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adiponectin ( arcp 30 , adipoq , apm1 or gbp28 ) , secreted by adipose tissue , is a 247-amino acid peptide which was discovered in 1995 ( 1 , 2 ) . the amounts of adiponectin account for 0.01% of total plasma protein . the concentration of adiponectin circulating in plasma is very high ( 2 to 20 g / ml ) ( 3 ) . plasma levels of adiponectin in the japanese population is about 5 to 10 g / ml ( 4 ) and serum adiponectin is lower in indo - asians when compared with caucasians ( median 3.3 vs. 4.9 g / ml ) ( 5 ) . adiponectin has gained particular interest on account of its relation with insulin sensitivity , atherosclerosis , and inflammation ( 6 ) . current projections estimate that cardiovascular disease , especially atherosclerosis , will be the leading cause of all disease in 2020 ( 7 ) . recent findings suggest that high adiponectin concentration is an independent predictor of mortality in chronic heart failure patients , chronic kidney patients , and elderly patients , who are at high risk for cardiovascular events ( 8 - 12 ) . obesity has been proven to be an independent risk factor for cad in both genders , and is a growing health problem in most developed and some developing countries ( 13 - 15 ) . body weight , body mass index ( bmi ) , waist circumference ( wc ) and whr are primary methods to determine obesity . while bmi reflects general obesity , waist circumference and waist hip ratio are related to central - type obesity , where body fat is primarily located in the abdomen . prospective epidemiological studies have revealed that central obesity ( determined by wc and whr ) is more relevant in cad risk compared to general obesity ( determined by bmi ) ( 16 - 18 ) . while whr is commonly used in reflecting central obesity , waist circumference is shown to have a better correlation with abdominal fat localization ( 19 - 22 ) . to our best knowledge , this is the first study performed in kosovo focusing on this particular issue . the study population consisted of 82 consecutive patients who underwent coronary angiography for suspected or known coronary atherosclerosis at the university clinical center of kosovo . with the exception of acute coronary syndromes , the patients had to present in a stable clinical condition without major concomitant non - cardiovascular disease . the study protocol was approved by the ethical committee of the faculty of medicine , university of prishtina , and research was conducted in accordance with declaration of helsinki guidelines . written informed consent cad was defined as more than 50% luminal stenosis identified in a minimum of two views , in the case of single - vessel involvement . gensini score assesses the severity of coronary artery disease : it grades narrowing of the lumen of the coronary artery and scores it as 1 for 1 - 25% narrowing , 2 for 26 - 50% narrowing , 4 for 51 - 75% , 8 for 76 - 90% , 16 for 91 - 99% and 32 for a completely occluded artery . this score is then multiplied by a factor , according to the importance of the coronary artery . the multiplication factor for a left main stem ( lms ) lesion is 5 , it is 2.5 for proximal left anterior descending artery ( lad ) and proximal circumflex artery ( cx ) lesions , 1.5 for a mid - lad lesion , and 1 for distal lad , mid / distal cx and right coronary artery lesions . gensini score was expressed as the sum of the scores for all three coronary arteries to evaluate the entire extent of coronary artery disease ( 23 ) . were taken after the patients had removed their shoes and upper garments and had donned an examining gown . waist height ratio ( whtr ) is measured by division of weight circumference by their height , always expressed in same units . body mass index ( bmi ) was calculated as weight ( kg ) divided by the square of height ( m ) . the wc was measured by tape in cm and was also categorized according to the world health organization ( who ) criteria ( 24 ) . blood pressure was measured by european society of hypertension - international protocol 2 ( esh - ip2 ) . with the whr , the waist is measured at the narrowest part of the waist , between the lowest rib and iliac crest , and the hip circumference is taken at the widest area of the hips at the greatest protuberance of the buttocks . the who defines the ratios of > 9.0 in men and > 8.5 in women as one of the decisive benchmarks for metabolic syndrome . the 12-h fasting blood samples were taken in the morning and the sera were stored at -70 c immediately after centrifugation until being assayed . the laboratory was monitored for the precision and accuracy of glucose and lipid measurements by the surveillance program . the primary predictor variable was serum adiponectin level as determined by immunoassay of thawed fasting serum samples . each sample was assayed in duplicate , and adiponectin level was calculated as the average of the two measurements . the continuous variables were primarily analyzed using a t - test , while the binary data were primary analyzed using a chi - squared test . the continuous variables are presented in the tables with their means sd ( standard deviation ) . additionally , single ( unadjusted ) and multiple ( adjusted ) binary logistic regressions were carried out with cad group as the dependent variable , and the other significant variables as independent predictors . the cut p value of 0.1 was used as a criterion to identify the variables which need to be included in the binary logistic regression models . adiponectin as the focusing variable of this study was included in the model regardless of its statistical significance . other adjusting variables included are : diastolic blood pressure ( dbp ) , whr , lvef , gender male , and smoking . the study population consisted of 82 consecutive patients who underwent coronary angiography for suspected or known coronary atherosclerosis at the university clinical center of kosovo . with the exception of acute coronary syndromes , the patients had to present in a stable clinical condition without major concomitant non - cardiovascular disease . the study protocol was approved by the ethical committee of the faculty of medicine , university of prishtina , and research was conducted in accordance with declaration of helsinki guidelines . written informed consent cad was defined as more than 50% luminal stenosis identified in a minimum of two views , in the case of single - vessel involvement . gensini score assesses the severity of coronary artery disease : it grades narrowing of the lumen of the coronary artery and scores it as 1 for 1 - 25% narrowing , 2 for 26 - 50% narrowing , 4 for 51 - 75% , 8 for 76 - 90% , 16 for 91 - 99% and 32 for a completely occluded artery . this score is then multiplied by a factor , according to the importance of the coronary artery . the multiplication factor for a left main stem ( lms ) lesion is 5 , it is 2.5 for proximal left anterior descending artery ( lad ) and proximal circumflex artery ( cx ) lesions , 1.5 for a mid - lad lesion , and 1 for distal lad , mid / distal cx and right coronary artery lesions . gensini score was expressed as the sum of the scores for all three coronary arteries to evaluate the entire extent of coronary artery disease ( 23 ) . were taken after the patients had removed their shoes and upper garments and had donned an examining gown . waist height ratio ( whtr ) is measured by division of weight circumference by their height , always expressed in same units . body mass index ( bmi ) was calculated as weight ( kg ) divided by the square of height ( m ) . the wc was measured by tape in cm and was also categorized according to the world health organization ( who ) criteria ( 24 ) . blood pressure was measured by european society of hypertension - international protocol 2 ( esh - ip2 ) . with the whr , the waist is measured at the narrowest part of the waist , between the lowest rib and iliac crest , and the hip circumference is taken at the widest area of the hips at the greatest protuberance of the buttocks . the who defines the ratios of > 9.0 in men and > 8.5 in women as one of the decisive benchmarks for metabolic syndrome . the 12-h fasting blood samples were taken in the morning and the sera were stored at -70 c immediately after centrifugation until being assayed . the laboratory was monitored for the precision and accuracy of glucose and lipid measurements by the surveillance program . the primary predictor variable was serum adiponectin level as determined by immunoassay of thawed fasting serum samples . each sample was assayed in duplicate , and adiponectin level was calculated as the average of the two measurements . the continuous variables were primarily analyzed using a t - test , while the binary data were primary analyzed using a chi - squared test . the continuous variables are presented in the tables with their means sd ( standard deviation ) . additionally , single ( unadjusted ) and multiple ( adjusted ) binary logistic regressions were carried out with cad group as the dependent variable , and the other significant variables as independent predictors . the cut p value of 0.1 was used as a criterion to identify the variables which need to be included in the binary logistic regression models . adiponectin as the focusing variable of this study was included in the model regardless of its statistical significance . other adjusting variables included are : diastolic blood pressure ( dbp ) , whr , lvef , gender male , and smoking . of the eighty - two study patients , 41 patients comprised the cad group ( study subject ) and 41 patients were classified as the non - cad group ( control subject ) . study group ( mean age 66.76 + 9.12 ) were on the average , 2 years older than the controls ( mean age 64.80 8.30 ) . the baseline characteristics of the two groups are depicted in table 1 . according to table 1 , in comparison with the controls , the cad group patients were older and were more likely to be male and smokers . the number of male patients in the study group was 29 ( 70.7% ) and in the control group was 17 ( 41.5% ) , which is significantly higher ( p=0.008 ) , also smokers were 27 ( 65.9% ) vs 13 ( 31.7% ) ( p=0.002 ) significantly more numerous in the study group than the controls . bmi : basal metabolic index , wc , whtr , whr , systolic blood pressure ( sbp ) , dbp . fifteen patients ( 18.2% ) had normal bmi ( mean 23.221.4 ) , 37 patients ( 45% ) were overweight , 30 patients ( 43.4% ) were obese . bmi in case group ( mean 28.17 3.32 ) and control ( mean 28.764.68 ) and wc in case ( 96.978.63 ) vs control ( 95.1311.52 ) they were no significant . whtr in case ( 1.76 7.56 ) vs control ( 0.570.08 ) , and whr is significant between case and control ( 0.930.06 ) vs ( 0.880.07 ) ( p - value : 0.0001 ) as seen in table 1 . cad patients had higher mean gensini scores than non - cad patients ( 53.16 33.46 vs. 1.21 5.66 , p<0.0001 ) , but there was no statistically significant difference detected between cad and non cad for plasma adiponectin levels , presented in table 2 . total cholesterol in the populations averaged ( 3.58 1.07 mmol / l ) in cases and ( 3.59 1.19 mmol / l ) in the control g biochemical characteristics of the two groups . cad , ast : aspartate aminotransferase , crp : c reactive protein , hdl - c : high density lipoprotein cholesterol , ldl - c : low density lipoprotein cholesterol , lvef . roups . baseline adiponectin concentrations correlated significantly in terms of the lipid parameters , positively with hdl cholesterol concentrations ( r= 0.365 , p=0.001 ) and serum triglyceride concentrations were correlated negatively ( r=-0.336 , p=0.001 ) . total cholesterol concentration and ldl cholesterol concentrations sixty - two patients ( 75% ) had hypertension , 29 patients ( 35% ) had diabetes , 23 patients ( 28% ) dyslipidemia . forty - two patients ( 51% ) had family history of coronary heart disease and sudden non - accidental death or a combination of these . correlations of adiponectin with other variables of all patients 14.6% had triple - vessel disease , 18.3% had double - vessel disease and 17% single - vessel disease . adiponectin levels show a non - significantly progressive decline as the angiographic severity of coronary artery stenosis increases ( value=0.335 ) as shown in table 4 cag : coronary angiography , svd : single vessel disease , dvd : double vessel disease , tvd : triple vessel disease . in control group 37 patients ( 45% ) have ( gensini score = 0 ) , 15 patients ( 18% ) had mild disease ( gensini score < 32 ) , 14 patients ( 17% ) had moderate disease ( gensini score= 32 - 58 ) , 16 patients ( 19.5% ) had severe disease ( gensini score 58 ) . bmi did not correlate with gensini score by severity of coronary arteries , as seen in table 5 . distribution of gensini in different bmi categories significant differences were seen in binary logistic regression models for cad analysis for adiponectin , whr , lvef , dbp , male gender and for smoker . whr ( or 14 ; 95% ci 0.94 - 207.60 ; p=0.06 ) , lvef ( or 4.61 ; 95% ci 1.49 - 14.21 ; p=0.01 ) , dbp ( or 2.75 ; 95% 2.75 ( 1.12 - 6.87 ; p=0.03 ) , smoking ( or 4.15 ; 95% ci 1.65 - 10.44 ; p=0.001 ) , male patients ( or 3.41 , 95% ci 1.37 - 8.52 ; p=0.01 ) , all presented in table 6 . binary logistic regression models for cad ( unadjusted and adjusted ) , dbp , whr , lvef . the present study found that there was a strong association between adiponectin and coronary artery disease . these findings provide further evidence for an adiponectin paradox in which higher levels of adiponectin may be secreted as a protective or a compensatory response to worsening cardiovascular disease . in our study , we found medium significant positive correlation between gensini and adiponectin ( r= 0.272 , p=0.01 ) . this is in compliance with the saphir study , which suggests that the role of adiponectin differs between early atherosclerosis and advanced stage vascular disease ( 25 ) . adiponectin protects against the development of disease , once the disease is established , adiponectin concentrations are elevated as a counter - regulatory response to protect against further inflammation and atherosclerosis ( 26 ) . findings of positive correlation between adiponectin and culprit lesion necrotic core ( nc ) content is consistent with the hypothesis that in advanced cad , serum adiponectin levels are elevated in a counter - regulatory response ( 27 ) . high serum adiponectin levels , instead of being protective , have been shown to be associated with increased mortality and adverse event rates . this paradoxical relation is further supported by study that included patients undergoing pci for symptomatic cad ( 28 ) . despite the potential association between heart failure and high plasma adiponectin shown by several clinical studies , other experimental studies have demonstrated a beneficial , protective effect of adiponectin on myocardium . elevated plasma levels of adiponectin among patients with heart failure furthermore , several statins are also effective for elevating plasma adiponectin ( 29 , 30 ) . there were no significant differences in plasma concentrations of adiponectin between patients with stable angina pectoris ( sap ) and control patients ( 11.3 v 12.8 g / ml ) ( 31 ) . our study findings were similar to this study , where plasma concentrations of adiponectin between patients with sap and control group ( 3.45 v 3.49 g / ml ) had no significant differences . in the present study , patients in our sap group had already been receiving some treatment with antianginal and antiatherosclerotic drugs . as certain medical treatments may confound interpretation of plasma concentrations of adiponectin measurements and there may not be significant differences in plasma concentrations of adiponectin between patients with sap and control patients . another important clinical finding showed that plasma concentrations of adiponectin were considerably increased in older and female patients with cad ( 31 ) . as a consequence of their findings they suggested that unstable plaque may stabilize as the patient grows older or that accumulation of adiponectin in atherosclerotic vascular walls may suppress its elimination half - life from plasma , resulting in an increase in plasma concentrations of adiponectin in older patients with cad . although a significant relation between serum adiponectin levels and age was not found in our study , it is known that age has an effect on serum adiponectin levels and that there is a positive relationship between age and serum adiponectin level ( 32 ) . including both individuals with and without cardiovascular disease , higher adiponectin has protecting role in those without cardiovascular disease and predictive of worse outcomes in those with existing disease ( 33 ) . moreover , it has been suggested that normal and even higher serum adiponectin levels may prevent the development of cardiovascular diseases and complications in healthy individuals ( 34 ) . male patients with hypoadiponectinemia ( < 4.0 g / ml ) had a two - fold increase in cad prevalence , independent of well - known cad risk factors ( 35 ) . similar to our study in male gender with hypoadiponectinemia ( < 2.0 g / ml ) increase the odds of having cad ( table 6 ) . our current study did not show a significant correlation between bmi and cad ( p - value 0.115 ) , despite the presence of significant association between whr and cad ( p - value= 0,0001 ) . this may be explained by the fact that bmi quantifies general adiposity , although person who is overweight or obese is likely to have excess fat but bmi does not give an indication as to how this fat is distributed in the body . however , fat distribution is an important determinant of cad independent of bmi and other classic risk factors of cad ( 36 ) . welborn and dhaliwal ( 37 ) and srikanthan , seeman , and karlamangla ( 38 ) confirmed , and cited several other investigations that show whr being the superior clinical measurement for predicting all cause and cardiovascular disease mortality . welborn and dhaliwal added that the hip circumference indicated a lower risk for body fat accumulation , and thus including it into the waist - to - hip equation enhances the accuracy of this measurement technique . relatively small sample of patients that were selected randomly from the general population , may have limited the ability to detect significant relationship between adiponectin and coronary artery disease , thus making it difficult to indicate the results to the general population . there is a significant positive correlation between waist - hip ratio and severity of coronary artery disease . also , there is a significant positive correlation between adiponectin and gensini score among kosovar patients .

background : this study aimed to investigate correlation between adiponectin and waist - hip - ratio with severity of coronary artery disease ( cad ) . there is uncertainty about the association between circulating concentrations of adiponectin and cad.methods:we enrolled eighty - two consecutive patients undergoing non - urgent coronary angiography for cad survey . according to the angiography results , the patients were divided into two groups in 1:1 ratio patients admitted with a diagnosis of cad and non - cad . we conducted hospital based research , involving study group with documented angiographically cad , and control group without evidence of cad . angiograms were also quantified for the extent and severity of cad by the gensini scoring system . we measured baseline adiponectin levels in stored serum samples of all patients , anthropometric and biochemical risk factors were assessed in both groups.results:the presence of cad was associated with current smoking , male gender , waist hip ratio ( whr ) and left ventricular ejection fraction ( lvef ) . baseline adiponectin concentrations correlated significantly in terms of the lipid parameters , positively with hdl cholesterol concentrations ( r=0.327 , p=0.028 , p<0.05 ) and serum triglyceride concentrations were correlated negatively ( r=-0.513 , p<0.001 ) . no significant difference between median adiponectin levels at baseline was observed between cases and controls.conclusion:there is a significant positive correlation between waist hip ratio and presence and severity of coronary artery disease . in conclusion , there is a significant positive correlation between adiponectin and gensini score among kosovar patients .

neurofibromatosis ( nf ) is an autosomal dominant disease , discovered in 1882 , by the german pathologist friedrich daniel von recklinghausen , characterized by disordered growth of ectodermal tissues , and is part of a group of disorders called phakomatoses ( neurocutaneous syndrome ) . neurofibromatosis type - i ( nf-1 ) , also known as von recklinghausen syndrome , is caused by the mutation of a gene on the long arm of chromosome 17 , which encodes a protein known as neurofibromin . it is characterized by spots of increased skin pigmentation ( caf au lait spots ) , combined with peripheral nerve tumors and a variety of other dysplastic abnormalities of the skin , nervous system , bones , endocrine organs , and blood vessels . the localized form of neurofibromatosis type - i , first described by gammel in 1931 , is very rare . crowe et al . proposed the term sectorial neurofibromatosis for this localized form of neurofibromatosis , and miller and sparkes modified the nomenclature to segmental neurofibromatosis ( sn ) the current term for neurofibromas of segmental distribution . the commonly affected sites for sn are the thorax and abdomen ( 55% ) , upper extremities ( 20% ) , and lower limb and face ( 10% each ) . only few cases ( less than 10 ) of segmental neurofibromatosis over the face have been described so far . a 26-year - old female patient reported to the dental department complaining about the unesthetic appearance of her face , since eight years [ figure 1 ] . history revealed that eight years back , the patient underwent surgery in the neck region for a cyst and one to two days after surgery the patient got a swelling on the face . the swelling was of the same size since then and there was no pain associated with the swelling . the unesthetic appearance of the face on examination , the face of the patient appeared asymmetrical , bilaterally , with a growth on the right side of the face . the skin over the right side of her face was thickened and hyperpigmented , with a soft and loose hanging overgrowth . there were numerous small and large , sessile and pedunculated nodular growths localized on the right side of her neck , with dark - colored underlying skin . the skin over the right cheek and chin area showed coffee - colored pigmentation ( caf - au - lait spots ) [ figure 2 ] . nodular growths localized on the right side of the neck with dark colored underlying skin there was a soft , compressible , nontender growth on the lower lip . the right ear showed disfigurement and hypertrophy , with thickened cartilage and overlying hyperpigmented skin [ figure 3 ] . disfigurement of the ear and hypertrophy with thickened cartilage and overlying hyperpigmented skin intraorally there were numerous soft tissue growths with a sessile base seen on the right buccal mucosa [ figure 4 ] and tongue [ figure 5 ] . soft tissue growths with sessile bases on right buccal mucosa soft tissue growths with sessile bases on the tongue laboratory findings revealed a raised erythrocyte sedimentation rate ( esr ) ( 70 mm in the first hour ) , a decreased hemoglobin level ( 7.5 gm% ) , and the enzyme - linked immunosorbent assay ( elisa ) was found to be nonreactive . a panoramic radiograph disclosed asymmetric enlargement of the right side of the mandible , with loss of cortication of the lower border in the midline area , expansion of the body of the mandible causing displacement of the teeth , seen on right side , expansion of the right inferior alveolar nerve canal , enlarged mental foramen , resorption of the roots of the mandibular right teeth , and loss of the right antegonial notch . a deficiency was seen on the right side of the maxilla , with crowding of the posterior teeth [ figure 6 ] . a panoramic radiograph the right lateral oblique radiograph revealed a deep sigmoid notch , loss of the antegonial notch , an enlarged and rounded coronoid process , with resorption of the mandibular right teeth [ figure 7 ] . a right lateral oblique radiograph subsequently , biopsy of one of the nodules was taken and sent for histopathological examination . the histopathological slide revealed a non - encapsulated tumor of the dermis with a normal overlying epidermis . the tumor consisted of loosely spaced spindle cells and wavy collagenous strands in a clear matrix [ figure 8 ] . a non - encapsulated tumor of the dermis , with a normal overlying epidermis these tumors are composed of schwann cells , fibroblasts , mast cells , and vascular components . the caf au lait spots are irregularly - shaped , evenly pigmented , brown macules . most individuals with neurofibromatosis have six or more spots that are 1.5 cm or greater in diameter . in young children , five or more caf au lait macules , greater than 0.5 cm in diameter , are suggestive of neurofibromatosis . less than 1% of the healthy children have three or more such spots , although one or two caf au lait macules are commonly encountered in healthy individuals without disease . bone involvement includes pseudoarthrosis of the tibia , bowing of the long bones , and orbital defects . treatment of neurofibromatosis is predominantly surgical . when neurofibromas increase in size or cause pain , malignant transformation must be suspected , and excision or biopsy must be performed . acoustic neuromas and tumors that cause tinnitus and vertigo must be excised with great caution . plastic surgeons may be included in the correction of deformities , especially those of the face . considering the autosomal dominant inheritance pattern of neurofibromatosis , genetic counseling

segmental neurofibromatosis is a rare disorder , characterized by neurofibromas or caf-au - lait macules limited to one region of the body . its occurrence on the face is extremely rare and only few cases of segmental neurofibromatosis over the face have been described so far . we present a case of segmental neurofibromatosis involving the buccal mucosa , tongue , cheek , ear , and neck on the right side of the face .

the zygomaticomaxillary complex is an important functional and aesthetic landmark of the midface , and it provides prominent cheek structure . however , unfortunately it is very vulnerable to injury because of its intrinsically prominent convexity . there are four bony attachments between zygoma and other facial bones : a superior attachment to the frontal bone ( frontozygomatic suture line ) , a medial attachment to the maxilla ( zygomaticomaxillary suture line ) , a lateral attachment to the temporal bone ( zygomaticotemporal suture line ) , and a deep attachment to the greater wing of the sphenoid bone ( zygomaticosphenoidal suture line ) . when blunt trauma to the zygomatic complex results in fractures of all four suture lines , it is referred to as a tetrapod fracture . closed reduction without surgical incisions has virtually been abandoned because judgment on the adequate reduction of fracture could not been made and it often resulted in disappointing cosmetic results . open reduction with surgical incisions has been accomplished through keen 's approach , gillies ' approach , bicoronal scalp flap approach or the more popular dingman 's approach ( 1 ) . this procedure has advantages in that it leaves no facial scars and is simple to perform . however , the fracture site is not directly visualized and access to the orbit is not possible , which may result in incomplete surgical reduction . however , it necessitates a lengthy scalp incision and provides inadequate exposure to the inferior , medial aspect of orbit , and may leave a conspicuous long scalp scar . also , because of its extensive dissection , the temporal branch of the facial nerve may be damaged . the dingman 's approach has become the most popular method in repairing zygomatic complex fractures . this procedure utilizes a lateral eyebrow incision line and a second incision across the entire lower eyelid in the subciliary line . this technique provides good exposure and direct visualization of the frontozygomatic area , inferior orbital rim and orbital floor . but , it provides limited exposure of the lateral orbital rim because it consists of two separate facial incisions , and it leaves external cutaneous scars . also , there is some risk of lower eyelid ectropion due to cicatrical contracture . since its first description for the removal of herniated orbital fat , there have been subsequent reports demonstrating the efficacy of transconjunctival approach not only for the lower eyelid blepharoplasty but also for the repair of orbital fractures ( 2 - 5 ) . mccord and moses ( 6 ) described the addition of the lateral canthotomy incision to gain access to the lateral orbital wall and inferior orbital rim , and nunery ( 1 ) reported a lateral superior cantholysis for the repair of trimalar fractures . since then , a few authors ( 7 - 9 ) have described the advantages of this type of incision , but there has been no report that describe the long term results of two - point fixation through the single transconjunctival incision for the treatment of zygoma fracture . between march 2002 and january 2006 , a total of 53 patients with zygomatic complex fractures , which were not severely comminuted , were treated with this surgical method . included were 48 men and five women , aged 15 to 88 yr ( mean age 33.2 yr ) . twenty eight patients ( 52.8% ) had associated blow out fracture , two patients ( 3.7% ) had associated nasal bone and mandible fracture , and one patient ( 1.9% ) had associated nasal bone fracture ( table 1 ) . upon knight and north classification , four patients ( 7.5% ) belonged to group i , twenty one patients ( 39.6% ) belonged to group ii , eight patients ( 15% ) belonged to group iv , eleven patients ( 20.7% ) belonged to group v , and nine patients ( 16.9% ) belonged to group vi . all patients were treated with two - point fixation through a single transconjunctival approach with a lateral canthal extension . if the reduction of zygoma was not complete , an additional small gingivobuccal incision ( less than 2 cm ) was made and subperiosteal dissection was performed to expose zygomaticomaxillary buttress and posterior surface of zygoma . through the small gingivobuccal incision , the follow - up period ranged from 8 to 46 months ( average 24.8 months ) . preoperative and postoperative clinical examinations were performed with special attention paid to features such as symmetry of zygoma and canthal ligaments , enophthalmos , diplopia , limited ocular function , scarring and ectropion . follow - up radiographs ( waters ' view and zygomatic arch view ) were routinely used to evaluate the adequacy of reduction . under general endotracheal anesthesia , a lateral canthal incision , about 1.0 - 2.0 cm , then , the conjunctiva is incised 2 - 3 mm below the tarsus extending from the commissure to a point just lateral to the punctum , and the transconjunctival incision is connected to the lateral canthal incision ( fig . the lower eyelid flap is pulled down by cutting the superficial lateral canthal tendon and inferior limb of deep lateral canthal tendon with fine scissors . dissection proceeds anteriorly and inferiorly between the orbicularis oculi muscle and orbital septum until the periosteum of the inferior orbital rim is exposed . through the extended lateral canthal incision , the lateral aspect of the upper lid is retracted superiorly , and the periosteum of lateral orbital rim is exposed . the periosteum is incised and a subperiosteal dissection is done to expose the orbital floor , the inferior orbital rim , and the lateral orbital rim including the zygomaticofrontal suture area ( fig . , the dingman 's elevator is placed posteriorly to the body of the zygoma along the zygomatic arch and a reduction of zygoma is done . if the reduction of zygoma is not complete or unsatisfactory , an additional small gingivobuccal incision ( less than 2 cm ) is made and a limited subperiosteal dissection is performed to expose the zygomaticomaxillary buttress and posterior surface of zygoma . dingman 's elevator is inserted through the small gingivobuccal incision and an accurate reduction can be accomplished . in all cases , complete reduction was confirmed by the accurate alignment of the lateral and inferior orbital wall . then , the two - points ( frontozygomatic suture and inferior orbital rim ) are fixated with plates and screws ( fig . resorbable 2.0-mm miniplates systems , macrosorb ( macropore , san diego , ca , u.s.a . ) were used in 35 patients ( 87.5% ) and titanium miniplates ( osteomed , dallas , tx , u.s.a . ) were used in 5 patients ( 12.5% ) . if there is an associated impure blow - out fracture , orbital floor reconstruction with porous polyethylene implant ( medpor ) is performed . the periosteum is closed with a 4 - 0 absorbable suture , and the conjunctiva is closed by a 7 - 0 absorbable suture . in case of old patient with lid laxity , the canthopexy is accomplished by a 5 - 0 absorbable suture fixation in the direction of posterior - superiorly to the periosteum of lateral orbital rim to prevent the postoperative ectropion . in other cases , finally , the skin and subcutaneous layer at the lateral canthal incision site is closed with a 6 - 0 suture . between march 2002 and january 2006 , a total of 53 patients with zygomatic complex fractures , which were not severely comminuted , were treated with this surgical method . included were 48 men and five women , aged 15 to 88 yr ( mean age 33.2 yr ) . twenty eight patients ( 52.8% ) had associated blow out fracture , two patients ( 3.7% ) had associated nasal bone and mandible fracture , and one patient ( 1.9% ) had associated nasal bone fracture ( table 1 ) . upon knight and north classification , four patients ( 7.5% ) belonged to group i , twenty one patients ( 39.6% ) belonged to group ii , eight patients ( 15% ) belonged to group iv , eleven patients ( 20.7% ) belonged to group v , and nine patients ( 16.9% ) belonged to group vi . all patients were treated with two - point fixation through a single transconjunctival approach with a lateral canthal extension . if the reduction of zygoma was not complete , an additional small gingivobuccal incision ( less than 2 cm ) was made and subperiosteal dissection was performed to expose zygomaticomaxillary buttress and posterior surface of zygoma . through the small gingivobuccal incision , the follow - up period ranged from 8 to 46 months ( average 24.8 months ) . preoperative and postoperative clinical examinations were performed with special attention paid to features such as symmetry of zygoma and canthal ligaments , enophthalmos , diplopia , limited ocular function , scarring and ectropion . follow - up radiographs ( waters ' view and zygomatic arch view ) were routinely used to evaluate the adequacy of reduction . under general endotracheal anesthesia , a lateral canthal incision , about 1.0 - 2.0 cm , is made down to the periosteum of lateral orbital rim . then , the conjunctiva is incised 2 - 3 mm below the tarsus extending from the commissure to a point just lateral to the punctum , and the transconjunctival incision is connected to the lateral canthal incision ( fig . the lower eyelid flap is pulled down by cutting the superficial lateral canthal tendon and inferior limb of deep lateral canthal tendon with fine scissors . dissection proceeds anteriorly and inferiorly between the orbicularis oculi muscle and orbital septum until the periosteum of the inferior orbital rim is exposed . through the extended lateral canthal incision , the lateral aspect of the upper lid is retracted superiorly , and the periosteum of lateral orbital rim is exposed . the periosteum is incised and a subperiosteal dissection is done to expose the orbital floor , the inferior orbital rim , and the lateral orbital rim including the zygomaticofrontal suture area ( fig . , the dingman 's elevator is placed posteriorly to the body of the zygoma along the zygomatic arch and a reduction of zygoma is done . if the reduction of zygoma is not complete or unsatisfactory , an additional small gingivobuccal incision ( less than 2 cm ) is made and a limited subperiosteal dissection is performed to expose the zygomaticomaxillary buttress and posterior surface of zygoma . dingman 's elevator is inserted through the small gingivobuccal incision and an accurate reduction can be accomplished . in all cases , complete reduction was confirmed by the accurate alignment of the lateral and inferior orbital wall . then , the two - points ( frontozygomatic suture and inferior orbital rim ) are fixated with plates and screws ( fig . resorbable 2.0-mm miniplates systems , macrosorb ( macropore , san diego , ca , u.s.a . ) were used in 35 patients ( 87.5% ) and titanium miniplates ( osteomed , dallas , tx , u.s.a . ) were used in 5 patients ( 12.5% ) . if there is an associated impure blow - out fracture , orbital floor reconstruction with porous polyethylene implant ( medpor ) is performed . the periosteum is closed with a 4 - 0 absorbable suture , and the conjunctiva is closed by a 7 - 0 absorbable suture . in case of old patient with lid laxity , the canthopexy is accomplished by a 5 - 0 absorbable suture fixation in the direction of posterior - superiorly to the periosteum of lateral orbital rim to prevent the postoperative ectropion . in other cases , finally , the skin and subcutaneous layer at the lateral canthal incision site is closed with a 6 - 0 suture . during the average 24.8 months of follow up , most patients had functionally and esthetically satisfactory results . all patients showed satisfactory facial symmetry , no lateral canthal displacement , and no functional impairment . none of them had persistent diplopia , enophthalmos or alteration of visual acuity . upon postoperative radiography ( skull waters view and zygomatic arch view ) , one patient ( 1.9% ) had persistent scleral show and was corrected three months after the repair of zygomatic complex fracture . this case was 78 yr - old patient who had lid laxity preoperatively and showed ectropion postoperatively , which may be due to scar contracture . and two patients ( 3.7% ) had mild hyperpigmentation at the lateral canthal incision site even after six months postoperatively . but most of patients showed hardly visible scar in the lateral canthal area ( fig . although subciliary incision with lateral eyebrow incision ( dingman 's approach ) is the most common approach to the zygomatic complex ( 7 , 10 ) , it can leave a visible scar and ectropion of the lower eyelid . factors predisposing to an eyelid retraction and ectropion include hematoma , eyelid edema , adhesions of the orbital septum , scar contracture , horizontal laxity of the eyelid margin , weakening of the pretarsal muscle , and wide dissection of the anterior periosteum ( 11 ) . to decrease the complication rates of the subciliary approach , many authors have tried to treat zygoma complex fracture repair using the transconjunctival approach ( 1 , 6 - 9 ) . they emphasized the advantage of this approach over the dingman 's approach , in terms of minimal ectropion , hidden scars , and minimal lid lymphedema . in the transconjunctival approach , the dissection can be performed in one of two separate planes ; preseptal approach and retroseptal approach . some authors ( 12 , 13 ) advocated that the retroseptal approach is preferred because it can be performed easily and the integrity between orbital septum and orbicularis oculi muscle is not disturbed . however , the retroseptal approach has increased risk of injury to the inferior oblique muscle and prolapse of orbital fat into the surgical field ( 10 , 11 ) . hence , all our patients were treated with the transconjunctival preseptal approach . in the management of the lateral canthal extension site , manson et al . ( 9 ) advocated that the canthal reattachment is not required in an acute fracture treatment but is necessary in fractures when treatment is delayed , in patients with established canthal deformities , or in patients in whom a simultaneous coronal incision is employed . according to them , the anterior limb of the lateral canthal tendon which is also referred to as the superficial lateral canthal tendon by other authors ( 14 ) , is continuous to the temporal fascia and galea aponeurotica and the diffuse connections of this limb facilitate proper positioning of the canthus after mobilization of the posterior limb ( deep lateral canthal tendon ) . the current authors think that in addition to some indications mentioned above , patients of old age with lid laxity also needs lateral canthal reattachments or canthopexy . its significance began to be considered following our case of a 78 yr - old patient who developed persistent ectropion when canthal reattachment had not been performed . the precise physical stability of the zygoma with respect to the number and location of miniplates applied is not known , because it depends on fracture anatomy , extent , and the amount of displacement . some laboratory studies , using human cadaver heads , have been conducted to investigate the stability of fixated zygoma . in 1989 , rinehart et al . ( 15 ) simulated noncomminuted zygoma fractures in eight human cadaver heads and the stability of fracture fixation was studied for various fixation patterns . in their study , double - miniplate fixation across the zygomaticofrontal and zygomaticomaxillary fracture lines is sufficient to withstand static and oscillating loading similar to physiologic masticatory forces . in 1990 , davidson et al . ( 16 ) analyzed different methods of internal fixation of simple displaced fractures of the zygoma in an attempt to define the simplest method of achieving postreduction stability . in their report , the three - point fixation ( frontozygomatic suture , inferior orbital rim , and zygomaticomaxillary buttress ) using either miniplates alone or interfragmentary wiring conferred the greatest stability . in addition , they proposed that the two - point fixation using miniplate alone conferred a degree of stability comparable to most methods of three - point fixation regardless of the site in which the miniplates were applied . we concur with the above assertions and think that two - point miniplate fixation at the infraorbital rim and zygomaticofrontal suture would suffice , provided the comminution of the zygoma is not severe . although not the focus of our attention in this study , in case of severely comminuted fracture , three - point fixation through the transconjunctival incision and gingivobuccal incision is preferably performed . to fixate the fracture site and provide stability , we used four hole resorbable 2.0-mm miniplates systems , macrosorb ( macropore , san diego , ca , u.s.a . ) in most patients , and titanium miniplates ( osteomed , dalllas , tx , u.s.a . ) only when there was a segmental fracture at the infraorbital rim or when requested by the patient due to the lighter financial burden . in addition to the inconspicuous scarring , the main advantage of this method is a direct and full visualization of the lateral and inferior orbital wall to be served as a guidance for an accurate reduction , which makes the reduction more successful . the only drawback we experienced in our method is a little difficulty in reducing the fracture - displaced zygoma because of limited space and a slightly inadequate vector for the elevator . usually a fractured zygoma is displaced and rotated posteriorly and medially , so an anterior - lateral pull is required . however , the lateral canthal incision is located medially to the zygoma body and lateral pulling of the zygoma body may be a little difficult . in conclusion , we believe that the proposed technique is very useful for the treatment of zygomatic complex fracture which is not severely comminuted , because it provides excellent exposure and postoperative stability of the zygoma with a lower incidence of complications , including visible scarring and ectropion .

the ultimate goal in treating zygomatic complex fracture is to obtain an accurate , stable reduction while minimizing external scars and functional deformity . the present authors present our experiences with a single transconjunctival incision and two - point ( inferior orbital rim and frontozygomatic suture ) fixation in 53 patients with zygomatic complex fracture which were not comminuted . all patients had transconjunctival approaches with lateral canthal extensions , and six out of 53 patients also had an additional small ( about less than 2 cm ) gingivobuccal incision to achieve an accurate reduction . there were 3 minor complications , and the overall esthetics and functional results were satisfactory with a long term follow - up . our method has the following advantages in the reduction of zygomatic complex fracture ; it leaves only an inconspicuous lateral canthal scar . in addition , it provides excellent simultaneous visualization of the inferior orbital rim and frontozygomatic suture area . hence , two - point fixation through a single incision can be performed with a satisfactory stability .

the gram - negative coccobacillus pasteurella multocida is normally found in the oral cavity of dogs and cats . it is a recognised cause of wound infection as a result of bites , licking or scratching . bacteraemia and endocarditis are uncommon complications and , from literature reviews , are found to be associated with prior animal trauma , a second source of infection ( e.g. cellulitis ) and high mortality rates . we herein report a case in which a pet owner developed p. multocida endocarditis and was subsequently treated via opat ( outpatient parenteral antimicrobial therapy ) service with an excellent clinical outcome . species identification of p. multocida was performed using traditional microbiological methods and confirmed with 16s rrna sequencing and matrix - assisted laser desorption / ionization time - of - flight mass spectrometry ( maldi - tof ) . a 38-year - old man was admitted to our hospital with a 2 days history of fever , malaise and cellulitis around a mitrofanoff abdominal catheter . multiple comorbidities included marfan syndrome ( with metallic aortic valve inserted sixteen years before ) and congenital urological abnormalities that led to the mitrofanoff appendicovesicostomy and a recent renal transplant , five years and six months before admission respectively . he was on steroids and tacrolimus as immunosuppressive therapy . on presentation , the patient was febrile , hypotensive and tachycardic , had a normal leukocyte count ( 5.0910/l ) but raised creactive protein ( crp ) 45 mg / l with mild anaemia ( hb of 10.1 g / dl from a baseline of 14.6 g / dl ) and renal impairment ( creatinine 163 umol / l and urea 8.1 mmol / l ) . the cellulitis around the mitrofanoff abdominal catheter was noted and a swab was taken for culture . his chest x - ray did not show any relevant consolidation and abdominal ultrasound noted two small abdominal collections that had already been noted post - transplant and had since decreased in size . the patient was documented to be penicillin allergic ( previous skin rash ) , so was initiated on ertapenem iv 1 gr / od . the cellulitis improved and the patient was clinically well within 48 hours of commencing antibiotics . the blood culture set ( aerobic and anaerobic bottles ) taken on admission was positive with p. multocida but the abdominal swab grew only mixed anaerobes . urine culture was negative and all subsequent blood cultures taken after starting ertapenem were negative after 5 days of incubation . transesophageal ( tee ) echocardiography revealed the presence of a vegetation ( 0.57 0.2 cm ) above the atrium of the right coronary artery . a previous transthoracic echocardiogram ( tte ) , around one month prior to admission , was reported as negative . therefore , the patient was treated following our local protocol for infective endocarditis : he was switched to ceftriaxone iv 2 gr / od , a picc line was inserted in the right arm and he was discharged under the opat ( outpatient parenteral antimicrobial therapy ) service to complete six weeks iv antibiotics as an outpatient . on follow - up , two repeat ttes showed clear aortic valve . we reviewed all the previous reported cases of p. multocida endocarditis in the literature through pubmed and cross - references search . to our knowledge this is the twenty - first reported case of endocarditis but the first to be reported in a penicillin allergic patient . as with previous cases , the diagnosis of infective endocarditis was made according to duke criteria : one major criterion ( evidence of endocardial involvement ) and three minor criteria ( predisposing heart condition , fever and microbiological evidence with positive blood culture ) . laboratory isolation and identification of p. multocida was performed using standard microbiological techniques : api-20ne , api-20e ( biomerieux ) and the phoenix automated system ( bd ) . interestingly , both api profiles reported a low confidence value for identification as p. multocida to the species level ( cv 53% ) and the phoenix assay only identified the isolate to genus level as pasteurella spp . thus , confirmation of species identification was performed using 16s rrna sequencing and maldi - tof protein profiling : 16s rrna sequence was performed using pcr parameters detailed in maskell na et al . ( 2006 ) and we obtained a 584bp fragment sharing 99% sequence homology to that of p. multocida sub species septica . maldi - tof profiling was performed using a bruker daltonics microflex platform with recommended -cyano-4-hydroxycinnamic acid matrix under pre - defined conditions as specified by bruker . the maldi - tof mass spectra for the clinical isolate was analysed against maldi - tof mass spectra for 3,438 species of bacteria located in the bruker daltonics flexanalysis 3.0 database . the clinical isolate was identified as p. multocida with a confidence value of 2.321 indicating highly probable species identification , with no differential bacterial species being reported . sensitivities were as follow ( eucast breakpoints ) : penicillin mic ( mg / l ) was 0.19 , ciprofloxacin 0.016 , doxycycline 0.5 , co - trimoxazole 0.032 , ertapenem 0.016 and ceftriaxone < 0.016 , all being susceptible . of note the accuracy of previous reports in the literature has been questioned as their standard microbiological description was incomplete and pasteurella species may be misidentified as haemophilus spp . they both belong to the pasteurellaceae family and based on the genomic sequence it appears they diverged approximately 270 million years ago . are gram - negative coccobacilli generally present in the oral cavity of cats and dogs . to date twenty - one species have been identified and the species p. multocida can be further divided into three different subspecies ( subspecies multocida , septicum and gallicida ) . the spectrum of infections varies from skin and soft tissue to central nervous system infections . ten out of twenty - one ( 47.6% ) endocarditis cases described in the literature had close contact with animals detailed in the clinical history . our patient did not have a clear history of bites or scratches but he did have two cats at home and close contact with dogs at his sister 's house . in fact , of the cases of p. multocida endocarditis reviewed , two out of twenty - one ( 9.5% ) had cirrhosis as underlying condition . penicillin still remains the best antimicrobial agent for the treatment of virtually all forms of pasteurella infection . a fluoroquinolone , doxycycline , or co - trimoxazole should be considered for skin and soft tissue infections as an alternative for patients with intolerance to -lactams . there are no clear guidelines about the treatment of p. multocida endocarditis in penicillin - allergic patients . the patient was known to be penicillin allergic ( previous rash ) and ciprofloxacin and doxycycline were contraindicated as causing interaction with his anticoagulant treatment . we decided then to switch to ceftriaxone as this antibiotic is already vastly used in the treatment of endocarditis caused by other gram - negative organisms and is more cost effective . aminoglycosides have moderate to poor in vitro activity against pasteurella and probably should not be used , particularly given the paucity of clinical experience . finally , our patient improved within 48 hours of commencing antimicrobial therapy and surgical intervention was unnecessary . from the literature , only three other cases have been successfully treated with solely antibiotics and valve replacement was mandatory in seventeen out of twenty - one ( 81% ) of cases described . clinicians may face various challenges , from achieving the correct species identification to chosing treatment regimens in penicillin - allergic patients . in our case new diagnostic techniques such as 16s rrna sequencing and maldi - tof were demonstrated to be invaluable tools for species confirmation . we propose that maldi - tof is a rapid , accurate method for the identification of p. multocida from bacterial cultures and can significantly reduce the time to diagnosis of p. multocida bacteraemia . furthermore we have shown that cephalosporins and carbapenems are probably good substitutes for penicillin in case of allergy .

pasteurella multocida is a rare cause of infective endocarditis with only a few cases described . this report involves a 38-year - old penicillin - allergic patient in an immunocompromised state with several co - morbidities . two molecular microbiological techniques , 16s rrna sequencing and matrix - assisted laser desorption / ionization time - of - flight mass spectrometry were used to confirm the species identification as p. multocida . previous reports in the literature are also reviewed .

patient aged 64 was admitted to the county hospital urology ward in september 2010 and diagnosed with tumor of the left kidney . ultrasonographic exam revealed the structure with a diameter of 45 mm in the central field of the left kidney . ct showed a solid focal lesion of the left kidney ( 30 x 40 x 32 mm ) located below the renal stalk and covering the renal pelvis and the paranephric section of the left ureter , apart from that no changes were observed ( figure 1 ) . radiological image of the chest did not reveal any pathological changes . in order to make the results of pre operational diagnosis sided ureterorenoscopy was conducted and a urine sample was taken for cytology to exclude epithelial nature of the tumor . after undergoing the above mentioned test , the patient was qualified for transperitoneal radical nephrectomy . with general anesthesia applied , the left kidney was removed through an incision below the left costal margin . during the operation , a significant peripheral infiltration was discovered which hindered access to the renal stalk of the left kidney . operative course passed without complications and the patient was discharged in good general condition 8 days after the surgery . left kidney tumor in ct exam . on pathological examination ( samples no 12399241239928 ) in all samples after detailed hematological work up which did not show any other tumors , the patient was qualified for radiation treatment and currently remains under permanent oncological supervision . medical records and literature mention several cases of malignant kidney lymphoma [ 1 , 2 ] . some authors claim that this entity accounts for 3% of all solid renal tumors among adults . lymphomatous renal involvement can have three different causes : most commonly it appears along with the generalized disease and enlarged lymph nodes . renal involvement develops without any symptoms then.it may be related to organ transplantation and infection with epstein such cases are rare and difficult to diagnose which may lead to mistakes in treatment . such cases are rare and difficult to diagnose which may lead to mistakes in treatment . the case presented in the report illustrates diagnostic and therapeutic difficulties that a urologist might encounter during treatment of patients with possible renal tumor . results provided by clinical imaging of focal lesions are inconsistent . what must always be taken into account is an atypical tumorous process , autoimmunological disorders as well as inflammatory condition . in this particular case control appointments in the department of hematology have been scheduled to take place every four months and the prognosis for the patient has been described as good . they develop from lymphocytes and , depending on the variety of b and t lymphocytes , those processes can have different clinical outcome . some of them are located solely within the lymph nodes or tissues and internal organs , while others are accompanied by leukemia . it might seem that when it comes to lymphomas , the pathogens initiating the course of the disease could be both , the epstein barr viruses which attack the b lymphocytes , and by htlv1 viruses dwelling in t lymphocytes and causing a subacute form of leukemia among adults . there can also be some co factors of environmental character : chloro organic compounds , ionization energy , benzene like substances , states of immunosuppression and autoimmunological diseases . the malignant lymphoma morbidity rate can also be increased by extensive exposition to herbicides , and is even 35 times higher among patients with a kidney transplant . it usually dwells within the lymphoid tissue of waldeyer 's ring , spleen and thymus . extranodal lymphomas can most often be found in the gastrointestinal tract , other types can be located in skin , bones , eye kidney might be at the stage of involvement which spreads through lymphatic system from the nodes in retro peritoneum . if the renal involvement occurs at an advanced stage of the disease , as yet another location of it , it is relatively easy to diagnose . once a patient is diagnosed with lymphoma of kidney , further examinations are required in order to rule out a systemic disease . the necessary test in such cases include : blood tests , bone marrow biopsy , radiological examination , pathomorphological evaluation and histo immunological evaluation of enlarged lymph nodes . the malignant types , however , build up rapidly , are resistant to treatment and show no signs of improvement within a short period of time . how long the patient will live depends on the histopathological diagnosis of the tumor and its stage of development . non hodgkin lymphomas are treated with chemotherapy , radiation , and , in some cases , surgeries . low malignant , originally extranodal , tumors are usually surgically removed and treated with radiotherapy . cell lymphoma associated with chronic inflammation arising in a renal pseudocyst with barely noticeable clinical symptoms . publications in english present several cases of spontaneous rupture of the ureter as primary symptom of malignant lymphoma . risk of their occurrence increases from 1.2% in 5 years after the operation , to 6.8% in 20 years after the transplantation , which is related to epstein barr virus infections . some authors also report on cases of co existence of inflammatory intestines disease , including the crohn disease , and malignant lymphomas but then changes can most frequently be located in the vicinity of intestines or other adjacent sections of the digestive tract . the presented case leads to a conclusion that each focal lesion of unclear etiology found on imagining studies should be treated as a possible malignant lymphoma and ought to be histopathologically verified as quickly as possible .

we present a 64year old patient suffering from extranodal malignant lymphoma . the patient was admitted to the county hospital urology ward with suspicion of the left kidney tumor . as part of pre operational diagnosis , ultrasonographic examination and computed tomography of kidneys were conducted . the results confirmed the initial diagnosis . after undergoing radical surgical treatment , the patient is currently under the care of hematology ward of oncology centre .

assessment of myocardial viability in patients with coronary artery disease is of great clinical importance , as dysfunctional but viable myocardium has the ability to regain contractility after coronary revascularization with subsequent improvements in cardiac function and prognosis . the perfusable tissue index ( pti ) can be used as a marker of myocardial viability , having been validated in patients with ischaemic heart disease [ 27 ] . however , pti has never been used in clinical practice , primarily due to the complex imaging protocol consisting of dynamic [ o]h2o and [ o]co scans , together with a transmission scan , and the lack of high - quality , clinically usable parametric images . recently , however , a method was developed to derive the pti from a single [ o]h2o pet / ct scan . given the current potential of clinical [ o]h2o - based perfusion imaging and the rapid growth in the availability of cardiac pet / ct systems , pti viability measurements could become incorporated into clinical practice [ 9 , 10 ] . parametric pti images derived from [ o]h2o pet / ct scans , however , have not yet been validated . the purpose of this study was to compare these novel parametric pti images with late gadolinium - enhanced cardiovascular magnetic resonance ( lge - cmr ) imaging , an established method for quantifying scar size and a marker of viability . the study population comprised 46 patients with documented or suspected coronary artery disease who had been studied using both pet / ct and cmr within a 2-month period . all patients were in a stable clinical condition and no ischaemic events or revascularizations had occurred during the period between the two examinations . patients with contraindications for pet / ct or cmr ( e.g. pacemaker , claustrophobia , atrial fibrillation ) were excluded . the study was approved by the institutional medical ethics review committee , and all participants gave written informed consent prior to inclusion . [ o]h2o scans were acquired using a gemini tf-64 ( philips healthcare , best , the netherlands ) pet / ct scanner . [ o]h2o ( 370 mbq ) was injected as a 5-ml bolus ( 0.8 ml s ) followed by a 35-ml saline flush ( 2 ml s ) , simultaneously starting a 6-min list mode emission scan . this scan was followed immediately by a respiration - averaged slow low - dose ( ld ) ct scan ( 55 mas , rotation time 1.5 s , pitch 0.825 , collimation 16 0.625 , acquiring 20 cm in 37 s ) during normal breathing . all scans were checked for misalignment between the ld ct scan and the [ o]h2o scan ; in none of the patients were corrections needed . dynamic [ o]h2o images were reconstructed into 22 frames ( 1 10 , 8 5 , 4 10 , 2 15 , 3 20 , 2 30 , 2 60 s ) using the three - dimensional row action maximum likelihood algorithm and applying all appropriate corrections for the scanner , normalization , dead time , decay , scatter , randoms and attenuation based on the corresponding ld ct scan . using capp software ( siemens / cti , knoxville , tn ) , regions of interest ( rois ) of 1 cm diameter were placed over the ascending aorta in at least ten transaxial image planes of the frame showing the first pass of the injected bolus . these rois were combined into one volume of interest ( voi ) for the ascending aorta . a second set of rois were placed over the right ventricle ( rv ) cavity in at least five transaxial planes , with roi boundaries at least 1 cm from the rv wall to avoid spill - in from myocardial activity . both vois were then projected onto all dynamic [ o]h2o images , thereby generating arterial ( ca(t ) ) and rv ( crv(t ) ) time activity curves . parametric pti images were calculated as previously described . in brief , parametric images of myocardial blood flow ( mbf ) , perfusable tissue fraction ( ptf ) , and arterial and venous blood volume fractions were calculated using a basis function implementation of the standard single - tissue compartment model for [ o]h2o [ 1114 ] . parametric images of arterial and venous blood volume fractions were subtracted from normalized ct transmission images , resulting in parametric anatomic tissue fraction ( atf ) images . finally , parametric pti images were calculated as the ratio of ptf and atf images ( fig . 1 ) . finally , 16 myocardial vois were defined manually on parametric ptf images , according to the 16 segments model of the american heart association , after which this voi template was projected onto the parametric pti images.fig . ( b ) atf ( g ml ) , ( c ) ptf ( g ml ) , and ( d ) pti in a patient without coronary artery disease example of short axis images of ( a ) blood volume , ( b ) atf ( g ml ) , ( c ) ptf ( g ml ) , and ( d ) pti in a patient without coronary artery disease cmr studies were performed on a 1.5-t whole - body scanner ( magnetom sonata or avanto ; siemens , erlangen , germany ) , using a six - channel phased - array body coil . after survey scans , a retrotriggered , balanced steady - state free precession gradient - echo sequence was used for cine imaging . image parameters included : slice thickness 5 mm , slice gap 5 mm , temporal resolution < 50 ms , repetition time 3.2 ms , echo time 1.54 ms , flip angle 60 , typical image resolution 1.3 1.6 5.0 mm . the cardiac cycle consisted of 20 phases . after obtaining four- , three- and two - chamber view cines , stacks of 10 to 12 short - axis slices were acquired to fully cover the left ventricle ( lv ) . contrast images were acquired 10 to 15 min after administration of 0.2 mmol kg gadolinium - dtpa in the same views as in the cine images , using a two - dimensional segmented inversion - recovery prepared gradient echo sequence ( te 4.4 ms , tr 9.8 ms , inversion time 250 to 300 ms , typical voxel size 1.3 1.6 5.0 mm ) . images were analysed off - line using the software package mass ( mr analytical software system ; medis , leiden , the netherlands ) . the endocardial and epicardial borders of the lv were outlined manually in both end - diastolic and end - systolic phases of all short - axis images . end - diastolic volume , end - systolic volume and ejection fraction were computed using these analyses . the amount of fibrosis was calculated using the full - width at half - maximum method , which defines fibrosis as myocardial tissue with a signal intensity 50 % of the maximum hyperenhancement intensity . if no enhancement was found in a slice , the maximum signal of the nearest slice with enhancement was used . all areas of enhancement were quantified by computer - assisted planimetry on each of the short - axis images and the segmental extent of enhancement was expressed as a percentage of the segmental area . cmr images were analysed according to the same 16-segment model as used for the parametric pet images . finally , myocardial segments were graded as viable or nonviable using the previously defined cut - off value of 50 % lge per segment [ 15 , 17 ] . continuous variables are presented as means sd , and categorical data are summarized as frequencies and percentages . multiple datasets were compared using analysis of variance ( anova ) , and specific differences were identified using student s t - test with bonferroni inequality adjustment . receiver operating characteristic curves were generated for ptf , pti and mbf for the prediction of myocardial viability assessed by lge cmr . the area under the curve ( auc ) was considered a measure of accuracy to discriminate between viable and nonviable myocardium . the study population comprised 46 patients with documented or suspected coronary artery disease who had been studied using both pet / ct and cmr within a 2-month period . all patients were in a stable clinical condition and no ischaemic events or revascularizations had occurred during the period between the two examinations . patients with contraindications for pet / ct or cmr ( e.g. pacemaker , claustrophobia , atrial fibrillation ) were excluded . the study was approved by the institutional medical ethics review committee , and all participants gave written informed consent prior to inclusion . [ o]h2o scans were acquired using a gemini tf-64 ( philips healthcare , best , the netherlands ) pet / ct scanner . [ o]h2o ( 370 mbq ) was injected as a 5-ml bolus ( 0.8 ml s ) followed by a 35-ml saline flush ( 2 ml s ) , simultaneously starting a 6-min list mode emission scan . this scan was followed immediately by a respiration - averaged slow low - dose ( ld ) ct scan ( 55 mas , rotation time 1.5 s , pitch 0.825 , collimation 16 0.625 , acquiring 20 cm in 37 s ) during normal breathing . all scans were checked for misalignment between the ld ct scan and the [ o]h2o scan ; in none of the patients were corrections needed . dynamic [ o]h2o images were reconstructed into 22 frames ( 1 10 , 8 5 , 4 10 , 2 15 , 3 20 , 2 30 , 2 60 s ) using the three - dimensional row action maximum likelihood algorithm and applying all appropriate corrections for the scanner , normalization , dead time , decay , scatter , randoms and attenuation based on the corresponding ld ct scan . using capp software ( siemens / cti , knoxville , tn ) , regions of interest ( rois ) of 1 cm diameter were placed over the ascending aorta in at least ten transaxial image planes of the frame showing the first pass of the injected bolus . these rois were combined into one volume of interest ( voi ) for the ascending aorta . a second set of rois were placed over the right ventricle ( rv ) cavity in at least five transaxial planes , with roi boundaries at least 1 cm from the rv wall to avoid spill - in from myocardial activity . both vois were then projected onto all dynamic [ o]h2o images , thereby generating arterial ( ca(t ) ) and rv ( crv(t ) ) time activity curves . parametric pti images were calculated as previously described . in brief , parametric images of myocardial blood flow ( mbf ) , perfusable tissue fraction ( ptf ) , and arterial and venous blood volume fractions were calculated using a basis function implementation of the standard single - tissue compartment model for [ o]h2o [ 1114 ] . parametric images of arterial and venous blood volume fractions were subtracted from normalized ct transmission images , resulting in parametric anatomic tissue fraction ( atf ) images . finally , parametric pti images were calculated as the ratio of ptf and atf images ( fig . 1 ) . finally , 16 myocardial vois were defined manually on parametric ptf images , according to the 16 segments model of the american heart association , after which this voi template was projected onto the parametric pti images.fig . ( b ) atf ( g ml ) , ( c ) ptf ( g ml ) , and ( d ) pti in a patient without coronary artery disease example of short axis images of ( a ) blood volume , ( b ) atf ( g ml ) , ( c ) ptf ( g ml ) , and ( d ) pti in a patient without coronary artery disease cmr studies were performed on a 1.5-t whole - body scanner ( magnetom sonata or avanto ; siemens , erlangen , germany ) , using a six - channel phased - array body coil . after survey scans , a retrotriggered , balanced steady - state free precession gradient - echo sequence was used for cine imaging . image parameters included : slice thickness 5 mm , slice gap 5 mm , temporal resolution < 50 ms , repetition time 3.2 ms , echo time 1.54 ms , flip angle 60 , typical image resolution 1.3 1.6 5.0 mm . the cardiac cycle consisted of 20 phases . after obtaining four- , three- and two - chamber view cines , stacks of 10 to 12 short - axis slices were acquired to fully cover the left ventricle ( lv ) . contrast images were acquired 10 to 15 min after administration of 0.2 mmol kg gadolinium - dtpa in the same views as in the cine images , using a two - dimensional segmented inversion - recovery prepared gradient echo sequence ( te 4.4 ms , tr 9.8 ms , inversion time 250 to 300 ms , typical voxel size 1.3 1.6 5.0 mm ) . images were analysed off - line using the software package mass ( mr analytical software system ; medis , leiden , the netherlands ) . the endocardial and epicardial borders of the lv were outlined manually in both end - diastolic and end - systolic phases of all short - axis images . end - diastolic volume , end - systolic volume and ejection fraction were computed using these analyses . the amount of fibrosis was calculated using the full - width at half - maximum method , which defines fibrosis as myocardial tissue with a signal intensity 50 % of the maximum hyperenhancement intensity . if no enhancement was found in a slice , the maximum signal of the nearest slice with enhancement was used . all areas of enhancement were quantified by computer - assisted planimetry on each of the short - axis images and the segmental extent of enhancement was expressed as a percentage of the segmental area . cmr images were analysed according to the same 16-segment model as used for the parametric pet images . finally , myocardial segments were graded as viable or nonviable using the previously defined cut - off value of 50 % lge per segment [ 15 , 17 ] . continuous variables are presented as means sd , and categorical data are summarized as frequencies and percentages . multiple datasets were compared using analysis of variance ( anova ) , and specific differences were identified using student s t - test with bonferroni inequality adjustment . receiver operating characteristic curves were generated for ptf , pti and mbf for the prediction of myocardial viability assessed by lge cmr . the area under the curve ( auc ) was considered a measure of accuracy to discriminate between viable and nonviable myocardium . a p value of < 0.05 was considered statistically significant . lge was seen in the cmr images of 34 patients ( 74 % ) . of the 736 myocardial segments , 364 ( 49 % ) showed some degree of lge.table 1patient characteristics ( n = 46)characteristicvalueage ( years)65 10gender ( male)36 ( 78 % ) previous myocardial infarction34 ( 74 % ) lv end - diastolic volume ( ml)226 65lv end - systolic volume ( ml)135 71lv ejection fraction ( % ) 42 16lv mass ( g)132 39 patient characteristics ( n = 46 ) pet / ct and cmr data are summarized in table 2 . there was a gradual decrease in ptf , pti and mbf values with increasing degree of lge on cmr images ( p < 0.001 by anova ) . atf values remained relatively constant , except for a significant decrease in the ( near ) transmurally enhanced segments ( p < 0.001 by anova).table 2segmental pet / ct and lge dataextent of lge ( % ) control ( n = 372)025 ( n = 190)2550 ( n = 83)5075 ( n = 54)75 ( n = 37)p value ( anova)atf0.76 0.090.77 0.090.75 0.110.73 0.080.68 0.11**<0.001ptf ( g ml)0.72 0.080.72 0.090.70 0.090.60 0.08**0.51 0.12***<0.001pti0.91 0.080.89 0.090.89 0.090.77 0.10**0.70 0.16***<0.001mbf ( ml g min)1.02 0.300.91 0.26 * 0.85 0.30 * 0.83 0.24 * 0.67 0.30**<0.001*p < 0.05 vs. control ; * * p < 0.05 vs. control , 025 % lge and 2550 % lge ; * * * p < 0.05 vs. control , 025 % lge , 2550 % lge and 5075 % lge . segmental pet / ct and lge data * p < 0.05 vs. control ; * * p < 0.05 vs. control , 025 % lge and 2550 % lge ; * * * p < 0.05 vs. control , 025 % lge , 2550 % lge and 5075 % lge . figure 2 shows a concordant pattern between parametric ptf , pti and lge cmr images in a patient with ischaemic cardiomyopathy after an anterior myocardial infarction.fig . 2long axis images ( a d ) and polar maps ( e h ) in a patient with an anterior myocardial infarction : a , e cmr with lge ( arrow , % transmurality ) ; b , f ptf ( g ml ) ; c , g pti ; d , h mbf ( ml g min)fig . 3long axis images in a patient with a nontransmural anterior myocardial infarction : a cmr with lge ; b pti long axis images ( a d ) and polar maps ( e h ) in a patient with an anterior myocardial infarction : a , e cmr with lge ( arrow , % transmurality ) ; b , f ptf ( g ml ) ; c , g pti ; d , h mbf ( ml g min ) long axis images in a patient with a nontransmural anterior myocardial infarction : a cmr with lge ; b pti using cmr as a reference , 91 of 364 ( 25 % ) segments showing some degree of lge were judged to be nonviable ( lge > 50 % ) . as shown in fig . 4 , the values of ptf and pti for predicting myocardial viability in all 736 segments were comparable ( auc 0.87 , ci 0.830.90 , and 0.86 , ci 0.820.91 , respectively , p = 0.541 ) . mbf was able to predict myocardial viability with less accurate ( auc 0.69 , ci 0.630.75 , p < 0.001 ) . optimal cut - off values of ptf , pti and mbf for predicting ( near ) transmural lge on cmr were 0.69 g ml , 0.80 , and 0.78 ml min g with sensitivities of 69 % , 91 % and 72 % , and specificities of 87 % , 73 % and 56 % , respectively . figure 3 shows an example of a patient with a nontransmural scar and a corresponding relatively high pti.fig . 4receiver operator characteristics curves for the abilities of ptf , pti and mbf to differentiate between viable and nonviable segments based on late gadolinium enhancement receiver operator characteristics curves for the abilities of ptf , pti and mbf to differentiate between viable and nonviable segments based on late gadolinium enhancement lge was seen in the cmr images of 34 patients ( 74 % ) . of the 736 myocardial segments , 364 ( 49 % ) showed some degree of lge.table 1patient characteristics ( n = 46)characteristicvalueage ( years)65 10gender ( male)36 ( 78 % ) previous myocardial infarction34 ( 74 % ) lv end - diastolic volume ( ml)226 65lv end - systolic volume ( ml)135 71lv ejection fraction ( % ) 42 16lv mass ( g)132 39 patient characteristics ( n = 46 ) pet / ct and cmr data are summarized in table 2 . there was a gradual decrease in ptf , pti and mbf values with increasing degree of lge on cmr images ( p < 0.001 by anova ) . atf values remained relatively constant , except for a significant decrease in the ( near ) transmurally enhanced segments ( p < 0.001 by anova).table 2segmental pet / ct and lge dataextent of lge ( % ) control ( n = 372)025 ( n = 190)2550 ( n = 83)5075 ( n = 54)75 ( n = 37)p value ( anova)atf0.76 0.090.77 0.090.75 0.110.73 0.080.68 0.11**<0.001ptf ( g ml)0.72 0.080.72 0.090.70 0.090.60 0.08**0.51 0.12***<0.001pti0.91 0.080.89 0.090.89 0.090.77 0.10**0.70 0.16***<0.001mbf ( ml g min)1.02 0.300.91 0.26 * 0.85 0.30 * 0.83 0.24 * 0.67 0.30**<0.001*p < 0.05 vs. control ; * * p < 0.05 vs. control , 025 % lge and 2550 % lge ; * * * p < 0.05 vs. control , 025 % lge , 2550 % lge and 5075 % lge . segmental pet / ct and lge data * p < 0.05 vs. control ; * * p < 0.05 vs. control , 025 % lge and 2550 % lge ; * * * p < 0.05 vs. control , 025 % lge , 2550 % lge and 5075 % lge . figure 2 shows a concordant pattern between parametric ptf , pti and lge cmr images in a patient with ischaemic cardiomyopathy after an anterior myocardial infarction.fig . 2long axis images ( a d ) and polar maps ( e h ) in a patient with an anterior myocardial infarction : a , e cmr with lge ( arrow , % transmurality ) ; b , f ptf ( g ml ) ; c , g pti ; d , h mbf ( ml g min)fig . 3long axis images in a patient with a nontransmural anterior myocardial infarction : a cmr with lge ; b pti long axis images ( a d ) and polar maps ( e h ) in a patient with an anterior myocardial infarction : a , e cmr with lge ( arrow , % transmurality ) ; b , f ptf ( g ml ) ; c , g pti ; d , h mbf ( ml g min ) long axis images in a patient with a nontransmural anterior myocardial infarction : a cmr with lge ; b pti using cmr as a reference , 91 of 364 ( 25 % ) segments showing some degree of lge were judged to be nonviable ( lge > 50 % ) . as shown in fig . 4 , the values of ptf and pti for predicting myocardial viability in all 736 segments were comparable ( auc 0.87 , ci 0.830.90 , and 0.86 , ci 0.820.91 , respectively , p = 0.541 ) . mbf was able to predict myocardial viability with less accurate ( auc 0.69 , ci 0.630.75 , p < 0.001 ) . optimal cut - off values of ptf , pti and mbf for predicting ( near ) transmural lge on cmr were 0.69 g ml , 0.80 , and 0.78 ml min g with sensitivities of 69 % , 91 % and 72 % , and specificities of 87 % , 73 % and 56 % , respectively . figure 3 shows an example of a patient with a nontransmural scar and a corresponding relatively high pti.fig . 4receiver operator characteristics curves for the abilities of ptf , pti and mbf to differentiate between viable and nonviable segments based on late gadolinium enhancement receiver operator characteristics curves for the abilities of ptf , pti and mbf to differentiate between viable and nonviable segments based on late gadolinium enhancement the present study was conducted to validate the use of a parametric myocardial viability imaging technique using [ o]h2o pet / ct in patients with ischaemic heart disease . viability assessed using parametric ptf and pti imaging was in good agreement with that assessed using lge cmr . furthermore , these images and myocardial perfusion imaging are obtained simultaneously allowing both myocardial viability and ischaemia to be evaluated in a single scanning session . [ o]h2o is generally considered to be the gold standard for absolute quantification of myocardial perfusion in vivo [ 10 , 18 , 19 ] . apart from mbf , [ o]h2o also provides estimates of the extent of myocardium within a roi that is able to exchange water rapidly , i.e. ptf . subsequently , ptf can be corrected for partial volume effects by dividing it by its anatomical counterpart atf , resulting in pti . it has been shown that both ptf and pti can identify myocardial scarring and thus can act as markers of viability . only recently , however , a method has been developed that enables generation parametric atf , ptf and pti images from a single [ o]h2o pet / ct scan . in addition , the traditional ( long ) transmission scan can be replaced by a ( rapid ) low - dose ct scan , thereby shortening the total scanning time substantially . now that the method has been shown to produce high - quality parametric images , its implementation into clinical practice needs investigation . using the described parametric imaging approach , the present study demonstrated that atf was relatively constant independent of tissue characteristics of the myocardium . only in ( near ) transmurally infarcted segments was atf significantly reduced , most likely due to wall thinning , as atf is prone to partial volume effects . in contrast , ptf and pti progressively decreased with increasing extent of scarring , as shown by lge cmr . these results are in line with those of previous studies in which reductions in both ptf and pti were observed in nonviable scarred myocardium . pti in control segments appeared to be somewhat lower than the expected value of unity for normal segments ( 0.91 0.08 ) . this phenomenon has previously been observed in cardiomyopathy in both animal experiments and human studies [ 6 , 20 ] . slight misalignment between pet and cmr segments may have occurred to additionally account for this reduction in pti . furthermore , the presence of interstitial fibrosis in dilated cardiomyopathy may also explain this reduction in normal segments that remains undetected on lge cmr [ 21 , 22 ] . taking lge as a reference , the optimal cut - off values for discriminating between viable and nonviable myocardium although this would reduce the data processing time , it would not affect the scanning protocol , as for both parameters a dynamic [ o]h2o pet scan in combination with a low - dose ct scan are required . in addition , the sensitivity of pti exceeded that of ptf ( 91 % and 68 % , respectively ) , whereas for specificity the opposite pattern was observed ( 73 % and 78 % , respectively ) . although further study is need to determine the cause of this discrepancy , as a potential clinical marker of viability pti may be favoured over ptf to reduce false - negative findings in patients who might benefit from revascularization . it is of interest to note that , compared to ptf and pti , mbf performed relatively poorly in distinguishing viable from nonviable tissue , rendering it less suitable for viability imaging . previous studies have indicated that the optimal cut - off value for pti is in the range 0.70.9 . lge was used as a surrogate end - point of myocardial viability instead of functional recovery of dysfunctional myocardium after revascularization . lge has been shown to be a good , but not perfect , marker of myocardial viability [ 15 , 17 ] . therefore , the results should be interpreted with caution , and more studies are warranted to establish the value of parametric pti as a viability marker . ptf and pti , obtained from a single [ o]h2o pet / ct scan , can be used as markers of myocardial viability in patients with coronary artery disease . ptf and pti , obtained from a single [ o]h2o pet / ct scan , can be used as markers of myocardial viability in patients with coronary artery disease . this article is distributed under the terms of the creative commons attribution license which permits any use , distribution , and reproduction in any medium , provided the original author(s ) and the source are credited .

purposethe perfusable tissue index ( pti ) is a marker of myocardial viability . recent technological advances have made it possible to generate parametric pti images from a single [ 15o]h2o pet / ct scan . the purpose of this study was to validate these parametric pti images.methodsthe study population comprised 46 patients with documented or suspected coronary artery disease who were studied with [ 15o]h2o pet and late gadolinium - enhanced ( lge ) cardiac magnetic resonance imaging ( cmr).resultsof the 736 myocardial segments included , 364 showed some degree of lge . pti and perfusable tissue fraction ( ptf ) diminished with increasing lge . the areas under the curve of the pti and ptf , used to predict ( near ) transmural lge on cmr , were 0.86 and 0.87 , respectively . optimal sensitivity and specificity were 91 % and 73 % for pti and 69 % and 87 % for ptf , respectively.conclusionpti and ptf assessed with a single [ 15o]h2o scan can be utilized as markers of myocardial viability in patients with coronary artery disease .

while advances in free trade and globalization increase the movement and accelerate the accumulation of invasive species ( lockwood et al . 2005 ) , it is still unclear how introduced populations can successfully establish . as elton ( 1958 ) pointed out , for every successful invasion that occurs , there are enormously more invasions that never happen , or fail quite soon or even after a good many years ( page 109 ) . this modern biological paradox can not readily be reconciled , especially in the characteristic case where the founder population is small , as such populations are definitely in a precarious position ( mayr 1965 ; page 42 ) . introductions of populations at low density and/or small size are often faced with inverse density dependent effects , attributed to demographic stochasticity or reduced cooperative interactions ( courchamp et al . allee ( 1931 ) first proposed that under these conditions , populations may suffer a decrease in the per - capita rate of increase , from here on referred to as the allee effect . upon arrival in a novel environment , individuals need to overcome a series of challenges in order to reduce the population 's risk of extinction . the time period in which this occurs is generally considered the initial establishment phase , and is thought to be a common feature and general pattern of invasion and the process of growth and expansion ( shigesada and kawasaki 1997 ; sakai et al . the occurrence of a lag phase that precedes a noticeable increase in population growth and density can result from ecological and/or evolutionary phenomena ( sakai et al . small populations that undergo logistic growth slowly increase through the initial phase of the exponential curve , leading to the perception of a time lag . where this time lag is more pronounced , populations may be recovering from inverse density dependent effects ( i.e. , allee effects ) . individuals may suffer a reduction in fitness at low densities for many reasons ( reviewed by courchamp et al . even when the initial population is large , rapid dispersal required for expansion could be suicidal as the population density decreases , thereby enhancing inverse density dependent effects ( lewis and kareiva 1993 ; drake et al . component allee effect where the population size and density affects the mean overall fitness or some component of individual fitness , respectively ( stephens et al . 1999 ; gascoigne et al . it is often difficult to decipher the exact mechanism that manifests allee effects ( and it is not always the case that component allee effects lead to demographic allee effects ) . nevertheless , we focus our attention on population level demographic allee effects with the underlying assumption that a component allee effect led to the demographic allee effect . essentially , it is the case that introduced individuals may be maladapted to small population sizes where their survival and reproductive ability are significantly impacted , and these impacts on individual fitness combine to produce an overall decrease in abundance ( i.e. , demographic allee effects ) . as allee effects impact individual fitness , the underlying traits that influence these effects ( i.e. , component allee effects ) may be subject to natural selection ( courchamp et al . 2008 ) . whereas propagule pressure is an emerging explanation for the establishment of invasive species ( lockwood et al . 2005 ) , it relies on an obvious relationship between the number and size of introduction events and the probability of success , as safety in numbers helps combat allee effects and stochastic extinction . in the event that propagule size is not large enough to overcome inverse density dependent effects , a population traits that may be responsible for reproductive success and survival at small population densities and sizes include mate finding cues ( pheromones and vocal / visual signals ) , dispersal / aggregation behavior , habitat preferences , mating synchronicity , and gamete morphology and performance ( see courchamp et al . direct evidence for the evolution of these traits as functional adaptations to allee effects is limited , but we can infer an adaptive evolutionary origin of these traits from studies of sexual selection ( courchamp et al . 2008 ; gascoigne et al . an evolutionary response to intensive selection pressure imposed by density dependent survival and reproduction relies on genetic variants for adaptive evolution . according to neutral quantitative genetic theory , a loss of genetic variation is expected from population bottlenecks and founder effects ( nei et al . however it is not neutral variation that matters , but rather evolvability depends on the variation relevant to selection . maintenance or even increases of this ( additive ) genetic variation has been theoretically and empirically observed following a bottleneck or in small founder populations ( bryant et al . 1986 ; goodnight 1988 ; willis and orr 1993 ; cheverud and routman 1996 ; briggs and goldman 2006 ; turelli and barton 2006 ) . additionally , many recent studies suggest that there is actually no significant reduction in genetic diversity in most successful invaders ( lee 2002 ; allendorf and lundquist 2003 ; roman and darling 2007 and references therein ) , and that evolution can occur on contemporary timescales ( reznick and ghalambor 2001 ; carroll et al . 2007 ; kinnison and hairston 2007 ) . our purpose here is to explore the feasibility of small populations that may adaptively respond to overcome allee effects in order to establish , given any amount of genetic variation . in this paper , we present evidence for the enhanced potential for growth and spread of a small introduced population of organisms faced with allee effects when adaptation occurs . we model the growth and spread of this population according to a reaction - diffusion equation , and allow evolution to influence inverse density dependent effects through a genetic subsystem that provides the opportunity for successful invasion when otherwise ( under the current , ecological paradigm ) the population would go extinct . the deterministic model that we explore in this paper broadly describes population dynamics with density - mediated growth ( i.e. , an allee effect ) and diffusive dispersal . this type of demographic model has been used as a compact and tractable representation of invasion ( e.g. , skellam 1951 ; lewis and kareiva 1993 ) . specifically , it has been applied to systems such as introductions of nonindigenous freshwater and marine species through ballast water discharge ( drake et al . 2005 ; drury et al . 2007 ) . using this approach , drake et al . ( 2005 ) report acceptable volumes of discharge for various organisms ( with differing reproductive rates ) for a range of invasion risk tolerances . here , we consider populations that are introduced below the invasion risk threshold , but nonetheless succeed if evolutionary dynamics are considered in conjunction with the ecological system . the growth and spread of an introduced population of organisms is represented by a reaction - diffusion equation described by ( lewis and kareiva 1993 ) : which determines the rate of change in the local population density relative to the carrying capacity [ u which denotes u(x , t ) ] over time , at a point in space . this equation models the growth of the population ( the first term on the right hand side of eqn . 1 ) at a spatial location that is subject to an allee effect in addition to migration ( which depends on the second term on the rhs of eqn . 1 ) . the diffusion coefficient ( d ) scales the rate of population spread , in this case across a one - dimensional habitat x. the reproductive rate is regulated by r , and a ( which is a function of space and time , derived below ) is the local critical density or allee threshold that determines if population growth is positive or negative ( fig . growth dynamics of the model population ( a ) , and the solution of eqn . ( 1 ) without diffusion ( b ) with reproductive rate , r = 0.6 , and allee threshold , a = 0.3 . trajectories for population size ( b ) are given for initial densities from 0 to 1 in increments of 0.05 . there are many variations of single - species models of population dynamics that exhibit allee effects ( see table 1 of boukal and berec 2002 ) , however the growth function of eqn . the behavior of this verhulst ( 1838 ) logistic model modified to include a nonlinear cubic term ( based on the fitzhugh - nagumo equations ; fitzhugh 1960 ; nagumo et al . 1962 ) , is bistable , with equilibria at u = 0 ( extinction ) , u = a ( unstable threshold ) , and u = 1 ( carrying capacity ) . figure 1a shows this behavior in terms of the growth of the population ( change in population density with respect to time ) versus the population density . at densities below the critical threshold ( a ) there is negative population growth declining to extinction ( from here on the population is considered extinct below a cutoff density of 0.0001 , as a declining population trajectory will only asymptotically approach zero in a deterministic model ; gomulkiewicz and holt 1995 ) ; otherwise the population will reach carrying capacity . 1b with the graph of the solution of the growth function ( population size versus time ) at various initial densities . when diffusion is added to this model of population growth , there are two critical elements that emerge based on the solution to the partial differential equation ( pde ) . the first is the wave speed , which is determined by the allee threshold ( a ) . as we are considering a strong allee effect in this model ( i.e. , 0 < a < 1 , where the population below this threshold exhibits negative growth versus reduced positive growth from a weak allee effect ) , there exists a unique wave speed of the invasion front that is a result of being pushed from the inside out , as opposed to being pulled by the leading edge ( lewis and kareiva 1993 ) . this velocity can be derived through the solution of the pde ( 1 ) : ( lewis and kareiva 1993 ; murray 1993 ) . this result suggests that in order for a wave to maintain a positive velocity of advance , the magnitude of the allee threshold ( a ) must be less than half of the maximum value of the population density relative to the carrying capacity . in addition to the velocity of the wave front , the region occupied by the invading population must exceed a certain critical size for positive growth to occur ( skellam 1951 ; kierstead and slobodkin 1953 ) . this phenomenon is clearly explained by okubo ( 1980 ) by noting that whereas reproduction takes place within a region or patch , diffusion takes place at the boundaries resulting in a loss of organisms , thus reducing the density within the patch . this tradeoff in the ratio of inner region volume to outer surface area will either allow a population to grow or decline with an inverse relationship of diffusivity to rate of growth . this relationship gives a minimum region within which reproduction can not compensate for loss due to diffusion , especially when allee effects influence population growth . thus , lewis and kareiva ( 1993 ) derive a minimum size condition ( i.e. , the radius of the initial beachhead ) based on the wave speed that is required for the population to establish and radially expand . we address this critical size threshold qualitatively , as the analytical solution ( i.e. , ; lewis and kareiva 1993 ) is for two - dimensional spread , while we work with a simpler one - dimensional model . the minimum critical radius is proportional to the ratio of diffusivity ( i.e. , diffusion coefficient , d ) to the reproductive rate ( controlled by r ) . the inclusion of diffusion in the model provides a spatially explicit understanding of how all of the components interact to affect invasion / establishment success . the diffusion process has been extensively analyzed in invasion processes ( e.g. , fisher 1937 ; skellam 1951 ; okubo 1980 ) . in order to incorporate evolutionary factors that may influence invasion success , this genetic subsystem is coupled to the ecological model to explore the effects of selection and genetic variance on traits that may increase a population 's likelihood of survival . specifically , we allow the allee threshold to become a dynamic parameter that is considered to be a fitness related trait ( e.g. , a trait impacting the component allee effect ) . from here on , except in the absence of evolution , referring to the allee threshold implies that that value is the initial value , as it changes over time . this quantitative trait influences an organism 's ability to survive and reproduce in a small population . the results reveal the possibility that an introduced population that would fail to persist in the ecological context of this model has the potential to succeed through evolutionary means . including evolution within the context of ecological invasions can serve to provide more robust predictions for management strategies . therefore , it is important to investigate the possibility of evolution in the analysis of invasions . the framework that is used to link evolutionary change with ecological processes involves developing a relationship between the fast , ecological and slow , evolutionary timescales in order to make these rates comparable ( kondrashov and khibnik 1996 ) . in the coupled evolutionary ecology model , the reaction - diffusion eqn . ( 1 ) describes the change in the population density over time and is tied into a genetic subsystem that allows the organismal response to population density to evolve in terms of the selection gradient and genetic variance . as the population dynamics vary across space , the genetic subsystem describes the rate of change of the trait mean ( i.e. , the allee parameter ) at each location x by : ( pease et al . 1989 ; garca - ramos and kirkpatrick 1997 ; kirkpatrick and barton 1997 ; hare et al . the first term on the right hand side reflects the force of local directional selection , where the selection gradient for frequency - independent selection is the rate of change of the mean malthusian fitness function ( i.e. , per - capita growth rate : ) with respect to the trait , a ( lande 1976 ; falconer 1989 ) . thus , , where we assume that individual fitness approaches the population mean fitness , as most individuals are close to the average phenotype ( webb 2003 ) . this suggests that the genetic variance ( ) is small ( and constant in this model ) . this small parameter for the genetic variance can be used to couple the fast ecological timescale , t , with the slow evolutionary timescale , = t ( kondrashov and khibnik 1996 ; webb 2003 ) . combing these two components of genetic variance and selection , quantifies the effect of natural selection on the local mean value of the quantitative trait ( the allee parameter ; lande 1976 ; falconer 1989 ) . in order to account for the influence of migration on the trait 's local mean , the latter two terms in eqn . the middle term takes into account asymmetrical gene flow caused by the variation of density across space ( pease et al . 1989 ; garca - ramos and kirkpatrick 1997 ; kirkpatrick and barton 1997 ; hare et al . this captures the influence of the mean trait value ( i.e. , genetic contribution ) from more abundant populations to less abundant neighboring locations due to the spatial gradient , as more individuals migrate from areas with relatively high population densities . the last term mirrors the diffusion term from the ecological model , and describes the homogenizing effect of random dispersal . we solved the spatially explicit system numerically using matlab 7.0 ( 2004 , the mathworks , natick , ma , usa ) using a finite difference method to incorporate diffusion and gene flow ( adapted from garvie 2007 ) . by iterating eqns ( 1 ) and ( 2 ) forward in time , the population density and allee threshold at each location are updated with diffusion following growth and selection , respectively , while incorporating the spatial gradient . the simulated populations , with and without evolution , behaved as we expected from the model eqns ( 1 ) and ( 2 ) , and adequately approximate / represent the critical conditions that govern this dynamical system . the growth and spread of an introduced population of organisms is represented by a reaction - diffusion equation described by ( lewis and kareiva 1993 ) : which determines the rate of change in the local population density relative to the carrying capacity [ u which denotes u(x , t ) ] over time , at a point in space . this equation models the growth of the population ( the first term on the right hand side of eqn . 1 ) at a spatial location that is subject to an allee effect in addition to migration ( which depends on the second term on the rhs of eqn . 1 ) . the diffusion coefficient ( d ) scales the rate of population spread , in this case across a one - dimensional habitat x. the reproductive rate is regulated by r , and a ( which is a function of space and time , derived below ) is the local critical density or allee threshold that determines if population growth is positive or negative ( fig . growth dynamics of the model population ( a ) , and the solution of eqn . ( 1 ) without diffusion ( b ) with reproductive rate , r = 0.6 , and allee threshold , a = 0.3 . trajectories for population size ( b ) are given for initial densities from 0 to 1 in increments of 0.05 . there are many variations of single - species models of population dynamics that exhibit allee effects ( see table 1 of boukal and berec 2002 ) , however the growth function of eqn . the behavior of this verhulst ( 1838 ) logistic model modified to include a nonlinear cubic term ( based on the fitzhugh - nagumo equations ; fitzhugh 1960 ; nagumo et al . 1962 ) , is bistable , with equilibria at u = 0 ( extinction ) , u = a ( unstable threshold ) , and u = 1 ( carrying capacity ) . figure 1a shows this behavior in terms of the growth of the population ( change in population density with respect to time ) versus the population density . at densities below the critical threshold ( a ) there is negative population growth declining to extinction ( from here on the population is considered extinct below a cutoff density of 0.0001 , as a declining population trajectory will only asymptotically approach zero in a deterministic model ; gomulkiewicz and holt 1995 ) ; otherwise the population will reach carrying capacity . 1b with the graph of the solution of the growth function ( population size versus time ) at various initial densities . when diffusion is added to this model of population growth , there are two critical elements that emerge based on the solution to the partial differential equation ( pde ) . the first is the wave speed , which is determined by the allee threshold ( a ) . as we are considering a strong allee effect in this model ( i.e. , 0 < a < 1 , where the population below this threshold exhibits negative growth versus reduced positive growth from a weak allee effect ) , there exists a unique wave speed of the invasion front that is a result of being pushed from the inside out , as opposed to being pulled by the leading edge ( lewis and kareiva 1993 ) . this velocity can be derived through the solution of the pde ( 1 ) : ( lewis and kareiva 1993 ; murray 1993 ) . this result suggests that in order for a wave to maintain a positive velocity of advance , the magnitude of the allee threshold ( a ) must be less than half of the maximum value of the population density relative to the carrying capacity . in addition to the velocity of the wave front , the region occupied by the invading population must exceed a certain critical size for positive growth to occur ( skellam 1951 ; kierstead and slobodkin 1953 ) . this phenomenon is clearly explained by okubo ( 1980 ) by noting that whereas reproduction takes place within a region or patch , diffusion takes place at the boundaries resulting in a loss of organisms , thus reducing the density within the patch . this tradeoff in the ratio of inner region volume to outer surface area will either allow a population to grow or decline with an inverse relationship of diffusivity to rate of growth . this relationship gives a minimum region within which reproduction can not compensate for loss due to diffusion , especially when allee effects influence population growth . thus , lewis and kareiva ( 1993 ) derive a minimum size condition ( i.e. , the radius of the initial beachhead ) based on the wave speed that is required for the population to establish and radially expand . we address this critical size threshold qualitatively , as the analytical solution ( i.e. , ; lewis and kareiva 1993 ) is for two - dimensional spread , while we work with a simpler one - dimensional model . the minimum critical radius is proportional to the ratio of diffusivity ( i.e. , diffusion coefficient , d ) to the reproductive rate ( controlled by r ) . the inclusion of diffusion in the model provides a spatially explicit understanding of how all of the components interact to affect invasion / establishment success . the diffusion process has been extensively analyzed in invasion processes ( e.g. , fisher 1937 ; skellam 1951 ; okubo 1980 ) . in order to incorporate evolutionary factors that may influence invasion success , we develop a quantitative genetic subsystem . this genetic subsystem is coupled to the ecological model to explore the effects of selection and genetic variance on traits that may increase a population 's likelihood of survival . specifically , we allow the allee threshold to become a dynamic parameter that is considered to be a fitness related trait ( e.g. , a trait impacting the component allee effect ) . from here on , except in the absence of evolution , referring to the allee threshold implies that that value is the initial value , as it changes over time . this quantitative trait influences an organism 's ability to survive and reproduce in a small population . the results reveal the possibility that an introduced population that would fail to persist in the ecological context of this model has the potential to succeed through evolutionary means . including evolution within the context of ecological invasions can serve to provide more robust predictions for management strategies . therefore , it is important to investigate the possibility of evolution in the analysis of invasions . the framework that is used to link evolutionary change with ecological processes involves developing a relationship between the fast , ecological and slow , evolutionary timescales in order to make these rates comparable ( kondrashov and khibnik 1996 ) . in the coupled evolutionary ecology model , the reaction - diffusion eqn . ( 1 ) describes the change in the population density over time and is tied into a genetic subsystem that allows the organismal response to population density to evolve in terms of the selection gradient and genetic variance . as the population dynamics vary across space , the genetic subsystem describes the rate of change of the trait mean ( i.e. , the allee parameter ) at each location x by : ( pease et al . 1989 ; garca - ramos and kirkpatrick 1997 ; kirkpatrick and barton 1997 ; hare et al . the first term on the right hand side reflects the force of local directional selection , where the selection gradient for frequency - independent selection is the rate of change of the mean malthusian fitness function ( i.e. , per - capita growth rate : ) with respect to the trait , a ( lande 1976 ; falconer 1989 ) . thus , , where we assume that individual fitness approaches the population mean fitness , as most individuals are close to the average phenotype ( webb 2003 ) . this suggests that the genetic variance ( ) is small ( and constant in this model ) . this small parameter for the genetic variance can be used to couple the fast ecological timescale , t , with the slow evolutionary timescale , = t ( kondrashov and khibnik 1996 ; webb 2003 ) . combing these two components of genetic variance and selection , quantifies the effect of natural selection on the local mean value of the quantitative trait ( the allee parameter ; lande 1976 ; falconer 1989 ) . in order to account for the influence of migration on the trait 's local mean , the latter two terms in eqn . the middle term takes into account asymmetrical gene flow caused by the variation of density across space ( pease et al . 1989 ; garca - ramos and kirkpatrick 1997 ; kirkpatrick and barton 1997 ; hare et al . this captures the influence of the mean trait value ( i.e. , genetic contribution ) from more abundant populations to less abundant neighboring locations due to the spatial gradient , as more individuals migrate from areas with relatively high population densities . the last term mirrors the diffusion term from the ecological model , and describes the homogenizing effect of random dispersal . we solved the spatially explicit system numerically using matlab 7.0 ( 2004 , the mathworks , natick , ma , usa ) using a finite difference method to incorporate diffusion and gene flow ( adapted from garvie 2007 ) . by iterating eqns ( 1 ) and ( 2 ) forward in time , the population density and allee threshold at each location are updated with diffusion following growth and selection , respectively , while incorporating the spatial gradient . the simulated populations , with and without evolution , behaved as we expected from the model eqns ( 1 ) and ( 2 ) , and adequately approximate / represent the critical conditions that govern this dynamical system . the dynamics of the evolutionary ecology model can be interpreted using the idea of fast and slow timescales ( kondrashov and khibnik 1996 ; webb 2003 ) . earlier , we assumed that the genetic variation ( ) was small ( to use mean fitness as a proxy for individual fitness ) , which can subsequently be taken advantage of for our analysis of the coupled evolutionary ecological dynamics . when = 0 , the situation without evolution , the genetic subsystem is frozen and the population moves towards a stable equilibrium of the ecological subsystem ( carrying capacity or extinction ) depending on its initial density ( greater than or less than the allee threshold respectively ; fig . when > 0 but small , the allee threshold evolves relatively slowly and influences the ecological system . whenever the population is below its carrying capacity ( u = 1 for each spatial coordinate x when space is explicit ) , eqn . ( 2 ) is negative , and decreases the mean allee threshold ( a ) , as the intensity of selection is density dependent . thus , fitness increases as allee effects are suppressed , and selection drives the allee threshold towards zero . if the population density is greater than the allee threshold , but still below the carrying capacity , it will progress towards carrying capacity more rapidly than it would without evolution as a decreases ; as the rate at which the population density changes ( eqn . 1 ) is proportional to the difference between u and a. the ecological dynamics are reversed when the population density is below the allee threshold as the population declines towards extinction , but more slowly than it does without evolution . when u < a , eqn . ( 1 ) is negative , and the population density approaches extinction more rapidly with a constant ( as the difference between u and a increases ) , than it does with evolution as a decreases ( revealing a more pronounced time lag to extinction ) . during this time lag , as the population slowly declines , the opportunity for evolution to overcome inverse density dependent effects occurs . if the rate of evolution is fast enough , the allee threshold can fall below the population density , causing the rate of change of population density to become positive ( where u > a ) and the population grows and can successfully invade . the chance that evolution can rescue the population from extinction depends on the relative rates of genetic change in the quantitative trait ( i.e. , allee threshold ) and of population decline ( gomulkiewicz and holt 1995 ) ; hence the amount of genetic variance greatly impacts the ability to adapt and survive . a nonspatial example of this process , referred to as evolutionary rescue ( gomulkiewicz and holt 1995 ) , is shown in fig . 2 , where a population is introduced below the allee threshold . without evolution , the population declines to extinction ( fig . 2a , solid line ) as the allee threshold remains constant ( fig . 2b , solid line ) . when the population can evolve ( fig . 2 , dotted line ) , it declines at first until it can overcome the magnitude of inverse density dependence , and is then able to successfully establish . as it is difficult to measure the allee effect empirically ( tobin et al . 2007 ) , we use an extreme value that exaggerates density dependent effects in order to investigate the worst case scenario ( a = 0.3 , where the population exhibits deterministic decline when its density is less than 30% of its carrying capacity ) . when evolution is included , we used a small value for the genetic variance , = 0.02 , in order to remain consistent with fast - slow dynamics , unless otherwise indicated . comparison of an invading population introduced at a density below the allee threshold , a = 0.3 ( u = 0.25 , r = 0.6 ) . the solid line represents the nonspatial system ( d = 0 ) described by eqns ( 1 ) and ( 2 ) without evolution ( = 0 ) which results in extinction ( a ) and a constant allee threshold ( b ) . the dotted line indicates population growth ( a ) when evolution ( = 0.02 ) acts to reduce the allee threshold ( b ) . in general , there is a range of parameter space that permits persistence for a population below the allee threshold in the nonspatial model with evolution ( instead of simple decline to extinction ) . as genetic variance increases , we are essentially relaxing the assumption of fast - slow timescales and allow evolution to occur more rapidly . 3 where initial population densities below the allee threshold require a minimum amount of genetic variance in order to avoid extinction . in this case , the rate of reproduction , r , also influences the potential for evolutionary rescue , as it impacts both population growth and rate of evolution ( eqns 1 and 2 , respectively ) . as we relax the assumption of fast - slow timescales , the behavior remains qualitatively the same as that described analytically under a strict fast - slow timescale assumption . parameter combinations of reproductive rate : r ; genetic variance : , and initial population density : u , that result in extinction or evolutionary rescue . in this nonspatial scenario , initial population densities greater than the allee threshold ( a = 0.3 ) always succeed , thus the focus is on the parameter space that allows for evolutionary rescue ( i.e. , where the population growth changes from negative to positive ) . as the reproductive rate increases from 0.1 to 1 , there is less genetic variance needed for a population to evolve to overcome inverse density dependence as increased reproduction will contribute to suppressing allee effects . nonetheless , the additional complexities result in qualitatively similar behavior to the nonspatial model . in this case , not only will evolution influence population growth , it affects the wave speed and the critical size threshold , rmin . as the population overcomes allee effects with a decreasing allee threshold , the wave speed accelerates and the critical patch size becomes smaller . thus , in addition to the initial density of the introduced population and the genetic variance , the initial radius or patch size of the initial invasion area , the ratio of diffusion to reproduction , and gene flow will factor into successful establishment and give rise to a wider range of interactions between the ecology and evolution of this system . ( 1 ) ( without the evolutionary subsystem ) in one - dimensional space , with an initial population density below the allee threshold , declines to extinction ( fig . this is contrasted by the results when the evolutionary subsystem is included . with the initial population density below the allee threshold , fig . the same type of rescue occurs for a population that starts near carrying capacity , but occupies an initial spatial size below that which is necessary for a population to successfully establish . figure 5a shows a rapidly declining population that goes extinct . under the same circumstance , but where evolution of the allee threshold occurs , fig . 5b shows the population density at first beginning to shrink and then growing and expanding . in addition to the time evolution of population density across space in figs 4 and 5 , the evolution of the mean trait value across space illustrates how gene flow and the density dependent selection gradient influences its rate of change and distribution ( figs 4c and 5c ) . as the intensity of selection is density dependent ( and we assume constant genetic variance ) , locations with smaller populations can evolve the trait value more rapidly compared to other areas where allee effects may not be as strong and experience weaker selection . the trait distribution over time , figs 4c and 5c , therefore reflect the population density distribution , but are also influenced by the trait values of the migrants . as individuals disperse out to new locations and push the boundaries of the species range , their trait values are averaged to determine the demographic allee threshold for that spatial coordinate . this demographic allee threshold combines with their local population density to influence individual fitness and population growth ( where the distance between the density and mean trait value is the initial degree of maladaptation ) . diffusive dispersal of an introduced population at an initial density ( bold dashed line ) below the initial allee threshold , a = 0.3 ( u = 0.25 , r = 1 , d = 0.1 ) across a linear , one dimensional habitat . the population collapses over time to extinction ( a ) where there is no evolution ( = 0 ) , and succeeds ( b ) after an initial decline with evolution ( = 0.02 ) . ( c ) the evolution of the mean value of the allee threshold across space ( where the initial distribution is given by the bold dashed line ) . the population density distribution and corresponding trait values ( i.e. , allee threshold ) are plotted at equal time increments ( every 20 of 1200 model iterations ) . population density of a diffusion dispersed population across one dimensional space . the initial population density ( bold dashed line ) is near carrying capacity ( u = 0.95 , a = 0.3 , r = 1 , d = 0.5 ) , but introduced below the minimum radius of area determined to be critical for invasion success . ( a ) is collapsing to extinction without evolution ( = 0 ) , whereas ( b ) shows success of an invader with evolution ( = 0.02 ) after initial decline . ( c ) the evolution of the mean value of the allee threshold across space ( where the initial distribution is given by the bold dashed line ) . the population density distribution and corresponding trait values ( i.e. , allee threshold ) are plotted at equal time increments ( every 20 of 1200 model iterations ) . we explored when evolutionary rescue occurred across a range of parameter values for the spatially explicit model . according to drake et al . ( 2005 ) , variability among locations and over time makes it unreasonable to determine precise estimates for the diffusion coefficient , d. we therefore explored a range of values , and present those that best illustrate breadth of behavior . the parameter that controls the reproductive rate , r , was also varied substantially , but as the spatial dynamics depend on the ratio of diffusion to rate of reproduction ( resulting in a measure of length ) ; we fixed r and varied d , unless otherwise noted . this was justified as the results of the spatial simulations are qualitatively identical for equivalent ratios . the effects of the critical patch size , initial population density , ratio of diffusion to growth and genetic variation on evolutionary rescue and population dynamics are shown in fig . the ratio of the diffusion coefficient ( d ) to the reproductive rate ( r ) determines whether the population will expand or collapse according to the initial radius of the introduced population . the areas under the curves denote combinations of genetic variance and initial population density that result in extinction . areas above the curves are combinations of genetic and/or demographic conditions that produce inevitable persistence . the parameter space between the vertical dashed lines refers to the different ways population survival is influenced . to the left of the initial allee threshold , the initial population density will either go extinct due to density dependent effects ( below the d / r curve ) , or given enough genetic variation , will be evolutionarily rescued ( above the d / r curve ) . the area to the right of the initial allee threshold [ and between the dashed lines in ( b ) and ( c ) ] is the case where the initial population density is greater than the allee threshold but due to the initial spatial size and the ratio of diffusion to reproduction , the population may go extinct without sufficient genetic variance ( below the d / r curve ) , otherwise it will evolve to overcome the critical patch size effect . for initial population densities thus , the area between the dashed lines in ( b ) and ( c ) truly delineates evolutionary rescue when d / r = 1 . the rightmost vertical line moves slightly to the left to the point of intersection of the d / r curve and the x - axis for other values of d / r . graphs ( a ) , ( b ) , and ( c ) represent different radii of the linear habitat that the introduced population initially occupies . when the size of the initial population is too small ( i.e. , a radius of 1 ) , a population at carrying capacity ( i.e. , u = 1 ) will go extinct without evolution due to the relative effect of diffusion to reproduction ( fig . if evolution occurs rapidly enough ( i.e. , > 0.02 ) , the population can overcome inverse density dependent effects and compensate for the loss due to diffusion and rebound from low densities . when the initial radius of the population is increased ( fig . 6b , c ) , the chance of survival and establishment ( growth and expansion ) of populations above or below the allee threshold increases with initial density and genetic variance . therefore , the initial radius of the population can significantly impact the likelihood of evolutionary rescue for populations with the same amount of genetic variance . this is demonstrated further in fig . 7a , where the rate of recovery ( i.e. , the inverse of the time lag before growth becomes positive and the population reaches carrying capacity ) for a population near carrying capacity depends on its initial size / radius and genetic variance . where size and variance are small , rescue never occurs . as these parameters increase , the rate of recovery gradually becomes faster until it essentially plateaus ( although with greater variance and initial radius , the rate of recovery may slow slightly if the initial spatial extent is large enough for the population to experience early growth before diffusion causes decline prior to recovery ) . if the population occupies a large enough spatial extent , it will succeed without evolution ( where the genetic variance is zero ) , however the lag time may be more pronounced depending on the ratio of diffusion to reproduction through the tradeoff between growth and spread ( e.g. , if spread is relatively fast compared to reproduction , d / r = 1 ) . the population density may thus initially decline across space until reproduction can sufficiently overcome the loss due to diffusion , and the population can grow to carrying capacity . similar to the nonspatial case , a population ( greater than the allee threshold ) above the spatial threshold will grow to carrying capacity more rapidly with evolution than without . rate of recovery in terms of the inverse of the time lag before population growth becomes positive , where one timestep equals 24 iterations of the model . in ( a ) , the initial population density is near carrying capacity ( u = 0.95 , a = 0.3 , d / r = 1 ) , and the initial radius and genetic variance , , varies . where the rate of recovery is zero , the population goes extinct as it initially occupies an area smaller than the critical patch size ( in this case , a radius of 1.4 ) or does not have sufficient genetic variance to evolve quickly enough to be rescued prior to extinction . increasing the genetic variance and initial radius will decrease this time lag until the population no longer experiences any negative growth ( in this case , for initial radii 3.8 and 0.036 ; for initial radii > 2.7 , the rate of recovery slows slightly due to early growth followed by a transient decline that precedes ultimate recovery ) . when the initial population density varies ( indicating the initial degree of maladaptation where a = 0.3 ) , ( b ) shows the rate of recovery with the initial radius fixed ( as in fig . 5a where the radius = 1 and d / r = 1 ) . in this case , extinction will occur without evolution not only for an initial density below the initial allee threshold , but for any density as the initial radius is below the critical patch size . hence , a nonzero rate implies evolutionary rescue and a zero rate means extinction . as shown in fig . 7b , when evolutionary rescue is possible , the initial level of maladaptation ( a0u0 ) and the genetic variance ( ) also determine the rate at which evolutionary rescue proceeds . figure 7b uses parameters ( i.e. , radius and ratio of d to r ) for a population that would decline and go extinct without evolution regardless of the initial density . hence , it is clear that the amount of time required for a population to begin growing depends on its initial level of maladaptation ( to both the critical density and spatial thresholds ) and/or genetic variance . as the rate at which this rescue occurs depends on the amount of genetic variance ( eqn . 2 ) , it may take an extremely long time ( as 0 , the rate of recovery 0 ) for the allee threshold to fall below the population density . in this circumstance , as the population density becomes very close to zero , the rate of change of the allee threshold is greater than that of the population density ( as u 0 , u/t 0 and a/t -2ra ) . thus , theoretically , rescue would always occur ( gomulkiewicz and holt 1995 ) . however , to maintain biological realism , when solving this system numerically , we always considered the population extinct when the maximum density ( across space , when diffusion is included ) becomes reasonably close to zero ( i.e. , u = 0.0001 ; we chose this protocol instead of the total population across space due to the diffusion dynamics based on the gaussian dispersal kernel and the pushed wave front behavior ) . overall , the numerical results qualitatively hold for a wide range of dimensional parameter values and initial conditions with and without diffusion and in one- and two - dimensional space . results for two - dimensional space are not shown as they are qualitatively similar to the simpler , one dimensional model . from these results , it is apparent that adaptations that enable organisms to overcome the negative effects of low densities can allow the population to rebound from a trajectory toward extinction to grow to reach carrying capacity . current management strategies ( e.g. , reducing population density or size ) are based on ecological theory ( e.g. drake et al . 2005 ) , but this evolutionary ecology model suggests that adaptive evolution can enable successful establishment and that ecological considerations alone may not be sufficient . under the assumptions of an allee effect and diffusive dispersal , the idea of ecological size thresholds fits well with the ecological evidence that a large founding population is a primary cause of successful establishment ( lockwood et al . however , by incorporating evolution , we see that the situation is not quite this simple because ecological size thresholds and genetic variance can interact to determine successful establishment . as the ratio of diffusion to reproduction decreases , the spatial constraint on population growth becomes weaker , and less genetic variance is needed to rescue populations with densities below the allee threshold . as the initial spatial radius of introduction increases , furthermore , the rate of this rescue depends on the initial genetic load or maladaptation ( i.e. , how far the population density is from the allee threshold ) , as well as the amount of genetic variance . because bottlenecks during founding events do not always result in highly reduced genetic variability , even small founding populations may have sufficient genetic variation to evolve to overcome allee effects and establish , contrary to solely ecologically based predictions . additionally , we can draw several general insights about how dispersal impacts selection and evolution of allee effects in an invasion context . as species are transported from their native environment into novel habitats or simply disperse on their own , it is clear that the genetic composition of the local population can influence the rate of evolution and adaptation to the new local conditions . given enough genetic diversity , local populations can adapt to their local environment , but dispersal may hinder survival across ecological clines as dispersers tend to be maladapted to the new local environment . essentially , local population persistence depends on the race between the rate of evolution and the degree of maladaptation ( gomulkiewicz and holt 1995 ) . in this case , gene flow will play a major role in determining the outcome . as kirkpatrick and barton ( 1997 ) and garca - ramos and kirkpatrick ( 1997 ) demonstrate , individuals moving from one selection regime from the center of their species range to the periphery can introduce enough maladaptation that the new area becomes a sink environment . on the other hand , holt et al . ( 2003 , 2004 ) show that immigration can have a positive influence on adaptation to sink environments , in some circumstances . resolving the disparity between these perspectives requires understanding what is contributing to the severity of maladaptation and the population 's ability to overcome it . in our model , dispersal impacts survival ecologically through the critical patch size , and genetically , as individuals may move from areas where they are well adapted ( i.e. , the population density is greater than the allee threshold or mean trait value ) to sink regions , where they are maladapted . as individuals disperse across space , they may be contributing positively in an ecological sense to the quality of their new local environment ( by increasing the local population density ) . however , dispersers are more likely to come from higher density areas where allee effects , and hence selection , are locally weak . these dispersers potentially introduce more maladaptation to their new location , because they increase the average phenotype ( allee threshold ) in the new location where density is likely to be lower . interestingly , the evolutionary impacts of migration in this model do not dramatically influence the dynamics . changes to the local mean phenotype through local selection and simple mixing ( i.e. , diffusion ) actually slightly hastens the evolutionary rescue effect over a model that considers only the impact of local selection . as the selection intensity is density dependent and proportional to u 1 for each point in space , the peripheral individuals faced with stronger selection with lower trait values have a small positive influence on the more dense neighboring populations . the gradient term accounts for asymmetric gene flow due to differential migration from areas of relatively high population densities . however , this term does not alter the overall evolutionary dynamics based on local selection any more than adding the diffusion term , as the negative effects of gene flow and the local rate of evolution ( which is relatively fast , based on the selection intensity ) essentially cancel each other out . in this context , similar to that of holt et al . ( 2003 , 2004 ) , the immigrants simply contribute to the local population density , which helps prevent extinction long enough for evolutionary rescue to occur locally ( i.e. , positive population growth ; note that whereas holt et al . ( 2003 ) attribute the main effect of immigration to the contribution of variation , this is not the case in our model , as we assume constant genetic variance ) . ( 2004 ) where immigration has a demographic effect on increasing fitness that can essentially outweigh the the primary determinant of invasion success depends on positive population growth at the center of the introduced range . this result comes from the allee effect [ and the solution to the pde ( 1 ) ] by forcing a pushed travelling wave front ( lewis and kareiva 1993 ) , where the wave speed causes population expansion , contraction , or propagation failure ( i.e. , pinning ; keitt et al . 2001 ) . intuitively , aggregation - like behavior emerges based on the strength of the allee effects . individuals that disperse too far from the whole are likely to die before they can pull others in their vicinity . in this regard , growth occurs from the inside out , where the population seemingly spills out and overflows to expand its range . consequently , in this study , and for biological invasions that exhibit similar dynamics , it is more important to focus on the center of the invader 's range and whether the initial beachhead can survive ( through evolutionary rescue ) , than the fate of peripheral populations at the wave front when determining the importance of evolution on invasion success . the overall dynamics are qualitatively similar in the parameter space that allows for evolutionary rescue to occur . even though gene flow and spatial structure do not dramatically influence the establishment of an introduced population , additional invasion criteria need to be considered . when analyzing the model behavior in a spatially explicit context , there is an ecological tradeoff between growth and spread that affects establishment and the rate of recovery . as previously mentioned , reproduction needs to compensate for the loss due to diffusion . including evolution and suppressing allee effects , actually contributes to the acceleration of the wave front ( i.e. , enhancing dispersal speed ) . a population then can more rapidly disperse as it evolves , and may become more of an invasion threat as long as this range expansion does not reduce their density too quickly . whereas this increasing wave speed can lead to a slightly longer lag phase prior to positive population growth , the population will likely be inevitably rescued because this effect primarily influences the dynamics at the periphery and is offset by the reduction in the critical invasion area ( rmin ) . although there is no range contraction ( as there is always a positive wave velocity with unbounded expansion due to the parameter values and absence of environmental heterogeneity or range limitations ; filin et al . 2008 ) , as the critical patch size ( rmin ) becomes smaller with the decreasing allee threshold , rescue occurs more readily at the range center as the critical patch size threshold criteria weakens and the behavior approaches that of the nonspatial model . this may seem like an oversimplification of the global dynamics ; however these conclusions are valid in the context of this investigation which focuses on the establishment phase rather than subsequent range expansion and spread . recognizing that evolution can significantly affect the establishment success of invasive species is becoming more widely accepted , influencing the ways in which invasion biologists conduct their research ( see the other articles in this issue ) . specifically , adaptations that diminish allee effects and evolutionary responses to density dependence are beginning to emerge as viable explanations for sustaining vulnerable populations at low density and size ( gascoigne et al . as it is difficult to conclusively support this claim empirically ( as the origin of the adaptation or the associated cost may be unknown ; courchamp et al . 2008 ; gascoigne et al . 2009 ) , mathematical models that incorporate evolution and compare the effects of various strategies ( e.g. , mitigating component allee effects ) can help decipher the mechanisms that both limit and facilitate population growth . two such models that incorporate adaptations to component mate - finding allee effects compare the efficiency and survival of populations at various densities that attract mates with or without a sexual pheromone ( jonsson et al . another study suggests that broadcast spawners that evolve their gamete morphology and performance under sperm limitation ( at low density ) bear a cost of decreased fitness at high density due to hybridization and competition ( levitan 2002 ) . in these cases , particular strategies are shown to influence population viability in addition to an associated tradeoff , whereas our investigation provides broad , albeit simplistic , results dealing with generalized demographic allee effects and evolution . in order to understand how the results of this simplistic model extend to more realistic and complex evolutionary scenarios , spatially explicit , individually - based stochastic simulation of the introduced populations should be developed to investigate more closely the mechanisms that allow these population level dynamics to emerge . in particular , tracking the mean value of a component allee effect is sufficient to illustrate how evolution can overcome inverse density dependence and result in invasion . however , this approach may not be sufficient to make the specific quantitative predictions necessary for management of invasive species . this is due to the simplifying assumption of constant genetic variance based on mutation - selection balance ( lande 1976 ) . complex simulations could relax this assumption and permit genetic variation to change via mutation , selection , and drift , in tandem with the demographic processes in a heterogeneous environment , and explicitly investigate the costs associated with avoiding allee effects . hence , future models should incorporate how propagule pressure ( size and frequency of introduction events ) impacts genetic variation and how more realistic genetic architectures contribute to the evolutionary trajectory of invasive species . although there is still much more work to be done to elucidate the factors that determine establishment success of founder populations , this theoretical approach has the promise to provide evidence in support of our working hypothesis that adaptive evolution can mitigate allee effects and be an important driver of biological invasions .

