochondria failed to share the activated pagfpmt . gfp fi remained unchanged ( n = 5 ; p = 0.46 ) in hfg - treated cells 50 min after photoactivation , compared with a significant decrease observed in control cells ( n = 6 ; p < 0.001 ) . these data reveal that fusion is compromised in fragmented mitochondria observed under hfg and that the reduction in fusion activity is measurable long before apoptosis . fusion activity was also monitored in cells exposed to palmitate or varying levels of glucose for 24 h. we found that similar to hfg , hf ( 11 mmol / l glucose and 0.4 mmol / l palmitate ) inhibits the mitochondrial fusion ability , whereas hg ( 20 mmol / l glucose ) and hflg ( 5 mmol / l glucose and 0.4 mmol / l palmitate ) leave fusion intact ( fig . we sought to determine whether the observed decrease in fusion activity is specific to hfg - induced toxicity as opposed to a physiological response to glucose challenge that is part of the glucose - stimulated insulin secretion ( gsis ) cascade ( fig . ins1 cells were kept at 2 mmol / l glucose for 2 h before exposure to two different stimulatory conditions : 8 and 15 mmol / l glucose . in these experiments glucose concentration was switched immediately after a group of mitochondria had been labeled , and thus the effect of acute glucose challenge on fusion activity was tested within the first 30 min of the exposure . we find that neither of these conditions resulted in statistically significant changes in mitochondrial fusion capacity of the ins1 cells when compared with 2 mmol / l glucose . these data suggest that mitochondrial fragmentation and loss of fusion capacity are specific to noxious external stimuli and are not altered during normal -cell function . we hypothesized that the fragmented mitochondrial morphology that accompanies hfg is mediated by alterations in the balance between fusion and fission processes . because these processes typically occur in a 1:1 ratio ( 17 ) , our hypothesis predicts that reduction in fusion activity may be counteracted by reduced fission . we chose to reduce the expression levels of fis1 because it plays a rate - limiting function in the recruitment of drp1 . we were able to inhibit the expression of fis1 using a shrna sequence described previously ( 18,19 ) . protein levels are reduced by 90% , and rna transcripts are reduced by an average of 83% ( n = 5 ; fig . it is notable that in ins1 cells the knockdown of fis1 does not cause noticeable alterations in mitochondrial morphology ( supplemental figs . s5 and s6a ) ( 17,20 ) . a : western blot and qpcr analysis of ins1 cells infected with control or fis1 rnai lentivirus . fis1 protein level was reduced by 8090% , and rna transcripts were reduced by an average of 83% ( n = 5 ) . b : ins1 cells expressing mitochondrially targeted dsred were stained for drp1 ( green ) . after 3.5 h of hfg incubation , drp1 puncta are abundant in the control rnai cells but not in the fis1 rnai cells . c : quantification of drp1 recruitment to mitochondria after 3.5-h exposure to hfg ( n = 7 for each group ) . d : western blot analysis indicates that the changes in drp1 recruitment observed were not due to changes in drp1 expression levels because these remained the same in all the treatments tested . ( a high - quality digital representation of this figure is available in the online issue . ) we have previously reported that -cell mitochondria exist as individual units that can exhibit differences in membrane potential ( 14 ) . with exposure to hfg , membrane potential heterogeneity increased due to the appearance of depolarized mitochondria . interestingly , the loss of fission ability by fis1 knockdown or by the expression of drp1 dn did not prevent the increase in membrane potential heterogeneity ( supplemental fig . s6 ) . to determine the efficiency of fis1 knockdown on the inhibition of fission machinery colocalization of drp1 puncta to mitochondria was visualized and quantified by immunofluorescence in control and lentivirally transduced fis1 rnai ins1 cells exposed to 4 h of hfg . this time period corresponds to the duration required to achieve full fragmentation of the mitochondrial web without apoptosis ( fig . control cells transduced with empty vector exhibited numerous drp1-positive puncta along every mitochondrial network after 4 h of hfg . colocalization of drp1 puncta with mitochondria was significantly higher in the empty vector control group compared with fis1 rnai ( p < 0.001 ; fig . a western blot was performed to test whether hfg , fis1 knockdown , or a combination of both affected the expression of drp1 . we find that drp1 expression levels did not change under these conditions , thereby supporting that fis1 knockdown and hfg affected puncta formation alone without altering drp1 expression ( fig . , immunostaining for drp1 in control ins1 cells exposed to hfg for 4 h revealed the same staining pattern as cells not exposed to hfg ( supplemental fig . s4 ) . inhibition of drp1 recruitment to mitochondria suggests that fis1 shrna can effectively prevent mitochondrial fission in -cells . we next determined whether fis1 knockdown can prevent hfg - induced fragmentation by performing morphometrical analysis of mitochondrial structures in ins1 cells infected with fis1 shrna and control shrna viruses . ins1 cells infected with control virus were cultured in either 11 mmol / l ( normal growth / culture conditions ) glucose or with 20 mmol / l glucose and 0.4 mmol / l palmitate for 24 h and stained with tmre . under hfg , we observed a change from elongated and elaborate networks to fragmented and punctate mitochondria ( fig . the aspect ratio ( ar ) of all mitochondria in each cell was calculated by measuring the length and width of best fit ellipses ( see research design and methods ; an ar of 4 represents a mitochondrion that is four times longer than it is wide ) . organelles were classified according to ar values into those with short ( ar 2 ) , intermediate ( 2<ar4 ) , and long ( ar > 4 ) morphology . hfg treatment reduced the fraction of long mitochondria fivefold and doubled the fraction of short mitochondria . it is notable that fis1 rnai alone did not alter the distribution of short , intermediate , and long mitochondria within a cell 's mitochondrial population ( fig . fis1 rnai restores mitochondrial morphology and dynamics under hfg in ins1 cells . a : mitochondrial morphometry . mitochondria were classified according to ar into short ( ar < 2 ) , intermediate , ( 2<ar<4 ) , and long ( ar > 4 ) length ( n = 8 cells per group ) . b : whole - cell mitochondrial fusion assays ( pagfpmt dilution ) in fis1 rnai ins1 cells exposed to hfg for 24 h ( ) . a hyperbolic fitting yielded t50 = 7.6 min . c : mitochondrial movement was calculated by measuring the velocity of single mitochondria over time . control rnai with and without hfg treatment are represented by and , respectively . fis1 rnai cells with and without hfg treatment are represented by and , respectively . the last image zooms to one mitochondrion and the movement over 10 min is depicted , in this case 4 m over 10 min . the velocity over time is plotted and indicates a peak around 250 s. when the distance from the origin is plotted over time , it is evident that mitochondria do not move at a constant speed . ( a high - quality digital representation of this figure is available in the online issue . ) in addition to rescuing mitochondrial architecture , fis1 knockdown also preserved the capability of mitochondria to fuse after 24-h hfg treatment . whereas cells transduced with control lentiviral treatment were unable to diffuse gfp after hfg treatment , those transduced with fis1 rnai lentivirus reduced gfp intensity by 31% , similar to control cells not treated with hfg that exhibited a 29% decrease in gfp intensity ( fig . fis1 knockdown alone also caused a mild decrease in mitochondrial fusion ability , exhibiting a 14% intensity decrease after 50 min . it is reasonable to infer that with decreased fission , mitochondrial dynamics may occur more slowly although the network morphology remains unchanged . it is possible that alterations in mitochondrial dynamics that we report are caused by changes in mitochondrial movement or velocity . using pagfpmt and time - lapse imaging of z - stacks , we were able to tag and quantify the velocity of mitochondria under a variety of conditions . we found that the velocity ( m / min ) of mitochondria remains unchanged by hfg or fis1 knockdown compared with controls ( fig . note that one mitochondrion remains relatively immobile during the time of tracking , whereas the other ( shown in greater detail ) moves 4 m over the 10-min period . rather , we observe spikes in the rate of movement over time similar to what has been reported in other cell types ( 21 ) . this is also evident when looking at the distance from the origin at various time points . in the example provided , the maximal distance from the origin is reached between 120250 s. previous studies have demonstrated that hfg induces -cell apoptosis ( 10 ) . control and fis1 rnai ins1 cells were exposed to hfg for 24 h and tested for various molecular markers of apoptosis ( fig . c ) . immunostaining for cleaved caspase-3 as well as the 17 kda and 19 kda proapoptotic active forms of caspase-3 revealed a reduction back to basal levels in the fis1 rnai cells exposed to hfg . the percentage of control ins1 cells exhibiting staining for cleaved caspase-3 staining ( 78 4% ) was significantly greater compared with fis1 rnai treated cells ( 25 3% ) when exposed to 24-h hfg ( fig . similar results were found when apoptosis was analyzed using transferase - mediated dutp nick - end labeling staining ( fig . fis1 knockdown cells did not possess the dark nuclear staining , indicating that they are not undergoing apoptosis . consistent with the immunofluoresence and tunel data , fluorescence - activated cell sorter analysis using annexin v , another apoptotic marker , revealed a dramatic reduction in apoptotic cells in fis1 rnai infected cells exposed to hfg ( fig . a : immunostaining for cleaved caspase-3 in fis1 rnai and control rnai ins1 cells exposed to hfg for 24 h. lower panels show the experiment repeated but with cells treated to secondary antibody only . b : transferase - mediated dutp nick - end labeling staining of cells treated with the same conditions described in a. c : fluorescence - activated cell sorter analysis for the apoptotic marker annexin v shows a reduction in cell death in fis1 rnai cells compared with control rnai . d : western blot analysis of akt and its downstream target pakt ; -actin serves as a loading control . e : insulin secretion measurements were performed on ins1 cells infected with control or fis1 rnai lentivirus . cells were incubated for 4 h in 11 mmol / l glucose or hfg media , sufficient time to achieve mitochondrial fragmentation and impaired fusion capacity . after a 1-h washout period , cells were preincubated with 3 mmol / l glucose for 30 min . insulin was measured after 30-min exposure to 3 mmol / l glucose ( ) , 15 mmol / l glucose ( ) , or 15 mmol / l glucose with 40 mmol / l kcl ( ) . incubation of control cells in hfg media resulted in a decrease in gsis ( p = 0.023 ) , which is not restored by knockdown of fis1 ( p = 0.43 ) . ( a high - quality digital representation of this figure is available in the online issue . ) phosphorylated akt ( pakt ) has been shown to protect a variety of cell types , including -cells , from apoptosis through the inhibition of proapoptotic proteins including bad and caspase-9 ( 22,23 ) . previous studies have found that -cell survival under elevated glucose or fatty acid is dependent on akt phosphorylation ( 2426 ) . to determine whether fis1 rnai was upstream or downstream of akt phosphorylation we examined the effect of hfg - induced apoptosis on the activation state of akt ( fig . control ins1 cells exposed to hfg , but not hg or hf , showed a marked decrease in the amount of pakt ( ser 473 ) , consistent with their progression through apoptosis . fis1 rnai expressing cells also exhibited a dramatic decrease in pakt because akt signaling in apoptosis is primarily upstream of the mitochondrial machinery and fis1 action ( 27 ) . expression levels of akt remained unchanged in both empty vector and fis1 rnai expressing cells under basal , hg , hf , and hfg culture conditions . although restoration of mitochondrial fusion improved cell viability under hfg , it did not rescue gsis ( fig . we found that under hfg , insulin secretion in response to 15 mmol / l glucose is significantly decreased by an average of 31% ( p = 0.023 ) . fis1 knockdown cells exposed to similar hfg treatment exhibit the same levels of insulin secretion compared with control cells ( p = 0.43 ) . the relative change in fis1 expression level after 24-h hfg exposure measured by western blot yielded results with high variability ( sd = 0.552 ) and insignificant induction compared with control ( n = 3 , p = 0.97 ; data not shown ) . a three - dimensional reconstruction of mitochondria within an entire -cell has been projected onto a single plain to illustrate the density of these organelles ( fig . this morphology suggests the presence of either large continuous networks throughout the cell or , alternatively , small discrete networks juxtaposed with one another . to decipher this complex architecture to its individual components , because individual mitochondria are often in apposition with one another , tagging individual mitochondria with pagfpmt targeted to the matrix allows morphological and biophysical studies of mitochondria within a complex web . in addition , this methodology takes into account organellar movement and mitochondrial dynamics that continuously change the mitochondrion 's size , shape , and location ( 6 ) . with adenoviral delivery , pagfpmt is expressed in all -cell mitochondria but remains in its inactive form ( nonfluorescent ) . c : summary of mitochondrial size distribution performed by 90 photoactivation steps in 9 cells . d : tagging and tracking individual mitochondria in primary -cells reveals fusion events ( transfer of activated pagfpmt to juxtaposed unlabeled mitochondria ) that are then followed by fission events ( separation of previously connected mitochondria ) . ( a high - quality digital representation of this figure is available in the online issue . ) precise ( 0.5 m ) photoactivation of pagfpmt molecules was achieved using a 2-photon laser . equilibration of activated pagfpmt was immediate and revealed the boundaries of matrix continuity of individual mitochondria . figure 1b shows five sequential photoactivations revealing the boundaries of multiple individual mitochondrial networks in a mouse -cell . whereas tmre staining may show continuous mitochondrial structures , the spread of matrix pagfpmt is limited to short segments , indicating that these segments do not form a continuous lumen . in 90 photoactivation events performed in nine different cells , we found that most ( > 95% ) individual networks are rod - like in shape with 76% exhibiting a length < 2 m ( fig . a series of control studies confirmed that laser intensity was kept at levels that did not affect mitochondrial function or morphology . these included time - lapse studies of mt in cells exposed to sequential laser scanning and dose - response studies with the 2-photon laser ( experiments are described in an online appendix at http://diabetes.diabetesjournals.org/cgi/content/full/db07-1781/dc1 and in ref . the small network size of -cell mitochondria raises the possibility that mitochondrial fusion is absent in these cells or , alternatively , that fusion events are transient and brief . to address this issue , fusion events were identified by the transfer of pagfpmt from a labeled mitochondria to a previously unlabeled one when the two came in contact as shown in fig . fusion events were commonly followed by fission ( 11 of 12 events ) in a manner that reverted to prefusion size and morphology of the involved mitochondria . we undertook a series of experiments designed to manipulate the balance between fusion and fission to assess the consequence on mitochondrial architecture in primary and clonal -cells . of particular interest is the fusion protein opa1 because transcriptom databases suggest its expression levels in the -cell is uniquely high ( www.t1dbase.org ) . to test the significance of opa1 in modulating -cell mitochondrial architecture , we overexpressed the protein by adenoviral transduction . overexpression of opa1 in primary -cells caused mitochondria to fragment into even smaller units ( fig . in ins1 cells , transduction with the same number of opa1 adenoviral particles caused mild overexpression and increased density in mitochondrial architecture . increasing the amount of virus further increased the expression level and led to fragmentation of the mitochondrial network ( fig . these findings suggest that the increased degree of fusion may promote elongation ; however , further increases in opa1 levels can lead to fragmentation . gfp infections were performed to control for potential effects of viral transduction and normalized so that cells were exposed to the same number of viral particles . we found that expression of gfp did not affect mitochondrial morphology in any of the experiments performed . a : in primary -cells , overexpression of the fusion protein opa1 leads to mitochondrial fragmentation . overexpression was achieved by adenoviral transduction of gfp or opa1 at equal concentrations of viral particles . b : level of opa1 overexpression in ins1 cells was controlled by the dose of viral particles used . mild overexpression causes mitochondria to take on a dense and elaborate morphology compared with controls . c : in primary -cells , overexpression of dn drp1 to reduce mitochondrial fission leads to mitochondrial swelling . d : in ins1 cells , overexpression of dn drp1 leads to network superfusion and a limited amount of mitochondrial swelling . the number and concentration of viral particles used in a , c , and d was identical . ( a high - quality digital representation of this figure is available in the online issue . ) an alternative approach to examining the consequences of a pro - fusion state is to prevent fission . we found that manipulating the pro - fission protein drp1 also caused dramatic changes in mitochondrial architecture . disruption of fission in primary -cells by overexpressing dominant negative ( dn ) drp1 resulted in swelling of the mitochondria ( fig . 2c ) . in ins cells , similar treatment with . the overall rate of mitochondrial fusion activity within an isolated -cell was assessed by photoactivating a subpopulation of mitochondria and monitoring the spread as well as the dilution of active pagfpmt throughout the rest of the cell 's mitochondria ( fig . the gradual spread of pagfpmt signal to other parts of the -cell , which were not photoactivated , is accompanied by dilution of pagfpmt ( fig . 3a and b ) . as a result , the average pagfpmt fluorescence intensity in mitochondria that contain photoactivated pagfpmt gradually decreases as long as the process of spreading is in progress . a : fusion of the photoactivated fraction ( 10% ) with the rest of the mitochondrial web dilutes activated pagfpmt and leads to a reduction in fluorescence intensity . b : dilution of gfp fi is shown for individual cells ( n = 6 ; gray lines ) and their average se ( ) . the average dilution values were fitted ( r = 0.99 ) to a hyperbolic function yielding t50 of 12.1 min . c and d : -cells within an intact islet also exhibit mitochondrial dynamics as revealed by the diffusion of the activated pagfpmt signal . ( see research design and methods and supplemental material for imaging and image analysis methodology ) . e : fusion activity in primary -cells is reduced after 24 h in hfg media . images taken during the assay show that the number of pagfpmt - positive mitochondria and the fi of pagfpmt remained unchanged . the hfg - treated cells possess significantly brighter gfp intensity ( p = 0.04 ) than controls , indicating reduced mitochondrial fusion activity . ( a high - quality digital representation of this figure is available in the online issue . ) to rule out the possibility that decay in pagfpmt fi was the result of either nonspecific leak of activated pagfpmt from tagged mitochondria or bleaching of pagfp by the laser , two experiments were conducted : gfp fi was measured over time 1 ) in individual mitochondria that did not engage in fusion events and 2 ) in the presence of fccp , a mitochondrial inner membrane uncoupler that collapses m and arrests mitochondrial dynamics . in both experiments gfp fi remained unchanged and stable over a period of 20 min , indicating a negligible component of nonspecific pagfpmt leak or laser - induced bleach ( see mitochondrial fusion assay in supplemental material ) . individual -cells within an islet were tested for mitochondrial fusion activity using pagfpmt that is under the insulin promoter ( fig . 3c ) . we found that fusion activity is functional and comparable with that in isolated -cells . interestingly , there is greater heterogeneity in the overall fusion activity among individual cells within intact islets exhibiting varied rates of gfp dilution ( fig . exposure to increased levels of glucose and fatty acids has been shown to compromise -cell function and viability ( 10 ) . incubation in 20 mmol / l glucose along with the fatty acid palmitate ( 0.4 mmol / l complexed with 0.5% bsa ) for 48 h resulted in fragmentation of mitochondrial morphology and inhibition of mitochondrial dynamics in -cells within mouse islets ( fig . the mitochondrial fusion capability of -cells decreased significantly under hfg ( 20 mmol / l glucose and 0.4 mmol / l palmitate ) which was revealed by the decreased ability of cells to dilute the gfp signal 2 h after photoactivation ( fig . el assaad et al . ( 10 ) showed that in ins1 cells , exposure to 20 mmol / l glucose and > 0.2 mmol / l palmitate for 24 h leads to apoptosis and that caspase inhibitors do not rescue the cells from death ( 10 ) . we used confocal imaging of mitochondria to examine alterations in morphology caused by alterations in nutrient level ( fig . cells were assessed visually , and those possessing > 50% punctate mitochondria were considered fragmented . exposure to 0.4 mmol / l palmitate in combination with 5 , 11 , or 20 mmol / l glucose ( hfg ) caused fragmentation in 40 , 75 , and 88% of cells after 4 h and in 51 , 61 , and 79% of cells after 24 h , respectively ( fig . 4a ) . only 16 and 28% of cells exposed to 20 mmol / l glucose without palmitate for 4 and 24 h , respectively , exhibited fragmented mitochondrial architecture . these data indicate that increasing levels of glucose in the presence of palmitate is deleterious to mitochondrial architecture culturing ins1 cells in media with high levels of fatty acids and glucose impairs mitochondrial morphology and dynamics . a : confocal images of ins1 cells stained with tmre after 24 h in control ( 11 mmol / l glucose ) , hg ( 20 mmol / l glucose ) , hflg ( 5 mmol / l glucose and 0.4 mmol / l palmitate ) , hf ( 11 mmol / l glucose and 0.4 mmol / l palmitate ) , and hfg ( 20 mmol / l glucose and 0.4 mmol / l palmitate ) media . hf and hfg induce mitochondrial fragmentation within 4 h as opposed to incubation with control , hflg , and hg media , which induce little or no effect on morphology . a group of mitochondria were labeled using a 2-photon laser ( , inset ) . through fusion events , photoactivated gfp distributed itself throughout the mitochondrial network within 50 min in cells treated with control media . redistribution is accompanied by dilution of the photoactivated form of pagfpmt , revealed by the decreased pagfpmt fi . in cells pretreated with hfg for 24 h ( right panel ) , the activated pagfpmt remained segregated in the mitochondria where it was initially photoactivated ; dilution does not occur and pagfp fi remains high . c : quantitative summary of pagfpmt dilution within the mitochondrial population after 24 h of hfg . , cells exposed to hfg for 24 h ; , cells incubated in normal growth media for the same amount of time . average gfp dilution values were fitted ( r = 0.99 ) to a hyperbolic function yielding t50 of 10.1 min only for the normal media group . d : mitochondrial fusion activity , measured by the ability to dilute pagfpmt after 50 min . histogram shows steady - state values of gfp fi obtained 50 min after photoactivation , when the pagfpmt dilution reached equilibrium . hfg- and hf - treated cells show reduced fusion activity compared with control and hflg- and hg - treated cells ( p < 0.05 ) . e : a 4-h hfg treatment is sufficient to reduce the fusion activity of ins1 cell mitochondria to levels similar to those found with 24-h hfg treatment . mitochondrial fusion activity is not affected by 30-min challenge with 8 and 15 mmol / l glucose . glucose media was changed from 2 to 8 or 15 mmol / l 10 min prior to photoactivation . the plateau gfp fi level after 50 min is similar to that of the ins1 cells that remained in 2 mmol / l glucose . ( a high - quality digital representation of this figure is available in the online issue . ) to determine the effect of hfg on mitochondrial fusion , we measured the spread of pagfpmt across the mitochondrial population by quantifying its dilution . 4b and c , 24-h hfg treated cells with fragmented mitochondria failed to share the activated pagfpmt . gfp fi remained unchanged ( n = 5 ; p = 0.46 ) in hfg - treated cells 50 min after photoactivation , compared with a significant decrease observed in control cells ( n = 6 ; p < 0.001 ) . these data reveal that fusion is compromised in fragmented mitochondria observed under hfg and that the reduction in fusion activity is measurable long before apoptosis . fusion activity was also monitored in cells exposed to palmitate or varying levels of glucose for 24 h. we found that similar to hfg , hf ( 11 mmol / l glucose and 0.4 mmol / l palmitate ) inhibits the mitochondrial fusion ability , whereas hg ( 20 mmol / l glucose ) and hflg ( 5 mmol / l glucose and 0.4 mmol / l palmitate ) leave fusion intact ( fig . we sought to determine whether the observed decrease in fusion activity is specific to hfg - induced toxicity as opposed to a physiological response to glucose challenge that is part of the glucose - stimulated insulin secretion ( gsis ) cascade ( fig . ins1 cells were kept at 2 mmol / l glucose for 2 h before exposure to two different stimulatory conditions : 8 and 15 mmol / l glucose . in these experiments glucose concentration was switched immediately after a group of mitochondria had been labeled , and thus the effect of acute glucose challenge on fusion activity was tested within the first 30 min of the exposure . we find that neither of these conditions resulted in statistically significant changes in mitochondrial fusion capacity of the ins1 cells when compared with 2 mmol / l glucose . these data suggest that mitochondrial fragmentation and loss of fusion capacity are specific to noxious external stimuli and are not altered during normal -cell function . we hypothesized that the fragmented mitochondrial morphology that accompanies hfg is mediated by alterations in the balance between fusion and fission processes . because these processes typically occur in a 1:1 ratio ( 17 ) , our hypothesis predicts that reduction in fusion activity may be counteracted by reduced fission . we chose to reduce the expression levels of fis1 because it plays a rate - limiting function in the recruitment of drp1 . we were able to inhibit the expression of fis1 using a shrna sequence described previously ( 18,19 ) . protein levels are reduced by 90% , and rna transcripts are reduced by an average of 83% ( n = 5 ; fig . it is notable that in ins1 cells the knockdown of fis1 does not cause noticeable alterations in mitochondrial morphology ( supplemental figs . s5 and s6a ) ( 17,20 ) . a : western blot and qpcr analysis of ins1 cells infected with control or fis1 rnai lentivirus . fis1 protein level was reduced by 8090% , and rna transcripts were reduced by an average of 83% ( n = 5 ) . b : ins1 cells expressing mitochondrially targeted dsred were stained for drp1 ( green ) . after 3.5 h of hfg incubation , drp1 puncta are abundant in the control rnai cells but not in the fis1 rnai cells . c : quantification of drp1 recruitment to mitochondria after 3.5-h exposure to hfg ( n = 7 for each group ) . d : western blot analysis indicates that the changes in drp1 recruitment observed were not due to changes in drp1 expression levels because these remained the same in all the treatments tested . ( a high - quality digital representation of this figure is available in the online issue . ) we have previously reported that -cell mitochondria exist as individual units that can exhibit differences in membrane potential ( 14 ) . with exposure to hfg , membrane potential heterogeneity increased due to the appearance of depolarized mitochondria . interestingly , the loss of fission ability by fis1 knockdown or by the expression of drp1 dn did not prevent the increase in membrane potential heterogeneity ( supplemental fig . s6 ) . to determine the efficiency of fis1 knockdown on the inhibition of fission machinery colocalization of drp1 puncta to mitochondria was visualized and quantified by immunofluorescence in control and lentivirally transduced fis1 rnai ins1 cells exposed to 4 h of hfg . this time period corresponds to the duration required to achieve full fragmentation of the mitochondrial web without apoptosis ( fig . control cells transduced with empty vector exhibited numerous drp1-positive puncta along every mitochondrial network after 4 h of hfg . colocalization of drp1 puncta with mitochondria was significantly higher in the empty vector control group compared with fis1 rnai ( p < 0.001 ; fig . a western blot was performed to test whether hfg , fis1 knockdown , or a combination of both affected the expression of drp1 . we find that drp1 expression levels did not change under these conditions , thereby supporting that fis1 knockdown and hfg affected puncta formation alone without altering drp1 expression ( fig . , immunostaining for drp1 in control ins1 cells exposed to hfg for 4 h revealed the same staining pattern as cells not exposed to hfg ( supplemental fig . inhibition of drp1 recruitment to mitochondria suggests that fis1 shrna can effectively prevent mitochondrial fission in -cells . we next determined whether fis1 knockdown can prevent hfg - induced fragmentation by performing morphometrical analysis of mitochondrial structures in ins1 cells infected with fis1 shrna and control shrna viruses . ins1 cells infected with control virus were cultured in either 11 mmol / l ( normal growth / culture conditions ) glucose or with 20 mmol / l glucose and 0.4 mmol / l palmitate for 24 h and stained with tmre . under hfg , we observed a change from elongated and elaborate networks to fragmented and punctate mitochondria ( fig . the aspect ratio ( ar ) of all mitochondria in each cell was calculated by measuring the length and width of best fit ellipses ( see research design and methods ; an ar of 4 represents a mitochondrion that is four times longer than it is wide ) . organelles were classified according to ar values into those with short ( ar 2 ) , intermediate ( 2<ar4 ) , and long ( ar > 4 ) morphology . hfg treatment reduced the fraction of long mitochondria fivefold and doubled the fraction of short mitochondria . it is notable that fis1 rnai alone did not alter the distribution of short , intermediate , and long mitochondria within a cell 's mitochondrial population ( fig . fis1 rnai restores mitochondrial morphology and dynamics under hfg in ins1 cells . a : mitochondrial morphometry . mitochondria were classified according to ar into short ( ar < 2 ) , intermediate , ( 2<ar<4 ) , and long ( ar > 4 ) length ( n = 8 cells per group ) . b : whole - cell mitochondrial fusion assays ( pagfpmt dilution ) in fis1 rnai ins1 cells exposed to hfg for 24 h ( ) . a hyperbolic fitting yielded t50 = 7.6 min . fis1 rnai cells not exposed to hfg are also plotted ( ) . c : mitochondrial movement was calculated by measuring the velocity of single mitochondria over time . control rnai with and without hfg treatment are represented by and , respectively . fis1 rnai cells with and without hfg treatment are represented by and , respectively . the last image zooms to one mitochondrion and the movement over 10 min is depicted , in this case 4 m over 10 min . the velocity over time is plotted and indicates a peak around 250 s. when the distance from the origin is plotted over time , it is evident that mitochondria do not move at a constant speed . ( a high - quality digital representation of this figure is available in the online issue . ) in addition to rescuing mitochondrial architecture , fis1 knockdown also preserved the capability of mitochondria to fuse after 24-h hfg treatment . whereas cells transduced with control lentiviral treatment were unable to diffuse gfp after hfg treatment , those transduced with fis1 rnai lentivirus reduced gfp intensity by 31% , similar to control cells not treated with hfg that exhibited a 29% decrease in gfp intensity ( fig . fis1 knockdown alone also caused a mild decrease in mitochondrial fusion ability , exhibiting a 14% intensity decrease after 50 min . it is reasonable to infer that with decreased fission , mitochondrial dynamics may occur more slowly although the network morphology remains unchanged . it is possible that alterations in mitochondrial dynamics that we report are caused by changes in mitochondrial movement or velocity . using pagfpmt and time - lapse imaging of z - stacks , we were able to tag and quantify the velocity of mitochondria under a variety of conditions . we found that the velocity ( m / min ) of mitochondria remains unchanged by hfg or fis1 knockdown compared with controls ( fig . note that one mitochondrion remains relatively immobile during the time of tracking , whereas the other ( shown in greater detail ) moves 4 m over the 10-min period . rather , we observe spikes in the rate of movement over time similar to what has been reported in other cell types ( 21 ) . this is also evident when looking at the distance from the origin at various time points . in the example control and fis1 rnai ins1 cells were exposed to hfg for 24 h and tested for various molecular markers of apoptosis ( fig . c ) . immunostaining for cleaved caspase-3 as well as the 17 kda and 19 kda proapoptotic active forms of caspase-3 revealed a reduction back to basal levels in the fis1 rnai cells exposed to hfg . the percentage of control ins1 cells exhibiting staining for cleaved caspase-3 staining ( 78 4% ) was significantly greater compared with fis1 rnai treated cells ( 25 3% ) when exposed to 24-h hfg ( fig . similar results were found when apoptosis was analyzed using transferase - mediated dutp nick - end labeling staining ( fig . fis1 knockdown cells did not possess the dark nuclear staining , indicating that they are not undergoing apoptosis . consistent with the immunofluoresence and tunel data , fluorescence - activated cell sorter analysis using annexin v , another apoptotic marker , revealed a dramatic reduction in apoptotic cells in fis1 rnai infected cells exposed to hfg ( fig . a : immunostaining for cleaved caspase-3 in fis1 rnai and control rnai ins1 cells exposed to hfg for 24 h. lower panels show the experiment repeated but with cells treated to secondary antibody only . b : transferase - mediated dutp nick - end labeling staining of cells treated with the same conditions described in a. c : fluorescence - activated cell sorter analysis for the apoptotic marker annexin v shows a reduction in cell death in fis1 rnai cells compared with control rnai . d : western blot analysis of akt and its downstream target pakt ; -actin serves as a loading control . e : insulin secretion measurements were performed on ins1 cells infected with control or fis1 rnai lentivirus . cells were incubated for 4 h in 11 mmol / l glucose or hfg media , sufficient time to achieve mitochondrial fragmentation and impaired fusion capacity . after a 1-h washout period , cells were preincubated with 3 mmol / l glucose for 30 min . insulin was measured after 30-min exposure to 3 mmol / l glucose ( ) , 15 mmol / l glucose ( ) , or 15 mmol / l glucose with 40 mmol / l kcl ( ) . incubation of control cells in hfg media resulted in a decrease in gsis ( p = 0.023 ) , which is not restored by knockdown of fis1 ( p = 0.43 ) . ( a high - quality digital representation of this figure is available in the online issue . ) phosphorylated akt ( pakt ) has been shown to protect a variety of cell types , including -cells , from apoptosis through the inhibition of proapoptotic proteins including bad and caspase-9 ( 22,23 ) . previous studies have found that -cell survival under elevated glucose or fatty acid is dependent on akt phosphorylation ( 2426 ) . to determine whether fis1 rnai was upstream or downstream of akt phosphorylation we examined the effect of hfg - induced apoptosis on the activation state of akt ( fig . control ins1 cells exposed to hfg , but not hg or hf , showed a marked decrease in the amount of pakt ( ser 473 ) , consistent with their progression through apoptosis . fis1 rnai expressing cells also exhibited a dramatic decrease in pakt because akt signaling in apoptosis is primarily upstream of the mitochondrial machinery and fis1 action ( 27 ) . expression levels of akt remained unchanged in both empty vector and fis1 rnai expressing cells under basal , hg , hf , and hfg culture conditions . although restoration of mitochondrial fusion improved cell viability under hfg , it did not rescue gsis ( fig . 7e ) . we found that under hfg , insulin secretion in response to 15 mmol / l glucose is significantly decreased by an average of 31% ( p = 0.023 ) . fis1 knockdown cells exposed to similar hfg treatment exhibit the same levels of insulin secretion compared with control cells ( p = 0.43 ) . the relative change in fis1 expression level after 24-h hfg exposure measured by western blot yielded results with high variability ( sd = 0.552 ) and insignificant induction compared with control ( n = 3 , p = 0.97 ; data not shown ) . a number of studies have established that mitochondrial dysfunction plays a role in the development of type 2 diabetes and that exposure of -cells to high levels of glucose and fatty acids may contribute to the development of the disease ( 11,28,29 ) . most notably , superoxide generation is increased and mitochondrial morphologies are short and swollen compared with those of nondiabetic littermates ( 30 ) . in this article , we report that exposure to toxic nutrient levels induces -cell mitochondrial fragmentation that is caused by alterations in mitochondrial dynamics . previous studies in a variety of cells have demonstrated the existence of elaborate networks of mitochondria that frequently connect through fusion events . given the dense appearance of -cell mitochondria and the relatively short unit size , it was unclear whether fusion and fission occur in -cells and whether these play any role in the development of diabetes . here , we demonstrate for the first time that mitochondria in -cells continuously undergo fusion and fission and that these interactions may function to negate the detrimental effects of long - term exposure to high levels of nutrients . under normal condition , the dynamic activity of mitochondria is evident in clonal -cells , dispersed -cells , and -cells within an intact islet . individual whole - cell mitochondrial fusion assays performed within intact islets indicate that fusion rates vary from cell to cell . the intercellular variation appears to be greater between -cells within an islet compared with dispersed -cells . this may correlate to previous studies by marsh and colleagues suggesting that differences in the level of mitochondrial branching between -cells in a islet may reflect differences in secretory capacity ( 31 ) . indeed , it is conceivable that cells with more branched mitochondrial networks will have a different pagfpmt fusion rate than cells with less branched mitochondria . tagging individual mitochondria with matrix - targeted pagfpmt enables quantitative dissection of mitochondrial architecture and dynamics . mitochondria in primary -cells are densely packed , which can lead to an overestimation of mitochondrial size and the impression of a continuous structure . however , when the matrix boundaries of each mitochondrial unit are determined by photoactivation of pagfpmt , it is clear that many of the larger convoluted webs actually represent short strands of mitochondria juxtaposed with one another without matrix continuity . for comparison , cos cells possess very elaborate mitochondrial structures that often appear to be interconnected within a complex web . despite the appearance of connectivity , the average cos cell mitochondrion is only about 1% of the size of the entire web , which is revealed by pagfpmt photoactivation of individual units ( 6 ) . interestingly , it has been reported that mitochondria are unconnected and can have distinct functional properties ( 32 ) . with the pagfpmt - based mitochondrial fusion assays , we have been able to observe that -cell mitochondria undergo transient fusion events . the relatively small size of these units is not the result of the inability of these mitochondria to undergo fusion . we show that these mitochondria frequently go through fusion , shown by complete equilibration of pagfpmt throughout the web in as little as 1 h. compared with other cell types , the time to equilibrium we report is similar to what has been observed in hela cells and several other cell types including rat hipoccampal neurons ( 16 ) . interestingly , ins1 cell mitochondria assemble into more elongated and convoluted networks than primary -cells . our data suggest that the mechanism underlying differences in mitochondrial morphology between cell types may be related to the levels of opa1 expression . similarly , overexpression of another mitochondrial fusion protein , mfn2 , leads to the clustering of mitochondria into short fragments ( 33 ) . in ins1 cells , it takes larger increases in opa1 to achieve similar fragmentation of the mitochondrial network . these findings suggest that ins1 cell mitochondria are able to maintain networking ability when faced with disruptions in the balance between fusion and fission . altering mitochondrial fission by expressing dn drp1 leads to the formation of very elongated mitochondrial tubules along with the appearance of swollen sections . in primary -cells , expression of dn drp1 leads to the formation of large swollen mitochondria that likely represent a superfused state . hfg , the synergistic combination of elevated glucose and fatty acid , is an in vitro model of type 2 diabetes described previously ( 34 ) and has been shown to cause progressive -cell dysfunction and cell death ( 35 ) . previous studies have shown that hfg leads to an increased level of mt heterogeneity and the appearance of a growing population of depolarized mitochondria ( 14 ) . it is conceivable that these changes may be mediated by alterations in mitochondrial dynamics and network morphology . indeed , we found dramatic differences in mitochondrial morphology between control ( basal glucose ) and hfg - treated groups . hfg caused the appearance of numerous punctate mitochondria and overall fragmentation of the mitochondrial network . when combined with palmitate , increasing concentrations of glucose ( 5 , 11 , and 20 mmol / l ) lead to increasing levels of fragmentation in a dose - dependent manner . however , in the absence of palmitate , even 20 mmol / l glucose does not affect mitochondrial architecture . these data are congruent with the synergistic effects of palmitate and glucose that have been reported previously ( 10 ) . interestingly , with higher levels of glucose alone and longer exposure , expression of the mitochondrial fission protein drp1 is increased ( 36 ) . this finding supports an important role for mitochondrial fission in apoptosis because the induction of drp1 expression increased high glucose induced apoptosis in drp1 wild - type cells . our results indicate that with the combination of high fat and glucose , mitochondrial fragmentation occurs within 4 h. furthermore , we report that alterations in mitochondrial fusion mediate mitochondrial fragmentation , whereas drp1 translocation is unaffected . fragmentation of the mitochondrial network may be mediated by increased fission , decreased fusion , or conditions characterized by uniquely high expression levels of fusion proteins such as opa1 or mfn2 . to determine which of these possibilities occurs under hfg , we quantified mitochondrial dynamics by studying the spread of photoconverted pagfpmt across the mitochondrial web . we found significant abrogation of mitochondrial fusion in cells exposed to hfg compared with control ( basal glucose ) . the disruption of mitochondrial dynamics in the presence of palmitate appears at 11 mmol / l glucose but is not apparent at 5 mmol / l glucose . as shown in the mitochondrial morphometry analysis , after 24-h hfg , there remains a small population of mitochondria that retains a relatively elongated morphology . indeed , certain cells retain complex mitochondrial morphology that is comprised of a mixture of elongated and punctuate structures . future work will address whether these cells represent a population that is less susceptible to apoptosis . cells under hfg that do not exhibit mitochondrial fragmentation are still capable of mitochondrial fusion , shown by pagfpmt dilution ( see supplemental material ) . however , the kinetics of pagfpmt sharing in these cells is reduced compared with control cells , indicating that fusion activity is compromised even before fragmentation is apparent . the contribution of fission machinery to fragmentation was determined by analyzing the effect of hfg on fis1 and drp1 . analysis of fis1 transcript as well as protein levels in cells treated with hfg did not reveal a statistically significant induction of this protein . although expression does not change , we show that hfg is accompanied by recruitment of drp1 to mitochondrial fission sites . this was inhibited in cells treated with fis1 rnai , indicating an essential role for fis1 . we rationalized that the inhibition of fission may balance reduced fusion activity observed under hfg . indeed , fis1 rnai restored mitochondrial dynamics and morphology in hfg - treated cells , determined by the spreading and dilution of matrix pagfpmt through the mitochondrial network . in our studies , knockdown of the fission protein fis1 did not result in robust morphological changes in ins1 cells ( 37 ) . this finding suggests that ins1 cells are able to counteract the effects of reduced fission by balancing the rate of fusion . indeed , our data indicate that fis1 knockdown is accompanied by a reduction in the fusion rate reported by the pagfpmt dilution assay . because fusion and fission events are paired , it is conceivable that decreased fission activity may render the mitochondrial webs more static , suggesting that a certain level of fission is required for an optimal mitochondrial dynamics rate . additionally , the lack of change in gross mitochondrial morphology with fis1 knockdown may be due to the packed and entangled appearance of mitochondria in ins1 cells compared with other cell types such as fibroblasts . the density of mitochondria leads to a large percentage of juxtaposed mitochondria that are not necessarily fused . under these conditions , alterations in the level of connectivity the mechanism by which hfg leads to mitochondrial fragmentation may be related to an increase in ros . hfg has been shown to increase superoxide levels ( 38 ) . in a separate study , treatment of ins1 with h2o2 resulted in fragmentation of the mitochondrial network , suggesting that nutrient - induced ros production can lead to mitochondrial fragmentation ( 39 ) . on the other hand , other studies have shown that dynamic changes in mitochondrial morphology may influence glucose - induced overproduction of ros ( 7 ) . in h9c2 and crl-1439 cell lines , inhibition of mitochondrial fission with dn drp1 attenuates ros production and mitochondrial fragmentation induced by acute exposure to 50 mmol / l glucose . in neurons , inhibition of fission was found to mitigate no - induced mitochondrial fragmentation and cell death ( 40 ) . thus , it is conceivable that the effect of hfg on mitochondrial morphology may be due to increased ros damage . the decrease in ros production in fis1 rnai cells under hfg may be at least partially responsible for the protection from fragmentation and apoptosis . in a previous study , we demonstrate that fis1 knockdown does not lower ros levels in ins1 cells under growth conditions inhibition of mitochondrial fission through fis1 rnai markedly reduced apoptosis compared with cells infected with control virus . this result demonstrates the importance of mitochondrial networking in -cell physiology because preventing hfg - induced fragmentation through fis1 rnai reduces -cell apoptosis . indeed , it has been shown that overexpression of hfis1 in ins1 cells only leads to nominal levels of apoptosis . moreover , fragmentation caused by the expression of dn mfn1 does not result in apoptosis in ins1 cells ( 41 ) . although it is possible to induce fragmentation and apoptosis by fis1 overexpression in other cell types , it is also possible to prevent apoptosis without affecting mitochondrial fragmentation , which has been demonstrated with the overexpression of bcl - xl ( 42 ) . previous studies have raised a number of potential mechanisms by which inhibition of mitochondrial fragmentation can affect apoptosis ( 43,44 ) . fragmentation occurs early in the cell death pathway ( 43,45 ) and is thought to contribute to the release of cytochrome c into the cytosol , culminating in caspase - mediated apoptosis ( 45 ) . it is notable that although a decrease in the translocation of bax to mitochondria and the subsequent release of cytochrome c have been reported with fis1 knockdown , dn drp1 left bax translocation unaffected ( 46 ) . an article by martinou and colleagues ( 47 ) has shown that inhibition of fission machinery can prevent mitochondrial fragmentation and reduce cytochrome c release . however , bax / bak - mediated apoptosis still occurs along with the release of smac / diablo . a more recent article by bossy - wetzel and colleagues ( 48 ) has reported that mitochondrial fission occurs upstream of bax translocation . revealing the relevance and timing of these events in hfg - induced apoptosis in -cells is a topic for future investigation . activated akt provides protection from apoptosis through phosphorylation and inhibition of proapoptotic proteins such as bad ( 24,49 ) . previous studies have indicated that -cell survival under elevated glucose or fatty acid is dependent on akt phosphorylation and that reduced level of pakt is associated with -cell loss ( 2426 ) . interestingly , in hfg - treated cells where fis1 has been knocked down , reduced pakt was not accompanied by cell death . -cell survival enhanced by fis1 knockdown indicates that the effect of fis1 occurs downstream of the akt signaling pathway and that the reduction in mitochondrial fragmentation bypasses the reduction in akt activity . recent studies have also indicated that increased calcium influx plays a role in palmitate - mediated -cell apoptosis ( 50 ) . activation of calcineurin by calcium may regulate mitochondrial morphology by dephosphorylation of drp1 ( 51,52 ) . however , our results indicate that hfg - mediated mitochondrial fragmentation is not accompanied by an increased level of drp1 assembly on mitochondria and increased fission but rather a decrease in the level of fusion activity , demonstrated by mtpagfp diffusion assays . our data indicate mitochondrial fusion rate is unchanged by acute stimulation by 15 mmol / l glucose . previous studies by our group and by wollheim and colleagues indicate that altering mitochondrial dynamics proteins mfn1 and fis1 does not improve gsis ( 17,41,53 ) . it is interesting to note that both fis1 overexpression and fis1 knockdown lead to a decrease in gsis . together , these data suggest that any disturbance in the balance of fusion and fission can lead to -cell dysfunction .

food allergy ( fa ) is an important public health problem that affects children and adults worldwide . fa carries the risk of severe allergic reactions and even death , but there are no curative treatments and no effective prevention methods for fa yet . fa can only be managed by avoidance of the allergen or by treatment of symptoms as they arise . the national institute of allergy and infectious diseases ( niaid ) in the united states ( us ) has recently published fa guidelines , including a consensus definition for fa , a discussion of comorbid conditions often associated with fa , and information on the epidemiology , natural history , diagnosis , and management of fa , including recommendations for treatment of severe symptoms and anaphylaxis1 ) . the european academy of allergy and clinical immunology has created a task force to develop guidelines for the diagnosis and management of fa , and japan has published a japanese guideline for fa2 ) . food - induced anaphylaxis and accidental exposure to food allergens in day care centers are primary reasons for hospital emergency department visits by children in korea3,4 ) . in addition , nutritional problems caused by dietary avoidance of allergens have become an important public health issue5 ) . fa is an adverse health effect arising from a specific immune response that occurs reproducibly on exposure to a given food . food intolerance is a non - immunologic adverse reaction to toxic contaminants or pharmacologic properties of the food ; it may be due to characteristics of the host , including metabolic disorders ( i.e. , lactase deficiency ) or idiosyncratic responses . food is defined as any substance - whether processed , semi - processed , or raw - that is intended for human consumption , and includes drinks , chewing gum , food additives , and dietary supplements . food allergens are defined as those specific components of food or ingredients within food ( typically proteins , but sometimes also chemical haptens ) that are recognized by allergen - specific immune cells and elicit specific immunologic reactions , resulting in characteristic symptoms . cross - reactivity is the reaction between an antibody and an antigen that differs from the immunogen . cross - reactivity may occur when an antibody reacts not only with the original allergen , but also with a similar allergen . in fa , cross - reactivity occurs when a food allergen shares structural or sequence similarity with a different food allergen or an aeroallergen , which may then trigger an adverse reaction similar to that triggered by the original food allergen . allergic sensitization is the presence of allergen - specific immunoglobulin e ( sige ) to allergen . because individuals can develop allergic sensitization to food allergens without having clinical symptoms on exposure to those foods , a sige mediated fa requires both the presence of sensitization and the development of specific signs and symptoms on exposure to the food . food allergic disorders can be broadly divided into those that are mediated by ige antibodies and those that are not ( table 1 ) . fa may coexist with asthma , atopic dermatitis , eosinophilic esophagitis , and exercise - induced anaphylaxis ( table 2 ) . children with moderate to severe persistent atopic dermatitis have a higher prevalence of ige - mediated fa , estimated at about 35%6,7 ) . in korea , the prevalence of fa is 5.3% in infants , and the mean lifetime prevalence of fa is 4.7% for children aged 6 to 12 and 5.1% for those aged 12 to 158,9 ) . one of the reasons is that studies of fa incidence and prevalence are difficult to compare due to inconsistencies in study design and variations in the definition of fa . furthermore , there are more people who believe they have fa that those who actually do , so that prevalence of self - reported fa is overestimated . a recently published meta - analysis regarding prevalence of fa included data from 51 publications and provided separate analyses of the prevalence of fa for 5 foods : milk , egg , peanut , fish , and crustacean shellfish12 ) . the investigators reported an overall prevalence of self - reported fa of 12% for children and 13% for adults , while overall prevalence of self - reported symptoms plus sensitization by double blind placebo - controlled food challenge for any of these 5 foods was 3% for adults and children . over 90% of fa results from exposure to egg , milk , peanut , tree nut , fish , shellfish , soy , and wheat13 ) . the most common offending foods are egg , milk , soy , and peanut in children , and wheat , and shellfish , tree nuts , and peanuts in adults . the order of importance of specific allergens varies by country , reflecting the interaction of culture and dietary habits . peanut , buckwheat , mustard , and sesame are good examples of this . despite similar levels of peanut consumption , there is a difference in the prevalence of peanut allergy between the us and china , and it is generally agreed that this discrepancy stems from the effects of different cooking methods on the allergenicity of peanuts ; i.e. , roasting using higher temperatures apparently increases the allergenic properties of peanut proteins14 ) . buckwheat is an important food causing anaphylaxis in korea and japan , but this is very rare in other countries15 ) . sesame seed allergy is more commonly observed in israel than elsewhere , and mustard allergy is mainly observed in france16,17 ) . the prevalence of shellfish allergy is 0.5% in canada , but 4% in singapore and the philippines18 ) . patients with fa developed during infancy tend to become tolerant and able to eat causative foods with ageing , so children with fa should have periodic evaluation for remission . remission rates vary according the offending foods , although the reason for this is still unknown . high remission rates are observed for eggs and cow 's milk , while low remission rates are reported for peanut and tree nut . in a retrospective study from korea including 106 children with atopic dermatitis and egg allergy diagnosed before 2 years of age , 60% became tolerant to egg by age 5 , and the age at the diagnosis of the egg allergy was the significant prognostic factor for tolerance19 ) . a study from spain reported that 66% of children with early fa became tolerant by age 520 ) . in a retrospective study from the us , the rate of egg allergy remission was 26% by age 6 , and risk factors for persistence of egg allergy included a high initial level of egg sige , the presence of other atopic disease , and the presence of an allergy to other foods21 ) . a study at a us university referral hospital reported that 80% of all infants who developed milk allergy in the first year of life became tolerant by their fifth birthday13 ) , while a more recent us study has reported a much lower rate ( 5% at age 4 and 21% at age 8) of fa remission22 ) . bock and atkins23 ) reported that most patients with peanut allergy did not develop tolerance . until recently , peanut allergy was considered life - long , but a small percentage of children tolerated peanut several years after their initial diagnosis24,25 ) . traditional or class 1 food allergens , such as egg , are heat- , enzyme- , and low ph - resistant water - soluble glycoproteins ranging in size from 10 to 70 kd . class 1 food allergens induce allergic sensitization via the gastrointestinal tract and are responsible for systemic reactions ( traditional or class 1 fa)13 ) . class 2 food allergens , such as apple and celery , are heat - labile , susceptible to digestion , and highly homologous with proteins in pollens . class 2 fa ( oral allergy syndrome , oas ) is typically the result of sensitization to labile proteins , such as pollens , encountered through the respiratory route . cross - reactivity is largely determined by structure : 2 proteins are cross - reactive if they share structural features . phylogenetically related mammals express similar milk proteins with significant amino acid sequence homology , resulting in clinical cross - reactivity among cow , sheep , and goat milk27,28 ) . clinical cross - reactivity between peanut and other legumes is extremely rare , despite the high degree of cross - sensitization based on ige binding and results of skin prick tests29 ) . oas is a form of contact allergy to raw fruits and vegetables ( table 3 ) . oas affects approximately 50% of pollen allergic adults and represents the most common adult fa . it results from the cross - reactivity between the allergenic proteins in pollens and plant foods26,30 ) . thermal denaturation of globular proteins disrupts the tertiary structure , leading to random - coiled aggregation and insolubility . in one study , heating for 15 minutes at 95 reduced ige binding with ovalbumin and ovomucoid , and egg white proteins boiled for 5 or 60 minutes showed greatly decreased allergenicity , especially after prolonged boiling for 60 minutes31,32 ) . lemon - mule et al.33 ) reported that 64 of 117 subjects tolerated egg baked in muffins ( 176 for 30 minutes ) and waffles ( 260 for 30 minutes ) , but only 23 tolerated regular scrambled egg or french toast . however , other studies have shown that the ige binding capacity of soy 2s - globulin is strengthened by heating , and , as noted above , roasting using higher temperatures apparently increases the allergenic properties of peanut proteins14 ) . the allergenicity of the major peanut allergens ara h 1 , ara h 2 , and ara h 3 was decreased by vinegar treatment , suggesting that the extent of allergenicity varies with ph34 ) . traditional or class 1 food allergens , such as egg , are heat- , enzyme- , and low ph - resistant water - soluble glycoproteins ranging in size from 10 to 70 kd . class 1 food allergens induce allergic sensitization via the gastrointestinal tract and are responsible for systemic reactions ( traditional or class 1 fa)13 ) . class 2 food allergens , such as apple and celery , are heat - labile , susceptible to digestion , and highly homologous with proteins in pollens . class 2 fa ( oral allergy syndrome , oas ) is typically the result of sensitization to labile proteins , such as pollens , encountered through the respiratory route . cross - reactivity is largely determined by structure : 2 proteins are cross - reactive if they share structural features . phylogenetically related mammals express similar milk proteins with significant amino acid sequence homology , resulting in clinical cross - reactivity among cow , sheep , and goat milk27,28 ) . clinical cross - reactivity between peanut and other legumes is extremely rare , despite the high degree of cross - sensitization based on ige binding and results of skin prick tests29 ) . oas is a form of contact allergy to raw fruits and vegetables ( table 3 ) . oas affects approximately 50% of pollen allergic adults and represents the most common adult fa . it results from the cross - reactivity between the allergenic proteins in pollens and plant foods26,30 ) . thermal denaturation of globular proteins disrupts the tertiary structure , leading to random - coiled aggregation and insolubility . in one study , heating for 15 minutes at 95 reduced ige binding with ovalbumin and ovomucoid , and egg white proteins boiled for 5 or 60 minutes showed greatly decreased allergenicity , especially after prolonged boiling for 60 minutes31,32 ) . lemon - mule et al.33 ) reported that 64 of 117 subjects tolerated egg baked in muffins ( 176 for 30 minutes ) and waffles ( 260 for 30 minutes ) , but only 23 tolerated regular scrambled egg or french toast . however , other studies have shown that the ige binding capacity of soy 2s - globulin is strengthened by heating , and , as noted above , roasting using higher temperatures apparently increases the allergenic properties of peanut proteins14 ) . the allergenicity of the major peanut allergens ara h 1 , ara h 2 , and ara h 3 was decreased by vinegar treatment , suggesting that the extent of allergenicity varies with ph34 ) . currently , the only way to manage fa is avoidance of the allergen and prompt treatment of symptoms when they arise . accidental exposure to food allergens is inevitable and patient and family education regarding cross - contamination , label reading , and prompt recognition and treatment of food allergic reactions is a cornerstone of fa management . because avoidance of specific allergens can limit the availability of nutritious food choices , nutritional counseling and regular growth monitoring are recommended for all children with fa , especially during infancy . elimination of causative foods should be minimized to prevent nutritional disorders and improve the quality of dietary life . even if a food is positive for sige antibodies and skin prick test , it should not be eliminated if an oral challenge test is negative . tolerance to foods , especially egg and milk , should be checked periodically , because children tend to outgrow the allergies . strict avoidance of offending allergen - containing food products has been the standard of care for children with fa . however , as noted , lemon - mule et al.33 ) have recently demonstrated that ingestion of extensively heated egg products was well - tolerated and safe for some patients with egg allergy , and they have suggested that strict dietary avoidance of heated egg might not be necessary for all patients with egg allergy . because even trace amounts of food allergens may trigger severe reactions in highly sensitive food - allergic individuals , strict avoidance is very important in these individuals . it is essential to educate patients and families regarding cross - contamination and label reading , because many patients and their caregivers do not recognize the risk of exposure to trace amounts of food allergen . in korea , food labeling regulations announced by the korean food & drug administration ( effective june 17 , 2006 ) require food manufacturers to indicate if a product contains any of 12 food allergens : milk , eggs , peanuts , soy , wheat , buckwheat , shrimp , crab , mackerel , pork , tomato , and peach . however , this regulation is not sufficient to prevent fa accident because it excludes tree nut and fish , which are also important allergens that can cause anaphylaxis . another danger faced by food - allergic patients is occult contamination with trace amounts of food from shared of equipment . all equipment and utensils used to prepare allergenic food should be cleaned with hot soapy water before being used to prepare allergen - free food . the us niaid guidelines recommend nutritional counseling and regular growth monitoring for all children with fa . children with fa may be at risk for inadequate nutritional intake or poor growth without apparent nutritional problems35 - 37 ) . flammarion et al.37 ) assessed food intake and nutritional status of children ( mean age , 4.72.5 years ) with fa following an elimination diet and reported that children with food allergies were smaller for their age than controls , even when they received similar nutrients . young children who are sensitive to multiple major food allergens are at risk for protein and calorie deficiency and may require a hypoallergenic formula to meet their needs . hypoallergenic formulas available in korea are based on extensively hydrolyzed casein derived from cow 's milk ( i.e. , babywell ha , pregestimil , neutramigen , alimentum ) or on a mixture of single amino acids ( neocate ) . soy protein formula is not an appropriate substitute for patients with cow milk allergy because soy allergy is highly prevalent in patients with cow milk allergy38,39 ) . sheep and goat milk - based formulas are also discouraged because of cross - reactivity with cow milk28 ) . children may be at risk for accidental exposure to food allergens and fa reactions , including anaphylaxis , in schools and daycares , where they spend many of their days . in korea , 99.6% of all schools provide school lunches , and 8 million students , representing approximately 20% of the korean population , are offered school lunches during school days40 ) . presently , most students with fa must rely solely on self - care , because no emergent measures for fa provided by the schools are in place . other developed countries , including the us and japan , have introduced school - wide or government - supported plans for management fa in schools41,42 ) . government - supported management and treatment action plans for fa at schools should also be established in korea . for families of patients in the us with fa who need practical information and a comprehensive curriculum regarding fa management , www.foodallergy.org provides advice on dietary avoidance , along with survival strategies for schools , restaurants , and camps , manufacturers ' updates , and special support programs for teenagers and fa experts43 ) . in korea , www.foodallergy.or.kr supplies food allergic families and supervisors with similar information . in summary , fa is an emerging worldwide health problem requiring increased attention from primary care clinicians in korea . although fa has a relatively low mortality and an almost continual absence of physical symptoms , patients with fa face the possibility of potentially severe , and even fatal , reactions and must maintain dietary vigilance . it is essential to offer information and support to children with fa and their families . general practitioners should tailor this information to the specific needs of each fa patient and parents and pediatricians should be aware of psychosocial problems in children with allergic diseases .

pilomatricoma ( pmx ) also known as calcifying epithelioma of malherbe or pilomatrixoma was first described as epithelioma cutis necrotic calcification malherbe by malherbe and chenantais in 1880 as a benign , subcutaneous tumor arising from sebaceous glands . however , the term pilomatrixoma now called pmx was proposed by forbis and helwig in 1961 , thus avoiding the word epithelioma , which may erroneously be described as malignancy by some . pmx is an uncommon tumor , which is sparsely reported in literature and is limited to a few case reports . the purpose of this study is to better describe the diagnostic features of this skin tumor cytologically and correlate these findings with histopathology in order to familiarize the clinicians with this uncommon entity . the present study was conducted in the department of pathology in our institute retrospectively over a period of 4 years . out of the total 7600 cases only 12 were diagnosed as pmx emphasizing the rarity of this benign yet an uncommon tumor . all of these lesions were superficially located with bluish discoloration [ figure 1 ] and adherent to overlying skin , non - tender , firm too hard on palpation with the duration of < 8 months in all cases . fine - needle aspiration cytology ( fnac ) was performed in all cases using a 23-guage needle attached to a 20 ml disposable syringe . the slides prepared were air - dried for may - grunwald - giemsa and wet fixed for h and e staining using the standard staining procedures . all the surgical biopsies were fixed in 10% buffered formalin , routinely processed and embedded in paraffin . sections were stained with h and e. clinical picture of pilomatricoma in a young female showing bluish discolouration of overlying skin cytological and histopathological features such as the presence of basaloid cells , shadow cells , giant cells and calcified deposits were studied in detail in each case separately . histopathological diagnosis was considered final and cytological findings were reviewed that were missed on cytology . all the relevant clinical data including were obtained from the medical records for each case and documented on microsoft excel sheet . data analysis was performed with statistical package for social science ( spss ) for windows software package ( spss ) , release 17 , chicago , usa . all the relevant clinical data including were obtained from the medical records for each case and documented on microsoft excel sheet . data analysis was performed with statistical package for social science ( spss ) for windows software package ( spss ) , release 17 , chicago , usa . in our study , out of the 7600 cases , only 12 cases were diagnosed as pmxs constituting 0.15% of the total cases emphasizing the rarity of this tumor . moehlenbeck reviewed 140,000 skin tumors and noted that pmx represented 0.12% of these cases . the median age of the presentation was 9.5 years ( mean age 13.58 years ) with age ranging from 3 to 58 years . out of the 12 cases , the most common site of predilection was head and neck ( 7/12 ) , followed by the upper limb ( 4/11 ) . size of the lesions varied from 0.4 to 1.2 cm ( mean 0.9 cm ) with the largest lesion measuring 1.2 cm located on the right thigh and the smallest lesion measuring 0.4 cm located on the forehead . several characteristics were studied on cytology [ figure 2 ] including the presence of aggregates of small cells having high nucleocytoplasmic ratio ( basaloid cells ) , cells with eosinophilic cytoplasm with a central clear area ( shadow cells ) , giant cells , squamous cells and calcium deposits . basaloid cells ( 6/12 ) were the most persistent findings in most cases followed by shadow cells ( 4/12 ) [ graph 1 ] . fine needle aspiration cytology picture showing shadow cells and basaloid cells ( may - grunwald - giemsa 100 ) cytological and histopathological features in 12 cases of pilomatricoma two cases showed only presence of macrophages leading to an erroneous diagnosis of a benign cystic lesion while in 2 cases fnac was non - contributory owing to a cellularity , which may have been due to the presence of calcium deposits [ table 1 ] . cytological and histopathological features of pilomatricomas the most persistent histopathological findings in all cases were the presence of basaloid and ghost cells with areas of apparent evolution from the former to the latter [ figure 3 ] . correct clinical diagnosis was given in only one case further emphasizing the need to incorporate this lesion in the differential diagnosis . the cytological diagnosis was reached in only four cases , whereas on histopathology , all cases conformed with the histological features of a pmx . cytology was reviewed and occasional shadow cells and basaloid cells were seen which were earlier missed . in this series of pmx , the median age of the presentation was 9.3 years with one patient presenting at the age of 58 years . lan et al . in his study concluded that pmxs is a disease of the children affecting the age group of 1 - 20 years . julian et al . in their study observed bimodal presentation of pmx in 1 and 6 decades of life . our study showed a female preponderance ( 3:1 ) which is in consonance with the majority of other studies on pmx . the most frequent anatomical location for pmx is the head and neck region followed by the upper extremities , trunk and the lower extremities . involvement in the face has been reported in cheek , preauricular , periorbital , frontal and temporal areas . this was seen in our study also as these lesions were misdiagnosed clinically as epidermal inclusion cyst , monomorphic adenoma and benign cystic lesion or as benign fibrous histiocytomas . clinically these lesions can be suspected based on two pathognomonic signs the tent sign in which the stretching of the overlying skin causes multiple facets and angles of the lesion and the teeter - totter sign in which pressing of one edge of the lesion causes the other edge to protrude from the skin . in our study , all the cases were solitary lesions although multiple occurrences and familial cases are also known in a small percentage of patients generally in association with disorders such as gardner syndrome , steinert disease , sarcoidosis , rubinstein - taybi syndrome and turner syndrome . the role of fnac as a pre - operative diagnostic investigation has been described . in our study , the characteristic cytological features were graded using a scale of + 1 to 3 + as was done by bansal et al . only 4 cases ( 33.33% ) were correctly diagnosed on fnac showing varying proportions of most of the described characteristic cytological features , which is a little lower than as seen in other studies . two cases on fna revealed cystic macrophages only based on which a presumptive diagnosis of a benign cystic lesion was made . the cause of the misdiagnosis could be attributed to the absence of ghost cells , dominance of squamous cells , interobserver variability and calcified nature of the lesion and also due to inadequate sampling . this was true in our study as well in which all of the cases were correctly diagnosed on histopathology . histopathologically , pmx is seen in nests composed of two cell types basaloid cells and shadow cells . shadow cells are a result of tumor maturation resulting due to loss of nuclei and acquiring the central area of the lesion , which may later calcify imparting firm to hard consistency . pmx is a rare tumor , which is mostly found in the pediatric age group . the lesion should be included in the differential diagnosis while examining superficial lesions in this age group mostly in the head and neck region . the main purpose of this article is to raise awareness among clinicians and also to highlight the cytological features of this benign tumor , the diversity of which may prove to be a diagnostic pitfall for the cytopathologists .

for patients with st - segment elevation myocardial infarction ( stemi ) , primary percutaneous coronary intervention ( pci ) is the preferred therapy [ 1 , 2 ] . guidelines have been composed to suggest optimal antithrombotic and antiplatelet regimens in order to prevent new ischaemic events while balancing the risk of bleeding events [ 2 , 3 ] . however , due to the frailty that comes with advanced age , the higher prevalence of diverse comorbidities in the elderly , and the fact that large clinical trials often list age 80years as an exclusion criterion , it is currently unclear if following guideline recommendations will also result in acceptable outcomes regarding ischaemic and bleeding endpoints in elderly patients undergoing primary pci . the mean age of the population in the developed world is increasing rapidly . in the european union , the proportion of people aged 80years is expected to triple from 4.8 to 12.0 % between 2010 and 2060 . an increase in the absolute and relative number of octogenarians undergoing primary pci has been described . a small number of observational studies have reported mortality rates after primary pci for stemi in patients aged 80 years [ 69 ] . however , there is a paucity of data on the long - term occurrence of recurrent ischaemic events and bleeding in the elderly after primary pci . we therefore investigated a cohort of stemi patients undergoing primary pci in a large tertiary care centre to provide more insight into ischaemic and bleeding outcomes after primary pci for stemi in patients aged 80 years and older . data analysed in this study were obtained from stemi patients who were accepted for primary pci at the academic medical center the study complied with the declaration of helsinki , and the local ethics committee approved the study protocol . in general , patients qualified for primary pci if they had typical ischaemic chest pain and at least 1 mm st - segment elevation in two or more contiguous leads or a new left bundle branch block ( lbbb ) . the primary pci and adjunctive pharmacological treatment were performed according to the american college of cardiology / american heart association and the european society of cardiology ( esc ) guidelines . if a coronary stent was implanted , clopidogrel was prescribed for at least one month to patients with a bare metal stent ( bms ) and for 6 to 12 months to patients with a drug - eluting stent ( des).patients were routinely pretreated with 300500 mg aspirin and 5000 iu unfractionated heparin ( ufh ) . glycoprotein iib / iiia inhibitors ( gpis ) were used in a bail - out setting at the discretion of the operator . a detailed description of the study protocol has been previously published [ 1012 ] . in short , procedural and angiographic data were prospectively collected by interventional cardiologists and specialised nurses in a dedicated database . we obtained follow - up of clinical outcome , including recurrent mi , stroke , stent thrombosis and bleeding , by reviewing inpatient and outpatient charts in the tertiary pci centre and referring hospitals between 2011 and 2012 for consecutive stemi patients with available activated partial thromboplastin time ( aptt ) measurements in the context of a study designed to investigate the relationship between aptt and clinical outcome in stemi patients treated with primary pci . patients whose whereabouts were unknown were considered lost to follow - up from the date of last known medical contact . follow - up information regarding vital status was obtained from computerised , long - term mortality records from the national death index between 1 december 2012 and 30 april 2012 . the study cohort consisted of all stemi patients included in our study database who were alive at the end of the procedure . clinical outcomes included major adverse cardiac events ( mace ; a composite of cardiac death , recurrent myocardial infarction ( mi ) , stroke and target lesion revascularisation ( tlr ) ) , all - cause , cardiac and non - cardiac mortality , recurrent mi , ischaemic and haemorrhagic stroke , stent thrombosis , tlr and bleeding . cardiac death , recurrent mi , stent thrombosis and tlr were defined according to the academic research consortium ( arc ) criteria . bleeding complications were defined according to the bleeding academic research consortium ( barc ) bleeding classification and the thrombolysis in myocardial infarction bleeding classification [ 14 , 15 ] stroke was defined as an irreversible neurological deficit , as classified by the treating neurologist , on the basis of supporting information , including brain images and neurological evaluation . normally distributed continuous variables are reported as the mean with standard deviation and compared with the one - way anova test , skewed distributed variables are presented as the median with interquartile range ( iqr ) and compared with the kruskal - wallis test . missing covariate data were imputed with the use of multiple imputations and were assumed to be missing at random . cox proportional hazards models were first fit separately in the 10 imputed datasets and subsequently pooled according to rubin s protocol . multivariable cox proportional hazards regression was used in the following manner : first , univariable hazard ratios were calculated for all variables in tables 1 and 2 . then , multivariable backward stepwise cox regression models were constructed using variables with p < 0.10 in univariate analysis as candidate variables . entry and exit criteria were set at p = 0.10 . in order to plot the graphic relationships between age and probability of death , mace , barc type 3 bleeding and stroke at 3 years , we calculated the predicted probability of these events by incorporating the coefficients returned by the unadjusted and adjusted cox models in the following equation : \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document } $ $ s\left ( t \right)={{e}^{\left ( -{{h}_{o}}\left ( t \right)*\left({{x}_{1}}{{\beta } _ { 1}}+{{x}_{2}}{{\beta } _ { 2}}+\ldots{{x}_{k}}{{\beta } _ { k } } \right ) \right)}}$$ \end{document } where s(t ) is the predicted event - free survival at timepoint t , h0(t ) is the baseline hazard at time t , x1 , 2 , k is the vector of the covariates , and 1 , 2 , k the coefficients determined from the cox regression models . the risk of adverse events was calculated as 1 min s(t ) . for the present analysis , we assumed the predicted probabilities were pooled over the imputed datasets utilising rubin s protocol after applying the logit transformation to the predicted probabilities . the relationship between age and the probability of death , mace , stroke and bleeding were plotted graphically using a model fitting approach involving cubic polynomials ( splines ) as previously employed by the gusto - i ( global utilization of streptokinase and tissue plasminogen activator for occluded coronary arteries ) trial investigators . all analyses were performed with statistical package for social sciences software ( spss version 19.0 , chicago , illinois ) . the study cohort consisted of all stemi patients included in our study database who were alive at the end of the procedure . clinical outcomes included major adverse cardiac events ( mace ; a composite of cardiac death , recurrent myocardial infarction ( mi ) , stroke and target lesion revascularisation ( tlr ) ) , all - cause , cardiac and non - cardiac mortality , recurrent mi , ischaemic and haemorrhagic stroke , stent thrombosis , tlr and bleeding . cardiac death , recurrent mi , stent thrombosis and tlr were defined according to the academic research consortium ( arc ) criteria . bleeding complications were defined according to the bleeding academic research consortium ( barc ) bleeding classification and the thrombolysis in myocardial infarction bleeding classification [ 14 , 15 ] stroke was defined as an irreversible neurological deficit , as classified by the treating neurologist , on the basis of supporting information , including brain images and neurological evaluation . normally distributed continuous variables are reported as the mean with standard deviation and compared with the one - way anova test , skewed distributed variables are presented as the median with interquartile range ( iqr ) and compared with the kruskal - wallis test . missing covariate data were imputed with the use of multiple imputations and were assumed to be missing at random . cox proportional hazards models were first fit separately in the 10 imputed datasets and subsequently pooled according to rubin s protocol . multivariable cox proportional hazards regression was used in the following manner : first , univariable hazard ratios were calculated for all variables in tables 1 and 2 . then , multivariable backward stepwise cox regression models were constructed using variables with p < 0.10 in univariate analysis as candidate variables . entry and exit criteria were set at p = 0.10 . in order to plot the graphic relationships between age and probability of death , mace , barc type 3 bleeding and stroke at 3 years , we calculated the predicted probability of these events by incorporating the coefficients returned by the unadjusted and adjusted cox models in the following equation : \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document } $ $ s\left ( t \right)={{e}^{\left ( -{{h}_{o}}\left ( t \right)*\left({{x}_{1}}{{\beta } _ { 1}}+{{x}_{2}}{{\beta } _ { 2}}+\ldots{{x}_{k}}{{\beta } _ { k } } \right ) \right)}}$$ \end{document } where s(t ) is the predicted event - free survival at timepoint t , h0(t ) is the baseline hazard at time t , x1 , 2 , k is the vector of the covariates , and 1 , 2 , k the coefficients determined from the cox regression models . the risk of adverse events was calculated as 1 min s(t ) . for the present analysis , we assumed the predicted probabilities were pooled over the imputed datasets utilising rubin s protocol after applying the logit transformation to the predicted probabilities . the relationship between age and the probability of death , mace , stroke and bleeding were plotted graphically using a model fitting approach involving cubic polynomials ( splines ) as previously employed by the gusto - i ( global utilization of streptokinase and tissue plasminogen activator for occluded coronary arteries ) trial investigators . all analyses were performed with statistical package for social sciences software ( spss version 19.0 , chicago , illinois ) . of 2009 stemi patients recorded in our database who were treated with primary pci in our institution between 1 january 2003 and 31 july 2008 , 2002 were alive at the end of the procedure . median follow - up duration in this cohort of patients was 4.9 years ( iqr 3.46.4 years ) . a total of 196 patients were aged 80 years or older ( 9.7 % ) , 921 patients were aged 6079 years ( 46.0 % ) , and 885 patients were aged < 60 years ( 44.2 % ) . the proportion of female patients increased with age ; among patients aged 80 years the majority of patients were female . patients aged 80 years were more likely to have diabetes mellitus , prior coronary artery bypass graft surgery ( cabg ) , a history of stroke or transient ischaemic event , peripheral artery disease and malignant disease compared with younger patients . moreover , they more often had anaemia and an estimated creatinine clearance of < 60 ml / min/1.73 m. on the other hand , patients aged 80 years were less likely to be smokers , less often had a positive family history of coronary artery disease and had lower rates of hypercholesterolaemia at admission compared with younger patients . patients aged 80 years had longer ischaemic times , a higher prevalence of cardiogenic shock at admission , lower rates of post - procedural timi 3 flow , a higher rate of coronary calcification , multivessel disease and chronic total occlusions in non - infarct related vessels compared with younger patients . figure 1 displays a kaplan - meier time - to - event curve for mace . figure 2 displays kaplan - meier time - to - event curves for recurrent mi , mortality , barc bleeding 3 , and stroke . table 3 shows kaplan - meier estimates of the incidence of adverse events at 3-year follow - up . rates of mace , mortality , recurrent mi , stroke and barc bleeding 3 increased with age . conversely , rates of stent thrombosis and target lesion revascularisation decreased non - significantly with age.fig . 2kaplan - meier curve for recurrent myocardial infarction , mortality , barc bleeding 3 , and stroke . barc bleeding academic research consortium kaplan - meier curve for major adverse cardiac events kaplan - meier curve for recurrent myocardial infarction , mortality , barc bleeding 3 , and stroke . barc bleeding academic research consortium table 1 of the supplementary appendix shows the results of multivariable cox regression analyses for 3-year clinical outcomes . relative to patients aged < 60 years , patients aged 6079 years and those aged 80 years were found to have an increased risk of cardiac and non - cardiac mortality , stroke and non - access site bleeding after correction for confounding variables . in addition , age 6079 years and age 80 years was associated with a borderline non - significant increase in risk of barc type 3 bleeding . on the other hand , age age as a continuous variable was an independent predictor of all - cause mortality ( 1.43 per 10 year increase ; 95 % ci 1.271.61 ; p < 0.001 ) , cardiac mortality ( hr 1.34 ; 95 % ci 1.171.53 , p < 0.001 ) , non - cardiac mortality ( hr 1.55 ; 95 % ci 1.241.95 , p < 0.001 ) , stroke ( hr 1.46 ; 95 % ci 1.181.80 ; p < 0.001 ) and barc type 3 bleeding ( hr 1.15 ; 95 % ci 1.041.26 ; p = 0.006 ) , but not of mace ( hr 1.06 ; 95 % ci 0.981.16 ; p = 0.17 ) , or recurrent mi ( hr 1.04 ; 95 % ci 0.931.17 , p = 0.49 ) ( supplementary appendix , table 2 ) . figure 3 shows a graphic representation of the unadjusted ( left panel ) and adjusted ( right panel ) relation between age as a continuous variable and the hazard of mace , mortality , barc bleeding 3 , and stroke up to 3-year follow-up.fig . 3probability of 3-year mortality , major adverse cardiac events , stroke , and barc bleeding 3 according to age . mace major adverse cardiovascular event , barc bleeding academic research consortium probability of 3-year mortality , major adverse cardiac events , stroke , and barc bleeding 3 according to age . the current study extends previous reports by providing insight into the occurrence of a large number of clinically relevant endpoints in up to 3 years of follow - up in patients undergoing primary pci according to age . we found higher rates of ischaemic and haemorrhagic endpoints with increasing age , illustrating the frailty of this rapidly growing high - risk patient group . in a recent large registry , velders et al . found an increased risk of early mortality and heart failure in patients aged 80 as compared with patients younger than 80 . however , among the elderly patients who survived the first month(s ) , the prognosis was similar compared with their younger counterparts . this indicates that the difference in mortality between patients aged 80 and their younger counterparts can be explained by the direct adverse consequence of the stemi itself , such as arrhythmia , heart failure , mechanical complications and procedural complications such as bleeding . interestingly , consistent with previous studies , we found that elderly patients were less likely to develop stent thrombosis or to undergo target lesion revascularisation [ 19 , 20 ] . the fact that the rate of recurrent mi was higher among elderly patients suggests that recurrent mi in elderly patients is the result of plaque rupture of non - target lesions , and is likely explained by the more extensive atherosclerosis among the elderly . the incidence of both access site and non - access site bleeding strongly increased with age in this study . well - known risk factors for bleeding such as female gender and chronic kidney disease were more prevalent in the elderly . moreover , advanced age is associated with increased vascular fragility and calcification , which are known risk factors for access site bleeding [ 22 , 23 ] . in the current study , age 80 years was independently associated with non - access site bleeding , an important finding given the fact that especially non - access site bleeding is associated with adverse outcome [ 21 , 24 ] . in a pooled patient - level analysis involving 14,180 patients recruited in 7 randomised trials pci treatment with a gp iib / iiia inhibitor was associated with a significant 27 % higher risk of non - access site bleeding . a large observational study in 10,469 patients aged 80 years included in the cathpci registry suggests that bleeding avoidance strategies such as radial access and use of vascular closure devices were independently associated with lower bleeding . a number of recommendations to reduce bleeding complications in the elderly may be hypothesised : ( 1 ) the radial approach would seem to be the preferred method of vascular access in patients aged 80 years ; ( 2 ) routine proton pump inhibition might reduce gastrointestinal bleeding in patients aged 80 years ; and ( 3 ) use of gp iib / iiia inhibitors in the elderly may lead to increased bleeding and should be avoided where possible . by observing the kaplan - meier curves for bleeding , recurrent mi , and stroke , it appears that bleeding predominantly occurs during the first month after primary pci , whereas the incidence of recurrent mi and stroke ( of the ischaemic type in the vast majority of patients ) continues to increase during the 3-year follow - up period . studies investigating novel p2y12 inhibitors such as prasugrel and ticagrelor have reported superior outcomes compared with clopidogrel with regards to ischaemic events , at a cost of increased bleeding events [ 27 , 28 ] . no studies investigating strategies to reduce mi and stroke after primary pci for stemi in patients 80 years have been conducted yet . it is therefore currently still unclear whether there is a role for novel p2y12 inhibitors in this population . recently , a small number of observational studies have reported results of primary pci for stemi in patients aged 80 years , showing 30-day mortality rates of around 28 % [ 68 ] . the high rates of mortality in patients aged 80 years can largely be explained by a higher burden of comorbidities , a higher rate of haemodynamic instability during primary pci , and high rates of bleeding and recurrent mi as shown in the current study . while acknowledging that advanced age itself is an important and unmodifiable predictor of mortality , a strategy aimed at reducing early bleeding complications , and late ischaemic complications might potentially lead to improved mortality in patients aged 80 years . recent developments in primary pci associated with superior outcomes such as the radial rather than the femoral approach becoming the default vascular access strategy in primary pci , and the introduction of more effective antiplatelet agents such as prasugrel and ticagrelor instead of clopidogrel may have the greatest absolute benefit in patients aged 80 years , as these patients also have the highest absolute event rates of adverse events . nonetheless , the novel p2y12 inhibitors that are relatively more effective in inhibiting platelet aggregation compared with clopidogrel may also cause more bleeding events in the elderly , potentially offsetting the reduction in ischaemic events observed in randomised controlled trials . ideally , this should be investigated in a randomised clinical trial enrolling stemi patients aged 80 years . first , only a relatively small cohort of patients aged 80 years were included in the present analysis . nonetheless , this is the largest cohort of very elderly patients to date with detailed reporting of a multitude of 3-year ischaemic and bleeding events . moreover , due to the high event rates , this cohort was sufficiently powered to conduct multivariable analyses . finally , as this was an observational study we can only speculate about treatment strategies to improve the clinical outcome of very elderly patients undergoing primary pci . first , only a relatively small cohort of patients aged 80 years were included in the present analysis . nonetheless , this is the largest cohort of very elderly patients to date with detailed reporting of a multitude of 3-year ischaemic and bleeding events . moreover , due to the high event rates , this cohort was sufficiently powered to conduct multivariable analyses . finally , as this was an observational study we can only speculate about treatment strategies to improve the clinical outcome of very elderly patients undergoing primary pci . patients 80 years experienced high rates of ischaemic and bleeding complications , especially in this high - risk patient group individualised therapy is needed to optimise clinical outcomes . this work was supported by the nuts ohra foundation , the netherlands [ sno - t-0702 - 61 ] this work was supported by the nuts ohra foundation , the netherlands [ sno - t-0702 - 61 ]

the clinical management of lesions suspicious of malignancy relies not only on diagnosis of benign versus malignant entity but also on tumor grading , immunohistochemical findings , and genetic information . hence , a carefully performed biopsy with low risk of complication is crucial . compared with open biopsy , image - guided biopsies are minimally invasive and confer several advantages including low morbidity , low complication rate , and cost savings . pet / ct has been used in initial staging , treatment response evaluation , and follow - up . pet / ct - guided biopsies may allow early histological diagnosis and staging before morphologic changes are evident . pet / ct biopsy can rule out malignancy and it can correctly stage different types of cancer . nonreal - time pet / ct biopsies use co - registration of a prior pet image with a procedural ct image . however , this method is inaccurate in time and space , and requires special software . the aim of this study is to report the initial experience of utilizing the real - time pet / ct - guided biopsies , and explore feasibility and technical requirements of the procedure . four real - time intra - procedural pet / ct biopsies were performed at our institution . patient records / information was anonymized and de - identified prior to analysis . in all cases , the patients had a prior whole body pet / ct scan showing abnormal focal uptake , but no ct correlation , and needed further histological confirmation . patients underwent overnight fasting before an 18f - fluorodeoxyglucose ( fdg)-pet / ct biopsy procedure . limited one bed pet / ct imaging was performed over the selected lesion following an uptake period of 60 min after intravenous administration of 5 mci of fdg or naf . imaging was performed in a pet / ct scanner ( ge ste 64 slice ct scanner , ge healthcare , waukesha , wi ) . imaging parameters were 140 kvp , 90120 ma , 1.25 mm collimation with reconstruction as 3.75 mm thick sections by using a 512 512 matrix and a filtered back projection algorithm . immediately after ctac , pet images were obtained in two - dimensional mode for 5 min acquisitions for one - bed position . pet images were reconstructed by using a 128 128 matrix and an iterative - ordered subset expectation maximization algorithm . pet / ct images therefore were obtained in a limited one - bed position over the region of the lesion . once the lesion was identified and assessed , a biopsy needle was guided , real time , to the area of focal uptake . a recent complete blood count ( cbc ) , history of allergies , prothrombin time ( pt ) , and international normalized ratio ( inr ) , co - morbidities and medications were reviewed prior to the procedure . a 12 cm jamshidi bone biopsy needle was used for bone biopsies ( 3 cases ) and a mesenteric biopsy was performed with an 18 gauge introducer needle and a 20 gauge core biopsy needle ( 1 case ) . the coaxial ( introducer ) needle allows the radiologist obtain several samples with a single needle stick . the patients were not moved in and out of the gantry for each manipulation of the needle . patients were positioned and immobilized in the correct position , depending on the location of the lesion . patients were placed in the biopsy position prior to limited pet / ct.local anesthesia with 1% lidocaine was administered.the biopsy needle was guided , real time , by using pet / ct imaging to the targetorgan showing the focal uptake . perfect intra - lesion placement of the tip of the biopsy needle was confirmed by pet / ct imaging.the core of the coaxial needle was pulled out and an 18 gauge biopsy needle was inserted.after removal of the needle , manual compression was performed for 2 - 3 min at the puncture site.the specimens obtained were fixed in 10% formalin and presented to the pathologist , who was in the same room , for quick histopathological examination.the patients were observed for 2 - 6 h and then discharged . patients were positioned and immobilized in the correct position , depending on the location of the lesion . patients were placed in the biopsy position prior to limited pet / ct . local anesthesia with 1% lidocaine was administered . the biopsy needle was guided , real time , by using pet / ct imaging to the targetorgan showing the focal uptake . perfect intra - lesion placement of the tip of the biopsy needle was confirmed by pet / ct imaging . the core of the coaxial needle was pulled out and an 18 gauge biopsy needle was inserted . after removal of the needle , manual compression was performed for 2 - 3 min at the puncture site . the specimens obtained were fixed in 10% formalin and presented to the pathologist , who was in the same room , for quick histopathological examination . in all cases , the patients had a prior whole body pet / ct scan showing abnormal focal uptake , but no ct correlation , and needed further histological confirmation . patients underwent overnight fasting before an 18f - fluorodeoxyglucose ( fdg)-pet / ct biopsy procedure . limited one bed pet / ct imaging was performed over the selected lesion following an uptake period of 60 min after intravenous administration of 5 mci of fdg or naf . imaging was performed in a pet / ct scanner ( ge ste 64 slice ct scanner , ge healthcare , waukesha , wi ) . imaging parameters were 140 kvp , 90120 ma , 1.25 mm collimation with reconstruction as 3.75 mm thick sections by using a 512 512 matrix and a filtered back projection algorithm . immediately after ctac , pet images were obtained in two - dimensional mode for 5 min acquisitions for one - bed position . pet images were reconstructed by using a 128 128 matrix and an iterative - ordered subset expectation maximization algorithm . pet / ct images therefore were obtained in a limited one - bed position over the region of the lesion . once the lesion was identified and assessed , a biopsy needle was guided , real time , to the area of focal uptake . a recent complete blood count ( cbc ) , history of allergies , prothrombin time ( pt ) , and international normalized ratio ( inr ) , co - morbidities and medications were reviewed prior to the procedure . a 12 cm jamshidi bone biopsy needle was used for bone biopsies ( 3 cases ) and a mesenteric biopsy was performed with an 18 gauge introducer needle and a 20 gauge core biopsy needle ( 1 case ) . the coaxial ( introducer ) needle allows the radiologist obtain several samples with a single needle stick . the patients were not moved in and out of the gantry for each manipulation of the needle . patients were positioned and immobilized in the correct position , depending on the location of the lesion . patients were placed in the biopsy position prior to limited pet / ct.local anesthesia with 1% lidocaine was administered.the biopsy needle was guided , real time , by using pet / ct imaging to the targetorgan showing the focal uptake . perfect intra - lesion placement of the tip of the biopsy needle was confirmed by pet / ct imaging.the core of the coaxial needle was pulled out and an 18 gauge biopsy needle was inserted.after removal of the needle , manual compression was performed for 2 - 3 min at the puncture site.the specimens obtained were fixed in 10% formalin and presented to the pathologist , who was in the same room , for quick histopathological examination.the patients were observed for 2 - 6 h and then discharged . patients were positioned and immobilized in the correct position , depending on the location of the lesion . patients were placed in the biopsy position prior to limited pet / ct . local anesthesia with 1% lidocaine was administered . the biopsy needle was guided , real time , by using pet / ct imaging to the targetorgan showing the focal uptake . perfect intra - lesion placement of the tip of the biopsy needle was confirmed by pet / ct imaging . the core of the coaxial needle was pulled out and an 18 gauge biopsy needle was inserted . after removal of the needle , manual compression was performed for 2 - 3 min at the puncture site . the specimens obtained were fixed in 10% formalin and presented to the pathologist , who was in the same room , for quick histopathological examination . all pet / ct positive lesions were successfully biopsied real - time without the need for repeating the biopsies . assessing the intra - lesion placement of the needle tip was very easy despite extreme windowing of the transmission images . the pathology report revealed that two lesions ( 50% ) were malignant and two lesions ( 50% ) were benign . pet / ct guided biopsy and stained biopsy samples from patient 3 [ figures 1 and 2 ] led to the identification of a benign lesion . pet / ct images and stained biopsy sample from patient 4 [ figures 3 and 4 ] identified the lesion as malignant . clinical and histological data of patients 56-year - old male with a complaint of difficulty in urinating was diagnosed with prostate cancer . ( a ) low power view of the marrow biopsy ( 20 ) shows no abnormalities . ( b ) high power view of the biopsy sample ( 200 ) shows normal hematopoietic cells , including megakaryocytes , visible within the adipose tissue of bone marrow ( arrows ) . 56-year - old male with a complaint of difficulty in urinating was diagnosed with prostate cancer . axial pet / ct images ( a ) increased fdg uptake in the left 8 rib ( arrows ) and ( b ) 12 cm jamshidi bone biopsy needle ( arrows ) in the metabolically active region of the left 8 rib . 83-year - old male with elevated prostate - specific antigen ( psa ) was diagnosed with prostate cancer . ( a ) low power view of fna cell block material ( 10 ) shows abundant tumor tissue ( arrows ) . scale bar = 100 microns ( b ) high power view ( 100 ) of an area of dyshesive neoplastic cells demonstrates esophageal adenocarcinoma of signet ring cell morphology . signet ring morphology is described as abundant in cytoplasm with the nucleus pushed to one side ( arrows ) scale bar = 20 microns ( c ) hematoxylin and eosin stained sample ( 50 ) shows clusters of neoplastic cells ( arrows ) and ( d ) immunohistochemical staining of the sample shows cells positive for pancytokeratin ( arrows ) ( d ) . 83-year - old male with elevated prostate - specific antigen ( psa ) was diagnosed with prostate cancer . ( a ) hybrid pet / ct guided the needle ( arrow ) to the exact location of the hypermetabolic lesion in the right iliac bone ( b ) the tip of the needle ( arrow ) in the lesion suspected for metastasis . this is the first study to report the intra - procedural real - time pet / ct biopsy procedure and to report the biopsy of a mesenteric mass using pet / ct . most pet / ct - guided biopsy studies published thus far use prior pet images to fuse with intra - procedural ct to guide the biopsy needle . success of biopsy and accurate diagnosing depends upon adequate tissue being obtained at the right location . the inaccuracy of diagnosis prior to surgery can lead to significant morbidity and increased mortality . most of the time , before performing a biopsy , magnetic resonance imaging ( mri ) or ct image is used to identify the mass . on mri , benign soft tissue lesions are usually small ( < 5 cm ) , well circumscribed , and have homogeneous signal intensity , whereas malignant lesions tend to be large , inhomogeneous and have poorly defined margins . tumors without these features are difficult to classify on mri and there are many exceptions to this classification . it is difficult to consistently distinguish benign from malignant areas within a heterogeneous mass ; more importantly , to define the most malignant area . for fdg avid tumors , fdg - pet can not only detect malignancy but also help to locate the most active areas within the malignancy . hain et al . , found that the pet scan can guide the needle to the best biopsy site . one method uses previously acquired pet / ct images to co - register with intra - procedural ct images , using computer software . a rigid image registration algorithm is required to achieve bimodal fusion between a pre - acquired pet scan and an intra - procedural ct scan . image fusion takes longer time , special hardware , and may require software add - ons . image fusion is the overlay of two or more imaging data sets together as one display , whereas image registration consists of aligning or matching the two imaging data sets spatially to each other . in the method employed for this paper , the emission images are therefore in the absence of movement , always matching the intra - procedural ctac images . the spatial accuracy of this technique is necessary to display clinically relevant image guidance information during biopsy . , has shown that relying on anatomic imaging alone was not adequate to detect cancer . even with the use of costly 3-t mri scanner , singh et al . , found cancer in only 3% of the cases . furthermore , artifacts increase with increasing magnetic field strength . in mri - guided biopsy , which is complex to perform , patients may have to slide in and out of the scanner several times during the procedure and the biopsy needle may have to be inserted several times until it is in the desired target area . also , commonly used titanium biopsy needles are not directly visible on mri and an artifact forms in the neighborhood of the needle . larger needles have higher diagnostic yield and accuracy , however , they cause larger artifacts on mri images . in a study by johnson et al . , mri - guided biopsy could not visualize 40% of the lesions 10 mm in size . some mri - guided biopsies require contrast injection and consequently , the biopsy time is limited to about 45 min before the contrast washes out . relative to ultrasound ( us)- and ct - guided biopsy , mri - guided biopsy generally requires longer procedure time . prior et al . , reported that ct missed small cancerous lesions ( 10 - 20 mm ) in their study and the lesions were visualized only by pet . in fact , the accuracy of ct - guided biopsy for cancerous lesions ( 0.71.5 cm ) in size could be as low as 35% . in a study by davis et al . , the ability of ct - guided biopsy to obtain adequate samples for solid tumors ( < 1 cm ) was 25% and for cystic tumors was 0% . with the us - guided biopsy , the diagnostic yield can be as low as 46% for lesions less than 20 mm in diameter . us - guided biopsy is not optimal for bone , bowel , and lesions deep inside the body . the field of view of us is also limited and nearby critical structures may not be visualized . the 2-dimentional us images commonly used today for real - time guidance make proper needle placement challenging when dealing with complex 3d targets . us - guided biopsy is operator dependent and it is difficult to visualize the needle tip . not surprisingly , the specificity of us - guided biopsy can be as low as 67% . in large tumor masses , most of the tissue can be necrotic and only a small portion can be metabolically active . pet / ct biopsy can pinpoint the most active tissue within the large mass and this decreases the need to re - biopsy . in the case of multiple lesions , pet / ct can guide the operator to the most active lesion . in the present study , there was no need for repeat biopsies and the diagnostic yield was 100% . fused real - time us to co - registered ct and fdg - pet scans . in that study , malignancy was found in 50% ( 18/36 ) biopsies and 2 biopsies were not successful . we also found malignancy in 50% of the cases and we have similar or better results compared with the us / pet / ct multimodality fusion study . in an article by servois et al . it is very important to rule out malignancy in suspicious lesions ; especially in patients with cancer . pet / ct biopsy is clearly better than ct - guided biopsy because in some cancers , morphological changes are not apparent . segaert et al . , found that integrated pet / ct is superior to both pet and ct in detecting cancerous lymph nodes and it had a sensitivity of 100% . pet / ct biopsy can be utilized for any part of the body including the bone and bone marrow . in addition , fdg dosage injected in our procedures was far less than the dosage for diagnostic pet / ct exams . combining pet / ct exam and pet - guided biopsy will minimize radiation by requiring only one fdg injection . tatli et al . , used an average of 20.6 mci of fdg in their pet / ct biopsies . , reported that pet / ct - guided biopsy is feasible and may optimize the diagnostic yield of image - guided interventions fdg - pet / ct - guided biopsy has been utilized for soft tissue tumors and for mucormycosis . fdg - pet / ct has much higher diagnostic accuracy than ct or mri in characterizing tumor . in addition to biopsy , pet / ct has been used in ablation and surgical procedures . werner et al . , wrote that bone scintigraphy can not reliably detect malignant bone marrow infiltration . bone scintigraphy shows only bone metabolism , not malignancy , and hence may not be specific enough to separate bone remodeling and healing fractures , which are common after treatment of bone metastases from live tumor cells . according to werner et al . , pet / ct - guided biopsy is the best approach for detecting bone and bone marrow abnormalities . the veteran patients are mostly male and the patients in this study were all males . the tracers used for the real - time pet / ct biopsy were naf18 and 18f - fdg . cancers such as thyroid cancer , renal cancer , and skeletal metastasis of prostate cancer can have low fdg avidity . in contrast , naf18 is not ideal for osteolytic bone lesions such as breast cancer metastases . the veteran patients are mostly male and the patients in this study were all males . the tracers used for the real - time pet / ct biopsy were naf18 and 18f - fdg . cancers such as thyroid cancer , renal cancer , and skeletal metastasis of prostate cancer can have low fdg avidity . in contrast , naf18 is not ideal for osteolytic bone lesions such as breast cancer metastases . pet / ct - guided biopsy is feasible and there is rarely a need for repeat biopsies with this technique . the radiation dosage required for pet / ct biopsy is less than routine pet / ct exams . pet / ct biopsy requires no additional equipment and can be done with equipment already available for regular examinations . we found that real - time pet / ct biopsy has benefits over conventional biopsy techniques in terms of accuracy of locating the correct biopsy site .