the mechanisms that facilitate success of an invasive species include both ecological and evolutionary processes . investigating the evolutionary dynamics of founder populations can enhance our understanding of patterns of invasiveness and provide insight into management strategies for controlling further establishment of introduced populations . our aim is to analyze the evolutionary consequences of ecological processes ( i.e. , propagule pressure and threshold density effects ) that impact successful colonization . we address our questions using a spatially - explicit modeling approach that incorporates dispersal , density dependent population growth , and selection . our results show that adaptive evolution may occur in small or sparse populations , providing a means of mitigating or avoiding inverse density dependent effects ( i.e. , allee effects ) . the rate at which this adaptation occurs is proportional to the amount of genetic variance and is a crucial component in assessing whether natural selection can rescue a population from extinction . we provide theoretical evidence for the importance of recognizing evolution in predicting and explaining successful biological invasions .

genital infection due to chlamydia trachomatis is one of the most prevalent bacterial sexually transmitted infections ( stis ) . according to the who estimates , globally 92 million new cases of c. trachomatis infection occur each year and about two - thirds of these cases occur in the developing world , where diagnostic and treatment services are scarce [ 2 , 3 ] . most epidemiological data on chlamydia trachomatis infection ( cti ) is from industrialized nations and reliable data from the resource poor developing nations is not available where the disease burden is concentrated . however , it is important to document laboratory - confirmed incidence and prevalence of cti from the developing world as well . the available indian data show a wide variation in ct prevalence with infection rates in indian women ranging from 3.3% to 33% depending on the population sampled [ 413 ] . infection with this agent is usually asymptomatic in up to 80% of women which makes diagnosis and detection all the more difficult . left undetected and untreated the infection may evolve into pelvic inflammatory disease and may result in serious sequelae , such as ectopic pregnancy and infertility [ 14 , 15 ] . cti in women has also been linked to adverse pregnancy outcomes like recurrent miscarriage and preterm labor and may cause conjunctivitis , nasopharyngitis , and pneumonia in newborns by vertical transmission . because the infection is easily treatable with antibiotics , early detection and treatment of infected individuals are the key to prevent adverse sequelae among those infected and reduce c. trachomatis transmission . thus , it is important to screen adolescents and sexually active women for cti even if they are asymptomatic [ 17 , 18 ] . but , in the developing countries with the exception of sporadic testing , screening for chlamydia is rarely done . epidemiological studies have also shown that untreated genital chlamydia infection can lead to an increased risk for heterosexual acquisition of hiv . hence , screening for cti done in high risk populations can assist in designing hiv risk reduction strategies . on the other hand , immunosuppression due to hiv may lead to more aggressive chlamydia disease conditions like pid in hiv seropositive women . thus , screening for cti in hiv seropositive women is highly recommended to prevent morbidity associated with the disease and devastating clinical consequences . different diagnostic modalities for detection of cti like serology , culture method , elisa for antigen and antibody , direct fluorescence assay ( dfa ) and nucleic acid amplification tests ( naats ) have been used in the last 20 years but none of them are 100% sensitive . table 1 shows the prevalence of cti detected by using different diagnostic techniques in new delhi population . polymerase chain reaction is an accurate , rapid , and reliable method for the detection of chlamydia trachomatis . real - time pcr has increasingly been used and is easier to perform and faster , and since it is performed in a closed system it is less prone to contamination than the conventional pcr . keeping the above background in mind this study was undertaken to generate reliable data regarding prevalence of cti in hiv - infected and hiv - uninfected women by real - time pcr , the most sensitive and specific test available currently for diagnosing genital chlamydia infection . the primary objective of this study was to establish the need for screening hiv seropositive women for cti . study subjects were recruited as follows : thirty adult hiv seropositive women with symptoms suggestive of rtis ( study group a1),thirty adult hiv seropositive women without symptoms suggestive of rtis ( study group a2),thirty age and sex matched adult hiv seronegative women with symptoms suggestive of rtis ( control group b1),thirty age and sex matched adult hiv seronegative women without symptoms suggestive of rtis ( control group b2 ) . thirty adult hiv seropositive women with symptoms suggestive of rtis ( study group a1 ) , thirty adult hiv seropositive women without symptoms suggestive of rtis ( study group a2 ) , thirty age and sex matched adult hiv seronegative women with symptoms suggestive of rtis ( control group b1 ) , thirty age and sex matched adult hiv seronegative women without symptoms suggestive of rtis ( control group b2 ) . all the study subjects were enrolled from the integrated counseling and testing center ( ictc ) for hiv / aids , department of microbiology , maulana azad medical college , which is attached to the lok nayak hospital , new delhi . this study was conducted prospectively between july 2010 and january 2011 at the hiv molecular laboratory of the department of microbiology , maulana azad medical college . this was a cross - sectional analysis to determine the prevalence of chlamydia trachomatis infection by using real - time pcr in hiv seropositive and seronegative , symptomatic , and asymptomatic women visiting the ictc of our department . subjects were enrolled in this study following institutional ethical committee clearance and a written informed consent of all participants . each participant was interviewed using a questionnaire concerning general sociodemographic information , personal details , and clinical symptoms . subjects having one or more of the following symptoms were considered symptomatic for rti : vaginal discharge , vesicular and/or nonvesicular genital ulcers , inguinal bubo , lower abdominal pain , genital skin conditions , urinary burning or frequency , dysmenorrhea , menorrhagia , and intermenstrual bleeding . vesicular and/or nonvesicular genital ulcers , lower abdominal pain , genital skin conditions , urinary burning or frequency , dysmenorrhea , menorrhagia , and intermenstrual bleeding . each study subject then underwent a general physical , per abdomen , per speculum , and per vaginum examination . diagnosis of hiv infection was done by following the standard protocol at our ictc that employs pretest and posttest counseling and obtains informed consent before hiv testing . three different rapid tests were used to detect hiv-1 and hiv-2 antibodies ( combaids ( span diagnostics ltd . ) , retrocheck hiv ( qualpro diagnostics ) , and tri - line ( rapid diagnostics ) ) following the manufacturer 's instructions . one endocervical swab was collected from all participants according to the instructions provided in the specimen collection and transport kit ( amplicor std swab collection and transport set ) for detection of chlamydia trachomatis by real - time pcr . genital c. trachomatis infection was diagnosed by using cobas taqman ct test , v2.0 , an in vitro nucleic acid amplification test for the qualitative detection of chlamydia trachomatis dna in female endocervical swab specimens . specimens were processed using the amplicor ct / ng specimen preparation kit for manual specimen preparation and the cobas taqman 48 analyzer for automated amplification and detection ( roche diagnostics ) . the age and gender profile of all the participants is shown in table 2 . in both the hiv seropositive study group and the hiv seronegative control group the mean age of hiv - infected cases was found to be 30.92 5.7 years and in hiv - uninfected controls 28.52 6.9 years . table 3 shows the presenting complaints of the symptomatic women in both the hiv seropositive study group and the hiv seronegative control group . vaginal discharge and lower abdominal pain were the most common presenting complaints amongst the symptomatic participants . table 4 shows the sti / rti syndromes diagnosed in study subjects on per speculum examination . vaginitis was the most common syndrome diagnosed in both the study and the control groups . table 5 shows the correlation between presence of symptoms and cti in the study and the control groups . chlamydia trachomatis infection was more commonly diagnosed in the asymptomatic hiv seropositive study subjects as compared to the symptomatic hiv seropositive study subjects . in our study the prevalence of cti was higher in hiv seropositive women as compared to hiv seronegative women ( or 4.214 ; 95% ci 0.45738.865 ) and among the hiv positive asymptomatic as compared to the hiv negative asymptomatic ( or 2.111 ; 95% ci 1.6062.776 ) , although the differences were not found to be statistically significant ( table 6 ) . in india stis / rtis and hiv / aids are major public health problems . incidence and prevalence data have a key role in control strategies for hiv and stis . comprehensive baseline information on the epidemiology of stis is essential for the design , implementation , and monitoring of successful control programs to reduce their incidence . routine surveillance of stis / rtis is not carried out in our country due to the lack of laboratory diagnostic facilities , limited resources , stigma , and discrimination associated with stis and poor attendance of sti patients , especially women , in sexually transmitted disease ( std ) clinics . taking the above facts into consideration , it is meaningful to have genuine laboratory - confirmed data on the incidence / prevalence of rtis / stis in india . as the asymptomatic nature of stis is well known , there is a need to adopt a specific strategy for the screening of the sexually active population in india to reduce the overall rate of stis , which would , in turn , reduce the risk of hiv infection . our study presents an insight into the prevalence data of genital chlamydia infections in hiv - infected and hiv - uninfected women visiting the ictc of new delhi 's largest tertiary care hospital . the mean age of the hiv seropositive subjects in our study was 30.92 5.7 and that of the hiv seronegative control group was 28.52 6.9 . this compares well with the findings of another study done in baroda , india , to look for the prevalence of rtis in hiv - infected women wherein the mean age for hiv positive women was 30 and that for hiv negative women was 27 . the predominant age group in the hiv positive participants in our study was 2635 years . this association of chlamydia infection with younger age is consistent with studies from other developing countries [ 32 , 33 ] . this finding supports the fact that young sexually active adults should constitute a priority target group in the sti control program . however , the findings of our study can not be generalized to all sexually active adults as the study has its limitations of a small sample size and the study participants belonging to a high risk group being recruited from the ictc of our department . 80% of our hiv positive study participants were married which is quiet similar to what has been reported by another study from sub - saharan africa in which 85.3% of the hiv - infected women were married . this finding highlights the importance of concurrently screening and treating spouses / sexual partners to decrease the sti burden in the country but this is challenging due to the lack of knowledge and cooperation from husbands especially in the indian setup . in the present study , vaginal discharge and lower abdominal pain were the 2 most frequently reported symptoms by the symptomatic women in both the study and the control groups , confirming the data reported by some previous indian studies [ 22 , 29 ] . another notable observation of our study was that vaginitis was the most common clinical finding detected on per speculum examination in both the hiv positive ( 50% ) and the hiv negative groups ( 56.67% ) which is in accordance with the observations of garg et al . and balamurugan and bendigeri where a majority of women on clinical examination had vaginitis of 94.6% and 36.9%% , respectively [ 22 , 35 ] . the present study also validates the fact that chlamydia infections are usually asymptomatic as 10% of the asymptomatic women were diagnosed with cti while only 3.3% of symptomatic women had lab - confirmed cti in the hiv positive study group . once again this emphasizes the importance of routine screening of at risk young sexually active women . in our study cti was detected in 6.67% ( 4/60 ) hiv - infected women and in 1.67% ( 1/60 ) hiv - uninfected women by real - time pcr although this difference was not statistically significant but the odds ratio was 4.214 . a study from cuba reported c. trachomatis infection in 10% of hiv - infected cases and 6.6% of hiv - uninfected cases by nested pcr but the difference was not statistically significant while the odds ratio was 3.39 which is in concordance with our study . studies conducted by natividad - villanueva et al . in usa and seck et al . in senegal reported c. trachomatis infection in 3.33% and 2.1% of hiv - infected females , respectively [ 38 , 39 ] . larger studies are required to validate our observation as our study was restricted to a small number of cases . this study clearly shows that cti is more prevalent among hiv - infected females ( with or without symptoms of rti ) as compared to hiv - uninfected females . our study also stresses the usefulness of screening asymptomatic hiv - infected and hiv - uninfected females for cti by risk assessment and diagnostic testing periodically to prevent the occurrence of adverse outcomes associated with the disease and also to check further spread of infection in the community . for countries like india that still do not currently have an active chlamydia screening program in place , randomized controlled trials are required to delineate the benefits of screening in the sexually active population as our study participants represent a high risk group . it is also important that any test adopted in a national screening program be used in the primary care setting by practitioners without the need for expensive training and equipment . however , since use of real - time pcr is not feasible in most hospitals in developing countries efforts should be made to develop a simple , cost - effective , sensitive , and specific point of care test to identify and treat women with cti for prevention of sequelae and hiv transmission .

reproductive tract infection ( rtis)/sexually transmitted infections ( stis ) are recognized as a major public health problem , particularly due to their relationship with hiv infection . early detection and treatment of chlamydia trachomatis infection ( cti ) among hiv - infected and hiv - uninfected women may impact heterosexual hiv transmission . a total of 120 participants were enrolled : 30 hiv seropositive women with symptoms of rtis , 30 hiv seropositive women without symptoms of rtis , 30 hiv seronegative women with symptoms of rtis , and 30 hiv seronegative women without symptoms of rtis . one endocervical swab was collected from all participants and cti was detected by real - time pcr ( cobas taqman ct test , v2.0 ) . cti was detected in 4 ( 6.67% ) hiv - infected women and in 1 ( 1.67% ) hiv - uninfected woman ( or 4.214 ; 95% ci 0.45738.865 ) . vaginal discharge was present in almost half of hiv - infected and hiv - uninfected women ; lower abdominal pain was present in 11 ( 18.3% ) of hiv - infected and in 9 ( 15% ) of hiv - uninfected women . this study showed that cti is more prevalent among hiv - infected females as compared to hiv - uninfected females . as the use of real - time pcr is not feasible in most hospitals , efforts should be made to develop a simple , sensitive , and specific test to identify women with cti for prevention of sequelae and hiv transmission .

multilevel cervical spondylotic myelopathy ( mcsm ) is a serious disease which can lead to spinal cord dysfunction and a substantial decrease in quality of life . in addition , mcsm requiring multilevel cervical spine surgery can be accompanied by extensive blood loss . as spinal surgery has become increasingly complex , so control of perioperative bleeding has become an important clinical issue for spine surgeons . excessive blood loss can lead to a range of comorbidities such as anemia , hypotension , hematoma formation , and inadequate oxygenation of organs , thereby affecting patient outcome . excessive blood loss often requires allogeneic blood transfusion , the risks of which are numerous and among which immunological reactions and transmission of viruses are considered the most serious . in addition , hematoma formation within a few millimeters of the spinal canal can cause considerable neurological damage . although occurrences of postoperative spinal hematoma formation requiring emergency surgery are rare , it is nevertheless important to control perioperative bleeding to decrease its incidence . tranexamic acid ( txa ) is a synthetic derivative of the amino acid lysine which operates through competitive inhibition of the activation of plasminogen to plasmin by binding specific sites on both plasminogen and plasmin , thereby retarding fibrinolysis , the degradation of blood clots . it has been widely used in various medical fields such as cardiac surgery , gynecology , dentistry , urological surgery , and liver transplantation to reduce the perioperative blood loss , as it is relatively inexpensive and has not been cited in the literature as causing any significant untoward side effects . in addition , txa has been reported to reduce blood loss and transfusion requirements during orthopedic surgery , most commonly in knee and hip joint replacement . although the benefits of txa in spinal surgery have been reported indeed , there are no studies of txa in cervical laminectomy with lateral mass screw fixation and bone grafting ( clf ) so far . clf has been widely used for many years for treating mcsm caused by multilevel cervical spinal cord compression . although some authors suggest that laminoplasty is superior to laminectomy with lateral mass screw fixation and bone grafting regarding preserved range of motion , indications for the use of clf are broader than that of laminoplasty and recent studies showed good outcomes for clf . thus , the aim of this study is to evaluate the efficacy and safety of txa in controlling blood loss during clf for the treatment of mcsm . a retrospective comparative analysis was performed in patients with mcsm undergoing clf of vertebrae c3 to c6 . patients with cirrhosis of the liver , serious cardiac disease , chronic renal failure , cancer , allergy to txa , a history of thromboembolic disease ( deep vein thrombosis , ischemic heart disease , pulmonary embolism , transient ischemic attack , strokes , or subarachnoid hemorrhage ) , bleeding disorders , hypercoagulation status , disseminated intravascular coagulation , pregnancy , combined anterior and posterior spinal fusions , patients receiving antiplatelet and/or anticoagulant therapy at the time of the study , and treatments of vertebrae outside of c3 to c6 were excluded from the study . between november 2014 and april 2016 , 119 clfs fitting the inclusion criteria were performed in our center . each surgeon ( hao , liu , he , wu , wang , zheng , and zhao ) in this study had more than 20 years of experience in spinal surgery . the control group consisted of 46 patients who underwent clf for the treatment of mcsm in our institution without being given txa . in both groups , we analyzed patient demographic trait ( age at surgery , gender , body weight , height , and body mass index ) and duration of surgery which was defined as the time from the initial incision to the completion of wound closure . the quantity of intraoperative and postoperative blood loss and the preoperative and day 1 postoperative hematological data for each patient were obtained in the 2 groups . total blood loss was calculated as the sum of intraoperative and postoperative blood loss ( the quantity during the 1st 16 hours ) . the spinous processes , lamina , and lateral mass facet complexes were exposed using a standard posterior midline opening after longitudinally dividing the nuchal fascia in line with a midline skin incision . laminectomy was performed using a high speed matchstick burr to drill troughs in the bone at the lateral edge of the lamina on each side , prior to complete removal of the entire lamina and associated ligamentum flavum of the target vertebrae ( c3c6 ) . the excised lamina and spinous processes were cleaned of soft tissue and cut into pieces for use as autograft material . lateral mass screws were placed bilaterally on vertebrae c3 to c6 then fixed with rods using the margerl technique . all patients in the txa group were given a dose of 15 mg / kg of txa ( transamin ; daiichi pharmaceutical , tokyo , japan ) before a skin incision was made , followed immediately with a maintenance dose of 100 mg / hour , and continued until wound closure . no patient in the control group received intraoperative administration of txa or any other antifibrinolytic drug . continuous variable data were presented as mean and standard deviation while categorical variable data were presented as a number and its specific value . statistical differences between the 2 experimental groups were compared using a chi - square test or fisher exact test for categorical variables and student t test for continuous variables . all analyses were performed using ibm spss statistics for windows , version 19.0 ( ibm corp . , united states of america ) . the spinous processes , lamina , and lateral mass facet complexes were exposed using a standard posterior midline opening after longitudinally dividing the nuchal fascia in line with a midline skin incision . laminectomy was performed using a high speed matchstick burr to drill troughs in the bone at the lateral edge of the lamina on each side , prior to complete removal of the entire lamina and associated ligamentum flavum of the target vertebrae ( c3c6 ) . the excised lamina and spinous processes were cleaned of soft tissue and cut into pieces for use as autograft material . lateral mass screws were placed bilaterally on vertebrae c3 to c6 then fixed with rods using the margerl technique . all patients in the txa group were given a dose of 15 mg / kg of txa ( transamin ; daiichi pharmaceutical , tokyo , japan ) before a skin incision was made , followed immediately with a maintenance dose of 100 mg / hour , and continued until wound closure . no patient in the control group received intraoperative administration of txa or any other antifibrinolytic drug . continuous variable data were presented as mean and standard deviation while categorical variable data were presented as a number and its specific value . statistical differences between the 2 experimental groups were compared using a chi - square test or fisher exact test for categorical variables and student t test for continuous variables . all analyses were performed using ibm spss statistics for windows , version 19.0 ( ibm corp . , united states of america ) . there was no statistical difference in demographic values ( patients age , gender , body weight , height , and body mass index ) between the 2 groups ( table 1 ) . no statistically significant difference in duration of surgery was observed between the 2 groups , with a mean time of 155.72 15.59 and 153.52 11.91 minutes in the control and txa groups , respectively ( p = 0.387 ) . there was significantly less intraoperative blood loss in the txa group compared to the control ( fig . 1 ) , at 179.66 81.45 and 269.13 94.68 ml , respectively ( p < 0.001 ) . the txa group had significantly less postoperative blood loss during the 1st 16 hours compared to the control group ( 108.08 44.31 vs 132.83 49.39 ml , p = 0.005 ) . total blood loss in the control group ( 401.96 127.88 ml ) was significantly higher than that in the txa group ( 287.74 115.40 ml , p < 0.001 ) . changes in blood hemoglobin ( hgb ) content and hematocrit ( hct ) were not statistically significantly different between the 2 groups : preoperative hgb : 138.28 9.12 g / l ( control ) and 140.42 10.05 g / l ( txa ) ; postoperative hgb : 122.35 10.74 g / l ( control ) and 125.21 11.29 g / l ( txa ) ; preoperative hct : 41.78 2.62% ( control ) and 42.07 2.08% ( txa ) ; and postoperative hct : 36.50 3.22% ( control ) and 37.32 2.41% ( txa ) . although the hgb and hct values in the txa group were all higher than those of the control group , the differences were not statistically significant ( fig . no patient required an allogeneic blood transfusion during or after the surgery in either group and no serious intra- or postoperative complications , for example , dural tear , infection , epidural hematoma formation , deep - vein thrombosis , pulmonary embolism , allergic reaction , renal failure , or cardiopulmonary complications were observed in either group . furthermore , no minor side effects associated with the use of txa such as nausea , vomiting , headache , or diarrhea occurred in either of the groups . demographic parameters of patients in each group . the txa ( tranexamic acid ) group had significantly less intraoperative , postoperative , and total blood loss compared to the control group . there were no significantly different in blood hgb hct between the control and txa groups . hgb = hemoglobin , hct = content and hematocrit , txa = tranexamic acid . numerous studies have supported the use of txa in orthopedic surgery . although it has been evaluated in many studies , the safety and efficacy of txa for blood loss reduction in spinal surgery has not been clearly demonstrated , especially for clf . clf for the treatment of mcsm has proved to be a successful technique for restoring normal cervical lordotic alignment , recovering neurological function , and decreasing the morbidity of c5 palsy and axial pain . the principal disadvantage of this approach is a significant decrease in motion due to the fixation of the target vertebrae . laminoplasty allows a better range of neck motion although it becomes more restricted in flexion and extension over time . for patients with mcsm combined with simple instability or correctable kyphosis , clf is a more suitable method than laminoplasty , even if the consequence is neck stiffness . fibrinolysis increases transiently when patients undergo surgery , and it has been shown that it contributes to perioperative blood loss during spinal surgery . txa inhibits fibrinolysis by blocking the lysine - binding sites of plasminogen , plasmin , and tissue plasminogen activator . since fibrinolysis is activated immediately during and after surgery , txa should be administered before surgery begins . the half - life of txa is approximately 80 minutes in patients with normal renal function . pharmacokinetic evidence suggests a loading dose of 10 to 15 mg / kg followed by a maintenance dose of 1 mg / kg / hour or repeated dosing . li et al conducted a meta - analysis of 6 randomized controlled trials and believed that higher txa dosage ( 15 yang et al conducted a meta - analysis of 9 randomized controlled trials which had a similar sample to li 's study . the 2 meta - analyses arrived at a similar conclusion although the quantity of blood loss and incidence of blood transfusion were different . elwatidy et al used a high single dose ( 30 mg / kg ) of txa and found that it reduced total blood loss and incidence of blood transfusion . raksakietisak et al reported that 2 doses of txa ( 15 mg / kg ) reduced perioperative blood loss and incidence of blood transfusion among low - risk adult patients undergoing elective complex thoracolumbar spine surgery . , we evaluated the effects of txa with a dose of 15 mg / kg followed by a maintenance dose of 100 mg / hour . the txa treatment group lost significantly less blood than that of the control group , including during the intra- and postoperative periods . total blood loss in the txa group was 28% less than that of the control group . although the hgb levels and hct in the txa group were higher than that of the control group , the differences were not significant , possibly because the sample size was too small and the perioperative blood loss in clf relatively minor . a theoretical anxiety associated with the use of txa is its potential for inducing thromboembolic complications . however , many studies have shown that the administration of txa does not increase this risk . in our study , there were no clinical symptoms or signs of thromboembolic events , such as deep - vein thrombosis or symptomatic pulmonary embolism . thus , the use of txa in patients undergoing clf should be regarded as safe . additionally , we are unsure whether the complexity of the surgery has an effect on the benefit of txa . a prospective randomized controlled study would better determine the safety and efficacy of txa during laminectomy with lateral mass screw fixation and bone grafting . in this study , blood loss ( both intra- and postoperative ) in the txa group was significantly lower than that in the control group , and no major intraoperative complications occurred . our results indicate that the use of intravenous txa is both safe and effective in reducing blood loss in clf . prophylactic txa may provide the benefit of limiting excessive blood loss in posterior approach cervical spinal surgery . a future prospective randomized controlled trial will provide superior evidence of the efficacy and safety of txa .

abstractto assess the safety and efficacy of tranexamic acid ( txa ) for decreasing perioperative blood loss in cervical laminectomy with lateral mass screw fixation and bone grafting ( clf ) , in which all surgical procedures are identical.from november 2014 to april 2016 , we performed a retrospective comparative analysis of 119 patients with multilevel cervical spondylotic myelopathy who had undergone a clf from c3 to c6 in our center . all surgeries were performed on the patients using a consistent , standard procedure . patients were divided into control ( 46 ) and txa ( 73 ) groups according to whether or not they had received txa treatment before and during surgery . demographic profiles of patients such as gender , age , body weight , height , and body mass index were collated and differences between the 2 groups compared . preoperative and postoperative hematological data in addition to intraoperative and postoperative blood loss were compared between the 2 groups . additionally , any complications of txa were also evaluated to assess safety.there was no statistically significant difference in demographic traits between the 2 groups . intraoperative blood loss in the txa group ( 179.66 81.45 ml ) was significantly lower than that of the control group ( 269.13 94.68 ml , p < 0.001 ) , as was postoperative blood loss ( 108.08 44.31 and 132.83 49.39 ml , respectively ; p = 0.005 ) . total blood loss in the txa group ( 287.74 115.40 ml ) was also significantly lower than that of the control group ( 401.96 127.88 , p < 0.01 ) . no major intraoperative complications occurred in any of the cases.txa significantly reduced perioperative blood loss in clf with no major side effects .

hip fracture is a moderate musculoskeletal trauma that mainly affects the older population with comorbid conditions . the number will increase markedly in coming years due to the ageing of the population . comorbidity and the double trauma may dispose them to serious postoperative adverse outcomes and a high mortality dominated by cardiovascular events [ 25 ] . myocardial injury may be difficult to diagnose because of impaired communication , limitations of clinical manifestation and non - specific electrocardiographic ( ecg ) changes [ 6 , 7 ] . the isoenzyme myocardium - specific creatine kinase ( ck - mb ) is expressed in the myocardium and ck in the skeletal muscle cells . these two enzymes have traditionally been analysed in plasma to distinguish myocardial injury and skeletal muscle injury [ 8 , 9 ] . troponins have been shown to be more specific and sensitive to cardiac injury [ 10 , 11 ] . increased plasma levels of troponin have also been reported in pulmonary embolism , septicaemia and following major orthopaedic and cardiac surgery . in those conditions high plasma troponin levels have been associated with severe adverse outcomes and increased mortality [ 7 , 1215 ] . in this study we wanted to test the hypothesis that fatal outcome following hip fracture the study was approved by the regional ethics committee and national medical authorities and conducted in accordance with the helsinki declaration . a total of 302 consecutive patients over 75 years of age with dislocated hip fractures were enrolled in the study at elverum ( ech ) and buskerud ( bch ) central hospitals during the period 20052009 . comorbidity was routinely assessed according to the classification of the american society of anesthesiologists ( asa ) . a hemiprosthesis was inserted through a lateral incision and fixed with or without bone cement ( landos titan or landos corail , depuy , warsaw , in , usa ) . thromboprophylaxis ( low molecular weight heparins ) was administered routinely preoperatively ( on hospital admission ) with dalteparin 5000 iu s.c . blood samples were obtained from an antecubital vein , collected preoperatively ( 1 ) and postoperatively [ within 24 hours ( 0 ) ] and on days one [ 2448 h ( + 1 ) ] and four ( + 4 ) . troponin t ( tnt ) and ck - mb were measured by electrochemiluminescence immunoassay ( elica , roche , basel , switzerland and abbott , abbott park , il , usa ) . ck was measured by absorption photometry ( roche , basel , switzerland and abbott , abbott park , il , usa ) . values for tnt were only measured at the ech study centre ( n = 146 ) . differences between mortality groups were tested using a two - sample t test or chi - square test for continuous or categorical data , respectively . non - parametric mann - whitney tests were done for tnt due to skewed distribution . a linear mixed model for repeated measurements with a random intercept and bonferroni adjusted pairwise post hoc comparisons were used to analyse the biochemical markers with respect to mortality and repeated venous blood analyses . univariate and stepwise multivariate logistic regression analyses were used to estimate unadjusted and adjusted odds ratios with respect to survival at the three month follow - up . results from multivariate logistic regression were based on models including only significant terms from stepwise regression to maximise the number of patients included . there were 229 women and 72 men , with a mean age of 84.7 ( sd 5.1 ) and 83.7 ( sd 4.7 ) years , respectively . by three months , 59 of 302 ( 19.5% ) had died , 62% women and 38% men . table 1demographic and clinical characteristics of patients at 3-month follow - upcharacteristicsalive ( n = 243)dead ( n = 59)p valuesage , years ( mean sd)84.1 ( 5.1)86.2 ( 4.6)0.004sex female193 ( 79.4%)37 ( 62.7%)0.005 male50 ( 20.6%)22 ( 37.3%)mobility not mobile37 ( 15.8%)10 ( 17.2%)0.109 living aid23 ( 9.8%)5 ( 8.6% ) crutches51 ( 21.8%)21 ( 36.2% ) no aid123 ( 52.6%)22 ( 37.9%)asa score i10 ( 4.2%)0 ( 0.0%)<0.001 ii103 ( 42.9%)9 ( 15.3% ) iii116 ( 48.3%)38 ( 64.4% ) iv11 ( 4.6%)12 ( 20.3%)asa american society of anesthesiologistsminor deviations between total number of patients in categories compared to number alive or dead are due to missing data . missing data are assumed to be completely at random demographic and clinical characteristics of patients at 3-month follow - up asa american society of anesthesiologists minor deviations between total number of patients in categories compared to number alive or dead are due to missing data . missing data are assumed to be completely at random asa , male sex and age were significantly associated with mortality within three months ( table 1 ) . within one day after surgery , the plasma levels of ck and ck - mb increased nearly threefold ( inverse for the ratio ) . on days 1 and + 1 , ck and ck - mb values were significantly higher among those who died compared to those who survived ( p = 0.001 and p = 0.031 , respectively ) ( fig . 1 ) . on the fourth postoperative day , tnt plasma levels rose twofold in the mortality group and remained unchanged in the alive group . the plasma levels were significantly higher ( p < 0.05 ) in the mortality group at all sampling times except at day 0 ( fig . 1mean and 95% confidence intervals for troponin t , creatine kinase ( ck ) , myocardium - specific creatine kinase ( ck - mb ) and ratio of ck - mb to ck before surgery , perioperatively and 1 and 4 days after surgery for 3-month mortality . statistical significance ( p < 0.05 ) between mortality groups is indicated by an asterisk . statistically significant ( p < 0.05 ) differences between before surgery and during follow - up are indicated by the connecting lines . day 1 1 day before surgery , day 0 within 24 h after the operation , day + 1 2448 h postoperatively , day + 4 4 days after surgery mean and 95% confidence intervals for troponin t , creatine kinase ( ck ) , myocardium - specific creatine kinase ( ck - mb ) and ratio of ck - mb to ck before surgery , perioperatively and 1 and 4 days after surgery for 3-month mortality . statistical significance ( p < 0.05 ) between mortality groups is indicated by an asterisk . statistically significant ( p < 0.05 ) differences between before surgery and during follow - up are indicated by the connecting lines . day 1 1 day before surgery , day 0 within 24 h after the operation , day + 1 2448 h postoperatively , day + 4 4 days after surgery the tnt plasma concentrations were split into three equal - sized data subsets , i.e. 0.01 , 0.010.04 and > 0.04 g / l , and the ratios of ck - mb to ck concentrations were split into two equal - sized data subsets , i.e. below and above 0.02 . / l before surgery correlated with three month mortality [ odds ratio ( or ) 10.9 , 95% confidence interval ( ci ) 2.254.0 , p = 0.003 ] ( table 2 ) . stepwise multivariate logistic regression with age , sex , asa category , levels of tnt and ratio of ck - mb to ck concentrations were performed . high tnt plasma concentration was associated with increased mortality ( or 6.1 95% ci 623.1 , p = 0.008 ) at day four after surgery . no statistically significant association was found for the ratio of ck - mb to ck during the entire sampling period when adjusted for age , sex and asa ( table 3 ) . similar regression analyses were done for ck and ck - mb . on day + 1 , the ck - mb was associated with mortality ( or 1.1 , 95% ci 1.021.2 , p = 0.012 ) . we found that our hypothesis , stating no predictive value from cardio - muscular plasma enzymes with regard to early mortality in patients with hip fracture , was false . table 2results from univariate logistic regression models predicting 3-month mortality ( death)variablesunadjusted or ( 95% ci)p valuesage1.1 ( 1.01.1)0.004if male2.4 ( 1.34.4)0.006asa3.7 ( 2.26.1)<0.001if ck - mb / ck > 0.02 day 11.6 ( 0.83.4)0.225 day 00.7 ( 0.31.6)0.413 day + 11.5 ( 0.82.9)0.239 day + 41.2 ( 0.62.4)0.620tnt day 10.011.0 ( reference)0.010.041.5 ( 0.46.0)0.532>0.0411.0 ( 2.254.6)0.0030.011.0 ( reference ) day 00.010.041.5 ( 0.45.4)0.556>0.042.9 ( 0.711.0)0.1270.011.0 ( reference ) day + 10.010.041.6 ( 0.47.4)0.512>0.046.0 ( 1.721.1)0.0050.011.0 ( reference ) day + 40.010.043.3 ( 0.715.0)0.126>0.047.0 ( 1.925.6)0.003or odds ratio , ci confidence interval , asa american society of anesthesiologists , ck - mb / ck ratio of myocardium - specific creatine kinase to creatine kinase , day 1 1 day before surgery , day 0 within 24 h after surgery , day + 1 2448 h postoperatively , day + 4 4 days after surgerytable 3results from multivariate stepwise logistic regression models predicting 3-month mortality ( death)variablesday 1day 0day + 1day + 4adjusted or ( 95% ci)p valuesadjusted or ( 95% ci)p valuesadjusted or ( 95% ci)p valuesadjusted or ( 95% ci)p valuesage1.1 ( 1.01.2)0.0061.1 ( 1.01.2)0.0061.1 ( 1.01.2)0.0011.1 ( 1.01.2)0.027if male2.4 ( 1.24.6)0.0132.4 ( 1.24.60.0132.7 ( 1.45.1)0.002asa3.3 ( 2.05.6)<0.0013.3 ( 2.05.6)<0.001ck - mb / cktnt 0.011.0 ( reference) 0.010.041.9 ( 0.49.4)0.450 > 0.046.1 ( 1.623.1)0.008or odds ratio , ci confidence interval , asa american society of anesthesiologists , ck - mb / ck ratio of myocardium - specific creatine kinase to creatine kinase , day 1 1 day before surgery , day 0 within 24 h after surgery , day + 1 2448 h postoperatively , day + 4 4 days after surgery results from univariate logistic regression models predicting 3-month mortality ( death ) or odds ratio , ci confidence interval , asa american society of anesthesiologists , ck - mb / ck ratio of myocardium - specific creatine kinase to creatine kinase , day 1 1 day before surgery , day 0 within 24 h after surgery , day + 1 2448 h postoperatively , day + 4 4 days after surgery results from multivariate stepwise logistic regression models predicting 3-month mortality ( death ) or odds ratio , ci confidence interval , asa american society of anesthesiologists , ck - mb / ck ratio of myocardium - specific creatine kinase to creatine kinase , day 1 1 day before surgery , day 0 within 24 h after surgery , day + 1 2448 h postoperatively , day + 4 4 days after surgery this prospective study on 302 elderly patients with hip fracture disclosed that 19.5% had died within three months . preoperatively obtained basic patient information was shown to be of particular importance to assess the risk of postoperative mortality . asa score on comorbidity , male sex and age correlated significantly with three month mortality , also described by other investigators [ 1719 ] . autopsy studies have shown that cardiovascular events are the main cause of death after hip fracture surgery [ 20 , 21 ] . autopsies are rarely done today and the direct cause of postoperative death has not been possible to establish in this or in other recently conducted studies . in the elderly perioperative myocardial ischaemia is often clinically silent , without haemodynamic or notable ecg changes [ 2225 ] . over the years , biochemical plasma markers have been used to distinguish myocardial injury from skeletal muscle damage , an approach also adopted in this study . potential heart muscle damage was investigated in this study with ck - mb and tnt analyses . we found small and inconsistent differences for ck and ck - mb plasma levels between those who died and those who survived . the ratio of ck - mb to ck was decreased from hospital admission ( day 1 ) to day 0 and stabilised , indicating that skeletal muscle damage dominated and that any myocardial injury remained undetected . this analysis suggests that ck and ck - mb are unspecific enzymes that are not distinct for skeletal and cardiac muscle damage following a hip fracture and are not feasible as prognostic markers of mortality , a finding consistent with other investigators . release of troponins into the circulation is considered to specifically reflect cardiac injury . in our study we analysed tnt and found that the plasma concentration was significantly higher on the fourth postoperative day in patients that subsequently died compared to those who were alive . g / l had a six times higher risk of dying vs those with normal plasma levels . this calculation was robust when correction was done for age , sex and comorbidity ( asa score ) . these results fit with other reports that showed a second wave of troponin elevation several days after surgery which correlated with postoperative mortality [ 13 , 29 ] . in summary , this study showed that basic clinical information on sex , age and comorbidity ( asa score ) and a high postoperative plasma concentration of tnt > 0.04 g / l are robust predictors of three month postoperative mortality in the elderly undergoing hip fracture surgery . this information may be of importance for therapeutic and post - hospital health care intervention .

hip fracture , a moderate musculoskeletal trauma , is associated with a high postoperative mortality . most patients are elderly , with comorbid conditions and often with heart disease . the objective of this study was to find out if clinical parameters and analyses of specific muscle enzymes could predict three month postoperative mortality . a total of 302 patients above 75 years of age with hip fracture were consecutively enrolled . baseline information on age , sex and comorbidity assessed with the american society of anesthesiologists ( asa ) score was obtained before surgery . creatine kinase ( ck ) , myocardium - specific creatine kinase ( ck - mb ) and troponin t ( tnt ) were analysed from venous blood , collected the day before surgery ( 1 ) and postoperatively , within 24 hours ( 0 ) and on days one ( + 1 ) and four ( + 4 ) . the overall three month mortality was 19.5% . multivariate analyses showed that age , male sex and comorbidity ( asa ) correlated with mortality ( p = 0.027 , p = 0.002 , p < 0.001 , respectively ) . surgery induced a two- to threefold increase of ck and ck - mb but without any correlation with mortality . however , high tnt levels > 0.04 g / l correlated significantly with death ( days 1 , + 1 and + 4 , p = 0.003 , p = 0.005 and p = 0.003 , respectively ) . multivariate analyses , adjusted for age , sex and asa category , confirmed this correlation ( day + 4 , p = 0.008 ) . thus , in elderly patients with comorbidities undergoing hip fracture surgery information on sex , age , asa category and postoperative laboratory analyses on tnt provide the clinicians with useful information on patients at risk of fatal outcome .

a young man was referred with a chief complaint of blurred vision and pain of the left eye 1 month after herpetic encephalitis in the left frontotemporal lobe . the patient had multiple foci of retinitis in the retinal periphery associated with vitritis , blot retinal hemorrhage and retinal arteriolitis . the impression of acute retinal necrosis was confirmed by polymerase chain reaction of aqueous humor by detecting herpes simplex virus type 2 ; therefore , the patient received intravenous acyclovir . herpetic encephalitis may be a risk factor for acute retinal necrosis . the virus may reach the eye by the trans - axonal route . acute retinal necrosis ( arn ) is one of the clinical presentations of herpes simplex virus ( hsv ) . this disease may present several years after a primary infection , or it may occur following systemic herpetic infection such as herpetic dermatitis . the prevalence of the disease is equal in both sexes and it occurs in the 5th-7th decade of life . american uveitis society criteria for the diagnosis of arn syndrome are : one or more foci of retinal necrosis with discrete borders located in the peripheral retina , rapid progression in the absence of antiviral therapy , circumferential spread , occlusive vasculopathy with arteriolar involvement , prominent vitritis , and anterior chamber inflammation . optic neuropathy , scleritis , and pain are supportive but not required . in most patients , the diagnosis is made clinically . polymerase chain reaction ( pcr ) analysis of the aqueous humor can detect the cause of arn specifically . pcr is very helpful in the detection of the varicella zoster , hsv type 1 and 2 . patients with arn due to hsv-1 and varicella zoster virus tend to be older , whereas those with arn due to hsv-2 tend to be younger [ 3 , 4 ] . in this article , we report one case of unilateral arn 1 month after the herpetic encephalitis . the patient is a 25-year - old man who was admitted with a chief complaint of decreased vision and pain in left eye for the previous 4 days . the patient also complained of perception of floating objects in the visual field of the left eye . the patient had no past medical history indicating an immunocompromised state ; he had been admitted to the neurology ward due to herpetic encephalitis 1 month previous to the occurrence of visual symptoms . herpetic encephalitis diagnosis was made by brain mri ( fig . 1 ) and pcr analysis of the cerebrospinal fluid . he did not report any underlying disease or drug consumption except oral acyclovir 800 mg every 8 h. at physical examination , the right eye was normal . in the left eye , visual acuity was 0.1 , papillary reflex was normal and marcus gunn sign was negative . slit lamp examination of the left eye revealed conjunctival hyperemia and ciliary injection , diffuse fine keratic precipitates in the corneal endothelium , 3 + cellular reaction in the anterior chamber , 3 + cellular reaction in the vitreous without snowball or snowbank opacities , or vitreous hemorrhage . in fundoscopic examination , multiple foci of retinitis and retinal necrosis 2 ) associated with arteriolar involvement ( arteriolitis ) , blot retinal hemorrhage and optic disc hyperemia . pcr analysis of aqueous humor revealed hsv-2 , so the patient was admitted with the diagnosis of arn and received intravenous acyclovir for 10 days ( 10 mg / kg / day ) . after 48 h of antiviral therapy , a systemic corticosteroid ( prednisolone 1 mg / kg / day ) was introduced and subsequently tapered over several weeks to treat active inflammation . the patient also received aspirin to treat an associated hypercoagulable state . with this treatment , no new lesion developed and opacity of the media decreased ; so prophylactic barrier laser photocoagulation in the areas of healthy retina at the posterior borders of the necrotic lesion prevented retinal detachment . intravitreal antiviral agents such as ganciclovir and foscarnet were not used in this patient because the disease was controlled by intravenous acyclovir . after discharge from hospital , oral acyclovir 400 mg every 5 h was started . during follow - up , the inflammation decreased and the patient had no vitreous traction or retinal detachment or other eye involvement . the brain and retinal involvement were on one side ; after 1 month of left frontotemporal lobe involvement of the brain , the retina was involved on the left side . some of the studies report arn several years after neonatal and infantile herpetic encephalitis [ 5 , 6 ] ; therefore , herpetic encephalitis may be a risk factor for arn development . other articles report that the virus accesses the retina from the brain by the trans - axonal route ; consequently , this virus can cause recurrent episodes of arn [ 8 , 9 ] . these reports may necessitate a prophylaxis with antiviral agents after herpetic encephalitis to prevent arn . in a study by pavsio et al . , the role of long - term prophylaxis with acyclovir in children who had herpetic encephalitis is an important issue . prophylaxis could be considered after hsv encephalitis to prevent arn or after arn to prevent second eye involvement . acyclovir used as prophylaxis for recurrent genital hsv infection in adults for 5 or more years has been associated with minimal toxicity and the selection of resistant strains has not been demonstrated , but there is little experience with the duration of prophylaxis that would have been necessary to prevent arn in our patient . with this report we reemphasize the correlation between these two diseases . on the other hand , arn may be an indication of possible central nervous system involvement and neuroimaging may be necessary in all cases of arn to rule out herpetic encephalitis .

purposeto report a case of unilateral acute retinal necrosis after herpetic encephalitiscase reporta young man was referred with a chief complaint of blurred vision and pain of the left eye 1 month after herpetic encephalitis in the left frontotemporal lobe . the patient had multiple foci of retinitis in the retinal periphery associated with vitritis , blot retinal hemorrhage and retinal arteriolitis . the impression of acute retinal necrosis was confirmed by polymerase chain reaction of aqueous humor by detecting herpes simplex virus type 2 ; therefore , the patient received intravenous acyclovir.conclusionherpetic encephalitis may be a risk factor for acute retinal necrosis . the virus may reach the eye by the trans - axonal route . prophylaxis with acyclovir may be necessary after herpetic encephalitis to prevent acute retinal necrosis .

inspection of the database of clusters of orthologous groups of proteins ( cogs ) revealed only one family of such proteins that is represented in most of the sequenced bacterial , archaeal and eukaryotic genomes . the prototype of this family is the rhomboid ( rho ) protein from drosophila melanogaster , a developmental regulator involved in epidermal growth factor ( egf)-dependent signaling pathways [ 2 - 4 ] . not only were homologs of rhomboid detected in prokaryotes and eukaryotes , but the pattern of sequence conservation in this family appeared uncharacteristic of nonenzymatic membrane proteins , such as transporters . specifically , several polar amino - acid residues are conserved in nearly all members of the rhomboid family , suggesting the possibility of an enzymatic activity . as three of these conserved residues were histidines , it has been hypothesized that rhomboid - family proteins could function as metal - dependent membrane proteases . recently , however , it has been shown that rho cleaves a transmembrane helix ( tmh ) in the membrane - bound precursor of the tgf-like growth factor spitz , enabling the released spitz to activate the egf receptor , and that a conserved serine and a conserved histidine in rho are essential for this cleavage . thus , it appears that rhomboid - family proteins are a distinct group of intramembrane serine proteases . altogether , the genome of drosophila encodes seven rho paralogs ( now designated rho1 - 7 , with the original rhomboid becoming rho-1 ) , at least three of which are involved in distinct egf - dependent pathways , apparently through proteolytic activation of diverse ligands of the egf receptor . the newly discovered intramembrane proteolytic activity of rho places the rhomboid family within the framework of regulated intramembrane proteolysis ( rip ) , a new paradigm of signal transduction , which appears to be prominent in all forms of life . under rip , signaling proteins undergo site - specific proteolysis within tmh , resulting in the release of active fragments , which are the actual effectors in signal tranduction cascades . until recently , the only characterized cases of rip in eukaryotes involved presenilin-1 , an aspartyl protease , which cleaves a transmembrane helix in type-1 membrane proteins such as amyloid -precursor protein ( app ) , notch and ire1 , and the metalloprotease s2p , which cleaves a tmh in a type-2 transmembrane protein , the sterol - dependent transcription factor srebp . notably , s2p has highly conserved bacterial homologs , and the protease domain of presenilins also might be homologous to bacterial and archaeal type iv prepilin peptidases , although , in this case , the sequence similarity is low . in the case of the rhomboid family , the existence of homologs of rho in most prokaryotes is particularly remarkable because animal rho proteins are involved in signaling pathways that are not found outside metazoa , which seems to make functional conservation in prokaryotes a remote possibility . the only prokaryotic protein of the rhomboid family that has been characterized experimentally in considerable detail is aara from the bacterium providencia stuartii . this protein is involved in the export of a quorum - sensing peptide , a function that , in physiological terms , resembles that of rho , although the signaling molecules , other than rho and aara , are obviously unrelated . in a striking recent development , two independent research groups have shown that several bacterial rhomboid - family proteins , including aara , can cleave the egf receptor ligands ( spitz , keren and gurken ) that are normally cleaved by rho paralogs . the cleavage depended on the conserved serine and histidine residues and , moreover , transgenic flies that expressed aara developed a phenotype indistinguishable from that induced by overexpression of rho , whereas rho could substitute for aara in providencia stuartii . these unexpected findings demonstrated the conservation of a rip mechanism producing extracellular signals in eukaryotes and prokaryotes . eukaryotic rhomboid family proteins seem to show considerable functional variability ; in particular , cross - talk might exist between different rip pathways . a distinct representative of the rhomboid family has been shown to physically interact with presinilins 1 and 2 , and was accordingly named presenilins - associated rhomboid - like protein ( parl ) . the yeast ortholog of parl has been suggested to participate in the processing of cytochrome c peroxidase precursor during its import into the mitochondrion . the near ubiquity of the rhomboid family among bacteria , archaea and eukaryotes , along with the remarkable functional conservation , suggests that a signaling mechanism mediated by rhomboids might have functioned already in the last common ancestor of all extant life forms , with subsequent loss in several lineages . to address this possibility although the sequence similarity between eukaryotic and prokaryotic rhomboid family proteins is relatively low ( around 10 - 15% identity in the conserved region ) , the entire superfamily could be retrieved from the protein sequence databases within three iterations of the psi - blast program with a high statistical significance and without any false positives . the conserved core of the rhomboid family consists of six conserved tmhs ( figure 1 ) . the predicted catalytic serine is located in tmh5 , whereas the predicted catalytic histidine is in tmh7 ; tmh3 contains two additional histidines and an asparagine , which are conserved in the great majority of the rhomboid - family proteins ( figure 1 ) . the roles of these conserved residues are not known , but , given the remarkable evolutionary conservation , it seems likely that they also contribute to catalysis ; indeed , it has been shown that the conserved asparagine is required for the cleavage of spitz by rho . when examining the multiple alignment of the rhomboid superfamily proteins , we noticed that several eukaryotic members appear to be inactivated proteases , as indicated by the loss of the predicted catalytic serine or histidine ( figure 1 , and data not shown ) ; these inactivated forms could be regulators of active rhomboid proteases . several other proteins lack one or more of the conserved residues in tmh3 ; it remains unclear whether or not these are active proteases . bacterial and archaeal members of the rhomboid superfamily contain six tmh , whereas the eukaryotic members typically have an additional seventh tmh , which may be attached to the core either from the amino terminus or from the carboxyl terminus as discussed below . the phyletic distribution pattern of the rhomboid family shows that this intramembrane protease is extremely common in all three kingdoms of life , but is not necessarily essential for cell function . rhomboids are missing in the microsporidian encephalitozoon cuniculi , a eukaryotic intracellular parasite with a highly degraded genome , the archaea methanothermobacter thermoautotrophicus and thermoplasma volcanium , and several bacterial species , primarily parasites with small genomes but also species with moderately sized genomes , such as xylella fastidiosum ( see cog0705 at ) . in two instances , a representative of the rhomboid family is present in only one of a pair of relatively close genomes ( present in t. acidophilum but missing in t. volcanium ; present in the spirochete treponema pallidum but missing in the related bacterium borrelia burgdorferi ) , which suggests relatively recent , repeated losses of this gene . most of the prokaryotic species have a single gene coding for a rhomboid - family protein , although some have two or three paralogs ( see cog0705 ) ; in contrast , eukaryotes show expansion of the rhomboid family , with seven members in drosophila , and as many as 13 in arabidopsis . the multiple alignment of the 6-tmh core of the rhomboid family ( figure 1 ) was employed to construct a phylogenetic tree using the least - squares algorithm with subsequent optimization using the maximum likelihood ( ml ) method ( see materials and methods ) . only the conserved regions including the tmh and short adjacent stretches shown in figure 1 were used as the input for tree building , whereas the poorly conserved intervening regions were omitted to avoid noise from potentially misaligned residues ( except for the bayesian analysis , which used the complete alignment ; see materials and methods ) . the phylogenetic tree of the rhomboid family presents a complex and unexpected picture ( figure 2 ) . neither the eukaryotic nor the archaeal subsets of the family appear to form monophyletic clades . instead , the eukaryotic rhomboids are split between two major subfamilies , which are positioned in the midst of different prokaryotic branches ( figure 2 ) . the first subfamily , which includes six of the seven drosophila rhomboids , clusters with a distinct prokaryotic assemblage , consisting primarily of gram - positive bacteria as well as a subset of archaea ; this clade is strongly supported by bootstrap analysis ( figure 2 ) . the proteins in this group of eukaryotic rhomboids , which we designated the rho subfamily , typically have an extra tmh added carboxy - terminally to the 6-tmh core ; some of these proteins also contain ef - hand calcium - binding domains amino - terminally of the core ( figure 2 ) . the second eukaryotic subfamily , which we designated the parl subfamily , after parl , the human ortholog of drosophila rho7 , resides within a large , heterogeneous prokaryotic cluster ( figure 2 ) . within this subfamily , parl and its orthologs from other animals and from fungi have distinct domain architecture , with an extra tmh added to the amino terminus of the core , whereas the rest have only the core ( a carboxy - terminal tmh and a ubiquitin - associated domain are appended in one arabidopsis protein ; figure 2 ) . thus , the existence of two distinct subfamilies of eukaryotic rhomboids is supported by features of domain architectures that appear to comprise shared derived characters . within these two major eukaryotic subfamilies , several lineage - specific expansions of paralogs are noticeable , in insects , mammals and plants ( figure 2 ) . archaeal rhomboids are scattered over the phylogenetic tree , with two major clusters and , in addition , three isolated proteins joining different bacterial branches ( figure 2 ) . there is no indication of an affinity between any of the archaeal and eukaryotic rhomboids . although many of the bacterial rhomboids form phylogenetically coherent clusters corresponding to the established bacterial lineages , there are also several clusters that have an odd composition , such as the grouping of proteobacterial and gram - positive species ; some of these clusters are well supported by bootstrap ( see clusters 1 - 4 in figure 2 ) . this concern is particularly serious for highly divergent families of membrane proteins , such as the rhomboids , in which parallel amino - acid substitutions are likely . therefore we investigated the phylogeny of the rhomboid family in greater detail using several independent phylogenetic methods and the corresponding statistical tests . first , we assessed the robustness of the topology of the tree shown in figure 2 using the kishino - hasegawa ( kh ) test whereby the clade of interest is forced into various positions on the tree and the likelihoods of the resulting topologies are estimated . specifically , the kh test was used to evaluate two alternative topologies , in which the rho and parl subfamilies formed a clade , and two topologies , in which the rho subfamily formed a clade with archaeal rhomboids ( figure 2 and table 1 ) . each of these alternative topologies had a significantly lower likelihood than the original topology shown in figure 2 ( see table 1 ) . in addition , a tree of the rhomboid family was constructed using the bayesian inference method , which has recently become a practical alternative to the more traditional methods of phylogenetic analysis . the tree produced using the mrbayes package showed the same major clades as the tree in figure 2 ( data not shown ) ; moreover , clustering of the rho and parl subfamilies of eukaryotic rhomboids with the respective prokaryotic clades was supported by high posterior probabilities ( figure 2 ) . we also attempted to construct a phylogenetic tree of the rhomboid family by using the maximum parsimony method . the resulting tree contained the same major clades as the trees constructed using ml and mrbayes ; however , the number of parsimony - informative sites was insufficient to obtain high bootstrap support with this approach ( data not shown ) . the alternative phylogenies reflected two distinct hypotheses : first , clustering of the rho and parl subfamilies of eukaryotic rhomboids with the prokaryotic rhomboid families as suggested by the tree topology in figure 2 ; and second , monophyly of the eukaryotic rhomboids ( figure 3 ) . the phylogenies corresponding to these alternative hypotheses were compared to the best phylogeny using three statistical tests ( table 2 ) . the hypothesis 1 tree was not significantly different from the best tree under any of these tests whereas the hypothesis 2 tree was significantly ( p < 0.05 ) worse than the best tree according to each of the tests ( table 2 ) . the concordance of the results obtained with several independent methods for phylogenetic tree construction and statistical analysis specifically aimed at testing the alternative hypothesis of monophyletic origin of eukaryotic rhomboids shows strong support for the major aspects of the tree topology in figure 2 and , in particular , for the polyphyly of eukaryotic rhomboids . the phylogenetic tree of the rhomboid family shown in figure 2 and supported by the additional tests described above follows neither the ' standard model ' scenario , with the major split between the archaeo - eukaryotic and bacterial lineages nor the ' mitochondrial ' scenario , which postulates acquisition of a gene by eukaryotes from the pro - mitochondrial endosymbiont . neither can this tree be explained by postulating a small number of lineage - specific gene losses . the parsimonious interpretation of the rhomboid family tree seems to be that the evolutionary history of this family had been replete with horizontal gene transfer ( hgt ) and lineage - specific gene loss events . in particular , in spite of the presence of rhomboids in the majority of modern life forms from all three primary superkingdoms , phylogenetic analysis suggests that this family has not been inherited from the last universal common ancestor ( luca ) . instead , the tree topology seems to indicate that this family emerged in some bacterial lineage and afterwards had been widely disseminated by hgt , and then lost in some lineages . in particular , at least two hgt events seem to have contributed to the origin of eukaryotic rhomboids , one of them yielding the rho subfamily and the other one the parl subfamily , with a possible additional hgt in plants ( figures 2,3 ) . given the broad phyletic representation of both subfamilies of eukaryotic rhomboids , both the rho subfamily and the parl subfamily must have been acquired through hgt at an early stage of eukaryotic evolution , definitely before the divergence of the major crown - group lineages . this early epoch in eukaryotic evolution is thought to have been dominated by hgt from multiple bacterial symbionts . an alternative to this multiple - hgt scenario is that luca already had multiple , paralogous rhomboids , which evolved by a series of ancient gene duplications , and the odd topology of the phylogenetic tree is due primarily to differential loss of these ancient paralogs . although this can not be ruled out formally , this hypothesis implies the existence of an elaborate signaling system in luca and , accordingly , suggests that luca was a complex organism , which might have had as many genes as modern bacteria . theoretical analysis of evolutionary scenarios constructed on the basis of the phyletic patterns of cogs by applying the parsimony principle shows that the complexity of the inferred gene set of luca critically depends on the relative rates of gene loss and hgt at the early stages of evolution . a complex luca with around 2,000 genes is predicted only when one assumes that the rate of gene loss is an order of magnitude greater than the rate of hgt . however , explicit reconstruction of the gene set of luca under the assumption of equal rates of gene loss and hgt leads to a hypothetical genome that consists of only around 600 genes but appears to be ' compatible with life ' , that is , it includes genes responsible for most , if not all , essential cellular functions . we currently believe that this is the most realistic , albeit inevitably imprecise , reconstruction of luca 's gene set . with respect to the rhomboid family and other families whose phylogenetic trees show similar patterns , this makes the multiple - hgt interpretation the scenario of choice . further theoretical , comparative - genomic and experimental analyses aimed at determining relative rates of gene loss and hgt will help in a more objective assessment of the validity of this argument . the multiple - hgt interpretation of the evolutionary history of the rhomboid family , while supported by the above argument , seems , at least at first glance , distinctly counter - intuitive , given that this family is nearly ubiquitous among extant life forms . indeed , when attempts are made to construct parsimonious evolutionary scenarios on the basis of phyletic patterns alone [ 31 - 33 ] , there is no chance that such a widespread family is not assigned to luca . it should be realized , however , that these approaches are inherently probabilistic , and extensive hgt can fool them . for the rhomboid family , the multiple - hgt mode of evolution seems to be particularly plausible . it seems likely that the ultimate ancestor of the rhomboid family evolved from a nonenzymatic integral membrane protein , probably a transporter that might have been involved in an early primitive form of export of signaling peptides in bacteria . the protease active center might have evolved in such a transporter by chance emergence of the suitable catalytic amino acids within two or three of the tmhs ( figure 4 ) . this would enable the transition from simple transport to the rip mode of controlled export of signaling molecules . emergence of rip could have conferred a major selective advantage on the respective bacteria and might have resulted in an evolutionary sweep whereby the gene carrying this trait was repeatedly fixed , rather than eliminated , after hgt . in terms of the evolution of sequence itself , the requirements for the conservation of the protease activity apparently ' locked ' the rhomboid family in a regime of relatively slow evolution , which ensures significant sequence similarity between all family members ( figure 1 ) . the scenario of origin from non - catalytic transporters might potentially apply to other integral membrane enzymes , including intramembrane proteases involved in rip , such as presenilins and their homologs and the archaeo - eukaryotic signal peptide peptidase . although the sequence similarity between eukaryotic and prokaryotic rhomboid family proteins is relatively low ( around 10 - 15% identity in the conserved region ) , the entire superfamily could be retrieved from the protein sequence databases within three iterations of the psi - blast program with a high statistical significance and without any false positives . the conserved core of the rhomboid family consists of six conserved tmhs ( figure 1 ) . the predicted catalytic serine is located in tmh5 , whereas the predicted catalytic histidine is in tmh7 ; tmh3 contains two additional histidines and an asparagine , which are conserved in the great majority of the rhomboid - family proteins ( figure 1 ) . the roles of these conserved residues are not known , but , given the remarkable evolutionary conservation , it seems likely that they also contribute to catalysis ; indeed , it has been shown that the conserved asparagine is required for the cleavage of spitz by rho . when examining the multiple alignment of the rhomboid superfamily proteins , we noticed that several eukaryotic members appear to be inactivated proteases , as indicated by the loss of the predicted catalytic serine or histidine ( figure 1 , and data not shown ) ; these inactivated forms could be regulators of active rhomboid proteases . several other proteins lack one or more of the conserved residues in tmh3 ; it remains unclear whether or not these are active proteases . bacterial and archaeal members of the rhomboid superfamily contain six tmh , whereas the eukaryotic members typically have an additional seventh tmh , which may be attached to the core either from the amino terminus or from the carboxyl terminus as discussed below . the phyletic distribution pattern of the rhomboid family shows that this intramembrane protease is extremely common in all three kingdoms of life , but is not necessarily essential for cell function . rhomboids are missing in the microsporidian encephalitozoon cuniculi , a eukaryotic intracellular parasite with a highly degraded genome , the archaea methanothermobacter thermoautotrophicus and thermoplasma volcanium , and several bacterial species , primarily parasites with small genomes but also species with moderately sized genomes , such as xylella fastidiosum ( see cog0705 at ) . in two instances , a representative of the rhomboid family is present in only one of a pair of relatively close genomes ( present in t. acidophilum but missing in t. volcanium ; present in the spirochete treponema pallidum but missing in the related bacterium borrelia burgdorferi ) , which suggests relatively recent , repeated losses of this gene . most of the prokaryotic species have a single gene coding for a rhomboid - family protein , although some have two or three paralogs ( see cog0705 ) ; in contrast , eukaryotes show expansion of the rhomboid family , with seven members in drosophila , and as many as 13 in arabidopsis . the multiple alignment of the 6-tmh core of the rhomboid family ( figure 1 ) was employed to construct a phylogenetic tree using the least - squares algorithm with subsequent optimization using the maximum likelihood ( ml ) method ( see materials and methods ) . only the conserved regions including the tmh and short adjacent stretches shown in figure 1 were used as the input for tree building , whereas the poorly conserved intervening regions were omitted to avoid noise from potentially misaligned residues ( except for the bayesian analysis , which used the complete alignment ; see materials and methods ) . the phylogenetic tree of the rhomboid family presents a complex and unexpected picture ( figure 2 ) . neither the eukaryotic nor the archaeal subsets of the family appear to form monophyletic clades . instead , the eukaryotic rhomboids are split between two major subfamilies , which are positioned in the midst of different prokaryotic branches ( figure 2 ) . the first subfamily , which includes six of the seven drosophila rhomboids , clusters with a distinct prokaryotic assemblage , consisting primarily of gram - positive bacteria as well as a subset of archaea ; this clade is strongly supported by bootstrap analysis ( figure 2 ) . the proteins in this group of eukaryotic rhomboids , which we designated the rho subfamily , typically have an extra tmh added carboxy - terminally to the 6-tmh core ; some of these proteins also contain ef - hand calcium - binding domains amino - terminally of the core ( figure 2 ) . the second eukaryotic subfamily , which we designated the parl subfamily , after parl , the human ortholog of drosophila rho7 , resides within a large , heterogeneous prokaryotic cluster ( figure 2 ) . within this subfamily , parl and its orthologs from other animals and from fungi have distinct domain architecture , with an extra tmh added to the amino terminus of the core , whereas the rest have only the core ( a carboxy - terminal tmh and a ubiquitin - associated domain are appended in one arabidopsis protein ; figure 2 ) . thus , the existence of two distinct subfamilies of eukaryotic rhomboids is supported by features of domain architectures that appear to comprise shared derived characters . within these two major eukaryotic subfamilies , several lineage - specific expansions of paralogs are noticeable , in insects , mammals and plants ( figure 2 ) . archaeal rhomboids are scattered over the phylogenetic tree , with two major clusters and , in addition , three isolated proteins joining different bacterial branches ( figure 2 ) . there is no indication of an affinity between any of the archaeal and eukaryotic rhomboids . although many of the bacterial rhomboids form phylogenetically coherent clusters corresponding to the established bacterial lineages , there are also several clusters that have an odd composition , such as the grouping of proteobacterial and gram - positive species ; some of these clusters are well supported by bootstrap ( see clusters 1 - 4 in figure 2 ) . this concern is particularly serious for highly divergent families of membrane proteins , such as the rhomboids , in which parallel amino - acid substitutions are likely . therefore we investigated the phylogeny of the rhomboid family in greater detail using several independent phylogenetic methods and the corresponding statistical tests . first , we assessed the robustness of the topology of the tree shown in figure 2 using the kishino - hasegawa ( kh ) test whereby the clade of interest is forced into various positions on the tree and the likelihoods of the resulting topologies are estimated . specifically , the kh test was used to evaluate two alternative topologies , in which the rho and parl subfamilies formed a clade , and two topologies , in which the rho subfamily formed a clade with archaeal rhomboids ( figure 2 and table 1 ) . each of these alternative topologies had a significantly lower likelihood than the original topology shown in figure 2 ( see table 1 ) . in addition , a tree of the rhomboid family was constructed using the bayesian inference method , which has recently become a practical alternative to the more traditional methods of phylogenetic analysis . the tree produced using the mrbayes package showed the same major clades as the tree in figure 2 ( data not shown ) ; moreover , clustering of the rho and parl subfamilies of eukaryotic rhomboids with the respective prokaryotic clades was supported by high posterior probabilities ( figure 2 ) . we also attempted to construct a phylogenetic tree of the rhomboid family by using the maximum parsimony method . the resulting tree contained the same major clades as the trees constructed using ml and mrbayes ; however , the number of parsimony - informative sites was insufficient to obtain high bootstrap support with this approach ( data not shown ) . the alternative phylogenies reflected two distinct hypotheses : first , clustering of the rho and parl subfamilies of eukaryotic rhomboids with the prokaryotic rhomboid families as suggested by the tree topology in figure 2 ; and second , monophyly of the eukaryotic rhomboids ( figure 3 ) . the phylogenies corresponding to these alternative hypotheses were compared to the best phylogeny using three statistical tests ( table 2 ) . the hypothesis 1 tree was not significantly different from the best tree under any of these tests whereas the hypothesis 2 tree was significantly ( p < 0.05 ) worse than the best tree according to each of the tests ( table 2 ) . the concordance of the results obtained with several independent methods for phylogenetic tree construction and statistical analysis specifically aimed at testing the alternative hypothesis of monophyletic origin of eukaryotic rhomboids shows strong support for the major aspects of the tree topology in figure 2 and , in particular , for the polyphyly of eukaryotic rhomboids . the phylogenetic tree of the rhomboid family shown in figure 2 and supported by the additional tests described above follows neither the ' standard model ' scenario , with the major split between the archaeo - eukaryotic and bacterial lineages nor the ' mitochondrial ' scenario , which postulates acquisition of a gene by eukaryotes from the pro - mitochondrial endosymbiont . neither can this tree be explained by postulating a small number of lineage - specific gene losses . the parsimonious interpretation of the rhomboid family tree seems to be that the evolutionary history of this family had been replete with horizontal gene transfer ( hgt ) and lineage - specific gene loss events . in particular , in spite of the presence of rhomboids in the majority of modern life forms from all three primary superkingdoms , phylogenetic analysis suggests that this family has not been inherited from the last universal common ancestor ( luca ) . instead , the tree topology seems to indicate that this family emerged in some bacterial lineage and afterwards had been widely disseminated by hgt , and then lost in some lineages . in particular , at least two hgt events seem to have contributed to the origin of eukaryotic rhomboids , one of them yielding the rho subfamily and the other one the parl subfamily , with a possible additional hgt in plants ( figures 2,3 ) . given the broad phyletic representation of both subfamilies of eukaryotic rhomboids , both the rho subfamily and the parl subfamily must have been acquired through hgt at an early stage of eukaryotic evolution , definitely before the divergence of the major crown - group lineages . this early epoch in eukaryotic evolution is thought to have been dominated by hgt from multiple bacterial symbionts . an alternative to this multiple - hgt scenario is that luca already had multiple , paralogous rhomboids , which evolved by a series of ancient gene duplications , and the odd topology of the phylogenetic tree is due primarily to differential loss of these ancient paralogs . although this can not be ruled out formally , this hypothesis implies the existence of an elaborate signaling system in luca and , accordingly , suggests that luca was a complex organism , which might have had as many genes as modern bacteria . theoretical analysis of evolutionary scenarios constructed on the basis of the phyletic patterns of cogs by applying the parsimony principle shows that the complexity of the inferred gene set of luca critically depends on the relative rates of gene loss and hgt at the early stages of evolution . a complex luca with around 2,000 genes is predicted only when one assumes that the rate of gene loss is an order of magnitude greater than the rate of hgt . however , explicit reconstruction of the gene set of luca under the assumption of equal rates of gene loss and hgt leads to a hypothetical genome that consists of only around 600 genes but appears to be ' compatible with life ' , that is , it includes genes responsible for most , if not all , essential cellular functions . we currently believe that this is the most realistic , albeit inevitably imprecise , reconstruction of luca 's gene set . with respect to the rhomboid family and other families whose phylogenetic trees show similar patterns , this makes the multiple - hgt interpretation the scenario of choice . further theoretical , comparative - genomic and experimental analyses aimed at determining relative rates of gene loss and hgt will help in a more objective assessment of the validity of this argument . the multiple - hgt interpretation of the evolutionary history of the rhomboid family , while supported by the above argument , seems , at least at first glance , distinctly counter - intuitive , given that this family is nearly ubiquitous among extant life forms . indeed , when attempts are made to construct parsimonious evolutionary scenarios on the basis of phyletic patterns alone [ 31 - 33 ] , there is no chance that such a widespread family is not assigned to luca . it should be realized , however , that these approaches are inherently probabilistic , and extensive hgt can fool them . for the rhomboid family , the multiple - hgt mode of evolution seems to be particularly plausible . it seems likely that the ultimate ancestor of the rhomboid family evolved from a nonenzymatic integral membrane protein , probably a transporter that might have been involved in an early primitive form of export of signaling peptides in bacteria . the protease active center might have evolved in such a transporter by chance emergence of the suitable catalytic amino acids within two or three of the tmhs ( figure 4 ) . this would enable the transition from simple transport to the rip mode of controlled export of signaling molecules . emergence of rip could have conferred a major selective advantage on the respective bacteria and might have resulted in an evolutionary sweep whereby the gene carrying this trait was repeatedly fixed , rather than eliminated , after hgt . in terms of the evolution of sequence itself , the requirements for the conservation of the protease activity apparently ' locked ' the rhomboid family in a regime of relatively slow evolution , which ensures significant sequence similarity between all family members ( figure 1 ) . the scenario of origin from non - catalytic transporters might potentially apply to other integral membrane enzymes , including intramembrane proteases involved in rip , such as presenilins and their homologs and the archaeo - eukaryotic signal peptide peptidase . the rhomboid family might be the most widespread and conserved group of integral membrane proteins . in and by itself , this would suggest that this family is part of the gene repertoire of luca . however , phylogenetic analysis suggests a different scenario , one of emergence in a bacterial lineage with subsequent multiple , independent hgt events and gene losses . although caution is due in the evolutionary interpretation of phylogenetic trees for large families , particularly when membrane proteins with a relatively small number of conserved positions , such as the rhomboids , are involved , the multiple - hgt scenario seemed to be supported by several methods of tree analysis and statistical tests . eukaryotes probably acquired their two major rhomboid subfamilies , rho and parl , as the result of two independent , early hgt events . these events , which might have introduced rip as a means of intercellular communication , could have been pivotal in the evolution of eukaryotic multicellularity along the lines discussed previously with regard to the apparent bacterial origin of key components of eukaryotic programmed cell death machinery . subsequent evolution of rhomboids in eukaryotes proceeded by lineage - specific expansion of paralogs followed by diversification through the addition of an extra tmh in different positions relative to the catalytic core , some limited domain accretion ( see figure 2 ) and sequence divergence . phylogenetic analysis of the rhomboid family described here carries a general message for studies aimed at the reconstruction of ancestral life forms , particularly luca . although most of the ( nearly ) ubiquitous protein families probably do derive from luca , explicit phylogenetic analysis is required to ascertain this in each case . the nonredundant ( nr ) protein sequence database at the national center for biotechnology information ( nih , bethesda ) was searched iteratively using the psi - blast program with multiple starting queries . psi - blast was normally run with expectation ( e ) value of 0.01 as the cut - off for inclusion of sequences into the position - specific scoring matrix . multiple alignments of protein sequences were constructed using the clustalw program and manually adjusted on the basis of the examination of psi - blast search outputs and the superposition of the predicted tmhs , which were identified using the programs tmpred and tmap . phylogenetic trees were built using the least - squares method implemented in the fitch program of the phylip package , with subsequent local rearrangement using the protml program of the molphy package to obtain the maximum likelihood tree . the reliability of the tree topology was assessed using the rell ( resampling of estimated log - likelihoods ) bootstrap method of molphy , with 10,000 replications . alternative placements of selected clades in maximum - likelihood trees were compared by using the rearrangement optimization method ( kishino - hasegawa test ) as implemented in the protml program [ 43 - 45 ] . maximum parsimony trees were constructed using the heuristic search option of paup * . in addition , trees were constructed by bayesian inference using the markov chain monte carlo method as implemented in the mrbayes package . the complete alignment information , including columns with gaps , was used for the mrbayes analysis . constraint trees were imported into paup * and subjected to neighbor - joining search to generate the phylogenies corresponding to alternative hypotheses . these phylogenies were compared using the kh , templeton ( wilcoxon signed - ranks ) and winning - sites ( sign ) tests implemented in paup*. l.p . is supported by a grant from the natural sciences and engineering research council of canada . the alignment includes the majority of the detected rhomboid family proteins ; some closely related sequences were omitted . only the six conserved ( predicted ) transmembrane helices ( tmh ) and short surrounding regions are shown . the boundaries of the predicted tmh are indicated by gray shading and overline and they are numbered 1 - 6 . the number of amino - acid residues in the omitted terminal and internal regions are indicated . the consensus shows amino - acid residues present in at least 90% of the aligned sequences ; h stands for hydrophobic residues ( a , c , i , l , v , m , f , y , w in the single - letter amino - acid code ) and s for small residues ( g , a , s , d , n , v ) . the proposed catalytic serine ( tmh4 ) and histidine ( tmh6 ) as well as conserved residues in tmh2 with possible ancillary roles in catalysis are highlighted in color . the proteins are identified with the gene identification ( gi ) number from the nonredundant database and an abbreviated species name . bacterial species are color - coded green , eukaryotic species blue and archaeal species yellow . species name abbreviations : aerpe , aeropyrum pernix ; agrtu , agrobacterium tumefaciens ; anoga , anopheles gambiae ; arath , arabidopsis thaliana ; arcfu , archaeoglobus fulgidus ; bacsu , bacillus subtilis ; brume , brucella melitensis ; caeel , caenorhabditis elegans ; caucr , caulobacter crescentus ; chlte , chlorobium tepidum ; cloac , clostridium acetobutilicum ; corgl , corynebacterium glutamicum ; deira , deinococcus radiodurans ; dicdi , dictyostelium discoideum ; drome , drosophila melanogaster ; escco , escherichia coli ; haein , haemophilus influenzae ; halsp , halobacterium sp . ; homsa , homo sapiens ; lacla , lactococcus lactis ; lisin , listeria innocua ; metja , methanoccocus jannaschii ; metka , methanopyrus kandleri ; metma , methanosarcina mazei ; meslo , mesorhizobium loti ; mycle , mycobacterium leprae ; myctu , mycobacterium tuberculosis ; neucr , neurospora crassa ; nossp , nostoc sp . ; prost , providencia stuartii ; pyrab , pyrococcus abyssi ; pyrae , pyrobaculum aerophilum ; ralso , ralstonia solanaraceum ; sacce , saccharomyces cerevisiae ; schpo , schizosaccharomyces pombe ; sinme , sinorhizobium meliloti ; strco , streptomyces coelicolor ; strpn , streptococcus pneumoniae ; sulso , sulfolobus solfataricus ; sulto , sulfolobus tokodaii ; synsp , synechocystis sp . ; theac , thermoplasma acidophilum ; thema , thermotoga maritima ; thete , thermus thermophilus ; vibch , vibrio cholerae ; xanca , xanthomonas campestris ; xylfa , xylella fastidiosa . the sequences and their regions used to construct the tree are exactly those shown in figure 1 . the color coding and abbreviations are as in figure 1 . the two major eukaryotic subfamilies are denoted as rho and parl ( see text ) and four clusters containing unexpected ( from a phylogenetic viewpoint ) sets of species are denoted 1 - 4 . the clades that were investigated in the kh test are denoted a through d. although the tree is shown in a pseudorooted form for convenience , this is an unrooted tree . internal nodes with at least 70% rell bootstrap supported are denoted by black circles and nodes with a 50 - 70% support by blue circles . the posterior probabilities reported by the mrbayes program are indicated for some key internal branches . the rho and parl subfamilies are denoted ; the remaining clusters include prokaryotic rhomboids designated as in figure 2 ( with ' a ' added to the gi number ) . within each cluster , the trees are unrooted , although shown in a pseudorooted form . a hypothetical scenario for the origin and dissemination of the rhomboid family proteases . the figure schematically shows the proposed three stages of evolution of the rhomboid family . in ( a ) , the progenitor of the rhomboid family functions as a transporter for a regulatory peptide in some bacterial lineage . in ( b ) , the catalytic site of the intramembrane protease evolves , allowing the switch to rip as the mechanism of the regulatory peptide release . in ( c ) , the emergence of rip is followed by a burst of hgt . the transmembrane helices of rhomboid are designated as in figure 1 ; their topology in the membrane is based on that proposed in . the catalytic histidine and serine are shown and connected by a dotted line to indicate the proposed charge - relay system of the protease ; possible ancillary catalytic residues are not shown . log - likelihood analysis of possible placements of selected branches of maximum likelihood trees for the proteins analyzed * a - d , clades that were subjected to local rearrangements in the tree as indicated in figure 2 and discussed in the text . bootstrap probability of the given tree calculated using the rell method ( resampling of estimated log - likelihoods ) . statistical comparisons of the best neighbor - joining tree with the hypothesis 1 and hypothesis 2 trees * probability of getting a more extreme test statistic under the null hypothesis of no difference between the two trees ( two - tailed test ) .

the near - universal presence of the rhomboid family in bacteria , archaea and eukaryotes appears to suggest that this protein is part of the heritage of the last universal common ancestor , phylogenetic tree analysis indicates a likely bacterial origin with subsequent dissemination by horizontal gene transfer .

an infliction in the life expectancy figure of patients with sickle cell disease ( scd ) occurred around the middle of the 1970s ( fig . this minimal increase in life expectancy coincided with my appointment at thomas jefferson university as the associate director of the newly created adult sickle cell center . the number of adults at that time was small and the transition from pediatrics to adult programs was at the age of 18 years . the trickle of patients increased gradually and we were faced with adolescent and young adult african american patients who were in a state of confusion . stripped from the protective sphere of the pediatric world and the empathy of their pediatric hematologists and the pediatric ancillary staff , they were in a state of fear , anxiety , depression and , worst of all , severe pain . the fact that most patients were barely educated , many without a high school degree , unemployed , mediocre health coverage , and dysfunctional family structure conferred a logarithmic dimension to the problem . the arrow indicates the infliction point where life expectancy of patients with sickle cell disease began to increase . the steady stream of admissions of patients with acute painful vaso - occlusive crises ( vocs ) to the emergency department ( ed ) and hospital were not welcome by most providers , hospital administration , the house and nursing staffs . there was subtle resentment of the patients that sometimes extended to the hematologists who showed compassion to the patients . soon labels such as drug addicts , drug - seeking behavior , and hospital hopping and frequent flyer emerged . listening to and believing the patients and keeping detailed records of ed and hospital admissions and the analgesics prescribed , revealed that most patients genuinely do not respond to a certain analgesic or a certain dose . increasing the doses of an analgesic or switching to another drug solved the problem in most patients . accordingly and with the approval of the institutional review board ( irb ) , i issued an identification wallet - sized , plasticized card that was carried by patients and presented to the provider treating their voc in the ed , hospital or any other medical facility . information printed on both sides of the card included : 1 ) demographic data and a recent photograph ; 2 ) hematological data including reticulocyte count ; 3 ) medical data including the type of scd , its complications and co - morbidities if present ; 4 ) all medications being taken by the patient and the recommended treatment of vocs including the name , dose , and the route of administration of the analgesics in question ; and 5 ) my name and contact information for answering questions if needed . it was not expensive to issue these cards . a polaroid camera available at that time and a laminator were the only equipments needed to issue these cards . later on , information on the card was computerized and a printed copy was given to the patient . the patients were very compliant in carrying it as faithfully as they carry their medical cards . some providers liked it very much because it facilitated having a concise history about the patients . while this controversy was brewing , interesting developments in basic science were in progress to understand the pharmacodynamics and pharmacokinetics of opioids . in the 1970s , it was hypothesized that opioids have receptors to bind to and activate in order to relieve pain by blocking or minimizing the transmission of painful stimuli and raising the pain threshold . it did not take long after that to identify opioids as ligands that bind to stereospecific and saturable receptors in the central nervous system and other tissues [ 3 , 4 ] . in addition , recent elegant studies [ 6 - 10 ] have revealed a helical structure of the opioid receptors , which forms pockets in which the corresponding opioid ( ligand ) fits snugly ( fig . recognition is highly specific , such that only l - isomers of certain opioids exert analgesic activity . physiologically , by binding to receptors , opioids initiate a series of biochemical events including activation of g proteins , inhibition of adenylate cyclase , and extrusion of potassium ions , resulting in hyperpolarization of cell membranes [ 14 - 16 ] ; this delays or prevents transmission of painful stimuli . thus , the riddle why some patients respond to one opioid but not another had a pathophysiologic explanation . ( a ) morphine - like molecule ( yellow ) in the deep pocket ( blue ) of the -opioid receptor . ( b ) -opioid receptors from an intimate pair when crystallized with a ligand ( yellow ) such as morphine . knowing how an opioid molecule ( yellow ) sticks in the pocket of its receptor ( blue ) parallel to the progress in the pharmacodynamics of opioids mentioned above , a concomitant advance in the pharmacokinetics of opioids was bubbling to the surface . phase i involves the cyp enzymes and phase ii metabolism conjugates the drug to hydrophilic substances , such as glucuronic acid , sulfate , glycine , or glutathione . morphine , hydromorphone and oxymorphone are metabolized by glucuronidation , whereas the majority of the other opioids are metabolized by the cytochrome p450 isoenzyme system . the net effect of an opioid depends on the availability of enzyme(s ) to convert it into metabolites that could be active or inactive . briefly , the cyp2d6 genotypes are categorized into phenotypes based on the activity of the variant enzymes . ultrarapid metabolizers ( ums ) have greater than normal activity due to duplication or triplication , of active alleles [ 20 - 23 ] , extensive metabolizers ( ems ) have normal enzyme activity , intermediate metabolizers ( ims ) have decreased enzyme activity , and poor metabolizers ( pms ) have absent or little enzyme activity . patients who are ums of fentanyl would rapidly convert it into inactive metabolites with minimal or absent analgesic effect requiring increasing the dose of fentanyl . on the other hand , patients who are pms of fentanyl would experience prompt relief with relatively small doses of fentanyl but higher doses could be toxic due to the accumulation of unmetabolized fentanyl . the cyp3a4 enzyme metabolizes more than 50% of all drugs ; consequently , opioids metabolized by this enzyme have a high risk of drug - drug interactions . together , current data on the pharmacodynamics and pharmacokinetics of opioids show great variability of genotypes among patients and extreme variability in individual responses to opioids . determining the pharmacogenetics profile of each patient facilitates the choice of drugs that would be efficacious for that patient and avoid those drugs associated with harmful drug - drug interaction . this approach in diagnostics and therapeutics ushers in the dawn of a new field for the management of individual patients based on their unique pharmacogenetics , phenotypic and biomarker characteristics . this future approach is referred to as personalized medicine or , more recently , precision medicine . we hope that this methodology would be approved and sponsored by the insurance companies for patients with scd . in the meantime , listening , believing and respecting the patient with sickle cell pain should be maintained for now as the approach to individualized therapy .

in the 1970s , sickle cell pain was treated with trial and error approach by increasing or decreasing the dose of an opioid or switching from one analgesic to another . this approach was controversial with criticism and doubt about its usefulness . since then , advances in determining the structure of opioid receptors and the role of the cyp450 enzymes in metabolizing opioids revealed that these anatomic and metabolic findings are not the same in all persons , thus explaining the variability in response to opioids among patients . thus , the trial and error approach has a scientific basis after all .

the potential of rnai to silence any gene has made it an attractive therapeutic modality . however , the main obstacle to rnai in the clinic is delivery . to be effective , sirnas must be transported through the body , bind , and be taken up by target cells where they must traverse the plasma membrane and gain access to the cytosolic compartment , where the rnai machinery resides . to be useful , ease of formulation and administration , overall cost , and any associated toxicities are essential considerations . dcs are heterogeneous , with subsets defined phenotypically , functionally and by location ( reviewed in ref . 2 ) . a delivery vehicle that knocks down expression of specific genes in a distinct dc population(s ) would be a valuable tool for targeting diverse diseases including cancers , infectious diseases , autoimmunity and as a vaccine component . therefore , a platform that delivers sirnas to specific dc subsets in situ would be useful for inhibiting or activating immune responses . lipid nanoparticles ( lnps ) are one of the most advanced platforms for sirna delivery to hepatocytes , and are under clinical evaluation for conditions that require hepatic gene silencing . these lnps typically contain ionizable cationic lipids ( pka ~6.5 ) that bind nucleic acids via electrostatic interactions at low ph , but are charge neutral at ph 7.4 . as a well - perfused organ furthermore , lnp uptake by hepatocytes is mediated by association with serum apoe leading to efficient uptake via low - density lipoprotein receptors in the liver . following cellular uptake of the lnp , the ionization of the lipid within acidic endosomes is thought to promote sirna escape to the cytosol . importantly , these lnps are associated with minimal toxicity , including little induction of proinflammatory cytokines following administration of physiologically relevant doses . in contrast to the liver , sirna delivery to extra - hepatic cells is challenging . in particular , immune cells such as dcs are relatively resistant to sirna uptake in vitro and in vivo . although designed for hepatic gene silencing , the efficacy of these lnps and their derivatives has been assessed for rnai - mediated gene silencing in macrophages ( mos ) and dcs in vitro and in vivo . lnp uptake and modest gene silencing was achieved at high doses of lnps ( ~ 5mg / kg ) . together with a complementary study , this work demonstrates the feasibility of gene silencing in immune cells using lnp technology . clearly , however , lnp formulations must be modified to achieve optimal gene silencing in immune cells . enhanced uptake of lnps by primary hepatocytes is mediated by apolipoprotein e binding to the neutral lnps . this results in recognition by receptors including the low - density lipoprotein receptor and scavenger receptors largely expressed on hepatocytes . the mechanism of uptake strongly suggests that retargeting of these particles to other receptors or other tissues is possible . indeed , akinc et al . demonstrated that lnps modified with n - acetylgalactosamine ( galnac ) were retargeted to the asialoglycoprotein receptor ( asgpr ) expressed by hepatocytes in vivo . for delivery to nonhepatic cells , we have shown in vitro that anti - transferrin receptor aptamers can be used to redirect similar lnps . more recently , liang et al . described a similar approach using aptamer - coated lnps to target osteoblasts in vivo . for uptake by immune cells , a recent study coated lnps with a full - length anti - cd4 antibody for sirna delivery to primary cd4 t cells . lnp uptake and gene silencing was observed in cd4 t cells in various organs including the spleen , lymph nodes , and blood . these studies demonstrate the potential for attaching exogenous ligands to lnps for delivery to hepatic , and importantly nonhepatic primary cells in vivo . drawing from these works , we reasoned that this lnp platform could be a useful foundation for the delivery of sirnas to dcs to modulate immune responses . to achieve this , we modified lnps using a single chain antibody ( scfv ) specific for the dc receptor dec205 , a c - type lectin expressed at high levels on cd8 dcs . dec205 dcs mediate cross - presentation of antigen resulting in modulation of cd8 t - cell responses . therefore , inhibition of gene expression by dec205 dcs is a potentially powerful approach for regulating cd8 t - cell activation . using this approach , we assessed the ability of dec - lnps ( lnps coated with scfv specific for murine dec205 , containing sirnas specific for costimulatory molecules ) to inhibit immune responses . injection of dec - lnps resulted in preferential uptake of the dec - lnps by splenic dec205 dcs . furthermore , when coadministered with adjuvant , lnps containing sirna targeting cd40 , cd80 , and cd86 reduced expression of these costimulatory molecules to levels similar to those observed in immature dcs . most importantly , dcs isolated from mice injected with a low dose ( ~0.6 mg / kg ) of these dec - lnps , were able to suppress a robust mixed lymphocyte reaction ( mlr ) , demonstrating the functional efficacy of this approach . interestingly , when we performed experiments using 2 ' fluoro ( 2'f ) modified sirna , a formulation reported to be nonimmunostimulatory and nonimmunogenic , significant immune activation of the targeted dcs was detected . this effect was not observed in assays performed with human peripheral blood mononuclear cells ( pbmcs ) or monocyte - derived dcs ( modcs ) and could be mitigated through selective 2-o - methyl ( 2ome ) modification of the sirna . overall , our study demonstrates the functionality of lnps modified for receptor targeting for sirna delivery to dcs . additionally our results suggest that specific cell targeting can mediate immunological responses to sirna formulations . while care needs to be taken in designing and evaluating targeted approaches using lnps , we found that 2ome modification of the sirna was sufficient to negate immune activation . our approach enhances the efficacy of sirna - mediated gene silencing by ~10-fold when compared to nontargeted delivery . therefore , this targeted strategy should prove effective for regulating multiple immune - mediated diseases . lnps containing sirnas specific either for cd80 , cd86 , cd40 , or a control ( nontarget ) sequence were synthesized by extrusion using a mixture of 1,2-distearoyl - sn - glycero-3-phosphocholine ( dspc):dlindma : dspe - peg : cholesterol at a ratio of 15:40:5:40 ( based on refs . murine anti - dec205 scfv with a c - terminal cysteine was attached to the lipid dspe - peg by a maleimide group ( figure 1a , b ) . following synthesis , we subjected lnps to quality control to ensure consistency between batches . dynamic light scattering was used to determine diameter and polydispersity of lnps , and cryo - electron microscopy confirmed lnp size and their unilamellar structure ( figure 1c , d ) . the efficiency of sirna incorporation and scfv binding to dspe - peg was also assessed ( figure 1c , e ) . to show specificity of binding to dec205 , scfv - lnps containing dy547-labeled sirna were cultured with either parental ( dec205 ) or dec205 cho cells . as seen in figure 2a , at 4 c dec205 cells bind dec - lnps approximately fourfold better compared with nontargeted lnps ( nt - lnps : lnps not coated with any scfv ) , or lnps coated with an isotype scfv ( iso - lnps ; cho - dec205 panel ) . when similar assays were performed at 37 c to allow cell uptake , cell staining increased by at least fivefold . little uptake was observed when lnps ( targeted or nontargeted ) were incubated with parental cho cells ( cho panel ) . similar results were obtained when bone marrow - derived dcs ( bmdcs ) , derived from wild - type mice ( b6 ) were incubated with dec - lnps . we note that as dec205 is expressed at lower levels on bmdcs compared with cho - dec205 , less dec - lnp binding and uptake was observed in bmdcs ( ~2-fold enhancement compared with nt- or iso - lnps ; b6 bmdc panel ) . to further confirm target - specific uptake we performed experiments using bmdcs derived from dec205 mice , and no specific cell binding or uptake the dec205 antibody is internalized via receptor - mediated endocytosis , and is targeted to late endosomes and lysosomes . confocal microscopy confirmed that similar to dec205 antibody , dec - lnps were targeted to lamp-1 late endosomes or lysosomes ( figure 2b ) . having demonstrated dec205-mediated uptake of dec - lnps in vitro , we investigated their localization in vivo . b6 mice were injected systemically ( intravenous , i.v . ) with fluorescently labeled scfv - lnps . as the dec205 receptor is endocytosed when it binds its ligand , we could not use this molecule to identify dec205 dcs . therefore , we used cd8 , a protein that is coexpressed on dec205 dcs , as a surrogate marker for tracking these cells . more than 50% of cd11ccd8 cells took up dec - lnps , and uptake was largely restricted to the cd8 dc population , with little uptake observed by cd8 dcs ( figure 3a , b ) . specificity was demonstrated by comparison of uptake of dec - lnps and iso - lnps . significant uptake was only observed in dec205 dcs following dec - lnp injection , and other splenic immune cells took up little dec- or iso - lnps ( figure 3a , c ) . when dec - lnps were injected into dec205 mice , no uptake was detected ( figure 3d ) . to show that dec - lnps were competent for specific gene silencing , we generated dec - lnps containing a sirna specific for cd80 ( having identified the most effective sirna sequence ; see supplementary figure s1 ) and injected into b6 mice . cd11cdec205 cells took up similar amounts of lnps following injection with dec - lnp containing either cd80- or control - sirna ( lnp uptake panel , mean fluorescence intensity ( mfi ) : dec - lnp - control = 443 versus dec - lnp - sicd80 = 464 ) . in mice injected with dec - lnp encapsulating cd80-specific sirna , cd80 protein expression approached basal levels , and was reduced by ~2-fold when compared with dec - lnp containing control - sirna ( figure 4a ) . a 75% reduction in cd80 mrna was observed in dec205 dcs that had taken up dec - lnps containing cd80-specific sirna ( compared with dec - lnps containing control sirnas ; figure 4b ) . we confirmed that gene knockdown was rnai - mediated using 5 race to detect sirna - directed mrna cleavage products ( figure 4c ) . sequencing of the polymerase chain reaction ( pcr ) fragment verified that it was derived from cd80 mrna and that the cut site corresponded with nucleotide positions 1011 of the sirna guide strand ( not shown ) . interestingly , while the data in figure 4 demonstrate the ability of our dec - lnps to effectively knockdown cd80 expression in dcs in vivo , we observed significant dc activation following injection of dec - lnps containing control sirnas ( figure 4a , right panel ) . this observation was surprising considering these experiments were performed using sirnas containing 2f modified rna , previously reported to have reduced immunostimulatory activity . to better assess this effect , we synthesized a series of luciferase - specific control sirnas ( luc ) containing 2oh rna ( unmodified ) , 2f pyrimidines or selected 2ome modifications ( see methods for modification strategy ; based on refs . lnps were generated and their ability to stimulate immune responses using multiple assays was examined . first , b6 mice were injected with dec - lnps that contained either unmodified , 2f or 2ome modified luc - specific sirnas in the absence of any adjuvant . one day later , splenic dcs were analyzed for the expression of costimulatory receptors ( supplementary figure s2a ) . as expected , dcs isolated from mice that had received unmodified sirnas demonstrated significant upregulation of cd40 , cd80 , and cd86 . consistent with our previous results ( figure 4a ) , animals treated with dec - lnps containing 2f modified sirnas also upregulated these costimulatory molecules . importantly , and similar to work by other groups , we found that 2ome modified sirnas induced minimal immune activation . to attempt to circumvent the need for a time and resource intensive in vivo assay , we wanted to determine whether an in vitro assay could be used to detect immunostimulatory sirnas . however , when we cultured b6-derived bmdcs with dec - lnps ( containing cd80-specific sirnas ) no difference between 2oh modified sirna , 2f or 2ome modified sirna was detected ( supplementary figure s2b ) . we also used nt - lnps to assess the effects of various sirna formulations on human pbmcs and modcs using standard preclinical assays ( supplementary figure s2c ) . pbmcs or modcs were cultured for 24 hours at which time cells were assessed for induction of apoptosis and culture supernatants were tested for the presence of proinflammatory cytokines ( see methods ) . apoptosis was not observed under any conditions tested ( data not shown ) . as expected , pbmcs cultured with the highest concentration of lnps containing unmodified sirnas elicited the secretion of several cytokines . 2f and 2ome luc sirnas induced production of one cytokine ( il8 ) , also at the highest concentration tested . no cytokine production was observed following culture of modcs with lnps encapsulating unmodified , 2f or 2ome sirnas . taken together , these experiments suggest that the assays tested show marked differences in sensitivity . surprisingly , bmdcs and modcs were the least receptive for detecting immunostimulatory sirnas . conversely , dec - lnp injection was useful for the identification of sirnas with immune - activating potential . from these results , we used sirnas containing selective 2ome substitutions for all subsequent studies . to target autoimmune diseases , reducing the expression of several costimulatory molecules therefore , we next determined the ability of dec - lnps that encapsulated 2ome modified cd86 specific sirnas , to reduce gene expression following i.v . administration ( optimal sirna identified as previously ; supplementary figure s1 ) . as the sirnas were 2ome modified , mice were coinjected with lps to stimulate expression of costimulatory molecules . analysis of splenic dec205 dcs 1 day following dec - lnp injection showed reduction of cd86 protein to near steady - state levels ( figure 5a ) . total rna was isolated from dec205 dcs that had taken up dec - lnps encapsulating either cd86 or control sirnas . an approximately 70% reduction in cd86 mrna levels was observed from dcs isolated from mice that received dec - lnps containing cd86 sirna in comparison to dcs derived from control sirna treated mice . to verify knockdown occurred via the rnai pathway , we next tested whether combining sirnas in dec - lnps would induce gene silencing equivalent to that observed with single sirnas . a combination of 2ome modified sirnas targeting cd80 , cd86 and cd40 were incorporated into dec - lnps and injected into b6 mice along with adjuvant ( lps ) to activate the dcs . reduction of each of the targeted costimulatory molecules was observed , with cd80 and cd86 knocked down to basal levels ( figure 5c ) . gene silencing was similar to that achieved following treatment with dec - lnps containing single sirnas ( e.g. , ~50% for cd86 : compare cd86 dec - lnp , figure 5a with mix dec - lnp , figure 5c right panel ) . to determine functional relevance of this knockdown , we used the robust mlr assay . b6 mice were injected with dec - lnps encapsulated with either a mix of cd80 , cd86 , and cd40 or control sirnas . after 24 hours , splenic cd8 dcs that had taken up dec - lnps were isolated , irradiated , and cultured with splenic t cells derived from a balb / c mouse . maximal proliferation was observed when dcs were isolated from mice injected with lps only , or lps plus dec - lnps containing control sirnas . in contrast , proliferation of t cells cultured with dcs derived from mice injected with lps plus dec - lnps containing cd80 , cd86 , and cd40 sirnas was significantly reduced ( figure 5d ) . currently , the most advanced lnp platforms use the ionizable cationic lipid dlindma , or its dlin - kc2-dma and dlin - mc3-dma derivatives . following systemic administration , these lnps accumulate in first pass organs , i.e. , liver and spleen , making them attractive vehicles for hepatic gene knockdown . furthermore , these lnps have been shown to safely and effectively reduce expression of hepatocyte genes in clinical trials . however , there is only very limited data demonstrating targeted delivery of potentially clinically relevant lnps to nonhepatic cells . we have shown that conjugation of a scfv , specific for the dc receptor dec205 , is sufficient to direct delivery of dlindma - formulated dec - lnps to splenic dcs . similar to conventional nt - lnps , dec - lnps incorporated sirnas with high efficiency ( > 80% ) , and were of uniform diameter ( ~100 nmol / l ) . we found that coating lnps with only ~50 scfv was sufficient for dc delivery ( figures 1 , 2 , and 3 ) . we showed that dec - lnp uptake correlated well with dec205 receptor density : dcs > b cells , t cells and macrophages ( dec205 expression is 1050-fold less than on dcs ) . use of dec205 mice further confirmed that uptake occurred via the dec205 receptor ( figures 2a and 3d ) . we also showed that the intracellular pathway accessed by dec - lnps was consistent with the dec205 pathway ( figure 2b ) . importantly , we demonstrated that targeted delivery resulted in effective rnai - mediated gene silencing of one , or several genes , to essentially basal expression levels ( figures 4 and 5a c ) . this reduction in gene expression was sufficient to inhibit a robust mlr ( figure 5d ) . coated lnps formulated with the cationic lipid ddab with full - length dec205 antibody for sirna delivery and demonstrated the ability to reduce expression of the costimulatory molecule cd40 in dec205 dcs . while some gene knockdown was achieved , this lipid is known for its adjuvant qualities , and is being developed for applications that require immune response stimulation . the lnps also proved relatively inefficient at sirna loading ( 10% compared with 85% for dlindma lnps ) . the sirnas used were also unmodified , which could serve as another possible source of immune stimulation . therefore , the potential for this formulation appears to be limited . more recently , ramishetti et al . utilized a full - length antibody targeting cd4 to enhance t - cell uptake and induce sirna - mediated gene silencing using a clinically relevant lnp formulation similar to the one we employed in our work . interestingly , both this study and the work from zheng et al . chose to utilize full - length antibodies for lnp targeting , which may limit their utility when considering the immunogenicity of whole antibodies and their rapid clearance from the circulation by fc - mediated uptake by macrophages . in fact , in preliminary studies , we coated lnps with full - length dec205 antibody and failed to observe uptake by dec205 dcs . using our dec - lnps mg / kg ) comparable to that study which used n - acetylgalactosamine ( galnac)-coated lnps to target hepatocytes via the asialoglycoprotein receptor ( asgpr ) in apoe knockout mice . in contrast , when the ability of nt - lnps to silence antigen presenting cells ( apcs ) , including dcs , was investigated a dose of 5 mg / kg was required to achieve partial silencing : ~10-fold higher when compared with our dec - lnps . furthermore , the authors used the dlindma derivative , dlin - kc2-dma , which displays improved silencing ability in hepatocytes ( relative ed50 dlin - kc2-dma : 0.1 mg / kg versus dlindma : 1 mg / kg ) . as these lnps were formulated for effective gene silencing in hepatocytes , biodistribution studies show that nt - lnps are found in the spleen , although at ~50-fold less than in the liver . while targeting ligands have been shown to have little effect on the overall biodistribution of nanoparticle formulations , they can enhance cell - specific uptake . therefore , while high doses of nt - lnps can confer partial gene silencing in apcs , at low lnp concentrations , we find that a targeted approach is required to achieve knockdown in a specific dc subset . injection of low - dose nt - lnps failed to result in detectable uptake / gene silencing . as the rnai pathway is resident in the cytoplasm , the intracellular pathway used following receptor ligation is an important consideration . we chose the well - characterized dec205 receptor for targeting our lnps . following binding , the internalized receptor is routed to late endosomes and associated cargo gains access to the cytoplasm . the dlindma lipid fuses with anionic phospholipids present in the endosomal membrane resulting in release of encapsulated cargo into the cytoplasm . however , it is likely that different routes of uptake will affect the efficiency of delivery and subsequent gene silencing . thus , a logical extension to our approach is to determine whether we observe improved gene silencing using other dc specific targeting agents and/or more effective dlindma derivatives . we are currently investigating the efficacy of dec - lnps formulated with the dlin - mc3-dma ( ed50 0.03 mg / kg ) . having demonstrated that we could efficiently target sirnas to dcs , the lack of toxicity of these lnps and their cargo had to be established . we observed significant dc activation following injection of targeted lnps containing unmodified and 2f modified sirnas , and incorporation of 2ome modifications at distinct residues was required to maintain costimulatory molecules analyzed at basal levels ( figure 4a and supplementary figure s2a ) . as injecting mice to determine immune stimulation is time and resource intensive , we assessed immune stimulation in murine and human tissue culture systems . murine bmdcs were refractory to lnp stimulation suggesting they would not serve as a useful surrogate for gauging immunogenicity of lnps . taken together , these assays demonstrate a requirement for in vivo analysis to uncover the stimulatory capacity of the lnps . recently , a human whole blood cytokine assay has been described that showed similar activation profiles elicited by lnps when compared with in vivo injection , potentially circumventing a requirement for cumbersome in vivo analyses . for the purposes of inhibiting a multifactorial immune response , such as that observed in graft rejection or autoimmunity , the simultaneous knockdown of several ( co)stimulatory genes this approach has been used to demonstrate that combining antibodies targeting cd80 and cd86 can reduce disease severity in experimental autoimmune encephalomyelitis and in antigen - induced arthritis . transfer of dcs treated ex vivo with cd40 , cd80 , and cd86 antisense oligonucleotides ( as - odn ) into nod mice delayed type 1 diabetes ( t1d ) onset . a follow - up study demonstrated that multiple injections of microspheres containing cd40 , cd80 , and cd86 as - odns were sufficient to prevent t1d , possibly due to reduced expression of the costimulatory molecules by splenic dcs . we achieve significant reduction in expression of cd40 , cd80 , and cd86 on splenic dcs using our dec - lnps following systemic delivery , and ongoing studies are addressing the durability of gene knockdown and the potential use of these dec - lnps for treating autoimmune diseases . in summary , we have shown that a clinically relevant lnp can be modified for cell - specific delivery of sirnas . in vivo administration did not produce overt inflammatory responses , and we achieved significant rnai - mediated gene - specific knockdown at low sirna doses . these attributes make this an attractive platform to warrant further investigation as a potential therapeutic agent for various autoimmune diseases . furthermore , judicious selection of the ligand coating the lnp opens up the potential for targeting multiple cell types . mice . c57bl/6 ( b6 ) and balb / c mice were purchased from taconic ( hudson , ny ) . dec205 mice were bred to homozygosity on the b6 background ( dec205 ) , and validated by pcr and flow cytometry . mice were housed in the barrier facility at the institute for animal studies , albert einstein college of medicine ( bronx , ny ) according to institutional animal care and use committee ( iacuc ) guidelines . the single - chain fragment variable ( scfv ) antibodies constructed for dec205 ( clone nldc145 ) and isotype ( clone gl117 ) were cloned from antibody constructs , as previously described . the variable regions of the heavy and light chain sequences were fused with a flexible linker ( g4s)6 separating them and cloned into a pet28a ( emd millipore , billerica , ma ) bacterial expression vector . further modifications to the constructs included a strep - tag - ii ( wshpqfek ) at the n - terminus and a 10-residue histidine tag ( h10 ) with a short flexible linker ( g4s ) near the c - terminus . a c - terminal unpaired cysteine residue was introduced for scfv conjugation to the lipid dspe - peg - mal . the scfv constructs were transformed into bl21 ( de3 ) plyss competent cells ( promega , fitchburg , wi ) . the scfv dec205 or isotype were expressed in escherichia coli strain bl21 ( de3 ) plyss as insoluble inclusion bodies . following bacterial growth in luria broth ( lb ) medium and induction of protein expression with isopropyl 1-thio - d - galactopyranoside cells were lysed , and insoluble protein pelleted by centrifugation . solubilized inclusion bodies were passed over a ni - nta column and the bound protein was eluted with 8 m guanidine hydrochloride . the purified proteins were refolded by rapid dilution in refolding buffer , and purified by size - exclusion chromatography using superdex 200 ( ge healthcare life sciences , pittsburgh , pa ) . proteins were buffer - exchanged in phosphate - buffered saline ( pbs ) and concentrated . scfvs were tested for endotoxin contamination using a kinetic chromogenic limulous amoebocyte lysate ( lal ) assay ( kinetic - qcl lal assay , lonza , walkersville , md ) . the free cysteine engineered onto the n - terminus of the scfv was reduced using tris-(2-carboxyethyl ) phosphine hydrochloride ( life technologies , carlsbad , ca ) . following buffer exchange into pbs , the reduced scfv was reacted with a 10-fold molar excess of alexafluor488 ( af488)-c5-maleimide according to manufacturer instructions ( life technologies , carlsbad , ca ) . polyacrylamide gel electrophoresis ( sds - page ) and coomassie staining and storm molecular phosphorimager scanning of the stained gel ( ge healthcare , piscataway , nj ) . preparation of lnps . lnps were formed using the following lipids at a 15:40:5:40 mol % : dspc ( avanti polar lipids , alabaster , al ) , cholesterol ( chol ; sigma - aldrich , mo ) , 1,2-distearoyl - sn - glycero-3-phosphatidylethanolamine - n-(maleimide-(polyethylene glycol)-2000 ) ( dspe - peg - mal , avanti polar lipids , alabaster , al ) , and 1,2-dilinoleyloxy-3-dimethylaminopropane ( dlindma ) synthesized by the chemical biology core facility at albert einstein college of medicine as previously described . a fluorophore conjugated to a lipid , 1,2-dioleoyl - sn - glycero-3-phosphoethanolamine - n-(lissamine rhodamine b sulfonyl ) ( dope - rhodamine b ; avanti polar lipids , alabaster , al ) was incorporated ( 0.1 % ) to monitor lnp delivery . lnps were formed using a modified version of the spontaneous vesicle formation by ethanol dilution method . the lipid mixture was extruded ( mini - extruder and 1 ml gas - tight syringes , avanti polar lipids , alabaster , al ) sequentially with polycarbonate 200 and 80 nm pore size membranes . the extruded lipid mixture was mixed via rapid pipetting and vortexing with sirna , followed by dialysis into pbs . for attachment of scfv , lnps were mixed with reduced scfv at room temperature for 1 hour , and free maleimide groups were then quenched with -mercaptoethanol ( me ) . tangential flow filtration ( microkros filter module ) with a 500 kda mwco ( spectrum laboratories , rancho dominguez , ca ) was used for buffer exchange into pbs and concentration of lnps . lnps were assayed for size and polydispersity by dynamic light scattering ( dynapro plate reader , wyatt technology , santa barbara , ca ) . cryo - electron microscopy confirmed lnp size , geometry and lamellarity ( analytical imaging facility ; albert einstein college of medicine , bronx , ny ) . sirna incorporation was determined by quant - it ribogreen rna assay ( life technologies , carlsbad , ca ) . to determine scfv conjugation efficiency , lnps were resolved on sds - page gels . a 3 kda difference in migration of the scfv was indicative of binding to dspe - peg - mal ( unconjugated scfv ~30 kda , scfv - dspe - peg - mal ~33 kda ) . a cholesterol quantification kit ( abcam , cambridge , ma ) was use to determine the total lipid concentration . based on lipid concentration , the surface area per lipid head group ( ~0.6 nm ) , lnp diameter and their unilamellar structure , ~5060 scfvs are conjugated to each lnp under the conditions outlined above . sirna synthesis . modified and unmodified individual sirna sequences were synthesized on an expedite 8909 dna synthesizer ( biolytic , fremont , ca ) and purified by standard protocols , as described . dylight 547-phosphoramidite was attached to the 5 end of the sense strand for synthesis of fluorescently labeled sirna ( dy547-sirna ; glen research , sterling , va ) . sirnas were modified by 2f substitutions at every pyrimidine or by 2ome substitutions at the bolded nucleotides : cd40 sense 5-gaagauuaucccggucauauu-3 ; cd40 anti - sense 5-uaugaccg ; ggauaaucuucuu-3 ; cd80 sense 5-gaauuaccuggcaucaauauu-3 ; cd80 anti - sense 5-uauugaugccagguaauucuu-3 ; cd86 sense 5-caacuggacucuacg acuuuu-3 ; cd86 anti - sense 5-aagucguagaguccaguuguu-3 ; control sense 5-uagcgacuaaacacaucaauu-3 ; control anti - sense 5-uugauguguuuaguc gcuauu-3. luciferase sense 5-gauuauguccgguuauguauu-3 ; luciferase anti - sense 5-uacauaaccggacauaaucuu-3. lnp binding , uptake , and intracellular localization . cho cells , cho cells that stably express dec205 ( cho - dec ) , a dec205 b lymphoma line ( a20 cells ) and bmdcs , derived from b6 and dec205 mice , were used to assess lnp binding and uptake . cells were grown in complete medium ( dulbecco 's modified eagle 's medium , 5% fbs , 10 mmol / l 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid , 20 mmol / l l - glutamine ) . bmdcs were generated from bone marrow derived from the femur and tibia as previously described . adherent cells ( a20 , cho ) were harvested using ethylenediaminetetraacetic acid to preserve receptor expression and were incubated at 4 c , 30 minutes with 500 nmol / l scfv - lnps ( containing either 0.1% dope - rhodamine b or cy3-labeled sirnas ) . for uptake , cells were washed with fluorescence - activated cell sorting buffer ( pbs containing 0.5% bsa , 0.1% na n3 , 2 mmol / l edta ) and analyzed by flow cytometry ( lsrii , becton dickinson , franklin lakes , nj ; flowjo , ashland , or ) . to monitor intracellular localization , a20 cells were incubated with scfv - lnps , containing sirnas labeled with dylight 547 ( ge dharmacon , lafayette , co ) , 1 hour , 37 c . following one wash with pbs , cells were pipetted onto poly - l - lysine coated coverslips and allowed to adhere . adhered cells were fixed with 4% paraformaldehyde , washed with pbs and stained with anti - lamp1 ( clone 1d4b ; ebioscience , san diego , ca ) and anti - eea1 ( clone c45b10 ; cell signaling technology , danvers , ma ) . stained cells were mounted with vectashield mounting media containing 4,6-diamidino-2-phenylindole ( dapi ) ( vector laboratories , burlingame , ca ) and samples were visualized on a leica sp5 aobs confocal microscope ( leica , buffalo grove , il ) . image processing ( linear adjustments to brightness and contrast ) was performed on image files using nih imagej software ( bethesda , md ) . lnps ( 0.61 mg / kg ) were injected ( retroorbital ( r.o . ) ) into b6 mice and 25 ng ultrapure lps ( invivogen , san diego , ca ) was injected r.o . one day later , spleens were harvested , collagenase treated to maximize dc yield , and single cell suspensions subjected to red cells lysis . cells were stained with cell surface markers and analyzed by flow cytometry to determine lnp uptake and expression of costimulatory molecules . antibodies were used for detection of the following cell surface markers : cd11c , cd8 , dec205 , cd40 , cd80 , cd86 , cd19 , b220 , cd3 , tcr , f4/80 , cd11b , h2k , dcir2 , cd49b , cd45.1 . cell viability was monitored using a live / dead exclusion dye , i.e. , live / dead blue ( life technologies , carlsbad , ca ) . cells were acquired using an lsrii cytometer and data analyzed using flowjo software . to measure gene knockdown , cd11ccd8 splenocytes that had taken up lnps ( i.e. , sirna - dy547 ) were sorted by flow cytometry ( facs aria iii , becton dickinson , franklin lakes , nj ) into rnaprotect ( qiagen , valencia , ca ) to stabilize rna . total rna was isolated using the rneasy rna isolation kit ( qiagen , valencia , ca ) , reverse transcribed using superscript iii ( life technologies , carlsbad , ca ) and random hexamers ( idt , coralville , ia ) , according to manufacturer 's instructions . quantitative pcr was performed using platinum taq polymerase ( life technologies , carlsbad , ca ) and sybr green to detect amplified products on an iq5 icycler ( biorad , hercules , ca ) . reactions were performed in triplicate with the following primers : u6 forward : 5-ctcgcttcggcagcacatatacta-3 ; u6 reverse : 5-acgaatttgcgtgtcatccttgcg-3 ; cd40 forward : 5-cggctgtgcgcgctatg-3 ; cd40 reverse : 5-ggctgagaattcgcctgagtc-3 ; cd80 forward : 5-ctactctcttatcatcctgggcct-3 ; cd80 reverse : 5-cccggaagcaaagcaggtaatcct-3 ; cd86 forward : 5-gccacccacaggatcaattatcct-3 ; cd86 reverse : 5-aaagagagaggctgttggagatac-3. relative amounts of mrna were normalized to u6 mrna and primer specificity verified by melt curve analysis . to detect rnai - mediated cleavage products , total rna was isolated from cd11ccd8 splenocytes that had taken up lnps , i.e. , sirna - dy547 ( as for gene knockdown by qpcr ) , ligated at the 5 end to an adapter rna oligonucleotide and first strand cdna was synthesized using a gene - specific primer ( gsp ) . pcr amplification using an adapter - specific primer and a nested gsp primer will amplify the cleavage product and identify the cut site which corresponds to position 10/11 of the sirna guide strand if mediated via rnai . the primers and oligonucleotide sequences used : adapter rna : 5-cgacuggagcacgaggacacugacauggacugaaggaguagaaa-3 ; gsp forward : 5-cgactggagcacgaggacactga-3 ; cd80 gsp reverse : 5-tgcgccgaatcctgccccaa-3 ; cd80 reverse nested : 5-atcaggagggtcttctgggg-3 ; cd86 gsp reverse : 5-agtaactgaagctgtaatctccttccaata-3 ; cd86 reverse nested : 5-ctgtgacattatcttgtgatatctgcatgt-3. analysis of immune stimulation . cd11c murine bmdcs ( day 6 ; macs purified , miltenyi biotec , san diego , ca ) were cultured with dec - lnps and monitored for activation ( cd40 , cd80 , cd86 ) 1 day later by flow cytometry . in vivo stimulation was assessed by injection of dec - lnps r.o . into b6 mice , followed by analysis of activation markers ( cd40 , cd80 , cd86 ) on splenic dcs after 24 hours by flow cytometry . the pbmc fraction was used directly or following purification of cd14 cells ( stemcell technologies , vancouver , british columbia , canada ) , monocytes were cultured for 5 days in the presence of granulocyte - macrophage colony - stimulating factor and il4 to generate modcs . nt - lnps ( 5 , 1 , 0.2 , 0.04 , 0.008 mol / l ) were cultured with pbmcs or modcs overnight and cell culture supernatants collected for cytokine analysis ( ifn , il1 , il2 , il4 , il5 , il6 , il7 , il8 , il10 , il12 , il13 , tnf , granulocyte - macrophage colony - stimulating factor ) using a luminex multiplex format ( life technologies carlsbad , ca ) . cd11ccd8 splenic dcs were isolated by flow cytometry from b6 mice injected the previous day with dec - lnps containing either a 1:1:1 mix of cd40 , cd80 , and cd86 sirnas or control sirnas . splenic t cells from balb / c mice were isolated by macs using a pan t cell isolation kit ii ( miltenyi , san diego , ca ) . macs sorted balb / c t cells were carboxyfluorescein succinimidyl ester labeled according to published protocols , and carboxyfluorescein succinimidyl ester - labeled t cells were cocultured with the irradiated b6 dcs . three days later , dilution of carboxyfluorescein succinimidyl ester was monitored by flow cytometry to determine t - cell activation .

due to their ability to knock down the expression of any gene , sirnas have been heralded as ideal candidates for treating a wide variety of diseases , including those involving undruggable targets . however , the therapeutic potential of sirnas remains severely limited by a lack of effective delivery vehicles . recently , lipid nanoparticles ( lnps ) containing ionizable cationic lipids have been developed for hepatic sirna delivery . however , their suitability for delivery to other cell types has not been determined . we have modified lnps for preferential targeting to dendritic cells ( dcs ) , central regulators of immune responses . to achieve directed delivery , we coated lnps with a single - chain antibody ( scfv ; dec - lnps ) , specific to murine dec205 , which is highly expressed on distinct dc subsets . here we show that injection of sirnas encapsulated in dec - lnps are preferentially delivered to dec205 + dcs . gene knockdown following uptake of dec - lnps containing sirnas specific for the costimulatory molecules cd40 , cd80 , and cd86 dramatically decreases gene expression levels . we demonstrate the functionality of this knockdown with a mixed lymphocyte response ( mlr ) . overall , we report that injection of lnps modified to restrict their uptake to a distinct cell population can confer profound gene knockdown , sufficient to inhibit powerful immune responses like the mlr .

sevoflurane does not sensitise the myocardium to arrhythmogenic effects of catecholamines and has the least effect on cardiac conduction system . despite all the research demonstrating its safety , adverse cardiac arrhythmias such as nodal rhythm with bradycardia , prolongation of qtc interval , precipitation of torsade de pointes and isorhythmic atrioventricular dissociation ( isravd ) have been reported with sevoflurane anaesthesia . a 60-year - old woman weighing 60 kg with carcinoma in the right breast was scheduled for elective modified radical mastectomy . she was a known hypertensive since 4 years , on treatment with oral metoprolol 50 mg twice daily and amlodipine 10 mg once daily , with no other comorbidities . preoperative pulse rate ( pr ) was 60/min and blood pressure ( bp ) was 140/70 mmhg . preoperative 12 lead electrocardiograms ( ecg ) showed sinus rhythm with a rate of 60/min . diazepam 10 mg was administered orally the night before and 60 minutes before surgery , along with morning dose of metoprolol and amlodipine . anaesthesia was induced with 8% sevoflurane in 100% oxygen after administering 1.5 mg morphine intravenously . during induction , sevoflurane concentration was reduced to 5% and satisfactory proseal laryngeal mask airway insertion ( size 3 ) was accomplished with suxamethonium 100 mg . capnograph showed a square wave form . immediately following induction , ecg trace in lead ii showed dissociated upright p waves which remained within qrs for several beats , then reappeared and recede from qrs complex . ecg also showed intermittent electrical alternans [ figure 1 ] and bp dropped to 86/50 mmhg . meanwhile , anaesthesia was maintained with sevoflurane 2% and 50% nitrous oxide in 50% oxygen . end - tidal carbon dioxide ( etco2 ) was maintained between 35 and 37 mmhg . pr ( 40 - 50 beats / min ) and rhythm ( isravd ) remained unaltered throughout the procedure that lasted for 3 hours . atropine 0.6 mg and mephenteramine 3 mg were administered intravenously when the pr dropped below 40/min and systolic bp below 90 mmhg . a discrepancy was observed between the hr recorded by the ecg and pr recorded by the pulse oximeter . hr was 50 to 60 beats / min but pr was 38 to 40 beats / min . at the end of surgery , sevoflurane was discontinued and neuromuscular blockade was reversed with a mixture of atropine ( 1.2 mg ) and neostigmine ( 2.5 mg ) . few minutes after removal of the lma , rhythm reverted to sinus with pr of 50/min and bp was 118/79 mmhg . metoprolol and amlodipine were discontinued and enalapril 5 mg once daily was started by the cardiologist postoperatively . patient was observed in intensive care unit for the next 48 hours . throughout the postoperative course , hr and rhythm remained normal . real - time picture from the monitor shows electrical alternans with absent p waves in ecg trace . the difference in heart rate ( ecg trace ) and pulse rate ( pulse oximetry trace ) is seen due to electrical alternans . real - time picture from the monitor shows the inspired and expired concentration of sevoflurane a 5-year - old boy weighing 15 kg was scheduled for left inguinal hernia repair . premedication administered was 7 mg of oral midazolam , 30 minutes before surgery . in the operating room , baseline pr was 88/min with sinus rhythm ; spo2 , 99% on room air and nibp , 96/62 mmhg . inhalation induction of anaesthesia was performed with sevoflurane in 100% oxygen ( fresh gas flow 5 l / min ) through t piece circuit with child breathing spontaneously at a rate of 20 to 24/min . inspired concentration of sevoflurane was increased in 2% increments every minute up to 8% concentration . the etco2 was between 31 and 33 mmhg , as measured through the angle piece interfaced between the face mask and the breathing circuit . after breathing 8% sevoflurane for 2 minutes sudden drop in hr to 56/min was noticed and the ecg revealed the presence of junctional rhythm with absent p waves and regular narrow qrs complexes . at that moment , spo2 was 99% and bp was 66/36 mmhg . intravenous fentanyl ( 30 g ) was administered and anaesthesia was maintained with 1.5% isoflurane in a mixture of 50% nitrous oxide in oxygen ( fresh gas flow rate 5 l / min ) . a 60-year - old woman weighing 60 kg with carcinoma in the right breast was scheduled for elective modified radical mastectomy . she was a known hypertensive since 4 years , on treatment with oral metoprolol 50 mg twice daily and amlodipine 10 mg once daily , with no other comorbidities . preoperative pulse rate ( pr ) was 60/min and blood pressure ( bp ) was 140/70 mmhg . preoperative 12 lead electrocardiograms ( ecg ) showed sinus rhythm with a rate of 60/min . diazepam 10 mg was administered orally the night before and 60 minutes before surgery , along with morning dose of metoprolol and amlodipine . anaesthesia was induced with 8% sevoflurane in 100% oxygen after administering 1.5 mg morphine intravenously . during induction , sevoflurane concentration was reduced to 5% and satisfactory proseal laryngeal mask airway insertion ( size 3 ) was accomplished with suxamethonium 100 mg . capnograph showed a square wave form . immediately following induction , ecg trace in lead ii showed dissociated upright p waves which remained within qrs for several beats , then reappeared and recede from qrs complex . ecg also showed intermittent electrical alternans [ figure 1 ] and bp dropped to 86/50 mmhg . meanwhile , anaesthesia was maintained with sevoflurane 2% and 50% nitrous oxide in 50% oxygen . end - tidal carbon dioxide ( etco2 ) was maintained between 35 and 37 mmhg . pr ( 40 - 50 beats / min ) and rhythm ( isravd ) remained unaltered throughout the procedure that lasted for 3 hours . atropine 0.6 mg and mephenteramine 3 mg were administered intravenously when the pr dropped below 40/min and systolic bp below 90 mmhg . a discrepancy was observed between the hr recorded by the ecg and pr recorded by the pulse oximeter . hr was 50 to 60 beats / min but pr was 38 to 40 beats / min . at the end of surgery , sevoflurane was discontinued and neuromuscular blockade was reversed with a mixture of atropine ( 1.2 mg ) and neostigmine ( 2.5 mg ) . few minutes after removal of the lma , rhythm reverted to sinus with pr of 50/min and bp was 118/79 mmhg . metoprolol and amlodipine were discontinued and enalapril 5 mg once daily was started by the cardiologist postoperatively . patient was observed in intensive care unit for the next 48 hours . throughout the postoperative course , hr and rhythm remained normal . real - time picture from the monitor shows electrical alternans with absent p waves in ecg trace . the difference in heart rate ( ecg trace ) and pulse rate ( pulse oximetry trace ) is seen due to electrical alternans . a 5-year - old boy weighing 15 kg was scheduled for left inguinal hernia repair . premedication administered was 7 mg of oral midazolam , 30 minutes before surgery . in the operating room , baseline pr was 88/min with sinus rhythm ; spo2 , 99% on room air and nibp , 96/62 mmhg . inhalation induction of anaesthesia was performed with sevoflurane in 100% oxygen ( fresh gas flow 5 l / min ) through t piece circuit with child breathing spontaneously at a rate of 20 to 24/min . inspired concentration of sevoflurane was increased in 2% increments every minute up to 8% concentration . the etco2 was between 31 and 33 mmhg , as measured through the angle piece interfaced between the face mask and the breathing circuit . after breathing 8% sevoflurane for 2 minutes , the child did not show any motor response to jaw thrust . sudden drop in hr to 56/min was noticed and the ecg revealed the presence of junctional rhythm with absent p waves and regular narrow qrs complexes . at that moment , spo2 was 99% and bp was 66/36 mmhg . intravenous fentanyl ( 30 g ) was administered and anaesthesia was maintained with 1.5% isoflurane in a mixture of 50% nitrous oxide in oxygen ( fresh gas flow rate 5 l / min ) . american college of cardiology / american heart association recommends using volatile agents during non - cardiac surgery for the maintenance of general anaesthesia in haemodynamically stable patients at risk for myocardial ischemia . anticipating a difficult airway and considering the patient 's cardiac status , and associated hypertension , isravd was recorded during induction of anaesthesia with sevoflurane which reverted back to sinus rhythm after discontinuation of sevoflurane during emergence . 2 pacemakers ( sinus and av nodes ) fortuitously discharge at the same or nearly similar rate , without antegrade or retrograde conduction across the av node . either slowing of the sinus node discharge rate or the emergence of a slightly faster subsidiary pacemaker controlling the ventricles is the common initiating event . p waves approaches the narrow qrs complex , disappears within it for several beats , then reappears and recedes from the qrs and this sequence may be repeated . the same was observed in the ecg recording in the present case [ figure 1 ] . from the limited number of experimental studies , the development of isravd and the particular electrocardiographic patterns resulting from there are dependent upon relative discharge rate of the dominant atrial and subsidiary pacemakers , presence or absence of retrograde conduction , chronotropic response to atrial stretch , baroreceptor - mediated autonomic nervous system output and responsiveness of the sinus node . high incidence of nodal rhythm ( 20% ) and bradycardia with use of sevoflurane has been reported in unpremedicated infants . the onset of nodal rhythm was significantly earlier in the high - concentration technique than with incremental induction technique . on the other hand , nakaigawa et al . demonstrated that up to 2 mac ( minimal alveolar concentration ) sevoflurane does not affect cardiac conduction system significantly . in both our patients , 8% sevoflurane was used for induction that possibly resulted in isravd and junctional rhythm . isravd was possibly precipitated by the volatile agent affecting ca release from the sarcoplasmic reticulum , resulting in depression of ca slow inward current in the myocardium . in patients on ca channel and -adrenergic blocker therapy , the arrhythmogenic potential of sevoflurane is likely to be enhanced as ca channel blockers inhibit the entry of ca into the cell or its mobilisation from intracellular stores , while b - adrenergic blockers depresses sinus rate and av node conduction . reported a case of intraoperative torsades de pointes ventricular tachycardia and ventricular fibrillation during sevoflurane anaesthesia , which persisted with dc shock but reverted to sinus rhythm after 10 minutes of discontinuation of sevoflurane and after the patient was awakened . a discrepancy was observed between the hr and the pr due to intermittent electrical alternans in the ecg . it is therefore wise to rely on the pr rather than the hr in such cases to treat bradycardia . rarely , sevoflurane - induced suppression of baroreceptor reflex activity might result in desynchronisation between the atria and ventricle causing haemodynamic instability which may require electrical pacing . to conclude , sevoflurane in high concentration ( 8% ) should be used with caution in children and in conditions where primary pacemaker activity is suppressed , as in elderly . caution should also be exercised with its use in patients on ca channel and b blocker therapy .

when inhalational induction is indicated , sevoflurane is the most popular choice in both children and adults . higher inspired concentrations of sevoflurane can cause adverse cardiac arrhythmias that are seen to disappear once the agent is discontinued . we report the occurrence of isorhythmic atrioventricular dissociation and junctional rhythm with absent p waves during sevoflurane anaesthesia in an adult and a child , respectively .

according to the mca , only a compromised ability to make a decision in accordance with certain procedural norms , loosely defined with reference to the ability to understand , retain and use or weigh relevant information , or an inability to communicate a decision , properly grounds judgments of incapacity ( s. 3(1 ) ) . in addition , the identified problems of decision - making must be attributed to an impairment of , or disturbance in the functioning of , mind or brain ( s. 2(1 ) ) . this law aims to protect the individual 's right to pursue their own ends by adopting a capacity test that focuses on the decision - making process rather than the substance of the patient 's decision . influential liberal thinking holds that it is not the place of the law to pursue certain ends rather , the law should aim to provide a space for individuals to pursue their own ends ( plant , 2011 ) and this position is reflected in english common law relating to mental capacity:[in assessments of capacity ] it is most important that those considering the issue should not confuse the question of mental capacity with the nature of the decision made by the patient , however grave the consequences . the view of the patient may reflect a difference in values rather than an absence of competence and the assessment of capacity should be approached with this firmly in mind.whether the patient 's decision is consistent with proposed substantive norms in relation to what they should pursue for example that people should pursue good health , or that they should want to live is said to be beyond the proper scope of the incapacity test . in adopting this position , english law allows that even the most controversial treatment decisions might be explained by the fact that the patient 's evaluation of the situation diverges from a clinical perspective , and perhaps many people 's evaluation of the situation . [ in assessments of capacity ] it is most important that those considering the issue should not confuse the question of mental capacity with the nature of the decision made by the patient , however grave the consequences . the view of the patient may reflect a difference in values rather than an absence of competence and the assessment of capacity should be approached with this firmly in mind. in practice then , questions of mental capacity might be thought of as boiling down to a question about whether a decision can be explained in terms of the patient 's particular motivating commitments ( their desires , values , projects , ideas about a good life ) ; or whether the decision is properly explained in terms of a problem in decision - making , which is due to a dysfunction of mind or brain . is the patient 's controversial decision understandable in the light of their commitments ? or is the patient 's ability to understand , retain , and use or weigh what the likely costs and benefits of treatment will be for them , currently compromised ? scrutinizing a patient 's commitments in the context of a capacity assessment raises moral concerns because the possibility that a choice can be explained by idiosyncratic commitments plays a significant role in protecting the patient 's right to make controversial decisions . perhaps the most straightforward approach to conceiving of commitments , and the one most likely to protect this right , is to say that the person 's commitments are their current commitments those they recognize at the time of the decision . the problem with conceiving of commitments in this way is that it fails to recognise certain problems of practical decision - making to do with motivational coherence over time . someone with bipolar disorder , for example , might clearly be choosing or acting in accordance with their current desires during a manic phase , but this perspective fails to capture what seemingly goes wrong with practical decision - making in such cases . as a result , if this understanding of commitments is adopted in assessments of mental capacity the reach of this area of law arguably does not extend as far as it should a person may be judged to have mental capacity where this seems doubtful . psychopathologies characterised by this kind of problem seem better accounted for in a legal structure that conceives of mental capacity , and in particular , commitments , in diachronic terms . a diachronic perspective might also help illuminate some other hard cases that are not normally characterised in terms of problems of motivational coherence over time . for example , someone with anorexia nervosa may be choosing rationally in accordance with their current commitments especially a strong desire not to gain weight , or to exert extreme control over what they eat and so may be judged to have capacity on these grounds . however , an assessment of their evaluative capacities in diachronic terms might yield the conclusion that their decision - making ability is seriously compromised ( craigie , 2011 ) . their future self may come to the view that they should have been motivated in ways that they could not recognise or could not act on at the time , and this might be a fairly robust feature of people with a diagnosis of anorexia nervosa . this perspective in relation to questions of mental capacity chimes with the suggestion that particularly in psychiatry , colloquialisms [ such as ] you will thank me later sometimes become the unwritten rules within which the merits of a patient 's consent are assessed ( gostin , 1981 , p. 742 ) . such a perspective may also offer a solution in cases where the patient does not have a psychiatric diagnosis . mackenzie and watts discuss a case of a woman who was judged to have the capacity to refuse life - saving treatment , and subsequently died , in a situation where she had recently been left by her husband , following the amputation of both her legs . they argue that the trauma of these events and her subsequent inability to imagine a life without her partner clouded the woman 's judgment in relation to treatment:the tragedy is that in this case therapy or treatment might have corrected her assumptions regarding future quality of life , and a decision to accept the lifesaving treatment might have resulted. ( 2011 , p. 33 ; for a full description of the case , see halpern , 2012)differences between these cases suggest that taking a diachronic perspective in assessments of mental capacity allows for decision - making to be compromised in at least two ways . in the bipolar disorder and anorexia nervosa cases a diachronic perspective is used to argue that a person 's evaluative capacities may be compromised the capacities that determine the desires or values that motivate a decision . in the emotional trauma case , which might equally be applied to cases of depression , a diachronic perspective is used to identify a problem in the person 's ability to assess whether what they want or value is achievable . according to jodi halpern 's original analysis of the emotional trauma case , the woman should have been judged to lack capacity on grounds that her beliefs about the future were unresponsive to evidence , rendering her unable to think through alternatives ( halpern , 2012 , pp . the tragedy is that in this case therapy or treatment might have corrected her assumptions regarding future quality of life , and a decision to accept the lifesaving treatment might have resulted. ( 2011 , p. 33 ; for a full description of the case , see halpern , 2012 ) one problem with adopting a diachronic perspective in assessments of mental capacity is that whether questions of procedural rationality are properly understood in diachronic terms remains a contested issue in the fields of economics , psychology and philosophy . the functional element of the mca 's incapacity test concerns problems in meeting certain procedural requirements problems in the ability to understand , retain , use or weigh relevant information . sceptics deny that at least some norms derived from a diachronic perspective fall into this category . they hold that such requirements are substantive rather than procedural in nature , so i turn now to this theoretical concern . much of the normative and descriptive work on a diachronic perspective in personal decision - making has taken place in the fields of economics and psychology , but these fields have tended to adopt contrasting positions on the normative question . economists often treat a person 's weighting of the future as a preference like any other , in a framework that assumes that preferences are not available to rational criticism . prudence to refer to taking a diachronic perspective in decision - making:prudence can not on this view be explained merely by the perception that something is in one 's future interest ; there must be a desire to further one 's future interests if the perception is to have an effect there seems little doubt that most people have the desire that makes prudence possible [ however , according to this view we are not ] in any sense required to possess the desires in question : consequently we are not required to act on the specified considerations . if one lacks the relevant desire , there is nothing more to be said . ( nagel , 1970 , p. 28)psychologists , on the other hand , often assume that people are rationally required to give significant weight to the future personal consequences of choices , the strongest version being that people ought to be temporally neutral in their decision - making . it is widely accepted within this camp that there are at least some good reasons to give less weight to future consequences , particularly in order to take into account an expected depreciation in their future value . . however , foreseeable changes in one 's circumstances can also be relevant : 50 might be much more valuable to a final year law student than 100 will be for them in the following year , when they reasonably expect to be working for a corporate lawyer . on these grounds it might be rational for the student to choose 50 now , foregoing 100 in a year 's time . likewise , it is argued that uncertainly with respect to future consequences can provide a good reason to choose a certain outcome now , over a more valuable but uncertain outcome in the future ( baron , 1994 , pp . i am using the term temporally neutral to refer to decisions that give equal weight to future and present utility , excepting the above kinds of considerations . the pressing question is why people ought to be impartial with respect to all parts of their lives once these kinds of consideration are taken into account . prudence can not on this view be explained merely by the perception that something is in one 's future interest ; there must be a desire to further one 's future interests if the perception is to have an effect there seems little doubt that most people have the desire that makes prudence possible [ however , according to this view we are not ] in any sense required to possess the desires in question : consequently we are not required to act on the specified considerations . if one lacks the relevant desire , there is nothing more to be said . ( nagel , 1970 , p. 28 ) the arguments used to defend this position in the psychological literature tend to take the form that a temporally neutral orientation advances the goals of practical decision - making . choosing an outcome merely because it will happen sooner is said to be irrational because if one option achieves your goals better , then you should choose that one , regardless of when you decide ( baron , 1994 , p. 514 ) . using the classic marshmallow experiments as an example , if a child would rather eat two marshmallows than one , then they should choose to wait and be given two marshmallows sometime later rather than one marshmallow now because overall this achieves the child 's goals better . this kind of argument considers the question of goal satisfaction from a position that is not situated at a particular point in time , and prescribes that people ought to take this perspective in their decision - making : they should be just as concerned about themselves a year from now as they are about themselves this minute ( p. 516 ) . individuals should give weight to the implications for their future self , so the argument goes , for their own overall good . nagel argues that prudence is a requirement of practical reason , which is secured because the personal consequences of a man 's action concern his future ( 1970 , p. 42 ; his italics ) . according to nagel , this constraint on practical decision - making follows from the person 's awareness that they persist over time . it reflects the individual 's conception of himself as a temporally persistent being : his ability to identify with past and future stages of himself and to regard them as forming a single life . failure to be susceptible to prudence entails radical dissociation from one 's future , one 's past , and from oneself as a whole , conceived as a temporally extended individual ( p. 58 ) . it is a mistake in practical reasoning because it comes at the cost of dissociation from one 's temporally extended self ( p. 69 ) . the conception of self as extended throughout a human lifetime is powerful in the context of questions of procedural rationality because it offers grounds for claiming that a person ought to give weight to considerations that they may not currently care about . it offers an answer to what i have characterised as the economists ' view , that if a person has no interest in the future personal consequences of their choice there is nothing more to be said , rationally speaking . applied to the context of treatment decision - making , this kind of perspective is powerful because it offers grounds for calling a patient 's mental capacity into question , with reference to their ability to consider their future and value it appropriately . rejections of this kind of rational requirement are therefore often based on a rejection of the idea of the self as something that necessarily extends throughout a lifetime , and derek parfit 's account of personal identity is often used to make this argument ( baron , 1994 , p. 516 ; frederick , 2006 ; maclean , 2006 ) . parfit holds that the persistence of a person over time must be understood in terms of their psychological continuity the connectedness of psychological features such as memories , character , interests and preferences across a lifetime ( parfit , 1983 ; 1984 ) . on this view , whether the future inhabitant of one 's body will be the same person can be a matter of degree . psychological features sometimes change so radically over a human lifetime that a person now will not necessarily be the same person who occupies that body in the future . and not giving weight to the interests of a future self is irrational only to the degree that one shares the relevant psychological characteristics with that self . more recently , galen strawson has defended a similar position in his rejection of narrative accounts of the self . strawson argues that although experiencing oneself as one person throughout a lifetime what he calls a diachronic self - experience may be more common , some people experience themselves in what he describes as a more episodic way . unlike the more diachronic person , the more episodic person has little or no sense that the self that one is was there in the ( further ) past and will be there in the future , although one is perfectly aware that one has long - term continuity considered as a whole human being ( strawson , 2005 , p. 65 ) . [ predominantly ] episodic individuals may sometimes connect to charged events in their pasts in such a way that they feel those events happened to them embarrassing memories are a good example and anticipate events in their futures in such a way that they think those events are going to happen to them thoughts of death can be a good example however , the episodic person has no great or special interest in [ their ] past , and nor do they have a great deal of concern for [ their ] future ( p. 67 ) . strawson holds that even a strongly episodic self is within the normal range of human experience , and that it is normatively on a par with a more diachronic self - experience . on his view , theorists such as nagel assert that selves extend over a lifetime , and diachronic norms of procedural rationality are derived from this claim . parfit and strawson dispute this assumption , and conclude that disregard for the future personal consequences of a choice may simply reflect an alternative way of living a human life , at least when we consider the question of reasons for action in non - moral terms . however , all of the theorists canvassed thus far reason from premises about the extension of self over a lifetime , to conclusions about what reasons individuals have , and it will be argued here that this conception of the relationship between the requirements of practical rationality and the metaphysics of self is the wrong way around . as described above , the kind of requirement in question is often defended by nagel and others on the grounds that not giving weight to the future undermines one 's own interests . however , at times a different kind of argument is suggested in nagel 's work . the suggestion seems to be that a unified self over a lifetime is not something that is automatically present , but rather something that is generated by giving weight to future personal consequences in decision - making . the proposed requirement is justified , not because it deploys the correct conception of self , or because one 's future self will otherwise regret the decision , but because adherence plays a role in bringing the self into being , by enabling the decision - maker to reach towards something outside himself ( nagel , 1970 , p. 43 ) . michael bratman is particularly clear about the role that he proposes giving weight to the future plays in the generation of the self , and how this in turn grounds a diachronic perspective on questions of procedural rationality , so it is his view that i will focus on here . self - governing policies which are integral to temporally extended agency lie at the core of human agency because it is through these that a person 's identity emerges . these processes are essential for understanding what it is for an agent to take a stand what it is for an agent to recognise certain desires as one 's own and it is this feature in particular that distinguishes human agency from the agency of other purposive creatures ( bratman , 2000 , pp . adopting broadly the same approach to personal identity as parfit begins with , bratman suggests that the forward - looking psychological ties that play a role in constituting a unified self over time can be generated through reflective and planning processes : i can help ensure appropriate psychological continuities and connections by sticking with and executing my prior plans and policies , and by monitoring and regulating my motivational structures in favor , say , of my continued commitment to philosophy such processes play an important role in the constitution and support of continuities and connections characteristic of the identity of the agent over time. indeed, bratman suggests , this is what plans and policies are for ( 2000 , p. 47 by emphasising the role that planning and policy - making play in generating the psychological ties that are proposed to underpin personal identity over time , bratman describes a picture of human agency in which the agent plays an active role . the temporally extended self develops through the agent 's exercise of certain psychological capacities . on this point bratman makes a significant departure from parfit and strawson , who seem to hold that the degree to which the relevant psychological ties extend over time is a fixed fact about the individual , from which rational requirements on their action may be derived . the claim that engaging in planning and policy - making processes is rationally required is defended primarily on the non - instrumental grounds that these processes play a central role in developing and sustaining the self . bratman 's argument takes the form of an answer to the problem that agents who reflect on their first - order desires and form second - order desires do not yet exhibit what is distinctive about human agency , because there is nothing to distinguish these new desires as those that the agent identifies with ; second - order desires seem to be just one more wiggle in the psychic stew ( bratman , 2000 , p. 38 ) . the solution that bratman offers is that where an agent stands with respect to a particular first - order desire is established through the exercise of planning and policy - making practices : self - governing policies might , so to speak , crystallize pressures from various elements of one 's psychic stew into a more decisive attitude that can , in the relevant context , establish where one stands ( 2000 , p. 51 ) . these mental processes that underpin temporally extended agency give authority to particular desires , so it is through these that the self the distinctive feature of human agency emerges . on this view , a compromised ability to engage in planning and policy - making processes threatens not the interests of the future self , but the very constitution of the self . this is what grounds the procedural requirement that one must give weight to the future personal consequences of actions . and this , we might suggest , is what makes the ability to consider and value the future an appropriate consideration to include in a mental capacity test . bratman 's account offers a story about the self that fits well with a developmental understanding of personhood that is often appealed to in contemporary medical law and ethics . his account of the relationship between temporally extended agency and the self also fits well with the difficulties associated with certain psychopathologies . it is no accident , i suggest , that disorders such as dementia , bipolar disorder and depression are associated with seriously compromised capacities for diachronic agency . among many other factors , in a complex picture , problems of diachronic agency surely make a contribution to our sense that these constellations of behavioural and experiential features are appropriate targets for the label of disorder . a sense of the self as compromised in the context of problems of diachronic agency is also found in first - person reports of dementia ( addis and tippett , 2004 ; however , see caddell and clare , 2012 ) and depression ( fuchs , 2008 ; ratcliffe , 2012 ) . it would seem that the functioning of capacities for diachronic agency is connected to our sense of others as having well - functioning human agency , as well as our subjective sense of selfhood , and for these reasons i find it implausible that a strongly episodic self - experience is just a less common way of living a human life . strawson defends a view along these lines , claiming that he himself is strongly episodic ( in his sense ) . yet he has a successful academic career which could not have been achieved without meeting a wide range of commitments that each extended over considerable periods of time , for example in completing academic qualifications and writing books . it seems to me that these achievements evidence considerable skills in diachronic agency . while for strawson a diachronic self - experience is connected to a narrative conception of the self , diachronicity in the sense concerned here a life lived in the moment could still be a life lived with diachronic agency , as i will argue at the end of the following section . i now turn back to questions of mental capacity and what implications a diachronic perspective such as bratman 's would have . the motivation for investigating these theoretical questions about procedural rationality was the idea that a diachronic perspective might enable a mental capacity test such as that adopted in the mca to deal with certain hard cases , particularly in the context of mental disorder , without undermining its value - neutral approach . the arguments above suggest that a diachronic perspective on questions of mental capacity is consistent with a procedural understanding of the functional element of the mca 's incapacity test . given this , could a diachronic understanding of what it is to have mental capacity help by justifying the inclusion of a requirement such as the ability to consider the future as a part of what it means to be able to understand , retain , use or weigh information in assessments of mental capacity ? something like this requirement already seems to be assumed in the condition that the patient must understand the consequences of the treatment options . the relevant information necessarily concerns the future as well as the present , due to the fact that understanding the likely effects of deciding one way or the other is a part of this requirement ( mental capacity act 2005 code of practice , s. 4.16 ) . for example , a person with a severe learning disability might be judged to lack the capacity to consent to a blood test on grounds that they do not understand that the test is necessary for their future physical well - being . the same might be said in a case of a delirious patient who is resisting treatment for a serious injury . in such cases the person 's connection to the world , in the sense of their ability to understand how things are likely to be in the future , what to do is limited in a way that can be compared to john stuart mill 's paradigm case of justified paternalistic intervention , where a person is about to cross a bridge that unbeknownst to them is unsafe . the person in the example does not understand that they risk falling into water if they attempt to cross . the widely accepted conclusion is that the person is not in a position to assess whether to cross the bridge , and this justifies the use of proportional physical force to stop them from crossing , when this is necessary to inform them about the state of the bridge . the same reasoning might be applied in the case of the woman who has recently suffered a serious emotional trauma , on grounds that her ability to imagine a range of future possibilities is severely limited ( halpern , 2012 ) . the patient can not currently conceive of alternative futures to the desperately unhappy one she imagines , and perhaps this justifies taking the decision out of her hands until she is seeing things more clearly . the problem that returns here is that understanding concerns not simply how things are likely to be , as in the fact that an attempt to cross the bridge is likely to result in a fall into water , or that a certain proportion of people who have both legs amputated learn to walk again using prosthetic limbs . it concerns understanding how the relevant state of affairs will be for this particular patient . this question about the future necessarily involves an evaluative judgment that , according to a value - neutral approach , only the patient is in a position to make . so while it might sometimes be clear that a patient 's ability to consider the future is compromised , for example when someone denies that their condition is life - threatening when it clearly is , in many cases assessing understanding in relation to the future will have no equivalent point of reference . in such a context there seems to be no standard against which the functional element of the mental capacity test the part concerning the patient 's ability to understand , retain , use or weigh information can be directly assessed . therefore , any conclusions drawn about this capacity would seem to be based on the fact that the patient has suffered an emotional trauma . removing a patient 's right to make their own treatment decision under these circumstances may seem justified given what we know about how people who lose limbs adapt , as reflected in self - reported measures of well - being , and given the known psychological effects of emotional trauma . however , an appeal to this kind of knowledge in an assessment of mental capacity would involve using generalised observations to draw a conclusion about a particular person 's capacity to assess how things will be for them in the future . the legislation is clear that an assessment of decision - making ability should not be based solely on features such as a patient 's diagnosis . it must be shown that this particular person 's mental capacities are relevantly impaired , not merely that they belong to a diagnostic category . however , this requirement can not be met in the case of this capacity , because there is no way to directly assess it while remaining value neutral in accordance with the aspirations of this area of law . what about the possibility raised at the beginning of this article , that a person 's evaluative capacities might be called into question when viewed from a diachronic perspective ? drawing on bratman 's account , planfulenss and self - governing policies are rationally required because of the role they play in developing and sustaining the self . caring about the interests of the future inhabitant of one 's body , and having at least some grip on what those interests will be , seem like prerequisites for engaging successfully in these processes . and perhaps this offers part of an explanation about how decision - making can be compromised in the context of disorders such as depression and bipolar disorder . very crudely , the idea would be that as part of a much bigger picture the capacities necessary for diachronic agency are not functioning properly and this undermines the constitution of the self . i have argued that , understood in this way , these considerations are procedural rather than substantive in nature . but would the implementation of these considerations in practice nonetheless come into conflict with the goal of preserving patient autonomy ? assessing a person 's grip on what interests the future inhabitant of their body will have runs into the same difficulties that occurred in the context of assessing understanding in the emotional trauma case . without making substantive assumptions judging that a person is likely to lack these capacities in the context of a manic episode may seem justified , because of what we know about the trajectory of bipolar disorder that fairly reliably people 's commitments during a manic episode radically undermine their overall interests . but whether this kind of consideration provides grounds for a judgment of incapacity in english law depends on what evidence is considered appropriate for assessing the functional element of the incapacity test . however , as genevra richardson points out , existing case law indicates that courts will take a flexible approach to the legal definition [ of capacity ] to enable them to reach their preferred outcome ( richardson , this volume , p. 00 ) . this may mean that in practice significant weight is given to a person 's diagnosis . however , this solution undermines the protections of patient autonomy conferred by making an individual assessment of functional capacities , rather than diagnosis , the proper grounds for infringements on self - determination . a related possibility considered at the beginning of this article was that a diachronic perspective might be used to justify giving weight to a reasonable prediction of what the patient 's future self would want in retrospect , in assessments of mental capacity . however , it does not follow from the view offered here , even given the sure knowledge that a patient 's future self would retrospectively endorse a medical intervention , that their present self is irrational in refusing it . in the imagined case there is an unambiguous conflict between what the patient currently wants ( no treatment ) and what the future self will want in retrospect ( treatment ) . using this conflict to doubt the patient 's current capacity to make the treatment decision assumes that the future self 's perspective should be given greater weight in the current self 's decision - making . however , there is nothing in the present view that would justify resolving the conflict in this way . the proposed account requires that the agent exercise planfulness and self - governing policies in decision - making , which would seem to involve giving equal weight at most , not greater weight , to one 's expected future desires . moreover , humans have a well - established time preference for negative experiences to be in the past rather than the present or future , and this makes it easier for the patient to endorse involuntary treatment when it is in the past ( doherty , 2011 ) . this would seem to weigh in favour of privileging the perspective of the self who is facing the infringement on liberty . in the absence of any reasons to believe that the future self 's perspective should be privileged , this form of justification for doubting mental capacity looks to be question - begging . its favouring of the future self 's perspective already assumes that the current self 's decision - making capacity is relevantly impaired . favouring the future self 's perspective might seem justified on grounds that , for example , evaluative capacities are often compromised in the context of certain diagnoses or during experiences such as emotional trauma . however , at least in theory , this evidence alone does not justify a judgment of incapacity under current english law , for reasons of preserving patient autonomy as argued above . finally , i turn to the question of caring about future personal interests , especially in the context of life - threatening choices . a central difficulty associated with including a consideration of this kind in questions of mental capacity is the problem of how to assess caring simpliciter , independently of caring about a particular outcome . according to the account offered here , procedural rationality requires that we care about the future personal consequences of choices , but what in the future we should give weight to is left open . specifying this in assessments of mental capacity would mean that the test was not value neutral . the case of the committed smoker ( exemplified by david hockney ) can be used to illustrate the point . one criticism often levelled at smokers is that they are irrational because they do not care sufficiently about the future personal consequences of their smoking . however , this criticism assumes that future health , or a long life , is what the person should care about . considering a person 's decision to keep smoking from a value - neutral perspective , which makes no assumptions of this kind , are they being short - sighted ? or do they care about things other than health and long life in a way that makes their decision rational ? bratman 's account shows how asking this second question can be consistent with a diachronic perspective on rationality . it is clear that the committed smoker is very likely undermining the health of the future inhabitant of his body . however , it does not follow that he is not engaging in the psychological processes that underpin temporally extended agency . it may be a central feature of the person 's life plan that he should not be overly concerned about living a long life . his deep and enduring commitment might be to a hedonistic lifestyle that is incompatible with being concerned about health in old age . for such a person , not living fully enough is a more likely source of future regrets than health - endangering behaviours . caring about one 's future might even require , in such a case , life - threatening or health - threatening choices . parallel arguments could be made in the case of a decision by a jehovah 's witness to refuse a blood transfusion ; for a political activist 's hunger strike ; for a person who risks their life in an attempt to save a loved one ; and a freediver 's commitment to their dangerous extreme - sports lifestyle . these kinds of decision do not seem characterized by a lack of planfulness about the future . rather than undermining the sense of oneself as a temporally persisting agent , decision - making in line with these kinds of commitment looks likely to contribute to the overlapping webs of cross - temporal connections and continuities that are proposed to play a role in constituting human agency ( bratman 2000 , pp . the future self taken into consideration in these cases may be a nearer self than in the case of a person who is invested in living a long life because the artist who is a committed smoker , for example , might not expect to or want to live a long life nothing in the above account enjoins us to aim for a life of a particular length . what it requires is that , at least to some degree , choices be made with a reflectivity about the future , such that insofar as we are agents that agency is temporally extended . this perspective allows us to reconcile the ideas that mental capacity requires that we care about the future personal consequences of our choices , and that life - ending decisions can be made with mental capacity . however , against this theoretical background , resolving questions about whether a patient 's capacity to care about the future is compromised would seem to require looking beyond their procedural capacities . answers to this question will rest on views about whether or not the life - threatening decision is consistent with the person 's commitments , understood from a diachronic perspective . these factors are taken into account in an assessment of best interests once it is decided that a patient lacks mental capacity ( mca s. 4(6 ) ) . however , if the ability to care about the future is included as part of what it means to have mental capacity , then consideration of a patient 's commitments from a diachronic perspective will also be central to this prior decision . it has been argued here that a diachronic perspective on mental capacity is relevant to questions about when an infringement on treatment decision - making liberty is justified . however , including considerations derived from a diachronic perspective in assessments of capacity comes into conflict with central patient autonomy - preserving features of the mca . it 's worth reflecting , then , on why a diachronic perspective might be morally relevant in this context . i will suggest that the answer lies in a common view about what makes patient autonomy valuable what makes it the case that for the most part , people 's treatment decisions , especially their refusals , ought to be respected . the importance of allowing patients to be self - determining in relation to their own medical care is often justified along lines that it protects people 's general capacity to lead their lives out of a distinctive sense of their own character , a sense of what is important to and for them ( dworkin , 1994 , p. 224 ) . different theorists argue for the value of patient autonomy on importantly different grounds , but a common theme is that treatment decisions should be respected because of their assumed connection to the self . however , in a case where human agency is seriously compromised in the way described by bratman , selfhood is undermined . capacities for diachronic agency that play a central role in establishing what 's important for the person where the person stands are compromised . and in the context of such problems , the importance placed on patient autonomy in liberal societies would seem to give medical professionals a reason to do what they can to restore selfhood , perhaps even if this requires an infringement on personal liberty . this might involve overriding a refusal of treatment , for example , though the justification for doing so would only warrant interventions of a particular kind those that are likely to restore the general capacity that the principle of patient autonomy aims to protect . this suggests that in some cases , especially in the context of mental illness , the importance placed on patient autonomy in contemporary medical ethics presents the law with a dilemma . on the one hand , it seems right that laws which license infringements on decision - making liberty should equally apply to everyone , not just to particular groups ; and that if a patient has mental capacity in relation to a particular treatment decision then they should be free to direct the course of their medical care . consequently , it seems we ought to do away with dedicated mental health laws that legalise detention and involuntary treatment without reference to mental capacity only in the context of a mental disorder . on the other hand , the way in which diachronic agency is compromised in some instances especially in the context of mental disorder seems to justify or perhaps even require an infringement on liberty to restore the self . these grounds are difficult to take into consideration in assessments of mental capacity without compromising features of the mca that are designed to preserve patient autonomy . so while it might be possible to implement the mca in a way that sufficiently accounts for the problems of decision - making and selfhood that are sometimes present in mental disorders , doing so comes at the price of what is seen to be a central virtue of the legislation . in this article my focus was requirements of rationality derived from a diachronic perspective , and the question of what kinds of consideration this perspective would enable mental capacity assessments to include . it was an attempt to explore the limits of the mca 's incapacity test , given its value - neutral approach . it was argued that considerations derived from a diachronic perspective are relevant to questions of treatment decision - making liberty , and are consistent with a procedural understanding of the functional element of the test . however , in practice , appeal to these considerations in assessments of mental capacity undermines the role played by the functional element of the test in preserving patient autonomy . while involuntary treatment without reference to mental capacity is often assumed to undermine the principle of respecting patient autonomy , it was argued here that this is not necessarily so . when human agency is seriously compromised , preserving patient autonomy may sometimes require overriding a treatment decision to restore selfhood , on grounds that are not easily accounted for under the mca without compromising the value - neutral aspirations of this area of law . in considering a move to abandon dedicated mental health law , legislators must therefore resolve a bind . the central motivation for such a move is concern about unjustified infringements on liberty in the context of psychiatric diagnoses . the worry is that the protections of patient autonomy in the mca do not currently extend to the treatment of mental disorders , because mental health legislation allows people to be treated for a mental disorder even if they refuse with mental capacity . however , considerations derived from a diachronic perspective that seem relevant to when an infringement on decision - making liberty is justified are especially present in mental disorder . the inclusion of these considerations seems necessary for adequately caring for those with a mental disorder under universal capacity legislation . yet including these factors in assessments of mental capacity undermines the protections of patient autonomy that supporters of a shift to universal capacity legislation hope to gain .

calls for the adoption of a universal capacity approach to replace dedicated mental health law are motivated by the idea that the measures designed to protect patient autonomy in legislation such as the mental capacity act 2005 should apply to everyone , including people with a psychiatric diagnosis . in this article it is argued that a diachronic perspective on questions of mental capacity is necessary if capacity law is to play this broader role , but that employing this perspective in assessments of capacity undermines central patient autonomy preserving features of the legislation , which presents a moral dilemma .

type iii dysbetalipoproteinaemia is a kind of lipid metabolism disorders , caused by apo - e deficiency , which leads to accumulation of chylomicrons and very low density lipoprotein remnants in the plasma.123 lipid metabolism disorders are mostly manifested by xanthomas , which are known as yellowish cholesterol - rich material in large foam cells accumulating in the skins and tendons.14 these yellowish lesions may appear all over the body , on the palm of the hands , sole of the foot , tendons , and even on the eyelids.5 these lesions firstly described with or without hyperlipidaemia in association with monoclonal immunoglobulin . based on this fact , there are three forms of xanthoma : hyperlipaemic xanthoma , normolipaemic xanthoma and necrobiotic xanthogranuloma.6 xanthomatosis is usually associated with hyperlipidaemia , and morbidity and mortality of this condition are related to atherosclerosis and pancreatitis.7 hyperlipaemic xanthoma lesions are more polymorphic and can include tuberous , tendinous , palmar or eruptive xanthoma . verruciform xanthoma usually presents as a hyperkeratotic , cauliflower like , verrucous or papillomatous lesion.6 it initially was described as a unique clinicopathologic lesion of the oral mucosa and was subsequently reported in the skin . the epidermal - mucosal changes of hyperparakeratosis , neutrophilic infiltrate and dermal - submucosal foam cell reaction are distinctive features of these xanthomas.8 hereby , we present a rare type of xanthomas calling cauliflower xanthoma in a 43-year - old man with dysbetalipoproteinaemia . a 43-year - old man was presented to outpatient endocrine clinic of tabriz university of medical sciences , tabriz , iran , by his sister for evaluation of skin lesions . he had multiple lesions on shoulders and back , lateral and medial part of dorsal surface of the foot , medial and lateral malleus of both feet , and dorsal and palmar surface of both hands . siblings of the patient have been followed up in that clinic for clinically diagnosed type - iii dysbetalipoproteinaemia . this diagnosis was based on characteristic palmar , eruptive , tuberous and trauma site xanthomas with typical high levels of both total cholesterol and triglycerides in the plasma . history of coronary artery or other atherosclerotic disorders were negative in index case and siblings . there were not any other complaint , and as a greengrocer , he had an active lifestyle . he had a pathology report of skin lesion biopsy that was performed by a dermatologist few months ago , with presence of lipid laden macrophages consistent with diagnosis of xanthoma . fasting serum lipid levels reported as : total cholesterol = 507 mg / dl , triglycerides = 470 mg / dl and high density lipoprotein ( hdl ) cholesterol = 41 mg / dl . he had normal fasting blood sugar , and normal thyroid , renal and liver function tests . physical examinations revealed numerous xanthomas resembling cauliflower on both knees [ figure 1 ] , unusual eruptive , tuberous xanthomas same as previous lesions , on the lateral malleus of both feet [ figure 2 ] . cauliflower xanthomas on the knees and lateral malleus of both lower extremities cauliflower xanthomas on the lateral malleus of lower extremities there were lots of smaller xanthomas on the right shoulder [ figure 3 ] , and cauliflower like xanthomas on the extensor side of the left upper extremity , especially on the elbow [ figure 4 ] . based on available information , the patient diagnosed as familial dysbetalipoproteinaemia ( fdl ) and the unique lesions on the lower limb called cauliflower xanthoma . although siblings of this case had the same disorder , they did nt develop such lesions . xanthomas on the shoulder and back of the neck xanthomas on the extensor side of the hand by searching in the medical data bases , we can not find a previous such a lesion . therefore , we decided to present this case without revealing patient 's name , after taking his consent . considering the fact that the treatment with high dose statin was effective in correction of lipid abnormality and regression of skin lesions in his three siblings , we wanted to try the treatment procedure in this patient but he refused any intervention . fdl , also known as hyperlipoproteinemia type - iii or broad beta disease , is a rare inherited disorder characterised by improper metabolism of certain lipids , specially plasma cholesterol , triglyceride rich chylomicron and very low density lipoproteins ( vldl ) remnants.1 presence or absence of the symptoms of this disease depends on two major risk factors : genetic and diet.910 mutations in the gene for apolipoprotein e ( apo e ) are the main cause of this disease . replacement of an arginine by a cysteine in position 158 of the 299-amino acid chain of apo e5 is responsible for the defective binding of chylomicron and vldl remnants to cell receptors . thereafter , slower plasma clearance of these particles occurs , and results in the abnormal accumulation of lipids in the body.9 on the other side , diet has an essential role in the development of the disease . this means that with standard cholesterol diet , symptoms of fdl will not appear , even in the genetically susceptible person.10 xanthomas can be a symptom of fdl . xanthomas may also be the symptoms of a generalised histiocytosis , or a local fat phagocytosing storage process.11 they are yellowish lesions on the skins and tendons , macroscopically . these macrophages are filled with lipid droplets , which are dissolved and removed from tissue during histologic processing.8 fdl is mostly diagnosed by combination of clinical and laboratory findings.12 most cases are inherited as autosomal recessive trait . men are more susceptible for fdl probably because of protective impact of estrogen in women . most of the diagnosed patients were typically young males , with strong family history , characteristic skin lesions , high serum levels of cholesterol and triglycerides and confirming skin histology.113 most of these cases have premature atherosclerosis and other signs of ischaemic disorders.9 the patient in this article was a middle aged man with family history of dysbetalipoproteinaemia and high levels of serum lipids as follows : total cholesterol = 507 mg / dl and triglycerides = 470 mg / dl . he had a rare form of xanthoma all over his body , causing social problems for him . nicotinic acid , clofibrate , statins or gemfibrozil properly reduce cholesterol and triglycerides in people affected with dysbetalipoproteinaemia.1 the patient mentioned in this article refused receiving any medical intervention . he has just been advised to have low lipid diet , fish oil and regular moderate exercise in order to reducing serum lipid levels . there should be distinctive monitoring of fdl patients including regular checking of their serum lipids . controlling underlying disorders , and reducing excess calories , saturated fat and cholesterol , is the main aim of treating these patients . according to this article , cauliflower xanthomas could be a symptom of fdl , and it should be considered as a differential diagnosis while approaching to these lesions .

familial dysbetalipoproteinaemia ( fdl ) is an inherited disorder in which both cholesterol and triglycerides are elevated in the plasma , pre - disposing the people to coronary artery disease and peripheral vascular disease . the disease is mostly manifested by xanthomas , which have variable forms according to lipid amounts in the plasma of the blood . hereby , we report a 43-year - old man with fdl , presenting with a rare form of xanthomas calling cauliflower xanthoma all over the body .

exacerbation is an important life - threatening event for patients with copd , and can lead to hospitalization and death.14 patients who suffer frequent and repeated exacerbations within 1 year have a poor prognosis,5 characterized by worsening of health - related quality of life ( hrqol),6,7 a rapid decline in lung function,810 and high mortality.11 frequent exacerbators also carry a high risk of further exacerbation and hospitalization.11,12 however , it has been suggested that japanese patients with copd may have fewer exacerbations , and they also may have a higher proportion of elderly patients , those with emphysema , and those with a lower body mass index in comparison to westerners.1215 the prognosis of japanese patients with copd who suffer frequent and repeated exacerbations is unclear . we conducted a 1-year prospective observational trial in a daily - life setting involving 90 japanese patients with copd to investigate whether previous moderate - to - severe exacerbations are associated with future exacerbations in this patient population . we conducted a 1-year prospective observational trial in accordance with good clinical practice ( gcp ) guidelines and approved by the ethics committee of kurume university and chikugo city hospital ( gcp 11 - 127 , september 2012august 2014 ) . consecutive patients for whom medical records were available covering a period of at least 1 year since provision of informed consent were selected for the study ; information on previous annual copd - related exacerbations and hospitalizations was collected on the basis of those medical records . copd patients were divided into three groups , based on the total number of moderate and severe exacerbations within the last year before enrollment in the study , ie , non- ( previous moderate and severe exacerbations , 0/year ) , infrequent ( one exacerbation / year ) , and frequent ( two or more exacerbations / year ) exacerbator groups , in accordance with a previous report.16 in addition , patients with previous hospitalizations were classified as having a subphenotype with severe exacerbation ( severe exacerbators ) . the data collected for each patient included baseline data for previous moderate and severe exacerbations and hospitalizations ; clinical parameters included age , sex , body mass index , smoking habits , smoking index , comorbidities , duration of copd , 5-grade modified medical research council ( mmrc ) dyspnea scale score,17 total copd assessment test ( cat ) score,18,19 frequency scale for symptoms of gastroesophageal reflux disease ( gerd ) ( fssg),20 center for epidemiologic studies depression ( cesd ) scale score,21 medications , blood pressure and heart rate , lung function and blood parameters , and chest computed tomography . duration of copd was defined as the period ( years ) since the patient had been diagnosed by a physician as having copd , emphysema , and/or chronic bronchitis.22,23 after stable status for at least 4 weeks had been confirmed , each patient was required to regularly visit the hospital every 2 months , and to request emergency admission when the symptoms worsened . regular respiratory medications were not changed during the period of the study , which was conducted in a daily - life setting . the diagnosis of copd was based on age 40 years , smoking index > 10 pack - years , forced expiratory volume in 1 second ( fev1)/forced vital capacity ( fvc ) ratio of < 0.7 after bronchodilator administration , and the spirometric gold ( global initiative for chronic obstructive lung disease)-stage classification , ie , stage i ( fev1 80% predicted ) , ii ( 50% fev1 < 80% predicted ) , iii ( 30% fev1 < 50% predicted ) , and iv ( fev1 < 30% predicted).1 chest computed tomography confirmed that all patients had low - attenuation areas . to exclude any patients with asthma , patients who had past symptoms of repeated spasmodic wheezes and medications for asthma and a classification of fev1 > 200 ml or > 12% after bronchodilation were excluded.24 diagnosis of asthma copd overlap syndrome ( acos ) was made on the basis of a history of dyspnea and wheezing attacks at rest , large variations in daily symptoms , a fixed fev1/fvc ratio of < 0.7 , marked reversibility of fev1 after administration of bronchodilators ( > 15% and > 400 ml ) , and a peripheral eosinophil count of > 600/mm , in accordance with a previous report.25 baseline data for the mmrc scale , total cat , fssg scale , and cesd scale scores were obtained only once , based on a self - report completed by each patient after written informed consent had been obtained.1721 information about comorbidities was obtained from the patients by interview , and the diagnoses were confirmed by physicians . however , patients who had moderate - to - severe comorbidities associated with poor prognosis , such as active malignancies , depression ( cesd scale > 16 points),21 liver cirrhosis , digestive ulcers , persistent arrhythmia , congestive heart disease , coronary artery disease , lung fibrosis and bronchiectasis , chronic renal failure requiring dialysis , and central nervous system disorders , including palsy and dementia , were excluded , in accordance with previous studies.26,27 hypertension ( systolic > 140 mmhg or diastolic > 90 mmhg blood pressure or use of medications ) , dyslipidemia ( serum low-[14 ] or high- [ < 4 ] 15 mg / l , or use of medications ) , diabetes ( blood hemoglobin a1c 6.5% national glycohemoglobin standardization program value or use of medications)28 without triopathy ( neuropathy , retinopathy , and nephropathy ) , and gerd ( fssg scale > 7 points)20 were accepted as comorbidities . previous and prospective exacerbations documented in the medical records made by physicians were accepted as moderate and severe events . moderate exacerbations that required a prescription for antibiotics and/or systemic corticosteroids were defined on the basis of symptom - based diagnosis , such as increased cough and sputum production , a change in sputum color , and worsening of dyspnea from a stable state and beyond normal day - to - day variations , ie , showing acute onset and necessitating a change in regular medication , in accordance with previous reports.1,29,30 copd - related and other causes of death and hospitalization were prospectively followed for 1 year . hospitalization was decided by each examining physician when hypoxemia required additional or intensive oxygen and/or assisted ventilation therapy , performance status was 3 , and unconsciousness occurred with copd exacerbation.1,23,31 however , pneumonia was also recognized as exacerbation . mild and unreported exacerbations were not considered to have been equal in severity to previous and prospective exacerbations . however , mild exacerbations were defined as those that improved naturally without any medication or administration of inhaled short - acting bronchodilators . unreported exacerbations were considered to be those to which patients had been insensitive , or those that had been self - controlled in spite of worsening of respiratory symptoms . data are expressed as mean standard deviation ( sd ) and number ( percentage ) of non- , infrequent , and frequent exacerbators at baseline . characteristics were compared using analysis of variance , fisher s exact test , -test for trend , and tukey subsequent moderate and severe exacerbation events during 1 year of prospective observation were recognized as future risk indicators , and baseline parameters were chosen and modified in accordance with a previous report.16 kaplan meier analyses and log - rank tests for subsequent moderate and severe exacerbations were performed in all three groups of patients . for patients who were observed throughout the study period , the odds ratio ( 95% confidence interval [ ci ] ) of baseline parameters was determined to predict factors for future risk ( subsequent exacerbations once or more and twice or more ) using univariate and logistic multivariate regression analyses . medians of age , body mass index , smoking index , and duration of copd were 68 years , 21.4 kg / m , 53.5 pack - years , and 4 years , respectively , and each median was used as the cutoff value for the analysis . statistical analysis was performed by the jmp version 9.0 statistical software package ( sas institute inc , cary , nc , usa ) . we conducted a 1-year prospective observational trial in accordance with good clinical practice ( gcp ) guidelines and approved by the ethics committee of kurume university and chikugo city hospital ( gcp 11 - 127 , september 2012august 2014 ) . consecutive patients for whom medical records were available covering a period of at least 1 year since provision of informed consent were selected for the study ; information on previous annual copd - related exacerbations and hospitalizations was collected on the basis of those medical records . copd patients were divided into three groups , based on the total number of moderate and severe exacerbations within the last year before enrollment in the study , ie , non- ( previous moderate and severe exacerbations , 0/year ) , infrequent ( one exacerbation / year ) , and frequent ( two or more exacerbations / year ) exacerbator groups , in accordance with a previous report.16 in addition , patients with previous hospitalizations were classified as having a subphenotype with severe exacerbation ( severe exacerbators ) . the data collected for each patient included baseline data for previous moderate and severe exacerbations and hospitalizations ; clinical parameters included age , sex , body mass index , smoking habits , smoking index , comorbidities , duration of copd , 5-grade modified medical research council ( mmrc ) dyspnea scale score,17 total copd assessment test ( cat ) score,18,19 frequency scale for symptoms of gastroesophageal reflux disease ( gerd ) ( fssg),20 center for epidemiologic studies depression ( cesd ) scale score,21 medications , blood pressure and heart rate , lung function and blood parameters , and chest computed tomography . duration of copd was defined as the period ( years ) since the patient had been diagnosed by a physician as having copd , emphysema , and/or chronic bronchitis.22,23 after stable status for at least 4 weeks had been confirmed , each patient was required to regularly visit the hospital every 2 months , and to request emergency admission when the symptoms worsened . regular respiratory medications were not changed during the period of the study , which was conducted in a daily - life setting . the diagnosis of copd was based on age 40 years , smoking index > 10 pack - years , forced expiratory volume in 1 second ( fev1)/forced vital capacity ( fvc ) ratio of < 0.7 after bronchodilator administration , and the spirometric gold ( global initiative for chronic obstructive lung disease)-stage classification , ie , stage i ( fev1 80% predicted ) , ii ( 50% fev1 < 80% predicted ) , iii ( 30% fev1 < 50% predicted ) , and iv ( fev1 < 30% predicted).1 chest computed tomography confirmed that all patients had low - attenuation areas . to exclude any patients with asthma , patients who had past symptoms of repeated spasmodic wheezes and medications for asthma and a classification of fev1 > 200 ml or > 12% after bronchodilation were excluded.24 diagnosis of asthma copd overlap syndrome ( acos ) was made on the basis of a history of dyspnea and wheezing attacks at rest , large variations in daily symptoms , a fixed fev1/fvc ratio of < 0.7 , marked reversibility of fev1 after administration of bronchodilators ( > 15% and > 400 ml ) , and a peripheral eosinophil count of > 600/mm , in accordance with a previous report.25 baseline data for the mmrc scale , total cat , fssg scale , and cesd scale scores were obtained only once , based on a self - report completed by each patient after written informed consent had been obtained.1721 information about comorbidities was obtained from the patients by interview , and the diagnoses were confirmed by physicians . however , patients who had moderate - to - severe comorbidities associated with poor prognosis , such as active malignancies , depression ( cesd scale > 16 points),21 liver cirrhosis , digestive ulcers , persistent arrhythmia , congestive heart disease , coronary artery disease , lung fibrosis and bronchiectasis , chronic renal failure requiring dialysis , and central nervous system disorders , including palsy and dementia , were excluded , in accordance with previous studies.26,27 hypertension ( systolic > 140 mmhg or diastolic > 90 mmhg blood pressure or use of medications ) , dyslipidemia ( serum low-[14 ] or high- [ < 4 ] 15 mg / l , or use of medications ) , diabetes ( blood hemoglobin a1c 6.5% national glycohemoglobin standardization program value or use of medications)28 without triopathy ( neuropathy , retinopathy , and nephropathy ) , and gerd ( fssg scale > 7 points)20 were accepted as comorbidities . previous and prospective exacerbations documented in the medical records made by physicians were accepted as moderate and severe events . moderate exacerbations that required a prescription for antibiotics and/or systemic corticosteroids were defined on the basis of symptom - based diagnosis , such as increased cough and sputum production , a change in sputum color , and worsening of dyspnea from a stable state and beyond normal day - to - day variations , ie , showing acute onset and necessitating a change in regular medication , in accordance with previous reports.1,29,30 copd - related and other causes of death and hospitalization were prospectively followed for 1 year . hospitalization was decided by each examining physician when hypoxemia required additional or intensive oxygen and/or assisted ventilation therapy , performance status was 3 , and unconsciousness occurred with copd exacerbation.1,23,31 however , pneumonia was also recognized as exacerbation . mild and unreported exacerbations were not considered to have been equal in severity to previous and prospective exacerbations . however , mild exacerbations were defined as those that improved naturally without any medication or administration of inhaled short - acting bronchodilators . unreported exacerbations were considered to be those to which patients had been insensitive , or those that had been self - controlled in spite of worsening of respiratory symptoms . data are expressed as mean standard deviation ( sd ) and number ( percentage ) of non- , infrequent , and frequent exacerbators at baseline . characteristics were compared using analysis of variance , fisher s exact test , -test for trend , and tukey subsequent moderate and severe exacerbation events during 1 year of prospective observation were recognized as future risk indicators , and baseline parameters were chosen and modified in accordance with a previous report.16 kaplan meier analyses and log - rank tests for subsequent moderate and severe exacerbations were performed in all three groups of patients . for patients who were observed throughout the study period , the odds ratio ( 95% confidence interval [ ci ] ) of baseline parameters was determined to predict factors for future risk ( subsequent exacerbations once or more and twice or more ) using univariate and logistic multivariate regression analyses . medians of age , body mass index , smoking index , and duration of copd were 68 years , 21.4 kg / m , 53.5 pack - years , and 4 years , respectively , and each median was used as the cutoff value for the analysis . statistical analysis was performed by the jmp version 9.0 statistical software package ( sas institute inc , cary , nc , usa ) . ninety of 110 patients who provided informed consent were finally analyzed ; 32 ( 35.6% ) patients had suffered previous moderate and/or severe exacerbations during the last year . the numbers of patients with non- , infrequent , and frequent exacerbations were 58 ( 64.4% ) , 12 ( 13.3% ) , and 20 ( 22.2% ) , respectively ( figure 1 ) . at baseline , frequent exacerbators had a significantly lower body mass index , were less likely to be male or current smokers , had higher mmrc - scale grades and higher total cat score , had a higher proportion of spirometric stages iii and iv , gerd , and use of inhaled corticosteroid ( ics)/long - acting muscarinic antagonist or ics / long - acting 2-agonist combination therapy , and lower lung - function parameters , including fev1 , fvc , and fev1/fvc ratio before and after bronchodilator use compared with nonexacerbators , but not in comparison with infrequent exacerbators ( table 1 ) . frequent and infrequent exacerbators had significantly higher mmrc - scale grades and total cat scores compared with nonexacerbators ( table 1 ) . there was no change in the frequency of previous annual hospitalizations per patient between those with frequent and infrequent exacerbations ( table 1 ) . during the 1-year prospective observation period , one frequent exacerbator , one infrequent exacerbator , and two nonexacerbators died , with causes of death respiratory failure with copd exacerbation , cerebrovascular attack , and malignancies ( small - cell lung cancer and colon cancer ) , respectively , whereas one frequent exacerbator and six nonexacerbators dropped out for private reasons . as a result , 78 patients ( 17 frequent , eleven infrequent , and 50 nonexacerbators ) completed the study ( figure 1 ) . figure 2 also shows that among the frequent exacerbators , the number of patients who subsequently suffered severe exacerbations ( requiring one or more hospitalizations ) was two ( 11.8% ) , and frequent ( two or more exacerbations / year ) , infrequent ( one exacerbation / year ) , and no moderate or severe exacerbations were seven ( 42.1% ) , six ( 35.3% ) , and four ( 23.5% ) , respectively , whereas seven ( 63.6% ) of eleven infrequent and 37 ( 74.0% ) of 50 nonexacerbators experienced no further exacerbation . the proportions of frequent exacerbators who subsequently experienced frequent and infrequent exacerbations were significantly higher than those of nonexacerbators ( odds ratio [ 95% ci ] 2.94 [ 1.217.17 ] , p=0.0340 ; 2.94 [ 1.725.03 ] , p=0.0004 , respectively ) , but not in comparison with infrequent exacerbators ( 1.51 [ 0.494.63 ] , p>0.05 ; 2.01 [ 0.924.80 ] , p=0.053 , respectively ) . among five ( 5.6% ) of the severe exacerbators , two ( 40.0% ) had subsequently suffered severe exacerbations . the number of patients who had subsequent frequent , infrequent , and no moderate or severe exacerbations was two ( 40% ) , 0 , and three ( 60% ) , respectively ( figure 2 ) . in subanalysis , there was no difference in the proportion of patients who had subsequently suffered severe exacerbations between the frequent and severe exacerbator groups ( p>0.05 ) . in addition , one severe exacerbator died due to copd - related respiratory failure during the 1-year prospective observation period . the numbers of patients who reported pneumonia ( n=78 ) and those who regularly used ics ( n=15 ) were seven ( 9% ) and three ( 20% ) , respectively . patients who regularly used ics had higher but not significant contraction of pneumonia than those who did not use ics ( p>0.05 ) . figure 3a shows that the mean annual frequencies ( sd , exacerbations / year ) of future total ( 1.41.2 , p=0.0020 ) and moderate ( 1.31.1 , p=0.0057 ) exacerbations in frequent exacerbators were significantly higher than those in nonexacerbators ( 0.40.8 and 0.40.7 , respectively ) , but not in infrequent exacerbators ( 0.91.4 and 0.81.4 , respectively ) . figure 3b shows the kaplan meier analysis of the period until the first moderate and severe exacerbations . the median ( mean sd ) periods ( days ) until the first moderate and severe exacerbations for frequent , infrequent , and nonexacerbators were 266 ( 23528 ) , 365 ( 29330 ) , and 365 ( 32313 ) days , respectively ( log - rank test , p=0.0012 ) . there was no significant difference in the annual frequencies of severe exacerbation ( hospitalization ) among non- ( 0.00.3 ) , infrequent ( 0.10.39 ) , and frequent ( 0.10.3 ) exacerbators ( p>0.05 ) . the median ( mean sd ) periods until the first severe exacerbation were 365 ( 3632 ) , 365 ( 3650 ) , and 365 ( 3605 ) days , respectively ( p>0.05 ; data not shown ) . univariate analysis revealed that a low fev1 ( < 50% ) had the highest odds ratio for risk of future exacerbation ( one or more exacerbations / year ) , followed in order by regular use of ics , two or more previous exacerbations in the last year , presence of gerd , pneumococcal vaccination , one or more previous exacerbations in the last year , low body mass index ( 21.4 kg / m ) , a high total cat score ( 10 points ) , a high mmrc scale grade ( 2 ) and older age ( 68 years ) , whereas in patients who suffered two or more exacerbations , the highest odds ratio was observed due to the presence of gerd , followed in order by regular use of ics , low fev1 predicted , pneumococcal vaccination , older age , a high mmrc - scale grade , long duration of copd ( 4 years ) , and two or more and one or more previous exacerbations in the last year ( table 2 ) . however , 13 ( 86.7% ) of 15 patients who had received ics had also received pneumococcal vaccination . the independent risk factors for both one or more and two or more future exacerbations were low fev1 predicted , presence of gerd , and regular use of ics , whereas a low body mass index was an independent risk factor for one or more but not for two or more future exacerbations ( table 3 ) . ninety of 110 patients who provided informed consent were finally analyzed ; 32 ( 35.6% ) patients had suffered previous moderate and/or severe exacerbations during the last year . the numbers of patients with non- , infrequent , and frequent exacerbations were 58 ( 64.4% ) , 12 ( 13.3% ) , and 20 ( 22.2% ) , respectively ( figure 1 ) . at baseline , frequent exacerbators had a significantly lower body mass index , were less likely to be male or current smokers , had higher mmrc - scale grades and higher total cat score , had a higher proportion of spirometric stages iii and iv , gerd , and use of inhaled corticosteroid ( ics)/long - acting muscarinic antagonist or ics / long - acting 2-agonist combination therapy , and lower lung - function parameters , including fev1 , fvc , and fev1/fvc ratio before and after bronchodilator use compared with nonexacerbators , but not in comparison with infrequent exacerbators ( table 1 ) . frequent and infrequent exacerbators had significantly higher mmrc - scale grades and total cat scores compared with nonexacerbators ( table 1 ) . there was no change in the frequency of previous annual hospitalizations per patient between those with frequent and infrequent exacerbations ( table 1 ) . during the 1-year prospective observation period , one frequent exacerbator , one infrequent exacerbator , and two nonexacerbators died , with causes of death respiratory failure with copd exacerbation , cerebrovascular attack , and malignancies ( small - cell lung cancer and colon cancer ) , respectively , whereas one frequent exacerbator and six nonexacerbators dropped out for private reasons . as a result , 78 patients ( 17 frequent , eleven infrequent , and 50 nonexacerbators ) completed the study ( figure 1 ) . figure 2 also shows that among the frequent exacerbators , the number of patients who subsequently suffered severe exacerbations ( requiring one or more hospitalizations ) was two ( 11.8% ) , and frequent ( two or more exacerbations / year ) , infrequent ( one exacerbation / year ) , and no moderate or severe exacerbations were seven ( 42.1% ) , six ( 35.3% ) , and four ( 23.5% ) , respectively , whereas seven ( 63.6% ) of eleven infrequent and 37 ( 74.0% ) of 50 nonexacerbators experienced no further exacerbation . the proportions of frequent exacerbators who subsequently experienced frequent and infrequent exacerbations were significantly higher than those of nonexacerbators ( odds ratio [ 95% ci ] 2.94 [ 1.217.17 ] , p=0.0340 ; 2.94 [ 1.725.03 ] , p=0.0004 , respectively ) , but not in comparison with infrequent exacerbators ( 1.51 [ 0.494.63 ] , p>0.05 ; 2.01 [ 0.924.80 ] , p=0.053 , respectively ) . among five ( 5.6% ) of the severe exacerbators , two ( 40.0% ) had subsequently suffered severe exacerbations . the number of patients who had subsequent frequent , infrequent , and no moderate or severe exacerbations was two ( 40% ) , 0 , and three ( 60% ) , respectively ( figure 2 ) . in subanalysis , there was no difference in the proportion of patients who had subsequently suffered severe exacerbations between the frequent and severe exacerbator groups ( p>0.05 ) . in addition , one severe exacerbator died due to copd - related respiratory failure during the 1-year prospective observation period . the numbers of patients who reported pneumonia ( n=78 ) and those who regularly used ics ( n=15 ) were seven ( 9% ) and three ( 20% ) , respectively . patients who regularly used ics had higher but not significant contraction of pneumonia than those who did not use ics ( p>0.05 ) . figure 3a shows that the mean annual frequencies ( sd , exacerbations / year ) of future total ( 1.41.2 , p=0.0020 ) and moderate ( 1.31.1 , p=0.0057 ) exacerbations in frequent exacerbators were significantly higher than those in nonexacerbators ( 0.40.8 and 0.40.7 , respectively ) , but not in infrequent exacerbators ( 0.91.4 and 0.81.4 , respectively ) . figure 3b shows the kaplan meier analysis of the period until the first moderate and severe exacerbations . the median ( mean sd ) periods ( days ) until the first moderate and severe exacerbations for frequent , infrequent , and nonexacerbators were 266 ( 23528 ) , 365 ( 29330 ) , and 365 ( 32313 ) days , respectively ( log - rank test , p=0.0012 ) . there was no significant difference in the annual frequencies of severe exacerbation ( hospitalization ) among non- ( 0.00.3 ) , infrequent ( 0.10.39 ) , and frequent ( 0.10.3 ) exacerbators ( p>0.05 ) . the median ( mean sd ) periods until the first severe exacerbation were 365 ( 3632 ) , 365 ( 3650 ) , and 365 ( 3605 ) days , respectively ( p>0.05 ; data not shown ) . univariate analysis revealed that a low fev1 ( < 50% ) had the highest odds ratio for risk of future exacerbation ( one or more exacerbations / year ) , followed in order by regular use of ics , two or more previous exacerbations in the last year , presence of gerd , pneumococcal vaccination , one or more previous exacerbations in the last year , low body mass index ( 21.4 kg / m ) , a high total cat score ( 10 points ) , a high mmrc scale grade ( 2 ) and older age ( 68 years ) , whereas in patients who suffered two or more exacerbations , the highest odds ratio was observed due to the presence of gerd , followed in order by regular use of ics , low fev1 predicted , pneumococcal vaccination , older age , a high mmrc - scale grade , long duration of copd ( 4 years ) , and two or more and one or more previous exacerbations in the last year ( table 2 ) . however , 13 ( 86.7% ) of 15 patients who had received ics had also received pneumococcal vaccination . the independent risk factors for both one or more and two or more future exacerbations were low fev1 predicted , presence of gerd , and regular use of ics , whereas a low body mass index was an independent risk factor for one or more but not for two or more future exacerbations ( table 3 ) . the frequent - exacerbator phenotype is important to consider in the management of copd patients , who require future exacerbation - related hospitalization associated with high mortality.16 a previous western study suggested that frequent exacerbators tend to have had exacerbations during the previous year , a lower fev1 , more severe hrqol , a history of gerd , and a higher peripheral white blood - cell count in comparison with infrequent exacerbators and nonexacerbators.12 another western study demonstrated that the characteristics of frequent exacerbators include a more severe mmrc - scale grade , lower fev1 predicted , comorbid cardiovascular disease , depression or osteoporosis , and female sex as independent risk factors.32,33 we conducted the present study to observe moderate and severe exacerbations 1 year before and after baseline to investigate the characteristics of japanese copd patients who were frequent exacerbators . based on exacerbations during the previous year , we found that frequent exacerbators were more likely to be female , have a lower body mass index , have a significantly lower fev1 predicted , have a higher mmrc - scale grade ( lower exercise tolerance ) , and have a lower total cat score ( lower hrqol ) . we also found that the characteristics of frequent exacerbators were similar between japanese and westerners , except for body mass index , as reported previously.12,16,32 interestingly , univariate analysis did not show that previous frequent exacerbators would become future frequent exacerbators ( table 2 ) . indeed , 60% of previous frequent exacerbators did not suffer subsequent exacerbations , whereas conversely 14% of patients who had not previously suffered exacerbations subsequently did so ( figure 2 ) . investigation of factors predicting the change in frequency of exacerbations is critically important , as they are still unclear.34 a total of 78 ( 86.7% ) of our 90 patients completed the 1-year prospective study period . among five severe exacerbators , one ( 20% ) died , two ( 40% ) again developed severe exacerbation , and two ( 40% ) therefore , severe exacerbators appear to have a poor prognosis . however , our study - sample size was small and the observational period short . frequent exacerbators had a significantly higher frequency of future exacerbations and a shorter period until the next exacerbation than nonexacerbators , and also had a significantly poorer prognosis than the latter , thus confirming the findings of a previous study.12 previous studies have demonstrated that over half of copd patients have unreported exacerbations . such unreported exacerbations are thought to be an important component of hrqol decline.7,35,36 in our present study , to make the conditions for previous and future exacerbations uniform , daily journals for symptoms were not accepted . previous studies have demonstrated that the frequency of annual moderate or severe exacerbations per patient in japanese individuals may be lower than that in the usa and europe.13,14,3537 in our study , the mean ( sd [ range ] ) of previous annual total and severe exacerbations were 0.841.48 ( 06 ) and 0.060.23 ( 01 ) exacerbations / year , respectively for all patients ( data not shown ) , whereas those of future total and severe exacerbations were 0.711.08 ( 04 ) and 0.060.28 ( 02 ) exacerbations / year , respectively . our data for the frequency of exacerbations seem to indicate a slightly lower incidence in japanese than in westerners.1215,31,32,38 previous japanese reports on the frequency of exacerbations have been scarce.13,14 japanese copd patients may be slightly older and thinner on average than westerners.1215,31,32,38 although the discrepancy between japanese and westerners is still unclear , the difference in the frequency of exacerbations may be associated with different populations of phenotypes with emphysema , locality , and understanding of both the physician and the patient about diseases and exacerbations , such as convenient use of ics , and in definition of exacerbation . patients with copd have a wide variety of numerous comorbidities , which are strongly associated with mortality and exacerbation.1,12,26 we assessed baseline comorbidities based on interviews with patients and the physicians diagnosis , and partly through examinations or questionnaires . several comorbidities , such as depression21,39 assessed by the cesd questionnaire , acos27,40 based on previous criteria , and congestive status with heart failure and cor pulmonale based on history or medical signs,41 were carefully excluded . gerd , although not moderate to severe , was a common major comorbidity , and the proportion of frequent exacerbators with gerd was significantly higher than that of nonexacerbators . gerd was an independent , and the highest , risk factor for future frequent exacerbations , although all patients with gerd were receiving proton - pump inhibitors . our results confirmed previous reports.12,42 a low fev1 predicted was an independent risk factor for future exacerbations . the guidelines1,43 recommend long - term treatment with ics and pneumococcal vaccination for patients with severe and very severe copd , and frequent exacerbations may not be adequately controlled by long - acting bronchodilators . in our study regular use of ics , but not pneumococcal vaccination , was an independent risk factor for future frequent exacerbations . sixteen ( 17.8% ) of our patients used ics , although none had received ics without long - acting bronchodilators at baseline . users of ics included four nonexacerbators with gold stage ii , seven patients with frequent exacerbations , and the remaining five had severe or very severe disease . the risk of ics for respiratory infection including pneumonia is a concern.4446 among four nonexacerbators with gold stage ii and users of ics during the prospective observation period , two patients had frequent exacerbations and one had infrequent exacerbations . this risk must be weighed against the benefits when prescribing ics to patients with copd . first , the study population was small and the observation period rather short for analyzing severe exacerbations and mortality . balcells et al demonstrated that female sex was the highest risk factor for exacerbation,33 although in the present study there was no difference in the frequency of exacerbations between males and females . both physicians and patients were aware of the severity and frequency of previous exacerbations at baseline . we carefully removed severe comorbidities , such as cardiovascular disease and depression , in accordance with previous reports.26,27 however , patients with asthma and acos may have been included , because we did not investigate airway responsiveness and inflammation or serum total immunoglobulin ( ig ) e levels , although we carefully excluded patients with asthma based on symptoms and spirometry . however , no patients had received medications for osteoporosis and low peripheral lymphocyte counts , although latent osteoporosis and hiv infections were not tested for . fourth , the contents of medicines could not be ethically unified in a clinical setting in japan , and in our study adherence to pharmacological medicines and compliance with inhalation techniques were not assessed or included . the effects of respiratory medications , including inhaled medicines , on previous and future exacerbations were investigated . the presence of gerd , regular use of ics , and low fev1 may be associated with frequent exacerbations .

backgroundthe prognosis of japanese patients with copd who suffer repeated exacerbations is unclear , although westerners with such episodes have a poor prognosis.materials and methodswe conducted a 1-year prospective observational trial involving 90 japanese patients with copd : 58 nonexacerbators , 12 infrequent exacerbators , and 20 frequent exacerbators classified on the basis of exacerbation frequency ( zero , one , and two or more exacerbations / year ) , respectively , during the previous year were observed prospectively for 1 year . the characteristics of frequent exacerbators , the frequency of exacerbation , and the period until the first event were then compared among the groups.resultsa total of 78 patients completed the study . frequent exacerbators had a significantly higher risk of frequent exacerbation in the following year than the case for nonexacerbators ( odds ratio [ 95% confidence interval ] 2.94 [ 1.217.17 ] , p=0.0340 ) , but not in comparison with infrequent exacerbators ( 1.51 [ 0.494.63 ] , p>0.05 ) . the mean annual frequency of exacerbations in the following year was significantly ( p=0.0020 ) higher in the frequent exacerbators ( 1.4 exacerbations / year ) than in the nonexacerbators ( 0.4 ) , but not in the infrequent exacerbators ( 0.9 , p>0.05 ) . the mean period until the first exacerbation was significantly shorter in the frequent exacerbators than in the infrequent or nonexacerbators ( p=0.0012 ) . independent risk factors for future frequent exacerbation included the presence of gastroesophageal reflux disease , more severe airflow obstruction , and use of inhaled corticosteroids.conclusionour present results indicate that japanese copd patients suffering frequent exacerbation have a poor prognosis . the characteristics of japanese and western copd patients suffering frequent exacerbation are similar .