mato grosso comprises 903,357.9 km and has an estimated population of 2,803,274 inhabitants living in 141 municipalities . nineteen municipalities have reported cases of hps , mainly near brazil s br-364 highway , located between the north and southwestern sections of the state . the climate is equatorial subhumid , with an annual rainfall of 1,700 mm , and temperature range 2440c ; the landscape consists of savannah ( cerrado ) and pre - amazon rainforest . hps was diagnosed in 24 persons who were igm positive for lnv during 20012006 in the municipalities of barra do bugres ( n = 1 ) , campo novo do parecis ( n = 13 ) , diamantino ( n = 3 ) , nova olmpia ( n = 1 ) , santo afonso ( n = 1 ) , so jos do rio claro ( n = 1 ) , and tangar da serra ( n = 4 ) ( figure 1 ) . detailed information of patient samples submitted for nucleotide sequencing is provided in the table . state of mato grosso , brazil , indicating municipalities where hanta pulmonary syndrome cases occurred . during a 2001 ecological epidemiologic study conducted in the municipalities of tangar da serra and campo novo do parecis , researchers obtained blood and viscera samples from wild rodents ( 10 ) . the researchers followed brazilian institute for the environment and renewable natural resources guidelines for the capture and handling of rodents and using biosafety level 3 protocols . the samples were tested for hantavirus ; animals with positive test results were identified taxonomically by morphometry and molecular analysis of mitochondrial dna ( cytochrome b gene ) ( 10,11 ) . for hantavirus detection , we conducted reverse transcription pcr to synthesize complementary dna with generic hantavirus primers as described ( 1,2 ) . we obtained n gene partial nucleotide sequences by using the sanger method with the same primers ( 3,4,6,9 ) . at least 3 amplicons per sample were sequenced in both directions to improve coverage and confidence for results . the obtained sequences were aligned with other hantavirus sequences available at the genbank database ( www.ncbi.nlm.nih.gov ) with clustalw software in bioedit version 7.1.3 ( www.mbio.ncsu.edu/bioedit/bioedit.html ) . we implemented the maximum - likelihood and bayesian methods by using phyml ( www.atgc-montpellier.fr/phyml/versions.php ) and mr . we used modeltest version 3.7 ( http://gel.ahabs.wisc.edu/mauve ) to determine the best nucleotide substitution model . we analyzed 2 million replicates , with the sample fixed at every 1,000 trees generated , and used tracer ( www.evolve.zoo.ox.ac.uk ) to determine whether the bayesian analysis reached appropriate convergence ( 3,6,9,12 ) . we obtained amplicons from 20 of the 24 samples from persons with hps and partial sequence of the n gene ( 434 bp ) from 16 of the 24 samples from patients who were symptomatic at the time of sampling . during the ecologic study , 126 rodents were captured : 68 ( 53.9% ) commensal synanthropic species , 49 ( 38.8% ) wild rodents [ calomys callidus ( n = 46 ) , proechimys sp . ( n = 1 ) , and necromys lasiurus ( n = 2 ) ] , and 9 ( 7.1% ) unidentified species . igg was detected in 8 ( 17.4% ) c. callidus rodents ( 2 captured in campo novo do parecis , 6 in tangar da serra ) . amplicons were produced in lung / heart samples from 7 of the 8 igg - positive rodents ; 3 of those were selected for nucleotide sequencing of the n gene ( table ) . all strains recovered from human and c. callidus rodent specimens were related and formed a monophylogenetic cluster with the lnv ( genbank accession no . these strains were included in subclade ii , which comprises anajatuba , rio mamore , rio mearim , and alto paraguay viruses ( figure 2 ) . the genetic distance between strains recovered from rodents and humans was 5.5% , whereas the genetic distance between the human strains was 6.8% . analysis of homology showed no difference between the partial amino acid sequences of human and rodent strains and lnv ( 100% homology ) . the homology of nucleotide sequences between the lnv strains was 89.9%93.4% ( table a1 ) . most changes were silent mutations in the nucleotide sequences , indicated by the genetic divergence between lnv strains ( div = 0.2%9.8% ) . phylogenetic comparison of the partial nucleotide sequences of nucleoprotein ( n ) gene of the small ( s ) rna segment of different hantavirus strains from the old world and new world by using the maximum - likelihood method and bayesian analysis , with detail of the phylogenetic relationship between lnv strains isolated from humans and rodents in the state of mato grosso , brazil . apv , alto paraguay ; anajv , anajatuba , andv , andes ; bmjv , andes bermejo , nemv , andes neembuco , lecv , andes lechiguanas ; ornv , andes oran ; arav , araraquara ; bayv , bayou ; bccv , black creek canal ; canov , cano delgadito ; casv , castelo dos sonhos ; chov , choclo ; emcv , el moro canyon ; htnv , hantaan ; juqv ; juquitiba - araucaria ; lnv , laguna negra ; lscv , limestone canyon ; mulv , muleshoe ; nyv , new york ; prnv , pergamino ; puuv , puumala ; riomv , rio mamor ; rimev , rio mearim ; riosv , rio segundo ; seov , seoul ; snv , sin nombre ; tulv , tula . lnv was initially confirmed in 1997 by serologic testing of a patient with hps who died . the patient lived in santiago , chile , but was probably infected in santa cruz , bolivia ( 13 ) . in 1999 , molecular analysis of the small n gene and medium gn and gc gene segments of the hantavirus genome in samples from hps patients from bolivia , western paraguay , and chile facilitated the genetic characterization of lnv and its association with the small vesper mouse calomys laucha , which is considered the primary host of lnv . subsequent studies in argentina have also demonstrated the circulation of lnv in patients with hps and in the large vesper mouse calomys callosus ( 4,9,1315 ) . our phylogenetic analysis of partial sequences of the n gene showed lnv as the cause of hps , and the possible association of the organism with c. callidus rodents in western brazil . these findings highlight the intense circulation of lnv in matto grosso municipalities located near the br-364 highway . the vegetation and the equatorial climate of the area provide an excellent microenvironment for the maintenance of c. callidus rodents , as do areas in bolivia paraguay , and northern argentina , where hps caused by lnv has been reported ( 4,9,1315 ) . the high nucleotide and amino acid homology between strains recovered from humans and the c. callidus rodent in matto grosso and the lnv prototype detected in paraguay and argentina suggest that lnv was transmitted by the rodent host c. callidus and led to the hps cases that occurred in the vicinity of the highway br-364 in southwestern matto grosso . no correlation was observed between the human lnv strains and year , geographic distribution , or between the severity of disease and the genetic diversity of lnv found in brazil . the genetic data obtained in this study provide a better understanding of the molecular characterization of lnv and its association with hps in southwestern matto grosso . finally , on the basis of the phylogenetic analysis , the rodent species c. callidus is suggested as a potential reservoir for lnv . further analyses of complete genome data are needed to confirm this result and to assess whether the c. callidus rodent is the sole carrier of of lnv in matto grosso .

rodents were captured in a rural area 22.8 km north of the town of real de catorce in the municipality of catorce , san luis potos , mexico ( figure 1 ) . a total of 400 live - capture traps were set each night on 2 consecutive nights in the first week of august 2005 . samples of whole blood , samples of kidney and other solid tissues , and the skins and skeletons of the rodents were deposited into the museum of texas tech university . the filled circle in southern texas indicates the locality in which catarina virus is enzootic . the star in san antibody ( immunoglobulin g ) to whitewater arroyo virus previously was found in white - toothed woodrats ( neotoma leucodon ) , a mexican woodrat ( n. mexicana ) , and deer mice ( peromyscus spp . ) captured in nuevo len ; white - throated woodrats ( n. albigula ) and white - toothed woodrats captured in san luis potos ; and deer mice ( peromyscus spp . ) and a southern plains woodrat ( n. micropus ) captured in tamaulipas ( m.l . the blood samples were tested by elisa ( 10 ) for antibody ( immunoglobulin [ ig ] g ) to whitewater arroyo virus strain av 9310135 ( 7 ) . samples of spleen and kidney from white - toothed woodrats tk133448 and tk133451 , 7 other white - toothed woodrats , 2 antibody - positive nelson s pocket mice ( chaetodipus nelsoni ) , and an antibody - positive merriam s kangaroo rat ( dipodomys merriami ) were tested for arenavirus by cultivation in monolayers of vero e6 cells ( 11 ) . samples of kidney from the antibody - positive rodents were tested for arenavirus rna by using heminested pcr assays . the first - strand cdna for the pcr was synthesized by using superscript ii reverse transcriptase ( invitrogen life technologies , inc . , carlsbad , ca , usa ) in conjunction with oligont 19c - cons ( 2 ) . the nucleotide sequence alignments were analyzed by using mrbayes 3.1.2 ( 12 ) in the computer software package paup * , version 4.0b10 ( http://paup.csit.fsu.edu ) . ( details of the assays for igg , arenavirus , and arenavirus rna and the analyses of the amino acid sequence data and nucleotide sequence data are available from c.f.f . ) nine white - toothed woodrats , 3 northern grasshopper mice ( onychomys leucogaster ) , 19 deer mice ( peromyscus spp . ) , 11 harvest mice ( reithrodontomys spp . ) , 13 nelson s pocket mice , 45 merriam s kangaroo rats , and 16 other kangaroo rats ( dipodomys spp . ) were captured during 800 trap - nights ; overall trap success rate was 116/800 ( 14.5% ) . igg against whitewater arroyo virus was found in 2 ( 22.2% ) of the white - toothed woodrats , 2 ( 15.4% ) of the nelson s pocket mice , 1 ( 2.2% ) of the merriam s kangaroo rats , and none of the 49 other rodents . the sequence of the 3,352-nt fragment of the s genomic segment of arenavirus av h0030026 ( genbank accession no . the 3,352-nt fragment included the complete glycoprotein precursor ( gpc ) gene and complete nucleocapsid ( n ) protein gene of av h0030026 . nonidentities between the amino acid sequences of the gpc and n protein of av h0030026 and the amino acid sequences of the gpc and n proteins of the 10 other north american viruses included in the analyses of amino acid sequence data ranged from 28.3% to 35.2% and from 11.6% to 21.0% , respectively ( table 2 ) . the results of the bayesian analyses of complete gpc gene sequences ( figure 2 , panel a ) and complete n protein gene sequences ( figure 2 , panel b ) indicated that av h0030026 represents a unique phylogenetic lineage but failed to resolve the relationship of av h0030026 to the other north american viruses analyzed . * numbers above and below the diagonal are the nonidentities among the glycoprotein precursors and nucleocapsid proteins , respectively . ef619035 ) ; bcnv , bear canyon virus strain av a0070039 ( ay924391 ) ; ctnv , catarina virus strain av a0400135 ( dq865244 ) ; sktv , skinner tank virus strain av d1000090 ( eu123328 ) ; tamv , tamiami virus strain w 10777 ( af512828 ) ; ttcv , tonto creek virus strain av d0150144 ( ef619033 ) ; wwav , whitewater arroyo virus strains av 9310135 ( af228063 ) , av 96010024 ( eu123331 ) , av 96010151 ( eu123330 ) , and av d1240007 ( eu123329 ) . the results of a previous study indicated that av 96010024 , av 96010151 , and av d1240007 are strains of wwav or strains of arenaviruses that are phylogenetically closely related to wwav ( 5 ) . nonidentities among the amino acid sequences of the glycoprotein precursors and among the amino acid sequences of the nucleocapsid proteins of av 9310135 , av 96010024 , av 96010151 , and av d1240007 in this study ranged from 16.0% to 25.8% and 7.3% to 10.3% , respectively . phylogenetic relationships among 11 north american arenaviruses based on bayesian analyses of a ) glycoprotein precursor gene sequences and b ) nucleocapsid protein gene sequences . the number(s ) at the nodes are clade probability values , a single 1.00 indicates that the clade probability values for both analyses were 1.00 , and clade probability values < 0.93 were not included in the phylograms . the branch labels include ( in the following order ) virus , host species , and state . ef619035 ) ; bcnv , bear canyon virus strain av a0070039 ( ay924391 ) ; ctnv , catarina virus strain av a0400135 ( dq865244 ) ; sktv , skinner tank virus strain av d1000090 ( eu123328 ) ; tamv , tamiami virus strain w 10777 ( af512828 ) ; ttcv , tonto creek virus strain av d0150144 ( ef619033 ) ; wwav , whitewater arroyo virus strain av 9310135 ( af228063 ) ; arenaviruses av 96010024 ( eu123331 ) , av 96010151 ( eu123330 ) , and av d1240007 ( eu123329 ) ; gtov , guanarito virus strain inh-95551 ( ay129247 ) ; pirv , pirital virus strain vav-488 ( af485262 ) ; lcmv , lymphocytic choriomeningitis virus strain we ( m22138 ) . az , arizona ; ca , california ; co , colorado ; fl , florida ; nm , new mexico ; slp , san luis potos ; tx , texas ; ut , utah ; por , portuguesa ( venezuela ) . nalb , neotoma albigula ( white - throated woodrat ) ; nleu , n. leucodon ( white - toothed woodrat ) ; nmex , n. mexicana ( mexican woodrat ) ; nmic , n. micropus ( southern plains woodrat ) ; pcal , peromyscus californicus ( california mouse ) ; sals , sigmodon alstoni ( alston s cotton rat ) ; shis , s. hispidus ( hispid cotton rat ) . pirital virus and gtov are south american tacaribe serocomplex viruses and were selected to represent south american lineages a and b , respectively . the lcmv strain we is a member of the lymphocytic choriomeningitis - lassa ( old world ) serocomplex and was included in the analyses to enable inference of the ancestral node among the north american arenaviruses . scale bars indicate 0.1 substitutions per site . the nucleotide sequence of a 687-nt fragment of the s genomic segment of arenavirus av h0030028 was determined from rna isolated from a kidney of white - toothed woodrat tk133451 . the sequence of this fragment ( genbank accession no . gu137350 ) was 99.9% identical to the sequence of the homologous region of the n protein gene av h0030026 . results of assays for arenavirus rna in the kidneys of the antibody - positive nelson s pocket mice and the antibody - positive kangaroo rat were negative . tacaribe serocomplex viruses have been isolated from white - throated woodrats ( n. albigula ) , large - eared woodrats ( n. macrotis ) , mexican woodrats ( n. mexicana ) , southern plains woodrats ( n. micropus ) , and a bushy - tailed woodrat ( n. cinerea ) captured in the western united states ( 25,7,8 ) , and antibody to whitewater arroyo virus has been found in woodrats captured in san luis potos ( m.l . our results indicate that an arenavirus is naturally associated with the white - toothed woodrat ( n. leucodon ) and that tacaribe serocomplex viruses are enzootic to mexico . the results of the bayesian analyses indicated that av h0030026 is phylogenetically distinct from other north american arenaviruses . sequence nonidentities between the gpc of av h0030026 and the gpc of the other north america arenaviruses ( table 2 ) were substantively greater than sequence nonidentities between the gpc of strains of phylogenetically closely related south american arenavirus species ( 3 ) . furthermore , sequence nonidentities between the n protein of av h0030026 and the n proteins of the other north american arenaviruses ( table 2 ) were comparable to the amino acid sequence nonidentities between the n proteins of strains of phylogenetically closely related south american arenavirus species ( 3 ) . thus , av h0030026 may represent a novel species ( proposed name real de catorce ) in the family arenaviridae , genus arenavirus ( 9 ) . nelson s pocket mouse ( c. nelsoni ) and merriam s kangaroo rat ( d. merriami ) are members of the rodent family heteromyidae . the anti - arenavirus antibody in the pocket mice and kangaroo rat in this study may be a consequence of infection with an arenavirus other than real de catorce virus . alternatively , the results of the elisa on these rodents are inaccurate ( i.e. , falsely positive ) . five tacaribe serocomplex viruses are known to naturally cause severe febrile disease in humans : gtov , junn , machupo , sabi , and chapare viruses . the diseases caused by these viruses range from sporadic cases to small outbreaks to hyperendemic episodes . humans usually become infected with arenaviruses by inhalation of virus in aerosolized droplets of secretions or excretions from infected rodents . for example , white - throated woodrats are consumed by humans in the potos - zacatecan mexican plateau ( 14 ) , and mexican woodrats ( n. mexicana ) , mexican deer mice ( peromyscus mexicanus ) , and other cricetid rodents are consumed by the tzeltzal indians in chiapas ( 15 ) . future studies on arenaviruses native to north america should include assessments of whether humans who consume woodrats or live or work in close association with woodrats are infected by the tacaribe serocomplex viruses associated with these rodents .

diabetes prevalence for blacks and latinos in the us are nearly double that of whites ( 11% of blacks , 10% of mexican americans , and 5% of whites have diabetes ) and blacks and latinos have higher obesity prevalence than whites [ 1 , 2 ] . physical activity is proven to help prevent and control diabetes , both independently , and through weight control . east harlem , new york is a predominantly low - income , black and latino community at the epicenter of the intertwined epidemics of obesity and diabetes . east harlem has the highest prevalence of obesity and the highest diabetes mortality rate in new york city and its residents have insufficient resources to cope with the myriad of environmental , social , nutritional , and stress - related forces that fuel these epidemics . the built environment is believed to play a large role in generating health disparities by creating differential opportunities to engage in recreational physical activity , resulting in obesogenic environments [ 57 ] . neighborhood safety and aesthetics including maintenance , cleanliness , and open space have been shown to influence the utilization of space for both recreational and routine physical activity [ 810 ] . perceptions of neighborhood safety may be particularly salient among those residents in lower - income urban settings , where crime is more prevalent . indeed , surveys find that racial / ethnic minorities and those of lower socioeconomic status are the most likely to rate their neighborhoods as unsafe . however , research to date on the relationship between perceived neighborhood safety and physical activity levels has generated mixed results , with many studies finding no relation . indeed , in low - income neighborhoods with limited personal transportation options , routine physical activity like walking may be a necessity of life in spite of safety concerns and is the least expensive , most accessible , proven effective form of physical activity . in addition to perceived safety , other perceived barriers might also influence physical activity levels . research in a variety of communities including older adults , high school , and college students have assessed the degree to which perceived barriers act as cognitive constraints on physical activity behavior . for instance , in a study of diverse rural older women , endorsing more personal barriers was negatively associated with physical activity and perceived physical environmental sets of variables were significant predictors of physical activity in a similar study [ 18 , 19 ] . few quantitative studies have assessed the relationship between perceived and physical activity levels in predominately african american and hispanic communities , but qualitative research on african american women 's attitudes towards physical activity suggests specific barriers that may differ from white populations . for instance , feelings about their physical appearance during physical activities and issues related to personal care after activities have been identified as factors that influence african american 's attitudes toward physical activity ( i.e. , not wanting to mess up ones hair by sweating ) [ 2124 ] . a positive body image despite their actual weight may also lower african american women 's desire to engage in physical activity for weight loss . one qualitative study of physical activity in latina women in north carolina found that gender roles , support from family / husband , child care issues , language , and isolation in the community served as barriers to engaging in physical activity . few quantitative studies have assessed perceived barriers in predominately hispanic populations with many first - generation immigrants such as east harlem [ 27 , 28 ] . few , if any , studies have assessed perceived barriers in low - income men . using community - based participatory research methods this study evaluated perceived safety and other perceived barriers to physical activity with self - reported data from a 2-census tract area of east harlem , new york . in 2007 , a new coalition , communities impact ( inspired and motivated to prevent and control ) diabetes formed . this centers for disease control and prevention ( cdc)-funded center aimed to use community - based participatory approaches to implement and disseminate interventions that build on local community resources and comprehensively combat the rising tide of diabetes and diabetes - related disparities . impact leaders organized a community - academic taskforce to identify specific elements of the community , built , social , and medical environments that may be altered to foster improved diabetes prevention and control as described elsewhere [ 29 , 30 ] . as suggested by partners during the grant - writing phase of the partnership , they selected a subneighborhood ( two census tracts ) within east harlem ( known as the sector of excellence to eliminate disparities , or seed ) for intensive focus . the taskforce reasoned that local concentrated efforts could harness disparate local resources and bring together diverse clinical , community , service , and religious leaders to take action . after discussing many potential interventions to enhance physical activity , healthy eating , and diabetes detection , prevention , and management , they decided they first needed to better understand local barriers to preventing and controlling diabetes . to achieve this goal , the partnership surveyed local community residents to gain insight into the ways characteristics of the neighborhood , particularly safety and perceived barriers , influence physical activity levels . an evaluation subcommittee of the taskforce , with community and academic members , developed , piloted , and conducted the survey to assess sociodemographics , medical history , diabetes risk perception , physical activity , diet and weight , community resources , and diabetes knowledge and to gather anthropometric measures of weight and height from willing participants . sociodemographic data was designed to be comparable to the data for these same 2 census tracts in the 2000 census . inclusion criteria consisted of living in the seed , age 18 and older , and the ability to speak either english or spanish . in contrast with population surveys generated from random digit dialing that may undersample hard to reach lower - income residents , recruitment for the community member survey took place on the sidewalk via street intercept . potential survey sites were selected based on the following criteria : ( 1 ) publicly accessible building or space or permission to use space obtained from building supervisor ; ( 2 ) space for a semiprivate interview . at each selected site , interviewers approached individuals who appeared to be adults over the age of 18 in front of the venue and invited them to participate . we recruited and surveyed 300 willing residents of the seed who consented to participate in the survey . nine participants were excluded as they did not have full data to assess residency within the seed area , thus , data for 291 individuals were included in the study . the final sample was predominately female ( 62.7% ) , latino ( 56.0% ) , and low income ( < 15,000/year ) ( 63.0% ) . physical activity and diet are notoriously difficult to measure via self - report with no true gold standard . as such , we chose to limit the respondent burden in these areas and target our queries towards items most likely to provide the community coalition with targets for intervention . physical activity was measured using 4 self - report items assessing average daily and weekly walking time and recreational activities modified from the international physical activity questionnaire ( ipaq ) . to capture both routine physical activity and physical activity specifically for recreation first , we divided respondents between those who met the cdc 's recommendation of engaging in at least 2.5 hours per week of moderate physical activity ( see : http://www.cdc.gov/physicalactivity/everyone/guidelines/adults.html ) , in this case , those who reported walking for at least 2.5 hours per week or approximately 20 minutes a day and those who did not meet this standard . second , to capture recreational physical activity , we asked respondents what do you like to do to be physically active ? for respondents reporting a physical activity other than walking ( specifically running / jogging , bicycling , playing a team sport and going to the gym ) , we coded them as engaging in a leisure time physical activity . respondents could choose more than one category , but respondents were coded according to whether they participated in a leisure time physical activity at all , not according to how many . six - item dichotomous measures of perceived barriers to physical activity were developed with input from the community coalition concerning perceptions of barriers to physical activity believed to be widely held within the community and are summarized in table 4 . respondents were asked whether or not they agreed with each of the six barriers ( yes / no ) . the barriers were then summed to create a composite measure of the number of barriers endorsed . in addition , a battery of six items was asked about perceptions of neighborhood safety in relation to neighborhood social capital . this measure was adapted from a measure used in a previous study of perceived neighborhood safety and physical activity . respondents were asked to rate how safe from crime they considered their neighborhood to be and to rate how safe they feel in a series of locations and situations ( e.g. , walking alone on a street in your neighborhood at night / day ; taking the public bus during the night / day ) using a 4-point likert scale . we generated a composite measure of neighborhood safety by dichotomizing the individual questions into generally perceived safe / unsafe categories and then summing the results . because one 's excessive weight can affect perceptions of barriers to physical activity including pain with exertion , we controlled for participants current body mass index ( coded as overweight or obese from our anthropometric measures of height and weight ) . in addition , current weight - loss efforts were measured using self - report including whether individuals were using physical activity or exercise to lose weight or maintain their current weight . we controlled for a variety of additional demographic factors : sex , age , race / ethnicity , education , and acculturation ( if respondents speak spanish at home and if they were born in the us ) . two multivariate logistic regression models with perceived barriers and concerns about neighborhood safety were modeled separately as predictors of engaging in recommended levels of exercise and recreational physical activity , controlling for respondent weight and sociodemographic characteristics . we first modeled the relationship between the number of perceived barriers to physical activity and reported physical activity levels ( time spent walking and participation in recreational physical activity apart from walking ) , controlling for overweight / obese , age , race / ethnicity , gender , acculturation , and whether or not individuals were trying to lose weight . we stratified the analysis by race / ethnicity and gender to detect group specific differences . with regards to self - reported physical activity , at a median of 7 hours per week or about 1 hour per day , respondents to the survey reported walking a great deal . the overwhelming majority of the sample ( 80.0% ) met the weekly recommendation of at least 2.5 hours of moderate intensity exercise , 88.0% of men and 75.0% of women . in regards to recreational physical activity , walking was also the most commonly reported physical activity that respondents liked to do to be physically active ( 65.2% ) . apart from walking , 72.0% of the sample reported engaging in at least one other form of recreational physical activity including cycling ( 20.6% ) , playing sports ( 17.9% ) , dancing ( 14.1% ) , going to the gym ( 13.4% ) , and running ( 11.7% ) ( table 2 ) . while there were many self - reported barriers to physical activity , the most commonly reported was being too tired or having little energy ( 45% ) . pain with exercise ( 35% ) , lack of time ( 30% ) , dislike of exercise ( 30% ) , and difficulty being active ( 28% ) were also commonly identified barriers . lack of safe spaces for exercise was a relatively less commonly cited barrier , reported by only 20% of the sample , despite nearly half those surveyed ( 45% ) considering the neighborhood to be somewhat or very unsafe ( table 3 ) . multivariate regression of perceived barriers found that reporting a greater number of perceived barriers was associated with a lower odds of meeting the weekly recommendation of 2.5 hours of moderate exercise ( or = 0.72 , p < 0.01 ) . additionally , individuals who perceived more barriers to physical activity were less likely to report engaging in some form of recreational physical activity other than walking ( or = 0.72 , p < 0.01 ) . perceived safety of the neighborhood , on the other hand , was not associated with significantly lower amounts of time spent walking or engaging in recreational physical activity ( table 4 ) . men spent more time walking and were more likely to engage in leisure time physical activity compared with women , although safety concerns were not a significant barrier to physical activity for either men or women ( analysis not shown ) . overall , stratified analysis between men and women and hispanic and black participants did not affect the direction or significance of the results ( analysis not shown ) . in disadvantaged neighborhoods such as east harlem , characteristics of the built and social environment , such as perceived safety , are often suggested as the greatest obstacles to increasing physical activity [ 513 ] . our results did not support the supposition that concerns about safety serve as a primary barrier to recreational and routine physical activity . nearly half of the sample perceived their neighborhood as very or somewhat unsafe . yet , safety concerns were reported as serving as a barrier to physical activity by only 20% of the sample and believing your neighborhood was unsafe was not associated with physical activity levels . the most commonly reported barriers to physical activity identified by nearly half of the sample were motivational lack of time and pain with exertion were also commonly cited barriers and multivariate regression found that endorsing a greater number of individual barriers was associated with reduced time spent walking and engaging in physical activity for sport controlling for covariates . this finding contrasts with other studies that have found support for the role of perceptions of neighborhood safety in predicting physical activity levels . a study from boston , for instance , that used a similar measure of perceived neighborhood safety found that female residents of neighborhoods with lower perceived safety at night walked less than similar women in neighborhoods perceived as safer . other studies have found mixed results with regards to the role of safety in reducing physical activity levels . some studies have found a significant inverse association between perceived neighborhood safety and physical activity levels [ 3341 ] , but others have found no significant association [ 4252 ] . another striking finding from this study was the percent of individuals that met the recommendation for moderate - intensity exercise of 2.5 hrs / wk : 80% of the sample met this recommendation through walking alone . moreover , the median of 7 hours per week of walking was well above the recommended amount and translates into an hour a day of moderate intensity exercise . this contrasts with studies from other parts of the country finding low levels of physical activity in predominately latino communities . for instance , a study of latina women in chicago found much lower rates of physical activity , with only 36% meeting current recommendations for moderate or vigorous physical activity . although it is possible that respondents exaggerated the actual amount of walking per day , it is also plausible that low - income areas of new york are more walkable than low - income areas in other cities . alternatively , respondents , particularly men , may have active jobs that require a great deal of walking . in addition to walking , 72.0% of the sample reported engaging in some other form of physical activity including team sports , cycling , dancing , running , and going to the gym . collectively , these findings contradict typical expectations regarding the need for unique interventions in socially deprived neighborhoods like east harlem and provides a rich foundation to launch community - based interventions that focus on addressing motivational barriers . in the face of these cognitive barriers , changes to the built environment given the strong preference for walking , these findings suggest that community - level efforts need to support ways for building activity into daily routines so they are not perceived as adding time to the day and requiring tremendous new sources of energy and motivation . for example , rather than designing a walking path or indoor walking trail in a random location in the neighborhood , efforts may be more successful if that path is mapped along a highly used route such as from a central or high traffic bus stop to a housing project or to a supermarket . furthermore , markers of distance or calories expended could be added to reinforce a sense of accomplishment to community residents as they walk these routes and to encourage more frequent use of them . in addition , efforts should be made to reinforce the notion that engaging in regular , routine exercise can increase energy levels and reduce fatigue . nearly 70% of the sample was either overweight or obese based on our anthropometric measures . this suggests that routine exercise , even in high dosages , may not be adequate to stave off obesity in the absence of changes in the food environment . despite the rich data afforded by this unique community resident assessment , several limitations should be noted . although substantial efforts were made to replicate a random sample through random selection of pre - selected block faces where recruitment was most feasible , the final sample was ultimately a convenience sample of willing residents that may not be representative of the community as a whole . the use of self - reported physical activity behaviors is inherently subject to significant recall and social desirability biases . the generalizability and comparability of results from this survey are limited by the design , which focused on a single , disadvantaged neighborhood . the findings with regards to the role of perceptions of safety and barriers to physical activity , therefore , pertain to differences among individuals within a single neighborhood rather than between neighborhoods . the objective of this study was to assess levels and types of physical activity in an area with high rates of obesity and diabetes and to analyze determinants of physical activity . although safety concerns are often believed to deter physical activity in low - income , urban settings we did find this in east harlem .

early childhood caries ( ecc ) is a common childhood disease in the united states ( us ) and worldwide . until recently , the standard of care for ecc has been primarily surgical and restorative treatment with relatively little emphasis on the prevention and management of disease . in the us , young children who are not cooperative are commonly sedated or treated under general anesthesia ( ga ) . however , despite receiving costly treatment under ga , such as in the operating room ( or ) [ 35 ] , children all too often develop new and recurrent caries [ 610 ] . it is now accepted that surgical repair alone does not address the underlying etiology of the disease . unless the caries balance is altered , new and recurrent caries are likely to occur . on the other hand , a successful rebalance of risk and protective factors may slow down or completely halt the disease process , resulting in caries arrest , if not also preventing the onset of new disease . contemporary approaches to caries prevention and management modeled after medical management of chronic conditions such as diabetes , obesity and asthma , have been described in the scientific literature and are herein known as chronic disease management ( dm ) [ 11 , 1315 ] . dm differs from the traditional approach of oral health care providers relying on a surgical treatment model in response to the disease , while telling the patients what to do . instead , it assumes that patients have a central role in determining the care of their chronic condition . a close collaboration between the healthcare provider and patient is required , ideally in a culturally and linguistically appropriate manner . in practice , healthcare providers coach patients and parents about the factors that lead to and protect against dental disease and assist them in selecting self - management goals to improve their own and their children 's risk for disease . treatment decisions are based on the latest evidence - based guidelines that are customized to patients ' individual needs . risk - based dm of ecc requires significant patient and family engagement and empowerment from the provider and care team in effective day - to - day behavior modifications ( e.g. , tooth brushing , topical fluorides , and dietary control ) that address disease etiology . family - centered behavior plans lead to real behavior change and maintenance of oral health behaviors in the child 's home . at the same time , the dental practice has a reciprocal role in tracking and managing the care of patients . phase 1 in 2008 , a risk - based dm approach to address preschool children with ecc was implemented and tested as a quality improvement ( qi ) demonstration project at boston children 's hospital in boston , massachusetts ( bch ) , and st . joseph health services of rhode island in providence , ri ( sjh ) . the clinical protocol and project results have been previously published . thirty months of results found that children in the ecc group experienced lower rates of new cavitated lesions , pain , and referrals for restorative treatment under general anesthesia in the or as compared to baseline historical controls ( table 1 ) . at bch , the ecc group experienced a 62% lower rate of new cavitation compared to the historical control group . structured interviews with phase 1 parents revealed that most believed the dm approach to be helpful for their children ; almost all parents appreciated given reasons as to why their children may have developed ecc . the collaborative approach allowed clinicians to engage parents or caregivers to better understand that they have a voice in the care their child receives . in 2008 , a risk - based dm approach to address preschool children with ecc was implemented and tested as a quality improvement ( qi ) demonstration project at boston children 's hospital in boston , massachusetts ( bch ) , and st . joseph health services of rhode island in providence , ri ( sjh ) . the clinical protocol and project results have been previously published . thirty months of results found that children in the ecc group experienced lower rates of new cavitated lesions , pain , and referrals for restorative treatment under general anesthesia in the or as compared to baseline historical controls ( table 1 ) . at bch , the ecc group experienced a 62% lower rate of new cavitation compared to the historical control group . structured interviews with phase 1 parents revealed that most believed the dm approach to be helpful for their children ; almost all parents appreciated given reasons as to why their children may have developed ecc . the collaborative approach allowed clinicians to engage parents or caregivers to better understand that they have a voice in the care their child receives . phase 2building upon the promising results of phase 1 , the project was expanded in 2011 to include five additional teams in the us . phase 2 further tested the feasibility and effectiveness of the dm approach to reduce ecc in more diverse settings . the five additional teams were in the following locations across the us : holyoke health center ( holyoke , ma ) ; native american health center ( san francisco , ca ) ; nationwide children 's hospital ( columbus , oh ) , neighborcare health ( seattle , wa ) ; and university pediatric dentistry ( buffalo , ny ) . the purpose of this report is to describe the phase 2 project and experiences , present the results after 18 months , and discuss the implications of what was learned . building upon the promising results of phase 1 , the project was expanded in 2011 to include five additional teams in the us . phase 2 further tested the feasibility and effectiveness of the dm approach to reduce ecc in more diverse settings . the five additional teams were in the following locations across the us : holyoke health center ( holyoke , ma ) ; native american health center ( san francisco , ca ) ; nationwide children 's hospital ( columbus , oh ) , neighborcare health ( seattle , wa ) ; and university pediatric dentistry ( buffalo , ny ) . the purpose of this report is to describe the phase 2 project and experiences , present the results after 18 months , and discuss the implications of what was learned . phase 2 was implemented as an 18-month qi learning collaborative . using established qi methods , a nationwide collection of staff , experts , and faculty provided training and technical assistance to the seven participating teams , which included the two teams that were part of phase 1 . teams were required to attend three on - site learning sessions where each of the seven teams received didactic education and training on qi concepts and activities . the learning session curriculum focused on the use of logic models , measurement plans , plan - do - study - act ( pdsa ) cycles , dm of ecc such as caries risk assessment ( cra ) , self - management goals ( smgs ) , effective patient - provider communication , and fluorides and other remineralizing modalities . the learning sessions provided invaluable opportunities for synergy as teams exchanged approaches to dm during consultations with experts , faculty , and staff , who provided coaching and support . qi has been defined as the combined and unceasing efforts of everyone to make changes that will lead to better patient outcomes ( health ) , system performance ( care ) , and professional development ( learning ) . qi is intended to support the redesign of care processes based on a system of learning , incremental change , and the incorporation of empirically supported best practices from evaluating performance and outcome measures . unlike a protracted randomized trial , qi uses systematic , data - guided activities designed to bring about immediate improvements in health care delivery in particular settings and can be considered as the scientific method used for action - oriented learning . the model for improvement developed by associates in process improvement was used as the essential framework to guide changes made by each team 's care delivery system in order to use a dm approach to address ecc . the collaborative developed a driver diagram outlining three main outcomes of interests : ( 1 ) new cavitation ; ( 2 ) pain related to untreated caries ; and ( 3 ) referral to the or , along with primary and secondary drivers affecting those outcomes ( figure 1 ) . pdsa cycles are small - scale tests of change in real work settings by planning a test , trying it , observing the results , and acting on what is learned . pdsa cycles promote creativity , offer quick results , and empirically support approaches to dm that are specific to the clinical teams . for example , pdsas served as learning opportunities for phase 2 teams to use evidence to determine how to perform activities such as cra , smgs , and patient scheduling to support the additional dm visits required . the phase 2 clinical dm protocol ( shown in figure 2 ) cra allows for a customized prevention and maintenance plan to be developed that is appropriate for the child and the family . cra involves asking parents a few questions to assess each child 's risk for caries at the initial visit and every visit thereafter . this form was adapted from the american academy of pediatric dentistry ( aapd cra ) form and the pediatric caries management by risk assessment ( cambra ) form . teams were able to customize forms for use with their specific patient populations and organizational context provided that they included at least the basic questions seen in figure 3 . children who had at least one tooth with clinical manifestation of caries tooth decay ( including demineralization)or who had a history of carious lesions was considered an ecc patient . at the initial and recall visits , a clinical examination and charting were performed to allow for the tracking of caries presence and activity by tooth and surface , since decay may progress and become inactive at different sites of the dentition at the same time . parents of ecc patients were engaged and coached about the factors that lead to caries and tooth decay by dentists , hygienist , dental assistants , and/or support staff . parents learned about the caries process as they were informed of the ways that tooth decay can be prevented and stopped . in addition , parents learned that without a change in diet and home care , new cavities and broken fillings will likely result . providers and care team members worked with parents to select smgs to improve their child 's disease risk . such goals include basic caries control strategies such as more frequent tooth brushing , using topical fluorides at home , and modifying one 's diet to include fewer and less frequent intake of sugary products . the frequency of return dm visits for patients and parents in - office and at the clinic site was based on their caries risk . whenever possible , the dm activities were coordinated with restorative treatment . table 2 shows the dm protocol with return visit intervals based on the most recent caries risk status in conjunction with restorative care as needed and as desired by the parent and dental provider . the in - office dm protocol was based on the assumption that children who initially presented as high caries risk may improve their risk over time . children who were assessed to be high caries risk were advised to return in 13 months for a dm visit . medium or moderate risk children returned in 36 months , while low risk children returned in 612 months . in some cases , accurate clinical assessment was hampered by the presence of heavy plaque and/or a lack of patient cooperation . as a result , a one - month follow - up visit for a child assessed to be high caries risk allowed for a more accurate assessment of demineralized , cavitated , and remineralized tooth surfaces . during each recall or subsequent dm visit , a cra was performed . providers asked parents to report on their experiences with the smgs in order to assess the level of compliance and the utility of the agreed - upon smgs . a clinical examination was also performed , reassessing for the presence of new demineralization and cavitation along with caries remineralization . parents were given the full range of options for restorative treatment , which included pharmacologic management ( i.e. , use of nitrous oxide , sedation , or ga / or ) as needed by the patient and as desired by the parent . restorative options included conventional treatment and minimally invasive restorative treatment ( i.e. , interim therapeutic restorations ( itr ) ) . if the destruction of the tooth structure by the caries process was minimal , caries arrest was possible with remineralization of the tooth structure . the restorative treatment was then deferred in patients if the caries process was stabilized , especially in a child unable to cooperate for restorative treatment . however , close follow - up and preventive care based on caries risk were essential to safeguard from relapse . if the decay had progressed into dentin and caries arrest was not achieved , itr was offered as an alternative treatment with early cavitated lesions . a secondary gain from more frequent visits for preventive care was usually a reduction in a child 's fears and a gain in trust between the dental provider and the child over time , allowing for restorative treatment to be completed with greater ease at a later time . in order for teams and their sites to support risk - based disease prevention and management of ecc , dentists , staff , patients , and families who were accustomed to conventional surgical and restorative care were educated about and guided to accept a contemporary approach that emphasizes risk assessment , individualized disease prevention , and management and maintenance of health . for example , scheduling systems , typically set up to accommodate recall preventive visits every six months as allowed by insurance , had to be adjusted to allow for more frequent preventive return visits for high caries risk patients . before and during the implementation of the project , senior leaders and clinical champions of each team provided training to their dental providers and staff about the dm protocol . they shared what they learned from the learning sessions and monthly calls on dm and qi methods with those who were involved in the day - to - day work of implementing the ecc dm protocol . teams were expected to hold regular meetings to address questions about the protocol and care management of patients , review project progress , plan pdsas , and institute change . most teams chose to begin the protocol initially with a few providers , followed by spreading to additional providers over time . in phase 2 , teams collected process and outcome measurement data for the purpose of evaluating improvement trends in care processes and patient outcomes over time . each month , teams randomly selected 20 patient records ( charts ) of their ecc patients to record the results of some measures . meanwhile , on a quarterly basis , they selected 30 charts of ecc patients to record results of other measures . the deidentified data were collected and managed using redcap ( research electronic data capture ) electronic data capture tools hosted at bch . redcap is a secure , web - based application designed to support data capture for research studies providing ( 1 ) an intuitive interface for validated data entry ; ( 2 ) audit trails for tracking data manipulation and export procedures ; ( 3 ) automated export procedures for seamless data downloads to common statistical packages ; and ( 4 ) procedures for importing data from external sources . bch staff retrieved and processed the data , screened for errors , and managed the deidentified data . each month , run charts were produced by bch and sent to the collaborative staff . in turn , the collaborative sent run charts to each team for use in monitoring progress toward improvement . during monthly collaborative calls , trends in the run charts were reviewed . in addition , any questions and concerns from the teams were addressed by the collaborative staff , faculty , and improvement advisor . although this collaborative was designed as a qi initiative that aimed to identify positive trends in process and outcome measures which would signify improvements in care and outcomes , a trends analysis would not necessarily infer causality . therefore , there was a need to compare the project outcome data to baseline data derived from historical controls ( i.e. , patients treated by the teams prior to the start of phase 2 ) . in the last several months of the collaborative , after obtaining irb approval , teams collected data , on the three outcomes of interest ( percentage of patients with new cavitation , pain , and unplanned referrals to the or ) by randomly selecting 50 charts of their ecc patients and 50 charts of baseline historical control patients . at each site , a computer generated randomized scheme identified the potential ecc and control patients . qualifying ecc patients were those who had been ( a ) in phase 2 for a minimum of 6 months and ( b ) had at least one formally scheduled preventive visit ( recall visit ) whereby caries charting was performed and documented . all sites reviewed their phase 2 records ( i.e. excel spreadsheets or billing generated report ) and randomly selected 50 ecc patients ( one site had only 46 patients ) . qualifying historical control patients were children who were younger than 60 months of age and had ( 1 ) a history of decay ; ( 2 ) at least one recall visit six months after the initial visit ; and ( 3 ) a last visit that was at least six months prior to the start of the phase 2 . for each team , a computer generated randomized scheme identified potential patients based on age and whose billing records were reviewed to determine whether they had a recall visit at least 6 months prior to the start of phase 2 . these patient records were selected for further review to determine those patients who met the qualifying criteria . the following information was documented for both ecc and historical control patients by visit date : ( 1 ) type of visit ( preventive , restorative , sedation , or , missed , or canceled ) , ( 2 ) new cavitation identified , ( 3 ) pain identified , and ( 4 ) referral to or . the first visit was determined for each patient as that which decay was initially charted or documented in the patient 's clinical notes . pain and or referral at first visit , including pain unrelated to untreated decay at any visit were excluded . from this information , the percentage of patients with new cavitation , pain identified , and referral to or were determined . the ecc and historical control data were collected by most teams onto paper collection forms . figure 4 shows some of the trend data for the seven phase 2 teams . over time , the teams demonstrated a highly consistent level of performance with their providers performing cras and smgs . most teams saw a reduction of ecc children deemed as high caries risk and an increase in ecc children with improved caries risk from the first visit . table 3 shows a comparison of the rates of new cavitation , pain , and referral to or between ecc patients and the baseline patients for phase 2 . these results reflect a random sample of 316 historical control children and 344 ecc children drawn from a total phase 2 population of 3,030 . in the aggregate , children in the ecc dm group experienced lower rates of new cavitated lesions , pain , and referrals for restorative treatment under general anesthesia in the operating room ( or ) as compared to baseline historical controls , although there was variability from site to site . in phase 2 of the ecc learning collaborative , by using qi methods to change their systems of care , teams were able to efficiently implement the dm protocol into their clinical practice . in the aggregate for phase 2 , fewer ecc children experienced new cavitation , pain , and referrals to the or compared to baseline historical controls . there was discrepancy from team to team in the degree of improvement in the process measures and outcomes achieved . the variation in outcomes among teams , as expected , may be attributed to differences in each team 's use of the dm protocol or to distinct cultural and socioeconomic differences in patients and families among the sites . similarly , phase 1 also found imbalance in terms of outcomes achieved at the two sites . the phase 1 team that demonstrated a relatively less dramatic improvement in new cavitation rates had predominantly english speaking providers serving predominantly latino populations who spoke spanish as their native language . unfortunately , demographic data were not collected in phase 2 . in terms of limitations , 50 randomly selected charts for each of the ecc and historical control groups at each site may be insufficiently representative of the groups for each team . at the same time , an 18-month follow - up period may be an inadequate length of time to evaluate the clinical outcomes of some children who were enrolled as ecc patients over time . in addition , although dental providers at all sites received training on the dm protocol to chart decay ( by using a modified icdas system ) , they did not receive calibration to chart new cavitated or precavitated lesions . however , by protocol , ecc patients were seen more frequently for dm visits , during which time they were examined for new cavitation and thus may have received increased opportunities for new cavitated lesions to be identified . during their participation in phase 2 , teams shared their experiences including their knowledge and skills gained , lessons learned , and tools developed with other collaborative participants . examples of new skills include training of support staff and employing them to assist with cra and patient education , collaborating with pediatric medical providers to enhance the referral of young children for early preventive dental visits , scheduling differently to accommodate the more frequent return needs of the ecc children for dm visits , and managing no - show appointments by using a registry to track patient visits . two teams were located in community health centers that initially saw a limited number of young children . these teams developed pdsas to specifically focus on increasing referrals from pediatricians within their centers , and they were successful in their efforts . one site had a baby 's clinic already in place prior to joining phase 2 and a hygienist to see children specifically younger than age three years for infant oral health visits ; this hygienist was already using a cra tool . this site incorporated the dm protocol , smgs , and more frequent visits first into their baby 's clinic and later spread the dm protocol to other providers in their main dental clinic over time . tools developed , enhanced , and willingly shared among the collaborative teams included forms for conducting and documenting carious lesions , cra , smgs , and tracking of the ecc patients . at the conclusion of phase 2 , team leaders convened for a final summary conference . all teams agreed that the dm approach to address ecc was a logical change in practice , albeit not easy to implement . challenges included having to accommodate the additional dm visits and the time required for each visit . in some cases initially , most providers and teams struggled with having to fit in the dm visits , especially if their schedules were booked out in advance . since most dental insurance plans do not cover more than two diagnostic / preventive visits each year , the additional dm visits and lost time for reimbursable restorative care posed as additional obstacles . changes that worked were implemented across the practice and continually improved upon through further testing . providers received training and coaching to be able to perform cra , explain the causes of the caries process , and work with the caregivers to select smgs more effectively and with greater efficiency . providers and families began to accept a paradigm shift that addressed disease etiology in lieu of relying solely on a surgical model of treatment . the use of qi methods was useful in facilitating the adoption of dm to address ecc , first by getting buy - in from the early adopters at each site , followed by later adopters . some teams had spread the protocol to more providers within their primary site , while others had spread it to all their providers . one team successfully spread the protocol beyond their primary site to other dental sites that were a part of their community center network . most sites embedded their dm protocols into their systems of care such that dismantling those systems would require effort . what impact did the collaborative have on you ? responses included it made me a better provider , a better teacher ; and i no longer view children 05 the same way ( i do not pick up the hand - piece first ) . we demonstrated the feasibility of an innovative approach to address ecc utilizing dm protocols that can be successfully implemented into dental practice using qi methods in a learning collaborative model . although not easy to implement , after 18 months , all teams reported that the dm approach resulted in overall improved care delivery and patient outcomes ( new cavitation , pain , and referrals to the or ) . teams recognized that while a dm model can be implemented into practice , policy and payment reforms are needed to facilitate a wider - scaled adoption of the dm protocols . elements to be addressed include compensation for providers ' time and efforts to perform cra , smgs , patient education and engagement , and the dm visits . future demonstration should quantify opportunities for cost savings to be realized by avoiding more costly restorative treatment . future policy changes are necessary to support a paradigm shift from surgical treatment of caries toward an individualized risk - based disease prevention and management model as the new standard of care . at the same time , the use of qi methods may help accelerate the adoption and spread of dm protocols into any dental practice .

it is evident that infant s umbilical cord is essential for its survival and growth . in the past , it was not considered as a necessary tissue . however , nowadays , it has attracted lots of attention and its relation to different diseases has been demonstrated . in iranian traditional medicine , caring for newborn s umbilical cord has been frequently recommended and it has been associated with different diseases . herein , iranian traditional medicine resources such as qanun - fil - tibb , exir - e - azam , and tib - e - akbari were studied . in addition , recent developments in classical medicine were reviewed using search engines including google scholar , pubmed , sid , and iranmedex . it has been revealed that there is a direct relation between early cord clamping and oxidative stress , anemia , and low cardiac output . in iranian traditional medicine , it is believed that the umbilical cord length , which should be cut off , is important . furthermore , if the umbilical cord is not squeezed efficiently , its content may enter the bladder , testis , and stomach leading to various diseases . it seems that the remarks by the iranian traditional medicine on caring for newborn s umbilical cord can be useful for the prevention of different diseases .

although kidney disease patients can survive without kidney function , dialysis is a life - saving procedure . however , many complications related to chronic kidney disease ( ckd ) have not been resolved , including cardiovascular disease ( cvd ) , mineral and bone disorders ( ckd - mbd ) , and infection . nephrology is a relatively new sub - specialty in the field of internal medicine , and we are still learning the extent of how the kidneys support the body . the social and economic burdens of dialysis are growing worldwide as the number of patients increases . thus , preventing end - stage kidney disease ( eskd ) is of the utmost importance . early detection and treatment is recommended because late referral is common , with most ckd patients remaining asymptomatic until a late stage . according to the annual report from the japanese society for dialysis therapy ( jsdt ) , three - quarters of dialysis patients initiated dialysis therapy within 1 year after referral to the facility . ckd is clinically defined by the presence of albuminuria and/or a decrease in kidney function for > 3 months . since its introduction in 2002 , the definition of ckd has been widely accepted not only by nephrologists but also other medical specialties , such as cardiologists and general practitioners . japan has a long history of universal screening for school children , university students , and employees of companies and government offices . the urine test for proteinuria and hematuria is popular among japanese people ; however , the outcomes have not been well studied . chronic dialysis therapy was started in okinawa in 1971 , several years after it was initiated in other parts of japan [ 35 ] . the number of dialysis patients per million population ( pmp ) is increasing faster in okinawa than the national average ( fig . 1 ) . the number was 1,982 in 1990 and 5,246 in 2000 when the population was 1.2 million ( 1990 ) and 1.3 million ( 2000 ) , respectively . initially , the objective of the okids was to determine the relative risk of cvd , including stroke and acute myocardial infarction , in dialysis patients . the strengths of the study are that all of the medical facilities have cooperated , and the data for the incidence of cvd in the general population were available at the same time in okinawa . we found that the relative risk of stroke , in particular cerebral hemorrhage was very high , but not as high as acute myocardial infarction . the incidence of cerebral hemorrhage was higher than in the general population , even for normotensive dialysis patients [ 6 , 7].fig . 1prevalence of chronic dialysis patients per million population in okinawa and japan ( cited from ref . ) prevalence of chronic dialysis patients per million population in okinawa and japan ( cited from ref . ) we examined the effects of clinical and laboratory data from several sources on survival [ 818 ] . among them , serum albumin was a strong predictor of death , suggesting the importance of nutritional management . our data suggest that factors other than atherosclerotic heart disease lead to heart failure in the dialysis population . the overall survival was higher for those with a higher blood pressure and total serum cholesterol , which contradicts data from the general population . table 1 summarizes the factors related to poor survival in chronic dialysis patients .table 1risk factors for death in chronic dialysis patients ( modified from iseki et al . cen2004 ) patient demographics age sex primary renal disease ( diabetes , nephrosclerosis ) predialysis comorbid conditions ( cardiovascular disease , malignancies)laboratory variables hypertension hypotension hypoalbuminemia hypocholesterolemia high crp high coronary artery calcification score ckd - mbd hyper- and hypophosphatemia hypercalcemia electrolyte disturbance hyperpotassemia hyponatremia risk factors for death in chronic dialysis patients ( modified from iseki et al . cen2004 ) several randomized controlled trials , such as the treatment of anemia using an erythropoietin - stimulating agent [ 21 , 22 ] and statin treatment [ 23 , 24 ] , have failed to show an improvement in survival . hypertension is a major risk factor for death and cardiovascular disease in dialysis patients , but the effect of lowering blood pressure in this high - risk patient group is uncertain . we examined the effect of an angiotensin receptor blocker on survival . in a multicenter prospective , randomized , open - label , blinded - endpoint trial , we assigned 469 patients on chronic hemodialysis ( hd ) with hypertension to receive the angiotensin receptor blocker olmesartan ( n = 235 ) or a treatment other than an angiotensin receptor blocker or angiotensin - converting enzyme inhibitor ( n = 234 ) . lowering blood pressure with an angiotensin receptor blocker did not significantly lower the risk of major cardiovascular events or death among patients with hypertension on chronic hd . two community - based registries for eskd patients and general screening have been available to us [ 27 , 28 ] . the okinawa general health maintenance association ( oghma ) has been performing universal screening annually in okinawa . since 1983 , they have filed records in the computer registry . with full collaboration of the physicians and medical staff , we were able to match subjects who participated in the screening and later developed eskd . because the area consists of sub - tropical islands , the eskd or ckd stage 5 patients reside exclusively in okinawa . after verifying the databases from 1983 ( n = 106,182 ) and 1993 ( n = 143,948 ) , we analyzed the relationship between commonly measured laboratory variables and eskd [ 2740 ] . the total number of identified eskd patients was 420 from 1983 to 2000 . among the variables examined , dipstick proteinuria had the strongest association ; the greater the dipstick proteinuria , the higher the risk of developing eskd ( fig . 2 ) . although the dipstick test is semi - quantitative , the test is clearly dose - dependent. serum creatinine was tested in 14 % of patients in 1983 and 35 % in 1993 . in addition to dipstick proteinuria , hematuria , blood pressure , body mass index ( bmi ) , serum creatinine , uric acid , and anemia were significant predictors of developing eskd . other factors , such as smoking , plasma glucose , dyslipidemia , and metabolic syndrome , also played a role in the development of ckd and eskd , suggesting the necessity of a multidisciplinary approach . the risk factors related to the development of eskd are summarized in table 2 .fig . table 2risk factors for the development of eskd ( modified from iseki et al . cen2005 ) patient demographics age sex race past history of cardiovascular disease family history of cardiovascular diseaseclinical and laboratory variables proteinuria hematuria hypertension diabetes ( hyperglycemia ) hyperuricemia anemia low egfrlifestyle smoking obesity , metabolic syndrome sleep disturbance risk of developing eskd based on dipstick proteinuria ( cited from ref . ) risk factors for the development of eskd ( modified from iseki et al . cen2005 ) only a few studies outside japan have examined the effect of microhematuria on developing eskd . compared to proteinuria , the risk of microhematuria was significant , but showed a weak dose the absolute risk of microhematuria was low but was a statistically significant predictor of eskd . notably , microhematuria is a risk factor for developing proteinuria ; if combined with proteinuria , the risk of developing eskd is even higher compared to having proteinuria alone . the jsdt has been conducting a nationwide survey on chronic dialysis therapy and reporting annually as an overview of regular dialysis treatment in japan. according to the 2011 report , the total number of dialysis patients was 304,592 ( 2,383 pmp ) , and the leading cause of eskd was diabetes ( 44.2 % ) ( fig . 3 ) . the mean age has increased steadily and was 67.8 years in incident and 66.5 years in prevalent patients ( fig . 4 ) . this result is most likely explained by the delay in ckd progression and better survival among the japanese . the number of patients with chronic glomerulonephritis has decreased linearly since 1998 , and the mean age at the start of dialysis has increased from 60.5 years in 1997 to 67.5 years in 2011.fig . 3causes of primary kidney disease among hemodialysis patients in japan ( cited from ref . ) fig . causes of primary kidney disease among hemodialysis patients in japan ( cited from ref . ) since 1983 , the outcomes of dialysis patients have been investigated . as shown in the okids data , hypoalbuminemia is a significant predictor of death regardless of the pre - dialysis blood pressure and use of anti - hypertensive drugs ( fig . 5 ) . survival among japanese dialysis patients is better than patients in europe and the united states , yet the reasons for this difference remain to be determined . the most common vascular access is an arteriovenous fistula . a relatively small body size , with a mean bmi of approximately 21 kg / m , might be advantageous for receiving adequate dialysis . renal transplantation is performed in approximately 1,0001,200 patients , and cadaveric donation is stable at approximately 200 annually.fig . 5annual mortality rate of dialysis patients based on pre - hemodialysis blood pressure and serum albumin ( cited from ref . ) annual mortality rate of dialysis patients based on pre - hemodialysis blood pressure and serum albumin ( cited from ref . ) the early initiation of dialysis has been practiced worldwide , and the mean initial estimated glomerular filtration rate ( egfr ) is becoming higher than ever before [ 4547 ] . the egfr threshold for starting dialysis is not available . according to the jsdt , the survival was best at around egfr 46 ml / min/1.73 m [ 48 , 49 ] . the effect of confounding variables other than age and diabetes is unknown , and we need more data to determine the egfr threshold . most japanese nephrologists rely on the research group criteria supported by the ministry of health , welfare , and labor , which use egfr and the presence of uremic symptoms . the jsn has published the clinical practice guidebook for diagnosis and treatment of chronic kidney disease in 2007 , 2009 , and 2012 . the evidence - based practice guideline for the treatment of ckd was published in 2009 and will be updated in 2013 . the jsn has been raising awareness of ckd on world kidney day , which is on the second thursday in march . therefore , an egfr 60 ml / min/1.73 m is considered to be normal for someone who is otherwise healthy . albuminuria can only be measured and reimbursed for patients with early - stage diabetic kidney disease in japan . instead , the jsn advocates using dipstick proteinuria or measuring the daily amount of proteinuria . frontier of renal outcome modifications in japan ( from - j ) . to prevent or halt ckd and eskd , general practitioners and medical staff , such as dieticians and public health nurses , must be involved . the jsn referral criteria for nephrologists were published to facilitate comprehensive care for ckd patients ( table 3 ) . additionally , the asian forum of ckd initiatives ( afckdi ) was started to exchange information on ckd at the inaugural 50th jsn meeting in hamamatsu in 2007.table 3jsn criteria for referring ckd patients to a nephrologist ( cited from ref . ) proteinuria ( 2 + by dipstick proteinuria)combined proteinuria and hematuria ( both 1 + and over by dipstick proteinuria)low egfr ( < 50 ml / min/1.73 m ) : < 60 ml / min/1.73 m ( if age < 40 years ) and < 40 ml / min/1.73 m ( if age 70 years ) jsn criteria for referring ckd patients to a nephrologist ( cited from ref . ) since 2008 , the special health check system ( so - called tokutei - kenshin ) has been used to detect subjects with metabolic syndrome and direct them towards a healthy lifestyle . the new kidney disease : global outcomes improving outcomes ( kdigo ) ckd classification prevalence of hypertension was clearly dependent on egfr and proteinuria ( fig . thus , the jsn is negotiating for a better screening system for ckd in japan . the jsn has launched web - based registries for ckd and kidney biopsy recipients [ 54 , 55 ] . 6prevalence of hypertension based on the new kdigo ckd classification ( cited from ref . ) prevalence of hypertension based on the new kdigo ckd classification ( cited from ref . ) since the introduction of the concept of ckd , the definition has been challenged with several criticisms : ( 1 ) the classification was too simple , ( 2 ) lack of key outcomes of ckd , and ( 3 ) significance of testing egfr and albuminuria . in this setting , the kdigo - controversies conference was held from october 46 , 2009 in london . we offered the dataset , including serum creatinine and dipstick proteinuria , for the conference . after the conference , the ckd classification was slightly modified and expressed as the ckd heat map. the clinical impacts of egfr and albuminuria were investigated for several major outcomes [ 5761 ] . to further examine the significance of the classification , the kdigo ckd prognosis consortium ( pc ) was organized . we are privileged that the okinawa 1983/1993 cohorts were involved in the kdigo - pc . the phase 2 analyses have already been completed for seven major topics , such as hypertension , diabetes , gender , ethnicity , age , ckd epidemiology collaboration , and cystatin c [ 6264 ] . the significance of a low egfr and albuminuria was confirmed for all - cause mortality and cardiovascular mortality . the relative risks of these markers were similar , but the absolute risks were different based on age , sex , and the presence of diabetes or hypertension . currently , there will be an additional 13 topics in the phase 3 step to be studied soon . ckd is common but treatable if detected early and properly managed . at an early ckd stage , patients are usually asymptomatic ; therefore , regular health checks using a urine dipstick and serum creatinine are recommended . the intervals for follow - up , however , are debatable due to the cost . in this regard , subjects with hypertension , diabetes , anemia , and/or metabolic syndrome have the highest risk of ckd ( fig . other factors , such as dyslipidemia , hyperuricemia , gout , cvd and/or a family history of ckd or eskd , also have a high risk for ckd . such people should have serum creatinine and albuminuria ( proteinuria ) assessed at least annually.fig . 7complications by baseline egfr among the screened population ( unpublished observation ) complications by baseline egfr among the screened population ( unpublished observation ) ckd patients are at risk of developing acute kidney injury due to contrast media , nephrotoxic drugs , surgery , and dehydration . ckd is a strong risk factor for developing cvd and death and also plays an important role in infection and malignancies , particularly in elderly people . japan is a front runner in the new society of a world where the elderly population ( 65 years ) is the most prevalent , reaching 30 % in 2020 . japan is the leader of medicine for an aged society and the science of ageing . high - quality observational studies could promote basic science and stimulate the invention of new treatments for ckd . the mechanisms of age - related gfr decline are entirely unknown , and we have no way to delay the process . further research on the role of ckd along with other medical conditions , such as infection , mental disorders , ckd - mbd , and malignancies is needed , especially among the elderly population . ckd campaigns in public and medical communities should be continued in order to delay , if not prevent , the development of eskd . many cases of ckd are left unrecognized , but the condition can be treated even at late stages , so screening is always beneficial .

a recent us study found increasing demand for blood transfusion associated with the increased aging of the population . blood transfusion is an important supportive treatment of cancer patients most of whom are anemic , and it is a critical issue for patients with chronic diseases such as heart failure and chronic kidney disease [ 3 , 4 ] . anemia should be considered as a factor that reduces quality of life and a risk for early death . the aim of anemia management should be to restore patient functionality and quality of life by restoring effective red cell volume . one of the considerations for decision of blood transfusion is the possibility of adverse reactions [ 710 ] . the most common complication of transfusion is febrile and chill - rigor reactions , nonhemolytic and the most serious complications are acute hemolytic reaction due to abo incompatibility . febrile nonhemolytic transfusion reaction ( fthtr ) is an acute ( < 24 hours ) immunological transfusion reaction . early recognition of symptoms suggestive for a transfusion reaction and prompt reporting to the blood bank are essential . in view of the increasing needs of blood transfusion in geriatric patients , we decided to examine all adverse blood transfusion reactions in hospitalized patients during one - year period . patients are admitted from the community , nursing homes , and general hospitals to the acute , rehabilitation , or long - term care wards . all the patients who received packed red blood cells transfusion during the study year were included in this study . we defined blood transfusion reactions such as objective symptoms , recorded by staff , fever , chills , shortness of breath , and vomiting within 24 hours after packed red blood cells transfusion was administrated . fever was defined as unexpected temperature rise ( 38 or 1c above baseline , if baseline 37 ) during or shortly after transfusion . vital signs such as blood pressure , pulse , peripheral oxygen saturation , and breaths number were measured five minutes after beginning of blood transfusion and every 15 minutes during first hour thereafter according to guidance of blood transfusion . the clinical and laboratory data were coded and analyzed by using the spss software ( spss 14 ) . two hundred forty - two patients received 382 blood units during the study year ( 2008 ) . most patients were female ; the median age was 82 9 ; fifty percent were hospitalized in acute geriatric departments , and 48% of them were demented and 45% bed ridden . 151 patients ( 63% ) received only one unit of packed cells and 67 patients ( 28% ) two units . the causes of anemia were as follows : the most common type was anemia of chronic disease ( 63% ) followed by iron deficiency anemia ( 19% ) , folic acid ( 17% ) , and vitamin b 12 ( 9% ) deficiency . decision for blood transfusion was related to the hemodynamic state of the patients that although treated with ferrum , folate , or vitamin b 12 according to the kind of their deficiency , remained anemic and clinically necessitated it . the data on the transfusion reactions is presented in table 2 . in 40 ( 11% ) cases , fever was the most common reaction ; 29 ( 72% ) of those 40 cases were with reactions . chills and vomiting were observed in three cases each ( 8% ) ; four ( 10% ) had shortness of breath . there were no lethal cases in the first 24 hours following blood transfusions . in all cases of febrile reaction , the blood sample was examined for contamination and no cases of pathogenic bacteria were identified . during one year in our multilevel geriatric hospital , 382 packed red blood cells transfusion were performed . blood transfusion reactions occurred in 40 ( 11% ) cases within 24 hours after transfusion . we were surprised of the relatively low rate of adverse blood transfusion reactions in this geriatric debilitated population . 's study shows potential usefulness of databases in assessment of blood utilization , transfusion - related complication , and risk factors among united states ' elderly in the outpatient setting . our study is the first one that surveys blood transfusions reactions in a geriatric inpatient setting . therefore , comparing data was performed with that of general hospitals and it shows comparable incidence of blood transfusion reactions [ 1416 ] . an overall incidence of transfusions reactions of 8.7% was reported , with febrile nonhemolytic transfusion reaction in 65% of these . reports that febrile reactions ( 47% ) are the main reaction in their study on 100 general hospitals . narvios et al . reported minor adverse reactions including low grade fever in 19% cases and itching in 41% . they suggest that careful evaluation of any suspected event of transfusion reaction should be referred to the transfusion medicine physicians of blood bank who will evaluate each case and discuss it promptly with the attending physician . most patients hospitalized in our multilevel geriatric medical center are frail elderly with a high rate of comorbidities , immobilization , and cognitive impairment . since many such patients are noncooperative , they do not report minor reactions such as itching , nausea , dizziness , and others . therefore , noncomplaining initial minor symptoms related to adverse blood transfusion reactions may endanger these patients . hypotension can be a manifestation of several transfusion reactions : acute hemolytic , bacterial contamination , transfusion - related acute lung injury , and anaphylaxis . in the rare cases , we suppose that this sign may be silent and not recognized in demented patients such as our study population . the main limitation of this study is its being a retrospective analysis of data of only one year . for better detection and understanding of the reactions associated with blood transfusion in elderly debilitated inpatients ,

class ii malocclusions are of interest to practicing orthodontists because they constitute a significant percentage of the cases they treat.1 myriad treatment modalities for class ii malocclusions have been investigated and published.2 some authors attempt to correct the underlying skeletal imbalance through growth modification by either extraoral traction or functional appliances , whereas others focus on dental camouflage of the jaw discrepancy . the twin block ( tb ; clark , 1982 ) appliance is one of the widely used removable functional appliances to correct class ii dentoskeletal disharmony . it was found to be the preferred functional appliance in the united kingdom ; more than 75% of british orthodontic society members claimed it is their first choice.3 one of the unique features of this appliance is that it is constructed in two separate parts : the upper and lower appliances . forward mandibular posturing is achieved by incorporating buccal blocks with interlocking inclined planes of approximately 70 , with the lower blocks engaging in front of the upper blocks.46 the effects of the tb are well established in the literature.4,5,7,8 obrien et al8 investigated the changes in the anteroposterior relationship of the maxilla to the mandible and the overjet during treatment for growing patients ( 810 years ) . the mean overjet correction was 6.6 mm , whereas the net effect of treatment in terms of overjet correction when compared with the untreated group was 6.9 mm . the skeletal contribution to the overjet correction was 27% ; the remaining 73% was a result of dentoalveolar changes . the tb , however , is highly dependent on patient compliance . to overcome this limitation , a new generation of fixed functional appliances these appliances are more streamlined than the removable types and do not interfere with speech . the herbst appliance2 and its different modification was one of the early compliance - free appliances . recently , the idea of incorporating nickel - titanium push coil springs as part of the appliances has received a lot of interest . these appliances include saif spring,9 jasper - jumper,9 klapper spring,9 eureka spring appliance,10 the adjustable bite corrector,11 and xbow.12 the more advanced and recently popularized designs include the twin force bite corrector13 and the forsus fatigue resistant device ( frd).1416 the frd ( 3 m unitek corp , monrovia , ca , usa ) is a semirigid telescoping system incorporating a superelastic nickel - titanium coil spring that can be assembled chair - side and that can be used in conjunction with complete fixed orthodontic appliances . the frd attaches at the maxillary first molar and onto the mandibular archwire , distal to either the canine or the first premolar bracket . many papers were published on the frd , but only a few were clinical studies1519 reporting variable effects . franchi et al16 found that the frd protocol is effective in correcting class ii malocclusion with a combination of skeletal and dentoalveolar modifications , although a recent cephalometric study20 of class ii correction with the sabbagh universal spring and the frd found that both appliances did not induce significant skeletal effects . the correction was mainly dentoalveolar , with more mandibular incisors proclination in the frd than with the sabbagh universal spring2 . the objective of this study was to evaluate the dentoskeletal changes with frd and tb in class ii patients in comparison with untreated controls . this is a retrospective comparative study of class ii malocclusion subjects treated with tb or frd in comparison with a matched sample of untreated class ii controls . a total of 92 patients were included : 30 in the frd group ( 12 girls and 18 boys ; mean sd of 12.91.1 years ) , obtained from one private practice , and 37 in the tb group ( 24 girls and 13 boys ; 11.21.6 years ) , selected from the private practice of the developer of the appliance , dr william clark ( scotland , united kingdom ) . this tb sample was previously used in other studies to investigate the tb therapy effects;6 25 untreated class ii subjects ( 12 girls and 13 boys ; 11.91.9 years ) obtained from the university of michigan growth study and matched with the experimental groups for skeletal age ( at the start or during the growth spurt ) , sex , and craniofacial morphology . this study was approved by the state university of new york at buffalo institutional review board . the inclusion criteria were : caucasian healthy boys and girls who were starting or within the period of their skeletal growth spurt , as indicated by the cervical vertebral maturation method ; class ii division 1 malocclusion , with the canines and molars in at least an end - to - end relationship ; in the late mixed or early permanent dentitions ; normal growth pattern ( frankfort to mandibular plane angle = 2135 ) ; a point - nasion - b point angle 4.5 ; retrognathic mandible ( sella - nasion - b point angle 76 , sella - nasion - a point angle 80 ) ; records of sufficient quality for accurate identification of landmarks on cephalograms ; exclusive treatment with frd or tb for at least 6 months ; having the appliance not removed prematurely because of breakage ; and nonextraction treatment . patients with unfavorable growth patterns , craniofacial anomalies , in the early mixed dentition , or who had an anterior open bite of more than 2 mm were all excluded . the tb sample received the original design and treatment protocol suggested by dr clark.6 no brackets were bonded during the tb therapy . the bite registration was taken by advancing the mandible sagittally , with no deviation , by 7 mm , with 35 mm interocclusal clearance in the first bicuspid region . although the frd sample received the clip - on design , before frd insertion , the maxillary and mandibular arches were bonded with pre - adjusted edgewise appliances ( 0.022-inch bracket slot ) until the upper and lower 1925-inch ss wires were reached . for the maxillary dentition , the archwire management varied according to the individual treatment goals in terms of upper molar distalization . the bracket torques were 17 and 10 in the maxillary central and lateral incisors and 6 in the mandibular incisors . two lateral cephalograms for each subject were used , pretreatment , with the functional appliances ( t1 ) , immediately before the insertion of the frd appliance in fully bonded upper and lower arches , and before any treatment in the tb group , and with postfunctional appliance removal ( t2 ) . clark cephalometric analysis was used , which includes 27 angular and linear measurements.6 nine randomly selected radiographs were digitally retraced and remeasured 3 weeks apart by a single evaluator . an intraclass correlation coefficient was used to evaluate intraobserver reliability , and the dahlberg formula was used to calculate the random method error . one investigator who was blind to the type of group digitally traced and analyzed the cephalograms of the frd and the controls , using the dolphin system ( dolphin digital imaging system , version 11 ; chatsworth , ca , usa ) . dr clark , using quick ceph ( quick ceph systems ; san diego ca , usa ) , traced the tb sample , and the intraexaminer reliability was reported in a previous study.6 the magnification for the radiographs was standardized at 8% . maxillary superimpositions were made along the palatal plane , registered at an a point - nasion - b point angle , whereas the mandibular superimpositions were made on the inner contour of the inferior symphysis , the inferior mandibular canal , and the germ of the third molar , if present . maxillary and mandibular dentoalveolar changes were evaluated using reference lines perpendicular on the palatal plane and mandibular plane , respectively . the data were analyzed using spss software ( version 21 ; ibm corporation , armonk , ny , usa ) . descriptive statistics were used to present the baseline data of each of the three groups . the levene test was used to check for the homogeneity of variances at t1 of all three groups . paired t - tests were used to compare pre- and posttreatment measurements in each group . analysis of variance ( anova ) was used to compare the mean changes between the three groups , followed by tukey hsd ( honestly significant differences ) for determining homogeneous subsets whenever appropriate . two lateral cephalograms for each subject were used , pretreatment , with the functional appliances ( t1 ) , immediately before the insertion of the frd appliance in fully bonded upper and lower arches , and before any treatment in the tb group , and with postfunctional appliance removal ( t2 ) . clark cephalometric analysis was used , which includes 27 angular and linear measurements.6 nine randomly selected radiographs were digitally retraced and remeasured 3 weeks apart by a single evaluator . an intraclass correlation coefficient was used to evaluate intraobserver reliability , and the dahlberg formula was used to calculate the random method error . one investigator who was blind to the type of group digitally traced and analyzed the cephalograms of the frd and the controls , using the dolphin system ( dolphin digital imaging system , version 11 ; chatsworth , ca , usa ) . san diego ca , usa ) , traced the tb sample , and the intraexaminer reliability was reported in a previous study.6 the magnification for the radiographs was standardized at 8% . maxillary superimpositions were made along the palatal plane , registered at an a point - nasion - b point angle , whereas the mandibular superimpositions were made on the inner contour of the inferior symphysis , the inferior mandibular canal , and the germ of the third molar , if present . maxillary and mandibular dentoalveolar changes were evaluated using reference lines perpendicular on the palatal plane and mandibular plane , respectively . the data were analyzed using spss software ( version 21 ; ibm corporation , armonk , ny , usa ) . descriptive statistics were used to present the baseline data of each of the three groups . the levene test was used to check for the homogeneity of variances at t1 of all three groups . paired t - tests were used to compare pre- and posttreatment measurements in each group . analysis of variance ( anova ) was used to compare the mean changes between the three groups , followed by tukey hsd ( honestly significant differences ) for determining homogeneous subsets whenever appropriate . the mean ages at t1 and treatment / observation periods are presented in table 1 . the results of the reliability testing between the repeated measurements revealed good agreement ( r=0.830.99 ) . dahlberg s variance results showed that the random method error was within 1 mm/1. the sex distribution between the three groups did not show statistically significant differences . although there were more men than women in the frd group , this 20% difference did not achieve statistical significance ( p=0.36 ) . overall , the groups were comparable at baseline ( t1 ) with the exception of ; the incisor overjet , the posterior facial height , the mandibular length , and the ramus height , which showed significant differences between the treatment groups and the untreated controls . significant skeletal changes were observed at t2 in all three groups ( table 2 ) . posterior facial height increased by 2.72 mm in the control group , 3.22.4 mm in the tb , and 1.61.4 mm in the frd . the mean total mandibular length significantly increased by 3.132.7 mm in the controls , 6.33.9 mm in the tb , and 1.62.1 mm in the frd . a similar finding was noted for the ramus height ( mean increase of 2.31.8 mm in controls , 4.22.5 mm in tb , and 1.31.3 mm in frd ) . the upper incisors retroclined significantly in the tb ( 12.46.5 ) and frd ( 3.94.5 ) groups , with no significant change in the controls . the mandibular incisors flared by 2.15.7 in the tb and by 3.94.6 in the frd groups , respectively . the mean decrease in the overjet was 62% in the tb and 56% in the frd groups , respectively . the mean change of the frankfort to mandibular plane angle when compared by anova was statistically different between the three groups ( p<0.01 ) . however , tukey analysis showed no evidence that any of the two experimental groups significantly differed from the controls ( table 3 ) . there was no significant difference between the three groups in many skeletal variables such as cranial base angle , facial depth , facial - axis angle , condyle - axis angle , maxillary plane angle , and mandibular corpus length . the net increases in the mandibular length ( basion - pogonion ) and ramus height in the frd group did not differ significantly from the controls . however in the tb group , the increases of both variables were two times larger than in frd and controls ( p<001 ) . the maxillary position to nasion / vertical did not show a significant difference ( p=0.08 ) . midfacial length ( condylion to a - point ) was significantly different between tb and frd ( p=0.04 ) ; however , neither of the two experimental groups was different from the controls . there is insufficient evidence to support that either of the two appliances had a significant headgear effect . the mean change of the lower incisor angle was significantly different between the three groups ( anova p=0.042 ) . statistically significant lower incisors proclination has occurred in the frd group when compared with the controls . however , the difference between the frd and the tb groups was not statistically significant , as confirmed by tukey hsd test . significant skeletal changes were observed at t2 in all three groups ( table 2 ) . posterior facial height increased by 2.72 mm in the control group , 3.22.4 mm in the tb , and 1.61.4 mm in the frd . the mean total mandibular length significantly increased by 3.132.7 mm in the controls , 6.33.9 mm in the tb , and 1.62.1 mm in the frd . a similar finding was noted for the ramus height ( mean increase of 2.31.8 mm in controls , 4.22.5 mm in tb , and 1.31.3 mm in frd ) . the upper incisors retroclined significantly in the tb ( 12.46.5 ) and frd ( 3.94.5 ) groups , with no significant change in the controls . the mandibular incisors flared by 2.15.7 in the tb and by 3.94.6 in the frd groups , respectively . the mean decrease in the overjet was 62% in the tb and 56% in the frd groups , respectively . the mean change of the frankfort to mandibular plane angle when compared by anova was statistically different between the three groups ( p<0.01 ) . however , tukey analysis showed no evidence that any of the two experimental groups significantly differed from the controls ( table 3 ) . there was no significant difference between the three groups in many skeletal variables such as cranial base angle , facial depth , facial - axis angle , condyle - axis angle , maxillary plane angle , and mandibular corpus length . the net increases in the mandibular length ( basion - pogonion ) and ramus height in the frd group did not differ significantly from the controls . however in the tb group , the increases of both variables were two times larger than in frd and controls ( p<001 ) . the maxillary position to nasion / vertical did not show a significant difference ( p=0.08 ) . midfacial length ( condylion to a - point ) was significantly different between tb and frd ( p=0.04 ) ; however , neither of the two experimental groups was different from the controls . there is insufficient evidence to support that either of the two appliances had a significant headgear effect . the mean change of the lower incisor angle was significantly different between the three groups ( anova p=0.042 ) . statistically significant lower incisors proclination has occurred in the frd group when compared with the controls . however , the difference between the frd and the tb groups was not statistically significant , as confirmed by tukey hsd test . the tb and frd groups showed favorable reduction in skeletal convexity ( 1.71.4 mm and 1.020.85 mm , respectively ) . this reduction was in agreement with previous frd16,18 and tb studies21,22 in which the a point - nasion - b point angle angle reduction was used to represent the relative skeletal convexity correction . the difference between the three groups in terms of convexity reduction was highly significant ( anova p<0.001 ) . there was a significant difference between the three groups in the total mandibular length and maxillary - mandibular ( mx - md ) differential lengths ( anova p<0.001 ) . the total mandibular length increased in the tb group two times more than in the controls ( 6.33.9 mm ) . similarly , the mx - md differential length increased by 4.62.1 mm in the tb group compared with the controls ( 1.81.9 mm ) . these findings confirm previous investigations21,23 that suggest a mandibular growth enhancement effect by this appliance . in contrast , the amount of change in the frd group was not significantly different from in the controls , which parallels the findings of the late puberty frd group in a previous study18 but is in disagreement with another.16 the differences between previous reports may not be easily explained because they could be associated with the treatment protocol and duration , wires , and slot dimension ; fixed appliance torque differences;16 the age factor;18,20 and possible different neuromuscular responses.24 there was a highly significant increase in the corpus length of all three groups ( student s t - test p<0.001 ) but no significant difference between them ( anova p=0.70 ) , which suggests it was only a result of the growth effect . the upper incisors retroclination was statistically and clinically significant in both experimental groups ( student s t - test p<0.001 ) , with the highest change seen in the tb group . this finding is in contradiction with some previous studies25,26 and is in agreement with many others.22,27,28 the considerable upper incisors retroclination may be attributed to the appliance design , particularly when the labial bow is incorporated.22 it may also be attributed to the treatment period and treatment modality . in a previous study,23 semirapid maxillary expansion and alignment of the upper arch were performed before tb therapy , which may have influenced the amount of retroclination . pretreatment dentoskeletal characteristics and the variation in appliance - wearing time among patients also have to be taken into consideration . in addition , variability among clinicians adds an operator factor to the effect of this appliance . the upper incisor retroclination in the frd group was smaller than in the tb group . the presence of fixed brackets on the upper incisors may have limited the amount of retroclination in this group . the 3.9 retroclination in the frd group was similar to previous findings,20 but it should be noted that direct comparison is not possible because of the different reference lines used in the clark cephalometric analysis . at t2 , the lower incisors proclined by an average of 2.1 and 3.9 in the tb and frd groups , respectively , but the difference between the groups was not statistically significant . the lower incisors proclination in the tb group was less than that reported in previous studies.21,22,25,28 many factors that were discussed as possible contributors for the difference in the upper incisors retroclination are similarly applicable . in some studies , the acrylic of the lower part of the tb appliance was extended to cover the lower incisal edges . this acrylic extension aimed to limit the lower incisors tipping.27,28 the mean changes in the molar relationship in both groups differed significantly from the controls ( 4.4 and 4.9 mm in the frd and tb groups , respectively ; anova p<0.001 ) . the molar relationship correction was found to be a result of differential anterior / posterior movements of both upper and lower molars in the frd group with more contribution from lower molar mesialization.29 overjet correction was significantly different between the three groups ( anova p<0.001 ) . the overjet reduction in the frd group was about half of that in the tb group , which could be partially explained by the small initial overjet , which was 7.1 and 11.3 mm in the frd and tb groups , respectively . a similar finding was reported in a recent investigation,29 where there was slightly larger overjet correction with tb ( ~6 mm ) than frd ( 5 mm ) , but interestingly , the authors found no statistically significant difference between the two appliances in the percentage change of overjet . patient compliance with the use of the tb removable appliance did not seem to play a major rule because the appliance was able to normalize the class ii malocclusion into class i. this study has several limitations . the results are applicable for short - term observation periods and may differ if a long - term follow - up is carried out . a randomized clinical trial is always recommended , as it has the highest level of evidence when investigating the efficacy of orthodontic appliances . however , it alone is not enough for a comprehensive understanding of the functional orthodontic treatment . an investigation of three - dimensional soft tissue changes and temporomandibular changes / remodeling during and after treatment completion in prospective tomographic studies is recommended . the frd and tb are effective in the treatment of patients with class ii malocclusion . both appliances were able to induce favorable changes in the sagittal relation , but the type of change differed significantly between the groups . the frd induced dentoalveolar changes , and the contribution to the final overjet correction was a result of an equal combination of upper incisor retroclination and lower incisor proclination .