lipoma is a rare tumor but can be found at various sites in the abdominal cavity ( fig . 2 ) . if it is of a large size , it can compress adjacent tissues or organs and become heterogeneous . in such cases , imaging is incapable of distinguishing it from liposarcoma.lipoblastoma is a type of a benign tumor with embryonic fat . it is usually found in the extremities and torso whereas abdominal cavity belongs to its rather rare sites . in us , such tumors are echogenic . lipoblastomatosis is a form of this tumor characterized by aggressive localized growth.hibernoma is a benign tumor , mainly composed of brown fat , in whose cells the numbers of mitochondria responsible for enhanced metabolic activity are impressive . these tumors are rarely encountered in the retroperitoneal space but are also echogenic with well - or ill - defined margins . their characteristic feature is evident flow in color doppler.other benign tumors with a fat component , such as myolipoma , angiomyolipoma and myelolipoma , apart from teratoma ( which is usually benign ) , tend to be homogeneous with echogenicity increased to various degrees ( fig . 3 , 4 ) . only gonadal germ cell tumors , such as teratoma and dermoid cyst that arise from several germ layers , frequently have irregular echotexture due to the presence of fluid sebum , hair , soft tissues and bony elements . that is why they are encountered in solid , cystic or solid - cystic forms ( fig . mature fat tissue can also be found in choristoma which , however , is not a neoplasm . lipoma is a rare tumor but can be found at various sites in the abdominal cavity ( fig . 2 ) . if it is of a large size , it can compress adjacent tissues or organs and become heterogeneous . it is usually found in the extremities and torso whereas abdominal cavity belongs to its rather rare sites . in us , such tumors are echogenic . hibernoma is a benign tumor , mainly composed of brown fat , in whose cells the numbers of mitochondria responsible for enhanced metabolic activity are impressive . these tumors are rarely encountered in the retroperitoneal space but are also echogenic with well - or ill - defined margins . other benign tumors with a fat component , such as myolipoma , angiomyolipoma and myelolipoma , apart from teratoma ( which is usually benign ) , tend to be homogeneous with echogenicity increased to various degrees ( fig . 3 , 4 ) . only gonadal germ cell tumors , such as teratoma and dermoid cyst that arise from several germ layers , frequently have irregular echotexture due to the presence of fluid sebum , hair , soft tissues and bony elements . that is why they are encountered in solid , cystic or solid - cystic forms ( fig . mature fat tissue can also be found in choristoma which , however , is not a neoplasm . two views show lipoma ( arrows ) as a slightly echogenic lesion in the supraperitoneal fat three lipomas ( l ) in the small bowel mesentery , which in computed tomography showed density ranging from 57 to 74 hounsfield units two views show angiomyolipoma ( l ) located entirely in the adipose capsule of the right kidney . an arrow points to the site of regrowth in the form of the beak sign myelolipoma in the right suprarenal field ( arrow ) as a visible hyperechoic mass a heterogeneous mass arising from the retroperitoneal space is a mature teratoma with slight calcifications ( arrows ) liposarcoma is the only malignancy deriving from adipose tissue that is relatively frequently encountered in the retroperitoneal space ( it constitutes nearly 1/3 of sarcomas at this site and 1015% of all neoplasms of this type ) . according to the who , these tumors can be divided into five histological subtypes depending on the grade of their differentiation ( well - differentiated , dedifferentiated , myxoid , round cell , pleomorphic ) , which is relevant in prognosis . its presence can be indicated only by uneven margins and heterogeneous enhancement upon contrast agent administration . poorly differentiated subtypes account for even 40% of local relapses , and metastases are observed in 17% of cases . as has already been mentioned , well - differentiated liposarcoma is difficult to distinguish from lipoma also in sonography . one of the differential criteria can be tumor 's reaction to compression with a transducer ( fig . more aggressive forms of this tumor , however , tend to be unevenly delineated , present heterogeneous echotexture and infiltrate adjacent tissues ( fig . myxoid liposarcoma is characterized by a cystic structure and should be distinguished e.g. from lymphangioma , teratoma or cystic mesothelioma . no lesion compressibility when pressure is applied with the transducer ( arrow ) heterogeneous retroperitoneal liposarcoma infiltrates into adjacent tissues ( arrows ) mesenteric liposarcoma with pathological vascularity finally , immature teratoma must be mentioned . it is a primary germinal tumor that can contain adipose tissue and occurs at similar sites . they are characterized by the predominance of a solid component , and the only sign suggesting a malignancy is poorly circumscribed margins . a chance for malignant transformation cystic lesions can correspond to mesenteric cysts , lymphatic or vascular angiomatosis and even a cystic form of peritoneal mesothelioma , which is considered a benign lesion ( fig . the differential diagnosis of these pathologies should include ovarian cystic tumors , endometrial cysts and cystic tumors growing beyond organs , e.g. of the pancreas , kidneys or liver . solid tumors found at these sites include : leiomyoma and rhabdomyoma , angiomas as well as stromal , neurogenic and desmoid tumors ( fig . desmoid tumors , also called fibromatosis , are an interesting group of benign tumors that exhibit local aggressiveness and have considerable tendency to recur ( in approximately 50% of cases ) . all lesions were hypoechoic with slightly irregular echo patterns , 59% of cases were well - defined and 41% had irregular margins . the differential diagnosis should include sarcomas , mainly the two types that are most frequently found in soft tissues : histiocytic fibrosarcoma and liposarcoma , i.e. lesions characterized by higher echogenicity . it is of note that desmoid tumors frequently coexist with familial adenomatous polyposis , which is estimated at 918% of cases . of all imaging modalities , some authors recommend punch or even surgical biopsy in the event of doubts . in this case , mesenteric lymphangioma as a multilocular lesion without flow ( arrow ) retroperitoneal neuroblastoma in a 13-year - old ( arrows ) . aorta , v inferior vena cava desmoid , poorly vascularized tumor ( d ) in the small bowel mesentery these changes are rarely encountered but they represent a rich spectrum of mainly mesenchymal neoplasms : fibrosarcoma , malignant fibrous histiocytoma , hemangiopericytoma , leiomyosarcoma , rhabdomyosarcoma and gastrointestinal stromal tumors ( fig . an epithelioid malignant neoplasm encountered at this site is malignant peritoneal mesothelioma , a lesion that almost exclusively develops in males in the fifth and sixth decade of life . it can assume two morphological forms : irregular peritoneal infiltration without ascites ( dry appearance ) ( fig . 13 ) and nodular thickening of the peritoneum and greater omentum with ascites ( wet appearance ) . lesions of this type must be distinguished from papillary serous carcinoma of the peritoneum ( a rare neoplasm found in elderly women ) , abdominal carcinomatosis , in which ascites prevails over implants , and peritoneal tuberculosis , in which there are no tumorous lesions ( but ascites is present , the greater omentum is thickened with preserved smooth surface and the mesenteric lymph nodes are enlarged ) . lymphomas , however , are characterized by considerable abdominal lymphadenopathy , rare and slight ascites and no omental involvement . rhabdomyosarcoma with rich vascularity in the retroperitoneal space ( arrows ) two views present poorly vascularized malignant mesothelioma of the greater omentum ( m ) this is the most numerous group with prevailing carcinoma foci , mainly deriving from abdominal organs , such as pancreas , stomach , large bowel or ovaries . these lesions spread by direct extension , via the lymphatic system or blood stream and through the peritoneal cavity . their echotexture can vary . 14 ) and thickened parietal peritoneum ( its normal thickness is up to 1.5 mm ) ( fig . echogenic lesions that only slightly differ from the surrounding tissues are the hardest to find ( fig . peritoneal sites at which carcinoma implants tend to develop are : the recto - uterine pouch , right iliac fossa , right paracolic gutter and the region of the upper sigmoid mesentery . moreover , ovarian carcinoma cells frequently migrate to the supra- and subhepatic area ( fig . well - designed prospective studies have proven ultrasound imaging to be highly useful in detecting abdominal carcinomatosis . it occurred that lesions in the greater omentum were the easiest to detect ( 91% ) whereas those in the small bowel mesentery were the hardest to find ( 67% ) . moreover , ultrasound guidance enables one to obtain diagnostically valuable material for cytological and histological analysis in the vast majority of such cases . metastasis of malignant melanoma ( m ) from the interscapular space to the small bowel mesentery peritoneal carcinomatosis of ovarian carcinoma . thickened hypoechoic parietal peritoneum with signs of vascular flow ( arrows ) two views show a hyperechoic implant of ovarian carcinoma in the gastrocolonic ligament ( arrows ) ovarian carcinoma implants in the pouch of morison ( arrows ) another rare neoplasm encountered in fatty bodies of the abdominal cavity is non - hodgkin lymphoma . the involvement of the small bowel mesentery , with prominent , large mesenteric lymph nodes , is observed to occur in a half of patients with this neoplasm . these lymph nodes , together with the superior mesenteric vessels , create a so - called sandwich image ( fig . it is usually a small neuroendocrine neoplasm located in the small bowel . sometimes , the first morphological sign of its existence is the presence of mesenteric abnormalities in the form of enlarged lymph nodes or a hypoechoic mass . mesenteric thickening or contraction , which can lead to small bowel obstruction , is more rarely a predominant element of the image . any doubts in the assessment of these lesions require verification in computed tomography and , sometimes , magnetic resonance imaging . ultrasound imaging , however , enables precise , percutaneous sampling for cytological and histological analysis , which is worth remembering and applying . sandwich sign in the small bowel mesentery ( arrows ) created by enlarged lymph nodes surrounding the superior mesenteric vessels a manifestation of non - hodgkin lymphoma extranodal location of follicular lymphoma in the small bowel mesentery ( arrow ) lesions in the lymph nodes , which are relatively frequently affected by various neoplastic and non - neoplastic processes , have been presented only briefly since these issues are too broad to be included in this review . authors do not report any financial or personal connections with other persons or organizations , which might negatively affect the contents of this publication and/or claim authorship rights to this publication .

this article focuses on various cancerous lesions that are found beyond organs in the intra - abdominal fat and can be visualized with ultrasonography . these lesions are divided into five groups . the first group includes primary benign tumors containing adipocytes , such as lipoma , lipoblastoma , hibernoma and other lesions with an adipose tissue component , such as myolipoma , angiomyolipoma , myelolipoma and teratoma . the second group comprises primary malignant adipocytecontaining tumors , including liposarcoma and immature teratoma . the third group contains primary benign tumors without an adipocyte component that are located in intra - abdominal fat . this is a numerous group of lesions represented by cystic and solid tumors . the fourth group encompasses primary malignant tumors without an adipocyte component that are located in intra - abdominal fat . these are rare lesions associated mainly with sarcomas : fibrosarcoma , malignant fibrous histiocytoma , hemangiopericytoma and leiomyosarcoma . an epithelioid tumor at this site is mesothelioma . the last but not least group includes secondary malignant tumors without an adipocyte component located in intra - abdominal fat . this is the most numerous group with prevailing carcinoma foci . for each of these groups , the authors present ultrasound features of individual lesions and discuss their differential diagnosis . in the vast majority of cases , the material for cytological and histological analysis can be obtained during ultrasound - guided procedures . this is the advantage of this imaging modality .

the national cancer institute ( nci ) of the united states of america has proposed a reporting system for thyroid fine needle aspiration ( fna ) cytology , so - called bethesda system , which became an international standard of thyroid cytology [ 1 , 2 ] . following this recommendation , the uk royal college of pathologists ( the uk system ) [ 3 , 4 ] and italian societies of endocrinology and the italian society for anatomic pathology and cytology joint with the italian division of the international academy of pathology ( the italian system ) [ 5 , 6 ] updated their diagnostic schema comparable with the bethesda system . in 2013 , the japan thyroid association ( jta ) published guidelines for clinical practice for the management of thyroid nodules , including its diagnostic system for reporting thyroid fna cytology as shown in table 1 [ 79 ] , using criteria similar to those used in the papanicolaou society recommendation , the bethesda system [ 1 , 2 ] , the uk system [ 3 , 4 ] , and the italian system [ 5 , 6 ] . the jta reporting system of thyroid cytology further recommends risk stratification of follicular neoplasms ( fn ) into favor benign ( low risk : lr ) , borderline ( moderate risk : mr ) , and favor malignant ( high risk : hr ) , which was adopted from the practice of high - volume thyroid centers in japan . this study examined observer variation in the subclassification of fn among 4 thyroid experts to validate the usefulness and limitations of this characteristic risk stratification of fn recommended in the jta guidelines . it is not our purpose to address in detail all the morphological issues of thyroid cytology . conventional smear samples of indeterminate diagnosis ( n = 389 ) were selected from files ( n = 3843 ) from the year 2005 at ito hospital , tokyo , japan , by one ( k. kameyama ) of the authors . inadequate samples or those with poor preparation and cases under clinical follow - up ( no final histologic diagnosis ) were excluded . there were 91 ( 23.4% ) surgically treated patients with thyroid nodule under indeterminate cytological diagnoses . these patients visited ito hospital in 2005 and underwent diagnostic surgery or curative surgery in the subsequent years between 2005 and 2008 . there were 48 cases of benign diagnoses , including 29 follicular adenomas ( fas ) , 19 adenomatous nodules ( an ) , and 43 malignant diagnoses , including 13 follicular carcinomas ( ftcs ) , 24 papillary carcinomas ( ptcs ) , 1 poorly differentiated carcinoma , 2 c - cell carcinomas , and 3 malignant lymphomas . twenty cases of cytological smear samples with follicular patterned lesions were randomly selected by one ( k. kameyama ) of the authors and they were further analyzed for subclassification of fn and diagnoses by 4 reviewers , as shown in table 2 . the present study is a reproducibility study undertaken by 4 reviewers ( kennichi kakudo , kaori kameyama , mitsuyoshi hirokawa , and ryohei katoh ) who have special interest in thyroid pathology , all of whom are members of the clinical guideline committee of the jta . they were requested to subclassify fn following the new jta reporting system of thyroid cytology as shown in table 1 . those 20 cases of smear samples ( one representative smear sample each stained by the papanicolaou method ) were circulated among the 4 reviewers without clinical information . the 4 reviewers examined cytological samples independently without exchanging opinions and were unaware of the original cytological diagnoses and final histologic diagnoses . because this study was conceived as an audit of cytology performance and the results are anonymized , institutional ethical committee permission was not required for its conduct . informed consent of all 4 reviewers was obtained for this study protocol . for statistical analysis of observer concordance , was first introduced as a measure of the level of agreement between pairs of raters and extended to multiple raters , known as composite [ 11 , 12 ] . composite statistical analysis was performed by using data analysis and statistical software , version 13 ( stata press , college station , texas , usa ) . values of can be interpreted as follows : 0.000.20 , slight or very weak agreement ; 0.210.40 , weak to fair agreement ; 0.410.60 , moderate agreement ; 0.610.80 , substantial or good agreement ; and values over 0.81 , optimal or almost perfect agreement . there were 8 malignant cases ( 2 ftcs , widely invasive ; 4 ftcs , minimally invasive ; and 2 ptcs ) and 12 benign cases ( 1 an and 11 fas ) . all diagnoses made by the 4 reviewers on the 20 follicular pattern lesions are shown in table 2 . there were only 6 ( 30% ) cases with a consensus diagnosis among all 4 of the reviewers : 3 in fa and 3 in ftc . diagnoses of fn and hr were made in only 7 cases ( 35% ) , including 4 malignant and 3 benign cases . composite statistical analysis clearly demonstrated good concordance for the hr diagnosis in malignant cases ( = 0.7714 ) ( table 3 ) , while this was not found for the other subcategories in both benign and malignant lesions ( < 0.4 ) . it is of note that there was no interobserver variation in ftcs , widely invasive ( n = 2 ) , and all 4 reviewers classified the 2 cases into the hr subcategory . it is clear that ftcs , widely invasive , have different degrees of cellular abnormality in our study . in contrast , there were significant disagreements among other types of malignancy and benign lesions , which clearly confirmed that the subclassification of fn is not a definite diagnosis but risk stratification useful for triage patients . concerning ftc , minimally invasive ( n = 4 ) , there was only one concordant case among the 4 reviewers and all reviewers made a mr diagnosis in 1 case . there were disagreements between mr and hr in 1 case and between lr and mr in 2 cases . as for ptc ( n = 2 ) , the 4 reviewers gave 2 diagnoses each , which included 3 lrs , 3 mrs , 1 hr , and 1 suspicious for ptc . however , it is of note that there was no single case in our 20 cases whose subclassification varied among all 3 subcategories . in benign final histologic diagnosis ( n = 12 ) , there were 9 split diagnoses , including 6 cases between lr and mr and 3 cases between mr and hr , but no case between lr and hr . the incidence of lr diagnoses by the 4 reviewers in the 12 benign lesions was 31.3% ( 15/48 ) and that of lr diagnoses in the 6 cases of ftc was 16.7% ( 4/24 ) ( p = 0.186 ) . the incidence of hr diagnosis in the 12 benign lesions was 10.4% ( 5/48 ) and that of hr in the 6 cases of ftc was 47.1% ( 10/24 ) ( p = 0.002 ) . the incidence of malignancy of the hr was high at 60% for reviewer a , 75% for b , 75% for c , and 75% for d ( 6075% ) . the incidence of malignancy in the mr was lower than that of hr and was 33.3% for reviewer a , 35.7% for reviewer b , 25% for c , and 30.8% for d ( 2535.7% ) . the incidence of malignancy in the lr was lower than that in the hr and was 33.3% for reviewer a , 0% for b , 37.5% for c , and 33.3% for d ( 037.5% ) . although the risks of malignancy in the subcategories of fn differed among the 4 reviewers , it is of note that hr cytological diagnosis was significantly correlated with malignant histological diagnosis , and the risk of malignancy ( 6075% ) was significantly higher than that in the other 2 subcategories ( 035.7% ) in our study ( p = 0.002 ) . although the standardization of terminology and diagnostic criteria is important for accurate communication among patients , clinical doctors , and cytopathologists [ 110 ] , there are still differences among reporting systems in thyroid cytology as to how to interpret cytological diagnoses and how to decide on the clinical management of patients . in japan , cytopathologists attempted to use an internationally accepted reporting system , but it had to be modified to fit our practice . in japan , all patients with indeterminate cytology undergo further diagnostic procedures , without immediate surgery , to search for higher risk patients who should undergo surgery [ 79 , 1315 ] . it is because the majority of thyroid carcinomas in indeterminate cytology are indolent and more conservative approaches , other than immediate diagnostic surgery , usually do not create any harm to the patient with malignancy [ 1620 ] and diagnostic surgery to all patients with indeterminate cytology results in risks of unnecessary surgery to the patients with benign nodules , more than 80% of the patients [ 79 , 1922 ] . the proportion of malignancy found at thyroidectomy from patients with indeterminate cytology in this clinical setting will increase in number , and the malignancy rate of patient with indeterminate cytology in our study was calculated as 47.3% as shown in materials and methods . takezawa et al . from japan retrospectively analyzed their 1606 cytological samples using bethesda system , although it was written in japanese with english abstract , and they identified 115 ( 7.9% ) cases of aus / flus ( atypia of undetermined significance / follicular lesions of undetermined significance ) and 61 ( 4.2% ) cases of fn / sfn ( follicular neoplasms / suspicious for follicular neoplasms ) . the resection rate of their aus / flus nodules was 30.4% ( 35/115 cases ) and its malignancy rate was 88.6% ( 31/35 cases ) , and the resection rate of fn / sfn nodules was 36.0% ( 22/61 cases ) and its malignancy rate was 72.4% ( 16/22 cases ) , which were very different from the ranges reported in most of the literatures from western countries using the bethesda system or the uk system [ 1 , 2 , 2023 ] . as it is clear , further triage of patients with indeterminate nodules reduces resection rates and increases malignancy rates in any diagnostic systems including the bethesda system . this clinical management was also true in some other countries [ 2427 ] , as crippa and dina commented in a letter to an editor that thyroid cytology is the most important but not the only important deciding factor and therefore it must be integrated with other diagnostic procedures . we propose that future thyroid cytology classification schemes should reconsider clinical managements of indeterminate categories and how to reduce unnecessary surgeries for patients with benign thyroid nodules [ 79 ] . the risk stratification of follicular pattern lesions into 3 subcategories ( cellular follicular lesion , fn favor benign , and fn favor malignant ) was suggested by the papanicolaou society in 1996 , but it did not become popular in thyroid cytology worldwide , apart from japan [ 8 , 9 ] . there have been some reports in the literature on risk stratification of fn [ 10 , 2834 ] . kelman et al . reported that 31/52 ( 60% ) nodules with nuclear atypia consistent with fn were malignant and it was 4/9 ( 44.4% ) in fn with atypia by goldstein et al . . reported that atypical proliferation was more often malignant than follicular group ( 53% versus 19% ) in their indeterminate category ( tir3/thy3 ) . some researchers pointed out that the malignancy rate of fn without atypia is low and assessment later on could be an alternative approach [ 31 , 32 ] and patients with high - risk cytological features such as nuclear overlapping ( crowding ) should be advised to have a surgical intervention [ 3133 ] . gerhard and da cunha santos using the papanicolaou society guidelines studied reproducibility between 2 observers in 97 diagnoses . they reported a substantial level of diagnostic interobserver ( = 0.71 ) and intraobserver ( = 0.66 ) reproducibility , although interobserver disagreement in the cytological diagnosis occurred in 23 cases ( 24.7% ) and 18 ( 41.7% ) of them were for fn . in an interobserver reproducibility study using the uk system , kocjan et al . reported that the statistic was very poor ( 0.11 ) for the thy3a category and that for thy4 was 0.17 , in contrast to moderate to good agreement for thy1 ( 0.69 ) , thy2 ( 0.55 ) , thy3f ( 0.51 ) , and thy5 ( 0.61 ) . the observer variation of fn in our study occurred more often between lr and mr ( 9 cases , 45% ) , followed by between mr and hr ( 5 cases , 25% ) , but it is remarkable to note that none occurred between lr and hr . in other words , discordance is limited to between lr and mr or mr and hr , so we may conclude that the mr subcategory has an essential role in minimizing discordance between lr and hr . another choice of subclassification of follicular neoplasms would be two categories ( low cancer risk and high cancer risk ) instead of three categories ( lr , mr , and hr ) , and this modification ( two categories ) is also described as acceptable in the reporting system recommended by the japan thyroid association [ 7 , 8 ] . the second conclusion we may draw is that hr in the jta system is a powerful cytological subcategory to be used for the triage of patients for diagnostic surgery because the risk of malignancy of hr is high ( 6075% ) equivalent to suspicious for malignancy category in the bethesda system , with good concordance among the 4 reviewers ( = 0.7714 ) . abele and levine reported their rate of indeterminate category to be 5% of 51,000 adequate fnas and they suggested that the national rate of 15% was in large part due to overdiagnosis . this significant difference in ratio of indeterminate categories may be due to experience of thyroid cytology and not patients ' background . clary et al . commented in their interobserver variability study that some pathologists make greater use of indeterminate categories such as follicular lesion , favor nonneoplastic or follicular lesion , and favor neoplastic lesion , whereas others show more definitive categorization into benign and neoplastic groups . cibas et al . also stated in their report on interobserver variability that cytopathologists with experience of thyroid cytopathology are more likely to make a definitive interpretation ( i.e. , benign or malignant ) . this tendency was seen in our present study in which reviewers b and d ( whose indeterminate diagnosis rates are about 15% in their practice ) made mr diagnosis more often ( 70% and 65% , resp . ) and lr diagnosis less frequently ( 10% and 15% , resp . ) than those of reviewers a and c ( whose indeterminate diagnosis rates are about 5% in their practice ) . the difference in the prevalence of benign and fn / sfn may explain the different rates of lr ( low risk ) and mr ( moderate risk ) among reviews in our study , because incidence of one category may expand or contract depending on the rates of other categories . some fn / sfn lesions with benign pattern would be classified as benign by different authors and the incidence of fn / sfn of bethesda system in recent 7 series varied from 1.5 to 9.7% and that of benign category was between 54 and 77.4% summarized by ohori and schoedel . rapid development of molecular analyses on thyroid cytology may lead us possibly in the near future to more accurately identify patients who should be referred to surgery . until that time comes , thyroid fna cytology remains a main stay in the management of patients with thyroid nodules integrated with other clinical tests , such as ultrasound image diagnosis . as a conclusion the hr subcategory has a high predictive value of malignancy and good agreement among the 4 reviewers which is clinically helpful to triage patients for surgery . we believe that patients with cytological diagnosis of hr subcategory of fn in the jta system should be surgically treated especially if other risk factors coexisted . on the other hand , patients with lr or mr category therefore , surgical resection rate of indeterminate category is low in japan usually less than 50% , particularly in cases with fn .

background . the japan thyroid association recently published guidelines for clinical practice for the management of thyroid nodules , which include a diagnostic system for reporting thyroid fine needle aspiration cytology . it is characterized by the subclassification of follicular neoplasms , which is different from other internationally accepted reporting systems . materials and methods . this study examined observer variability in the subclassification of follicular neoplasms among 4 reviewers using papanicolaou - stained smear samples from 20 surgically treated patients with indeterminate cytology . results . the favor malignant subcategory had high predictive value of malignancy ( risk of malignancy : 6075% ) and good agreement among the 4 reviewers ( = 0.7714 ) . conclusion . these results clearly confirmed that the risk stratification of follicular neoplasms , which was adapted from cytology practice of high - volume thyroid centers in japan , can provide clinically helpful information to estimate the risk of malignancy and to triage patients for surgery .

it is not uncommon to see bleeding symptoms in patients in outpatient or hospital - based practice . postpartum hemorrhage complicates pregnancy and accounts for significant morbidity and mortality , particularly in underdeveloped countries.1,2 menorrhagia is a common clinical challenge and is often associated with secondary anemia , excessive fatigue , and a negative effect on health - related quality of life.3 postoperative bleeding is one of the more common complications of surgery . trauma is a leading cause of morbidity and mortality in the younger population.4 while bleeding symptoms may be commonly seen by physicians of multiple specialties , it is unclear how frequently these symptoms belie an underlying undiagnosed congenital or acquired bleeding disorder . in the us , the most common congenital bleeding disorders include von willebrand disease , which affects approximately 1% of the population ( males and females equally),5 and hemophilia a and b combined , which affect approximately 20,000 persons ( essentially all males , with rare exception).6 medications can also affect coagulation or platelet function,7 as can certain herbal supplements.8 trauma and surgery can lead to blood loss , and critical reduction in coagulation factors can lead to additional non - surgical bleeding complications ( coagulopathic bleeding).9 the most ubiquitous method for evaluating coagulation is prothrombin time ( pt)/international normalized ratio ( inr ) and activated partial thromboplastin time ( aptt ) . typically , they are ordered to monitor anticoagulant therapy ( pt / inr for warfarin , aptt for heparin ) , to evaluate coagulation preoperatively , or in response to hemorrhagic symptoms . the pt / inr provides an assessment of the extrinsic ( tissue factor - dependent ) and final common pathways of the coagulation cascade , while the aptt provides an assessment of the intrinsic ( tissue factor - independent ) and final common pathways.10 an example of a potentially life - threatening cause of unexplained recent onset or acute bleeding associated with a prolonged aptt is acquired hemophilia , with an incidence of approximately 1 to 4 per million / year.11 acquired hemophilia primarily affects older adults,12 an ever - growing segment of the population that presents for medical evaluation and care , but may also occur during pregnancy as postpartum hemorrhage or in association with other underlying diseases , including cancer and autoimmune disorders.13 prompt diagnosis is a primary determinant of prognosis in acquired hemophilia14 because initiation of definitive therapy ( ie , hemostatic and immunosuppressive ) is delayed until the diagnosis is made . acquired hemophilia - related bleeding does not respond to the typical management algorithms used for hemorrhaging in a patient and , therefore , is associated with high morbidity and mortality . severe bleeds occur in up to 90% of patients with acquired hemophilia,11 and the reported overall mortality rate in these patients ranges from 8% to 22%.1517 given the rarity of acquired hemophilia , combined with the general lack of familiarity of nonhematologists with this condition , the diagnosis of acquired hemophilia poses a clinical challenge , even in patients presenting with straightforward bleeding and an isolated , prolonged aptt . a survey was conducted of physicians across a number of specialties to identify potential barriers to the effective recognition and management of this rare but important cause of serious bleeding . the survey , based on an actual case found to be the result of acquired hemophilia , focused on participants stepwise evaluation and management of a case patient who presented to the hospital with recent - onset bleeding . the survey also assessed participants history with regard to consulting hematologists , discovering or diagnosing underlying bleeding disorders , and encountering acquired hemophilia in clinical practice . findings pertaining to the interpretation and follow - up of abnormal coagulation studies , enlistment of hematology consultation , and the diagnostic decision process in an actively bleeding case patient were the primary areas assessed in this analysis . results were evaluated across specialties to determine any specialty - specific practice trends that might hinder effective recognition and management of an actively bleeding patient with coagulopathy , including acquired hemophilia . physicians within the specialties of hematology , hematology / oncology , emergency medicine , geriatrics , internal medicine , rheumatology , obstetrics and gynecology , critical care medicine , and general surgery were randomly sampled from the american medical association physician masterfile . physicians who were part of the harris interactive online physician panel were invited via email to participate in the survey , while physicians who were not part of the panel were invited via first class mail . the invitation provided a uniform resource locator address and password for one - time use to log on to the survey site , where invitees first encountered questions to determine eligibility to participate in the survey . eligible physicians were required to be actively practicing , with additional specialty - specific requirements . in particular , hematologists , hematologist / oncologists , general surgeons , and emergency medicine and critical care physicians were required to be practicing at an acute care hospital . for internists and geriatricians , at least 60% of their practice had to consist of patients older than 60 years of age . obstetricians / gynecologists were required to have an active obstetrics practice . upon confirmation of eligibility , participants completed the online survey , the average duration of which was approximately 10 minutes . each specialty was recruited to a final sample of 50 complete responses to ensure a sufficient number of responses to generate reasonable hypotheses about specialists behavior to test in face - to - face interviews . findings from the surveys completed by the physicians specializing in hematology , hematology / oncology , emergency medicine , geriatrics , internal medicine , rheumatology , and critical care medicine are presented herein . general surgeons and obstetrics and gynecology physicians each completed different surveys from the aforementioned specialties ; the findings of those surveys are not included here . survey questions focused on the diagnostic and general therapeutic approach to the management of a case patient ( figure 1 ) whose presentation and clinical course were based on those of a real patient who experienced significant delays in diagnosis despite the involvement of multiple specialists . at each juncture in the case , participants were given lists of actions that included diagnostic tests ( laboratory , radiology , other ) , consultations , and potential treatments addressing bleeding or anemia ( local hemostasis , transfusion ) . since familiarity with the upper limits of normal of the pt / aptt tests was being tested in this study , upper limits of normal were not specified , and aptts were 25% to 30% above the upper limits of normal and set at a value that respective specialties would recognize as abnormal based on their experience with ordering and interpreting this laboratory test . the survey did not specify whether laboratory testing , imaging studies , or consultations in this hypothetical case had to be available immediately or locally , and the assumption was that respondents would answer based on their best medical judgment in an ideal practice situation . rheumatologists were only asked about an initial emergency department / urgent care presentation because it was expected that if a bleeding disorder developed in one of their patients it would present in an outpatient or emergency department / urgent care setting . conversely , critical care specialists started at presentation 2 , since their involvement in a recurrent epistaxis workup would be unlikely . vital signs were adjusted to make it more likely that a potential intensive care unit admission would require their evaluation . since emergency medicine physicians would most likely have ordered an imaging study initially to define the patient s retroperitoneal hematoma , we allowed for the option of a general surgery consultation even though the computed tomography findings were never presented . hematology / oncology respondents were given the option of ordering additional specialty coagulation laboratory tests ( eg , mixing studies ) . participants were additionally questioned about their experiences diagnosing underlying bleeding disorders and , in the case of nonhematologist specialists , consulting hematologist colleagues . conversely , hematology and hematology / oncology specialists were asked about being on the receiving end of consultations , specifically from emergency medicine personnel , and the reasons for those consultations . a total of 302 physicians ( 5051 per specialty ) participated in the case - based survey reported herein . demographics of the specialty groups were consistent : mean age ranged from 45.6 to 50.6 years , and mean years of practice experience ranged from 14.5 to 19.3 . faced with an older adult female patient with recurrent epistaxis , nearly 90% of physicians in each of the surveyed specialties indicated they would have ordered a complete blood count and coagulation studies ( pt / inr and aptt ) as part of the initial evaluation ( figure 2a ) . despite abnormal results of the aptt at 42 seconds ( with an upper limit of normal typically ranging from 35 to 39 seconds , although not specified in the case ) , less than half the physicians in most specialties would have chosen to repeat the coagulation studies as one of the next steps ( figure 2b ) . in contrast , 67% of hematologists would have repeated these studies . less than 45% of surveyed physicians in all nonhematology specialties would have consulted a hematologist after reviewing the initial coagulation study results ( figure 2b ) . rheumatologists were most likely to obtain a consult ( 43% ) , although they were presented with an initial aptt that was more abnormal ( 50 seconds ) than the aptt initially presented to the other specialists . after the patient s second presentation several weeks later with bruising and abdominal / back pain , again nearly 90% or more of respondents ( including critical care specialists , who began their case review at this juncture in the patient s clinical course ) would have ordered both a complete blood and coagulation studies as part of their initial evaluation ( figure 2c ) . when these results revealed a clearly abnormal and markedly changed aptt of 63 seconds , the majority of respondents indicated they still would not have repeated coagulation studies ( figure 2d ) . approximately 75% of internal medicine and geriatrics physicians would have consulted a hematologist at this point , compared with 47% and 50% of emergency medicine and critical care specialists , respectively ( figure 2d ) . sixty percent of hematologists and hematologists / oncologists surveyed would have evaluated the patient s peripheral blood smear at this point . in addition to the aforementioned hematologic laboratory tests , other diagnostic tests and consultations that participants would have recommended from a series of options as part of their evaluation after the patient s second presentation are shown in figure 3 . participants across specialties clearly preferred computed tomography of the abdomen ( 80%84% ) over abdominal ultrasound ( 9%28% ) , upper gastrointestinal endoscopy ( 2%16% ) , or colonoscopy ( 0%16% ) . emergency medicine physicians demonstrated the greatest breadth of testing , with 82% additionally recommending urinalysis and 92% recommending a stool guaiac test . requests for gastroenterology consultations ranged from 10% to 43% and were highest for the internal medicine group , which also had the highest proportion of physicians recommending endoscopy ( 16% each for upper gastrointestinal endoscopy and colonoscopy ) . for the purposes of assessing multiple specialties , the next 12 hours of observation in the case patient s clinical course were described as having occurred in the emergency department . by the end of this observation period , the aptt had further increased to at least twice the upper limit of normal , while the hemoglobin level had decreased . internists and geriatricians were most inclined to repeat the laboratory studies at this juncture ( figure 4 ) . in contrast with previous time points , by this point in the patient s clinical presentation , 73% to 94% of respondents would have consulted a hematologist . the majority of hematologists would have ordered 1:1 mixing studies of patient and normal plasma to rule out coagulation inhibitors ( 97% ) and fibrinogen / fibrin split products testing ( 75% ) in response to the laboratory results obtained after 12 hours of observation , as part of a diagnostic evaluation of the prolonged aptt . emergency medicine physicians were given the additional option of consulting general surgery practitioners ; 2% would have done so after the first set of laboratory results at the second presentation , and 20% would have done so in response to the second set of laboratory results . the percentages of physicians in each specialty who would have recommended admission of this case patient to a general hospital floor or back to a skilled nursing facility or nursing home after initial presentation with subsequent abnormal aptt of 42 seconds are shown in figure 5a . across the majority of specialties , less than 20% of physicians would have recommended overnight admission to a general hospital floor , while more than 50% of physicians in these specialties would have recommended discharge to a skilled nursing facility or a nursing home . in contrast with respondents from other specialties , a slightly higher proportion of hematologists and hematologist / oncologists would have endorsed a higher level of care at this point ; 36% of physicians in this group indicated they would have favored overnight hospital admission over discharge to a skilled nursing facility . the majority of physicians faced with the second presentation would have recommended that the patient be admitted to the hospital . the differences among specialties were most noticeable in allocation to a general hospital floor versus an intensive care unit after review of initial laboratory results or the second set of laboratory results obtained after 12 hours of observation ( figure 5b ) . while approximately 80% or more of physicians in each specialty would have recommended hospital admission in response to both sets of laboratory results , the proportion recommending admission specifically to the intensive care unit increased as the laboratory values deteriorated ( worsening anemia and coagulopathy ) . based on the laboratory results obtained after 12 hours of observation , nearly 50% or more of physicians in each specialty would have recommended admission to the intensive care unit in lieu of a general hospital floor , compared with approximately 40% or less across specialties after the initial laboratory results obtained during the patient s second presentation . the difference was most noticeable for physicians in emergency medicine ( 35%73% ) , the specialty most likely to refer the patient to an inpatient medical / hospitalist or critical care service . more than 85% of emergency medicine and critical care physicians reported having ever discovered an underlying bleeding disorder , while 100% of hematologists and hematologist / oncologists reported having ever diagnosed one ( figure 6a ) . the percentage of physicians in other specialties who had ever discovered an underlying bleeding diathesis ranged from 47% to 65% . among those physicians who had previously diagnosed or discovered a bleeding disorder , 14% to 77% had specifically encountered acquired hemophilia ( figure 6b ) , although this quantitative survey did not assess whether they understood this disorder . hematologists and hematologists / oncologists accounted for the highest percentage in this group , while internal medicine specialists accounted for the lowest percentage . table 2 shows the frequencies with which various specialties had ever consulted a hematologist when they encountered a patient with an abnormal pt / inr and aptt and no history of a bleeding diathesis or medications that affect coagulation . the distribution of reasons for which respondents would have consulted a hematologist is also shown in table 2 . in the majority of specialties ( emergency medicine , geriatrics , and internal medicine ) , the highest percentage of physicians had consulted a hematologist 1 to 2 times for a patient with an unexplained prolonged pt / inr or aptt . in contrast , more than 80% of rheumatologists had consulted a hematologist 3 , 4 , or 5 or more times for the same reason . the highest percentage ( 39% ) of critical care physicians the most common reason for consulting a hematologist was abnormal coagulation study results in the setting of clinical bleeding ( table 2 ) , which was parallel to the findings for the 100 additional surgeons and obstetricians surveyed ( data not shown ) . the most common response among critical care specialists to the open - ended question , what are the circumstances around which you would call for a hematology consult ? was an unexplained abnormal lab or bleeding ( 33 like or similar mentions ) , followed by any unexplained bleeding disorder conversely , the frequency with which hematologists had ever been consulted by an emergency medicine provider for an abnormal pt / inr or aptt in a patient with no history of bleeding diathesis or medications that affect coagulation is outlined in table 2 . the majority ( 57% ) of hematologists had been consulted a minimum of 5 times by their colleagues in emergency medicine . the majority of these consults pertained to significantly abnormal coagulation studies with or without clinical bleeding ( table 2 ) . faced with an older adult female patient with recurrent epistaxis , nearly 90% of physicians in each of the surveyed specialties indicated they would have ordered a complete blood count and coagulation studies ( pt / inr and aptt ) as part of the initial evaluation ( figure 2a ) . despite abnormal results of the aptt at 42 seconds ( with an upper limit of normal typically ranging from 35 to 39 seconds , although not specified in the case ) , less than half the physicians in most specialties would have chosen to repeat the coagulation studies as one of the next steps ( figure 2b ) . in contrast , 67% of hematologists would have repeated these studies . less than 45% of surveyed physicians in all nonhematology specialties would have consulted a hematologist after reviewing the initial coagulation study results ( figure 2b ) . rheumatologists were most likely to obtain a consult ( 43% ) , although they were presented with an initial aptt that was more abnormal ( 50 seconds ) than the aptt initially presented to the other specialists . after the patient s second presentation several weeks later with bruising and abdominal / back pain , again nearly 90% or more of respondents ( including critical care specialists , who began their case review at this juncture in the patient s clinical course ) would have ordered both a complete blood and coagulation studies as part of their initial evaluation ( figure 2c ) . when these results revealed a clearly abnormal and markedly changed aptt of 63 seconds , the majority of respondents indicated they still would not have repeated coagulation studies ( figure 2d ) . approximately 75% of internal medicine and geriatrics physicians would have consulted a hematologist at this point , compared with 47% and 50% of emergency medicine and critical care specialists , respectively ( figure 2d ) . sixty percent of hematologists and hematologists / oncologists surveyed would have evaluated the patient s peripheral blood smear at this point . in addition to the aforementioned hematologic laboratory tests , other diagnostic tests and consultations that participants would have recommended from a series of options as part of their evaluation after the patient s second presentation are shown in figure 3 . participants across specialties clearly preferred computed tomography of the abdomen ( 80%84% ) over abdominal ultrasound ( 9%28% ) , upper gastrointestinal endoscopy ( 2%16% ) , or colonoscopy ( 0%16% ) . emergency medicine physicians demonstrated the greatest breadth of testing , with 82% additionally recommending urinalysis and 92% recommending a stool guaiac test . requests for gastroenterology consultations ranged from 10% to 43% and were highest for the internal medicine group , which also had the highest proportion of physicians recommending endoscopy ( 16% each for upper gastrointestinal endoscopy and colonoscopy ) . for the purposes of assessing multiple specialties , the next 12 hours of observation in the case patient s clinical course were described as having occurred in the emergency department . by the end of this observation period , the aptt had further increased to at least twice the upper limit of normal , while the hemoglobin level had decreased . internists and geriatricians were most inclined to repeat the laboratory studies at this juncture ( figure 4 ) . in contrast with previous time points , by this point in the patient s clinical presentation , 73% to 94% of respondents would have consulted a hematologist . the majority of hematologists would have ordered 1:1 mixing studies of patient and normal plasma to rule out coagulation inhibitors ( 97% ) and fibrinogen / fibrin split products testing ( 75% ) in response to the laboratory results obtained after 12 hours of observation , as part of a diagnostic evaluation of the prolonged aptt . emergency medicine physicians were given the additional option of consulting general surgery practitioners ; 2% would have done so after the first set of laboratory results at the second presentation , and 20% would have done so in response to the second set of laboratory results . the percentages of physicians in each specialty who would have recommended admission of this case patient to a general hospital floor or back to a skilled nursing facility or nursing home after initial presentation with subsequent abnormal aptt of 42 seconds are shown in figure 5a . across the majority of specialties , less than 20% of physicians would have recommended overnight admission to a general hospital floor , while more than 50% of physicians in these specialties would have recommended discharge to a skilled nursing facility or a nursing home . in contrast with respondents from other specialties , a slightly higher proportion of hematologists and hematologist / oncologists would have endorsed a higher level of care at this point ; 36% of physicians in this group indicated they would have favored overnight hospital admission over discharge to a skilled nursing facility . the majority of physicians faced with the second presentation would have recommended that the patient be admitted to the hospital . the differences among specialties were most noticeable in allocation to a general hospital floor versus an intensive care unit after review of initial laboratory results or the second set of laboratory results obtained after 12 hours of observation ( figure 5b ) . while approximately 80% or more of physicians in each specialty would have recommended hospital admission in response to both sets of laboratory results , the proportion recommending admission specifically to the intensive care unit increased as the laboratory values deteriorated ( worsening anemia and coagulopathy ) . based on the laboratory results obtained after 12 hours of observation , nearly 50% or more of physicians in each specialty would have recommended admission to the intensive care unit in lieu of a general hospital floor , compared with approximately 40% or less across specialties after the initial laboratory results obtained during the patient s second presentation . the difference was most noticeable for physicians in emergency medicine ( 35%73% ) , the specialty most likely to refer the patient to an inpatient medical / hospitalist or critical care service . more than 85% of emergency medicine and critical care physicians reported having ever discovered an underlying bleeding disorder , while 100% of hematologists and hematologist / oncologists reported having ever diagnosed one ( figure 6a ) . the percentage of physicians in other specialties who had ever discovered an underlying bleeding diathesis ranged from 47% to 65% . among those physicians who had previously diagnosed or discovered a bleeding disorder , 14% to 77% had specifically encountered acquired hemophilia ( figure 6b ) , although this quantitative survey did not assess whether they understood this disorder . hematologists and hematologists / oncologists accounted for the highest percentage in this group , while internal medicine specialists accounted for the lowest percentage . table 2 shows the frequencies with which various specialties had ever consulted a hematologist when they encountered a patient with an abnormal pt / inr and aptt and no history of a bleeding diathesis or medications that affect coagulation . the distribution of reasons for which respondents would have consulted a hematologist is also shown in table 2 . in the majority of specialties ( emergency medicine , geriatrics , and internal medicine ) , the highest percentage of physicians had consulted a hematologist 1 to 2 times for a patient with an unexplained prolonged pt / inr or aptt . in contrast , more than 80% of rheumatologists had consulted a hematologist 3 , 4 , or 5 or more times for the same reason . the highest percentage ( 39% ) of critical care physicians had consulted a hematologist at least 5 times for such a patient . the most common reason for consulting a hematologist was abnormal coagulation study results in the setting of clinical bleeding ( table 2 ) , which was parallel to the findings for the 100 additional surgeons and obstetricians surveyed ( data not shown ) . the most common response among critical care specialists to the open - ended question , what are the circumstances around which you would call for a hematology consult ? was an unexplained abnormal lab or bleeding ( 33 like or similar mentions ) , followed by any unexplained bleeding disorder conversely , the frequency with which hematologists had ever been consulted by an emergency medicine provider for an abnormal pt / inr or aptt in a patient with no history of bleeding diathesis or medications that affect coagulation is outlined in table 2 . the majority ( 57% ) of hematologists had been consulted a minimum of 5 times by their colleagues in emergency medicine . the majority of these consults pertained to significantly abnormal coagulation studies with or without clinical bleeding ( table 2 ) . bleeding is commonly encountered in outpatient and hospital - based medical practice and can have a wide variety of underlying causes . while bleeding may be relatively common , most acquired and congenital bleeding disorders are uncommon , and some , such as acquired hemophilia , are rare . nevertheless , the consequences of failure to recognize promptly and treat properly a bleeding disorder may be significant.18 in the case of acquired hemophilia , morbidity and mortality rates are particularly high : severe bleeding is experienced by up to 90% of affected patients , and mortality rates are as high as 22%.11 this survey provided a step - wise methodology to tease out specialty - specific patterns of interpretation of clinical data to identify barriers to the diagnosis and treatment of underlying bleeding disorders . the sample size obtained across specialties was sufficient to generalize these findings , at least to the point of identifying specific issues for education and development of clinical decision - making pathways . when presented with a clinical picture that includes a recent history or symptoms of active bleeding , clinicians typically obtain coagulation times , such as the pt / inr and aptt , as part of the initial diagnostic evaluation . proper interpretation of laboratory test results includes recognition of abnormal values and , more important , the potential clinical significance of such results . a common pitfall in the interpretation of coagulation times is failure to appreciate that even mildly abnormal values may represent a serious underlying coagulation deficit . another important observation is to identify how an abnormal laboratory value may have changed over time , which can be facilitated by the ability of electronic medical record systems to display data trends . in the absence of iatrogenic causes , even a mildly elevated pt / inr or aptt may be indicative of true coagulopathy and should not be ignored or dismissed , particularly when there is evidence of bleeding , as was the case with this patient , even at initial presentation . after excluding laboratory error , the differential diagnosis of an isolated prolongation of aptt includes heparin effect , lupus anticoagulant , and deficiency of or antibody against an intrinsic pathway factor ( viii , ix , xi , or xii).19,20 a detailed history , focusing on factors such as heparin exposure,19,20 history of thromboembolism ( lupus anticoagulant),21 and prior personal or family history of bleeding,22 may provide diagnostic clues . laboratory testing should include a 1:1 mixing test of patient plasma with control plasma to determine whether a prolonged aptt is the result of an intrinsic pathway factor deficiency or an inhibitor that continues to block the activity of the intrinsic system even in the presence of control plasma . the majority of inhibitor antibodies identified in this manner will turn out to be lupus anticoagulants . although far less common , this is the same diagnostic pathway that leads to the identification of the antifactor viii antibodies associated with acquired hemophilia.23 unlike acquired hemophilia , lupus anticoagulants typically do not present with bleeding , and the abnormal aptt is due to interference with phospholipid - dependent coagulation reactions . once an acquired antifactor viii antibody is suspected , confirmatory testing includes measuring factor viii activity , which should be significantly reduced , and the bethesda assay,24 which is used to quantitate antifactor viii antibodies inhibitor activity . we found a general lack of appropriate consideration and response to the presenting symptom of bleeding and the prolonged aptt throughout this case study . this is consistent with data from the european acquired haemophilia registry ( each-2 ) , which reported a median delay of 3 days between onset of bleeding symptoms and the diagnosis of acquired hemophilia and a median delay of one day between the first abnormal aptt test in those same patients and the established diagnosis.23 in addition , we found that emergency medicine and critical care physicians were reluctant to consider a bleeding disorder as the primary explanation for this patient s clinical presentation . the disposition of a patient with active hemorrhage and evidence of coagulopathy should be based on several factors , including the patient s current condition and anticipated clinical course , taking into account the presenting vital signs and evolving laboratory findings . at the time of the patient s second presentation , vital signs were notable for mild tachycardia and a pulse pressure at the upper limit of normal , and subsequent laboratory findings indicated a decreasing hemoglobin level and an increasing aptt . these findings alone prompted hospital admission , although the exact location ( general floor versus intensive care unit ) of admission may vary , based on the level of monitoring and nurse - to - patient ratios in a particular hospital . another important variable is the anticipated potential for clinical deterioration , which is based in large part on clinician appreciation of the seriousness of the diagnosis . we found a consistent tendency to consider admission to a general floor bed with the second presentation , even though this was ultimately an unstable , critically ill patient with an undiagnosed bleeding disorder . we also found that the physicians who participated in this survey were reluctant to consult a hematologist as they worked through this case scenario , particularly given that options for additional testing ( liver function , disseminated intravascular coagulation ) were not available to evaluate for common causes of coagulopathy . relative to their reported historical experience with hematology consultations during an average practice experience of approximately 20 years , this survey finding was somewhat surprising . rheumatologists and critical care specialists reported a greater frequency of hematology consultation relative to the other specialties ( table 2 ) . we would expect the emergency medicine physicians to be most likely to consult a hematologist , yet 16% of them reported never having consulted a hematologist . the highest percentage ( 46% ) had only consulted a hematologist 1 or 2 times , and almost one quarter of geriatricians and internists had never consulted a hematologist , even though these specialists would be expected to first encounter patients with undiagnosed bleeding disorders , including acquired hemophilia . one potential reason for not seeking hematology consultation might be the lack of availability of hematology / oncology specialists with expertise in coagulation disorders , including in rural and community hospitals . a limitation of this survey was that one can not interpret the thinking behind the responses of the individual participants . therefore , 31 qualitative 45-minute interviews were conducted subsequent to the quantitative study and focused particularly on critical care ( n = 7 ) , emergency medicine ( n = 6 ) , hematology / oncology ( n = 4 ) , or hematology ( n = 2 ) physicians to understand the reasoning behind their decisions and depth of knowledge ( unpublished data ) . we found that the physicians focus was generally on finding the source ( location ) of bleeding and not on finding the underlying reason for bleeding . this could potentially lead to surgical intervention in the face of an underlying bleeding disorder , with subsequent adverse outcomes . in a series of 67 patients with acquired hemophilia at a single center in bonn , germany , 4 of 5 deaths were the result of surgical intervention for bleeding at outside hospitals in the setting of a delayed diagnosis of acquired hemophilia.13 when queried about their experience encountering and/or diagnosing underlying bleeding disorders , particularly acquired hemophilia , more than 85% of physicians in hematology , hematology / oncology , emergency medicine , and critical care medicine reported having ever discovered or diagnosed an underlying bleeding disorder , compared with 65% , 54% , and 47% of rheumatologists , internists , and geriatricians , respectively . while reports of ever specifically having encountered acquired hemophilia were high , it is unclear from this study whether the participants truly understood the diagnosis . this seems unlikely , given the rarity of acquired hemophilia , relative to the reported frequency of having encountered it . except for hematology and/or oncology ( 77% ) and critical care ( 36% ) specialists , approximately one quarter of surveyed physicians had ever encountered acquired hemophilia . although they accounted for the highest percentage of physicians who had ever encountered this condition , nearly one quarter of hematologists had never encountered acquired hemophilia . subsequent unpublished data from the aforementioned qualitative research further suggest that , compared with hematology practitioners , specialists in hematology / oncology , who likely practice mostly oncology , might be able to identify mixing studies and inhibitors but might not fully understand the underlying pathophysiology that constitutes acquired hemophilia , making it hard for them to recognize the condition . given the survey findings reflecting the infrequency with which most physicians have encountered these conditions , consultation with a hematologist may facilitate the diagnostic evaluation and proper management of a hemorrhaging patient suspected of having an underlying bleeding diathesis , particularly acquired hemophilia . the consulting hematologist can provide specific guidance , leading to the prompt diagnosis and optimal management of an actively bleeding patient with acquired hemophilia , including initiation of immunosuppression , which is usually necessary to eradicate the inhibitor and to prevent additional bleeding episodes . however , this requires a level of familiarity and expertise in treating acquired hemophilia and other rare bleeding disorders that is not often seen outside of an academic hematology practice . this represents yet another barrier to the effective diagnosis and management of this rare yet serious bleeding diathesis . for the hospitalist or intensivist charged with the care of a bleeding patient , immediate stabilization is the initial priority and should take precedence over determination of a specific etiology of bleeding . however , in the presence of underlying coagulopathy , particularly acquired hemophilia or other rare disorders , traditional measures of stabilization may not be as effective as expected . a prolonged pt / inr or aptt in the absence of iatrogenic causes should never be ignored , even with only minimally prolonged values , and determination of the cause of an abnormal coagulation study should carry at least equal weight to looking for the anatomic site of bleeding . any delay in establishing the diagnosis of a bleeding diathesis such as acquired hemophilia can result in significant morbidity or even death . while hospitalists and intensivists should be able to conduct a thorough differential of bleeding and eliminate most common etiologies , consultation with a hematologist ( particularly one with specific expertise in coagulation disorders ) may facilitate the evaluation of coagulopathic patients and subsequent interpretation of diagnostic findings , as well as initiation of appropriate treatment . given the rarity of acquired hemophilia , as exemplified by the findings of this survey , physicians must harbor a high index of suspicion to diagnose this condition promptly in patients who present with recent - onset or acute bleeding . given the high morbidity and mortality the insights from this survey highlights knowledge and practice gaps that could be the focus of targeted educational initiatives , including diagnostic algorithms , to ensure proper and efficient workup of the abnormal laboratory studies that characterize acquired hemophilia .

background : bleeding symptoms commonly seen by multiple physician specialties may belie undiagnosed congenital or acquired bleeding disorders . acquired hemophilia is a potentially life - threatening cause of unexplained acute bleeding manifested by an abnormal activated partial thromboplastin time ( aptt ) that does not correct with 1:1 mixing with normal plasma.methods:practicing physicians ( hematology / oncology , emergency medicine , geriatrics , internal medicine , rheumatology , obstetrics and gynecology , critical care medicine , and general surgery ) completed an online survey based on a hypothetical case scenario.results:excluding surgeons and obstetrician / gynecologist respondents , 302 physicians ( about 50 per specialty ) were presented with an older adult woman complaining of recurrent epistaxis . nearly 90% ordered a complete blood count and coagulation studies ( aptt , prothrombin time [ pt]/international normalized ratio [ inr ] ) . despite a prolonged aptt of 42 seconds , < 50% of nonhematologists would repeat the aptt , and < 45% would consult a hematologist ; emergency medicine physicians were least likely ( 10% ) and rheumatologists were most likely ( 43% ) to consult . after presentation weeks later with bruising and abdominal / back pain , 90% of physicians within each specialty ordered a complete blood count or pt / inr / aptt . despite an aptt of 63 seconds , the majority did not repeat the aptt . at this point , approximately 75% of internal medicine and geriatric physicians indicated they would consult a hematologist , versus 47% in emergency medicine and 50% in critical care . all participants preferred abdominal computed tomography ( 80%84% ) . after 12 hours of additional observation , 73% to 94% of respondents consulted a hematologist . complete blood count revealed anemia and an aptt twice the upper limit of normal ; emergency medicine physicians remained least likely to request a consult.conclusion:determining the cause of an abnormal coagulation study result should carry equal weight as looking for the site of bleeding and could be facilitated by consultation with a hematologist . insight from this survey highlights knowledge and practice gaps that could be the target of focused educational initiatives .

plant cells need to maintain the functional integrity of their walls during cell morphogenesis and exposure to biotic / abiotic stress . the available evidence suggests that a dedicated plant cell wall integrity ( cwi ) maintenance mechanism exists ( wolf et al . , 2011 ) . while our understanding of the mechanisms regulating stress responses and morphogenesis has increased significantly , our knowledge regarding the processes maintaining cwi is still limited . in the last years several reviews have been published on the plant cwi maintenance mechanism illustrating the increased interest in this area ( humphrey et al . , 2007 ; hematy et al . , 2009 ; ringli , 2010 ; seifert and blaukopf , 2010 ) . a recently published review focuses on cwi maintenance during plant cell wall morphogenesis ( wolf et al . , 2011 ) . similarities between the yeast and plant cwi maintenance mechanisms have also been reviewed ( hamann and denness , 2011 ) . therefore , the available knowledge regarding cwi maintenance during plant development and in yeast will be covered here only briefly to provide a conceptual framework regarding cellular processes involved and to illustrate the degree of functional conservation between species . this review will focus on recent developments regarding the role of cwi maintenance during biotic stress responses . it will discuss how cwi maintenance could have a previously unrecognized role in the perception of and response to biotic stress . while both plant and yeast cells are enveloped by cell walls , certain important differences exist that affect the biological role and function of the plant cwi maintenance mechanism . in addition , plant cell walls are structurally and chemically more complex than the yeast cell wall . this means that in plant cells the sheer number of cell wall - related signaling events during development and plant environment interaction could disguise the activity of a dedicated plant cwi maintenance mechanism . the yeast cwi monitoring and maintenance network is quite complex , providing an indication of the possible complexity of the plant cwi maintenance network . by combining inputs from a turgor pressure sensor ( sln1 ) , mechano - perception ( mid1/cch1 ) , and dedicated cell wall damage ( cwd ) sensors ( wsc1 , 2 , 3 , mid2 , mtl1 ) the yeast cwi maintenance network generates signals that permit highly specific responses to any challenge that impairs the functional integrity of the yeast cell wall . the available phenotypic and genetic data also implicate turgor pressure , mechano - perception , and cwd detection in plant cwi maintenance ( hamann et al . interestingly , arabidopsis histidine kinase1 ( ahk1 ) and cytokinin receptor1/arabidopsis histidine kinase4 ( cre1/ahk4 ) can at least partially rescue a yeast strain with a loss of function allele in sln1 ( urao et al . in addition , expression of the arabidopsis thaliana mid1 complementing activity 1 and 2 ( mca1,2 ) genes rescues a mid1-deficient yeast strain suggesting that they could function as stretch - activated calcium channels ( nakagawa et al . no functional homologues for the yeast cwd sensors have been identified in plants . in yeast , the signals generated by the sensors are relayed to the response genes via different signaling cascades involving calcineurin ( mid1/cch1 ) ; rho1/mpk1 ( wsc1 , mid2 ) , and ypd1 ( sln1 ) ( levin , 2005 ) . transcription factors mediating the response are skn7 , rlm1 , and swi4/6 ( levin , 2005 ) . the response can involve activation of genes required for cell wall biosynthetic processes , remodeling of the cytoskeleton , and cell cycle progression . the available data suggest both organizational and functional similarities between the yeast and plant cwi maintenance mechanism while also highlighting how signals from mechanical and chemical sensors regulate jointly the cwd response . the plant cell wall is comparable to an exoskeleton surrounding the plant cell and providing both structural support and protection from biotic as well as abiotic stresses . it consists of cellulose microfibrils , pectin , hemicelluloses , proteins , and in certain cases lignin ( somerville et al . , 2004 ) . plant cell walls are divided into primary ( laid down during cell elongation / differentiation ) and secondary ( formed after cell morphogenesis is concluded ) walls . in parallel , dependent on the presence of certain polysaccharides type i and type ii cell walls are distinguished ( popper et al . , 2011 ) . cellulose microfibrils are the main load bearing elements , which are cross - linked to hemicelluloses and ( in vitro ) to pectin ( dick - perez et al . , 2011 ) . hemicelluloses and pectin also form direct links creating a matrix in which the microfibrils are embedded like the steel mesh in a concrete wall . pectic polysaccharides like homogalacturonan ( hg ) are connected by calcium bridges between dimethyl - esterified parts of the molecules or through borate ester linkages in the case of rhamnogalacturonan ii ( rg ii ) . they are targeted by pathogen - derived cell wall degrading enzymes ( polygalacturonases ) , which generate oligogalacturonides ( ogas ) from hg ( hahn et al . , 1981 ; kars et al . , 2005 ) . biologically active ogas consist of chains of 915 galacturonic acid ( gala ) monomers and can function as signaling molecules ( see below ) . during secondary cell wall formation monolignols ( precursors for lignin ) are secreted into the cell wall space and randomly cross - linked ( vanholme et al . , 2010 ) . the cross - linking is dependent on the availability of reactive oxygen species ( ros ) generated by laccases and peroxidases . this process reinforces the wall against pathogen infection , waterproofs it , and increases structural integrity ( tronchet et al . , 2010 ; reduction of cellulose biosynthesis during primary cell wall formation through genetic or chemical means leads to lignin production ( ellis et al . , 2002 ; hamann et al . , this highlights the ability of the cell to adapt to changes in cell wall composition and provides evidence for the existence of a cwi maintenance mechanism . plant cell walls are capable of adjusting their composition and structure in response to pathogen infection ( dong et al . , 2008 ) . a mutation in ( cellulose synthasea3 , cesa3 ) a subunit of the cellulose synthase complex leads to ectopic production of lignin , i.e. , replacement of a missing load bearing cell wall component by another one ( cano - delgado et al follow up studies found that inhibition of cellulose biosynthesis during primary cell wall formation either through mutations like constitutive expression of vsp1 ( cev1 ) and ectopic lignification1 ( eli1 ) or inhibitors such as isoxaben results in transcriptional activation of stress response mechanisms ; ectopic production of ethylene ( et ) , salicylic acid ( sa ) , jasmonic acid ( ja ) , callose , and ros as well as changes in cell wall composition and structure ( ellis et al . , 2002 ; cano - delgado et al . , 2003 ; manfield et al . , 2004 ; hamann et al . , the experiments also showed that the response to cwd consists of early and late stages reminiscent of the response to pathogen infection ( denness et al . , 2011 ) . during the early stage , ros- and calcium - based signaling cascades are required for initiating the response to cwd ( denness et al . , 2011 ) . interestingly , 1-aminocyclopropane-1-carboxylic acid ( acc , an et precursor ) and not et itself seems to be acting as signaling substance during the early response to isoxaben with inhibition of cell expansion being an active process and not simply an automatic consequence of cellulose biosynthesis inhibition ( tsang et al . , 2011 ) . during the late stage , responses to cwd - like lignin deposition are initiated and the extent of lignin formation is apparently modulated by a negative feedback loop formed by ros and ja ( denness et al . , 2011 ) . a combination of genetic and phenotypic analysis has implicated the nadph oxidases respiratory burst oxidase homologd and f ( rbohd , f ) , the serine / threonine kinase oxidative signal inducible1 ( oxi1 ) , mca1 , the receptor - like kinase ( rlk ) theseus1 ( the1 ) as well as the ja biosynthesis genes allene oxide synthase ( aos ) and jasmonic acid resistant1 ( jar1 ) in the signaling mechanism mediating the response to cwd in arabidopsis seedlings . interestingly , the cev1 mutation that affects cellulose biosynthesis during primary cell wall formation also causes enhanced resistance to infection by different powdery mildews ( erysiphe orontii , e. cichoracearum , and oidium lycopersicum ; ellis and turner , 2001 ) . a screen for mutants causing resistance to powdery mildew infection provides further evidence of the close relationship between plant cell walls and pathogen resistance ( vogel and somerville , 2000 ) . three of the powdery mildew resistance ( pmr ) mutants that have been identified on the molecular level affect genes involved in cell wall biosynthetic processes . pmr4 encodes a callose synthase , pmr5 a gene of unknown function required for pectin production and pmr6 a pectate lyase ( vogel et al . pmr4 resistance seems to be mediated via hyper - activation of sa signaling , whereas pmr5 and 6 resistance phenotypes are independent of ja , sa , and et signaling . mutations in irregularxylem1 ( irx1/cesa8 ) , 3 ( irx3/cesa7 ) , and 5 ( irx5/cesa4 ) impair cellulose biosynthesis during secondary cell wall formation and cause enhanced resistance to the soil borne bacterium ralstonia solanacearum and the necrotrophic fungus plectosphaerella cucumerina ( hernandez - blanco et al . , 2007 ) . mutations affecting cellulose biosynthesis during primary cell wall formation ( cesa1 , 3 , 6 ) or other components of the secondary cell wall ( pmr5 , pmr6 ) did not cause enhanced resistance to the same pathogens . genetic analysis showed that the enhanced resistance in cesa4 , 7 , 8 is independent of ja , sa , and et - based signaling mechanisms . results from expression profiling experiments and genetic analysis using different abscisic acid ( aba)mutants(aba insensitive 1 - 1;2 - 1;aba1 - 6 ) suggest that aba is mediating developmental and pathogen resistance phenotypes caused by the irx mutants . however , it remains to be determined if the aba involvement is direct or a secondary effect due to water stress caused by problems with xylem cell wall formation in the mutants . to summarize , these results suggest distinct resistance signaling cascades are induced by defects in primary and secondary cell wall formation as well as for different secondary cell wall components . they also highlight the direct impact of changes in cell wall composition / structure on the response to pathogen infection . the mode of action of the plant cell wall maintenance mechanism is not well understood . based on the knowledge from yeast , chemical and physical signals could act as indicators for the functional integrity of the plant cell wall either individually or jointly . by combining these different types of signal the plant cell would receive precise information regarding the state of its cell wall and the exact type of cwi impairment occurring . physical signals could be generated by stretching of the plasma membrane due to a weakened cell wall that can not resist the high turgor pressure levels within a plant cell or a plasma membrane that is displaced relatively to the cell wall . these events could be detected by stretch - activated or mechanosensitive channel proteins that lead to calcium influx into the cytoplasm , indicating cwd . sensor candidates could be encoded by members of the mechanosensitive channels of small conductance ( mscs)-like ( msl ) gene family like msl 9 and 10 affect mechano - perception in protoplasts derived from arabidopsis root cells ( haswell et al . , 2008 ) . another candidate of interest is the putative stretch - activated calcium channel mca1 is required for cwd - induced lignin deposition . interestingly , all isoxaben - induced cwd phenotypes can be suppressed by provision of osmotic support suggesting that changes in turgor pressure due to a weakened cell wall could result in signal generation via turgor pressure sensors ( hamann et al . , 2009 ; denness et al . , 2011 ) . while ahk1 and 4/cre1 can function as osmosensors in yeast and have been implicated in abiotic stress responses , no clear evidence exists implicating them in cwd perception in plants ( urao et al . , 1999 ; inoue et al . , 2001 ; tran et al . , 2007 ) . in addition , ahk4/cre1 has been shown to function as a cytokinin receptor(inoue et al . , 2001 ) . therefore , the question that needs to be resolved at this point is if turgor pressure is a passive element in the process ( generating cell wall fragments due to a weakened cell wall ) or an active component that is being monitored and provides input into the process . the plant cell wall contains a large number of components that could generate chemical signals ( ligands ) indicative of cwd or general danger signals . the term damage associated molecular patterns ( damps ) has been coined to describe such ligands and the number of possible damps originating in plant cell walls is rather large ( zipfel , 2009 ) . here i will focus on the best - characterized group of signals , which are probably ogas . they can be generated through degradation of hg by pathogen - derived enzymes ( kars et al . ogas have been shown to induce gene expression changes , stomatal closure , production of et , and ros as well as cell wall reinforcement ( denoux et al . , 2008 ; ferrari et al . , 2008 ) . a hybrid kinase consisting of the extra cellular domain of wall - associated kinase1 ( wak1 ) and the intracellular domain of elongation factor tu receptor ( efr ) kinase can bind ogas and activate defense responses ( brutus et al . , 2010 ) . wak1 belongs to a family of five wak genes encoding plasma membrane - localized ser / thr kinases that have been implicated in response to pathogen infection and regulation of cell elongation ( kohorn et al . , 2011 ) . the effects of the chimeric wak1 kinase on pathogen resistance suggest that ogas and waks represent an in vivo ligand receptor pair . results from the analysis of a dominant active wak2 allele suggest the cwd signals perceived by waks could be relayed to downstream response genes through mapkinase6 ( mpk6 ) ( kohorn et al . interestingly , wak2 has also been implicated in regulation of invertase activity and turgor pressure during cell elongation ( kohorn et al . , 2006 ) . however , there is currently no confirmation that waks are actively involved in cwi maintenance . in arabidopsis , more than 600 rlks have been identified and a large number of them have been implicated in developmental and stress response processes ( shiu and bleecker , 2001 ) . i will focus here on several kinases that have been implicated in cwd perception and/or pathogen response . most of the rlks implicated in cwi maintenance [ the1 , hercules1 ( herk1 ) , feronia ( fer ) ] belong to the catharanthus roseus rlk1 ( crrlk1)-like protein family , which has 17 members in arabidopsis . the1 was isolated as a suppressor of the cellulose - deficient cesa6 procuste ( pre ) mutant , which exhibits a hypocotyl elongation defect ( hematy et al . , 2007 ) . subsequently it has been shown that the1 is required for cellulose biosynthesis inhibition - induced ros production and lignification in the root elongation zone ( denness et al . , 2011 ) . the1 , herk1 , and fer have been implicated in brassinosteroid - induced cell elongation ( guo et al . , 2009 ; deslauriers and larsen , 2010 ) . both fer and nortia / mildew resistance locus o 7 ( ml07 ; a seven - transmembrane domain protein involved in powdery mildew resistance ) are required for successful fertilization and resistance to infection by golovinomyces ( syn . interestingly , ropgef ( guanine - exchange factors ) proteins have been identified as targets of fer activity ( duan et al . , 2010 ) . ropgefs are required for the activation of rho gtpases , which in turn activate nadph oxidases like rbohd / f . these results suggest the same molecular components could mediate cell - cell interaction during development and plant heterotrimeric g - proteins ( g , g , g ) form a highly conserved signaling complex that has been implicated in signal transduction during development and stress responses in mammals , yeast , and plants ( digby et al . , 2006 ; temple and jones , 2007 ) . in arabidopsis , five genes gpa1 ( g ) , agb1 ( g ) , agg1 , 2 , 3 ( g1 , 2 , 3 ) encode the subunits of the complex ( thung et al . , 2011 ) . recently , it has been reported that mutations in agg1 , 2 , and agb1 apparently cause enhanced susceptibility to infection with p. cucumerina ( delgado - cerezo et al . , 2011 ) . a combination of metabolomic and microarray - based expression profiling studies of the mutants established that the pathogen phenotype is independent of sa , ja , aba , and et signaling cascades . interestingly , a large number of cell wall biosynthetic / modifying genes are mis - regulated in agbl and aggl2 plants . analysis of cell wall composition / structure in these plants found reduced xylose contents in the mutants compared to wildtype . to summarize , the available evidence supports the notion that the plant cell wall is an integral component contributing to pathogen response mechanisms and illustrates the influence of cell wall defects on infection . specific signaling cascades seem to mediate the response to particular cell wall defects , which in turn affect the response to necrotrophic or biotrophic pathogens . more importantly the data presented above allow correlation between certain types of cell wall defects , and not only signaling cascades but also resistance phenotypes . mutations in cesa4 , 7 , 8 , agg1 , 2 , and agb1 affect resistance to necrotrophs and are independent of phytohormone - based signaling cascades . pmr5 , 6 plants exhibit resistance to biotrophs and also do not rely on phytohormone - based signaling cascades . pmr4 affects resistance to biotrophs and resistance depends on the integrity of the sa signaling cascade . the cell wall composition / structure changes could prevent pathogen colonization simply because the infection machinery of the pathogen is too specialized to breach the chemically modified cell wall . another option is that the cell wall mutants cause defects similar to those occurring during infection by particular pathogens , i.e. , simulate infection by necrotrophs or biotrophs . this would cause early / constant activation of the cwi maintenance / defense mechanism , which primes plant immunity thus making successful infection more difficult . the latter would explain both the specificity of the responses observed and dependence on particular signaling mechanisms . therefore , studies focusing on the effects of particular cell wall defects on pathogen resistance and the mode of action of the cwi maintenance mechanism could facilitate research into biotic stress response . the reason being , that by removing the potentially multiple effects of the pathogen during infection , they reduce the complexity of the interaction and should therefore allow novel insights into the mechanisms responsible for detection of infection and/or physical damage . the author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest .

plant cell walls provide structural support during development and represent together with the cuticle the first line of defense against biotic and abiotic stress . in recent years , evidence has accumulated that a dedicated plant cell wall integrity ( cwi ) maintenance mechanism exists . this mechanism monitors and maintains functional integrity of the cell wall during different biological processes . the available data suggest that it may represent a component of the stress response mechanisms underlying biotic and abiotic stress responses , which has not been identified previously as a distinct mechanism . here i will review the available evidence regarding the mode of action of the cwi maintenance mechanism and discuss its role in the context of biotic plant stress response mechanisms .

genomic dna hypomethylation has been observed in the peripheral blood mononuclear cells of leukemia patients and in tumor cells of patients with b - cell lymphoproliferative diseases ( 1 ) . dna methylation may affect karyotypic stability , may influence euchromatin - heterochromatin interactions , and has been correlated with disease progression ( 2 ) . on the other hand , for example , some patients with lymphomas do not express tumor suppressor genes because the promoters of these genes are methylated . , bone marrow produces a large number of abnormal white blood cells , which overwhelm the other types of blood cells , including red blood cells and platelets , thus impairing the production of normal white blood cells . in clinical classification , leukemia can be classified as acute myeloid leukemia ( aml ) and chronic myeloid leukemia ( cml ) . cml is a clonal disease of stem cell origin that is characterized by the presence of the philadelphia chromosome ( ph+ ) , which has been named t(9,22)(q34:q11 ) . its fusion gene product , bcr - abl , is a constitutively active tyrosine kinase . nevertheless , the differential factors through which they produce a different type of leukemia are not yet completely understood . in hematopoietic malignancies , hypermethylation of several genes including e - cadherin , dap kinase , estrogen receptor ( er ) alpha , and p15 are associated with gene inactivation ( 7 - 10 ) . genes , such as dab2ip , dlc-1 , h - cadherin , id4 , integrin 4 , runx3 , sfrp1 , and shp1 , has been identified as being implicated in aberrant dna methylation during development of human malignancy ( 11 - 18 ) . in order to gain insight into the differential epigenetic alterations in leukemia , we investigated the methylation statuses at selected locus of these genes in aml and cml patients using a methylation - specific polymerase chain reaction ( msp ) . diagnostic bone marrow samples were obtained from 23 patients with aml and 21 patients with cml . the samples were gathered by the division of hematology / oncology ( department of internal medicine , korea university medical center , seoul , republic of korea ) and analyzed by pathologist . institutional review board approval and informed consent were obtained ( kumc - irb-2006011-p-1 , kumcirb-2006012-p-2 ) . of the aml patients , 17 males and 6 females were included , with ages ranging from 26 to 78 yr , at a median age of 45.61 ( sd , 15.56 ) yr . of the cml patients , 11 males and 10 females were included , with ages ranging from 18 to 75 yr ( meansd , 49.9116.53 ) . as controls , 22 normal peripheral bloods were obtained from healthy volunteers ( 11 males and 10 females ) ranging from 20 to 78 yr of age ( meansd , 45.3620.64 ) . chemical modification was performed as described previously , with minor modifications ( 19 ) . in brief , 1 g of genomic dna was denatured by incubation with 0.2 m naoh for 10 min at 37 , followed by the addition of 550 l of 3 m sodium bisulfite ( ph 5.0 ) ( sigma , st . louis , mo , u.s.a . ) and 10 mm hydroquinone ( sigma ) , which was brought to a final volume of 600 l . the mixtures were incubated at 55 for 16 hr , and the modified dna was then desalted with the wizard clean - up system ( promega corp . , we performed polymerase chain reaction ( pcr ) using specific pcr primers capable of distinguishing between methylated and unmethylated dna sequences . the primers for the unmethylated and methylated dna sequences , pcr product size , and annealing temperature are shown in table 1 . the msp primer sets were selected at the 5'-cpg island regions of genes using the methprimer software ( www.urogene.org ) . the pcr conditions were as follows : initial denaturation and hot start at 95 for 5 min , and cycles consisting of 30 sec at 95 , 30 sec at the annealing temperature , and 30 sec at 72. in addition , sck , a human cholangiocarcinoma cell line , was used as positive control for integrin 4 gene ( 20 ) . msp results were analyzed as a dichotomous variable based on the presence or absence of gene methylation . the msp results of tumor and normal samples were compared and analyzed with the pearson chi - square test ( version 12 ; spss inc . , diagnostic bone marrow samples were obtained from 23 patients with aml and 21 patients with cml . the samples were gathered by the division of hematology / oncology ( department of internal medicine , korea university medical center , seoul , republic of korea ) and analyzed by pathologist . institutional review board approval and informed consent were obtained ( kumc - irb-2006011-p-1 , kumcirb-2006012-p-2 ) . of the aml patients , 17 males and 6 females were included , with ages ranging from 26 to 78 yr , at a median age of 45.61 ( sd , 15.56 ) yr . of the cml patients , 11 males and 10 females were included , with ages ranging from 18 to 75 yr ( meansd , 49.9116.53 ) . as controls , 22 normal peripheral bloods were obtained from healthy volunteers ( 11 males and 10 females ) ranging from 20 to 78 yr of age ( meansd , 45.3620.64 ) . chemical modification was performed as described previously , with minor modifications ( 19 ) . in brief , 1 g of genomic dna was denatured by incubation with 0.2 m naoh for 10 min at 37 , followed by the addition of 550 l of 3 m sodium bisulfite ( ph 5.0 ) ( sigma , st . louis , mo , u.s.a . ) and 10 mm hydroquinone ( sigma ) , which was brought to a final volume of 600 l . the mixtures were incubated at 55 for 16 hr , and the modified dna was then desalted with the wizard clean - up system ( promega corp . , we performed polymerase chain reaction ( pcr ) using specific pcr primers capable of distinguishing between methylated and unmethylated dna sequences . the primers for the unmethylated and methylated dna sequences , pcr product size , and annealing temperature are shown in table 1 . the msp primer sets were selected at the 5'-cpg island regions of genes using the methprimer software ( www.urogene.org ) . the pcr conditions were as follows : initial denaturation and hot start at 95 for 5 min , and cycles consisting of 30 sec at 95 , 30 sec at the annealing temperature , and 30 sec at 72. in addition , sck , a human cholangiocarcinoma cell line , was used as positive control for integrin 4 gene ( 20 ) . msp results were analyzed as a dichotomous variable based on the presence or absence of gene methylation . the msp results of tumor and normal samples were compared and analyzed with the pearson chi - square test ( version 12 ; spss inc . , the multiple genes were found to be methylated in bone marrow from patients with aml or cml . specifically , the frequencies of promoter hypermethylation at selected locus in the 23 aml samples were : 78.3% ( 18/23 ) for shp1 , 65.2% ( 15/23 ) for id4 and sfrp1 , 26.1% ( 6/23 ) for h - cadherin , 8.7% ( 2/23 ) for dlc-1 , and 4.3% ( 1/23 ) for dab2ip and runx3 . the frequencies of dna hypermethylation at selected locus in the 21 cml samples were : 28.6% ( 6/21 ) for shp1 , 19.0% ( 4/21 ) for h - cadherin , 14.3% ( 3/21 ) for id4 , 9.5% for ( 2/21 ) for sfrp1 , and 0% ( 0/21 ) for dab2ip , dlc-1 , integrin 4 , and runx3 ( fig . however , promoter hypermethylation of the 22 normal peripheral bloods was observed less frequently ( table 2 ) . there was a statistically significant difference between normal peripheral bloods and aml with respect to the frequencies of methylation of id4 , sfrp1 , and shp1 ( pearson chi - square test ; p<0.0001 , p<0.0001 , and p<0.0001 , respectively ) and between normal peripheral bloods and cml with respect to the frequencies of shp1 methylation ( pearson chi - square test ; p=0.007 ) . furthermore , there was a statistically significant difference between the dna methylation frequencies of aml patients and cml patients . the frequencies of dna methylation of id4 , sfrp1 , and shp1 were higher in aml compared to those in cml ( p=0.001 , p<0.0001 , and p=0.001 , respectively ) ( table 2 ) . in contrast , no statistical differences between aml and cml were detected in other genes such as dlc-1 , dab2ip , h - cadherin , integrin 4 , and runx3 . promoter methylation results at selected locus of eight genes with msp method were shown in fig . the results of the present study demonstrate the substantially increased frequency of promoter hypermethylation in id4 , sfrp1 , and shp1 genes in aml compared to cml . leukemia , a heterogeneous group of hematopoietic malignancies that occur worldwide , includes acute and chronic myeloid leukemia . despite many important advances in understanding the different biological and cytogenetic aspects of acute and chronic leukemia , it is understood that some kinds of leukemia present specific cytogenetic alterations ( 21 ) . different types of leukemia usually have specific epigenetic modifications that cause the activation of oncogenes and , in particular , the formation of abnormal fusion genes such as aml1-eto ( 22 , 23 ) , a fusion protein resulting from t(8,21 ) translocation that commonly occurs in aml . another typical example is the fact that bcr - abl is a constitutively active , cytoplasmic tyrosine kinase that is generated by t(9;22 ) translocation in more than 95% of cml ( 24 ) . the central role of epigenetic modification of these genes in leukemia development promoted them as reasonable targets for understanding the differential mechanism between acute and chronic leukemia . in the present study , id4 , sfrp1 , and ship1 these results suggest that the epigenetic modification of these genes may play specific roles in the development of aml rather than cml and , thus , promoted them as ideal targets for drug development to treat aml . in the future , it is clearly imperative to understand the patho - physiological differential mechanisms of these genes in aml , and to finally explore novel therapeutic strategies . in present study , methylation frequencies of shp1 gene were the high detected in cml as well as aml . shp1 is a member of the shp family of proteins , cytoplasmic protein tyrosine phosphatase ( ptp ) , and has been known as a candidate tumor suppressor gene in lymphoma , leukemia and other cancers ( 25 ) . also , the hypermethylation of shp1 gene was frequently detected in several human cancers and the reduced expression of the shp1 gene in various types of leukemias and lymphomas mainly occurred by promoter methylation ( 26 - 28 ) . therefore , these results indicate that aberrant dna methylation of shp1 gene may be related to the tumorigenicity of myeloid leukemia . the methylation frequencies of the dab2ip , dlc-1 , and runx3 genes were detected at different levels in acute and chronic leukemia patients ; however , the difference was negligible . in some genes like integrin 4 gene , however , hypermethylation of these genes was frequent event in esophageal squamous cell carcinomas , lung cancers , and gastric cancers ( 11 , 12 , 15 , 16 ) . thus , dna methylation of these genes may be not related to tumorigenicity of aml / cml in contrast with other cancers . in summary , we have identified that aberrant dna methylation of shp1 is a frequent event in aml and cml . also , the frequencies of dna methylation of several methylation - controlled genes , including id4 , sfrp1 , and shp1 , were higher in aml patients compared to those in cml patients . although these results should be confirmed , more comprehensive studies are necessary involving various known risk factors such as smoking and occupational carcinogens , and genetic susceptibilities . our results suggest that aberrant dna methylation of shp1 may be related to the tumorigenicity of aml and cml and hypermethylation of id4 , sfrp1 , and shp1 genes may contribute to the pathogenesis mechanism of aml specifically .

to gain insight into the differential mechanism of gene promoter hypermethylation in acute and chronic leukemia , we identified the methylation status on one part of 5'cpg rich region of 8 genes , dab2ip , dlc-1 , h - cadherin , id4 , integrin 4 , runx3 , sfrp1 , and shp1 in bone marrows from acute myeloid leukemia ( aml ) and chronic myeloid leukemia ( cml ) patients . also , we compared the methylation status of genes in aml and cml using methylation - specific pcr ( msp ) . the frequencies of dna methylation of id4 , sfrp1 , and shp1 were higher in aml patients compared to those in cml patients . in contrast , no statistical difference between aml and cml was detected for other genes such as dlc-1 , dab2ip , h - cadherin , integrin 4 , and runx3 . taken together , these results suggest that these methylation - controlled genes may have different roles in aml and cml , and thus , may act as a biological marker of aml .

acute patellar dislocation is a common injury usually associated with a significant traumatic mechanism resulting in lateral displacement . vertical axis rotation following dislocation is a rare variant of this type of injury and can prevent closed reduction in the acute setting . a 32-year old gentleman presented with an irreducible patella dislocation following an unusual atraumatic mechanism . following attempts at closed reduction under sedation and regional nerve block , eventual open reduction and soft tissue reconstruction was required under general anesthetic . during the open reduction procedure , it was noted that the patella had dislocated into a lateral extra - articular position and rotated around its vertical axis . a review of the literature suggests dislocations such as the current presentation , are extremely rare and although have been described to occur with minor trauma , have never been described to occur following a largely atraumatic event . in such cases , closed reduction may be impossible even with adequate analgesia due to patella position and soft tissue obstruction . acute patellar dislocation is defined as the abrupt disruption in the relationship of the patella within the femoral groove . patellar dislocation is a relatively common occurrence and is usually managed in the emergency department with closed manipulation under sedation . however , when the patella itself rotates around its vertical axis during dislocation , surgical intervention is usually required . a 32-year old gentleman was admitted to our trauma department with a painful , immobile right knee . he sustained the injury externally rotating his knee whilst in slight flexion while he was moving from the driver s seat to the passenger s seat of his car . the first event was 15 years prior , when his knee twisted in flexed position while pushing a car . the second event was four years prior , when he sustained an injury while pushing a van , this time with the right knee fully extended . on this occasion , our patient had no other significant past medical or surgical history and explained his right knee was completely asymptomatic prior to this current event . on examination , there was an obvious deformity suggesting lateral displacement of the patella with a sulcus in the skin evident over the femoral groove . furthermore , the knee was held fixed in 15 degrees of flexion . due to patient positioning difficulties we were unable to obtain true ap and lateral views on x - ray . the radiographs demonstrated a laterally displaced and a mal - rotated patella in the vertical plane ( see figs . 1 and 2 ) . lateral radiograph of the right knee demonstrating rotation of patella our patient was subsequently taken to theatre where one final attempt of closed reduction was carried out under general anaesthetic and muscle relaxation . ultimately , this failed and an open reduction was performed . complete rupture of the medial patellofemoral ligament ( mpfl ) was identified with the patella situated lateral to the lateral femoral condyle , everted by approximately 100 degrees . the knee joint was washed out with normal saline and the medial patella retinaculum was repaired . after the repair , post - operative radiographs confirm the patella in a satisfactory position in the antero - posterior and lateral planes ( see figs . 3 and 4 ) . the patient was placed in an extension splint for comfort purposes immediately post - operatively . early mobilization was encouraged after 5 days and the patient was referred for early physiotherapy . at 3 months follow - up , our patient had no further episodes of dislocation , full range of knee extension and flexion , and normal patella tracking . a hypermobility assessment at this stage revealed a beighton score of 2 with extension beyond 10 degrees of both elbows only . intraoperative antero - posterior florous copy image following successful reduction lateral radiograph of the right knee following surgery in knee splint patellar dislocations can vary widely in their pattern of presentation however vertical axis rotation complicates only a small number of these cases . in 1844 , cooper described the earliest case of patellar dislocation with vertical axis rotation . since then , only a handful of case reports describing vertical axis rotation of the patella have been published with the majority describing the location of the patella within the femoral trochlea ( intra - articular ) [ 2 - 11 ] . our case is rare as our patient had a less common variant of extra - articular patellar dislocation complicated by vertical axis rotation and by the fact , that the causative mechanism was largely atraumatic . previous descriptions postulate that the prominence of the lateral femoral condyle acts as a pivot point , which can cause the patella to rotate around on its vertical axis . in this case , however , the patient denies any trauma , and instead describes externally rotating the leg whilst extended as the mechanism of injury . it is almost certain that the previous dislocations in our patient s case had resulted in significant damage of the mpfl . with the patient reporting his injury taking place with the knee slightly flexed , the function of the mpfl in this case as the primary medial restraint during the first 20 degrees of flexion was likely to be inadequate . previous authors have described similar cases of extra - articular lateral patellar dislocation with vertical axis rotation and have noted in their subsequent reviews of the limited literature that intra - aritcular dislocations were usually related to far more significant trauma than extra - articular dislocations [ 3 - 10 ] . this was also the finding in a previous report highlighting the relative minor force resulting in an extra - articular dislocation . we feel this may be an indication of the chronic deficiency in the mpfl likely contributing to cases of extra - articular dislocation . on reviewing the previous cases of extra - articular dislocation in the literature , none had described a completely atraumatic mechanism and none had discussed previous dislocations or previous surgical history in their patients . detailed imaging can be difficult to obtain in the acute scenario ; however in these cases ct imaging may clarify patella position and demonstrate mechanical engagement of the patella on the lateral femoral condyle . in one case report , ct imaging was utilized and confirmed avulsions of the vastusmedialis muscle and medial crus of the patella tendon with resultant impaction of the medial border on the patella onto the lateral femoral condyle . in this case , imaging led to the decision for open reduction being required , minimizing further attempts at closed reduction which could in theory result in further damage . it is difficult to be absolutely sure why attempts at closed reduction had failed in our particular case , but this is likely to be multifactorial . vertical axis rotation results in impaction of the lateral edge of the patella on the lateral femoral epicondyle which may result in a corresponding femoral defect resulting in a lodging effect . furthermore patient factors , namely obesity and large muscle mass around the knee made manipulation technically difficult in our case . open reduction under general anaesthetic is rarely necessary for routine patellar dislocations however there are some obvious advantages . these include ; the possibility of direct visualisation of the obvious damage to the soft tissue structures contributing to or resultant from the dislocation . in our case it was likely the mpfl had been damaged previously and repair is likely to help in restoring central patella tracking and overall stability . in this case , the patient was noted as having a shallow trochlea which might have accounted for his increased tendency to dislocations . direct visual inspection may also help determine patients who are at risk of recurrent dislocations due to factors relating to their bony morphology and allow the surgeon to plan further management accordingly . lastly , visualisation of the articulating surfaces allows the surgeon to identify and address any osteochondral defects and corresponding loose bodies which may otherwise have contributed to long term future problems requiring eventual surgical intervention . from our experience with this case , difficulty in reducing a dislocated patella should alert surgeons to the possibility of an extra - articular dislocation with possible vertical axis rotation . in these scenarios , multiple attempts at reduction we were able to achieve adequate analgesia and sedation using local and regional anaesthesia , without successful reduction of the dislocation . although some authors have demonstrated successful closed reduction with the use of local anaesthetic and muscle relaxants , this is not always effective . furthermore , attempted closed reduction under general anaesthesia in our case was also unsuccessful , demonstrating how closed reduction can be futile in certain cases . we would recommend a low threshold for open reduction in such cases by a specialist knee surgeon who may assess and reconstruct damaged soft tissue damage in one operation vertical axis rotation is a rare entity complicating patella dislocations . in this rare event emergency physicians should be aware of this complication and refer to the relevant specialist for open reduction under general anaesthesia in order to minimize patient discomfort and potential articular surface damage .

introduction : acute patellar dislocation is a common injury usually associated with a significant traumatic mechanism resulting in lateral displacement . vertical axis rotation following dislocation is a rare variant of this type of injury and can prevent closed reduction in the acute setting.case presentation : a 32-year old gentleman presented with an irreducible patella dislocation following an unusual atraumatic mechanism . following attempts at closed reduction under sedation and regional nerve block , eventual open reduction and soft tissue reconstruction was required under general anesthetic . during the open reduction procedure , it was noted that the patella had dislocated into a lateral extra - articular position and rotated around its vertical axis . following patella reduction , the medial patellar retinaculum was repaired . examination under anesthetic revealed satisfactory tracking of patella following repair.conclusion:a review of the literature suggests dislocations such as the current presentation , are extremely rare and although have been described to occur with minor trauma , have never been described to occur following a largely atraumatic event . in such cases , closed reduction may be impossible even with adequate analgesia due to patella position and soft tissue obstruction . open reduction is essential in these situations .

graphene oxide ( go ) solution was synthesized from natural graphite powder by a modification of hummers method.45 cu2o / rgo composites were fabricated by a microwave - assisted hydrothermal reaction . firstly , cu(no3)2 was added to a mixture of ethanol and water in the ratio of 64:36 . then , a calculated amount of go solution and formic acid ( 3 ml ) were added . for optimization , cu2o / rgo composites with different amounts of rgo were also synthesized , which include cu2o/0.25 % rgo , cu2o/0.5 % rgo , and cu2o/1 % rgo , in which x % represents the calculated weight ratio of the go added to cu2o . after stirring for 2 h , the homogeneous solution was heated with stirring in the microwave system at 150 c for 3 h. after the product was cooled to rt , the final product was collected by centrifugation , washed with water five times , and dried at 70 c . blank cu2o was synthesized through the same procedure , except for the addition of go solution . xrd was performed by using a rigaku rint 2100 diffractometer at a voltage of 40 kv . the morphologies of the products were characterized by field - emission scanning electron microscopy ( fesem , jeol-6701f ) and tem ( jeol-2010f ) . uv / vis spectra were recorded by using a shimadu uv / vis 2550 spectrophotometer . pl emission spectra were measured at rt by using a fluorescence spectrophotometer ( f-4500 , hitachi ) . photocatalyst ( 5 mg ) and nafion solution ( 10 l , 5 wt % ) were dispersed in a water / isopropanol mixture ( 1 ml , 3:1 v / v ) by at least 30 min sonication to form a homogeneous catalyst colloid . for the measurements , the catalyst colloid ( 100 l ) was deposited onto an area of approximately 1 cm of the fto conductive glass to form the working electrode . a pt wire was used as a counter electrode , and an ag / agcl electrode was the reference electrode in the three - electrode photo - electrochemical system . eis were recorded under an alternating current perturbation signal of 10 mv over the frequency range of 1 mhz to 100 mhz . the potential ranged from 1.0 to 0 v with a potential step of 10 mv at a frequency of 1 khz . the co2 reduction reaction was performed in batches by using a septum - sealed glass chamber with a volume of 120 ml , which was heated at 160 for 1 h prior to measurement . to remove possible trace organic contaminants , photocatalysts were treated at 200 c for 3 h in a tubular furnace under the protection of ar or in air ( denoted cu2o treated in ar or air ) . a typical photocatalytic experiment was conducted by using 0.5 g of photocatalysts and 3 ml of deionized water in a co2-purged 120 ml reactor . excess ( 0.7 m ) sodium sulfite was added to each batch as a hole scavenger.15 , 46 a 150 w xe lamp ( newport ) was used as a light source . the light output was measured by using a newport 1918-r high - performance optical power meter fitted with a newport 918-d calibrated photodetector equipped with an integrated attenuator . the reaction product was monitored by periodical sampling of the gas phase from the glass chamber by using a gas - tight syringe and analyzed by gc ( varian gc-450 ) with a thermal conductivity detector ( tcd , connected to a molecular sieve column ) to detect h2 , o2 , and n2 and a flame ionization detector ( fid , connected to a cp - sil 5cb capillary column ) to detect hydrocarbons . a methanizer was installed to enable the fid to detect co with 1000 higher sensitivity . for the isotope - tracer experiment , after the addition of cu2o / rgo ( 0.5 g ) into co2-saturated water ( 10 ml ) , the septum - sealed reactor was purged by ar gas for 10 min . the sample was irradiated with a 150 w xe lamp for 30 min , and then 0.5 ml of the reaction product taken from the vessel headspace was analyzed by gc graphene oxide ( go ) solution was synthesized from natural graphite powder by a modification of hummers method.45 cu2o / rgo composites were fabricated by a microwave - assisted hydrothermal reaction . firstly , cu(no3)2 was added to a mixture of ethanol and water in the ratio of 64:36 . then , a calculated amount of go solution and formic acid ( 3 ml ) were added . for optimization , cu2o / rgo composites with different amounts of rgo were also synthesized , which include cu2o/0.25 % rgo , cu2o/0.5 % rgo , and cu2o/1 % rgo , in which x % represents the calculated weight ratio of the go added to cu2o . after stirring for 2 h , the homogeneous solution was heated with stirring in the microwave system at 150 c for 3 h. after the product was cooled to rt , the final product was collected by centrifugation , washed with water five times , and dried at 70 c . blank cu2o was synthesized through the same procedure , except for the addition of go solution . xrd was performed by using a rigaku rint 2100 diffractometer at a voltage of 40 kv . the morphologies of the products were characterized by field - emission scanning electron microscopy ( fesem , jeol-6701f ) and tem ( jeol-2010f ) . uv / vis spectra were recorded by using a shimadu uv / vis 2550 spectrophotometer . pl emission spectra were measured at rt by using a fluorescence spectrophotometer ( f-4500 , hitachi ) . photocatalyst ( 5 mg ) and nafion solution ( 10 l , 5 wt % ) were dispersed in a water / isopropanol mixture ( 1 ml , 3:1 v / v ) by at least 30 min sonication to form a homogeneous catalyst colloid . for the measurements , the catalyst colloid ( 100 l ) was deposited onto an area of approximately 1 cm of the fto conductive glass to form the working electrode . a pt wire was used as a counter electrode , and an ag / agcl electrode was the reference electrode in the three - electrode photo - electrochemical system . eis were recorded under an alternating current perturbation signal of 10 mv over the frequency range of 1 mhz to 100 mhz . the potential ranged from 1.0 to 0 v with a potential step of 10 mv at a frequency of 1 khz . the co2 reduction reaction was performed in batches by using a septum - sealed glass chamber with a volume of 120 ml , which was heated at 160 for 1 h prior to measurement . to remove possible trace organic contaminants , photocatalysts were treated at 200 c for 3 h in a tubular furnace under the protection of ar or in air ( denoted cu2o treated in ar or air ) . a typical photocatalytic experiment was conducted by using 0.5 g of photocatalysts and 3 ml of deionized water in a co2-purged 120 ml reactor . excess ( 0.7 m ) sodium sulfite was added to each batch as a hole scavenger.15 , 46 a 150 w xe lamp ( newport ) was used as a light source . the light output was measured by using a newport 1918-r high - performance optical power meter fitted with a newport 918-d calibrated photodetector equipped with an integrated attenuator . the reaction product was monitored by periodical sampling of the gas phase from the glass chamber by using a gas - tight syringe and analyzed by gc ( varian gc-450 ) with a thermal conductivity detector ( tcd , connected to a molecular sieve column ) to detect h2 , o2 , and n2 and a flame ionization detector ( fid , connected to a cp - sil 5cb capillary column ) to detect hydrocarbons . a methanizer was installed to enable the fid to detect co with 1000 higher sensitivity . after the addition of cu2o / rgo ( 0.5 g ) into co2-saturated water ( 10 ml ) , the septum - sealed reactor was purged by ar gas for 10 min . the sample was irradiated with a 150 w xe lamp for 30 min , and then 0.5 ml of the reaction product taken from the vessel headspace was analyzed by gc as a service to our authors and readers , this journal provides supporting information supplied by the authors . such materials are peer reviewed and may be re - organized for online delivery , but are not copy - edited or typeset . technical support issues arising from supporting information ( other than missing files ) should be addressed to the authors

a facile one - step microwave - assisted chemical method has been successfully used for the synthesis of cu2o / reduced graphene oxide ( rgo ) composites . photocatalytic co2 reduction was then investigated on the junction under ambient conditions . the rgo coating dramatically increases cu2o activity for co2 photoreduction to result in a nearly six times higher activity than the optimized cu2o and 50 times higher activity than the cu2o / ruox junction in the 20th hour . furthermore , an apparent initial quantum yield of approximately 0.34 % at 400 nm has been achieved by the cu2o / rgo junction for co2 photoreduction . the photocurrent of the junction is nearly double that of the blank cu2o photocathode . the improved activity together with the enhanced stability of cu2o is attributed to the efficient charge separation and transfer to rgo as well as the protection function of rgo , which was proved by xrd , sem , tem , x - ray photoelectron spectroscopy , photo - electrochemical , photoluminescence , and impedance characterizations . this study further presents useful information for other photocatalyst modification for efficient co2 reduction without the need for a noble - metal co - catalyst .