there are some recent reports indicating a high failure rate ( 50% ) of the modified tension band technique despite previous good results . several modifications have been used to increase the stabilisation of the tension band technique along with other techniques [ 4 , 7 , 9 ] . although difficulties concerning the modified tension band technique are described and defined in textbooks and the literature in detail , technical inadequacies are often underestimated . this study aimed to explore the relationship between patellar anatomy , tension band and kirschner wiring . although the tension band is the main factor in achieving stability , kirschner wiring designates tension band projection on tendon and bone . first of all , we explored this relationship with a magnetic resonance imaging ( mri ) study . with the data obtained , technical requirements and anatomical and dynamic specifications sagittal sections of patellae that pass the thickest point were obtained from 30 knee mri studies ; patellar diameter , tendon insertion , localisation and thickness were measured ( fig . 1 ) . 1patellar section of mri patellar section of mri the modified tension band technique was used according to the ao manual of internal fixation . templating the tension band technique was performed on mri ( kirschner wire drawn 56 mm posteriorly from the anterior surface of the patella ) ( fig . 2 ) . kirschner wiring position in relation to tendinous tissue was evaluated with three parameters : outside - anterior ( a ) , inside - median ( m ) and outside - posterior ( p ) . 2kirschner wire templating on mri section kirschner wire templating on mri section seven cow patellae with tendinous tissue were used as biomechanical test material . cow patellae were cut transversely 2 cm from the pole to simulate human patellar fractures . prepared cow patellar fragments were fixed on a traction apparatus by two 2-mm kirschner wires , which passed 5 mm posteriorly from the anterior surface of the patella and through tendinous tissue . with a 1.2-mm tension band wire , four configurations were simulated : vertically oriented figure - of - eight passed anteriorly to the tendon ( a ) , horizontally oriented figure - of - eight ( b ) , vertically oriented figure - of - eight passed posteriorly to the tendon ( c ) and to eliminate the effect of the tendon a control group ( d ) was established as vertically oriented figure - of - eight without tendinous tissue . 3tension band configurations are vertically oriented figure - of - eight passed anteriorly to the tendon ( a ) , horizontally oriented figure - of - eight ( b ) , vertically oriented figure - of - eight passed posteriorly to tendon ( c ) and to eliminate the effect of the tendon a control group ( d ) was established as vertically oriented figure - of - eight without tendinous tissue tension band configurations are vertically oriented figure - of - eight passed anteriorly to the tendon ( a ) , horizontally oriented figure - of - eight ( b ) , vertically oriented figure - of - eight passed posteriorly to tendon ( c ) and to eliminate the effect of the tendon a control group ( d ) was established as vertically oriented figure - of - eight without tendinous tissue distractions on the tension band wire were performed by a compression - distraction test device ( ag - i 10 kn , shimadzu , kyoto , japan ) with 3 mm / min traction speed to a maximum load of 200 n and then ten cyclic loadings were performed between 100 and 200 n ( fig . 4photograph showing application of the cyclic loading photograph showing application of the cyclic loading the data on force - displacement variation were evaluated with the kruskal - wallis test using spss 11.0 for windows . statistical analyses were performed using a mann - whitney u test to estimate group differences . sagittal sections of patellae that pass the thickest point were obtained from 30 knee mri studies ; patellar diameter , tendon insertion , localisation and thickness were measured ( fig . 1 ) . 1patellar section of mri patellar section of mri the modified tension band technique was used according to the ao manual of internal fixation . templating the tension band technique was performed on mri ( kirschner wire drawn 56 mm posteriorly from the anterior surface of the patella ) ( fig . 2 ) . kirschner wiring position in relation to tendinous tissue was evaluated with three parameters : outside - anterior ( a ) , inside - median ( m ) and outside - posterior ( p ) . cow patellae were cut transversely 2 cm from the pole to simulate human patellar fractures . prepared cow patellar fragments were fixed on a traction apparatus by two 2-mm kirschner wires , which passed 5 mm posteriorly from the anterior surface of the patella and through tendinous tissue . with a 1.2-mm tension band wire , four configurations were simulated : vertically oriented figure - of - eight passed anteriorly to the tendon ( a ) , horizontally oriented figure - of - eight ( b ) , vertically oriented figure - of - eight passed posteriorly to the tendon ( c ) and to eliminate the effect of the tendon a control group ( d ) was established as vertically oriented figure - of - eight without tendinous tissue . 3tension band configurations are vertically oriented figure - of - eight passed anteriorly to the tendon ( a ) , horizontally oriented figure - of - eight ( b ) , vertically oriented figure - of - eight passed posteriorly to tendon ( c ) and to eliminate the effect of the tendon a control group ( d ) was established as vertically oriented figure - of - eight without tendinous tissue tension band configurations are vertically oriented figure - of - eight passed anteriorly to the tendon ( a ) , horizontally oriented figure - of - eight ( b ) , vertically oriented figure - of - eight passed posteriorly to tendon ( c ) and to eliminate the effect of the tendon a control group ( d ) was established as vertically oriented figure - of - eight without tendinous tissue distractions on the tension band wire were performed by a compression - distraction test device ( ag - i 10 kn , shimadzu , kyoto , japan ) with 3 mm / min traction speed to a maximum load of 200 n and then ten cyclic loadings were performed between 100 and 200 n ( fig . 4 ) . 4photograph showing application of the cyclic loading photograph showing application of the cyclic loading the data on force - displacement variation were evaluated with the kruskal - wallis test using spss 11.0 for windows . statistical analyses were performed using a mann - whitney u test to estimate group differences . patellar size measurements were taken with the following findings : mean patellar lengthening 34 3 mm ( 30.642.9 mm ) , mean patellar thickness 19.6 2.3 mm ( 15.526.1 mm ) , mean patellar tendon insertion thickness 4.8 1 mm ( 36.8 mm ) and mean quadriceps tendon insertion thickness 6.5 1 mm ( 3.98.1 mm ) . the correlation between patellar length and thickness was 0.70 , but a low correlation was observed between tendon thickness and patellar diameter ( table 1 ) . templating the modified tension band technique on the mri slide indicated that all kirschner wiring has to pass through tendinous soft tissue . table 1mri results for 30 patellae : patellar length ( pl ) , patellar thickness ( pt ) , quadriceps tendon thickness ( qtt ) , patellar tendon thickness ( ptt ) and correlation between them plptqttpttpl / ptptt / ptqtt / pt134.521.65.74.81.5972220.2222220.263889235.219.46.85.91.8144330.3041240.350515337.918.93.93.32.0052910.1746030.206349436.622.96.85.71.5982530.2489080.296943535.620.36.73.31.7536950.1625620.330049635.21664.52.20.281250.375733.9186.95.41.8833330.30.383333833.919.26.86.11.7656250.3177080.354167942.926.186.81.6436780.2605360.3065131035.217.15.84.72.058480.2748540.3391811131.817.66.93.91.8068180.2215910.3920451231.7205.45.71.5850.2850.271330.616.26.33.91.8888890.2407410.388889143115.58.14.820.3096770.5225811538.822.85.93.61.7017540.1578950.2587721633.520.88.161.6105770.2884620.3894231733.919.34.33.61.7564770.1865280.2227981837.519.76.85.71.9035530.289340.3451781940.320.35.74.81.9852220.2364530.2807882039.923.65.55.51.6906780.2330510.2330512132.8196.84.31.7263160.2263160.3578952230.617.66.531.7386360.1704550.3693182331.419.57.65.41.6102560.2769230.3897442433.219.56.84.71.7025640.2410260.3487182534.619.57.26.51.7743590.3333330.3692312633.919.76.84.81.7208120.2436550.3451782733.620.57.86.31.6390240.3073170.3804882835.319.97.24.21.7738690.2110550.3618092933.917.96.54.51.8938550.2513970.3631283031.919.45.13.61.644330.1855670.262887mean34.7033319.593336.494.8433331.7824330.2480850.335262standard deviation3.0015492.2676031.0243081.04310.154950.0494920.065327correlation0.6966170.3976790.061432 mri results for 30 patellae : patellar length ( pl ) , patellar thickness ( pt ) , quadriceps tendon thickness ( qtt ) , patellar tendon thickness ( ptt ) and correlation between them biomechanical study results are shown in table 2 . at the first 200 n loading , the mean displacement in method 1 was 0.23 mm ( sd : 0.07 ) , in method 2 it was 0.16 mm ( sd : 0.14 ) , in method 3 it was 0.23 mm ( sd : 0.06 ) and in method 4 it was 0.14 mm ( sd : 0.04 ) . the mean displacement after the cyclic loading test in method 1 was 0.89 mm ( sd : 0.31 ) , in method 2 it was 0.57 mm ( sd : 0.17 ) , in method 3 it was 0.46 mm ( sd : 0.11 ) and in method 4 it was 0.29 mm ( sd : 0.09 ) . table 2tension band cyclic loading test resultstension band methodfirst displacementtotal displacementa0.230 0.0700.893 0.313b0.228 0.0560.461 0.114c0.164 0.1380.572 0.170d0.141 0.0410.275 0.086tension band configurations are vertically oriented figure - of - eight passing anteriorly to tendon ( a ) , horizontally oriented figure - of - eight passing anteriorly to tendon ( b ) , vertically oriented figure - of - eight passing posteriorly to tendon ( c ) and vertically oriented figure - of - eight without soft tissue ( d ) . total displacement shows total displacement after ten cyclic loadings tension band cyclic loading test results tension band configurations are vertically oriented figure - of - eight passing anteriorly to tendon ( a ) , horizontally oriented figure - of - eight passing anteriorly to tendon ( b ) , vertically oriented figure - of - eight passing posteriorly to tendon ( c ) and vertically oriented figure - of - eight without soft tissue ( d ) . total displacement shows total displacement after ten cyclic loadings the results indicate that there was a significant difference between all groups compared with the kruskal - wallis test ( p = 0.000 ) . using the mann - whitney test , there was a significant difference between group 1 and group 2 ( p = 0.012 ) , group 1 and group 3 ( p = 0.04 ) , group 1 and group 4 ( p = 0.02 ) and group 2 and group 4 ( p = 0.02 ) . yet there are high complication rates with the tension band technique and early mobilisation requires more rigid fixation methods . several biomechanical studies and several modifications of the tension band technique indicate that the relationship between the tension band and soft tissue around the patella affect stability in treatment of patellar fractures [ 110 ] . the tension band technique is important for patellar fractures ; yet these techniques are more functional in the knee flexion position . horizontally oriented figure - of - eight for tension band configuration is found to be stronger than the vertically oriented ones , which our test results support . improved wire twisting and flexible tension band material are helpful for good force distribution , thus decreasing failure rate [ 6 , 8 ] . other techniques are effective in the knee extension position , but their combination with the tension band technique is best and most effective in any position [ 2 , 3 ] . according to the templating study of the tension band technique on the mri section , the kirschner wire must pass through tendinous tissue and tension band material must turn over this soft tissue . our anatomical study shows that there is no statistical correlation between patellar length and thickness to confirm similar knowledge in the literature . it is a fact that the expected result of the tension band techniques is affected by many factors such as variety of patellae , surgical practice and traumatic damage . but the kirschner wire end point is placed deeply in some practices so that more interposed tendinous tissue can diminish stability . many treatment methods are discussed in the literature with clinical results and biomechanical studies , and numerous results indicate the importance of the relationship between tension band and soft tissue . for the biomechanical study the cow patella is too big to correlate with a human patella so a biomechanical test was designed to use only one part of the cow patella . the pole part of the cow patella with tendinous insertion was prepared similar in length to a human patellar fragment . the test was performed on one part of the patella only to eliminate practical error , as we were only interested in the tension band and soft tissue relationship . in our study , we preferred cyclic loading test at 100200 n because loading at over 200 n causes kirschner wire bending . if this test were repeated in the flexion position , the kirschner wire could not bend so we could reach a physiological loading force . besides this , compression on the tension band is initially too high to prevent the progressive instability resulting in loss of reduction and angulation deformities . in our study , the tension band adjacent to bone surface had more stability than the tension band technique turned over tendinous tissue , but cyclic loading tests have shown that all tension band applications in this study lost their initial stability . in conclusion , it is well known that the tension band is the main stability factor in patellar fractures in flexion . more stability and durability can be achieved in patellar fracture treatment , by transferring the tension force directly on to bone or at least choosing material that protects the tendon .

surfaceenhanced raman scattering ( sers ) is a novel and powerful characterization technique and slowly developing toward maturity as a tool for heterogeneous catalysis.1 many effects play a role in the actual sers spectra that are observed,2 and full understanding of the reasons for spectral fluctuations is important for practical application of reactivity studies on the scale of single catalytic particles.3 the large enhancement effects that are associated with the phenomenon opens doors to nanoscale molecular characterization that is otherwise impossible . this resolution is present both in quantity , or the number of molecules required for characterization , and in spatial resolution , especially with techniques such as tipenhanced raman scattering ( ters).2e , 4 however , single hotspot measurements require consideration in their interpretation , due to the small measurement volume , but also because of their timedependent variability.5 spectral fluctuations are a common feature in these timeresolved measurements.4a , 2 g , 6 due to the small number of molecules and extreme enhancement effect involved in these measurements , rare events are more readily observed where they are not ( or less ) visible in long time scale or bulk measurements , due to averaging over many more modes . the interpretation of shortterm variations in the spectra has been the subject of debate , as the highenhancement field at a hotspot can also lead to photodecomposition of molecules within that hotspot.2e , f , i however , the spectral fluctuations are commonly explained through local changes in chemical environment around the sensed molecules , or orientation effects . 2h , 6 this socalled blinking is at any rate a dynamic process , dependent on temperature and mobility of the molecules on the surface.7 in the case of heterogeneous catalysis , shortterm fluctuations might actually give valuable information about , for example , the orientations of molecules or the presence of reaction intermediates . to study these phenomena in detail a few selected locations will have a very intense nearfield , where other locations on the surface will hardly cause a raman enhancement effect . this makes it an ideal technique to study any shortlived reaction intermediates that are otherwise insignificant in a bulk observation , which is best studied in fewhotspot samples to limit ensembleaveraging . sufficiently long measurements will eventually capture the signature spectrum of shortlived species , as it arises at the location of an intense hotspot . all the above effects result in spectral differences between the spectral fluctuations from the regular ( bulk ) spectra . therefore , the use of chemometrics can help to separate spectra of large datasets in an effective manner . various groups have already linked sers measurements to multivariate analysis techniques.8 but where signal intensity fluctuations are no problem for multivariate analysis , spectral fluctuations on various time scales can give problems in these methods . here , we present the use of multivariate analysis tools as an approach to separate reactivity , related to bulk changes , from shortterm blinking , related to orientations , intermediate states and temporary conditions . both are important to understand the dynamics at the catalyst particle and the developed methodology allows analysis of both effects without removal of any spectra . the chemometric method used here is a combination of principalcomponent analysis ( pca ) and multivariate curve resolution ( mcr ) . in pca , the variance in a dataset is decomposed in a set of orthogonal principal components . using only a limited number of these principal components , a large amount of variance of the input spectra can be described.9 with mcr it is possible to decompose a mixture of spectra into their individualcomponent spectra.10 the main difference between pca and mcr lies in the components they calculate . pca components describe variance and can thereby also result in components with both negative and positive peaks . it is typically used as a tool to gain insight into the variance within a dataset . mcr with nonnegativity constraints decomposes a set of spectra into a predefined number of spectral components , which relate to the actual spectra of the related compound . this description of a dataset with a limited number of components usually works well for the description of bulk changes . however , shortterm fluctuations , like reaction intermediates , will be ignored as they are not prominent enough in the full dataset . we use pca to analyze the full dataset and separate reaction kinetics from shortterm variations . these separated datasets are subsequently examined with mcr , for direct comparison to normal raman spectra . we present here a novel chemometric method based on pca for time filtering ( see figure 1 ) . chemical reactions can hereby be separated from shortterm variations , enabling better analysis of either dataset . to present this analysis method , we use a selfassembled monolayer ( sam ) of pnitrothiophenol ( pntp ) on a flat au substrate , as illustrated in figure 2 . sers activity , and related reactivity , is introduced by deposition of ag nanoparticles , similar to other experiments described in literature.11 the monolayer of pntp can be reduced to p , pdimercaptoazobisbenzene ( dmab ) via photocatalysis over plasmonic nanoparticles , and typically uses green laser excitation and ag nanoparticles.3 , 12 rather than using green laser excitation , an excitation wavelength of 785 nm is chosen . this excitation excites the coupled plasmons of au and ag and gives sufficient enhancement effect to observe dynamics over single nanoparticle hotspots on the flat au substrate . the choice of this lowenergy excitation wavelength also ensures that the catalytic reaction is sufficiently slow to observe reactivity during the acquisition of 15 000 consecutive 1 s sers spectra . workflow for the chemometric analysis of the timeresolved sers spectra measured . a onecomponent principalcomponent analysis ( pca ) is used to create a filter , separating shortterm spectral blinking from reaction kinetics . both sets of spectra consists of single ag nanoparticles deposited on a selfassembled monolayer ( sam ) of pnitrothiophenol ( pntp ) assembled on a flat gold substrate . obtaining sers over single hotspots is achieved by deposition of single ag nanoparticles over a pntpfunctionalized flat au surface , as illustrated in figure 2 . a set of 15 000 raman spectra were recorded at an excitation wavelength of 785 nm , with a 1 s integration time . preprocessing was done via a cosmicray remover ( wire 3.2 , renishaw ) to remove the spikes in individual raman spectra . these narrow peaks occur only in single raman spectra and their removal has no effect on the timeresolved scale . the raman spectra are analyzed for the spectral region of 10001635 cm , where most vibrations characteristic for both reactant and product are found . a weighted leastsquares ( wls ) baseline correction is calculated , after which the absolute value of this dataset is taken . the main raman peaks of pntp and dmab are clearly visible in the color plot ( figure 3 a ) , as well as in the first and last raman spectra of the series ( figure 3 b ) . a simple method for tracking the reaction progress is to plot peak areas of reactant and product as a function of time . figure 3 c shows the results of this approach , for the raman peaks at 1335 ( sym . no2 stretch)13 and 1440 cm ( n = n stretch).14 the main trends are visible : the dmab signal comes up with time , whereas the pntp signal appears to be less affected by the reaction and suffers more from sersintensity variations . the noise level in the signals is rather high , most likely due to the noise in the spectra themselves , though peak shifts also contribute to the noise level . this method is sufficient for monitoring the ( irreversible ) reaction in time , but shows quite some noise in the overall signal . c ) pntp ( green ) and dmab ( blue ) peak area as a function of time . e ) calculated mcr components , component 1 ( green ) describes 62 % of the variance in the dataset , component 2 ( blue ) 32 % , resulting in a total of 94 % of the variance explained by the twocomponent mcr model . mcr is a more reliable method , as it takes the full spectrum into account , rather than just a single peak . for this dataset , an unrestrained twocomponent mcr analysis results in a model that describes 94 % of the total signal variance . component 1 ( 62 % , see figure 3 e ) resembles the reactant very well , while the second component ( 32 % ) comprises a mixture of dmab with a smaller contribution of pntp . the corresponding scores in time ( figure 3 f ) show more detail and less noise respect to the peak area analysis method ( figure 3 c ) . nonetheless , the spectral residuals after mcr ( figure 3 d ) still show many spectral features . blinking is a prominent feature in this dataset and occurs in spectral patterns that do not overlap with those of pntp and dmab . as this photoreaction encompasses multiple reaction steps,15 these spectra can contain valuable information regarding the mechanism . in fact , when time and spatial resolution of sers is high enough , these spectral fluctuations are expected to be the way to identify possible reaction intermediates . to separate shortterm ( reversible ) fluctuations from the longterm reaction progress , a new method of data processing is introduced . the photoreduction of pntp to dmab can largely be characterized by the disappearance of the pntp spectrum , and the rise of the dmab spectrum . after a simple spike removal , pca is used to separate longterm trends from shortterm trends using a 200 s time filter . the exact window required for the time filter a onecomponent pca on the time series ( after spike removal ) results in the component and score shown in figure 4 a , b. the spectral pattern of the component shows great resemblance to the spectrum of dmab , and describes 56 % of the variance in the dataset . the score plot of this component in time is similar to that expected for a reaction , with a slow increase in loading as time progresses . a ) loading of component 1 ( 56 % variance captured ) , b ) score of component 1 in time , c ) separation of spectra into the three categories , black and green spectra describe the bulk reaction , red spectra are spectral fluctuations and will be analysed separately later . the qresiduals in time ( figure 4 d ) show the sum of squares of the residual spectra , not explained by the model . these show a stable ( low ) baseline , but with quite some shortterm deviation from this baseline . the pca component does not describe those spectra very well as they are present only for short periods during the total 15 000 s. in other words , they show some form of spectral deviation from the bulk reaction . to separate these spectral fluctuations for closer examination , any qresiduals with a value higher than the mean value + 0.1the standard deviation of all qresiduals are selected , unless they consist of a series of spectra that are above this norm for 200 s consecutively . at the start of the reaction , the pca does not always fit the full trend of reactivity , since changes due to the reaction can result in relatively large spectral changes . however , these first spectra are only a minor part of a large dataset and thus are not accurately described by the one component pca model . therefore the qresidual criterion of the first 100 spectra is a fivefold higher value to ensure no reactivity information is lost . in figure 4 d , all black data points represent spectra that have a sufficiently low qresidual value , and are taken for further analysis of bulk reaction . in this first separation they are above the set qresidual criterion , but persistent enough to be considered as bulk processes . red data points are below the qresidual criterion as the corresponding spectra are only present during short time intervals . the color coding in figure 4 d is also applied to figure 4 b , c to indicate the relation between these different parameters . figure 4 c shows the breakdown of the full set into the different sets of spectra . green spectra do not meet up to the qresidual criterion , but make it through the time filter . they are likely an orientation effect due to changes in the monolayer , and are included in the analysis of the bulk reaction in figure 5 . the spectral pattern of these three sets is quite clear . with respect to the black spectra , the green spectra are only small deviations . in contrast , the red spectra show much more vibrational bands than observed in the black spectra . component 1 ( green ) represents 27 % of the calculated model , component 2 ( blue ) represents 70 % of the calculated model , with a total of 97 % variance explained . c ) all input spectra for the mcr model and d ) the spectral residuals that are not explained by the mcr components . a ) the four calculated mcr components . the variance capture with the components is for black 52 % , for red 24 % , for blue 13 % , and for green 5 % . b ) score plot in time for these four components , colourcoded as for ( a ) . having separated the photocatalytic reduction from shortterm variations , each dataset can now be analyzed separately . the reaction spectra are taken through a twocomponent mcr , after preprocessing of the spectra by a wls baseline correction and taking the absolute value of the resulting spectra . other than assuming two components to describe the reaction , no other constraints are put onto the mcr . the calculated mcr components are shown in figure 5 a and depict pntp ( in green , 27 % variance ) and a mixture of pntp and dmab ( in blue , 70 % variance ) . the score as a function of time is shown in figure 5 b , and , like the components , rather similar to the results obtained in figure 3 e , f. the main difference is the level of noise in the score , which is lower in figure 5 b. the removal of spectral fluctuations and subsequent analysis of the pure reaction has led to better description of this reaction via mcr . as can be expected , the variance explained by two mcr components within this filtered dataset is slightly higher ( 97 % ) than in the nonfiltered dataset ( 94 % ) . this is further emphasized by the input spectra ( figure 5 c ) and spectral residuals ( figure 5 d ) . less spectral fluctuations are put into the model , so less are found in the spectral residuals , when comparing figure 5 d to figure 3 d. overall , the time filter benefits the mcr model . but for application of this model to the description of reaction kinetics , some additional steps will have to be taken . the mixture of pntp and dmab in component 2 makes it difficult to determine the fraction of pntp in the total signal . dmab is simpler , as that is only expressed in component 2 . though the actual contribution of dmab will only be a fraction of the score for component 2 , this will be a matter of scaling the intensity and will not affect the calculation of reaction rate . most important in this novel chemometric method is its ability to analyze shortterm spectral fluctuations in large dataset . these fluctuations are expected to give information on shortlived events on the surface of the ag substrate , which could be either due to reorientation of pntp or dmab , or a reaction intermediate . figure 4 showed how variations present for less than 200 s were separated from the longterm effects of the bulk reaction . the separated spectra that exhibit these shortterm fluctuations are shown in figure 6 c. many spectral features are present , and at varying intensities . a fourcomponent mcr analysis was found to describe the majority ( 94 % ) of the spectral components in this dataset . the largest component ( figure 6 a , b , black , 52 % ) ) has a maximum contribution at around 4 000 s in the dataset . it is very similar to component 1 in figure 5 a , and is also most prominent at the beginning of the reaction . in comparison to the reactant component in figure 5 , a slight variation in peak ratios is observed , which is most likely caused by a different orientation or local environment for the reactant . similarly , the second component ( figure 6 a , b , red , 24 % ) has similarities to component 2 in figure 5 a , and is present at the end of the reaction . again , the differences are in peak ratios , likely caused by molecular orientation or variations in the local environment . the third ( figure 6 a and b , blue , 13 % ) and fourth ( figure 6 a , b , green , 5 % ) components have a different behavior in their score in time both are rather low in intensity , apart from one instance at 11 000 for component 3 , and 12 500 for component 4 . this is a known vibration of dmab,14 and appears to be strongly enhanced at that specific moment . the fourth component is hard to place in the context of either pntp or dmab , and could be a reaction intermediate . reaction intermediates will have a similar molecular structure as pntp and dmab , but with a different functional group . the characteristic vibrations at for example , 1335 and 1440 cm would not be expected at exactly that location in an intermediate spectrum . also , reorientation of pntp or dmab molecules should not result in new vibrations with respect to the normal raman spectrum . though beyond the scope of this work , it is anticipated that future density functional theory ( dft ) calculations should be able to shed more light on the chemical information in these spectral components . though most deviating spectra will probably be explained via orientation effects , there is a small chance that intermediates will also be present at the intense enhancement field in a sers hotspot , if the measurement runs long enough . normally shortlived spectra are disregarded in the analysis of a 15 000 sers spectra dataset , but now these spectra can be identified for the study of the surface reaction dynamics . we have presented a new approach to the chemometrics of sers spectra , which can find widespread use in this important field of research . it was found that with a onestep time filter with a pca approach , the photocatalytic reduction of pntp monitored by sers could be analyzed in great detail . more specifically , with this pca analysis , the photocatalytic reduction process ( > 200 s ) was separated from strong spectral deviations occurring over shorter time intervals ( < 200 s ) . both sets of data were then separately analyzed . the subsequent mcr analysis on the reaction data yields less noise , compared to simple peak area analysis and to regular mcr without pca filter . additionally , the blinking sers spectra separated from the bulk reaction process can now be analyzed separately . here where the bulk reaction data cleans up nicely with the use of this filter , the real advantage is in the blinking spectra . here , we have shown that the developed approach is able to isolate these events . if sers is indeed going to reveal reaction intermediates , it will be most probably during sparse events . it is important to note that sers has this inherent capability due to the inhomogeneous enhancement effect over the sersactive substrate . ag nanoparticles were synthesized according to lee and meisel16 in short , 0.04908 g silver nitrate ( sigma aldrich , acs reagent , 99.0 % ) was dissolved in 10 ml mq water . 3.667 ml of this stock solution was added to 100 ml mw water in a roundbottomed flask equipped with a teflon stirrer bar and reflux column . after heating the solution to reflux temperature , 2 ml of 0.09 m sodium citrate tribasic dihydrate ( sigma , reagent grade , 99 % ) was added . after 5 min at reflux temperature , this sol was left to cool and stored in a refrigerator until required for experiments . a flat au substrate of 100 nm au over a 0.51.0 cm si wafer ( philips innovation services ) was cleaned with uv / ozone and anhydrous ethanol . subsequently , the substrate was immersed into a 10 mm ethanolic solution of pntp ( fluka , technical grade ) for 24 h , followed by threefold rinsing in 10 ml ethanol . a tenfold dilution of ag nanoparticle sol was directly dropcasted onto the substrate and left to dry in ambient conditions . raman measurements were performed on a renishaw invia microscope , using 785 nm diode laser excitation , through a 50 longworkingdistance objective . the described experiment was done using 0.25 mw laser excitation power , for 15 000 consecutive spectra at a 1 s integration interval . chemometric analysis was performed using the pls toolbox 6.71 ( eigenvector co. ) in combination with matlab 2012a ( mathworks ) . unless mentioned otherwise , raw sers spectra were loaded directly into matlab and smoothed with a secondorder ( 9 pixel ) savitsky golay filter . a quick spike removal was done through a homewritten routine : a onecomponent pca ( mean centre preprocessing ) was analysed on the basis of the qresiduals in time . any spectrum with a qresidual value higher than the mean + 0.1 standard deviation of all qresiduals was removed if the datapoint was three times larger than its direct neighbours . a five times higher filter was used for the first 100 spectra , to cope with the start of the reaction . the result of this filtering is the separation of the total dataset in categories that relate to spectral variations on different time scales that van be linked to different physical and chemical changes on the surface .

globally an estimated 2.5 million children are living with hiv / aids , 10,000 becoming infected daily and 2,60,000 deaths of children under 15 occur due to aids - related illnesses . the government of india estimates that about 2.40 million indians are living with hiv ( 1.933.04 million ) with an adult prevalence of 0.31% ( 2009 ) . children ( < 15 years ) account for 3.5% of all infections whereas 83% are the in age group 1549 years . india 's highly heterogeneous epidemic is largely concentrated in only a few states in the industrialized south and west , and in the north - east . the four high prevalence states of south india ( andhra pradesh 500,000 , maharashtra 420,000 , karnataka 250,000 , tamil nadu 150,000 ) account for 55% of all hiv infections in the country . although efficacious , ( antiretroviral therapy [ art ] ) highly active antiretroviral treatment ( haart ) is not available to more than half with hiv in india and those who receive it develop neuropsychiatric side - effects . art , along with comorbid medical conditions and substance abuse are associated with new - onset psychosis whose prevalence among patients with hiv infection is reported to range from 0.23% to 15.2% . side - effects have been described in patients without psychiatric antecedents , but those with a previous psychiatric history are more vulnerable and should be monitored closely . these problems are very debilitating when they occur in children who acquire hiv , unfortunately , from infected mothers . although world health organization guidelines on art prophylaxis reduces hiv transmission from mother to fetus , the united nations guidelines on primary prevention in health care settings on universal precautions in needle use practices are equally important and together helps to reduce childhood hiv . safe needle practices only reduce the risk of mother - to - child hiv transmission to 2% . sadly , not surprisingly breaches in safe needle practices during childhood or adolescence we report a case where there was initially a breach of safe needle practice in a health care setting that had led a 16-year - old boy to accidently acquire hiv infection from his hiv - positive father . a 16-year - old boy was brought to our psychiatric outpatient setting by his parents with 5 days history of mutism , grimacing , excitement , echopraxia and withdrawal symptoms in addition to poor sleep . he was admitted into the child and adolescent inpatient psychiatric unit of our hospital for further management with the consent of the parents . detailed history and mental state examination provided a definitive diagnosis of catatonia with bush francis rating scale score of 12 . there was no past psychiatric history . both his parents were illiterates , and there was no family history of any neurodegenerative disorders , mental illness , and epilepsy except for the father who had a history of alcohol abuse . antenatal history was uneventful but perinatal period was complicated with birth asphyxia and forceps delivery in a hospital with birth weight of 2.7 kg and he was breastfed and weaned normally . although clinically relevant differential diagnosis such as wilson 's disease , thyroid disorders , mental retardation were considered , only iq assessment could be done which was in the average range . hiv serology was positive and negative for hepatitis b and syphilis . a plain computed tomography scan of the brain revealed gliotic changes in the left temporal lobe , which the radiologist reported as a possible consequence of birth asphyxia . past medical history showed that hiv seropositivity was diagnosed in 2011 when the boy was admitted for pulmonary tuberculosis ( ptb ) at government general hospital , vizhupuram and had received intensive 6 months treatment for ptb as per revised national ptb programme ( rntcp ) . both his parents also tested positive for hiv but the other two children showed hiv negative results . subsequently , the boy and his parents have been taking art which consisted of tablet zidovudine ( zdv ) 250 mg 2 times a day , tablet abacavir ( abc ) 300 mg one in the morning and two at night with tablet efavirenz ( efv ) 300 mg at night from the integrated counseling and treatment centre ( ictc ) , vizhupuram . further , it was evident from the old hospital records that the daily dosage of zdv , abc and efv were increased 2 months before the onset of catatonic symptoms . in consultation with the ictc , all the art medications were stopped , tablet risperidone 1 mg twice daily was started , and the boy was monitored for resolution of symptoms . within 5 days , all the catatonic symptoms resolved completely , and he was communicating relevantly and taking his feeds and sleeping well . the patient returned for review after 3 weeks and there was no evidence of any psychiatric symptomatology . he was still asymptomatic after 6 weeks follow - up on art . during the inpatient admission , repeat serology of other family members showed that our patient 's elder sister and younger brother tested negative for hiv as before while both his parents tested positive . this gap in seropositivity among the children was explored further , and it was found that the father had acquired hiv through unsafe extramarital sexual contact , but after having three children with his wife and remained ignorant of it . further exploration revealed a history that our patient had acquired hiv infection in 2009 , when he and his father had attended a rural health care centre , for treatment of loose stools and that is when the same needle was allegedly used on both the father and the son . firstly the mandatory need for safe needle use practices always which could have prevented hiv infection in our patient and secondly the neurotoxic side - effects of art in children . world health organization 's new guidelines ( 2010 ) , reports that early art for a larger group of hiv - positive pregnant women , longer provision of art prophylaxis for hiv - positive pregnant women with relatively strong immune systems to benefit both the health of the mother and provision of arv prophylaxis to the mother or child can prevent hiv transmission to her child during pregnancy and breastfeeding . illiteracy , low socioeconomic status , rural population , unsafe sex practices and nonavailability of art still remain risk factors for hiv transmission in children in developing countries like india and sub - saharan africa . inadequate precautions in rural health care settings in developing countries sometimes become a matter of medical negligence and unacceptable reason for hiv transmission in children . safer use of the same needle in a health care setting is definitely an easily preventable primary prevention method of hiv across all ages . although effective against central nervous system ( cns ) infection , antiretrovirals are themselves increasingly recognized as a source of neuropsychiatric disorders and the combination of at least three antiretroviral drugs also known as haart has created a large potential for drug interactions and subsequent toxicity . several factors interact , including the direct effect of the virus , immune mediators and medications . among the art 's , abc does not cause prominent psychiatric side - effects , although when used in combination , it may induce psychosis and catatonia . efv notably has been reported to induce a variety of cns side - effects in up to 50% of patients that may be severe and sudden in onset . symptoms include twilight states , personality changes , with increased hostility , and cognitive disturbances . severe cases may require switching or discontinuing the haart regimen , which could be reinstated ( often with different medication ) once the patient improves . our patient was receiving a combination of abc , didanosine and efv at therapeutic dosages , and the catatonic symptoms noted possibly due to a recent dose increase and the neurological effects of hiv infection . this case stresses the need for imparting better knowledge to the vulnerable population about unsafe sex practices , stricter adherence to universal precautions of safe needle usage and to all medical professionals about neurotoxic side - effects of art .

pull - down experiments using a tagged version of slra established that it is able to interact with both sinr and slrr . these 2 protein interaction activities would appear to be in conflict because slra would both promote and repress biofilm formation simultaneously by interacting with and inhibiting the functions of sinr and slrr , respectively . using a combination of surface plasmon resonance and isothermal titration calorimetry , a high affinity interaction between slra and sinr but not between slra and slrr was found , necessitating a reassessment of the role of slra as a dedicated antagonist of sinr . while this reassessment would simplify the role of slra in the circuitry of the switch , the questions would arise as to why 2 dedicated antagonists are required and how the activities or functions of these 2 antagonists differ . one possible answer to these questions may lie in the genetic contexts of the 2 proteins : slra is under the transcriptional control of the tetr family repressor ywcc , whereas sini is expressed from a locus immediately upstream of sinr , which has a complex transcriptional profile that includes a significant amount of bicistronic sini - sinr mrna . this may exert its effects in 2 ways : first , by allowing further environmental signals to enter the system through the ywcc repressor ; second , because the expression of sini and sinr are temporally and spatially coupled , sini would be better positioned to interact with sinr prior to its engagement with dna , whereas slra may be in the better position to compete in the cytoplasmic pool with slrr for sinr binding ( fig . 1 ) . support for this hypothesis comes from the fact slra mutants are only mildly repressed at the biofilm loci in comparison to sini mutants , despite the similar overall affinities of the slra - sinr and sini - sinr complexes ( 10 nm and 2 nm , respectively ) . furthermore , it has been noted by 2 independent studies that it is not possible to displace directly the sinr tetramer from dna in vitro , indicating that temporal , non - equilibrium effects may play an important role in repression . schematic diagram illustrating the different roles of the antagonists sini and slra in modulating the activity of sinr . ( a ) sinr , when expressed in the absence of antagonists , must first form the sinr tetramer before binding and repressing the activity of the nearby tapa - sipw - tasa operon . ( b ) sini is located immediately adjacent to sinr in a bicistronic sini - sinr operon , and as such is well positioned to immediately form a 1:1 complex with sinr , thereby blocking tetramerization , and the ability to repress the matrix operons . ( c ) while slra is able to bind sinr with a similar affinity , its expression from a distant chromosomal location means that it would have to compete with other agents in the cytoplasmic pool for the binding of the slrr tetramer , and as such is less efficient at antagonising the dna binding activity of slrr . levels of expression of the genes in the schematic are indicated by the thickness of the arrows that represent each gene . it has been established that slrr is subject to degradation in vivo , appearing to degrade proteolytically with a half - life of approximately 100 min , leading to the suggestion that slrr undergoes autocleavage in a manner similar to the lexa family of repressors . while it is beyond doubt that slrr is subject to proteolytic degradation , it would seem highly unlikely that slrr contains autoendopeptidase activity . though a lexa - like consensus motif is present in the slrr sequence , a catalytic domain would also be required for this activity , which is certainly not present in slrr . moreover , the lexa - like motif is located within the first of 2 consecutive helical hook regions , the first of which appears to have diverged more significantly than the second , with the closest homologs being sini and slra at 33% and 50% identities , respectively . in lexa , the cleavage site is in a flexible and accessible loop , whereas the motif in slrr ( based on its likely homology to sinr ) will form an ordered -helical structure , a site that is unlikely to be a protease substrate . here central to this model is the fact that slrr uniquely contains 2 helical hooks , which are capable of participating in both heterodimeric protein protein interactions and oligomeric self - interactions . assuming that both helical hook domains are active and , in accordance with the situation for sinr , sini and slra , the hetero - oligomeric interactions are of higher affinity than the self - interactions , slrr possesses the potential to form both open and closed complexes ( fig . 2 ) . the open complexes would contain the potential to form aggregates in a concentration dependent manner ( fig . 2 ) , which would then be targeted for degradation by clp - type proteases that are specific to mis - folded or aggregated proteins . this model agrees with the concentration dependent aggregation behavior of the purified protein in vitro , and also the increased stability of slrr in a clpc - deficient background in vivo . furthermore , mutation of the putative vqag consensus motif to vqvv increases significantly the stability of slrr . it is most likely that this substitution , being within the helical hooks , affects the ability of the protein to form these oligomeric interactions . the experimental validation of these ideas as well as an investigation of the dna binding activities of the sinr - slrr complex will be the subject of further work . schematic diagram showing the basis of an alternative model to explain the instability of slrr , based on the assumption that the 2 interaction domains present within slrr are able to associate with each other . the result of this is the possibility to form both closed and open ( with unfulfilled interaction potential ) complexes , the latter containing the obvious capacity to form large aggregates in a concentration dependent manner .

stroke is ischemic or hemorrhagic brain damage that causes neurological deficits , such as a sensory , motor , cognitive , speech , and visual disabilities1 . more than 85% of stroke patients experience unilateral paralysis , and more than 69% of them suffer damage to upper extremity function2 . a range of interventions , such as constraint - induced movement therapy , functional electrical stimulation , biofeedback , and transcranial magnetic stimulation , have been used for stroke patients3,4,5,6 . many studies have reported that intensive training of the upper limb is needed to recover the nervous system and motor control . with the recent technological advances related to improvement of upper limb function for stroke patients , upper limb robot - assisted therapy has the features of automated repetition , high intensity and interactive feedback , and it was found to be more effective in improving the motor function of stroke patients7,8,9 . these robot therapies with objective and reliable means to monitor the condition of the patients reported that robot therapy for acute stoke patients was effective in improving upper limb function and decreasing the level of movement damage10 . a systematic review on the effectiveness of robot therapy reported a positive effect in improving the upper limbs8 , 11 . robot - assisted therapy offers motor practice to relearn motor skills without a therapist s assistance . most robot - assisted therapies for motor practice not only allow assistance in movement and guidance but also provide accurate feedback information . most robot therapy is based on the assumption that brain recovery involves activity - dependent plasticity12 . to provide a further understanding of neural plasticity , it is important to consider the options of new technologies regarding the motor function of stroke patients . most randomized controlled studies have shown the benefits of upper limb robot - assisted therapy for acute stroke patients . these studies have reported that stroke patients who received robot - assisted therapy showed significant gains in motor control and the activities of daily livings13 , 14 . the improvement in the movement skills of the upper limbs has an important effect on functional recovery and independent performance . therefore , this study examined the effects of upper limb robot - assisted therapy in the rehabilitation of stroke patients . fifteen stroke patients with no visual or cognitive problems ( mini - mental state examination score > 24 points ) were enrolled in this study . the subjects were required to demonstrate at least a trace muscle contraction in the wrist extensors and a score of > 10 in the fugl - meyer assessment ( fma ) . all subjects gave their written informed consent to participation in the experiments in accordance with the ethical standards of the declarations of helsinki . table 1table 1.general characteristics of the subjectscharacteristicsn%gendermale1386.67female213.33age4049853.335059320.0060 and above426.67causehemorrhage426.67infarction1173.33affected sideright853.33left746.67time since stroke ( months)061066.677 and above533.33 lists the general characteristics of the subjects . the patients were admitted to the same hospital and assigned to the same team of rehabilitation professionals for the intervention . the experimental group received conventional therapy and an additional half hour per weekday of robot therapy . the patients participated in a total of 20 sessions , each lasting 60 minutes ( conventional therapy 30 min , robot - assisted therapy 30 min ) , which were held 5 days a week for 4 weeks . the novel inmotion shoulder - elbow robot , mit - manus ( interactive motion technologies , inc . , cambridge , ma , usa ) , which was been designed for clinical neurological applications , was used . this robot that was capable of sensing and recording the position and velocity in the horizontal plane with a 2 of freedom ( dof ) robotic device for the upper limb that provides shoulder - elbow training in the horizontal plane with a supported forearm . smoothness is the patient s ability to quickly and accurately move the arm from a center position to targets in each of eight compass directions . the reach error is the difference between the patients actual reach endpoints and the endpoint targets . the hold deviation is the average deviation distance when attempting to maintain the arm against resistance across the eight directions . the displacement is the patients ability to move the arm against resistance in each of eight compass directions . the spss 18.0 statistical software ( spss , chicago , il , usa ) , was used for statistical analyses . table 2table 2.comparison of scores for upper limb motion from before and after the interventionbeforeafterpoint to pointsmoothness0.450.070.530.05***reach error0.030.040.010.00*circlecircle size0.020.000.020.00***independence0.750.120.810.09***playback statichold deviation0.060.020.040.02***round dynamicdisplacement0.130.010.120.02*p<0.05 ; * * * p<0.001ratio of mean velocity / max velocity ( optimal score 0.53 ) . accuracy of reaching the middle of the target ( perfect score=0 ) . measured area of circle ( the higher the score , the better ) . measured ratio between the x and y axes ( max score=1 ) . measured displacement from center point ( optimal score=0 ) . measured average distance to target with resistance ( max score=0.14 m ) lists the scores for upper limb motions before and after the intervention . the patients showed a significant difference in smoothness and reach error of the point to point test , circle size and independence of the circle in the circle test , and hold deviation of the playback static test between before and after the intervention ( p<0.05 , p<0.001 ) . on the other hand , no significant difference was observed in the displacement of the round dynamic test . the patients also showed significant improvement in the fma and modified barthel index ( mbi ) after the intervention ( p<0.001 ) ( table 3table 3.comparison of the changes in fma and mbi between before and after the interventionbeforeafterfugl - meyer assessment ( fma)39.3312.6844.1312.86***modified barthel index ( mbi)63.6718.0268.1318.11******p<0.001 ) . measured area of circle ( the higher the score , the better ) . measured ratio between the x and y axes ( max score=1 ) . measured displacement from center point ( optimal score=0 ) . the main aim of this study was to determine if robot - assisted therapy is an effective intervention for the recovery of upper limb function of stroke patients . the results showed that there are positive effects on upper limb function after robot therapy . the results support the primary hypothesis that robot - assisted therapy would improve the upper limb motor function of stroke patients10 , 14 . the smoothness value of the point to point test was significantly different from that before the intervention . this result is similar to that of a previous study , which reported improved smoothness of motor function after robot - assisted therapy15 . the inmotion robot in this study has a back - drivable impedance - controlled system that allows for almost frictionless smoothness . therefore , repetitive task training through robot - assisted therapy can help improve the smoothness of patients motor function . in particular , rohrer et al . reported that the improved smoothness of motor function resulted in more natural recovery rather than training of learning16 . on the other hand , the reach error of the point to point test showed a reduction after the intervention , but there was no significant difference from that before the intervention15 . the circle test is an indicator of the shoulder and elbow active range of motion . the circle size and independence value of the circle test were significantly different compared with those to that before the intervention . this result is similar to that of a previous study and indicates that a possible increase in the movement range and more accurate movement of the shoulder and elbow17 , 18 . these studies reported that the point to point test was related to the retention task and that the circle test was related to the transfer task ( generalization of skill)19 . these values of the kinematic evaluation will help clinicians understand the information pertaining to patients motor recovery . the effect of the measured value of the hold deviation and displacement on the upper limb function after robot therapy has not been reported19 . all the hold deviations and displacements showed a decrease after the intervention , but only the hold deviation showed a statistically significant difference between before and after the intervention . this means that the paretic hand improved or maintained its ability to hold its position against the provided robot power . on the other hand , these findings support the results of previous studies on the effects of robot - assisted therapy . on the other hand , there is no a consistent conclusion that robot - assisted therapy has an effect on the activities of daily living9 , 10 , 14 , 15 . in fact , it is believed that the activities of daily living are promoted by the improvement in the learning motor skills because the mbi measures a common disorder of daily living and not arm function . the limitations of this study were the small sample and the lack of a control group . on the other hand , this study is a worthy pilot study because movement analysis was performed through an evaluation of the affected factors . these kinematic factors can provide good information when analyzing the upper limb function of stroke patients in robot - assisted therapy .

rheumatic fever ( rf ) is a nonsuppurative late autoimmune inflammatory complication of streptococcus pyogenes infection in the upper airway . the clinical picture consists mainly of episodes of migratory polyarthritis , sydenham 's chorea , and rheumatic heart disease . antiphospholipid syndrome ( aps ) is an autoimmune disease characterized by thrombotic events and/or recurrent abortions in the presence of antiphospholipid antibodies ( apls ) . rf and aps share clinical and pathophysiological manifestations , among which we highlight the carditis , chorea , arthritis , and thromboembolic events ( mainly represented by stroke ) . furthermore , apls have been related to valve manifestations which are similar in both pathologies , as much from the standpoint echocardiographic as pathophysiological ( this paper showed a considerable overlap of humoral immunity in rf and aps , supporting the hypothesis of common pathogenic mechanisms between both diseases ) . previous studies also were able to demonstrate that apls are associated with the onset of chorea in aps [ 4 , 5 ] . on the other hand , when the scientific literature is analyzed , the relationship between aps and rf has been little studied , and there is no article which conducted a review about this association . therefore , the aim of this study was to evaluate the clinical associations between rheumatic fever and antiphospholipid syndrome and the impact of coexistence of these two diseases in an individual . this is a systematic review of scientific papers that deal with the relationship between rheumatic fever and antiphospholipid syndrome . a search was performed in electronic databases : pubmed , medline , lilacs , scielo , and cochrane database , regarding the period from 1983 ( when the antiphospholipid syndrome was described ) to october 2012 . a combination of the keywords rheumatic fever , antiphospholipid syndrome , antiphospholipid antibody syndrome , and their respective translations was used . the combination search of keywords was performed by two authors independently and showed 22 scientific articles . a full reading of each of these papers and their references reduced the universe of work for 11 articles that evaluated the relationship of antiphospholipid syndrome with rheumatic fever . of these , only 7 showed clinical cases of association between aps and rf , being composed of 2 cross - sectional studies and 5 case reports . however , we have used only one cross sectional study since they describe the same patients . therefore 11 cases described in the literature about the association of rheumatic fever and antiphospholipid syndrome were identified [ 611 ] . the demographic , clinical , and laboratorial data of patients are summarized in table 1 . the age of patients with aps and rf ranged from 22 to 45 years , with a predominance of females ( 10/11 patients ) and white ( 6/7 ) . the duration of illness ranged to aps from 5.6 to 24 years and for rheumatic fever from 1.5 to 35 years . clinical presentation of rheumatic fever was characterized by the predominance of carditis ( 11/11 ) and chorea ( 7/11 ) . arthritis was observed in 3 of 11 patients and only one individual presented subcutaneous nodule ( 9% ) . no patient had erythema marginatum . serum levels of aso ( antistreptolysin - o ) were elevated in most cases ( 9/11 ) , with values ranging from 451 to 883 ui / m . in patients in whom it was studied for antidnase , this antibody was also positive and with high titers . the prophylaxis for rheumatic fever was reported in all cases , being the benzathine penicillin used as the first choice ( 8/11 ) , followed by sulfadiazine ( 1/11 ) and oral penicillin ( 1/11 ) . regarding the manifestations of aps , stroke was observed in 7/11 ( 63.6% ) , being one of them from probable embolic origin . three of eleven ( 27.3% ) patients had venous event characterized by deep vein thrombosis ( dvt ) . thrombocytopenia was observed in 3 of 11 cases and no article described the presence of livedo reticularis . acute myocardial infarction ( ami ) was present in 2 of 10 patients , also being reported the occurrence of splenic infarction in one patient . the antibodies research showed that anticardiolipin ( acl ) igm was the most found antibody and was positive in 5/7 patients , followed by anti-2gpi in 4/6 and lupus anticoagulant ( lac ) in 5/8 . already the acl igg antibody was present in 3/8 . in only two cases manifestations of systemic lupus erythematosus ( sle ) were presented , characterized by pericarditis and positive antibodies ( ana and anti - dsdna ) . in two of the patients the presence of anti - dsdna ( antidouble stranded dna ) without other manifestations of sle and other presented positive ana ( antinuclear antibody ) was reported singly . there was no report of another autoimmune disease , besides rf , aps , or sle . common clinical manifestations of aps and rf were distributed as follows : carditis with valvular disease in 100% of cases , chorea in 63.6% , and arthritis in 18.2% . the present work performed the first systematic review of all clinical cases published in the literature which presented an association between aps and rf . the major advantage of the present study was carrying out an extensive and complete literature review , for the first time , on the main world databases ( pubmed , medline , lilacs , scielo , and cochrane ) regarding the period of 1983 to october 2012 , with the total of 11 cases of association between aps and rf . in the scientific literature , there is some evidence of the relationship between aps and rf . in fact , the clinical and experimental study published by blank et al . , which evaluated the serum of 90 patients with rf and of 42 patients with aps , showed that 24% of patients with rheumatic heart disease had positivity for anti-2gpi ( antibeta-2-glycoprotein i ) and that patients with aps had antistreptococcal activity , recognizing the m protein in 16.6% of cases . interestingly , this study concluded that there is some overlap in the activity of anti-2gpi and anti - m protein / n - acetylglucosamine , suggesting common mechanisms in development of heart valve and neurological manifestations in aps and rf . the same authors , in a previous study , had also shown a possible infectious origin of the saf , where several infectious agents , including streptococcus pyogenes , are able to induce the formation of apls , responsible for the clinical manifestations of this condition ( this work showed a possible infectious origin of the saf , where several infectious agents , including streptococcus pyogenes , are able to induce the formation of antiphospholipid antibodies ) . the study by figueroa et al . showed that during the active phase of fr , 80% of patients had positive anticardiolipin antibodies ( acl ) . on the other hand , during the inactive phase of the disease , only 40% of these patients had such antibodies ( this study showed that 80% of rf patients were positive for anticardiolipin antibodies during active phase of the disease ) . in the same line of study , carvalho and goldenstein - schainberg simultaneously evaluated the presence of three antiphospholipid antibodies routinely used ( acl , lupus anticoagulant , and anti-2gpi ) in a group of nine patients with long - term fr ( about eleven years ) . in contrast to other studies , no patient showed positivity to any of these antibodies . this systematic review has highlighted that all patients with aps and rf had carditis , constituting the most frequent manifestation . in fact , the literature indicates that about 3045% of patients with rf develop carditis , its most serious complication , responsible for permanent valvular lesions ( this paper highlights a possible link between the mechanisms of heart afflictions in rf and aps ) . in the places of cardiac injury , a predominance of cd4 + t cells and macrophages exists , besides deposition of complement and expression of vcam-1 ( vascular cell adhesion protein 1 ) in the endothelium valve . cardiac involvement can also be explained by cross - reactivity of anti - m protein with myocardial proteins such as myosin , tropomyosin , and protein valvular endocarditis triggering feature of fr . the valve involvement in aps affects about 3238% of patients and is associated with high levels of antiphospholipids antibodies . the cardiovascular disorder in this syndrome occurs due to deposition of immune complexes , which may lead to formation of vegetations and valvular dysfunction , characterized by thickening of the valve leaflets and getting the name of aseptic endocarditis libman - sacks . the mitral valve is the most affected , followed by the aortic and tricuspid valves . therefore , although known cross - reactivity between molecules of 2gpi and m - protein that can contribute to heart damage , aps and fr have different pathogenic mechanisms with regard to the valve involvement . regarding chorea , whose prevalence in the aps is around 1.3% , this study found that 63.6% of our patients had aps associated chorea . this neurological manifestation has been evaluated in a few studies in the literature , although , in the same series , patients with sle or other autoimmune diseases were included . in order to evaluate chorea in a group of patients with primary aps , appenzeller et al . studied 88 patients with aps and identified 4 patients with chorea . interestingly , the presence of chorea in patients with aps was associated with rf and thrombocytopenia ( this study suggests a link between aps patients presented with chorea and rheumatic fever ) , suggesting the need for echocardiography ( echo ) in all patients with aps and chorea . the presence of chorea may be explained from the same mechanisms proposed for rheumatic carditis , when occurs cross reactivity against self - antigen ( basal ganglia cells ) and thus generating the characteristic movement disorder . moreover , antiphospholipid antibodies have also been associated with chorea , both in aps and in rf and sle . one feature that caught the attention in aps manifestations of this study was the prevalence of stroke , present in 63.6% of cases . this finding is confirmed in the literature by the work of camargo et al . , where they compared 5 patients with aps associated with rf with 68 patients presented with primary aps . remarkably , the presence of stroke was significantly higher in the first group ( 80% versus 25% ) ; however , the echocardiogram in these patients showed no intracardiac thrombi . these results suggest a possible formation of thrombus in situ ( this paper suggests that aps patients associated with rf have higher stroke risk when compared with primary aps patients ) . the achievement of this paper has made the design of the profile of a patient with aps and rf possible . this individual is typically female , white , and aged between 22 and 45 years . the duration of disease in this patient is around 5 years for aps and can vary from 1.5 to 35 for rf , and usually the fr appears as the first manifestation . in relation to clinical manifestations of rf , these individuals will follow the pattern of involvement of this condition ; in other words , they will have more carditis and chorea with high aso and will receive antibiotic prophylaxis . however , for the manifestations of aps , interestingly , arterial thrombosis was more common than vein thrombosis , as discussed above . dvt , as main venous event of this syndrome , which afflicts about 55% of patients , occurred in only 27.3% of the subjects in our study . no description of the presence of livedo reticularis was reported , although it is the most common cutaneous manifestation of aps . this study provides information that the association between saf and fr is quite rare , however , is of great clinical importance because the isolated treatment of one of these diseases , such as lack of anticoagulation in an individual with aps hidden and lack of prophylaxis of streptococcal infection in an individual with rf undiagnosed , can lead to serious consequences . this study also warns the doctors who deal with the rf , condition still very common in our environment , such as rheumatologists , pediatricians , cardiologists , cardiac surgeons , and general practitioner , to include in their differential diagnosis the aps , especially in the presence of stroke in patients with rf and whose echocardiogram did not show intracavitary thrombi . the relationship between aps and rf has been little studied.11 cases described in the literature about the association of rheumatic fever and antiphospholipid syndrome were identified.common clinical manifestations of aps and rf were distributed as follows : carditis with valvular disease in 100% of cases , chorea in 63.6% , and arthritis in 18.2%.previous study showed that 24% of patients with rheumatic heart disease had positivity for anti-2gp and that patients with aps had antistreptococcal activity , recognizing the m protein in 16.6% of cases.this systematic review has highlighted that all patients with aps and rf had carditis , constituting the most frequent manifestation.regarding chorea , whose prevalence in the aps is around 1.3% , this study found that 63.6% of our patients had aps associated chorea.about aps manifestations , interestingly , arterial thrombosis was more common than vein thrombosis.this study provides information that the association between saf and fr is quite rare , however , is of great clinical importance.this study warns clinicians to include in their differential diagnosis the aps , especially in the presence of stroke in patients with rf and whose echocardiogram did not show intracavitary thrombi . 11 cases described in the literature about the association of rheumatic fever and antiphospholipid syndrome were identified . common clinical manifestations of aps and rf were distributed as follows : carditis with valvular disease in 100% of cases , chorea in 63.6% , and arthritis in 18.2% . previous study showed that 24% of patients with rheumatic heart disease had positivity for anti-2gp and that patients with aps had antistreptococcal activity , recognizing the m protein in 16.6% of cases . this systematic review has highlighted that all patients with aps and rf had carditis , constituting the most frequent manifestation . regarding chorea , whose prevalence in the aps is around 1.3% , this study found that 63.6% of our patients had aps associated chorea . about aps manifestations , interestingly , this study provides information that the association between saf and fr is quite rare , however , is of great clinical importance . this study warns clinicians to include in their differential diagnosis the aps , especially in the presence of stroke in patients with rf and whose echocardiogram did not show intracavitary thrombi .

primitive basilar carotid anastomoses form early during fetal embryogenesis . in the 4-mm human embryo , the internal carotid arteries extend from the paired dorsal aortic arches and anastomose at 3 major sites with the paired longitudinal neural arteries that constitute the primitive vertebro - basilar system in the hindbrain.6 ) from cephalic to caudal , these anastomoses affect the trigeminal , otic , and hypoglossal arteries , named according to adjacent structures.10 ) ordinarily , these arteries regress and disappear by the fourth week of embryogenesis , beginning with the otic artery , followed by the hypoglossal , and finally , trigeminal arteries . failure of these vessels to regress during embryonic development results in various persistent carotid - basilar anastomoses . persistent primitive trigeminal artery ( ppta ) is the most common carotid - basilar anastomosis , with an incidence between 0.1% and 0.6%.14 ) the least common anastomotic connection is persistent primitive otic artery ( ppoa ) , which occurs in roughly 0.001% of the population.10)16 ) we reported a case of ppta that appeared similar to ppoa on cerebral angiography . emergent cerebral angiography was performed , which led to a diagnosis of a ruptured aneurysm in the right middle cerebral artery bifurcation and 2 other unruptured aneurysms in the left middle cerebral artery bifurcation and left distal anterior cerebral artery bifurcation ( a2 - 3 ) . later , surgical clipping and coil embolization were performed for the unruptured cerebral aneurysms . on cerebral angiography , we found a carotid basilar anastomosis that seemed to arise from the petrous segment and join with the middle basilar trunk ( fig . this vessel supplied blood to the regions of the distal basilar artery , contralateral posterior cerebral artery , and both superior cerebellar arteries . the ipsilateral posterior cerebral artery was supplied by a fetal type of posterior communicating artery . we first diagnosed this condition as persistent primitive otic artery because of its appearance on cerebral angiography . however , on brain computed tomography angiography ( cta ) performed to identify the origin and course of the abnormal artery ( fig . 2a , b ) , we identified the origin of the abnormal artery as the petrocavernous junction . on cta , this vessel was seen to originate from the lateral wall of the carotid artery , course posterolaterally with the trigeminal nerve through meckel 's cave , and insert into the basilar artery in parallel with the internal acoustic meatus . based on the brain cta images , we made a diagnosis of lower - lying ppta . ppta was initially described by quain.13 ) the trigeminal artery is most commonly associated with carotid basilar anastomoses . their incidence is between 0.1% and 0.6%.14 ) the most common site of origin of ppta is the posterior bend or lateral wall of the intracaverous carotid artery . a ppta runs along the trigeminal nerve , enters the posterior fossa through meckel 's cave or the isolated dural foramen , and directly supplies blood to the cerebellum . the proximal basilar artery ( ba ) often shows some degree of hypoplasia with or without hypoplasia of the vertebral artery.2 ) proximal hypoplastic ba below the point of abnormal communication and enlargement above is caused by hemodynamic changes in the posterior circulation distal to the connection point between the ppta and the ba . ppta may be associated with intracranial aneurysms in 14 - 32% of cases.1)5 ) the reason for the frequent coexistence of intracranial aneurysms with pta has been explained by the presence of structural defects in the walls of the cerebral arteries . also , other associated vascular anomalies , such as vb insufficiency , arteriovenous anomalies , arteriovenous fistulas , and moyamoya disease have been reported.3)7)8)11 ) our case of sah due to a ruptured aneurysm associated with a lower - lying ppta and concomitant multiple aneurysms is rare . the existence of ppoa is still controversial , because ppoa has never been identified in lower organisms.9 ) nevertheless , there have been a few reports of ppoa . ppoa , if it exists , is thought to arise from the petrous internal carotid artery within the carotid canal , course laterally through the internal auditory canal , and anastomose with the proximal basilar artery.10 ) it is unclear whether previously reported cases of ppoa are true ppoa or , instead , lower - lying ppta or stapedial artery remnants because the poor quality of images and the presence of a single angiographic picture in the published reports makes it difficult to be sure of the origin , course , and junction site of the vessels . croft suggested that a true otic artery has not yet been reported.4 ) in our case , a carotid basilar anastomosis with coexisting multiple aneurysms was identified on cerebral angiography . at first , this vessel appeared to arise from the lateral petrous internal carotid artery and course parallel to the internal auditory canal . however , using brain cta , we concluded that this vessel originated from the internal carotid artery at the outlet of the carotid canal . thus , brain cta is useful in evaluating the relationship between the vascular anatomy and surrounding bony structures . finally , we were able to diagnose the abnormal artery as a lower - lying ppta and not ppoa . several previous case reports on ppoa did not clearly demonstrate the origin , course and termination of anastomoses.4)12)15 ) therefore , based on our experience , it seems that brain cta is needed to definitively diagnose the type of carotid - basilar anastomoses .