in recent years , the concept of minimal intervention ( mi ) has prevailed in dentistry . therefore , the importance of diagnosing caries at an early stage has increased . in conventional procedures , the diagnosis of caries has mainly consisted of visual inspection and tactile assessment with probing . however , lussi reported that the sensitivity of detecting caries was 0.62 by visual inspection and 0.82 by probing . in addition , the pressure of probing can damage the demineralized fissure and increase the risk that caries progress [ 3 , 4 ] . to promote mi , diagnosis without a probe has been recommended . the laser fluorescence - based caries detection device diagnodent ( kavo , germany ) has been introduced as an alternative . however , no single detection method for caries is sufficient ; therefore , the combination of some detection methods has been recommended [ 57 ] . recent improvements in the personal computer have made the process of digital imaging more efficient and convenient . if the shape of caries can be quantified , and the relationship between the numerical value and the condition of the lesion can be demonstrated , this information would be helpful to diagnose dental caries . the authors of this paper have previously shown that the fractal dimension and proportion of the area of pit - and - fissure discoloration to the area of occlusal surface obtained by digital imaging were significantly correlated with the depth of the caries and the diagnodent values in extracted teeth . for assessment of the method as a diagnostic system , the ability of the diagnosis , such as the sensitivity , the specificity , and the accuracy , should be researched in clinical situation . the aim of this study was to assess the possibility of the clinical application of the diagnosis of occlusal caries using digital imaging by examining the sensitivity , the specificity , and the accuracy in comparison with the diagnodent values and the dentists ' diagnoses . one hundred teeth ( 36 premolars and 64 molars ) with pit - and - fissure discoloration from 19 outpatients were examined at the clinic of oral diagnosis and general dentistry , dental hospital , tokyo medical and dental university . the occlusal surface of each tooth was washed with the robinson brush to remove dental plaque without any abrasive paste . then , pit - and - fissure discoloration was dried by air and measured three times using diagnodent . the mean scores were used as the diagnodent values of the teeth ( dd ) . next , the occlusal surface of each tooth was photographed as large as possible with an intraoral digital camera ( penscope , morita , japan ) . each image was stored in a personal computer using a video capture interface ( pc - mdvd / u2 , buffalo , japan ) . without knowing the dd , a dentist preliminarily diagnosed each tooth using visual inspection and tactile examination to decide which treatment plan would be appropriate ( preventive or operative ) . the clinical diagnosis of preventive treatment for teeth was classified as co. on the other hand , carious lesions requiring operative treatment were removed in a conventional clinical way . if the resulting cavity preparation was limited in the enamel , then the clinical diagnosis was classified as c1 . if the resulting cavity preparation reached the dentin , and sound tissue still remained between the cavity and the pulp chamber , then the clinical diagnosis was classified as c2 . five dentists ranging from 3 to 15 years of professional experience examined the teeth after calibration of the criteria of the caries assessment conducted before the study ; the calibration was done as follows ; first , the five dentists examined 30 extracted teeth and decide which treatment plan would be appropriate ( preventive or operative ) . at that time then , the teeth were sliced pallarel to the teeth axis and to the depth of lesion for each tooth was determined . at last , the dentists discussed to accord the treatment planning for each tooth referring the depth of the lesion . the digital photographs obtained were processed and analyzed using image analysis software ( image j , nih , usa ) . first , each image was converted to an 8-bit gray - scale image , in which the density of grayness of each pixel was linearly scaled from min 0 ( black ) to max 255 ( white ) . then , the occlusal surface in the image was isolated from the background using a density histogram of the image , and the area was measured . pit - and - fissure discoloration was also isolated from the occlusal surface using the density histogram , and the area was measured . the proportion of the area of pit - and - fissure discoloration to the area of the occlusal surface was calculated ( pa ) . next , the image of the isolated pit - and - fissure discoloration was converted into a binary image , in which the density of pit - and - fissure discoloration was 0 , and its background was 255 , followed by calculating the fractal dimension of pit - and - fissure discoloration ( fd ) . differences in fd , pa , and dd between each clinical diagnosis were analyzed using two - way anova and games - howell test to reveal the clinical diagnosis and the effect of the examining dentists . the correlation between the clinical diagnosis and each fd , pa , and dd was analyzed using spearman 's correlation coefficient . discriminant formulas were obtained using discriminant analysis with the treatment plan ( preventive / operative ) as the objective variable and fd , pa , and dd as explanatory variables . sensitivity and specificity were calculated by applying fd , pa , and dd to each discriminant formula . the accuracy , ratio of the number of teeth showing accordance between the treatment plan decided by the dentists and the predictive treatment plan decided using the discriminant formula to the number of all the teeth , was also obtained . the entire process was approved by the ethics committee of the faculty of dentistry , tokyo medical and dental university ( no . 317 ) . fd , pa , and dd values corresponding to each clinical diagnosis are shown in table 1 . the two - way anova revealed that the fd , pa , and dd were different among the clinical diagnosis ( p < .01 ) . on the other hand , the difference of the examining dentists did not affect the fd , pa , and dd . spearman 's correlation coefficients between the clinical diagnosis and each fd , pa , and dd were 0.743 , 0.700 , and 0.652 , respectively ( p < .01 ) . there were also significant correlations among fd , pa , and dd ( p < .01 ) . table 2 shows the discriminant formula , sensitivity , specificity , and accuracy of each explanatory variable . based on the discriminant formula of each explanatory variable , the thresholds of fd , pa , and dd between preventive and operative treatments were 1.20 , 0.012 , and 28.8 , respectively . the sensitivity of fd was greater than that of pa , dd , and the combination of fd and pa . the specificity of pa was greater than that of fd , dd , and the combination of fd and pa . the accuracy of the combination of fd and pa was greater than that of fd , pa , and dd . previously , it was reported that the fractal dimension and the proportion of the area of pit - and - fissure discoloration to the area of occlusal surface were significantly correlated with the depth of the caries and the diagnodent values in extracted teeth . in this study , the fractal dimensions for c0 , c1 and c2 in the former study were 0.97 , 1.30 , and 1.52 , respectively . these results indicate that image analysis of molar pit - and - fissure discoloration was clinically useful for the diagnosis of caries . an increase of the proportion of the area of discoloration corresponded to a change of the volume of caries lesion , while an increase of the fractal dimension corresponded to a change of the shape of the lesion caused by caries progression . a fractal is a geometric shape , possessing characteristics of self - similarity or self - affinity , and widely observed in nature [ 10 , 11 ] . recently , fractals have been in the spotlight in the field of medicine , and research has been introduced regarding its use in the field of diagnosis [ 1214 ] . for example , a point is described as the zero dimension ; a straight line is described as the first dimension , and a plane is described as the second dimension . such decimal dimensions can be obtained by expanding the definition of the dimension as the rate at which the perimeter ( or the surface area ) of an object increases , and the measurement scale is reduced . several ways to measure the fractal dimension have been introduced . in this study , the authors used a simple way to determine the fractal dimension called box counting . in this method , a grid of squares is placed over the object , and the number of squares through which any part of the object passes is counted . this process is repeated with different grids having different sizes . the number of squares placed over the object versus the length of the side of the square are then plotted on log - log scale . when a regression line is obtained from the plots , the degree of uneven complexity of a boundary or a coast can be quantified using this approach . in this research , the fractal dimension of discoloration increased from 0.8 to 1.6 as the depth of the caries increased , which corresponded to a change in the shape of the discolored area from a point or a line to an area based on the progression of the caries . generally , the addition of valuables into discriminant formula is one of the ways to improve the accuracy , however , in this study , the accuracy of the combination of fd and pa was similar to that of single fd . therefore , further study to find other valuables is needed to improve the accuracy of this method by combination of valuables . thus , the accuracy of the diagnosis of occlusal caries using digital images of discolored areas was comparable to that of diagnodent ; therefore , its clinical application as a diagnostic tool is possible . because this study was clinical , the final diagnosis of each examined tooth was not confirmed by a histological procedure but by a dentist 's clinical examination . as mentioned above , , the results of two - way anova showed that the effect of the examining dentist on the dd , pa , and fd values was not significant . therefore , we considered that difference of the diagnosis among the dentists would be small . in the present study , to examine the possibility of digital imaging in the diagnosis of occlusal caries , diagnodent was used as a comparative pre - existing dental caries detection tool . there are other caries detection tools , such as fiber optic transillumination ( foti ) , digital imaging fiber optic transillumination ( difoti ) , quantitative laser or light fluorescence ( qlf ) , and electrical conductive measurements ( ecm ) . foti , difoti , and qlf have been tested in vivo , however , the number of clinical studies has still been small [ 15 , 16 ] . on the other hand , a comparatively long time has passed since diagnodent was introduced to the market , and many findings have been reported . sheehy et al . reported that diagnodent had greater sensitivity and specificity than ecm . the correlation coefficient between diagnodent readings and the depth and the volume of caries lesions was reported to be 0.47 . several researches have pointed out that diagnodent measurements are affected by other factors , such as hypomineralization , plaque , debris , staining and wetness [ 1921 ] , while a high correlation between inter and intraobserver agreements was also mentioned [ 22 , 23 ] . we employed diagnodent as a comparison because the diagnosis was provided as a number , the handling was easy , and , moreover , its clinical use has been discussed in other studies . in the present study , diagnosis using digital images of pit - and - fissure discoloration depended on the statistical relationship between the shape of discoloration and the depth of caries . the shape of the discolored area in the occlusal surface is , however , possibly affected by medication history , individual history , and lifestyle . consequently , diagnosis using digital images of pit - and - fissure discoloration is rather experimental . additionally , the procedure can not be applied to colorless lesions , such as acute caries . therefore , diagnosis using digital images of pit - and - fissure discoloration should not be used for definitive diagnosis . rather , initial diagnosis , screening such as mass examination would be suitable because of its good sensitivity of fd and , the convenience of the procedure . actually , the core of the diagnostic system is digital imaging processing by computers . if computer programing for all procedures was achieved , then screening of hundreds of examinees would be automated after photograph taking , which would make mass examination less time - consuming with low cost . for such automated uses of the computer , the process of extracting colors from the image must be improved ; in this study , the threshold for the colored area was decided one by one by an observer 's visual inspection . how to calculate the fractal dimension is also open to discussion . we used the box - counting method attached in the image analysis software , image j. the box - counting method is only suitable for self - similar profiles , not for more general , self - affine cases , that is , the fractal dimension using the box - counting method might be the approximate value . a special computer program must be developed to measure the fractal dimension more accurately , for example , the minkowski method or the richardson method [ 8 , 10 ] . as mentioned above , the shape of the discolored area on an occlusal surface is affected by many factors . therefore , the thresholds or the discriminant formulas acquired from this research are not universal . further research is needed to determine the discriminant formulas to diagnose caries using the image analysis of molar pit - and - fissure discoloration .

the aim of this study was to assess the possibility of digital image analysis of pit - and - fissure discoloration in order to diagnose caries . digital images showing pit - and - fissure discoloration in 100 teeth of 19 patients were analyzed to obtain the fractal dimension ( fd ) and the proportion of the area of pit - and - fissure discoloration to the area of occlusal surface ( pa ) . diagnodent values were measured ( dd ) , and dentists ' diagnoses were also obtained . the sensitivity and specificity of fd , pa , dd , and the combination of fd and pa compared to the dentists ' diagnoses were calculated . the sensitivities of fd , pa , dd , and the combination of fd and pa were 0.89 , 0.47 , 0.69 , and 0.86 , respectively , and the specificities were 0.84 , 0.95 , 0.91 , and 0.86 , respectively . although further research is needed for the practical use , it is possible to use the analysis of digital images of pit - and - fissure molar discoloration as a diagnostic tool .

craniofacial defects are rare , disfiguring facial anomalies , with an incidence of about 1.4 to 4.9 cases per 100,000 live births . craniofacial clefts may be caused by failure of the maxillary processes to fuse , external pressure , amniotic bands , oligohydramnios , central disorganization of the neural crest , and hematomas . in 1973 , the median cleft of the upper lip , or tessier 0 class , has a variety of presentations , ranging from a minimal notching of the lip , vermilion , and nose to a wide cleft that divides craniofacial structures . tessier 0 clefts result from failure of the two medial nasal processes to fuse at midline . median clefts are broadly classified into true and false ; false clefts are due to agenesis of the medial nasal process , while true clefts are due to failure of the medial nasal process to fuse . treatment of median clefts depends on the clinical presentation and may vary from simple alignment of orbicularis oris and vermilion , to reconstruction of the cupid 's bow and philtrum for true median clefts , and complex craniofacial procedures in case of false median clefts . various techniques have been described to repair mild or moderate true median cleft lips . here we report using a pfeifer incision to correct a moderate true median cleft of the upper lip . pfeifer incision includes wavy lines , which elongate the incised tissues as the waves are straightened to close in a straight line , and also provide extra tissue for a tension free closure . a five - year - old boy with a facial cleft presented to our clinic . on examination , there was a median cleft of the upper lip involving the white roll , with no bony involvements . the highest- ( points a , a ) , and the deepest - points ( points b , b ) of the white roll were marked on both sides . subsequently , a wavy incision line was made starting from the deepest point and extending over the philtrum just above the cleft ( fig . a diamond incision was made over the vermilion and the labial mucosa , intra - orally extending just beyond the cleft margin . incision was made and the mucosal tissue covering the area medial to the incision site ( sterile zone ) was removed . undermining of the orbicularis oris muscle was performed and it was approximated using 4 - 0 vicryl . the cupid 's bow was properly aligned , with equal height on both sides ( fig . vermilion form was satisfactory and the fullness and continuity of the orbicularis oris were maintained . incision marking for pfeifer technique ( a , a : highest points on white roll ; b , b : deepest points on white roll ) postoperative result after suture removal a median cleft can also be called midline cleft or vertical cleft through the centre of the upper lip , and is a rare anomaly , the exact developmental origin of which is not clear . tessier 0 clefts occur during the third week of gestation due to failure of the two medial nasal processes to close in midline . it can occur in isolation or be a part of a syndrome such as orofacial - digital syndrome . false clefts are associated with abnormalities of the forebrain and are categorized under the category of holoprosencephaly . in 1937 , veau categorized median clefts to notch of the lip , median cleft extending to the columella , and defects due to atrophy of the midline facial structures . median cleft face syndrome , frontonasal dysplasia , and tessier 0 clefts are various terms used to describe abnormalities associated with true median clefts that are not accompanied by forebrain abnormalities . the tessier 0 anomaly may present as a small notch in the soft tissue , or in association with hypertelorism , midline craniofacial osseous defects , and hairline abnormalities . various treatment options are present for mild deformities which do not involve the white roll . when developing the treatment plan , reconstruction of the cupid 's bow , labial philtrum , vermillion , and buccal mucosa should be kept in mind . urata and kawamoto described using a v - y flap , while weimer et al , used a diamond incision to repair these anomalies . da silva frietas and colleagues described a mucosal z transposition technique to treat mild cases . for moderate defects involving the philtrum , these waves are subsequently approximated in a straight line , which help expanding the length and width of the tissue . this incision has previously been used to correct other tessier clefts with a high success rate . the above - mentioned studies prove the versatility of the pfeifer incision . in our case we were able to achieve adequate symmetry of the philtrum and the cupid 's bow , which is important in cases like this . the postoperative results were excellent and proper approximation of the orbicularis oris muscle and the vermilion was achieved with a symmetrical cupid 's bow and philtrum . the only disadvantage of this technique is that the final closure line is placed directly over the philtrum . since the presentation of midline cleft deformities varies widely , each case should be individually considered and treated . pfeifer incisions can successfully provide mucosal length , vermilion fullness , and lip symmetry in patients with moderate median cleft lip .

median cleft is the midline cleft of the lip . it develops due to incomplete or failed fusion of the median nasal prominence . it can present with minimal deformities such as involvement of the vermilion border , or complex clefting of the midline structures and brain . median clefts are broadly classified as true and false clefts . this case report describes a rare case of median cleft of the upper lip involving the white roll , which was not associated with any other deformities . treatment included reconstruction of the philtrum and the cupid 's bow while maintaining vermilion fullness and continuity , and minimizing scar formation . various techniques have been advocated for treatment of this type of median upper lip cleft . here we describe a technique using pfeifer incision to correct our patient 's defect . pfeifer incision consists of wavy lines and its use has been advocated for correction of various craniofacial abnormalities .

pseudomonas aeruginosa is an important gram - negative opportunistic pathogen that has the capability to create variable clinical infections , including , but not limited to , wound infections , urinary tract infections ( utis ) , and blood stream infections ( bsi ) , particularly in hospitalized and immunocompromised patients ( 1 , 2 ) . pseudomonas aeruginosa was the second most common cause of hospital - acquired pneumonia , as well as the third and fifth common cause of hospital - acquired utis in usa and europe , respectively ( 2 - 4 ) . evidently , p. aeruginosa due to low permeability of its outer membrane in combination with efflux pump overexpression is intrinsically resistant to a variety of antibiotics ( 5 ) . expression of different classes of extended - spectrum -lactamas enzymes along with aminoglycoside - modifying enzymes ( aminoglycoside phosphoryltransferases , aminoglycoside acetyltransferases , and aminoglycoside nucleotidyltransferases ) , and mutation are other well - characterized mechanisms for antibiotic resistance in p. aeruginosa ( 6 , 7 ) . data extracted from several independent studies in iran showed that p. aeruginosa was responsible for 11%-32.4% of utis , 17.2%-32.4% of pneumonia , 36.7% of bsi , and 47% of wound infections ( 8 - 12 ) . moreover , based on previous studies performed in iran , treatment of p. aeruginosa infections is a major concern for health - care setting due to its high resistant rate to different antibiotics ( 12 , 13 ) . since determining bacterial isolates relatedness is essential for understanding the transmission routes , different typing methods that can be divided into two major categories , phenotypic and genotypic methods , have been established ( 14 ) . phenotypic methods such as serotyping , pyocin typing , and antimicrobial susceptibility typing depend on the environmental factors , therefore , have low discriminatory power ( 14 , 15 ) . genotypic methods have been performed by different typing techniques such as restriction fragment length polymorphism ( rflp ) , pulsed field gel electrophoresis ( pfge ) , multilocus sequence typing ( mlst ) , enterobacterial repetitive intergenic consensus - pcr ( eric - pcr ) , variable number tandem repeat ( vntr ) , dna hybridization and random amplified polymorphic dna ( rapd ) ( 16 - 21 ) . although mlst and pfge have more discriminatory power , they are expensive , especially in developing countries ( 22 ) . rapd - pcr has the advantage of being fast , reproducible , simple , and low - cost , thus , it is an appropriate approach for primary screening of epidemic strains in large number of isolates ( 23 , 24 ) . to our knowledge , limited data are available about genetic diversity of p. aeruginosa recovered from different clinical specimens in isfahan ; the purpose of the current study was to determine the antibiotic susceptibility profiles and genetic relatedness in p. aeruginosa isolated from patients admitted to a referral hospital in isfahan , iran . this study was conducted at a teaching referral hospital of isfahan , iran , from february 2013 to november 2013 . during the mentioned period , a total of 150 non - duplicate samples ( urine , sputum , wound , blood , and eye discharge ) taken from hospitalized - patients admitted to different wards of hospital were analyzed . primary identification of p. aeruginosa was done based on the standard conventional biochemical tests , including gram staining , catalase , oxidase , oxidative - fermentative ( of ) tests , pigment production and growth at 42c . then , primary identification was further confirmed with species - specific pcr using its ( 16s-23s rrna internal transcribed spacer ) primer ( 25 , 26 ) . the study was approved by the ethics committee of isfahan university of medical sciences ( no . 392063 ) . resistance to antibiotics was evaluated by kirby - bauer s disk diffusion method according to clsi ( clinical laboratory standard institute ) recommendation ( 27 ) . the following disks ( mast , uk ) were applied : ceftazidime ( caz , 30 g ) , imipenem ( imp , 10 g ) , meropenem ( mem , 10 g ) , ciprofloxacin ( cip , 5 g ) , aztreonam ( atm , 30 g ) , polymyxin b ( pb , 300 units ) , and amikacin ( amk , 30 g ) . p. aeruginosa standard strain ( atcc 27853 ) was used as the quality control . optimized rapd reactions mixtures comprised 2.5 l 10x pcr buffer , 2.5 mm mgcl2 , 300 m of dntps , 1.7 u taq dna polymerase ( cinnagen , iran ) , 3 l genomic dna ( 40 ng ) , and 10 pm of 272- agcgggccaa primer ( 21 ) ( metabion , germany ) in 25 l final volume . dna amplification was carried out using biometra thermocycler ( germany ) and following a two - step program , 1 ) denaturation 5 min at 95c , annealing 5 min at 36c , elongation 5 min at 72c , for 4 cycles , and 2 ) 31 cycles consisted of 94c for 1 min , 45c for 1 min , 72c for 2 min , followed by a final extension at 72c for 10 min ( 21 ) . electrophoresis was carried out using 2% agarose gel ( w / v ) and 0.5x tbe ( tris - boric acid - edta , ph = 7.5 - 8 ) buffer at 7 v / cm for 3h . we also used 100 bp dna ladder ( cinnagen , iran ) as the standard molecular size ( 100 bp-3000 bp ) . separated bands stained with ethidium bromide ( 0.5 g / ml ) and visualized picture was captured on gel - documentation system ( uvitec , uk ) . rapd - fingerprints were recorded as present ( 1 ) or absent ( 0 ) for each band . by means of freetree and treeview softwares , dice similarity coefficient and unweighted average pair group method ( upgma ) were used for similarity matrix calculation and cluster analysis , respectively ( 28 , 29 ) . only major reproducible bands we used cut - off value of 80% for determination of potential clonal relatedness ( 31 - 33 ) . this study was conducted at a teaching referral hospital of isfahan , iran , from february 2013 to november 2013 . during the mentioned period , a total of 150 non - duplicate samples ( urine , sputum , wound , blood , and eye discharge ) taken from hospitalized - patients admitted to different wards of hospital were analyzed . primary identification of p. aeruginosa was done based on the standard conventional biochemical tests , including gram staining , catalase , oxidase , oxidative - fermentative ( of ) tests , pigment production and growth at 42c . then , primary identification was further confirmed with species - specific pcr using its ( 16s-23s rrna internal transcribed spacer ) primer ( 25 , 26 ) . the study was approved by the ethics committee of isfahan university of medical sciences ( no . 392063 ) . resistance to antibiotics was evaluated by kirby - bauer s disk diffusion method according to clsi ( clinical laboratory standard institute ) recommendation ( 27 ) . the following disks ( mast , uk ) were applied : ceftazidime ( caz , 30 g ) , imipenem ( imp , 10 g ) , meropenem ( mem , 10 g ) , ciprofloxacin ( cip , 5 g ) , aztreonam ( atm , 30 g ) , polymyxin b ( pb , 300 units ) , and amikacin ( amk , 30 g ) . optimized rapd reactions mixtures comprised 2.5 l 10x pcr buffer , 2.5 mm mgcl2 , 300 m of dntps , 1.7 u taq dna polymerase ( cinnagen , iran ) , 3 l genomic dna ( 40 ng ) , and 10 pm of 272- agcgggccaa primer ( 21 ) ( metabion , germany ) in 25 l final volume . dna amplification was carried out using biometra thermocycler ( germany ) and following a two - step program , 1 ) denaturation 5 min at 95c , annealing 5 min at 36c , elongation 5 min at 72c , for 4 cycles , and 2 ) 31 cycles consisted of 94c for 1 min , 45c for 1 min , 72c for 2 min , followed by a final extension at 72c for 10 min ( 21 ) . electrophoresis was carried out using 2% agarose gel ( w / v ) and 0.5x tbe ( tris - boric acid - edta , ph = 7.5 - 8 ) buffer at 7 v / cm for 3h . we also used 100 bp dna ladder ( cinnagen , iran ) as the standard molecular size ( 100 bp-3000 bp ) . separated bands stained with ethidium bromide ( 0.5 g / ml ) and visualized picture was captured on gel - documentation system ( uvitec , uk ) . rapd - fingerprints were recorded as present ( 1 ) or absent ( 0 ) for each band . by means of freetree and treeview softwares , dice similarity coefficient and unweighted average pair group method ( upgma ) were used for similarity matrix calculation and cluster analysis , respectively ( 28 , 29 ) . only major reproducible bands we used cut - off value of 80% for determination of potential clonal relatedness ( 31 - 33 ) . out of 150 samples , 54 ( 36% ) were positive for p. aeruginosa culture . distribution of the 54 isolates were as follow , urine 39 ( 72.2 % ) , sputum 7 ( 13% ) , wound 5 ( 9.3% ) , blood 2 ( 3.7% ) , and eye discharge 1 ( 1.9% ) . the most frequent involved wards were icu 28 ( 51.9% ) , followed by graft 12 ( 22.2% ) , internal 5 ( 9.3% ) and surgery 9 ( 16.7% ) . thirty - six isolates ( 66.7% ) belonged to male patients and 18 ( 33.3% ) belonged to female patients . the highest percentage ( 55.6% ) of resistance was observed against ceftazidime and imipenem with 30 of the isolates being resistant ; all isolates were sensitive to polymyxin b ( figure 1 ) . twenty - eight ( 51.8% ) isolates tested in the study revealed resistance to all applied antibiotics except for polymyxin b. electrophoresis patterns of rapd - pcr for some isolates of studied p. aeruginosa are shown in figure 2 . genetic relatedness of clinical isolates of the detected p. aeruginosa has been carried out using rapd - pcr assay , which showed 55% to 100% similarity ( figure 3 ) . the number of bands in rapd typing varied from 2 to 12 , with the length of 150 bp to 4300 bp . rapd fingerprinting results of p. aeruginosa based on 80% showed 39 different groups ( figure 3 ) . although the majority of isolates had unique fingerprint , group one composed of 3 isolates ( patients s48 , s29 , s31 ) . isolate s29 was fully sensitive to applied antibiotics , whereas isolates s31 and s48 were resistance to applied antibiotics ( figure 3 ) . groups 22 , 31 , 35 , and 36 , each consisted of two isolates , were also obtained from the same ward ( figure 3 ) . based on 65% similarity level , eight main cluster , with most of the strains belonging to cluster 1 were detected ( figure 3 ) . line 1 , 14 dna ladder ( 3000 - 100 bp ) , lines 2 - 13 showed 12 different rapd types . abbreviations : amk , amikacin ; azt , aztreonam ; caz , ceftazidime ; cip , ciprofloxacin ; imp , imipenem ; mp , meropenem ; sp , sputum ; uc , urine ; wo , wound . constant monitoring of antibiotic susceptibility profiles and genetic relatedness among bacterial infectious agents are essential steps for infection control ( 34 ) . to improve our understanding about p. aeruginosa antibiotic resistance and their distribution , 54 non - duplicate isolates of p. aeruginosa by using kirby - bauer s disk diffusion and rapd - pcr methods p. aeruginosa involved in this study were highly resistant to different antibiotic families like , monobactams , cephalosporins , quinolones , and carbapenems ( figure 1 ) . our isolates had the highest resistance rate to imipenem and meropenem ( 55.6% ) , which is higher than similar reports from iran ( 11 - 13 ) . in addition , increased percentage of resistance to ciprofloxacin and amikacin ( 53.7% and 48.1% , respectively ) were observed in comparison with previous study ( 29% and 17% ) that have been performed in the same hospital ( 35 ) . although , in our investigated hospital , carbapenems ( imipenem , meropenem ) along with amikacin are the most currently used antibiotics for the treatment of p. aeruginosa infections , results of this study revealed that prescription of them should be limited . in order to optimize treatment of p. aeruginosa infections in our hospital ( also it can be mentioned as a limitation of this study ) , determination of resistance to other antibiotics like piperacillin , piperacillin - clavulanic acid , ticarcillin , ticarcillin - clavulanic acid , gentamicin , and cefepime , are essential to be evaluated in future studies . the finding of present study corresponds with the data presented by haeili and associates , which showed the lowest percentage of resistance ( 0% ) to polymyxin b ( 11 ) . there are reports that pfge and mlst are more reliable methods for establishing clonal relatedness among p. aeruginosa strains , but because of the high cost , their utilization is limited , especially in developing countries ( 36 , 37 ) . allegedly , rapd typing is a valuable and useful method for the evaluation of genetic diversity among p. aeruginosa isolates ( 21 , 23 , 24 ) . out of various primers applied for rapd - pcr , primer 272 was reliable because of its higher discriminatory power and reproducible profiles ( 38 , 39 ) . in the present study , all 54 isolates were typeable by rapd - pcr ; moreover , rapd typing allowed us to reveal 54 unique finger prints among 54 clinical isolates of p. aeruginosa . previous studies performed in iran showed different distribution of genotypes . for instance , in the study of salimi et al . , by using rapd typing , only 8 different groups among 129 isolates of p. aeruginosa were reported ( 40 ) . in addition , in another study conducted by nanvazadeh et al . 9 groups among 50 clinical samples of p. aeruginosa were observed ( 41 ) . using limited source for recovering p. aeruginosa although in a previous study the correlation between rapd type and p. aeruginosa infection was suggested , our finding is not in agreement with these data ( 42 ) . furthermore , our data demonstrated a weak correlation between rapd and antibiotic resistance profiles ; it is supported by the results of other studies ( 34 , 43 ) . in a number of cases , the reason for this diversity may be attributed to the involved different antibiotic resistance mechanisms ( 5 - 7 ) . moreover ; results of the independent studies showed that determination of antibiotic resistance pattern as phenotypic method for p. aeruginosa typing has low discriminatory power ( 34 , 43 ) . according to our data , the majority of isolates probably originated from host itself , but cross infection of p. aeruginosa is possible to occur in studied hospital . for instance , isolates s48 , s29 , and s31 were recovered from urine of the patients in different wards or isolates 26 and 33 from the same ward and sample had genetic similarity ( based on rapd fingerprinting results ) . before taking any decision , we think that clonal relatedness of obtained isolates should be confirmed by more discriminatory methods , such as pfge or mlst . in conclusion , prescription of common antibiotics ( imipenem and amikacin ) in our hospital due to high resistance rate must be restricted . according to our data , considerable genetic diversity exists among isolated p. aeruginosa ; it is possible that different sources of p. aeruginosa be involved in our hospital that can lead to host colonization . in order to accurately control the infection , other p. aeruginosa isolated from hospital environment

background : pseudomonas aeruginosa is a well - known opportunistic pathogen , which affects hospitalized patients in different wards due to its natural resistance to drugs.objectives:the purpose of the current study was to determine the antibiotic susceptibility profiles and genetic relatedness in p. aeruginosa isolated from patients admitted to a referral hospital in isfahan , iran.materials and methods : out of 150 analyzed samples , 54 p. aeruginosa isolates were recovered and were subjected to antibiotic resistance patterns and genetic diversity determination by kirby - bauer s disk diffusion method and rapd - pcr , respectively.results:the highest percentage of resistance was observed against ceftazidime and imipenem with 30 ( 55.6% ) isolates ; meanwhile all isolates were sensitive to polymyxin b. twenty - eight ( 51.8% ) isolates revealed resistance to all applied antibiotics . rapd - pcr ( random amplified polymorphic dna- polymerase chain reaction ) results showed 54 unique genotypes , which were divided into 39 clusters.conclusions:although different source of p. aeruginosa may involve in patient colonization , genetically related strains were isolated from different wards and or the same ward of the hospital . our results pointed to the restriction of currently used antibiotics in studied hospital . we hope that our results cast light on the control and transmission of the infection in the investigated hospital .

polyarginine peptides are known as one of the widely used classes of cell - penetrating peptides ( cpps ) and cellular delivery tools . it has been reported that the presence of the guanidine group in the side chain of arginine plays a key role for improved ability of arginine - rich peptides to cross the cell membrane . various systematic structural investigations have been performed to determine the required number of arginine residues and the length of the peptide for the optimization of cellular uptake . short polyarginine peptides containing less than six arginine residues did not exhibit significant cellular uptake in several previously reported investigations . thus , the presence of more than six arginine residues in the structure of polyarginine peptides is critical for their efficient cell - penetrating functions . however , several investigations were conducted to increase the cellular uptake of polyarginines by attaching the fatty acid to the n - terminal of the peptide . it has been previously reported that the acylation of the n - terminal by fatty acids can facilitate the intracellular uptake of polyarginines . for instance , katayama et al . synthesized acylated octa - arginines and discovered that the introduction of hydrophobic fatty acid enhanced the intracellular uptake of octa - arginine peptide and its conjugated ubiquitin . furthermore , lee et al . designed a class of lipopeptides carrying 715 arginine residues . among them , myristoylated - hendeca - arginine ( c14r11 ) was found to be the most efficient cell - penetrating peptide . however , the fatty acylated polyarginine peptides that contain 715 arginine residues can potentially cause toxicity , and they can be easily degraded by proteases . moreover , linear peptides carrying l - form are not stable in serum and therefore have a limited application for in vivo studies ( figure s2 , supporting information ) . replacing l - form amino acids with d - form to improve the peptide stability leads to high cost production . thus , the synthesis and development of cyclic cpps containing short amino acid sequence with less toxicity is desired . herein , we designed acylated cyclic polyarginine peptides ( acpps ) containing five arginine residues and investigated their ability as cell - penetrating peptides . we compared acpps with a corresponding acylated linear polyarginine peptide ( alpp ) and a nonacylated cyclic polyarginine as controls . we hypothesize that the combination of acylation and cyclization of short polyarginine peptides having less than six arginine residues will increase the intracellular uptake and can generate peptides with molecular transporter properties . for convenience , square brackets [ ] and parentheses ( ) phosphopeptide ptyr - glu - glu - ile ( pyeei ) is an optimal peptide ligand for binding to the src tyrosine kinase sh2 domain . in this study , we used negatively charged fluorescein - labeled phosphopeptide f-gpyeei as a model cell - impermeable compound . all peptides were synthesized by fmoc / tbu solid - phase peptide synthesis strategy either manually or using rainin ps3 synthesizer ( protein technologies inc . ) . manual reactions were carried out in a glass reaction vessel with a sintered glass frit by mixing under nitrogen bubbling at room temperature . fmoc - l - amino acid building blocks , fatty acids , and preloaded h - arg(pbf)-2-chlorotrityl resin were used as starting materials . 2-(1h - benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate ( hbtu ) , hydroxybenzotriazole ( hobt ) , and n , n - diisopropylethylamine ( dipea ) in n , n - dimethylformamide ( dmf ) were used as coupling and activating reagents , respectively , for manual synthesis . in the case of using peptide synthesizer , 0.4 m n - methylmorpholine in dmf was used instead of dipea . piperidine in dmf ( 20% ) was employed to deprotect fmoc group at each step . to cleave the linear peptide from the resin , a mixture of trifluoroacetic acid ( tfa)/triisoproylsilane ( tis)/water ( 92.5/5/2.5 , however , in the synthesis of cyclic peptides , the side chain protected linear peptides were first cleaved from the resin by using a cleavage cocktail , containing 2,2,2-trifluoroethanol ( tfe)/acetic acid / dichloromethane ( dcm ) ( 2:1:7 , v / v / v ) . cyclization reaction was performed by employing a mixture of 1-hydroxy-7-azabenzotriazole ( hoat ) and n , n-diisopropylcarbodiimide ( dic ) in anhydrous dmf / dcm for 12 h. after solvent evaporation , the peptide was deprotected and cleaved from the resin by using a cleavage cocktail reagent r , containing tfa / thioanisole/1,2-ethanedithiol ( edt)/anisole ( 90:5:3:2 , v / v / v / v ) for 23 h. the crude peptides were precipitated and washed with cold diethyl ether . to purify the crude peptides , we used a reversed - phase high pressure liquid chromatography ( rp - hplc ) system using shimadzu lc-8a preparative liquid chromatography on a phenomenex gemini c18 column ( 10 m , 250 21.2 mm ) with a gradient 0100% of acetonitrile ( ch3cn ) containing 0.1% tfa ( v / v ) and water containing 0.1% tfa ( v / v ) for 1 h with a flow rate at 15.0 ml / min at the wavelength of 214 nm . as a representative example , the synthesis of dodecanoyl-[r5 ] is described here . h - arg(pbf)-2-chlorotrityl resin ( 660 mg , 0.35 mmol , 0.53 mmol / g ) was swelled in dmf for 40 min by n2 . fmoc - arg(pbf)-oh ( 681 mg , 1.05 mmol , 3 equiv ) was coupled to the n - terminal of the resin , using hbtu ( 398 mg , 1.05 mmol , 3 equiv ) , hobt ( 142 mg , 1.05 mmol , 3 equiv ) , and dipea ( 366 l , 2.1 mmol , 6 equiv ) in dmf ( 15 ml ) by agitating the resin for 1 h using n2 . after the coupling , the resin was washed with dmf , followed by fmoc - deprotection with piperidine in dmf ( 20% ) . the subsequent three fmoc - arg(pbf)-oh couplings and one dde - lys(fmoc)-oh ( 559 mg , 1.05 mmol , 3 equiv ; dde = 1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl ) coupling was carried out in the same manner , respectively . after removing the fmoc group in side chain of lysine , dodecanoic acid ( 210 mg , 1.05 mmol , 3 equiv ) was coupled using hbtu , hobt , and dipea . then dde protection group at n - terminal of peptide was removed by 2% hydrazine in dmf , followed by washing with dmf and dcm . the side chain protected linear peptides were cleaved from the resin by using a cleavage cocktail , tfe / acetic acid / dcm ( 2:1:7 , v / v / v ) , for 1 h. the filtrate was evaporated , and the residue was dried overnight in a vacuum . the cyclization was conducted under a dilute condition with anhydrous dmf / dcm ( 5:3 , v / v , 250 ml ) , using hoat ( 190 mg , 1.4 mmol , 4 equiv ) and dic ( 240 l , 1.54 mmol , 4.4 equiv ) , and stirred for 12 h under nitrogen atmosphere . after cyclization , the solvent was evaporated , and the side chain deprotection was carried out by the addition of reagent r for 2 h. the crude dodecanoyl-[r5 ] was precipitated and washed with cold diethyl ether and purified by preparative rp - hplc system as described above . fluorescein - labeled peptides were synthesized with the same protocol before the attachment of fatty acid . we used fmoc-12-aminododecanoic acid instead of dodecanoic acid , and after removing fmoc group , 5(6)-carboxyfluorescein diisobutyrate ( cfdi ) was attached using 7-azabenzotriazol-1-yl - oxytris(pyrrolidino)phosphonium hexafluorophosphate ( pyaop ) , hoat , and dipea . as an example , we started fluorescein - labeled peptide synthesis in smaller scale with h - arg(pbf)-2-chlorotrityl resin ( 208 mg , 0.11 mmol , 0.53 mmol / g ) . fmoc - arg(pbf)-oh ( 214 mg , 0.33 mmol , 3 equiv ) , dde - lys(fmoc)-oh ( 176 mg , 0.33 mmol , 3 equiv ) , and fmoc-12-aminododecanoic acid ( 144 mg , 0.33 mmol , 3 equiv ) were used to couple each building block to the resin using hbtu ( 125 mg , 0.33 mmol , 3 equiv ) , hobt ( 45 mg , 0.33 mmol , 3 equiv ) , and dipea ( 115 l , 0.66 mmol , 6 equiv ) in dmf . cfdi ( 170 mg , 0.33 mmol , 3 equiv ) was coupled with amino group of 12-aminododecaonic chain using pyaop ( 172 mg , 0.33 mmol , 3 equiv ) , hoat ( 45 mg , 0.33 mmol , 3 equiv ) , and dipea . after removal of dde protecting group with 2% hydrazine in dmf and washing with dmf and dcm , side chain protected fluorescein linear peptides were cleaved from resin using tfe / acetic acid / dcm ( 2:1:7 , v / v / v ) . the molecular weights of final products were confirmed by an axima performance matrix - assisted laser desorption / ionization - time - of - flight ( maldi - tof ) mass spectrometer ( shimadzu corporation ) . dodecanoyl-(r6 ) : maldi - tof ( m / z ) c48h96n22o8 calcd . 1108.7781 ; found 1109.7308 [ m + h ] . f-dodecanoyl-(r5 ) : maldi - tof ( m / z ) c69h107n23o14 calcd . 1481.8368 ; found 1482.7586 [ m + h ] . w - dodecanoyl-[r5 ] : maldi - tof ( m / z ) c61h107n25o9 calcd . human ovarian adenocarcinoma ( sk - ov-3 ) , leukemia ( ccrf - cem ) , and embryonic kidney ( hek 293 t ) cells were purchased from american type culture collection . the sk - ov-3 and hek 293 t cells were grown in eagle s minimum essential medium ( emem ) , and rpmi-1640 medium ( atcc , manassas , va ) was used for ccrf - cem cells in a humidified atmosphere of 5% co2 at 37 c . both media were supplemented with fetal bovine serum ( fbs , 10% ) and penicillin streptomycin solution ( 1% , 10,000 units of penicillin and 10 mg of streptomycin in 0.9% nacl ) . cytotoxicity of peptides was examined by mts proliferation assay in two human cancer cell lines ( sk - ov-3 and ccrf - cem ) and one human normal cell line ( hek 293 t ) . cells were seeded into 96-well plates ( sk - ov-3 ( 5 10 cells / well ) , ccrf - cem ( 1 10 cells / well ) , and hek 293 t ( 1 10 cells / well ) ) . then , the cells were incubated with complete media ( 100 l ) overnight . different concentrations ( 0600 m ) of peptide solution ( 10 l ) were added to cells and incubated at 37 c with 5% co2 for 24 h. then , celltiter 96 aqueous solution ( 20 l ) was added to each well and incubated for 14 h. the absorbance was measured at 490 nm using microplate reader . sk - ov-3 cells were grown in 6-well plates ( 2 10 cells / well ) with complete emem media 24 h prior to the experiment . the fluorescein - labeled peptide stock solution ( 1 mm ) was prepared in water and diluted in gibco opti - mem i reduced serum medium to obtain the final concentration of 5 m . the media were removed , and the mixture containing fluorescein - labeled peptide solution ( 5 m ) was added . after 1 h incubation , trypsin edta solution was added to detach cells from the plate s surface and remove cell surface binding peptides . after 5 min , the complete media ( 2 ml ) were added to neutralize the trypsin . finally , the cells were analyzed by bd facscalibur or facsverse flow cytometer using fitc channel . 5(6)-carboxyfluorescein ( fam ) was used as a negative control . for examination of the cellular uptake mechanism of f-dodecanoyl-[r5 ] at low temperature , the assay was carried out at 4 c to inhibit the energy - dependent cellular uptake pathways . the sk - ov-3 cells were preincubated at 4 c for 15 min and incubated with the fluorescein - labeled peptide for 1 h at 4 c . cells were collected and analyzed using flow cytometry with the previously described protocol above . the data collected at 37 c were used as the control . to perform the atp - depletion assay , cells were incubated with sodium azide ( 10 mm ) and 2-deoxy - d - glucose ( 50 mm ) for 1 h before adding the fluorescein - labeled peptide . during the incubation time ( 1 h ) , the fluorescein - labeled bicyclic peptide ( 5 m ) was prepared in the opti - mem i reduced serum medium in the presence of sodium azide ( 10 mm ) and 2-deoxy - d - glucose ( 50 mm ) . then , the cells were incubated with this solution for 1 h. the following sample preparation and flow cytometry analysis protocol was the same as described above . sk - ov-3 cells were seeded with complete emem on coverslips in 6-well plate ( 1 10 cells / well ) and kept until 50% confluency . the media were removed , and cells were incubated with f-dodecanoyl-[r5 ] ( 10 m ) and f-dodecanoyl-(r5 ) ( 10 m ) in gibco opti - mem i reduced serum medium ( life technologies , grand island , ny ) for 1 h at 37 c . then cells were washed with 1 phosphate buffered saline with calcium and magnesium ( pbs ) for three times . the coverslips were mounted on microscope slides , and images were obtained using carl zeiss lsm 700 system with a 488 nm argon ion laser excitation and a bp 505530 nm band - pass filter . sk - ov-3 and ccrf - cem cells were seeded in 6-well plates ( 2 10 cells / well for sk - ov-3 and 1 10cells / well for ccrf - cem ) and grown with complete emem media ( rpmi-1640 for ccrf - cem ) overnight . a mixture of fluorescein - labeled phosphopeptides f-gpyeei ( 5 m ) and peptides ( 10 m ) were prepared in opti - mem i reduced serum medium at room temperature and incubated for 15 min . then the cells were incubated with the premixed solution at 37 c with 5% co2 for 1 h. the sample preparation for facs analysis was carried out by previously mentioned protocol described before . in this assay , the acylated cyclic polyarginine peptides were synthesized by fmoc / tbu solid - phase peptide synthesis method . fmoc - l - arg(pbf)-oh was coupled on h - arg(pbf)-loaded 2-chlorotrityl resin in the presence of hbtu , hobt , and dipea in dmf . then dde - lys(fmoc)-oh was attached , and a fatty acid was coupled to the side chain of lysine . dde protecting group was removed by 2% hydrazine in dmf , and a cleavage cocktail containing tfe / acetic acid / dcm ( 2:1:7 v / v / v ) was used for 1 h to cleave the side - chain protected linear peptides from the resin . cyclization of linear peptides was carried out in the presence of a mixture of hoat and dic in anhydrous dmf / dcm for 624 h. the side - chain deprotection of cyclic peptide was carried out by a cleavage cocktail reagent r for 2 h. the crude peptides were precipitated and purified with rp - hplc as described above . as a representative example , the synthesis of dodecanoyl-[r5 ] is shown in scheme 1 . a corresponding acylated linear polyarginine peptide ( alpp ) was synthesized for comparative studies with the cyclic peptide ( acpp ) . moreover , a cyclic polyarginine without fatty acid [ r5 ] was also synthesized to investigate the effect of the fatty acid on cyclic peptide and its effect on molecular transporting efficiency . to investigate whether the peptide alone can enter into cells , fluorescein - labeled f-dodecanoyl-[r5 ] and f-dodecanoyl-(r5 ) , where f = fluorescein , were synthesized for facs and microscopy investigations . chemical structures of synthetic peptides used in this study ( f , fluorescein - labeled ; [ ] , cyclic peptide ; ( ) , linear peptide ) . the cytotoxicity of all peptides were tested in two different cancer cell lines , adherent ( sk - ov-3 ) and nonadherent ( ccrf - cem ) cells , and a normal cell line ( hek 293 t ) using mts assay ( figure 2 ) . cyclic polyarginine [ r5 ] without fatty acid was used to explore the effect of n - terminal acylation on cytotoxicity and cellular uptake . alpp ( dodecanoyl-(r5 ) ) and [ r5 ] peptides showed consistently less cytotoxicity in all three cells compared to acpps ( dodecanoyl-[r5 ] and dodecanoyl-[r6 ] ) . after 24 h incubation , acpps showed approximately 20% toxicity in cells at a concentration of 25 m in ccrf - cem cells . however , dodecanoyl - linear ( r5 ) and [ r5 ] did not exhibit significant cytotoxicity at 25 m and showed less than 20% toxicity at the concentration of 100 m . in sk - ov-3 cells , dodecanoyl-(r5 ) and [ r5 ] showed more than 80% cell viability at the concentration of 25 m . in normal cells ( hek 293 t ) , all peptides exhibited less than 5% toxicity at 25 m . this differential behavior of the peptides in normal , and cancer cells can be possibly rationalized through the interaction between polyarginine peptides and cell membranes . the membrane of cancer cells holds more negative charges compared to that in normal cells because of the presence of anionic lipids such as phosphatidylserine . therefore , polyarginine peptides can be interacted with cancer cells more effectively compared to normal cells . these data indicated that acpps are more toxic than alpp and a nonacylated cyclic peptide [ r5 ] , especially at concentration of 25 m . thus , a noncytotoxic concentration of 510 m was used in cell - based assays . comparison of cytotoxicity between cyclic and linear acylated polyarginine peptides and nonacylated cyclic peptide [ r5 ] at various concentrations against ccrf - cem , sk - ov-3 , and hek 293 t after 24 h. the intracellular uptake studies of fluorescein - labeled acylated cyclic and linear pp , f-dodecanoyl-[r5 ] and f-dodecanoyl-(r5 ) , was carried out in sk - ov-3 cells by using flow cytometry and confocal laser scanning microscopy ( clsm ) methods . fluorescein ( fam , f ) alone was selected as a negative control . as it is shown in figure 3 , the f-dodecanoyl-[r5 ] and f-dodecanoyl-(r5 ) showed approximately 13.7- and 10.3-fold higher cellular uptake than that of control 5,6-carboxyfluorescein ( fam ) , respectively , in sk - ov-3 cells . the cellular uptake of f-dodecanoyl-[r5 ] was confirmed by clsm images ( figure 4 ) . f-dodecanoyl-[r5 ] showed higher fluorescence intensity compared to that of f-dodecanoyl-(r5 ) in sk - ov-3 cells . therefore , acpp f-dodecanoyl-[r5 ] was found to be a more efficient cell - penetrating peptide compared to its linear counterpart . as shown in figure 4 , the fluorescence signal is extended through the whole cells , suggesting that f-dodecanoyl-[r5 ] can get localized in the nucleus as well as cytoplasm . comparative cellular uptake of f-dodecanoyl-[r5 ] and f-dodecanoyl-(r5 ) ( 5 m ) in sk - ov-3 cells ( 1 h ) . confocal laser scanning microscope image of ( a ) f-dodecanoyl-[r5 ] and ( b ) f-dodecanoyl-(r5 ) . the peptides were incubated for 1 h in sk - ov-3 cells at 10 m concentration . the mechanism of the cellular internalization of f-dodecanoyl-[r5 ] was investigated by a temperature control assay at 4 c along with atp depletion assay . these two assays have been widely used to examine the energy - dependent endocytosis . facs results showed that the intracellular uptake of f-dodecanoyl-[r5 ] was significantly reduced at 4 c , indicating that the mechanism of internalization was mainly dependent on the endocytosis pathways ( figure 5 ) . furthermore , atp depletion assay was performed to investigate receptor - mediated endocytosis . to induce atp depletion , sk - ov-3 cells were preincubated with sodium azide ( 10 mm ) and 2-deoxy - d - glucose ( 50 mm ) for 1 h prior to the experiment , and a similar concentration was maintained during the incubation ( 1 h ) with f-dodecanoyl-[r5 ] . the results showed that the cellular uptake of f-acpp was inhibited in the presence of sodium azide and 2-deoxy - d - glucose , suggesting that receptor - mediated endocytosis is involved for the cellular uptake of f-acpp . in atp depletion assay , the basic cellular uptake of fam was higher compared to that in temperature control assay . however , this is evident that the intracellular uptake of f-dodecanoyl-[r5 ] was inhibited , and there was no significant difference between the cells treated with fam compared to those treated with f-dodecanoyl-[r5 ] in temperature control and atp depletion assays , suggesting that endocytosis is the major pathway for the cellular uptake of f-dodecanoyl-[r5 ] . cellular uptake of f-dodecanoyl-[r5 ] ( 5 m ) in sk - ov-3 cells in temperature control assay at 37 and 4 c , and atp depletion assay with nan3 ( 10 mm ) and 2-deoxy - d - glucose ( 50 mm ) analyzed by flow cytometry . cells with no treatment were used as control . the ability of acpps as a molecular transporter was evaluated and compared by selecting a fluorescein - labeled phosphopeptide , f-gpyeei , as a molecular cargo . phosphopeptide , pyeei ( ptyr - glu - glu - ile ) is an optimal peptide template for the sh2 domain of src tyrosine kinase . several analogues of this peptide have been synthesized as potent ligands for this target . because of the presence of the negatively charged amino acid residues in the structure of the peptide including phosphorylated tyrosine moreover , the internalization of the negatively charged phosphopeptide in cancer cells by diffusion is more difficult because cancer cell membranes are composed of more negatively charged lipids . thus , cellular delivery of cell - impermeable negatively charged phosphopeptides is significantly challenging . we have previously reported different peptide - based carriers for the intracellular delivery of negatively charged phosphopeptides as model cell - impermeable drugs in several cell lines . in this study , the intracellular uptake of f-gpyeei was monitored in the presence and absence of synthetic peptides after 1 h incubation by flow cytometry . as it is exhibited in figure 6 , the acpps ( dodecanoyl-[r5 ] and dodecanoyl-[r6 ] ) delivered the phosphopeptide more efficiently compared to alpps , dodecanoyl-(r5 ) and -[r5 ] . the intracellular uptake of f-gpyeei in the presence of dodecanoyl-[r5 ] and dodecanoyl-[r6 ] was enhanced by 3.4- and 5.5-fold higher than the uptake in the absence of acpps . however , dodecanoyl-(r5 ) and -[r5 ] only improved 1.3- and 1.4-fold intracellular uptake , respectively . the results showed that acylated and cyclized polyarginine peptides can deliver the phosphopeptide effectively . however , the intracellular uptake of the phosphopeptide did not improve significantly in the presence of either acylated linear polyarginine peptide or cyclic [ r5 ] . these data suggest that a combination of acylation and cyclization would improve the molecular transporting efficiency of the polyarginine - based peptide ( containing less than six arginines ) for the intracellular delivery of a cell - impermeable phosphopeptide . this has been previously reported that the acylated linear octa - arginine increased the cellular uptake of molecular cargoes by just adding fatty acid to the n - terminal of octa - arginine . however , we discovered that both cyclization and acylation in a short penta - arginine can significantly improve the delivery of a cell - impermeable phosphopeptide in sk - ov-3 cells . cellular uptake of f-gpyeei ( 5 m ) in the presence of dodecanoyl-[r5 ] and -[r5 ] , dosecanoyl-(r5 ) , and dodecanoyl-[r6 ] ( 10 m ) in sk - ov-3 cell line . phosphopeptide delivery efficiency of dodecanoyl-[r5 ] were compared with known cpps ( r7 , crrrrrrr ; tat , ygrkkrrqrrr ) . the major driving forces for the intracellular delivery are presumed to be structural rigidity through cyclization of the peptide and the interaction of the fatty acid with the cell membrane . it has been previously reported that the cellular uptake of the peptide can be increased due to the structural rigidity by cyclization of arginine - rich peptides . they proposed that the maximal distance between guanidine groups of arginine residue can lead to an efficient transduction of arginine - rich peptides . our investigations showed that dodecanoyl-[r6 ] is able to deliver more efficiently by 1.6-fold higher f-gpyeei uptake compared to that of dodecanoyl-[r5 ] . increasing the number of positively charged arginine residues can enhance the cellular uptake through ionic interactions with the negatively charged phosphopeptide and/or phospholipid in the cell membrane through ionic interactions . however , the higher number of arginine residue is not the only responsible element for the efficient cellular internalization . for example , it has been reported that polyarginine containing 11 amino acids ( r11 ) showed higher cellular uptake compared to the polyarginine containing 13 amino acids ( r13 ) . at the same time , r11 was found to be a more potent transporter compared to r9 in prostate cancer cells . these investigations showed that an optimal number of arginine residues are required for the highest degree of functionality . however , the greater number of amino acid residues in cyclic peptides can decrease the structural rigidity , which lowers the ability of the peptide to get into cells . dodecanoyl-[r5 ] was also compared with several commonly cpps , such as cr7 and tat ( ygrkkrrqrrr ) peptides . the acpp improved the cellular uptake of the phosphopeptide by 1.4- and 1.8-fold higher than those of cr7 and tat , respectively ( figure 6 ) . these results revealed that although other peptides containing arginine can deliver the molecular cargo , acpp dodecanoyl-[r5 ] with a shorter peptide sequence than cr7 and tat can work as a molecular transporter with higher efficiency . chemical structures of acpps with different length of fatty acid chains ( c8 , c12 , and c16 ) . to investigate the effect of the chain length on the cell penetration potency , we synthesized octanoyl-[r5 ] , dodecanoyl-[r5 ] , and hexadecanoyl-[r5 ] ( figure 7 ) . acpps showed less than 20% toxicity in cells at the concentration of 25 m ( figure 8a ) . the in vitro toxicity results showed that increasing the fatty acid chain length caused enhanced toxicity in cells as hexadecanoyl-[r5 ] was more cytotoxic than dodecanoyl-[r5 ] and octanoyl-[r5 ] . these data indicate that the fatty acid chain length could alter the interaction with the cell membrane and disturb the membrane integrity . on the basis of the cytotoxicity data , ( a ) cytotoxicity assay of cyclic polyarginine peptide - fatty acid conjugates against sk - ov-3 cells ( 24 h incubation ) . ( b ) cellular uptake of a phosphopeptide , f-gpyeei ( 5 m ) , in the presence of peptide - fatty acid conjugates ( 10 m ) in sk - ov-3 cells . it has been previously reported that there was a relationship between the length of fatty acid in polyarginines and their cellular uptake , meaning that the optimal length of fatty acid is required for optimal functionality based on the peptide sequence and cell type . the results exhibited that the cellular uptake of f-gpyeei was improved in the order of octanoyl-[r5 ] < dodecanoyl-[r5 ] < hexadecanoyl-[r5 ] ( figure 8b ) . the cellular uptake of f-gpyeei in the presence of hexadecanoyl-[r5 ] was 9.3- and 6.0-fold higher than that in the presence of octanoyl-[r5 ] and dodecanoyl-[r5 ] , respectively . these data suggest that the length of the attached fatty acid chain in the structure significantly influences the efficiency of the peptide as a molecular transporter . sixteen - carbon chain length ( c16 ) was found to be an optimized length for the intracellular delivery of f-gpyeei in sk - ov-3 cells . after acpps were found to act as cpp and molecular transporter , a systematic investigation was performed to modify the fatty acid to another hydrophobic moiety . w4-[r5 ] , the whole fatty acid chain was replaced with four tryptophan residues . the second conjugate w - dodecanoyl-[r5 ] had one more tryptophan at the end of the dodecanoyl fatty acid chain . w4-[r5 ] enhanced intracellular delivery of f-gpyeei by 4.1-fold higher compared to that of dodecanoyl-[r5 ] in sk - ov-3 . however , w - dodecanoyl-[r5 ] improved the uptake of f-gpyeei by 1.3-fold higher compared to that of w4-[r5 ] in ccrf - cem cells ( figure 9 ) . thus , it is not straightforward to compare the cellular uptakes in sk - ov-3 and ccrf - cem . however , the results could be assessed indirectly by relative comparison with the cellular uptake by w4-[r5 ] . these data suggest that the presence of hydrophobic tryptophan moieties could enhance the molecular transporting efficiency . in other words , appropriate modification of hydrophobic moiety in cpps can increase the drug delivery ability of acpp . thus , cyclic nature , short length of polyarginine , and hydrophobic segments were found to be critical elements to generate a peptide with an optimized molecular transporter efficiency . cellular uptake assay of a phosphopeptide , f-gpyeei ( 5 m ) , in the presence of w4-[r5 ] and w - dodecanoyl-[r5 ] ( 10 m ) against sk - ov-3 and ccrf - cem cell lines ( 1 h incubation ) . cells with no treatment were used as control . to evaluate whether the presence of peptides can enhance the pharmacological effect of a molecular cargo , the antiproliferative activity of doxorubicin ( dox ) was examined in the presence of dodecanoy-[r6 ] ( [ r6]-c12 ) in mcf-7 cells in a time - dependent assay . as it is shown in figure 10 , the antiproliferative potency of dox ( 5 m ) was enhanced by 7% , 11% , and 14% in the presence of [ r6]-c12 ( 5 m ) when compared with that of the drug alone after 24 , 48 , and 72 h incubation , respectively . these results showed that the cell proliferation of mcf-7 cells was inhibited in a time - dependent manner suggesting a sustained drug release by peptide leading to a higher inhibitory effect . the presence of the peptide possibly improved the cellular uptake of dox and enhanced the antiproliferative activity . time - dependent antiproliferative activity of dox ( 5 m ) in the presence and absence of [ r6]-c12 ( 5 m ) . in conclusion , acylated cyclic polyarginine peptides were synthesized and examined as cpps and potential molecular transporters . acpps showed higher potency as molecular transporter compared to the corresponding linear counterpart and cyclic polyarginine without fatty acid . the mechanism of the peptide internalization was found to be energy - dependent endocytosis . cyclization and acylation reactions on the structure of the peptide enhanced the intracellular uptake of polyarginine peptides although they carry a short length of sequence . this intracellular delivery property of acpps can be optimized by modifying the length of fatty acid chain . to the best of our knowledge , this is the first report of cyclic fatty acylated polyarginine peptide as molecular transporter of a cell - impermeable phosphopeptide . this study provided insights about how a combination of the cyclic nature and acylation can improve the cell internalization of polyarginines . further investigations are undergoing to determine whether conjugation of cell - impermeable hydrophobic drugs to acylated cyclic polyarginines can be an efficient method for designing novel drug delivery systems .

many of the reported arginine - rich cell - penetrating peptides ( cpps ) for the enhanced delivery of drugs are linear peptides composed of more than seven arginine residues to retain the cell penetration properties . herein , we synthesized a class of nine polyarginine peptides containing 5 and 6 arginines , namely , r5 and r6 . we further explored the effect of acylation with long chain fatty acids ( i.e. , octanoic acid , dodecanoic acid , and hexadecanoic acid ) and cyclization on the cell penetrating properties of the peptides . the fluorescence - labeled acylated cyclic peptide dodecanoyl-[r5 ] and linear peptide dodecanoyl-(r5 ) showed approximately 13.7- and 10.2-fold higher cellular uptake than that of control 5,6-carboxyfluorescein , respectively . the mechanism of the peptide internalization into cells was found to be energy - dependent endocytosis . dodecanoyl-[r5 ] and dodecanoyl-[r6 ] enhanced the intracellular uptake of a fluorescence - labeled cell - impermeable negatively charged phosphopeptide ( f-gpyeei ) in human ovarian cancer cells ( sk - ov-3 ) by 3.4-fold and 5.5-fold , respectively , as shown by flow cytometry . the cellular uptake of f-gpyeei in the presence of hexadecanoyl-[r5 ] was 9.3- and 6.0-fold higher than that in the presence of octanoyl-[r5 ] and dodecanoyl-[r5 ] , respectively . dodecanoyl-[r5 ] enhanced the cellular uptake of the phosphopeptide by 1.42.5-fold higher than the corresponding linear peptide dodecanoyl-(r5 ) and those of representative cpps , such as hepta - arginine ( cr7 ) and tat peptide . these results showed that a combination of acylation by long chain fatty acids and cyclization on short arginine - containing peptides can improve their cell - penetrating property , possibly through efficient interaction of rigid positively charged r and hydrophobic dodecanoyl moiety with the corresponding residues in the cell membrane phospholipids .

as of 2014 , the worldwide prevalence of type 2 diabetes mellitus ( t2 dm ) was estimated to be 9% among adults aged 18 years with great impact on mortality , particularly in low- and middle - income countries ( lmic ) [ 1 , 2 ] . moreover , globally , approximately 25% to 75% of diabetes cases remain undiagnosed [ 3 , 4 ] , until further complications , especially at the macro- and micro - vascular level , manifest clinically . in latin america , an important strategy to prevent or delay t2 dm complications is the early identification of those with undiagnosed diabetes ; yet , universal screening for diabetes at the population level is not practical in resource - limited settings . the american diabetes association recommends the use of glucose test as t2 dm screening in people with overweight and obesity as well as in those with other risk factors . as a result , risk assessment scores have been developed to address this problem in a simple and inexpensive way . most of the available algorithms for diabetes screening have been developed in caucasian [ 79 ] and asian populations [ 1013 ] and very few in other ethnic groups [ 14 , 15 ] . to date , one diabetes risk score has been developed and validated in latin america so far which was derived from one urban area in brazil , thus bearing limited generalizability to the wider region . furthermore , it is well established that before adopting existing risk scores as screening tools in different populations and ethnic groups , their performance needs to be evaluated , calibrated , or validated in local settings . as the american diabetes association , the peruvian ministry of health recommends diabetes screening in general population with fasting glucose in adults aged 40 to 70 years with risk factors . however , fasting glucose is not always available in primary care settings , especially in semiurban and rural areas . as a result , a major challenge to be overcome in many countries is the implementation of a simple , fast , and laboratory - free based screening method . consequently , we aimed to develop a simple laboratory - free risk score to identify people with undiagnosed diabetes and incident diabetes in peru , a latin american country that spans coastal , andean , and rainforest settings . in order to do so , this work benefited from two large - scale population - based surveys : the first one , representative at the national level , was used to develop the score , and the second one , a cohort study , was utilized for external validation . the national survey of nutritional and biochemical indicators for noncommunicable diseases ( eninbsc in spanish ) , conducted by the peruvian national institute of health , was used to develop our predictive model . this was complemented with the cronicas cohort study , whose baseline and longitudinal information was used to validate the risk score . the eninbsc is a national population - based survey carried out in peru between august 2004 and april 2005 , designed to estimate the prevalence of hypertension , type 2 diabetes mellitus , and other risk factors for noncommunicable diseases at the national and regional level . potential participants were those aged 20 years , habitual residents in the study area , and able to provide consent for their participation in the study . pregnant women and those currently breastfeeding were excluded from the study . as per design , the eninbsc sample was stratified according to peru 's five major regions of the country : lima , rest of the coast , urban highlands , rural highlands , and jungle . in each stratum , cluster of blocks the cronicas cohort study is an ongoing cardiopulmonary project aimed to estimate the prevalence and incidence of hypertension , diabetes mellitus , and obesity in four different settings in peru that differ in terms of their urbanicity and altitude : pampas de san juan de miraflores , in the highly urbanized lima , puno in the altitude ( 3,825 meters above the sea level ) contributing with rural and urban areas , and tumbes , a semiurban area in the northern coast of peru . the study started in september 2010 and a follow - up visit was completed in march 2014 . a sex- and age - stratified sample was selected at random for each of the settings and all participants aged 35 years , full time residents in the study area , and able to consent , were enrolled . follow - up data used for this analysis was collected , on average , at 30 months after baseline . the first one lasted on average 40 minutes and was carried out to apply a face - to - face questionnaire regarding data about household characteristics , demographics , lifestyles behaviors , risk factors , and blood pressure measurements . the second visit lasted 30 minutes on average and was planned to have an appropriate period of fasting for blood sampling for glucose , total cholesterol , hdl - cholesterol , and the remaining anthropometric measures ( height , weight , and waist circumference ) using standard procedures . similarly , the procedures of the cronicas study has been published elsewhere . in brief , participants responded to a face - to - face questionnaire applied by trained community health workers . data collected comprised risk factors for cardiovascular disease based on a modified version of the who step approach questionnaire for surveillance of noncommunicable disease . a period of 8 to 12 hours of fasting was required for blood sampling to collect fasting glucose , total cholesterol , and hdl - cholesterol . height , weight , and waist circumference were also assessed , and blood pressure was measured in triplicate after five minutes of resting using an automatic monitor ( omron hem-780 ) previously validated in adult 's population . in both studies , diabetes was defined as any of the following conditions : fasting glucose 7.0 mmol / l ( 126 mg / dl ) and/or self - report of physician diagnosis . fasting glucose was assessed by an enzymatic colorimetric method ( glucose oxidase god - pap ) in both studies . after excluding individuals without known diabetes , undiagnosed diabetes variables included in the analyses were built to guarantee similarities between both studies : sex ; age ( < 55 and 55 years ) ; education ( in years ) ; self - reported smoking ( current versus never / former smoker ) ; alcohol use ( user versus never user ) ; self - reported diabetes in first - degree relatives ( participant 's parents and/or siblings ) ; and levels of physical activity ( low versus moderate / high levels , based on the transport - related domain of the ipaq ) . anthropometric measurements included in the analysis were body mass index ( ( bmi ) , < 25 , 2529.9 , and 30 kg / m ) , waist circumference ( < 90 , 9099.9 , and 100 cm ) , waist - to - height ratio ( < 0.50 , 0.500.59 , 0.600.69 , and 0.70 ) , and hypertension ( measured or previously diagnosed ) . a total of 4,206 participants were enrolled in the eninbsc , but only 2,472 were included in this analysis . reasons for exclusion were 1,524 because of age < 35 years to make both databases comparable , 129 because of no data about fasting plasma glucose levels being available , and 81 because of known diagnosis of diabetes . in the cronicas study , 3,601 participants were enrolled at baseline but only 2,948 records were analyzed as 465 had no data about glucose levels , and 188 were excluded because of previous diagnosis of diabetes . in addition , only data from 2,577 participants was used in the longitudinal assessment of the risk score ( comparison of baseline characteristics among those included and excluded from longitudinal analysis is shown in online supplement : e - table 1 ; see supplementary material available online at http://dx.doi.org/10.1155/2016/8790235 ) . initially , population characteristics of both studies were tabulated using proportions in the case of categorical variables and means and standard deviation ( sd ) with numerical variables . then , the prevalence and 95% confidence intervals ( 95% ci ) of total diabetes and undiagnosed diabetes were estimated in each study . the risk score was derived from data of the eninbsc survey taking into account the multistage sampling strategy of the study . each potential risk factor ( i.e. , sex , age , family history of diabetes , etc . ) was assessed in bivariate models using logistic regression and undiagnosed diabetes as the dependent variable . then , risk factors with a p value < 0.10 in the bivariate analysis were included in a multiple logistic regression model using stepwise backward elimination with a significance level of 5% . the hosmer - lemeshow goodness - of - fit test was used to assess how well the predicted prevalence matched the observed prevalence of undiagnosed diabetes ( i.e. , p values over 0.20 indicate that model fits well ) . as we sought for an easily applicable and implementable algorithm , the risk factors in the final model were each assigned a weighted score by rounding up all regression coefficients in the final model to the nearest integer as in a previous report . for the evaluation of the risk score , the area under the receiver operating characteristic ( roc ) curve , sensitivity , specificity , and positive and negative predictive values ( ppv and npv ) were calculated . the optimal cut - point was determined using the youden index , a single statistic that captures the performance of a diagnostic test ( i.e. , sensitivity + specificity 1 ) . as one of the main aims of a nonlaboratory risk score is to identify people who warrant having a blood test ( i.e. , fasting glucose , glycated haemoglobin , etc . ) , the cut - point with the highest sensitivity was also estimated and described . we assessed the performance of our score using bootstrap techniques as well as carrying out an external validation using the cronicas cohort study . the resulting 1,000 prediction models were then assessed to estimate the bootstrap auc using the bias - corrected version of the confidence intervals . in addition , using baseline data from the cronicas cohort study , validation measures ( auc , sensitivity , specificity , predictive values , and likelihood ratios ) were estimated . to evaluate the performance of our algorithm , the peruvian risk score was compared to previously published models for undiagnosed diabetes including the brazilian risk score , the qingdao score , the indian risk score , the kuwaiti risk score , the patient self - assessment score , and the rotterdam risk score using the c - statistic . finally , using the follow - up data of the cronicas cohort study , the risk score was also evaluated to detect incident cases of t2 dm by excluding those with diabetes diagnosis at baseline . analyses were performed using stata 13.0 ( statacorp , college station , tx , usa ) . the protocol and informed consent forms of the eninbsc study were reviewed and approved by the instituto nacional de salud and the centro nacional de alimentacin y nutricin , both part of the ministry of health in lima , peru . in the case of the cronicas cohort study , protocol and consent forms were reviewed and approved by the institutional review boards of the universidad peruana cayetano heredia and the ngo asociacin benfica prisma in lima , peru , and the johns hopkins university in baltimore , usa . overall , participants from the cronicas study were 5 years older , reported consuming lower levels of alcohol , and were less physically active than those from the eninbsc survey . the overall prevalence of diabetes was 5.1% ( 129/2538 ; 95% ci : 4.2%5.9% ) in the eninbsc survey and 8.7% ( 272/3135 ; 95% ci : 7.7%9.7% ) in the cronicas cohort study 's baseline . after excluding those with known diabetes , undiagnosed diabetes was present in 2.0% ( 48/2457 ; 95% ci : 1.4%2.5% ) in the eninbsc survey and in 2.9% ( 85/2948 ; 95% ci : 2.3%3.5% ) in the cronicas cohort study . after stepwise backward logistic regression , age , diabetes in first - degree relatives , and waist circumference were independently associated with undiagnosed diabetes ( table 2 ) . the hosmer - lemeshow test showed that the final model fitted relatively well ( p = 0.21 ) . the peruvian diabetes risk score was constructed based on the coefficients of that final regression model . the score gave an auc of 0.73 ( 95% ci : 0.650.78 ) , and the optimal cut - point for undiagnosed diabetes using the youden index was 2 ( figure 1 ) . with this cut - point , about 34.8% of participants were categorized as at high risk of diabetes : sensitivity 69.6% , specificity 65.8% , and ppv and npv of 3.9% and 99.1% , respectively . with a cut - point 1 , 69.8% of participants would be at high risk of diabetes with improved sensitivity ( 93.5% ) but lower specificity ( 30.6% ) . table 3 shows the performance of the risk score for detecting undiagnosed diabetes at different cut - points . when bootstrap was used , the performance of our risk score was similar to the obtained in the development model ( auc = 0.72 ; 95% ci : 0.650.78 ) . in addition , when the risk score was evaluated by applying the score to the cronicas cohort study 's population , the auc for undiagnosed diabetes was 0.68 ( 95% ci : 0.620.73 ) . at the suggested cut - point of 2 , 42% would be categorized as undiagnosed diabetes with sensitivity , specificity , ppv , and npv of 70.2% , 58.9% , 4.8% , and 98.5% , respectively ( table 4 ) . on the other hand , with a cut - point 1 , 80% would be categorized as undiagnosed diabetes with sensitivity , specificity , ppv , and npv of 94.0% , 20.0% , 3.3% , and 99.1% , respectively . when previous published algorithms for undiagnosed diabetes were applied to the cronicas cohort study , the performance of the rotterdam score ( p < 0.001 ) , indian score ( p < 0.001 ) , and qingdao score ( p < 0.01 ) was poorer than our score ; however , our algorithm performed similar to the other assessed models , such as the brazilian risk score ( p = 0.93 ) , the kuwaiti score ( p = 0.26 ) , and the patient self - assessment score ( p = 0.74 ) , but having only three variables . the performance of this risk score was also assessed to predict incident cases of diabetes using the longitudinal data from the cronicas cohort study . one hundred twenty - one new cases of diabetes were found accounting for 6,207 person - years at risk , with an overall incidence of 1.95 ( 95% ci : 1.632.33 ) cases per 100 person - years of risk . the auc of the score was 0.66 ( 95% ci : 0.610.71 ) . with a cut - point 2 , 42.5% of participants were categorized as at high risk of developing diabetes : sensitivity , specificity , ppv , and npv were 69.4% , 58.9% , 7.8% , and 97.4% , whereas , for a cut - point 1 , the respective values were 79.9% , 91.9% , 20.7% , 5.5% , and 98.1% . using a national population - based survey , a simple nonblood based risk score based on age , history of diabetes in first - degree relatives , and waist circumference was built and shown to perform moderately in detecting undiagnosed diabetes when externally validated . moreover , the performance of the score was almost similar for detecting incident cases of diabetes in the peruvian population . a relatively recent systematic literature search found 23 different blood - free prevalent diabetes risk scores : ten from europe , nine for asian populations , two from the united states , and two from middle east . in addition , and not included in the aforementioned review , only one risk score was developed in latin america using brazilian urban population . the same systematic review reported that auc for these predictive models was greater in the development studies ( range : 0.65 to 0.88 ) than in the validation studies ( range : 0.63 to 0.80 ) , similar to our findings . another systematic review found that several noninvasive algorithms were created using variables such as age , gender , waist circumference and/or bmi , and family history of diabetes in the final model . as impracticality due to use of the algorithms was a common barrier to the uptake of risk scores by healthcare staff and individuals , our model , created with three of these more common variables , reached a moderate - to - high sensitivity depending on the used cut - point . moreover , two of these variables are easily evaluable during medical appointment or through individual 's self - assessment , and only a measuring tape and no calculations are required to be implemented in clinical practice or at the population level . from a cross - sectional point of view , with a cut - point 2 , from 1000 participants assessed by the peruvian diabetes risk score , a total of 420 would be classified as undiagnosed diabetes with the detection of 20 cases and only 6 will be missing . on the other hand , with a cut - point 1 , from 1000 screened individuals , a total of 804 would be categorized as having undiagnosed diabetes with the detection of 27 cases and only 7 will be missing . thus , the reduction of the cut - point of the risk score would increase sensitivity but reducing the specificity and imposing the need of performing a confirmatory test ( i.e. , fasting glucose ) to almost the double of individuals , with the benefit of having only 7 more people diagnosed . longitudinally , the same risk score would detect an important number of participants at risk of developing diabetes : 43% of screened individuals would be classified at high risk of diabetes , and of them , 8% would develop diabetes in the next 2.5 years . according to a previous study , 17 reports described a noninvasive model to predict the development of diabetes and included a median of six risk predictors , ranging from 2 to 11 . although our score did not perform as good as other well - known longitudinal models in the literature such as the findrisc or the aric scores [ 35 , 36 ] , it only included three variables and was built using cross - sectional information . in addition , some variables used in the aforementioned studies are difficult to standardize within a country as peru , that is , food portions , physical activity , or sedentarism , limiting therefore its use on a wider scale and in a simple pragmatic fashion . our algorithm performed better than the rotterdam , the indian , and the qingdao risk scores in our population , which highlights the need of calibration and/or development of a specific score for different ethnic groups before its adoption . as there are ethnic differences in risk factors for diabetes and peru is considered a multiethnic country , it is necessary to create specific scores or recalibrate existing algorithms before applying in specific contexts . in addition , with only three variables included , the performance of our predictive model was similar to the other assessed scores included in the analyses . taken together , the score developed has the potential to augment , in a pragmatic manner , initial rapid screening for diabetes , especially at various nonspecialized primary healthcare services . our findings also demonstrate that approximately 35% of cases of t2 dm ( 39% in the eninbsc survey and 33% in the baseline of the cronicas cohort study ) are not aware of their disease . results are similar to those reported in previous studies in our context and in similar settings in latin america . as the developed risk score is simple , it does not require a blood test or laboratory services , and it might be easily implemented in clinical practice . moreover , because our score asks for general information in the form of age and diabetes in first - degree relatives and is complemented by a simple anthropometric measure of waist , there is potential for the score to be self - administered . according to our results , any patient aged 55 years and above and having at least one first - degree relative with t2 dm has greater probability of having undiagnosed diabetes but also is at risk of developing diabetes in the future . in addition , a greater central obesity , that is , 100 cm or more , independent of the other terms of the score is alone a good predictor of diabetes as reported in previous studies . our algorithm included waist circumference instead of body mass index as other risk scores , providing a better indicator of accumulation of visceral fat and metabolic dysfunction in our context . recently , the peruvian ministry of health has published the guide of clinical practice for diagnosis , treatment and control of diabetes mellitus in primary care and only recommends screening in general population with plasma glucose among adults between 40 and 70 years with obesity or overweight as suggested by the american diabetes association . as in other lmic , plasma glucose is not always available in primary care , especially in semiurban and rural areas ; therefore , a major challenge to be overcome in many countries is the implementation of a simple , fast , and laboratory - free based screening method . moreover , within the peruvian context , no risk score has been proposed as part of the aforementioned guide . thus , our algorithm might fill a gap to facilitate further specialized assessment of high risk individuals for diabetes , an approach that may be of utility to various other countries facing similar challenges . the strengths of this study include the use of a national population - based survey , including urban and rural areas across major geographical regions , to develop the peruvian diabetes risk score , as well as its validation using bootstrap but also an independent longitudinal cohort study . additionally , it is only based on three variables ensuring its simplicity to be used and implemented . however , the study has also some limitations . first , we have utilized fasting plasma glucose as the gold standard for diagnosing diabetes instead of an oral glucose tolerance test ( ogtt ) . although the ogtt is more sensitive and specific than the fasting plasma glucose , more cases would have been detected with the overload of glucose ; it is rarely performed as part of the routine clinical practice . second , the cronicas cohort study did not include information from the amazon rainforest as did the eninbsc survey . when a sensitivity analysis was performed excluding individuals from the jungle from eninbsc data , in addition , the score was created using a national survey to be applicable to the entire peruvian population . third , some variables were not assessed in our logistic regression model such as dietary intake or history of gestational diabetes as such data was not available . as a result , some caution should be made when our algorithm is compared to other risk scores . fourth , our model is based on the idea of risk stratification instead of individualisation ; for instance , variables were categorized instead of being preserved as numerical . nevertheless , the performance of our score did not change when age and waist circumference were treated as numerical variables ( data not shown ) . moreover , our idea was to develop a simple and easily applicable score instead of a complex algorithm for predicting undiagnosed and incident diabetes . finally , as other diabetes risk scores , the model warrants further scrutiny before it can be used in other populations . the peruvian diabetes risk score , built using age , self - reported diabetes in first - degree relatives , and waist circumference , proves to be a simple pragmatic screening tool for undiagnosed and incident cases of diabetes in peru . this experience in generating such simple , easy - to - use approaches for the identification of t2 dm can serve to inform other similar lmic efforts who are on early stages of diabetes prevention . this tool , due to its simplicity , can facilitate various initiatives oriented to introduce and scale up early preventative and management strategies on a wider scale .

objective . to develop and validate a risk score for detecting cases of undiagnosed diabetes in a resource - constrained country . methods . two population - based studies in peruvian population aged 35 years were used in the analysis : the eninbsc survey ( n = 2,472 ) and the cronicas cohort study ( n = 2,945 ) . fasting plasma glucose 7.0 mmol / l was used to diagnose diabetes in both studies . coefficients for risk score were derived from the eninbsc data and then the performance was validated using both baseline and follow - up data of the cronicas cohort study . results . the prevalence of undiagnosed diabetes was 2.0% in the eninbsc survey and 2.9% in the cronicas cohort study . predictors of undiagnosed diabetes were age , diabetes in first - degree relatives , and waist circumference . score values ranged from 0 to 4 , with an optimal cutoff 2 and had a moderate performance when applied in the cronicas baseline data ( auc = 0.68 ; 95% ci : 0.620.73 ; sensitivity 70% ; specificity 59% ) . when predicting incident cases , the auc was 0.66 ( 95% ci : 0.610.71 ) , with a sensitivity of 69% and specificity of 59% . conclusions . a simple nonblood based risk score based on age , diabetes in first - degree relatives , and waist circumference can be used as a simple screening tool for undiagnosed and incident cases of diabetes in peru .

cancer is one of the leading causes of death worldwide and accounted for 7.6 million cases ( 13% of all deaths ) in 2008 ; in 2013 in the united states there are 12,340 estimated new cases and 4,030 deaths by cervical cancer [ american cancer society : cancer facts and figures 2013 ] . meanwhile in romania , based on statistical data from 2012 , cervical cancer is ranked third place taking in account the women cancer incidence , after the breast and colorectal cancer . cervical cancer develops in a multi - stage process , successively passing from a cervix with a normal epithelium to cervical intraepithelial neoplasia and finally leading to invasive cervical cancer . a relevant epidemiologic risk circumstance for the progress of the cervical cancer is represented by the presence of high risk human papillomavirus infections . consequently , considerable importance has been given to the molecular premise which leads to the progression of cervical cancer . rnai ( rna interference ) is a natural mechanism able to specific inhibit a target gene . the prospective exploitation as a therapeutic has lately been strengthened by the small interfering rna ( sirna ) delivery to human tumours . rnai has been demonstrated as a relevant investigation device for particular target genes by using exogenous sirnas . sirnas are single - stranded non - coding rnas with 2023 nucleotides length , being able to negatively modulate gene expression profile at post - transcriptional level . sirnas represent an encouraging novel type of medicaments for cancer therapy , as a result of targeting the mutated tumour suppressor genes or activated oncogenes . many recent investigations have shown that sirna can efficiently inhibit oncogene expression in cancer cells . various investigations demonstrated clearly that the deactivation of p53 constitutes a crucial step in carcinogenesis . the present study was designed to investigate the potential use of p53 rnai to block p53 expression , as well as the subsequent effect on cell invasion and gene expression on the human cervical cancer cell . hela is a human cervical carcinoma cell line ; this was incubated in a 5% co2 incubator at 37c . for maintenance , cells were cultured in dmem medium , 10% fcs ( foetal bovine serum ) , 100 u / ml penicillin and 100 mg / ml streptomycin , 2 mm l - glutamine , 1% nonessential amino acids , all purchased from sigma - aldrich , bucharest , romania . for this investigations 510 hela cells / well were used for reverse - transfection , based on a preliminary optimised protocol for p53sirna , using siport neofx as transfection agent ( ambion , usa ) . a validated p53sirna pool ( sc-29435 , santa cruz ) was used at a concentration of 50 nm in serum starvation condition using optimem medium ( gibco , invitrogen , romania ) , in accordance with the producer recommendations . siport neofx 0.25% concentration was able to achieve the maximum inhibition of target gene with no effects on cell viability . we performed the invasion assays on hela cells to evaluate the impact of p53sirna treatment using the cim - plate 16 with the xcelligence rtca dp instrument ( roche applied science ) according to the described protocol by . the total rna for the cells treated with 50 nm p53sirna andthe control group respectively at 24 hours post treatment was extracted using trireagent ( sigma - aldrich , bucharest romania ) based on the producer recommended protocol for each triplicate specimen . all of the samples had a rin ( rna integrity number ) higher than 7.5 . the 8 genes selected were assessed using specific primer and upl based on in silico design . the total rna ( 500 ng ) from all the samples was reverse transcribed using the first strand cdna synthesis kit for rt - pcr ( roche , bucharest romania ) . for the gene amplification we used taqman universal pcr master mix , in a 20 l volume in a 96-well plate using the roche lightcycler 480 system . the qrt - pcr reaction amplification program was as follows : 10 minutes at 95c for enzyme activation followed by 45 cycles of 15 seconds at 95c and 1 minute at 60c for the amplification step . the data analysis was carried out to compare the gene expression values for the treated and untreated groups using ct method . as housekeeping gene the evaluation of the vegf protein expression at 48 hours post treatment was done using human vegf quantikine elisa kit ( r&d , catalog no . hela is a human cervical carcinoma cell line ; this was incubated in a 5% co2 incubator at 37c . for maintenance , cells were cultured in dmem medium , 10% fcs ( foetal bovine serum ) , 100 u / ml penicillin and 100 mg / ml streptomycin , 2 mm l - glutamine , 1% nonessential amino acids , all purchased from sigma - aldrich , bucharest , romania . for this investigations 510 hela cells / well were used for reverse - transfection , based on a preliminary optimised protocol for p53sirna , using siport neofx as transfection agent ( ambion , usa ) . a validated p53sirna pool ( sc-29435 , santa cruz ) was used at a concentration of 50 nm in serum starvation condition using optimem medium ( gibco , invitrogen , romania ) , in accordance with the producer recommendations . siport neofx 0.25% concentration was able to achieve the maximum inhibition of target gene with no effects on cell viability . we performed the invasion assays on hela cells to evaluate the impact of p53sirna treatment using the cim - plate 16 with the xcelligence rtca dp instrument ( roche applied science ) according to the described protocol by . the total rna for the cells treated with 50 nm p53sirna andthe control group respectively at 24 hours post treatment was extracted using trireagent ( sigma - aldrich , bucharest romania ) based on the producer recommended protocol for each triplicate specimen . the rna concentrations and quality were assessed by the nanodrop-1100 agilent 2100 bioanalyzer spectrophotometer . all of the samples had a rin ( rna integrity number ) higher than 7.5 . the 8 genes selected were assessed using specific primer and upl based on in silico design . the total rna ( 500 ng ) from all the samples was reverse transcribed using the first strand cdna synthesis kit for rt - pcr ( roche , bucharest romania ) . for the gene amplification we used taqman universal pcr master mix , in a 20 l volume in a 96-well plate using the roche lightcycler 480 system . the qrt - pcr reaction amplification program was as follows : 10 minutes at 95c for enzyme activation followed by 45 cycles of 15 seconds at 95c and 1 minute at 60c for the amplification step . the data analysis was carried out to compare the gene expression values for the treated and untreated groups using ct method . as housekeeping gene the evaluation of the vegf protein expression at 48 hours post treatment was done using human vegf quantikine elisa kit ( r&d , catalog no . xcelligence system is an innovative device that allows the scanning of cellular response via an impedance - based technology in real time , lacking any exogenous labels . the cim - plate 16 furnishes a kinetic cell - response profile to p53sirna throughout an investigation , specifying the commencement and ratio of invasion and migration of hela cells . this data can facilitate to comprehend the response to treatment in dynamic . in figure 1 we can observe a delay and a reduction of the cell migration after the p53sirna treatment . taqman qrt - pcr assay was used to examine the effect of p53sirna on a panel of 8 genes related to apoptosis and angiogenesis . relative gene expression quantification using ct method leads to the downregulation of the selected gene , presented in the figure 2 . after 48h post transfection with p53 sirna inhibition in hela cell line , vegf protein was found dowregulated in the culture medium than in the control group ( figure 3 ) . xcelligence system is an innovative device that allows the scanning of cellular response via an impedance - based technology in real time , lacking any exogenous labels . the cim - plate 16 furnishes a kinetic cell - response profile to p53sirna throughout an investigation , specifying the commencement and ratio of invasion and migration of hela cells . this data can facilitate to comprehend the response to treatment in dynamic . in figure 1 we can observe a delay and a reduction of the cell migration after the p53sirna treatment . taqman qrt - pcr assay was used to examine the effect of p53sirna on a panel of 8 genes related to apoptosis and angiogenesis . relative gene expression quantification using ct method leads to the downregulation of the selected gene , presented in the figure 2 . after 48h post transfection with p53 sirna inhibition in hela cell line , vegf protein was found dowregulated in the culture medium than in the control group ( figure 3 ) . although at this moment cervical cancer is considered as a preventable disorder there is a significant risk of disease recurrence causing a persuasive necessity to research new therapeutic targets for this disease management . it is now well recognized that the tumour progression of all cancers is characterized by intensified proliferation and invasion rate and diminished in apoptosis . at the same time the apoptosis and angiogenesis are interconnected as can be observed from figure 4 , using string.9 database . the idea of this study is in agreement with the previous studies which are based on the hypothesis that once mutated , p53 exercised oncogenic role . by using sirna the role of the present study is to emphasize the cooperation between oncogenic mechanisms , confirming the crosstalk between apoptotic and angiogenic mechanisms . this has a significant therapeutic relevance based on the fact that mutated p53 is related to cancer aggressiveness or to promoting metastasis . in a similar study was observed that , by using sirna targeting p53/p73 , tumoral cells were sensitized to chemotherapy . in a recent study , pinx1 was displayed as a novel target gene of p53 , proposing the suppression of p53/pinx1 pathway as a novel mechanism to enhance the telomerase activity in cervical cancer cells , and leading to reduction of cell proliferation . targeting oncogenic signals such as the mutated p53 gene that gains oncogenic role , we observed a downregulation of specific genes involved in apoptosis but also in angiogenesis , connected with a reduction of the invasion rate in the case of p53sirna therapy . after 24 hours of treatment with p53sirna we can observe downregulations of important genes involved in apoptosis and angiogenesis , as displayed in figure 2 . this study confirmed our hypothesis that p53 plays a central role in carcinogenesis as displayed in figure 3 , as important therapeutic target . p53 could promote the convergence of the apoptotic and angiogenic pathways in cervical cancer cells . it has been proved that p53 is able to indirectly downregulate vegf gene expression , via the retinoblastoma ( rb ) , in a p21-dependent mode , confirming its function in cell - cycle regulation as displayed by recent studies . the complete comprehension of the cross - talk between p53 and angiogenic pathways represents a challenge in cancer therapy [ 2426 ] . another study reveals that mutant p53 correlates with overexpression of vegf . in a similar study the inhibition of mutant p53 represents a significant therapeutic target , a fact emphasized by walerych as rebel angel . p53 gene knockdown in hela cells plays an important role not only in apoptosis but also in angiogenesis through various mechanisms that regulate tumour cell development and migration . the angiogenic phenotype related to p53 targeted by sirna in human cancers is frequently associated with resistance to conventional genotoxic anticancer agents . therefore , p53 inhibitions become dependent and rely on angiogenesis for growth and metastasis . the results in the current research may also have an important significance outside the context of cervical cancer , by using specific inhibitors for p53 for increasing therapeutic response in a wide range of tumoral pathology .

background and aimssirnas represent an encouraging novel alternative in cancer therapy as a result of targeting the mutated tumour suppressor genes or activated oncogenes . targeting oncogenic signals , as the mutated p53 gene that gains oncogenic role , we observed inhibition of migration , a downregulation of specific genes involved in apoptosis but also in angiogenesis , connected with a reduction in invasion rate in the case of p53sirna therapy.methodsthe study was designed to assess the role of p53 by using rnai ( rna interference ) in hela in vitro cell culture model . therefore cell migration rate was assessed by using xcelligence systems , gene expression for a panel of genes involved in apoptosis and angiogenesis , and validation of gene expression data at protein level.resultson the selected in vitro model p53 sirna therapy was correlated with the reduction of cell migration . the downregulation of p53 , pten , tnf , nfkb , bcl-2 , icam-2 , vegf , and fgfb was evidenced as response to p53 inhibition.conclusionrnai may be a valuable technology in order to restore the normal cellular phenotype . the results in the current research may also have an important significance outside the context of cervical cancer , by using specific inhibitors for p53 for increasing the therapeutic response in a wide range of tumoral pathology .

atherosclerosis is a coronary heart disease ( chd ) , and is the most common cause of death in the industrialized world . high blood pressure ( bp ) , hypercholesterolemia , diabetes mellitus ( dm ) and smoking are considered as the major risk factors for the development of atherosclerosis . in addition , increasing age , male sex , family history and genetic factors predispose patients to chd.1 there is some data that have suggested the possible potential role of some infectious agents in the pathogenesis of atherosclerosis.26 the possibility of infectious agents may trigger the process of atherosclerosis by direct or indirect inflammatory effects , especially at younger ages.5 although different studies have been carried out to prove this hypothesis some infections may contribute in the pathogenesis of atherosclerosis ; nothing has ever been conclusively proven . the possible role of some agents such as chlamydophila pneumoniae,6 hepatitis c. virus,7 human immunodeficiency virus,7 hepatitis b virus,8 cytomegalovirus,9 herpes virus,9 epstein - barr virus,10 mycobacterium tuberculosis,11 and helicobacter pylori,3 have been considered in the pathogenesis of atherosclerosis . among these pathogens , c. pneumoniae is important , because it could be effectively treated by antibiotics.12 c. pneumoniae is an obligatory intracellular organism , and is responsible for at least 10% of community - acquired pneumonias . this bacterium has also been associated with pharyngitis , bronchitis , otitis , influenza - like illness , sinusitis and myocarditis.1214 conflicting results have been reported about the relationship between c. pneumonia and atherosclerosis . west et al.15 apfalter et al.,16 and zibaeenezhad et al.17 reported no association between c. pneumoniae and atherosclerosis but others found significant association between c. pneumoniae and atherosclerosis.61819 the results of jha et al.,6 dabiri et al.18 and sessa et al.19 support the idea that c. pneumoniae may have a role in the development of atherosclerosis . this controversy about the role c. pneumoniae in the pathogenesis of atherosclerosis emphasizes the importance of more research works . the aim of this study was to demonstrate whether there is any difference between two group of patients with coronary atherosclerotic plaque and subjects without coronary atherosclerosis for the presence of c. pneumoniae dna by polymerase chain reaction ( pcr ) method . this case - control study was carried out on 60 formalin - fixed paraffin - embedded ( ffpe ) coronary artery biopsies in the molecular pathology laboratory , ghaem hospital , mashhad , iran in 2010 . coronary artery tissues were selected from another study that was performed previously by our colleagues for determining prevalence of atherosclerotic plaques in autopsy cases with non - cardiac death in northeast iran . in this study , left coronary artery ( lca ) , right coronary artery ( rca ) and left circumflex artery ( lcx ) had been evaluated grossly and microscopically . our materials included 30 coronary artery tissues with atherosclerotic plaques that were defined as study group , and 30 coronary artery tissues without atherosclerotic plaques that served as the control group . archived slides of the two groups were reviewed by two pathologists for confirmation of diagnosis , tissue adequacy and selection of the best paraffin blocks for dna extraction . for all subjects , the weight and height were determined , and the body mass index ( bmi ) was calculated . five to seven , 5-m - thick sectionswere cut from each ffpe specimen under sterile conditions , and then the dna was extracted by using proteinase k and non - heating dna extraction method.20 dna concentration was determined by using the thermo scientific nanodrop 2000 spectrophotometer , and specimens with low dna content ( < 20 ng/l ) were excluded from the study . pcr was performed for detecting c. pneumoniae dna by using the pcr kits ( dna technology ( jsc ) , pcr kit , moscow , russia ) . 10 l of pcr master mix and 0.5 l unit of taqpolymerase ( hot start taq dna polymerase ) were added into each paraffin sealed tube and were mixed , then 5 l ( 100 ng ) of dna samples were added ( except for positive and negative controls ) and were spun at 1,000 rpm for 3 - 5 s. the tubes were placed into applied biosystems ( abi ) veriti thermal cycler and pcr was performed with the program of 180 s at 94c for the first step and then 45 cycles was run as follows : 50 s at 94c , 50 s at 64c , 50 s at 72c . amplified pcr products were electrophoresed on a 2% agarose gel , stained with ethidium bromide , and photographed under ultraviolet light by gel documentation instrument . pcr was interpreted as positive when the dna band corresponds to the band of the positive control ( 254bp ) in addition to the internal control band ( ic:370 bp ) . results of pcr in control and atherosclerotic groups were analyzed with spss version 11.5 by a statistician . we used fisher 's exact test for comparison of categorical variables and independent sample t - test for continuous variable in patients and control groups . the age range in the control group was 20 - 46 years with a mean ( standard deviation ) age of 26.0 ( 6.41 ) years . in the study group , it ranges from 18 to 50 years with a mean ( sd ) age of 32.0 ( 9.46 ) years and the t - test showed no significant difference between the ages of patients in the study and control groups ( p=0.97 ) . twenty - five patients ( 83.3% ) were males and five patients ( 16.7% ) were females . the male to female ratio was 5:1 and no significant difference was seen between study and control groups for gender ( p = 1.00 ) by fisher 's exact test . the mean bmi of the patients in the study and control groups were 24.4 ( 3.77 ) and 23.9 ( 2.62 ) kg / m respectively [ table 1 ] . the frequency of atherosclerotic plaques in the different vessels of heart of the patients in the study is as shown in table 2 . as a result , the possibility of the appearance of advanced atherosclerotic plaques s in lca was higher than the two other vessels . concurrent occurrences of different stages of atherosclerosis were seen in our patients . only one vessel involvement with fibro - fatty morphology ( mild ) was seen in five patients ( 16.7% ) , one vessel with advanced plaque was observed in three patients ( 10.0% ) , seven patients ( 23.3% ) had one vessel with fibro - fatty and one vessel with advanced plaque , two vessels with advanced plaque were noticed in six patients ( 20.0% ) , three vessels with advanced plaque were observed in five patients ( 16.7% ) and four patients ( 13.3% ) had two vessels with advanced plaque and one fibro - fatty morphology . comparisons between patients with coronary atherosclerosis and controls for weight , height and body mass index the frequency of atherosclerotic plaques in different vessels of the heart in the study group positive pcr result for c. pneumonia was seen in one ( 3.3% ) sample among the 30 coronary artery tissues with atherosclerosis ( advanced plaque ) , while all the control samples were negative . fisher 's exact test showed no significant difference for detection of c. pneumoniae between samples of coronary artery with and without atherosclerotic plaque ( p = 1 ) . ischemic heart disease could be observed in the absence of its major risk factors such as increasing age , male gender , hypertension , hyperlipidemia , smoking and dm . inflammatory cells are seen during all steps of atherogenesis , and some infectious agents may trigger this inflammation.113 although some studies showed that c. pneumoniae can increase the risk of atherosclerosis , 61819 but we did not find any significant difference in the frequency of c. pneumoniae dna isolation in the coronary arteries with and without atherosclerosis by pcr method . we believe that at least two important factors can account for this discrepancy in the results from previous studies : methodology and epidemiology . c pneumonia infection can be diagnosed by various methods such as serology with an increase in serum antibodies against the organism , dna detectionby pcr , immunocytochemistry , electron microscopyand c. pneumonia isolation by culture.14 in other studies , the most widely used method for identifying of c. pneumoniae infection was based on antibody detection . the frequency of antibody against c. pneumoniae starts to rise in children and is observed approximately in 50% of adolescents.1214 somesero - epidemiologic studies have shown a relationship between c. pneumoniae and atherosclerosis . saikku et al.,21 podsiady et al.22 and romano et al.23 detected a higher level of antibody against c. pneumoniae in chd compared to control group but zibaeenezhad et al.,17 romeo et al.,24 and ericson et al.,25 found that the presence of antibodies was unable to predict coronary artery events . we used pcr for detection of c. pneumonia ; this assay appears to be more sensitive than cell culture.1426 our finding was in concordance with those of west et al.,15 jantos et al.27 and satpathy et al.28 similarly , reszka et al.,29 studied presence of c. pneumonia dna in aortic vessels , voorend et al.30 in cerebral vessels , and kwon et al.31 in carotid arteries and could not establish any significant difference in pcr results between the study and control groups . these findings suggest that serology could detect past as well as present infection , whereas pcr detects current c. pnuemoniae infection . considering the study of west et al.15 and satpathy et al.28 who showed a higher c. pnuemoniae igg antibody in patients with chd compared to control group but could not detect the organism by nested pcr assay in any specimen strengthens our hypothesis . we believe that the lack of standardized methods for the detection of c. pnuemoniae infection used in the aforementioned studies is probably one of the important reasons for the discrepancies in their results . c. pnuemoniae is a common human pathogen ; the majority of infected subjects have few or no symptoms . the frequency of antibodies to c. pneumoniae is approximately 50% in the northern hemisphere.1432 exposure to c. pneumoniae is probably common in iran . moghaddam et al.33 reported 38% seropositivity for c. pneumonia antibodies ( igg and igm ) in healthy subjects by elisa method in tehran , iran . studies carried out in iran on the association between c. pneumonia infection , and atherosclerosis revealed conflicting results ; while some studies showed the association between c. pneumonia infection , and atherosclerosis1834 others didnt demonstrate any significant associationbetween c. pneumonia and atherosclerosis.1735 the anatomical localization of vessels may also be a factor influencing the results from previous studies . jha et al.6 showed that c. pneumoniae may be associated with chd and coronary artery was more susceptible to c. pneumoniae as compared with carotid artery . bahrmand et al.25 and ericson et al.34 examined the relationship of c. pneumoniae infection to the severity of coronary atherosclerosis by pcr and serology respectively . they examined coronary artery samples with two different methods , and both revealed a high rate of reactivity to this bacterium in severe atherosclerosis and a much lower rate in mild atherosclerosis ; therefore , they concluded that severity ( stage ) of atherosclerosis can be presumed as an important factor in diagnosis of c. pneumoniae infection . our study was performed on 30 archived ffpe coronary artery tissues of patients with atherosclerosis . this sample size may be too small for final decision ; therefore , future study with a larger number of case groups and also using fresh specimens are helpful . in conclusion , we did not find any significant association between c. pneumoniae infection and coronary artery atherosclerosis by pcr method . further studies involving a greater number of patients and also using more specific methods for detecting living c. pneumonia organisms by culture can be helpful .

background : atherosclerosis is a coronary heart disease , andis the most common cause of death in the industrialized world . some studies suggested that atherosclerosis may be triggered by infectious agents , mostly chlamydophila pneumoniae . however , the role of c. pneumoniae in the pathogenesis of coronary atherosclerosis is still controversial.objectives:this study was performed to evaluate whether there is a significant association between coronary artery atherosclerosis and c. pneumoniae by the polymerase chain reaction ( pcr ) method.materials and methods : this case - control study was carried out on formalin - fixed paraffin - embedded tissue biopsies of the coronary arteries obtained from 30 patients with coronary atherosclerosis and 30 subjects without atherosclerosis living in northeast of iran . all subjects ' weight and height were determined , and the body mass index was calculated . we also reviewed the medical history and previous laboratory reports of patients . deoxyribonucleic acid ( dna ) was extracted , and c. pneumonia dna was amplified and detected using pcr assay.results:the age of the patients in the study group was from 18 to 50 years , and the male to female ratio was 5:1 . only oneout of the 30 coronary tissue samples had positive pcr for c. pneumoniae ( 3.3% ) , while it was negative for patients in the control group.conclusions:this study showed that c. pneumoniae infection is not strongly associated with coronary artery atherosclerosis in northeast of iran .

confrontation with the consequences of diabetes causes a crisis in physical , mental , and spiritual dimensions . sometimes the spiritual crisis can be tremendous . since spiritual health coordinates different aspects of human life , this study aimed to identify the spiritual health of patients with defects caused by diabetes this was a qualitative - phenomenological and descriptive study and the participants were selected from rehabilitation centers in isfahan and valiasr hospital in zanjan . outcome of this phase of the study was 173 codes and 2 groups that included hindering factors in spiritual health and the promotion of the relation with god . the concepts that patients had experienced as hindering factors of the treatment process were disappointment and hopelessness , guilt , feeling distant from god , quitting obligatory acts and knowing god as cruel . the concepts that patients had experienced as contributing factors to the healing process were resorting to imams , god s ordering the disease as a reward , fear of god s punishment , believing in miracles , being closer to god , believing in the mercy of god , returning to religious practice , feeling of enjoying life and knowing that the disease is the atonement of sins . with regard to the importance of spiritual and religious care as one of the tasks of nurses , as the key members of health team , they should respect the patients beliefs and values in addition to considering their physical and mental conditions . although the mortality rate in diabetic patients due to ketoacidosis and infection is declining , deaths from diabetes complications including diabetic foot complications has increased dramatically . according to some reports , its rate in the first year after amputation is 1340 percent , in the third year , 3565 percent and in the fifth year it is 3980 percent , which figures are comparable to death rates in malignancies . in addition , the loss of job and the need for medical and nursing care , reduction in social and family interactions , and lifestyle changes are the major problems that influence the family and socioeconomic status of these patients . the absence of the feet and lower limbs can be extremely problematic for these patients and exerts too much energy for walking ; therefore , amputation of higher limbs or development of ulcers may occur . ultimately , these patients with amputated legs need long - term hospitalization , rehabilitation , home care and social support . organ amputation due to diabetes causes crisis in the physical , mental , and spiritual life of the patients . in this case , the spiritual health is the unique element that can harmonize physical , mental and social aspects of the human life and is necessary for coping with the disease . as a matter of fact , when the spiritual health is seriously at risk , a person may experience emotional disturbances such as loneliness , depression and loss of meaning in life . the spirit of prayer is a deep human instinct , which is the center of humanity , and where the passion and knowledge to communicate with the world comes from . forms and statements of people who pray are different : speaking , listening , waiting and wailing . it exposes them to severe stress and they seek different approaches to cope and adapt to life . koenig believes that religion creates a positive attitude towards the world and makes the person powerful against unfortunate events in life such as diseases and helps to improve the life with motivation and energy . this increases tolerance and acceptance of situations that can not be changed . in many cases of emergency in which science is unable to help a person , this issue is of vital importance especially in serious cases . the increasing interest of medical scientists to the impact of praying in the treatment of other diseases can be indicative of treatment of some diseases in case of modern medicine failure . on the other hand , medical researchers also acknowledge the importance of medical procedures of traditional and complementary therapies including prayer to treat the diseases . even the western scientific community has recently published articles showing that doctors can not be indifferent to the religious beliefs of patients for treatment . this study was conducted to understand the spiritual experiences of the patients with organ amputation due to diabetes . the aim of phenomenological studies is to understand the phenomena of human experience through the analysis of participants verbal explanations of experiences . the study population consisted of patients with defects , caused by diabetes , who had referred to rehabilitation centers in esfahan and zanjan provinces . sampling was purposive and the inclusion criteria for participants were the injury caused by diabetes and the willingness to participate in the interview . there were 15 participants entered the study ( 8 men and 7 women at 5070 years of age ) . all interviews were fully recorded and all participants were given a code based on the interviews . in this study , the researcher tried to be cautious with the information collected to be free from any kind of bias and to avoid persons with poor memory and those who could not provide the correct information . in the interviews , the researcher attempted to withdraw his assumptions about the phenomenon under study and thus help to strengthen the data . they were asked that how has your life changed compared to before the disease ? in response , they described their life experiences with this disability , and how their performance was changed due to the disease . during the interview , the patients focused on cases where the disease led to their functioning . when it was necessary to clarify the data in specific areas , the questions were asked in details . finally , participants were asked if they wanted to provide alternative explanations in relationship with their disease . the researcher wrote down the participants statements through repeated listening to in - depth interviews . after the formation of concepts and ideas from each interview , the next interview was conducted . the above process was repeated for each interview ( second stage of colaizzi s method ) . after the all interviews , the concepts were formulated in particular subjects and were classified into categories ( third stage ) and eventually all ideas were combined so that a complete description of the phenomenon under the study was derived and presented ( the fourth , fifth and sixth stages ) . finally , the concepts were returned to the participants and were analyzed ( the seventh ) . in this study , the criteria for validity and reliability of data were lincoln and guba . in order to make the study believable , the participants and the research partners also reviewed all the data . to achieve reliability and objectivity of the research data , the account access was used . to increase the portability of the present study , the complete research project was introduced . there were 15 patients participating in this study , comprised of 7 women and 8 men . the age range of participants was between 50 - 75 years with mean age of 59.7 years . length of diabetes was between 530 years and the length of organ amputation caused by diabetes also varied from 2 weeks to 11 years with mean of 4.2 years . the education level comprised of uneducated to junior high school . regarding employment , 53.3 percent of the participants ( 8 people ) lost their jobs and were unemployed due to organ amputation caused by diabetes . about 66.6 percent ( 10 people ) , a leg was amputated below the knee and 26.6 percent of the participants ( 4 people ) both legs were amputated below the knee area . in one patient both legs below the knee and the middle finger were amputated due to diabetes . the concepts derived from patients experiences as factors interfering spiritual health in the treatment process were disappointment and hopelessness , guilt , feeling distant from god , quitting obligatory acts and considering god cruel . factors promoting the healing process were deemed by god , fear of god punishment , belief in miracles , being closer to god , believing god to be merciful , returning to religious practice , enjoying life and indicating the disease as the atonement of sins . most of the participants , encounteringdiabetes - related organ amputatio , said that in such circumstances , intellectual protections , religious sources , and having a strong relationship with a higher power could improve the quality of life , and support the individuals and reduce the severity of symptoms . one of the participants expressed his experiences in turning to practice religion as follows : i was deemed to cut my foot to know god , i am sure there is a wisdom behind this , i ve been weak in a prayer . i was wavering , but when my leg was cut , i celt much closer to god , i know god took my foot , but after this incident , i feel that god wanted to try me , before this , if my prayer was late , i did not become upset . but now , if i do not perform my prayer on time , i will get upset , i feel that i did not do an important job , i become worried with no reason ( participant 1 ) . another participant stated when my uncle came to visit me , he said the disease is the expiation of sins . whenever this is said , my heart becomes calm , but i did not commit a sin to deserve such a punishment . even , i had not bothered anything in my life . i do not remember , i wish , everything could be finished in this world ( participant 3 ) . since my foot ( right foot ) has been cut off , i rub it every day . as i can not bend i m afraid ; it may give testimony against me in the doomsday and tells god that i had been overeating because our hands and legs have rights and we must consider this ( participant 5 ) . our tenant is a believer who always reads the quran . when she saw that i was confused , she said , let me read the koran for you to be calm . although i am illiterate and i do not understand anything from the quran , when i hear the peaceful verses of the quran , i forget my sorrows i participate in the middle of moharram mourning ceremony every year and in mourning ceremonies every thursday , but when my leg was cut , i did not go there anymore because i feel i m too far from god and as a result , these ceremonies can not help when the wounds of the foot were fine after surgery , at fast ; i performed my prayer in sitting position , but i realized that i do not feel good in just sitting and praying , i just hurt myself ( participant 15 ) . the prayer and vow are likely to enhance the human s tolerance against diseases and problems communication with the source of existence and asking him to help would repair the physical and mental powersof man and relieves a lot of disorders and diseases . therefore , the appeal to the imams in patients with organ amputation due to diabetes is high . several studies have also shown that when the spiritual health is at a serious risk , prayers in acute cases may face difficulty . in these cases , praying for the patient can be an important spiritual intervention . one of the most useful sentences in the form of prayers could be the short ones , asking god for fulfillment of the needs , melting away the fears and giving hopes to the patient and ultimately remembering that god is able to meet the needs of the patients . if the patient is depressed , angry or upset or suffering from extreme pain , the expression of praying sentences is like the salt to the wound . nurses should pay close attention in the implementation of these provisions , and if the patients are upset , they should leave them alone . in this regard , a research by tracy et al . was conducted on two thousand nurses in critical care units in which 55% of nurses reported that patients and families were asking them to pray . another study which studied the effects of prayer on blood infection , randomly employed 1691 experimental and 1702 control patients . parameters studied included duration of fever in patients , duration of hospitalization and mortality in the hospital . the results of this study showed a significant difference between the duration of fever and length of patients stay that was lower in the intervention group compared to the control group . nowadays , some of the organizations that evaluate health care systems and are responsible for granting accreditation to them , suggest that the spiritual needs of the patients in health care should be also evaluated . finally , the importance of spiritual and religious care , which is today considered as one of nurses tasks , urges them as the key members of health teams to respect patients beliefs and values as well as their physical and mental aspects .