triple - negative breast cancers ( tnbcs ) lack expression of the steroid receptors estrogen ( er ) and progesterone ( pr ) and the tyrosine kinase human epidermal growth factor receptor 2 ( her-2 ) . therefore , tnbcs are a diagnosis of exclusion , typically characterized by upregulation of cytokeratins 5 , 14 , and 17 and elevation of the epidermal growth factor receptor ( egfr ) [ 13 ] . studies estimate that approximately 1520% of breast cancers meet these criteria [ 46 ] . compared to other breast cancer subtypes , tnbcs are typically aggressive , invasive ( ductal , medullary , or metaplastic ) , grade iii tumors with high rates of mitotic division , of which approximately half contain a high rate of p53 mutations . for these reasons , they account for a disproportionately high percentage of metastases , distant recurrence , and death among patients with breast cancer . metastases in tnbcs are most common to visceral organs including liver , lungs , and central nervous system . as a diagnosis of exclusion , tnbc overlaps considerably with basal - like breast cancer ( blbc ) although differences between the two subtypes exist , especially at a genetic level . other molecular subtypes defined by gene expression patterns include luminal a , luminal b , her-2-enriched group , and claudin - low , all of which may include tnbcs to some extent [ 8 , 9 ] . tncbs are most common among premenopausal women , especially those of african american descent [ 46 , 10 ] . in addition , tnbcs are common among patients with brca1 mutations [ 11 , 12 ] . since the first molecular characterization of tnbcs in the literature in 2005 , the topic has quickly emerged as an active area of research . while initial studies focused on molecular and clinical characterizations of patients with the diagnosis , more recent studies have identified subgroups of patients with tnbc , proposed molecular mechanisms that may contribute to tumorigenesis , and explored potential therapeutic interventions for patients . in this paper , we examine the connection between tnbc and the metabolic syndrome , which consists of central obesity , insulin resistance , impaired glucose tolerance , dyslipidemia , and hypertension . our analysis of the literature will encompass in vitro and in vivo studies in cell lines and mouse models of tnbc , respectively , as well as clinical studies examining epidemiology and treatment of tnbc . obesity , which is associated with insulin resistance and type 2 diabetes mellitus ( dm ) , is an established risk factor for cancer incidence . in a meta - analysis of 141 articles , body mass index ( bmi ) was positively associated with an increased incidence of postmenopausal breast cancer , along with colon , endometrial , esophageal , gallbladder , pancreas , renal , thyroid cancers , leukemia , multiple myeloma , and non - hodgkin 's lymphoma in women . the results were less clear , however , for premenopausal breast cancer as a positive association between obesity and premenopausal breast cancer was found in asia - pacific women ( risk ratio ( rr ) = 1.16 ; 95% ci , 1.011.32 ) , while inverse relations were reported in north american women ( rr = 0.91 ; 95% ci , 0.850.98 ) and european and australian women ( rr = 0.89 ; 95% ci , 0.840.94 ) these findings suggest that different subpopulations of women possess different risk factors for breast cancer . it may also suggest that bmi is not an ideal measure of adiposity . instead , other measures such as waist - to - hip ratio ( whr ) or waist circumference , which are specific measures of central or abdominal adiposity , may be preferential to assess cancer risk . two meta - analyses that examined a correlation between elevated whr and risk of breast cancer in premenopausal women reported positive associations [ 15 , 16 ] . the study by connolly et al . reported that elevated whr was associated with a 79% ( summary risk ( sr ) = 1.79 ; 95% ci , 1.222.62 ) increased risk of breast cancer for premenopausal women and a 50% ( sr = 1.50 ; 95% ci , 1.102.04 ) increased risk for postmenopausal women . similarly , the study by harvie et al . reported that small whr was associated with a 37% decreased risk ( rr = 0.63 , 95% ci , 0.450.88 ) in premenopausal women only after adjusting for bmi . the authors hypothesized that general obesity may not modulate risk , but central obesity increases risk in premenopausal women . in contrast , the authors reported that general obesity and not central obesity increased cancer risk in postmenopausal women . this interesting result led the authors to hypothesize that insulin resistance and insulin - like growth factors , which are associated with central obesity , may play a larger role in modifying breast cancer risk for premenopausal women , while estrogen may play a greater role in postmenopausal breast cancer . while the link connecting obesity and incidence of all types of breast cancers is well established , the data examining obesity and tnbc are much less prevalent . in the carolina breast cancer study , whr was compared between the highest ( 0.84 ) and lowest ( < 0.77 ) groups in relation to blbc . across all women , there was an increased risk ( odds ratio ( or ) = 2.3 ; 95% ci , 1.43.6 ) for developing blbc with higher whr . premenopausal women ( or = 1.8 ; 95% ci , 1.03.4 ) and postmenopausal women ( or = 2.7 ; 95% ci , 1.35.4 ) with high whr both had elevated risk of developing breast cancer compared to the lowest whr group . weight gain in women as reported since fifth grade was highest in african american women in this sample . in contrast , no significant trend was reported for bmi and risk of breast cancer . a 2008 study examining 620 predominantly white women in rural appalachia , 117 of whom had tnbc , reported a significant association between obesity and incidence of tnbc . in this sample , approximately 50% of patients with tnbcs were obese as compared to 36% of non - tnbcs . the preponderance of evidence suggests an association between tnbc and obesity when obesity is defined as an elevated whr , but more contradictory evidence exists when using bmi as a measure of obesity . clearly , the conflicting results warrant additional research . future epidemiological studies would benefit from measurement of all three receptor markers and studies that concurrently examine multiple definitions of obesity . a common corollary of metabolic syndrome a 2007 meta - analysis of twenty studies estimated a 20% increased risk of breast cancer for women with type 2 dm ( rr = 1.20 ; 95% ci , 1.121.28 ) . for tnbc , one study reported a significant relation with 58% of patients with tnbc possessing a comorbid diagnosis of metabolic syndrome compared to 37% of patients without tnbc in a sample of 176 individuals using criteria of the national cholesterol education program and 52% compared to 34% using criteria of the american association of clinical endocrinologists . in addition , a 2011 study reported a 75% increase in the risk of postmenopausal breast cancer ( rr = 1.75 ; 95% ci , 1.372.22 ) for women who were found to have at least three of the four components of metabolic syndrome . however , the carolina breast cancer study reported no elevated prevalence of type 2 dm in tnbc compared to other breast cancer subtypes . recently , epidemiological studies have associated dyslipidemia and hypertension with breast cancer risk . in a prospective study examining all - cancer incidence of 1,189,719 korean men and women , kitahara et al . reported a positive association between total cholesterol and breast cancer risk in women ( hazard ratio ( hr ) = 1.17 ; 95% ci , 1.031.33 ) . the researchers compared individuals with total cholesterol 240 mg / dl to individuals with cholesterol < 160 mg / dl and adjusted for cigarette smoking , alcohol consumption , bmi , fasting serum glucose , hypertension , and physical activity . in addition , hypertension was independently predictive of breast cancer risk in a sample of 3,869 postmenopausal women with breast cancer as compared to 4,082 controls ( or = 1.19 ; 95% ci , 1.071.33 ) . another study reported a 23% increased risk of breast cancer for hypertensive women . however , after adjustment of confounders including bmi , the elevated risk was no longer significant ( hr = 1.14 ; 95% ci , 0.931.40 ) . epidemiological studies suggest a positive association between the metabolic syndrome as a whole , along with many of its individual components , and breast cancer risk . the many confounding variables that may mediate this effect need to be considered in order to determine whether this is a causative effect . further studies should also address how changes in components of metabolic syndrome , such as weight , affect incidence of disease , and treatment outcomes after initial diagnosis of tnbc . larger sample sizes will determine whether subpopulations of patients with tnbc ( e.g. , pre- versus postmenopausal women ) possess unique clinical and molecular characteristics . in addition to exploring risk factors that influence incidence of tnbc ( primary prevention ) , it is also essential to understand factors that influence recurrence of tnbc ( secondary prevention ) . compared to other subtypes of breast cancer , tnbcs are more often diagnosed as aggressive , invasive , grade iii , and lymph - node positive tumors . these outcomes are predictive of increased morbidity and mortality . in addition , tnbcs have a high rate of recurrence with visceral metastases compared to other subtypes of breast cancer , especially within the first five years after diagnosis . after five years , the risk of recurrence drops dramatically . obese patients with breast cancer have more frequent recurrence and worse prognosis as compared to lean patients . in a sample of 495,477 u.s . women , increasing bmi was significantly associated with increased death rates for breast cancer . as compared to the lowest bmi group ( 18.524.9 ) , there was an elevated risk of 34% for bmi of 25.029.9 ( rr = 1.34 ; 95% ci , 1.231.46 ) , 63% for bmi of 30.034.9 ( rr = 1.63 ; 95% ci , 1.441.85 ) , 70% for bmi of 35.039.9 ( rr = 1.70 ; 95% ci , 1.332.17 ) , and 112% for bmi 40.0 ( rr = 2.12 ; 95% ci , 1.413.19 ) of dying of breast cancer . furthermore , in a sample of 18,967 patients in denmark with early - stage breast cancer , bmi at diagnosis was correlated with disease prognosis . kg / m had a 46% higher risk of distant metastases ( hr = 1.46 ; 95% ci , 1.111.92 ) after 10 years and 38% increased risk of mortality from breast cancer ( hr = 1.38 ; 95% ci , 1.111.71 ) ) as compared to patients with bmi < 25 kg / m . the authors also suggested that adjuvant chemotherapy and endocrine therapy were less effective over time periods greater than 10 years for patients with bmi > 30 although it was unclear whether this effect was mediated by poor responsiveness to treatment or differences in biology . even though obese patients were more likely to present with advanced tumors in terms of size and spread to lymph nodes , obesity was still an independent predictor after controlling for these confounders . the study measured bmi after diagnosis of tnbc and then counted the number of recurrences and deaths . after controlling for clinically significant factors , no significant relation was found between bmi and overall survival ( hr = 0.94 ; 95% ci , 0.541.64 ) or recurrence - free survival ( hr = 0.81 ; 95% ci , 0.491.34 ) . in a sample of 1,169 patients diagnosed with invasive breast cancer , the relationship between general obesity and response to neoadjuvant chemotherapy was examined . when comparing overweight ( bmi 25 to < 30 kg / m ) and obese ( bmi 30 kg / m ) groups to the normal / underweight group ( bmi < 25 ) , a significant association was present for pathologic complete response to neoadjuvant chemotherapy ( or = 0.67 ; 95% ci , 0.450.99 ) in the normal / underweight group . while high bmi was associated with worse overall survival , no significant effects were seen for breast - cancer specific or progression - free survival . finally , although data linking risk of recurrence and mortality in patients with hypertension and tnbc are limited , a 2011 study retrospectively examined the use of beta blockers on prognosis for patients with breast cancer . after adjustment for a number of covariates , patients with tnbc who were taking beta blockers had significantly improved relapse - free survival ( hr = 0.30 ; 95% ci , 0.100.87 ) , and while overall survival was improved ( hr = 0.35 ; 95% ci , 0.121.00 ) , it only approached a significance level ( p = 0.05 ) . similar findings were also reported for non - tnbc subtypes . a number of epidemiological studies have suggested that physical activity and weight loss are inversely related breast cancer risk and recurrence . the women 's healthy eating and living ( whel ) study prospectively examined 1,490 women with breast cancer . the authors reported that performing exercise equivalent to walking 30 min , six days per week , and consuming 5 daily servings of fruits and vegetables decreased mortality by 46% ( hr = 0.56 ; 95% ci , 0.310.98 ) . while er+ tumors were associated with decreased mortality with these lifestyle interventions ( p < 0.05 ) , no significant effect was observed for er , pr tumors ( p = 0.40 ) . to the best of our knowledge , the largest study to date examining the link between physical activity and invasive breast cancer was a meta - analysis of 12,108 patients , which included six studies . while physical activity prior to diagnosis had no effect on breast cancer deaths across all patients , physical activity after diagnosis reduced breast cancer deaths by 34% ( hr = 0.66 , 95% ci , 0.570.77 ) and disease recurrence by 24% ( hr = 0.76 , 95% ci , 0.660.87 ) . postdiagnosis exercise only provided significant benefits for patients with bmi 25 kg / m . interestingly , physical activity after diagnosis reduced breast cancer deaths by 50% ( hr = 0.50 , 95% ci , 0.340.74 ) for er+ tumors with no significant effect for patients with er tumors . when looking at the individual studies that composed the meta - analysis , the studies that examined postdiagnosis physical activity were prospective , observational , and questionnaire - based studies , while those that examined prediagnosis physical activity had case - control designs [ 3237 ] . while the definition of physical activity varied somewhat from study to study , the studies generally defined physical activity as moderate recreational activity , and for the purpose of their analyses , the authors combined these forms of exercise into metabolic equivalent task ( met ) hours per week . examples of moderate physical activity included walking , jogging , running , biking , swimming , tennis , calisthenics / aerobics , and squash / racquetball . one study included in the meta - analysis specifically examined the relation between risk reduction of breast cancer and duration of exercise . in a sample of 2,987 women diagnosed with breast cancer compared to women who performed the equivalent of walking at an average pace less than 3 met - hours per week , there was a nonsignificant 20% risk reduction of death from breast cancer for 3 to 8.9 met - hours per week ( rr = 0.80 ; 95% ci , 0.601.06 ) , a significant 50% risk reduction for 9 to 14.9 met - hours per week ( rr = 0.50 ; 95% ci , 0.310.82 ) , 44% risk reduction for 15 to 23.9 met - hours per week ( rr = 0.56 ; 95% ci , 0.380.84 ) , and 40% risk reduction for 24 or more met - hours per week ( rr = 0.60 ; 95% ci , 0.400.89 ) . this study , however , did not find a significant effect for exercise , even for 9 or more met - hours per week , for er , pr tumors . these studies provide an insight on the role of physical activity as a potentially beneficial breast cancer treatment that may be used in conjunction with existing radiation and chemotherapy treatments [ 3237 ] . although studies explicitly targeting patients with tnbc have not been performed , a potential mechanism behind this link may be decreased concentrations of estrogen via reduction in body fat or decreased androgens via increase in globulins that bind testosterone . in addition to exercise , two large randomized studies have examined whether diet interventions are effective in reducing breast cancer recurrence and mortality [ 39 , 40 ] . the women 's intervention nutrition study ( wins ) examined 2,437 women with breast cancer . the randomized study involved a dietary intervention group with a goal of reducing calories from fat to 15% without compromising nutrition compared to control with median followup of 60 months . when comparing relapse events between the two groups , relapse was lower in the intervention group as compared to the control group ( hr = 0.76 ; 95% ci , 0.600.98 , p = 0.077 for stratified log rank and p = 0.034 for adjusted cox model analysis ) . the authors reported a trend for a stronger effect for dietary fat reduction for hormone receptor - negative cancers ( hr = 0.58 ; 95% ci , 0.370.91 ) compared to er+ tumors ( hr = 0.85 ; 95% ci , 0.631.14 ) , although no significant effect was found ( interaction test , p = 0.15 ) . one of the criticisms of the wins study was the fact that the intervention group lost about 6 pounds more than the control arm over the duration of the study ( p = 0.005 ) . as a result , it was unclear whether the outcomes were due to decreased weight or decreased fat intake . furthermore , the dietary intervention was relatively strict , making it hard to implement in everyday practice . in addition , the whel study evaluated the potential benefit of physical activity and a diet rich in vegetables and fruit in breast cancer survivors . the arm randomized to a diet rich in vegetables , fruit , and fiber , but low in fat did not have a significantly lower mortality ( hr = 0.91 ; 95% ci , 0.721.15 ) or a lower incidence of second invasive breast cancer ( hr = 0.96 ; 95% ci , 0.801.14 ) during a 7.3-year follow - up period . in this study , the intervention and comparison groups had an average weight difference of 1-kg or less based on measurements at baseline , 1 year , 2 or 3 years , 4 years , and 6 years . in an analysis of the comparison group only , consuming 5 daily servings of fruits and vegetables and performing exercise equivalent to walking 30 min , six days per week at baseline was associated with lower mortality . no effect , however , was reported in the randomized trial based on physical activity at baseline for additional breast cancer events or all - cause mortality . these conflicting findings warrant further research , especially to assess diet interventions for patients with tnbc . alcohol consumption also appears to moderate recurrence and mortality for breast cancer survivors . in a recent study of 1,897 individuals , consumption of three to four alcoholic drinks or more per week was associated with a 35% ( hr = 1.35 ; 95% ci , 1.001.83 ) increased risk of breast cancer recurrence and 51% ( hr = 1.51 ; 95% ci , 1.002.29 ) increased risk of death due to breast cancer . no difference was found between er+ versus er subgroups although the authors noted that this lack of effect may have been due to a small sample size of patients with er tumors . further studies will be important to assess whether different subtypes of breast cancer are affected differently by diet and alcohol in order to further probe the mechanism of these effects . in particular , upregulation of insulin has been hypothesized to directly increase proliferation of breast tissue and breast cancer cells . a 2009 study , which measured insulin at baseline and at 1 , 3 , and 6 years of followup , reported a hr of 2.22 ( 95% ci , 1.393.53 ) for incidence of breast cancer in postmenopausal women when comparing the highest baseline insulin concentration group to the lowest group . another study demonstrated that a high homeostatic model assessment score , which is associated with serum levels of insulin and glucose , was correlated with increased breast cancer mortality in a sample of 527 women . similarly , a 2011 study of 604 women in the health , eating , activity , and lifestyle ( heal ) study measured serum c - peptide , a marker of insulin secretion , three years after diagnosis . an increased c - peptide concentration of 1 ng / ml was associated with a 35% increased risk of death from breast cancer ( hr = 1.35 ; 95% ci , 1.021.87 ) . collectively , these data suggest that hyperglycemia and hyperinsulinemia are associated with poor prognosis for patients with breast cancer . in contrast , a 2007 case - control study examining blood samples in predominantly premenopausal women reported that increased levels of insulin and c - peptide were not risk factors for breast cancer . baseline hemoglobin a1c ( hba1c ) levels among 3,003 patients were examined for recurrence and all - cause mortality . the authors reported a significant increase in all - cause mortality after adjustment for confounders for women with hba1c 7.0% as compared to < 6.5% ( hr = 2.35 ; 95% ci , 1.563.54 ) . the actions of insulin may also occur indirectly via decreased availability of globulin and insulin - like growth factor- ( igf- ) binding proteins and increased blood concentration of testosterone , estrogens , or igfs . elevated concentrations of unbound estradiol and testosterone have been associated with increased breast cancer risk in pre- and postmenopausal women [ 4750 ] . insulin also inhibits sex hormone - binding globulin ( shbg ) production and increases the levels of igf - i in blood , which results in increased mitogenic activity . this link is consistent with approximately 50% of breast cancer tumors overexpressing igf - i receptor . a recent laboratory study found that seven cell lines that serve as models of tnbc expressed igf receptors . surprisingly , expression was at similar levels to er+ cell lines even though type i igf receptor levels are increased by estrogen in er+ cell lines . in all cases , although studies have reported a positive association between type 2 dm and breast cancer , a potential confounding variable in establishing this relation is treatment regimen . insulin has recently been implicated to have cancer promoting effects , while recent evidence suggests metformin to have cancer protecting effects in patients with type 2 dm . insulin glargine use , especially when prolonged , may increase the incidence of breast cancer . in one study , this effect was especially prominent for individuals who had received insulin for an average of 5.6 years before starting insulin glargine ( hr = 2.7 ; 95% ci , 1.16.5 ) . in contrast , metformin has been shown to inhibit proliferation and colony formation of tnbc cells in vitro . metformin resulted in decreased tumor growth if injected in tnbc tumor xenograft mice and decreased tumor incidence if added before injecting tnbc cells . while the molecular mechanism of how metformin reduces breast cancer incidence and survival is unclear , potential mechanisms include ( 1 ) acting as a general growth inhibitor , ( 2 ) reducing serum insulin levels , and ( 3 ) reducing body weight [ 54 , 57 ] . interestingly , the drug only exhibited an antiapoptotic effect in tnbc cell lines , an effect which was not present for luminal a , b , and her-2 subtypes . recently , observational studies were performed suggesting that metformin reduces the risk of breast cancer in humans . in one study , metformin use was associated with a 38% lower incidence of er+ , pr+ tumors in postmenopausal women with type 2 dm . no significant effect was demonstrated for er , pr tumors , however , although the sample size for tnbcs was limited . in addition , prospective studies are under way on the role of metformin in breast cancer recurrence . further studies are necessary to determine whether elevated levels of insulin and c - peptide are risk factors for women with tnbc , as well as to elucidate the mechanism behind this association . leptin is the product of the obesity ( ob ) gene and is primarily synthesized and secreted by adipose tissue , with increasing adiposity associated with higher circulating leptin levels . . leptin helps regulate food intake and metabolism via its actions on the arcuate nucleus of the hypothalamus . it is hypothesized that leptin resistance in obese individuals may be analogous to insulin resistance in diabetics . this resistance has been proposed to develop via impaired transport of leptin across the blood brain barrier and circumventricular organs and leptin receptor signal attenuation . clinical studies have reported a positive association between circulating blood leptin and breast cancer risk with particular elevation of mrna expression in adipocytes in close proximity to the tumor . on a molecular level , it has been hypothesized that elevated leptin expression in epithelial mammary cells may promote tumorigenesis via mechanisms including cell proliferation ( aromatase , mapk , stat3 , and cyclin d1 ) , angiogenesis ( vegf ) , apoptosis ( p53 and caspase 9 ) , cell - cycle regulation ( p21 ) , and cell survival ( akt ) in breast cancer cell lines . in tnbc cell lines , a study by saxena et al . reported that leptin directly increased activity of the igf - i receptor . in addition , bidirectional crosstalk between leptin and igf - i upregulated egfr promoting proliferation and migration of tnbc cells . the study further reported that using the egfr inhibitors , lapatinib and erlotinib , in an in vitro model system for metastasis after application of leptin and igf - i reduced invasion and migration of breast cancer cells . collectively , these data suggest a possible therapeutic route for treatment of tnbc with egfr inhibitors , because up to 70% of tnbcs overexpress egfr . in addition to leptin and igf - i , a 2011 study by burga et al . reported another potential mechanism for elevated levels of egfr protein . after rna knockdown of brca1 in mammary epithelial cells , egfr protein was upregulated due to transcriptional modification and posttranslational stabilization of egfr . this is important to our understanding of tnbcs , because brca1 mutations are highly correlated with tnbcs . interestingly , egfr inhibition with erlotinib in female brca1 knockout mice , in vivo , prevented or delayed development of er , but not er+ tumors . however , the treatment was not effective in shrinking the tumor after tumorigenesis . a causal link between leptin and breast cancer is supported by animal studies in which obese mice that overexpressed transforming growth factor - alpha ( tgf- ) , but were deficient in leptin , did not develop mammary tumors , while heterozygous and homozygous wild type leptin mice developed tumors in 50% and 67% of cases , respectively . however , these findings were difficult to interpret , because leptin deficient mice possessed limited mammary tissue . further studies in mouse models , in vivo , suggest a therapeutic potential for leptin receptor , antagonists . in a recent study of 69 tnbc tumors , 92% of breast tumors expressed leptin receptor and 86% expressed leptin . in this study , the peptide allo - aca , a leptin receptor antagonist , extended survival time by up to 80% in a tnbc mouse xenograft model , in vivo . clinical studies are needed to determine whether leptin antagonists may hold promise as a therapy in humans , especially in obese patients who overexpress leptin . clinical trials in humans are currently underway to test the efficacy of egfr inhibitors in tnbc . these studies have focused on using cetuximab , a humanized antiegfr igg1 antibody in conjunction with ixabepilone , cisplatin , carboplatin , or a taxane . 12 patients with metastatic tnbc were treated with either paclitaxel or docetaxel with cetuximab weekly . of the eleven patients assessable to followup , nine ( 82% ) exhibited decrease in size of metastasis , but three ( 27% ) developed brain metastasis during treatment ( 133 ) . other studies by carey et al . and o'shaughnessy et al . have reported therapeutic value of using egfr inhibitors in conjunction with other chemotherapy agents including ( 1 ) carboplatin plus cetuximab and ( 2 ) irinotecan and carboplatin , plus cetuximab [ 70 , 71 ] . the study by carey et al . compared cetuximab alone to carboplatin plus cetuximab in patients with tnbc metastases . of the 71 patients who received both drugs , 13 ( 18% ) responded to treatment as compared to only 2 of 31 ( 6% ) of patients who received cetuximab alone . in addition , the preliminary results of the randomized phase ii study of metastatic patients with tnbc by o'shaughnessy et al . reported no improvement in objective response rate ( orr ) , progression - free survival , and overall survival across all patients with metastatic disease when comparing cetuximab in conjunction with irinotecan and carboplatin as compared to irinotecan and carboplatin . however , subset analysis of revealed that orr was increased in metastatic patients with tnbc when using all three drugs ( 19 of 39 ; 49% ) as compared to only irinotecan and carboplatin ( 10 of 33 ; 30% ) . these findings may suggest a therapeutic benefit of using egfr inhibitors for a subset of patients with tnbc . larger experimental and control groups and increased number of follow - up years will benefit our understanding of the potential for these treatments . adiponectin , a protein secreted exclusively by adipose tissue , is an endogenous insulin sensitizer . after controlling for bmi , studies have reported that women with increased adiponectin concentrations possessed a 65% reduced risk for breast cancer [ 7274 ] . in another sample of 527 women diagnosed with stage i iiia breast cancer , adiponectin levels above 15.5 g / ml were associated with improved breast cancer survival ( hr = 0.39 ; 95% ci , 0.150.95 ) . the authors reported prognostic value of adipokines in er , pr tumors but not er+ , pr+ tumors ( p for trend = 0.027 ) . patients with low adiponectin levels as defined by the first quartile in the study had a significantly increased likelihood of cancer recurrence as compared to patients in the fourth quartile ( hr = 2.82 ; 95% ci , 1.037.68 ) . these results were significant even after adjustment for bmi and homeostasis model assessment scores for insulin resistance . we recently evaluated the role of adiponectin pathway single nucleotide polymorphisms ( snps ) in breast cancer risk . we performed a case - control study on 733 breast cancer cases and 839 controls and genotyped 10 haplotype - tagging snps of adiponectin ( adipoq ) and the type i adiponectin receptor ( adipor1 ) genes . we showed that two functional polymorphisms of adipoq , and one functional polymorphism which has been shown to alter mrna levels of adipor1 was significantly associated with risk of breast cancer . when categorized by signaling status , low adiponectin signalers had a 6.56-fold increase in breast cancer risk ( 95% ci , 0.7854.89 ) , and intermediate adiponectin signalers had a 4.16-fold increase in risk ( 95% ci , 0.4935.19 ) compared to high signalers ( p for trend = 0.001 ) . although these data are preliminary , they provide evidence for a significant role for adiponectin in predicting breast cancer risk . the mechanisms underlying the association between adiponectin and breast cancer risk have been studied by several investigators . components of the adiponectin signaling pathway have been implicated in breast tumorigenesis . more specifically , a number of compounds related to cell proliferation ( aromatase , mapk , and cyclin d1 ) , apoptosis ( bcl2 and caspase 8) , cell - cycle regulation ( ampk ) , and cell survival ( akt ) have been implicated to mediate tumorigenesis in breast cancer cell lines . while adiponectin has been shown to have an antiproliferative effect on cell growth in both er+ and er cell lines , the dominant mechanisms responsible for these effects in er+ and er cell lines are likely different . for example , in mcf-7 cells , 24 hour treatment with adiponectin resulted in an antiproliferative effect lasting up to 96 hours . whether adiponectin induces cell apoptosis is controversial and depends on the particular breast cancer cell line and the duration of the adiponectin incubation period . one study reported that increased cleavage of poly ( adp - ribose ) polymerase ( parp ) , which serves as an early apoptotic biomarker , was only detected in er+ cell lines . other studies have reported that adiponectin inhibits aromatase and estrogen receptor activity , mechanisms which would primarily act on er+ tumors . collectively , these data suggest that adiponectin acts via multiple signaling pathways with different mechanisms predominating in er+ and er cell lines . animal studies have demonstrated that overexpression of adiponectin , both locally and systemically , reduces mammary tumor size . the proposed mechanisms linking low adiponectin levels and breast carcinogenesis are ( 1 ) interaction with insulin [ 60 , 81 ] , ( 2 ) interaction with leptin , ( 3 ) inhibition of tnf- in macrophages , ( 4 ) binding of fibroblast growth factor and platelet - derived growth factor - beta polypeptide , ( 5 ) inhibition of nuclear factor b , and ( 6 ) promotion of angiogenesis . further research exploring the link between adiponectin levels over time and breast cancer risk is needed in order to elucidate dominant mechanisms in different breast cancer subtypes . furthermore , monitoring changes in adiponectin levels in conjunction with different pharmacological and/or behavioral modifications such as diet or exercise in human patients may contribute to a better understanding of its role in tnbc . finally , treatments aimed at increasing adiponectin levels should be explored for their potential therapeutic and preventive benefit in breast cancer . considerable evidence links the components of metabolic syndrome , including central obesity , insulin resistance , glucose intolerance , dyslipidemia , and hypertension , with the different breast cancer subtypes . although data on the connection between tnbc and the metabolic syndrome are limited , several studies have provided evidence for this association . studies have reported an association between elevated abdominal obesity , as defined by a high whr , and increased incidence of tnbc , but the evidence for bmi is more contradictory [ 17 , 18 ] . in addition , while type 2 dm and insulin resistance are associated with elevated breast cancer incidence , early evidence suggests that tnbcs do not have increased prevalence of type 2 dm compared to non - tnbcs . in terms of disease progression , obesity is associated with worse prognosis and increased recurrence across all breast cancer subtypes [ 25 , 26 , 28 ] . hyperglycemia and hyperinsulinemia have also recently been associated with increased incidence and poor prognosis [ 4244 ] . additionally , behavioral modifications including moderate physical activity , a diet rich in fruits , vegetables , and micronutrients , and reduced alcohol consumption show promise across all breast cancer subtypes [ 3237 , 39 , 41 ] . it remains to be seen whether these alternative therapies may prove useful in conjunction with chemotherapy for patients with tnbc . molecular mechanisms of how these components of metabolic syndrome may mediate tumorigenesis and disease progression have been proposed . insulin may mediate breast cancer risk via both direct and indirect effects , resulting in increased concentration of androgens and estrogens , along with increased concentration of igf - i [ 4753 ] . leptin and adiponectin , which are both secreted by adipose tissue and often by breast tumors , act via a number of downstream signaling pathways involved in cell proliferation , apoptosis , cell - cycle regulation , angiogenesis , and cell survival . it is likely that normal cells must maintain a fine balance between leptin and adiponectin in order to maintain proper cell and tissue homeostasis , and the components of metabolic syndrome appear to disrupt this balance by increasing leptin and decreasing adiponectin levels [ 61 , 62 , 64 ] . in addition , insulin , igf - i , and egfr may play a pivotal role in mediating the potential interactions between these two hormones [ 65 , 66 ] . we propose that components of the metabolic syndrome and the insulin - leptin - adiponectin axis play a pivotal role in the pathogenesis and progression of tnbc ( figure 1 ) . at present , treatments for tnbc are limited compared to other subtypes of breast cancer , because these tumors are resistant to hormone therapy and drugs that target the her-2 protein . clinical trials have shown efficacy of treatments such as chemotherapy , anti - egfr drugs , antiangiogenic drugs , and parp inhibitors in the treatment of tnbc . lifestyle factors including diet , reduced alcohol consumption , and physical activity , which may modulate components of the metabolic syndrome , may also play a pivotal role in decreasing incidence and risk of recurrence of tnbc . trials that incorporate agents such as metformin or leptin antagonists as well as other therapies that modify the insulin - leptin - adiponectin axis may prove very beneficial for prevention and treatment of tnbc .

patients with a history of sexually transmitted infections ( stis ) are at increased risk for the development of ectopic pregnancies because of postinflammatory fallopian tube scarring and deformity . unilateral twin ectopic pregnancy is a rare condition , first described in 1891 by de ott . live twin ectopic pregnancies are thought to occur at a frequency of 1/125 000 . unilateral twin ectopic gestation is thought to occur in approximately 1 in 200 ectopic gestations . one would therefore calculate a frequency of 1 in 20 000 pregnancies with a 1% ectopic rate in the united states . simultaneous bilateral tubal pregnancy is the rarest form of double - ovum twin pregnancy . a 24-year - old primigravida with an unknown last menstrual period presented to the emergency room with a 3-month history of lower abdominal and lower back pain that radiated into her right thigh . the pain occurred two to three times daily , lasting 5 minutes with each episode and worsening during the few days prior to presentation . during the three previous months the patient described irregular vaginal bleeding and watery vaginal discharge . she was evaluated two months previously at another hospital and was diagnosed as having a cyst . she was discharged with a narcotic prescription that she noted had improved her pain , but did not relieve it . coitarche began at 14 years of age and she noted a minimum of 15 previous sexual partners . hospital medical records listed positive toxicology screen results for illegal drugs on multiple emergency room visits . during the previous 6 years , culture and serology results documented four prior separate infections with chlamydia trachomatis , neisseria gonorrhoeae , herpes simplex virus 2 , and treponema pallidum , respectively . current testing for c trachomatis and n gonorrhoeae by dna probe was negative for both species . the patient did not demonstrate any clinical sequelae of herpes simplex virus and thus testing for this sti was not performed on the current admission . on physical exam , pelvic examination revealed blood in the vagina , a closed cervical os , uterine tenderness , and a palpable right adnexal mass . rectovaginal exam concurred with the pelvic exam and stool guaiac testing for occult blood was negative . diagnostic testing showed a quantitative serum -hcg titer of 263 miu / ml , a hematocrit of 38% , and a serum progesterone level of 2.2 ng / ml . additionally , ultrasonographic examination displayed a 10 10 8 cm right adnexal mass containing a nonviable twin pregnancy with each fetal crown - rump length corresponding to a gestational age of 9.3 weeks . a separating linear echo was seen in the mass between the two fetuses ( figure 1 ) . an exploratory laparotomy revealed a hemoperitoneum containing 300 ml and a 10 cm right - sided pelvic mass incorporating the right fallopian tube and ovary . small intestine and the appendix were adherent by fibrous adhesions to the mass . a right salpingoophorectomy , an appendectomy , and an extensive lysis of adhesions were performed . pathological evaluation of the surgical specimen showed a twin pregnancy within the fallopian tube ( figure 2 ) . during more detailed histopathologic examination , the finding of a dividing membrane established the pregnancy to be monochorionic diamnionic , and measurement of the twin fetuses estimated their gestational age at 7 weeks . pelvic inflammatory disease is associated with the greatest increase in risk , but other associated factors include operative trauma , congenital anomalies , tumors , adhesions , and advancing maternal age . arey in 1923 suggested that anything that interferes with the passage of the ovum through the tube increases the risk of an abnormal placental implantation site . additionally , studies suggest a delay in ovum transport and in implantation also increase the risk of monozygotic twinning . minor trauma to the blastocyst during assisted reproductive technology may also lead to an increased incidence of monozygotic twinning . correspondingly , the majority of twin ectopic pregnancies have been monozygotic . as of 1990 , 95% of twin ectopic pregnancies were reported as monozygotic . despite these potential sources for ectopic twin gestation , goker in 2001 reported only the one hundred and first case of unilateral twin ectopic pregnancy , and the sixth to be diagnosed preoperatively . the current case is the eighth in the literature to be diagnosed preoperatively . one reason for the low incidence may stem from the fact that fetal wastage in monozygotic twins is high and malformations are common . in the current case , the patient 's prior stis may have had a direct causal link to her ectopic twin gestation . current studies show that c trachomatis and n gonorrhoeae interfere with the function of the ciliated cells of the tubal mucosa , thereby slowing ovum transport and increasing the risk for tubal implantation . grard reported that 7 of 10 tubes with ectopic pregnancies were found to have c trachomatis . in addition to these findings , it has been demonstrated with laparoscopy that one episode of pid resulted in a 12.8% incidence of bilateral tubal occlusion , two episodes with a 35% incidence , and three episodes with a 75% incidence of bilateral tubal occlusion . this case illustrates one of the long - term consequences of stis with respect to reproductive function . patients with a history of multiple stis are known to be at risk for development of chronic pelvic infection and its associated fallopian tube scarring . the tubal anatomy distortion that ensues potentially increases the risk for a presentation of ectopic pregnancy that is considered uncommon .

the runt domain ( rd ) is the dna - binding domain of p53 type transcription factors known as core binding factor ( cbf ) subunits . the cbfs are expressed by the three related genes runx1 , runx2 and runx3 ( 1 ) . the three runx genes are important for developmental processes ; runx1 is important for hematopoiesis , runx2 is essential for skeletal development ( osteogenesis ) and runx3 is required for the development of gastric epithelium ( 24 ) . runx1 and runx2 also have been shown to be important for vascular development or angiogenesis and their activity is augmented by angiogenic growth factors ( 5,6 ) . mutation of the runx1 gene is associated with human leukemias , whereas mutation of the runx3 gene is involved with the development and progression of gastric cancers and testicular yolk sac tumor ( 1,7,8 ) . mutation of the runx2 gene is associated with the inherited human skeletal disorder known as cleidocranial dysplasia ( 9 ) . a related protein , known as cbf , binds to the rd of the cbfs and enhances the dna - binding affinity of the rd . the rd is the site of both dna binding and heterodimerization with cbf. because of the involvement of cbfs in many developmental processes and cancerous diseases , understanding rd dna and rd cbf interactions may shed light on the control of these processes . in addition , the involvement of runx genes with angiogenesis also makes the rd a potential target in the development and treatment of many types of cancers . the 3d structures of the rd in the free state , bound to dna , cbf and both dna and cbf have been determined using nuclear magnetic resonance ( nmr ) spectroscopy and/or x - ray crystallography ( 1014 ) . biochemical and biophysical studies have been performed characterizing the binding of rd to dna in the presence and absence of cbf ( 15,16 ) . in the presence of cbf , the dissociation constant ( kd ) for rd dna decreases 6- to 10-fold ( 16 ) . several studies have identified rd residues important for both dna and cbf binding via mutational analysis ( 1719 ) . the mutational analysis usually involved mutations to alanine and analysis of its effect on rd dna and rd cbf-binding activities , as determined using yeast hybrid assay analysis . in addition , a recent nmr - based study has identified a variety of residues involved in the cooperativity associated with cbf binding to rd ( 20 ) . while these important studies have given insights into the role of individual residues to rd dna and rd cbf interactions , the interpretation of the mutational analysis may be complicated by the influence of the mutation on protein stability , protein folding and other localized changes , especially at protein protein interfaces , while structural and dynamic information from nmr and x - ray studies can not yield energetic information at an atomic level of detail . computational approaches can yield important details about the interaction of rd with dna and cbf ( 21 ) . for example , the energetic contribution of individual residues to both dna and cbf binding can be determined . by doing such analysis in systems with all wild - type residues present , the risk of mutation induced conformational change , including changes in protein stability , is avoided . computational analysis can also account for the contribution of all atoms in a given residue , including the backbone atoms , whereas mutational analysis can only detect the importance of side chain atoms . thus , computational analysis can fill gaps in understanding the rd dna , rd cbf and cbfrd dna ternary complex interactions . in the present work , the rd was subjected to molecular dynamic ( md ) simulations in the free state and in several complexes . complexes include the binary rd dna and rd cbf complexes , and the cbfrd dna ternary complex . in addition , two dna simulations and a cbf monomer simulation were performed . from the md simulations , in combination with free energy component analysis ( 2224 ) based on the generalized - born model ( 25,26 ) , overall binding energies , desolvation effects and detailed interaction energies these results , along with previous structural analysis and observations , were used to interpret a variety of experimental data involving rd dna and rd cbf interactions ( 1619,27 ) . md simulations and all calculations were performed using the program charmm ( 28 ) . the all - hydrogen charmm22 protein ( 29 ) and charmm27 nucleic acid ( 30,31 ) , the tip3p water model ( 32 ) , along with published sodium and chloride parameters were used ( 33 ) . coordinates from the cbfrd dna ternary complex x - ray crystal structure ( pdb accession code 1h9d ) ( 13 ) were used as starting structures of the seven simulation systems ( supplementary table s1 ) . the only exception was the second dna simulation , which was initiated with the dna in the canonical b form ( 34 ) , as obtained from the program quanta ( accelrys inc . ) . in the crystal structure terminal residues 5053 and 179183 in rd are not observed experimentally as are residues 7179 of cbf. accordingly , in the simulations these residues were omitted . omission of residues 7179 of cbf , which are spatially removed from the rd in the experimental structure ( figure 1 ) , was performed by creating a peptide bond between residues 70 and 80 and relaxing the structure during the minimization and md simulation protocols described below . root - mean - square ( rms ) difference analysis of the residues adjacent to the deleted residues in cbf ( i.e. residues 6670 and 8084 ) showed significant structural changes to occur only in the one or two residues directly adjacent to the deleted residues ( supplementary table s2 ) . the dimensions of each complex ( solute ) were determined , and a box of solvent was prepared to overlay the solute . the edge of the solvent was extended to a minimum of 8 beyond the solute in each dimension . then each solute was overlaid with the solvent water box that also contained either sodium or chloride ions , depending on the total charge of the solute . all water molecules whose oxygen atom was within 2.5 of any solute non - hydrogen atom were then deleted and excess ions were deleted to make the systems electrically neutral . all subsequent calculations were performed using periodic boundary conditions , with images generated using the crystal module of charmm . equilibration of each simulation system was carried out by performing 500 steps of steepest descent energy minimization followed by a 10 ps md nvt simulation with harmonic restraints of 5 kcal/(mol ) on the solute non - hydrogen atoms . additional minimization using the adopted basis newton raphson ( abnr ) algorithm was performed for 100 steps without any restraints . the systems were then subjected to a 5 ns md simulation in the npt ensemble at 300 k with the leap frog integrator , and a time step of 0.002 ps . particle mesh ewald ( pme ) was used to treat long - range electrostatic interactions ( 37 ) . pme calculations were carried out using real space electrostatic and lennard jones interaction cutoffs at 10 , with utilization of heuristic non - bond list update out to 12 and smoothing of the lj interactions from 8 to 10 using the force switch method ( 38 ) . coordinates were saved every 5 ps , and by the end of the 4.5 ns production md simulation , 900 sets of structural coordinates were available for analysis . structural figures were generated using the program vmd ( 39 ) and rendered by raster3d ( 40 ) . calculation of binding free energies via free energy component analysis : binding free energies , gbind , were calculated as follows : 1gbind = gcomplexgpartneragpartnerb , where the overall free energy of each complex was described using ( 24 ) 2g = emm+gsolvationtsmm . in equation 2 emm describes the molecular mechanical energy of the system consisting of all components in the charmm potential energy function , as shown in the following equation 3 : 3emm = ebond+eangle+eurey - bradley+edihedral+eimproper+evdw+eelec . for complexes , emm , can be separated into interaction energy , eie and strain , estrain components : 4emm , complex = eie+estrain , where eie is the sum of the vdw and electrostatic interaction energy between individual molecules in a complex and estrain is the sum of emm terms for each molecule in the complex ( i.e. all mm energy contributions for each species ignoring contributions from the other molecules in the complex ) . the gsolvation term describes the solvation free energy of a system and includes both electrostatic , gelec and non - polar , gnp , components . the electrostatic portion of the solvation free energy was calculated using the generalized - born molecular volume ( gbmv ) method ( 25,26 ) . the non - polar portion of the solvation energy ( i.e. due to cavity formation and hydrophobicity ) was calculated using the still equation , enp = sa , where is an empirical atomic solvation parameter , 7.2 cal / , and sa is the solvent accessible surface area calculated with a solvent probe radius of 1.4 ( 25,41 ) . the final term in equation 2 , tsmm , describes the entropic part of the energy as a sum of translational , rotational and vibrational entropy terms calculated via the harmonic approximation ( 42 ) for the temperature 300 k. vibrational analysis was performed on the solute molecules extracted out of the solvent box . the solute structure was minimized using the conjugate - gradient method to an rms gradient of 10 kcal/(mol ) with a distance - dependent dielectric of 4r applied to approximate the screening of electrostatic interactions by solvent , where r is the distance between two atoms , as performed previously ( 22 ) . to better understand the contributions of the various energetic terms to complex formation this typically involved calculation of the difference between a selected term for the complex and those of the individual molecules comprising the complex . in the case of the interaction energies their contribution was simply the sum of the contribution between the individual molecules in each complex ( i.e. for the cbfrd dimer the eie between cbf and rd was included ) . however , in the case of the trimer formation via dimer + monomer complexation the eie contribution includes those between the monomer being added and the two molecules in the original dimer along with the change in the interaction energy between the molecules in the original dimer upon going to a trimer . for example , eie , cbf-rd + dna = eie , cbf-dna + eie , rd - dna eie , cbf-rd(dimer to trimer ) . this term accounts for gains or losses in the interaction energy of the molecules in a dimer when the third molecule binds to form a trimer . interaction energies , einter , and strain energies , estrain , were calculated for each individual species using an infinite cutoff in order to mimic the long - range electrostatic interactions treated via pme in the md simulations . energy calculations were performed on the 900 structures from each simulation system with statistical analysis performed by dividing each 4.5 ns simulation into nine 500 ps time blocks . statistical analysis was performed by obtaining the averages from each time block , with those averages treated as independent data points , allowing calculation of the overall averages , and standard errors as described previously ( 43 ) . the entropy calculations were performed on 30 structures extracted from the simulation trajectories at 150 ps time intervals , with the statistical analysis derived by dividing the 30 observations into ten 450 ps independent time blocks from which the averages and the standard errors were obtained . overall rms differences of all simulated structures versus the crystallographic structure of the trimer are shown in supplementary table s3 . md simulation studies were undertaken on the rd to understand the nature of interactions with the dna and with the binding partner , cbf. average rms differences for all non - hydrogen atoms with respect to the crystallographic ternary complex structures were 1.62 0.04 , 3.56 0.10 , 2.15 0.04 , 3.23 0.16 , 3.63 0.11 and 2.81 0.05 for the dna , rd , cbf , rd dna , rd cbf and cbfrd dna structures , respectively , indicating that the overall structures were maintained in the simulations . the structure of the trimer after a 4.5 ns simulation is illustrated in figure 1 . analysis focused on the overall binding as calculated using equation 1 followed by investigation of the contributions of different components to the overall binding ( equations 2 and 4 ) . this was followed by identifying contributions of different regions of the proteins and dna to binding , ultimately analyzing the role of individual amino acids . emphasis was placed on using the present results to interpret the body of experimental data available on rd . free energies of complex formation for the dimers and the trimer were calculated using equation 1 based on the data presented in supplementary table s4 . the resulting free energies of binding for the various complexes are presented in table 1 and are included in the thermodynamic cycle shown in figure 2 . two free energies of binding are reported , gbind and gbind , ts , that exclude and include the configuration entropy , tsmm , respectively . for gbind the calculated energies are all favorable , although the interactions involving addition of cbf are extremely exothermic . in contrast , upon inclusion of the entropy the events that involve dna binding become unfavorable . in addition , the calculations predict that rd cbf binding is more favorable than rd dna binding , in contrast to experimental observations ( 16 ) . the presence of these discrepancies is not surprising due to a variety of assumptions in the calculations , including the omission of energetic contributions , such as the solvation and entropic contributions of the added counterions ( 23 ) , limitations in the potential energy function and the force field ( 44 ) along with possible limitations in the sampling of conformational space . dna interaction energies in the force field by < 4% would account for the prediction that binding of rd to cbf is more favorable than rd binding to dna . with respect to the tsmm , the use of gas phase minimized structures may be problematic , especially in the case of the polyanionic dna where the electrostatic screening due to the 4r dielectric is expected to be insufficient when compared with the proteins . however , when the two types of binding free energy are compared , the trends are similar . moreover , in both scenarios , the binding of dna to the rd cbf dimer to form the trimer is more favorable than the binding of rd and dna to form the rd thus , it appears that the present results , although not in quantitative agreement with experiment due to assumptions and limitations in the theoretical models , are representative of the experimental regimen , allowing for atomic details of events driving binding to be elucidated . in addition , the emphasis in this work on differences in energies and structures between the systems rather than absolute energies should allow for increased confidence in the observations and the conclusions based on those observations . concerning the convergence of the present results , the energies calculated from the two independent dna simulations are within the estimated errors ( supplementary table s4 ) , suggesting that the present calculations have adequately converged . analysis of the thermodynamic cycle in figure 2 predicts that binding of both cbf and dna are cooperative . these results are consistent with previous studies showing the binding of dna to rd to be enhanced by the binding of cbf as well as the binding of cbf to rd to be enhanced by dna based on electrophoretic mobility shift assay ( 10,16,18 ) . as discussed , such cooperativity may be important for maintaining binding of rd to dna via stabilization of the ternary complex to insure adequate transcriptional activation . tighter protein protein interactions due to dna binding have been observed in other types of transcription factors ( 45 ) . contributions from solvation , strain and interaction energies to the calculated binding energies are shown in table 1 . as expected , the solvation energies are all unfavorable upon binding , with the unfavorable contributions being significantly larger for the interactions involving the highly solvated polyanionic dna . the strain energies are all also unfavorable with the exception of formation of the cbfrd dimer . typically , it would be expected that the protein protein or protein dna interactions would lead to an unfavorable strain contribution upon binding ( 23 ) . ongoing studies in our laboratory are investigating the unusual behavior in the cbfrd dimer interaction . countering the unfavorable solvation and clearly , the free energies of binding are driven by the large , opposing forces of solvation and , typically , strain energies versus interaction energies between the macromolecules in the complexes . data at the bottom of table 1 addresses the contributions to the calculated gain in binding energy associated with cooperativity upon going from the dimers to the trimer . contributing to the cooperativity is a decrease in the unfavorable solvation that occurs upon binding , evidenced by the trimer the more favorable change in interaction energy occurred with both the rd dna and the rd cbf interactions in the trimer ( table 1 ) ; details of the contributions of different regions of the proteins to this will be discussed below . countering these contributions are unfavorable increases in the strain energy , consistent with gains in interaction energies , as discussed above . thus , the cooperativity of dna and cbf binding to rd is predicted to be dominated by gains in the favorable interactions between the macromolecules and with lowering of the solvation penalty to complex formation . a more detailed understanding of the atomic determinants of cooperativity associated with rd binding to cbf and dna was obtained via interaction energy partitioning analysis between different regions of the rd and both cbf and dna . this was facilitated by partitioning rd into a variety of segments as specified in table 2 and shown in figure 1 . several regions of the protein have been previously indicated to contribute to either rd dna or rd cbf interactions ( 13 ) . residues adjacent to the respective binding partner but not included in a defined segment are referred to as other - dna and other - cbf and all residues not previously defined are referred to as non - interacting - cbf , allowing for the quantification of their contributions to binding . of note are the wing residues , which are primarily important for dna binding , while residues in wing1 are proximal to the wing residues , contacting both cbf and the wing residues . for all these segments of rd , interaction energies were calculated between the respective monomers in the dimers as well as in the trimer . as discussed above , cooperativity exists between both dna and cbf with respect to their binding to rd . dna interaction energies in both the trimer and the dimer complexes along with the change in interaction energy upon going from the rd dna dimer to the trimer , with those contributions broken down into the segments presented in table 2 . upon trimer formation the total rd dna interaction energy becomes more favorable , with three of the segments contributing significantly to the more favorable interaction energy in the trimer . these include wing , a loop and other - dna segments . the largest contribution is from the wing segment , consistent with its location in the vicinity of both the dna and the cbf. this location allows for interactions of cbf to be communicated to wing via wing1 ( see below for additional details ) , as discussed previously ( 13 ) . interestingly , the other - dna region makes a significant contribution to cooperativity by its unfavorable contribution in the rd such a change indicates that the binding of cbf leads to a conformational change in the core region of rd that favors interactions with the dna and , due to the other - dna region not being in direct contact with the dna , suggests that long - range interactions contribute to binding cooperativity . to better understand the role of the wing segment in facilitation of dna binding upon trimer formation , the contribution of this region was broken down into a per residue basis ( table 4 ) . overall , the majority of the favorable interactions are made by arg139 , arg142 and lys144 , as expected due to their positive charge and the polyanionic nature of dna . with all three residues in addition , more favorable interactions occur with ser140 , gly143 and ser145 , with the contribution of gly143 being relatively large . thus , specific residues in the wing contribute to more favorable interactions with the dna upon formation of the ternary complex . details of the role of cbf and the wing1 segment to these contributions will be discussed below . similar to rd dna binding , the rd to cbf interaction energy is increased upon trimer formation . the overall cooperativity gain of 60 kcal / mol is due to the wing1 and n - terminal segments . the contribution of the wing segment to cbf binding becomes less favorable whereas the contribution of this segment becomes more favorable to dna binding upon trimerization ( table 3 ) . such a scenario suggests a subtle balance between interactions of the wing and wing1 segments that contribute to the cooperative binding . interestingly , as with the other - dna segment discussed above for rd dna binding , the n - terminal segment makes a favorable contribution to the cooperativity of rd cbf binding although it is spatially remote from the dna in the ternary complex . this indicates that binding of dna to form the trimer leads to global conformational changes in rd that favor rd cbf interactions , further indicating the contribution of long - range interactions to cooperativity . residue breakdown of the interaction energy contributions of the wing1 and n - terminal regions to cbfrd interactions is presented in table 6 . the energetic contribution of a number of the residues changes significantly upon going from the cbfrd dimer to the trimer , with both favorable and unfavorable contributions occurring . the most significant contributions , both favorable , occur with glu116 in wing1 and asp66 in the n - terminal segment . the importance of glu116 for the binding of cbf has not previously been described , although perturbed chemical shifts of glu116 have been observed in nmr studies in the presence of either cbf or dna ( 13,17,46 ) and a significant decrease in the order parameter of glu116 has been observed upon going from the rd the interaction energies between rd glu116 and cbf in the binary and ternary complexes are 20 8 and 67 9 kcal / mol , respectively . in the present study , it was found that rd glu116 interacts with cbf residue arg33 . interestingly , the experimental change in kd was not accounted for in terms of any residue(s ) in the rd . to understand this omission , distances between the two residues in the binary and ternary complexes as a function of time were obtained ( supplementary figure s1 ) . it is observed that in the ternary complex during the md simulation , the sidechains of rd glu116 and cbf arg33 move to within 23 , in contrast to the oe2-nh2 distance being 9.4 in the crystallographic structure of the ternary complex ( 13 ) . in contrast , in the binary complex crystal structure the oe2-nh2 distance is 12 ; this distance initially decreases to shorter values during the md simulation , but upon further simulation time assumes a stable conformation with a distance of 12 between the oe2-nh2 atoms . to better understand the significant changes in the rd glu116 and cbf arg33 distances for the different systems the experimental structure along with final time frames from the 4.5 ns rd cbf binary and ternary simulations were obtained ( figure 3 ) . in the rd cbf dimer , rd glu116 and cbf arg33 are relatively far apart ( figure 3a ) . upon going to the ternary complex in the md simulation it is clear that a salt bridge forms between glu116 and arg33 ( figure 3b ) . the formation of this salt bridge is associated with a shift in the position of the arg33 sidechain towards the rd , which is suggested to be associated with the electrostatic field due to the presence of dna in the ternary complex as evidenced by the favorable interactions between the cbf arg33 and the dna ( 252.2 6.1 kcal / mol ) . in addition , energy partitioning analysis shows that arg33 of cbf overall disfavors rd cbf binding in the trimer with this effect significantly increased in the rd cbf dimer by 40 kcal / mol [ arg33-rd(trimer ) = 11.0 4.9 and arg33-rd(dimer ) = 50.3 3.3 ] , further disfavoring the rd glu116 to cbf arg33 interaction in the dimer . however , the question remains why is the rd glu116 and cbf arg33 interaction not observed in the ternary complex crystal structure ( figure 3c ) . although speculative , it may be due to the presence of higher salt concentrations in the experimental study ( 200 mm nacl ) ( 13 ) , which may partially screen the arg33dna interactions . this screening combined with competing interactions involving other residues , such as the rd tyr113 to cbf arg33 interaction that occurs in the crystal and rd cbf dimer md structures , may lead to the subtle difference between the two systems . supporting this possibility is the nmr observed decrease in the glu116 order parameter in the ternary complex ( 20 ) , suggesting that in solution and at lower salt concentration the proposed salt bridge may be present . other experimental results indicate a 5-fold increase in the dissociation constant upon formation of the ternary complex from the rd dna dimer ( 18 ) , which may be related to tyr113 properly orienting arg33 for the actual binding event . regardless , the rd glu116 to cbf arg33 salt bridge along with the additional interactions leads to the more favorable interaction energy between cbf and rd in the ternary versus the binary complex ( table 3 ) . thus , it is predicted that glu116 of rd has a significant impact on the binding of cbf to rd as well as on the cooperativity of rd binding to dna and cbf. notably , binding of cbf to the rd dna dimer has been shown via nmr to decrease the conformational variability of glu116 ( 20 ) , supporting the impact of rd interactions with its binding partners on this residue . the interaction energies for met106 , ala107 , asn109 , asn112 , tyr113 and ser114 ( table 6 ) are consistent with the experimental mutation data . mutation of these residues decreases rd cbf binding in the experimental study ( 17,18 ) , consistent with the favorable interaction energies of these residues with cbf. however , mutation of asn109 to alanine decreased the rd cbf binding by 60-fold while the calculated interaction energy is relatively small . this discrepancy may be due to mutation induced structural perturbations of the rd ( i.e. partial unfolding ) , leading to the experimentally observed decrease in binding ( 18 ) . recently , it has been shown that polar residues at protein protein interfaces provide structural rigidity so as to maintain specificity and to reduce the entropic cost upon binding ( 47 ) . thus , asn109 may play an important role in maintenance of the structural integrity of rd with a modest contribution to affinity associated with direct interactions with cbf. the n - terminal segment makes a significant , favorable gain in interaction energy with cbf upon going from the rd cbf binary to the ternary complex ( table 5 ) . as shown in table 6 , the contribution was dominated by asp66 . however , experimental mutation of asp66 to alanine only moderately affected the rd cbf binding strength ( 19 ) . visual inspection of the binary md , ternary md and crystal structures ( figure 4 ) , along with energy partitioning analysis shows asp66 to interact favorably , via its backbone carbonyl , with lys11 of cbf , with this interaction becoming a salt bridge involving the sidechains in the trimer . cbf dimer md and crystal structures and the trimer md structure appears to be due to a competitive interaction between asp66 with his163 in rd ( figure 4a and c , respectively ) . consistent with the results discussed above , this suggests that the electrostatic field imposed by the polyanionic dna in the ternary simulations disrupts the asp66-his163 interaction seen in the dimer and helps in orienting asp66 toward lys11 ( figure 4c ) , with the presence of 200 mm nacl in the experimental structure potentially damping this interaction . his163 was treated as neutral in the md simulations ; hypothetically , a different result might be obtained if his163 was treated as being protonated . presumably this would create a stronger interaction between rd asp66 and his163 , such that this interaction would dominate in both the ternary and the binary md simulations , with the asp66 backbone carbonyl oxygen interacting with the lys11 sidechain amino group in both cases . such a scenario where the interaction of asp66 with lys11 involves the formers backbone atoms could explain why a significant contribution of this residue to binding was not observed experimentally . area2 residues make favorable contributions to the interaction energy between rd and cbf ( table 5 ) . although this region does not contribute to the cooperativity , as evidenced by the similar contribution in the binary and the ternary complexes , the large favorable interaction energy contribution makes it of interest to investigate the role of individual residues in this region to binding ( table 6 ) . the importance of thr161 is thought to be due to its interaction with asn104 of cbf. indeed , visual inspection of the rd cbf binary md , ternary md and crystal structures shows two hydrogen bonds between these residues [ asn(nh2)-thr(og1 ) and asn(od1-thr(hn ) ] ( figure 5 ) . however , calculation of the residue residue interaction energy between rd thr161 and cbf asn104 yields a value of 4.9 0.4 kcal / mol , including the contribution from the thr hn atom , which does not correlate well with the change in dissociation constant in the t161a mutation . alternatively , calculation of the contribution of thr161 to the strain energy of rd yields values of 21.0 1.8 and 26.5 0.2 kcal / mol in the rd monomer and rd cbf dimer , respectively . interestingly , it has been reported that the mutation of thr161 to alanine leads to decreased exchange broadening in rd alone in nmr experiments , indicating a decrease in the conformational flexibility of residue 161 and surrounding residues ( 18 ) . this observation combined with the present energetic results suggests that thr161 may play a role in maintaining the conformation of the local region of rd , thereby impacting binding , rather than contributing directly to protein protein interactions . such a role of thr161 is consistent with the idea that conserved polar residues at protein protein binding sites provide structural rigidity ( 47 ) . the role of such conserved residues is primarily structural rather than functional . as discussed previously ( 13 ) and as emphasized above , the region of rd that includes the segments wing and wing1 appears to be of central importance for binding cooperativity . presented in table 7 are the interaction energies between wing and wing1 , including a breakdown of the interaction of wing residues with all wing1 residues and vice versa . upon going from rd to the rd wing1 total interaction energy becomes significantly more favorable , even though favorable intermolecular interactions occur between wing1 and cbf in the rd this suggests that the binding of cbf to rd , while forming a favorable interaction of cbf with wing1 also induces a conformational change in wing1 that favors the wing wing1 interaction . indeed , figure 6 shows that two salt bridges , arg142-glu111 and lys144-asp110 , are present in the rd cbf dimer ( figure 6b ) ; this represents a gain in interaction energy over the hydrogen bonds between lys144 and asp110 and glu111 in rd alone ( figure 6a ) . indeed , these favorable interactions contribute to the gain in favorable strain energy upon formation of the rd cbf dimer discussed above ( table 1 ) . moreover , nmr experiments indicate all four of these residues to undergo changes in mobility upon going from the rd thus , these intramolecular interactions make a significant contribution to the binding of rd to cbf. in contrast to the rd cbf interaction , upon going from rd alone to the rd this is consistent with the significantly favorable interaction energy between rd and dna in the binary complex , such that when the wing residues interact with the dna , the wing wing1 interactions are diminished ( table 7 ) . similarly , the wing wing1 interaction energy in the trimer is not significantly different as that in rd alone . visual analysis of the rd structure in the monomer ( figure 6a ) , the rd dna complex ( figure 6c ) and the ternary complex ( figure 6d ) , reveals the spatial relationships of residues asp110 , glu111 , arg142 and lys144 to be similar , consistent with the interaction energy contributions in these systems ( table 7 ) . in the rd dna binary and ternary complexes , it is evident that the negatively charged dna attracts arg142 away from the wing1 residues , contributing to the less favorable interactions . in the rd monomer , the lack of salt bridges between arg142 and asp110 or glu111 may be related to repulsion between the two positively charged residues , arg142 and lys144 , such that the system gains more stabilization by orienting arg142 toward the solvent . the balance of attraction and repulsion taking place between closely situated positively and negatively charged residues suggests a subtle balance of interactions , such that their relative orientations are sensitive to the influence of solvent and the presence of dna and cbf. as stated above , formation of the ternary complex leads to a wing energy partitioning analysis ( table 7 ) shows significantly less favorable interaction energies involving arg142 , asp110 and glu111 as compared with the cbfrd binary complex . the less favorable interactions of asp110 and glu111 are consistent with the loss of salt bridges with arg142 , although more favorable interactions with lys144 occur ( figure 6d ) . thus , a subtle balance of intramolecular and intermolecular interactions appear to contribute to the observed cooperativity . interaction of rd with dna leads to favorable intermolecular interactions ( table 3 ) with a simultaneous loss of wing however , upon formation of the ternary complex , the binding of cbf leads to the favorable wing interestingly , the majority of residues predicted to make the largest contributions to cooperativity are charged ( i.e. rd residues asp66 , asp110 , glu111 , glu116 , arg139 , arg142 and lys144 ) . consequently , the long - range character of electrostatic interactions may be an important factor in the observed cooperativity . for example , asp to glu mutations , or vice versa , of the acidic residues suggested to be important for cooperativity may alter the subtle balance of intermolecular and intramolecular interactions contributing to the cooperativity . in addition , mutation of any of the charged residues discussed above to neutral species would be expected to impact cooperativity . asp110 and glu111 may be the most interesting as their impact on cbf binding would be expected to be minimal while their impact on cooperativity should be large . md simulations of rd along with its binding partners cbf and dna , as well as the cbfrd and rd dna binary complexes and the cbfrd dna ternary complex have been performed to better understand the cooperative nature of the binding of these residues . in accordance with the experiment , the simulation data show cooperative binding of dna in the presence of cbf as well as predict that binding of dna to rd facilitates rd cbf binding , indicating cooperativity for this interaction . these observations are consistent with the experimental data ( 16,18 ) , supporting that ability of the applied simulation models to represent the experimental regimen . energetic and structural analysis allow for contributions to the binding and cooperativity to be understood in terms of different regions of the rd protein as well as the contribution of individual residues . of note these interactions are , to a large extent , dominated by charged residues suggesting that long - range interactions may contribute to cooperativity . from these results a number of residues of interest for mutational analysis are suggested . ribbon image of the cbfrd dna after 4.5 ns dynamics simulation including coloring of the rd segments used for analysis ( table 2 ) . dna ( green ) , cbf ( tan ) and residues surrounding omitted residues 7179 in cbf ( red ) . overall binding pathways rd dna , rd cbf and rd cbfdna . free energies before and after the / represent gbind and gbind , ts , as reported in table 1 . cbf interactions involving rd residues tyr113 , glu116 , arg118 and arg139 , and cbf residues arg33 and arg35 . images from snapshots at 4.5 ns from the ( a ) rd cbf binary and ( b ) ternary md simulations and ( c ) from the x - ray crystal structure . images from snapshots at 4.5 ns from the ( a ) rd cbf binary and ( b ) ternary md simulations and ( c ) from the x - ray crystal structure . rd cbf interactions involving rd residue thr161 and cbf residue asn104 . images from snapshots at 4.5 ns from the ( a ) rd cbf binary and ( b ) ternary md simulations and ( c ) from the x - ray crystal structure . wing1 interaction in the md simulation structures at 4.5 ns for ( a ) rd , ( b ) rd cbf , ( c ) rd dna and calculated binding energies and component contributions energies in kcal / mol calculated using equations 14 from the data reported in supplementary table s4 . free energy of binding based on the differences in the free energy of each monomer or complex , g , in supplementary table s4 . it should be noted that the sum of the terms gsolvation , estrain and eie are not exactly equivalent to gbind due to rounding errors . based on the sums , free energies of binding of 1.3 , 81.4 , 86.5 , 6.4 , 87.8 and 5.2 kcal / mol were obtained for the rd + dna , cbf + rd , rd dna + cbf , rd cbf + dna , trimer total and trimer definitions of regions of the rd interacting with dna or cbf color as shown in figure 1 . dna interaction energy energies in kcal / mol as mean se , ie = cbfrd dna rd dna . interaction energy of rd wing residues with dna energies in kcal / mol as mean se , ie = cbfrd dna rd dna . cbf interaction energy energies in kcal / mol as mean se , ie = cbfrd dna cbfrd . interaction energy of selected rd residues with cbf energies in kcal / mol as mean se , ie = cbfrd dna cbfrd . interaction energies between individual wing and wing1 residues in rd alone and in the three complexes energies in kcal / mol .

metabolism of propionyl - coa to succinyl - coa involves three successive enzymatic reactions ( fig . 1 ) . inherited defects in the enzymes catalysing the first and third steps of this pathway , and in the metabolism of their respective cofactors , cause well - characterised , clinically significant diseases , , . in contrast , the physiological significance of the second enzymatic step , catalysed by methylmalonyl - coa epimerase ( mce ; also known as methylmalonyl - coa racemase ) , has remained less clear . there is evidence that this reaction may be partially bypassed in vivo , by a shunt pathway proceeding via free methylmalonic acid , . one patient presented with acute metabolic decompensation ; an older sibling had developmental delay attributable to co - existing hydrocephalus , but was otherwise asymptomatic . two unrelated patients had progressive neurological symptoms ; however , each was also affected by a second inherited disorder , sepiapterin reductase deficiency , which fully explained the clinical course , , . all four patients presented with methylmalonic aciduria which was persistent but usually mild or moderate . two additional cases were identified by sequencing of the mcee gene in fibroblasts banked from over 200 patients with methylmalonic aciduria of unknown cause . one of these two had non - specific neurological symptoms ; the other was asymptomatic . of the six cases reported overall , five were homozygous for a single nonsense mutation , ( c.139c > t , p.arg47ter , p.r47 * ) in mcee , implying a complete loss of mce activity , and showed moderately decreased propionate incorporation in cultured fibroblasts . we describe a new case of mce deficiency , which contributes to the clinical , biochemical and molecular characterisation of this condition . the proband is a french - canadian female , born at term from an uncomplicated pregnancy . parents were not consanguineous and the family history was unremarkable . at the age of 5 years , following a previously unremarkable medical history , she presented acutely with vomiting , dehydration , confusion and visual hallucination . there was severe metabolic acidosis with increased anion gap ( ph = 6.96 , bicarbonate 3.5 mmol / l and pco2 15.9 mm hg , anion gap 25.5 mmol / l ) , accompanied by mild hyperammonemia ( 107 mol / l ; ref . biochemical testing at this time suggested a disorder in the pathway of propionyl - coa metabolism , as described below in section 3 . following the initial acute episode , she was treated with carnitine supplementation ( 100 mg / kg / day ) and a protein restricted diet . subsequently , the protein intake was gradually increased to a near - normal diet , only avoiding excessive protein intake . during intercurrent illnesses , the patient was treated with protein restriction , increased caloric intake and other basic measures . however , at the age of 9 years , she manifested one episode of acute and transient diplopia , which occurred in the context of a fever and lasted about a week . organic acid profiles were analysed by gas chromatography mass spectrometry following trimethylsilyl derivatisation , using selective ion monitoring for a panel of 58 metabolites . the related method for dedicated quantitation of methylmalonic acid in serum included stable isotope dilution . serum acylcarnitine profiles were analysed by tandem mass spectrometry with multiple reaction monitoring , following derivatisation by butylation . during the patient 's acute presentation , the organic acid profiles in urine , serum and cerebrospinal fluid ( csf ) were dominated by massive elevations of ketones . the urine profile was complex , including marked elevations of many metabolites considered secondary to the acute catabolic state . table 1 presents the results for methylmalonic acid ( mma ) and for several by - products of propionyl - coa , namely methylcitric acid , propionylglycine and 3-hydroxypropionic acid ( tiglylglycine was not analysed as part of the organic acid profile ) which were observed in samples collected during the acute episode . during subsequent follow - up , with the patient in a stable condition , the organic acid profiles were always unremarkable except for elevations of mma , methylcitric acid and 3-hydroxypropionic acid . the results obtained over a six - year period of follow - up are also summarised in table 1 . ms / ms method , showed unremarkable profiles , including essentially normal results for propionylglycine and tiglylglycine ( data not shown ) . the serum acylcarnitine profile during the acute episode showed a marked elevation of c3 ( propionyl ) carnitine : 21.6 mol / l ( reference range < 0.58 ) . c5:1 carnitine , representing the sum of tiglylcarnitine and methylcrotonylcarnitine , was moderately elevated : 0.19 mol / l ( ref . carnitine ( the sum of 3-hydroxybutyrylcarnitine and 3-hydroxyisobutyrylcarnitine ) was elevated : 2.13 mol / l ( ref . the acute profile showed some other mild elevations , considered secondary to the catabolic state . however , c4dc carnitine , representing the sum of methylmalonylcarnitine and succinylcarnitine , was well within reference range : 0.05 mol / l ( ref . < 0.19 ) . during the six - year follow - up , seven further serum acylcarnitine profiles showed a persistent moderate elevation of c3 carnitine , ranging from 1.53 to 5.82 mol / l ( median , 3.96 ; ref . the acylcarnitine profiles during follow - up were otherwise unremarkable : there was sometimes a mild elevation of free carnitine , attributable to dietary supplementation , and occasional mild elevations of a few other acylcarnitines considered non - specific and non - significant . plasma total homocysteine and serum cobalamin were not measured at the time of the acute episode . during follow - up , the initial impression , at the time of the patient 's acute presentation , was of a likely propionic acidemia , i.e. a primary deficiency of propionyl - coa carboxylase ( pcc ) . this working diagnosis was based chiefly on the first urine organic acid profile and the first serum acylcarnitine profile , and the clinical presentation . the serum acylcarnitine profile , with substantial elevation of c3 carnitine , moderate elevation of c5:1 carnitine and normal c4dc carnitine , was typical of profiles seen in propionic acidemia patients ; whereas patients with severe methylmalonic acidemia due to defects in methylmalonyl - coa mutase often show elevation of c4dc , reflecting increased methylmalonylcarnitine , particularly during episodes of acute decompensation . in urine , the propionyl - coa metabolites methylcitric acid , propionylglycine and 3-hydroxypropionic acid were all clearly elevated . although there was also a mild increase of methylmalonic acid ( mma ) in urine , its fold - elevation was less than that of any of the propionyl - coa metabolites . while one would not a priori expect patients with propionic acidemia to show any elevation of mma in urine , within the context of an acute clinical state and an overall complex profile , the significance of such a mild elevation of mma was not immediately obvious . such mild elevations of mma in urine are often seen as secondary findings in various contexts including disturbances of gastro - intestinal structure or function and states of nutritional deficiency , including decreased intake of vitamin b12 ( cobalamin ) . it is also worth noting , however , that 3-hydroxypropionic acid , which was much more dramatically elevated than the other propionyl - coa metabolites , can also be elevated due to several causes unrelated to inherited metabolic disorders , including production by gut bacteria . in the serum and csf samples collected during the acute episode , however , the overall profiles were less complex than in urine and the relative elevations of mma were more striking than in the urine ; the potential diagnostic significance of the mma therefore could not be readily dismissed . during follow - up , urine mma remained persistently elevated , but the elevations were always relatively mild . the same was true of 3-hydroxypropionic acid , in contrast to its striking elevation during the initial episode of acute decompensation . the normal results for serum cobalamin and total homocysteine argued against a nutritional deficiency of vitamin b12 ( cobalamin ) and also against most defects of cobalamin uptake , transport or metabolism . considering together all the biochemical findings at the time of acute decompensation and during long - term follow - up , the overall biochemical profile was not typical of a defect acting at the level of pcc ; either a primary propionic acidemia or a deficiency of multiple carboxylases due to defective biotin metabolism . nor , however , was it typical of a disorder at the level of methylmalonyl - coa mutase ( mut ) ; either a primary deficiency of this enzyme or a defect related to its cofactor , cobalamin . while there was clearly evidence implying a disorder acting within the propionyl - coa metabolic pathway ( fig . 1 ) , the overall impression was of an intermediate biochemical phenotype perhaps reflecting a block at the intervening step between pcc and mut . we thus came to suspect a deficiency of methylmalonyl - coa epimerase ( mce ) as the most likely underlying cause . as there was no suitable mce enzyme assay available for direct application to patient cells , two lines of investigation were pursued : molecular analysis of the mcee gene and studies on cultured fibroblasts . the three aims of the fibroblast studies were : to seek confirmation of diminished capacity for flux through the propionyl - coa metabolic pathway ; to test the hypothesis of primary mce deficiency ; and to rule out the possibility of diminished activity of pcc or mut . assay of pcc enzymatic activity on the patient 's fibroblasts gave results within the reference range : 811 pmol / min / mg protein ( control mean 1123 , range 2951837 ) . moreover , no mutations were detected in the pcca or pccb genes encoding the subunits of pcc . activities of other biotin - dependent enzymes , pyruvate carboxylase and 3-methylcrotonyl - coa carboxylase , were also normal in fibroblasts ( data not shown ) . m. ugarte , universidad autonoma de madrid ) effectively ruled out propionic acidemia and related disorders of biotin metabolism . the synthesis of cobalamin coenzyme derivatives from [ co]cncbl ( assay method described in ) yielded results within reference ranges . synthesis of adenosylcobalamin by the patient 's fibroblasts was 9.4% of total ( control mean , 15.3% , standard deviation 4.2 ) , while synthesis of methylcobalamin was 60.0% ( control mean 58.0% , sd 6.7 ) . incorporation of [ c]propionate was somewhat lower than control values , and did not respond to the presence of hydroxycobalamin in the culture medium . in absence of hydroxycobalamin , h ( control mean 10.8 , sd 3.7 ) ; in presence of hydroxycobalamin , the patient 's cells incorporated 3.3 nmol propionate / mg protein/18 the moderately decreased propionate incorporation was confirmed in two successive experiments , in which complementation studies were also performed . fusion of cultured fibroblasts from the patient with cells of the mut , cbla and cblb complementation classes respectively brought about an increase in propionate incorporation , indicating that the patient did not belong to any of those classes . however , fusion of cells from the patient with cells from each of two known patients in the mcee complementation class did not cause any increase in propionate incorporation . this indicated that this patient belongs to the mcee class , i.e. that she is deficient in activity of methylmalonyl - coa epimerase , mce . organic acid profiles were analysed by gas chromatography mass spectrometry following trimethylsilyl derivatisation , using selective ion monitoring for a panel of 58 metabolites . the related method for dedicated quantitation of methylmalonic acid in serum included stable isotope dilution . serum acylcarnitine profiles were analysed by tandem mass spectrometry with multiple reaction monitoring , following derivatisation by butylation . during the patient 's acute presentation , the organic acid profiles in urine , serum and cerebrospinal fluid ( csf ) were dominated by massive elevations of ketones . the urine profile was complex , including marked elevations of many metabolites considered secondary to the acute catabolic state . table 1 presents the results for methylmalonic acid ( mma ) and for several by - products of propionyl - coa , namely methylcitric acid , propionylglycine and 3-hydroxypropionic acid ( tiglylglycine was not analysed as part of the organic acid profile ) which were observed in samples collected during the acute episode . during subsequent follow - up , with the patient in a stable condition , the organic acid profiles were always unremarkable except for elevations of mma , methylcitric acid and 3-hydroxypropionic acid . the results obtained over a six - year period of follow - up are also summarised in table 1 . ms / ms method , showed unremarkable profiles , including essentially normal results for propionylglycine and tiglylglycine ( data not shown ) . the serum acylcarnitine profile during the acute episode showed a marked elevation of c3 ( propionyl ) carnitine : 21.6 mol / l ( reference range < 0.58 ) . c5:1 carnitine , representing the sum of tiglylcarnitine and methylcrotonylcarnitine , was moderately elevated : 0.19 mol / l ( ref . carnitine ( the sum of 3-hydroxybutyrylcarnitine and 3-hydroxyisobutyrylcarnitine ) was elevated : 2.13 mol / l ( ref . the acute profile showed some other mild elevations , considered secondary to the catabolic state . however , c4dc carnitine , representing the sum of methylmalonylcarnitine and succinylcarnitine , was well within reference range : 0.05 mol / l ( ref . < 0.19 ) . during the six - year follow - up , seven further serum acylcarnitine profiles showed a persistent moderate elevation of c3 carnitine , ranging from 1.53 to 5.82 mol / l ( median , 3.96 ; ref . the acylcarnitine profiles during follow - up were otherwise unremarkable : there was sometimes a mild elevation of free carnitine , attributable to dietary supplementation , and occasional mild elevations of a few other acylcarnitines considered non - specific and non - significant . plasma total homocysteine and serum cobalamin were not measured at the time of the acute episode . during follow - up , the initial impression , at the time of the patient 's acute presentation , was of a likely propionic acidemia , i.e. a primary deficiency of propionyl - coa carboxylase ( pcc ) . this working diagnosis was based chiefly on the first urine organic acid profile and the first serum acylcarnitine profile , and the clinical presentation . the serum acylcarnitine profile , with substantial elevation of c3 carnitine , moderate elevation of c5:1 carnitine and normal c4dc carnitine , was typical of profiles seen in propionic acidemia patients ; whereas patients with severe methylmalonic acidemia due to defects in methylmalonyl - coa mutase often show elevation of c4dc , reflecting increased methylmalonylcarnitine , particularly during episodes of acute decompensation . in urine , the propionyl - coa metabolites methylcitric acid , propionylglycine and 3-hydroxypropionic acid although there was also a mild increase of methylmalonic acid ( mma ) in urine , its fold - elevation was less than that of any of the propionyl - coa metabolites . while one would not a priori expect patients with propionic acidemia to show any elevation of mma in urine , within the context of an acute clinical state and an overall complex profile , the significance of such a mild elevation of mma was not immediately obvious . such mild elevations of mma in urine are often seen as secondary findings in various contexts including disturbances of gastro - intestinal structure or function and states of nutritional deficiency , including decreased intake of vitamin b12 ( cobalamin ) . it is also worth noting , however , that 3-hydroxypropionic acid , which was much more dramatically elevated than the other propionyl - coa metabolites , can also be elevated due to several causes unrelated to inherited metabolic disorders , including production by gut bacteria . in the serum and csf samples collected during the acute episode , however , the overall profiles were less complex than in urine and the relative elevations of mma were more striking than in the urine ; the potential diagnostic significance of the mma therefore could not be readily dismissed . during follow - up , the same was true of 3-hydroxypropionic acid , in contrast to its striking elevation during the initial episode of acute decompensation . the normal results for serum cobalamin and total homocysteine argued against a nutritional deficiency of vitamin b12 ( cobalamin ) and also against most defects of cobalamin uptake , transport or metabolism . considering together all the biochemical findings at the time of acute decompensation and during long - term follow - up , the overall biochemical profile was not typical of a defect acting at the level of pcc ; either a primary propionic acidemia or a deficiency of multiple carboxylases due to defective biotin metabolism . nor , however , was it typical of a disorder at the level of methylmalonyl - coa mutase ( mut ) ; either a primary deficiency of this enzyme or a defect related to its cofactor , cobalamin . while there was clearly evidence implying a disorder acting within the propionyl - coa metabolic pathway ( fig . 1 ) , the overall impression was of an intermediate biochemical phenotype perhaps reflecting a block at the intervening step between pcc and mut . we thus came to suspect a deficiency of methylmalonyl - coa epimerase ( mce ) as the most likely underlying cause . as there was no suitable mce enzyme assay available for direct application to patient cells , two lines of investigation were pursued : molecular analysis of the mcee gene and studies on cultured fibroblasts . the three aims of the fibroblast studies were : to seek confirmation of diminished capacity for flux through the propionyl - coa metabolic pathway ; to test the hypothesis of primary mce deficiency ; and to rule out the possibility of diminished activity of pcc or mut . assay of pcc enzymatic activity on the patient 's fibroblasts gave results within the reference range : 811 pmol / min / mg protein ( control mean 1123 , range 2951837 ) . moreover , no mutations were detected in the pcca or pccb genes encoding the subunits of pcc . activities of other biotin - dependent enzymes , pyruvate carboxylase and 3-methylcrotonyl - coa carboxylase , were also normal in fibroblasts ( data not shown ) . m. ugarte , universidad autonoma de madrid ) effectively ruled out propionic acidemia and related disorders of biotin metabolism . the synthesis of cobalamin coenzyme derivatives from [ co]cncbl ( assay method described in ) yielded results within reference ranges . synthesis of adenosylcobalamin by the patient 's fibroblasts was 9.4% of total ( control mean , 15.3% , standard deviation 4.2 ) , while synthesis of methylcobalamin was 60.0% ( control mean 58.0% , sd 6.7 ) . incorporation of [ c]propionate was somewhat lower than control values , and did not respond to the presence of hydroxycobalamin in the culture medium . in absence of hydroxycobalamin , the patient 's cells incorporated 4.1 nmol propionate / mg protein/18 h ( control mean 10.8 , sd 3.7 ) ; in presence of hydroxycobalamin , the patient 's cells incorporated 3.3 nmol propionate / mg protein/18 h ( control mean 10.9 , sd 3.5 ) . the moderately decreased propionate incorporation was confirmed in two successive experiments , in which complementation studies were also performed . fusion of cultured fibroblasts from the patient with cells of the mut , cbla and cblb complementation classes respectively brought about an increase in propionate incorporation , indicating that the patient did not belong to any of those classes . however , fusion of cells from the patient with cells from each of two known patients in the mcee complementation class did not cause any increase in propionate incorporation . this indicated that this patient belongs to the mcee class , i.e. that she is deficient in activity of methylmalonyl - coa epimerase , mce . extracted dna from blood was sequenced to screen for mutations in the three coding exons and splice site junctions of mcee , by the sanger method using a 3730xl dna analyzer ( applied biosystems ) , at the mcgill university and genome quebec innovation center . a heterozygous variant ( c.139c > t , p.arg47ter , refseq nm_032601.3 ) was found in exon 2 ( fig . this mutation was previously reported in the homozygous state in several unrelated patients with mce deficiency , as described above in the introduction . deletion / duplication analysis by exon array ( mitomet v3.0 oligonucleotide microarray , performed by bcm medical genetics laboratories ) yielded normal results . we then searched for an intronic sequence variation that could result in an aberrant splicing of the transcript . total rna was extracted from whole blood and reverse transcribed to cdna using a standard protocol ( superscript vilo cdna synthesis kit , life technologies ) . pcr - based amplification of mcee transcript isoforms was performed , as previously described , at the rnomics platform of universit de sherbrooke . this showed expression of an additional transcript that included a 92 bp sequence between exons 2 and 3 , compared to control ( fig . the splicing index of this alternate transcript suggests that its abundance is equal to that of the wild type transcript . cdna sequencing of this alternative transcript confirmed that the additional 92 bp sequence mapped to intron 2 ( genomic position hg19 chr2 : 71,337,89771,337,988 ) . this sequence corresponds to a putative 96 bp untranslated exon in a known spliced est ( ca427623 ) , but missing the last 4 nucleotides at the 3 end . dna sequencing of the 3 end of this putative untranslated exon showed a heterozygous variation ( c.379 - 644 a > g , genomic position hg19 ch2 : 71,337,896 ) creating a new consensus donor splice site gtaa ( fig . 2 ) . all the three alternative reading frames result in a premature stop codon . there is no sequencing coverage in this region for the exac or evs databases to allow an estimation of population frequency . sequencing of the mother 's dna revealed that the intronic variation c.379 - 644 a > g was maternally inherited . > t ( p.arg47ter ) was absent in the mother 's dna , and was thus presumed to be paternally inherited . while several previously reported mce deficiency patients did not present with an acute metabolic decompensation or with acidosis , and the clinical significance of this biochemical disorder has remained unclear , our patient is now the second recognised with an acute metabolic presentation . mce deficiency should therefore be considered as a condition with potentially significant clinical implications . following the initial episode our patient subsequently has not required aggressive ongoing management to maintain normal development , and long - term clinical course remains relatively uneventful on a essentially normal diet . the severity of the initial presentation led us to maintain a careful approach by treating undercurrent illness with caloric supplementation and protein restriction . while there is very little data available on similar cases , we advise prudence for clinicians encountering patients with this disorder . five of the six cases described in previously published articles were homozygous for the nonsense mutation c.139c > t , p.arg47ter . comparing the observed propionate incorporation in fibroblasts from our patient with data from those five patients , the splicing mutation identified in our patient is predicted to result in a null mutation , as the incorporation of the intronic sequence between exon 2 and 3 would result in a premature stop codon . in accordance with this , results from our in vitro method suggest that the relative abundance of this alternate transcript containing the intronic insertion is ~ 50% of total , with the other allele likewise accounting for ~ 50% . we can not exclude the possibility that up to 5% of wild type transcript could be produced by the intronic mutation allele , permitting some residual mce enzyme activity and possibly contributing to a relatively late - onset presentation . however , it is also important to note that , even in the presence of homozygosity for an mcee nonsense mutation , there is some residual propionate incorporation . this is presumably due to the existence of alternative shunt pathways allowing some conversion of propionyl - coa to succinyl - coa in the situation where there is no mce activity . this provides a plausible explanation why complete mce deficiency is clinically less severe than the classic forms of propionic acidemia and methylmalonic acidemia . the sixth previously reported case of mce deficiency was homozygous for a missense mutation ( c.178 a > c , p.k60q , p.lys60gln ) . in that case , there was a clear elevation of methylmalonic acid in urine , but observed propionate incorporation in fibroblasts was within reference range . this patient was described as having deterioration of motor function , ataxia , mild spastic paraplegia and dysarthria . according to sift , it is tolerated , and using polyphen 2 it is considered benign , whereas with provean it is considered deleterious . notably , 8 homozygotes are reported in the exac database ( ~ 60,000 individuals ) and allele frequency is 1.5% in south asians . this population - based data tends to argue against pathogenicity , or at least to imply that this mutation would give rise to a very mild condition with normal survival and reproduction rate . nonetheless , one can not exclude the possibility that partial mce deficiency could be a factor putting individuals at increased risk of neurologic disease , but with incomplete penetrance . there is currently no mce enzyme assay available which would be directly applicable to human cells in the clinical setting . direct assays of the recombinant human enzyme and of recombinant mce enzymes from other species have been described , , , providing a possible option for in vitro expression analysis of missense mutant mce proteins if required , although these methods require the isolation of partially purified proteins . our patient is the first to be described who is compound heterozygous for two different mutations in mcee rather than homozygous for a single mutation . in the previously reported study of a series of cell lines from patients with methylmalonic aciduria , there were two in which only a single heterozygous mcee sequence variation ( c.427c > t , p.r143c , p.arg143cys in both cases ) was found by sequencing of exons . we have now tested both of those cell lines for the novel intronic splicing mutation identified in our patient ; with negative results . the significance , if any , of the variation found in those two cases thus remains unclear , particularly considering that propionate incorporation was within reference range in both heterozygous cell lines . in silico predictions for this variant are as follows : polyphen2 , probably damaging ; provean , deleterious ; sift , damaging . however , in the exac dataset : 10 homozygotes are reported , and the allele frequency in finnish populations is 2.6% . this population data leads to a similar interpretation as for the variant p.lys60gln . while we can not exclude the possibility that simple heterozygosity for certain mcee mutations could cause mild elevation of mma in urine or blood under certain conditions , for example by a dominant negative effect another remote possibility could be that heterozygosity for an mcee mutation together with a heterozygous mutation at another locus relevant to mma metabolism could cause elevation of mma in body fluids . in the meantime , however , these two heterozygous p.arg143cys cases remain unclassified and they do not contribute to characterisation of the mce deficiency clinical or biochemical phenotype . conversely , it is also possible that some missense mutations allowing significant residual activity of mce might not cause methylmalonic aciduria , even in homozygosity or compound heterozygosity , thus contributing to the apparent rarity of this condition . in addition to the six cases of mce deficiency previously reported , and the patient described in our present article , the identification of a further nine cases was indicated in an abstract . these patients were identified by sequencing of mcee in dna from 150 fibroblast cell lines banked from patients with mild or moderate methylmalonic aciduria . eight of these cases were homozygous for the same previously - described nonsense mutation , p.arg47ter , while the ninth was homozygous for a novel missense mutation . one other abstract briefly describes a single patient , homozygous for p.arg47ter , who presented with an episode of acute metabolic acidosis . the initial working diagnosis for our patient , at the time of her acute presentation , was propionic acidemia . we previously flagged this potential pitfall in a preliminary abstract , and now provide a detailed description of the biochemical phenotype , its evolution during long - term follow - up and the further biochemical and molecular investigations which were needed to confirm the true diagnosis of mce deficiency for this patient . another recent abstract refers to a patient originally suspected to have propionic acidemia but for whom subsequent next - generation gene panel analysis instead revealed homozygosity for the p.arg47ter mutation in mcee . such reports suggest that mce deficiency might thus far be underdiagnosed , particularly considering that the associated mma elevations are often relatively mild even in patients with genotypes predicting complete loss of mce activity . on the other hand , the overall allele frequency of p.arg47ter in the exac database is only 0.025% ( 0.042% in europeans ) , with no homozygotes listed . the organic acid profiles of our patient were in fact not typical of propionic acidemia , nor of classical methylmalonic acidemias , nor of other it is possible that the observed disproportionate elevation of 3-hydroxypropionic acid could be a particular feature of mce deficiency , as in this situation 3-hydroxypropionic acid might potentially be formed by alternative metabolism of the accumulating d - methylmalonyl - coa and/or of free methylmalonic acid . this would represent a route different from the pathway by which 3-hydroxypropionic acid is generated from propionyl - coa in propionic acidemia . certain aspects of our patient 's organic acid profiles during decompensation , namely ketoacidosis with marked elevation of 3-hydroxypropionic acid , considerable elevation of methylcitric acid and propionylglycine , and relatively mild elevation of mma , together may represent a characteristic pattern to be seen in other patients with mce deficiency . in summary , we have identified and characterised a new case of the rare disorder mce deficiency , presenting with acute metabolic decompensation . we have described the biochemical phenotype of this patient , highlighting a possibility of diagnostic confusion in the context of an initial acute presentation , and emphasising that the observation of persistent mild or moderate methylmalonic aciduria is a clue which warrants consideration of mce deficiency in the differential diagnosis . the finding in this case of a novel intronic splicing mutation in mcee also shows that sequencing of exons alone is not sufficient to diagnose this condition in all cases , or to exclude it .

extramedullary hematopoiesis ( eh ) refers to the hematopoiesis that occurs in organs other than bone marrow . a classic example of eh is the increased ectopic erythropoiesis in a liver or spleen in hypoxia due to increased erythropoietin production.1 eh can be further classified into active or passive categories . normal hematopoiesis , which occurs in the fetal yolk sac , liver , and spleen , is an example of active eh ; it is programmed as an essential process for routine fetal development . another example of active eh is that which occurs in the spleen and liver during immune responses following infection . by contrast , eh also occurs as a result of failed marrow hematopoiesis in peripheral organs such as the liver and spleen and this is considered a passive form of eh . both active and passive eh produce blood cells such as antigen - presenting cells , granulocytes , nk cells , red blood cells , and/or platelets for the growth or survival of the host . inadequate eh leads to insufficient production or maturation of blood cells , while excessive eh leads to inflammatory diseases ( figure 1 ) . hematopoiesis occurs in specialized tissue sites ( eg , hematopoietic niches ) conducive for the maintenance and differentiation of stem and progenitor cells . for example , hematopoietic stem cells , which are quiescent and self - renewing , are present in the marrow stem cell niche , which is low in oxygen and favorable to maintaining their identity as stem cells.2 the marrow stem cell niche is composed of various cells and their products which positively and negatively regulate the process.3,4 key regulatory cells include osteoblasts , cxc chemokine ligand 12 ( cxcl12)-expressing reticular cells , and vascular endothelium cells.58 moreover , signals from the sympathetic nervous system and osteoclasts regulate the hematopoietic stem cell egress from the bone marrow through regulation of a critical stem cell homing and retention factor termed cxcl12.9,10 the requirement of a specialized niche is a limiting factor for eh in the periphery . as a consequence , a number of molecular pathways including wnt , calcium - sensing receptors , angiopoietin 1 , tie-2 , and extracellular matrix components are involved in the process to finely control the stem cell niche.6,7,1113 although incompletely understood , these pathways are thought to be involved not only in the maintenance of the stem cell niche but also in renewal and differentiation of hematopoietic stem cells within the niche . various cells produce hematopoietic cytokines such as stem cell factor ( scf ) , notch ligands , bone morphogenic proteins , transforming growth factor , thrombopoietin ( tpo ) , fibroblast growth factors , and insulin - like growth factor 2 , which maintain and regulate the primitive hematopoietic stem cells.14 other cytokines , such as granulocyte colony stimulating factor ( g - csf ) , interleukin-3 ( il-3 ) , il-7 , erythropoietin ( epo ) , granulocyte macrophage ( gm)-csf , and macrophage ( m)-csf , play important roles in the differentiation of hematopoietic stem and progenitor cells to committed cell lineages.15 in addition to the aforementioned cells and factors , hematopoiesis can be regulated by a number of other means , such as toll - like receptor ligands , metabolic / physiological products , various inflammatory mediators , and hormones.2,1618 many of the cytokines produced in inflammation act as myelopoietic factors,19 certain toll - like receptor ligands promote myelopoiesis,20 and hypoxia is a well known inducer of erythropoiesis.21 also , parathyroid hormone ( pth ) and the insulin - like growth factors ( igf ) control the hematopoietic stem cell niche ; pth can increase the number of bone marrow stem and progenitor cells and igf can regulate the survival and expansion of hematopoietic stem and progenitor cells.18 many of the cell types produced in the marrow , such as monocytes and b cells , require further maturation in the periphery to become fully functional immune cells . monocytes will migrate into various tissue sites to become macrophages or dendritic cells and b cells must be activated in the periphery to become memory and plasma cells . nave t cells are made in the thymus from progenitors that originated in the marrow and they undergo further differentiation in response to antigens proffered by antigen - presenting cells . thus , the term extramedullary hematopoiesis refers to a broad range of hematopoietic activities from the early stages of lineage commitment to late stages of hematopoietic cell maturation . bone marrow becomes functional as a site of hematopoiesis in the fetus from 45 months in human pregnancy . in mice , the developing fetus needs hematopoietic cells for the supply of oxygen and other less obvious reasons . therefore , nonmarrow tissues serve as sites of hematopoiesis before the bone marrow takes over the role as the major hematopoietic site . in early embryo , the yolk sac serves as the primary site of hematopoiesis.22,23 during mid - gestation , yolk sac cells colonize the umbilical cord , the aorta - gonad - mesonephros ( agm ) region , and subsequently the embryonic liver . the yolk sac produces hematopoietic stem cells and red blood cells initially , but later produces myeloid cells as well.24 in this regard , the yolk sac is an important place , harboring primitive erythroblasts and essentially all definitive hpc . later in embryonic development , some of these cells seed the fetal liver.25 as mentioned , the next hematopoietic tissue site after the yolk sac is agm and later , the fetal liver becomes a major hematopoietic site . following birth , the number of colony - forming hematopoietic progenitors in the spleen increases , peaking at two weeks of age in mice.26 in fact , the spleen remains a hematopoietic organ in mice throughout their lives , albeit at low levels . it is thought that the liver ceases to act as a site of hematopoiesis after birth . however , the liver maintains hematopoietic stem cells , erythropoiesis , and myelopoiesis at low levels during adult life.27 moreover , the liver is considered a maturation site for unconventional t cells including nkt cells , cd8 t cells , cd4cd8 double - negative t cells , and t cells . following birth in mammals and rodents , extramedullary hematopoiesis occurs when immune responses occur in the periphery ( figure 1 ) . the liver and spleen are the main sites of extramedullary hematopoiesis . other organs such as the lungs , kidney , and the peritoneal cavity can also become the sites of hematopoiesis when in diseased states . the increased extramedullary hematopoiesis in the spleen and liver of fas ( cd95)-deficient mice provides a useful insight into the cause of eh.28 in fas - deficient mice , hematopoietic cells are resistant to apoptosis , allowing these cells to grow in organs which are normally not conducive for growth and differentiation of hematopoietic stem and progenitor cells . also , immune cells are greatly expanded in these mice , producing large volumes of cytokines that can feed the growth of hematopoietic progenitor cells in the periphery . another example of induced eh is the increased myelopoiesis found in many organs of foxp3-null scurfy mice . scurfy mice display increased cd11b myeloid cells in the spleen and the liver , which is consistent with increased expression of gm - csf and il-3.29 in scurfy mice , t cells producing gm - csf and il-3 are overly activated in the absence of foxp3 t cells , driving excessive eh in the spleen and liver . by contrast , the transfer of functional foxp3 t cells into newborn scurfy mice completely suppresses eh and scurfy - related inflammatory disease.30 an important function of the foxp3 t cells is to suppress the differentiation of nave t cells into hematopoietic cytokine - producing t effector t cells ( figure 2).30 foxp3 t cells function mainly in the t - ell zone of the spleen rather than in the red pulps where eh occurs . nk cells appear to play a negative role in the regulation of eh in the spleen . depletion of nk cells with the anti - nk1.1 antibody in postnatal mice increased myeloid progenitor cells by 310 times in the spleen.31 this study , however , did not determine if the increase was the outcome of cell mobilization from the bone marrow or the product of the eh in the spleen . in vitro , nk cells can conversely decrease the numbers of progenitor cells.32,33 while the mechanism remains to be determined , nk cells could perhaps regulate the hematopoiesis with soluble inhibitory factors or through their cell - killing activity . toxic shock syndrome toxin-1 ( tsst-1 ) is a superantigen produced by most staphylococcus aureus strains . supe - rantigens can conjugate the mhc ii molecules of antigen - presenting cells and certain chains of t cell receptors for polyclonal activation of t cells . t cells are a good source of hematopoietic factors such as gm - csf , il-3 and oncostatin m.30,34,35 in this regard , tsst-1 can induce the production of hemopoietic factors.36 staphylococcus enterotoxin b ( seb ) is another superantigen produced by staphylococcus aureus . seb can activate nave t cells and induce their differentiation into effector t cells that produce gm - csf and il-3.30 microbial components such as tlr ligands can affect hematopoiesis in both the marrow and spleen . tlr ligands such as lipopolysaccharides ( lps , a tlr2 ligand ) and pam3csk4 ( a tlr4 ligand ) can directly activate hematopoietic progenitor cells through their receptors.37 consequently , differentiation of progenitors into myeloid cells , such as macrophages and dendritic cells , is greatly enhanced in vitro while myeloid cells in marrow and spleen are increased following the injection of lps in vivo . thus , tlr ligands are effective regulators of extra - medullary hematopoiesis.37 this suggests that infection may significantly affect the hematopoiesis in the marrow and peripheral organs , such as the spleen . the function of tlr ligands in regulation of hematopoiesis suggests that infection is an important element for eh to occur . as an example , it has been determined that leishmania major infection increases colony - forming unit cells or myeloid progenitors in the spleen.38 interestingly , the response in balb / c mice was greater than that in c57bl/6 mice . this could be due to the lesser ability of balb / c mice to clear the infection by intracellular pathogens . one of the mechanisms for the increased myelopoietic activity appears to be the production of gm - csf and tnf-,39 which can synergistically support the growth and maturation of myeloid progenitors . infection with plasmodium berghei ( ie , malaria ) provided more insights into the kinetics of hematopoietic shift from the bone marrow to spleen.40 infection with plasmodium berghei increased numbers of cfu - gm in the bone marrow during the first week of infection , which was followed by a rise of cfu - gm in the peripheral blood and finally in the spleen around the end of the second week . the marrow progenitor numbers were normalized within two weeks , but those of the spleen remained elevated for a more prolonged time period . it is unclear how the hematopoiesis in the marrow and the spleen is sequentially regulated . eh in an infection would generate sufficient numbers of mature myeloid cells to help clear pathogens , however , increased myelopoiesis without proper maturation of the myeloid progenitors could induce cd11b gr-1 myeloid - derived suppressor cells.41,42 myeloid - derived suppressor cells emerge in the spleen and other organs impacted by infection and cancer . these cells are highly heterogeneous and are considered normal constituents of myelopoiesis.41 thus , incomplete eh could ultimately hamper immune responses to pathogens . primary myelofibrosis is a form of philadelphia - negative chronic myeloproliferative syndrome.43 in primary myelofibrosis , displacement and mobilization of stem and progenitor cells occur . as a consequence , hematopoietic stem and progenitor cells occupy the liver and spleen as alternative sites of hematopoiesis . at the same time , the bone marrow stem cell niche is altered so that it no longer supports normal hematopoiesis.4446 thus , in primary myelofibrosis , the site of hematopoiesis changes from the marrow to the spleen and liver . primary myelofibrosis and related philadelphia - negative chronic myeloproliferative syndrome are associated with a point mutation in the tyrosine kinase jak2 ( jak2v617f).4750 it is thought that this mutation makes hematopoietic stem and progenitor cells more sensitive to growth factors , alters the marrow stem cell niche , and causes the cells to mobilize to the spleen and liver . another notable feature of primary myelofibrosis is the high production of inflammatory cytokines such as sdf-1 , hgf , il-6 , il-8 , scf , and vegf . likewise , the factors that promote fibrosis and angiogenesis ( bfgf , tgf- , pf4 , vegf , etc ) are increased.5153 these molecules are produced mainly by hematopoietic cells and contribute to the regulatory humoral changes occurring within the medullar and spleen niches . in addition to the jak2 mutation , activating mutations affecting the thrombopoietin receptor mpl ( mplw515l and mplw515k ) have been found in small numbers of patients with philadelphia - negative chronic myeloproliferative syndrome.54,55 eh , particularly eh in myelofibrosis , can never fully replace the marrow hematopoiesis in production of necessary blood cells , however . while the bone marrow is the major site of hematopoiesis , it can occur in many other tissues both during fetal development and after birth . extramedullary hematopoiesis can occur as long as there are appropriate supporting cells , accommodation of hematopoietic progenitors , and local production of soluble and cell - bound hematopoietic factors that maintain and induce differentiation of the stem and progenitor cells . it plays an essential role during fetal development , namely the survival of the fetus before formation of the functional marrow hematopoietic niche . as a normal response to infection and inflammation , myelopoiesis occurs in the spleen and liver to produce phagocytic cells and antigen - presenting cells . in malignant conditions , such as various forms of myelofibrosis , the marrow hematopoietic niche becomes inhabitable , and hematopoietic stem and progenitor cells move out to the periphery . eh can not fully supplant the marrow hematopoiesis in terms of the production of necessary hematopoietic cells , rather it is imperative for the maturation of hematopoietic cells which are produced as immature cells in the marrow . while programmed extramedullary hematopoiesis is required to supplement the hematopoietic activity in the bone marrow , excessive and disease - associated extramedullary hematopoiesis can occur and mediate chronic inflammation . more research is required to discover useful strategies for controlling unwanted extramedullary hematopoiesis and chronic inflammation .

the incidence of malignant melanoma is increasing worldwide , and upon dissemination has a very poor prognosis . only two systemic treatments are approved for disseminated disease and encompass il-2 based immunotherapy ( 16% response rate and 6% complete responses ) and dacarbazine ( 6%15% response rate with no improved survival ) . however , results from clinical trials of til - based immunotherapy conducted at two centres has shown 50% response rates in patients with advanced disease , and responses were long lasting [ 4 , 5 ] . tils were reported to be dominated by cd8 t - cells and mediate specific killing of autologous tumor in most patients . information on taa - derived peptide specificities in til has mainly shown the occasional large frequency of mart-1 and gp100 specific t - cell populations . on the other hand , results on the clonotypic and phenotypic composition has been scarce ; one publication has revealed a mixed clonal content of til by facs analysis , and two recent studies report surface markers identical to memory like effector t - cells from a limited patient material [ 8 , 9 ] . in our study , we have analysed til characteristics from 17 melanoma patients , whereof five have undergone lymphodepletion and til - based act with low - dose il-2 . patients referred to surgery for primary or recurrent stage iii - iv malignant melanoma were eligible for the study . the study protocol was approved by the local ethics committee , and all patients were included after signing informed consent . tumor material from the patients was obtained from the surgically removed tumour within 30 minutes after surgery . the til culturing method was adapted from dudley et al . constituting a two - step expansion process : ( i ) initiating bulk cultures and ( ii ) rapid expansion of selected bulk cultures with a proliferative potential . following surgical removal of tumor tissue from patients with mm fragments were subsequently placed individually in 24-well culture plates ( nunc , denmark ) and maintained in 2 ml of culture medium ( cm ) containing rpmi1640 ( invitrogen ) , penicillin , streptomycin , fungizone ( bristol - myers squibb ) , 10% human serum ( sigma ) and 7300 or 6000 iu / ml il-2 ( aldesleukin , novartis ) . each fragment initiated an individual til culture which was maintained separately during subsequent expansion and activation . bulk cultures were selected for further expansion according to a rapid expansion protocol ( rep ) . til were cocultured with irradiated ( 40 gy ) allogeneic pbmcs serving as feeder cells in a ratio of 1 : 200 in a 1 : 1 mixture of cm and aim - v ( invitrogen ) initially with 10% hs , and containing 30 ng / ml okt-3 ( cilag ag , suisse ) and 7300 or 6000 iu / ml il-2 ( aldesleukin , novartis ) in upright t - flasks . reps for preclinical purposes generally were initiated from 1 10 til per flask , while 1 10 til were used per flask in clinical scale reps . on day 5 , half of the medium was replaced with fresh medium containing aim - v , cm with 10% hs and 7300 iu / ml il-2 . from there on , the til concentration were maintained at 1 10 cells / ml by adding aim - v supplemented with fungizone and 7300 or 6000 iu / ml il-2 . half of the patients til where cultured in 7300 iu / ml il-2 , while the other half received 6000 iu / ml il-2 during culturing . cells were stained with trypan blue followed by counting of live and dead cells in a haemocytometer . bulk and rep cultures were intermittently sampled for microbiological testing of fungal and bacterial contamination . we used the following hla - a2 restricted peptides : sur1m2 ( lmlgeflkl ) , htert p540 ( ilakflhwl ) , cyclin b1 204 ( ilidwlvqv ) , mart-1 2735 ( aagigiltv ) , and ny - eso 1 157165 ( sllmwitqc ) . autologous tumor cell lines were established from tumor fragments by outgrowth in 24 well or 6 well plates ( nunc ) in medium consisting of rpmi1640 ( invitrogen ) , penicillin , streptomycin , fungizone , 10% fetal calf serum ( invitrogen ) , and solucortef ( pfizer ) . tumor cells were cryopreserved in 90% fcs and 10% dmso ( hospital pharmacy , regionh , copenhagen , denmark ) and stored at 140c . phenotyping were conducted using a facs - aria with diva software ( from bd ) and fluorescence conjugated monoclonal antibodies ( mab ) against cd3 apc - cy7 , cd4 apc , cd8 percp , cd25 pe , cd27 pe , cd45ra fitc , cd45ro pe , cd56 pe ( all from bd ) , ccr7 fitc ( r&d systems ) , cd16 fitc ( dako ) , cd28 fitc ( immunotech ) , cd62ligand pe ( bd pharmingen ) , and cd57 fitc ( bd pharmingen ) along with corresponding isotypes . rna was extracted using the nucleospin rna ii ( macherey - nagel , germany ) . cdna synthesis and quantitation of cdna in each sample was carried out as previously described . for tcr clonotype mapping , cdna was amplified using a primer panel covering the 24 bv region families of the tcr . amplifications were carried out in a total volume of 45 l containing 1xpcr buffer ( 50 mm kcl , 20 mm tris ph 8.4 , 2.0 mm mgcl2 , 0.2 mm cresol red , 12% sucrose , 0.005% ( wt / v ) bsa ( boehringer - mannheim , mannheim , germany ) ) , 2.5 pmol of each primer , 40 mm dntps ( pharmacia lkb , uppsala , sweden ) and 1.25 units of amplitaq polymerase ( perkin elmer cetus corporation , emeryville , calif , usa ) . , as described , in [ 11 , 12 ] . for dgge 10 l aliquots were loaded onto a denaturing gradient gel containing 6% polyacrylamide and a gradient of urea and formamide ranging from 20% to 80% . gels were run at 160 v for 4.5 h in 1x tae buffer kept at a constant temperature of 56c . after electrophoresis , the gel was stained with sybr green i ( molecular probes , oregon , usa ) and visualized using the fla-3000 fluorescence detection system ( fuji film , science imaging scandinavia , sweden ) . , nitrocellulose bottomed 96 well plates ( multiscreen maip n45 ; millipore , denmark ) were coated with inf capturing antibody ( 1-dik ; mabtech , sweden ) and further washed and blocked with rpmi 1640 . a maximum of 1 10 effector cells per well were either added alone when stimulated by peptides , or in coculture with target cells ( 1 10 cells per well ) consisting of autologous tumor cells . after a four - hour or overnight incubation period , the medium was discarded and wells washed followed by application of secondary biotinylated antibody ( 7-b6 - 1-biotin ; mabtech ) . the plates were incubated for one hour , further washed , and avidin - enzyme ( streptavidin ; mabtech ) conjugate , were added to each well followed by one - hour incubation at room temperature . succeedingly , the wells were washed and the enzyme substrate nbt / bcip ( nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate ; mabtech ) were added into each well . the plates incubated at room temperature for 2 to 10 minutes , while emerging purple spots developed . spots were counted with the immunospot series 2.0 analyzer ( ctl analyzers ) and the frequency of tumor specific til could be calculated from the numbers of spot forming cells . the assays were preferably done in triplets or in duplicates in case of low cell numbers . a standard cr - release assay was used to quantify the specific cytotoxic ability of selected til cultures . in brief , 5 10 cr - labeled tumor cells ( duplicates or triplicates ) were cocultured with til ( maximum e : t ratio of 100 : 1 and titrated ) in rpmi containing 10% fcs for a 4 hour incubation period . thereafter , cr - release was measured and percentage of tumor lysis calculated as ( # count min count)/(max count min count ) 100% . we utilized graphpad prism statistical software to analyse for statistical differences , using a paired two - tailed t test . tumor material were obtained from 17 patients with either locally advanced or advanced disease from metastasis localized either in lymph nodes ( majority of specimens ) or subcutaneously . a minimum requirement of 1 cm of tumor was needed to ensure sufficient material for til expansion . 12 of the patients had only been treated surgically prior to inclusion , while five patients who were included in our recent established clinical pilot trial had previously received il-2 and/or dc vaccination based immunotherapies . patients showed the following distribution of hla - a types : one hla - a1 + , two hla - a1/a3 + , one hla - a3 + , two hla - a3/a11 + , one hla - a11 + , one hla - a3+/a2 + , four hla - a2 + , one hla - a2/24 + , and four non - hla - a1/a2/a3/a11/a24 . lymphocytes migrated out from the fragments within two - to - five days and expanded into a confluent layer before splitting the wells . til bulk cultures expanded to at least 5 10 cells were considered sufficiently expanding . this was obtained in 15 out of 17 patients ( 88% ) in 6% to 100% of the bulk cultures ( mean 58% ) within 35 weeks . we found that growth rates varied markedly even between cultures from the same patient , and there was no difference in success rate of til growth from ln or sc tumor material , nor between the il-2 concentrations ( data not shown ) . we next tested the proliferative potential of a range of bulk cultures from 12 of the 15 patients with sufficiently growing til . this rapid expansion procedure ( rep ) involves the addition of allogeneic feeder cells and a cd3 antibody and has shown to increase til expansion rates considerably , in previously reports in both melanoma and head and neck squamous cell carcinoma . again , the kinetics could vary between cultures from the same patient ; however , the procedure could efficiently expand til bulk cultures to over a 1000-fold in more than half of the cultures in 2 weeks ( figure 1 ) . til were visualized in the microscope , showing a blasted morphology related to actively dividing lymphocytes laying either as single cells or in clusters / clones . in acquisition of cells by flow cytometry , gating of viable cells t - cells ( cd3 ) predominated the cultures , while nk cells ( cd16/56 ) ( figure 2 ) were consistently absent . in bulk cultures , we observed a heterogeneous cd4 and cd8 t - cell distribution among cultures inter- and intraindividually . there was , however , an overall skewing towards a cd8 ( mean = 74% 24% , range 30%94% ) t - cell predominance in relation to cd4 t - cells ( mean = 19.5% 23.5% , range 1%64% ) . next , we investigated the occurrence of surface markers identifying t - cell memory subsets , or alternatively , a differentiation path of effector cells , in the overall cd3 population , and among cd4 and cd8 t - cell subsets in comparison to til after two weeks of rep ( figure 2 ) . overall , there was a distinct predominance of cd45ro and ccr-7 t - cell populations before and after rep identifying the cells as t effector memory like . til were further characterized by surface markers according to a proposed model of effector cd8 t - cell differentiation stages by gattinoni et al . . expression of the lymphoid homing marker cd62l was significantly reduced after rep in the cd3 population and the cd8 subsets and remained unchanged among the cd4 subsets . concerning expression of costimulatory markers , we observed a significantly higher expression of cd27 among cd8 bulk til compared to cd4 cells , while cd4 cells had sustained higher cd28 expression in bulk cultures and after rep . although there was a relatively high percentage of cd27/28 double positive cells in a few bulk til , they were downregulated after rep . finally , there was a significant increase in the high - affinity il-2 receptor ( cd25 ) after rep in the cd4 population . in conclusion , the cd8 population express surface markers ( cd45ro , ccr-7 , cd62l , cd27 , cd28 , and cd57 ) resembling intermediate to late - stage effector cells as reported by other groups [ 8 , 9 , 16 ] . selected expanded cultures were analyzed for the presence of clonally expanded t cells by rt - pcr / dgge - based tcr clonotype mapping . analysis revealed the presence of at least 10 different t - cell clonotypes in bulk cultures as well as in rapidly expanded cultures ( data not shown ) . the results support our previous findings in expanded til from head and neck cancer patients , that expansion by high - dose il-2 and cd3 antibody seems to support the continued expansion of bulk t - cell clones . til cultures from eight patients were selected to scrutinize the presence of specific t - cell populations in bulk cultures and after rep . peptides derived from over expressed ( telomerase , survivin , and cyclin b1 ) , differentiation ( mart-1 ) and cancer testis antigens ( ny - eso-1 ) served as known targets , while autologous tumor cell lines presented a panel of unknown antigen specificities . elispot detection of inf release upon antigenic stimulation revealed a sustained functionality of til after rep . due to the high sensitivity of the assay , we could follow the presence and loss of low - frequency single - peptide - specific t - cell populations ( figure 3(a ) , occurring as a consequence of an increase or decrease in cell number of a given specific cell population , during the unspecific expansion procedures provided by il-2 and anti - cd3 . autologous tumor cell lines were available in four patients , and all patients contained til showing antitumor activity in elispot ( figure 3(b ) and data not shown ) . the presence of autologous tumor - specific t - cell populations was more resistant during rep and showed a sustained functional capacity . although t - cell - specific antitumor activity was predominating in til , we observed lak / nk cell activity in a few cultures ( figure 3(b ) ) by unspecific engaging the cell lines k562 and daudi . finally , we confirmed a sustained tumoricidal capacity of til after rep ( figure 3(c ) ) indicating that til expanded to clinical relevant numbers ( 2400- and 4000-fold ) can engage and kill autologous tumor . we were able to establish sufficiently expanding til bulk cultures in five weeks from the majority of included melanoma patients . further expansion by rep generated a mean expansion fold of 1400 in two weeks , ensuring the feasibility to reach clinical relevant quantities for clinical testing . based on earlier studies of t - cell therapy of melanoma patients were as low as 1,3 10 infused cells containing 30% mart-1-specific cd8 t - cells mediated a complete clinical response , we estimate that a minimum of 3 10 cells are required to obtain a therapeutic effect . cell - based analysis revealed an oligoclonal composition of t effector memory cells , predominated by cd8 cells showing an intermediate to late stage of differentiation after rep . til retained the functional capacity measured by inf release and lytic activity against autologous tumor . notably , we did not find differences between the two doses of il-2 used during til culturing , and even further lowering of il-2 dose to 3000 iu / ml is now the standard used in til expansion at other centres . finally , there were no significant influence on til expansion kinetics or phenotypes by pretreatment , age or performance status of the patients . in a recently initiated clinical trial of til - based act , low - dose il-2 , and lymphodepletion preconditioning , one out of five treated melanoma patients has obtained an ongoing partial response ( + 13 months ) . we are currently screening the til cultures for the occurrence of tumor associated antigen ( taa ) specificities by measuring inf in elispot . this enables us to identify the specific combination of taa specificities in each patient , which potentially can be identified during immune monitoring of the patient samples . in addition , we are establishing and validating a flow cytometry - based method of identifying taa - specific t - cell populations and obtain information on the kinetics of t - cell memory and effector stages before and after treatment .

well - differentiated systemic mastocytosis ( wdsm ) is a rare , recently recognized provisional subvariant of systemic mastocytosis ( sm ) . we report a case of wdsm that showed excellent clinical and cutaneous response to imatinib in the absence of known molecular genetic abnormalities . we present a 24-year - old woman with childhood onset of skin manifestations that progressed to mediator - related systemic events , and a gastrointestinal tract mastocytoma . treatment with imatinib resulted in complete resolution of cutaneous lesions and systemic symptoms , which relapsed with the discontinuation of the drug . targeted next - generation sequencing - based mutation analysis did not demonstrate any mutations in the coding regions of kit or other genes commonly associated with myeloid neoplasms . the diagnosis of wdsm is challenging in the absence of spindle - shaped mast cells , cd2 or cd25 expression , and kit d816 mutation . this case illustrated the need for recognizing this unique variant of sm for diagnostic and therapeutic implications . well - differentiated systemic mastocytosis ( wdsm ) is a rare , recently described provisional subvariant of systemic mastocytosis ( sm ) , with distinct morphologic , immunophenotypic , and molecular features . wdsm accounts for approximately 5% to 7% of sm , shows a female predominance , often childhood onset and tendency for familial predisposition . based on the proposed spanish network on mastocytosis ( rema ) algorithm for the diagnosis and classification of mastocytosis , diagnosis of wdsm could be made according to the following criteria : skin involvement , compact bone marrow ( bm ) mast cell aggregates ( > 15 mast cells ) , mast cells with round - shape and larger cd25 negative mast cells , elevated serum tryptase level ( > 20 ng / ml ) , despite lacking kit d816v mutation . however , patients with wdsm usually present with a low mast cell burden that progresses over time , and have a prolonged disease course . due to the low disease burden , especially early in the disease course , some cases with low serum tryptase levels ( minor criteria ) may not meet the 2008 world health organization ( who ) diagnostic criteria for sm , since the other 3 minor criteria ( spindle - morphology , aberrant cd2/cd25 expression , kit d816 mutation ) are missing in this subvariant sm . however , recognition of this entity is of significance , not only for diagnosis but also for treatment . the responders often showed kit mutations not involving the d816v loop . here , we report a case of wdsm and highlight the clinical , pathologic , and immunophenotypic findings of this entity , and demonstrate excellent clinical and cutaneous responses to imatinib even in the absence of kit mutation by next - generation sequencing ( ngs)-based testing . a 24-year - old woman presented with a 7-year history of intermittent nausea , vomiting , abdominal pain , joint pains , and progressive weight loss . the patient was diagnosed with a gastric mastocytoma ( 5 cm ) on endoscopic biopsy 7 months before this presentation and had a partial gastrectomy afterward . the diagnosis of mastocytoma was based on the presence of sheets of mature , nonatypical mast cells involving the submucosa ; no increased number of mitoses was seen . the patient had a long - standing history of maculopapular skin rash over the chest and back since the age of 4 . on admission , serum tryptase level was elevated at 47.1 ( normal < 11.5 ng / ml ) . a bm biopsy showed loose aggregates and interstitial infiltrate of round mast cells with abundant cytoplasmic granules , representing about 20% of the marrow cellularity . flow cytometry immunophenotypic ( fci ) analysis on the bm showed an aberrant mast cell population representing 0.2% of total analyzed cells , positive for cd117 , and cd63 ( partial ) ; but negative for cd2 , cd25 , and cd69 . mutation - specific quantitative real - time pcr for d816v mutation of the kit gene was negative . a diagnosis of sm was rendered using the 2008 who classification , based on the presence of 1 major criterion ( multifocal mast cell aggregates ) and 1 minor criterion ( elevated serum tryptase levels ) . the patient was treated with imatinib mesylate 400 mg daily , and the symptoms resolved over the course of 8 months . unfortunately , the patient had poor compliance and started to miss doses after 8 months of continuous therapy , and developed intermittent constitutional symptoms over time . repeat serum tryptase levels were elevated , ranging between 31.7 and 36.1 ng / ml . bm aspirates showed numerous intact round and well - granulated mast cells , representing about 4% to 5% of the cellularity . this time , many mast cells showed erythrophagocytosis ( fig . 1 a and b ) . bm biopsy showed extensive paratrabecular and interstitial aggregates of round mast cells with abundant cytoplasm and coarse granules without spindle or atypical morphology , involving about 50% of the cellularity ( fig . fci studies on bm showed a discrete mast cell population representing 0.5% of total analyzed cells that were positive for cd22 ( dim ) , cd123 ( dim ) , cd117 ( bright ) , hla - dr ( dim ) , and cd30 ( 66% , dim ) ; and negative for cd2 , cd19 , cd25 , and cd34 ( fig . ngs - based analysis for the detection of somatic mutations in the coding regions of a total of 28 genes related with myeloid neoplasms ( abl1 , asxl1 , braf , dnmt3a , fgfr , ezh2 , flt3 , gata1 , gata2 , hras , idh1 , idh2 , ikzf2 , jak2 , kit , kras , mdm2 , mll , mpl , myd88 , notch1 , npm1 , nras , ptpn11 , runx1 , tet2 , tp53 , and wt1 ) was performed on the dna extracted from bm aspirate in our clia - certified molecular diagnostic laboratory . no mutation was detected including kit ( exons 121 ) . similar to the previous sample , no fip1l1pdgfra gene fusion transcript was detected by qualitative nested rt - pcr analysis . ( a ) aspirate smear , wright giemsa stain 500 magnification ; ( b ) clot section : erythrophagocytosis , hematoxylin , and eosin stain 500 magnification ; ( c ) clot section , mast cell tryptase immunohistochemistry , 500 magnification ; ( d ) clot section , cd30 immunohistochemistry , 500 magnification . flow cytometry immunophenotype shows that mast cells are positive for cd30 ( 66% ) and cd117 , and negative for cd2 and cd25 . here , we present a case of wdsm that manifested initially at childhood , with skin lesions that progressed to mediator - related systemic events including musculoskeletal complaints and constitutional symptoms , and a gastric mastocytoma diagnosed at adult age . although childhood - onset mastocytosis is usually a skin - limited disease that commonly spontaneously regresses before puberty , a small subset of patients show progression to aggressive disease . in this patient , the diagnosis of systemic involvement by mastocytosis , wdsm subvariant was made at 24 years of age , based on the bm examination , however , bm involvement before this can not be excluded . the findings met 1 major and 1 minor diagnostic criteria of sm , reportedly elevated serum tryptase levels over 20 at the time of diagnosis and relapse . the relapsed bm had a higher degree of bm mast cell infiltration by morphology and flow cytometry ( 0.5% ) than when performed at diagnosis ( 0.2% by flow ) . however , the degree of the serum tryptase level elevation , measured at multiple time points over a period of 18 days before restarting therapy , was lower than the diagnostic sample . this illustrates that the serum tryptase level reflects the degree of mast cell activation , and not merely quantitative mast cell burden . hence , correlation between bm mast cell burden and serum tryptase levels may not be absolute . one of the reasons is sampling error due to patchy mc infiltration in the marrow . secondly , serum tryptase levels correspond to mast cell activation of the total body mast cells in various organs . in this patient , the extent of skin and bm involvement at 2 different time points could have been different . further , the degree of mast cell activation , and thereby serum tryptase levels can be dependent on time , external stimuli , etc . in this case , unlike serum tryptase testing at the time of relapse , serum tryptase level was performed only at 1 time point . another interesting finding at relapse was overt erythrophagocytosis observed in mast cells while the patient was neither anemic nor being transfused . erythrophagocytosis in mast cells has been reported in both neoplastic and normal mast cells from human and animal species , but the significance is unknown . the mast cell immunophenotype of the current case is similar to previous studies that have shown the neoplastic mast cells in wdsm to show variable expression of cd22 , cd30 , cd63 , cd117 , cd123 , and very dim hla - dr , while they show negativity for cd2 , cd19 , cd25 , and cd34 . in this case , this is important since wdsm is often negative for cd2 and cd25 , and aberrant cd30 expression may be a surrogate marker of mast cell clonality for this variant sm . cd30 is a transmembrane glycoprotein receptor found on certain hematopoietic cells and activated t - cells . as we learn from this current case and previous reports , cd30 is expressed in the cytoplasm and on the cell surface of the neoplastic mast cells and is therapeutically targetable . brentuximab vedotin ( bv ) is a cd30-directed antibody drug conjugate ( adc ) , that selectively induces cell cycle arrest and apoptotic death of the cd30-positive tumor cells . in vitro studies in sm have confirmed the effectiveness of this drug in targeting neoplastic mast cells with cd30 expression . this is currently being evaluated in a phase ii trial in patients with cd30-positive advanced sm ( clinicaltrials.gov identifier nct01807598 ) . however , the utility of this drug has not yet been demonstrated in wdsm patients , and could be of value and worth exploring . imatinib is the only sm treatment currently approved by the food and drug administration ( fda ) . it is an effective target agent that inhibits activated tyrosine kinase , and has been shown to be effective in sm with kit mutations occurring outside exon 17 , but not with mutations in the kit d816v loop . kitd816v mutation causes conformational changes in the enzymatic domain of the protein , thereby preventing binding of imatinib . from the literature review , we identified 5 reported cases of wdsm with excellent response to imatinib : 4 of 5 cases had kit non - d816 mutation involving phe522cys and k509i ; 1 case showed absence of kit mutation in exons 10 , 11 , and 17 . our patient showed excellent clinical and cutaneous response to imatinib with complete resolution of cutaneous lesions and mast cell - mediated release symptoms ( summarized in table 1 ) . following the discontinuation of the drug , the patient developed progression of symptoms , and bm confirmed relapse . furthermore , our patient had a normal karyotype , and did not show any mutations in the other myeloid - related genes included in our ngs panel . recently , mutations in tet2 , srsf2 , asxl1 , cbl , and runx1 have been reported in sm patients who also had kit d816v mutation , and are associated with a worse overall survival . the observed clinical response to imatinib in the absence of a mutation causing kinase activation is intriguing . the mechanism behind the response of imatinib may be related with a nonspecific inhibition of tyrosine kinase due to yet undefined mechanisms . the diagnosis of wdsm is challenging in the absence of spindle - shaped mast cells , cd2 or cd25 expression , and kit d816 mutation . in such cases , aberrant cd30 expression can help in the diagnosis and may serve as a potential therapeutic target . the excellent clinical and cutaneous responses to imatinib treatment as observed in this case of wdsm suggest the need for diagnosis of this variant of sm and empirical treatment with tyrosine kinase inhibitors , despite no identifiable molecular genetic alterations .

accordingly , interest in the lives and welfare of the disabled is also improving which requires execution of a systematic and effective policy for the disabled . in order to operate it , guidelines to rate the impairments objectively and scientifically is required ( 1 - 3 ) . this is due to diagnostic errors , bogus disabled , and the fact that guidelines for the impairment of the digestive system does not even exist ( 2 , 7 ) . there are many people unable to carry on normal lives due to serious digestive system diseases , and yet no help is given from the nation and society due to lack of proper guidelines . the united states has already a scientific impairment rating guideline prepared by the american medical association ( ama ) . therefore , we have decided to develop our own scientific and objective digestive system impairment rating guidelines fitting the given conditions of korea based on social and cultural realities . the research committee was organized by specialists who have knowledge and experience in rating digestive system impairments under the supervision of the korean academy of medical sciences ( kams ) . the committee consists of the medical doctors of internal medicine , general surgery , family medicine , and medical law and ethics . the committee members received education on the background and purpose , basic concept , rating methods , and principles of impairment of the rating impairment guidelines . the members analyzed the systems of european union , u.s.a . and many countries in asia including the digestive system impairment rating . especially ama guides ( 8) that was reformed and put into practice from the year 2000 was heavily referred . we used these guidelines positively in a way by making them satisfy the conditions of korea , and then developed the digestive system impairment evaluation guidelines under the management of kams . in this evaluation guideline of the digestive system was divided into three parts : the upper and lower gastrointestinal tracts and the liver . we examined the lives of people with digestive diseases to see whether or not they could carry on with normal activities . we evaluated the loss of function in the digestive system as 100% impairment due to the possibility of death from function loss . we fixed the impairment rate in proportion to the impairment state with the relative rate to the loss before proper functioning . we paid careful attention to the kams guidelines to satisfy the requirements of science , objectivity , convenience , rationality , and actuality . impairment evaluation should be conducted when the symptoms are in a fixed state , but if there is an expected change of symptoms , then another evaluation should be made two years later . in principle , the subject of impairment for medical evaluation should be as fixed symptoms that are left without recovery upon completion of treatment . the source of impairment does not have to result from traumas , but may also come from congenital diseases and just as a disease itself . the impairment should be evaluated by specialists of the appropriate fields of which it belongs to . we evaluated the loss of function in the digestive system as 100% impairment due to the possibility death from the loss of functions . we fixed the impairment rate in proportion to the impairment state with the relative rate to the loss before functioning . the upper digestive tract includes the esophagus , stomach , duodenum , small intestine , and pancreas . useful objective methods for confirming upper gastrointestinal tract impairments are : 1 ) fluoroscopy , contrast media using radiological tests and imaging studies such as a computed tomography ( ct ) or an magnetic resonance imaging ( mri ) , 2 ) cytology test or endoscopy including a biopsy , 3 ) esophageal manometry , 4 ) gastric acid secretory studies , 5 ) absorption abnormality test , 6 ) stool studies , and 7 ) helicobacter pylori urea breath test . also , fat content in the stool and intestinal malabsorption may be examined . the impairment rate , as signified in percentage , reflects the anatomical , physiological , and functional abnormality occurring in an organ or system and the ability to perform daily activities . patients belong to the normal scope of gastrointestinal impairments if patients are able to perform daily activities ; show regular and intermittent gastrointestinal manifestations without need of specific dietary treatment or medications ; and are able to keep normal weight with the necessary nutrition . we divided the upper gastrointestinal tract impairments into the stomach , the duodenum , the esophagus , and upper gastrointestinal tract after surgery ( table 1 - 3 ) . the lower gastrointestinal tract impairments include the colon , the rectum , and the anus . the signs and symptoms of the lower gastrointestinal tract impairment are : abdominal pain , pelvic pain , perineal pain , difficulty of defecation , tenesmus , stool incontinence , hematochezia , abscess , fissure , and fistula . in general findings , fever , weight loss , weakness , anemia , etc . useful objective methods to confirm colon , rectum , and anus impairment are the following tests such as : 1 ) digital rectal examination , proctoscopy , sigmoidoscopy , colonoscopy , 2 ) biopsy , 3 ) microscopic examination of the stool and cultivation , 4 ) fluoroscopy and radiological test using contrast media , and 5 ) ct and mri examinations . the impairment rate , as signified in percentage , reflects the anatomical , physiological , and functional abnormality occurring in an organ or system and the ability to perform daily activities . patients belong to the normal scope of gastrointestinal impairments if patients are able to perform daily activities ; show regular and intermittent gastrointestinal manifestations without need of specific dietary treatment or medications ; and are able to keep normal weight with necessary nutrition . we divided the lower gastrointestinal impairments evaluation into the colon , rectum , anus , intestinal stomas and after surgery of the lower gastrointestinal tract ( table 4 - 7 ) . the symptoms of hepatic bile impairment are pain , nausea , vomiting , anorexia , general weakness , fever , jaundice , and itching . the symptoms of progressive liver disease complications are edema , ascites , esophageal varix , portal hypertension which generates bleeding , hepatic encephalopathy , metabolic impairment , and the loss of kidney function . useful objective methods to confirm liver impairments are 1 ) an abdominal sonogram , 2 ) radiological examination such as percutaneous and endoscopic cholangiography , 3 ) ct and mri , 4 ) liver isotope studies , 5 ) liver biopsy & fine needle aspiration biopsy , and 6 ) a laboratory test for diagnosis of bile duct and other liver functions . the determination of impairment degree occurred by liver diseases is executed by specialists noting the clinical symptoms , results of the liver function test , and the results of the image test . we paid careful attention to the kams guidelines to satisfy the requirements of science , objectivity , convenience , rationality , and actuality . impairment evaluation should be conducted when the symptoms are in a fixed state , but if there is an expected change of symptoms , then another evaluation should be made two years later . in principle , the subject of impairment for medical evaluation should be as fixed symptoms that are left without recovery upon completion of treatment . the source of impairment does not have to result from traumas , but may also come from congenital diseases and just as a disease itself . the impairment should be evaluated by specialists of the appropriate fields of which it belongs to . we evaluated the loss of function in the digestive system as 100% impairment due to the possibility death from the loss of functions . we fixed the impairment rate in proportion to the impairment state with the relative rate to the loss before functioning . the upper digestive tract includes the esophagus , stomach , duodenum , small intestine , and pancreas . useful objective methods for confirming upper gastrointestinal tract impairments are : 1 ) fluoroscopy , contrast media using radiological tests and imaging studies such as a computed tomography ( ct ) or an magnetic resonance imaging ( mri ) , 2 ) cytology test or endoscopy including a biopsy , 3 ) esophageal manometry , 4 ) gastric acid secretory studies , 5 ) absorption abnormality test , 6 ) stool studies , and 7 ) helicobacter pylori urea breath test . also , fat content in the stool and intestinal malabsorption may be examined . the impairment rate , as signified in percentage , reflects the anatomical , physiological , and functional abnormality occurring in an organ or system and the ability to perform daily activities . patients belong to the normal scope of gastrointestinal impairments if patients are able to perform daily activities ; show regular and intermittent gastrointestinal manifestations without need of specific dietary treatment or medications ; and are able to keep normal weight with the necessary nutrition . we divided the upper gastrointestinal tract impairments into the stomach , the duodenum , the esophagus , and upper gastrointestinal tract after surgery ( table 1 - 3 ) . the lower gastrointestinal tract impairments include the colon , the rectum , and the anus . the signs and symptoms of the lower gastrointestinal tract impairment are : abdominal pain , pelvic pain , perineal pain , difficulty of defecation , tenesmus , stool incontinence , hematochezia , abscess , fissure , and fistula . in general findings , fever , weight loss , weakness , anemia , etc useful objective methods to confirm colon , rectum , and anus impairment are the following tests such as : 1 ) digital rectal examination , proctoscopy , sigmoidoscopy , colonoscopy , 2 ) biopsy , 3 ) microscopic examination of the stool and cultivation , 4 ) fluoroscopy and radiological test using contrast media , and 5 ) ct and mri examinations . the impairment rate , as signified in percentage , reflects the anatomical , physiological , and functional abnormality occurring in an organ or system and the ability to perform daily activities . patients belong to the normal scope of gastrointestinal impairments if patients are able to perform daily activities ; show regular and intermittent gastrointestinal manifestations without need of specific dietary treatment or medications ; and are able to keep normal weight with necessary nutrition . we divided the lower gastrointestinal impairments evaluation into the colon , rectum , anus , intestinal stomas and after surgery of the lower gastrointestinal tract ( table 4 - 7 ) . the symptoms of hepatic bile impairment are pain , nausea , vomiting , anorexia , general weakness , fever , jaundice , and itching . the symptoms of progressive liver disease complications are edema , ascites , esophageal varix , portal hypertension which generates bleeding , hepatic encephalopathy , metabolic impairment , and the loss of kidney function . useful objective methods to confirm liver impairments are 1 ) an abdominal sonogram , 2 ) radiological examination such as percutaneous and endoscopic cholangiography , 3 ) ct and mri , 4 ) liver isotope studies , 5 ) liver biopsy & fine needle aspiration biopsy , and 6 ) a laboratory test for diagnosis of bile duct and other liver functions . the determination of impairment degree occurred by liver diseases is executed by specialists noting the clinical symptoms , results of the liver function test , and the results of the image test . we developed an evaluation guidelines for digestive system impairments under the supervision of kams suitable to the conditions of korea by referring to the ama guides . the disabled people in korea , social environment , and hospital environment were taken into consideration while drawing up the guidelines . we worked to make it easy and convenient to use for both the subjects who receive the impairment evaluation and the doctors who give the evaluation . the guidelines of the impairment rating are the synthesis of science and public opinion ( 8) . the rating of digestive system impairments of kams are the clinically evaluated ratings of physical impairments . the physical impairment of ama guides are also the ratings of physical impairments . in order to satisfy the scientific characteristics , the impairment rate of ama has scientific characteristics and public trust to be used as a " global standard " ( 4 ) . to satisfy objectivity we considered the patient 's signs but did not consider the patient 's symptoms as much . for this we rated the impairment according to the objective signs and the results of examinations . we avoided excessively detailed evaluation for convenience , introducing a comprehensive evaluation method so that the evaluation of overall functions will be achieved . we made a sum total of the varied impairments of a specific area not to be greater than the total functional loss of the appropriate organ . this impairment evaluation is divided into the upper and lower gastrointestinal tracts and liver . in regarding the upper gastrointestinal tract of the stomach , duodenum , and esophagus , we also determined the impairment rate after surgery for the upper gastrointestinal tract , unlike the ama guides . the decision to include the impairment rate after surgery is because many people , who undertook surgical operation for the upper gastrointestinal tract , complain of the difficulty in performing daily life tasks , but no objective evaluation guidelines had existed in the u.s.a . as well as in korea for this situation . accordingly , the impairment evaluation after surgery for the digestive system would be useful . regarding the lower gastrointestinal tract , we made consensus on the evaluation guidelines for impairment of constructed intestinal stomas . stool incontinence generated from anal sphincter impairment was given special consideration . in choosing evaluation guidelines for the upper gastrointestinal tract , we considered weight loss . the measurement of weight loss is economical and easy in that both patients and doctors would agree objectively and it would be a useful evaluation guideline for upper gastrointestinal tract impairment . if the impairment can be cured with surgical treatment , the patient is to be reexamined after a year to receive an objective and rational evaluation . the child - pugh classification should be followed objectively in the case of liver impairment , with leftover function of the liver . complications such as ascites , spontaneous bacterial peritonitis , hepatic encephalopathy should be regarded , and then we made the entire impairment guidelines of liver disease objectively and easily . considering the reality , the present impairment guidelines needs supplementary and periodic improvements . with respect to the hospital environment , close examination might be needed for improved scientific evaluation . therefore , doctors should be able to rate impairments objectively with general equipment . in some patients that need more attention , referral system to a hospital with special equipment the evaluation of digestive system impairment is applied to a medically permanent impairment , a fixed physical state but not a temporary state of impairment . permanent impairment means a fixed impairment which has not changed a year after evaluation ( 5 , 6 , 8) . a certain time interval was set up for evaluation of the digestive system impairment after it is fixed . the fixation of symptoms is generally judged after completion of treatment , but it is not always the case . if it does not get worse or there is no possibility of getting better , it could be considered as a fixed symptom , even during treatment . if the symptom or impairment gets worse , another team of interval could be set up the patient . regarding the symptom without an objective evaluation tool with which most people would agree , such as pain , the committee agreed to defer the impairment evaluation until a useful evaluation tool is developed ( 9 , 10 ) .

in this study , we provide an overview of the korean burden of disease ( kbod ) study , which was the first study of its type to assess the national burden of disease in an advanced asian country , using disability - adjusted life years ( dalys ) as measures of disability . burden of disease measurements that employ dalys have been previously utilized by several studies conducted at international and national levels ( 1 - 5 ) . the dalys concept was initially developed by murray and lopez , and was applied for the first time in their authoritative global burden of disease ( gbd ) project ( 6 , 7 ) . moreover , its results have been published in a variety of forms , and researchers in many countries involved , and continue to make , recommendations at the national level and expedite programs that properly address the burden of disease issues raised by the gbd ( 3 , 8) . in korea , changing disease trends have been observed over recent years . specifically , the natures and magnitudes of the threats posed by contagious diseases have receded , whereas the prevalences of non - contagious and chronic diseases have increased ( 9 ) . moreover , such changes weigh heavily on the national healthcare system , and make the process of resource allocation a difficult proposition ( 10 ) . in this article , we describe some of the key findings of the kbod study , which was initiated in the early 2000s and continues today . in general , the kbod study followed the protocols of the original gbd study ( 6 ) . however , the kbod study differs in terms of the detailed methodology used with respect to ; the disease classification and epidemiological data estimation methods used , and the relative weighting of disabilities . in order to evaluate burden of disease and health care performance , a measure is needed that can be used to quantify scientific health status and to create a systematically organized classification system capable of comprehensibly including every important disease entity . daly is a summary measure that represents health status . using dalys , who arranged icd codes into 3 major groups , 16 sub - categories , and 93 third - level categories . however , decisions concerning the diseases that should be measured and the units of estimation used are as important as the need for a measurement index . in the present study , we developed a new disease classification that can be used to fully adjust the structure of diseases in korea and health care performance . first , we collected and addressed the disease classifications used by who and other dalys yielding procedures . third , the project units of public health activity and public health - related medical services that are in need of the evaluation of public health performances were comprehensibly grasped . fourth , representative data concerning the disease structure in korea such as major causes of death and frequent medical service utilizing diseases were investigated . fifth , based on the above , we developed a new disease classification , which included several added disease entities , and then consulted a team of clinical and public health specialists about the new disease classification system . our disease classification is based on the global burden of disease as defined by who , and adheres to the principle that a disease classification should be exhaustive and exclusive . on the other hand , considering the ease of approach offered by intervention , and attempt was made to avoid overly detailed disease classifications . in addition , we added diseases included in the korean national infectious disease surveillance system and the national cancer registration project . the following diseases ( all of which are currently included in the national communicable disease surveillance system ) were added to group i of the gbd classification ( communicable diseases ) ; herpes genitalia , cholera , typhoid and paratyphoid , shigellosis , mumps , rubella , and chicken pox . these were added because they were described as ' lower respiratory infections ' in the previous gbd classification . moreover , these diseases are included in the korean contagious disease prevention act and thus need to be monitored separately . in group 2 ( non - communicable diseases ) , gallbladder cancer , thyroid cancer , kidney cancer , brain cancer , and bone and cartilage cancer were added to the ' malignant neoplasm ' subcategory . these disease entities have been under continuous surveillance by the korean national cancer registry project , and are also included in the list of the 236 most frequent causes of death in korea . benign brain neoplasm was added to the ' other neoplasms ' subcategory , because the disease process and final results associated with brain neoplasms are similar to those of other malignancies . however , neuro - psychiatric diseases were addressed in a somewhat different manner . instead of simply adding locally important diseases to the current gbd classification consequently , we added a new disease group , which includes 19 disease entities , namely , 8 communicable diseases , 6 neoplastic diseases , and 5 neuro - psychiatric conditions . in order to determine the incidences and prevalence of all disease categories , we constructed a large normative cohort , representing the korean population in 1998 as a cooperative project with the national health insurance corporation of korea . the korean national health insurance system is an obligatory program that covers all 47 million koreans . korean national health insurance and related data sources have been previously described in detail ( 11 ) . we randomly sampled 1,209,693 persons , and stratified them for age into 5-yr age groups , sex , area of residence ( seoul / large city / other ) , and type of insurance ( employee / self - governor / medical aid ) . the korean normative cohort includes 2.5% of all koreans , with and overrepresentation of elderly , to add stability to estimated parameters ( table 1 ) . for the cohort , morbidity and mortality outcomes were followed using data links to the national health insurance ( knhi ) medical claim and korean national statistical office ( nso ) databases during the period 1998 until june 2002 . icd-10 codes which are used by both the knhi and nso were reclassified according to disease classification . prevalences ( per 1,000 persons per year ) were calculated after averaging the total number ( person - base ) of those who had been diagnosed during the follow - up period ( 1998 - 2002 ) . incidences were calculated for 2000 and 2001 after excluding patients who had visited hospital during the first two years ( i.e. , 1998 - 1999 ) . to reduce possible over - estimations of morbidity rates caused by using medical claim data , an operational definition for each disease category for example , cancer cases were counted only when the diagnosis was made in a general hospital , and there were two or more claims for the same disease category . moreover , all bona vide cancer cases deemed to have required more than one admission event . in terms of mortality , causes of death as reported by the nso were used as was . to further refine morbidity rates , 3,678 cases in 9 typical disease groups ( asthma , epilepsy , stroke , myocardial infarction , herniated intervertebral disk , rheumatoid arthritis , systemic lupus erythematosus , lung cancer , and common surgical conditions ) the concordance rate , which was defined based on " probable cases " and " confirmed cases " , was as low as 52.6% for asthma ( 298 acceptable cases out of 567 claims ) and as high as 84.1% for a herniated intervertebral disk ( 132 acceptable cases out of 157 claims ) . several factors were found to be associated with validity , i.e. , admission / outpatient , patient age , type of hospital , whether operations were performed or not , total cost , and length of admission . models were constructed to predict the probability of validity of each case , using the above medical utilization factors . original prevalence and incidences were recalculated by summing predicted probabilities to yield adjusted morbidity rates . adjusted morbidity rates were compared to those in previous independent korean reports when possible . in order to determine disability duration and average age at onset , we used the dismod ii model ( 12 ) . prevalence , incidences , and specific mortality rates were required to estimate disability duration and average age at onset . when developing our new classification system , it was found necessary to standardize and quantify a number of social preferences , in order to accurately deduce a set of disability weightings . multiple measurement methods were used to perform this task , these included person trade off ( pto ) , time trade off , visual analogue scale , and standard gamble . in the gbd study , however , a protocol was developed based on the pto measurement scheme ( 6 ) . in the present study , we selected 16 indicator conditions that fit the korean context , using three selection criteria as follows : health state should be meaningful in terms of public health , health status is most popular , and health status can be represented on a scale from 0 ( representing a good state ) to 1 ( representing a fatal state of health ) . after conducting a pilot test of the method using a group of medical students , the pto protocol was revised in order to render it more appropriate for this study . the 16 indicator conditions are as follows : chronic back pain , colon and rectum cancers , down syndrome , diabetes mellitus , peptic ulcer disease , stomach cancer , hepatitis b and hepatitis c , influenza , pneumonia , unipolar major depression , dementia , low birth weight , schizophrenia , iron - deficiency anemia , ischemic heart disease , epilepsy , and rheumatoid arthritis . the first measurement group contained one female doctor and the 2nd and 3rd groups two apiece . second , each of the three panels was then assigned preference weighting for the 16 indicator conditions , each group taking a different date . third , panel members took approximately 30 min to assign preference weightings to each of the health states , using a form developed for this study and by following a coordinator 's instructions , were based on pto1 and pto2 protocols , respectively . panel members recorded values to the questions asked in these variants , provided reasons for their answers , and discussed results with other members . after this process had been repeated three times , each of the members provided final values for pto 1 and pto2 variants . forth , disability weightings ( dw ) were calculated for each of the health states using the pto values obtained from the panels , as follows : dw=1 - 1000/pto1=1000/pto2 . fifth , median disability weights were recorded on the disability scale , for each of the 16 indicator conditions . then , for health states other than those covered by the 16 indicator conditions , each panel member was presented with a set of 37 health states ; 30 of which were selected from different categories and 7 from a list of common core diseases . panel members then assigned preference weights to a total of 123 health states , including the indicator conditions , by interpolation , using the disability scale that had been developed for the indicator conditions . finally , the study employed generalizability theory , which is commonly used in the reliability behavior theory , to estimate the relative magnitudes of various components of measurements ( generalizability study or g study ) , by analyzing the reliability coefficient ( g coefficient ) and ensuring the desired level of reliability ( decision study or d study ) ( 13 , 14 ) . the results of this g study revealed that the degree of contribution made by the 16 indicator conditions was 78.3% . this indicates that the total variance could be explained by the indicator conditions , and that the measurement errors associated with panels and panel members were essentially insignificant . in terms of total variance , the d study showed that the generalizability coefficient was greater than 0.9 in all nine , which suggests a high level of reliability . the generalizability coefficient associated with research design ( i.e. , indicator conditions , taking triplicorte measurements , 10 respondents for each panel ) was also found to be high , at 0.973 . after completing this process in general , the kbod study followed the protocols of the original gbd study ( 6 ) . however , the kbod study differs in terms of the detailed methodology used with respect to ; the disease classification and epidemiological data estimation methods used , and the relative weighting of disabilities . in order to evaluate burden of disease and health care performance , a measure is needed that can be used to quantify scientific health status and to create a systematically organized classification system capable of comprehensibly including every important disease entity . daly is a summary measure that represents health status . using dalys , who arranged icd codes into 3 major groups , 16 sub - categories , and 93 third - level categories . however , decisions concerning the diseases that should be measured and the units of estimation used are as important as the need for a measurement index . in the present study , we developed a new disease classification that can be used to fully adjust the structure of diseases in korea and health care performance . first , we collected and addressed the disease classifications used by who and other dalys yielding procedures . third , the project units of public health activity and public health - related medical services that are in need of the evaluation of public health performances were comprehensibly grasped . fourth , representative data concerning the disease structure in korea such as major causes of death and frequent medical service utilizing diseases were investigated . fifth , based on the above , we developed a new disease classification , which included several added disease entities , and then consulted a team of clinical and public health specialists about the new disease classification system . our disease classification is based on the global burden of disease as defined by who , and adheres to the principle that a disease classification should be exhaustive and exclusive . on the other hand , considering the ease of approach offered by intervention , and attempt was made to avoid overly detailed disease classifications . in addition , we added diseases included in the korean national infectious disease surveillance system and the national cancer registration project . the following diseases ( all of which are currently included in the national communicable disease surveillance system ) were added to group i of the gbd classification ( communicable diseases ) ; herpes genitalia , cholera , typhoid and paratyphoid , shigellosis , mumps , rubella , and chicken pox . these were added because they were described as ' lower respiratory infections ' in the previous gbd classification . moreover , these diseases are included in the korean contagious disease prevention act and thus need to be monitored separately . in group 2 ( non - communicable diseases ) , gallbladder cancer , thyroid cancer , kidney cancer , brain cancer , and bone and cartilage cancer were added to the ' malignant neoplasm ' subcategory . these disease entities have been under continuous surveillance by the korean national cancer registry project , and are also included in the list of the 236 most frequent causes of death in korea . benign brain neoplasm was added to the ' other neoplasms ' subcategory , because the disease process and final results associated with brain neoplasms are similar to those of other malignancies . however , neuro - psychiatric diseases were addressed in a somewhat different manner . instead of simply adding locally important diseases to the current gbd classification consequently , we added a new disease group , which includes 19 disease entities , namely , 8 communicable diseases , 6 neoplastic diseases , and 5 neuro - psychiatric conditions . in order to determine the incidences and prevalence of all disease categories , we constructed a large normative cohort , representing the korean population in 1998 as a cooperative project with the national health insurance corporation of korea . the korean national health insurance system is an obligatory program that covers all 47 million koreans . korean national health insurance and related data sources have been previously described in detail ( 11 ) . we randomly sampled 1,209,693 persons , and stratified them for age into 5-yr age groups , sex , area of residence ( seoul / large city / other ) , and type of insurance ( employee / self - governor / medical aid ) . the korean normative cohort includes 2.5% of all koreans , with and overrepresentation of elderly , to add stability to estimated parameters ( table 1 ) . for the cohort , morbidity and mortality outcomes were followed using data links to the national health insurance ( knhi ) medical claim and korean national statistical office ( nso ) databases during the period 1998 until june 2002 . icd-10 codes which are used by both the knhi and nso were reclassified according to disease classification . prevalences ( per 1,000 persons per year ) were calculated after averaging the total number ( person - base ) of those who had been diagnosed during the follow - up period ( 1998 - 2002 ) . incidences were calculated for 2000 and 2001 after excluding patients who had visited hospital during the first two years ( i.e. , 1998 - 1999 ) . to reduce possible over - estimations of morbidity rates caused by using medical claim data , an operational definition for each disease category for example , cancer cases were counted only when the diagnosis was made in a general hospital , and there were two or more claims for the same disease category . moreover , all bona vide cancer cases deemed to have required more than one admission event . in terms of mortality , causes of death as reported by the nso were used as was . to further refine morbidity rates , 3,678 cases in 9 typical disease groups ( asthma , epilepsy , stroke , myocardial infarction , herniated intervertebral disk , rheumatoid arthritis , systemic lupus erythematosus , lung cancer , and common surgical conditions ) the concordance rate , which was defined based on " probable cases " and " confirmed cases " , was as low as 52.6% for asthma ( 298 acceptable cases out of 567 claims ) and as high as 84.1% for a herniated intervertebral disk ( 132 acceptable cases out of 157 claims ) . several factors were found to be associated with validity , i.e. , admission / outpatient , patient age , type of hospital , whether operations were performed or not , total cost , and length of admission . models were constructed to predict the probability of validity of each case , using the above medical utilization factors . original prevalence and incidences were recalculated by summing predicted probabilities to yield adjusted morbidity rates . adjusted morbidity rates were compared to those in previous independent korean reports when possible . in order to determine disability duration and average age at onset prevalence , incidences , and specific mortality rates were required to estimate disability duration and average age at onset . when developing our new classification system , it was found necessary to standardize and quantify a number of social preferences , in order to accurately deduce a set of disability weightings . multiple measurement methods were used to perform this task , these included person trade off ( pto ) , time trade off , visual analogue scale , and standard gamble . in the gbd study , however , a protocol was developed based on the pto measurement scheme ( 6 ) . in the present study , we selected 16 indicator conditions that fit the korean context , using three selection criteria as follows : health state should be meaningful in terms of public health , health status is most popular , and health status can be represented on a scale from 0 ( representing a good state ) to 1 ( representing a fatal state of health ) . after conducting a pilot test of the method using a group of medical students , the pto protocol was revised in order to render it more appropriate for this study . the 16 indicator conditions are as follows : chronic back pain , colon and rectum cancers , down syndrome , diabetes mellitus , peptic ulcer disease , stomach cancer , hepatitis b and hepatitis c , influenza , pneumonia , unipolar major depression , dementia , low birth weight , schizophrenia , iron - deficiency anemia , ischemic heart disease , epilepsy , and rheumatoid arthritis . the first measurement group contained one female doctor and the 2nd and 3rd groups two apiece . second , each of the three panels was then assigned preference weighting for the 16 indicator conditions , each group taking a different date . third , panel members took approximately 30 min to assign preference weightings to each of the health states , using a form developed for this study and by following a coordinator 's instructions , were based on pto1 and pto2 protocols , respectively . panel members recorded values to the questions asked in these variants , provided reasons for their answers , and discussed results with other members . after this process had been repeated three times , each of the members provided final values for pto 1 and pto2 variants . forth , disability weightings ( dw ) were calculated for each of the health states using the pto values obtained from the panels , as follows : dw=1 - 1000/pto1=1000/pto2 . fifth , median disability weights were recorded on the disability scale , for each of the 16 indicator conditions . then , for health states other than those covered by the 16 indicator conditions , each panel member was presented with a set of 37 health states ; 30 of which were selected from different categories and 7 from a list of common core diseases . panel members then assigned preference weights to a total of 123 health states , including the indicator conditions , by interpolation , using the disability scale that had been developed for the indicator conditions . finally , the study employed generalizability theory , which is commonly used in the reliability behavior theory , to estimate the relative magnitudes of various components of measurements ( generalizability study or g study ) , by analyzing the reliability coefficient ( g coefficient ) and ensuring the desired level of reliability ( decision study or d study ) ( 13 , 14 ) . the results of this g study revealed that the degree of contribution made by the 16 indicator conditions was 78.3% . this indicates that the total variance could be explained by the indicator conditions , and that the measurement errors associated with panels and panel members were essentially insignificant . in terms of total variance , the d study showed that the generalizability coefficient was greater than 0.9 in all nine , which suggests a high level of reliability . the generalizability coefficient associated with research design ( i.e. , indicator conditions , taking triplicorte measurements , 10 respondents for each panel ) was also found to be high , at 0.973 . after completing this process the burdens imposed by major disease groups ( dalys lost per 100,000 of the population ) are shown in fig . 1 . cancer was found to be the most prominent cause of disease burden , with a score of 1,525 , followed by cardiovascular diseases ( 1,492 ) , digestive diseases ( 1,140 ) , diabetes mellitus ( 970 ) , respiratory diseases ( 951 ) , and neuro - psychiatric conditions ( 883 ) . the mortality burden of cancer ( ylls lost per 100,000 of the population ) was determined to be the leading cause of premature mortality , with a score of 1,222 , and this was followed by cardiovascular diseases ( 768 ) , digestive diseases ( 368 ) and diabetes mellitus ( 291 ) . digestive diseases were determined to be the leading causes of disability in korea ( injuries not included ) , with a yld of 853 per 100,000 individuals , followed by respiratory diseases ( 841 ) , neuro - psychiatric conditions ( 768 ) , and cardiovascular diseases ( 735 ) ( fig . 1 ) . a list of the top twenty leading causes of disease burden among men ( dalys lost per 100,000 of the population ) diabetes mellitus was found to be associated with the highest burden of disease with 1,020 dalys , followed by cva ( 937 ) , cirrhosis of the liver ( 671 ) , asthma ( 663 ) , and ischemic heart disease ( 601 ) . by individual disease , in men , diabetes mellitus exhibited the highest yld , at 801 per 100,000 individuals , followed by asthma ( 610 ) and peptic ulcer disease ( 547 ) ( fig . the top twenty leading causes of disease burden among women ( dalys lost per 100,000 of the population ) are shown in fig . 3 . diabetes mellitus was associated with the highest daly rate , with a score of 919 , followed by cardiovascular disease ( 900 ) , peptic ulcer disease ( 794 ) , and asthma ( 755 ) ( fig . peptic ulcer disease had the highest yld , at 788 , followed by diabetes mellitus ( 740 ) and asthma ( 710 ) ( fig . the burden attributable to unipolar major depression was determined to be twice as high in women than in men , whereas the disability burden associated with cirrhosis of the liver was determined to be 3.7 times as high in men than in women ( fig . 2 , 3 ) . in this study , the authors attempted to measure the burden of disease in korea , using dalys . this study represents one of the first examples of such calculations using epidemiologic data and disability weights derived in an asian country . daly , as applied in this study , is a single measurement that consists of a summation of time lost as a result of premature death and time lived under disabling conditions . the primary reason why this indicator has attracted the attentions of researchers in the public health area is that it can be used as a tool for simultaneously measuring the level of death and prevalence ( 8 , 15 ) . the developed classification described above has been found to be a useful tool for many health related studies in korea and allows comparative studies to be conducted between countries . who recommends that a summary measure should correctly reflect the characteristics of a country or region , and that to achieve an intensive study concentrated on a targeted nation should take priority over any other procedures . the results are expected to provide us with supportive data for evidence - based decision - making and health resource allocation . the results obtained reveal that the burden of disease in korea originates primarily from cancers , cardiovascular disease , digestive disease , diabetes mellitus , and neuro - psychiatric conditions ( fig . 1 ) . our findings in this regard are comparable with , but clearly different from , a similar study conducted in australia ( 3 ) . in australia , the burden of disease ranking in men per 100,000 persons using daly ( not including injuries ) was found to be cardiovascular disease , cancer , mental disorders , nervous system diseases , and chronic respiratory diseases ( 3 ) . our findings also clearly differ from those of the gbd study for the eme ( 3 , 6 ) , in which the top ranked diseases using dalys ( not including injuries ) were ischemic heart disease , cerebrovascular disease , dementia , tracheal , bronchial and lung cancer , unipolar major depression , and osteoarthritis ( 3 , 6 ) , whereas in korea , the top leading causes were diabetes mellitus , cerebrovascular disease , asthma , peptic ulcer disease , ischemic heart disease , and cirrhosis of the liver . these results indicate that the burden of digestive diseases , such as , peptic ulcer disease and cirrhosis of the liver , are higher in korea than in australia . korea 's higher level of digestive disease burden , especially its higher mortality is explained by a high prevalence of hepatitis b infection and the dietary habits of koreans , who traditionally consume great quantities of salty , highly spiced food ( 16 , 17 ) . first , some degree of uncertainty was introduced by estimating epidemiologic parameters using medical utilization data . especially , this study did not have exact information regarding utilization data of oriental medicine , pharmacy . however , we found that our results are in line with previous studies on estimated disease incidence in korea ( 8 , 18 ) . nevertheless , no prevalences or incidences were available for most disease categories , and thus , there remains a possibility that morbidity rates could have been over- or under- estimated ( 8) . especially , the possibility remains that morbidity rates of older age group could have been overestimated due to the over - sampling method in this study ( table 1 ) . moreover , the cohort approach used in the present study provided direct estimates and representative morbidity rates for all disease categories , with a certain degree of reliability . this is because death certificates are not always issued by physicians , and even when a diagnosis is rendered by a physician , discrepancies sometimes exist between recorded causes of death and actual causes ( 19 ) . third , it has also been found to be impossible to determine the burden of disease attributable to injuries in korea because resulting insurance claims do not contain details of injury type . moreover , given that the attributable burden of injuries is substantial , this failure to incorporate the influence of injuries into daly calculations constitutes a major limitation of this study . finally , the korean burden of disease study is limited by its lack of adjustment for comorbidities , which are likely to affect diseases like diabetes and ischemic heart disease and thus increase the burden of disease ( 5 ) . in conclusion , despite the limitations of the data utilized in this study , we are confident that daly is an appropriate and reasonably accurate tool in the public health area , as has been found in the netherlands and australia . moreover , the present study also provided the basic epidemiologic data to establish an evidence - based health policy .

a three month - old baby girl was admitted to the emergency department because of a seizure which occurred the previous day . there was no fever , and no definite history of trauma . she was a term baby with no perinatal problems such as birth trauma , and the findings of physical and laboratory examinations were unremarkable . mri performed on the day of admission showed left frontal subdural hematoma ( sdh ) . the sdh signal was heterogeneous ( high and low ) on t1-and t2-weighted images , suggesting rebleeding in chronic sdh . an eight - month - old baby boy was admitted to the emergency department with a seizure which occurred 40 minutes earlier . for three days he was a term baby with no perinatal problems , and the results of physical and laboratory examinations were unremarkable . this showed a mainly low signal on t1-weighted images and a high signal on t2-weighted images . on the right side day 22 , subdural catheter drainage was performed and about 400cc of chronic hematoma was evacuated . a two - month old baby girl was admitted to the emergency department on account of a seizure 16 hours earlier , and for three days she had had a fever . she was a term baby with no perinatal problems , and the findings of physical and laboratory examinations were unremarkable . mri performed on the day of admission showed sdh in both cerebral hemispheres and the posterior fossa . the hematoma showed a low signal on t1-weighted images and a high signal on t2-weighted images . a bilateral focal high signal was also noted on both t1- and t2-weighted images of occipital areas , suggesting rebleeding or hemorrhage of a different stage , and in addition , diffuse dural thickening and enhancement were observed . a three month - old baby girl was admitted to the emergency department because of a seizure which occurred the previous day . there was no fever , and no definite history of trauma . she was a term baby with no perinatal problems such as birth trauma , and the findings of physical and laboratory examinations were unremarkable . mri performed on the day of admission showed left frontal subdural hematoma ( sdh ) . the sdh signal was heterogeneous ( high and low ) on t1-and t2-weighted images , suggesting rebleeding in chronic sdh . an eight - month - old baby boy was admitted to the emergency department with a seizure which occurred 40 minutes earlier . for three days he was a term baby with no perinatal problems , and the results of physical and laboratory examinations were unremarkable . this showed a mainly low signal on t1-weighted images and a high signal on t2-weighted images . on the right side day 22 , subdural catheter drainage was performed and about 400cc of chronic hematoma was evacuated . a two - month old baby girl was admitted to the emergency department on account of a seizure 16 hours earlier , and for three days she had had a fever . she was a term baby with no perinatal problems , and the findings of physical and laboratory examinations were unremarkable . mri performed on the day of admission showed sdh in both cerebral hemispheres and the posterior fossa . the hematoma showed a low signal on t1-weighted images and a high signal on t2-weighted images . a bilateral focal high signal was also noted on both t1- and t2-weighted images of occipital areas , suggesting rebleeding or hemorrhage of a different stage , and in addition , diffuse dural thickening and enhancement were observed . sbs was first described by caffey in 1972 as whiplash baby syndrome , and acceleration - deceleration stress is known to be the cause of the injury ( 1 ) . approximately two - thirds of shaken babies suffer permanent brain damage or die ( 6 ) . violent shaking can produce significant damage , especially to the brain or head and neck ; this includes intracranial hemorrhage , retinal hemorrhage , and hemorrhage in the high cervical cord ( 4 , 5 ) . because their neck muscles are not strong enough to support their relatively large head compared with adults , and because their brains are not completely myelinated , infants are very susceptible to shaking ( 7 ) . sdh is the most common manifestation of head injury in sbs , and since sdh , especially chronic sdh , is a very rare cause of accidental injury among infants ( 3 , 8 , 9 ) , radiologists should suggest the possibility of sbs and child abuse . in young babies , whose csf space is larger than in adults , bridging veins can easily be torn during shaking ( 1 ) . if repeated shaking leads to injury , sdh can be chronic and hemorrhage may occurr during both the acute and chronic stages , as in our cases ( 9 ) . the presence of sdh of different ages is indicative of injuries from repeated abuse ( 2 ) . differentiation between acute and chronic sdh on the sole basis of a t1 or t2 signal can be difficult , and - as in our cases - dural enhancement observed at contrast - enhanced mri and probably due to a granulation tissue membrane can be a helpful finding for diagnosing chronic sdh ( 9 ) . other manifestations , such as subarachnoid hemorrhage , cerebral contusion and diffuse axonal injury can also occur in sbs , but only sdh was noted in our three cases . except for subarachnoid hemorrhage , mri is superior to ct in diagnosing these head injuries ( 9 ) . retinal hemorrhage is one of the characteristic signs of sbs and is known to result from an abrupt rise in intracranial pressure during shaking ( 7 ) . subacute bacterial endocarditis , anemia or thrombocytopenia can also lead to retinal hemorrhage , but these can be ruled out by clinical findings such as the results of laboratory tests ( 10 ) . bilateral retinal hemorrhage was found in two of three cases in this report ; in neither was there a history of birth trauma , and nor were the laboratory findings remarkable . conditions which can cause retinal hemorrhage , such as subacute bacterial endocarditis , anemia or thrombocytopenia , were ruled out by the clinical and laboratory findings in all our three cases . many reports have indicated that in the absence of any disease process , retinal hemorrhage is indicative of sbs ( 1 ) . in case three , fundoscopic examination was not performed , but a history of child abuse was revealed by the interview with the social worker ; adequate counseling of the patient and her family was thus possible . in none of the three cases was there a history of trauma and the infants ' parents or carers did not admit to any physical insults to the patients . many people do not know , however , that shaking a baby can cause severe injury , and thus fail to mention it during history taking ; in many instances , unless there is external evidence of injury , a diagnosis of sbs may therefore be delayed or even impossible . in detecting abnormal injuries and suggesting to the clinician the possibility of sbs , the radiologist 's role is thus very important and he or she can help prevent further damage due to repeated shaking . in summary , we have described three cases of sbs shown by mri to be chronic sdh . when radiologists interprete the images in cases involving head injuries to young basies , they should bear in mind the possibility of sbs , and by suggesting to the clinician that a particular case may involve this and child abuse , may be able to prevent further harm to a patient .

a 67-year - old female patient visited the hospital for acute debakey type i aortic dissection surgery . echocardiography showed 25% ejection fraction with a right ventricular wall motion abnormality , and the blood tests revealed creatine kinase - mb ( ck - mb ) at 220 mcg / l . for treatment of the acute myocardial infarct , the patient underwent emergency coronary stent insertion into the proximal right coronary artery ( rca ) stenosis lesion , which revascularized after the procedure ( fig . , the cause of the chest pain was thought to be acute myocardial infarction ( mi ) , but echocardiography and angiography showed the aortic dilatation , which was suggestive of the membrane of aortic dissection , and computed tomography ( ct ) was performed , which revealed acute debakey type i aortic dissection from the ascending aorta to the suprarenal artery . the mi appeared to be associated with acute debakey type i aortic dissection involving the proximal rca ( fig . 1c ) , and the ascending aorta and the descending aorta measured 52.0 and 30.8 mm in diameter , respectively . the right renal artery originated from the true lumen , and the left renal artery originated from the false lumen ( fig . the patient was then transferred to gangnam severance hospital , yonsei university health system . upon admission , however , there was a definite pupil reflex response , and ck - mb was at 13.35 mcg / l . a median sternotomy was performed , and dual cannulation was performed at the left femoral artery and the right axillary artery , which was anastomosed with an 8 mm vascular graft . after venous cannulation of the right atrium , cardio - pulmonary bypass ( cpb ) was started . when we open the aorta after unilateral scp , there was a big intimal tear at the distal ascending aorta . the aortic arch and the branches of the aorta were removed with the patient in a low temperature state with antegrade selective cerebral perfusion ( scp ) . we inserted a set with a 32 mm diameter , and 20 cm length ( 5 + 15 ; s&g biotech inc . , seongnam , korea ) to the descending aorta 1 to 2 cm posterior of the left subclavian artery through the introducer . during the operation , through trans - esophageal echocardiography the cpb time was 190 minutes , the aortic cross - clamping time was 69 minutes , the total cardiac arrest time was 50 minutes , and the scp time was 20 minutes . the patient 's postoperative state was stable , and she was followed up with ct . in debakey type i acute aortic dissection , emergency surgery is inevitable and the prognosis can not be ensured . to produce better results , the role of surgical treatment is the most critical . conventionally , in debakey type i acute aortic dissection , one of the following operations has been performed : ascending aorta graft replacement , total arch replacement , or hemiarch replacement . the ultimate goal of the operation is the complete resection of the intimal tear and the obliteration of the false lumen in order to prevent more dilatation of the remaining dissected descending aorta . due to this , the conventional approach has limitations . the conventional ( frozen ) elephant trunk technique was next introduced , but complications such as kinking and flapping emerged . extensive surgery has a high risk . the optimal treatment for acute debarkey type i aortic dissection has not been established . because of the need for a safe and comprehensive treatment , the technique of total arch replacement with a stented elephant trunk ( set ) graft has been introduced . a bare graft portion of an set ( s&g biotech inc . ) and a 4-branched graft were used for total arch replacement after set insertion using a specific introducer at 0.5 to 1 cm distal to the left subclavian artery . according to a german study in 2008 , the hybrid operation of total arch replacement with an set was found to be equivalent to the conventional operation ( total arch replacement only ) with respect to in - hospital mortality and morbidity . the set procedure has advantages such as false lumen obliteration of the descending aorta . in primary intimal tear of descending aorta , direct insertion of the stented graft into the descending aorta which is difficult to approach using mid - sternotermy , the stented graft could cover the tear and make false lumen obliteration by thrombus formation , as a result , the blood flow improves , which makes space for the true lumen that is compressed by the false lumen . also sealing with thrombus formation at the tearing focus can prevent progressive bleeding and dissection , and thrombus obliteration without tearing of the false lumen can prevent more dilatation of the aorta , and because of this , gradual normalization of the aorta can be expected . as a result , thrombus obliteration of the residual false lumen of the descending aorta lowers the risks of later dilatation and dissection , and can lower the reoperation rate , which in turn can reduce late mortality . jacob et al . found that 10% of their patient group had a high false lumen obliteration rate , which ranged between from 77% to 100% in review paper of murzi et al . and studies of sun et al . the usual indications for total arch replacement with set debakey i acute dissection are retrograde dissection involving the distal aortic arch or proximal descending thoracic aorta , and in young patients , it is critical to have a proper size and length of the stent to cover the aorta and sleeve dissection showing the detached adventitia . this hybrid operation can result in spinal cord injury due to the prompt thrombosis of the intercostal arteries of the descending aorta , and can result in the potential for false lumen reentry of the distal stent part in patients with marfan disease , who have a small true lumen , thus limiting the potential for surgery . when the true lumen aorta is too small and the tortuous aorta and aneurysmal aorta are more than 50 mm in size , staged surgery should be considered first . during preoperative decision making , it is critical to have the proper size and length of the stent and to check the shape of the true lumen of the aorta to avoid the aforementioned complications . liu et al . explained that for acute cases , the diameter of the stented graft should be 10% bigger than the diameter of the native proximal descending aorta , and that the proper location of the stent should be between t7 and t8 to avoid injury to the vital intercostal arteries . as a combination of the interventional procedure and conventional approach , total arch replacement with an set graft is feasible for repairing intimal tears and for promoting thrombus formation in the false lumen , and this procedure is expected to reduce the rate of late mortality and morbidity . this procedure can also be an alternative treatment for patients with acute debakey type i aortic dissection who have an intimal tear in the aortic arch and descending aorta .

it can be single or multiple , unilateral or bilateral , malformed morphologically or normal in size and shape , and erupted or impacted.[24 ] in the primary dentition , the incidence is said to be 0.3% to 0.8% and in the permanent dentition 1.5% to 3.5% . the prevalence of supernumerary teeth in the general caucasian population ranges between 1% to 3% , with prevalence of 2.7% and 3.4% among japanese and hong kong chinese populations , respectively . there appeared to be an increased frequency for males of nearly 2 : 1 , and more than 90% of all supernumeraries occur in the maxilla with a strong predilection for the premaxilla . phylogenetic process of atavism , the dichotomies of the tooth bud , hereditary , and a combination of genetic and environmental factors - unified etiologic explanation have been suggested . a hyperactive dental lamina is where the localized and independent hyperactivity of dental lamina is the most accepted cause for the development of the supernumerary teeth . however , the presence of supernumerary teeth may be part of developmental disorders such as cleft lip and palate , cleidocranial dysostosis , gardner 's syndrome , ellis - van creveld syndrome , ehlers - danlos syndrome , incontinentia pigmenti , and tricho - rhino - phalangeal syndrome . supplemental teeth have morphology similar to a tooth of the normal dentition , while rudimentary teeth are small , conical , or tuberculate . single supernumeraries occur in 76% to 86% of cases , double supernumeraries in 12% to 23% of cases , and multiple supernumeraries in less than 1% of cases . however , mesiodens that remains unerupted causes over - retained maxillary primary incisors , delayed or ectopic eruption of permanent central incisors , displacement and rotation of adjacent teeth , crowding , development of median diastema , eruption into the floor of the nasal cavity , and less frequently formation of primordial or follicular cysts with significant bone destruction , and root resorption of adjacent teeth . when these complications are anticipated , surgical removal of the supernumerary tooth followed by orthodontic treatment is indicated . early surgical intervention is preferred treatment to prevent clinical problems and to minimize further complications . the case report documented four unusual cases of supernumerary teeth presented as multiple non syndromic supernumeraries in the premaxilla , supernumerary tooth with macrodontia of permanent incisor , inverted mesiodens , and supernumerary causing concomitant extrusion and intrusion of adjacent teeth . a 12-year - old boy reported to the department of pedodontics and preventive dentistry , rohtak , with the chief complaint of delayed eruption in relation to upper left front teeth and irregular arrangement of those present . a thorough general examination was carried out to rule out the presence of any syndrome . intraoral examination revealed permanent dentition with a supernumerary tooth , made up of more than one cusp or tubercle present palatal to 11 displacing it in a labial direction . furthermore , a soft tissue buldge was seen associated with unerupted 21 labially [ figure 1a ] . radiographic examination revealed presence of another supernumerary in relation to 21 hindering its eruption [ figure 1b ] . buccal object rule was applied and tooth was found to be in palatal position . on consultation , it was decided to remove both the supernumerary , surgically wait till eruption of 21 , followed by orthodontic alignment of remaining upper teeth . orthodontic treatment was started to align the incisors [ figure 1d ] . the patient is now under regular review regarding future - fixed orthodontic treatment . preoperative view showing upper anterior supernumerary ( tuberculate type ) opg showing impacted midline supernumerary hindering eruption of 21 surgical exposure of impacted supernumerary postoperative view showed well - aligned incisors a 12-year - old boy presented with a chief complaint relating to unesthetic appearance of upper front teeth . supernumerary tooth ( conical in shape ) was located in between two central incisors displacing 11 labially and distally resulted in slight palatal displacement of 12 [ figure 2a ] . crown structure of 11 appears somewhat larger than its normal - appearing counterpart tooth , i.e. , 21 . radiographic examination disclosed presence of a mesiodens ( conical ) with completed root formation causing displacement of 11 distally [ figure 2b ] . treatment plan consisted of removal of mesiodens and retained right primary canine followed by orthodontic intervention . initially , orthodontic treatment consisted of bracketing erupted upper teeth 15 - 25 , except for 12 . on further follow ups when 11 had moved mesially sufficiently , creating space for 12 , it was banded and bracketed subsequently . patient was maintained on follow ups and showed a satisfactorily outcome without complications [ figures 2c and 2d ] . preoperative view showing conical supernumerary resulted in displacement of 11 iopa showing midline supernumerary postoperative view showing aligned incisors a 9-year - old boy presented with displaced upper front teeth . intraoral examination showed mixed dentition with a mesiodens , conical in type caused rotation and impaction of upper left and right central incisors , respectively . on palpation , a prominent soft tissue elevation was seen associated with unerupted 11 , labially as well as palatally . right lateral incisor was erupting in rotated position in palatal direction from its mesial half [ figure 3a ] . radiographic examination revealed another supernumerary tooth with completed root formation , inverted in alignment wedged between a clinically noted mesiodens and 11 , hindering its eruption [ figure 3b ] . using buccal object rule , treatment was instituted to surgically extract both the supernumeraries [ figure 3c ] and to facilitate alignment and space closure using upper arch 2 by 4 fixed orthodontic therapy . after banding and bracketing 11 , 12 , 21 , and 22 , initially a ligature wire was placed in between 11 and 21 followed by continuous elastic chain to allow space closure [ figure 3d ] . the early treatment goals were achieved without complication and the patient is under regular review . preoperative view showing rotation and impaction of upper central incisors maxillary occlusal view showing inverted mesiodens showing surgical removal of supernumerary a 12-year - old boy reported with complaint of an extra tooth in between two upper front teeth causing an unesthetic appearance . intraoral examination revealed permanent dentition with the presence of a mesiodens deviating 21 labially and cervically . there was mild extrusion in relation to 11 and palatal inclination of mesial half of 22 [ figure 4a ] . radiographic examination showed presence of conical supernumerary wedged between upper two central incisors [ figure 4b ] . treatment involved extraction of the mesiodens , subsequently followed by upper 2 by 4 fixed orthodontic therapy . brackets were placed on 11 , 12 , 21 , and 22 , followed by banding over 16 and 26 [ figure 4c ] . also , continuous elastic chain was applied over bracketed teeth , changed on follow - up visits . six to eight months postoperatively , both central incisors were well aligned ; closing diastema [ figure 4d ] and patient was kept on follow up with removable retention appliance . preoperative view showing midline supernumerary causing concomitant extrusion and intrusion maxillary occlusal view showing mesiodens showing fixed 2 by 4 orthodontic therapy a 12-year - old boy reported to the department of pedodontics and preventive dentistry , rohtak , with the chief complaint of delayed eruption in relation to upper left front teeth and irregular arrangement of those present . a thorough general examination was carried out to rule out the presence of any syndrome . intraoral examination revealed permanent dentition with a supernumerary tooth , made up of more than one cusp or tubercle present palatal to 11 displacing it in a labial direction . furthermore , a soft tissue buldge was seen associated with unerupted 21 labially [ figure 1a ] . radiographic examination revealed presence of another supernumerary in relation to 21 hindering its eruption [ figure 1b ] . buccal object rule was applied and tooth was found to be in palatal position . on consultation , it was decided to remove both the supernumerary , surgically wait till eruption of 21 , followed by orthodontic alignment of remaining upper teeth . orthodontic treatment was started to align the incisors [ figure 1d ] . the patient is now under regular review regarding future - fixed orthodontic treatment . preoperative view showing upper anterior supernumerary ( tuberculate type ) opg showing impacted midline supernumerary hindering eruption of 21 surgical exposure of impacted supernumerary postoperative view showed well - aligned incisors a 12-year - old boy presented with a chief complaint relating to unesthetic appearance of upper front teeth . medical and family histories were unremarkable . dental history revealed exfoliation of 63 four days back . supernumerary tooth ( conical in shape ) was located in between two central incisors displacing 11 labially and distally resulted in slight palatal displacement of 12 [ figure 2a ] . crown structure of 11 appears somewhat larger than its normal - appearing counterpart tooth , i.e. , 21 . radiographic examination disclosed presence of a mesiodens ( conical ) with completed root formation causing displacement of 11 distally [ figure 2b ] . treatment plan consisted of removal of mesiodens and retained right primary canine followed by orthodontic intervention . initially , orthodontic treatment consisted of bracketing erupted upper teeth 15 - 25 , except for 12 . on further follow ups when 11 had moved mesially sufficiently , creating space for 12 , it was banded and bracketed subsequently . patient was maintained on follow ups and showed a satisfactorily outcome without complications [ figures 2c and 2d ] . preoperative view showing conical supernumerary resulted in displacement of 11 iopa showing midline supernumerary postoperative view showing aligned incisors medical and family histories were unremarkable . intraoral examination showed mixed dentition with a mesiodens , conical in type caused rotation and impaction of upper left and right central incisors , respectively . on palpation , a prominent soft tissue elevation was seen associated with unerupted 11 , labially as well as palatally . right lateral incisor was erupting in rotated position in palatal direction from its mesial half [ figure 3a ] . radiographic examination revealed another supernumerary tooth with completed root formation , inverted in alignment wedged between a clinically noted mesiodens and 11 , hindering its eruption [ figure 3b ] . using buccal object rule , treatment was instituted to surgically extract both the supernumeraries [ figure 3c ] and to facilitate alignment and space closure using upper arch 2 by 4 fixed orthodontic therapy . after banding and bracketing 11 , 12 , 21 , and 22 , initially a ligature wire was placed in between 11 and 21 followed by continuous elastic chain to allow space closure [ figure 3d ] . the early treatment goals were achieved without complication and the patient is under regular review . preoperative view showing rotation and impaction of upper central incisors maxillary occlusal view showing inverted mesiodens showing surgical removal of supernumerary a 12-year - old boy reported with complaint of an extra tooth in between two upper front teeth causing an unesthetic appearance . intraoral examination revealed permanent dentition with the presence of a mesiodens deviating 21 labially and cervically . there was mild extrusion in relation to 11 and palatal inclination of mesial half of 22 [ figure 4a ] . radiographic examination showed presence of conical supernumerary wedged between upper two central incisors [ figure 4b ] . treatment involved extraction of the mesiodens , subsequently followed by upper 2 by 4 fixed orthodontic therapy . brackets were placed on 11 , 12 , 21 , and 22 , followed by banding over 16 and 26 [ figure 4c ] . also , continuous elastic chain was applied over bracketed teeth , changed on follow - up visits . six to eight months postoperatively , both central incisors were well aligned ; closing diastema [ figure 4d ] and patient was kept on follow up with removable retention appliance . preoperative view showing midline supernumerary causing concomitant extrusion and intrusion maxillary occlusal view showing mesiodens showing fixed 2 by 4 orthodontic therapy multiple tuberculate supernumerary teeth occurring in the absence of any syndrome have rarely been reported . they have more than one cusp ; appear either alone or in pairs usually palatal to the permanent incisors and very often obstructs their eruption . it is speculated that they represent a third dentition and thus are different in origin from supplemental teeth . yusof , in a literature review of multiple supernumerary teeth occurring in the absence of a syndrome , found the anterior maxilla to be an unusual site for this occurrence as found . similar findings were reported in case 1 where two supernumerary teeth ; both tuberculate in type , one erupted and other impacted , were found in upper anterior region that resulted in delayed eruption and displacement of 21 and 11 , respectively . . unusual larger or bulbous crown structure of 11 as depicted in case 2 might have been because of fusion between a supernumerary and a permanent tooth bud . rajab and hamdan and liu et al . stated that supernumerary teeth are frequently normally orientated . our findings were supported by tay et al . who studied unerupted maxillary anterior supernumerary teeth and observed that most cases are in an inverted position . furthermore , in case 4 , concomitant intrusion as well as extrusion was noted in relation to upper central incisors by a mesiodens , often not reported . it is essential to enumerate and identify the teeth present clinically and radiographically before a definitive diagnosis and treatment plan regarding supernumerary teeth can be formulated . radiographs played an important role to rule out the presence of impacted supernumerary teeth or other associated anomalies . furthermore , buccal object rule serves as an essential criteria and helpful in defining an accurate position of a supernumerary tooth , either buccal or palatal , as used in case 1 and 3 . timing of interceptive treatment should be as soon as possible following clinical detection of an abnormal eruption pattern . if a significant delay , i.e. , more than six months in the eruption of maxillary central incisors with respect to its antimere was noted , the presence of mesiodens should be suspected and investigated radiographically . a panoramic radiograph is a most useful screening radiograph in such situations as it shows all areas of the maxilla and mandible . the options include removal of the supernumerary only , removal of the supernumerary , and orthodontic treatment to re - establish sufficient space , with or without surgical exposure of the unerupted tooth at the time of supernumerary tooth removal . gomes et al . verified that the most common treatment of choice was surgery followed by orthodontic therapy , i.e. , in 62.0% of cases . it is in the best interest of the patient to have the tooth removed immediately on discovery unless the surgery would be unjustifiably hazardous to the adjacent structures . furthermore , an immediate surgical removal was indicated after diagnosis because intra- or postoperative complications were less likely to occur . in the present cases , there was a very low risk of iatrogenic damage to adjacent permanent incisors root according to the clinical and radiographic findings since root development of the central incisors was complete . also , surgical procedure was simple ; patients were cooperative and are more receptive to surgical management under local anesthesia and thus easier to manage . however , when unerupted teeth are symptom less , do not appear to affect dentition in any way , it is best to be left in place and kept under observation . the potential disadvantages associated with this deferred surgical plan include loss of eruptive force of adjacent teeth , loss of space and crowding of the affected arch , and possible midline shifts . in their study , 23 children aged less than 11 years and 17 aged greater than 11 years at the time of supernumerary removal , hogstrum and andersson found no evidence of root resorption , loss of vitality , or disturbance of root development during the three - year follow - up period . however , clinical complications were seen in 88.5% of the patients with st , with displacement and failure of eruption being the most frequent . moreover , a possible relationship between st and other developmental anomalies , such as dens evaginatus and talon cusps , has been proposed . st premolars appeared to be more prevalent in chinese children with dens evaginatus than in the general population . in the present case reported , supernumerary teeth resulted in varying degrees of orthodontic problems , i.e. , rotation , crowding , displacement , and delayed eruption of permanent incisors necessitating surgical removal ; facilitate spontaneous eruption of the permanent central incisors . spontaneous eruption following supernumerary removal is suggested to be in the range of 54% to 75% . in case 1 and 3 dibiase suggests that most teeth experiencing delayed eruption will spontaneously erupt within 18 months of supernumerary removal alone , provided the delayed tooth is not excessively displaced and have a potential to erupt . furthermore , if the roots of the incisors are completely or nearly formed , there may be diminution of eruptive potential that will necessitate orthodontic treatment , as carried out in the present cases . whenever supernumerary teeth are diagnosed , single or multiple , a decision regarding the appropriate management should be made carefully . in our opinion , therefore , it is important to initiate appropriate consultation and each case should be individually assessed . . the clinician should have thorough knowledge of signs suggesting the presence of supernumerary teeth including non- or delayed eruption , alterations in the eruptive pattern , diastema formation , midline shift , and crowding . on appropriate diagnosis , early intervention is required in the form of surgical or orthodontic treatment and combination in order to minimize unwanted side effects to the developing dentition . the cases described above represent a small sample of the possible presentations for cases involving supernumerary teeth .