background : confrontation with the consequences of diabetes causes a crisis in physical , mental , and spiritual dimensions . sometimes the spiritual crisis can be tremendous . since spiritual health coordinates different aspects of human life , this study aimed to identify the spiritual health of patients with defects caused by diabetes.materials and methods : this was a qualitative - phenomenological and descriptive study and the participants were selected from rehabilitation centers in isfahan and valiasr hospital in zanjan . a purposive sample of 15 participants underwent deep interviews . colaizzi s method of analysis was used to analyze the data.findings:outcome of this phase of the study was 173 codes and 2 groups that included hindering factors in spiritual health and the promotion of the relation with god . the concepts that patients had experienced as hindering factors of the treatment process were disappointment and hopelessness , guilt , feeling distant from god , quitting obligatory acts and knowing god as cruel . the concepts that patients had experienced as contributing factors to the healing process were resorting to imams , god s ordering the disease as a reward , fear of god s punishment , believing in miracles , being closer to god , believing in the mercy of god , returning to religious practice , feeling of enjoying life and knowing that the disease is the atonement of sins.conclusions:with regard to the importance of spiritual and religious care as one of the tasks of nurses , as the key members of health team , they should respect the patients beliefs and values in addition to considering their physical and mental conditions .

vitamin d deficiency was once thought to exclusively affect bone metabolism , but now there is ample evidence of its role in many other conditions including metabolic syndrome , autoimmune diseases , and cancer . vitamin d receptors are recognized to be in numerous extraskeletal tissues , such as pancreas and muscle . a systematic review by pittas and colleagues reported that vitamin d may have a beneficial effect on the action of insulin , either directly or indirectly . several observational studies in adults , including the framingham heart study , have reported an inverse association between vitamin d status and insulin resistance . however , data from children and adolescents do not consistently report this inverse relationship [ 510 ] . even in studies reporting this association in adolescents and children , further , none of the studies in adolescents have studied the effect of puberty on this association . a fall in insulin sensitivity with compensatory increase in insulin secretion has been reported in puberty [ 11 , 12 ] . high prevalence of cardiometabolic risk factors , including insulin resistance ( 64.8% of normal weight children had at least one cardiometabolic abnormality ) , even in healthy young children of normal weight , has been reported in indian children and adolescents . south - asian adolescents , including those with normal bmi , have a higher prevalence of insulin resistance when compared with age and bmi matched european adolescents [ 14 , 15 ] . this ethnic difference in insulin sensitivity was attributed to the higher body fat percentage in south - asian children . an alternative hypothesis to explain the ethnic differences in insulin sensitivity is vitamin d deficiency . despite plenty of sunlight available throughout the year , a high prevalence of vitamin d deficiency has been reported in all age groups including toddlers , school children , pregnant women and their neonates , and adult males and females residing in rural and urban india . vitamin d deficiency has been reported in 8492% children in the age group of 1018 . in view of high prevalence of both , insulin resistance and vitamin d deficiency in asian - indian children and adolescents , we sought to examine the relationship between vitamin d status and insulin resistance . we also sought to determine if puberty affected the relationship of vitamin d status and parameters of insulin resistance and sensitivity in asian - indian children and adolescents . the study was conducted as a prospective observational study after approval from the ethics committee of the all india institute of medical sciences ( aiims ) , new delhi . subjects were selected from the children and adolescents attending the endocrinology outdoor services at aiims for evaluation of obesity . the inclusion criteria included children between the ages of 617 years with obesity as per international obesity taskforce ( iotf ) criteria . the exclusion criteria included subjects with diagnosed diabetes mellitus or taking metformin or any weight reducing drugs , subjects with any known systemic illness or endocrine or metabolic disorders , known to be associated with obesity , or subjects with symptoms to suggest hypothalamic obesity were excluded from the study . blood samples were collected in fasting state ( minimum 9 hours fasting ) , followed by oral glucose tolerance test ( anhydrous glucose = 1.75 gm / kg bodyweight , maximum of 75 gram , dissolved in 250 ml of water , ingested over 5 minutes ) with sample collection at 60 and 120 minutes after glucose ingestion . height was measured with holtain 's stadiometer ( holtain inc . , crymych , pembs . waist circumference was measured ( nonelastic measuring tape ) at the end of normal expiration at the midpoint between the iliac crest and the lower edge of ribs in the midaxillary line . blood pressure ( bp ) was measured in the right upper limb in sitting position with a mercury sphygmomanometer after 5-minute rest with an appropriate size cuff . total body fat was measured with dual energy x - ray absorptiometry ( dxa , hologic qdr 4500 with pediatric software , hologic inc . , fat mass index ( fmi , fat mass in kg / height in meter ) and fat - free mass index ( ffmi , lean body mass+ bmc)/(height in meter ) were calculated . bone mineral area , content ( bmc ) and areal density ( abmd ) at the femoral neck , lumbar spine ( l1 - 5 ) , and forearm were measured , using a hologic qdr 4500a fan beam dxa machine . hologic spine phantom ( hologic spine phantom number 21373 ) was scanned daily before subject evaluation . the measured phantom bone mineral density was stable throughout the study period at 0.9150.945 gm / cm . similarly , whole body phantom ( hologic wb number 1252 ) was also scanned before subject evaluation and remained stable during study period . the in vivo precision error for adults was 0.62% for femoral neck abmd , 0.65% for lumbar spine abmd , and 0.77% for forearm abmd . however , in view of additional radiation exposure , the reproducibility of these scans was not assessed among children . pubertal stage was assessed by a single endocrinologist , based on breast stage and pubic hair development in girls and genitalia development in boys . b - mode ultrasonography was used to measure cimt ( 7.510 mhz probe , philips envisor ultrasound machine ) using standard protocol . insulin resistance ( ir ) was calculated by computer - based model called homeostasis assessment model ( homa - ir ) utilizing fasting blood glucose and fasting serum insulin levels .the whole body insulin sensitivity index ( wbisi ) or matsuda index was calculated by the formula suggested by matsuda and defronzo .area under curve ( auc ) was calculated for blood glucose ( 0 , 60 , and 120 minutes ) and serum insulin ( 0 , 60 , and 120 minutes ) by using trapezoidal rule . insulin resistance ( ir ) was calculated by computer - based model called homeostasis assessment model ( homa - ir ) utilizing fasting blood glucose and fasting serum insulin levels . the whole body insulin sensitivity index ( wbisi ) or matsuda index was calculated by the formula suggested by matsuda and defronzo . area under curve ( auc ) was calculated for blood glucose ( 0 , 60 , and 120 minutes ) and serum insulin ( 0 , 60 , and 120 minutes ) by using trapezoidal rule . complete blood counts , liver function tests , renal function test , serum calcium ( corrected calcium with serum albumin ) , phosphate , alkaline phosphatase , uric acid , and blood glucose were measured in all subjects with an automated chemistry analyzer ( roche hitachi 912 chemistry analyzer , gmi inc . glycosylated hemoglobin ( hba1c ) was measured in whole blood using ion - exchange high performance liquid chromatography ( bio - rad laboratories inc . , ca , us ) . serum insulin was measured on an autoanalyzer ( roche elecsys 2010 ) using electrochemiluminometric assay . serum total cholesterol ( tc ) , triglyceride ( tg ) , and hdl cholesterol ( hdl - c ) levels were estimated directly with automated analyzer , while ldl cholesterol ( ldl - c ) was estimated by using the friedewald equation . vitamin d deficiency was defined as a serum 25(oh)d levels less than 20 ng / ml . this was further subdivided to severe , moderate , and mild vitamin d deficiency if serum 25(oh)d levels were < 5 ng / ml , 5<10 ng / ml , and 10<20 ng / ml respectively . statistical analysis was carried out using spss ( spss version 11.5 ; spss inc . , continuous variables not showing normal distribution were treated with appropriate log transformations before any parametric analysis . student 's t - test was used to determine statistical difference between subjects with serum 25(oh)d < 10 ng / ml or more . pearson 's correlation was used to assess association between continuous variables and partial correlations were used to determine relationship after adjusting for relevant covariates . stepwise linear regression analysis was used with serum 25(oh)d as dependent variable in the model . values for serum 25(oh)d , serum insulin , homa - ir , and matsuda isi were not normally distributed ; therefore , they were logarithmically transformed which yielded normal distribution . a total of 62 subjects participated in this cross - sectional study from june 2008 to may 2009 . the mean age of subjects was 13.0 3 years ( 35 boys ; 27 girls ) . assessment of pubertal status by tanner 's method showed 19 subjects in stage 1 , 11 in stage 2 , 6 in stage 3 , 3 in stage 4 , and 23 subjects in puberty stage 5 . no significant difference was observed between boys and girls except higher whr in boys than girls ( 0.93 0.05 versus 0.89 0.05 ; p = 0.013 ) . all study subjects were classified as vitamin d deficient ( mean sd 8.5 4.2 ng / ml ; maximum minimum : 3.919.2 ng / ml , median = 6.9 ng / ml ) . severe vitamin d deficiency ( < 5 ng / ml ) was seen in 11 subjects ( 17.7% ) while 30 subjects had serum 25(oh)d levels between 5<10 ng / ml ( 48.3% ) . serum 25(oh)d equal to or more than 10 but less than 20 ng / ml was present in 21 subjects ( 33.8% ) . pearson correlation coefficient analyses showed significant inverse relationship between serum 25(oh)d and total body fat percentage ( r = 0.31 , p = 0.01 ) and serum 25(oh)d and fmi ( r = 0.33 , p = 0.01 , figure 1 ) . partial correlation analysis showed persistence of significant correlation between serum 25(oh)d and total body fat percentage ( r = 0.37 , p = 0.005 ) and between serum 25(oh)d and fmi ( r = 0.36 , p = 0.006 ) even after adjustment for age , sex , pubertal stage , and bmi . although blood glucose and serum insulin levels showed inverse relationship trends with serum 25(oh)d , but these were not statistically significant ( data not shown ) . similarly , no statistically significant correlation was seen between serum 25(oh)d and parameters of insulin sensitivity or resistance . fmi showed positive correlation with homa - ir ( r = 0.26 ; p = 0.04 ) , auc insulin ( r = 0.41 ; p = 0.001 ) , auc glucose ( r = 0.31 ; p = 0.018 ) , and negative correlation with wbisi ( r = 0.42 ; r = 0.001 ) . however , total body fat did not show any correlation with parameters of glucose and insulin parameters . on the basis of serum 25(oh)d , subjects were divided into two groups , group a with serum 25(oh)d level < 10 ng / ml ( n = 41 ) and group b with serum 25(oh)d level 10 ng / ml ( n = 21 ) ( an arbitrary cutoff to compare subjects with mild vitamin d deficiency with moderate and severe vitamin d deficiency , 25 ) . no statistical significant difference was seen in blood glucose , serum insulin , and auc for both , glucose and insulin between group 1 and group 2 . trends for higher insulin resistance ( homa - ir ) and lower insulin sensitivity ( wbisi ) were seen in subjects with lower serum 25(oh)d concentrations but not statistically significant . total body fat percentage and fmi were significantly higher in group 1 in comparison to group 2 ( p = 0.015 and 0.007 , resp . ) . however , no significant difference was seen in waist and hip circumference and waist hip ratio . similarly , ffmi , lipid parameters , or thyroid profile were also not different between the two groups . group 1 ( prepubertal group ; tanner stage 1 ) had 19 subjects , group 2 ( peripubertal group ; tanner stage 2 , 3 , and 4 ) had 20 , while group 3 ( postpubertal group ; tanner stage 5 ) had 23 subjects . no significant correlation was seen between vitamin d status and any other parameters including total body fat percentage , fmi and parameters of insulin resistance and sensitivity in the prepubertal ( group 1 ) or peripubertal groups ( group 2 ) . partial correlations after adjusting for age , sex , and bmi were also nonsignificant for both groups ( data not shown ) . in postpubertal subjects ( group 3 , n = 23 ) , a significant inverse correlation was seen between serum 25(oh)d and homa - ir ( r = 0.41 , p = 0.03 , figure 2 ) . this association between serum 25(oh)d and homa - ir persisted even after control for age , sex , and bmi ( r = 0.505 , p = 0.023 ) . positive trends of association between serum 25(oh)d and wbisi were also seen , but this was not significant ( r = 0.29 , p = 0.17 ) , even after adjusting for age , sex , and bmi ( r = 0.35 , p = 0.13 ) . serum 25(oh)d showed significant inverse correlation with total body fat percentage ( r = 0.58 , p = 0.003 ) and fmi ( r = 0.49 , p = 0.016 ) which improved after adjusting for age sex and bmi ( r = 0.63 , p = 0.002 for total body fat percentage ; r = 0.582 , p = 0.007 for fmi ) . serum 25(oh)d also showed significant inverse relationship with fasting serum insulin levels ( r = 0.44 , p = 0.034 ) which improved after adjusting for age , sex , and bmi ( r = 0.53 , p = 0.016 ) . when prepubertal subjects ( group 1 ) were compared with the prepubescent subjects ( group 2 ) , no significant difference was seen in any of the parameters including blood glucose , serum insulin , homa - ir , and wbisi . similarly , when prepubertal subjects were compared with all other subjects ( subjects in group 2 and 3 combined ) , significantly higher insulin sensitivity ( wbisi , mean sd 6.07 5.7 versus 3.42 2.7 , p < 0.01 ) and lower insulin resistance ( homa - ir , mean sd 3.68 2.7 versus 5.22 4.0 , p = 0.04 ) were seen in prepubertal subjects . this is the first study examining the relationship between serum 25(oh)d levels and parameters of insulin resistance in obese asian - indian children . our study confirms a significant inverse relationship between serum 25(oh)d levels and body fat indices as reported in other studies in children [ 6 , 10 ] . we demonstrated trends for that higher serum 25(oh)d levels are associated with better insulin sensitivity and less insulin resistance though this correlation did not reach statistical significance . one of the main reasons for the absence of significance could be the universal presence of vitamin d deficiency ( < 20 ng / ml ) resulting in very narrow range of serum 25(oh)d . we also show , for the first time , that the association between serum 25(oh)d and insulin resistance and sensitivity is influenced by pubertal staging . when subjects were stratified by pubertal stage , only subjects in tanner stage 5 exhibited a significant correlation between serum 25(oh)d and homa - ir , while prepubertal subjects did not show statistically significant correlation . high prevalence of vitamin d deficiency has been reported across the age range in pediatric , adolescent , and adult in obese individuals [ 5 , 6 , 2631 ] . this may be just reflection of very high prevalence of vitamin d deficiency in asian - indians [ 16 , 17 ] . however , high prevalence of vitamin d deficiency has been reported in obese children and adolescents from populations where vitamin d deficiency is not that prevalent [ 5 , 6 , 10 ] . reis et al . , in a study of 3577 adolescents in the age group of 1219 years , found significantly lower serum 25(oh)d levels in adolescents with bmi > 95th centile in comparison to subjects with bmi < 95th centile ( p < 0.001 ) . it has been postulated that increased sequestration of vitamin d in fat tissues , leading to decreased vitamin d bioavailability , low dietary vitamin d intake due to poor nutritional habits , and minimal sun exposure due to sedentary indoor lifestyle are important factors associated with high prevalence in the obese population [ 29 , 32 ] . it has also been suggested that vitamin d deficiency is a cause of common obesity and can account for the secular trends in the prevalence of obesity and for individual differences in its onset and severity . the inverse relationship between serum 25(oh)d and insulin resistance in adults have been reported in most studies , though some have failed to find this association . in comparison , reports in children and adolescents have shown conflicting results . delvin et al . , in a study of 1745 french - canadian children , reported modest but significant negative associations between serum 25(oh)d and homa - ir . each 10-nmol / l ( 4 ng / ml ) increase in serum 25(oh)d was associated with lower glycemia and homa - ir . did not find significant correlation between serum 25(oh)d levels and insulin , homa - ir and homa - b % . in another study based on nhanes data of 3577 adolescents from 20012004 showed that serum 25(oh)d levels were inversely associated with plasma glucose concentration ( p = 0.01 ) . lenders et al . , in a study of 58 obese adolescents , did not found any correlation between insulin indices and serum 25(oh)d in both , adjusted and unadjusted models . in our study , we did not find a statistically significant association between serum 25(oh)d and parameters of insulin sensitivity and resistance . however , trends for higher blood glucose and serum insulin levels were seen in subjects with low serum 25(oh)d level . no significant difference was seen in insulin resistance and sensitivity between group 1 and group 2 . this is in contrast to the observations made by ashraf et al . who reported in 51 african - american obese female adolescents that higher serum 25(oh)d concentrations were associated with significantly higher wbisi ( p = 0.018 ) but no difference in homa - ir . these differences may be due to marked difference in severity of obesity as mean bmi of study subjects was 43.3 9.9 in study by ashraf et al . , while it was 29.3 9.8 kg / sqm in our study subjects . studies in adult population have shown that parameters of insulin sensitivity and insulin resistance as calculated by hyperinsulinemic euglycemic clamp study ( m value ) correlated better with serum 25(oh)d levels than indirect parameters like homa - ir . studies with direct measures of insulin sensitivity like clamp studies are required to further investigate these issues in pediatric and adolescent population . the phenomenon of pubertal insulin resistance has been well described in cross - sectional as well as longitudinal studies . a longitudinal study of insulin resistance in american children showed that during puberty , insulin sensitivity decreased by 50% , with a compensatory increase in plasma insulin which is independent of the changes in body fat . these changes are thought to be mediated by interaction of various hormones during puberty including increased growth hormone secretion . no studies have yet reported the relationship between serum 25(oh)d concentrations and insulin sensitivity and resistance in pediatric and adolescent population and the potential influence of puberty except one . lenders et al . reported no significant association between serum 25(oh)d and insulin indices , even after adjusting for tanner stage . however , our study , for the first time , shows that the relationship between serum 25(oh)d concentrations and insulin resistance is influenced by puberty . when subjects with all stages of puberty were analyzed , no significant correlation was found , but when subjects in tanner stage 5 were analyzed alone , significant inverse correlation was seen between serum 25(oh)d concentrations and homa - ir ( r = 0.41 , p = 0.03 ) . this association persisted / improved after controlling for age , sex , and bmi ( r = 0.505 , p = 0.023 ) but disappeared when subjects were controlled for fmi ( r = 0.27 , p = 0.24 ) . most of the evidence of an association between adiposity and vitamin d status comes from adult studies [ 4 , 33 ] . a recently published study of 382 healthy subjects aged 621 years with dxa scans showed that serum 25(oh)d concentrations were more likely to be lower in those with greater bmi z - scores and fm , but not ffm . when this association was adjusted for possible covariates in the model , it became nonsignificant . . showed that obese adolescents with and without vitamin d deficiency differed according to fm and fm percentage , but not lean mass and this relation of fm percentage to serum 25(oh)d remained significant after potential confounding variables were adjusted for . showed that vitamin d deficiency was associated with higher visceral adipose tissue in white and greater subcutaneous adipose tissue in black american children and adolescents . in agreement with these studies , our findings indicate that body fat indices ( total body fat percentage and fmi ) are inversely correlated with serum 25(oh)d level . this correlation persisted even after adjustment for possible covariates like age , sex , and bmi . the differences in study findings may be explained in part by differences in study design and underlying sample characteristics like bmi and variation in serum 25(oh)d concentrations . the major strengths of our study are the use of robust measures of insulin sensitivity and resistance based on ogtt rather than fasting sample and all our study subjects belonged to one ethnic group . we have used dxa for assessment of body fat as opposed to several studies which have used bmi as a surrogate for body fat . the limitations of our study included , small sample size , presence of vitamin d deficiency in all study subjects which gave us very narrow range of serum 25(oh)d concentrations thereby limiting comparisons . absence of pth measurement was another important limitation , since it has been implicated in pathogenesis of insulin resistance and metabolic syndrome . another important limitation was inability of dxa technique to differentiate between subcutaneous and visceral adipose tissue . in conclusion , our study demonstrates a significant inverse relationship in obese asian - indian children between body fat indices and serum 25(oh)d concentrations which persists even after adjustment of covariates . the association between serum 25(oh)d and parameters of insulin sensitivity and resistance is influenced by puberty as a significant association was found between these two variables in postpubertal subjects ( tanner stage 5 ) only which remained significant even after adjustment for covariates . as association is not equal to causation , future longitudinal research studies are required in determining role of puberty on association between serum 25(oh)d and insulin resistance in both , obese as well as subjects with normal bmi .

to study the effect of puberty on the relationship between serum 25-hydroxyvitamin d ( 25(oh)d ) and parameters of insulin kinetics in obese asian - indian children . material and methods . the study population included 62 obese asian - indian children and adolescents in the age group of 617 years . blood glucose , serum insulin , and serum 25(oh)d were measured . total body fat was measured by dual energy x - ray absorptiometry . indices of insulin resistance ( homa - ir , auc for insulin ) and sensitivity ( wbisi ) were calculated after oral glucose tolerance test . result . a total of 62 subjects ( 35 boys ; mean age = 13.0 3 years ; bmi = 29.3 4.8 kg / sq m ; 19 subjects in tanner stage 1 , 11 in stage 2 , 6 in stage 3 , 3 in stage 4 , and 23 subjects in tanner stage 5 ) were studied . all study subjects were vitamin d deficient with a mean serum 25(oh)d of 8.5 4.2 ng / ml . no significant relationship was observed between serum 25(oh)d and parameters of insulin kinetics in prepubertal children . however , a significant inverse correlation was seen between serum 25(oh)d and homair ( r = 0.41 , p = 0.03 ) in postpubertal subjects . conclusion . the relationship between vitamin d status and parameters of insulin kinetics are affected by puberty .

a 21-year - old man ( height = 180 cm , weight = 95 kg , body mass index = 29.3 kg / m ) presented to the outpatient department with a long history of frequent and severe snoring . on physical exam , after a diagnosis of obstructive sleep apnea was made , a bilateral tonsillectomy and uvulopalatopharyngoplasty was planned for treatment . prior to surgery , the patient had normal laboratory results with hemoglobin ( hb ) of 15.8 g / dl and no systemic or hematological diseases or history of bleeding disorders . on the day of the surgery , the patient received no premedication due to the history of sleep apnea . intraoperative monitors included electrocardiogram , non - invasive blood pressure ( bp ) , pulse oximetry ( spo2 ) , and end - tidal co2 . pre - anesthetic vital signs were bp 120/82 mmhg , hr 68 bpm , and spo2 96% . general anesthesia was induced with remifentanil 0.5 g / kg / min and propofol 180 mg iv at 10:10 am . at loss of eyelash reflex , cisatracurium 16 mg was administered , and the trachea was intubated after a short period of facemask ventilation . anesthesia was maintained with sevoflurane 2 vol% in 50% oxygen - enriched air and remifentanil 0.2 g / kg / min . the neuromuscular block was antagonized with glycopyrrolate 0.6 mg and pyridostigmine 15 mg iv without neuromuscular monitoring upon the completion of the surgical procedure at 11:25 am . postoperatively , a physical examination confirmed the presence of acute bleeding in the left inferior pole of the tonsillectomy site in the post anesthetic care unit . the surgeon requested an emergent re - operation under general anesthesia for the control of bleeding . a rsi for a second round of general anesthesia was performed with propofol 120 mg , midazolam 5 mg , and rocuronium 100 mg at 11:55 am . the surgeon confirmed active bleeding due to an aberrant arterial blood supply in the left inferior pole of the tonsillectomy site . after the completion of the surgical procedure , no twitch was shown after train - of - four ( tof ) stimulation ( tof = 0 ) with neuro - stim ( model ns-3cc , houston , tx , usa ) at 12:40 pm . the neuromuscular block was then antagonized with sugammadex 200 mg iv and the rocuronium - induced nmb was completely reversed within 2 min . the patient was transferred to the recovery room , and there were no specific findings on physical exam . a post - operative laboratory assessment showed hb of 14.6 g / dl ( table 1 ) . after 6 h and 20 min , the surgeon again requested emergent anesthesia and re - operation for control of bleeding . the surgeon attempted to control bleeding at the ambulatory otolaryngology clinic but was unable to do so . when the patient arrived at the operating room ( or ) , vital signs were within normal limits and hb was found to be 13.2 g / dl . a third round of general anesthesia was induced with propofol 120 mg and rocuronium 40 mg by the on - call resident at 19:00 pm . the neuromuscular block was antagonized with sugammadex 200 mg iv and a tof = 0 at 20:00 pm . the patient was again extubated successfully and transferred to the recovery room with no specific findings ( table 1 ) . after 2 h and 35 min , when the patient arrived at the or , vital signs were stable and hb was 11.6 g / dl . a fourth induction of general anesthesia was performed with propofol 120 mg and rocuronium 40 mg by the same resident at 22:35 pm . during tof stimulation , 1 twitch was seen , and an additional dose of rocuronium 10 mg was given due to bucking 20 min after the induction of anesthesia . at the end of the procedure , the neuromuscular block was antagonized with glycopyrrolate 0.4 mg and pyridostigmine 10 mg iv with a tof = 1 at the end of the surgical procedure at 0:20 am . extubation was carried out after confirming the recovery of consciousness , regular respiration , and the absence of fade on tof monitoring . after extubation , the patient vomited a large amount of blood on the operating table ( approximated to be more than 200 ml ) . however , the patient was alert and aware of his surroundings and had regular respirations ( table 1 ) . after 20 min , the surgeon requested a re - induction of general anesthesia for additional bleeding control . an rsi for a fifth round of general anesthesia was performed with propofol 120 mg and rocuronium 100 mg at 0:40 am . the neuromuscular block was antagonized with glycopyrrolate 0.4 mg and pyridostigmine 10 mg iv with a tof = 1 after the surgical procedure was completed at 1:30 am . approximately 15 min passed after the nmb was antagonized as above , but no evidence of recovery from the rocuronium - induced nmb was seen . therefore , we injected a bolus of iv sugammadex 200 mg to reverse the residual nmb . once again , the nmb was completely reversed within 2 min , and tracheal extubation was performed in the or uneventfully ( table 1 ) . a decision by surgeon was made to perform angiography of the left external carotid artery with subsequent embolization of the bleeding vessel . embolization of the left ascending pharyngeal artery was performed with polyvinyl alcohol particle ( 350 m ) and tornado embolization microcoil ( cook , bloomington , usa ) ( 3/2 mm ) ( fig . the patient 's postoperative course was uneventful , and hb was found to be 10.5 g / dl . pth occurs in approximately 1 in 20 adults ( 5.1% ) , and more than half of patients who bleed are likely to require a procedure of some type to control their hemorrhage . aberrant arterial blood supply to the tonsillar region derived from the internal carotid artery or the carotid bulb may be present . to control bleeding , electrocauterization , packing of the pharynx , or angiographic embolization of the feeding artery may become necessary . the endovascular embolization due to refractory bleeding after tonsillectomy proved to be a valuable treatment method as an effective alternative to surgical intervention . it is a safe and permanent treatment option in this potentially life - threatening complication . in a case such as this , the aberrant arterial blood supply in the left inferior pole of tonsillectomy site caused continuous bleeding . the anesthesiologist has experienced difficulties , because an experienced surgeon has embarrassed due to repetitive bleeding . in the first induction of anesthesia , no problems were encountered with conventional anesthesia , although neuromuscular monitoring was not performed . the most common anesthetic used for rsi during the management of children with pth is succinylcholine . since the introduction of sugammadex , rsi with rocuronium followed by reversal with sugammadex allowed for earlier establishment of spontaneous ventilation than with succinylcholine , which has many adverse side events . during the second round of anesthesia , rocuronium 100 mg ( 1 mg / kg ) was injected for rsi , followed by reversal with sugammadex 200 mg ( 2 mg / kg ) at deep nmb ( tof = 0 ) . during the operation , nmb monitoring was difficult due to problems attaching leads to the arms during the operation . tof stimulation was applied at the ulnar nerve using conventional instrumental monitoring rather than quantitative monitoring due to convenience . deep nmb was maintained for bleeding control in an attempt to not disturb the procedure . during nmb reversal , we found an absence of fade on tof monitoring just 2 min after sugammadex was administered . re - administration of 0.6 mg / kg rocuronium could then be given after sugammadex at the recommended waiting time of 6 h . during the third anesthesia induction , an on - call resident used rocuronium 40 mg for muscle relaxation during intubation followed by reversal with sugammadex 200 mg at deep nmb ( tof = 0 ) . an absence of fade on tof monitoring was shown 2 min after sugammadex was given . for the fourth round of anesthesia , the same resident used rocuronium 40 mg for intubation 2 h and 35 min after the previous nmb reversal with sugammadex . however , we believe this decision was made without considering the recent sugammadex use . when rocuronium is used within a short time interval ( less than 6 h ) after sugammadex administration , the onset time of rocuronium may be prolonged and may also have an unpredictable duration of action . we suspect that the additional dose of rocuronium 10 mg due to bucking may be related to the short time interval between sugammadex and rocuronium administration . in situations such as these , the benzylisoquinoline class of medications are recommended instead of re - administering rocuronium within 6 h after sugammadex . when considering this case , cisatracurium for muscle relaxation and intubation may have been a better choice for the fourth round anesthesia . however , one drawback of cisatracurium is the slower onset time than rocuronium . reversal of the rocuronium - induced nmb with pyridostigmine at a moderate nmb ( tof = 1 ) was uneventful . after extubation , the patient vomited more than 200 ml of blood on the operating table ; however , he was alert and showed regular respirations . postoperative nausea and vomiting ( ponv ) after an operation for pth is common due to blood swallowed during the procedure . therefore , patient awareness and complete reflexes are important , but aspiration must be avoided and monitored closely during the recovery period . however , ponv increases the risk of primary hemorrhage and unexpected postoperative hospital admissions . in this case the patient did not aspirate , but did have an increased risk of pth due to ponv . for the fifth round of anesthesia , rocuronium 100 mg ( 1 mg / kg ) was injected for rsi after a time interval from sugammadex administration of 4 h and 40 min . it has been reported that re - administration of 1.2 mg / kg rocuronium could have been given after sugammadex 2 mg / kg without a waiting time . however , neuromuscular monitoring did not show the expected recovery , and the patient had residual paralysis after 15 min . it has been shown previously that the reversal of nmb with anticholinesterase drugs may not be complete after a large dose of a neuromuscular blocking agent . sugammadex resolved the residual nmb within 2 min , even after reversal with anticholinesterase agents , and is a safe alternative to reversal of nmb induced by steroidal non - depolarizing agents . in this case , deep rocuronium - induced nmb can be reversed successfully with sugammadex at doses of 2 mg / kg and in as little as 2 min . re - administration of rocuronium within a short time interval after sugammadex may result in unpredictable effects of the nmb agent . sugammadex made it possible to perform a rapid , complete reverse when the residual block was maintained by an incomplete reversal of anticholinesterase .

post - tonsillectomy hemorrhage ( pth ) is the most frequent complication of tonsillectomy , and occasionally results in a lethal outcome . a 21-year - old man ( height 180 cm , weight 95 kg ) was scheduled for a bilateral tonsillectomy and uvulopalatopharyngoplasty for treatment of obstructive sleep apnea . he required 5 rounds of general anesthesia due to recurrent pth . the anesthesiologist used sugammadex a total of 3 times to achieve the successful reversal of the deep neuromuscular blockade ( nmb ) induced by rocuronium . after sugammadex 2 mg / kg was administered , the nmb was reversed in 2 minutes each time . re - administration of rocuronium within a short time interval after sugammadex may result in unpredictable effects of neuromuscular blocking agents . sugammadex made it possible to perform a rapid , complete reverse when the residual block was maintained by an incomplete reversal of anticholinesterase .

infertility has been recognized as a public health issue worldwide ( 1 ) leading to an increasing need to the use of assisted reproductive technologies ( art ) , including in vitro fertilization ( ivf ) . after the first reported case of ivf in 1978(2 ) art enabled millions of people to have their own children in cases when pregnancy did not occur under natural circumstances . 700,000 cycles a year in the usa and europe together ( 3,4 ) . despite of evolving intracytoplasmatic sperm injection ( icsi ) technique a success rate of 25% and 28% has been reported in 2005(7 ) and 2008(8 ) , respectively . nowadays , this rate went up to 32% ( 9 ) , which can not be considered as a significant development . earlier clinical protocols preferred multiple embryo transfer , but multiple gestations can result in the increased risk of preterm delivery ( 10 - 16 ) . other studies report that multiple gestations also increased the risk of low birth weight cerebral palsy ( 17 ) . in the us alone , preterm births resulting from multiple pregnancies during ivf cause a 1 billion usd extra cost to the social insurance ( 18 ) . in order to exclude the discussed risk factors , it is imperative , however , that accurate and economical methods should be developed to ensure that the most viable euploid embryo is selected for transfer . ideally , such tests would be noninvasive , lessening the risks to the embryo and reducing costs and workload in the embryology laboratory ( 19 ) . the biggest issue with pre - implantation viability assessment is that due to ethical reasons any assay should be completely non - invasive because no one can predict what kind of interference would be the unwanted result in the later embryonic development . the most apparent and routinely applied - way of the assessment of viability is the morphological evaluation of in vitro fertilized embryos using microscopy . there are several morphological features described which could be used for viability assessment purposes , these are dependent on the time spent after fertilization . right after fertilization in the 1-cell embryo the size and symmetry of the two pronuclei can be examined . the time of the first cell division is also a good predictor of later implantation potential , as zygotes that divide early tend to develop more frequently to the blastocyst stage . criteria as cleavage rate and blastomere shape and symmetry , an adequate trophectoderm layer ( te ) and an inner cell mass ( icm ) is a morphological marker of the later stages ( 5,20 ) . not only can the morphology of the fertilized embryo be used for further prediction of implantation potential , but morphological defects of the retrieved oocyte as well . fertilization and pregnancy rate correlates with the grade of cumulus - oocyte complexes , and embryos originating from dysmorphic oocytes show a larger grade of pregnancy loss ( 21 - 23 ) . the cleavage stages of morulae and blastocysts or the symmetry and patterns of cell division are also notable and frequently used aspects , and are often examined during the prediction of embryo viability ( 23 ) . the biggest issue of morphological assessment is that it is still a highly subjective method ( 20 ) . the reason is partly due to the fact that the final decision is made by a clinician , and not by an objective test result , and secondly it is does matter how important are the individual morphological features in the final conclusion ( 24 - 26 ) . to overcome the different practice of laboratories worldwide in 2011 an international consensus ( istanbul consensus ) has been reached on embryo viability assessment ( 27 ) . the selected morphological markers of respective stage embryos , the weighing of individual features and a scoring system has been set up . the limitations due to static time - point observation , is now solved with the use of time - lapse microscopy ( 28,29 ) . time - lapse microscopy also enables the observation of dynamics of cytoplasmic movements and cytokinesis , reflecting the functionality of microtubule and actin cytoskeleton , which is critical for proper development . in our laboratory , we aimed to improve the success rate of implantation by adopting and further optimizing the istanbul consensus . this score has been called as the optimized criteria system ( ocs ) . according to this scoring , 3-day old embryos were divided into two subgroups : the subgroup with low blastomere number ( less than 7 ) and with high blastomere number ( 7 or more ) . symmetric position of blastomeres indicates the rate of symmetry of holoblastic cleavage along the embryo axis . it was classified as good ( full symmetry ) ; fair ( light asymmetry ) ; or poor ( evident asymmetry ) . the percent values of fragmentation are based on the ratio of fragmented to total cell numbers . as a further modification to the istanbul consensus , embryos were considered as good if the fragmentation rate was < 15% ( instead of the original 10% ) . this shift from 10 to 15% was the result of our observation that a fairly high proportion of the embryos between 10 - 15% appeared to be viable . in summary the optimized criteria system ( ocs ) highlights 3 modified or new parameters : fragmentation ( with a more permissive criterion of < 15% in the good category ) ; symmetry and the blastomere number . in addition , the blastomere size was evaluated according to the original istanbul consensus . a scoring - map was created to facilitate the evaluation ( table 1 ) as far as the 5-day old embryos are concerned , we modified the original istanbul consensus for blastocysts by leaving out the hatched stage from the evaluation . the istanbul consensus for the 5-day old embryos has a shortcoming , i.e. it does not express the viability of embryos with a single category ( good , fair , poor ) . we tried to overcome this by using a scoring map ( table 2 ) . in conclusion , we constructed a composite score for day-3 , as well as day-5 old embryos , based on morphological parameters . as it is evident from the results , this composite score is sensitive to evaluate viability ( figure 1 ) another possibility for non - invasive embryo viability assessment is the metabolomic examination of the culture medium surrounding the in vitro fertilized embryo . metabolomic , ( proteomic ) profiling of spent embryo culture ( sec ) offers an exceptional , non - invasive opportunity for the assessment of embryo viability ( 30,31 ) . the metabolomic profiling ( 32,33 ) of early embryo development might mean the analysis of the total metabolome by following the changes of several selected compounds , metabolomic analysis using unidentified , but significantly differing metabolomic changes , or by the analysis of a limited population of nutrients or end products . the common feature in all three concepts is that they are concentrating on the metabolomic alterations caused by differently developing embryos in the culture medium . very simple idea is the monitoring of glucose consumption or pyruvate formation , since this would directly indicate the metabolism of the developing embryo and it is an obvious conclusion that a metabolically active embryo would have higher implantation potential . some authors reported that the identification of these parameters resulted in successful prediction of embryo implantation potential , but other research groups describe contradictory results ( 34 , 35 ) . the amino acid profile of culture media is also used in the prediction of implantation potential , though not exclusively as an independent parameter , rather in combination with morphological features ( 36 ) . the detection of unidentified metabolomic changes using near infra - red ( nir ) or raman spectroscopy ( 37 , 38 ) is a very interesting and challenging possibility . more complicated is the concept when unknown , new biomarker molecules of embryo viability are searched for , assuming that these biomarkers were secreted by the embryo . the difficulty of the concept is that only 4 - 8 cells are present in the culture medium ; thus a very sensitive analytical tool is required . mass spectrometry ( ms ) has the potential of specific and sensitive quantification in a wide spectrum of molecular mass ranges and therefore suites well the needs of metabolomic or proteomic fingerprinting and quantification . in parallel to the spreading of mass spectrometry , proteomics is also an emerging field in the understanding of embryo development ( 39,40 ) . the analysis of the embryonic secretome ( 41,42 ) provides information of the total transcriptome of the developing embryos . mass spectrometry can be used both in targeted and discovery analysis with accurate quantification of identified biomarkers after molecular identification by bottom - up or top - down proteomics using tandem or multiple ms ( 43 - 46 ) . in a recent publication from our laboratory ( 47 ) using liquid chromatography coupled mass spectrometry ( lc - ms ) , a fragment of the human haptoglobin molecule was identified in the culture medium . rather than analyzing the embryonic secretome , the aim this experiment was to use preexisting molecules present in the cell culture media as biomarkers . haptoglobin - which was detected in the culture medium - is not a product of the developing embryo ; the polypeptide is a contaminant of the human serum albumin standard used to supplement the culture medium ( 47,48 ) . during the first three days of embryo development the formation of a subunit ( alfa-1 ) of the human haptoglobin molecule was observed . this subunit similar to the total haptoglobin molecule was detectable in the blank control medium samples as well . the differentiation of the viable and non - viable embryos was done using the observation that compared to blank controls the samples of embryos which later did not resulted in pregnancy contained the alpha-1 subunit in a much larger quantity than the samples of embryos which did ( figure 2 ) . clinical statistical analysis of the results revealed that the specificity of the diagnostic test was 64% , while the sensitivity was 100% . it is more informative that the positive predictive value of the assay was 51% and maybe more importantly the negative predictive value was 100% . receiver operating characteristic ( roc ) analysis provides tools to select possibly optimal models and to discard suboptimal ones . roc analysis is related in a direct and natural way to cost / benefit analysis of diagnostic decision - making . the roc curve of the morphological versus metabolomic approach in relation to the correct prediction of pregnancy outcome is illustrated in figure 3 . it is obvious that our biochemical investigation method enables a selection of the embryos by sorting out the non - viable ones . the test selected with 100% potential the embryos , which did not lead to successful implantation at all . one of the areas of collaboration between clinicians , the clinical laboratory and the research laboratory at the university of pcs is related to the research of infertility . since the clinical background gives the beauty and the medical importance of laboratory research , it was of outstanding importance for us to receive the eflm - abbott diagnostics award for excellence in outcomes research in laboratory medicine ( paris , 2015 ) , the award given to the best published paper ( 47 ) , as judged by an independent panel of experts , which demonstrates improved outcomes arising out of the application or improved utilization of an in - vitro diagnostics test .

human reproduction is a relatively inefficient process and therefore the number of infertile couples is high . assisted reproductive technologies ( art ) have facilitated the birth of over five million children worldwide . art , however , superimposes its own relative inefficiency on the preexisting inefficiency of normal reproduction . the efficiency ( expressed as pregnancy rate ) is generally not more than 30% . modern reproductive medicine is gradually moving from multiple embryo transfer to the transfer of a single embryo , mainly because of obvious and unwanted side effects of multiple embryo transfer ( e.g. epidemic multiple pregnancies ) . this concept , however , requires a fast , professional selection of the most viable embryo during the first few days of art . thus the aim of a modern art is the safe transfer of a healthy , viable , single embryo . accurate and rapid methods of quantifying embryo viability are needed to reach this goal . methodological advances have the potential to make an important contribution , and there has been a drive to develop alternative non - invasive methods to better meet clinical needs . metabolic and genetic profiling of spent embryo culture ( sec ) media should offer an exceptional opportunity for the assessment of embryo viability . the current review focuses on the latest non - invasive diagnostic approaches for pre - implantation viability assessment of in vitro fertilized embryos .

this study is part of a larger project examining the possible health effects of lifetime exposure to marine food and pollutants . the faroe islands constitute a unique setting at northern latitude in which the residents have an increased exposure to methylmercury and persistent organic pollutants , including polychlorinated biphenyls ( pcb ) , because of their intake of traditional marine food , including pilot whale meat and blubber . to examine subjects aged 7074 years all 1,131 faroese citizens born between 2 january 1934 and 31 august 1937 received a letter of invitation , 713 of whom gave consent and were examined , corresponding to a participation rate of 64% , taking into account 14 deceased subjects . furthermore , sufficient serum for determination of vitamin d status was not available from 44 subjects ; thus , 668 subjects are included in the current study . the participants were asked to fast and arrive before breakfast to undergo blood sampling and a thorough clinical examination . all participants traveled to the same examination location and were examined by the same research nurses and a single physician . the participants completed current health , past medical history , and lifestyle questionnaires , including summary information on the frequency of fish dinners during the last year . a fasting capillary blood sample was used to determine blood glucose concentration using a direct - reading glucose biosensor ( precision xceed and abbott precision xtra plus strips ; abbott diabetes care , copenhagen , denmark ) . because plasma has higher water content than whole blood , the blood values were transformed to plasma glucose concentration by multiplying by 1.12 as recommended by the manufacturer . fasting venous blood samples were collected by phlebotomy to perform hba1c analyses by high - pressure liquid chromatography . all hba1c values were given as relative concentration , % ( diabetes control and complications trial , dcct , aligned results ) . hba1c % values were converted to international federation of clinical chemistry and laboratory medicine ( ifcc ) units ( millimoles per moles ) by the following equation : hba1c ( dcct ) % = 0.0915 hba1c ( ifcc ) mmol / mol + 2.15 , provided by the laboratory . edta - plasma was used for determination of fasting plasma insulin concentration by time - resolved fluoroimmunoassay ( autodelfia ; perkinelmer life and analytical sciences , walla oy , turku , finland ) . serum hdl and triacylglycerides were measured by an enzymatic colorimetric reaction , using a modular p analyzer ( roche diagnostics , indianapolis , in ) . diabetic subjects were identified from self - reported doctor s diagnosis and the reported use of hypoglycemic medication ; subjects were also classified as diabetic if they reported use of diabetes medication without reporting a diagnosis of diabetes . in participants not previously diagnosed , type 2 diabetes was considered to be present if the fasting plasma glucose was 7.0 insulin resistance was estimated by the homeostasis model assessment ( homa - ir ) and calculated as fpi fpg / 22.5 ( 10 ) , where fpi is fasting plasma insulin and fpg is fasting plasma glucose . -cell dysfunction was determined by calculating the homa -index of ( 20 fpi)/(fpg 3.5 ) ( 10 ) . after storage at 80c , serum 25(oh)d3 concentrations were assessed using liquid chromatography - tandem mass spectrometry ( lc - ms / ms ) as described previously ( 11 ) . nmol / l , and the coefficient of variation was 9.6% at 20 nmol / l and 6.7% at 60 nmol / l . means ( sd ) were calculated for variables showing normal distribution and median and 2575th percentiles ( interquartile range [ iqr ] ) for those deviating from it . when transformations were insufficient to obtain normal distribution , we applied nonparametric spearman s correlation coefficient . when appropriate , multiple regression analysis was carried out . a logistic regression model was used to assess the association between serum 25(oh)d3 concentration and the risk of having type 2 diabetes . vitamin d status was used as a binary exposure variable , i.e. , dichotomized at above ( sufficient ) or below 50 nmol / l ( deficient ) , and in the simple model , sex was used as an obligate covariate . in the adjusted model , additional covariates were included , i.e. , bmi , serum triacylglycerides , serum hdl , pcb exposure ( sum of major pcb congeners cb-138 , cb-153 , and cb-180 multiplied by 2 ) , smoking , and month of blood sampling . in addition , we examined the association between vitamin d status and risk of having 1 ) newly diagnosed type 2 diabetes or 2 ) a known diagnosis of type 2 diabetes using the same models separately . finally , the same approach was used to estimate the influence of fish intake on risk of having type 2 diabetes using subjects reporting fish intake less than or equal to two times per week as a reference group . in the models , we further tested whether there was evidence of an interaction effect between vitamin d status and sex and vitamin d status and obesity . a value of p < 0.05 ( two - tailed ) was taken to indicate statistical significance . stata version 11.0 ( stata corporation , college station , tx ) was used for statistical analyses . a fasting capillary blood sample was used to determine blood glucose concentration using a direct - reading glucose biosensor ( precision xceed and abbott precision xtra plus strips ; abbott diabetes care , copenhagen , denmark ) . because plasma has higher water content than whole blood , the blood values were transformed to plasma glucose concentration by multiplying by 1.12 as recommended by the manufacturer . fasting venous blood samples were collected by phlebotomy to perform hba1c analyses by high - pressure liquid chromatography . all hba1c values were given as relative concentration , % ( diabetes control and complications trial , dcct , aligned results ) . hba1c % values were converted to international federation of clinical chemistry and laboratory medicine ( ifcc ) units ( millimoles per moles ) by the following equation : hba1c ( dcct ) % = 0.0915 hba1c ( ifcc ) mmol / mol + 2.15 , provided by the laboratory . edta - plasma was used for determination of fasting plasma insulin concentration by time - resolved fluoroimmunoassay ( autodelfia ; perkinelmer life and analytical sciences , walla oy , turku , finland ) . serum hdl and triacylglycerides were measured by an enzymatic colorimetric reaction , using a modular p analyzer ( roche diagnostics , indianapolis , in ) . diabetic subjects were identified from self - reported doctor s diagnosis and the reported use of hypoglycemic medication ; subjects were also classified as diabetic if they reported use of diabetes medication without reporting a diagnosis of diabetes . in participants not previously diagnosed , type 2 diabetes was considered to be present if the fasting plasma glucose was 7.0 insulin resistance was estimated by the homeostasis model assessment ( homa - ir ) and calculated as fpi fpg / 22.5 ( 10 ) , where fpi is fasting plasma insulin and fpg is fasting plasma glucose . -cell dysfunction was determined by calculating the homa -index of ( 20 fpi)/(fpg 3.5 ) ( 10 ) . after storage at 80c , serum 25(oh)d3 concentrations were assessed using liquid chromatography - tandem mass spectrometry ( lc - ms / ms ) as described previously ( 11 ) . nmol / l , and the coefficient of variation was 9.6% at 20 nmol / l and 6.7% at 60 nmol / l . means ( sd ) were calculated for variables showing normal distribution and median and 2575th percentiles ( interquartile range [ iqr ] ) for those deviating from it . when transformations were insufficient to obtain normal distribution , we applied nonparametric spearman s correlation coefficient . when appropriate , multiple regression analysis was carried out . a logistic regression model was used to assess the association between serum 25(oh)d3 concentration and the risk of having type 2 diabetes . control subjects were nondiabetic subjects , i.e. , subjects with fasting blood glucose 6 mmol / l . vitamin d status was used as a binary exposure variable , i.e. , dichotomized at above ( sufficient ) or below 50 nmol / l ( deficient ) , and in the simple model , sex was used as an obligate covariate . in the adjusted model , additional covariates were included , i.e. , bmi , serum triacylglycerides , serum hdl , pcb exposure ( sum of major pcb congeners cb-138 , cb-153 , and cb-180 multiplied by 2 ) , smoking , and month of blood sampling . in addition , we examined the association between vitamin d status and risk of having 1 ) newly diagnosed type 2 diabetes or 2 ) a known diagnosis of type 2 diabetes using the same models separately . finally , the same approach was used to estimate the influence of fish intake on risk of having type 2 diabetes using subjects reporting fish intake less than or equal to two times per week as a reference group . in the models , we further tested whether there was evidence of an interaction effect between vitamin d status and sex and vitamin d status and obesity . a value of p < 0.05 ( two - tailed ) was taken to indicate statistical significance . stata version 11.0 ( stata corporation , college station , tx ) was used for statistical analyses . in the current study , the cohort consisted of 327 women and 341 men with a mean age of 72.5 years . a total of 158 septuagenarians ( 24% ) had type 2 diabetes , of whom 88 ( 13% ) had been previously diagnosed with the disease and 70 new subjects ( 11% ) were identified in the current study . more than half of the subjects had vitamin d deficiency , as suggested by a serum 25(oh)d3 concentration below 50 nmol / l ( median , 47.6 nmol / l ; iqr , 29.864.8 nmol / l ) . baseline clinical and biochemical characteristics of 668 faroese septuagenarians by vitamin d status data are means ( sd ) or median ( 2575th percentile ) . * hba1c ( ifcc ) mmol / mol can be converted to hba1c ( dcct ) % by the following equation : hba1c ( dcct ) % = 0.0915 hba1c ( ifcc ) mmol / mol + 2.15 ; homa - ir = ( fasting plasma insulin fasting plasma glucose)/22.5 ; homa-% = ( 20 fasting plasma insulin)/(fasting plasma glucose 3.5 ) ; sum of major pcb congeners cb-138 , cb-153 , and cb-180 multiplied by 2 ( 12 ) . nmol / l , was associated with an 80% increase in the sex - adjusted odds of having diabetes ( newly diagnosed and previously diagnosed subjects ) compared with sufficient vitamin d status ( odds ratio [ or ] 1.80 [ 95% ci 1.232.64 ] ; p = 0.002 ) . after further adjustment for bmi , serum triacylglycerides , serum hdl , pcb exposure , smoking , and month of blood sampling , the association between vitamin d deficiency and type 2 diabetes remained , and the or was only marginally affected ( or 1.67 [ 1.112.50 ] ; p = 0.013 ) . in the separate analyses of previously undiagnosed and diagnosed subjects , we found that the sex - adjusted or for having newly diagnosed type 2 diabetes in the presence of vitamin d deficiency had more than doubled ( or 2.25 [ 95% ci 1.313.85 ] ; p = 0.003 ) . after further adjustment for bmi , serum triacylglycerides , serum hdl , pcb exposure , smoking , and month of blood sampling , the association between vitamin d deficiency and newly diagnosed type 2 diabetes remained unchanged ( or 1.99 [ 1.143.48 ] ; p = 0.022 ) , whereas the adjusted or for pre - existing diabetes in vitamin d deficiency was reduced considerably by similar adjustment ( or 1.48 ; p = 0.132 ) ( table 2 ) . we performed logistic regression analysis on the impact of fish dinner frequencies on the risk of type 2 diabetes . frequent fish intake ( more than twice a week ) did not appear to alter the odds of having type 2 diabetes ( or 1.09 [ 95% ci 0.641.86 ] ; p = 0.744 ) . obesity is a risk factor for vitamin d deficiency , and obese subjects had a lower serum 25(oh)d3 concentration than nonobese subjects ( 11 ) . furthermore , we have previously demonstrated in this cohort ( 11 ) that women have higher serum 25(oh)d3 concentration and tend to have lower bmi . however , we did not find any interaction between vitamin d status and sex ( p = 0.19 ) or vitamin d status and obesity ( p = 0.84 ) on risk of diabetes . statistically significant bivariate correlations were observed between serum 25(oh)d3 concentration and several indicators of glucose metabolism , including hba1c ( rs = 0.10 ; p = 0.01 ) , fasting plasma insulin ( rs = 0.10 ; p = 0.01 ) , homa - ir ( rs = 0.10 ; p = 0.01 ) , and homa - b ( r = 0.10 ; p = 0.01 ) , whereas the plasma glucose concentration ( rs = 0.01 ; p = 0.78 ) was not correlated with 25(oh)d3 concentration . however , because bmi was strongly associated with vitamin d status ( rs = 0.17 ; p < 0.0001 ) , the observed associations of 25(oh)d3 with fasting plasma insulin and the homa indexes were substantially reduced after adjustment for bmi , sex , smoking status , and serum triacylglyceride concentration , where none of them were statistically significant ( table 3 ) . however , hba1c decreased at higher serum 25(oh)d3 concentrations after adjustment ( = 0.026 , p = 0.026 ) . excluding subjects with physician - diagnosed type 2 diabetes resulted in a smaller effect that was no longer significant , probably because of reduced variation in hba1c concentration and fewer observations ( = 0.012 , p = 0.112 ) ( table 2 ) . finally , multiple regression analysis did not reveal any association between frequency of fish dinners and the hba1c concentration ( results not shown ) . unadjusted and adjusted or for type 2 diabetes in elderly faroese aged 7074 years data are or ( 95% ci ) expressed for binary traits . mmol / l ; the crude model is adjusted for sex and compares the odds of either overall type 2 diabetes ( all type 2 diabetic subjects ) , newly diagnosed type 2 diabetes ( new subjects ) , or known type 2 diabetes in subjects ( previously known subjects ) with 25(oh)d < 50 nmol / l ( reference group ) ; the adjusted model includes sex , bmi , total pcb , month of blood sampling , serum triacylglyceride concentration , serum hdl concentration , and smoking status . in this cross - sectional analysis of a population - based sample of septuagenarians , we found that the 25(oh)d3 concentration was inversely associated with the risk of newly diagnosed type 2 diabetes after adjustment for bmi and other established risk factors for type 2 diabetes . subjects with 25(oh)d3 concentration below 50 nmol / l had the doubled risk of newly diagnosed diabetes compared with those having higher serum concentrations . therefore , our data suggest an inverse association between the serum concentration of 25(oh)d3 and the odds of newly diagnosed type 2 diabetes , thereby suggesting a protective role of vitamin d against the development of the disease , as suggested previously ( 13 ) . in further support of this association , we observed an increasing concentration of hba1c with decreasing 25(oh)d3 concentration , and this association was independent of bmi , serum triacylglycerides , smoking status , and sex . vitamin d deficiency showed a much weaker or for type 2 diabetes previously diagnosed , as could perhaps have been anticipated , given the vigorous clinical management of the disease and the longer time interval between disease development and the assessment of vitamin d status . the confidence interval for the or for the two groups of type 2 diabetes continues to overlap considerably , and therefore , the slight difference should not be overinterpreted ( table 2 ) . linear regression coefficients for the association between 25(oh)d3 and markers of glucose metabolism in elderly faroese including or excluding subjects with known diabetes * numbers vary because of varying missing sample material for each biochemical analysis . is considered an indicator of average blood glucose concentrations during the preceding 2 to 3 months and , thus , a long - term marker of glucose homeostasis ( 14 ) . abnormalities may be a result of changes in insulin secretion and insulin - stimulated uptake of glucose in muscle and fat tissue . in vitro studies and laboratory animal studies suggest possible mechanisms for the effects of the active form of vitamin d , i.e. , 1,25(oh)2d , on both insulin secretion and insulin sensitivity as reviewed by pittas and dawson - hughes ( 15 ) . for instance , in mice expressing a functionally inactive mutant vitamin d receptor , blood glucose concentrations were increased and serum insulin was reduced , as was insulin mrna . pancreatic -cells express the vitamin d receptor and the enzyme 1--hydroxylase , thus suggesting that the active form of vitamin d has a functional role in these cells . even though the current study can not elucidate the likely mechanisms involved , our results appear highly plausible given the findings in experimental studies . increased fat mass adversely influences glucose metabolism , in part by increasing insulin resistance , and is also linked with lower 25(oh)d concentrations in serum ( 1618 ) . the results suggest that the negative impact of vitamin d deficiency on hba1c and newly diagnosed type 2 diabetes risk is independent of increased fat mass . however , because bmi does not distinguish visceral from subcutaneous fat , some misclassification of adiposity may have occurred , thereby possibly causing some uncertainty in regard to the relative influence of serum 25(oh)d3 and adiposity in diabetes development . our study results are comparable with studies on vitamin d status and type 2 diabetes in younger populations ( 4,16,19,20 ) , and the current study extends this association to 70 years of age and older . vitamin d status is known to be lower in the elderly compared with the young as the subcutaneous synthesis of vitamin d decreases with increasing age , due to a reduced concentration of 7-dehydrocholesterol in the skin ( 1 ) and reduced absorption of oral vitamin d ( 8) . hence , if the relationship between vitamin d status and glucose homeostasis is causal , the age - related decline in circulating concentration of 25(oh)d may augment the increase in risk of type 2 diabetes with age . in addition , our results suggest that this relationship is not the result of increased fish intake , in agreement with the existing , somewhat equivocal evidence in regard to this effect ( 21,22 ) . these differences may be a result of variations in fish intake to inherent differences in the populations examined and the study designs . among the strengths of our study , we examined a general population sample with a high prevalence of both vitamin d deficiency ( 11 ) and type 2 diabetes . however , the current study is limited by its cross - sectional design and its single measurement of vitamin d status . nonetheless , a recent study demonstrated excellent tracking and that the correlation of serum 25(oh)d3 concentrations sampled 14 years apart was similar to that seen for other cardiovascular risk factors and that most subjects with vitamin d deficiency were still deficient 14 years later ( 23 ) . patients with type 2 diabetes and elevated hba1c concentrations are at increased risk of cardiovascular disease and total mortality compared with patients with lower hba1c levels ( 24 ) . thus , clinical management focuses on glycemic control , including treatment of other modifiable cardiovascular risk factors to reduce the macro- as well as microvascular complications . the inverse association between serum 25(oh)d3 and hba1c suggests that vitamin d supplementation could be considered a possible means for supporting glycemic control of type 2 diabetes . however , some intervention studies have shown inconclusive results on the effect of vitamin d on hba1c and type 2 diabetes ( 15 ) . for example , the women health s initiative demonstrated that daily supplementation with 10 g of vitamin d3 for a median follow - up time of 7 years did not reduce the risk of type 2 diabetes in postmenopausal women ( 25 ) . however , the doses used were probably too low to bring about the necessary concentrations of circulating 25(oh)d3 . therefore , further randomized clinical intervention studies are needed to elucidate the impact of vitamin d on the risk of type 2 diabetes . in summary , our study extends previous findings that a high vitamin d status protects against type 2 diabetes in younger subjects to subjects older than 70 years . likewise , our findings suggest an inverse association between hba1c and 25(oh)d3 also in the elderly . because our study has a cross - sectional design , formal confirmation requires intervention studies with appropriate doses to elucidate whether vitamin d supplementation could be used to counter the worldwide epidemic of type 2 diabetes . in this regard , the need to identify the necessary daily intake of vitamin d should include the possible effect on reducing the risk of type 2 diabetes , with special emphasis on the dietary needs of the elderly .

objectivevitamin d deficiency is thought to be a risk factor for development of type 2 diabetes , and elderly subjects at northern latitudes may therefore be at particular risk.research design and methodsvitamin d status was assessed from serum concentrations of 25-hydroxyvitamin d3 [ 25(oh)d3 ] in 668 faroese residents aged 7074 years ( 64% of eligible population ) . we determined type 2 diabetes prevalence from past medical histories , fasting plasma concentrations of glucose , and/or glycosylated hemoglobin ( hba1c).resultswe observed 70 ( 11% ) new type 2 diabetic subjects , whereas 88 ( 13% ) were previously diagnosed . having vitamin d status < 50 nmol / l doubled the risk of newly diagnosed type 2 diabetes after adjustment for bmi , sex , exposure to polychlorinated biphenyls , serum triacylglyceride concentration , serum hdl concentration , smoking status , and month of blood sampling . furthermore , the hba1c concentration decreased at higher serum 25(oh)d3 concentrations independent of covariates.conclusionsin elderly subjects , vitamin d sufficiency may provide protection against type 2 diabetes . because the study is cross - sectional , intervention studies are needed to elucidate whether vitamin d could be used to prevent development of type 2 diabetes .

leaf senescence is the last phase of plant development and a highly coordinated process regulated by a large number of senescence associated genes ( sags ) ( 12 ) . leaf senescence can either be naturally induced during development stages , or stimulated by environmental factors including darkness , nutritional deficiency and various stresses ( 13 ) . premature senescence is an important factor leading to the decrease of crop yield and quality , which becomes an increasing concern due to the global climate change in the recent years . many advances in the understanding of leaf senescence at the molecular level had been achieved through the identification and characterization of hundreds of sags and senescence - related mutants in arabidopsis thaliana , lycopersicon esculentum and nicotiana tabaccum ( 14 ) . microarray expression profiling in arabidopsis revealed that more than 800 genes are up - regulated during the course of leaf senescence ( 5 ) . among them , more than 200 transcription factors , including wrky , nac , mads , myb , bzip and bhlh family members , are involved in the regulation of leaf senescence , indicating that leaf senescence is governed by complex transcriptional regulatory networks . molecular and genetic studies of leaf senescence in recent years led to the accumulation of a large volume of scattered information related to sags . the construction of a leaf senescence related database with wide - spread collection and systematic annotation of sags may provide a useful resource and a good starting point for the further study of the molecular aspects of leaf senescence . some initial efforts to this end had been made ; including the online website of plant senescence network ( sennet ) constructed by thomas and his colleagues ( http://www.sidthomas.net/plant_senescence/ ) and the corresponding senwiki web pages ( http://www.sidthomas.net/senwiki/ ) . sennet brings together various community information including meetings , laboratories , websites and useful links related to plant senescence , while senwiki provides general information and knowledge of senescence with texts and images . however , a wide - ranging compilation and detailed annotation of known sags that would be of great help and demand to the systemic study of leaf senescence at the molecular level has yet to be done . in this regard , we have developed a leaf senescence database ( lsd ) ( http://www.eplantsenescence.org/ ) by retrieving and integrating information from research papers and public databases . at present 1145 sags from 21 species were manually curated and categorized into several groups according to their function . users can browse the entries in the database to obtain information including literatures , mutants , phenotypes , expression profiles , mirna interactions , orthologs in other plants and cross links to protein domain and family databases . users can also search the database easily through the text search interface with locus names , keywords and author names , etc . we have implemented the blast tool kit for sequence similarity search against nucleotide or protein sequences of these sags in the lsd . a major feature of the lsd is the integration of a bioinformatics platform weblab we had developed previously ( 6 ) , which allows users to perform extensive sequence analysis of the sags they are interested in . all the sag sequences are freely available for downloading . help information including user guides , a tutorials and faqs are also available online . we plan to identify putative sags from completely sequenced genomes and add them into the database in the near future , and improve the user interface based on user feedback , provide more help documents with case studies , add more links to other useful sites , and update the database in time with more leaf senescence related data available . we hope that lsd could be a useful resource for the leaf senescence research community , as well as a gateway for the collaborative project we are working with both domestic and international colleagues . we made an extensive literature survey and collected approximately 200 leaf senescence related papers published before october 2010 on major plant biology journals including plant cell , plant physiology , plant journal , new phytologist , journal of experimental botany , among others . a total of 1145 sags from 21 species including arabidopsis , rice and so on , were identified and manually verified based on genetic , genomic , proteomic , physiological and other experimental evidence . among them , 96 and 58 genes were supported by mutational investigation or transgenic over - expression study , respectively ( table 1 ) . table 1.number of sags and mutants in 21 speciesspeciescommon namesagsmutantstransgenicarabidopsis thalianathale cress9499035oryza sativarice10439medicago truncatulabarrel clover3100brassica napusrape1500lycopersicon esculentumtomato803nicotiana tabacumtobacco503brassica oleraceabroccoli400pisum sativumpea410glycine maxsoybean401sorghum bicolorsorghum400solanum tuberosumpotato303zea maysmaize300hordeum vulgarebarley300astragalus sinicuschinese milk vetch101chenopodium rubrumred goosefoot110festuca pratensis huds.fescue110ipomoea niljapanese morning glory101medicago sativaalfalfa101rosa hybridrose100triticum turgidumwheat101triticum aestivumwheat100total2111459658 number of sags and mutants in 21 species the information of 154 leaf senescence related mutants such as name , ecotype and mutagenesis method were also retrieved from literatures . expression profiling data were acquired from a classical and systemic research paper ( 5 ) . in addition to manual curation , computational approaches were also employed to annotate these sags . we predicted the potential mirna targets for the sags using the rnahybrid method ( 7 ) . the orthologs of each sag in other plants were retrieved from the online database orthomcl - db ( 8) . finally , putative function domains of sags - encoding proteins were identified with interproscan ( 9 ) . to meet the general requirement of data analysis , we integrated the sequence similarity search tool blast ( 10 ) and the sequence analysis platform weblab ( 6 ) into our leaf senescence database . users can either retrieve the sequence from lsd , or upload their own sequences to search homologs against different divisions ( gene , mrna , cds and protein ) in the lsd . weblab is a web - based bioinformatics platform developed by our center and publicly available worldwide ( http://www.weblab.org.cn/ ) ( 6 ) . a user space is provided to save input data and analysis results for registered users . users may retrieve sequence data and submit to weblab directly to perform extensive analysis for dna , mrna and protein sequences of the sags they are interested in . the lsd database enables users to retrieve and analyze sags through the browse or search page . users may browse the entries by clicking the buttons of species , mutants or phenotypes at the main page . a tree - like structure was designed for both species and phenotypes , and a table was created for mutants . currently , the major source of sags was from the two model organisms a. thaliana ( 949 entries ) and oryza sativa ( 104 entries ) . the phenotypes of all sags were divided into the following groups : ( i ) natural senescence , ( ii ) dark induced senescence , ( iii ) nutrition deficiency induced senescence , ( iv ) stress induced senescence and ( v ) others . the text search interface allows users to make queries with three types of data : ( i ) locus name , genbank i d , alias , species and description of genes ; ( ii ) name , type and ecotype of mutants ; and ( iii ) title , author , journal and date of literature papers . figure 1 shows the annotation for a typical lsd entry ntl9 , a member of the nac transcription factor family and a membrane - associated gene that mediates osmotic stress signaling in leaf senescence ( 11 ) . general information such as locus name , alias , organism , taxonomy was retrieved from the literature . functional category , effect and evidence of senescence , as well as a brief description of this gene were manually annotated ( figure 1a ) . expression profiles generated from microarray data can be found for most arabidopsis sags ( figure 1c ) . we predicted potential mirna targets for some sags and added links to mirbase ( 12 ) for these mirnas ( figure 2a ) . orthologs from other plants are listed with links to the orthomcl database ( figure 2b ) . putative functional domains of proteins encoded by sags were identified and annotated using the interproscan program ( 9 ) , and matches were displayed with cross links to several protein domain and family databases such as prosite and pfam ( figure 2c ) . ( a ) basic information , ( b ) mutant information and ( c ) expression profile . figure 2.computational annotations for the arabidopsis nac transcription factor . ( a ) mirna targets , ( b ) ortholog groups and ( c ) cross links to other databases . a typical entry in lsd , the arabidopsis nac transcription factor . ( a ) basic information , ( b ) mutant information and ( c ) expression profile . ( a ) mirna targets , ( b ) ortholog groups and ( c ) cross links to other databases . in addition to sequence similarity search with the blast tool kit implanted in the database , users may also perform extensive analysis for sequence data retrieved from the lsd . for each entry , links to different sequence types ( genomic , mrna , cds and protein ) are provided . users can click these links to bring up the corresponding sequence and submit it to weblab for further analysis , such as predicting gene structures , making pairwise or multiple sequence alignment , generating sequence logos , constructing phylogenetic trees , finding sequence motifs , etc . we will update the database regularly with more leaf senescence related data available , and predict putative sags from completely sequenced plant genomes in the near future . we will improve the user interface with comments and suggestions from the user community and add more documents including case studies to help user to make thorough analysis of sags . we hope that lsd can be a platform not only for the domestic and international collaborators we are working with , but also for the research community of leaf senescence worldwide . national natural science foundation of china ( 31071160 to j.l . , 30625003 and 30730011 to h.g . ) ; ministry of science and technology of china ( 2009cb119101 to h.g . ) ; ministry of education of china ( ngi2008 - 108 - 3 to j.l . , ed20060047 to h.g . ) . funding for open access charge : national laboratory of protein engineering and plant genetic engineering .

by broad literature survey , we have developed a leaf senescence database ( lsd , http://www.eplantsenescence.org/ ) that contains a total of 1145 senescence associated genes ( sags ) from 21 species . these sags were retrieved based on genetic , genomic , proteomic , physiological or other experimental evidence , and were classified into different categories according to their functions in leaf senescence or morphological phenotypes when mutated . we made extensive annotations for these sags by both manual and computational approaches , and users can either browse or search the database to obtain information including literatures , mutants , phenotypes , expression profiles , mirna interactions , orthologs in other plants and cross links to other databases . we have also integrated a bioinformatics analysis platform weblab into lsd , which allows users to perform extensive sequence analysis of their interested sags . the sag sequences in lsd can also be downloaded readily for bulk analysis . we believe that the lsd contains the largest number of sags to date and represents the most comprehensive and informative plant senescence - related database , which would facilitate the systems biology research and comparative studies on plant aging .

female study participants were recruited from participants in a cross - sectional study assessing bmd in young women with type 1 diabetes and age - matched community control subjects ( 7 ) . cases for the cross - sectional study ( baseline ) were recruited from a regional tertiary hospital pediatric diabetes center and from endocrinology practices in western new york . at the time of the initial study , the diabetes center followed over 600 patients aged 221 years with type 1 diabetes . female patients aged 1321 years in that clinical center were offered the opportunity to participate in the baseline study ( n = 138 ) . recruitment of older women involved contacting former center patients ( aged > 21 years ) , patients from regional endocrinology practices , and affected relatives of subjects screened by our diabetes center for the diabetes prevention trial . the comparison group included young women recruited from the same region as the case subjects ; most were classmates or acquaintances of the case subjects or were volunteers who learned of the study through flyers posted at the university and hospital . the baseline study enrolled 72 case subjects ( diabetes duration > 2 years ) and 91 control subjects aged 1337 years who were at least 2 years postmenarchal ( 7 ) . all type 1 diabetic case subjects and nondiabetic control subjects who participated in the baseline study were recruited 2 years later to assess change in bmd over time . contact was maintained with study participants between the baseline and follow - up exam via phone and mail . inclusion criteria for the follow - up study were participation in the baseline study , current negative pregnancy test , and signed informed consent . for individuals younger than 18 years of age , exclusion criteria for both the baseline and follow - up study included systemic illness affecting bmd ( other than diabetes ) , other endocrine disorders ( except autoimmune thyroiditis ) , and diagnosis of juvenile osteoporosis or other bone disease . the study was approved by the institutional review boards of the women and children 's hospital of buffalo and the university at buffalo . at baseline , study participants completed questionnaires relating to personal and family health , lifestyle habits , dietary intake ( food frequency questionnaire ) , and medication intake including use of calcium supplements . participants also reported prior fracture history , menstrual history , and demographic information . for participants with type 1 diabetes , information on disease duration , insulin dose schedule ( injections versus continuous subcutaneous insulin infusion ) , and diabetes complications were reported . bmi was calculated as weight in kilograms divided by the square of height in meters . dexa ( hologic qdr-4500a ; hologic , waltham , ma ) was used to measure bmd in the anterior / posterior ( l14 ) spine , total hip , femoral neck , total forearm , and whole body . a single technician performed all baseline scans and 65% of the follow - up scans ; two additional technicians performed the remainder of the follow - up scans ( 27% and 7% , respectively ) . coefficients of variation ( cvs ) were determined for measures of all sites for all technicians throughout the study and were all < 1% throughout the study . the following biomarkers were measured in nontimed blood samples : serum osteocalcin , igf-1 , igf binding protein ( igfbp)-3 ( by radioimmunoassay ) , and a1c ( by high - performance liquid chromatography with bio - rad variant ; bio - rad , richmond , ca ) . random urinary n - teleopeptide levels were measured by enzyme - linked immuno - absorbent assay . serum for hormonal assays was frozen at 80c and stored until sent for analysis in batches to esoterix laboratory ( calabasas hills , ca ) . a1c ( fresh plasma ) was assayed in a sequential fashion at the women and children 's hospital of buffalo laboratory ( kaleida health ) . intra - assay cv was < 3% for n - telopeptide , 6% for igf-1 and osteocalcin , and 13% for igfbp-3 . interassay cv was < 7% for n - telopeptide , < 9.7% for igf-1 , 13% for osteocalcin , and < 17% for igfbp-3 ( esoterix laboratories ) . data from study participants were analyzed in stratum based on age at initial enrollment ( 7 ) . demographic , lifestyle , metabolic characteristics , and bmd were compared between diabetic case subjects and nondiabetic control subjects , stratified by age . comparisons were made for baseline differences , differences at follow - up , and percent change between the two time points . results were presented as both unadjusted comparisons and comparisons of adjusted means ( adjusted for age , bmi , and oral contraceptive [ oc ] use ) . unadjusted differences for continuous variables were examined using student 's t test and for categorical variables using the test . data analyses were performed using sas version 8 ( sas , cary , nc ) . post hoc power analysis of follow - up total hip bmd ( adjusted for age , bmi , and oc use ) indicated that our dataset of 63 case subjects ( n = 37 , < 20 years of age ) and 85 control subjects ( n = 36 , < 20 years of age ) gave our study 80% power to detect a difference between case and control subjects with an of 0.05 and a target effect size of 0.45 in women < 20 years of age and 0.63 in those > 20 years of age . table 1 includes baseline and follow - up characteristics of women who participated in this follow - up study . for comparative purposes , the follow - up group included 63 women with type 1 diabetes ( 58.7% < 20 years of age ) and 85 control subjects without diabetes ( 42.4% < 20 years of age ) at ages 1539 years at enrollment . of the participants in the baseline study with diabetes , 87.5% ( 63 of 72 ) participated in this follow - up study , and 93.4% ( 85 of 91 ) of control subjects participated in both examinations . more subjects from the baseline cohort 20 years of age were lost to follow - up ( 13.8 vs. 3.94% , cohort < 20 years of age ) , with the highest attrition being from individuals with diabetes 20 years of age ( 21.2% ) . participants in this follow - up study were predominantly non - hispanic caucasian ( 95% ) and were similar in age at menarche and years since menarche . participants 20 years of age with diabetes used oc therapy longer than control subjects ( p 0.05 ) . although few participants were smokers , more women with diabetes reported current smoking ; there were no smokers among the control subjects < 20 years of age . at follow - up , women with diabetes , particularly the older women , continued to have higher bmi than control subjects . weight change ( in percent ) was not significantly different over the follow - up period . as reported previously , more patients in the older cohort were using continuous subcutaneous insulin infusion ( p < 0.01 , data not shown ) . while a trend toward lower daily insulin requirement was present in the older cohort ( 0.86 0.42 vs. 0.75 0.22 units kg day ) , that difference was not statistically significant . subjects 20 years of age were more likely to have diabetes - related complications , although no statistical differences were found . table 2 presents bmd values at baseline and follow - up for each body site measured . unadjusted means and means adjusted for age , bmi , and oc use are shown stratified by age - group and diabetes diagnosis . in those < 20 years of age , both case and control subjects had an increase in bmd over the 2-year interval . the percent increase in bmd from baseline to follow - up was not statistically different between younger subjects and control subjects . for all participants in the population 20 years of age , the bmd from baseline to follow - up remained stable . significant differences in bmd were identified at the total hip , femoral neck , and whole body in the case subjects 20 years of age compared with the control subjects . as in the previous report , no differences in bmd were seen between groups in the younger cohort . in the adjusted model ( for age , bmi , and oc use ) , bmd values at the total hip ( baseline , p = 0.003 ; follow - up , p = 0.007 ) and femoral neck ( baseline and follow - up , p < 0.001 ) were significantly lower in older women with diabetes compared with control subjects . after adjustment , the whole - body bmd was no longer significantly different between case and control subjects ; however , both age - groups showed a trend toward lower whole - body bmd in the case subjects . further adjustment for dietary intake of calcium and vitamin d as well as physical activity did not alter the bmd results ( data not shown ) . when the analysis was restricted to nonsmokers , the adjusted means changed very little . for the older cohort , however , the p values were attenuated for the total hip ( p = 0.017 ) and whole body ( p = 0.215 ) . in nonsmokers > 20 years of age , the percent change difference at the total hip became more pronounced between case and control subjects and reached statistical significance ( p = 0.008 ) . overall , igf-1 levels were lower for all groups compared with baseline ( table 3 ) . this is consistent with a physiologic decrease in igf-1 in postteenage years ( esoterix normative data 11 ) . at the 2-year follow - up , however , the igf-1 level was significantly lower in the younger diabetic subjects than in the control subjects , although still within the normal range . igf-1 levels were not statistically different between diabetic participants and control subjects in the older cohort . within the older age - group , igfbp-3 levels were lower in participants with diabetes than in control subjects ( p < 0.05 ) . igfbp-3 levels were comparable for control and diabetic subjects in the cohort < 20 years of age . serum osteocalcin levels were lower at follow - up for all groups , even with increased bone mineral accrual in the group < 20 years of age ( table 3 ) . there was no difference in osteocalcin or n - teleopeptide levels between case and control subjects < 20 years of age ( table 3 ) . after adjusting for age and bmi , case subjects had significantly lower osteocalcin levels than control subjects in the population 20 years of age ( p < 0.04 ; table 3 ) . in addition , the percent change in osteocalcin level from baseline to follow - up was significantly different in diabetic women 20 years of age compared with control subjects after adjustment ( p < 0.03 ) . markers of bone formation did not correlate with bmd in our subjects . metabolic control , as measured by a1c , was poorer in the younger diabetic cohort , as we reported previously ( 7 ) . overall metabolic control was stable within a given age stratum between the baseline and follow - up studies . no correlation was found between a1c and any of the bmd measurements ( data not presented ) . when evaluating for associations between metabolic control and bmd measurements that were statistically lower in case than in control subjects at follow - up ( table 2 ) , the pearson correlation coefficient ( r value ) for a1c and bmd was between 0.121 ( total hip ) and 0.334 ( femoral neck ) ( p > 0.095 , data not shown ) . as in our previous study , there was no association found between bmd and diabetes duration , baseline a1c , or years since menarche . while longitudinal studies have examined the effect of insulin therapy on bmd in patients with long - standing type 1 diabetes ( 12 ) , few have reported on the natural history of metabolic bone disease in younger women . here we present bmd data for a well - characterized cohort of young women with type 1 diabetes at baseline and 2 years later . the baseline study demonstrated that the older cohort had significantly lower bmd at the femoral neck and lateral spine than age - matched control subjects ( 7 ) . the data presented here demonstrate that case subjects in the older cohort had persistently lower bmd than age - matched healthy control subjects . in this cohort , bmd at the total hip and femoral neck remained lower than in control subjects after adjusting for age , bmi , and oc use , even with no significant decrease in bmd from baseline to follow - up ( table 2 ) . similar to our initial study , there was no difference in bmd between women with diabetes aged < 20 years and control subjects at any of the sites measured . however , there was a trend toward lower bmd at the total hip and whole body in women with diabetes . this difference occurred in the face of bmd increases ( percent change ) at both the total hip and whole body in this population , suggesting that bone mineral accrual is altered in a subtle manner during late adolescence in women with type 1 diabetes . individuals with type 2 diabetes have higher than average bmd compared with control subjects ( 13 ) , in part due to the mechanical load of obesity . in our study , women with type 1 diabetes had higher bmi than control subjects . however , bmd continued to be lower in older women with type 1 diabetes , even after adjusting for bmi ; thus , overweight status does not confer protection against poor bone mineralization in these young women . although the younger cohort had poorer diabetes control than the older cohort , we were again unable to demonstrate an association between bmd measures and metabolic control , as measured by a1c . others have reported similar findings with respect to bmd in children and adolescents with diabetes ( 14 ) . a1c measures short - term diabetes control ; perhaps cumulative life - time glycemic control is a better indicator of osteoporotic risk . this hypothesis is supported by studies demonstrating that decreased lumbar spine and femoral neck bmd in adults with type 1 diabetes is associated with retinopathy , nephropathy , and peripheral neuropathy , all of which are long - term complications of poor metabolic control ( 1517 ) . in addition , our data did not find a correlation between diabetes duration and bmd , further supporting a possible relation to lifetime diabetes control . the adverse effects of chronic hyperglycemia related to osteoblast function , osteoclast activity , and formation of advanced glycosylated end products that may impact bone quality as well as bone quantity ( 18 ) are difficult to measure in a systematic fashion . the role of insulin as a direct anabolic agent in bone metabolism is unclear ( 19 ) . animal models of spontaneous and pharmacologically induced diabetes demonstrate that as insulinopenia develops , there is a suppression of osteoblast markers ( 20 ) . it has been postulated that in addition to insulinopenia , relative igf-1 deficiency , whether systemic or local , contributes to low bmd in diabetes . igf-1 levels are lower in individuals with type 1 diabetes , and poor glycemic control negatively impacts igf-1 production by the liver . in adults with type 1 diabetes , igf-1 levels were lower in individuals with osteopenia at the femoral neck ( 1 ) . additionally , in a study of 127 children ( aged 620 years ) with diabetes , low bone mineral content correlated with low igf-1 levels ( 9 ) . in our study , igf-1 levels of case subjects were significantly lower than those of control subjects only for the younger cohort . however , igf-1 levels of subjects with diabetes were lower in the group 20 years of age compared with the group < 20 years of age , and igfbp-3 levels in the older cohort were significantly lower compared with control subjects . given that no biochemical markers of bone metabolism correlated with bmd at the hip or femoral neck in our study , it could be hypothesized that a cumulative history of insulinopenia and low igf-1 levels are better correlates for low bone density . our data demonstrate significant differences in bmd at the femoral neck and total hip that are likely to be clinically significant and may explain why women with type 1 diabetes have an increased risk of hip fractures later in life ( 4,5 ) . our data also indicate that alterations in bone mineral accrual occur within years of achievement of peak bone mass and that there is not a later period of catch - up bone mineralization . the most clinically relevant factor is whether a decrease in bmd correlates with fracture risk , which can not be determined in this study . besides bmd , multiple factors impact fracture risk including , but not limited to , age , nutrition status , smoking history , degree of frailty , and fracture history ( 21 ) . the well - characterized population , the large percentage of women who participated in the follow - up study , and the standard procedures used in this longitudinal study all represent significant strengths . yet , we acknowledge certain limitations . the study population is small , limiting its ability to detect true differences that may still exist . study control subjects were community based and may not represent the general population of women . finally , the results presented should be interpreted keeping in mind that multiple comparisons were made . we are aware of the lack of unanimity of opinion regarding the statistical approach when adjusting the statistical testing for many comparisons ( 22 ) . because many of our hypotheses are nested , and not independent , we feel that using a bonferroni approach is overly conservative . however , we have interpreted our results with caution and believe they are stronger when considered as a whole rather than as independent statistical tests . our findings demonstrate lower bmd in young women with type 1 diabetes compared with control subjects . although bone density testing is not routinely performed in young women , these data suggest that screening may be important in young women with type 1 diabetes . in addition , these women should be counseled regarding lifestyle interventions that may improve bone health , including adequate intake of calcium and vitamin d , and exercise .

objective individuals with type 1 diabetes have decreased bone mineral density ( bmd ) , yet the natural history and pathogenesis of osteopenia are unclear . we have previously shown that women with type 1 diabetes ( aged 1335 years ) have lower bmd than community age - matched nondiabetic control subjects . we here report 2-year follow - up bmd data in this cohort to determine the natural history of bmd in young women with and without diabetes.research design and methods bmd was measured by dual - energy x - ray absorptiometry at baseline and 2 years later in 63 women with type 1 diabetes and in 85 age - matched community control subjects . a1c , igf-1 , igf binding protein-3 , serum osteocalcin , and urine n - teleopeptide were measured at follow-up.resultsafter adjusting for age , bmi , and oral contraceptive use , bmd at year 2 continued to be lower in women 20 years of age with type 1 diabetes compared with control subjects at the total hip , femoral neck , and whole body . lower bmd values were observed in cases < 20 years of age compared with control subjects ; however , the differences were not statistically significant . lower bmd did not correlate with diabetes control , growth factors , or metabolic bone markers.conclusionsthis study confirms our previous findings that young women with type 1 diabetes have lower bmd than control subjects and that these differences persist over time , particularly in women 20 years of age . persistence of low bmd as well as failure to accrue bone density after age 20 years may contribute to the increased incidence of osteoporotic hip fractures seen in postmenopausal women with type 1 diabetes .

many studies have shown that bracket debonding can cause enamel loss , particularly when the fracture occurs at the enamel - adhesive interface . enamel damage may be in the form of enamel cracks , which may propagate during debonding [ 13 ] . acid - etching , rinsing , drying and applying resin and bonding agents are the routine steps of conventional orthodontic bracket bonding . concurrent with studies on the efficacy of the conventional technique , many studies , aimed at reducing the number of steps necessary for adhesion , have examined new bonding techniques with clinically adequate bond strength but less etching depth [ 56 ] . traditional methods of bonding orthodontic brackets to teeth rely on acid etching to achieve adequate retention . however , maintaining a sound enamel surface after bracket removal is of primary concern to clinicians . bond failure at the bracket - adhesive interface or within the adhesive is generally considered safer than a fracture at the enamel - adhesive interface ; this is because studies have demonstrated that enamel fracture can occur during debonding . on the other hand , if a considerable amount of adhesive remains on the tooth surface after debonding , more chair time is required to remove the residual adhesive . in addition , the process of removing the residual adhesive results in further enamel loss . all - purpose or multi - purpose , self - adhesive resin cements are now commercially available in the market . they are capable of bonding to different substrates such as enamel , dentin , amalgam , metal and porcelain [ 910 ] . these cements require no surface pre - treatment due to their monomer and filler content and initiation technology . the phosphoric acid methacrylates react with basic fillers in the luting cement and the hydroxyapatite of the tooth structure [ 1112 ] . these self - adhesive cements do not require an etch and rinse phase as they are capable of conditioning the tooth surface and simultaneously preparing it for adhesion . this not only shortens the clinical application time , but also significantly reduces technique - sensitivity and risk of errors during application or manipulation . the aim of the current study was to assess the enamel cracks after debonding of brackets bonded with two different adhesive systems and also to evaluate enamel cracks after removing adhesive remnants and enamel polishing . sixty upper central incisors , extracted due to periodontal disease , were selected and stored in normal saline at room temperature until required . the study design was approved by the ethics committee of mashhad university of medical sciences . using a stereomicroscope ( blue light industry , waltham , ma , usa ) and digital camera ( exwave had , sony corporation , japan ) a magnified image ( 23.9x ) the number of enamel cracks and the length and direction of each crack were assessed with the aid of adobe photoshop cs software ( adobe systems incorporate , usa ) ( figure 1 ) . enamel cracks before bracket bonding ( a ) , after deboning ( b ) and after polishing ( c ) . new cracks are hatched . the number of visible cracks ( i.e. cracks visible to the naked eye ) was also recorded . the teeth were then randomly divided into two equal groups . in the first group , the buccal surface of each central incisor a standard edgewise twin metal bracket ( dentaurum , pforzheim , germany ) was bonded with transbond xt ( 3 m , usa ) to the center of the labial surface of each tooth . in the second group , the same bracket was bonded with self - adhesive composite cement ( maxcem elite , kerr , usa ) . in both groups , the adhesive was cured for 40 seconds ( 10 seconds on each side of the bracket ) . the teeth were then incubated in distilled water at 37c for 24 hours to allow the material to set . all the brackets were debonded with rmo i-546 remover pliers ( rmo , germany ) . the blades of the pliers were placed at the bracket - adhesive interface and a gentle squeezing force was applied to the pliers by a zwick / z250 at a speed of 0.5 mm / min until bond failure occurred . the present method of force application was modified from a diametric compression test for tension that indirectly measures tensile strength . this approach was used because it simulates the usual clinical method for applying debonding forces . the debonding force recorded by the zwick is not equal to the actual force applied at the bracket adhesive interface because the force was applied at a predetermined distance on the pliers beaks . these forces , however , are proportional and can be expressed in the following ratio : the actual force ( f ) equals the measured force ( f ) multiplied by ( a ) divided by ( b ) : f= f ( a / b ) . in this ratio , ( a ) is the distance from the point of force application to the fulcrum of the pliers and ( b ) is the distance from the point of actual force application to the fulcrum of the pliers . deboning force was measured in newton and was then converted to megapascal ( mpa ) . after debonding , the buccal surfaces of the teeth in each group were examined by the same stereomicroscope and the magnified images ( 23.9 x ) were taken with the same digital camera . the number of enamel cracks and the length and the direction of each crack were assessed with the aid of the same software . a : the distance from the point of force application to the fulcrum of the pliers . b : the distance from the point of actual force application to the fulcrum of the pliers the number of visible cracks in each tooth was also recorded ( figure 1 ) . the adhesive remnants on each tooth were then assessed with the ari in a 03 scale : 0 indciates no composite left on the tooth ; 1 indicates less than half of the composite left on the tooth , 2 indicates that more than half of the composite is left on the tooth surface and 3 shows that all the composite is left on the tooth surface with a distinct impression of the bracket base . the buccal surface of each tooth was polished with 12-blade tungsten carbide bur ( g&h , usa ) under water coolant . the enamel surface was examined for the third time in the same way ( figure 1 ) . the amount of shear bond strength in each group was compared with independent t - test . the enamel cracks in each group were compared before bonding , after debonding and after polishing using repeated measures anova . friedman and mann - whitney tests were applied in case of non - normal distribution of data . the result of the independent t - test indicated that the mean shear bond strength of maxcem elite ( the self - adhesive composite cement ) was significantly lower than that of transbond xt ( the adhesive composite ) ( p<0.001 ) . the mean sbs of maxcem elite and transbond xt was 10.291.14 and 11.481.09 , respectively . there were no significant inter - group differences in the number of enamel cracks before debonding . there were significantly more enamel cracks in the transbond xt group after debonding and polishing compared to the maxcem elite group . in the maxcem elite group , the difference in the number of enamel cracks before and after debonding was statistically significant ( p<0.001 ) . the difference , however , was not significant after debonding and after polishing ( p=0.06 ) . the number of visible enamel cracks followed a similar pattern . in each group , before and after debonding , a significant intra sd : standard deviation significant difference significant difference between a and b ( wilcoxon test ) however , before and after polishing , the difference was not significant ( table 2 ) . sd : standard deviation significant difference significant difference between a and b ( wilcoxon test ) there was no significant difference in the length of enamel cracks between the two groups . in each group , however , a significant increase in the length of enamel cracks was obvious after debonding . repeated measures test sd : standard deviation significant difference significant difference between a and b ( wilcoxon test ) the mann - whitney - u test showed that the differences were statistically significant among the groups ( p=0.0001 ) ( table 4 ) . the adhesive remnant index ( ari ) ( 0 , no composite left on the tooth ; 1 , less than half of the composite left on the tooth ; 2 , more than half of the composite left on the tooth ; 3 , all the composite left on the tooth with a distinct impression of the bracket base ) the result of the independent t - test indicated that the mean shear bond strength of maxcem elite ( the self - adhesive composite cement ) was significantly lower than that of transbond xt ( the adhesive composite ) ( p<0.001 ) . the mean sbs of maxcem elite and transbond xt was 10.291.14 and 11.481.09 , respectively . there were no significant inter - group differences in the number of enamel cracks before debonding . there were significantly more enamel cracks in the transbond xt group after debonding and polishing compared to the maxcem elite group . in the maxcem elite group , the difference in the number of enamel cracks before and after debonding was statistically significant ( p<0.001 ) . the difference , however , was not significant after debonding and after polishing ( p=0.06 ) . the number of visible enamel cracks followed a similar pattern . in each group , before and after debonding , a significant intra sd : standard deviation significant difference significant difference between a and b ( wilcoxon test ) however , before and after polishing , the difference was not significant ( table 2 ) . sd : standard deviation significant difference significant difference between a and b ( wilcoxon test ) there was no significant difference in the length of enamel cracks between the two groups . in each group , however , a significant increase in the length of enamel cracks was obvious after debonding . repeated measures test sd : standard deviation significant difference significant difference between a and b ( wilcoxon test ) the mann - whitney - u test showed that the differences were statistically significant among the groups ( p=0.0001 ) ( table 4 ) . the adhesive remnant index ( ari ) ( 0 , no composite left on the tooth ; 1 , less than half of the composite left on the tooth ; 2 , more than half of the composite left on the tooth ; 3 , all the composite left on the tooth with a distinct impression of the bracket base ) the main objective of this study was to evaluate the enamel cracks after debonding brackets , bonded with two different bonding systems followed by polishing . the sbs of maxcem elite , a self - adhesive cement , and transbond xt , a conventional adhesive composite , was also compared . the number and length of enamel cracks before bonding , after debonding and after polishing were also compared as well as the ari following the removal of orthodontic brackets . before bonding zacchrison et al . showed that debonding of brackets created enamel cracks , which were different in size and direction . reported a significant increase in the number and length of enamel cracks after debonding with different pliers . stated that 82% of the teeth studied did not show significant increase in the number of enamel cracks after debonding and the teeth showing increased number of cracks had significantly greater mean bond strength . after polishing , the number and length of enamel cracks increased in all groups , although there were no differences among the groups . according to zarrinnia et al . , debonding pliers cause resin fracture at the adhesive - bracket interface and using a tungsten carbide bur , accompanied by water coolant , effectively removes all adhesive remnants . they showed that the smoothest surface was obtained with the gloss polishing paste . a direct correlation between ari and bond strength the current study found significant differences among the ari scores and concluded that , in the transbond xt group , debonding was mostly of cohesive type , in contrast to the adhesive mode in the maxcem elite group . reported that self - adhesive cements had satisfactory bond strength to dentin and restorative materials , but that their bond strength to enamel was weak . vicente et al . showed that relyxunicem ( a self - adhesive resin cement ) possessed a significantly lower bond strength than transbond xt . reported low bond strength for relyxunicem and suggested that the sbs of this self - adhesive universal cement needs to be increased for its successful use in bonding orthodontic brackets . . showed that one - component adhesives ( relyxunicem and maxcem ) had significantly lower sbs than two- or three - component adhesives . bishara et al . reported that maxcem had significantly lower bond strength than transbond xt . in the current study , maxcem elite ( a new self - adhesive system ) the mean bond strength in the transbond xt group was significantly greater than that in the maxcem elite group , although the sbs of the latter was 10.291.14 mpa , which is clinically acceptable . debonding brackets bonded with maxcem elite resulted in less enamel cracks . removing remnants of adhesives and polishing with a 12-blade tungsten carbide bur did not increase enamel cracks . maxcem elite has clinically acceptable bond strength and can be used as a routine adhesive for orthodontic purposes .

objectives : the objective of this study was to assess the effect of new bonding techniques on enamel surface.materials and methods : sixty upper central incisors were randomly divided into two equal groups . in the first group , metal brackets were bonded using transbondxt and , in the second group , the same brackets were bonded with maxcem elite . the shear bond strength ( sbs ) of both agents to enamel was measured and the number and length of enamel cracks before bonding , after debonding and after polishing were compared . the number of visible cracks and the adhesive remnant index ( ari ) scores in each group were also measured.results:there were significantly more enamel cracks in the transbond xt group after debonding and polishing compared to the maxcem elite group . there was no significant difference in the length of enamel cracks between the two groups ; but , in each group , a significant increase in the length of enamel cracks was noticeable after debonding . polishing did not cause any statistically significant change in crack length . the sbs of maxcem elite was significantly lower than that of transbond xt ( 95% confidence interval).conclusion : maxcem elite offers clinically acceptable bond strength and can thus be used as a routine adhesive for orthodontic purposes since it is less likely to damage the enamel .