in recent years , the role of mutations in non - protein - coding regions has come to light in the pathogenesis of different neuromuscular diseases , so that the terms toxic rna and spliceopathy have been increasingly referred to pathological conditions in which accumulation of mutant rnas results in a deleterious gain - of - function deregulating transcript processing and protein synthesis in multiple metabolic pathways . one of the best studied examples of rna - dominant disease is myotonic dystrophy ( dm ) , an autosomal dominant disorder characterised by a variety of multisystemic features including muscular dystrophy with increased number of centrally located or clumped nuclei in muscle fibres , myotonia ( muscle hyperexcitability ) , dilated cardiomyopathy , cardiac conduction defects , insulin - resistance , cataracts , and disease - specific serological abnormalities . two forms of dm are presently known : the more severe dm1-steinert 's disease ( omim 160900 ) , caused by an expanded ( ctg)n nucleotide sequence in the 3 ' untranslated region of the dystrophia myotonic protein kinase ( dmpk ) gene ( omim 605377 ) on chromosome 19q13 , and the milder form dm2 ( omim 602688 ) , caused by the expansion of the tetranucleotidic repeat ( cctg)n in the first intron of the zinc finger protein ( znf)-9 gene ( omim 116955 ) on chromosome 3q21 . in the cell nucleus , the gene primary transcripts undergo molecular processing to generate mature rnas , which are finally exported to the cytoplasm : these mrna maturation events are chronologically and spatially ordered , and mostly occur on distinct rnp - containing structures . by combining biomolecular techniques with the analysis in situ of the nuclear organization and molecular composition , it has been demonstrated that the expanded - cug- or ccug - containing transcripts , in dm1 and dm2 cells respectively , are retained in the cell nucleus , and accumulate in the form of rnp - containing focal aggregates . these nuclear foci specifically sequester the alternative splicing regulators cug - binding protein 1 ( cugbp1 ) and muscleblind - like 1 ( mbln1 ) protein , which are necessary for the physiological processing of pre - mrna , especially for contractile protein synthesis . these focal aggregates are considered as a biomolecular feature of dms , and have been detected in several adult tissues as well as in cultured cells from dm patients . these foci also sequester hnrnps and snrnps , i.e. splicing factors involved in the early phases of the pre - mrna processing , thus strengthening the hypothesis that the multifactorial phenotype of dystrophic patients may result from a more general alteration of the pre - mrna post - transcriptional pathway . recently , it has been demonstrated that mbnl1 accumulate in the nuclear foci during interphase but , at mitosis , the foci relocate to the cytoplasm where they undergo degradation , while newly - formed foci develop in the nucleus of the daughter cells as a consequence of de novo accumulation of expanded rnas . therefore , in proliferating cells , the cyclic release from the nucleus of the foci and their cytoplasmic degradation would prevent the massive intranuclear sequestration of nuclear factors ; on the contrary , in non cycling cells , the nuclear foci do not undergo intracellular relocation / degradation and progressively increase in number and size . this dynamic behaviour of nuclear foci is compatible with the evidence that in dm patients the most affected organs or tissues are those where non - renewing cells are mainly present , such as the skeletal muscle , heart and the central nervous system , whereas cells from self - renewing tissues ( such as skin fibroblasts or layering epithelial cells ) are much less affected . accordingly , measuring intranuclear foci in skeletal muscle biopsies taken from patients at different times it has been demonstrated that the mbnl1-containing foci actually become larger with increasing patient 's age . in addition to the formation of intranuclear foci , dm1 and dm2 cells show an altered distribution of nuclear ribonucleoprotein ( rnp)-containing structures and molecular factors responsible for pre - mrna transcription and maturation . in particular , by means of ultrastructural immunocytochemistry on skeletal muscle biopsies from dm1 and dm2 patients , it has been shown that splicing and cleavage factors accumulate in the intranuclear functional sites where they usually localize , sometimes ectopically relocating also into nuclear rnp domains where they generally do not occur ( unpublished results ) . this accumulation could hamper the functionality of the splicing machinery and slow down the intranuclear molecular trafficking thus reducing the metabolic activity of myonuclei , consistent with recent findings demonstrating a reduced protein synthesis in dm1 and dm2 myoblasts . during ageing , the skeletal muscle undergoes a progressive loss of mass , strength and function , in the process known as sarcopenia . sarcopenia affects healthy , physically active subjects : the rate of muscle loss in humans has been estimated to range 1 to 2% per year after age of fifty . therefore , sarcopenia represents a great risk factor for frailty , loss of independence and physical disability in elderly , since it is associated with decreased functional performance , higher risk of falls and motor function impairment . the mechanisms underlying age - related skeletal muscle wasting and weakness are probably manifold and still remain to be fully elucidated ; however , although no specific therapy is presently available to counteract its onset or progress , studies performed on humans and other mammals have stressed the importance of physical exercise as an effective , although still debated , approach to prevent or limit the age - related muscle mass loss . interestingly , the sarcopenic process is characterised by structural and functional alterations of the skeletal muscle that are reminiscent of myotonic dystrophy . in fact , the aged muscle shows grouped atrophy , fibre size variability and centrally located nuclei . in addition , factors involved in the post - transcriptional processing of pre - mrna have been found to accumulate not only in the nucleoplasmic rnp - containing structures where they usually locate but also in ectopic nuclear domains . this intranuclear accumulation/ delocalization of rnp structures containing splicing and cleavage factors has been found not only in the skeletal muscle but also in other tissues ( e.g. , liver , brain ) of aged mammals . moreover , aged cells undergo malfunctions of the degradation systems both in the cytoplasm and in the nucleus with accumulation of crosslinked insoluble molecules ( including non - coding rnas ) which hampers the intracellular transport mechanisms . this suggests that in ageing cells the entire production chain of mrna , from its synthesis to the cytoplasmic export , becomes less efficient , likely contributing to the reduced capability of cells to positively react to metabolic stimuli , which typically occurs in elderly . this loss of responsiveness would have particularly severe effects in skeletal muscles , where a misregulated protein turnover would result in a structural imbalance between muscle protein degradation and the restoring protein synthesis . a recent in vitro study reported that satellite - cell - derived myoblasts from dm2 patients show cell - senescence alterations ( e.g. , cytoplasmic vacuolisation , reduction of the proteosynthetic apparatus , accumulation of heterochromatin and impairment of the pre - mrna maturation pathways ) earlier than the myoblasts from healthy patients ; moreover , when grown in a differentiation medium dm2 myoblasts fuse into multinucleated myotubes exhibiting structural defects similar to those observed in senescent myotubes from healthy patients . the early occurrence of senescence - related features in satellite cell - derived myoblasts suggests that satellite cells from dm2 patients have a reduced regeneration capability , which would contribute to the muscular dystrophic phenotype . the cytochemical and ultrastructural evidence demonstrates that the skeletal muscle of dm patients shares intriguing similarities with the muscle from aged individuals in several nuclear features , especially in the altered nuclear rnp - containing structures involved in pre - mrna transcription and splicing . this opens interesting perspectives on the role of the rnp nuclear components in the onset of muscle cell dysfunctions and encourages comparative studies aimed at detecting common cellular mechanisms at the basis of skeletal muscle wasting . finally , it is worth noting that the analysis in situ of the organization and molecular composition of nuclear domains is a powerful tool not only for getting information about the dna / rna pathways which govern cellular metabolism , but also for detecting the occurrence of cell dysfunctions related to pathological phenotypes .

various abdominal exercises are used in physical therapy for the treatment of low back injury and as a component of fitness training programs1,2,3,4 . clinicians should be knowledgeable about the types of exercise and training programs that would most effectively recruit abdominal muscles , as frail elderly individuals are unable to exert the effort required for strenuous exercise . the activities of the rectus abdominis ( ra ) , external oblique ( eo ) , and internal oblique ( io ) muscles during performance of abdominal exercises in combination with expiration have been investigated because abdominal muscles are the principal muscles involved in forced expiration5 , 6 . yoon et al.5 reported that the activities of the transversus abdominis and io ( tra / io ) are significantly greater during an abdominal curl - up with slow expiration than during an abdominal curl - up with quiet inspiration . ishida and watanabe6 showed that abdominal muscle activities ( ra , eo , and io ) increased when maximum expiration was held in the side bridge exercise compared with resting expiration . these studies used exercises requiring sub - maximal voluntary contraction of the abdominal muscles to quantify the additive effects of expiration . the capacity for muscular activity is related to the number of active motor units and their level of activation . if motor units are activated in a task - specific manner , a muscle s maximal activity will differ with the task . it is possible that the highest muscular activity would occur during a dual task condition ( maximum trunk flexion with expiration ) in which there would be a summation of the activities of two task - specific motor unit populations . to the best of our knowledge , no study has measured the muscle activities of abdominal muscles during maximum trunk flexion with expiration . one of the reasons for selecting the maximum trunk flexion exercise is its ease of performance compared to other abdominal strengthening exercises . the purpose of this study was to demonstrate the effects of expiration on abdominal muscle activity during maximum trunk flexion . twenty - one healthy university students ( 10 men , 11 women ) participated in this study . their age , height , and weight ( mean sd ) were 21.3 2.0 years , 164.6 8.4 cm , and 59.5 12.2 kg , respectively . the study was explained to the subjects , whose consent was obtained prior to their participation . the protocol for this study was approved by the ethics committee of the kawasaki university of medical welfare . electromyography ( emg ) was conducted using the mq8 system ( kissei comtec , japan ) . disposable silver / silver chloride surface electrodes with a recording diameter of 1 cm ( blue sensor p-00-s , ambu , denmark ) were used . the emg signals of the right ra , eo , and tra / io muscles were recorded . electrode placement was based on previous work that described the following positions for these muscles5 , 6 : ra , electrode positioned 2.5 cm lateral to the umbilicus ; eo , electrode positioned at the anterior end of the eighth rib ; and tra / io , electrode positioned horizontally within a triangle consisting of the inferior border made up of the line from the anterior - superior iliac spine ( asis ) to the pubic symphysis , and the superior border made up of the line from one asis to the other . wide straps secured the thorax , pelvis , and thighs during maximum voluntary trunk flexion in the supine position ( fig . subjects were asked to hold maximum trunk flexion for 5 s. during trunk flexion the following 2 respiration patterns were performed : ( 1 ) holding the breath after resting expiration with an open airway ( breath holding ) ; and ( 2 ) breathing out from the end of resting expiration to the maximum expiration level over 3 s in the middle of the task ( slow expiration ) . while the subjects performed each task , emg signals were measured for 3 s in the middle of the task . the emg signals were amplified , digitized at a sampling frequency of 1,000 hz , band - pass filtered ( 10500 hz ) , and full - wave rectified . the mean muscle activity values over the 3-s sample of the 3 trials performed for each of the 2 tasks were averaged and used in the statistical analysis . the paired t - test was used to examine the significance of differences in the abdominal muscle activities between maximum trunk flexion with breath holding and slow expiration . the mean muscle activity values are listed in table 1table 1.the mean sd values of the abdominal muscle activitybreath holding ( mv)slow expiration ( mv)ra0.15 0.110.14 0.11eo0.08 0.050.06 * : p < 0.05 . the eo muscle showed a significantly lower muscle activity during maximum trunk flexion with slow expiration than during breath holding . the ra and tra / io muscle activities were not significantly different between the tasks . to the best of our knowledge , this study is the first to investigate the effects of performing a maximum trunk flexion exercise with expiration on emg activity of the abdominal muscles . our results show that maximum trunk flexion combined with slow expiration did not change the ra and tra / io activities compared to breath holding . previous studies have shown that expiration activates abdominal muscles during trunk exercises ( side bridge exercise and abdominal curl - up exercise)5 , 6 . side bridge exercise elicited 17.6% maximum voluntary contraction ( mvc ) in the ra , 26.9% mvc in the eo , and 20.3% mvc in the io6 . to classify low and high muscle activities , a previous study categorized 0% to 20% mvc as low muscle activity , 21% to 40% mvc as moderate muscle activity , 41% to 60% mvc as high muscle activity , and greater than 60% mvc as very high muscle activity7 . the difference between the results of our study and those of other studies might be related to the intensity of the tasks , since a very high - load abdominal exercise was used in this study , compared to low to moderate - load abdominal exercises in previous studies . in this study , the difficulty in augmentation of muscle activities in healthy young subjects , who were capable of exerting effort during exercise requiring exertion , may be due to the activity level of the abdominal muscles during maximum trunk flexion being too high to recruit more motor units during slow expiration . in this study , maximum trunk flexion with slow expiration when a subject tries to move a body part , excitation of the cortical units responsible for the muscles moving that part is accompanied by the inhibition of the cortical units responsible for the neighboring muscles8 . because the ra is the principal muscle of trunk flexion and the deep abdominal muscles are principal muscles of slow expiration1 , 9 , 10 the limitation of this study lies in the inclusion of only healthy young subjects in the analysis . as part of the normal aging process , there is an overall decline in skeletal muscle mass and strength , and the elderly are unable to make the necessary effort during exercise requiring exertion . further investigation is necessary to determine whether expiration changes the abdominal muscle activity during maximum trunk flexion exercise in the elderly . future studies should also investigate more than one exercise to examine abdominal muscle activity and to generalize the findings .

metabolic and cardiovascular diseases are well known to be more prevalent in obese individuals than non - obese individuals ( 1 ) . more specifically , increased visceral adipose tissue , the fat that surrounds the internal organs ( e.g. , heart , intestines ) in the cavities of the body , predicts an unfavorable cardiovascular and metabolic risk profile ( 15 ) . for example , visceral adiposity is strongly correlated with diabetogenic features ( e.g. , impaired insulin sensitivity , increased insulin levels ) , atherogenic features ( e.g. , increased triglycerides , decreased high density lipoproteins ) , pro - thrombotic factors ( e.g. , increased fibrinogen , factor vii , plasminogen activator inhibitor 1 ) and pro - inflammatory cytokines ( e.g. , interleukin-6 [ il-6 ] and tumour necrosis factor - a [ tnf- a ] ) ( 15 ) . accurately and reliably allows for better cardiovascular and metabolic risk stratification of patients and may be beneficial at the community level . visceral adipose tissue quantification may also serve to measure the effectiveness of pharmacotherapy or physical activity regimens in targeting visceral adipose tissue , thus serving as a therapeutic endpoint . various methods to quantify visceral adipose tissue directly by expensive magnetic resonance imaging and radiation - exposing computed tomography , as well as indirectly by anthropometric measures ( e.g. , waist - circumference , body - mass index ) exist ( 69 , 27 ) . magnetic resonance imaging is the gold standard technique to accurately measure visceral adiposity although there is some concern about the accuracy of actual visceral adiposity content based on single slice sampling ( i.e. , whole body magnetic resonance imaging scan is the true gold standard ) ( 7 , 10 ) . waist - circumference as measure of visceral obesity may be less reliable in older persons ( 11 , 12 ) . also , waist - circumference may be a better measure of subcutaneous rather than visceral obesity ( 11 , 12 ) . anthropometric techniques requiring measurement of the waist ( e.g. , waist - circumference , waist - to - hip ratio ) may not account for possibly confounding subcutaneous adipose tissue especially in more obese individuals though waist circumference is considered an accepted measure of intra - abdominal adiposity ( 11 , 12 , 13 ) . body - mass index , an anthropometric measure of visceral adiposity is suggested to be a poorer indicator of cardiovascular risk than waist - circumference across ethnicities , suggesting that body - mass index may not be a very good measure of visceral obesity ( 14 ) . in light of the limitations , lack of practicality of existing methods and the recognition that more reliable measures of visceral adiposity are needed , iacobellis et al . proposed the direct measurement of epicardial adipose tissue thickness via echocardiography as a marker for visceral adiposity ( 15 ) . in this article , we review epicardial adipose tissue s basic characteristics , method of clinical assessment , and reliability in measuring visceral adiposity , as well as identify areas that need further study . adapted from a recent review on epicardial adipose tissue s relationship with the heart , figure 1 illustrates the macroscopic appearance of adipose tissue on a normal and hypertrophied heart ( 16 ) . typically , normal fat distributions in the heart are restricted to the grooves between the atrium and ventricles , between the ventricles and along the coronary arteries ( 16 ) . as the mass of epicardial adipose tissue increases , figure 2 shows the massive amount of epicardial adipose tissue that is deposited between the pericardium and heart in an obese patient undergoing coronary artery bypass . both tissues share the same coronary blood supply and no fascia - like structure separates the epicardial adipose tissue from myocardium ( 17 ) . the specific function of epicardial adipose tissue is yet to be determined though it is known to be very active ( e.g. , considered a secretory organ ) ( 18 ) . the tissue is not known to serve to protect the heart from blunt trauma ( 16 ) . interestingly , though the tissue is thought to be important , the tissue does not exist in laboratory mice or rats ( 19 ) . marchington s animal experimental studies showed that the principle difference between epicardial adipose tissue and other visceral adipose tissues is its greater capacity for free fatty acid release ( 19 ) . thus , epicardial adipose tissue may act as a local energy supply for the myocardium or buffer against toxic levels of free fatty acids ( 19 ) . epicardial adipose tissue is a very active endocrine organ and may have an effect on local coronary artery health . for example , epicardial adipose tissue is shown to express various inflammatory factors such as il-1beta , il-6 , mcp-1 ( monocyte chemotactic protein-1 ) , and tnf - alpha mrna in high risk cardiac patients ( e.g. , coronary artery disease ) ( 20 , 21 ) . the tissue also secretes adiponectin , a serum protein known to be secreted exclusively by adipose tissues , with anti - inflammatory and anti - atherogenic properties ( 18 , 22 ) . however , adiponectin secretion is markedly decreased in patients with coronary artery disease compared to those patients without the disease ( 23 ) . studies show that epicardial adipose tissue is associated with increased ventricular mass ( 17 ) . a recent study we conducted also demonstrates that atria enlargement and impairment in diastolic filling is associated with epicardial adipose tissue in morbidly obese subjects ( 24 ) . epicardial fat thickness is measured on the free wall of the right ventricle from both parasternal long- and short - axis views ( figure 3 ) ( 15 ) . the largest amount of epicardial fat is usually seen at this right ventricular free wall site . epicardial adipose tissue is usually seen as an echo - free or if it is massive , hyper - echoic space . it is important to ensure that epicardial fat thickness is not measured obliquely since it falsely increases measurements . since there is some variation in the longitudinal thickness of epicardial fat , three measurements are usually taken and averaged in various research studies that demonstrate the reliability of epicardial fat thickness as a marker of visceral adiposity . the reliability of epicardial adipose tissue thickness on the free wall of the right ventricle is good when comparing with baseline magnetic resonance imaging ( 25 ) . it is also relatively cost and time efficient since it may be part of the routine assessment that patients suspected to be at risk for cardiovascular or metabolic illness undergo . first , though epicardial adipose tissue thickness is not as clearly measured as in magnetic resonance imaging , as already mentioned , the method is highly reliable . second , at times it is possible to confuse pericardial fluid with adipose tissue since the fluid also shows up as relatively echo - free on an echocardiograph ( 15 ) . several studies have demonstrated the reliability of epicardial adipose tissue s thickness measurement via echocardiography as a marker for visceral adiposity ( and the related metabolic and cardiovascular risk ) ( 15 , 17 , 20 , 2427 ) . epicardial adipose tissue has been shown to be very closely related to intra - abdominal adiposity , a marker of entire body visceral adiposity , according to various magnetic resonance imaging studies ( 15 , 20 ) . for example , an excellent correlation between epicardial adipose tissue and waist circumference ( r = 0.895 , p = 0.01 ) and magnetic resonance image abdominal visceral adipose tissue ( r = 0.864 , p = 0.01 ) is reported ( 15 ) . in the same study , multiple regression analysis showed that epicardial adipose tissue thickness was the strongest independent variable correlated to magnetic resonance image visceral adipose tissue ( r2 = 0.442 , p = 0.02 ) ( 15 ) . visceral adiposity is associated with features of cardiovascular risk profile and metabolic syndrome ( 2527 ) . epicardial fat thickness has been shown to be directly associated with many of these features that include increased ldl cholesterol , fasting insulin ( r2 = 0.387 ; p = 0.03 ) , left ventricular mass ( r = 0.755 , p = 0.01 ) and diastolic blood pressure ( r2 = 0 . 387 ; p = 0.02 ) , as well as decreased adiponectin ( 25 , 26 ) . furthermore , waist - circumference , considered a good indicator of visceral adiposity , is confounded by subcutaneous abdominal fat thickness ( 28 ) . it is well known that visceral adiposity rather than subcutaneous adiposity is more responsible for the health risks associated with fat deposition in humans ( 29 ) . epicardial adipose tissue measurements are not confounded by sub - cutaneous fat ( 16 ) . thus , given the poor reliability of waist circumference as a measure of visceral adiposity , echocardiographic measurement of visceral adipose tissue likely provides a truer visceral fat content estimate . the epicardial adipose tissue measurement via echocardiography technique to predict visceral adiposity and their associated health risks is relatively young ( the technique was suggested in 2003 ) . thus far , various studies suggest it is an extremely useful and effective method , and one that is increasingly important as more and more anatomic , bimolecular , and clinical relationships are found with epicardial adipose tissue ( 26 ) . nevertheless , various questions remain unanswered or are in the process of being answered in various studies we are conducting . the use of the technique to measure epicardial adipose tissue thickness , as well as the relationship between epicardial adipose tissue thickness and health risks , has been mostly limited to european - caucasian subjects . whether or not epicardial adipose tissue allows for prediction of cardiometabolic alterations in non - caucasian subjects remains to be determined . the use of epicardial adipose tissue thickness as measured by echocardiography as an effective therapeutic or physical health endpoint remains to be determined . often , exercise regimens may not decrease a person s weight or fatty appearance . physical activity is shown to promote fat catabolism and muscle anabolism ( one reason for the constant weight ) . secondly , it is the visceral adiposity that is the important marker of health risks . indeed , measuring epicardial adipose tissue , a marker of visceral adiposity , may be more important than using change in weight or a less reliable measure of visceral adiposity as a marker of cardiometabolic fitness . many questions remain about the molecular relationships of epicardial adipose tissue that may support the importance of measuring epicardial adipose tissue thickness . additionally , studies on the relationship of thickness of epicardial adipose tissue to secretion of adipocytokines are limited ( 3031 ) . finally , various additional questions remain that will be answered as further studies regarding epicardial adipose tissue are conducted , including the reliability of the echocardiographic technique between users and the ability for widespread use of the techique .

major postoperative complications in intracranial lesions have greatly reduced with the introduction of modern neurosurgical devices and microsurgical techniques . however , cerebrospinal fluid ( csf ) leakage after craniotomy occurs not infrequently and has continued to be the significant cause of minor postoperative complications such as wound dehiscence , local infection and meningitis . most of the complications are well cured but in some cases , failure in infection control not only leads to prolonged hospitalization but also affects the patient 's quality of life . to prevent these complications , various techniques such as the use of fibrin glue , preparation of galeal or pericranial flaps and prophylactic lumbar drain have been described3,11,16,17 ) . in this study , we introduce a surgical technique , so called " galeal tack - up suture " for the prevention of csf collection in subgaleal space . this study included 87 patients who underwent craniotomy for various lesions such as brain tumor , vascular and traumatic lesions from december 2012 to may 2013 . the patients were randomly allocated to either one of the two groups , the wound closure with " galeal tack - up suture " ( group a ) or routine wound closure ( group b ) . there were 32 patients in group a and 55 patients in group b. we prospectively compared the frequency of postoperative subgaleal csf collection in two groups until the wound has been stitched out . all minor subgaleal csf collections which could be easily cured by compression with elastic bandage were also included . in all patients , routine dural tack - up sutures are placed at the craniotomy margin to hold the dura against the bone edge in attempt to prevent postoperative epidural hematoma . dural csf leak was checked with intraoperative valsalva maneuver in all cases to ensure the water - tight dural closure . after the bone flap has been fixed with cranial plate , central dural tenting suture passing through the small holes burred previously was made , to fix the dural flap to the bone flap . upon closing the scalp , one of the three methods of " galeal tack - up suture " was used ( fig . first method of " galeal tack - up suture " utilizes the pairs of small hole burred for central dural tenting suture . 4 - 0 vicryl attached to curved needle is passed through the hole from external side of bone flap to internal side , and then passed to external side again through the adjacent hole . for second method , pairs of v - shaped holes are burred on the surface of bone flap but not piercing through it . single piece of straight titanium plate can be installed independently to be used as an anchor . this anchor is especially useful in cases of suboccipital craniotomy or foramen magnum decompression where making of holes directly ( straight or v - shaped ) is difficult . three to five suture materials are prepared depending on the size of the bone flap . while closing the scalp in layer by layer fashion , galea aponeurotica is sutured with the bone flap using the previously prepared sutures ( fig . 2 ) . the chi - square test was used to test for an association between galeal tack - up suture and postoperative subgaleal csf collection . the association between age and sex , and the incidence of subgaleal csf collection was tested using the logistic regression test . routine dural tack - up sutures are placed at the craniotomy margin to hold the dura against the bone edge in attempt to prevent postoperative epidural hematoma . dural csf leak was checked with intraoperative valsalva maneuver in all cases to ensure the water - tight dural closure . after the bone flap has been fixed with cranial plate , central dural tenting suture passing through the small holes burred previously was made , to fix the dural flap to the bone flap . upon closing the scalp , one of the three methods of " galeal tack - up suture " was used ( fig . first method of " galeal tack - up suture " utilizes the pairs of small hole burred for central dural tenting suture . 4 - 0 vicryl attached to curved needle is passed through the hole from external side of bone flap to internal side , and then passed to external side again through the adjacent hole . for second method , pairs of v - shaped holes are burred on the surface of bone flap but not piercing through it . single piece of straight titanium plate can be installed independently to be used as an anchor . this anchor is especially useful in cases of suboccipital craniotomy or foramen magnum decompression where making of holes directly ( straight or v - shaped ) is difficult . three to five suture materials are prepared depending on the size of the bone flap . while closing the scalp in layer by layer fashion , galea aponeurotica is sutured with the bone flap using the previously prepared sutures ( fig . the chi - square test was used to test for an association between galeal tack - up suture and postoperative subgaleal csf collection . the association between age and sex , and the incidence of subgaleal csf collection was tested using the logistic regression test . a total of 87 cases of craniotomy for intradural lesions were done in 75 patients . six patients underwent repeated surgery for brain tumor , one of them for three times . two patients underwent revision surgery for persistent csf leakage , 1 patient underwent revision surgery for subdural empyema , and 2 patients underwent cranioplasty for craniectomy state in which dural tearing occurred while dissecting due to dura - galeal adhesion . there were 35 males and 40 females with mean age of 49 years old ( range : 1 - 82 ) . intradural lesions included 32 cases of vascular lesion , 36 cases of brain tumor , 13 cases of trauma , 3 cases of congenital lesion , and 3 cases of infection ( table 1 ) . galeal tack - up suture was performed in 32 cases and routine wound closure without galeal tack - up was done in 55 cases . the three different galeal tack - up suture techniques were applied according to the size , location , and shape of the bone flap where it is appropriate . twelve cases occurred in group b patients where only routine wound closure was done and there was 1 case of postoperative csf collection in group a patients with galeal tack - up suture . in supratentorial lesions , galeal tack - up suture tended to be effective ( p=0.051 ) but was not proven statistically significant . however , galeal tack - up suture applied in total sum of cases showed significant value statistically ( p=0.026 ) ( table 2 ) . among 13 patients with subgaleal csf collection , 8 patients were treated with percutaneous aspiration followed by compression with elastic bandage and 1 patient with lumbar drain . in 4 patients , routine treatment has failed to stop subgaleal csf collection so revision surgery had to be done . there was no statistical significance between age ( p=0.401 ) , sex ( p=0.154 ) and the incidence of subgaleal csf collection . csf collected in subgaleal space can be diagnosed by several methods such as brain computed tomography , magnetic resonance imaging but it can also be detected by simply touching the operation site which feels like water - filled balloon8 ) . it is usually considered one of the minor postoperative complications but if left untreated , it can cause severe brain infection such as meningitis14 ) . it also results in prolonged hospital stay , increased treatment costs , and revision surgery in some cases12,14 ) . usually , it can be treated by bed rest with head elevation , percutaneous aspiration followed by compression with elastic bandage , and installation of lumbar drain but sometimes requires repeated surgery for csf fistula6,9,14 ) . it has been accepted that careful watertight dural closure is imperative procedure for prevention of postoperative csf leakage after the operation for intradural lesion1,2,13 ) . application of fbrin glue on the dural suture line has also been known to be effective measure for prevention of postoperative csf leakage10,15,17 ) . but even with all the preventive measures , some cases of csf leakage incurs in the operation for intradural lesions . grotenhuis5 ) reported that in the series of 412 patients of nontraumatic neurosurgical cases , postoperative csf leakage occurred in 44 ( 10.7% ) patients including self - limiting subcutaneous minor csf collection . kwon et al.7 ) reported that in the series of 47 cases which underwent orbital roof craniotomy and superolateral orbital craniotomy , 13 ( 27.6% ) cases of periorbital csf collection had occurred . csf leaks through the dural suture lines and defects which were failed to close properly during the operation . leaked csf is pooled in epidural and subgaleal spaces and disrupts the adhesion between galea and cranial vault by creating dead space filled with csf . it can also prevent leaked csf to pool in the epidural space . by performing galeal tack - up suture , single large subgaleal space is removed and divided into small multiple subgaleal spaces and galea becomes tightly adhered to the cranial vault which prevents csf to pool . goel4 ) previously described ' tenting ' stitches for the scalp can effectively reduce subgaleal collection of csf in 1991 . they utilized pericranium - galeal tenting but we did not use it since the technique is more difficult than galeal - cranium tenting and also the suture strength is weak . kato et al . reported a csf leak rate of 0.5% by using galea - cranium suture method in their technical note in 1999 . they utilized v - shape holes and plate but the clinical data was not suggested . among the surgical techniques suggested in the two technical reports , we chose the appropriate methods to apply in clinical setting and the results were statistically analyzed . our study indicated that galeal tack - up suture is an effective and easy - to - perform technique for prevention of postoperative subgaleal csf collection which does not require additional costs . all three techniques are essentially the same that aims to attach galea to the cranium and remove the space for csf to pool . these different techniques can be chosen and applied in most of the craniotomies with different size , shape , and location of bone flaps where it is appropriate . even with only minor efforts , galeal tack - up suture can effectively prevent subgaleal csf collection in surgical wound closure . in various situations of craniotomy

the surgery is accepted as a predominant method in the treatment of erectile dysfunction ( ed ) . in the domestic literature about penile prosthesis implantation , however , not many comparative studies have been conducted to assess ejaculatory function and erectile function after the surgery . given this background , we examined ejaculatory function and erectile function after surgery in patients with ed who underwent penile prosthesis implantation . a total of 127 patients who underwent penile prosthesis implantation among patients who were diagnosed with organic ed from january 2000 to december 2008 were included . the patients had scores of 0 to 6 points on the erectile function domain of the international index of erectile function ( iief - ef ) before therapy , as determined by a retrospective chart analysis . after we excluded 2 patients ( 1.6% ) who had a mechanical failure postoperatively but continued their daily lives without specific treatments and 4 patients ( 3.1% ) who underwent removal of the penile prosthesis due to mechanical failure ( 2 patients ) or infection ( 2 patients ) , 121 patients were assigned to the surgery group . the patients ' mean age was 56.548.89 years , and the mean follow - up period was 68.2441.04 months ( table 1 ) . a single operator performed the surgery in all surgery groups , for which the ams 700 cxm ( ams , minneapolis , usa ) was used as an inflatable prosthesis and the ams 600 ( ams , minneapolis , usa ) was used as a malleable prosthesis . we also enrolled patients who were diagnosed with organic ed and who were prescribed to take viagra ( pfizer , new york , usa ) through the outpatient department of urology from january 2000 to december 2008 . the nonsurgical patients had scores of 7 to 24 points on the iief - ef before therapy and reported having sexual intercourse more than twice during the recent 6-month period . a total of 120 of these patients were randomly selected and then assigned to the nonsurgery group . for the first questionnaire survey tool , we used the iief - ef and the intercourse satisfaction domain of the iief ( iief - is ) . scores for each item as well as the total score were obtained . in all patients in the surgery group the questionnaire was consistently performed for all patients who were assigned to the surgery group during a period from july to december 2009 . in the nonsurgery group , as the second tool , we used the ejaculation domain of the male sexual health questionnaire ( mshq - ejd ) . with this tool , a questionnaire survey was performed preoperatively and at postoperative year 1 in 32 patients in the surgery group who underwent implantation of a penile prosthesis from january 2006 to december 2008 . the questionnaire was consistently performed for these 32 patients during a period from july to december 2009 . in the nonsurgery group , the questionnaire study was performed in 40 patients during the same period before drug administration and 1 year later . after the questionnaire survey , the differences in the iief - ef , iief - is , and mshq - ejd between before and after treatment were compared between the surgery group and the nonsurgery group . then , comparisons were made within the surgery group between the inflatable group and the malleable group . moreover , in the inflatable and malleable groups , the postoperative period was classified as 5 years and > 5 years , and comparisons were made of differences in the iief - ef and iief - is recorded preoperatively and from july to december 2009 . another comparison was made for the mshq - ejd recorded from july to december 2009 . after we excluded six patients who did not use the penile prosthesis or underwent removal of the penile prosthesis ( mechanical failure : 4 ; infection : 2 ) , we additionally evaluated postoperative complications in the surgery group . for comparisons between the surgery group and the nonsurgery group , the inflatable group and the malleable group , and by the postoperative period of each inflatable and malleable group , statistical analysis was performed by use of student 's t - test with spss for windows version 16.0 ( spss inc , chicago , usa ) . for comparisons between the surgery group and the nonsurgery group , the inflatable group and the malleable group , and by the postoperative period of each inflatable and malleable group , statistical analysis was performed by use of student 's t - test with spss for windows version 16.0 ( spss inc , chicago , usa ) . comparing the surgery group and the nonsurgery group , the mean differences in iief - ef and iief - is between before and after treatment were 10.922.47 and 8.032.95 in the surgery group and 6.841.14 and 5.361.37 in the nonsurgery group . the mean differences in iief - ef and iief - is between before and after treatment were 11.392.98 and 6.561.35 in the inflatable group and 10.582.61 and 6.141.18 in the malleable group . comparing the items on the iief - ef and iief - is , however , erection confidence and intercourse satisfaction were 2.190.48 and 2.470.37 in the inflatable group and 1.780.45 and 2.010.32 in the malleable group . these differences were statistically significant ( p<0.05 ) ( table 3 ) . in the inflatable group and the malleable group , comparison was made of the mean differences in iief - ef and iief - is between preoperatively and postoperatively . in this analysis , the mean differences in iief - ef and iief - is between preoperatively and postoperatively were 11.782.98 and 6.871.35 in cases of 5 years and 11.362.61 and 6.481.18 in cases of > 5 years in the inflatable group . in the malleable group , the respective values were 11.652.98 and 6.581.35 in cases of 5 years and 11.472.61 and 6.491.18 in cases of > 5 years . these results indicate that the mean differences in iief - ef and iief - is between preoperatively and postoperatively were lower in the subgroups with a postoperative time of 5 years , but this difference was not statistically significant ( table 4 ) . comparing the surgery group and the nonsurgery group , the mean difference in mshq - ejd between before and after treatment was 3.810.67 in the surgery group and 3.720.20 in the nonsurgery group . the surgery group was subdivided into the inflatable group and the malleable group and the mean difference in mshq - ejd was evaluated between before and after treatment . in this analysis , this difference was not statistically significant ( table 3 ) . in the inflatable group and the malleable group , the postoperative period was classified as 5 years and > 5 years and mshq - ejd was compared between periods . in the inflatable group , the mshq - ejd was 20.143.67 in cases of 5 years and 15.244.12 in cases of > 5 years . in the malleable group , it was 21.974.00 in cases of 5 years and 17.653.58 in cases of > 5 years . there were three cases of mechanical failure , three cases of infections , and one case of erosion in the inflatable group . in the malleable group , there were one case of infection and one case of postoperative pain . these results suggest that the incidence of complications was relatively higher in the inflatable group ( table 5 ) . comparing the surgery group and the nonsurgery group , the mean differences in iief - ef and iief - is between before and after treatment were 10.922.47 and 8.032.95 in the surgery group and 6.841.14 and 5.361.37 in the nonsurgery group . the mean differences in iief - ef and iief - is between before and after treatment were 11.392.98 and 6.561.35 in the inflatable group and 10.582.61 and 6.141.18 in the malleable group . comparing the items on the iief - ef and iief - is , however , erection confidence and intercourse satisfaction were 2.190.48 and 2.470.37 in the inflatable group and 1.780.45 and 2.010.32 in the malleable group . these differences were statistically significant ( p<0.05 ) ( table 3 ) . in the inflatable group and the malleable group , comparison was made of the mean differences in iief - ef and iief - is between preoperatively and postoperatively . in this analysis , the mean differences in iief - ef and iief - is between preoperatively and postoperatively were 11.782.98 and 6.871.35 in cases of 5 years and 11.362.61 and 6.481.18 in cases of > 5 years in the inflatable group . in the malleable group , the respective values were 11.652.98 and 6.581.35 in cases of 5 years and 11.472.61 and 6.491.18 in cases of > 5 years . these results indicate that the mean differences in iief - ef and iief - is between preoperatively and postoperatively were lower in the subgroups with a postoperative time of 5 years , but this difference was not statistically significant ( table 4 ) . comparing the surgery group and the nonsurgery group , the mean difference in mshq - ejd between before and after treatment was 3.810.67 in the surgery group and 3.720.20 in the nonsurgery group . the surgery group was subdivided into the inflatable group and the malleable group and the mean difference in mshq - ejd was evaluated between before and after treatment . in this analysis , this difference was not statistically significant ( table 3 ) . in the inflatable group and the malleable group , the postoperative period was classified as 5 years and > 5 years and mshq - ejd was compared between periods . in the inflatable group , the mshq - ejd was 20.143.67 in cases of 5 years and 15.244.12 in cases of > 5 years . in the malleable group , it was 21.974.00 in cases of 5 years and 17.653.58 in cases of > 5 years . there were three cases of mechanical failure , three cases of infections , and one case of erosion in the inflatable group . in the malleable group , there were one case of infection and one case of postoperative pain . these results suggest that the incidence of complications was relatively higher in the inflatable group ( table 5 ) . to examine erectile function after surgery , we compared the mean differences in iief - ef and iief - is between before and after treatment in the surgery group and the nonsurgery group . our findings showed that the mean differences were significantly higher in the surgery group . according to a study by martin - morales et al , the satisfaction rate was found to be 40% to 58% in patients who were given pde-5 inhibitors . in patients who underwent implantation of a penile prosthesis , the satisfaction rate was found to be 85% to 90% . according to mulhall et al , who compared the erectile function domain and satisfaction domain on the iief between preoperatively and postoperatively , dramatic improvement occurred within the first year after implantation of the penile prosthesis . in particular , regarding the satisfaction domain , improvement was found to be more significant in the latter half of the first year than in the former half . based on these studies of the differences between preoperatively and postoperatively , in patients who were depressed with their daily lives or sexual life because of ed , unless there was a mechanical failure of the prosthesis after the surgical treatment , persistent erections could occur at ordinary times . in addition to the postoperative degree of satisfaction , erectile confidence was shown to rise . this indicates that the mean differences in iief - ef and iief - is were significantly higher in the surgery group . the differences in iief - ef and iief - is between before and after treatment were compared in the inflatable group and the malleable group but no significant differences were found . when we compared each item on the iief - ef and iief - is , however , scores indicating erection confidence and intercourse satisfaction were significantly higher in the inflatable group . in our series , the inflatable prosthesis provided the advantages of not only making the erection possible in such a way that the natural appearance of the penis was maintained but also that the erection could be directly controlled at any time by the patients themselves . in addition , compared with the malleable prosthesis , the inflatable prosthesis was relatively more expensive and its characteristics in causing a feeling of superiority in patients might affect the results accordingly . we also classified the postoperative period as 5 years and > 5 years and then compared the differences in iief - ef and iief - is between preoperatively and postoperatively between the subgroups . in both the inflatable and the malleable group whose period of postoperative course exceeded 5 years , the mean values were lower but this was not statistically significant . in this regard , there have been reports about the postoperative natural history of penile prostheses suggesting that the survival rate between inflatable and malleable prostheses is significantly different . according to this study , we had speculated that there might be a significant difference in outcome with the inflatable prosthesis , but this was not the case . we propose that the reason for this lack of difference may be that in the recruitment of subjects , patients who did not receive the treatment despite the presence of a mechanical failure or those who had a prosthesis removed because of severe infection were excluded from the analysis . factors like mechanical failure and severe infection of penile prosthesis could not affect the results since the early stage . even in cases in which a revision surgery is performed , kava et al showed that revision surgery for a penile prosthesis is associated with a high rate of success and high degree of satisfaction . in the study by kava et al , even in cases in which a revision surgery was performed , its course and surgical outcomes were good and there was no significant difference . meanwhile , considering the surgical technique and the anatomical structure of the penis , it is possible that a penile prosthesis compresses the urethra and that this may impede ejaculation . in one case report , the inflatable prosthesis compressed the seminal vesicles and this caused ejaculatory pain . in another study , however , the implantation of a penile prosthesis did not interfere with ejaculation or orgasmic functioning . this might be associated with the findings in this study of no statistical significance in the comparison of the difference in mshq - ejd between before and after treatment . on the other hand , according to gan et al , the factors causing ejaculatory dysfunction include age , social impairment , and ed . in patients who had a prosthesis implanted during a long - term period , the increased frequency of lower urinary tract symptoms as well as the decreased ejaculatory function due to the natural course of aging could induce ejaculatory disorder ; accordingly , in cases of > 5 years , mshq - ejd was determined to be significantly lower . finally , concerning complications , the inflatable prosthesis had a higher complication rate than did the malleable type . with reference to other reports , the inflatable types had a higher complication rate and a lower survival rate than did the malleable types . in our series , the complication rate was relatively higher for the inflatable prosthesis than for the malleable prosthesis . in patients with the inflatable prosthesis minervini et al compared inflatable and malleable prostheses in 482 patients who underwent penile prosthesis implantation . the rate of mechanical failure was 13.7% and 0.5% , respectively , as major complications ; the postoperative infection rate was 15% and 5.1% ; and the erosion rate was 7.5% and 5.1% . according to this , the complication rate was also higher in the inflatable prosthesis . according to the results of the present study , subjective symptoms such as erectile confidence and erectile function were improved after penile prosthesis implantation compared with nonsurgical treatments , and these findings were marked for the inflatable prosthesis . on the other hand , our findings suggest that there was no significant difference in ejaculatory function depending on the treatment method , but that ejaculatory function decreased as time passed .

hydrogen gas has been extensively used in chemical field such as fuel processing , fertilizer production ( 3h2 + n2 2nh3 ) and so on . it is a colourless , odourless , non - metallic , tasteless , highly flammable diatomic gas which was considered as a physiological inert gas . found that inhalation of a mixture ( 2.5% o2 and 97.5% h2 ) at a total of 0.8 mpa for 2 weeks caused a marked regression of the skin squamous cell carcinoma in a mouse model , and they tried to elucidate the phenomenon with the possibility of h2 as a free radical decay catalyser . in 1988 , a paper by buxton et al . demonstrated that h2 could reduce hydroxyl radicals ( oh ) produced by radiolysis or photolysis of h2o in cell - free systems . in 2001 , it was demonstrated that schistosomiasis - associated chronic liver inflammation was significantly attenuated by one normal atmosphere supplemented with 0.7 mpa h2 in mice . however , these investigations did not draw attention from researchers . in 2007 , ohsawa et al . discovered that hydrogen gas has antioxidant and antiapoptotic properties that protect the brain against ischaemia reperfusion injury and stroke by selectively neutralizing hydroxyl and peroxynitrite radicals . since then , hydrogen gas has come to the forefront of therapeutic medical gas research . recent basic and clinical research has revealed that hydrogen is an important physiological regulatory factor with antioxidant , anti - inflammatory and antiapoptotic protective effects on cells and organs [ 510 ] , proving that hydrogen could down - regulate cytokines , including ccl2 , il-1 , il-6 , il-12 , tnf- , etc . we also have proposed and proved that hydrogen has radioprotective effects in cultured cells and mice [ 1117 ] . it also has been demonstrated that h2 is effective in the prevention of cerebral , myocardial , hepatic ischaemia since 2009 , hydrogen was applied on the field of organ transplantation including intestinal transplantation , lung transplantation , renal transplantation and heart transplantation . it was demonstrated that hydrogen could protect allograft function in those models [ 1923 ] . however , the potential effect of hydrogen gas on another transplantation type is largely ignored . allogeneic hsct is a potentially curative therapy for many malignant haematologic disorders , while it is restricted by its severe complications . one major complication associated with hsct is agvhd , which is a major cause of death following allo - hsct . stage ii involves activation of donor lymphocytes ( t cells ) . in stage iii , cellular and inflammatory factors are released , including tnf- , interleukin ( il)-1 and il-6 et al . these cytotoxic molecules directly attack various host tissues and underlie the clinical manifestations of agvhd [ 25 , 26 ] . the activated cells also produce a large number of harmful free radicals resulting in severe cell damage , which also play an important role in the development of agvhd . nowadays , the standard initial therapy for agvhd includes the use of high - dose steroids , which results in about 40% complete response ( cr ) rate . this cr rate is unsatisfactory , and in patients who have less than a cr , there is high mortality , both from the agvhd itself and from the steroid - related infectious complications . treatment of steroid - resistant agvhd is very difficult , with many institutions using monoclonal antibody treating steroid - resistant agvhd patients , including anti - tnf- monoclonal antibody(mab ) , anti - cd52 mab , anti - cdl47 mab ( abx.cbl ) , anti - cd3 mab , et al . various researchers have engaged in identifying novel , non - toxic , effective and convenient drugs to cure or alleviate agvhd . our hypothesis is based on the theory that hydrogen could down - regulate cytokines , ccl2 , il-1 , il-6 , il-12 , tnf- , et al . and selectively reduce hydroxyl and peroxynitrite radicals ( fig . , h2 has other advantages ; it has demonstrated that h2 has non - toxicity at any pressure . it can penetrate biomembranes and diffuse into the cytosol , mitochondria and nucleus to protect nuclear dna and mitochondria . cytotoxic oxygen radicals that hydrogen could selectively reduce . as those cytokines including il-6 , il-1 , tnf- , et al . and free radicals have been suggested to play great important roles in the formation and development of agvhd as discussed above , we suggested that hydrogen gas may be potentially effective for agvhd by down - regulating cytokines and selectively reducing hydroxyl and peroxynitrite radicals . for testing the hypothesis , hydrogen gas could be administered by two ways : first , it may be administered to patients via inhalation as room air at safe concentrations ( < 4.6% in air by volume ) . this may be more practical in daily life and more suitable for daily consumption for therapeutic use . hydrogen - rich drinking water can be generated by several methods including dissolving electrolysed hydrogen into pure water , dissolving hydrogen into water under high pressure and utilizing electrochemical reaction of magnesium with water . we propose the experimental study by detecting cbc , clinic signs of agvhd mice ( weight loss , posture , activity , fur change , skin integrity ) as described previously [ 31 , 32 ] . we also propose to detect the levels of tnf- , il-2 , il-1 , il-6 as described previously [ 33 , 34 ] , which have been demonstrated to play important roles in the pathogenesis of agvhd . and plasma malondialdehyde ( mda ) , 8-hydroxydeoxyguanosine ( 8-ohdg ) and endogenous antioxidants such as sod , gsh will also be detected in vivo . to discover potential mechanisms of the therapeutic effects of hydrogen on the agvhd model , we will examine gene - expression profiles , such as expression of bcl-2 , bax , caspase-3 , caspase-8 [ 35 , 36 ] . on the basis of these experimental designs , we preliminarily demonstrated the therapeutic effects of hydrogen on agvhd in a mice model . we demonstrated that hydrogen treatment could protect mice from lethal gvhd and improve clinic syndrome of agvhd mice and also could promote the recovery of white blood cells of agvhd mice . cytokines , such as tnf- , il-2 were also reduced by hydrogen , which play critical roles in the development of agvhd ( fig . 2 ) . cytokines that hydrogen could down - regulate ( ) in the pathogenesis of agvhd . adapted from ferrara . this study will open a new therapeutic avenue combining the field of therapeutic medical gases and agvhd . this theory is original and probably of importance , because therapeutic medical gases has never been used for agvhd previously . we believe that in vitro and in vivo work for hydrogen gas on agvhd would come in great numbers as soon as possible . in view of the high lethality rate of agvhd ,

recent world issues associated with fuel consumption and supply have turned attention towards biofuel production , especially cellulosic biofuel . perennial grasses provide an optimal source of cellulosic biomass due to their high yield potential . prairie cordgrass ( spartina pectinata bosc ex link ) is a perennial indigenous grass of north america and can be found as a native from texas to near the arctic circle . ongoing studies on prairie cordgrass in comparison with switchgrass ( panicum virgatum l. ) indicate that prairie cordgrass could produce more biomass than switchgrass . furthermore , results from the comparison of prairie cordgrass and switchgrass performed by boe and lee in 2007 indicated that prairie cordgrass has a wider environmental amplitude and is adapted to poorly drained wet areas which can have high salinity and be poorly aerated , regions not suitable for the production of conventional crops such as maize ( zea mays ) [ 2 , 3 ] . these results are indicative of the potential of prairie cordgrass as a source of biomass for cellulosic biofuel production . a research program at south dakota state university ( sdsu ) is underway to develop native prairie cordgrass into a viable cellulosic biomass crop . the development of a new crop species requires a multidisciplinary approach ; examining and validating each step before commercialization can occur . these steps include , but are not limited to , the assembling of a germplasm collection , the development of an accelerated breeding program , and the optimization of cultivation practices . a fundamental step to accelerate the breeding of prairie cordgrass is to determine and optimize a crossing protocol . prairie cordgrass has been intrinsically believed to be a protogynous outcrossing species , based on the mode of reproduction of its maritime relative smooth cordgrass ( spartina alterniflora ) [ 4 , 5 ] . from work performed upon other members of the spartina genus , it has been conjectured that prairie cordgrass will have a similar method of reproduction . in the majority of other graminaceae species , breeding is performed via the initial emasculation of the floret to ensure that only cross pollination can occur . prairie cordgrass has an inflorescence composed of between 0 and 31 short paracladia and 1113 long paracladia , bearing a total of 1080 fertile spikelets , of single florets . physical emasculation of prairie cordgrass is essentially impractical , but this technique may not be necessary as with protogyny and ascertaining the appropriate timing , directed cross pollination is feasible . self pollination has not been identified previously in prairie cordgrass and assumptions have been made that prairie cordgrass may be self incompatible , if this is the case then the classic mapping technique of producing recombinant inbred lines will be fundamentally impossible and alternate strategies will need to be utilized . an initial stage of the prairie cordgrass project is to develop a molecular map of the species . only limited molecular analyses have been performed upon prairie cordgrass , notably in contrast to its maritime relative s. alterniflora . in the national centre for biotechnology information ( ncbi ) , only 57 prairie cordgrass sequences have been deposited ( predominantly regions of the nuclear and organelle genomes utilized for diversity analysis , that is , waxy ( ay508655 and af372461 ) , its ( af019843 , aj489796 , and ef153082 ) and the chloroplast trnl - trnf intergenic spacer region ( ef137568 , eu056305 , and af372625 ) ) . a recent publication on the analysis of the transcriptome of prairie cordgrass has increased the available knowledge on the genome of the species . constructed from the analysis of the expression sequence tags ( ests ) and identified from the transcriptome of prairie cordgrass , a total of 26,302 contigs and 71,103 singletons additional molecular analysis of genetic diversity of prairie cordgrass in natural populations in minnesota with amplified fragment length polymorphism ( aflp ) has been performed . an optimal marker system for the development of a molecular map of prairie cordgrass are ssrs or microsatellites . microsatellites are highly utilized molecular markers and have been developed for the majority of agronomically and economically important crop species ; their efficacy arises predominantly due to their reproducibility , codominant inheritance , and abundance . modern techniques allow the rapid identification of microsatellite regions in species which have limited sequence information , and by using proprietary genomic library screening techniques , microsatellites have been developed for numerous non - model organisms , encompassing insects , birds , fish , mammals , and plants , including other species in the spartina genera . previous studies on other members of the spartina genera have developed 35 markers for microsatellite loci in s. alterniflora [ 16 , 17 ] . of these s. alterniflora markers , three have been found to amplify in prairie cordgrass . ssrs have been used extensively in numerous crop species , in wheat ( triticum aestivum l. ) , and another poaceae species ; ssrs have been used to enhance molecular maps begun with restriction fragment length polymorphism ( rflp ) and to identify genes of interest . furthermore , wilde et al . in 2007 found that the incorporation of mas with ssr markers into a traditional breeding program can result in a substantial increase in the incorporation of fusarium head blight resistance , a quantitative trait , in wheat with the shortest possible time . this paper details the first investigation into initial breeding and crossing of prairie cordgrass , specifically the production of f1 individuals and the validation of a crossing protocol . furthermore , the characterization of 35 microsatellite loci in prairie cordgrass from genomic dna is discussed . the validation of the microsatellite loci occurred in a prairie cordgrass germplasm collection and a reciprocal cross as an initial step in the development of a molecular map , which can be further utilized during marker - assisted selection of lines with traits desirable for the improvement of prairie cordgrass as a biofuel crop . genomic dna was extracted from four lines of prairie cordgrass using the method as described by karakousis and langridge in 2003 , with minor modifications . a mixture of the four random prairie cordgrass clones dna , totaling > 100 g , was sent to genetic identification services ( gis ) ( http://www.genetic-id-services.com/ ) for the development of genomic libraries . the genomic libraries were enriched for the four simple sequence repeat ( ssr ) motifs ( ca)n , ( ga)n , ( aag)n , and ( cag)n . subsamples of 100 clones were sequenced and primers were designed to flank the ssr regions using designerpcr , version 1.03 ( research genetics , inc . ) and primer 3 . the ssr regions were classified according to jones et al . as being pure repeats ( i.e. , [ n1n2]x ) , compound repeats ( i.e. , [ n1n2]x[n3n4]y ) , and interrupted repeats ( i.e. , [ n1n2]xn3[n4n5]y ) . wild germplasm has been collected from throughout the mid - western states of the united states , creating a core germplasm collection to be utilized in prairie cordgrass breeding ( unpublished data ) . a geographically diverse sample of sixteen plants , collected from south dakota , north dakota , minnesota , and iowa , was characterized using the identified ssr loci . a sample from the closely related species s. spartinae was also included to examine cross species amplification . crosses between and amongst these genotypes were performed . crossing between two plants of prairie cordgrass was performed in the following manner ; two inflorescence at the appropriate stage of development were placed inside a crossing bag for seven days , producing f1 plants ( figure 1 ) . a reciprocal cross between two genotypes ( designated rr2 and rr21 ) from the red river morphotype was produced . the reciprocal cross produced 45 f1 plants from the rr21 rr2 directed cross and 49 f1 plants from the rr2 rr21 directed cross , a total of 94 plants . two genotypes identified as clones ( designated sp1.1b and sp1.2a ) from the ssr evaluation were crossed via the same technique , producing a total of 46 f1 individuals ; a total of 20 f1 plants were produced when sp1.1b was used as the maternal parent and 26 f1 plants when sp1.2a was used as the maternal parent . dna was extracted from all examined plants using the method as described by karakousis and langridge in 2003 , with minor modifications . evaluation of the primers was performed in a pcr reaction consisting of 2 u taq dna polymerase ( promega gotaq ) , 1 pcr buffer ( promega gotaq ) , 2 mm mgcl2 , 0.6 mm dntps , and 0.6 mm of each primer , with a final volume of 20 l . pcr reactions were performed in a biorad mycycler ( biorad , hercules , ca ) . initially a gradient of annealing temperatures was used to determine optimal ta , as these primers were designed to be utilized in a prairie cordgrass breeding program , a robust and high - throughput protocol was developed . specific thermocycling conditions for the primers used in this study were as follows : an initial denaturation at 94c for 5 minutes , followed by 35 cycles of 94c for 1 minute , 53 or 55c for 1 minute , 72c for 1 minute , followed by an extension step of 72c for 10 minutes , and a 10c hold . pcr product was visualized on 8% nondenaturing page ; bands were scored and sized by comparison to a 100 bp ladder using alphaeasefc software ( alpha innotech , san leandro , ca ) . polymorphic bands in the two examined populations described above were scored on a presence ( 1 ) or absence ( 0 ) basis . an estimation of genetic distance was calculated with nei and li 's algorithm and the resulting matrix was clustered with unweighted pair group method with arithmetic mean ( upgma ) . the crossing of the germplasm collection was successful with the production of a total of 14,813 putatively viable seeds from 110 crosses , performed in lines and collected from 48 distinct collection locations ( table 1 ) . a seed was determined to be viable if the actual seed ( endosperm and embryo ) was visible within the glumes ( figure 2 ) . the number of viable seeds produced per head varied from 6 to 642 , with an average of 134 seeds per cross . the crosses can be grouped into two , dependent upon the parents , those that were outcrossed and those that were selfed . crosses that were designated out were between genotypes that were geographically diverse , while crosses designated self were produced from members of the population collected at the same location . the average number of seeds produced from outcrossed individuals was 143 , with a range of 12 to 250 , while selfed individuals produced an average of 112 seeds per cross , with a range of 6 to 642 ( figure 3 ) . of the total 100 genomic clones sequenced , 70 contained microsatellite motifs , 26 from the ( ca)n enriched library , 25 from the ( ga)n enriched library , 3 from the ( aag)n enriched library , and 16 from the ( cag)n enriched library . all 70 loci were examined and 35 were amplified with the standardized conditions ( table 2 ) . the repeat ssr structure that occurred most frequently was the pure repeat ( 27 ) , followed by the compound repeat ( 7 ) and only one interrupted repeat sequence was found ( table 2 ) . only two primers produced monomorphic profiles from the analysis of the sixteen lines , spsd004 from the ( ga)n library and spsd048 from the ( aag)n library . the remaining primers produced between 1 and 12 scorable bands . of the examined primers , 11 were amplified between 1 and 7 bands in s. spartinae ( table 2 ) . all sequences were examined using blastn on the national center for biotechnology information ( ncbi ) server for similarities to other recorded sequences . only two of the prairie cordgrass sequences displayed homology ( ee ) to recorded sequences , spsd003 to six sequences from rice , wheat , and field mustard ( brassica rapa ) ( accession numbers : ap008214.1 , ap005495.3 , nm_001067901.1 , ak111788.1 , eu660901.1 , and ac189183.2 ) and spsd050 to five sequences all from rice ( accession numbers : cr855236.1 , ap008210.1 , al606607.3 , nm_001059819.1 , and ak108706.1 ) . a total of 94 individuals from the rr2 rr21 and the rr21 rr2 crosses were examined with 35 ssr primers . the amplified bands were scored based on presence or absence , the resulting data was analyzed with nei and li 's coefficient producing a similarity matrix ( data not shown ) , and the data was clustered with upgma producing a dendogram ( figure 4 ) . the results of the dendogram indicate that each individual was a cross between the two parents . when the population derived from the cross between the two plants ( sp1.1b and sp1.2a ) and determined to be clones was examined , bands present in both parents were found to segregate in the progeny ( data not shown ) , indicative of sexual recombination ; this result provides validation that successful crossing occurred . the primary requirement of any breeding program is to ensure that accurate crosses are made ; in many other members of the poaceae this is achieved by physical or chemical emasculation . the prolific numbers of flowers per head in addition to the small size of the flowers make physical emasculation unfeasible . furthermore , due to limited knowledge about the nature of the fertility of this species , chemical emasculation has not been developed for prairie cordgrass . the results from the ssr analysis indicate that utilizing the inherent protogyny of prairie cordgrass allows successful crossing between two individuals without the need for emasculation , confirming the validity of the breeding methodology used . the presence of individuals in the f1 mapping populations which show limited genetic dissimilarity from the parents could be evidence of selfing ; further investigations are required . the presence of these potential selfed individuals indicates that future breeding and/or mapping populations should be examined with the molecular markers devised in this research to remove suspect individuals . subsequently , the successful crossing between two clones is indicative that prairie cordgrass may not be self - incompatible and that it may be possible to develop in this species conventional mapping populations , such as recombinant inbred lines . further studies into self - compatibility with investigations into potential apomictic prairie cordgrass plants are underway . the number of seeds observed in this research appears to be larger than what was previously described by clayton et al . . the variation between the two studies can be attributed to both environmental and genetic variations . genetic variation in seed set in prairie cordgrass , although at this stage not quantified , is demonstrated by the range in viable seed set observed in this research . the amplification of s. spartinae with the ssr primers developed in this research are indicative of the potential colinearity amongst the genomes of spartina spp . and other grass species ; the colinearity between prairie cordgrass and other poaceae is currently utilized to examine genes identified in related species in prairie cordgrass , specifically genes utilized to examine phylogeny ( i.e. , the waxy gene for granule bound starch synthase ) . variation in ssrs is not limited to their location , but also their motif , putative function , abundance , and repeat number . the results of this analysis indicate that the two dinucleotide repeats ( ca)n and ( ga)n are more prevalent in prairie cordgrass ( 31% and 46% , resp . ) , than the two trinucleotide repeats ( aag)n and ( cag)n ( 6% and 17% , resp . ) . the prevalence of the dinucleotide motif in prairie cordgrass is similar to what was observed in the characterization of ssrs in other spartina sp . , where blum et al . found 82% of isolated ssrs contained dinucleotide repeats and sloop et al . found 71% containing similar motifs . in all three studies based on the results found in this research , the genomic libraries enriched with the dinucleotide repeats have been extensively sequenced ; the resulting sequence information will be screened to isolate additional ssr regions , primers will be designed , and the resulting markers will be used to develop a molecular map of prairie cordgrass . the molecular map will then be used to find linkage between ssr markers and traits of interest allowing future mas to be performed .

what study is this and what is being shown ? what are the typical symptoms and causes?what is the best radiological study to confirm the diagnosis ? what study is this and what is being shown ? what are the typical symptoms and causes ? what is the best radiological study to confirm the diagnosis ? this is a colovesical fistula , seen at cystoscopy , in a man with diverticulitis . congested and erythematous urothelium is seen , with oedematous mucosa in the centre emitting mucus and gas . other causes include inflammatory bowel disease , large bowel tumors , radiation , surgical trauma , foreign body , and chronic specific infections such as actinomycosis and tuberculosis . an abdominopelvic ct scan with oral , rectal , and intravenous contrast is the imaging modality of choice to diagnose and characterize a colovesical fistula 1 , although recently the accuracy of mri has been reported to approach 100% 2 .

pseudoaneurysm of ascending aorta ( paa ) is a rare clinical entity , which may occur after thoracic trauma or following cardiac surgery . these aneurysms are reported to produce symptoms due to mass effect or fistulous communications with the cardiac chambers . we report two cases of postcardiac surgery paa , presenting as right heart dysfunction and discuss the possible challenges in anesthetic management . a 2-year - old child who was operated for closure of ventricular septal defect ( vsd ) a year previously , presented to our hospital with the complaints of pedal edema , puffiness of face , hoarseness of voice and shortness of breath for last 7 days duration . transthoracic echocardiography ( echo ) revealed a paa with fistulous communication to right atrium ( ra ) [ figure 1a ] and shift of interatrial septum to left side , moderate pulmonary hypertension and mild to moderate right ventricular ( rv ) dysfunction ( tapse-1.3 ) . a three - dimensional reconstruction computed tomography ( ct ) scan confirmed the paa with a size of 5.5 cm 4.6 cm 4.8 cm , neck of 1 cm and fistulous communication to ra [ figure 1b ] and multiple venous collaterals suggestive of obstruction of superior vena cava ( svc ) flow although no direct compression of svc could be demonstrated [ figure 1c ] . anesthesia was induced with titrated doses of fentanyl and midazolam so as to achieve a balance between hemodynamics and sympathetic stimulation , which may trigger a rise of pulmonary vascular resistance . due to impaired svc drainage , airway difficult airway cart was kept ready , and ease of ventilation with face mask was confirmed before administering the muscle relaxant . after institution of femorofemoral bypass and midline sternotomy , the patient was cooled to 25 , aorta was cross - clamped and cardioplegia was given . thereafter , the paa opening was closed with a synthetic patch under transient period of low flows and reinforced with a bovine pericardium ( st . injection milrinone 25 mcg / kg bolus over 10 min followed by infusion at 0.375 mcg / kg / min , dopamine 5 mcg / kg / min , and nitroglycerin 0.25 mcg / kg / min were initiated during rewarming . the child was extubated the next day and had an uneventful postop course with regression of the preoperative symptoms . transthoracic echocardiography with color flow doppler showing neck of pseudoaneurysm of ascending aorta ( paa ) and paa - right atrium fistula computed tomography scan showing neck of pseudoaneurysm of ascending aorta ( paa ) and paa - right atrium fistula maximum intensity projection computed tomography images shows the collaterals as a result of superior vena cava obstruction ( marked by white arrows ) this case describes a 17-year - old boy who was electively operated for vsd closure and aortic valve replacement for aortic regurgitation with a 25 st . he was readmitted 1 year later with gradually enlarging pulsatile swelling of the chest , off and on episodes of chest pain , pedal edema and raised liver enzymes suggestive of right heart failure . transthoracic echo showed a large paa with peripheral thrombus , and compression of rv outflow tract ( rvot ) [ figure 2a ] , mild to moderate rv dysfunction . ct angiography confirmed the presence of a 10.5 cm 5.9 cm 11.2 cm pseudoaneurysm arising from the anterior wall of ascending aorta with a small neck ( 7 mm ) [ figure 2b ] . it was abutting the ostioproximal right coronary artery ( rca ) with significant compression [ figure 2c ] . anesthesia was induced with fentanyl 200 mcg , midazolam 3 mg , sevoflurane and intubated with rocuronium 30 mg . femorofemoral bypass was established before commencing sternotomy so that in the event of paa rupture during sternotomy immediate circulatory arrest can be instituted . the patient was gradually cooled to 24 while avoiding ventricular fibrillation and thus ventricular distension . the ostium of the paa could be accessed within 5 min , and a finger was insinuated into the aneurysm cavity to occlude it . the aorta was cross - clamped and cardioplegia could be administered through both the coronaries as the aneurysm was decompressed . injection milrinone infusion was started in the rewarming period in view of the rv dysfunction . the patient was extubated on 2 postoperative day and was discharged on 10 postoperative day . transthoracic echocardiography , apical four chamber view shows the pseudoaneurysm of ascending aorta compressing the right ventricle and interventricular septum computed tomography scan showing pseudoaneurysm of ascending aorta , its neck and sternum erosion computed tomography scan showing compression of ostioproximal right coronary artery by pseudoaneurysm of ascending aorta a 2-year - old child who was operated for closure of ventricular septal defect ( vsd ) a year previously , presented to our hospital with the complaints of pedal edema , puffiness of face , hoarseness of voice and shortness of breath for last 7 days duration . transthoracic echocardiography ( echo ) revealed a paa with fistulous communication to right atrium ( ra ) [ figure 1a ] and shift of interatrial septum to left side , moderate pulmonary hypertension and mild to moderate right ventricular ( rv ) dysfunction ( tapse-1.3 ) . a three - dimensional reconstruction computed tomography ( ct ) scan confirmed the paa with a size of 5.5 cm 4.6 cm 4.8 cm , neck of 1 cm and fistulous communication to ra [ figure 1b ] and multiple venous collaterals suggestive of obstruction of superior vena cava ( svc ) flow although no direct compression of svc could be demonstrated [ figure 1c ] . anesthesia was induced with titrated doses of fentanyl and midazolam so as to achieve a balance between hemodynamics and sympathetic stimulation , which may trigger a rise of pulmonary vascular resistance . due to impaired svc drainage , airway difficult airway cart was kept ready , and ease of ventilation with face mask was confirmed before administering the muscle relaxant . after institution of femorofemoral bypass and midline sternotomy , the patient was cooled to 25 , aorta was cross - clamped and cardioplegia was given . thereafter , the paa opening was closed with a synthetic patch under transient period of low flows and reinforced with a bovine pericardium ( st . injection milrinone 25 mcg / kg bolus over 10 min followed by infusion at 0.375 mcg / kg / min , dopamine 5 mcg / kg / min , and nitroglycerin 0.25 mcg / kg / min were initiated during rewarming . the child was extubated the next day and had an uneventful postop course with regression of the preoperative symptoms . transthoracic echocardiography with color flow doppler showing neck of pseudoaneurysm of ascending aorta ( paa ) and paa - right atrium fistula computed tomography scan showing neck of pseudoaneurysm of ascending aorta ( paa ) and paa - right atrium fistula maximum intensity projection computed tomography images shows the collaterals as a result of superior vena cava obstruction ( marked by white arrows ) this case describes a 17-year - old boy who was electively operated for vsd closure and aortic valve replacement for aortic regurgitation with a 25 st . he was readmitted 1 year later with gradually enlarging pulsatile swelling of the chest , off and on episodes of chest pain , pedal edema and raised liver enzymes suggestive of right heart failure . transthoracic echo showed a large paa with peripheral thrombus , and compression of rv outflow tract ( rvot ) [ figure 2a ] , mild to moderate rv dysfunction . ct angiography confirmed the presence of a 10.5 cm 5.9 cm 11.2 cm pseudoaneurysm arising from the anterior wall of ascending aorta with a small neck ( 7 mm ) [ figure 2b ] . it was abutting the ostioproximal right coronary artery ( rca ) with significant compression [ figure 2c ] . anesthesia was induced with fentanyl 200 mcg , midazolam 3 mg , sevoflurane and intubated with rocuronium 30 mg . femorofemoral bypass was established before commencing sternotomy so that in the event of paa rupture during sternotomy immediate circulatory arrest can be instituted . the patient was gradually cooled to 24 while avoiding ventricular fibrillation and thus ventricular distension . the ostium of the paa could be accessed within 5 min , and a finger was insinuated into the aneurysm cavity to occlude it . the aorta was cross - clamped and cardioplegia could be administered through both the coronaries as the aneurysm was decompressed . injection milrinone infusion was started in the rewarming period in view of the rv dysfunction . the patient was extubated on 2 postoperative day and was discharged on 10 postoperative day . transthoracic echocardiography , apical four chamber view shows the pseudoaneurysm of ascending aorta compressing the right ventricle and interventricular septum computed tomography scan showing pseudoaneurysm of ascending aorta , its neck and sternum erosion computed tomography scan showing compression of ostioproximal right coronary artery by pseudoaneurysm of ascending aorta ascending aortic pseudoaneurysm is a rare , but life - threatening complication after cardiac surgery and thoracic trauma . poor surgical suture technique , pre - existing aortic wall disease , perioperative infection are predisposing factors of pseudoaneurysm . most common sites of origin of pseudoaneurysm include aortotomy or anastomotic suture lines , aortic and cardioplegia canulation sites , cross - clamp sites , bypass graft sites , and needle puncture holes . the size , site , anatomic location , and pressure effect on neighboring structures determines the symptoms of the paa . svc obstruction , aorto - pulmonary fistula , rv inflow obstruction , fistula into ra , rv and lv and angina are a few reported complications . pressure overload in most cases is a result of raised pulmonary vascular resistance , rvot obstruction or pulmonary stenosis whereas volume overload is mostly due to tricuspid regurgitation , pulmonary regurgitation , atrial septal defect or anomalous venous return . impaired rv contractility may be due to rv ischemia , inadequate protection during cardiopulmonary bypass , or coronary air . this report describes two cases of rv dysfunction , one presenting as volume overload ( paa - ra fistula ) , and the other as pressure overload ( rvot compression ) with intermittent ischemia ( rca compression by paa ) . this patient had a paa - ra fistula which imposes a large volume overload on the ra and rv . the flow through the paa fistula could have resulted in high ra pressure with a leftward shift of the atrial septum on the echo . this high ra pressure could also have caused impaired svc drainage in our patient despite lack of any direct mechanical obstruction . our patient presented with sudden onset severe puffiness of the face with hoarseness of voice suggestive of impaired svc drainage . the presence of hoarseness of voice as with our case should alert the anesthesiologist to plan airway access . our second patient presented with off and on angina due to rca compression which is another rare presentation of paa . the right coronary insufficiency along with rvot obstruction could have resulted in rv dysfunction in this patient . these patients are also at risk for postoperative deterioration of rv dysfunction due to insufficient myocardial protection during cardiopulmonary bypass . our second patient was specifically at risk of worsening of rv function due to circulatory arrest without cardioplegia for 5 min . early institution of an inodilator during management of our cases might have led to uneventful postoperative course . although percutaneous interventions have been reported as a management strategy for paa , literature is scarce and is not well established . a big danger in pseudoaneurysm surgery is the risk of intraoperative rupture and is a major cause of mortality . various techniques have been described for controlling the hemorrhage in paa during redo sternotomy such as temporary occlusion of pseudoaneurysm ostium using balloon of foley 's catheter or even a fingertip . as rapid exsanguination can occur , it is important to ensure availability of adequate blood and facilities for rapid transfusion . because of a rarity of the condition , most standard teaching and anesthetic literature do not highlight on these postoperative aortic complications of corrected cardiac lesions . thus , a thorough understanding of the underlying lesion , its pathophysiology , and anesthetic implications is essential for planning the management of these cases . to summarize , right heart dysfunction though unusual can occur with paa . these two reports aid in understanding the anatomical and pathophysiological consequences of paa and the implications for anesthetic management .

the timing of umbilical cord clamping , which separates the newborn from the placenta , has been the subject of much debate for decades ( mercer et al . , 2001 ) . immediate umbilical cord clamping ( icc ) remains the most commonly employed method and is performed in the third stage of labor , during the period extending from complete delivery of the infant to complete delivery of the placenta ( aflaifel et al . in a recent cochrane review ( mcdonald et al . , 2014 ) , early cord clamping was defined as covering a wide range from immediately following birth to less than 1 minute post - birth , whereas delayed cord clamping occurs one or more minutes after birth or when cord pulsation ceases . the benefits of delayed umbilical cord clamping ( dcc ) have been well documented and include lower risks of intraventricular hemorrhage ( all grades ) , lower risk for necrotizing enterocolitis , increased early hemoglobin concentration , increased iron stores , and increased cerebral oxygenation in preterm infants ( baenziger et al . while some contend that the prevalence of icc is simply because of custom , other reasons include reduced risk of post - partum hemorrhage , easier identification of placental detachment , minimized risk of rhesus iso - immunization , and time constraints faced by physicians in the busy environment of the delivery room ( hutchon , 2010 ; downey and bewley , 2012 ) . however , it is worth noting that recent studies have found no significant differences between early versus late cord clamping groups for the primary outcome of severe postpartum hemorrhage ( mcdonald et al . the benefits of dcc are primarily attributed to an increase in neonatal blood volume , secondary to placenta - fetal transfusion ( niermeyer and velaphi , 2013 ) . this transfusion has been suggested to follow an exponential decay curve with 25% being transferred within the first 15 seconds , 50% by 60 seconds , and flow ceasing in most infants by 23 minutes ( yao et al . , 1968 , 1969 ; yao and lind , 1974 ) . however , venous and arterial umbilical flow may occur for longer than previously described and placental transfusion appears to be complex and dependent on several factors ( boere et al . , 2014 ) . the transfer of umbilical cord blood is of particular interest in this review because of the various valuable stem cells contained such as hematopoietic stem cells , endothelial cell precursors , mesenchymal progenitors and multipotent / pluripotent lineage stem cells . human umbilical cord blood ( hucb ) plays a significant role as a reservoir of stem and progenitor cells ( chen et al . , 2005 ; watson et al . , 2015 these stem cells , which have infinite medical potential , are currently used in the treatment of several life - threatening diseases and are viewed by many as the stem cells source of choice for clinical and non - clinical research applications ( chakraborty et al . , 2014 ) . the hematopoietic progenitor cells ( hpc ) of umbilical cord blood have an extensive proliferative capacity that exceeds that of bone marrow hpc ( broxmeyer et al . , 1989 ; ballen et al . , 2013 ) , and even a single hucb sample can provide enough hematopoietic stem cells for both short- and long - term engraftment ( sirchia and rebulla , 1999 ) . while the first umbilical cord blood transplant occurred 26 years ago in france in a child with fanconi anemia ( gluckman et al . , 1989 ; ballen et al . , 2013 ) , one might argue that nature 's first stem cell transplant occurs at birth as the placenta and umbilical cord contract and pump blood toward the newborn ( sanberg et al . once the blood equilibrates in both compartments , the cord becomes pulseless and blood flow ceases . this is the natural course in most placental mammals , yet in humans this cord blood transfusion is curtailed by early clamping of the umbilical cord , thus depriving infants of additional stem cells . the existence of stem cells in fetal circulation indicates that a delay in cord clamping should increase stem cell supply to newborns . . the effects of dcc on diseases such as neonatal and adult diseases , is unknown but warrants further investigation . of note , transplantation of exogenous hucb has been shown to be therapeutic in animal models of cerebral palsy ( yasuhara et al . , 2010 ) , autoimmune diseases ( liu et al . , 2013 ) , acute injuries like traumatic brain injury ( acosta et al . , 2014 ; dela pena et al . , 2014a , b ) and myocardial infarction ( acosta et al . , 2013 ) , and adult - onset disorders , such as stroke ( borlongan et al ou et al . , 2010 ) , altogether implicating that prophylactic stem cell transplantation , as may be achieved with dcc , can afford benefits against diseases in neonates and adults . the postnatal transfer of hucb may be particularly important in preterm infants born between 24 to 31 weeks gestation because of the higher concentration of primitive hpc and long - term culture - initiating cells when compared with cord blood of infants born closer to term ( haneline et al . , 1996 ) . consequently , the timing of umbilical cord clamping may be especially important in preterm neonates . dcc may prove to be a safe and inexpensive practice that could potentially decrease morbidity and mortality associated with many newborn conditions , especially when there is no plan to harvest such important cells through cord blood banking . in 2010 , the international liaison committee on resuscitation ( ilcor ) recommended that cord clamping be delayed for at least 1 minute in healthy term infants , but stated that evidence was insufficient to recommend a time for clamping in those who require resuscitation ( perlman et al . , 2010 ) . ironically , it could be argued that these infants stand to receive the greatest benefit from dcc . several systematic reviews have suggested that dcc decreased incidence of intracranial hemorrhage in preterm infants ( committee on obstetric practice , 2012 ) . delaying cord clamping ( for at least 3060 seconds ) , with the infant maintained at or below the level of the placenta was associated with increased blood volume , reduced need for transfusion , and decreased frequency of iron deficiency anemia in term infants ( committee on obstetric practice , 2012 ) . the existence of stem cells in fetal circulation suggests that a delay in cord clamping should increase stem cell supply to the neonate , providing immediate benefits if neonatal disease is indicated ( table 1 ) . in fact , if cord clamping was delayed by 180 seconds , the newborn may receive an additional 75 ml of blood volume ( yao et al . , 1969 ; diaz - rossello , 2006 ) that could contain approximately 1,100 - 45,000 hematopoietic stem cells . early versus delayed cord clamping some studies have even suggested that physicians delay cord clamping until ventilation ( bhatt et al . , 2013 ) , relying on the infant 's physiology rather than proceeding in a simple time - dependent fashion . umbilical cord milking or stripping has also been put forth as a viable means of placental transfusion and been suggested to have beneficial effects for newborns ( hosono et al . , 2008 ; rabe et al . , 2011 ; erickson et al . , 2012 ; upadhyay et al . such milking likely increases stem cell supply to the neonate . in forming a consensus on the optimal timing for cord clamping , further investigation of the effects of dcc on concentrations of stem and/or progenitor cells in while dcc is likely to increase the supply of these valuable cells to the infant , it is unknown for what period of time cord clamping should be delayed for optimal benefit to the infant . alternative non - stem cell mechanisms may account for some of the therapeutic effects of dcc . of note , dcc has a significant impact on newborn hemodynamics , mainly because of increased blood volume and improved blood oxygenation ( yigit et al . , 2015 ) . for example , germinal matrix hemorrhage is known to occur with hypoxia , and dcc may protect against this hemorrhage by way of enhanced blood oxygenation and larger blood volume delivered to the baby . as previously stated , dcc is associated with less necrotizing enterocolitis and reduced incidence of intraventricular hemorrhage ( mercer et al . , 2006 ; aziz et al . , 2012 ) , which may be due to the increase of stem cells transferred to the baby ( sanberg et al . , 2010 ; tolosa et al . , 2010 likewise , dcc may improve cerebral oxygenation ( baenziger et al . , 2007 ) and may increase blood volume ( yigit et al . clinical and research - based evidence suggests that dcc may benefit neurodevelopment and ameliorate early neurological disorders , especially in preterm neonates ( mcadams , 2014 ) . dcc 's reduction of intraventricular hemorrhage incidence ( rabe et al . , 2012 ) is an implicated method of therapy . abnormal neurodevelopment often spurs infant iron - deficient anemia ( yager and hartfield , 2002 ) , and dcc is a seemingly effective intervention . with iron deficiency affecting a substantial portion of the world 's population and approximately 25% of global births ( de benoist et al . , 2008 ) , dcc could prove a very low - cost and easy to implement treatment . current research also suggests that dcc provides therapeutic relief beyond the neonatal period ( mcdonald , 2008 ) . andersson et al . ( 2013 ) produced a multi - year study investigating the neurodevelopmental benefits of dcc at various age increments . beginning two days after birth , infants were seen to have significantly higher hemoglobin levels as well as a decrease in neonatal anemia ( andersson et al . , 2015 ) . in conjunction dcc neonates were seen to have increased scores on a series of five different fine - motor tests at 4 years of age . such a long - term relief suggests that dcc may have a substantial impact in development . however , on the time intervals prior to the four - year mark , the data was not as promising ( andersson et al . , 2015 ) . interestingly enough at the 12-month checkup , dcc did not have large effect on the iron levels or neurodevelopment in the infant population . while these sporadic improvements have been documented , more research needs to be done to demonstrate the biological cause of this phenomenon ( andersson et al . , 2014 ) . another anemia therapy , immediate blood transfusion , has been shown to also produce neuroprotective effects . these transfusions significantly reduce early brain injury in preterm infants by altering the oxygen extraction demand within the body ( osborn , 2007 ) . an elevated cerebral fractional tissue oxygen extraction ( cftoe ) typically precedes intraventricular haemorrhage in very preterm infants ( verhagen , 2010 ; balegar , 2014 ; noori , 2014 ) . the transfusion of red blood cells ( rbc ) balances the low blood flow and the high oxygen demand , eliminating the risk of hypoxia - ischaemia ( altman , 1993 ) . additionally , research also suggests an improvement in cardiac output and cerebral tissue in late anaemia prematurity ( andersen et al . , 2015 ) . in summary , dcc can increase in neonatal blood volume , secondary to placenta - fetal transfusion . a larger blood volume may result in a higher stem cell supply to the neonate , which likely accompanies this hucb - transfusion . human umbilical cord blood is known to possess valuable stem and progenitor cells , which the newborn likely stands to benefit from . human umbilical cord blood is currently being evaluated for its efficacy in mitigating the effects of various diseases and the artificial loss of stem cells imposed by early or immediate clamping of the umbilical cord may negatively affect a child 's endogenous ability to combat various diseases . in conjunction with improved oxygenation and increased blood volume , the additional stem cells delivered to the baby following dcc may afford therapeutic effects against neonatal- and adult - onset diseases .

consequently , extrusion of infected material apically and destruction of the apical binding point for the root filling can affect the outcome of root canal treatment negatively . however , radiographs are subject to distortion , magnification , interpretation variability , and lack of a two - dimensional representation of a three - dimensional object . radiograph is a technique that provides information about the canal anatomy and surrounding tissues , but superimposition and anatomy interferences can be problematic and affect correct interpretation of the images . the results of a microscopic study showed that the major foramen may be located up to 3.5 mm from the radiographic apex . in such teeth , if the canal terminates in the plane of the film , the radiographic appearance will be short , and any adjustment will result in the wl being overextended recently , the use of electronic devices became popular and numerous devices have been introduced to the market . the advantages of electronic apex locators ( eals ) include reduction in radiation dosage and procedure time , both of which aid in maintaining patient cooperation . microscopic studies have shown the apical constriction to be located 0.5 - 1.0 mm from the major foramen . root zx mini ( j. morita corp . , kyoto , japan ) is a modified version eal based on the same technology of the root zx . besides the root zx mini offers 3 programmable memory settings , shock resistance and automatic calibration . however , there is a lack of information in the literature about the in vivo accuracy of root zx mini in determining the correct wl . the continuous monitoring of wl is important during canal preparation as the wl may vary during the procedure , especially in curved canals . the combinations of eal and low - speed endodontic hand - pieces have been introduced to achieve the accuracy of conventional eals during canal shaping . the purpose of this randomized clinical study was to evaluate the clinical accuracy of traditional radiographic method , eal and apex locating endodontic motor . a total of 120 patients with 283 root canals that referred for endodontic treatment were selected . informed written consent in full accordance with ethical principles was obtained from each patient before the treatment was initiated . the teeth with no apical patency or with the radiographic signs of resorption were excluded . all clinicians involved in this study had at least 5 year of experience with apex locators . in case of the disability of determination of proper wl with the selected method , such teeth were excluded from the study . under local anesthesia ( ultracain d - s , sanofi aventis , turkey ) and isolation with rubber dam , caries , and existing restorations were removed . standard access cavity was carried out using high - speed diamond round bur ( dentsply maillefer , ballaigues , switzerland ) under water coolant for each tooth and a straight - line access to the root canals was achieved . the entrances of the root canals were irrigated with 2.5% sodium hypochlorite solution and excess sodium hypochlorite was removed from the pulp chamber by using cotton pellets , no attempt was made to clean debris or pulp tissue remnants prior to introducing a size 15 k - file ( dentsply maillefer , ballaigues , switzerland ) into the canals . following the preparation of adequate access cavity , the steps followed for each group were as follows : the preoperative periapical radiography was taken using paralleling technique.a size 15 k - file was placed to the estimated length and wl radiograph was taken . the initial wl was recorded.canal preparation was carried out with protaper ( dentsply maillefer , ballaigues , switzerland ) nickel titanium ( ni - ti ) rotary file and an endodontic motor ( x - smart ; dentsply maillefer , ballaigues , switzerland ) to a size of f3.the master cone ( protaper f3 ) was inserted to the wl and radiograph was taken using paralleling technique . the preoperative periapical radiography was taken using paralleling technique . a size 15 k - file canal preparation was carried out with protaper ( dentsply maillefer , ballaigues , switzerland ) nickel titanium ( ni - ti ) rotary file and an endodontic motor ( x - smart ; dentsply maillefer , ballaigues , switzerland ) to a size of f3 . the master cone ( protaper f3 ) was inserted to the wl and radiograph was taken using paralleling technique . the measurement was determined as final obturation and recorded . the same as step 1 in group 1.the working file was placed to the estimated length after access cavity preparation according to preoperative radiograph . a size 15 k - file , attached the file clip of root zx mini ( j. morita co. , kyoto , japan ) , was advanced apically into the canal until the screen showed apex . according to the manufacturer , the reference point for measurement or enlargement can be set anywhere between 2 and apex . the length of the file was then measured at this point , and 0.5 mm was subtracted to get the initial wl.canal preparation was carried out with protaper ni - ti rotary file and endodontic motor to a size of f3.the master cone ( protaper f3 ) was inserted to the wl and radiograph was taken using paralleling technique . the measurement was determined as final obturation and recorded . the same as step 1 in group 1 . the working file was placed to the estimated length after access cavity preparation according to preoperative radiograph . a size 15 k - file , attached the file clip of root zx mini ( j. morita co. , kyoto , japan ) , was advanced apically into the canal until the screen showed apex . according to the manufacturer , the reference point for measurement or enlargement can be set anywhere between 2 and apex . the length of the file was then measured at this point , and 0.5 mm was subtracted to get the initial wl . canal preparation was carried out with protaper ni - ti rotary file and endodontic motor to a size of f3 . the master cone ( protaper f3 ) was inserted to the wl and radiograph was taken using paralleling technique . the measurement was determined as final obturation and recorded . the same as step 1 in group 1.the wl was determined using a size 15 k - file and multifunctional endodontic motor with integrated apex locator ( vdw gold , vdw , munich , germany ) was switched to the apex locator mode.the canal was prepared with protaper ni - ti file to a size of f3 with vdw gold which consists of a torque - controlled endodontic motor and an integrated eal . the ana ( safety for difficult anatomy ) and asr ( auto stop reverse ) functions were activated . the wl was controlled during the preparation procedures by continuous monitoring property of the device.the master cone ( protaper f3 ) was inserted to the wl and radiograph was taken using paralleling technique . the measurement was determined as final obturation and recorded . the same as step 1 in group 1 . the wl was determined using a size 15 k - file and multifunctional endodontic motor with integrated apex locator ( vdw gold , vdw , munich , germany ) was switched to the apex locator mode . the canal was prepared with protaper ni - ti file to a size of f3 with vdw gold which consists of a torque - controlled endodontic motor and an integrated eal . the ana ( safety for difficult anatomy ) and asr ( auto stop reverse ) functions were activated . the wl was controlled during the preparation procedures by continuous monitoring property of the device . the master cone ( protaper f3 ) was inserted to the wl and radiograph was taken using paralleling technique . for the roots obturated shorter than 2 mm or overfilled , the wls were reestablished and root canal obturations were carried out according to the corrected wl in all groups . root canal treatment was finished in one to three visits according to pathological status , time available , the difficulty of the case and patient cooperation . the master cone ( final ) radiographs were evaluated by two clinicians blinded to group allocation of each case and graded as follows : short ( shorter than 2 mm from radiographic apex ) , acceptable ( within 0 - 2 mm from radiographic apex ) , and over ( beyond the apex ) . the preoperative periapical radiography was taken using paralleling technique.a size 15 k - file was placed to the estimated length and wl radiograph was taken . the initial wl was recorded.canal preparation was carried out with protaper ( dentsply maillefer , ballaigues , switzerland ) nickel titanium ( ni - ti ) rotary file and an endodontic motor ( x - smart ; dentsply maillefer , ballaigues , switzerland ) to a size of f3.the master cone ( protaper f3 ) was inserted to the wl and radiograph was taken using paralleling technique . 15 k - file was placed to the estimated length and wl radiograph was taken . canal preparation was carried out with protaper ( dentsply maillefer , ballaigues , switzerland ) nickel titanium ( ni - ti ) rotary file and an endodontic motor ( x - smart ; dentsply maillefer , ballaigues , switzerland ) to a size of f3 . the master cone ( protaper f3 ) was inserted to the wl and radiograph was taken using paralleling technique . the same as step 1 in group 1.the working file was placed to the estimated length after access cavity preparation according to preoperative radiograph . a size 15 k - file , attached the file clip of root zx mini ( j. morita co. , kyoto , japan ) , was advanced apically into the canal until the screen showed apex . according to the manufacturer , the reference point for measurement or enlargement can be set anywhere between 2 and apex . the length of the file was then measured at this point , and 0.5 mm was subtracted to get the initial wl.canal preparation was carried out with protaper ni - ti rotary file and endodontic motor to a size of f3.the master cone ( protaper f3 ) was inserted to the wl and radiograph was taken using paralleling technique . the measurement was determined as final obturation and recorded . the same as step 1 in group 1 . the working file was placed to the estimated length after access cavity preparation according to preoperative radiograph . a size 15 k - file , attached the file clip of root zx mini ( j. morita co. , kyoto , japan ) , was advanced apically into the canal until the screen showed apex . according to the manufacturer , the reference point for measurement or enlargement can be set anywhere between 2 and apex . the length of the file was then measured at this point , and 0.5 mm was subtracted to get the initial wl . canal preparation was carried out with protaper ni - ti rotary file and endodontic motor to a size of f3 . the master cone ( protaper f3 ) was inserted to the wl and radiograph was taken using paralleling technique . the same as step 1 in group 1.the wl was determined using a size 15 k - file and multifunctional endodontic motor with integrated apex locator ( vdw gold , vdw , munich , germany ) was switched to the apex locator mode.the canal was prepared with protaper ni - ti file to a size of f3 with vdw gold which consists of a torque - controlled endodontic motor and an integrated eal . the ana ( safety for difficult anatomy ) and asr ( auto stop reverse ) the wl was controlled during the preparation procedures by continuous monitoring property of the device.the master cone ( protaper f3 ) was inserted to the wl and radiograph was taken using paralleling technique . the measurement was determined as final obturation and recorded . the same as step 1 in group 1 . the wl was determined using a size 15 k - file and multifunctional endodontic motor with integrated apex locator ( vdw gold , vdw , munich , germany ) was switched to the apex locator mode . the canal was prepared with protaper ni - ti file to a size of f3 with vdw gold which consists of a torque - controlled endodontic motor and an integrated eal . the ana ( safety for difficult anatomy ) and asr ( auto stop reverse ) functions were activated . the wl was controlled during the preparation procedures by continuous monitoring property of the device . the master cone ( protaper f3 ) was inserted to the wl and radiograph was taken using paralleling technique . for the roots obturated shorter than 2 mm or overfilled , the wls were reestablished and root canal obturations were carried out according to the corrected wl in all groups . root canal treatment was finished in one to three visits according to pathological status , time available , the difficulty of the case and patient cooperation . the master cone ( final ) radiographs were evaluated by two clinicians blinded to group allocation of each case and graded as follows : short ( shorter than 2 mm from radiographic apex ) , acceptable ( within 0 - 2 mm from radiographic apex ) , and over ( beyond the apex ) . the results of length adequacy in each group for root canal obturation are summarized in table 1 . in groups 1 , 2 and 3 adequate filling were recorded as 77 ( 81,9% ) , 80 ( 87,0% ) and 81 ( 83,5% ) , respectively . the lowest rate of short and over fillings were recorded in group 2 whereas , the highest rate of short and over fillings were recorded in group 1 . however , no statistically significant difference was found between the three tested methods ( p = 0.894 ) . both methods were used to investigate conventional radiographs and eals solely or in comparison to each other . however , neither in vitro nor in vivo results can be a true representative of clinical situations in which the whole treatment is done in the mouth . on the other hand , randomized clinical studies may provide high - level of evidence for clinical practice since they reflect a truly clinical condition . the apical constriction is the ideal and recommended end - point for instrumentation and canal filling . the foramen does not coincide with the anatomical apex ; it might be located laterally and in a distance of up to 3 mm from the anatomical apex . root canal filling 0 - 2 mm short of the radiographic apex was reported as the most favorable outcome for the prognosis of root canal treatment , so this reasonable criteria was chosen for our study for evaluating the length adequacy on radiographs . in our study , the length adequacy of master cone radiography was considered as the final outcome of root canal treatment . following the preparation of root canal with a tapered rotary ni - ti file , the use of a matched tapered single gutta percha cone for obturation became popular . tapered cones provide three - dimensional obturation of the root canal over its whole length without the requirements for accessory cones and the use of tapered single cone technique produces faster root canal obturation than the lateral compaction technique . previous studies reported the tapered single cone technique as an efficient alternative to the lateral compaction technique because of similar sealing ability , shorter application time and ease in the application . operator is the most significant factor affecting the prevalence of root filling extrusion when cold lateral compaction was used . therefore , in order to minimize operator 's procedural errors , the utilization of compatible single cone was preferred . radiography is the only universally accepted , available , and meaningful method of length adequacy assessment in the clinic . however , there is repeated expose to unwanted radiation before , during and immediately after the endodontic treatment . the adverse effect of radiation was investigated on areas in the path of ionizing radiation including , the oral tissues , periodontal tissues , human embryos , and fetuses in pregnant women . therefore , reducing the number of radiographs is crucial to prevent the patients from the repeated expose of ionizing radiation . similarly , ravanshad et al . reported that the results of endodontic treatment using eal are quite comparable to radiographic length measurement and the average number radiographs taken was recorded significantly lower when eals were used during wl determination . our results are in accordance with the previous clinical trial , in which the accuracy of eal on wl determination was reported as quite comparable to radiographic length measurement . the result of present study demonstrated that there was no statistical significant difference between the success of both tested eals and conventional radiographic method in terms of wl determination . many studies have addressed the advantages and clinical performance of electronic devices for root canal length determination and the use of eals became widespread in daily practice . past in vivo studies reported that measuring the location of the apical constriction using root zx apex locator was more accurate than radiographs and would reduce the risk of instrumentation and filling beyond the apical foramen . the accuracy of different locators working on the same principle was found to be similar . since root zx mini was built on the industry standard root zx technology and works on the same principle , root zx mini can measure in both wet or dry canal conditions . besides , the miniaturized size of root zx mini can be accepted as an advantage in clinical practice . although , this study is not in agreement with previous reports which reported that eal are more accurate than radiographs , if not superior , the results of wl determination of root zx mini was similar to conventional radiographs . previous studies reported that 75.0 - 83% of the determined wls were accurate and such rates were reported to be clinically acceptable . in this study , the acceptable root canal filling rates ranged from 81.9% to 87.0% . a laboratory study showed that the combination of eal and endodontic motor was accurate in terms of length control during root canal preparation . to our knowledge , the clinical accuracy of an endodontic motor in combination with a eal has not yet been described in literature . therefore , one of the objectives of the present study was to test this combination under clinical conditions with respect to its function and accuracy . the results revealed that the accuracy of multi - functional endodontic motor with integrated apex locator was acceptable and quite comparable to eal and conventional radiographs . besides , the continuous monitoring of the wl may help clinicians to prevent the procedural errors like loss of reference point and over instrumentation . according to the results of this study success of both apex locators were similar to the radiographic wl determination technique . in conclusion , root zx mini and vdw gold are clinically acceptable apex locators for determination of wl .

in modern cellular systems , rnas frequently associate with proteins to form ribonucleoprotein ( rnp ) complexes [ 15 ] . in these rnp complexes , biological functions ascribed to the main component ( rna or protein ) are regulated by the accessory component ( protein or rna ) . during molecular evolution , both main and accessory components often evolved from single- to multi - molecular systems to elaborate and diversify their functions . a typical example can be seen in ribonuclease p ( rnasep ) , which plays a pivotal role in trna processing [ 68 ] . most rnasep enzymes act as ribonucleoproteins ( rnps ) , which have been proposed to have evolved from a catalytic rna . while prokaryotic rnasep is composed of a single rna subunit and single protein subunit , eukaryotic rnasep has 9 - 10 protein subunits that decorate one rna subunit [ 68 ] . a different type of multicomponent system is seen in the evolutionary fragmentation of rna components in caulobacter and euglena , in which the tmrna and large ribosomal rna are reconstituted by assembling 2 and 14 fragments , respectively . for example , it is well known that some protein subunits of eukaryotic rnase p are shared with rnase mrp , which processes non - trna substrates [ 11 , 12 ] . more recently , the core proteins of telomerase rnp complex were reported to play novel roles by associating with nontelomerase rna [ 13 , 14 ] . these features of multicomponent rnp complexes are not only advantageous in natural molecular evolution but also attractive from the viewpoint of artificial design of functional biomacromolecular systems . previously , we reported self - folding rna ( figure 1(a ) , left ) and self - assembling rna ( figure 1(a ) , middle ) as platforms to design rna templates for assisting in chemical peptide ligation [ 1517 ] . previous molecular design for template rnas had limitations due to the use of the bacteriophage boxb motifs as a dual module for rna - rna interaction in the template rnas and also recognition of the substrate n - peptides ( prm1 in figure 1(a ) , left and middle ) . , we designed a new template system based on a two - piece derivative of the tetrahymena group i intron rna ( figure 1(a ) , right ) . the tetrahymena group i intron rna is a unimolecular rna with modular architecture that exhibits self - splicing activity . its structural modules can be classified into conserved core modules responsible for catalysis and nonconserved peripheral modules . peripheral modules , which are not directly involved in the mechanism of catalysis , stabilize the conserved core by organizing a tertiary interaction network that wraps and fixes the core modules ( figures 1(b ) and 1(c ) ) [ 1922 ] . the p5abc domain is a self - folding peripheral element playing crucial roles in stabilization of the core modules . these interactions are so strong that the function of p5abc is preserved in the two - piece format in which the p5abc domain rna ( p5 rna ) and the rest of the intron ( p5 rna ) are physically separated ( figures 1(b ) , 1(c ) , and 2(a ) ) . the resulting two rnas stably form a p5 : p5 complex ( figures 1(b ) , 1(c ) , and 2(a ) ) that is capable of conducting in vitro ribozyme functions . we employed this two - piece ( p5 : p5 ) complex as a platform to arrange two peptide recognition modules ( figure 1(a ) , right ) [ 24 , 25 ] . although the system designed in this study is still primitive , the rna design based on the p5 : p5 platform is potentially attractive for development of versatile rna - based template for chemical peptide ligation . molecular models were constructed from the coordinates of the crystal structure of tetrahymena group i intron ( protein date bank i d 1x8w ) and the nmr structures of the complex of n - peptide and boxb rna ( 1qfq ) , the complex of rev - peptide and rre rna ( 1etf ) , and the complex of p22n - peptide and p22 boxb rna ( 1a4 t ) . molecular modeling was performed by using discovery studio ( accelrys software inc . ) and the pymol molecular graphics system ( schrdinger , llc ) . protected fmoc aminoacids , n , n - diisopropylethylamine ( diea ) , hbtu , hobt , piperidine , ho - ch2-pam resin , and fmoc - ala - alko - peg resin were purchased from watanabe chemical ( hiroshima , japan ) . other reagents and solvents were purchased from nacalai tesque ( kyoto , japan ) , tci ( tokyo , japan ) , kishida chemical ( osaka , japan ) , or kanto chemical ( tokyo , japan ) . synthesis ( 0.1 mmol scale ) was manually carried out using ho - ch2-pam resin ( 1.0 mmol / g ) . fmoc - gly - oh ( 1.2 mmol ) and hobt ( 1.2 mmol ) were dissolved in dimethylacetamide ( dma ) ( 4 ml ) . then n , n-dicyclohexylcarbodiimide ( dcc ) ( 1.2 mmol ) was added to the solution with cooling on the ice for 10 min . to the resulting solution , ho - ch2-pam resin ( 0.3 mmol ) the resin was then washed with dmf ( 3 ml ) and ch2cl2/ch3ch2oh ( 1/1 ) ( 3 ml ) three times , respectively . the resin was further washed with ch2cl2 ( 3 ml ) and dmf ( 3 ml ) three times , respectively . the resin was added to benzoic anhydride ( 1.5 mmol ) dissolved in pyridine / dmf ( 3/1 ) ( 3 ml ) and agitated for 1 hour . finally , the resin was washed with dmf ( 3 ml ) and ch2cl2 ( 3 ml ) four times . after the introduction of gly , the second aminoacid was introduced by using hobt ( 3 equiv . ) , hbtu ( 3 equiv . ) , and diea ( 6 equiv . ) . repetitive removal of the fmoc groups followed by introduction of protected fmoc aminoacids was performed according to the standard procedure to give the protected peptide resin . protected peptide resin ( 0.033 mmol ) in 1.0 ml of dry ch2cl2 was stirred for 15 min under argon in a 20 ml flask . in a second flask , 0.67 ml of ( ch3)3al ( 2 m hexane solution , 0.67 mmol ) diluted with 2.33 ml of dry ch2cl2 was cooled to 0c under argon [ 26 , 27 ] . to this solution , 0.15 ml of ethanethiol ( 124 mg , 2.0 mmol ) was added dropwise , and the resulting mixture was allowed to stir for 15 min at 0c . this solution was added at once to the suspension of peptide resin in ch2cl2 . after stirring for 5 hr at room temperature , protected peptide thioester was treated with tfa ( 2.7 ml ) in the presence of m - cresol ( 60 l ) , thioanisole ( 60 l ) , ethanedithiol ( 60 l ) , phenol ( 60 g ) , and water ( 60 l ) for 2 hr at room temperature . the resulting crude peptide was repeatedly purified by hplc with cosmosil 5c18-ar - ii ( 20 25 mm and 4.6 25 mm ) under gradient conditions for ch3cn in 0.1% aqueous tfa . concentrations of peptide were determined and adjusted by using absorption of amide bonds at 220 nm as follows : h2n - mdaqtrrrerraekqaqwkaaaaggg - cosch2ch3 ( n - peptide1 ) ; maldi - tof - ms : m / z = 2944.282 [ m ] ( calcd . mw = 2944.38 ) ; h2n - aaaatrqarrnrrrrwrerqrg - cosch2ch3 ( rev - peptide 1 ) ; maldi - tof - ms : m / z = 2824.679 [ m ] ( calcd . mw = 2825.29 ) ; h2n - naktrrherrrklaierdtaaggg - cosch2ch3 ( p22n - peptide 1 ) ; maldi - tof - ms : m / z = 2766.51 ( calcd . h2n - mdaqtrrrerraekqaqwkaaaaggg - cosch2ch3 ( n - peptide1 ) ; maldi - tof - ms : m / z = 2944.282 [ m ] ( calcd . mw = 2944.38 ) ; h2n - aaaatrqarrnrrrrwrerqrg - cosch2ch3 ( rev - peptide 1 ) ; maldi - tof - ms : m / z = 2824.679 [ m ] ( calcd . mw = 2825.29 ) ; h2n - naktrrherrrklaierdtaaggg - cosch2ch3 ( p22n - peptide 1 ) ; maldi - tof - ms : m / z = 2766.51 ( calcd . mw = 2764.17 ) . solid - phase peptide synthesis ( 0.1 mmol scale ) was manually carried out using fmoc - ala - alko - peg resin ( 0.25 mmol / g ) . the resin was treated with 20% piperidine in dmf to remove fmoc group at the amino - terminus . then , protected fmoc aminoacid was introduced to the resin by using with hobt ( 3 equiv . ) , hbtu ( 3 equiv . ) , and diea ( 6 equiv . ) . repetitive removal of the fmoc groups followed by introduction of protected fmoc aminoacids was performed according to the standard procedure to give the protected peptide resin . after the deprotection of fmoc group of cysteine , the resin was dried under high vacuum . then , the protected peptide resin was treated with tfa ( 2.6 ml ) in the presence of m - cresol ( 150 l ) , thioanisole ( 150 l ) , and ethanedithiol ( 75 l ) for 1 the resulting crude peptide was purified by hplc with cosmosil 5c18-ar - ii ( 20 25 mm and 4.6 25 mm ) under gradient conditions for ch3cn in 0.1% aqueous tfa . concentrations of peptide were determined and adjusted by using absorption of amide bonds at 220 nm as follows : h2n - cggaaamdaqtrrrerraekqaqwka - cooh ( n - peptide2 ) ; maldi - tof - ms : m / z = 2949.034 [ m ] ( calcd . mw = 2950.38 ) ; h2n - cgaaatrqarrnrrrrwrerqraaaa - cooh ( rev - peptide2 ) ; maldi - tof - ms : m / z = 3096.007 [ m ] ( calcd . mw = 3095.54 ) . h2n - cggaaamdaqtrrrerraekqaqwka - cooh ( n - peptide2 ) ; maldi - tof - ms : m / z = 2949.034 [ m ] ( calcd . mw = 2950.38 ) ; h2n - cgaaatrqarrnrrrrwrerqraaaa - cooh ( rev - peptide2 ) ; maldi - tof - ms : m / z = 3096.007 [ m ] ( calcd . plasmid encoding the tetrahymena group i intron ( ptz - ivsu ) was used as a template for pcr amplification of dna fragments for transcription of the p5 , ez-5half , ez-3half , and tet l-30 rna . plasmid encoding the p5abc mutant intron ( pl21-p5abc ) was used as a template for pcr amplification of a dna fragment for transcription of the p5 rna . peptide recognition modules ( prm-1 and -2 ) were attached to the template dnas for the p5- and ez-5half rna , respectively by pcr with primers containing the sequence of prm . transcription reactions with t7 rna polymerase were performed according to the published protocol , and transcripts were purified on 6% denaturing polyacrylamide gels . the concentrations of rnas were determined from the intensities of uv absorption at 260 nm . chemical ligation of peptides in the presence of rna molecules was carried out as follows . the p5 rna , ez-5half rna , and ez-3half rna were separately denatured in water for 2.5 min at 80c and then mixed together . to this rna solution , concentrated reaction buffer ( final compositions of the reaction mixture were 40 mm hepes , ph 8.0 , 80 mm kcl , and 10 mm mgcl2 ) was added , and the mixture was warmed for 5 min at 37c . reaction was started by adding aqueous solution of substrate peptides ( 7.5 m ) and 2-mercaptoethanesulfonate ( mesna , final concentration was 1% ( w / v ) ) at 37c . samples ( 20 l ) were quenched with 2 l of 40% aqueous tfa , and the reaction mixtures were analyzed by hplc with cosmosil 5c18-ar - ii ( 4.6 25 mm ) . gradient conditions for ch3cn in 0.1% aqueous tfa were as follows : 5%32.5% in 22.5 min ; flow rate : 1 ml / min . the fractions corresponding to the ligated products were collected and their molecular weights were confirmed by maldi - tof - mass spectrometry . the mean values are shown in the figures in which error bars indicate the minimal and maximal values . for samples forming complexes , aqueous solutions of the p5 rna ( 5 pmol , final concentration 0.5 m ) , the 3 ends of which were labeled with bodipy fluorophore , nonlabeled ez-5half and ez-3half rna ( 10 pmol , final concentration 1.0 m ) were heated separately at 80c for 2.5 min . , a 10 concentrated folding buffer was added to adjust the mixture to 50 mm tris - oac ( ph 7.5 ) and 10 mm mg(oac)2 . the resulting mixture was incubated at 37c for 30 min . after adding 6 concentrated loading buffer consisting of glycerol and xc ( 0.1% ) , the samples were loaded onto a 5% nondenaturing polyacrylamide gel ( 29 : 1 acrylamide : bisacrylamide ) containing 50 mm tris - oac ( ph 7.5 ) and 10 mm mg(oac)2 . electrophoresis was carried out at room temperature with 200 v for the initial 5 min and then 75 v for 5 h. the resulting gels were analyzed with a fluoroimager pharos fx ( biorad , hercules , ca , usa ) . to modify the tetrahymena intron rna as a self - assembling template assisting chemical peptide ligation , a three - step redesign was carried out ( figures 1(b ) , 1(c ) , and 2(a ) ) . the first step involves conversion of the parent unimolecular rna ( tet l-30 ) to a two - piece derivative consisting of the p5 rna and the rest of the intron ( p5 rna ) as described in the introduction ( figures 1(b ) , 1(c ) , and 2(a ) ) . the second step involves the introduction of a break in the p5 region of the p5 intron rna ( figures 1(b ) , 1(c ) , and 2(a ) ) . the resulting two fragments ( ez-5half and ez-3half ) the reconstitution of p5 intron rna ( termed ez in this study ) from the two rna fragments is convenient for installation of the peptide - recognition motif ( prm ) . in the template dna for in vitro transcription , the introduction of prm to the ez-5half fragment of the bimolecular version of the p5 rna ( ez rna ) can be achieved simply by modifying the pcr primers . on the other hand , for the introduction of a prm sequence to the corresponding position ( the 92nd nucleotide from the 5 end ) of the unimolecular p5 rna ( 309 nucleotides ) , template dnas need to be prepared using either a reliable but laborious method ( plasmid construction using standard recombination techniques ) or a convenient but less reliable method ( preparation of a double - stranded dna using multistep pcr to assemble a set of dna oligonucleotides ) . gel mobility shift assay was carried out to confirm reconstitution of the tetrahymena group i intron ( tet l-30 ) by the three rnas ( p5 , ez-5half , and ez-3half ) ( figure 3(a ) ) . in the presence of the three rnas , mobility of the main band was similar to those of the parent unimolecular tet l-30 rna and the bimolecular complex ( p5 : p5 ) , indicating that the two - piece ( p5 and ez ) rna consisting of three rna fragments ( p5 , ez-5half , and ez-3half ) can be used as a platform rna ( termed the p5 : ez platform ) for chemical peptide ligation . the third step involves the introduction of two peptide - recognition motifs ( prms ) to the complex ( figures 1(b ) , 1(c ) , and 2(a ) ) . the one prm ( prm1 ) was introduced to the p5 rna and the other prm ( prm2 ) was introduced to the ez-5half fragment of the ez rna . to determine the versatility of the p5 : ez platform in chemical peptide ligation , we prepared six template rna complexes designed for peptide chemical ligations with six combinations of substrate peptides ( figure 2(a ) ) . to confirm that the introduction of prm1 and prm2 does not interfere with the complex formation between p5 and ez , we performed gel mobility shift assay with six template rna complexes , none of which showed significant reduction of the complex formation ( figure 3(b ) ) . since the binding between the p5 rna and the p5 rna was reported to be highly sensitive to misfolding , these results also indicate that neither the folding of p5 and ez nor their complex formation was disturbed severely by the introduction of prm1 and prm2 . structure - based design of the template rnas was carried out by installing two prms ( prm1 and prm2 ) on the platform p5 : ez complex ( figure 1 ) . each prm1 captures its target substrate ( peptide1 ) bearing the reactive thioester group in its c - terminus . each prm2 captures its target substrate ( peptide2 ) bearing cysteine in its n - terminus . to employ three rna - binding peptides ( n - peptide1 , p22n - peptide1 , and rev - peptide1 , figure 2(b ) ) as the substrate peptide-1 , we designed three p5 rna derivatives with prm1s for substrate peptides1 ( boxb , p22boxb , and rre ) . as the substrate peptides2 recognized by the ez rna , we used two peptides ( n - peptide2 and rev - peptide2 , figure 2(b ) ) . their target boxb and rre motifs were installed to the ez rna as prm2s ( figure 2(a ) ) . basal activities of six combinations of the substrate peptide-1 and -2 were investigated ( figure 4 ) . in the absence of template rnas , products yields in 7 h reaction of chemical ligations with six combinations of substrates ( 7.5 m each ) were around 1020% ( figure 4(b ) ) . to evaluate the ability of template rna derived from the platform complex ( p5 : ez ) , we designed and examined six combinations of peptide ligation reactions based on the model 3d structures of the substrate - template complexes ( figure 4(a ) ) . in the presence of stoichiometric amounts of the cognate template rna complex ( 7.5 m ) , product yields of the four reactions were improved to around 40% ( figure 4(b ) ) . the reaction between p22n - peptide1 and rev - peptide2 exhibited remarkable improvement of the product yield that reached 73% . on the other hand , the reaction between n - peptide1 and n - peptide2 was not promoted by the template rna ( figure 4(b ) ) . to determine whether the improvement of the reaction is due to the complex formation modeled in figure 4(a ) , control reactions were carried out using the parent p5 and ez rnas lacking prm1 and prm2 . in the reactions between rev - peptide1 and rev - peptide2 and between n - peptide1 and n - peptide2 ( figure 4(b ) ) , prms gave no positive effect ( figure 4(b ) ) . these results are predictable because these templates capture the two substrates in a productive manner ( peptide1/prm1 + peptide2/prm2 ) and inhibitive manners ( peptide2/prm1 + peptide1/prm2 , peptide1/prm1 + peptide1/prm2 , and peptide2/prm1 + peptide2/prm2 ) . since the yield of the reaction between p22n - peptide1 and rev - peptide2 ( 58% ) was close to the yield in the presence of the cognate template rna ( 73% ) , the template effect for this reaction was compared in the early phase of the reaction ( 1-h reaction , figure 4(c ) ) . the product yield of the cognate template rna was twofold higher than the yield with the control rna . on the other hand , the effect of the control rna on the reaction between p22n - peptide1 and rev - peptide2 was more efficient than the effect of the cognate template rna on the reaction of n - peptide1 and rev - peptide2 ( figure 4(c ) ) . among the six combinations of chemical peptide ligations , the reaction between n - peptide1 and rev - peptide2 was chosen for further investigation of the template ability of the self - assembling rna derived from the p5 : ez complex . reaction time courses indicated that the ligated product was produced through ( i ) intrinsic reactivity of the two peptides , ( ii ) assistance of rna in a prm - independent manner , or ( iii ) assistance of rna in a prm - dependent manner ( figure 5(a ) ) . to evaluate the contributions of ( ii ) and ( iii ) , we systematically prepared mutant complexes and investigated their effects . to determine the importance of two prms being a pair in the prm - dependent promotion of the ligation , we examined the ligation reaction in the presence of only the p5-boxb rna or the ez - rre rna ( figure 5(b ) ) . the product yields of the two reactions ( 15% and 13% ) were lower than the yield with cognate complex ( 44% ) and close to the yield with the platform p5 : ez complex ( 17% ) . to further investigate the effect of prm complex formation between p5 and ez was not affected by the number of prm in the p5 : ez complex ( figure 5(c ) ) . the resulting two complexes lacking either prm1 or prm2 served as templates much less effectively than the complex with two prms , and their effects were comparable to that of the platform complex ( p5 : ez ) ( figure 5(d ) ) . these results indicated that the prm - dependent enhancement was achieved by cooperative effects of prm1 ( boxb motif ) and prm2 ( rre motif ) . the product yields in the presence of the mutant complexes possessing one prm ( p5-boxb : ez and p5 : ez - rre in figure 5(d ) ) were slightly higher than those in the presence of p5-boxb or ez - rre alone ( figure 5(b ) ) . this difference may reflect the prm - independent enhancement contributed by the ez and p5 components in the mutant p5-boxb : ez and p5 : ez - rre complexes , respectively . in this study , we employed the two - piece derivative of the tetrahymena intron rna ( p5 : p5 complex ) as a self - assembling platform for a template rna to assist peptide ligation . the structural organization of the p5 : p5 platform ensures structural independence between the modules for rna - rna assembly and the modules for recognition of substrate peptides ( prm ) , suggesting the versatility and flexibility of the p5 : p5 platform in molecular design . this study confirmed the versatility of the p5 : p5 platform for template design because six types of template rna complex were designed and their template capabilities were tested experimentally . on the other hand , this study also revealed the current limitation of the p5 : p5 platform . in the case of the reaction between n - peptide1 and rev - peptide2 , the prm - dependent facilitation by the p5 : ez complexes possessing two prms was similar to the facilitation by the self - folding p4p6 rna with two prms . the prm - independent facilitation by the parent p5 : ez platform , however , was more efficient than those by the previous platforms based on the self - folding rnas . the negatively charged surface of 3d - rnas electrostatically accumulates positively charged substrate peptides [ 33 , 34 ] . this effect may be stronger in the p5 : p5 platform than in previous platforms because of the considerably larger surface area of the p5 : p5 platform . this property is undesirable if the p5 : p5-based rna is applied as a template to conduct prm - dependent ligation of the cognate substrate pair in the presence of non - cognate substrates . therefore , reduction of the surface size of 3d - rna may be a strategy to suppress prm - independent facilitation of peptide ligation . in the p5 : p5 platform , however , most of the core and peripheral modules contribute to self - assembly of p5 and p5 and also maintenance of its 3d structure . addition of cosolute molecule to the buffer solution may be an alternative approach [ 35 , 36 ] . positively charged small molecules or polymers may competitively block the substrate peptide to assemble with the platform region of the template rna , thus competitively suppressing the prm - independent reaction promotion . on the other hand , an appropriate amount of cationic cosolute may not inhibit specific binding between peptides and their target prms , resulting in preservation of prm - dependent reaction . an unexpected finding of the present study was the exceptionally efficient rna assistance of ligations between p22n - peptide1 and rev - peptide2 and between rev - peptide1 and rev - peptide2 without prms ( figure 4(b ) ) . in contrast to the two reactions , rna templates gave no positive effect on the reaction between n - peptide1 and n - peptide2 ( figure 4(b ) ) . no prm - independent facilitation was observed in the reaction between p22n - peptide1 and n - peptide2 ( figure 4(b ) ) . these results suggest that the prm - independent reaction promotion by the p5 : p5 platform depends on the combination of rna - binding peptides . rev - peptide and p22n - peptide seem more congenial to the rna - promoted chemical ligation than n - peptide ( figure 4 ) . although the molecular basis underlying these results remains to be elucidated , it is important to see whether apparently different results originate from the physical property of each peptide . n - peptide is less hydrophilic than p22n- and rev - peptides ( figure 2(b ) ) , and p22n - peptide has markedly higher affinity to cognate and noncognate rna motifs than n- and rev - peptides . thus , it is also important to determine whether the possible approaches to suppress prm - independent reaction promotion are effective on the reaction between p22n - peptide1 and rev - peptide2 .

intellectual disability ( i d ) , previously referred to as mental retardation , is most often associated with other medical and psychiatric conditions such as cerebral palsy , epilepsy , down syndrome , fragile x syndrome , attention deficit hyperactivity disorder ( adhd ) , autism , and other emotional and behavioral disorders . the study of intellectual disability falls within the field of psychiatry , in which dual diagnoses have historically been common . however , specifically investigating the coexistence of psychiatry disorders among people with i d has only recently begun [ 13 ] . compared with the general population , people with i d have a higher prevalence of psychiatry disorders [ 4 , 5 ] , ranging from 10% to 80% [ 6 , 7 ] . for the past few decades , the psychiatric community in developed countries , like england and australia , has done more to identify the psychiatric needs of people with i d , which has allowed doctors to provide these patients with appropriate mental health services [ 7 , 1416 ] . in india dual diagnosis of psychiatric disorders in people with i d has gradually become more common since implementation of the persons with disabilities act in 1995 . however , making further efforts to identify people in india with i d who also suffer from psychiatric disorders is necessary to plan for and provide comprehensive intervention for their well - being . identifying psychiatric disorders in people with i d is a highly specialized area for professionals because the symptoms of psychiatric disorders in people with i d do not always resemble the symptoms of the same psychiatric disorders in the general population . furthermore , poor cognitive abilities may overshadow other psychiatric disorders in children with i d , preventing accurate diagnoses of coexisting conditions [ 18 , 19 ] . objective . to determine the prevalence of coexisting psychiatry disorders in children with i d ranging in age from 3 to 18 years in barwani . the prevalence of coexisting psychiatric disorders among children with i d identified in a cross - sectional study as part of a community - based rehabilitation project in the barwani block of barwani district in madhya pradesh . ashagram trust ( agt ) is a nongovernment organization located in barwani that provides medical and rehabilitation services to the people of barwani and its surrounding districts . of the entire district , 53% of the population lives below the poverty line . in general , the tribal population is more disadvantaged in the district than the nontribal population . with financial help from action aid , agt implemented a community - based rehabilitation ( cbr ) project , spanning from 1999 to 2010 , in 63 villages of the barwani block . in 2001 , a total of 10,909 households , comprising 63,789 people ( 2001 census ) , in 51 villages were surveyed door - to - door to identify children with i d . all identified children were recruited so that they could receive proper medical , rehabilitation , educational , and vocational support . first , consent to conduct the screening survey was obtained from each village 's politically elected leader ( sarpanch ) . consent was also obtained from the heads of each family ( parents or grandparents ) to assess the child for i d and coexisting disorders and receive comprehensive intervention under the cbr project . an ethical committee comprising members of the funding organization action aid , agt , and the community approved the study . from this survey , 262 children ranging in age from 3 to 18 years were identified as having i d . children with i d who developed psychiatric disorders during the course of the study were also included in the study . children identified in the cross - sectional study were further evaluated by two professionals specializing in the assessment of mental retardation ( mr ) . these professionals were employed by the cbr project at the time ( one is the author of this paper ) . diagnoses of i d were made according to iq classification , following the criteria outlined in the icd-10 . the icd-10 criteria are popular and widely used across member countries of the world health organization to diagnose people with mental and behavioral disorders . two tests , a developmental screening test ( dst ) developed by the national institute for the mentally handicapped ( nimh ) in india and an indian adaptation of the vineland social maturity scale ( vsms ) , were used in the first round of assessment . the dst provided the developmental quotient ( dq ) value , while the vsms provided the social quotient ( sq ) value . the average of these two values ( dq and sq ) is considered a person 's iq . most of the study participants were also given an adaptation of the stanford - binet intelligence test , called the binet - kamat test , which better reflects conditions in india . a number of previous studies have supported the value of these tests , concluding that their psychometric properties are appropriate for making diagnoses [ 25 , 26 ] . furthermore , the nimh in india recommends these tests for diagnosing people with i d . all cases evaluated by mr professionals were referred to a psychiatrist for psychiatric evaluation and a physician for physical examination . laboratory testing was also done to determine chromosomal abnormalities . in each case , psychiatrists used clinical observations , mental state examinations , parental interviews , and reviews of previous consultations to make a psychiatric diagnosis , incorporating the icd-10 criteria . because epilepsy shares characteristics of both neurological and psychiatric disorders , we categorized it with other psychiatric disorders in this study . children with i d who developed psychiatric disorders during the time frame of the project were also included in the study . occasionally , communication barriers arose because of the tribal dialect . most professionals on the cbr team were able to understand tribal dialect , but they were unable to respond in a dialect that could be understood . communication was also difficult with some nontribal parents . in situations where communication was a problem , community - based rehabilitation workers ( cbrws ) interpreted between psychiatrists , mr professionals , and parents . most cases were brought to the agt psychiatric clinic , but a few could not reach the clinic because of inadequate transportation . in these situations , children were evaluated in rural camps within their own or nearby villages . statistical analysis . the prevalence of psychiatry disorders in the study 's 262 children with i d was reported using frequencies and percentages . chi - square tests were performed to compare the prevalence of psychiatric disorders with independent variables , such as the severity of i d ( severe intellectual disability ( sid ; iq 49 ) and mild intellectual disability ( mid ; iq 50 ) ) , gender , poverty level , and population group ( tribal and non - tribal ) . because the psychiatric group disorders had expected count values less than five , the chi - square test was performed with the yates correction . of the total i d population ( n = 262 ) , 17 ( 6.5% ) were found to have adhd , 11 ( 4.2% ) were autistic , 7 ( 2.7% ) showed anxiety , 3 ( 1.1% ) had bipolar affective disorder , 2 ( 0.8% ) had delusional disorder , 6 ( 2.3% ) had depression , 2 ( 0.8% ) had obsessive - compulsive disorder , 5 ( 1.9% ) had schizophrenia , 27 ( 10.3% ) had enuresis , 62 ( 23.7% ) had epilepsy , and 212 ( 80.9% ) showed behavioral problems . . however , children with profound i d appeared to only have epilepsy and behavioral problems . the number of disorders per child in each i d category was also examined . in the borderline however , the mild and moderate i d categories both had a ratio of 1.27 disorders per child , while the severe i d group was calculated to have 1.65 disorders per child . overall , behavioral problems were considerably more prevalent than other psychiatric disorders in all i d categories except borderline i d . for comparison purposes , the rarely occurring psychiatric disorders of adhd , autism , anxiety , bipolar disorder , delusional disorder , depression , obsessive - compulsive disorder , and schizophrenia were grouped together for analysis ( psychiatric group ) . all cases of i d were grouped into categories of sid and mid . upon comparing the sid and mid groups , we found that the prevalences of psychiatric group disorders = 0.3 * , p = .03 ) and epilepsy ( = 27.6 , p < .001 ) were significantly higher in children with sid , while the prevalences of enuresis ( = 0.5 , p = .31 ) and behavioral problems ( = 1.3 , p = .25 ) were not statistically different between the two i d groups . the prevalences of psychiatric group disorders ( = 0.2 * , p = .54 ) , enuresis ( = 1.7 , p = .13 ) , and behavioral problems ( = 0.1 , p = .47 ) were not statistically different between male and female participants , but epilepsy was found to be more prevalent among male participants than females ( = 7.4 , p = .005 ) . enuresis was found to be statistically more prevalent among poor children ( = 4.1 , p = .03 ) , but no differences in prevalence were found for psychiatric group disorders ( = 0.1 * , p = .91 ) , behavioral problems ( = 0.0 , p = .57 ) , and epilepsy ( = 0.8 , p = .45 ) between poor children and children who were not poor . the prevalences of enuresis ( = 7.2 , p = .006 ) and behavioral problems ( = 4.1 , p = .03 ) were higher among the tribal population , but psychiatric group disorders ( = 0.2 * , p = .50 ) and epilepsy ( = 0.1 , p = .43 ) showed no significant difference in prevalence between tribal and nontribal populations ( table 2 ) . in this study , we found a higher prevalence of psychiatric disorders in children with i d having an iq 49 , an outcome that is consistent with the findings presented in gillberg et al . ( 1986 ) . among children with an iq 49 , those with iq between 49 and 35 had the highest rates of psychiatric disorders . other than epilepsy and behavioral problems , none of the disorders from psychiatric group were found in children whose iq 20 . one possible explanation for this result is that diagnostic difficulty increases with the severity of a person 's i d [ 6 , 12 , 13 , 29 ] . the findings of our study related to psychiatric disorders in the tribal population are consistent with a prior study conducted on an indian population by kishore et al . that study found that children living in poor families and children belonging to the tribal community have a higher prevalence of enuresis , which may arise from improper care , improper training about personal care , activities of daily living , social exposure , and a lack of stimulation . our study also showed that behavioral problems are higher in the tribal population group , indicating that behavioral problems and enuresis could have similar contributing factors within this population . the prevalence of enuresis between males and females in this study was not found to be different . however , a previous study from 1986 indicated that males are likely to have a higher prevalence of enuresis than females , with 16% of males having the condition in that study , compared with 12% of females ( p < .01 ) . enuresis is approximately twice as common in boys as in girls in the normal population . family history of enuresis , lower socioeconomic status , poor self - image , and diminished academic achievements are considered to be risk factors for enuresis . in our study , factors like less gender discrimination , poverty , and simple lifestyles may have contributed to the equal prevalence of enuresis between boys and girls in the study population . the prevalence of epilepsy was not found to be affected by poverty level ( poor versus not poor ) and population group ( tribal versus non - tribal ) , but it was found to be significantly higher in males than females . this finding is consistent with the normal population , in which studies have reported a marginally lower prevalence of epilepsy in females . in our study , a significantly higher rate of epilepsy was found in children with severe and profound i d ( iq 49 ) , which has previously been reported as the most common disorder in people with severe i d . when people with i d have additional psychiatric disorders , they become even more dependent on others and may urgently require mental health services . treating coexisting psychiatric disorders may favorably affect rehabilitation intervention in people with i d . therefore , it is important to assess people with i d to identify any coexisting psychiatric disorders . a study by mohr et al . ( 2002 ) highlighted the benefits of diagnosing these coexisting conditions : they reported successful social rehabilitation of people with i d that had been concurrently diagnosed and treated for psychiatric disorders . in addition standardized tests were used for i d diagnoses , but in some cases standardized tests were not used to diagnose psychiatric disorders , resulting in diagnoses made solely on the basis of clinical judgments by psychiatrists . senior - level psychiatrists and lower - level trainee psychiatrists both provided their services in evaluating i d cases . however , the individual clinical abilities and biases of each psychiatrist might have impacted the results . furthermore , the language barrier between clients and professionals was also a potential factor , which might have affected the clinical decisions made by professionals . another limitation of the study was that the psychiatric unit could not maintain proper records in all cases ; some of the case files did not contain complete information about the subjects or their clinical examinations . thus , there exists the potential that some people who had psychiatric disorders were excluded in the study analysis . thus , it is very important to properly diagnose any coexisting psychiatric disorders so that the appropriate services can be provided .

brain tumors occur rarely in pregnant women but are nevertheless the third commonest cause of cancer death in women aged 15 to 39 years.1 treatment of brain tumors during pregnancy is a difficult task which presents a major dilemma how to save the life of the mother and that of the growing fetus ? specialists may be presented with a patient who has an overwhelming desire to continue the pregnancy , while decision making may be complicated by the involvement of a uniquely broad multidisciplinary team consisting of obstetricians , surgeons , oncologists , anesthesiologists , neonatologists , and other disciplines . we present a retrospective clinical case of a fourth ventricle anaplastic ependymoma with brain stem invasion in a woman who presented in the 20th week of a pregnancy initiated via in vitro fertilization and embryo transfer ( ivf - et ) . we discuss aspects of her treatment which included surgical resection of the tumor , resulting in a favorable outcome for mother and child . a 33-year - old patient was admitted to a neurosurgical tertiary referral center with constant , intense headaches , dizziness , nausea , and vomiting . she was in the second trimester of a long - awaited first pregnancy , which came after 11 years of attempts at conception and was the result of a third cycle of ivf - et . her symptoms began in the third week of pregnancy as pulsating headaches which were worse in the morning , responsive to oral analgesics and occasionally accompanied by nausea , and were attributed to early pregnancy toxemia . in the second trimester , deterioration in the form of worsened headache , nausea , dizziness , gait disturbance , photophobia , and recurrent vomiting ensued . magnetic resonance imaging ( mri ) , performed at a secondary hospital ( fig . 1a , b ) , revealed a 2 3 cm tumor arising in the region of the fourth ventricle , invading the brain stem and causing obstructive hydrocephalus . ( a ) a sagittal t1-weighted gadolinium - enhanced mri scan of the head of a pregnant 33-year - old woman , demonstrating a contrast - enhancing lesion in the posterior fossa invading the brain stem and causing mass effect and obstructive hydrocephalus . ( b ) a coronal mri scan of the same lesion . ( c ) a t1-weighted sagittal mri scan of the same patient 2 months after total resection of the lesion confirmed to be an anaplastic ependymoma arising from the fourth ventricle . a multidisciplinary meeting was held with neurologists , neurosurgeons , anesthetists , and obstetricians in which the strong desire of the patient to continue the pregnancy was taken into account . in light of the hydrocephalus , the deteriorating neurological condition of the patient and signs of intrauterine fetal distress , intervention in the form of removal of the tumor with intraoperative monitoring of the fetus was planned . informed consent was provided by the patient and she underwent a posterior fossa craniotomy and microsurgical resection of the tumor . we normally perform posterior fossa tumor excision in the seated position but , in this case , the patient was positioned in the left lateral position to reduce the risk of aortocaval compression and thromboembolic complications . general anesthesia was initiated using low - flow inhaled isoflurane ( mean alveolar concentration 0.40.6 ) and maintained with 200 mg of 2% propofol with 0.1 mg of 0.005% fentanyl intravenously . the surgery lasted 3 hours 40 minutes and intraoperative blood loss was approximately 300 ml . histological examination of the resected tumor confirmed a grade iii anaplastic ependymoma with extensive hemorrhage . the postoperative period was uneventful for mother and fetus , and the patient was discharged on the 11th postoperative day . follow - up mri with contrast was performed at 2 months with no evidence of tumor recurrence ( fig . the patient remained under the careful supervision of neurologists , neurosurgeons , and obstetricians for 4 months until giving birth to a healthy baby girl weighting 3,200 g at 39 weeks ' gestation by cesarean section . meningiomas have been observed in some pregnant women to become symptomatic during pregnancy , followed by postpartum remission and recurrence in subsequent pregnancies , arguably due to direct hormonal influence.2 similar symptomatic exacerbations have been observed in glial tumors.2 the literature on the incidence of brain tumors during pregnancy in the context of ivf is critically sparse , with a single case report of glioblastoma in the third trimester of an ivf pregnancy . malignant transformation of a histologically verified breast fibroadenoma following hormonal stimulation for ivf has also been documented . the physiological changes associated with pregnancy may mask the signs and symptoms of a brain tumor . symptoms such as headache , nausea , vomiting , seizures , fainting , and cognitive impairment are very similar to toxemia of early pregnancy , preeclampsia , and eclampsia and may , therefore , result in delay and difficulty in the diagnosis of the true cause of these symptoms . health professionals involved in the care of pregnant woman should maintain oncological vigilance in cases involving assisted reproductive techniques such as ivf , especially in those women with a previously diagnosed tumor of any kind . despite growing clinical experience in the treatment of cancer during pregnancy , knowledge about optimal management the need for multicenter studies to provide an evidence base for this multidisciplinary problem continues to grow and initiatives such as the international network on cancer , infertility and pregnancy3 can play a significant role in facilitating informed decisions . the diagnosis and treatment of brain tumors during pregnancy presents a challenge to clinicians as the safest approach for both mother and fetus may not always be clear . the case presented here demonstrates that the existence of neurosurgical pathology requiring urgent intervention is not an absolute contraindication to continuation of the pregnancy . success in such cases is based on close cooperation between members of the multidisciplinary team . the possibility of an effect of hormone stimulation on the growth and malignant transformation of neoplasms during ivf therapy should be borne in mind .

as a part of ten conditions ten morbidity measure study,13 this cross - sectional study invited 100 consecutive patients ( both male and female at a minimum age of 18 years ) diagnosed with ra , confirmed by a rheumatologist based on the american rheumatism association criteria.14 the patients were recruited between june and december 2006 to the outpatient rheumatology clinic of baqiyatallah hospital , tehran , iran . exclusion criteria were association with a necrotic vascular disease or a handicap prior to ra.15 eighty patients met the inclusion criteria . the study was approved by the ethics committee on human research of baqiyatallah university of medical sciences . written informed consents were obtained from all participants . a checklist containing questions on demographic data ( age , gender , marital status , living place , monthly family income , and education level ) was completed by every patient and was collected by a research nurse upon enrollment . the patients global assessment of disease activity ( patga ) and pain ( pain - vas ) over the previous month were recorded using a visual analogue scale ( vas ) . a 100-mm rating scale was applied , with one end bearing the statement no problem whatsoever and the other end the patients were instructed to place a vertical mark on the scale reflecting their own health status . all the patients underwent a medical examination by a board - certified rheumatologist , who also determined the duration of their disease and performed a standard 28-joint assessment for tender and swollen joints making use of pain score and swelling score described in the eular manual of clinical methods.16 a similar vas was utilized to determine the physician 's global assessment of the disease activity ( phyga ) . laboratory investigations comprised of determining erythrocyte sedimentation rate ( esr ) and c - reactive protein ( crp ) by westergren method and nephelometry , respectively.17 das indices , namely das - esr-3 , das - esr-4 , das - crp-3 , das - crp-4 , das28-esr-3 , das28-esr-4 , das28-crp-3 , das28-crp-4 , and simplified disease activity index [ sdai ( esr ) , sdai ( crp ) ] , were calculated using the formula described in previous reports.1718 the patients hrqol was measured via the medical outcomes study 36-item short form health survey ( sf-36).19 sf-36 is a generic multidimensional measure of hrqol that contains eight subscales representing physical functioning , social functioning , role limitations due to physical health problems , role limitations attributable to emotional problems , mental health , vitality , bodily pain , and general health perceptions . higher scores of each subscale ( which can range from 0 to 100 ) indicate a better hrqol . the physical and mental components of the eight scales are combined into physical ( pcs ) and mental ( mcs ) component summary scale scores . sf-36 has been proved reliable and valid in ra patients.20 a persian version of sf-36 was used to ensure face validity and maximize the acceptability in our participants , all of whom were persian speakers.2122 sf36 has been used for ra studies in iran.23 spss statistical software was employed in statistical analyses . the pearson correlation was utilized to examine 1 ) the correlations between the sf-36 scores and the das indices and 2 ) the correlations between different das indices . the correlation coefficients of the sf-36 scores and the das indices were compared using pass 2000 ( power analysis and sample size ) software . a checklist containing questions on demographic data ( age , gender , marital status , living place , monthly family income , and education level ) was completed by every patient and was collected by a research nurse upon enrollment . the patients global assessment of disease activity ( patga ) and pain ( pain - vas ) over the previous month were recorded using a visual analogue scale ( vas ) . a 100-mm rating scale was applied , with one end bearing the statement no problem whatsoever and the other end the patients were instructed to place a vertical mark on the scale reflecting their own health status . all the patients underwent a medical examination by a board - certified rheumatologist , who also determined the duration of their disease and performed a standard 28-joint assessment for tender and swollen joints making use of pain score and swelling score described in the eular manual of clinical methods.16 a similar vas was utilized to determine the physician 's global assessment of the disease activity ( phyga ) . laboratory investigations comprised of determining erythrocyte sedimentation rate ( esr ) and c - reactive protein ( crp ) by westergren method and nephelometry , respectively.17 das indices , namely das - esr-3 , das - esr-4 , das - crp-3 , das - crp-4 , das28-esr-3 , das28-esr-4 , das28-crp-3 , das28-crp-4 , and simplified disease activity index [ sdai ( esr ) , sdai ( crp ) ] , were calculated using the formula described in previous reports.1718 the patients hrqol was measured via the medical outcomes study 36-item short form health survey ( sf-36).19 sf-36 is a generic multidimensional measure of hrqol that contains eight subscales representing physical functioning , social functioning , role limitations due to physical health problems , role limitations attributable to emotional problems , mental health , vitality , bodily pain , and general health perceptions . higher scores of each subscale ( which can range from 0 to 100 ) indicate a better hrqol . the physical and mental components of the eight scales are combined into physical ( pcs ) and mental ( mcs ) component summary scale scores . sf-36 has been proved reliable and valid in ra patients.20 a persian version of sf-36 was used to ensure face validity and maximize the acceptability in our participants , all of whom were persian speakers.2122 sf36 has been used for ra studies in iran.23 spss statistical software was employed in statistical analyses . the pearson correlation was utilized to examine 1 ) the correlations between the sf-36 scores and the das indices and 2 ) the correlations between different das indices . the correlation coefficients of the sf-36 scores and the das indices were compared using pass 2000 ( power analysis and sample size ) software . a checklist containing questions on demographic data ( age , gender , marital status , living place , monthly family income , and education level ) was completed by every patient and was collected by a research nurse upon enrollment . the patients global assessment of disease activity ( patga ) and pain ( pain - vas ) over the previous month were recorded using a visual analogue scale ( vas ) . a 100-mm rating scale was applied , with one end bearing the statement no problem whatsoever and the other end the patients were instructed to place a vertical mark on the scale reflecting their own health status . all the patients underwent a medical examination by a board - certified rheumatologist , who also determined the duration of their disease and performed a standard 28-joint assessment for tender and swollen joints making use of pain score and swelling score described in the eular manual of clinical methods.16 a similar vas was utilized to determine the physician 's global assessment of the disease activity ( phyga ) . laboratory investigations comprised of determining erythrocyte sedimentation rate ( esr ) and c - reactive protein ( crp ) by westergren method and nephelometry , respectively.17 das indices , namely das - esr-3 , das - esr-4 , das - crp-3 , das - crp-4 , das28-esr-3 , das28-esr-4 , das28-crp-3 , das28-crp-4 , and simplified disease activity index [ sdai ( esr ) , sdai ( crp ) ] , were calculated using the formula described in previous reports.1718 the patients hrqol was measured via the medical outcomes study 36-item short form health survey ( sf-36).19 sf-36 is a generic multidimensional measure of hrqol that contains eight subscales representing physical functioning , social functioning , role limitations due to physical health problems , role limitations attributable to emotional problems , mental health , vitality , bodily pain , and general health perceptions . higher scores of each subscale ( which can range from 0 to 100 ) indicate a better hrqol . the physical and mental components of the eight scales are combined into physical ( pcs ) and mental ( mcs ) component summary scale scores . sf-36 has been proved reliable and valid in ra patients.20 a persian version of sf-36 was used to ensure face validity and maximize the acceptability in our participants , all of whom were persian speakers.2122 sf36 has been used for ra studies in iran.23 the pearson correlation was utilized to examine 1 ) the correlations between the sf-36 scores and the das indices and 2 ) the correlations between different das indices . the correlation coefficients of the sf-36 scores and the das indices were compared using pass 2000 ( power analysis and sample size ) software . median ( first and second quartiles ) disease duration was 60 ( 24 - 99 ) months . . different disease activity score ( das ) indices and formulas demographic and clinical characteristics of the studied population ( n = 80 ) mean serum levels of esr and crp were 2 6.0 19.6 mm / h and 1.14 0.34 mg the mean ( sd ) das4 score was 1.7 0.8 ( range , 0.2 to 4.2 ) , and the mean values of the das - esr-3 , das- crp -4 , and das - crp-3 crp scores were 1.9 0.8 ( range , 0.4 to 4.4 ) , 1.5 0.7 ( range , 0.5 to 4.3 ) , and 1.6 0.7 ( range , 0.7 to 4.5 ) , respectively . in addition , the mean ( sd ) das28-esr-4 score was 3.0 1.0 ( range , 0.5 to 5.6 ) , and the mean values of the das28-esr-3 , das28-crp-4 , and das28-crp-3 scores were 3.3 1.1 ( range , 0.6 to 6.0 ) , 2.4 0.9 ( range , 1.2 to 5.3 ) , and 2.7 0.9 ( range , 1.4 to 5.8 ) , respectively . also , the respective mean ( sd ) sdai and sdai ( crp ) scores were 36.5 23.3 ( range , 5.7 to 106.3 ) and 12.0 7.4 ( range , 1.8 to 39.2 ) . table 3 dpresents the correlations between different dass . correlations among different disease activity indices the mean ( sd ) sf-36 total score among all patients was 49.8 16.7 ( range , 18.1 to 91.5 ) . the correlation coefficients between hrqol and different das indices are depicted in table 3 . as it is seen in table 4 , the dass correlated with physical hrqol but they did not relate with mental hrqol . correlations between different disease activity indices and health - related quality of life in rheumatoid arthritis the use of pass 2000 software showed no significant difference in terms of correlation coefficients between different das indices and hrqol scores . median ( first and second quartiles ) disease duration was 60 ( 24 - 99 ) months . . different disease activity score ( das ) indices and formulas demographic and clinical characteristics of the studied population ( n = 80 ) mean serum levels of esr and crp were 2 6.0 19.6 mm / h and 1.14 0.34 mg the mean ( sd ) das4 score was 1.7 0.8 ( range , 0.2 to 4.2 ) , and the mean values of the das - esr-3 , das- crp -4 , and das - crp-3 crp scores were 1.9 0.8 ( range , 0.4 to 4.4 ) , 1.5 0.7 ( range , 0.5 to 4.3 ) , and 1.6 0.7 ( range , 0.7 to 4.5 ) , respectively . in addition , the mean ( sd ) das28-esr-4 score was 3.0 1.0 ( range , 0.5 to 5.6 ) , and the mean values of the das28-esr-3 , das28-crp-4 , and das28-crp-3 scores were 3.3 1.1 ( range , 0.6 to 6.0 ) , 2.4 0.9 ( range , 1.2 to 5.3 ) , and 2.7 0.9 ( range , 1.4 to 5.8 ) , respectively . also , the respective mean ( sd ) sdai and sdai ( crp ) scores were 36.5 23.3 ( range , 5.7 to 106.3 ) and 12.0 7.4 ( range , 1.8 to 39.2 ) . table 3 dpresents the correlations between different dass . correlations among different disease activity indices the mean ( sd ) sf-36 total score among all patients was 49.8 16.7 ( range , 18.1 to 91.5 ) . the correlation coefficients between hrqol and different das indices are depicted in table 3 . as it is seen in table 4 , the dass correlated with physical hrqol but they did not relate with mental hrqol . correlations between different disease activity indices and health - related quality of life in rheumatoid arthritis the use of pass 2000 software showed no significant difference in terms of correlation coefficients between different das indices and hrqol scores . median ( first and second quartiles ) disease duration was 60 ( 24 - 99 ) months . . different disease activity score ( das ) indices and formulas demographic and clinical characteristics of the studied population ( n = 80 ) mean serum levels of esr and crp were 2 6.0 19.6 mm / h and 1.14 0.34 mg the mean ( sd ) das4 score was 1.7 0.8 ( range , 0.2 to 4.2 ) , and the mean values of the das - esr-3 , das- crp -4 , and das - crp-3 crp scores were 1.9 0.8 ( range , 0.4 to 4.4 ) , 1.5 0.7 ( range , 0.5 to 4.3 ) , and 1.6 0.7 ( range , 0.7 to 4.5 ) , respectively . in addition , the mean ( sd ) das28-esr-4 score was 3.0 1.0 ( range , 0.5 to 5.6 ) , and the mean values of the das28-esr-3 , das28-crp-4 , and das28-crp-3 scores were 3.3 1.1 ( range , 0.6 to 6.0 ) , 2.4 0.9 ( range , 1.2 to 5.3 ) , and 2.7 0.9 ( range , 1.4 to 5.8 ) , respectively . also , the respective mean ( sd ) sdai and sdai ( crp ) scores were 36.5 23.3 ( range , 5.7 to 106.3 ) and 12.0 7.4 ( range , 1.8 to 39.2 ) . the mean ( sd ) sf-36 total score among all patients was 49.8 16.7 ( range , 18.1 to 91.5 ) . the correlation coefficients between hrqol and different das indices are depicted in table 3 . as it is seen in table 4 , the dass correlated with physical hrqol but they did not relate with mental hrqol . correlations between different disease activity indices and health - related quality of life in rheumatoid arthritis the use of pass 2000 software showed no significant difference in terms of correlation coefficients between different das indices and hrqol scores . our study failed to show any significant difference between correlation coefficients ( r ) regarding the association between various das scores and hrqol . on the other hand , although no association was found between das indices and mental hrqol , they were found to be linked with physical hrqol . to our knowledge , this is the first report on the comparison between the correlation coefficients ( r ) of various das scores and hrqol . one study in lithuania showed that patients with higher disease activity scores , as expressed by das - esr-28 , had worse physical function , more frequent comorbidities , and worse general health measured by sf-36.24 patients of certain races may report a more pronounced impact of das on their daily life.25 also , groups of ra patients with similar disease activity may report different health status across several dimensions of health . these show that the correlation between das and hrqol may be more complicated than it is thought . in our study , this may be due to the fact that variance of general hrqol questionnaires are attributable to non disease - related aspects , which are not measured by das indices.26 in this study , dass had a correlation with physical hrqol but not with mental hrqol . previous studies have demonstrated that disease related variables are strong determinants of physical disability , but not necessarily the mental health.2730 nevertheless , in some studies , disease activity has been shown to be correlated with both physical and psychological domains of hrqol . it is worth mentioning that in all of these studies , disease activity has a stronger correlation with physical health than with mental health.3133 to explain the mechanism of the correlation between dass and physical health , one should note that disease activity is closely related to the extent of joint destruction and that surrogates of inflammation include the degree of clinical disease activity , the major expressions of which are pain , stiffness , and swelling of the joints . furthermore , the variables of disease activity are rapid loss of working capacity , premature mortality , and a reduction of quality of life.2730 another finding of the present study was the almost perfect correlation between das28-esr-4 and das28-crp-4 ( r = 0.861 ) . one study had previously reported this correlation coefficient to be 0.946 . the strong correlation between das28-esr and das28-crp means that these can be used interchangeably in clinical settings.34 in contrast , some researchers believe that in comparison to das28-esr , das28-crp may underestimate the disease activity and overestimate the improvement in disease activity.35 this was a secondary analysis with its own limitations.3637 the hrqol of ra patients is influenced by several variables which were not measured in our study.1112 this was a single center study with limited sample size , and because of the cross - sectional design , illustrating a causal relationship was beyond the scope of this study . associations of different dass with other morbidity measures such as disability , physical condition , and pain need further research . , there is no superiority for one of the dass in comparison to others , to reflect the morbidity in ra . we believe that further prospective studies should be conducted to assess whether an improvement in the various das variables has a stronger correlation with the degrees of improvement in the hrqol of ra patients . one important point that should be borne in mind in the interpretation of our results is the fact that our patients were experiencing mild to moderate das and none of them was experiencing severe , incapacitating disease . the low disease severity of ra patients has , however , been reported elsewhere.3839 to conclude , in our ra patients the correlations between various das indices and hrqol were similar , and the das indices had correlations with physical hrqol but not with mental hrqol . all the authors have carried out the study , participated in the design of the study and acquisition of data performed the statistical analysis and wrote the manuscript .

the use of technetium-99 m ethylenedicysteine ( tc99m - ec ) as renal tracer agent had evolved on the observation that brain perfusion agent ethylenedicysteine dimer rapidly cleared through kidneys after being metabolized to ethylenedicysteine . since then , numerous studies have shown it to be a better renal agent than the already available renal agents like tc-99m - mercaptoacetyltriglycine ( mag3 ) and i-131 orthoiodohippurate ( oih ) because of its better excretion characteristics and image quality . it has a high first pass extraction of ~ 70% , low plasma protein ( ~30% ) and ~ 56% red blood cell binding . herein , we are reporting the unusual visualization of liver activity persisting throughout the dynamic as well in delayed phases in ec renal scan . we present a case of 3-year - old asymptomatic male child subjected to renal dynamic imaging following incidentally diagnosed as having single left kidney on ultrasonography ( usg ) abdomen . the usg abdomen demonstrated normal sized and normally positioned left kidney while right kidney was not localized in the abdomen / pelvic region . blood urea profile ( 26 mg / dl ) and serum creatinine ( 0.8 mg / dl ) were within normal limits . renal dynamic scintigraphy was performed in posterior view under the gamma camera after intravenous administration of 2 mciof tc99m - ec along with 10 mg frusemide . from the initial frame onward , tracer activity was localized in both renal fossae , more intense in the renal left fossa . the activity in both renal fossae appeared to decrease with time and 3-h and 5-h delayed images showed faint tracer activity in bilateral renal fossae [ figure 1a g ] . the scintigraphic study was interpreted as normal functioning left kidney with unobstructed drainage and impaired functioning enlarged right kidney . the single - photon emission computerized tomography / computerized tomography ( spect / ct ) abdominal imaging [ figure 2a d ] was performed because of the nonvisualization of right kidney on usg ; localized this tracer activity in the enlarged right lobe of liver . technetium-99 m ethylenedicysteine renal scintigraphy : ( a ) flow images showed normal perfusion in left renal fossa and impaired perfusion in right renal fossa , ( b ) cortical uptake images revealed normal uptake in left kidney with pseudo - impression of impaired uptake in right kidney . prevoid ( c ) , postvoid ( d ) , and delayed 3-h ( e ) static images showing the progressive tracer clearance from both renal fossa . posterior ( f ) and anterior ( g ) images at 5-h showed mild tracer retention in the right renal fossa delayed single - photon emission computerized tomography / computerised tomography trans - axial ( a and b ) and coronal ( c and d ) images revealed tracer in the right lobe of liver instead of right kidney . however , hepatobiliary clearance of the renal agent should be minimal for accurate estimation of the effective renal plasma flow as significant hepatobiliary excretion may lower renal clearance values . the oih is the ideal reference agent for renal clearance but the technetium based agent like mag3 is far from ideal replacement of oih because of its high protein binding and low plasma clearance in human . the tc99m - ec has efficient extraction fraction and excretion through the kidneys and is closer to oih in function . it provides the high quality images due to negligible liver accumulation and high kidney to background ratio . different authors had shown significantly low accumulation of ec in the liver and intestine than that of mag3 and oih in animals as well as in human . reported better kidney delineation and only faint liver activity with ec compared to mag3 in patients with severe renal failure . nonfasting state in the patients undergoing mag3 renal scintigraphy results in minimal or reduced gb uptake . apart from this , it has been shown that radiolabeled impurities are associated with hepatobiliary excretion of mag3 . presence of oxidizing agents ( sodium hypochlorite or hydrogen peroxide ) in the sodium pertechnetate solution reduces the labeling . photolytic degradation and increased time spent in steps for mag3 preparation may lead to impurities formation . similarly , it has been shown that reconstitution with 10 ml of saline is optimal for high labeling efficiency while reconstitution with lesser amount of saline would result in the reduced labeling efficiency of tc99m - mag3 . the presence of different stereoisomers of ec in different kits may result in different excretion characteristics of ec . it has been shown that the pretreatment with probenecid impairs the renal excretion of tc99m - ec with more shifting toward hepatobiliary system and slower clearance from blood . again there would be the presence of impurity in the ec kit , if labeling is done below ph of 12 . the unusual presence of tracer activity in the gall bladder ( gb ) has been described in a renal transplant patient undergoing ec renal scan . in the present case , the visualization of liver activity could not be attributed to radiochemical impurity as other renal scans done on the same day had not shown any altered bio - distribution . the patient in our case had normal renal function ruling out the reason of liver uptake due to impaired renal function . the initial perfusion images might have shown the liver activity in right abdominal region because of high blood flow to liver , but persistence of activity in the delayed image raised the suspicion for normally located impaired functioning enlarged right kidney . the careful evaluation of the renal scan and sometime additional imaging acquired with spect / ct is essential before making the final interpretation . the present case highlights the unusual and rare ec uptake in liver in spite of the child having normal renal function , no history of drug intake affecting the renal clearance and no evidence of faulty radiopharmaceuticals on that day . it is assumed that the control of factors affecting the labeling efficiency may reduce the hepatic uptake and its excretion . though we could not point out the exact reason for ec uptake in the liver , but have tried to point out the various reasons for the altered bio - distribution with review of the literature . this rare case report showing presence of liver activity on ec renal scintigraphy required careful evaluation of renal scan and correlation with spect / ct before making the final impression .

impaction of foreign bodies in the upper aerodigestive tract is a serious pathologic condition in ent practice and is particularly common in children , prisoners , and psychiatric patients . the potentially fatal complications include mediastinitis , tracheoesophageal fistula , and retropharyngeal abscess , which can lead to septicemia and shock and require immediate treatment . although the removal of the foreign bodies through endoscopic procedures is the preferred method of treatment , this is not always possible or uncomplicated . we present a case of iatrogenic complication with migration of an impacted foreign body into the prevertebral space , after an unsuccessful attempt of endoscopic removal . a 44-year - old female patient visited the emergency department of a local municipal hospital with severe symptoms of dysphagia and odynophagia after ingestion of chicken bone . she underwent flexible esophagoscopy for diagnostic and therapeutic reasons , during which the foreign body was initially visualized in the hypopharynx , but iatrogenically impacted in the posterior pharyngeal wall , in a position where it was no longer possible to be removed through the esophagoscope . the staff in the municipal hospital lacked the expertise of performing rigid esophagoscopy or open neck exploration and the patient was referred to our hospital at the same day for further treatment . on admission , however , she had an elevated temperature of 39.5c with a rise in white blood cell count indicating an inflammatory process . she was heavy smoker and her medical history was significant only for episodes of acute bronchitis , for which she occasionally received oral antibiotics and bronchodilators . on clinical examination pharyngoscopy , indirect laryngoscopy and direct laryngoscopy with the flexible laryngoscope plain cervical and thoracic x - rays did not reveal signs of the foreign body or retropharyngeal and mediastinal emphysema . the foreign body was finally visualized in the cervical ct scan impacted extraluminally in the retropharyngeal space between the right common carotid artery and jugular vein , in contact with the anterior surface of the c5 vertebral body . no signs of air in the surrounding tissues were present ( figure 1 ) . in view of the clinical signs and symptoms and in order to preclude a deterioration of the patients ' condition and a possible abscess formation a prompt removal of the bone under general anaesthesia was decided . a rigid esophagoscopy was not performed at this stage as it was considered unnecessary due to the extraluminal position of the foreign body and carried more risk of enlarging the perforation . through a right cervical incision , retraction of the sternocleidomastoid muscle , and the carotid sheath , the bone was palpated in the prevertebral space where it had further migrated and was removed ( figures 2 and 3 ) . the soft tissues were irrigated , a vacuum surgical drain was placed , and the incision was closed in layers with interrupted sutures . the drain was removed in the 2nd postoperative day and the patient was discharged home . impaction of foreign bodies in the upper food and airway passage is usual in otolaryngological practice . commonly found objects include fish bones , chicken bones , pieces of glass , dental prostheses , coins , and needles [ 26 ] . the morbidity of the impacted foreign objects relates to their size , shape , and site of impaction . although the majority of the small and round ones pass along the gastrointestinal tract spontaneously , the sharper and larger objects can lead to serious complications . perforation of the pharyngeal or esophageal wall is possible , and migration of the foreign body in the adjacent tissues can be facilitated by swallowing , coughing , and esophageal peristalsis , as well as by the weakening of the pharyngeal wall due to the local inflammation . complications from perforation of the hypopharynx and upper esophagus include retropharyngeal abscess [ 2 , 5 ] , mediastinitis , and even rare instances of esophagoarterial fistulas [ 9 , 10 ] , penetration of the common carotid and facial artery , thyroid abscess , and epidural abscess . most authors agree that delayed diagnosis and retrieval is associated with an increased complication rate [ 1517 ] . pain in the form of odynophagia is the most constant symptom , although retrosternal pain and painful cervical contracture can also be observed [ 15 , 17 ] . foul smelling expectoration and muffled voice are symptoms of abscess formation and subsequent clinical deterioration and are common in patients seeking late medical attention [ 12 , 17 ] . drooling , dysphagia , dysphonia , and dyspnea due to airway obstruction can also be observed , depending on the site of impaction [ 5 , 6 ] . increased body temperature , shiver , cervical crepitance , and cervical swelling are signs of complications of the foreign body and should alert the clinician for urgent treatment . in the present case , signs of bacterial complication ( leucocytosis and fever ) were evident , although the patient presented to our hospital within a few hours from the impaction . diagnosis is based on history and symptoms , but visualization of the foreign body and evaluation of the possible complications often require the use of a variety of diagnostic procedures . indirect laryngoscopy , soft tissue lateral neck x - rays , routine chest x - rays , oesophagography with gastrografin , barium swallow , ct scan , and endoscopy through flexible and rigid endoscopes can be used , each one having advantages and disadvantages [ 17 , 18 ] . since the majority of foreign bodies are impacted in the suprahyoid region , they are usually detected by indirect laryngoscopy alone and can be removed with local anesthesia in the outpatient setting . plain radiography has the benefit of evaluating the deeper soft tissues and revealing potential complications . the physician should look for abnormal calcifications in the cervical region , cervical or mediastinal emphysema , widening of the prevertebral soft tissue space , and presence of soft tissue swelling in the region of the base of the tongue [ 17 , 20 ] . on the other hand , ct scanning of the neck is considered the most accurate imaging modality for diagnosing the presence of any foreign body impaction and superior to the plain radiogram [ 21 , 22 ] . studies have shown that sensitivity and specificity of plain x - ray for the detection of fish bones range between 23.5% and 54.8% and 86.3 and 100% , respectively [ 19 , 23 ] , whereas non - contrast - ct has a sensitivity of over 90% and specificity of 100% [ 23 , 24 ] . some authors recommend its use in complicated cases , while others advocate that ct should be performed in all cases of suspected foreign body which can not be visualized through laryngeal mirror or laryngeal fiberscope . the high radiation dose compared to plain radiography is a serious disadvantage of the ct scan , and sound clinical judgment is required in order to be used in selected patients , under the do no harm principle . in the present case , the ct scan was the diagnostic method of choice , since the foreign body was impacted extraluminally in the posterior pharyngeal tissues . endoscopy has the advantage of direct visualization of the mucosal layer under magnification to assess for signs of trauma . a flexible endoscope with a lumen for insertion of grasping instruments is required in order to be used therapeutically , as well as diagnostically . in a recent study , the authors reported complications such as lacerations ( 6.7% ) and perforation ( 1% ) of the esophagus occurring during the endoscopic procedure . in our case , the foreign body was impacted further into the retropharyngeal space during the endoscopic procedure , in a position where it was no longer visible through the endoscope . the authors are unaware of a report of a similar complication in the published literature . clinical management of impacted foreign bodies in the hypopharynx and upper esophagus focuses on preserving the airway , removing the foreign body , and treating any complications that may arise . an open surgical procedure with neck exploration is mandated when endoscopic removal fails , as in the present case , and when infectious complications , such as abscess formation and mediastinitis , have developed . after removal of the foreign body , primary closure of a perforation can be attempted and the neck should be thoroughly irrigated and drained . postoperative antibiotic therapy is necessary in order to prevent further complications . impacted foreign bodies in the upper respiratory tract and upper esophagus are common in otolaryngology practice . endoscopic removal is the treatment of choice of the symptomatic cases , although not always possible or without complications . the open surgical procedure with careful neck exploration is an alternative approach when other treatments fail or when complications arise . a prompt diagnosis and early therapeutic intervention is required in order to minimize morbidity and mortality from the impacted foreign bodies .