sensitive skin syndrome is a common and challenging condition , yet little is known about its underlying pathophysiology . sensitive skin is a common term used by patients and clinicians , as well as the cosmetic industry , and represents a complex clinical challenge faced by dermatologists and other skin care professionals . patients with sensitive skin often present with subjective complaints that are out of proportion to the objective clinical findings . the patients complain of severe facial irritation , burning , and/or stinging after application of cosmetic products and toiletries such as sunscreens and soaps , yet they do not demonstrate the clinical stigmata of scaling , induration , and/or erythema that would be expected in known inflammatory or allergic processes . the condition is often worsened by specific climatic conditions and may affect areas other than the face . maibach described the cosmetic intolerance syndrome ( cis ) , which probably covers the bulk of the clinical presentation of the sensitive skin syndrome ( sss ) . fisher is credited with coining the term status cosmeticus to describe one extreme of the natural history of the cis in which the patient gradually becomes completely intolerant to the application of any cosmetic product . fundamentally , sss is a state of hyperreactivity to environmental stimuli , presenting with a clear clinical picture and resulting from a single underlying pathology or a combination of pathologies . defined as a self - diagnosed condition , sss is by definition difficult to quantify . some of the contradictions between investigators could be explained by flawed methodologies since the scientific community has yet to identify an acceptable objective screening test for sensitive skin . however , it is not only the subjective nature of the complaints that make sss difficult to diagnose . robinson , by performing patch testing with sodium dodecyl sulfate ( sds ) and looking for intra - individual response patterns , found that there is significant positive correlation between sds and other irritants but noted overall low correlation coefficients . he therefore deduced that it is inappropriate to define a subject 's reaction to a chemical based on his or her response to another irritant . she skin - tested 58 subjects with history of strong positive sds or lactic acid response . sds- or lactic acid - positive group , a reaction to one irritant could not predict a reaction with another . to further complicate matters there is evidence to suggest that no correlation exists between the lactic acid stinging test and the response to sds . this data resonate the idea that even the reference irritants commonly used in studies of this topic do not correlate with each other and with hyperreactivity tendencies . finally , judge tested 22 nonatopic adults with varying doses of sds and found marked inter - individual variation in the response threshold . this complexityreinforces the need for a thorough diagnostic algorithm and , specifically , the need to test patients with multiple , repeated and complete ( i.e. all cosmetics applied by the patient at home ) patch testing before making a diagnosis . in order to formulate a systematic clinical approach , the medical and cosmetic communities have attempted to characterize the condition . lacking an objective screening test , investigators resorted to epidemiological studies using patient surveys . in a large epidemiological study in the uk ( n = 2316 ) , a staggering 51.4% of women and 38.2% of men self - reported themselves as having sensitive skin . of note , interestingly , atopy did not appear to predict self - perceived sensitivity in the participating women . in addition , in the same study self - reports of sss symptoms were statistically over - represented in the self - reported sensitive cohort compared to the self - reported nonsensitive cohort . this finding validates the link between self - perception of sensitive skin and neurosensory discomfort . two recent studies in the us and europe made similar observations . in the us , females were significantly more concerned about sensitive skin than men ; however , no age or ethnic differences were found . the european study reported an overall sensitive skin prevalence of 38.4% and found no ethnic differences . the european study , once again , demonstrated that people who reported having sensitive skin were significantly more likely to experience sss symptoms . of note , both the american and european studies used large samples ( n = 994 and n = 4506 , respectively ) ; however , they were both limited by a phone survey methodology and the lack of any objective assessment . in light of the increased incidence of self - reported hypersensitivity in females , he compared the patch test responses of 384 patients to sds as the positive control and found increased reactivity in males compared to females . lammintausta tested seven males and seven females with sodium lauryl sulfate ( sls ) and then performed visual inspections , transepidermal water loss measurements , and dielectric water content measurements but like other investigators found no reactivity differences between males and females , adding to the overall controversy . female self - perception of sensitivity is consistently increased compared to that of males , yet when put through objective reactivity testing the trend is unclear . ethnic differences in skin reactivity have been explored through the years , leading to the clinical hypothesis that black skin is less reactive than caucasian skin , which is in turn less reactive than asian skin . however , an epidemiological telephone survey performed in the us ( n = 811 ) revealed similar incidence of self - reported sensitive skin across four major ethnic groups ( afro - americans , asians , euro- american , and hispanics ) . the incidence rate of 52% is comparable to the incidence found in the uk ( see above ) . the investigators did , however , find minimally statistically significant ethnic differences in the self - reported triggers and symptoms of sensitivity : afro - americans reacted less to environmental and alcoholic triggers ; hispanics reacted less to alcohol ; asians reacted more to wind , spices , and alcohol ; and euro - american reacted more to wind . in terms of symptomology , statistically significant increase in recurrent itching on the face was noted in the asian population , and fewer euro - american avoided certain cosmetics due to skin reactivity . these findings were supported in a critical literature review , which showed that the evidence supporting the ethnic difference hypothesis using objective tests rarely reaches statistical significance and is often biased by subjective endpoints . recent data looking into age - related differences in hyperreactivity are showing more consistent results . whorl performed patch testing with 34 irritants on 2776 patients and found that young patients were more reactive than old patients . robinson 's findings also corroboratedthe trend towards increased reactivity in the young subjects compared to the old subjects . the mechanism leading to the sss has been debated in the literature , but the leading hypothesis relates to increased stratum corneum permeability . there is a known inverse relationship between corneocyte size and stratum corneum thickness and the permeability of the skin , and changes in this mechanical barrier result in abnormal skin penetration by irritants . in addition , low levels of ceramides in the stratum corneum have been linked to increased severity of sls - induced irritant contact dermatitis , pointing toward barrier compromise in sensitive skin . seidenari linked self - reported hyperreactivity to decreased skin capacitance ( indicating decreased water content ) , objectively suggesting increased absorption of irritants by the sensitive - skin population . in the same study , increased erythema was objectively measured in hyperreacting subjects suggesting involvement of some baseline vascular component , and leading more recent investigators to propose the involvement of cutaneus sensory innervation in the pathogenesis of sss . as with many syndromes , the management scheme presented here emphasizes the diagnostic framework [ table 1 ] . the clinical approach to the sss demands a thorough process of eliminating obvious as well as more subtle diagnoses . once the diagnosis is made , deriving the appropriate treatment becomes a relatively simple task . note that since much of the mechanisms of sss are unknown , the clinician must be comfortable with a certain degree of ambiguity and uncertainty . underlying mechanisms of sensitive skin syndrome it is reasonable to organize the differential diagnosis by dividing sss into visible and invisible sss . some dermatoses that most clinicians find simple to diagnose may have atypical presentation and as a result lack the expected morphology . eczema , atopic dermatitis , and rosacea are likely the three most common causes of sss related to barrier defect . full discussion of these conditions is beyond the scope of this overview but we will highlight some common clinical features . careful history , including family history and occupational history , combined with a detailed physical exam will often reveal the diagnosis . in the history particular consideration should be given to culprits such as the masking effect of other topical agents applied by the patient . the physical exam should include scrutiny of the face and scalp for subtle signs of minor inflammation , which are often masked in sss with underlying endogenous dermatoses . in all of these conditions the patients are likely over - exfoliating their skin and thus exacerbating the barrier dysfunction . therefore , following specific treatment to halt the acute pathological process , preventing recurrence with a proper skin care regimen is indicated . in eczema and atopic dermatitis , the careful clinician may resort to short - term ( 2-week ) use of corticosteroids to stop the inflammatory process . calcineurin inhibitors are alternatively indicated for delicate areas on the face . in an effort to control histamine release in atopic dermatitis , antihistamines can be added and a relatively allergen - free environment should be created . in rosacea , the mainstay of treatment is oral and topical antibiotics . in seborrhoeic dermatitis , azoles are the mainstay of treatment and low potency corticosteroids and emollients can be added acutely to treat the inflammatory process . contact dermatitis ( cd ) and photocontact dermatitis ( pcd ) , as well as nonimmune contact urticaria ( nicu ) and immune contact urticaria ( icu ) , are all conditions that may elicit sss symptomology , with transient objective findings on physical exam . allergen - free products are now available and should be included as part of further skin care regimen in these patients . the main reason why nicu is often a missed diagnosis in patients presenting with the sss is the transient nature of the reaction . thus , after testing with small amounts of product on the skin , the patient should be carefully examined using minimal magnification : the lesions will be typically revealed within 20 minutes . common trigger agents are fragrances ( e.g. cinnamic aldehyde ) and preservatives such as sorbic and benzoic acids . once triggers icu can be demonstrated with open and occluded testing , followed by prick testing with appropriate positive and negative controls if no response is elicited . various foods , latex , parabens , and other chemicals have been implicated in the causation of icu . however , there are cases in which no clinical clue is provided by the physical exam . in these cases , a 2-week trial of appropriate - strength corticosteroid may solve the mystery by pointing to an eczematous process in the case of symptoms resolution . nonetheless , the careful clinician should consider avoiding prolonged use of topical corticosteroids since there is anecdotal clinical experience of topical corticosteroids inducing sss and , at least in the case of barrier dysfunction , earlier and complete steroid tachyphylaxis has been demonstrated . if no symptomatic relief is observed , the patient 's sss is likely due to an invisible underlying cause . both of these cases defined by maibach , no signs are present or can be elicited on the skin . in objective irritation the mechanism is unknown and this diagnosis likely bundles more than one pathological process in it . interestingly ( and clinically frustratingly ) , maibach considers subjective irritation to be the most common cause of cis . finally , body dysmorphic disorder ( bdd ) should always be considered by the dermatologist when assessing skin complaints without objective findings . a recent cross - sectional study found a prevalence of 14% among patients in a cosmetic dermatology clinic . in the context of sss , dermatologists should be aware that facial irritation complaints without any findings on exam are a common presentation in the dysmorphic patients and therefore extra caution is warranted . referral to a mental health professional is indicated since these patients are at risk of suicidal behavior . maibach notes that patients suspected of having bdd often require a detailed diagnostic process in order to build the trust needed to safely refer them to the mental health professional . table 2 summarizes the steps in the evaluation of a patient suspected to have sss . specific algorithms have been developed for the management of sss but they are the result of accumulated clinical experience and lack experimental evidence to support their use . nonetheless , algorithmic thinking may aid in ensuring complete assessment . these algorithms include four basic steps : discontinuation , assessment , testing , and slow re - introduction . discontinuation of all topical medication and/or products , as well as elimination of activities and clothing resulting in skin friction , should be initiated and may need to last up to 12 months . the patient should then be assessed ( as described above ) for the occurrence of any visible dermatoses . if no clear pathology has emerged , rigorous testing is the next step . testing should include patch and photopatch testing of routine allergens as well as of all of the patient 's skin care items . specific testing for contact urticaria should be a part of the testing protocol . finally , the next step in management is slow re - introduction of the minimally necessary skin care products . for females , recommend adding low - allergenic- potential cosmetic products one at a time in the following order : lipstick , face powder , and blush . for all products re- introduced , recommend testing by applying the product to a 2-cm area lateral to the eye for five consecutive nights and documenting the positive and/or negative result . this exhausting process is not only a rational solution from a clinical management standpoint but may also aid in diagnosing the specific culprit in cases of invisible sss . understanding the underlying mechanisms may lead to the development of a well - standardized screening test . this will allow clinical studies to rely on objective findings rather than self - reports and this , in turn , may result in more comprehensive and efficient management strategies . female self - perception of sensitivity is consistently increased compared to that of males , yet when put through objective reactivity testing the trend is unclear . it is reasonable to organize the differential diagnosis by dividing sss into visible and invisible sss .

sensitive skin syndrome ( sss ) is a common and challenging condition , yet little is known about its underlying pathophysiology . patients with sss often present with subjective complaints of severe facial irritation , burning , and/or stinging after application of cosmetic products . these complaints are out of proportion to the objective clinical findings . defined as a self - diagnosed condition lacking any specific objective findings , sss is by definition difficult to quantify and , therefore , the scientific community has yet to identify an acceptable objective screening test . in this overview we review recent epidemiological studies , present current thinking on the pathophysiology leading to sss , discuss the challenges sss presents , and recommend a commonsense approach to management .

spontaneous pneumothorax ( sp ) comprises the largest group and is classified into primary spontaneous ( psp ) and secondary spontaneous pneumothorax . psp usually occurs in young , tall , thin men , especially smokers , in the absence of an underlying lung disease . although , sp is a relatively common condition , bilateral primary spontaneous pneumothorax ( bpsp ) is a very rare clinical condition with an occurrence ranging from 1.3 to 1.9% of all cases of sp . herein , we present a case of bpsp in an overweight young male patient , which is an exceptionally rare event . an otherwise - healthy , non - smoker , 20-year - old , overweight ( bmi 29.8 kg / m ) greek male patient was presented to the emergency department complaining of low - intensity chest pain and breathlessness of progressive worsening over the preceding 7 days . on admission , blood pressure was 125/70 mmhg , heart rate 100 beats / min , sao2 97% on air and axillary temperature 36.8c . initial plain chest x - ray demonstrated bilateral pneumothoraces ( more prominent on the left side ) and no deviation of the trachea ( fig . 1 ) . plain chest x - rays on admission revealing bilateral pneumothorax . on emergency basis , the patient was managed with bilateral tube thoracostomy through the fifth intercostal spaces . during hospitalization , both lungs were gradually expanded ; the use of continuous aspiration was necessary for a couple of days , in order to achieve complete expansion of the left lung ( fig . 2 ) . next day the plain x - ray revealed recurrence of pneumothorax on the left side ( fig . the tube was removed after 5 days ; no recurrence took place at this time . during the next 3 days the patient remained under observation ; serial physical examinations and chest x - rays were normal and the patient was discharged home . recurrence of pneumothorax on the left side after removing the chest tube . during hospitalization a chest computed tomography ( ct ) was performed and revealed the presence of blebs at the apices of both upper lungs ( fig . the patient was referred to a tertiary center for an elective video - assisted thoracoscopic surgery intervention ( vats ) . three years after , no recurrence of pneumothorax took place and the patient remains healthy . psp usually occurs in the absence of any obvious underlying lung disease ; however , blebs and bullae apparently play a role in the pathogenesis of psp , as they are found on ct or during thoracoscopy or thoracotomy in 4879% of these patients . diagnosis of bilateral pneumothorax presents difficulties ; while unilateral pneumothorax is relatively easily suspected from the patient 's medical history , physical and radiological findings , the diagnosis of bilateral pneumothorax is usually delayed . the presence of equally diminished breath sounds in both sides and the presence of a not deviated trachea often mislead clinicians and delay chest drain insertion which is usually performed after the transaction of chest ct . although sp is a relatively common condition , bpsp is a very rare clinical condition . lee et al . , in their study of the 616 patients with 807 episodes of psp , found that the incidence of bpsp was 1.6% ( 13 patients ) . in univariate regression analysis , patients with psp compared with patients with bpsp had significantly lower body weight , bmi , higher body height / body weight ratio and higher incidence of bilateral blebs / bullae seen in hrct of the lung . however , in multiple regression analysis , only bmi and the presence of bilateral blebs / bullae retained statistically significant importance . huang et al . found that only lower bmi and smoking were significantly associated with the formation of bpsp . in their study , the proportion of bilateral blebs / bullae seen in hrct was higher in the bpsp group than that in the psp group ( 63 vs 53% , respectively ) , but the difference was not statistically significant ( p = 0.724 ) . the present report describes a case of bpsp in an overweight patient showing that psp is an existing condition in patients with bmi value higher than the normal . generally , if an sp affects < 20% of one lung , observation is efficient ; the absorption rate of the air is 1.25% ( 5075 ml / day ) . if pneumothorax affects > 20% of the lung or if it increases during observation , chest tube drainage may be required . air leakage can be diminished in 5 h and in 48 h in 52 and 82% of patients with tube thoracostomies , respectively . in patients with bpsp , one side should always be drained regardless of the extent of pneumothorax , whereas the other side can be managed by simple observation depending on the extent of the air in the pleura space . prolonged air leakage is the most common indication for operation in the first episode of pneumothorax . today compared with open thoracotomy , this procedure provides visualization of the entire thoracic cavity by video and causes less postoperative pain . single - stage bilateral vats procedure for bpsp has been advocated as an elective procedure to avoid subsequent anesthetic and operative procedures and longer hospital stays . if any underlying pulmonary disease is detected during surgical treatment , the existence of pleural communications should be investigated and mediastinal pleura should be examined carefully . in addition , apical pleurodesis further reduces the risk of recurrence . in our case , the patient was successfully managed initially with a bilateral tube thoracostomy and finally with an elective bilateral vats procedure . in conclusion , bpsp is a rare clinical condition and usually develops in patients with low bmi and bilateral blebs / bullae . bpsp in overweight - obese patients is an existing condition and its early diagnosis requires high suspicion index . bpsp needs urgent assessment and management , followed by bilateral single - stage vats treatment as a safe and effective procedure .

bilateral primary spontaneous pneumothorax ( bpsp ) is a rare condition , accounting for 1.6% of spontaneous pneumothoraces . patients with primary spontaneous pneumothorax have typically low body weight . bpsp in overweight / obese patients is an exceptionally rare event . the present report describes a case of an otherwise - healthy 20-year - old male patient with bmi 29.8 kg / m2 presented to our hospital owing to low - intensity chest pain and shortness of breath 7 days after the onset of symptoms . plain chest x - ray revealed the presence of bilateral pneumothorax . chest tubes were inserted on both sides . during hospitalization , a computed tomography demonstrated multiple blebs on the surface of the upper lobes of both lungs . hereupon , the patient referred to a tertiary center and underwent elective single - stage minimally invasive bilateral video - assisted thoracoscopic surgery . the present case report shows that bpsp in overweight / obese patients is an existing condition .

familial adenomatous polyposis account for 1% of colorectal cancers , and provides a model of apc inactivation as an early genetic event for the approximately 80%85% of cancers that develop from sporadic polyps . colorectal cancers arising in patients with familial adenomatous polyposis can be largely prevented by polyp surveillance and prophylactic colectomy . total proctocolectomy with construction of a conventional ileostomy or ileoanal anastomosis with preparation of an ileal pouch , has various effects on the function of the terminal ileum and the intestinal bacterial flora . this may deteriorate cholesterol metabolism , as absorption of cholesterol in duodenum and jejunum requires micellar solubilization with bile acids , fatty acids , monoglycerides , and phospholipids . hypothetically , ileal - pouch - anal anastomosis and ileostomy patients might differ with regard to the presence of various fatty acids in feces and their relationship to other reflections of lipoprotein metabolism , but we found no previous study focusing upon this issue . dietary fatty acids are incorporated into blood and tissues , and the fatty acid composition in these tissues are often used as biomarkers of fat intake . furthermore , the fecal amount and composition of fatty acids reflect fat ingestion , intestinal fatty acid absorption , and the activity of colonic bacteria . although some of the discrepancies between studies may be due to the use of different methods to analyze fatty acids , differences in diet , or the fact that assessments have been performed in different body compartments , modifications in the metabolism of fatty acids have been suggested in cancer patients [ 5 , 6 ] . it is not clear at what steps in the multistage carcinogenesis process a possible distorted fatty acid metabolism occurs . notably , if such alterations occur in the development of carcinogenesis , this may affect the biological functions of essential fatty acids and their derivates . very little is known about fatty acid metabolism in familial adenomatous polyposis , although chemoprevention affecting the fatty acid derivates and the cyclooxygenase enzymes is often administered to familial adenomatous polyposis patients . deregulation of the cyclooxygenase-2 pathway appears to affect tumorigenesis via a number of distinct mechanisms : promoting tumour maintenance and progression , encouraging metastatic spread , and perhaps even participating in tumour initiation . cyclooxygenase-1 and -2 are the rate limiting enzymes in the synthesis of prostaglandins and thromboxanes . arachidonic acid is the main substrate for these enzymes , leading to the synthesis of prostaglandins which have growth promoting effects . substituting arachidonic acid with omega-3 fatty acids has been shown to lead to the production of less potent prostaglandins . since cyclooxygenase-2 is a fatty acid metabolising enzyme , the relationships between cyclooxygenase-2 and fatty acid composition of different tissues is of interest . colectomized familial adenomatous polyposis patients had a deviant fatty acid profile with high levels of arachidonic acid and docosahexaenoic acid and low levels of linoleic acid and alpha - linolenic acid in serum phospholipids , which is in accordance with studies in patients with other types of cancers [ 5 , 1013 ] . in a previous familial adenomatous polyposis study , comparable treatment effects of a cyclooxygenase-2 inhibitor were observed on the fatty acid composition in serum phospholipids and duodenal lesions , presumably and most importantly the nonbeneficial effects involving essential fatty acids . the results describe the content of fatty acids in feces , and relate this to the proportions of fatty acids and levels of cyclooxygenase mrna expression in duodenal biopsies , diet , and levels of serum lipoproteins . because the effects of ileostomy construction may differ from that of ileal - pouch - anal anastomosis because of scarcity of the bacterial flora and different surface area of the terminal ileum , we did separate analyses for these two groups . previous familial adenomatous polyposis studies have focused upon faecal sterols and bile acids [ 1517 ] , but none has to the best of our knowledge analysed the content of fatty acids in feces , including very long chained fatty acids . data from the present study are taken from a randomized double - blind placebo - controlled intervention study with a cyclooxygenase-2 inhibitor [ 12 , 14 , 18 ] . the main aim was to compare the effect of rofecoxib treatment on duodenal lesions ( data in preparation ) . the present study comprises data from baseline for only the patients operated with ileal - pouch - anal anastomosis and ileostomy ( 77% of total study group ) . all of them had been colectomized , and duodenal lesions had been verified by endoscopy and histology . rikshospitalet university hospital is a highly specialized university hospital with national responsibilities in the area of complicated treatments , such as follow - ups on the norwegian familial adenomatous polyposis patients . inclusion criteria were verified familial adenomatous polyposis , colectomy , 1870 years of age , and documented duodenal lesions graded as spigelman i , ii , or iii and the largest adenoma10 mm . exclusion criteria were indications for surgical treatment , suspected or documented intestinal obstruction or stenosis , patients unwilling or unable to adhere to protocol , known cardiac failure requiring medical treatment , and pregnancy . as for the adenomas sampled , they were all assessed histologically as mild / moderate dysplasia , all below 10 mm and all flat , located in the descending part of the duodenum . samples of feces were transferred to tubes and mixed to make a homogeneous mass of which 100200 ul were used for lipid extraction by adding 50 ul butanol , 100 l h2o , 1 ml methanol , and 1 ml chloroform . after 10 min under n2 , the mixture was filtered and the filter washed with 1 ml chloroform + 1 ml chloroform / methanol ( 2 : 1 ) + 0.8 ml 0.73% nacl . after mixing for 15 s , the tubes were centrifuged for 30 min at 1000 g. the upper phase was discarded and the lower phase transferred to new tubes and evaporated under n2 at 65c using a heat block . then 0.4 ml 0.5 n naoh in methanol was added and the solution kept for 7 min at 100c under nitrogen . after cooling to room temperature , 0.5 ml 12% bf3 was added and the solution kept for 10 min at 100c under nitrogen . after cooling to room temperature , 1 ml heptan was added under nitrogen , and mixing for 30 s , 1 ml of saturated nacl solution was added . after mixing , the solution was centrifuged for 10 min at 1000 g , and the upper phase transferred to new tubes . finally , the heptan was evaporated and 300 l hexan added before continuing with the gas chromatography procedure referred to earlier . dietary intake was assessed by a validated food frequency questionnaire , designed to cover as much of the total habitual diet as possible . questions were related to habitual frequency of consumption and the amount of foods eaten during the last year . they were asked to identify their habitual choices of edible fats by pointing at specially prepared pictures , in order to increase the validity of the estimate of dietary fat . each biopsy was put into a tube containing 600 l trizol and immediately mixed on a mixermill ( retsch gmbh , germany ) . gene expression analyses were carried out on a 7900 ht real - time pcr machine from applied biosystems . based on the results from running a housekeeping gene test ( taqman human endogenous control plate , applied biosystems ) with rna from isolated human leucocytes ( data not shown ) . the primers and probes were initially designed as three assays per gene and validated for efficiency and specificity . the primers and probes for the cox-1 assay were : forward primer : 5-cttccaggagctcgtagga-3 , probe : 5-agaaggagatggcagcagagttggag-3 , and reverse primer : 5-acgcatcaatgtctccatacaat-3. cox-2 forward primer : 5-tggaacatggaattacccagt-3 , probe : 5-tgttgaatcattcaccaggcaaattgct-3 , and reverse primer : 5-tcctaccaccagcaaccct-3. gus forward primer : 5-gaaaatatgtggttggagagctcatt-3 , probe : 5-ccagcactctcgtcggtgactgttca-3 , and reverse primer : 5-ccgagtgaagatccccttttta-3. tbp forward primer : 5-ctggaaaagttgtattaacaggtgc-3 , probe : 5-agcagaaatttatgaagcatttgaaaacatctaccctatt-3 , and reverse primer : 5-cattacgtcgtcttcctgaatc-3. taqman universal pcr master mix ( applied biosystems ) was added as reaction mix . the reaction conditions were initiated by a step of 2 min . at 50c and 10 min . at 95c , followed by 40 cycles of denaturation at 95c for 15 sec . and annealing at 60c for 1 min . a combination of cdna from several samples were made and diluted in order to make a dilution curve that was included on each plate . the average of the three values for each gene was divided by the average of the corresponding tbp values , generating a normalized value of the gene expression which is a unit less value used to compare the relative amount of mrna for each gene in the different samples . patients were informed by a physician , and in addition thoroughly written information was given to all patients . the protocol was explained to the subjects that had to give their consent before inclusion . was approved by the norwegian health authorities and the regional committee of medical ethics 20/06/2002 ( reference : s-02127 ) . nonparametrical statistical methods were chosen , as some of the variables were skewed and the number of observations limited . age and fecal content of palmitolic acid , linoleic acid , and arachidonic acid differed between the ileostomy and the ileal - pouch - anal anastomosis patients ( table 1 ) . the content of linoleic acid in feces correlated negatively to the proportion of eicosaenoic acid in duodenal biopsies ( r = 0.6 , p < .05 ) . the content of arachidonic acid in feces was correlated negatively to the proportions of eicosapentaenoic acid ( r = 0.7 , p = .02 ) and docosahexaenoic acid ( r = 0.7 , p = .008 ) in duodenal biopsies . total cholesterol were negatively correlated to the content of palmitic acid ( r = 0.7 , p = .03 ) , palmitoleic acid ( r = 0.7 , p = .02 ) , stearic acid ( r = 0.7 , p = .02 ) , oleic acid ( r = 0.7 , p = .02 ) , linoleic acid ( r = 0.8 , p = .002 ) , and monounsaturated fatty acids ( r = 0.7 , p = .02 ) in feces . the levels of triglycerides was negatively correlated to the content of linoleic acid ( r = 0.8 , p = .003 ) and eicosaenoic acid ( r = 0.7 , p = .03 ) in feces . no significant correlations were found between the content of different fatty acids in feces and the fatty acid dietary intake ( data not shown ) . with the exception of the palmitoleic acid / palmitic acid ratio and levels of cyclooxygenase-2 expression in duodenal biopsies ( r = 0.8 , p = .003 ) , no relationships were found between fatty acids in feces and the levels of cyclooxygenase mrna expression ( figure 1 ) . significant correlations were found between the content of oleic acid in feces and the proportion of myristic acid ( r = 0.8 , p < .001 ) , oleic acid ( r = 0.5 , p < .05 ) , and eicosaenoic acid ( r = 0.5 , p = .03 ) in duodenal biopsies . moreover , the content of linoleic acid in feces correlated significantly to the proportion of myristic acid ( r = 0.8 , p < .001 ) in duodenal biopsies , whereas the content of alpha - linolenic acid in feces correlated inversely to eicosaenoic acid ( r = 0.6 , p < the content of palmitic acid in feces was positively correlated to levels of ldl - cholesterol ( r = 0.05 , p = .05 ) . no significant correlations were found between the content of different fatty acids in feces and the levels of cyclooxygenase mrna expression in duodenal biopsies ( data not shown ) . several different fatty acids in feces correlated positively to dietary intake of fatty acids , most predominantly monounsaturated fatty acids ( table 2 ) . as expected , we found a difference between ileal - pouch - anal anastomosis and ileostomy patients concerning associations between fecal fatty acid composition and other variables involved in lipoprotein metabolism . possibly , our results relate to metabolic differences caused by the different intestinal reconstructions , but the data are not suitable to explain the findings . we found no significant relationships between the levels of cyclooxygenase mrna expression in duodenal biopsies and the content of fatty acids in feces , except for the estimates of the content of delta-9-desaturase ( stearoyl - coa desaturase , scd ) activity , namely the ratio of palmitolic acid / palmitic acid [ 19 , 20 ] in the ileostomy group . stearoyl - coa desaturase is the central lipogenic enzyme catalyzing in vivo reactions in the synthesis of monounsaturated fatty acids , particularly oleic acid and palmitoleic acid , which are the major monounsaturated fatty acids of membrane phospholipids , triglycerides , wax esters , and cholesteryl esters . both delta-9-desaurase and cyclooxygenase-2 are ppar alpha regulated , although this possible link is only a speculation . a study in healthy subjects showed that human fecal water contains components can affect both the cyclooxygenase-2 protein level and enzymatic activity . one recent study claims that it is possible to detect cyclooxygenase-2 mrna in feces of colorectal cancer patients irrespective of clinical stage . notably , increased cyclooxygenase-2 expression in duodenal compared with colonic tissues in familial adenomatous polyposis was recently reported . the significant negative correlation between content of arachidonic acid in the feces and amounts of omega-3 fatty acids in duodenal biopsies which was found in the ileostomy group is interesting , since substitution of arachidonic acid with omega-3 fatty acids has been shown to lead to the production of less potent prostaglandins . a previous familial adenomatous polyposis study established a positive correlation between the reduction in tissue prostanoid levels and clinical response , as measured by the reduction in size and number of adenomas , when patients were treated with sulindac . however , this correlation only became consistently significant when prostanoids were assayed after tissue homogenates were first incubated with arachidonic acid . we may suspect transformation of linoleic acid to arachidonic acid in feces , but data are limited as with regard to feces from colectomized familial adenomatous polyposis subjects . however , the dietary assessment showed that the intake of such foods was normal among these familial adenomatous polyposis patients . dietary controlled intervention studies on ileostomy subjects have shown that dietary manipulation with fat and fiber may modify cholesterol absorption and sterol excretion . in the ileostomy group , levels of total cholesterol were negatively correlated to the content of palmitic acid , palmitolic acid , stearic acid , oleic acid , linoleic acid , and monounsaturated fatty acids ( < 18 c atoms ) in feces , which is interesting since the present levels of total cholesterol are lower than those among a healthy reference group . in the ileal - pouch - anal anastomosis group separately , the content of monounsaturated fatty acid in feces was positively correlated to dietary intake of monounsaturated fatty acids in particular . moreover , positive correlations were found between the content of oleic acid in feces and the proportion of myristic acid , oleic acid , and eicosaenoic acid in duodenal biopsies . the precise role of monounsaturated fatty acids synthesis in cell proliferation and programmed cell death remains unknown . the strong correlation of high levels of monounsaturated fatty acids and neoplastic phenotype may suggest that the regulation of stearoyl - coa desaturase must play a role in cancer development . this was an observational study , and not a metabolic balance study that was designed to investigate the metabolism of fatty acids in familial adenomatous polyposis patients . although some studies have found that the absorption rate of fatty acids are unaffected by age [ 26 , 27 ] , we can not exclude the possibility that some of the observed differences between the ileal - pouch - anal anastomosis and ileostomy patients might be due to age , rather than type of surgery , since lipoprotein clearance has been shown to be altered by age , and that might affect the relationship between dietary fatty acids and the fatty acid profiles found in tissues . moreover , no conclusions can be drawn from the present study because this is an observational study . . however , the study addresses the need to learn more about the dietary and lifestyle habits of this subset of at - risk patients . the present study includes a number of patients close to the maximum possible number of eligible norwegian familial adenomatous polyposis patients . we may , however , suspect that the low sample size and use of a food frequency questionnaire , may have weakened any associations between the content of fatty acids in feces and dietary intake . notably , these data do not contradict that the nutrient intake among these patients should at least meet the recommendations for healthy subjects . nevertheless , we suggest that these data are compelling enough to suggest that future familial adenomatous polyposis studies should investigate overall fatty acid metabolism , molecular mechanisms relevant to fatty acid metabolism , inflammation , and angiogenesis , in addition to nutrition requirements . in conclusion , we found a difference between ileal - pouch - anal anastomosis and ileostomy patients concerning associations between fecal fatty acid composition and other variables involved in lipoprotein metabolism . we may suggest that fatty acid content in feces is related to dietary intake , serum lipids , and fatty acid composition in duodenal biopsies , even in colectomized familial adenomatous polyposis patients . this observational study may represent hypothesis - generating suggestions for future familial adenomatous polyposis studies .

a few familial adenomatous polyposis studies have focused upon faecal sterols and bile acids but none has analysed the fecal content of fatty acids . we report here findings of an observational study on 29 colectomized familial adenomatous polyposis patients that describe the fecal content of fatty acids , and relate this to the proportions of fatty acids and levels of cyclooxygenase mrna expression in duodenal biopsies , levels of serum lipoproteins , and diet . in the ileostomy group separately ( n = 12 ) , the fecal content of arachidonic acid was correlated negatively to the proportions of eicosapentaenoic acid and docosahexaenoic acid in duodenal biopsies . total serum - cholesterol was negatively correlated to the fecal content of saturates and monounsaturates . the fecal palmitoleic acid / palmitic acid ratio was positively correlated to the levels of cyclooxygease-2 expression in duodenal biopsies.in the ileal - pouch - anal anastomosis group separately ( n = 17 ) , significant correlations were found between the fecal contents of oleic acid , linoleic acid , and alpha - linolenic acid , and the proportions of myristic acid , oleic acid and eicosaenoic acid in duodenal biopsies . dietary monounsaturates were positively correlated to different fecal fatty acids . future studies should focus on molecular mechanisms relevant to fatty acid metabolism , inflammation , and angiogenesis , in addition to nutrition .

local anesthesia and pain control is one of the most important elements of dentistry , and particularly prosthetic dentistry . according to the american dental association , fear of pain is the most important factor preventing patients from visiting their dentists . different kinds of fear related to previous clinical experience affect patients ' attitudes to local anesthesia or dentist20 . although local anesthesia techniques do not provide pain - free treatment , this pain is generally tolerable . pain can result from the mechanical trauma of needle introduction into the site of injection , or from the sudden distension of the tissues , resulting from a rapid discharge of the contents of the syringe . pain can also be caused by the stimulation of the first few drops of the local anaesthetic3,14 . contrary to prevalent ideas , needle penetration of tissue is not the primary reason for discomfort . volume and pressure of the local anesthetic being injected causes more distress and/or pain . administering local anesthetic injection may not only provoke anxiety in patients , but also in the dentist29 . despite that , administering local anesthesia by injection for pain relief is still the most common method used in dentistry . however , there are several ways to alleviate pain before dental procedures begin , or the often painful nature of the injection in local anesthesia . transcutaneous electrical nerve stimulation ( tens)24 , use of intraoral lidocaine patch11 and computerized local anesthesia7 ( the wand ) are newly developed methods to alleviate the pain and anxiety of dental patients , as alternatives to conventional local anesthesia . the wand ( milestone scientific , livingston , nj ) is a computer - controlled local anesthesia delivery system that drives local anesthetic from a conventional local anesthetic cartridge through plastic microtubing into a plastic handle to which a luer - lok needle is attached . the computer - controlled flow is initiated by exerting pressure on a foot pedal . the pump allows administration of local anesthetic at two slow but constant rates , and the computer compensates for variation in resistance to flow4 . during needle insertion , continuous positive pressure delivers an anesthetic drip that precedes the needle . the combination of an anesthetic pathway and controlled flow rate results in a virtually imperceptible injection and rapid onset of profound anesthesia . the wand system offers several advantages over conventional syringes , including excellent tactile sensation afforded by a lightweight plastic handle and the ability to rotate the needle as it is introduced into tissues , producing a coring penetration that minimizes needle deflection . wand devices automatically deliver the anesthetic more slowly and precisely than a conventional syringe , thereby reducing patient discomfort5,15 . anesthetic solution gently pumped into the soft tissue can be absorbed at the same rate by soft tissue in the wand system . the theory is that when the needle is advanced slowly , the drops of solution anesthetize the tissue ahead of the needle . all techniques of local anesthesia , such as maxillary and mandibular infiltration , mandibular block22 , intraligamentary26 , and anterior middle superior alveolar ( amsa ) injection8 , and anterior superior alveolar ( asa ) injection25 can be performed with the wand system . conventionally , maxillary teeth have been anesthetized by administering an infiltration injection on the buccal or labial aspects of the target tooth . palatal injections have also been administered by plastic syringe for this objective , but can be painful5,7 . the amsa derives its name from the injection 's ability to anesthetize both the anterior and middle superior alveolar nerves57 . the middle superior alveolar ( msa ) and asa nerves branch from the infraorbital nerve before they exit from the infraorbital foramen . the msa nerve is thought to innervate the maxillary premolars and plays some role in pulpal innervation of the mesiobuccal root of the first molar . the anterior , superior alveolar nerve supplies pulpal innervation to the central and lateral incisors and canines18 . the plexus where the 2 nerves join is the target site for the amsa injection . the 10 maxillary teeth extending from the second premolar to contralateral second premolar , and the associated palatal tissue , can be anesthetised with bilateral amsa injection57 . wand research is largely attributable to pediatric dentistry1,2,9 , restorative dentistry7 , endodontics14 , periodontology17,26,27 , maxillofacial surgery16 , comparison of pain perception to the wand and a traditional syringe21,28 , influence of anesthetic flow rate delivered by the wand on pain response to the palatal injection23 . the hypothesis of the present study was that the wand anesthesia technique reduces the pain at different phases ( needle insertion , delivery of local anesthetic and tooth preparation ) versus conventional buccal anesthesia with conventional plastic syringe . therefore , the study compared pain levels at needle insertion , during delivery of local anesthetic solution and during tooth preparation , for the conventional syringe technique on the buccal or labial aspect on one side , and the amsa technique with wand local anesthesia application on the other side of the maxilla . a 5-point verbal pain rating scale ( vrs)12,17 was used on 16 patients who were under prosthodontic treatment in the ondokuz mayis university clinic sixteen adult patients who had partially edentulous states of both left and right maxillary arch , and were to undergo fixed prosthodontic treatment , participated in this study . they ranged in age from 27 to 64 , were predominantly women ( 12 women and 4 men ) , and were in a good health . patients were not taking any medication that would alter their pain perception , as determined by a written medical health form and oral questioning . allergy to lidocaine , mepivacaine , sulphides or articaine , history of significant medical problems , taking central nervous system depressants within 48 hours , pregnancy , or inability to give informed consent , were the exclusion criteria . the human subjects review committee of ondokuz mayis university approved this study ( 28.11.2005 dated , and 328 numbered ) , and written , informed consent was obtained from each patient . all patients had previously experienced a conventional syringe , no patients had previously received the wand injection , and all patients had at least one tooth absent both on the left and right maxillary arch . the amsa technique with the wand for half the maxillary arch and conventional plastic syringe anesthesia on mucobuccal fold ( control group ) for opposite maxillary arch were administered by the same operator . the order of anesthesia techniques was randomly selected , and anesthesia procedures and tooth preparations were completed in the same session ( table 1 ) . vrs ( 0 no pain , 1 minimal pain , 2 slight pain , 3 moderate pain and 4 severe pain ) . the pain level descriptions were as follows : no pain , minimal pain , slight pain , moderate pain and severe pain ( 0 , 1 , 2 , 3 , 4 ) . patients were blindfolded with a commonly used sleeping mask so they would not distinguish which anesthetic delivery system was used . the wand produces audible beeps as the injection is administered . because the beeping tones can not be turned off with a switch , the sound were produced during both injection methods ( the wand or conventional - control ) so the patients were not aware of the method being used . immediately after needle insertion , delivery of local anesthetic and tooth preparation , patients were asked to remove mask and rate the pain for both conventional and wand injection techniques . no topical anesthetic was used because the wand manufacturer 's instructions suggest that topical application is optional . this study was conducted according to a split- mouth design , with both injections given to all patients . for each patient , the amsa technique with single wand injection was used on one side for central incisors , lateral incisors , upper canine , first and second premolars , and the traditional plastic aspirating syringe injection was used on the mucobuccal fold of the related tooth on the opposite side . the amsa injection site is located at a point that bisects the maxillary first and second premolars , and midway between the crest of the free gingival margin and mid - palatine suture . the needle is orientated at a 45 degree angle with the bevel facing the palatal tissue , while rotating the wand needle . all injections were given by the same operator who had had two months experience using the wand at the department of prosthodontics , faculty of dentistry , ondokuz mayis university , turkey . the ampul form of articaine hydrochloride with adrenalin ( hoechst marion roussel , deutschland , gmbh , germany ) was injected with conventional plastic syringe and 27gauge dental needle . the cartridge form of articaine hydrochloride with adrenaline ( hoechst ag , frankfurt , germany ) was administered with the computer - controlled anesthesia device ( the wand , milestone scientific , livingston , nj , usa ) , with 27gauge handpiece and needle ( milestone scientific ) . approximately 0.9 to 1.0 ml local anesthetic was used both for amsa with the wand on one side and control group for each tooth on the other side . groups were labelled as follows : group ( c ) , pain perception scores at needle insertion for the conventional technique ; group ( c ) , pain perception scores for local anesthetic delivery by the conventional technique ; group ( c ) , pain perception scores at tooth preparation for the conventional technique ; group ( w ) , pain perception scores at needle insertion by the wand technique ; group ( w ) , pain perception scores for local anesthetic delivery for the wand technique ; and group ( w ) , pain perception scores at tooth preparation for the wand technique . power analysis were performed to detect adequate sample size using statistical software ( pass 2008 , ncss , kaysville , ut , usa ) . since our variable was an ordinal level , the mann - whitney u test was used to determine the number of times a score from the conventional method is ranked than a score from the wand techniques at 1% significance level . statistical tests were performed with the statistical software ( spss version 12.0 ; spss inc , chicago , il , usa ) . differences in pain rating were analyzed at needle insertion , delivery of local anesthetic and during tooth preparation for conventional versus the wand techniques . sixteen adult patients aged from 27 to 64 ( 12 women and 4 men ) participated in this study . median scores , minimum and maximum pain rating values after needle insertion , and delivery of anesthetic and tooth preparation time for both conventional and wand techniques , are shown in table 3 . the wand technique registered a highly significantly lower pain level during the needle insertion phase ( z = - 4.064 , p<0.01 ) , with the wand technique having a lower pain level compared to the conventional . during the anesthetic solution delivery phase , the pain level for the wand technique was again highly significantly lower than for the conventional technique ( z = - 3.897 , p<0.01 ) . however , there was no difference between the wand and conventional techniques for pain level during tooth preparation ( z = - 0.671 , p>0.05 ) . the research hypothesis stated that the wand anesthesia technique reduces the pain at different phases ( needle insertion , delivery of local anesthetic and tooth preparation ) versus conventional buccal anesthesia with conventional plastic syringe . the wand anesthesia technique reduces the pain compared the conventional anesthesia for needle insertion and anesthetic solution delivery phases ( p<0.01 ) , with the exception of tooth preparation phase ( p>0.05 ) . the majority of literature on the computer - controlled injection system ( the wand ) has dealt with the pain of injection with the computer - assisted injection system , compared to injection using a conventional syringe1,2,8l0,l6,2l,27,28 . in general , the results have been favorable1,8,9,16,23,27 for the computer - assisted injection system , with only 2 studies showing no difference2,28 , and 1 study showing higher pain ratings10 . palatal injections are generally considered the most painful injections13 ; wahl , et al.30 showed that palatal injections caused significantly more pain than other intraoral injections . the authors of18,30 speculated that palatal injection pain might be mainly the result of pressure . the traditional route for administration of anesthesia to the maxillary teeth is supraperiosteal infiltration delivered in the mucobuccal fold near the apex of the tooth to be anesthetized . although effective in achieving pulpal anesthesia of the neurovascular bundle by diffusion of anesthetic through the porous maxilla , this approach routinely anesthetizes the surrounding tissues ( lips , surface of the face ) , and also affects muscle activity . the patient 's natural repose , as well as active facial expressions are temporarily affected , including an distortion of the smile . the patient also experiences the annoying sensation of facial numbness , and the smile line is temporarily invalidated as an esthetic reference for restorative and prosthetic dentistry . an alternative to maxillary infiltration is the infraorbital nerve block injection which , if properly performed , achieves anesthesia of the anterior superior alveolar branch of the trigeminal nerve . this injection site , however , is also close to muscles of expression , and anesthesia of cutaneous fibers of the infraorbital nerve results in a corresponding loss of sensation in the face and the upper lip29 . the palatal approach amsa nerve block can be attempted with a traditional manual syringe , but the precise pressure and volume ratios of the wand are virtually impossible to reproduce5,6 . the clinician can anesthetize several teeth in the maxillary arch , extending from the mesiobuccal root of the first molar to the central incisor , by means of a single palatal infiltration . in addition to profound pulpal anesthesia , palatal soft tissue anesthesia is realized , which also extends from the maxillary first molar to the central incisor and the associated palatal tissues , without numbness to the lips and face , and without interference with the muscles of facial expression for at least 60 min16,25 . during needle insertion with a coring motion , the drops of solution anesthetize the tissue ahead of the needle when the needle is advanced slowly7,15 . the combination of an anesthetic pathway and controlled flow rate results in virtually imperceptible needle insertion and injection , and rapid onset of profound anesthesia28 . the same efficacy of anesthesia was achieved with conventional buccal anesthesia and the palatal approach amsa technique with the wand device25 . the current study was in agreement for tooth preparation , but contradictory for needle insertion and delivery of local anesthetic . numbness of the lips and face was not observed in the current study for the palatal approach when using the amsa technique with the wand . another study using the vrs ( verbal rating scale ) compared computer - controlled and conventional local anesthesia delivery systems for performing scaling and root planing on patients with moderate periodontal disease ; amsa injections with the wand were considered less painful than the conventional syringe17 . two further benefits of the amsa injection technique are that it also eliminates the potential for an intravascular injection because no major vessels are encountered in this region of the palate5 , and the palatal bone is porous enough to permit the anesthetic solution to diffuse through the tissues and anesthetize both the anterior and middle branches of the superior alveolar nerve when the injection is deliberately slow and steady . for this reason , the successful deposition of the anesthetic solution through the fibrous tissue is said to be accomplished more easily with the computer - controlled delivery device that regulates the pressure and volume ratio of solution delivered5,7 . the wand system offers several advantages over conventional syringes , including excellent tactile sensation afforded by the lightweight plastic handle and the ability to rotate the needle as it is introduced into tissues , producing a coring penetration that minimizes needle deflection . presumably , the slow rate of anesthetic flow reduces patient discomfort compared with palatal injections administered with a traditional syringe4 . decreasing the total amount of anesthetic and vasoconstrictor necessary for maxillary anesthesia , shorthening the total anesthesia time , and diminishing patient - operator anxiety however , the wand 's beeping sounds make the injection seem longer and may make the patient feel uncomfortable . other negative aspects of the wand are frequent breakage of the anesthetic cartridge on activation of the unit , and a separate instrument is required for removing the cartridge from the unit10 . additionally , 1.4 ml anesthetic solution can be delivered in about 1 min in rapid injection mode , and in 4 min 45 s in slow injection mode9 . fukayama , et al.8 examined the efficacy of the amsa anesthesia technique using the wand system to assist with the electrical pulp test . electrical pulp stimulation indicated that the lateral incisors , canines , and first and second premolars were more anesthetized than the central incisors and first molars . they also stated that anesthetic applied via amsa with the wand has a slow onset and further studies are required to evaluate its effectiveness for this reason . however , amsa injection using the wand method seems to both preclude severe injection pain and be very effective for pulpal anesthesia of lateral incisors , canines , and premolars . pain scores were higher for amsa technique in the central tooth than in other teeth ( p<0.05 ) in another related study16 . in the present study , in two cases of amsa using the wand , pain was at the minimum level ( vrs score 1 ) during tooth preparation of central incisors . the reason for this pain rating could be that the central incisors were innervated from the opposite arch , or alternatively , fukayama , et al.8 and lee , et al.16 stated that the effectiveness of the wand is lower in the central incisors and first molar teeth than lateral incisors , canines , and premolars . friedman and hochman57 stated that careful needle insertion and slow anesthetic delivery could reduce the sensation of needle insertion . primosch and brooks23 showed that slow anesthetic delivery produces significantly lower pain scores than fast anesthetic delivery , and the wand decreases the pain for both slow or fast anesthetic delivery rate , but contrary to the manufacturer 's claims , it did not eliminate pain elicited by palatal injections in some patients . in the present study , a highly significant ( p<0.01 ) difference for pain rating was obtained between conventional buccal and amsa using the wand injection for needle insertion and anesthetic solution delivery . however , contrary to the manufacturer 's claims , pain was not eliminated at slow anesthetic flow rate in this study there have been limited prosthodontic or restorative studies for amsa using wand injection . in one study , amsa was bilaterally applied with the wand and 6 anterior maxillary teeth were prepared . incisal reduction of these teeth was achieved with reference to the lip , which is not affected by amsa . bilaterally , the amsa technique using the wand was repeated for tooth sensitivity during the cementation procedure5,6 . as in previous studies , amsa using the wand was also of benefit for lip guidance in the present study . another benefit of palatal amsa injection using the wand is that it reduces the number of injections , and also the amount of anesthetic solution compared to conventional buccal infiltration anesthesia that applies multiple injections to each tooth . in addition , more teeth can be anesthetized with a single injection , without numbness of lips and face , compared to the conventional technique . the amsa technique administered using the wand in a prosthodontic treatment reduced pain highly significantly more than conventional buccal anesthesia , both in inserting the needle and in the of delivery of local anesthetic . when comparing the anesthesia efficiency of amsa using the wand injection and conventional buccal infiltration techniques , no statistical difference was found . although the slow anesthetic flow rate proved superior in pain control , the pain score was not zero for the wand , contrary to the manufacturer 's claim . computer - controlled anesthesia using the wand appears advantageous for restorative procedures because more teeth are anesthetized with one palatal injection , and without numbness of lips and face , in contrast to multiple conventional buccal anesthetic injections for each tooth .

objective : the objective of this study was to compare the pain levels on opposite sides of the maxilla at needle insertion during delivery of local anesthetic solution and tooth preparation for both conventional and anterior middle superior alveolar ( amsa ) technique with the wand computer - controlled local anesthesia application.material and methods : pain scores of 16 patients were evaluated with a 5-point verbal rating scale ( vrs ) and data were analyzed nonparametrically . pain differences at needle insertion , during delivery of local anesthetic , and at tooth preparation , for conventional versus the wand technique , were analyzed using the mann - whitney u test ( p=0.01).results : the wand technique had a lower pain level compared to conventional injection for needle insertion ( p<0.01 ) . in the anesthetic delivery phase , pain level for the wand technique was lower ( p<0.01 ) . however , there was no difference between the wand and conventional technique for pain level during tooth preparation ( p>0.05).conclusions;the amsa technique using the wand is recommended for prosthodontic treatment because it reduces pain during needle insertion and during delivery of local anaesthetic . however , these two techniques have the same pain levels for tooth preparation .

chronic obstructive pulmonary disease ( copd ) is a progressive lung disease which is characterized by airway obstruction and lung parenchyma destruction . patients may experience dyspnoea or shortness of breath , a persistent cough with sputum production , decreased exercise tolerance and decreased quality of life [ 1 , 2 ] . globally , copd is the fourth most common cause of death and is predicted by the world health organisation to be a major health concern in the coming decade . in australia , 1.2 million people are estimated to have moderate - to - severe copd ( gold stages ii iv ) . however , the true number is potentially much higher as copd is commonly underdiagnosed [ 4 , 5 ] . the total cost of living with copd ( including nonmonetary costs ) is estimated to be $ 83 000 aud per person each year [ 6 , 7 ] . as the severity of copd greatly impacts the cost of care , providing early diagnosis and effective management of the individual is an essential component in reducing its economic impact [ 5 , 810 ] . pr is a multidisciplinary approach which incorporates self - management education with exercise training , psychosocial and nutritional support [ 11 , 12 ] . it is a fundamental part of managing people with copd and has been shown to be effective in improving exercise tolerance , reducing dyspnoea , improving , quality of life and reducing the severity of acute exacerbations [ 1 , 1117 ] . although pr has been shown to be effective in managing symptoms and reducing hospital admissions , poor participation and adherence are a current problem among outpatient programs . factors include difficulty travelling to the program 's location [ 1922 ] , inconvenience of hospital attendance , or difficulty accessing programs . australia is one of the least densely populated nations ( 2.7 people per square kilometre ) with up to 11.8% of the population residing in outer regional or remote locations . conducted a study in kyabram ( rural victoria ) to assess the efficacy of their pr program and noted that patients who lived outside the town centre were more likely to withdraw from pr as the program was conducted within the town hospital . this finding is also supported by de angelis and colleagues who found that transport , travel distance , and related costs were the main reason for nonattendance of a cardiac rehabilitation program . difficulty in accessing outpatient programs has led to the development of home - based programs . it is well established that home - based pr programs are safe and effective [ 13 , 2931 ] and are recommended by many international guidelines and reviews [ 2 , 11 , 12 , 17 ] . given the effectiveness of independent home - based pr programs one could ask what additional benefits supervised remote - based programs would provide . the main advantage of supervised remote - based programs is the ability to minimise anxiety in both patient and therapist . this benefit has been shown in remote - based cardiac exercise programs where the use of transtelephonic exercise monitoring ( tem ) has been used to relieve patients ' and trainers ' anxiety during exercise [ 3234 ] . it is anticipated that a person with copd could exercise to a higher intensity without the risk of undetected adverse events ( e.g. , exercise - induced oxygen desaturation ) . telerehabilitation is an emerging technology used to facilitate the delivery of rehabilitation services at a distance by combining telecommunication and electronic transmission of health information . it has the potential to provide equitable access for people living in rural or remote areas where access to health service is limited . for telerehabilitation to be successful , however , there must be accurate assessment and outcome measurement tools available to therapists [ 37 , 38 ] . in pr , the pulse oximeter is a noninvasive device which continuously monitors a person 's peripheral blood oxygen saturation ( spo2 ) and heart rate ( hr ) . it is used clinically to measure a person 's exercise intensity and warn of exercise - induced desaturation . the concept of remote monitoring of physiological data including spo2 and hr for exercise rehabilitation purposes has received attention in the literature . as early as the mid-1980s , physicians were successful at remotely monitoring a patient 's electrocardiogram ( ecg ) through the analogue telephone network during a home - exercise program [ 40 , 41 ] . however , the majority of electronic healthcare literature on pr has focused on telemonitoring systems [ 4246 ] or consultations services [ 36 , 47 ] . there has been little research using e - health to deliver real - time , live supervision of patients for treatment purposes . this study aimed to develop and verify the use of a remote pulse oximetry system on healthy individuals . specifically , it aimed to establish the technical feasibility of transmitting pulse oximetry data remotely , to determine the validity of remote measurements when compared to conventional face to face measurements , and to evaluate the participants ' perception on the usability of the technology . it was hypothesised that remote pulse oximetry would not only be technically feasible but would also be valid when compared to conventional units and would be easy for participants to use . a favourable result will enable the development of remote pulmonary rehabilitation programs for people living in rural or remote areas . this study was reviewed and granted ethical clearance from the appropriate medical research ethics committee at the university of queensland . thirty - seven healthy individuals were recruited to participate in a single remote cardiopulmonary exercise session . the following exclusion criteria were applied : cardiovascular , respiratory , metabolic , or orthopaedic conditions that would prevent participation in an exercise program , chronic infectious diseases , pregnancy , and mental or physical impairment . the exercise session was conducted using the ehab telerehabilitation system ( v2.0 , uniquest , brisbane , australia ) which is a computer - based videoconferencing and remote consultation system . two ehab devices were used , one with the participant and the other with the therapist . the participant and therapist were located in separate rooms . the ehab device with the participant was configured to receive data from a bluetooth pulse oximeter module ( onyx ii 9560 , nonin medical inc . , plymouth , mn ) ( figure 1 ) . this information was transmitted by the participant device at a frequency of one hertz via an encrypted data channel to an offsite server via an active server page ( asp ) web service . the data was subsequently stored in a mysql database on the server . a therapist device retrieved this data via an asp web service to produce a graph of the participant 's heart rate and spo2 recordings in near real time . for this study the ehab telerehabilitation system utilised a local wi - fi network connection ; however , the connection speed was throttled to 128 kbit / s to ensure compatibility with the 3g / hsdpa mobile network available in remote areas of australia . after written consent was obtained , participants were taken to a room with the ehab device , remote pulse oximeter module , headset , and treadmill . they were asked to remove any nail polish from their index finger on the nondominant hand or jewellery which may disrupt accurate recordings . participants were given an information sheet with instructions on how to affix the pulse oximeter module to the index finger of their nondominant hand and how to establish a connection with a therapist in another room via the ehab device . once a videoconference was established , the therapist guided the participant through an exercise session . the modified borg score ( mbs ) was used to assess the participant 's exercise intensity . as per current copd exercise protocols the session began with a warm - up phase ( mbs 2/10 ) for 5 minutes before gradually progressing to a moderate / somewhat severe intensity ( mbs 4 - 5/10 ) . the last 5 minutes consisted of a cool - down phase ( mbs 2/10 ) . if any adverse signs were observed , the exercise session would be ceased immediately . interrupted sessions due to technical difficulties were noted , and those participants were asked to repeat the exercise session at a later date . at the conclusion of the exercise session , participants completed a questionnaire about their experience with the remote pulse oximeter module and ehab device by marking on a 100 mm visual analogue scale ( vas ) and providing any open - ended comments . during each session , the participant ehab device logged heart rate ( hr ) and spo2 values from the pulse oximeter and time stamped the data . this data was saved into a database stored locally on the computer ( dataset # 1 ) . the participant device also transmitted the data to the remote therapist device where another time stamp was applied to enable the calculation of transmission latency . this data was saved into a database stored locally on the therapist device ( dataset # 2 ) . the mean data transmission rate was determined by measuring the bandwidth requirement ( kbit / s ) . the satisfaction questionnaire rated the comfort , quality , and usability of the ehab device and remote pulse oximetry module . the validity of remote pulse oximetry was assessed by comparing the remote measurements ( therapist data ) with conventional face - to - face measurements ( participant data ) . these were percentage agreement , mean absolute difference ( mad ) , and the bland and altman 's limits of agreement method [ 49 , 50 ] . percentage agreement was calculated as the proportion of data successfully transferred between participant and therapist . the limits of agreement method enabled the quantification of the agreement between the two data sets in terms of the clinical significance of the transmission error . specifically , it gave the upper and lower bounds which show the 95% confidence interval for the difference between the remote and conventional measurements ( i.e. , mean 1.96 sd ) . when applying the limits of agreement method comparing the two datasets , the average of 10 second intervals of data was used . in terms of clinical significance , the minimum clinically important difference ( mcid ) was used as a threshold value to represent meaningful and worthwhile change . remote measurements were classified as valid if the measurement error was lower than the mcid . the mcid for hr , however , varies depending on the individual 's exercise tolerance . for the purpose of this study , the manufacturer 's device accuracy of 3 beats per minute ( bpm ) was used as the mcid , which was a conservative estimate . data was analysed using statistical package for the social sciences ( v17.0 spss inc . , chicago , il ) . a p value of < 0.05 was considered to be significant , where applicable . thirty - seven healthy individuals ( 14 males and 23 females ) completed a total of 15 exercise hours . participants had a mean age of 30 years and standard deviation of 15 years ( range : 18 to 62 ) . there were 34 successfully completed sessions on first attempt confirming the feasibility of the method , while three had to be repeated due to technical difficulties with the bluetooth or wifi connection . the mean ( sd ) transmission latency of the pulse oximetry data ( not the videoconference ) between the computers was 2.94 ( 0.59 ) seconds . the mean ( sd ) bandwidth usage for data transmitted was 6.5 ( 1.0 ) kbit / s . of the remaining data , 9.0% was found to be omitted from the therapist device , while 11% was found to consist of duplicated numbers . the mad and limits of agreement between participant and therapist data are shown in table 1 . overall , participants rated their experience with the remote pulse oximeter and ehab device highly . the mean response to each of the eight questionnaire items is shown in figure 2 . positive written comments included the following : i would be comfortable using [ this device ] at home ( n = 4 ) and [ the ehab was ] simple and easy to use negative comments included worried [ remote pulse oximeter ] could dislodge during vigorous exercise ( n = 5 ) , remote pulse oximeter was uncomfortable ( n = 4 ) , and audio or video quality was jumpy ( n = 3 ) . however , inconvenience , distance , or availability of pr programs can lead to lack of participation and poor compliance . this study demonstrated the feasibility of a remote pulse oximeter for use during a pr program . remote measurements of spo2 and hr were valid when compared to conventional face - to - face measurements . these findings provide confidence for the development of a remote pr program . in our study , there was an unexpectedly high mean rate of data omission ( 9.0% ) and duplication ( 11% ) between participant and therapist data . although these error rates appear comparatively high , they do not necessarily indicate a failure in the data transmission . rather , the logging of data points by the participant device appears to not be perfectly synchronized with data retrieval from the therapist device . this may be a function of the variable delays that can be present with web service calls which were used in this study to transmit data . asynchrony in web service responses may have resulted in either repetition of data or omission of single values . moreover , a visual inspection of the data confirms a random distribution of error throughout the data file . despite this asynchrony , the mad analysis revealed a small difference ( spo2 0.04% ; hr 0.21 bpm ) in comparison to the mcid of 4% for spo2 and 3 bpm for hr . similarly , the limits of agreement demonstrated a small difference in comparison to the mcid for spo2 and hr . the upper and lower bounds for spo2 were 0.67 to 0.67% and for hr were 2.90 to 2.89 bpm . both are within the mcid , and this provides further evidence for the validity of remote measurements . unlike mad , which analyses the actual difference , the limits of agreement give a conservative estimation of difference through a 95% confidence interval . clinically , it means 95% of the time of the difference due to measurement error will be within the relevant limits for spo2 and hr . the mad and limits of agreement values show that remote measurements are sufficiently accurate and valid for real - time monitoring purposes . overall , as indicated by questionnaire responses , participants found the remote pulse oximeter easy to use ( mean vas 89/100 ) and felt confident that they would be able to use it at home ( mean vas 87/100 ) . this is similar to literature which has found the ehab device user friendly [ 5557 ] . however , some participants ( n = 4 ) stated that the remote pulse oximeter was uncomfortable . unlike a conventional pulse oximeter , the remote module has the sensor , display and battery unit all incorporated on the fingertip ( see figure 1 ) . this creates additional weight compared to a conventional sensor , and it could be a contributing factor to the feeling of instability experience by these participants . future studies should compare the comfort with other remote pulse oximeters that have the display and battery unit located at the wrist . furthermore , this needs to be investigated in the copd population as they may be more accustomed to pulse oximetry than healthy adults . five participants commented that they were worried the remote pulse oximeter could dislodge during vigorous exercise . this could be a result of the weight of the device and high intensity of exercise , where most participants needed to jog or run in order to reach an mbs of 5/10 . problems with video and audio quality have been identified as key issues in telehealth [ 5860 ] . during testing , there were occasional disruptions in audio and video transmissions during the exercise session , and on three occasions , the videoconference unexpectedly exited due to connectivity issues on the wifi network , requiring the call to be reestablished . the low frame rate is likely due to the low bandwidth available ( 128 kbit / s ) on the network connection . however , it should be noted that the remote pulse oximeter only uses a very small bandwidth ( 6.5 kbit / s ) for data transmission and , therefore , is unlikely to have attributed to the reduced video / audio quality . there were several limitations of this study ( 1 ) our sample size was small ( n = 37 ) and consisted primarily of young , healthy university students ( mean age 30 years ) . our healthy participants did not show any clinical changes ( e.g. , exercise - induced desaturation ) , and this may have affected the magnitude of error in mad and limits of agreement . furthermore , the young population may be more confident to use or learn about new technology , and this may have therefore influenced the result of the satisfaction questionnaire . ( 2 ) this study was conducted within the university clinic . although the participant and therapist were in separate rooms , they were in close proximity to each other , with the therapist on call if any difficulties occurred . whist the ehab system was bandwidth throttled to ensure compatibility with the 3g / hsdpa mobile network , other factors such as mobile network coverage , signal strength , and speed were not assessed , and this may have affected the performance and the latency of transmission . future studies should include a trial of the remote pulse oximetry system in the home environment with a copd population on the 3g / hsdpa mobile telephone network . a large sample size is required to more rigorously assess the validity and reliability of data transmissions . furthermore , it has been shown that patients demonstrate more positive views of telecare encounters than their healthcare providers . a therapist questionnaire is therefore required to assess their satisfaction and confidence in safely conducting a remote exercise session . our pilot study demonstrated that remote pulse oximetry was feasible , and the data transmission method produced valid data at the remote end when compared with conventional face - to - face measures . although this study was performed on healthy individuals , participants found the ehab device easy to operate and were confident it could be used at home . further studies are needed to address the technical feasibility and validity in a home environment and the therapist 's confidence in conducting a safe exercise program .

pulmonary rehabilitation is an effective treatment for people with chronic obstructive pulmonary disease . however , access to these services is limited especially in rural and remote areas . telerehabilitation has the potential to deliver pulmonary rehabilitation programs to these communities . the aim of this study was threefold : to establish the technical feasibility of transmitting real - time pulse oximetry data , determine the validity of remote measurements compared to conventional face - to - face measures , and evaluate the participants ' perception of the usability of the technology . thirty - seven healthy individuals participated in a single remote pulmonary rehabilitation exercise session , conducted using the ehab telerehabilitation system . validity was assessed by comparing the participant 's oxygen saturation and heart rate with the data set received at the therapist 's remote location . there was an 80% exact agreement between participant and therapist data sets . the mean absolute difference and bland and altman 's limits of agreement fell within the minimum clinically important difference for both oxygen saturation and heart rate values . participants found the system easy to use and felt confident that they would be able to use it at home . remote measurement of pulse oximetry data for a pulmonary rehabilitation exercise session was feasible and valid when compared to conventional face - to - face methods .

acute mountain sickness ( ams ) occurs during exposure to high altitude ( ha ) and is a clinical syndrome characterised by headache , insomnia , malaise , and gastrointestinal symptoms . it is common , developing in 1030% at 25003000 meters and in up to 60% of those ascending to around 4500 meters . it causes significant morbidity and is a challenging clinical condition in remote environments . a biochemical marker of ams , particularly one available as a point - of - care test ( poc ) , could have widespread clinical utility . the pathophysiology of ams is not clearly understood but involves alterations in fluid balance , endothelial function , vascular permeability , inflammation , and oxidative stress . the renal response to ha is an important factor in acclimatization , and ha exposure leads to renal arteriole constriction and relative hypoxia [ 3 , 4 ] . despite the relative renal hypoxia , ngal ( neutrophil gelatinase - associated lipocalin ) is a 25 kda peptide , part of the lipocalin family of small soluble proteins . it is produced in a number of human tissues , notably the distal nephron but also in the lung ngal rises rapidly in the nephron in response to a renal insult and an ngal 150 ng / ml following acute kidney injury ( aki ) is predictive of acute renal failure ( arf ) well before creatinine has risen . ngal is also an acute - phase protein , has a role in inflammation [ 8 , 9 ] , and is upregulated in the lung during inflammation [ 5 , 10 , 11 ] . ngal is also known to rise in conditions associated with oxidative stress [ 12 , 13 ] , and oxidative stress has been implicated in ams [ 14 , 15 ] . we therefore hypothesised that ngal would increase at ha secondary to these various stimuli and that the magnitude of any increase might relate to the presence of ams . we therefore studied a combined cohort of trekkers from 2 expeditions to ha . in order to clarify the relative contribution of ams , hypoxia or exercise to ngal levels , we also studied a cohort pre- and postexercise at near sea level , and a further cohort exposed to acute normobaric hypoxia . the potential role of inflammation in stimulating ngal was assessed by the measurement of highly sensitive c - reactive protein ( hscrp ) in a subset of participants . all study protocols were approved by the ministry of defence research ethics committee , whitehall , uk , and satisfied the requirements of the declaration of helsinki . in all studies informed , thirty - two subjects participating in a defence medical services ( dms ) trekking expedition ( trek 1 ) in the khumbu region of nepal were studied . blood samples were taken from the antecubital fossa at 3 study altitudes : on day 2 at 3400 m , day 6 at 4270 m , and day 10 at 5150 m ( following ascent to everest base camp at 5364 m ) . all samples in this study were collected immediately following a day trekking ( posttrek ) to the study altitude . twenty subjects from a further dms expedition ( trek 2 ) to nepal were also studied . blood samples were again taken at 3 study altitudes : on day 2 ( 3400 m ) , day 6 ( 4270 m ) , and day 10 ( 5150 m ) ( following ascent to kala patthar ( kp ) , 5643 m ) . samples in this study were again collected immediately following a day trekking ( posttrek ) . additional samples were taken at rest in kathmandu ( kat ) at 1300 m and at rest the next morning at the 3 study altitudes . as serving members of the military , all subjects were able to fulfil the fitness criteria of their relevant service . this broadly includes an age - adjusted ability to run 1.5 miles in under approximately 11 minutes and to perform an age - adjusted number of sits - ups and push - ups within two blocks of 2 minutes . fourteen subjects underwent a 3-hour exposure to normobaric hypoxia ( fio2 11.6% , equivalent to 4800 m altitude ) in a hypoxic chamber . this exposure included a 5-minute step test ( step height of 25 cm , 1 complete step every 2 seconds ) at 95 minutes . a group of 22 subjects had ngal assayed at rest and after exercise at sl in the uk following ascent from sea level to 1085 m over 6 hours ( an equivalent gain in altitude and duration of exercise similar to that experienced on a trekking day in nepal ) . two subjects from trek 2 were part of the sl exercise group , but data collection occurred several months apart . ngal was analysed in the field on a biosite triage point of care monitor ( alere ltd , stockport , uk ) using a triage ngal test kit . the triage ngal test is a point - of - care , fluorescence - based immunoassay used which gives a rapid ( 15 minutes ) quantitative measurement of ngal in a range from 60 to 1,300 ng / ml . oxygen saturation ( digitally on warm hands at rest ) was measured using a nellcor np-20 pulse oximeter ( covidian , ma , usa ) during trek 1 + 2 and in the hypoxic chamber study at the same time as blood samples were taken . during trek 1 + 2 , twice - daily ams scores were assessed using the lake louise score ( lls ) questionnaire . the lls allocates a score of 0 to 3 ( symptom not present to severe ) for symptoms of ams ( headache , gastrointestinal symptoms , fatigue / weakness , dizzy / light - headedness , and difficulty sleeping ) . a score of 3 or more in the presence of headache is consistent with ams , a score of 6 or more with severe ams . the commercially available , highly sensitive , immunoturbidimetric assay ( roche diagnostics ) was used to measure crp in trek 2 at the same time points as ngal . mg / l and a between - run coefficient of variation between 2.5 and 5.7% . for statistical calculations , the software package spss 14.0 was used . for subjects with a ngal below the limit of detection of the assay ( 60 ng / ml ) , a value of 60 ng / ml was assigned for the purposes of statistical analysis . all data were tested for gaussian distribution using the kolmogorov - smirnov test and shapiro wilks statistic . for the analysis of dependent variables that were normally distributed , changes were tested by student 's paired t - test . for independent variables that were normally distributed , a within - subjects anova was performed to investigate any serial changes in ngal with ascent at rest and post - trek . a two - way mixed anova with either resting or after trek ngal at each study altitude as the within - subjects factor and the presence of ams ( according to the ll score at multiple altitudes ) as the between - subjects factor if the mauchly sphericity test was significant , then p values were expressed after multiplication by the greenhouse - geisser epsilon . correlation analyses for normally distributed data were performed by calculating the pearson coefficient of correlation . a p value < 0.05 ( two - sided ) was considered significant . as the ascent profile and route were closely matched in trek 1 and trek 2 , data were combined and analysed as a whole . taking medication ( acetazolamide , dexamethasone ) had no apparent effect on ngal values , and therefore these subjects ( n = 11 ) were not excluded from the analysis . demographic data for the field study ( trek 1 + 2 ) , the controls , and the hypoxic chamber study are shown in table 1 . in the 22 subjects ascending to 1085 m in the uk , there was no significant ( p = 0.084 ) rise in ngal following exercise : resting sl ngal was 64 11 ( ng / ml , mean sd , range 60104 ) and postexercise ngal was 71 14 ( ng / ml , mean sd , range 60100 ) . of the 52 subjects , spo2 ( % , mean sem ) dropped from 97 2 at kat ( 1300 m ) to 84 5 and 79 7 at 4270 and 5150 m , respectively ( p < 0.001 ) . there was a moderate inverse correlation between ngal and spo2 at 5150 m ( r = 0.477 , p = 0.001 ) ( figure 1 ) with a weaker inverse correlation between ngal and spo2 at 4270 m ( r = 0.340 , p = 0.019 ) . within the subjects , anova demonstrated a significant change in ngal with ascent both at rest ( p = 0.007 ) and after trek ( p = 0.001 ) ( figure 2 ) . spo2 ( % , mean sem ) dropped from 99 0.4 at baseline to a nadir of 79 5 ( p < 0.001 ) . despite an equivalent drop in spo2 to that seen in trek 1 + 2 , ngal ( ng / ml , mean sd , range ) showed no change between baseline and 180 minutes : 63 26 ( 2980 ) versus 67 25 ( 2784 ) , p = 0.538 . in trek 2 , serum creatinine { mol / l , mean sem , ( range ) , ( p value versus baseline at kat ) } was 78 2 ( 6395 ) at baseline ; at 3400 , 4270 and 5150 m it was 87 3 ( 72120 ) ( p = 0.001 ) ; 84 2 ( 72104 ) ( p < 0.001 ) ; and 94 5 ( 76142 ) ( p < 0.001 ) . according to their ll scores at the highest study altitude ( 5150 m ) , there were 23 subjects with no ams , 16 subjects with mild ams , and 7 subjects with severe ams . there was a significant difference between ngal depending on the presence or absence of ams at 5150 m ( figure 3 ) with higher values in those with ams and severe ams . a two - way mixed anova revealed a significant change ( p = 0.003 ) in resting ngal with ascent and an interaction with ams at 4270 m ( p = 0.017 ) and 4910 m ( p = 0.002 for change in ngal , p = 0.027 for interaction with ams ) . hscrp was ( mean sem , range ) : 1.6 0.4 ( 0.337.53 ) at baseline ; at 3400 m , 4270 m , and 5150 m post - trek : 7 2.9 ( 0.7847.93 ) ( p = 0.002 versus baseline ) ; 25.7 8.1 ( 0.58104 ) ( p < 0.001 versus baseline ) ; 9 3.2 ( 0.5644.62 ) ( p = 0.003 versus baseline ) . at 3400 m , 4270 m , and 5150 m at rest : 6.2 2.9 ( 1.8725.1 ) ( p = 0.001 versus baseline ) ; 21.6 5.7 ( 0.4983.9 ) ( p < 0.001 versus baseline ) ; and 5.8 2.1 ( 0.5426.59 ) ( p = 0.012 versus baseline ) . ngal at 5150 m , after trek was moderately correlated with hscrp at 5100 m after exercise ( rho 0.526 , p = 0.036 ) . this is the first report to describe an association between ngal and both the presence and severity of ams at ha . the significant novel findings are that ngal rises in response to sustained hypobaric hypoxia but not acute normobaric hypoxia or near sl exercise and that this rise is related to ams at 5150 m. the rise in ngal following trekking ( by day 2 at 3400 m ) was to the levels normally associated with the subsequent development of arf ( > 150 ng / ml ) , but this did not occur . although creatinine rose significantly with altitude , the rise was very modest , and we suspect that a combination of factors other than a simple renal insult is responsible for the increase in ngal at ha . our data suggest an inverse correlation between spo2 and ngal at 5150 m ( and to a lesser extent at 4270 m ) . although no such correlation was found at 3400 m , this may still suggest that prolonged renal hypoxia could be a significant drive to ngal release . in addition to renal hypoxia , we suspect that other factors may also contribute to the rise in ngal at ha . the significantly greater ngal in those with severe or mild ams versus those without at 5150 indeed , ngal is an acute - phase protein with a role in inflammation [ 8 , 10 , 11 ] . exercise stimulates an immune response , and hypoxia is also known to cause a response in immune and endothelial cells with inflammatory markers such as hscrp increasing with ha [ 1922 ] . consistent with this , we saw a significantly higher hscrp at all altitudes compared to baseline . limited data have suggested hscrp may be associated with ams but we did not demonstrate any evidence to support this . there was a weak correlation between hscrp and ngal at 5150 m but this can not explain the rise in ngal as a whole . ngal also rises with oxidative stress [ 9 , 12 ] which is increased by exercise , and ha - induced oxidative stress has been implicated in ams . as such , it is interesting to note that we found a higher ngal following trekking and in those with ams at the highest altitude . in an attempt to clarify the relative influence of exercise and hypoxia on ngal , we measured ngal before and after exercise of a similar duration ( 6 hrs ) and similar incremental altitude ( 1085 m ) as that experienced daily in nepal and also in a hypoxic chamber . in neither scenario did ngal rise . this may reflect inadequate duration or severity of stimulus but may also reflect that the ngal response is not due to exercise or hypoxia alone but is multifactorial involving hypoxia , oxidative stress , an inflammatory response , and other , as yet unidentified , stimuli . we also acknowledge limitations such as a lack of serum markers of oxidative stress and a lack of resting ngal data in trek 1 . in addition , we did not measure ngal at sl before departure to nepal , although the ngal recorded as a baseline at kat ( 1300 m ) ( 68 ng / ml ) was no different to those recorded at sl in the uk ( 63 and 64 ng / ml ) . we also acknowledge the fact that although we measured creatinine in trek 2 , we did not continue to monitor it after the cessation of trekking . as a consequence of creatinine rising more slowly in response to a renal insult than ngal , in conclusion , there are several interesting and novel findings that are worthy of further exploration . ngal rises in response to prolonged hypobaric hypoxia ; marked increases in ngal may occur without concomitant arf and the degree of ngal rise at ha is associated with the presence or absence of ams . the fact that ngal does not appear to rise secondary to acute normobaric hypoxia or exercise in isolation suggests that the rise at ha and relation with ams may have common pathways , perhaps related to prolonged hypoxia and an inflammatory response . with the huge and increasing popularity of recreational sports undertaken at both moderate and high altitude assessment of ngal takes a matter of minutes using poc testing , and its use in identifying ams requires further evaluation .

acute mountain sickness ( ams ) is a common clinical challenge at high altitude ( ha ) . a point - of - care biochemical marker for ams could have widespread utility . neutrophil gelatinase - associated lipocalin ( ngal ) rises in response to renal injury , inflammation and oxidative stress . we investigated whether ngal rises with ha and if this rise was related to ams , hypoxia or exercise . ngal was assayed in a cohort ( n = 22 ) undertaking 6 hours exercise at near sea - level ( sl ) ; a cohort ( n = 14 ) during 3 hours of normobaric hypoxia ( fio2 11.6% ) and on two trekking expeditions ( n = 52 ) to over 5000 m. ngal did not change with exercise at sl or following normobaric hypoxia . during the trekking expeditions ngal levels ( ng / ml , mean sd , range ) rose significantly ( p < 0.001 ) from 68 14 ( 60102 ) at 1300 m to 183 107 ( 65519 ) ; 143 66 ( 60315 ) and 150 71 ( 60357 ) at 3400 m , 4270 m and 5150 m respectively . at 5150 m there was a significant difference in ngal between those with severe ams ( n = 7 ) , mild ams ( n = 16 ) or no ams ( n = 23 ) : 201 34 versus 171 19 versus 124 12 respectively ( p = 0.009 for severe versus no ams ; p = 0.026 for mild versus no ams ) . in summary , ngal rises in response to prolonged hypobaric hypoxia and demonstrates a relationship to the presence and severity of ams .

when deposited on various substrate surfaces , rod - like , -conjugated , small organic molecules are well - known for their tendency to form highly anisotropic crystal shapes , which are frequently called fibers or needles . whereas the epitaxial growth has been studied on various substrates , in particular anisotropic surfaces seem favorable to conserve the highly anistropic morphology and optical properties , for example , polarized emission or adsorption provided by a parallel molecular orientation obtained by self - assembly . consequently , cu(110 ) , tio2(110 ) and muscovite mica(001 ) are frequently chosen as a proper fundament to study the epitaxial growth of rod - like small molecules . in this paper , the epitaxial growth of 2,2:6,2-ternaphthalene ( nnn ) on muscovite mica(001 ) is reported . as indicated in figure 1a , the molecule is built from three naphthalene units , which are linked together by c the coexistence of needle - like structures and island - like crystallites is verified . structural analysis reveals two different crystal orientations . whereas island - like structures are built up by upright standing molecules orientated with a ( 001 ) contact plane relative to the muscovite mica substrate ( see figure 1b ) , needles consist of nnn molecules with a ( 111 ) lying orientation ( see figure 1c ) . both crystal configurations provide a well - defined azimuthal alignment , which is discussed based on force field simulations and a recently reported growth model . the azimuthal alignment of island like structures is explained by ledge directed ( b ) a side view of nnn molecules packed in the observed crystal structure . each unit cell houses two nnn molecules . molecules are approximately standing on the s ( 001 ) contact plane , which is indicated in blue . ( c ) a side view along the long molecular axis visualizing the edge - on / flat - on herringbone stacking of nnn . the blue area represents the orientation of the b ( 111 ) contact plane where molecules are aligned in almost in lying configuration . this all - aromatic compound could be obtained in high yield by coupling 2 equiv of 2-naphthaleneboronic acid ( 1 ) with 1 equiv of 2,6-dibromonaphthalene ( 2 ) , as described in the supporting information . the final product , 2,2:6,2-ternaphthalene ( nnn ) , was obtained as a colorless product , which appears to be highly insoluble in common solvents and could only be recrystallized from 1,2,4-trichlorobenzene ( colorless platelets ) . the material was checked with gas chromatography and mass spectroscopy and found to be > 99% pure before thermal sublimation . all samples have been fabricated on muscovite mica(001 ) substrates ( spi , structure probe , inc . ) . muscovite mica is a representative of sheet silicate minerals and provides a layered structure of aluminum silicate sheets weakly bound by layers of potassium ions . each layer is characterized by a high symmetry direction identified by parallel aligned surface grooves . between the individual sheets , the high symmetry direction alternates by 120 leading to a periodic stacking sequence along direction . immediately after cleaving , the mica substrates were transferred to the hot wall epitaxy ( hwe ) chamber . the hwe technique was applied for the deposition of the organic material , which allows us to perform the growth process close to thermodynamic equilibrium , and in further consequence relatively high vapor pressure of the organic deposit in the substrate region can be achieved . therefore , the requirements concerning vacuum conditions are reduced compared with , for example , molecular beam epitaxy . the source material nnn was purified twice by thermal sublimation before filling it into the quartz tube of the hwe reactor . muscovite mica substrates were transferred into the deposition chamber via a load lock and subsequently preheated at the deposition temperature ( 80 c ) for 30 min to ensure a stable temperature during the whole deposition process . the deposition was performed thereafter under a base pressure of 9 10 mbar . optical microscope images have been acquired by a nikon labophot 2a microscope in combination with a nikon type 115 digital camera . scanning force microscopy ( sfm ) studies of the deposited organic films were performed using a digital instruments dimension 3100 in the tapping mode . the 10 10 m images have been acquired at scan speeds of 46 m / s using sic tips ( masch , hq : nsc15/al bs ) exhibiting a cone angle of 40. nominal values for resonance frequency and tip radius are 325 khz and 10 nm , respectively . x - ray diffraction ( xrd ) measurements were carried out on a philips xpert x - ray diffractometer using cr k radiation ( = 2.29 ) and a secondary graphite monochromator . please note that the monochromator is transparent for , /2 , /3 , etc . , so despite the weak intensity of the bremsspektrum , it can give clear bragg peaks due to the scatting on the single crystalline mica substrate . specular scans were performed in bragg brentano configuration by varying the z - component of the scattering vector q. consequently it is possible to detect lattice planes that are parallel to the sample surface . pole figures were acquired by measuring at a constant length of q and only varying its direction . the unit cell parameters of nnn , which were used for analysis , are defined by a = 8.148 0.005 , b = 5.978 0.005 , c = 19.45 0.2 , and = 94.6 0.2 describing a monoclinic lattice ( p21/a ) . the unit houses two nnn molecules in planar configuration . the van der waals ( vdw ) interaction between the organic molecule and the dielectric substrate is modeled by lennard - jones - type potentials . corresponding parameters are taken from the universal force field implemented in a matlab program . the molecules and substrates are assumed to be rigid where the internal structure of an isolated nnn molecules is determined from the crystal structure . simulations were performed for the adsorption of a single nnn molecule as well as a crystal stack . by assuming that a single nnn molecule prefers to lie flat on the surface , the energy minimization procedure is simplified in the following way : we consider only four molecular degrees of freedom , the x- , y- , and z - positions of the molecular center of mass and the angle . the angle defines the azimuthal molecular alignment and is probed by rotating the nnn molecule around the z - axis ( surface normal ) . we perform a grid - based optimization to search for the best molecular adsorption geometry using a grid of 81 81 points for the lateral position . the adsorption angle was tested with a step size of = 1. the surface structure of the substrate has been assumed to be the same as in the bulk where the corrugation is about 0.2 . the substrate surface is assumed to be terminated by the tetrahedral layer of muscovite mica . for the simulation of the 7 2 nnn stack , the molecular packing has been deduced from the crystal structure of a ( 111 ) orientated nnn crystallite . due to the presence of flat and edge - on molecules , the adsorption distance has been optimized , yielding a distance of 1.6 of the lowest h atom of edge - on nnn molecules to the substrate surface . because energetic minima are significantly narrow for the molecular stack , an angular resolution of = 0.5 has been chosen for the calculations . in a first step , nnn was deposited by hot wall epitaxy ( hwe ) on muscovite mica(001 ) . whereas the substrate temperature was kept constant at 80 c , the deposition time was continuously increased . scanning force microscopy was chosen to study the sample morphology versus deposition time , and obtained images ( 10 10 m ) are depicted in figure 2 . as the maximum height scale z0 significantly changes with increasing deposition time , the corresponding values are indicated for each sample . the morphology of all samples is dominated by the presence of several micrometer long needle like structures that are aligned along multiple orientations . as exemplified by the cross - section ( 1 ) , these fibers reach height levels up to 200 nm and are characterized by similar dimensions in width . a more detailed analysis is provided in figure 2a , plotting the mean needle height versus growth time . as indicated by the solid line , the growth rate can be approximated by a linear fit , yielding a slope of 17.8 1.2 nmmin . interestingly , the fibers surface coverage is approximately constant for the whole sample series yielding a value of 12.5% . the sfm analysis reveals that flat islands start to nucleate at the side walls of the fibers and continuously fill the substrate surface between the fibers with increasing deposition time . the latter observation is underlined by analyzing the islands surface coverage , which is depicted in figure 2b as a function of growth time . the solid line , which represents a guide for the eye , indicates an asymptotic approach to 85% of the surface area . again , a representative sample position has been chosen to deduce a cross - section ( 2 ) , which is presented in the bottom right of figure 2 . as reported for other rod - like molecules , a steplike morphology with height levels in the range of 1.82 nm the obtained value corresponds to approximately one monolayer of upright standing nnn molecules . contrarily , the step size of the boundary , defined by the islands and the substrate surface , is significantly larger reaching values in the range of 15 nm . these steps are further characterized by straight extensions , which suggest the formation of well - defined crystal facets . scanning force microscopy ( sfm ) images showing the sample morphology versus deposition time . all samples are dominated by needle - like structures . with increasing growth time , island - like structures start to nucleate at the needle side walls covering continuously the substrate surface between the fibers . exemplary cross sections for both morphologies are indicated in the bottom part of the figure . as indicated in part a , the height of the fiber like structures linearly increases with growth time reaching values of some 100 nm . contrarily , the surface coverage by needles stays approximately constant ( part b ) . step heights in the center part of the islands approximately correspond to a monolayer of upright standing molecules . in a next step , x - ray diffraction ( xrd ) has been chosen to study the structural properties of the organic crystallites . in order to obtain sufficient diffraction intensity , a sample with 216 nm high nnn fibers has been selected . figure 3a reports the acquired specular xrd diffraction pattern , which is dominated by a series of { 00n } diffraction peaks . these peaks are characteristic for island - shaped crystal morphologies , built up by approximately standing nnn molecules , and are consequently abbreviated by s - orientations ( q001 = 0.324 ) . arrows in the upper part of figure 3a indicate the positions of ( 00.2n ) diffraction peaks stemming from the muscovite mica(001 ) substrate . additionally , a diffraction peak arises at qz = 1.36 , which correlates with ( 111 ) orientated nnn crystallites , abbreviated as b orientation . the orientation is characteristic for a nearly flat lying molecular configuration and thus explains the presence of needle - like crystallites as revealed by sfm analysis . a more detailed analysis is reported in the supporting information . in order to analyze the azimuthal alignment of the nnn crystallites relative to the muscovite mica(001 ) substrate , xrd - pf measurements have been performed and are reported in figure 3b . for a profound analysis , xrd - pf have been acquired with a maximum sensitivity to diffraction intensities stemming from the scattering at { 202 } and { 201 } netplanes and are depicted in the bottom leftand right part of figure 3b . the diffraction intensities show a distinct azimuthal distribution , which underlines a well - defined epitaxial relationship to the muscovite mica substrate . the obtained symmetry of the diffraction intensities further underlines the presence of an terminated muscovite mica surface , which is characterized by a mirror plane along the [ 110]mica orientation . the orientation of the substrate has been determined from diffraction patterns stemming from muscovite mica ( 001 ) and are indicated by black solid circles . xrd - pf patterns of the organic crystallites can be constructed by mirror operation from the top hemisphere , sketched by a gray shaded sector . moreover , the xrd - pf patterns reveal a 2-fold rotational symmetry , which can be understood by an approximately equivalent adsorption energy for 180 turned organic crystallites . consequently , discrimination between both crystal alignments has been omitted and simulated 2-fold symmetric diffraction spots are labeled identically . based on these geometrical considerations , only the diffraction spots of a single quadrant have to be analyzed and labeled . ( a ) specular x - ray diffraction ( xrd ) spectrum of nnn on muscovite mica(001 ) . scanning force microscopy images revealed a needle height of 216 nm for the chosen sample . the spectrum is dominated by a series of s ( 001 ) diffractions peaks , which are representative for island - like morphologies . ( b ) xrd pole figure ( xrd - pf ) analysis of { 202 } and { 201 } diffraction peaks , providing information about the azimuthal crystal orientation . as indicated by the simulated pole distribution ( bottom ) , all diffraction spots can be explained by the presence of three differently aligned ( 001 ) crystallites , labeled as s13 ( red ) . the mirror symmetry plane of the muscovite mica ( 001 ) surface is indicated by a horizontal line , which explains the presence of mirrored s13 * crystallites ( blue ) with a ( 001 ) contact plane . moreover , xrd - pf reveal a well - defined azimuthal orientation of b1 ( 111 ) crystallites ( red circles ) . again mirror symmetric crystallites b1 * ( 111 ) are indicated by blue symbols . diffraction intensities from the muscovite mica substrate are indicated by black solid filled circles . diffraction intensities that are characteristic for s - orientated crystallites are located at = 63 ( 74 ) in the left ( right ) xrd - pf , and their azimuthal distribution can be explained by the presence of three crystal orientations labeled as s13 . consistently , both xrd - pf measurements hint the strongest diffraction intensities originating from s1 crystallites . diffraction spots , which can be attributed to b * ( 111)/b ( 111 ) crystallites are expected to appear at 51 for both diffraction geometries and are marked by blue / red filled circles . as each quadrant reveals the presence of one diffraction spot , the azimuthal alignment of b orientated fibers again , mirror symmetric crystals are labeled as b1 * and are characterized by a ( 111 ) contact plane . based on the simulated xrd - pf diffraction peaks , the long needle axis ( lna ) and long molecular axis ( lma ) orientations of b orientated fibers have been deduced and are presented in figure 4 by solid filled arrows . whereas the lna coincides with the [ 110 ] orientation , the lma can be approximated by the alignment of [ 101 ] relative to the muscovite mica substrate . mirror symmetry of the muscovite mica substrate leads to the generation of two energetically equivalent crystallites . fibers that are built up by b1 * ( 111 ) orientated crystallites ( blue ) are aligned with their lna ( lma ) 59.5 ( 49 ) relative to the muscovite mica substrate s [ 110]mica crystallographic orientation . contrarily , their mirror symmetric twins ( b1 ) can be constructed by flipping the b1 * crystallites upside down ( red arrows ) , azimuthally aligned with their lna ( lma ) 59.5 ( 49 ) relative to [ 110]mica . in order to verify the lna alignment , which has been constructed based on xrd - pf measurements , optical microscopy has been chosen , and an image of a representative sample area is depicted in figure 4 . the sample morphology is dominated by fibers that are aligned in a v - shaped , herringbone fashion . as indicated by large red and blue arrows , which represent the expected b1 and b1 * lna orientations deduced by xrd - pf analysis nevertheless , additional needle orientations , which are present in minor fraction , can be observed and are marked by small red ( b2 ) and blue ( b1 * ) arrows , respectively . in order to gain better statistics , a microscopy image of a larger sample area has been chosen to perform a fast fourier transformation ( fft ) and the obtained pattern is depicted beside . the fft is dominated by two stripes , which are characterized by an enclosing angle that perfectly reflects the lna orientation of b1 and b*1 crystallites ( indicated by red and blue arrows ) . long needle axis ( lna ) ( left ) and long molecular axis ( lma ) ( right ) orientations of b1 ( red solid filled arrows ) and mirror symmetric b1 * ( blue solid filled arrows ) crystallites , deduced by x - ray pole figure ( xrd - pf ) measurements . in the outer ring of the lna polar plot , additionally the [ 110 ] crystallographic orientations of s13 ( red ) and it is visualized that the [ 110 ] crystallographic direction of s1 crystallites provides the same azimuthal orientation as the lna of b1 fibers . below , the obtained lna orientations are verified by the observed sample morphology using optical microscopy . beside b1 and b1 * fibers additionally minor fractions of approximately horizontally aligned crystallites are observed ( marked by small red and blue arrows ) . by using fast fourier transformation ( fft ) ( depicted beside ) , the dominant fraction of b1 and b1 * crystal orientations is further underlined . in a next step in particular , the azimuthal alignment of their [ 110 ] directions has been deduced and is depicted in the outer ring of figure 4 ( lna ) . because s1 and s1 * crystallites represent the major fraction , they are indicated by large arrows approximately aligned 60 relative to [ 110]mica . the azimuthal orientation perfectly coincides with the lna of b1 and b1 * crystallites , which already suggests an epitaxial relationship of both crystal types . in order to analyze the latter observation in more detail , the sfm image , which is shown in figure 5 , is dominated by two approximately vertically aligned fibers . between fibers , the presence of an s orientated island can be observed , which is terminated in the bottom part of the image by a sharp l - shaped boundary . the observed boundary shape perfectly correlates with the expected angle between the and [ 110 ] crystallographic orientations of an ( 001 ) orientated crystallite . the generation of the observed crystal shape can be understood by the formation of (11n ) and (11n ) side facets , which represent low index planes for n = 1 , 0 , or 1 . an extracted cross - section of the observed fiber is presented in the left part of figure 5 . the observed fiber is terminated in the right part by a flat plane , which is aligned parallel to the substrate at a height of approximately 100 nm . contrarily , the left side of the fiber shows a constantly decreasing height level . the slope of the side facet approximately correlates with a 25 nm decrease in height along 100 nm of the needle width ( 14 ) . the lna of b * type crystallites is defined by their [ 110 ] orientation and consequently all crystallographic planes (11n ) are aligned parallel to it . because the angular tilt of 13.8 between the low index planes ( 110 ) and ( 111 ) perfectly correlates with the observed sfm analysis , the theoretically expected cross - section of a b1 * fiber has been modeled and is depicted below the experimental data . although , the observed steep height decrease at the fiber side walls is below the resolution limit of the sfm , a termination of the fibers by ( 001 ) and ( 001 ) facets can be assumed . scanning force microscopy ( sfm ) image showing vertically aligned fibers and s orientated islands in between after the deposition of 60 min nnn at 80 c substrate temperature . the island s boundaries correlate with the geometrical alignment of and [ 110 ] orientations . the extracted fiber cross - section ( top , left ) can be explained by the formation of ( 111 ) , ( 110 ) and (001 ) facets . based on the observed crystal shapes , their crystallographic orientations relative to each other has been deduced and is visualized by a 3d model below . the epitaxial relationship between fibers and islands is consistent with the structural analysis and can be explained by a nucleation of nnn molecules at the fiber side walls , also called ledge directed epitaxy . based on the latter analysis , a three - dimensional model of both crystal types has been generated and is depicted in the bottom part of figure 5 . as indicated by the xrd - pf analysis , which is presented in figure 4 ( lna ) , b1 * and s1 * crystallites shared the same azimuth for their crystallographic [110 ] orientations . in that way , the tilt angle of standing nnn molecules within the s - type crystallite approximately correlates with the tilt angle of the fiber ( 001 ) low energy plane . analogous observations were demonstrated for 6 t fibers and are explained by the nucleation of islands at the sidewalls of already existing needles . moreover , the latter picture is perfectly consistent with the sfm analysis presented in figure 2 , which reports a continuously increasing island coverage for longer deposition times . such epitaxial alignment based on a geometrical fit between nucleating crystallites and already existing topographic features on the substrate is called ledge directed epitaxy . based on the latter analysis together with xrd - pf data presented in figure 4 ( lna ) , it can be concluded that the minor fraction of s23 crystallites has most likely nucleated at b - type fibers , which are orientated approximately 30 tilted relative to [ 110]mica of the muscovite mica substrate . this conclusion is further consistent with the microscopy image , presented in figure 4 , revealing the presence of a minor fraction of such fibers ( below the detection limit of xrd - pf measurements ) , which are subsequently labeled by b2 and b2*. in order to understand the observed growth behavior of b1 and b2 fibers on muscovite mica(001 ) , the left part of figure 6 depicts a planar nnn molecule in gas phase . analogous to 6 t , a planar molecule is characterized by a mirror plane h , which is aligned in the plane of the naphthalene rings , and a 2-fold rotational axis , which is aligned normal to h . , it should be assumed that single nnn molecules tend to adsorb lying flat on the muscovite mica substrate in order to maximize their contact area . in that way , h is orientated parallel to the substrate , and the molecule becomes chiral when adsorbed on an arbitrary surface . molecules that are intrinsically achiral but obtain a form of 2d chirality when adsorbed on a substrate surface are also called prochiral . analogous to 6 t , two mirror symmetric nnn enantiomers ( sketched as red and blue molecules ) can adsorb on the muscovite mica surface , which can not be brought into congruence by translation and rotation . taking a top view of the molecular stacking at the contact plane of b - type crystallites , which is depicted in the right panel of figure 6 , reveals that ( 111 ) orientated fibers are built up by an alternating assembly of only one enantiomer ( red ) and edge - on nnn molecules . contrarily , their twin crystallites b * ( 111 ) are built up by the mirrored molecular configuration ( blue ) only . the latter observation further explains the consistent choice of a red and blue color code for molecules and crystallites . beside a real space model for b1 crystallites , which is deduced by xrd - pf analysis , the right part of figure 6 further includes the proposed geometry of b2 fibers . based on a growth model that has been deduced for 6 t fibers , it is assumed that two needle orientations , for example , b1 and b2 * can originate from one molecular adsorption site . the existence of these two lna orientations is explained by a mirror symmetric molecular stacking during crystal nucleation . interestingly , epitaxially grown 6 t on muscovite mica showed a comparable fraction of both stacking types , which explains the observation of four lna orientations . the latter phenomenon is further explained by force field simulations for both crystal types , yielding a similar adsorption energy with a deviation of some millielectronvolts / molecule . contrarily , xrd - pf analysis revealed that nnn fibers are dominantly present only in one configuration , which reduces the observed lna orientations to two ( see the fft in figure 4 ) . consequently , it can be stated that both crystal types seem to significantly differ concerning their adsorption energy , which should be investigated and underlined by the discussion of force field simulations within the next paragraphs . molecular symmetry of a planar nnn in gas phase and when adsorbed flat on an arbitrary surface . due to the presence of a mirror symmetry plane h parallel to the naphthalene rings and a 2-fold rotational axis , aligned normal to it , the nnn molecule in gas phase can be described by the c2h point group . contrarily , when adsorbed flat on a substrate surface , nnn can form two mirror symmetric enantiomers ( sketched by red and blue molecules ) , which follow c2 symmetry . the right panel depicts a real space model of the discussed crystal orientations ( top view ) . the molecular alignment of nnn within the surface unit cell has been deduced from its bulk structure , oriented with a ( 111)/(111 ) contact plane for b / b * crystallites . the orientation of the long molecular axis ( lma ) or long needle axis ( lna ) is indicated by blue or red arrows . taking a closer look at the molecular stacking at the contact plane of b - type crystallites reveals that ( 111 ) orientated crystals are alternately assembled by red enantiomers and edge - on nnn molecules . contrarily , their mirror symmetric twins b * only consist of blue molecular configurations . the real space image of b1 ( b1 * ) and b2 * ( b2 ) the adsorption energy as a function of the long molecular axis ( lma ) orientation ( ) is depicted in the top panel in terms of a polar plot . red and blue arrows indicate the molecular orientation deduced by experiments . at the indicated positions , force field simulations reveal a broad maximum for the corresponding enantiomer . contrarily , the adsorption site seems less favorable for the mirror symmetric molecule ( e 20 mev ) . a real space model that sketches the lateral position for the molecular adsorption angle at = 57 simulations reveal a preferred alignment of the terminating naphthalene units in the surface corrugations of the muscovite mica substrate ( indicated by horizontal lines ) . below , an analogous analysis has been done for a 7 2 molecular stack representative for the contact plane of a b ( 111 ) crystallite . simulations reveal a strongly pronounced adsorption maximum at the experimentally observed adsorption angle ( red arrow ) and a significantly different adsorption energy for a b * crystal with opposite stacking sequence ( blue arrow , e 300 mev / molecule ) . a real space model of the optimized adsorption position is depicted in the right panel . by comparing the alignment of the nnn molecules with the muscovite mica unit cell , an approximately periodic alignment can be recognized along mica/[110]mica for an / terminated surface . in a first step , the optimal adsorption energy of a single nnn molecule has been deduced based on force - field simulations by selecting the most favorable adsorption site for each angle . the angle characterizes the azimuthal alignment of the lma relative to mirror symmetry plane of the muscovite mica substrate surface . the molecules are assumed to adsorb flat on the substrate and the adsorption energy , ead , is defined as the difference between the energies of the isolated subsystems and the energy of the combined system . therefore , maxima in the ead versus curves evidence the favorable adsorption geometries . to increase the readability , ead curves are presented in figure 7 in terms of a polar plot , where ead = e0 ead . the parameter e0 represents the adsorption energy at the least favorable angle , yielding a value of e0 = 2.49 ev for the isolated molecules . because nnn molecules can adsorb either in their left- or right - handed configuration , simulations have been performed for both enantiomers and are color coded by red- and blue - filled curves . simulations yield , for both molecular configurations , multiple adsorption maxima , which are located for the blue marked enantiomer at max , blue = 42 , 57 , 102 , 161 , and 178. due to 2-fold rotational symmetry of the nnn molecule , identical values are obtained for ead(+180 ) . moreover , optimized adsorption positions for the red molecular type are found at max , red = max , blue due to mirror symmetry of the substrate surface . experimentally obtained adsorption angles are further indicated by a blue ( red ) arrow at = 49 ( 131 ) . both adsorption angles correlate with the broadest maxima obtained by simulations and are importantly consistent with the simulations of the corresponding enantiomer . the fact that beside experimentally observed adsorption geometries force field calculations also yield additional maxima is attributed to the usage of empirical potentials , which in some cases may yield the wrong energetic ordering of competing structure solutions . nevertheless , it has to be underlined that simulations indicate a significant less favorable adsorption site for the mirror symmetric molecular configuration ( 20 mev ) . in general , adsorption energies for both molecular configurations significantly differ , which in further consequence leads to a nonequal distribution or even breakup of both enantiomers depending on the adsorption angle . contrarily , simulations as well as experimental data that are reported for 6 t indicate a significantly lower energetic splitting between both molecular configurations , which may result from a higher symmetry of the molecule . the latter statement can be understood by the fact that thiophene molecules with an odd ring number , for example , quinquethiophene or septithiophene , are characterized by a mirror symmetry plane when adsorbed on a surface and consequently do not show a prochiral character . contrarily , the asymmetric alignment of the c c bond between two naphthalene units of nnn inevitably leads to a prochiral behavior when adsorbed on a substrate surface and consequently plays an essential role concerning the energetic separation of both enantiomers at a defined adsorption angle . the experimentally confirmed adsorption position of nnn molecules is further depicted in the right part of figure 7 . for both molecules , an adsorption angle = analogous to calculations for p-6p and 6 t , nnn molecules tend to align their rings in the surface corrugations , which are indicated by vertical solid lines . in order to study the adsorption energetics of a b - type crystallite , a 7 2 molecular stack has been deduced from a ( 111 ) orientated crystallite . analogous to the force field simulations of an isolated molecule , the adsorption energy ead for the stack has been probed depending on the molecular orientation and the adsorption energy at the least favorable adsorption angle is given by e0 = 0.41 ev / molecule . because the curve calculated for the a b * ( 111 ) stack follows the same behavior as discussed for a single molecule ( mirror symmetric ) , only the results for a b contact plane are depicted in order to increase readability . interestingly , simulations reveal that not only the number of energetically favorable adsorption sites decreases but also the angular acceptance , which becomes visible by well pronounced peaks . simulations further indicate the presence of two adsorption maxima , which are located at = 12 and 48. again , the experimentally obtained adsorption angle for b1 crystallites is indicated by a red arrow and underlines a nearly perfect agreement . moreover , it can be recognized that the adsorption energy for a b2 crystal ( at + 48 ) becomes even more unfavorable than that for a single molecule , which is manifested by a much lower value of ead in the range of some 100 mev / molecule . consequently , force field simulations not only reflect the experimental observations but also explain the dominant fraction of b1 crystallites by a preferred nucleation of their stacking sequence in contrast to b2 crystallites . the observed behavior can be even better understood by analyzing the real space model of the simulated adsorption position at = 48. besides the molecular alignment , also the surface unit cells of the muscovite mica and b crystal have been indicated . obviously , the unit vector [ 110 ] of the nnn crystal stack , which also defines its lna , tends to align parallel to one surface unit vector of the muscovite mica crystal , which is defined by the mica, ( [ 110]mica, ) orientation for an ( ) terminated surface . the epitaxial growth of ternaphtalene ( nnn ) on muscovite mica(001 ) has been investigated by combining structural ( xrd - pf ) and morphological ( sfm ) methods . consistently , both methods reveal the formation of s ( 001 ) orientated nnn island - like structures which have nucleated at the sidewalls of b ( 111 ) orientated fibers . it is demonstrated that the latter nnn crystal types tend to align along two dominant directions , which leads to the formation of a v - shaped sample fiber morphology . because the tilt angle of nnn molecules within s - orientated crystallites correlates with the tilt angle of the fiber side facets , the island nucleation is explained by ledge directed epitaxy . based on this growth model , it can be understood that both crystal types provide a well - defined azimuthal orientation relative to the muscovite mica substrate . by use of force field simulations , the growth of the fibers is further analyzed . the epitaxial growth of sexithiophene ( 6 t ) on muscovite mica showed the formation of four well - defined fiber orientations , which can be explained by mirror symmetry of the muscovite mica substrate and two differently stacked 6 t crystallites , which can nucleate at a molecular adsorption position . contrarily , experimental investigations indicate that nnn crystallites tend to stack in a single configuration , which explains the dominant formation of only two fiber orientations . based on force field simulations , the latter observation is further investigated and explained by significantly different adsorption energies of both crystal types . it is further demonstrated that the observed behavior results from an interplay of the molecular adsorption and lattice match .

the morphology and structure of 2,2:6,2-ternaphthalene ( nnn ) deposited on muscovite mica(001 ) substrates was investigated by scanning force microscopy ( sfm ) and specular x - ray diffraction measurements . consistently , both methods reveal the coexistence of needle - like structures with a { 111 } contact plane and { 001 } orientated island - like crystallites , which are built up by almost upright standing nnn molecules . both orientations are characterized by a well - defined azimuthal alignment relative to the substrate surface , which is analyzed by x - ray diffraction pole figure ( xrd - pf ) measurements . based on xrd - pf and sfm analysis , the azimuthal alignment of { 001 } orientated crystallites is explained by ledge - directed epitaxy along the fibers sidewalls . these fibers are found to orient along two dominant directions , which is verified and explained by a doubling of the energetically preferred molecular adsorption site by mirror symmetry of the substrate surface . the experimental findings are confirmed by force - field simulations and are discussed based on a recently reported growth model .