the society s demand for energy and how it is currently satisfied interweaves strongly with anthropogenic co2 emissions and hence to the changing climate . it is evident that , to maintain present living standards , the energy sector needs to be altered drastically . new environmentally friendly ways of transforming energy have to be implemented on a large scale . this significant change of the energy sector is , however , still years from being fulfilled . near - term measures include the considerable reduction of co2 emitted by conventional power plants . to reduce co2 emissions of current power plants , co2 needs to be separated from , e.g. , the flue gas . besides carbon capture , co2 removal is also crucial for other technologies , e.g. , the purification of natural gas . a promising technology for the efficient separation of large quantities of co2 is the separation via solid adsorbents . in this context , mofs are a relatively new and versatile type of material with various possible application areas such as in gas separation , gas storage , gas and liquid separation , catalysis , sensing , drug delivery , microelectronics , and biotechnology . mofs are constructed of metal ions or clusters connected by organic linkers . in recent years , a tremendous number of new mofs has been synthesized and an almost infinitive number seems to be theoretically possible . by adjusting the combination of metal ions and the organic parts , the properties of mofs are widely tunable and materials with exceptionally large surface areas can be created . the pore geometry can be customized to enhance the separation of molecules due to the topology . in addition to the geometry , the chemical composition can be tuned to further improve separation performance . for instance , coordinatively unsaturated metal ions , so - called open - metal sites , can be embedded on the surface of the pore structure . these metal ions are accessible for guest molecules and therefore interact strongly with certain adsorbate molecules . understanding and predicting the interaction of open - metal sites with adsorbates is crucial for the design of new customized adsorbent materials . a challenge that is inherent with the enormous number of possibilities in the synthesis of mofs is the selection of the best one for a particular application . a large effort has been made on developing computational screening approaches to facilitate the selection . today , it is possible to predict adsorption properties for large sets of existing and hypothetical mofs based on molecular simulations . a prerequirement for this kind of computational screening is a force field that represents the molecular interactions reasonably well for all materials under investigation . unfortunately , the existing generic force fields do not fulfill this prerequirement for all mofs . especially , the promising class of mofs with open - metal sites has been shown to be poorly described by generic force fields and research has been focused on developing improved force fields for these materials . to illustrate the failure of generic force fields , figure 1 compares experimental measurements from herm et al . to the co2 uptake in mg - mof-74 calculated from grand - canonical monte carlo simulations applying the generic uff force field for mg - mof-74 and the trappe force field for co2 ( i.e. , standard generic force fields commonly used for porous materials ) . comparison between the experimental adsorption isotherm of co2 in mg - mof-74 from herm et al . and the simulated one using the uff force field for mg - mof-74 and the trappe force field for co2 at 313 k. the co2 uptake of mg - mof-74 predicted from molecular simulation for low fugacities with the uff force field is considerably lower than the experimentally determined one . in particular , this region is highly relevant to carbon capture . the distinct inflection in the adsorption isotherm is also not depicted , which suggests that the strong affinity of co2 close to the open - metal sites is not modeled correctly with the uff force field . to obtain accurate force fields , several studies have been conducted in which force fields for individual mofs are matched to interaction energies computed with quantum mechanical methods . in some of these studies , the applicability of the customized force fields was also investigated for mofs with very similar topology and composition . used this methodology to investigate the influence of the exchange of the metal ion for a mof without open - metal sites . designed an extension to the uff force field to capture the structure of mofs . moreover , vanduyfhuys et al . developed a software package called quickff to automatically derive force fields for mofs from ab initio input . the iso - structural m - mof-74 has been pointed out to be a well suited study case to investigate the influence of different metal ions on the adsorption properties of small molecules and thereby further improve force fields . the pore structure of m - mof-74 is only slightly influenced by the exchange of the metal ion , whereas the adsorption properties can change considerably . understanding and describing the underlying interactions of open - metal sites with guest molecules is of fundamental interest and can help to find trends and design even better adsorbent materials . m - mof-74 is built of one - dimensional hexagonal pores with a diameter around 11 and exhibits a particular high density of open - metal sites.figure 2 shows an extract from the periodic structure of mg - mof-74 . mg , c , o , and h atoms are represented in green , gray , red , and white , respectively . in recent studies , mg - mof-74 has been shown to be a promising candidate for carbon capture due to its high co2 uptake capacity at low partial pressures and for natural gas sweetening . subsequently , m - mof-74 has been extensively investigated for various gas separations . among others , experimental studies include adsorption measurements of co2 , co , ch4 , c2h6 , c2h4 , c2h2 , c3h8 , c3h6 , ar , o2 , and n2 . adsorption sites have been investigated via neutron and x - ray powder diffraction to determine the binding geometry . as a complement to experiments , various quantum mechanical studies have been conducted to theoretically investigate adsorption sites , adsorption energies , and the underlying contributions and mechanisms for a large number of guest molecules . moreover , these authors found that kinetic effects can play a significant role in the replacement of adsorbate molecules close to the open - metal sites . molecular simulations have been used to investigate the adsorption behavior at uptakes larger than one guest molecule per open - metal site and the hopping of guest molecules between open - metal sites . despite the significant progress , it is still a major challenge to accurately capture the change of interaction strength with varying metal ions in m - mof-74 in molecular simulations . several simulation studies have been conducted to reproduce the adsorption behavior of some of the m - mof-74 structures . in these studies , standard interaction potentials , it is suggested that guest molecules are polarized in the vicinity of the open - metal sites in m - mof-74 and that this interaction contributes to the enhanced co2 affinity . standard force fields do not include this effect directly and therefore separate adjustments of the force field parameters may be necessary for every new structure . in this context , polarizable force fields for porous materials have been suggested , due to the observation of polarization in several other mofs . however , we underline the potential of polarizable force fields for the study of adsorption in grand - canonical monte carlo simulations of m - mof-74 . we anticipate that considering polarization explicitly can help to create force fields that overcome the shortcomings of current generic force fields . in this manuscript , we evaluate the potential of explicit polarization to improve the issue of limited force field transferability using mofs with open - metal sites . in particular , we study the adsorption of co2 and ch4 in m - mof-74 with m = co , cr , cu , fe , mg , mn , ni , ti , v , and zn . we extend the previously developed polarizable force field for co2 in mg - mof-74 to structures based on nine more metal ions and ch4 without additional fitting parameters . subsequently , we conduct grand - canonical monte carlo simulations , and compare our results to results using other force fields and experiments . thereby , it is shown that polarizable force fields have the potential to improve the transferability of force fields describing porous materials . force fields describing intermolecular interactions are the foundation of molecular simulations . by definition , molecular simulations represent the behavior of a system for a given force field . the capability of the force field to describe the true molecular interactions determines its applicability . ideally , a force field should be able to describe the experimentally observed behavior for a preferably large set of systems . generic force fields like uff , dreiding , and opls have been designed for organic , biological , and inorganic materials . however , if the conditions of the system under investigation vary from the ones the force field was developed for , the resemblance of the real system behavior may be poor . nevertheless , the transferability of the created force fields is likely to be limited to structures with very similar topology and chemical composition . in addition , for each pairwise interaction and all point charges , new parameters are required which are all mutually dependent . repeatedly readjusting force field parameters for every new system the reason for the limited transferability of this approach could be attributed to the implicit consideration of the interactions that are exceptional for a particular system . another disadvantage is that the predictive potential of molecular simulations is largely lost when force fields become completely empirical and need to be readjusted for every new system . a more sustainable approach is to develop force fields with a broader applicability due to a physically motivated extension which considers these exceptional interactions . unfortunately , a force field with general applicability for the adsorption of small molecules in mofs does not exist . especially , the modeling of mofs with open - metal sites represents a challenge . quantum mechanical calculations of co2 adsorption in mg - mof-74 suggest that polarization of co2 in the vicinity of mg ions is important and significantly contributes to the interaction energy . in contrast , charge transfer between co2 molecules and the mof framework seems to be negligible . several methods have been proposed for considering polarization in molecular simulations , i.e. , the induced dipole method , the fluctuating charge method , and the shell method ( also known as drude oscillator and charge - on - a - spring model ) . for molecular dynamics simulations , however , the many - body nature of polarization makes these algorithms more suitable for molecular dynamics simulation in which all molecules are moved in every simulation step . this is in contrast to monte carlo simulations in which usually only one molecule is moved . hence , in monte carlo simulations , more steps are required to create independent configurations of the system . normally , this is unproblematic , since the interactions need to be computed only for the moved molecule . however , when considering polarization , the interactions between all molecules change and have to be recomputed due to the many - body nature of polarization for every step . this leads to a less frequent consideration of polarization in monte carlo simulations . as described in our initial study , we use the procedure developed by lachet et al . to mitigate this limitation . the procedure uses the induced dipole method in which the induction energy uind is expressed as1where i is the induced dipole , ei0 is the permanent electric field created by the static partial charges at interaction site i , and n is the total number of interaction sites in the system . the energy contribution of the induction energy has to be computed in every monte carlo step . in this way , the difference in induction energy to the previous configuration can simply be added as another energy term in the acceptance rule of the monte carlo algorithm . higher order induced multipoles are not explicitly incorporated in the induced dipole method . in a similar system , lachet et al . estimated the related error to be less than 5% of the total induction energy . is that it accounts solely for polarization between the framework and adsorbate molecules and that it neglects polarization caused by induced dipoles , so - called back - polarization . using these assumptions , eq 1 can be rearranged to2where i is the atomic polarizability of interaction site i and n is the number of interaction sites of the moved molecules . thereby , an iterative scheme is avoided and the computational costs of the method are drastically reduced . in fact , the computational costs can be similar to simulations without considering explicit polarization . in the case of , e.g. , a translation move of a single molecule only , the n interaction sites of this molecule have to be evaluated to determine the change in the induction energy . lachet et al . showed that the error in energy introduced by this assumption is around 6% in a xylene nay zeolite system . to verify the contribution of back - polarization in mg - mof-74 , in figure 3 , the total interaction energy of a co2 molecule approaching the mg ion with and without consideration of back - polarization for the developed polarizable force field is compared . total energy of a single co2 molecule in mg - mof-74 calculated using the developed polarizable force field as a function of the distance to the open - metal site . the influence of back - polarization increases with decreasing distance between the co2 molecule and the metal ion . for the most favorable position at approximately 2.4 , the difference in total energy is approximatively 7% . this deviation seems to be acceptable in comparison with the considerable speedup of the simulations . besides polarization , repulsion and dispersion interactions are considered via a standard lennard - jones potential and static charge distributions are modeled via point charges . when explicitly accounting for polarization , one has to ensure that the force field parameters describing the remaining interactions do not include an implicit polarization contribution which would have to be removed . otherwise , the contribution of polarization would be double counted , once implicitly and once explicitly . the removal of implicit polarization is necessary if a standard force field is used as the starting point for the development of a polarizable force field , because current force fields are likely to be calibrated to reproduce certain experimentally observed properties . for example , the trappe force fields for co2 and n2 are fitted to reproduce experimental vapor liquid equilibria of the pure components and their mixtures with alkanes without explicitly considering polarization . hence , in the fitting of these force fields , all present interactions are indirectly considered and the resulting potential parameters are effective parameters . as the starting point for our polarizable force field , we use the uff and trappe force fields . these are standard force fields frequently used for molecular simulations of porous materials . to remove the contribution of implicitly considered polarization to the interaction potential , a global scaling parameter is applied to all lennard - jones energy parameters developed without explicit polarization . a simple procedure is chosen to verify the applicability of polarizable force fields rather than attempt to perfectly reproduce experimental results . here , we reduce the lennard - jones energy parameters i taken from the uff and trappe force fields with respect to their atomic polarizabilities via3where i and max are the atomic polarizabilies of interaction site i and the largest atomic polarizability , respectively . thereby , it is assured that nonpolarizable interaction sites ( i = 0 ) are unchanged and that the potential energy parameters of the atoms with the largest polarizability are reduced the most . a more detailed description of the derivation and the algorithm of the presented method can be found in our previous publication . their values can differ significantly depending on the experimental procedure or the theoretical assumptions made . hence , a global scaling factor is used to adjust the magnitude of the atomic polarizabilities taken from the literature ilit with respect to the chosen interaction potential according to4thereby , the ratio between the individual atomic polarizabilities is not affected to ensure a reasonable relative contribution of polarization between the atoms . this kind of scaling procedure for atomic polarizabilities is frequently used in the literature , and the scaled polarizabilities adopted in this study have comparable magnitudes to previous molecular simulation studies . in this manuscript , the values of and are adjusted to reproduce the experimental adsorption isotherm for co2 in mg - mof-74 . in a first step , the low fugacity region of the simulated adsorption isotherm and the heat of adsorption for co2 in mg - mof-74 are tuned by scaling all atomic polarizabilities with . in the low fugacity region , co2 molecules adsorb close to the open - metal sites where polarization interactions are of particular importance . subsequently , the scaling parameter is adapted to remove the implicit contribution of polarization from the lennard - jones potential . therefore , the value of is lowered to match the high fugacity region of the experimentally determined adsorption isotherm . in this region , the centers of the channels of mg - mof-74 are filled with co2 molecules . the locations in the centers of the channels are further away from the open - metal sites , and therefore , polarization is less important . by applying this two - step procedure , we divide the interaction energy into the underlying physical contributions without using an elaborated approach . for the remaining m - mof-74 structures and for ch4 , the scaling factors determined for the mg structure with co2 are used . the procedure is chosen to verify if the polarizable force field has the potential to describe the difference between the different metal ions embedded in m - mof-74 . grand - canonical monte carlo simulations implemented in the raspa software package are conducted to compute the uptake and heats of adsorption of ch4 and co2 in the different structures of the m - mof-74 ( m = co , cr , cu , fe , mg , mn , ni , ti , v , and zn ) family . the uptakes are computed for varying fugacities , for pure components and mixtures at 298 k. dft - optimized , all atomic mof structures with atomic charges are taken from lee et al . in the simulations , lennard - jones parameters for ch4 and co2 are taken from the trappe force field . interactions between guest molecules are not modified and computed according to the trappe force field . the uff force field is used for the atoms of m - mof-74 . all lennard - jones energy parameters iscale used in the simulations are adjusted according to eq 3 with = 0.7 . the lennard - jones potential is truncated at a cutoff distance of 12.8 without tail corrections . to mimic the behavior of the continuous system , i.e. , a repetition of identical one - dimensional pores , periodic boundary conditions are applied in all directions ( see figure 2 ) . the simulated system is composed of multiple unit cells to ensure a minimum distance of more than twice the cutoff radius between periodic images . the ewald summation technique with a relative precision of 10 the required atomic polarizabilties i are taken from shannon and van duijnen and swart and are scaled with a value of 0.09 . all force field parameters are summarized in tables s1s11 ( supporting information ) . for the comparison with experimental results and simulation results of others , we use the peng robinson equation of state to convert pressures to fugacities . the dft calculations to determine the orbital interaction energy ( as explained below ) are performed with the amsterdam density functional ( adf ) package . the b3lyp - d3 exchange - correlation functional is used with a tzp - sto basis set and a large frozen core . a fragment analysis is performed between co2 and mg - mof-74 to assess the net interaction between these two fragments . using the energy decomposition analysis scheme by ziegler and rauk , the interaction energy eint between the two fragments is decomposed into5velstat comprises classical electrostatic interactions between unperturbed charge distributions of the deformed fragments . epauli describes the pauli repulsion energy and corresponds to the destabilizing interactions between occupied orbitals . as an initial step in the evaluation of the developed polarizable force field , it is important to investigate the role of polarization in the adsorption of guest molecules in m - mof-74 . in figure 4 , we compare the polarization energy of a co2 molecule approaching the mg ion of mg - mof-74 estimated with the developed polarizable force field to the orbital interaction energy calculated from adf . comparison of the polarization energy computed with the developed polarizable force field without considering back - polarization and the orbital interaction energy from dft calculations as a function of the distance between a co2 molecule and the mg ion . the orbital interaction energy should be a good approximation for the polarization energy , since no reaction is taking place and considerable charge transfer is not expected for very similar configurations of co2 inside mg - mof-74 . for relevant distances , the most relevant distance between the co2 molecule and the mg ion is where the total energy is the lowest ( i.e. , 2.32.5 , as shown in figure 3 ) . at this distance , the polarizable force field predicts that the polarization energy of a single co2 molecule in mg - mof-74 has a significant contribution of around 30% to the total energy . for larger distances , , we investigated the quality of the developed polarizable force field by comparing the total energy of random co2 positions inside mg - mof-74 with detailed dft calculations from lin et al . thereby , also less favorable positions further away from the mg ions are probed . these positions are occupied after all open - metal sites are saturated . in general , the polarizable force field describes most positions considerably better than the uff force field and with a quality comparable to a nonpolarizable force field that has been developed by readjusting the majority of force field parameters . the resulting adsorption isotherms for co2 in mg - mof-74 in comparison to experimental measurements , the uff force field , and the dft - derived nonpolarizable force field of mercado et al . are shown in figure 5a . ( open ) , queen et al . ( yellow ) , yu et al . ( orange ) , and dietzel et al . ( brown ) and simulation results using the developed polarizable force field ( black ) , the uff force field ( blue ) and the dft - derived nonpolarizable force field of mercado et al . , 313 k ) ; ( b ) heat of adsorption as a function of uptake . the simulation results with the polarizable force field clearly display the inflection of the experimental adsorption isotherm . the predicted behavior is significantly better than that with the uff force field . this is expected , because the scaling factors are adjusted to reproduce the experimental data . the overall agreement with the experimental measurements is comparable with the dft - derived nonpolarizable force field of mercado et al . both force fields can predict the low fugacity region which is particularly important for carbon capture . for higher fugacities , simulations with all compared force fields predict higher co2 uptakes in comparison to the experiments . as pointed out earlier , this can be attributed to the fact that a certain degree of inaccessibility due to diffusion limitation or defects in the crystal structure is inherent with experimental structures . in the limit of very high co2 uptakes , the guest host interactions become less important and the adsorption is dominated by the accessible volume for co2 . in the development of the used trappe force field , therefore , the uptake of co2 predicted using the polarizable and uff force field converges in the high fugacity region . it should be noted that mercado et al . scaled the calculated co2 uptakes with 0.85 to account for inaccessibility of open - metal sites . this scaling procedure mainly improves the agreement between experiments and computations for the high fugacity region . figure 5b shows the heat of adsorption as a function of co2 molecules per metal ion . the distinct inflection of the adsorption isotherm caused by the strong affinity of the co2 molecule toward the metal ions is reflected by the change of the heat of adsorption with increasing gas uptake . the calculated heat of adsorption has an inflection at exactly one co2 per metal ion . before and after the rapid decrease at one co2 molecule per metal ion , the heat of adsorption increases slightly . this increase can be related to a rise in the total number of adsorbed co2 and therefore a larger contribution of the co2co2 interactions to the total energy . the heat of adsorption is not measured directly but calculated according to qst / r = (ln p)/(1/t ) at constant loading and averaged over adsorption isotherms at different temperatures . the temperatures considered vary for all experimental studies . the experimental curves consistently show a drop in the heat of adsorption for lower ratios of the number of guest molecules and metal ions than the simulation results . different sets of experimental adsorption isotherms show inflections at different uptakes of co2 . this is another indication for defects in the crystal structure and the blocking of some of the metal ions of the experimental structures . further investigated the effect of blocking for the co , cu , mn , and ni based structures . these authors illustrate that varying levels of pore accessibilities can explain the discrepancy between different experimental studies . especially , the unavailability of open - metal sites can explain the drop in the heat of adsorption prior to the complete saturation of these sites . this can be caused by residual solvent molecules binding to the open - metal sites . according to haldoupis , these residual solvent molecules could reduce the number of accessible open - metal sites by 2030% , while only slightly affecting the accessible surface area and the accessible volume . previously , the good agreement between the experimental bet surface area and pore volume and the theoretical void space in the empty crystal structure made dietzel et al . suggest that their mof was fully activated . in contrast , haldoupis et al . concluded that a combined effect of crystalline defects and residual solvent molecules is most likely to cause the difference between simulations and experiments . the focus of this study is to evaluate the applicability of a polarizability force field for describing the interactions of guest molecules with different metal ions . in this regard , the consideration of residual solvent molecules and defects does not seem to be crucial . for the remaining m - mof-74 structures , the parameters adjusted for mg - mof-74 and co2 are used . thus , the calculated values are predictions based on the two global scaling parameters and adjusted for mg - mof-74 . to obtain an overview of the results , we divided the predictions for co2 in the m - mof-74 structures into three groups . the first group consists of co , cu , ni , and zn , the second of cr , ti , and v , and the third of fe and mn . in figure 6 , the computational results for the first group are presented and compared to experimental measurements of queen et al . and the uff force field . comparison between the experimental results of queen et al . ( yellow ) and simulation results using the developed polarizable force field ( black ) and the uff force field ( blue ) for co2 in the co ( ) , cu ( ) , ni ( ) , and zn ( ) based structures . ( a and c ) adsorption isotherms at 298 k ; ( b and d ) heats of adsorption as a function of uptake . for these structures , the developed polarizable force field is able to describe the experimental measurements well . this is most striking in comparison to the uff force field which is not able to model the differences between the metal ions . for the polarizable force field , the largest deviations in the adsorption isotherms can be observed for cu and co based structures . in agreement with the experimental data , the simulation results for co and ni based mofs show a less distinct inflection for the co2 adsorption isotherm than for mg - mof-74 . the experimental adsorption isotherms for cu and zn based structures do not show an inflection for co2 , which is also accurately predicted in the simulations applying the polarizable force field . the comparison to the experimental results of yu et al . and the simulation results of mercado et al . these authors did not develop a force field for cu - mof-74 , because of an elongation of the unit cell in the c - direction in comparison to the other m - mof-74 structures . although our results for the cr based structure deviate from the experimental results ( compare figure 6a ) , the elongation does not seem to be problematic for the general applicability of our approach . the calculated heats of adsorption shown in figure 6b and d have a similar quality as that for the mg based structure . the largest discrepancy between simulations and experiments can be observed for zn - mof-74 . this is surprising , because the calculated adsorption isotherm agrees very well with experiments and is very similar to the calculated adsorption isotherm for co - mof-74 with a similar heat of adsorption . similar to mg - mof-74 , the heat of adsorption derived from experiments shows an inflection significantly before an uptake of one co2 molecule per metal ion . as mentioned previously , residual solvent molecules are likely to cause this shift in the heat of adsorption , since less open - metal sites are accessible . the simulations predict a similar behavior for all structures after all open - metal sites are saturated with co2 . in this region , the geometry of the channels is almost identical for all types of m - mof-74 , and the co2 molecules are sufficiently far away from the metal ions to not be significantly affected by polarization . overall , the polarizable force field seems to have the potential to capture the different degrees of polarizations related to the different metal ions for these four structures . moreover , in contrast to the uff force field , the polarizable force field is able to predict the correct order of adsorption strength for the co , cu , ni , and zn based structures . the computationally predicted adsorption isotherms and heats of adsorption for the second group are compared to the computational results with the uff force field in figure 7 . comparison between the simulation results using the developed polarizable force field ( black ) and the uff force field ( blue ) for co2 in the ti ( ) , v ( ) , and cr ( ) based structures . ( a ) adsorption isotherms at 298 k ; ( b ) heats of adsorption as a function of uptake . for these structures , no experimental adsorption measurements are available . to the best of our knowledge , these structures belong to the group for which the experimental syntheses are still challenging . the simulations predict a very distinct inflection for the adsorption isotherms of ti- and v - mof-74 , while the one for cr - mof-74 does not show an inflection . the predictions for the ti and v based structures agree with theoretical predictions of park et al . these authors expect the structures to have even stronger interactions with co2 than mg - mof-74 which is supported by our simulations . the trend of the adsorption isotherms for the three mofs is reflected in the heats of adsorption . the possibility to predict large differences between adsorption behavior shows further that polarizable force fields have the potential to describe such significant differences in the adsorption behavior . again , the uff force field predicts a totally different adsorption behavior and a smaller difference between the metal ions ( compare figure 7 ) . the remaining m - mof-74 structures are based on fe and mn . a notably large discrepancy between simulations and experiments ( yellow ) and simulation results using the developed polarizable force field ( black ) and the uff force field ( blue ) for co2 in the fe ( ) and mn ( ) based structures . ( a ) adsorption isotherms at 298 k ; ( b ) heats of adsorption as a function of uptake . the experimental results for the mn and fe based structures are very similar . both structures show weaker interactions between the metal ions and the co2 molecules than for the mg based structure . as can be seen , the developed polarizable force field significantly overestimates these interactions . several reasons for the overestimation are possible , and we feel a combination of different effects is most likely . interestingly , mercado et al . also failed to obtain a reasonable force field for mn - mof-74 based on fitting the interaction potential to quantum mechanical energies . for example , ferromagnetic effects which are not considered in the polarizable force field may play a more important role for mn and fe than for the other structures . additionally , the initial force field parameters taken from the uff force field for the two metal atoms could be of particularly bad quality . a comparison between the values for fe from the dreiding ( /kb = 27.677 k , = 4.045 ) and the uff force field ( /kb = 6.542 k , = 2.59 ) shows the huge difference . simulations based on the dreiding parameters result in a totally different prediction of the adsorption behavior . this is illustrated by the henry coefficients of co2 in fe - mof-74 in the limit of infinite dilution condition we computed for both sets of force field parameters using widom test particle insertions ( supporting information ) . in addition , the quality of the selected level of theory for the structure optimization could be better for some of the metal ions than for others . the failure of the polarizable force field to predict the behavior of these two structures needs to be further investigated . nevertheless , it does not diminish the potential of polarizable force fields for the description of mofs with open - metal sites . to further verify the applicability of polarizable force fields , grand - canonical monte carlo simulations are performed for ch4 in the m - mof-74 series . additionally , ch4 is explicitly chosen to examine the suitability of the polarizable force field to capture the varying influence of different metal ions in m - mof-74 . both co2 and ch4 have a similar polarizability but show a totally different adsorption behavior in the series of m - mof-74 . previous studies explain the difference with a combination of electrostatic interactions caused by the permanent quadrupole of co2 and polarization . as an example , in figure 9 , the predicted adsorption isotherms for ch4 in the mg , co , ni , and zn structures are compared to experimental measurements , simulations of mercado et al . , and the uff force field . ( cyan ) and simulation results using the developed polarizable force field ( black ) , the uff force field ( blue ) , and the dft - derived nonpolarizable force field of mercado et al . ( a ) mg - mof-74 , ( b ) co - mof-74 , ( c ) ni - mof-74 , ( d ) zn - mof-74 . the data for the remaining structures and the heats of adsorption are provided in figures s12s21 ( supporting information ) . for low fugacities , simulations with the mn based structure show an unphysical behavior ( compare figure s17 ) . four adsorption sites close to mn ions are much stronger for ch4 than the remaining adsorption sites . these adsorption sites are occupied with ch4 for all fugacities . to investigate this unusual behavior , we conducted widom test particle insertions to compute henry coefficients of ch4 in mn - mof-74 in the limit of infinite dilution condition with and without blocking of these four adsorption sites . in addition , simulations were performed with lennard - jones parameters for mn from the uff force field and with parameters for zn from the dreiding force field . the results are presented in the supporting information and show that the four adsorption sites are responsible for the behavior . for the dreiding parameters , the locations of the ch4 molecules are different due to the different lennard - jones parameters and hence interactions are much smaller . overall , the uff force field performs better than the developed polarizable force field for ch4 . this is due to the very simple and crude procedure to determine the force field parameters . co2 and ch4 have a similar polarizability , but co2 is modeled with three interaction sides and ch4 with only one interaction side . hence , in our force field , ch4 has a larger assigned polarizability than co2 and the lennard - jones energy parameter for ch4 is more reduced . it is very reassuring that the adsorption isotherms of ch4 computed with the polarizable force field do not show a distinct inflection which is in agreement with experimental results . as expected by mishra et al . and observed in this study , the strong interactions in the case of co2 are caused by a superposition of static polarity and polarization . hence , the adsorption of the uncharged ch4 molecule is far less affected by the different metal ions and a similar behavior is observed for all m - mof-74 structures . in addition , the large ch4 molecules have a larger distance to the metal ions and hence almost no dipole is induced . it would be straightforward to improve the performance of the polarizable force field by introducing a molecule dependent scaling parameter to account for the unequal number of interaction sides . are better than the ones with the uff force field and the polarizable force field . however , the same procedure as that for co2 was performed to fit the force field parameters for all metal ions separately and the ch4 uptake is scaled with a factor of 0.85 to account for inaccessible open - metal sites and blocked pores . in comparison to experimental measurements , the uff , and the polarizable force fields , the curvature of the computed adsorption isotherms for ch4 of mercado et al . the reason for this behavior should be investigated to further improve the approach . finally , it is possible to make predictions for the adsorption of mixtures of ch4 and co2 with the developed polarizable force field . measuring the gas uptake of mixtures in mofs is more complicated experimentally and is not often done . therefore , computational prediction of mixtures is very useful to predict the capability of a material to separate gases . in figure 10 , the predictions of an equimolar mixture of ch4 and co2 are shown for the polarizable force field and compared to predictions based on the uff force field for mg - mof-74 and zn - mof-74 . comparison between the simulation results using the developed polarizable force field ( black ) and the uff force field ( blue ) for an equimolar mixture of co2 and ch4 in the mg ( up ) and zn ( down ) based structures . ( a and c ) adsorption isotherms at 298 k and ( b and d ) heats of adsorption as a function of uptake in mg - mof-74 and zn - mof-74 , respectively . for these structures , the single component adsorption isotherms are reproduced reasonably well and therefore the mixture adsorption isotherms are expected to be predicted reasonably . predictions of mixtures being adsorbed in the other m - mof-74 structures can be found in figures s22s31 ( supporting information ) . close to the open - metal sites , the interactions between individual guest molecules with the framework are predominant . inside the channels , the interactions between co2 and ch4 molecules should be dominant which are reproduced well by the trappe force field . due to the strong interactions for co2 with the framework , a significantly lower uptake of ch4 is predicted for mg - mof-74 with the polarizable force field . in contrast , the uff force field predicts an uptake of ch4 even for very low fugacities . the depicted heats of adsorption show the released heat of adsorption for the mixture as a function of co2 uptake per metal ion . for mg - mof-74 , the initial adsorption behavior is very similar to the one for pure co2 , because initially almost exclusively co2 is adsorbed . in contrast , in zn - mof-74 , ch4 is already adsorbed for low fugacities . the trend of the heat of adsorption for the mixture deviates from the ones for the pure components . the uff force field predicts an initially larger uptake of ch4 in comparison to the polarizable force field . this can partially be attributed to smaller interactions between ch4 and the framework for zn - mof-74 with the polarizable force field . the simulations using the developed polarizable force field agree reasonably well with experimental measurements for most of the investigated structures of the m - mof-74 family . the quality of the predictions for co2 is significantly better than with the uff force field and for most cases comparable to structure specific force fields developed with more elaborated schemes . the polarization energy computed with the polarizable force field shows a behavior similar to the orbital interaction energy determined from dft calculations . in principle , these energy contributions should be similar if no reaction and no charge transfer takes place . the conducted procedure of first scaling atomic polarizabilities and subsequently adjusting the lennard - jones interaction parameters including implicit polarization is simple and requires relatively little effort . the two global scaling factors used here are exclusively tuned for the mg based structure and co2 . hence , the results for the other structures and ch4 are predictions . for ch4 , the predictions for ch4 adsorption could be significantly improved by a molecule specific adjustment of the polarizability . the concept of only considering explicit polarization between guest molecules and the framework and neglecting back - polarization seems to be a well suitable approach to study adsorption phenomena in porous materials . the assumptions considerably enhance the computational performance of monte carlo simulations while using polarizable force fields . actually , the computational time can be similar to monte carlo simulations without a polarizable force field . this is an important assessment , because monte carlo simulations are the method of choice for the prediction of adsorption properties in porous materials . future work will focus on further developing polarizable force fields and deriving a consistent set of parameters from quantum mechanical calculations to avoid fitting to experimental data . we believe that this can lead to force fields with better physical justification and improved transferability . this is crucial for the usage of monte carlo simulations for material screening and to make meaningful predictions . polarizable force fields for monte carlo simulations are also promising for other systems with a significant polarization contribution , i.e. , water , systems including ions , or xylenes .

pancreatic ductal adenocarcinoma ( pdac ) is the fourth most common cause of cancer - related mortality in the western world . only 5%-20% of patients are candidates for curative pancreatectomy and most cases of pdac are diagnosed at advanced stages , leading to dismal prognosis . numerous chemotherapy drugs have been investigated alone and in combination for the treatment of advanced pdac but have shown little or no impact on disease progression or survival . in the two recent clinical trials , folfirinox ( 5-fluorouracil , leucovorin , irinotecan , and oxaliplatin ) and gemcitabine plus nanoparticle albumin - bound paclitaxel showed superior outcomes compared with gemcitabine alone . however , these new regimens are associated with considerable toxicities and the long - term survival remains unsatisfactory . such factors help patients to avoid unnecessary toxicity and complications from aggressive treatment and can further facilitate stratification for clinical trials . many studies have examined various preoperative / postoperative factors predictive of treatment outcomes . however , their significance is still questionable , as most have been identified based on small sample sizes with inadequate statistical power . widely suggested parameters are derived from functional and nutritional status , distant metastasis , and laboratory findings including c - reactive protein ( crp ) , carcinoembryonic antigen ( cea ) , and carbohydrate antigen 19 - 9 ( ca 19 - 9 ) . however , the role of these parameters is not affirmed , and the risk of bias subsists when the choice of cutoff for the continuous variables is random , leading to the use of different cutoff points across multiple studies , which impedes direct comparison . in addition , regarding advanced disease which is unresectable or metastatic , few risk scoring indices were available , and the majority focused on prediction after chemotherapy particularly gemcitabine . however , it is more important in the clinical field to discriminate patients who might benefit from chemotherapy from those who can not . therefore , the aim of this study is to determine prognostic values of clinic - laboratory parameters routinely evaluated at the time of diagnosis , leading to implementation of a scoring index for use in effective identification of different risk patient groups . from july 2006 to april 2014 , 482 patients were diagnosed with pancreatic cancer at gangnam severance hospital , seoul , korea . inclusion criteria for further analysis were as follows : ( 1 ) age > 18 years , ( 2 ) histologically confirmed diagnosis of pdac , ( 3 ) metastatic disease at the time of diagnosis or systemic recurrence after curative resection , and ( 4 ) available electronic medical records ( including treatment information ) . exclusion criteria were as follows : ( 1 ) brain or leptomeningeal metastasis ; ( 2 ) locally advanced disease who received definitive chemoradiotherapy ; and ( 3 ) other histology types such as cystic neoplasms , neuroendocrine tumors , or lymphomas ; or ( 4 ) synchronous advanced malignancies . the institutional review board approved this retrospective study and the requirement to obtain informed consent was waived . the following baseline data were recorded at the time of diagnosis : age , sex , previous pancreatectomy , tumor location , eastern cooperative oncology group ( ecog ) performance status ( ps ) , metastatic site(s ) , presence of diabetes mellitus , presence of thromboembolism , and body mass index ( bmi ) . hematological and blood chemistry values included hemoglobin , white blood cell ( wbc ) count , serum protein , albumin , total bilirubin , cholesterol , blood urea nitrogen ( bun ) , alkaline phosphatase ( alp ) , aspartate aminotransferase ( ast ) , alanine aminotransferase ( alt ) , crp , cea , and ca 19 - 9 . neutrophil - to - lymphocyte ratio ( nlr ) was calculated by dividing absolute neutrophil count by absolute lymphocyte count . computed tomography ( ct ) of chest and abdominopelvis , radionuclide bone scan , and f - fluorodeoxyglucose positron emission tomography were performed for evaluation of distant metastasis . radiological detection of peritoneal carcinomatosis relied on the following findings evaluated by independent radiologist(s ) : thickening and nodular enhancement of peritoneal reflections , multiple soft tissue nodules , stranding and thickening of the omentum ( omental cake ) , stranding and distortion of the small bowel mesentery , and ascites , especially if loculated . the presence of ascites was inferred from evidence of paracentesis and malignant cell detection in cytology examination , or from suspicious fluid collection on ct or ultrasonogram . hematological and blood chemistry values were initially recorded as continuous variables and later transformed into categorical variables according to the upper normal values ( hemoglobin , wbc , serum protein , albumin , bilirubin , bun , alp , ast , and alt ) or the best cutoff point ( nlr , crp , cea , and ca 19 - 9 ) determined using the contal and oquigley method , which calculates the maximizing hazard ratio ( hr ) based on log rank statistics and estimates the best cut - off value . overall survival ( os ) was defined as the time from the date of diagnosis until death from any cause . survival curves were generated using the kaplan - meier method and compared using the log - rank test . univariate analysis was performed to determine the association of the following prognostic factors with os : age , sex , previous pancreatectomy , tumor location , ps , diabetes mellitus , liver metastasis , peritoneal carcinomatosis , thromboembolism , bmi , hemoglobin , wbc count , nlr , serum protein , albumin , bilirubin , bun , alp , ast , alt , crp , cea , and ca 19 - 9 . a stepwise multivariate analysis with cox s proportional hazard model hrs , 95% confidence intervals ( cis ) , and chi - sqaure scores were obtained for all regressions . a risk scoring system was devised by summing the rounded chi - sqaure scores of the independent prognostic factors identified in the multivariate analysis . finally , patients were grouped according to the best cutoff value as calculated by k - adaptive partitioning , which calculates the best cutoff value by selection of maximizing the sum of pairwise log - rank statistics . 18.0 ( spss inc . , chicago , il ) , sas ver . 9.2 ( sas institute inc . , cary , nc ) , and r ver . 3.1.3 ( kaps add - on package ; institute for statistics and mathematics , vienna , austria ; http://www.r-project.org ) . from july 2006 to april 2014 , 482 patients were diagnosed with pancreatic cancer at gangnam severance hospital , seoul , korea . inclusion criteria for further analysis were as follows : ( 1 ) age > 18 years , ( 2 ) histologically confirmed diagnosis of pdac , ( 3 ) metastatic disease at the time of diagnosis or systemic recurrence after curative resection , and ( 4 ) available electronic medical records ( including treatment information ) . exclusion criteria were as follows : ( 1 ) brain or leptomeningeal metastasis ; ( 2 ) locally advanced disease who received definitive chemoradiotherapy ; and ( 3 ) other histology types such as cystic neoplasms , neuroendocrine tumors , or lymphomas ; or ( 4 ) synchronous advanced malignancies . the institutional review board approved this retrospective study and the requirement to obtain informed consent was waived . the following baseline data were recorded at the time of diagnosis : age , sex , previous pancreatectomy , tumor location , eastern cooperative oncology group ( ecog ) performance status ( ps ) , metastatic site(s ) , presence of diabetes mellitus , presence of thromboembolism , and body mass index ( bmi ) . hematological and blood chemistry values included hemoglobin , white blood cell ( wbc ) count , serum protein , albumin , total bilirubin , cholesterol , blood urea nitrogen ( bun ) , alkaline phosphatase ( alp ) , aspartate aminotransferase ( ast ) , alanine aminotransferase ( alt ) , crp , cea , and ca 19 - 9 . neutrophil - to - lymphocyte ratio ( nlr ) was calculated by dividing absolute neutrophil count by absolute lymphocyte count . computed tomography ( ct ) of chest and abdominopelvis , radionuclide bone scan , and f - fluorodeoxyglucose positron emission tomography were performed for evaluation of distant metastasis . radiological detection of peritoneal carcinomatosis relied on the following findings evaluated by independent radiologist(s ) : thickening and nodular enhancement of peritoneal reflections , multiple soft tissue nodules , stranding and thickening of the omentum ( omental cake ) , stranding and distortion of the small bowel mesentery , and ascites , especially if loculated . the presence of ascites was inferred from evidence of paracentesis and malignant cell detection in cytology examination , or from suspicious fluid collection on ct or ultrasonogram . hematological and blood chemistry values were initially recorded as continuous variables and later transformed into categorical variables according to the upper normal values ( hemoglobin , wbc , serum protein , albumin , bilirubin , bun , alp , ast , and alt ) or the best cutoff point ( nlr , crp , cea , and ca 19 - 9 ) determined using the contal and oquigley method , which calculates the maximizing hazard ratio ( hr ) based on log rank statistics and estimates the best cut - off value . overall survival ( os ) was defined as the time from the date of diagnosis until death from any cause . survival curves were generated using the kaplan - meier method and compared using the log - rank test . univariate analysis was performed to determine the association of the following prognostic factors with os : age , sex , previous pancreatectomy , tumor location , ps , diabetes mellitus , liver metastasis , peritoneal carcinomatosis , thromboembolism , bmi , hemoglobin , wbc count , nlr , serum protein , albumin , bilirubin , bun , alp , ast , alt , crp , cea , and ca 19 - 9 . a stepwise multivariate analysis with cox s proportional hazard model hrs , 95% confidence intervals ( cis ) , and chi - sqaure scores were obtained for all regressions . a risk scoring system was devised by summing the rounded chi - sqaure scores of the independent prognostic factors identified in the multivariate analysis . finally , patients were grouped according to the best cutoff value as calculated by k - adaptive partitioning , which calculates the best cutoff value by selection of maximizing the sum of pairwise log - rank statistics . 3.1.3 ( kaps add - on package ; institute for statistics and mathematics , vienna , austria ; http://www.r-project.org ) . the median follow - up period was 7.9 months ( range , 0.1 to 70.5 months ) from diagnosis . the median age was 66 years , and 198 patients ( 49.1% ) were men . sixty - two patients ( 15.4% ) underwent curative resection , 217 patients ( 53.8% ) had an ecog ps of 0 - 1 , and 178 ( 44.2% ) and 79 ( 19.6% ) patients had liver metastasis and peritoneal carcinomatosis at the time of diagnosis , respectively . the median value of nlr was 2.92 , and the median value of cea and ca 19 - 9 was 4.4 ng / ml and 224.5 u / ml , respectively . median os for total patients was 8.2 months ( 95% ci , 7.3 to 9.1 ) . the statistically determined best cutoff points were as follows : 3.0 for nlr , 6.0 mg / l for crp , 7.0 ng / ml for cea , and 453.0 u / ml for ca 19 - 9 . significantly extended survival was observed in patients with low nlr levels compared with high nlr ( median os , 11.3 months vs. 4.7 months ; p < 0.001 ) ( fig patients with low levels of crp , cea , or ca 19 - 9 , respectively , also showed superior survival ( median os ; crp : 11.3 months vs. 5.0 months , p < 0.001 ; cea : 10.9 months vs. 5.7 months , p < 0.001 ; ca 19 - 9 : 10.3 months vs. 6.5 months , p < 0.001 ) ( fig . 1b - d ) . in univariate analysis , age , previous pancreatectomy , tumor location , ps , liver metastasis , peritoneal carcinomatosis , bmi , hemoglobin , wbc count , nlr , serum albumin , bun , alp , ast , crp , cea , and ca 19 - 9 showed significant association with os ( table 2 ) . in multivariate analysis , ecog ps , hemoglobin , wbc count , nlr , and cea were significant in both forward and backward stepwise analyses ( table 3 ) . scores were calculated by summing the rounded chi - squre scores of these factors ( table 4 ) : 2 ( ecog ps=2 ) or 11 ( ecog ps 3)+1 ( hemoglobin < 12 g / dl)+2 ( wbc count 10,800/l)+4 ( nlr 3.0)+10 ( cea 7 ng / ml ) . the scores ranged from 0 to 28 , and the maximally discriminative cut - off values were 6 and 25 , respectively . patients were then categorized according to three subgroups based on their scores : low ( 198 [ 49.1% ] ) , intermediate ( 182 [ 45.2% ] ) , and high - risk groups ( 23 [ 5.7% ] ) . the median os was 11.7 months ( 95% ci , 10.2 to 13.2 ) , 6.2 months ( 95% ci , 5.0 to 7.4 ) , and 1.3 months ( 95% ci , 0.9 to 1.7 ) , respectively ( p < 0.001 ) ( fig . the internally validated harrell s c - index of the scoring system was 0.731 ( 95% ci , 0.696 to 0.766 ) . of the 403 patients , 315 ( 78.2% ) underwent palliative chemotherapy and 88 ( 21.8% ) refused chemotherapy , receiving supportive care only . palliative chemotherapy included gemcitabine monotherapy ( 21.6% ) , gemcitabine doublet ( 40.4% ) , and fluoropyrimidine - based chemotherapy ( 8.9% ) . radiotherapy was performed with palliative aim in 7.2% of patients . with these different kinds of chemotherapy regimens , patients receiving chemotherapy had significantly longer survival than patients who received best supportive care only ( median os , 10.3 vs. 2.5 months ; p < 0.001 ) . one hundred seventy - five ( 88.4% ) , 138 ( 75.8% ) , and only two patients ( 8.7% ) in the low , intermediate , and high - risk groups received chemotherapy , respectively . palliative chemotherapy provided patients with a survival benefit in the low - risk group ( median os , 12.5 months vs. 5.9 months ; p < 0.001 ) ( fig . 3a ) , and patients in the intermediate - risk group also benefitted from chemotherapy ( median os , 8.0 vs. 2.0 months ; p < 0.001 ) ( fig . however , no survival difference was observed with chemotherapy in the high - risk group ( median os , 3.7 vs. 1.2 months ; p=0.163 ) ( fig . median os for total patients was 8.2 months ( 95% ci , 7.3 to 9.1 ) . the statistically determined best cutoff points were as follows : 3.0 for nlr , 6.0 mg / l for crp , 7.0 ng / ml for cea , and 453.0 u / ml for ca 19 - 9 . significantly extended survival was observed in patients with low nlr levels compared with high nlr ( median os , 11.3 months vs. 4.7 months ; p < 0.001 ) ( fig patients with low levels of crp , cea , or ca 19 - 9 , respectively , also showed superior survival ( median os ; crp : 11.3 months vs. 5.0 months , p < 0.001 ; cea : 10.9 months vs. 5.7 months , p < 0.001 ; ca 19 - 9 : 10.3 months vs. 6.5 months , p < 0.001 ) ( fig . analysis , age , previous pancreatectomy , tumor location , ps , liver metastasis , peritoneal carcinomatosis , bmi , hemoglobin , wbc count , nlr , serum albumin , bun , alp , ast , crp , cea , and ca 19 - 9 showed significant association with os ( table 2 ) . in multivariate analysis , ecog ps , hemoglobin , wbc count , nlr , and cea were significant in both forward and backward stepwise analyses ( table 3 ) . scores were calculated by summing the rounded chi - squre scores of these factors ( table 4 ) : 2 ( ecog ps=2 ) or 11 ( ecog ps 3)+1 ( hemoglobin < 12 g / dl)+2 ( wbc count 10,800/l)+4 ( nlr 3.0)+10 ( cea 7 ng / ml ) . the scores ranged from 0 to 28 , and the maximally discriminative cut - off values were 6 and 25 , respectively . patients were then categorized according to three subgroups based on their scores : low ( 198 [ 49.1% ] ) , intermediate ( 182 [ 45.2% ] ) , and high - risk groups ( 23 [ 5.7% ] ) . the median os was 11.7 months ( 95% ci , 10.2 to 13.2 ) , 6.2 months ( 95% ci , 5.0 to 7.4 ) , and 1.3 months ( 95% ci , 0.9 to 1.7 ) , respectively ( p < 0.001 ) ( fig . the internally validated harrell s c - index of the scoring system was 0.731 ( 95% ci , 0.696 to 0.766 ) . of the 403 patients , 315 ( 78.2% ) underwent palliative chemotherapy and 88 ( 21.8% ) refused chemotherapy , receiving supportive care only . palliative chemotherapy included gemcitabine monotherapy ( 21.6% ) , gemcitabine doublet ( 40.4% ) , and fluoropyrimidine - based chemotherapy ( 8.9% ) . radiotherapy was performed with palliative aim in 7.2% of patients . with these different kinds of chemotherapy regimens patients receiving chemotherapy had significantly longer survival than patients who received best supportive care only ( median os , 10.3 vs. 2.5 months ; p < 0.001 ) . one hundred seventy - five ( 88.4% ) , 138 ( 75.8% ) , and only two patients ( 8.7% ) in the low , intermediate , and high - risk groups received chemotherapy , respectively . palliative chemotherapy provided patients with a survival benefit in the low - risk group ( median os , 12.5 months vs. 5.9 months ; p < 0.001 ) ( fig . 3a ) , and patients in the intermediate - risk group also benefitted from chemotherapy ( median os , 8.0 vs. 2.0 months ; p < 0.001 ) ( fig . however , no survival difference was observed with chemotherapy in the high - risk group ( median os , 3.7 vs. 1.2 months ; p=0.163 ) ( fig . pancreatic cancer is the only human malignancy for which patients ' survival has rarely improved during the past 30 years . however , current systemic treatments offer only a modest benefit in tumor - related symptoms and survival and these benefits , if any , are usually limited to patients even with a novel regimen of folfirinox with good ps and adequate nutritional status , and abrupt changes in clinical status may occur in debilitated patients with advanced disease despite systemic treatment . therefore , primary point was ( 1 ) how to predict survival of patients at the time of initial diagnosis and ( 2 ) how to differentiate patients who might benefit from chemotherapy from those can not . in general , pancreatic cancer patients are not clinically separated into prognostic groups , with the exception of surgical status , before treatment . a previous korean study reported liver metastasis , peritoneal seeding , high crp , and low serum albumin as poor indicators of os . a western study reported age , weight loss , and metastasis as poor prognostic factors . both studies focused on gemcitabine - treated patients , and are small retrospective analyses with questionable statistical powers . however , pancreatic cancer is a heterogeneous disease and recent introduction of the three - drug combination regimen or the new taxane represent key advances for better control of the disease over gemcitabine , which requires a new prognostic model . another debate was cutoff points of routinely measured tumor markers crp , cea , and ca 19 - 9 . using a chemiluminescence immunoassay , the standard diagnostic cutoff points for ca 19 - 9 and cea were 37 iu / ml and 5 ng / ml , respectively . however , the cut - off points for these variables have not been established in metastatic disease . some researchers advocate that ca 19 - 9 serum level of < 100 u / ml implies likely resectable disease , while levels > 100 u / ml may suggest unresectablity or metastatic disease . however , most studies used median values or normal ranges for dichotomization . for dichotomizing continuous variables , splits of some percentile or average are arbitrary and may not be useful in assessing the true prognostic value of variables . in our study , instead , cutoff point was derived using the contal and oquigley method , which selects the cut - off point that maximizes the model likelihood only after all possible cut - offs have been evaluated . in addition , k - adaptive partitioning for the survival model was used to determine minimum p - value . the purpose of implementing a scoring system is for clinical decision - making and patient risk stratification , using routinely measurable clinical parameters on admission . in this study ps , hemoglobin , wbc , nlr , and cea were identified as independent prognostic markers . for patients with a score of 6 , we may hypothesize that their survival can be extended up to 12.5 months by chemotherapy , which provides us with a rationale for recommendation of relatively aggressive treatment such as folfirinox . conversely , high - risk group ( score > 25 ) patients had an extremely poor prognosis , with a median survival duration of only 1.3 months and the 1-year survival rate was less than 1% . chemotherapy has no statistical benefit in these patients , but it is impetuous for now to declare that chemotherapy is contraindicated for the high risk group , in which only two patients who received chemotherapy were included for analysis . ultimately , these findings suggest that this scoring system can be helpful in selection of the " right " patients for the " right " treatment . several previous studies reported reduced survival in patients with elevated ca 19 - 9 levels . we believe increased false positive results in the presence of obstructive jaundice and inflammation20% of our patients accompanied obstructive jaundice and 12% with systemic leucocytosis could severely limit the universal applicability of serum ca 19 - 9 levels in pancreatic cancer management . hypothesis of the role of inflammation in development and prognosis of cancer has been examined and several studies have suggested that nlr and wbc count are indicative of survival . whether the systemic inflammation is malignancy - associated or is the result of any co - morbid conditions that cancer patients may suffer is still unclear , but here we assumed that systemic inflammation might also occur in commonly prevalent conditions . one plausible explanation is that cellular response of blood components might be mediated through endothelial dysfunction , leading to depletion of vasodilator , antithrombotic , and anti - atherogenic properties of the endothelium and may result in capillary leukocytosis and subsequent increased vascular resistance . in addition , high neutrophil counts reflect inflammation , whereas low lymphocyte counts reflect malnutrition . cytokine - mediated systemic inflammation has been shown to promote angiogenesis , dna damage response , and tumor invasion . nlr has recently been reported as a prognostic marker in colorectal and lung cancers , and its prognostic value in pancreatic cancer has also been reported . although an nlr cut - off value of 4.0 or 5.0 has been shown to have significant prognostic value in resectable cancers , a cutoff point of 3.0 may be reasonable for survival predication , which is consistent with a previous report [ 18 - 20 ] . cea functions as an adhesion molecule during metastasis , and hostetter et al . reported that cea increases the percentage of colorectal liver metastases from 2% to 48% in vivo . in a small study , cea was identified as prognostic in advanced pancreatic cancer ; superior survival outcome has been reported in patients with low cea levels ( < 2.5 ng / ml ) compared to those with high cea levels . although the cut - off value for cea in our study was 7 ng / ml , further evaluation is warranted to elucidate the role of cea in pancreatic cancer . another factor , hemoglobin level , has been reported as a poor prognostic factor and is reflective of nutrition or general condition . however , a prognostic model using clinical parameters only had a limitation in that tumor biology could not be reflected . in pancreatic cancer , k - ras mutations occur in 90% of cases , and overexpression of growth factors such as epidermal growth factor , transforming growth factor is common . in addition , expression of these growth factors and their receptor has been found to show association with decreased survival , thus , a combined approach using both clinical parameters and biologic markers is warranted for implementation of a more robust prognostic model . however , our system showed a harrell s c - index of 0.731 , indicating sufficient discrimination power for patients according to the risk , and it warrants prospective clinical utility . patients underwent heterogeneous treatments including gemicitabine monotherapy or doublet , fluoropyrimidines , oral chemotherapy or even concurrent chemoradiotherapy which might have affected differential influence on survival . in conclusion , we found that nlr and cea were independent prognostic factors and suggest 3.0 for nlr and 7 ng / ml for cea as statistically determinant cut - off values for prognostic dichotomization . in addition , our prognostic scoring system provides rationale for risk stratification of advanced pdac patients , leading to individualized treatment .

obesity is becoming a major health issue in patients with copd , as it is in the general population . several studies have reported an increased proportion of patients with copd who are either overweight or obese.14 considering the well - established link between obesity and hypertension , type 2 diabetes , cancer and cardiovascular diseases ( cvds)5 and the fact that copd is an independent risk factor for cvds,6 this increased prevalence of obesity in copd is a source of concern . consistent with these observations , all - cause mortality and cardiovascular mortality are also increased in patients with copd.7 depending on copd severity , cvds are the primary cause of mortality in 20%27% of patients with copd.8,9 one important issue to consider is that body mass index ( bmi ) is only a crude index of body composition while not allowing the quantification of ectopic fat accumulation . rather than body size , body shape may be important to consider in the interpretation of the relationship between obesity and copd . for instance , it is very well documented that the adverse metabolic and cardiovascular consequences of obesity are largely explained by the presence of unwanted adipose tissue ( at ) accumulation in the abdomen and in normally lean tissues such as the liver , the heart and the skeletal muscles ( collectively called ectopic fat tissue accumulation).10 individuals with a selective accumulation of ectopic fat are at increased risk of developing features of the metabolic syndrome , diabetes , an atherogenic dyslipidemia , or cvds compared to those who preferentially accumulate lipids in subcutaneous healthy at.10 we took advantage of the evaluation of copd longitudinally to identify predictive surrogate endpoints ( eclipse ) cohort with a 3-year follow - up to address the following questions : 1 ) is copd associated with increased ectopic fat accumulation within the abdomen and in the muscles ? 2 ) does ectopic fat accumulation modify the expression of copd in being associated with reduced functional and health status , as well as with forced expiratory volume in 1 second ( fev1 ) rate of decline , exacerbations , mortality and cardiovascular comorbidities ? our overarching hypothesis was that copd would be associated with increased ectopic fat accumulation compared to controls with normal lung function and that this would be associated with adverse clinical outcomes . eclipse ( clinicaltrials.gov , nct00292552 ) was a multicenter , observational and longitudinal study to identify surrogate markers of copd progression.11 it enrolled 2,164 current or ex - smokers ( 10 pack - years ) with global initiative for chronic obstructive lung disease ( gold ) stages 24 , 337 smokers with normal lung function and 245 never - smokers.12 all eclipse participants underwent a low - dose volumetric computed tomography ( ct ) scan of the chest at baseline . in a subset of them that constitutes the population for the current report , a valid ct scan slice at l2l3 could be identified , allowing to assess body composition from which fat and muscle densities , a reflection of tissue fatty infiltration , could be accurately quantified.13 patients characteristics ( age , race , sex , smoking history ) , chest ct scan , pulmonary function tests , st georges respiratory questionnaire ( sgrq ) score and 6-minute walking distance ( 6mwd ) were obtained at baseline and yearly for the 3-year study duration as previously reported.11,14,15 standardized questionnaires were used to record comorbidity at baseline . all patients provided written informed consent , and the study was approved by the ethics committees of the participating centers . the eclipse steering and scientific committees approved the protocol for this substudy ( table s1 ) . all eclipse participants underwent a low - dose volumetric ct scan of the chest at baseline with the following protocol : 120-kv peak , 40 ma , and 1.00- or 1.25-mm slice thickness , at full inspiration . for the purpose of this eclipse substudy , we analyzed all subjects with a thoracic ct scan that contained images at l2l3 intervertebral space . ectopic fat accumulation was quantified by measuring cross - sectional area ( csa ) of visceral adipose tissue ( vat ) and muscle tissue ( mt ) attenuation , a reflection of muscle fat infiltration . measures of fat and mt ( psoas , paraspinal and abdominal wall muscles ) csa and attenuation were performed by the same investigator ( mm ) using the body composition analysis platform of the institut universitaire de cardiologie et de pneumologie de qubec and a specialized image analysis software ( sliceomatic ; tomovision , montreal , qc , canada ) according to standardized techniques ( figure 1).13 csas of at and mt were measured using an attenuation range of 190 to 30 hounsfield units ( hus ) and 29 to 130 hus , respectively . the mean attenuation value in hu of each structure was generated . to avoid subjectivity in the separation between scat and vat , these two fat tissues were separated from each other midway in the thickness of abdominal wall muscles . subjects for whom a tissue surface area > 1 cm fell outside the field of view of the scope were excluded from further analysis . a random selection of 10% of all segmented images was redone by the same evaluator to assess for intraobserver variation . interobserver variation was evaluated on a random sample of 10% of the segmented images that were also analyzed by a second imaging expert . we also took advantage of the thoracic ct scan to quantify coronary artery calcification , a surrogate of coronary artery disease , using standardized techniques.16 characteristics were compared between patients with copd and controls ( smokers with normal lung function and never - smokers ) , using student s t - test statistics for continuous variables and chi - square statistics for discrete variables . coronary artery calcification scores were analyzed on a log10 scale because of their lognormal distribution . patients with copd were further classified according to the gold spirometric classification system.17 the overall test retest reproducibility of each body composition parameter was assessed by calculating a pearson s correlation coefficient between evaluation 1 and evaluation 2 for each ct body composition parameter . test retest reliability was evaluated using intraclass correlation coefficient ( icc ) as it reflects both systematic and random differences in test measures.18 an icc > 0.75 was judged to be excellent.19 ct body composition variables were compared between patients with copd and controls , and also across gold spirometric grades , using a multivariate analysis of covariance ( mancova ) model , with age , sex , bmi and smoking status included as covariates . when a significant effect of groups or subgroups were identified , univariate analysis of covariance ( ancova ) models were adjusted to investigate which body composition variables were different , and least significant difference - protected multiple comparisons were performed to differentiate the subgroups . the association between body composition parameters with clinical outcomes of interest ( 6mwd , exacerbation rate , quality of life , fev1 decline ) was addressed using the cochran armitage trend test to determine a dose response relationship between csa of vat , mean mt attenuation , csa of mt and each of the clinical outcomes . for this analysis , body composition parameters ( explanatory variables ) were expressed in four equal groups determined by their quartiles . in this analysis , quartile 1 was considered as the reference value , while quartiles 2 , 3 and 4 indicated progressive increase in ectopic fat accumulation or decrease in csa of mt . each patient - reported outcome was expressed as dichotomous variables using specific cut - points : 350 m for the 6mwd,20 more than or equal to two exacerbations per year,17 sgrq score > 2517 and fev1 decline > 40 ml / year.21 the impact of body composition on comorbidities was assessed by logistic regression models , while its relationship with survival was studied using a cox proportional hazards regression model . all analyses were controlled for age , sex , bmi , smoking status and fev1 . data analyses were performed using sas version 9.4 ( sas institute inc . , cary , nc , usa ) . eclipse ( clinicaltrials.gov , nct00292552 ) was a multicenter , observational and longitudinal study to identify surrogate markers of copd progression.11 it enrolled 2,164 current or ex - smokers ( 10 pack - years ) with global initiative for chronic obstructive lung disease ( gold ) stages 24 , 337 smokers with normal lung function and 245 never - smokers.12 all eclipse participants underwent a low - dose volumetric computed tomography ( ct ) scan of the chest at baseline . in a subset of them that constitutes the population for the current report , a valid ct scan slice at l2l3 could be identified , allowing to assess body composition from which fat and muscle densities , a reflection of tissue fatty infiltration , could be accurately quantified.13 patients characteristics ( age , race , sex , smoking history ) , chest ct scan , pulmonary function tests , st georges respiratory questionnaire ( sgrq ) score and 6-minute walking distance ( 6mwd ) were obtained at baseline and yearly for the 3-year study duration as previously reported.11,14,15 standardized questionnaires were used to record comorbidity at baseline . all patients provided written informed consent , and the study was approved by the ethics committees of the participating centers . the eclipse steering and scientific committees approved the protocol for this substudy ( table s1 ) . all eclipse participants underwent a low - dose volumetric ct scan of the chest at baseline with the following protocol : 120-kv peak , 40 ma , and 1.00- or 1.25-mm slice thickness , at full inspiration . for the purpose of this eclipse substudy , we analyzed all subjects with a thoracic ct scan that contained images at l2l3 intervertebral space . ectopic fat accumulation was quantified by measuring cross - sectional area ( csa ) of visceral adipose tissue ( vat ) and muscle tissue ( mt ) attenuation , a reflection of muscle fat infiltration . measures of fat and mt ( psoas , paraspinal and abdominal wall muscles ) csa and attenuation were performed by the same investigator ( mm ) using the body composition analysis platform of the institut universitaire de cardiologie et de pneumologie de qubec and a specialized image analysis software ( sliceomatic ; tomovision , montreal , qc , canada ) according to standardized techniques ( figure 1).13 csas of at and mt were measured using an attenuation range of 190 to 30 hounsfield units ( hus ) and 29 to 130 hus , respectively . the mean attenuation value in hu of each structure was generated . to avoid subjectivity in the separation between scat and vat , these two fat tissues were separated from each other midway in the thickness of abdominal wall muscles . subjects for whom a tissue surface area > 1 cm fell outside the field of view of the scope were excluded from further analysis . a random selection of 10% of all segmented images was redone by the same evaluator to assess for intraobserver variation . interobserver variation was evaluated on a random sample of 10% of the segmented images that were also analyzed by a second imaging expert . we also took advantage of the thoracic ct scan to quantify coronary artery calcification , a surrogate of coronary artery disease , using standardized techniques.16 characteristics were compared between patients with copd and controls ( smokers with normal lung function and never - smokers ) , using student s t - test statistics for continuous variables and chi - square statistics for discrete variables . coronary artery calcification scores were analyzed on a log10 scale because of their lognormal distribution . patients with copd were further classified according to the gold spirometric classification system.17 the overall test retest reproducibility of each body composition parameter was assessed by calculating a pearson s correlation coefficient between evaluation 1 and evaluation 2 for each ct body composition parameter . test retest reliability was evaluated using intraclass correlation coefficient ( icc ) as it reflects both systematic and random differences in test measures.18 an icc > 0.75 was judged to be excellent.19 ct body composition variables were compared between patients with copd and controls , and also across gold spirometric grades , using a multivariate analysis of covariance ( mancova ) model , with age , sex , bmi and smoking status included as covariates . when a significant effect of groups or subgroups were identified , univariate analysis of covariance ( ancova ) models were adjusted to investigate which body composition variables were different , and least significant difference - protected multiple comparisons were performed to differentiate the subgroups . the association between body composition parameters with clinical outcomes of interest ( 6mwd , exacerbation rate , quality of life , fev1 decline ) was addressed using the cochran armitage trend test to determine a dose response relationship between csa of vat , mean mt attenuation , csa of mt and each of the clinical outcomes . for this analysis , body composition parameters ( explanatory variables ) were expressed in four equal groups determined by their quartiles . in this analysis , quartile 1 was considered as the reference value , while quartiles 2 , 3 and 4 indicated progressive increase in ectopic fat accumulation or decrease in csa of mt . each patient - reported outcome was expressed as dichotomous variables using specific cut - points : 350 m for the 6mwd,20 more than or equal to two exacerbations per year,17 sgrq score > 2517 and fev1 decline > 40 ml / year.21 the impact of body composition on comorbidities was assessed by logistic regression models , while its relationship with survival was studied using a cox proportional hazards regression model . all analyses were controlled for age , sex , bmi , smoking status and fev1 . data analyses were performed using sas version 9.4 ( sas institute inc . , cary , nc , usa ) . we identified 585 patients ( 511 patients with copd and 74 controls ) for whom a ct slice at l2l3 could be analyzed for body composition assessment . patients with copd and controls had similar bmi but important differences were otherwise noted : patients with copd were older , had a higher proportion of men and were current / former smokers compared to controls . they also exhibited higher sgrq scores ( worse health - related quality of life ) , greater prevalence of cardiovascular comorbidities ( angina , heart attack , myocardial infarction , stroke and heart failure ) , hypertension and greater coronary artery calcification score than controls . during the 3-year follow - up , 50 deaths were recorded ( 49 patients with copd and one control ) , resulting in lower mean survival days in patients with copd . a total of 1,661 moderate - to - severe exacerbations were reported in patients with copd , with a mean exacerbation rate of 1.280.06/year / patient ( range : 09/year / patient ) . the pearson s coefficient and icc for each body composition parameter are reported in table 2 and show an excellent intra- and inter - observer reproducibility of our body composition measurements . ct body composition data adjusted for age , sex , smoker status and bmi are presented in figure 2 . for a similar bmi ( figure 2a ) , patients with copd exhibited higher csa of vat ( figure 2b ) , lower mt attenuation ( figure 2c ) and a tendency to have lower csa of mt ( figure 2d ) than controls . csa of scat could be quantified in 384 subjects ( 327 copd subjects and 57 controls ) in whom the abdomen external wall fell inside the field of view . the same results are also presented according to the severity of airflow obstruction from gold stages 2 to 4 . patients with gold 3 and 4 copds had lower bmi than gold 2 patients , while gold 4 patients were characterized by larger csas of vat and scat and less mt attenuation than the remaining groups . the associations between body composition parameters and copd - associated clinical outcomes are shown in figure 3 . progressively increasing csa of vat was not associated with worsening in functional status ( 6mwd ) , exacerbation rate and fev1 decline but it was associated with a reduced odds ratio of having sgrq scores > 25 ( indicating worse health status ) . in contrast , progressively decreasing mt attenuation and csa of mt were associated with increased probability of exhibiting low 6mwd and accelerated fev1 decline . in copd , the probability of having diabetes ( p=0.024 ) and gastroesophageal reflux ( p=0.0048 ) at baseline increased in parallel with vat accumulation , while mt attenuation predicted increased probability of cardiovascular comorbidities ( p=0.042 ; figure 4 ) . this eclipse substudy reports increased ectopic at accumulation in a large group of patients with copd compared to controls with normal lung function . patients with copd were characterized by increased csa of vat and evidence of muscle fat infiltration as assessed by ct as well as by an increased prevalence of cardiovascular comorbidities . our study is concordant and extends existing knowledge that patients with copd are associated with increased vat accumulation compared to controls7,22 by reporting , for the first time , decreased mt attenuation ( consistent with increased fat infiltration ) in copd and investigating the relationship between ectopic fat accumulation and copd - related outcomes and comorbidities . alterations in ct body composition parameters were particularly notable in patients with gold 4 copd . interestingly , progressively decreasing mt attenuation and csa of mt were associated with increased probability of experiencing worse functional status and fev1 decline and prevalence of cvds , while vat accumulation was associated with the prevalence of diabetes and gastroesophageal reflux at study entry . importantly , these associations remained after adjustment for age , sex , bmi , smoking status and fev1 . we are not aware of previously published study on intra - abdominal muscle attenuation and csa data in copd . similar to our findings involving the psoas , paraspinal and abdominal wall muscles , evidence of fat infiltration and atrophy of the quadriceps has been reported in copd.23,24 the same authors also reported inverse relationships between indices of quadriceps fat infiltration and walking capacity in copd . in this context , it is interesting that lower intra - abdominal mt density , a reflection of increased fatty content , and reduced muscle intra - abdominal csas were associated with increased probability of having a 6mwd < 350 m. it could be argued that the performance of the psoas and paraspinal muscles that are involved in walking was altered by a combination of fat infiltration and reduced area , together leading to impaired muscle contractility.24,25 alternatively , intra - abdominal muscle fat infiltration and atrophy may simply be a reflection of a generalized process involving several muscle groups , including those of the lower limbs . these phenomena have been associated with insulin resistance,26 resulting from a combination of factors , including age , genetics , poor diet , smoking and lack of vigorous physical activity , to only name a few.10 another potential contributor to ectopic fat accumulation in copd that was not addressed in the present study included repeated use of systemic corticosteroid to treat copd exacerbations.27 we also did not have direct measurement of physical activity and dietary habits.7 ectopic fat is metabolically active as evidenced by its production of a host of proinflammatory mediators , including tnf- , il-6 , leptin and adiponectin , whose effects may be seen not only locally within the at but also at the systemic level . considering systemic inflammation as a risk factor for cvds and possibly for copd progression,28,29 we speculated that ectopic fat accumulation would be associated with adverse clinical outcomes such as poor functional status and quality of life , increased exacerbation risk and faster fev1 decline as well as with cardiovascular comorbidities . although this interpretation is partly supported by our data , the reported association between ectopic fat accumulation , clinical outcomes and comorbidities should not be viewed as implying causal relationships . nevertheless , these associations point to the fact that ectopic fat accumulation could be detrimental to cardiovascular and metabolic health in copd . investigators from the copdgene study reported a significant association between the amount of vat and the risk of having a history of a previous myocardial infarction in patients with copd.30 in the present investigation , we found that csa of vat and muscle attenuation were , respectively , associated with increased prevalence of diabetes and cardiovascular comorbidities at study entry . these results are consistent with the bulk of knowledge recognizing ectopic fat accumulation as a risk factor for cardiovascular and metabolic diseases.10 we did not find , however , an association between the extent of coronary artery calcification and vat in this cohort . this may be an indication that the mechanisms by which vat contributes to cvd risk do not mainly involve a calcification process . it involves an appreciable number of patients with longitudinal follow - up , offering the possibility of studying the clinical consequences of ectopic fat accumulation . body composition analysis was performed rigorously , according to standardized and recommended procedures.13 there are however potential limitations that need to be considered in interpreting the data . despite the relatively large study sample and the 3-year follow - up , the number of deaths ( n=50 ) was likely too small to secure the notion of a possible relationship between ectopic fat accumulation and survival in copd . because eclipse ct scans were not performed purposely to determine body composition , the analysis had to be restricted to those participants with abdominal ct images , which would be valid for segmentation of at and muscle area and densities . we do not believe , however , that these issues compromise the validity of our findings because baseline characteristics of patients involved in this substudy were similar to those of the entire eclipse cohort.12 eclipse did not mandate to match control subjects with patients with copd . as such , important differences in age , sex distribution and smoking status were seen between the two groups . this issue was addressed by controlling for baseline differences for these variables in all analyses . in addition , physical activity levels were not assessed , making it difficult to comment on the potential impact of sedentary behaviors on the body composition differences observed in copd in this cohort . although eclipse was a considerable undertaking , it remains that 3 years of follow - up was likely too short to highlight the clinical impact of body composition abnormalities , particularly in this cohort of patients with advanced copd . finally , some important well - established cardiovascular risk factors such as lipid and glycemic profile were not assessed . in this regard , it could be argued that these traditional risk factors could at least partly capture the elevated cardiovascular risk in copd . our study adds to the existing knowledge by confirming the presence of increased ectopic fat accumulation in copd . in this patient population , body composition abnormalities in the form of increased csa of vat and fat infiltration of intra - abdominal muscle and reduced csa of intra - abdominal muscle were associated with cardiovascular comorbidities , diabetes and gastroesophageal reflux as well as with relevant clinical outcomes such as walking capacity and fev1 decline . as successfully accomplished in other patient populations , we submit that assessing the level of ectopic fat accumulation is relevant in copd . in addition , we suggest that we should also consider the development of lifestyle modification programs focusing on both nutritional quality and physical activity / exercise with the objective of improving at distribution / ectopic fat in patients with copd .

colorectal cancer ( crc ) is one of health problems in the world ( 1 ) . it was the third most common cancer with nearly 1.4 million new cases in 2012 . about 54 percent of colorectal cancer cases occurred in more developed countries ( 2 ) . people with a first - degree relative ( fdr ) ( parent , sibling , or offspring ) have 2 to 3-foldrisk of developing disease compared to individuals with no family history ( 3 ) . according to iranian annual of national cancer registration report , crc is the fourth most common cancer after skin , breast and gastric cancer and its incidence rate was estimated 6 to 7.9 in 100,000 people . furthermore , over the last 25 years there has been a growing trend ( 4 - 9 ) . because of family history or certain medical conditions , colorectal cancer screening should begin before age 50 . recent data indicate that fdrs of crc patients significantly under - use colonoscopy screening . the participation rate lying between 30 and 64% ( 10 - 13 ) . several studies showed factors such as sex , age , health status , knowledge and attitude , number of colorectal cancer affected cases in family , age and stage of diagnosis are important to refuse to participate in colonoscopy screening program . a low socioeconomic status ( low income , unemployment , low educational level , no health insurance coverage ) has been associated with lower screening participation in many studies ( 14 - 17 ) . one turkish study indicated fdrs of patients having a higher educational level and income had screening testing twice more than the patients having lower income ( 36% vs 18% , p < 0,001 ) ( 18 ) . the aim of this study was investigation of the association between socioeconomic factors and participation in colonoscopy screening program among first degree relatives of colorectal cancer affected cases . this cross sectional descriptive study was conducted on 200 first degree relatives of colorectal cancer cases . a clustered probability design was used to select representative samples among total 400 high risk fdrs who were consulted and recommended to participate in colonoscopy screening program by physicians between 2007 and 2013 in research institute for gastroenterology and liver disease of shahid beheshti university of medical sciences , tehran , iran . the questionnaire included first degree relatives demographic and socioeconomic status including educational level , employment , income , health insurance coverage and health status in terms of smoking , alcohol consumption , suffering from diabetes and hypertension diseases . finally they answered to these questions have you ever undergone colonoscopy screening test ? and would you like to participate in colonoscopy screening program ? we investigated the association between participation of fdrs in colonoscopy screening program , and socioeconomic and health status variables through using chi - square and fisher exact test by spss 19 . also , multiple logistic regression analysis used to predict for participation in colonoscopy screening program . odds ratios ( ors ) and its 95% confidence intervals ( cis ) were obtained . of total 200 participants , 57.5% were female , 51.5% , 50 years and older , 85% married , 87.1% living in tehran , 55.5% diploma and higher educational level , 83% were employed ( full or part time ) and 53.7% were with 10 million rials and more monthly income . the mean of age , monthly income and body mass index ( bmi ) was 51.4 years , 16,000,000 rials and 26.4 respectively . from interviewees population , 59% had undergone at least one colonoscopy screening test to the time of the interview . tables 1 and 2 shows results of bivariate analysis between participation in colonoscopy screening program to time of interview and decision to participate in future with demographic , socioeconomic and health status variables . as can be seen , in data analysis by chi - square method , there was no statistical significance between demographic , socioeconomic , health status variables and participation in colonoscopy screening program to time of interview . but in the same analysis , there is statistical significance between educational level and monthly income with decision to participate in future . analysis showed first degree relatives with academic educational level were more interested to participate in colonoscopy screening program ( p = 0.006 , ors = 2.7 and 95%ci = 1.3 - 5.8 ) . also , fdrs with monthly income 10 million rials and over participated more than others in this program ( p = 0.005 , ors = 2.4 and 95%ci = 1.2 - 4.5 ) . in the logistic regression analysis , high educational level ( diploma and higher ) was predictors for participating in colonoscopy screening program in first degree relatives ( p = 0.031 , ors = 2.9 and 95% ci = 1.10 - 7.67 ) . this study examined the association between socioeconomic status ( ses ) and participation in colonoscopy screening program in first degree relatives of colorectal cancer patients . we did not find association between demographic variables , socioeconomic status and participation in colonoscopy screening test to time of interview in bivariate chi - square analysis , whereas there was statistical significance in high level education and more income with decision to participate in same analysis . in this survey a recent study indicated fdrs female with age below 50 underwent more than males and age 50 and older ( 19 ) . another study indicated fdrs with higher educational level and income participated in screening program more frequently ( 18 ) . one survey in korea showed income disparities for participating in colorectal cancer screening among both males and females ( 20 ) . one older study in canada confirmed people in the highest - income quintile had higher odds of receiving colonoscopy screening ( adjusted or 1.50 ; 95% ci 1.48 - 1.53 ) ( 21 ) . in the current study we used multiple logistic regression for demographic and socioeconomic variables analysis to predict participation in colonoscopy screening program in fdrs . therefore , high educational level ( diploma and university degree ) was a predictor to participate in colonoscopy screening test . one published study in 2012 showed the frequency of screening in patients with higher educational level ( ninth class ) , below the ninth grade and none were 33% , 21% , and 17% respectively ( p = 0,017 ) . the fdrs of patients with higher income levels ( > 1000 turkish liras , equivalent to about 700 usd at the time of the study ) had screening twice more often than the patients having lower income ( 36% vs. 18% , p < 0,001 ) ( 22 ) . as a matter of fact , socioeconomic status ( ses ) is a powerful factor to participate in colonoscopy screening test . in this study there was not an association between participation in colonoscopy program and health insurance coverage but one study in australia notified fdrs with private insurance were at significantly greater odds of receiving colonoscopy testing ( 23 - 25 ) . it is obvious that socioeconomic status is an important variable for participation in colonoscopy screening test . therefore , when crc screening is planned , elderly with positive family history , low educational level and lower income should get special attention to be convinced for undergoing colonoscopy screening test . additionally , it is necessary that people with low income be covered by appropriate health insurance to participate in colorectal cancer screening tests . these data collected through interviewing via phone and we relied to interviewees answers . it is obvious that socioeconomic status is an important variable for participation in colonoscopy screening test . therefore , when crc screening is planned , elderly with positive family history , low educational level and lower income should get special attention to be convinced for undergoing colonoscopy screening test . additionally , it is necessary that people with low income be covered by appropriate health insurance to participate in colorectal cancer screening tests . these data collected through interviewing via phone and we relied to interviewees answers .

influenza a and b cause highly contagious , acute respiratory illness , associated with high morbidity and mortality among persons with underlying diseases . the causes of death in the more severe forms of influenza are ( a ) severe pneumonia with respiratory distress , ( b ) neurological complications , and ( c ) secondary bacterial pneumonia . outbreaks in children units , senior facilities , cancer centers , neonatal units , pulmonary rehabilitation centers , emergency departments , and others have been described . large nosocomial outbreaks in large tertiary referral hospitals that provide care to large numbers of patients with advanced aids have not been frequently described . roosevelt hospital is a large teaching , university tertiary referral hospital in guatemala city , with 850 beds capacity . more than 3,500 aids patients were in followups , and 1561 treated with antiretroviral drugs at the time when the outbreak was detected in march 2006 ; all of whom were at potential risk to develop severe forms of influenza . 35% of the patients have less than 50 cd4 cells / mm and 68% less than 200 cd4 cells / mm , at the time the hiv infection diagnosis is made at roosevelt hospital . the main admission diagnosis in the internal medicine department in the men wards is aids ( on average 25/66 beds are occupied by patients with aids ) . throughout the country mortality rate at the time of the first hospital admission in these advanced aids patients has decreased from 85% to 89% in the pre - haart era ( 19902000 ) , to 18%21% after the year 2000 . rapid dissemination of influenza virus is more likey in closed areas with poor ventilation ( air exchanges less than 6 per hour ) , no policies for health care worker vaccination and suboptimal awareness about the clinical presentation of severe influenza in immunocompromised persons may contribute to the magnitude of the outbreaks . the mortality rates in immunocompromised patient can be high , as we observed in this group of patients , with fulminant pneumonia and respiratory distress as the final cause of death . during the months of february to april 2006 , a large outbreak of influenza a was detected at roosevelt hospital ; health care workers and hospitalized patients were affected in this large teaching hospital in guatemala city . during the first 5 days of the initial suspicion of a possible respiratory outbreak , after 3 consecutive deaths in aids patients who were stabilized for different opportunistic infections , the first results of nasopharyngeal swabs were positive for influenza a virus , with no other pathogens found . the patients had been hospitalized for diagnosis and treatment , developed acute respiratory failure , and died within 1836 hours from the beginning of symptoms ; testing for legionella , rapid antigen test for influenza a , influenza b , and parainfluenza virus in regional or national labs , and cultures for common bacterial causes for nosocomial pneumonia in our microbiology laboratory were negative . an extensive triage and surveillance program in health care workers ( hcws ) was initiated . we could establish that the medical students and the internal medicine residents in the ward had a clinical diagnosis of influenza . we began to test hcw and patients in all internal medicine wards and 3 days after in all wards of the hospital . influenza diagnosis was established by clinical case definition and rapid test for influenza a ; viral cultures and histopathological examination of tissues from dead patients were also instituted . patients were considered as having nosocomially acquired infection if they had been admitted at least 72 hours before the onset of the symptoms . clinical triage was established in the emergency room for the early detection of new influenza cases from the community . interventions to interrupt the transmission were implemented and includedbarrier methods ( n95 masks , respiratory isolation measures , etc . ) and strengthening of hand hygiene , vaccination of healthy health care workers ( hcws ) and furloughing of sick hcw for 3 to 5 days , antiviral use for treatment or prophylaxis of hospitalized immunosuppressed patients , initially with amantadine , and after several failures , and the results of susceptibility tests obtained from collaboration with the us cdc , with oseltamivir , daily active surveillance for early detection of influenza cases in patients and hcw , daily active surveillance for early detection of influenza a cases in new patients before admission from the emergency rooms , no family visits to the internal medicines wards were permitted for 3 weeks , no elective surgery was permitted during the last 10 days of the outbreak . barrier methods ( n95 masks , respiratory isolation measures , etc . ) and strengthening of hand hygiene , vaccination of healthy health care workers ( hcws ) and furloughing of sick hcw for 3 to 5 days , antiviral use for treatment or prophylaxis of hospitalized immunosuppressed patients , initially with amantadine , and after several failures , and the results of susceptibility tests obtained from collaboration with the us cdc , with oseltamivir , daily active surveillance for early detection of influenza cases in patients and hcw , daily active surveillance for early detection of influenza a cases in new patients before admission from the emergency rooms , no family visits to the internal medicines wards were permitted for 3 weeks , no elective surgery was permitted during the last 10 days of the outbreak . patient 1 presented during the first week in march with acute respiratory failure and died within 24 hours . his diagnosis prompted a reevaluation of three recent deaths ; all of whom tested positive in histopathology , resulting in the recognition of the outbreak . in the period from february to april 2006 , 59 hospitalized patients were diagnosed with influenza a ; 19 of them were aids patients ( mortality rate : 71% ) while 5/40 ( 12.5% ) had other underlying diseases including cancer ( 3 ) , severe cardiac failure ( 1 ) , and severe malnutrition ( 1 ) . the final attack rate at one month was 21% in doctors and medical students and 10.5% in other hcws ( figures 3 and 4).a selective emergency vaccination plan was instituted in 5 days : 1,720 of 3,100 hcws ( 56% ) were reached ( figure 1).a daily active surveillance plan for early detection of influenza cases in hospitalized patients and hcw was implemented.after 3 weeks of the implementation of the control plan , deaths were stopped , and after 3 more weeks no more cases were detected in hcw ( figure 2 ) . a selective emergency vaccination plan was instituted in 5 days : 1,720 of 3,100 hcws ( 56% ) were reached ( figure 1 ) . a daily active surveillance plan for early detection of influenza cases in hospitalized patients and hcw after 3 weeks of the implementation of the control plan , deaths were stopped , and after 3 more weeks no more cases were detected in hcw ( figure 2 ) . influenza a can cause a very severe disease , with a high mortality rate in advanced aids patients . the clinical presentation was characterized by the accelerated evolution to death from the beginning of symptoms ( average 50 hours ) and affected mainly the more advanced aids cases , with a mortality rate among this group of 74% , compared with the 12.5% mortality in the non - aids patients . it is important to consider the possibility of influenza in the differential diagnosis in advanced cases of aids that are admitted to the general internal medicine wards , who are under treatment for opportunistic infections and develop acute respiratory distress . the rapid evolution to death and the risk of transmission to other immunosuppressed patients can be stopped , if we implement general isolation measures and use antiviral drugs like oseltamivir as prophylaxis or therapy early in the course of the disease . the protection conferred by the available vaccines is uncertain , and the information in the aids population is limited . all patients had concomitant severe opportunistic infections , but the histopathological findings showed in more than 90% of the patients in whom an autopsy was performed the typical pathology caused by influenza virus in the respiratory epithelium previously described . amantadine failed to protect patients exposed to influenza in the hospital , due to the resistance that was documented during the evolution of the outbreak . the influenza a virus in advanced aids patient could be considered an acute and severe opportunistic infection , that requires rapid actions to limit the negative impact in morbidity and mortality in this population . in addition to these points , several aspects of this outbreak are unique , and they deserved a discussion / comment . few outbreaks of severe influenza have been described in tropical or subtropical regions ; the fact that this one follows the seasonal pattern of the northern hemisphere season is worthy of a comment . this is one of the largest outbreaks in hospitalized patients with advanced aids and highlights the high risk of severe disease and mortality in these patients . several aspects of the control plan instituted have been described as potential control measures for the control of outbreaks . respirators , respiratory precautions , hand hygiene , and early diagnosis and furloughing of health care workers have been described . this plan shows that institution of all the measures may be necessary to control outbreaks . vaccination of health care workers has been advocated but only mandatory programs have achieved 100% compliance . it would be good to know what the rate of vaccination in health care workers in the years after the outbreak has been . it is very urgent to investigate about the role of vaccination in the control of this outbreak , since it takes a few weeks to develop immunity after vaccination . the role of oseltamivir treatment and prophylaxis in the control of this outbreak also deserves some comment . a rapid , comprehensive plan for the control of nosocomial epidemic influenza a outbreaks is essential to limit severe morbidity and mortality in hospitals who attend large immunocompromised populations , including aids patients . a multidisciplinary approach is important to prevent possible future outbreaks , especially with increasing reports of human infection with highly pathogenic avian influenza viruses or other novel viruses that could become pandemic in the near future .