a-38-year - old female patient consulted to our emergency outpatient clinic with complaints of fever , sore throat , coughing , shivering , chills , joint , and muscle pain , headache , chest pain continuing for nearly 2 weeks and she was hospitalized to investigate the etiology of fever . physical examination findings were as follows : bp 130/80 mm hg , body temperature , 39.8 c ; hr 120/min ; moderately well general physical status with full cooperation , markedly hyperemic pharynx , and tonsils , herpetic rashes around her lips , bilaterally diminished respiratory sounds heard over basal segments and tachycardia . her neck rigidity was evaluated however the kernig , and brudzinsky signs could not be elicited . laboratory test results were as follows : hb 11 g / dl ( 12.117.2 ) , mcv 80.8 fl ( 82.299 ) , wbc 22.10 k / ul ( 410 ) , neu 90.5% ( 3773% ) , esr 92 mm / s ( 020 ) , procalcitonin 0.8776 ng / ml ( < 0.1 ) , total protein 5.66 g / dl ( 6.48.3 ) , albumin 2.9 g / dl ( 3.55 ) , inr 1.15 ( 11.5 ) , ferritin 2889 ng / ml ( 14150 ) . glucose , creatinine , ast , alt , alp , na , k , complete urinalysis , ana , rf , and ena profile were unremarkable . on thoracal imagings , bilateral hilar lymph nodes of which the largest being 2 cm in diameter , bilateral pleural effusion , and atelectasic areas in the parenchyma surrounding the pleural effusion were observed ( figure 1 , 2 ) . on abdominal tomograms , no bacterial growth was detected in urine , throat , csf , and blood cultures . lumbar punction performed because of suspect neck rigidity did not reveal any abnormal cells in csf . cephtriaxone at daily parenteral doses of 2 g ) . during follow - up period , rashes , and swelling on her right , and left wrists were detected , so she was consulted to rheumatology clinic , and naproxen sodium therapy was started with the indication of suspect aosd . on physical examination dullness over traube s space however her hyperfebrile state persisted , echocardiograms obtained for the differential diagnosis of infective endocarditis . posteroanterior chest radiogram costodiaphragmatic sinuses are not distinctly visualized , high - lying diaphragms , and an enlarged mediastinum were observed . biochemical analysis of the fluid drained by thoracenthesis of the patient whose computed tomograms demonstrated bilateral pleural effusion revealed the presence of 2 gm / dl albumin , 3.34 g / dl protein , 483 u / l ldh ( concurrent serum albumin 2.9 gm / dl , total protein 5.99 g / dl , ldh 393 u / l ) . antibiotherapy of the patient with pleural effusion ( exudate ) was discontinued , and methylprednisolone treatment at daily doses of 16 mg was initiated . after initiation of steroid therapy , she maintained an afebrile state , her joint manifestations regressed , and her complaints decreased during her follow - up period . the patient whose pleural effusion completely regressed with steroid therapy discharged to be further followed up by rheumatology outpatient clinic ( figure 3 ) . adult onset still s disease is a disease whose diagnosis can be made by excluding infectious , malign , autoimmune , and autoinflammatory diseases , and in consideration of its characteristic clinical , and laboratory findings . for definitive diagnosis , yamaguchi criteria can be used . its major criteria consists of higher body temperature at 39c lasting for at least one week , marked nonpruritic macular / maculopapular salmon - colored rashes on the torso or extremities persisting for at least 2 weeks , leukocytosis ( > 10.000/ml ) , and neutrophilia ( > 80% ) . minor criteria include sore throat , lymphadenopathy , hepatomegaly or splenomegaly , abnormal hepatic function tests ( increases in especially ast , alt , and ldh levels ) , and ana , and rf negativities . for the establishment of diagnosis , 5 diagnostic criteria ( including at least 2 major criteria ) should be met . in our patient , during approximately 4 weeks up to initiation of steroid therapy , fever exceeding 39c every day , symptoms of arthralgia , marked arthritis of both wrists , leukocytosis , neutrophilia , sore throat , and lymphadenopathies fulfilled the required diagnostic criteria , and pleural exudate seen during the clinical course of the disease was assessed to be associated with the disease . in the adult onset still s disease pleural effusion , transient pulmonary infiltration , and pericarditis can be seen in 3040% of the patients . affected individuals may complain of mild episodes of coughing , pleuritic chest pain , and shortness of breath . even in some patients it may progress to acute respiratory distress syndrome ( ards ) [ 6 , 7 ] . rarely , myocarditis , arrhytmias , heart failure , and cardiac tamponade can be seen . myocarditis , and ards are more frequently seen especially in patients with mas . in the differential diagnosis , infectious diseases ( ebv , cmv , hbv , rubella , parvovirus , coxsackie , hiv , subacute bacterial endocarditis , meningococcemia , tuberculosis , syphilis ) ; vasculites ( takayasu arteritis , polyarteritis nodosa , serum disease , thrombotic thrombocytopenic purpura ) ; malign diseases ( leukemia , lymphoma ) , connective tissue diseases ( ra , sle , mixed connective tissue disease ) , granulomatous diseases ( sarcoidosis , crohn disease ) ; autoinflammatory diseases ( kikuchi disease , sweet syndrome ) , and drug hypersensitivity reactions should be considered . in our patient serological tests , cultures , and imaging modalities were used for differential diagnosis which led to the establishment of the diagnosis of aosd . higher ferritin levels are among the most important laboratory findings of aosd , however markedly lower glycosylated ferritin levels are detected ( < 20% ) . increased ferritin level is an indicator of the disease activity , and also a serologic marker of treatment response . in our patient ferritin levels may increase as a response of hepatocytes to cytokines as il-18 , and il-1b which may be thought to play a role in the pathogenesis of aosd . as the first - line therapy nsaids ( especially endomethacin ) , and corticosteroids are recommended . in cases of severe organ involvement pulsatile high doses of steroids for few days can be used , than daily dose is dropped down to 1 mg / kg . in cases of recurrence or with the intention to decrease steroid dose , methotrexate at a weekly dose of 7.525 mg can be given . hydroxychloroquine , sulphasalazine , azothioprine , cyclosporine can be used separately or in combination with methotrexate . in recent years , effectiveness of biological agents ( infliximab , etanercept , anakinra , and tocilizumab ) , and ivig therapies have been reported in the treatment patients with serious organ involvement , and mas . still many characteristic features of aosd are not known , and a standard follow - up or a treatment protocol of the disease is not available . in patients who met diagnostic criteria , pleural effusion which may be seen in the clinical course of the disease should be presumably associated with the disease . besides regression of pleural effusion can be expected with the treatment of underlying desease , additional locally invasive diagnostic , and/or therapeutic interventions should be avoided .

adult onset still s disease is a rare systemic inflammatory disorder . at the onset of the disease sore throat , pharyngitis ; which does not respond to antibiotics , one or two times peaking febrile episodes , marked salmon - colored rash on the trunk and extremities , arthralgia , arthritis , myalgia , fatigue , loss of appetite with nausea and weight loss ; hepatosplenomegaly and lymphadenopathy can be seen . among laboratory examinations levels of ferritin and other acute phase reactants distinctly rise , and neutrophilic leukocytosis ; ana and rf negativity are detected . pleural and pericardial effusions , transient pulmonary infiltration , and rarely myocarditis can be seen during the course of the disease . here we report a patient who was examined for fever of unknown origin and diagnosed with adult onset still s disease which is a rare etiology of pleural effusion .

dual - polymerizing resin cements have been extensively used for placement of indirect restorations and posts . the dual - polymerizing materials were developed to compensate for the lack of polymerization in the absence of light and to represent a combination of auto- and light - polymerizing components . in some clinical situations , such as in dark zones at the apical region and during the cementation of indirect restorations , the severe light attenuation results in low degree of conversion ( dc ) , which can compromise the mechanical properties and consequently the longevity of the indirect restorations.14 modifications in the viscosity of the resin cements allow their use in different clinical situations . the option for low viscosity versions offer some benefits , such as minor thickness of the pellicle that was formed following the restoration placement . the lowest film thickness generates smaller polymerization shrinkage , reducing the possibility of gaps formation and premature marginal leakage.5,6 the difference in the cement formulations that change the viscosity is related to the proportion between resin matrix and filler particle content.7 high volume fraction of fillers can increase the viscosity and the elastic modulus and strength of composites.7,8 low viscosity or flowable resins and resin cements present lower filler loading than regular restorative materials . most direct dental restorative composites use bisphenol - a - diglycidylether dimethacrylate ( bis - gma ) , which is considered a very viscous monomer , and when mixed with higher filler loadings , it becomes a nearly solid mass and unusable product . vinyl groups ( e.g. , ethylene glycol dimethacrylate ) are added as a thinner or diluent monomer for uncured pastes , which are considered another approach to change the viscosity of resin - based materials . the filler loading and the viscosity of composites may interfere in the monomer conversion , since they could restrict the mobility of monomers and the propagation of polymerization reaction.7,911 the restorative resin - based materials must reach a high degree of monomer conversion in order to present better clinical performance and longevity and also to reduce the early failures . for the dual - polymerizing resin cements , the self - cure mode should ensure the high level of conversion , especially in the cervical proximal areas , the root canal and in the internal and deep areas of the cavity preparations.1214 several methodologies have been used to analyze the dc of resin - based materials ; however , most of them use fourier transform infrared spectroscopy ( ftir ) . although many researchers have evaluated the polymerization effectiveness to determine the physical and mechanical properties of dental materials,9,15,16 little information is known about the influence of different viscosities of dual - cured resin cements on their physical properties , such as the degree of conversion in situations of self - and dual - polymerization . thus , the purpose of this study was to measure the dc of two commercially dual - cured resin cements in different viscosities ( low and high ) when they were light - activated or when the materials were allowed to self - cure solely , after 5 minutes and 24 hours from the mixture of pastes ( base and catalyst ) . the hypothesis tested was that curing mode , evaluation time and viscosity would affect the dc of the resin cements . two commercial dual - cure resin cements in high and low viscosities were evaluated : variolink ii ( ivoclar vivadent , schaan , liechtenstein ) and nexus 2 ( kerr corp . , orange , ca , usa ) . the resin cements consist of two paste components that were equally dispensed and mixed together according to manufacturers instructions . after mixing , resin cements ( n=5 ) were applied to the horizontal attenuated total reflectance znse crystal at 45 ( fourier transform infrared spectrometer , ft - ir spectrometer 520 , nicolet instrument corp , madson , wi , usa ) at room temperature . adhesive tape ( 3 m , sumar , sp , brazil ) was placed on the znse crystal surface to act as a spacer , ensuring standard thickness for all specimens ( 100120 mm ) . a mylar strip ( quimidrol com . ltda , joinville , sc , brazil ) was placed over the cement and was pressed flat to spread the material on the crystal surface . the spectrum of unpolymerized material was obtained and the cements were either light - cured ( dual - polymerizing mode ) or allowed to self - cure only ( autopolymerizing mode ) . each specimen was left on the crystal surface and further spectra were obtained 5 minutes and 24 hours after post mixing . for light - cured groups , a halogen light curing unit ( xl 3000 , 3 m espe , st . paul , mn , usa ) was used during 40 seconds ( 600 mw / cm ) . the light intensity was periodically checked with a radiometer ( curing radiometer , model 100 , kerr corp . , orange , ca , usa ) . spectra were obtained between 4000 cm and 750 cm at a resolution of 4 cm . monomer conversion was calculated ( % ) according to the changes in the ratio between the absorbance peaks corresponding to the aliphatic ( cc ) ( 1638 cm ) and aromatic ( 1608 cm ) carbon double bonds prior to and 5 minutes and 24 hours after polymerization initiation for both curing modes.10 the aromatic peak is used as an internal reference because the intensity does not change during the polymerization reaction.9,11 the effect of curing mode , viscosity and time intervals on the dc were evaluated for each material . three - way repeated measure analysis of variance ( anova ) ( curing mode , viscosities and evaluation time ) was performed and tukey s post - hoc test was used to detect pair wise differences among experimental groups . the data was analyzed using the statistical program sas 9.1 ( sas institute , cary , nc , usa ) and all statistical testing was performed at a pre - set alpha of 0.05 ( p<.05 ) . the statistical analysis of the data showed significant differences in curing mode , viscosity and evaluation time factors for both resin cements ( p=.01 ) . there was significant interaction between factors : the curing mode x evaluation time ( p=.01 ) . other double - interactions and the triple - interaction ( curing mode x viscosity x evaluation time ) were not significant ( p>.05 ) . the dc of resin cements was higher when the materials were light - cured and when the low - viscosity versions were used than when self - curing mode and the high - viscosity resin cements were tested , respectively ( p<.05 ) . the results of this study showed that the curing mode ( auto- and dual - polymerization ) , the viscosity ( low or high ) and the evaluation time ( 5 minutes or 24 hours ) influenced the dc of resin cements . thus , the research hypothesis stating that these factors would result in significant changes in the dc was accepted . as the dc is dependent on the monomeric composition and the concentration and type of the photoinitiator in each product,9,17 the statistical analysis comparing the dc between resin cements the main factors under study ( curing mode , viscosities and time intervals ) produced similar effects for the resin cements , which presented different formulations regarding the resin matrix composition and type and concentration of filler particles . the variations on the proportion of components of resin matrix and filler particles that produced the low and the high versions caused no difference in behavior between the two cements results.7 a high monomer conversion with cross - linked polymeric network formation is essential for the durability of the resin - based restorations.911 studies have shown the importance of a high dc of resinous materials in order to reach better mechanical properties , since the presence of a high amount of residual methacrylate monomers compromises the hardness , the abrasion and the fracture resistance . moreover , the low dc can increase the sorption and the solubility , interfering in the color stability and mass loss of the resin cement.2,4,9,14,1720 the resin cements showed significant difference between the evaluation times , 5 minutes and 24 hours from the polymerization initiation , independently of viscosity or curing mode . thus , the polymerization reaction for both resin cements continued after 5 minutes of the mixing of the base and catalyst pastes . the continuation of the chemical part of the polymerization reaction ( benzoyl peroxide + tertiary amines ) was primarily responsible for the increase of the dc after 24 hours . additionally , the percentage of dc increasing after 24 hours was higher for the self - cure mode than the dual cure mode , which ranged from 38.3 to 42.4% and from 26.6 to 28.4% , respectively . the continuation reaction 24 hours after polymerization initiation resulted in a reduction of absorbance peaks that corresponded to the aliphatic carbon double bonds10 and the dual polymerization increased dc , which corroborates with several studies that compared the polymerization modes for resin - based materials.2,11,21,22 for both resin cements used in this study , the direct light - polymerization provided dc that was higher and ranged from 10 to 30% higher than those observed for the self - curing mode . also , it was higher at 5 minutes ( 24 to 30% ) than for 24 hours ( 10 to 12% ) from the polymerization initiation . the auto - polymerization may be insufficient to result in an adequate dc for the long lasting restorations . clinically , the low viscosity version ensured higher dc , and 24 hours elapsed time after setting of restoration is appropriate for the final appointment , since the higher dc could better support the final oclusal adjustments , finishing , and polishing procedures . the direct light - polymerization can be considered a limitation of this study , since the light is applied directly to the resin cement layer only at the periphery of restorations . on the other hand , the inner part of indirect restorations is reached by an attenuated light and resin cement may depend only on self - cure activation . regarding the differences in the dc between high and low viscosities , higher mean values were found for low viscosity versions of both resin cements . low viscosity materials can be due to lower filler loading and/or higher addition low viscosity diluents monomers . the low viscosity monomer allows better mobility and distribution of free radicals inside the resin material , which can increase the polymerization reaction and the monomer conversion.9 the increase of dc promoted by the low viscosity versions in percentage , following the polymerization mode ranged from 0.9 to 3.1% for the dual - polymerizing groups and ranged from 1.5 to 3.9% for the auto - polymerizing mode , which was the highest percentage among the groups from nexus 2 resin cement . for the variolink ii , dual - polymerizing groups ranged from 1.4 to 2.8% , while the auto - polymerizing mode ranged from 1.7 to 2.4% . the direct light - activation and the low viscosity version were important to provide higher dc for the dual - polymerizing resin cements . twenty - four hours after seating the indirect restoration is appropriate for final oclusal adjustments , finishing , and polishing procedures , which are capable of generating stress to the resin cement layer .

objective : the purpose of this study was to evaluate the effects of the viscosities and curing modes on the degree of conversion ( dc ) of two resin cements.methods:eight experimental groups were evaluated ( n=5 ) , according to the dual - cured resin cements ( nexus 2/variolink ii ) , viscosity ( low and high ) and evaluation time ( 5 minutes and 24 hours ) . the resin cements were applied to surface of a horizontal attenuated - total - reflectance unit and were polymerized either with self - cure ( sc ) or light exposure ( xl3000/3 m espe ) for 40 seconds . infrared spectra were obtained after 5 minutes and 24 hours ( nicolet 520 ft - ir / thermo scientific inc . ) . dc was calculated according to changes in aliphatic - to - aromatic peak ratios pre- and post - curing . data ( % ) were analyzed by 3-way repeated measure anova ( curing mode , viscosity and time interval ) and tukey s post - hoc test ( p<.05).results : the dual - polymerizing mode provided higher dc than auto - polymerization . the dc mean values increased for both resin cements after 24 hours . the low - viscosity resin cements from light - activated or self - cured groups exhibited higher dc than high viscosity version.conclusion:the dc of resin cements was higher for the low viscosity version , following the light - polymerization and when were tested after 24 hours .

the task force on child health and maternal health for the united nations millennium development goals reiterated the challenges of achieving the goals in its report who 's got the power ? transforming health systems for women and children . while the technology and interventions exist to prevent or treat the vast majority of adverse health conditions that affect children and women of reproductive age , many continue to die and even more suffer from ill health [ 2 , 3 ] . the central challenge to achieving these goals is to provide known interventions to the full population and at the right time when most known adverse outcomes can be prevented . despite 15 years of the global safe motherhood maternal and child health is connected to social , economic , and environmental conditions . in resource - poor countries , aside from pre - existing risks for poor maternal and child health outcomes , global changes have introduced new risks to the health of women and pregnancy , including alcohol and tobacco use , over nutrition , and micronutrient malnutrition [ 5 , 6 ] . because not all the deficits in achieving adequate prenatal care are likely to be solved in the near future , and global factors that lead to increases in risk can not be reversed , new strategies and policies to improve pregnancy outcome remain a high priority . one emerging policy is the role of preconception care as a complement to prenatal care . this report highlights examples of national programs and projects indicating that such approaches are feasible and acceptable to the population . these case studies on preconception care that were presented at the first national summit on preconception care , atlanta , usa , in 2005 , range from a mature program in hong kong , to emerging programs in korea and belgium , and select program enhancement and recovery efforts in china . the family planning association of hong kong ( fpahk ) was founded in 1950 with an objective to reduce family size . the average number of children per woman declined from 4.5 in 1965 to 0.93 by 2004 . having achieved a low birth rate , a number of sexual and reproductive health programs have been launched with the objective of improving reproductive outcomes . the pre - pregnancy preparation service was launched in 1998 , in response to public demand for preconception care . the service integrates medical , counseling , and education services for couples planning conception to achieve the following outcomes : prevent and treat infections to safeguard the health of both partners and the fetus . thalassemia is an important hereditary disease in hong kong , because approximately 8% of persons in the local population are heterozygous carriers of mutations for thalassemia . couples with family history of hereditary diseases are referred for genetic counseling.all women are advised to consume folic acid.help couples understand their health and adjust their lifestyle to optimize pregnancy outcome.identify women who might have difficulty conceiving and to advise them to seek sub - fertility investigations early when indicated . for men , semen analysis is included in the package.help couples prepare for parenthood.help women with pre - existing medical illness understand the best timing for pregnancy , the effect of their illness on their pregnancy , the effect of pregnancy on their illness , the effect on her offspring , the need to adjust medication , and to avoid drugs with known or uncertain teratogenic effects.help women with gynecological diseases understand the impact of their diseases on conception and pregnancy . the role of assisted reproductive technology is discussed if appropriate.counsel couples with coital failure to help them consummate marriage . prevent and treat infections to safeguard the health of both partners and the fetus . thalassemia is an important hereditary disease in hong kong , because approximately 8% of persons in the local population are heterozygous carriers of mutations for thalassemia . identify women who might have difficulty conceiving and to advise them to seek sub - fertility investigations early when indicated . help women with pre - existing medical illness understand the best timing for pregnancy , the effect of their illness on their pregnancy , the effect of pregnancy on their illness , the effect on her offspring , the need to adjust medication , and to avoid drugs with known or uncertain teratogenic effects . help women with gynecological diseases understand the impact of their diseases on conception and pregnancy . at the first visit , the woman fills in a health assessment checklist that include preexisting medical conditions and medication being taken , adverse gynecological history and history of abnormal pregnancy outcomes , and risk behaviors . this disc provides detailed information about the objectives of the service ; the physical and genital checkup procedures ; pregnancy preparation , e.g. optimal body mass index , balanced diet , regular exercise , quit smoking and avoiding alcohol ; fertility regulation including contraception , conception and sub - fertility ; and information about the laboratory tests including hiv testing . , a nurse will explain all the normal reports first and then a physician performs the history , physical examination , and counseling to explain any abnormal results . the basic counseling and assessment is provided by non - specialists , although women with complicated problems are seen by specialists in obstetrics and gynecology or an internist . regular case conferencing is conducted to enhance knowledge of the non - specialists and improve their counseling skills . the service fee is $ 600 hong kong dollars ( usd $ 75 ) per couple which includes laboratory tests and one medical consultation . this low service fee is achieved through cost sharing with other sexual and reproductive health programs offered by the fpahk . the continuous flow of clients for the preconception service reflects public support and trust for the service . as may be seen in many emerging economies , south korea is experiencing decreasing birth rates ( from 16.3 per 1000 live births in 1994 to 9.8 per 1000 live births in 2004 ) and decreasing infant mortality rates ( from 9.9/1ive births in 1993 to 4.6/live births in 2004 ) . due to a high rate of unintended pregnancy , an increase in the rate of preterm delivery , a high rate of pregnancy termination following accidental exposure to potential teratogens during pregnancy , and low intake of folic acid , the society of maternal and fetal medicine made a decision to promote and enhance preconception care in south korea . the goals are to enhance primary prevention of birth defects and preterm birth , increase in intended pregnancy , decrease in unnecessary pregnancy termination , and recovery of a higher birth rate in korea to maintain population replacement levels . education includes lectures for obstetricians and gynecologists as well as for child - bearing age women attending maternity services . service delivery is clinic - based preconceptional counseling , medical protocol examination by a physician , and follow - up of women who want to have a baby . the preconception counseling clinic , based at the korean motherisk program at samsung cheil hospital , an affiliated university hospital , includes teratogen information service for pregnant and breast feeding women , as well as preconception counseling . the preconception care medical protocol is composed of a physical examination , laboratory tests , and a questionnaire . of the women served , 40% reported no history of adverse reproductive outcomes , about 30% had history of spontaneous abortion , 13% had maternal disease , 15% had a baby with birth defect , and 2% had family history of genetic disease . however , careful review of medical and behavioral history indicated that 92% of preconception care attendees have at least one risk factor for adverse outcomes . patients with specific problems such as a previous child with a birth defect are transferred to the department of maternal and fetal medicine . also , pre - implantation genetic diagnosis is performed in the department of infertility , a service that predates the introduction of preconception care . this service resulted in a substantial reduction in pregnancy loss from 91% in 1995 to 19% to 203 among 260 couples with balanced chromosomal translocation . the korea motherisk program is supported by the samsung chiel hospital and is staffed by three university faculty members and rotating fellows in clinical medicine . although no consultation fee is charged to the patients , laboratory expenses are paid for by the patients . depending on family history and other factors and various rationale used for laboratory examinations , the maximum cost of such laboratory examinations can be as high as 150 usd per person . the current challenge is to market it to all women who plan pregnancy , and to assure that the health care system has the capacity to match the demand and expansion of the service to other parts of the country . organization for birth and childhood ) , is a belgian governmental organization charged with the protection and promotion of health of women and children . given the low fertility rates and high rates of birth defects ( 2 to 3% of all live births ) in belgium , o.n.e launched a plan to assess the feasibility of implementation of preconception care in the french speaking part of belgium , the region with a population of 5 million people and about 50,000 births per year . this activity will be implemented through prenatal care , pediatric and gynecology clinics , as well as general practitioners , in partnership with genetic and counselling centers . an ad - hoc committee was formed to propose strategies , activities , financing options , and evaluation methods , starting with the development of evidence - based guidelines based on belgian epidemiological data . the committee proposed that the guidelines would be in accordance with the recommendations of the advisory committee of bioethics of belgium concerning genetic tests . the committee proposed also to organize a campaign targeting both the general public and health care providers to help market preconception care in primary care settings . the proposed guidelines include various serologic tests , screening of endocrine or genetic diseases , and preventive services including promotion of folic acid supplementation . the o.n.e . developed a sustainable campaign plan to inform and sensitize the population about the benefits of preconception care , directing their efforts to individuals aged 15 to 45 years , health professionals including general practitioners , gynaecologists , paediatricians , midwives and all medical social workers , members of the family planning centres , and school health promotion centers . the first step in the social marketing campaign was the creation of the tools including folders , posters , and letters to professionals . these materials were reviewed by the ad - hoc committee and field tested among a selected group of social workers and the public . the second step will be an information campaign among men and women of reproductive age using posters , folders , and radio and television advertisements . medical and social workers will also receive folders and posters for distribution to the target population and a letter to inform them about the purpose and methodology of the campaign . medical practitioners will then receive the guidelines to help them with information to be shared with patients and the laboratory examinations to be conducted . the final step will be the evaluation process to assess behavioral changes both within the community and at provider level . the indicators will include advice received from providers by clients in participating facilities , referral of relevant clients to preconception visits by providers , uptake of preconception visits by clients , and compliance with suggested interventions by clients . the early phase of implementation of preconception care in primary care settings in belgium was a challenge , as it highlighted the need for a strong partnership of all institutions involved in maternal and child health care . a three - pronged approach to reducing mother - to - child transmission of hiv has long been advocated by global initiatives to reduce the burden of hiv / aids , but to our knowledge , never been implemented anywhere . this approach includes reduction of hiv infection among young couples through primary prevention and voluntary hiv counseling and testing ( vct ) , voluntary avoidance of pregnancy by hiv infected couples , and finally antiretroviral treatment of pregnant women who are infected and subsequent management of outcomes in children . the unique premarital medical examination system prevalent in china and late onset of hiv transmission in china provided an opportunity to pilot implementation of the three - pronged strategy in guangxi province . babu county ( population 0.93 million ) of hezhou city , guangxi province ( population 48.9 million ) experienced the fastest increase in reported hiv / aids cases in guangxi province between 1998 ( 18 ) and 2002 ( 629 ) since the first case was detected in 1997 . the epidemic concentrated among the younger age group ( 90% , age 2049 years ) of the population and predominantly rural farmers ( 70% ) . given the demographics of the affected population and because the condom usage rate was low in this population ( std clients 5% , drug users 5% , sex workers 40% ) , the potential for mother - to - child transmission was evident . to reduce the impact of hiv on women , children and families , the county health department in 2001 embarked on a three - pronged approach embedded in the mandatory premarital medical examination system . a feasibility survey in the region had indicated that the majority of the population and public health community were supportive of the concept . the activities included family and community awareness , preconceptional voluntary hiv counseling and tests for couples ( pvct ) , and vct and antiretroviral therapy to hiv infected pregnant women . to facilitate pvct and the antenatal vct and antiretroviral treatment component , 40 antenatal and maternal / child care clinicians were trained . to facilitate the broad based interventions , 942 medical and public health professionals and barefoot doctors were trained on vct and other prevention approaches . to facilitate the community engagement process , training seminars using a combination of entertainment and educational films and media slides were conducted in middle schools and high schools for students as well as adults in the catchment 's area . an one - time pre- and post test ( 8800 respondents ) evaluation using structured questionnaire was conducted in selected locations . the students were required to take educational materials ( 100,000 copies ) to their homes to educate parents and neighbors as part of their homework requirements . information on hiv and vct opportunities were displayed on the community information blackboard , which is the usual tool for disseminating information on public health , legislative , and social policies to villagers and updated frequently by community volunteers . in the first quarter after launching the program ( october to december 2002 ) an average of 1099 ( 73% ) of the 1500 eligible couples each month sought pvct consultation , and 52% of them ( or 38% of eligible couples ) accepted hiv testing . by the end of 2003 , the pvct consultation rate among eligible couples increased to 77% and hiv testing rate among them reached 80% ( or 62% of all eligible couples ) . the main challenge to this approach was the change of legal requirement of premarital medical examination from mandatory to voluntary status in october 2003 . our pilot efforts indicate that screening for hiv in the preconception period is feasible and acceptable in this rural community . a combination of testing strategies implemented at various stages before and during pregnancy would help identify all women at risk for hiv - exposed pregnancies . given that the community is aware of the concept of premarital medical examination , social marketing techniques using family health enhancement as a core benefit , and efforts to regain the conceptual interest of population in preconception care are needed . in october 2003 , the longstanding mandatory chinese requirement for couples planning marriage to obtain a premarital health check , a routine physical examination , laboratory tests , and reproductive health education , was removed , and this visit became voluntary . reported rates of premarital examinations plummeted , and health officials voiced concern that women were not receiving adequate risk assessment and pre - pregnancy education , particularly regarding the benefit of folic acid in preventing neural tube defects . to assess the feasibility of increasing the rate of preconception care uptake , we conducted a pilot social marketing effort in a northern county ( mancheng , in hebei province ) during february and march , 2004 . we used a three - stage process : 1 ) ascertain the nature of the problem regarding delivery of preconception care through the use of a series of questionnaires and interviews , 2 ) develop a test plan to promote preconception care , and 3 ) evaluate and refine the plan . overall , 91% of 10,000 questionnaires were completed , and 140 subjects were interviewed , including women of reproductive age , maternal and child health workers , and local government officials . the major obstacles to preconception care included the following : a ) lack of systematic organization of these services within the health care system ; b ) poor coordination between governmental organizations involved in marriage , family planning and health care ; c ) unclear media messages ; and d ) the perception of the term voluntary by women to mean unnecessary . the implementation plan included the use of social marketing to solicit government support , coordinate with various government organizations , train maternal child health ( mch ) workers at all levels , and to market the concept of preconception health care to women of childbearing age . to solicit government support , we established a project coordination committee , including high - level officials from the county project office , the county women 's office , the bureaus of health , family planning , and civil administration , and the women 's federation . in addition , a series of face - to - face interviews were conducted with government officials , workshops were held in the county , and an official document was issued by the county government . recommendations were developed for improving coordination among government departments and organizations , including development of an education program that can be presented by the mch institute , the marriage registration office , and the family planning office , whenever women have contact with the system . such recommendations were shared with all stakeholders . following the workshop , 95% of women reported that they would be willing to pay up to rmb 100 ( us$12 ) for preconception health care services , and half indicated that more and better health messages were needed . improving coordination among government agencies , developing training materials targeted to different groups , and promulgating clear , consistent messages concerning the importance of preconception care that are delivered in all settings where women may have contact with the system before pregnancy can create demand for preconception services . the family planning association of hong kong ( fpahk ) was founded in 1950 with an objective to reduce family size . the average number of children per woman declined from 4.5 in 1965 to 0.93 by 2004 . having achieved a low birth rate , a number of sexual and reproductive health programs have been launched with the objective of improving reproductive outcomes . the pre - pregnancy preparation service was launched in 1998 , in response to public demand for preconception care . the service integrates medical , counseling , and education services for couples planning conception to achieve the following outcomes : prevent and treat infections to safeguard the health of both partners and the fetus . thalassemia is an important hereditary disease in hong kong , because approximately 8% of persons in the local population are heterozygous carriers of mutations for thalassemia . couples with family history of hereditary diseases are referred for genetic counseling.all women are advised to consume folic acid.help couples understand their health and adjust their lifestyle to optimize pregnancy outcome.identify women who might have difficulty conceiving and to advise them to seek sub - fertility investigations early when indicated . for men , semen analysis is included in the package.help couples prepare for parenthood.help women with pre - existing medical illness understand the best timing for pregnancy , the effect of their illness on their pregnancy , the effect of pregnancy on their illness , the effect on her offspring , the need to adjust medication , and to avoid drugs with known or uncertain teratogenic effects.help women with gynecological diseases understand the impact of their diseases on conception and pregnancy . the role of assisted reproductive technology is discussed if appropriate.counsel couples with coital failure to help them consummate marriage . prevent and treat infections to safeguard the health of both partners and the fetus . thalassemia is an important hereditary disease in hong kong , because approximately 8% of persons in the local population are heterozygous carriers of mutations for thalassemia . identify women who might have difficulty conceiving and to advise them to seek sub - fertility investigations early when indicated . help women with pre - existing medical illness understand the best timing for pregnancy , the effect of their illness on their pregnancy , the effect of pregnancy on their illness , the effect on her offspring , the need to adjust medication , and to avoid drugs with known or uncertain teratogenic effects . help women with gynecological diseases understand the impact of their diseases on conception and pregnancy . , the woman fills in a health assessment checklist that include preexisting medical conditions and medication being taken , adverse gynecological history and history of abnormal pregnancy outcomes , and risk behaviors . this disc provides detailed information about the objectives of the service ; the physical and genital checkup procedures ; pregnancy preparation , e.g. optimal body mass index , balanced diet , regular exercise , quit smoking and avoiding alcohol ; fertility regulation including contraception , conception and sub - fertility ; and information about the laboratory tests including hiv testing . , a nurse will explain all the normal reports first and then a physician performs the history , physical examination , and counseling to explain any abnormal results . the basic counseling and assessment is provided by non - specialists , although women with complicated problems are seen by specialists in obstetrics and gynecology or an internist . regular case conferencing is conducted to enhance knowledge of the non - specialists and improve their counseling skills . the service fee is $ 600 hong kong dollars ( usd $ 75 ) per couple which includes laboratory tests and one medical consultation . this low service fee is achieved through cost sharing with other sexual and reproductive health programs offered by the fpahk . the continuous flow of clients for the preconception service reflects public support and trust for the service . as may be seen in many emerging economies , south korea is experiencing decreasing birth rates ( from 16.3 per 1000 live births in 1994 to 9.8 per 1000 live births in 2004 ) and decreasing infant mortality rates ( from 9.9/1ive births in 1993 to 4.6/live births in 2004 ) . due to a high rate of unintended pregnancy , an increase in the rate of preterm delivery , a high rate of pregnancy termination following accidental exposure to potential teratogens during pregnancy , and low intake of folic acid , the society of maternal and fetal medicine made a decision to promote and enhance preconception care in south korea . the goals are to enhance primary prevention of birth defects and preterm birth , increase in intended pregnancy , decrease in unnecessary pregnancy termination , and recovery of a higher birth rate in korea to maintain population replacement levels . education includes lectures for obstetricians and gynecologists as well as for child - bearing age women attending maternity services . service delivery is clinic - based preconceptional counseling , medical protocol examination by a physician , and follow - up of women who want to have a baby . the preconception counseling clinic , based at the korean motherisk program at samsung cheil hospital , an affiliated university hospital , includes teratogen information service for pregnant and breast feeding women , as well as preconception counseling . the preconception care medical protocol is composed of a physical examination , laboratory tests , and a questionnaire . of the women served , 40% reported no history of adverse reproductive outcomes , about 30% had history of spontaneous abortion , 13% had maternal disease , 15% had a baby with birth defect , and 2% had family history of genetic disease . however , careful review of medical and behavioral history indicated that 92% of preconception care attendees have at least one risk factor for adverse outcomes . patients with specific problems such as a previous child with a birth defect are transferred to the department of maternal and fetal medicine . also , pre - implantation genetic diagnosis is performed in the department of infertility , a service that predates the introduction of preconception care . this service resulted in a substantial reduction in pregnancy loss from 91% in 1995 to 19% to 203 among 260 couples with balanced chromosomal translocation . the korea motherisk program is supported by the samsung chiel hospital and is staffed by three university faculty members and rotating fellows in clinical medicine . although no consultation fee is charged to the patients , laboratory expenses are paid for by the patients . depending on family history and other factors and various rationale used for laboratory examinations , the maximum cost of such laboratory examinations can be as high as 150 usd per person . since the establishment of preconception services in 2004 the current challenge is to market it to all women who plan pregnancy , and to assure that the health care system has the capacity to match the demand and expansion of the service to other parts of the country . the o.n.e ( office de la naissance et de lenfance = organization for birth and childhood ) , is a belgian governmental organization charged with the protection and promotion of health of women and children . given the low fertility rates and high rates of birth defects ( 2 to 3% of all live births ) in belgium , o.n.e launched a plan to assess the feasibility of implementation of preconception care in the french speaking part of belgium , the region with a population of 5 million people and about 50,000 births per year . this activity will be implemented through prenatal care , pediatric and gynecology clinics , as well as general practitioners , in partnership with genetic and counselling centers . an ad - hoc committee was formed to propose strategies , activities , financing options , and evaluation methods , starting with the development of evidence - based guidelines based on belgian epidemiological data . the committee proposed that the guidelines would be in accordance with the recommendations of the advisory committee of bioethics of belgium concerning genetic tests . the committee proposed also to organize a campaign targeting both the general public and health care providers to help market preconception care in primary care settings . the proposed guidelines include various serologic tests , screening of endocrine or genetic diseases , and preventive services including promotion of folic acid supplementation . the o.n.e . developed a sustainable campaign plan to inform and sensitize the population about the benefits of preconception care , directing their efforts to individuals aged 15 to 45 years , health professionals including general practitioners , gynaecologists , paediatricians , midwives and all medical social workers , members of the family planning centres , and school health promotion centers . the first step in the social marketing campaign was the creation of the tools including folders , posters , and letters to professionals . these materials were reviewed by the ad - hoc committee and field tested among a selected group of social workers and the public . the second step will be an information campaign among men and women of reproductive age using posters , folders , and radio and television advertisements . medical and social workers will also receive folders and posters for distribution to the target population and a letter to inform them about the purpose and methodology of the campaign . medical practitioners will then receive the guidelines to help them with information to be shared with patients and the laboratory examinations to be conducted . the final step will be the evaluation process to assess behavioral changes both within the community and at provider level . the indicators will include advice received from providers by clients in participating facilities , referral of relevant clients to preconception visits by providers , uptake of preconception visits by clients , and compliance with suggested interventions by clients . the early phase of implementation of preconception care in primary care settings in belgium was a challenge , as it highlighted the need for a strong partnership of all institutions involved in maternal and child health care . a three - pronged approach to reducing mother - to - child transmission of hiv has long been advocated by global initiatives to reduce the burden of hiv / aids , but to our knowledge , never been implemented anywhere . this approach includes reduction of hiv infection among young couples through primary prevention and voluntary hiv counseling and testing ( vct ) , voluntary avoidance of pregnancy by hiv infected couples , and finally antiretroviral treatment of pregnant women who are infected and subsequent management of outcomes in children . the unique premarital medical examination system prevalent in china and late onset of hiv transmission in china provided an opportunity to pilot implementation of the three - pronged strategy in guangxi province . pilot experience in babu county in guangxi province is outlined here . babu county ( population 0.93 million ) of hezhou city , guangxi province ( population 48.9 million ) experienced the fastest increase in reported hiv / aids cases in guangxi province between 1998 ( 18 ) and 2002 ( 629 ) since the first case was detected in 1997 . the epidemic concentrated among the younger age group ( 90% , age 2049 years ) of the population and predominantly rural farmers ( 70% ) . given the demographics of the affected population and because the condom usage rate was low in this population ( std clients 5% , drug users 5% , sex workers 40% ) , the potential for mother - to - child transmission was evident . to reduce the impact of hiv on women , children and families , the county health department in 2001 embarked on a three - pronged approach embedded in the mandatory premarital medical examination system . a feasibility survey in the region had indicated that the majority of the population and public health community were supportive of the concept . the activities included family and community awareness , preconceptional voluntary hiv counseling and tests for couples ( pvct ) , and vct and antiretroviral therapy to hiv infected pregnant women . to facilitate pvct and the antenatal vct and antiretroviral treatment component , 40 antenatal and maternal / child care clinicians were trained . to facilitate the broad based interventions , 942 medical and public health professionals and barefoot doctors were trained on vct and other prevention approaches . to facilitate the community engagement process , training seminars using a combination of entertainment and educational films and media slides were conducted in middle schools and high schools for students as well as adults in the catchment 's area . an one - time pre- and post test ( 8800 respondents ) evaluation using structured questionnaire was conducted in selected locations . the students were required to take educational materials ( 100,000 copies ) to their homes to educate parents and neighbors as part of their homework requirements . information on hiv and vct opportunities were displayed on the community information blackboard , which is the usual tool for disseminating information on public health , legislative , and social policies to villagers and updated frequently by community volunteers . in the first quarter after launching the program ( october to december 2002 ) an average of 1099 ( 73% ) of the 1500 eligible couples each month sought pvct consultation , and 52% of them ( or 38% of eligible couples ) accepted hiv testing . by the end of 2003 , the pvct consultation rate among eligible couples increased to 77% and hiv testing rate among them reached 80% ( or 62% of all eligible couples ) . the main challenge to this approach was the change of legal requirement of premarital medical examination from mandatory to voluntary status in october 2003 . our pilot efforts indicate that screening for hiv in the preconception period is feasible and acceptable in this rural community . a combination of testing strategies implemented at various stages before and during pregnancy would help identify all women at risk for hiv - exposed pregnancies . given that the community is aware of the concept of premarital medical examination , social marketing techniques using family health enhancement as a core benefit , and efforts to regain the conceptual interest of population in preconception care are needed . in october 2003 , the longstanding mandatory chinese requirement for couples planning marriage to obtain a premarital health check , a routine physical examination , laboratory tests , and reproductive health education , was removed , and this visit became voluntary . reported rates of premarital examinations plummeted , and health officials voiced concern that women were not receiving adequate risk assessment and pre - pregnancy education , particularly regarding the benefit of folic acid in preventing neural tube defects . to assess the feasibility of increasing the rate of preconception care uptake , we conducted a pilot social marketing effort in a northern county ( mancheng , in hebei province ) during february and march , 2004 . we used a three - stage process : 1 ) ascertain the nature of the problem regarding delivery of preconception care through the use of a series of questionnaires and interviews , 2 ) develop a test plan to promote preconception care , and 3 ) evaluate and refine the plan . overall , 91% of 10,000 questionnaires were completed , and 140 subjects were interviewed , including women of reproductive age , maternal and child health workers , and local government officials . the major obstacles to preconception care included the following : a ) lack of systematic organization of these services within the health care system ; b ) poor coordination between governmental organizations involved in marriage , family planning and health care ; c ) unclear media messages ; and d ) the perception of the term voluntary by women to mean unnecessary . the implementation plan included the use of social marketing to solicit government support , coordinate with various government organizations , train maternal child health ( mch ) workers at all levels , and to market the concept of preconception health care to women of childbearing age . to solicit government support , we established a project coordination committee , including high - level officials from the county project office , the county women 's office , the bureaus of health , family planning , and civil administration , and the women 's federation . in addition , a series of face - to - face interviews were conducted with government officials , workshops were held in the county , and an official document was issued by the county government . recommendations were developed for improving coordination among government departments and organizations , including development of an education program that can be presented by the mch institute , the marriage registration office , and the family planning office , whenever women have contact with the system . following the workshop , 95% of women reported that they would be willing to pay up to rmb 100 ( us$12 ) for preconception health care services , and half indicated that more and better health messages were needed . improving coordination among government agencies , developing training materials targeted to different groups , and promulgating clear , consistent messages concerning the importance of preconception care that are delivered in all settings where women may have contact with the system before pregnancy can create demand for preconception services . as illustrated by the six examples highlighted above , it is evident that preconception care is not just a conceptual debate but a primary approach used to address various health issues and emerging national challenges . as noted so cogently by atrash et al . a healthy baby and a healthy mother are valued hopes and dreams of families and cultural heritages across the world . further , in the countries such as south korea and hong kong , and others in similar demographic transition , this although adequate prenatal , obstetric , and primary care services can reduce the infant and maternal mortality in high - mortality - developing countries , such countries are not free from emerging risks to maternal and child outcomes . tobacco and alcohol use rates among women in many developing countries have increased . however , as more women than before have access to education and information , are employed , have personal income and decision making power , and delay pregnancy , there are many opportunities to inform them about the need for preconception care and a healthy reproductive life . indeed , even a perfectly organized preconception care system is not likely to address other deficits in the reproductive health care system or in an inadequate prenatal care system in developing countries ; neither will it lead to rapid positive changes in related indicators . similar to the arguments in 1990s for and against hiv voluntary counseling services amidst the lack of availability of antiretroviral treatment , or against the attempts to market cell phones where land phones services are inadequate , advocating preconception care as a component of maternal child care services will remain controversial . as such , establishment of preconception care services in fact , introduction to preconception care can complement the efforts to improve prenatal care uptake . consistent with the conceptual framework of the 2005 bangkok charter on health promotion , preconception care should be seen as a program for the future , a development agenda that is aimed at overall health of the family . making preconception care available can have transgenerational impact on some women ; those who are aware of such opportunities and can access such services . our responsibility is to provide that opportunity .

globally , maternal and child health faces diverse challenges depending on the status of the development of the country . some countries have introduced or explored preconception care for various reasons . falling birth rates and increasing knowledge about risk factors for adverse pregnancy outcomes led to the introduction of preconception care in hong kong in 1998 , and south korea in 2004 . in hong kong , comprehensive preconception care including laboratory tests are provided to over 4000 women each year at a cost of $ 75 per person . in korea , about 60% of the women served have known medical risk history , and the challenge is to expand the program capacity to all women who plan pregnancy , and conducting social marketing . belgium has established an ad hoc - committee to develop a comprehensive social marketing and professional training strategy for pilot testing preconception care models in the french speaking part of belgium , an area that represents 5 million people and 50,000 births per year using prenatal care and pediatric clinics , gynecological departments , and the genetic centers . in china , guangxi province piloted preconceptional hiv testing and counseling among couples who sought the then mandatory premarital medical examination as a component of the three - pronged approach to reduce mother to child transmission of hiv . hiv testing rates among couples increased from 38% to 62% over one year period . in october 2003 , china changed the legal requirement of premarital medical examination from mandatory to voluntary . this change was interpreted by most women that the premarital health examination was unnecessary and overall premarital health examination rates dropped . social marketing efforts piloted in 2004 indicated that 95% of women were willing to pay up to rmb 100 ( us$12 ) for preconception health care services . these case studies illustrate programmatic feasibility of preconception care services to address maternal and child health and other public health challenges in developed and emerging economies .

pleura is divided into a parietal layer which lines the inner aspect of the chest wall and a visceral layer which covers the interlobar fissures . fluid collection within the pleural cavity can be assessed with clinical and radiological means . when pleural effusion is detected , the characteristics of the fluid ( exudate or transudate ) must be revealed using thoracocentesis . pneumonias and congestive heart failure are the most frequently encountered causes of transudative and exudative effusions , respectively . accumulating knowledge about cytokines demonstrated their important roles in the pathogenesis of most of the infectious diseases . interleukins ( ils ) mediate the reactions of the our organism against foreign antigens and harmful agents . the ils act through autocrine or paracrine rather than endocrine pathways . in various infectious diseases significant alterations in il concentrations of blood , as well as of body fluids , occur . although il levels in serum and various body fluids have been studied in various diseases , very few researches involving il levels in pleural effusions of children have been conducted [ 3 , 4 ] . it is already acknowledged that established criteria used for the differentiation between exudative and transudative pleural effusions in adults are not valid for children and further studies are required to determine diagnostic criteria for children . the aim of this study is to compare il levels in exudative and transudative pleural fluids by measuring il levels in pleural effusions developed owing to various etiological factors in childhood . we aimed to examine whether the changes in pleural fluid protein , glucose , and lactate dehydrogenase ( ldh ) parameters , as well as in il-1 , il-2 , il-6 , and il-8 levels , were significant in differential diagnosis of childhood pleural effusions . the study group consisted of 26 patients admitted in the department of pediatrics , medical faculty , firat university with a diagnosis of pleural effusions . thoracal ultrasonograms were obtained from patients thought to have pleural effusions in consideration of medical history , physical examination , and chest x - rays . the presence and the amount of pleural fluids were determined and then thoracocentesis was performed . all samples were obtained by thoracentesis at the diagnosis time ( table 1 , figure 1 ) . the first samples were centrifuged immediately for the measurement of il concentration and the supernatant layers were stored at 70c until testing for cytokines . the second samples were inoculated in aerobic , anaerobic , and lwenstein - jensen media and stained with gram , giemsa , and ziehl - nielsen dyes . the third samples were analysed for protein , glucose , and ldh . as indicators of infection , peripheral leukocyte counts ( wbc ) , erythrocyte sedimentation rate ( esr ) , c - reactive protein ( crp ) measurements , blood culture , and purified protein derivate ( ppd ) tests were performed . pleural effusions were categorized as exudates and transudates according to the clinical and laboratory findings . the cutoff values for the differentiation between the pleural transudates and exudates were determined as follows : protein , 3 g / dl ; ldh , 200 iu / l ; pleural fluid / serum protein ratio , 0.5 ; and pleural fluid / serum ldh ratio , 0.6 . pleural effusions with values above these cutoffs were classified as exudates and those below as transudates . empyematous effusions were characterised by the presence of gram - positive bacteria in pleural fluid smears , attendant pneumonia with or without bacterial growth in the cultures of pleural fluid samples , and typical glucose ( < 40 mg / dl ) and/or protein ( > 5 g / dl ) and/or ldh ( > 1000 the samples of pleural fluids with higher glucose concentrations ( > 40 mg / dl ) whose gram - stained smears did not reveal any specific characteristics or bacterial growth were considered as parapneumonic effusions . the patients whose pleural fluid smears revealed afb which could be cultured in lwenstein - jensen media and also responded favorably to the antituberculostatic treatment were acknowledged as cases with tuberculous pleurisy [ 5 , 6 , 7 ] . pleural fluids preserved at 70c were thawed and il-1 , il-2 , il-6 , and il-8 levels in the samples were measured using enzyme - linked immunosorbent assay ( elisa ) and il-1 , il-2 , il-6 , and il-8 bender medsystems commercial kits . the clinical and laboratory characteristics of the patients were given as means ( standard deviation ( sd ) ) . statistical evaluations between different groups were done with kruskal - wallis variance analysis and post - hoc tukey - scheffe tests using spss 10.0 software programs . in our study group pleural effusions were of exudative ( n = 27 , 75% ) and transudative ( n = 9 , 25% ) type . the ages of the study subjects ranged between 9 months and 14 years . twelve parapneumonic ( 44% ) , 12 empyematous ( 44% ) , and 3 ( 12% ) tuberculous pleural effusions were detected in the exudate group ( table 1 ) . a statistically significant difference was found between crp values of the exudate ( 140.2 92.4 mg / dl ) and the transudate ( 9.6 3.2 mg / dl ) groups ( p < .001 ) ( figure 2 ) . protein levels in pleural fluids were measured as 5.1 1.2 g / dl and 1.6 0.5 g / dl in the exudate and the transudate groups , respectively ( p < .001 ) . glucose levels in pleural fluids were measured as 53.3 36.6 mg / dl in the exudate group and 83.8 14.7 mg / dl in the transudate group ( p < .001 ) ( figure 3 ) . il-1 levels of pleural fluids in the exudate and the transudate groups were detected as 304.1 127.7 pg / ml and 70.1 23.4 pg / ml , respectively ( p < .001 ) . il-1 levels were demonstrated as 210 43.5 pg / ml in the parapneumonic effusion group , 430.3 69.9 pg / ml in the empyematous effusion group , and 175.3 36.3 pg / ml in the tuberculous effusion groups . statistically significantly higher values were found in the empyematous group when compared with the parapneumonic and tuberculous pleural effusion groups ( p < .001 ) . il-2 concentrations of pleural fluids were ascertained to be at undetectable levels in 12 ( 44.4% ) and 176.6 173.6 pg / ml in 15 ( 55.6% ) patients in the exudate group . statistically significant difference was uncovered between the exudate and the transudate groups ( p < .001 ) . statistically significant differences were not revealed between the empyematous group and the other two groups ( p > .05 ) . statistically significantly higher values were recognized in the tuberculous pleural effusion group rather than the parapneumonic effusion group ( p < .01 ) . il-6 levels were detected to be 18589.5 3631.8 pg / ml in the exudate group and 65.6 16.0 pg / ml in the transudate group ( p < .001 ) . the corresponding values were 15295.0 22678.1 pg / ml in the parapneumonic effusion group , 20518.0 1410.9 pg / ml in the tuberculous pleural effusion group . in the empyematous group , statistically significantly higher values were obtained when compared with those of the parapneumonic effusion group , while the corresponding values detected in the tuberculous pleural effusion group were also significantly higher than those found in the parapneumonic effusion group ( p < .001 ) . pg / ml in the exudate group and 108.8 92.0 pg / ml in the transudate group ( p < .001 ) . the concentrations of il-8 were 1884.5 366.7 in the parapneumonic effusion group , 4789.8 1013.2 pg / ml in the empyematous effusion group and 919.0 9454.5 pg / ml in the tuberculous pleural effusion group . the values were statistically significantly higher in the empyematous group when compared with the other two groups ( p < .001 ) ( figure 4 ) . pleural effusion can be seen due to the intrusion of an infectious agent or an irritating foreign substance inside the pleural cavity or due a direct access of harmful materials or neoplastic cells into the pleural cavity via hematogenous route . it can be observed following pleural trauma or in association with asbestosis related pleural diseases . pleural effusions are known to develop secondary to trauma , cardiac , renal , collagenous diseases and malignancies besides pneumonia [ 9 , 10 , 11 ] . from 61% to 80% of the cases with pleural effusions empyema is also an important cause of mortality in the developing countries . in the remaining 20%39% of the cases , empyema develops secondary to trauma , malignancies , and renal diseases [ 5 , 12 , 13 , 14 , 15 ] . in our study group many studies have been conducted concerning the levels of il-1 , il-2 , il-6 , and il-8 in body fluids in various infections . although many studies investigating the alterations in levels of il according to the exudative or transudative nature of pleural effusions in adults have been performed , similar studies pertaining to children are relatively few [ 16 , 17 , 18 ] . for that reason many studies have analysed il-1 levels in patients with meningitis , sepsis , urinary tract infections , and empyema . variable results have been obtained dependent on the presence of different etiological agents [ 17 , 18 ] . in patients with bacterial and tuberculous meningitis , il-1 levels of cerebrospinal fluids were found to be significantly higher than those with aseptic meningitis and in the control group . umblical plasma levels of il-1 of infected newborns with early - onset sepsis were found to be higher than those of the control group . il-1 levels in synovial fluids were detected to be higher than those in the pediatric cases with suppurative arthritis due to other causes . silva - mejias et al have found comparatively higher il-1 levels in the empyema group . similar to our findings , alexandrakis et al revealed that il-1 levels in pleural effusions were higher in the exudate group compared with those of the transudate group . naito et al ascertained that il-1 levels in pleural effusions were higher than those found in bacterial infections due to other causes . in accordance with the literature in our study , il-1 levels were nearly 4.5 times higher in the exudate group . il-2 levels in pleural effusions due to malignancies were reported to be lower than those with tuberculous pleural effusions . shimokata et al stated that il-2 levels were at undetectable levels in 25% of tuberculous pleural effusions and 75% of cancerous pleural effusions . in our study il-2 levels in pleural fluids were at undetectably lower levels in 44% of the patients in the exudate group while the il-2 levels of the remaining 56% were assessed as 176.6 33.4 pg / ml . however all the patients in the transudate group demonstrated undetectable il-2 levels . in our study a statistically significant difference was not present between the empyema group and the other two groups . however a statistically significant difference was detected between the parapneumonic and the parapneumonic pleural effusion groups . various studies have been performed concerning the levels of il-6 which is the mediator and the regulator of inflammatory responses in miscellaneous inflammatory processes . blood il-6 levels were found to be higher in children with mycoplasma pneumoniae infections who stayed febrile for more than 3 days compared with those having shorter febrile episodes . they were also statistically significantly higher in pediatric cases with urinary tract infections when compared with the control groups . il-6 levels of cerebrospinal fluid in the patients with bacterial meningitis were reported to be significantly higher than the aseptic meningitis and in the control groups . levels above 100 pg / ml were measured in pediatric cases with viral ( 14% ) but especially with bacterial meningitis ( 53% ) . xirouchaki et al and yokoyama et al reported the levels of il-6 in pleural fluids to be significantly higher in the exudate group rather than the transudate group . they also found il-6 concentrations significantly higher in the tuberculous group rather than the parapneumonic effusion group . in our study not only the levels of il-6 were different between the groups with parapneumonic and empyematous effusions , but at the same time statistically significant differences were found between the empyematous and the parapneumonic or tuberculous pleural effusion groups . alexandrakis et al measured il-6 levels in serum and pleural fluids and ascertained that serum il-6 levels did not differ significantly between the exudate and the transudate groups . however pleural fluid il-6 levels were detected to be meaningfully higher in the exudate rather than the transudate group . a definite proportion between the serum and pleural fluid il-6 concentrations could not be assessed . however we found il-6 levels in the pleural fluid to be 285 times higher than those detected in the exudate group . il-8 is the mediator and the regulator of chemotaxis of leukocytes in inflammatory processes . in adults il-8 levels were found to be higher in cases with infectious pleural effusions compared with the patients with noninfectious effusions [ 3 , 26 , 27 , 28 , 29 , 30 , 31 , 32 ] . ceyhan et al reported higher levels in empyematous / parapneumonic effusion groups rather than the tuberculous group . however antony et al detected higher levels in cases with parapneumonic effusions unlike those found in the group with tuberculous effusions . il-8 concentrations in the empyematous group were reported to be higher than those measured in the parapneumonic group . in accordance with this study , in our study the highest levels of il-8 were detected in the empyematous , parapneumonic , and tuberculous pleural effusions in decreasing order . dlugovitzky et al discovered statistically significantly higher il-8 values in the tuberculous pleural effusion group in contrast with findings in the parapneumonic pleural effusion group . ashitani et al and broaddus et al found higher levels in the empyema group compared with the other groups . miller and idell have detected significantly higher levels in the exudate group rather than the transudate group . in this study , similar to the findings of other investigations , il-8 levels in the exudate group were reported to be 28 times higher than those found in the transudate group . this study have shown that for the differentiation between the exudative and transudative pleural effusions , in addition to the parametres such as protein , glucose and ldh , pleural fluid il-1 , il-2 , il-6 , and il-8 levels could be used . etiological factors can be differentiated by determining pleural fluid il levels . taking these levels into consideration accordingly , a definitive diagnosis , a successful treatment and reduction in mortality can be achieved .

the aim is to examine whether the changes in pleural fluid interleukin ( il)-1 , il-2 , il-6 , and il-8 levels were significant in differential diagnosis of childhood pleural effusions . il-1 , il-2 , il-6 , and il-8 levels in pleural fluids of all 36 patients were measured . the levels of il-1 , il-2 , il-6 , and il-8 in pleural fluids were statistically significantly higher in the transudate group compared with those of the exudate group . the levels of il-1 , il-6 , and il-8 were also found to be statistically significantly higher in the empyema group compared with both the parapneumonic and the tuberculous pleural effusion groups . the levels of il-2 and il-6 were detected to be statistically significantly higher in the tuberculous pleural effusion group in comparison with those of the parapneumonic effusion group . the results showed that pleural fluids il-1 , il-2 , il-6 , and il-8 could be used in pleural fluids exudate and transudate distinction .

literature analysis showed few articles about this complication , but no publication has described the management of open patella fracture around total knee arthroplasty . we report a unique case of an open patellar fracture above a total knee arthroplasty , sustained by a 56-year - old female patient . despite the poor outcome of operative management in patellar periprosthetic fracture , this approach should be considered for acute and post traumatic fractures in young patients with a good remaining bone stock . the demographic changes with a high rate of aging and active population , as well as the improved life expectancy during the last decades have led to an increase in the number of total knee arthroplasties ( tka ) [ 1 , 2 , 3 , 4 ] , and consequently a raise of post - operative complications . among these complications , periprosthetic fracture after tka is the most frequent cause of post - operative pain , and revision surgery . they may occur in the supracondylar region of the femur , followed by the patella , then the proximal tibia [ 1 , 2 , 3 , 4 , 5 , 6 , 7 ] . this entity can be very challenging to manage , with unpredictable and poor outcome , especially for displaced fracture or in cases with implant loosening [ 1 , 2 , 3 , 4 , 5 , 8 ] . in this article , we report a unique case of an open periprosthetic patellar fracture that was managed with operative treatment . it also involves a literature analysis with description of the risk factors and treatment options of this uncommon entity . a 56-year - old female underwent a cemented postero - stabilized condylar tka with fixed tibial plateau through an antero - medial approach , in our department . she had severe osteo - arthritis of the left knee with a major stiffness ( irreducible flexum at 25 ) . initial ( 1 ) and post operative(2 ) knee radiographs before , and after the arthroplasty . three weeks later , the patient tripped and fell off with all her standing height striking the anterior aspect of the left knee with hyper flexion . she was taken to the emergency room with left knee pain and a large skin wound . physical examination showed a large , deep and longitudinal wound measuring 15 cm in length due to a total breakdown of the suture line ( the antero - medial approach performed during the tka ) ( fig . 3 , 4 ) . fig 3 . a clinical picture showing the open patellar fracture a clinical picture showing the open patellar fracture . lateral knee radiograph showing the patellar fracture above a total knee arthroplasty , without any signs of implants loosening . lateral knee radiograph showing the patellar fracture above a total knee arthroplasty , without any signs ofimplants loosening . the knee radiographs showed a short oblique fracture at the inferior pole of the patella without any loosening of the implants or dislocation of the knee : this fracture is classified as type i according to ortigueraet d.j . she urgently received intravenous antibiotic ( augmentin , gentamicin ) , as well as tetanus vaccination . her fracture was temporarily stabilized in a long - leg plaster splint before she was transported to the operating room . she underwent wound irrigation , debridement of non - viable soft tissue from the wound , open reduction and fixation with a figure - eight cerclage wiring , without any revision of the implants . the wound was closed without any skin tension , after placing an intra - articular drain . complementary immobilization with a removable knee pad at full extension , for six weeks was prescribed . post operative knee radiographs post operative knee radiographs she received intra venous antibiotics : augmentin for 2 weeks associated with gentamicin during the first five days , as well as subcutaneous heparin . she was discharged from the hospital after she was instructed to remain non - weight bearing with a follow - up in 2 weeks . at 6 months follow - up , physical examination did not reveal any signs of local or deep infection . her knee was pain free and she was able to walk without crutches , but she had a mild stiffness : the knee range of motion was 0/60. no antero - posterior or frontal laxities were noted . clinical pictures at last follow - up showing a clean and dry wound ( 7 ) , and knee range of motion ( 8 , 9 ) clinical pictures at last follow - up showing a clean and dry wound ( 7 ) , and knee range of motion ( 8 , 9 ) . the patient achieved union of the fracture : knee radiographs showed a healed patellar fracture , with hardware in place . since the late 1960s , total knee arthroplasty has been the most effective treatment for knee osteoarthritis , providing good functional outcomes and improvement in the patient s quality of life . constant advances in the joint replacement surgery , and especially the total knee arthroplasties have led to an increase in the number of complications following this type of surgery , such as periprosthetic fractures . [ 1 , 2 , 3 , 4 ] this entity is very challenging to treat even for the most skilled and experienced surgeons [ 7 , 8 , 10 ] . the femur is the most frequent fracture site , followed by the patella and the tibia [ 1 , 2 , 3 , 4 , 5 , 6 , 7 ] . patella fracture after tka can occur after trauma , or result from a stress fracture due to patella osteonecrosis , which is the most common cause of this type of fracture [ 1 , 2 , 3 , 4 , 5 , 8 , 10 , 11 ] . chalidis et al ( 2007 ) have reported in a systematic review of a 752 periprosthetic patella fracture that in 88.3% of the cases , the fracture was not associated with a traumatic event , asymptomatic , and discovered during routine follow - up . many risk factors of these stress fractures have been described in the previous literature [ 1 , 2 , 3 , 4 , 5 , 7 , 8 , 9 , 10 , 11 ] including : poor blood supply for patella after tka especially for antero - medial approach , and in case of lateral release or unwarranted fat pad removal : knee replacement surgery is likely to cause disruption of the vascular peri- patellar network especially the medial and the superior lateral genicular arteries.mal-alignment of the femoral and tibial implantspatella resurfacing arthroplastiesan excessive ( thickness < 15 mm ) or asymmetric bone resection during patella resurfacingosteonecrosis due to bone cement polymerization : the heat of polymerization of the polymethylmethacrylate may exceed the coagulation temperature , causing thermal necrosis of the bonethe use of large central fixation peg designs for patellar implant , instead of small peripheral peg design.revision surgeryosteoporosis and bone losshyperflexion of the kneerhumatod arthritis poor blood supply for patella after tka especially for antero - medial approach , and in case of lateral release or unwarranted fat pad removal : knee replacement surgery is likely to cause disruption of the vascular peri- patellar network especially the medial and the superior lateral genicular arteries . mal - alignment of the femoral and tibial implants patella resurfacing arthroplasties an excessive ( thickness < 15 mm ) or asymmetric bone resection during patella resurfacing osteonecrosis due to bone cement polymerization : the heat of polymerization of the polymethylmethacrylate may exceed the coagulation temperature , causing thermal necrosis of the bone the use of large central fixation peg designs for patellar implant , instead of small peripheral peg design . osteoporosis and bone loss hyperflexion of the knee open patellar fracture above a total knee arthroplasty is a very uncommon injury , and challenging to treat . to our knowledge , reported a unique case of patellar fracture associated with open knee dislocation after a total knee arthroplasty . it was treated with open reduction and internal fixation of the patella with band tension wiring . greco et al , reported an open tibial shaft fracture below an ipsilateral tka , managed with intramedullary nailing . some factors should be considered to succeed in the management of this type of fracture such as displacement of the fracture , function of the extensor mechanism , stability of the patellar implant , remaining bone stock [ 1 , 2 , 3 , 4 , 5 , 8 , 9 , 10 , 11 , 13 ] . it includes immobilization by a brace locked in full extension or a cylinder cast in case of non - operative treatment ; open reduction and internal fixation , implant revision in case of loosening , partial or total patellectomy , repair of the quadriceps tendon or patellar ligament in case of surgical treatment [ 4 , 5 , 7 , 8 ] . the periprosthetic patellar fractures are usually managed by a non - operative treatment especially for type i and ii , because of the high infectious risk of open reduction and internal fixation [ 13 , 14 ] , and consequently a high risk of arthroplasty failure . literature analysis showed that conservative or non - operative treatment was more successful for the management of non - displaced patella fracture with stable implant and an intact extension mechanism . ortiguera and berry reported good results in 37 out of 38 peri - prosthetic patellar fractures managed with conservative treatment . dennis reported a complication rate of 39% of operative treatment ( in a series of thirty one patients who sustained a peri- prosthetic patellar fracture ) . chalidis s systematic review has shown that open reduction and internal fixation failed in 92% of the cases . in this particular case , we were forced to opt for an internal fixation with a cerclage wiring after intensive wound irrigation , debridement and soft tissue repair . although no conclusion can be established from a single case report , we think that the successful result in our case can be explained by the relative young age of the patient ( 56 years old ) , the good bone stock , the acute and post - traumatic characteristics of the fracture , the early and adequate management necessary to avoid infectious complication . the surgical management may reduce the ability of patella to heal because of the evacuation of peri - fractural hematoma as well as the supplementary disruption of the vascular network around the patella after a knee replacement surgery . nevertheless , the high failure rate of the operative treatment in the patellar peri - prosthetic fracture can be explained by : the high proportion of stress , atraumatic fracture ( 88% ) due to osteonecrosis which suggests a poor remaining bone stock.the stress fracture , unlike the acute and post traumatic ones , has a higher risk of non-union.the poor outcome of partial or complete patellectomy , which is a type of operative treatment.in our point of view , the post traumatic fractures in young patients are more likely to heal than stress fractures in the elderly . the high proportion of stress , atraumatic fracture ( 88% ) due to osteonecrosis which suggests a poor remaining bone stock . the stress fracture , unlike the acute and post traumatic ones , has a higher risk of non - union . the poor outcome of partial or complete patellectomy , which is a type of operative treatment . in our point of view , the post traumatic fractures in young patients are more likely to heal than stress fractures in the elderly . peri - prosthetic patella fracture is a very uncommon entity , and challenging to treat , especially the open ones , because of the high risk of infectious complications . although several previous studies report a high rate of failure and unpredictable results of open reduction for periprosthetic patellar fracture , this approach ( especially the internal fixation with a strong and dynamic construct ) should be considered in young patients with a good remaining bone stock , who sustained an open or a displaced fracture after a high energy trauma . orthopaedic surgeons should consider open reduction and internal fixation as a good management alternative for periprosthetic patellar fracture , in case of a post traumatic and acute fracture in young patients with a good bone stock .

introduction : periprosthetic patellar fracture after total knee arthroplasty ( tka ) is a rare complication . their management can be very challenging with unpredictable results . literature analysis showed few articles about this complication , but no publication has described the management of open patella fracture around total knee arthroplasty.case presentation : we report a unique case of an open patellar fracture above a total knee arthroplasty , sustained by a 56-year - old female patient.conclusion:despite the poor outcome of operative management in patellar periprosthetic fracture , this approach should be considered for acute and post traumatic fractures in young patients with a good remaining bone stock .

near - infrared ( nir ) radiation can penetrate the skin and the sclera of the eye . the high permeability of nir radiation also allows it to affect tissues deeper within the eye , such as muscles , the lens , and retina . nir radiation can induce various biological effects,19 and intensive or long - term exposure to nir radiation is a factor in premature aging . despite the wide prevalence of a variety of ultraviolet ( uv ) blocking materials , such as sunblock , sunglasses , glasses , films , and umbrellas , that are useful in protecting our tissue against uv exposure , nir can not be blocked sufficiently.8 consequently , in the absence of suitable protection , nir radiation can induce various kinds of tissue damage and diseases , such as cataracts and photoaging.7,8 the human cornea plays a critical role in refracting light onto the retina and also protects the eye against external agents . since the epithelial layer of the cornea provides the first line of defense against environmental insults , the structural integrity of this layer is a key component of corneal function.10 although uv - induced corneal damage has been described in many previous studies,1115 the effects induced by nir radiation on the cornea have not been thoroughly investigated . we hypothesized that nir irradiation simulating solar nir radiation that reaches human tissues can induce changes in gene expression . to test this hypothesis , a three - dimensional reconstructed human corneal epithelial model with multilayered , corneal epithelium - like structure was used to simulate the human eye , and we evaluated dna microarray and real - time polymerase chain reaction ( pcr ) analysis results from normal human corneal epithelial cells exposed to water - filtered broad - spectrum nir irradiation to simulate solar nir radiation that reaches the eye . nir irradiation was performed with a broadband nir source ( titan ; cutera , brisbane , ca , usa ) . the nir device emits an nir spectrum between 1,100 nm and 1,800 nm , with water filtering to remove wavelengths between 1,400 nm and 1,500 nm , and simulates solar nir radiation that reaches the skin of humans on the earth s surface . to avoid thermal effects , the sapphire contact cooling tip was set to a fixed temperature of 20c . in our previous in vitro study , ten rounds at 10 j / cm using continuous energy single irradiation pulses of 4.3 seconds achieved drastic reduction in cell count . the three - dimensional reconstructed human corneal epithelial model ( labcyte cornea - model ) prepared from enzymatically digested normal human corneal epithelial tissues was purchased from japan tissue engineering corporation , aichi , japan as an in vitro model of corneal tissue.16 cells were cultured in media ( assay medium ; japan tissue engineering corporation ) , which was changed every 2 days until the cultures reached subconfluence.16 the subconfluent corneal cells were then subcultured with trypsin and seeded on a cell culture insert containing a microporous membrane with a 0.4 m pore size . corneal cells were cultivated between the air and liquid interface to form a multilayered , corneal epithelium - like structure . total rna from the corneal epithelial cells was extracted in qiazol reagent ( qiagen nv , venlo , the netherlands ) and spin - column purified using a rneasy mini spin column ( qiagen nv ) . the rna was extracted from control and irradiated groups , which consisted of six whole individual three - dimensional reconstructed human corneal epithelial models . the quantity and quality of the rna samples were then measured using uv absorbance ( nanodrop technologies , wilmington , de , usa ) , and the quality was also assessed using an agilent 2100 bioanalyzer series ii ( agilent technologies , santa clara , ca , usa ) . cy3-labeled crna samples were synthesized using the low input quick amp labeling kit ( agilent technologies ) according to the manufacturer s instructions . for each time point , 50 ng of total rna was used to generate first - strand cdna . after the denaturation ( 10 minutes at 65c ) and crna synthesis ( 2 hours at 40c ) steps , the reactions were incubated at 70c for 15 minutes to inactivate the affinity - script enzyme ( agilent technologies ) . for labeling reactions , crna samples were each mixed with 6 l of transcription master mix cocktail containing cy3-ctp and incubated at 40c for 2 hours . labeled crna was quantified on a nanodrop nd-1000 ( nanodrop technologies ) , and quality was also checked by agilent 2100 bioanalyzer series ii . slides were scanned using the microarray scanner ( agilent technologies ) , and images were processed by the feature extraction software ( agilent technologies ) with background correction . data were imported into genespring , and normalization was performed with a 75 percentile shift . after normalization and filtering of the raw data output files , log - fold changes in gene expression in the nir - irradiated group compared with the control group were analyzed using the genespring gx 11.0 software ( agilent technologies ) to determine the upregulated and downregulated genes , using a twofold change as the cutoff . upregulated and downregulated genes were further clustered into functional gene groups using the ontology analysis function with the genespring gx 11.0 software . microarray data were deposited into the minimum information about a microarray experiment ( miame)-compliant gene expression omnibus ( geo ) database ( accession number : gse76500 , http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=gse76500 ) . quantitative real - time pcr analysis was achieved using the dna engine opticon 2 system ( bio - rad laboratories inc . all six whole individual three - dimensional reconstructed human corneal epithelial tissue culture models were used for quantitative real - time pcr analysis . the primer sequences of epidermal growth factor receptor ( egfr ) , tubulin tyrosine ligase - like family member 5 ( ttll5 ) , and pq loop repeat containing 2 ( pqlc2 ) are given in table 1 . since pqlc was ubiqitously and highly expressed in all samples , pqlc2 was used for normalization . the variant - specific primer sequences for the four egfr variants are given in table 2 . cdna templates were prepared for each rna sample by reverse transcription reaction using superscript iii ( thermo fisher scientific , waltham , ma , usa ) . each quantitative pcr ( 25 l ) contained 1 l of cdna template , 1.5 l of forward and reverse primer ( 10 m each ) , 7.5 l of 2 master mix , and 3.5 l of nuclease - free water . the thermal cycling conditions included one cycle at 95c for 5 minutes and 40 cycles at 95c for 10 seconds , 60c for 30 seconds , and 68c for 30 seconds . all statistical data are presented as the mean sem , and a p - value of < 0.05 was considered to be statistically significant . nir irradiation was performed with a broadband nir source ( titan ; cutera , brisbane , ca , usa ) . the nir device emits an nir spectrum between 1,100 nm and 1,800 nm , with water filtering to remove wavelengths between 1,400 nm and 1,500 nm , and simulates solar nir radiation that reaches the skin of humans on the earth s surface . to avoid thermal effects , the sapphire contact cooling tip was set to a fixed temperature of 20c . in our previous in vitro study , ten rounds at 10 j / cm using continuous energy single irradiation pulses of 4.3 seconds achieved drastic reduction in cell count . the three - dimensional reconstructed human corneal epithelial model ( labcyte cornea - model ) prepared from enzymatically digested normal human corneal epithelial tissues was purchased from japan tissue engineering corporation , aichi , japan as an in vitro model of corneal tissue.16 cells were cultured in media ( assay medium ; japan tissue engineering corporation ) , which was changed every 2 days until the cultures reached subconfluence.16 the subconfluent corneal cells were then subcultured with trypsin and seeded on a cell culture insert containing a microporous membrane with a 0.4 m pore size . corneal cells were cultivated between the air and liquid interface to form a multilayered , corneal epithelium - like structure . total rna from the corneal epithelial cells was extracted in qiazol reagent ( qiagen nv , venlo , the netherlands ) and spin - column purified using a rneasy mini spin column ( qiagen nv ) . the rna was extracted from control and irradiated groups , which consisted of six whole individual three - dimensional reconstructed human corneal epithelial models . the quantity and quality of the rna samples were then measured using uv absorbance ( nanodrop technologies , wilmington , de , usa ) , and the quality was also assessed using an agilent 2100 bioanalyzer series ii ( agilent technologies , santa clara , ca , usa ) . cy3-labeled crna samples were synthesized using the low input quick amp labeling kit ( agilent technologies ) according to the manufacturer s instructions . for each time point , 50 ng of total rna was used to generate first - strand cdna . after the denaturation ( 10 minutes at 65c ) and crna synthesis ( 2 hours at 40c ) steps , the reactions were incubated at 70c for 15 minutes to inactivate the affinity - script enzyme ( agilent technologies ) . for labeling reactions , crna samples were each mixed with 6 l of transcription master mix cocktail containing cy3-ctp and incubated at 40c for 2 hours . labeled crna was quantified on a nanodrop nd-1000 ( nanodrop technologies ) , and quality was also checked by agilent 2100 bioanalyzer series ii . slides were scanned using the microarray scanner ( agilent technologies ) , and images were processed by the feature extraction software ( agilent technologies ) with background correction . data were imported into genespring , and normalization was performed with a 75 percentile shift . after normalization and filtering of the raw data output files , log - fold changes in gene expression in the nir - irradiated group compared with the control group were analyzed using the genespring gx 11.0 software ( agilent technologies ) to determine the upregulated and downregulated genes , using a twofold change as the cutoff . upregulated and downregulated genes were further clustered into functional gene groups using the ontology analysis function with the genespring gx 11.0 software . microarray data were deposited into the minimum information about a microarray experiment ( miame)-compliant gene expression omnibus ( geo ) database ( accession number : gse76500 , http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=gse76500 ) . quantitative real - time pcr analysis was achieved using the dna engine opticon 2 system ( bio - rad laboratories inc . all six whole individual three - dimensional reconstructed human corneal epithelial tissue culture models were used for quantitative real - time pcr analysis . the primer sequences of epidermal growth factor receptor ( egfr ) , tubulin tyrosine ligase - like family member 5 ( ttll5 ) , and pq loop repeat containing 2 ( pqlc2 ) are given in table 1 . since pqlc was ubiqitously and highly expressed in all samples , pqlc2 was used for normalization . the variant - specific primer sequences for the four egfr variants are given in table 2 . cdna templates were prepared for each rna sample by reverse transcription reaction using superscript iii ( thermo fisher scientific , waltham , ma , usa ) . each quantitative pcr ( 25 l ) contained 1 l of cdna template , 1.5 l of forward and reverse primer ( 10 m each ) , 7.5 l of 2 master mix , and 3.5 l of nuclease - free water . the thermal cycling conditions included one cycle at 95c for 5 minutes and 40 cycles at 95c for 10 seconds , 60c for 30 seconds , and 68c for 30 seconds . all statistical data are presented as the mean sem , and a p - value of < 0.05 was considered to be statistically significant . to elucidate changes in genetic expression induced by exposure to nir irradiation that simulates solar nir radiation that reaches human eye tissues , dna microarray analysis was carried out using rna isolated from control and nir - irradiated corneal epithelial cells . using ~6.310 probes , we identified 52 upregulated genes and 98 downregulated genes in the nir - irradiated group compared with the control group . gene ontology analysis of these 150 genes identified 41 upregulated genes that encode proteins with receptor - binding properties ( table 3 ) . quantitative real - time pcr analysis was then performed for the two genes showing the highest degree of upregulation in the dna microarray analysis : egfr and ttll5 . egfr showed a high degree of upregulation ( 19.4-fold ) relative to control cells , although statistically significant upregulation of egfr and ttll5 was not observed ( p=0.5127 and p=0.8273 , respectively ) ( figure 1 ) using the primer sequences listed in table 1 . meanwhile , when the expression of egfr variants was assessed in a dna microarray analysis , expression increased by 1.0-fold and 1.3-fold for egfr variants 1 and 3 , respectively . upon inclusion of variant - specific primer sequences for egfr ( table 2 ) , quantitative real - time pcr analysis revealed that statistically significant upregulation of variants 1 and 3 was observed ( p<0.05 ) , whereas there was no statistically significant upregulation of variants 2 and 4 ( p=0.1266 and p=0.5127 , respectively ) ( figure 2 ) . to the best of our knowledge , this is the first study that examines changes in genetic expression in three - dimensional reconstructed corneal epithelial tissue after nir irradiation to simulate solar nir radiation that reaches human eye tissues . the biological effects of sun and uv exposure have been extensively investigated . however , over half of the solar energy is in the nir range , and most of the uv - blocking materials can not block nir radiation . moreover , humans are continuously exposed to artificial nir radiation from sources , such as electrical appliances.17,18 despite the prevalence of nir radiation , the need for protective measures against this type of radiation has received less attention than that of uv . because solar nir radiation is filtered by atmospheric water,19,20 water - filtering in experimental settings is indispensable to provide an accurate simulation of solar nir radiation that reaches human tissues.5 cooling is also recommended when investigating the effects of nir radiation , as nir radiation can increase surface temperatures and induce thermal effects.5 indeed , without water filter or contact cooling , nir irradiation immediately increases the temperature of the superficial layer of culture fluid in a laboratory dish or on skin , as nir radiation is predominantly absorbed by hydrogen bond - containing molecules , such as water and hemoglobin . meanwhile , as the nir radiation penetrates deeper into the target , its energy can dissipate to the point where insufficient amounts of energy reach target cells in the base of culture dishes or deeper tissues in skin samples . thus , to accurately investigate the biological effects of solar nir radiation that reaches human tissues , a water - filter that excludes wavelengths between 1,400 nm and 1,500 nm and a cooling system were used in this study.5 a three - dimensional reconstructed human corneal epithelial model was used as an in vitro model of corneal tissue . the corneal epithelial model was proven to have a fully differentiated corneal epidermal tissue including all major corneal epithelial cell layers , such as the basal cell layer , the wing layer , and the superficial cell layer . cytokeratin-3 is a specific marker of corneal epithelium , and it was expressed in all layers of the corneal epithelial model.16 muc-1 and muc-16 , which compose the transmembrane glycoproteins in the surface of the corneal epithelium , were well expressed in the superficial layer of the corneal epithelial model.16 cells in the corneal epithelium are connected by desmosomes , tight junctions , and adherence junctions.16 claudin-1 , which is a marker of a tight junction , desmograin-3 , which is a marker of desmosome , and e - cadherin , which is a marker of an adherence junction , are localized in the interface between cells at all cell layers , including the superficial layers.16 lamin is an important constituent in the basement membrane at the basal corneal epithelium junction , which is expressed continuously in basal cells of the corneal epithelial model basal layer.16 the basement membrane was smooth like that of the human eye.16 therefore , the corneal epithelial model reproduced many of the characteristics of the native human corneal tissue , and it provides a morphologically relevant means to assess nir effects as an alternative to animal testing . the corneal epithelium , like other epithelial barriers in the human body , is continuously subjected to physical , chemical , and biological insults , which can produce a wound and/or loss of barrier functions . proper healing of corneal wounds is vital for maintaining a clear , healthy cornea and preserving vision . corneal epithelium responds rapidly to injury , wherein wound healing occurs by cells migrating as a sheet to cover the defect and reestablish barrier functions.21 in wounded cornea , the epithelium plays a central role , not only as a key cell type in corneal repair , but also as a growth factor source.22 as in other tissues , several growth factors are suggested to play a role in regulating corneal epithelial function and wound healing.21,23 following nir irradiation that simulates solar radiation , microarray analysis of corneal epithelial cell dna in this study showed that egfr was highly expressed , and statistically significant upregulation of egfr variants 1 and 3 was observed ( p<0.05 ) by quantitative real - time pcr . egfr is a prototypic tyrosine kinase receptor that is part of a larger family of erbb receptors . erbb2 , erbb3 , and erbb4 share with egfr characteristics that include extracellular ligand - binding sites , intracellular kinase domains , and tyrosine - rich regions . egfr activated in response to injury serves as a powerful mediator of corneal epithelial wound healing10 by coordinating multiple extracellular signals generated in response to cell injury.2427 in turn , the levels of egfr and tyrosine kinase activity are major determinant factors for epithelial cell function in tissues and organs.22 echoing this concept is the finding that in some cancer patients , treatment with egfr - specific monoclonal antibodies cetuximab and gefitinib ( an egfr kinase inhibitor ) resulted in ocular abnormalities , including diffuse punctate keratitis and corneal erosion.2830 thus , maintaining a proper level of egfr signaling is critical for corneal homeostasis.22 ttll5 gene has 32 exons with high expression in heart and skeletal muscle and lower expression in many other tissues , including the eye and brain.31,32 it encodes a 1,281-amino acid protein that is localized to the cytoplasm and nucleus.31 ttll5 is thought to be a key initiator of polyglutamylation in -tubulin33 and has been previously reported to be essential for the correct function of sperm flagella.34 ttll5 plays a role in polyglutamylation of primary cilia in vitro.35 notably , genes involved in the polyglutamylation and deglutamylation of -tubulin have been associated with photoreceptor degeneration in mice.35 in this study , ttll5 showed the high degree of upregulation in the dna microarray analysis , whereas statistically significant upregulation of ttll5 was not observed in quantitative real - time pcr analysis . further studies are needed to determine if a higher power output or increased treatment frequency may promote significant upregulation of ttll5 . the findings here that egfr expression is significantly upregulated following exposure of corneal epithelial cells to nir irradiation support that solar nir radiation that reaches human tissues can induce corneal cell injury . although significant egfr expression upregulation occurred after just one treatment at the tested power output , further studies are needed to determine if a higher power output or increased treatment frequency may promote even larger changes in expression . furthermore , this was a preliminary study based on a fairly small number of samples , so a large - scale study will be needed . nir irradiation simulating solar nir radiation that reaches human tissues induced upregulated expression of egfr in human corneal cells . nir radiation represents more than half of solar energy and can not be sufficiently blocked by eyewear . moreover , thus , the results of this study indicate that enhanced protection from and avoidance of both uv and nir radiation should be considered to prevent eye damage .

background and objectivehumans are increasingly exposed to near - infrared ( nir ) radiation from both natural ( eg , solar ) and artificial ( eg , electrical appliances ) sources . although the biological effects of sun and ultraviolet ( uv ) exposure have been extensively investigated , the biological effect of nir radiation is still unclear . we previously reported that nir as well as uv induces photoaging and standard uv - blocking materials , such as sunglasses , do not sufficiently block nir . the objective of this study was to investigate changes in gene expression in three - dimensional reconstructed corneal epithelial tissue culture exposed to broad - spectrum nir irradiation to simulate solar nir radiation that reaches human tissues.materials and methodsdna microarray and quantitative real - time polymerase chain reaction analysis were used to assess gene expression levels in a three - dimensional reconstructed corneal epithelial model composed of normal human corneal epithelial cells exposed to water - filtered broad - spectrum nir irradiation with a contact cooling ( 20c ) . the water - filter allowed 1,0001,800 nm wavelengths and excluded 1,4001,500 nm wavelengths.resultsa dna microarray with > 62,000 different probes showed 25 and 150 genes that were up- or downregulated by at least fourfold and twofold , respectively , after nir irradiation . in particular , epidermal growth factor receptor ( egfr ) was upregulated by 19.4-fold relative to control cells . quantitative real - time polymerase chain reaction analysis revealed that two variants of egfr in human corneal epithelial tissue were also significantly upregulated after five rounds of 10 j / cm2 irradiation ( p<0.05).conclusionwe found that nir irradiation induced the upregulated expression of egfr in human corneal cells . since over half of the solar energy reaching the earth is in the nir region , which can not be adequately blocked by eyewear and thus can induce eye damage with intensive or long - term exposure , protection from both uv and nir radiation may prevent changes in gene expression and in turn eye damage .

hypertension is a common chronic medical condition affecting people in pakistan and the rest of the world . it is an important modifiable risk factor for cardiovascular morbidity and mortality , particularly for stroke ( accounting for 51% of all stroke deaths worldwide ) , ischemic heart disease ( 45% of all deaths ) , chronic kidney disease , congestive heart failure , aortic aneurysm , and peripheral arterial disease . prevalence of hypertension ( systolic blood pressure > 140 mm hg or diastolic blood pressure > 90 mm hg , or on antihypertensive medications ) in pakistan has increased from 17% in 1980 to 35% in 2008 in adults aged 18 years and older . the increasing prevalence of hypertension together with a deficient control makes this one of the frequent conditions that require urgent medical attention . the prevalence of uncontrolled hypertension varies around the world , with the lowest prevalence in rural india ( 3.4% in men and 6.8% in women ) and the highest prevalence in poland ( 68.9% in men and 72.5% in women ) . however , the control of hypertension was 23% from a community based data in urban population from karachi , pakistan . uncontrolled hypertension can progress to hypertensive crisis defined as a systolic blood pressure > 180 mm hg or a diastolic blood pressure > 120 mm hg . hypertensive crisis can be further classified as a hypertensive urgency or hypertensive emergency depending on end - organ involvement including cardiac , renal , and neurologic injury . hypertensive urgency refers to severe hypertension without evidence of new or worsening end - organ injury while hypertensive emergency refers to a severe hypertension that is associated with new or progressive end - organ damage . hypertensive crises ( 76% urgencies , 24% emergencies ) represented more than one - fourth of all medical urgencies / emergencies . hypertensive urgencies frequently present with headache ( 22% ) , epistaxis ( 17% ) , and psychomotor agitation ( 10% ) and hypertensive emergencies frequently present with chest pain ( 27% ) , dyspnea ( 22% ) , and neurological deficit ( 21% ) . the reason for uncontrolled hypertension in pakistan is high due to lack of awareness , knowledge , adherence , and attitudes of pakistani patients with hypertension . however there is no data on patients with hypertensive crisis from tertiary care hospitals in pakistan . additionally the number of patients who complicate towards stroke , myocardial infarction , and chronic kidney disease is also not known . hence , it is essential to have figures on prevalence and clinical presentation from our setup . therefore , we conducted this study to determine the prevalence of hypertensive crisis , its management , and its outcome in patients presenting to a tertiary care center in karachi . this was a retrospective study conducted at the aga khan university , karachi , pakistan . the aga khan university hospital ( akuh ) has 563 beds in operation and provides services to over 50,000 hospitalized patients and to over 600,000 outpatients annually . ethical approval from the ethics review committee of the aga khan university ( 1985-med - erc-11 ) was taken for conduct of the study . adult inpatients ( > 18 yrs ) presenting to the er who were known hypertensive and had uncontrolled hypertension were included . controlled blood pressure was defined as systolic blood pressure ( sbp ) < 140 mm hg or diastolic blood pressure ( dbp ) < 90 mm hg . uncontrolled hypertension was defined as sbp > 140 mm hg and dbp > 90 mm hg in both diabetic and nondiabetic patients who were either aware of their problem or under pharmacological treatment . the sample was drawn using computerized medical record system international classification of diseases-9-coordination and maintenance ( icd-9-cm ) at health information management system in the hospital . patients admitted with primary diagnosis of hypertension crisis , uncontrolled hypertension , hypertensive emergency , and hypertensive urgency were selected through the icd-9-cm ( i-10 : essential ( primary ) hypertension , i-11 : hypertensive heart disease , i-12 : hypertensive renal disease , i-13 : hypertensive heart and renal disease , and i-15 : secondary hypertension ) . patients whose medical records did not contain minimal clinical information to allow case classification ( hypertensive urgency or emergency ) were excluded from the study . data over a period of 5 years , from year 2005 till year 2010 , was used . a sample of 1336 consecutive patients fulfilling the inclusion criterion was selected . all patients gave a general consent on admission ; however informed consent was not taken as data was extracted later through icd-9-cm . data on demographics , comorbid conditions , clinical symptoms , blood pressure readings at subsequent time intervals , length of stay , and antihypertensive drug therapy was recorded by trained data collectors . a history of physician - diagnosed diabetes mellitus ( dm ) , chronic kidney disease ( ckd ) , ischemic heart disease ( ihd ) , and stroke was noted from the patient 's medical record file . dm was defined as fasting plasma glucose 126 mg / dl at a prior visit . ckd was defined as rise in serum creatinine of > 1.2 mg / dl for 3 months . blood pressure readings , at different time intervals , were recorded from vital sheets for nursing services . hypertensive crisis was defined as a systolic blood pressure > 180 mm hg or a diastolic blood pressure > 120 mm hg . management of patient was assessed by recording the list of medication from the computer generated pharmacy sheet attached inside the medical record file . antihypertensive treatment was divided into two types : medication given per oral and medications given intravenously . i values where available were recorded using the patient profile viewer , an online hospital database software . mean length of stay was recorded by calculating the time interval between the patient 's admission date / time and discharge date / time from er / ward . various complications like myocardial infarction , stroke , aortic dissection , acute renal failure , and pulmonary edema developed during the hospital stay were recorded using the discharge summary notes filled by the primary consultant . myocardial infarction was diagnosed when blood levels of sensitive and specific biomarkers such as cardiac troponin or ckmb are increased in the clinical setting of acute myocardial ischemia with electrocardiographic changes . according to who definition , stroke was defined as a rapidly developing clinical sign of focal ( at times global ) disturbance of cerebral function , lasting more than 24 hours or leading to death with no apparent cause other than that of vascular origin . aortic dissection was referred as the condition when a separation has occurred in aortic wall intima as diagnosed on ct scan , causing blood flow into a new false channel composed of the inner and outer layers of the media . acute renal failure was diagnosed when the plasma urea nitrogen ( pun ) or serum creatinine did not stabilize within 72 hours . acute pulmonary edema was defined as alveolar or interstitial edema verified by chest x - ray and/or with o2 saturation < 90% on room air prior to treatment accompanied by severe respiratory distress , with crackles over the lungs and orthopnea . a minimum sample size of 237 patients was required to estimate a proportion of 19% of patients with hypertensive crises presenting to er , with bound on error of 5% and alpha of 5% . mean and standard deviation were used for qualitative variables and frequency and percentage for qualitative variables . chi - square test was used to compare categorical variables and student 's t - test was used to compare quantitative variables . a total of 73,063 hypertensive patients presented to the er between years 2005 and 2010 out of which a sample of 1336 ( 1.8% ) patients was taken for this study . the prevalence ( % ) of uncontrolled hypertension was 28.9% ( 387 ) . prevalence of hypertensive crisis overall was 16.3% ( 218 ) and among those with uncontrolled hypertension is 56.3% ( 218 ) . mean age ( sd ) of patients presenting to the er was 56.7 ( 14.7 ) and 175% ( 45.2 ) of patients were male . overall , dyslipidemia was the most common comorbidity in patients presenting with uncontrolled hypertension to the er with the prevalence of 43.2% ( 167 ) followed by diabetes mellitus , 36.9% ( 143 ) , and ischemic heart disease , 21.4% ( 83 ) , and 13.9% ( 54 ) of them were smokers . the baseline characteristics of patients overall , with hypertensive crisis and without hypertensive crisis , are given in table 1 . headache was the most common presenting symptom , 35.7% ( 138 ) , followed by dyspnea , 32.6% ( 126 ) , chest pain , 21.4% ( 83 ) , dizziness , 21.2% ( 82 ) , vomiting , 17.3% ( 67 ) , epistaxis , 5.2% ( 20 ) , and neurologic deficit , 3.6% ( 14 ) . on comparison of patients with hypertensive crisis with those with no hypertensive crisis the clinical symptoms that were statistically significant were as follows : headache was present in 42.2% ( 92 ) versus 27.2% ( 46 ) p value = 0.002 and chest pain was present in 17.4% ( 38 ) versus 26.6% ( 45 ) p value = 0.02 . the mean ( sd ) systolic blood pressure ( sbp ) recorded in patients with hypertensive crisis versus no hypertensive crisis in er was 202 ( 17.971 ) and 158 ( 13.387 ) ( p value 0.001 ) . the mean ( sd ) diastolic blood pressure in patients with hypertensive crisis versus no hypertensive crisis in er was 108 ( 17.429 ) mm hg and 87 ( 14.984 ) mm hg , respectively , ( p value 0.001 ) . the trend of blood pressure recorded in the er is shown in figures 1 and 2 . antihypertensive medications used in management of patients with hypertensive crisis . calcium channel blocker was the most widely used oral antihypertensive medication in the er , 35.4% ( 137 ) . intravenous ( iv ) nitrate was the most commonly administered iv medication in er , 22.7% ( 88 ) . the mean ( sd ) drop in sbp in patients with hypertensive crisis who received intravenous medications versus oral medications was 53.1 ( 29 ) mm hg and 43 ( 27 ) , respectively ( p value = 0.01 ) . the mean drop in dbp in patients with hypertensive crisis who received intravenous versus perioral medications was 25.8 ( 19 ) and mm hg and 17.8 ( 22 ) mm hg , respectively ( p value = 0.006 ) . comparison of blood pressure trends in patients with and without hypertensive crisis on intravenous or per oral medication is shown in table 2 . the maximum drop in systolic blood pressure and diastolic blood pressure was achieved by sodium nitroprusside : 80 ( 15 ) mm hg and 37.5 ( 7.77 ) mm hg . types of intravenous medications and drop in sbp and dbp are shown in table 3 . the total length of stay ( in days ) for patients with hypertensive crisis was 2.46 ( 0.164 ) whereas the total length of stay ( in days ) for patients without hypertensive crisis was 2.20 ( 0.158 ) . overall prevalence ( % ) of complications was 47.7% ( 104 ) in patients with hypertensive crisis . acute renal failure was the most common complication with the prevalence ( % ) of 41.3% ( 43 ) followed by myocardial infarction 28.8% ( 30 ) and pulmonary edema 18.26% ( 19 ) . stroke accounted for 6.5% ( 12 ) of complications . on comparison of patients with hypertensive crisis and patients without it , myocardial infarction occurred in 7.2% ( 15 ) versus 9% ( 15 ) , stroke occurred in 2.4% ( 5 ) versus 4.2% ( 7 ) , and acute kidney injury occurred in 11.5% ( 24 ) versus 11.4% ( 19 ) patients , respectively . on further categorization of patients with hypertensive crisis who received intravenous antihypertensive in er versus not receiving intravenous medication , there was no significant difference in the complications in both groups . we have shown in this study on patients presenting to er with hypertensive crisis that the prevalence of hypertensive crisis is high ( 56.3% ) . mean length of stay was longer in patients with hypertensive crisis and acute renal failure was the most common complication in these patients . the incidence of hypertensive crisis was 47.22% in a study conducted in bosnia and 16% in a study conducted in brazil [ 22 , 23 ] . the reasons for high prevalence of patients with hypertensive crisis in our setup are multiple . lack of knowledge about control of hypertension and poor compliance to antihypertensive medications is a major issue in pakistan . also , lack of proper health infrastructure in public sector leading to inability of the poor population to access healthcare adds onto the severity of hypertension in these patients . in previous studies [ 25 , 26 ] , the most frequent clinical sign of patients presenting to the er was headache ( 22% and 42% ) and dizziness was reported among 30% of emergency department patients . this study showed ( 36% ) headache as the most common clinical sign followed by dizziness ( 21% ) which supports the previous findings about the characteristics of hypertensive crisis patients . complications of hypertensive crisis reported in previous studies conducted in bahrain and italy were acute coronary syndrome ( 32% ) , left ventricular heart failure ( 38% ) , and stroke ( 29.3% ) and cerebral infarction ( 24% ) , pulmonary edema ( 23% ) , and hypertensive encephalopathy ( 16% ) . however acute renal failure , myocardial infarction , and pulmonary edema are the most common complications in our setup . the reason for high prevalence of acute renal failure in our setup may be the higher number of patients with hypertensive emergency . the most common intravenous medication used to control hypertensive crisis in this study was nitrates . recommendation for use of intravenous nicardipine was reported by a study conducted in 2005 whereas sodium nitroprusside , because of its direct vasodilator effect and immediate onset of action , was recommended for hypertensive emergency in a subsequent study in 2006 . we observed in this study that sodium nitroprusside was the most potent intravenous antihypertensive in dropping blood pressure . however it was not the most commonly used drug in this study . in a similar study , intravenous labetalol was reported as the most frequently used antihypertensive medication for emergency department patients presenting with hypertensive urgency . this indicates that the use of intravenous nitrate as the most common antihypertensive for management of hypertensive crisis ( apart from use in patients with cardiac cause ) is not completely in accordance with the recommendations . however in some studies , oral labetalol , beta blockers , diuretics , ace inhibitors , and calcium channel blockers have been recommended . calcium channel blockers have also been found to be the most common antihypertensives used for management of hypertensive in this population and have been found to have good control rates . calcium channel is now recommended as a first line antihypertensive in the recent nice guidelines . the reduction in systolic and diastolic blood pressure reported in our study shows a smooth decline in order to avoid risks of potential side effects of a much rapid decline . it is recommended that the initial reduction in mean arterial pressure ( map ) in case of hypertensive emergency should not be more than 20%25% below the pretreatment blood pressure or that map be reduced within the first 3060 minutes to 110115 mm hg . this can be comparable to our study which reported an overall decrease of blood pressure of about 52 mm hg in sbp from intravenous medication from the time of admission until 2 hours after admission in er . a rapid decline in blood pressure is associated with acute deterioration in renal function , ischemic , cardiac , or cerebral events , and occasional retinal artery occlusion and acute blindness . the goal is to reduce bp by 1015% over a period of 3060 minutes , with the exception of the patient that presents with aortic dissection or acute intracranial bleed . however treatment should be individualized to each patient based on the type and extent of end - organ damage , degree of bp elevation , and the specific side effects that each medication could have on a patient 's preexisting comorbidities . the strength of this study is that it is the first study from this region to report figures on prevalence of hypertensive crisis , as well as prevalence of complications as a result of hypertensive crisis among patients . moreover , this study also reports management of these patients in terms of the treatment received in the er and into the ward and their mean length of stay . however there are limitations in this study ; firstly it has limited external validity as the sample is not representative for an entire population . it represents population visiting a single - tertiary care hospital and , hence , it is not representative of the entire population of pakistan . secondly , compliance issues with the medication regimen have not been taken into consideration ; hence , the medications administered to the patients may be misjudged . thirdly , some patients may also have gotten discharged against medical advice ; hence , the optimal length of stay for these patients may not have been achieved . fourth , this retrospective study can not strongly give a cause and effect association and we have not reported the hypertensive urgencies and emergencies separately . we did not report data separately on hypertensive emergency and urgency and retinopathy could not be examined and reported in all patients . the prevalence of hypertensive crisis is high in our subjects . per oral calcium channel blocker and intravenous nitrate are the most commonly administered medications in the er and ward .

objectives . hypertension , if uncontrolled , can lead to hypertensive crisis . we aim to determine the prevalence of hypertensive crisis , its management , and outcome in patients presenting to a tertiary care center in karachi . methods . this was a cross - sectional study conducted at the aga khan university , karachi , pakistan . adult inpatients ( > 18 yrs ) presenting to the er who were known hypertensive and had uncontrolled hypertension were included . results . out of 1336 patients , 28.6% ( 387 ) had uncontrolled hypertension . the prevalence of hypertensive crisis among uncontrolled hypertensive was 56.3% ( 218 ) . per oral calcium channel blocker ; 35.4% ( 137 ) and intravenous nitrate ; 22.7% ( 88 ) were the most commonly administered medication in the er . the mean ( sd ) drop in sbp in patients with hypertensive crisis on intravenous treatment was 53.1 ( 29 ) mm hg and on per oral treatment was 43 ( 27 ) mm hg . the maximum mean ( sd ) drop in blood pressure was seen by intravenous sodium nitroprusside ; 80 ( 51 ) mm hg in sbp . acute renal failure was the most common complication with a prevalence of 11.5% ( 24 ) . conclusion . the prevalence of hypertensive crisis is high . per oral calcium channel blocker and intravenous nitrate are the most commonly administered medications in our setup .

a basic set of proteins and mrnas are differentially expressed among cell types , temporally and spatially , generating a vast assortment of cell phenotypes and/or activation states within a single tissue . outlining this protein / mrna portrait is thus crucial for understanding not only the uniqueness characterizing cells , but especially their distinguished functions . this becomes of major relevance when the balance between cell - intrinsic properties and identity cues received and provided by each cell to its neighboring cells then shapes the cell - to - cell cross talk during physiopathological conditions . in the cns , neuroinflammation is the typical consequence of the interchange among different cell types , particularly neurons , astrocytes , oligodendrocytes and microglia , of a variety of cues as neurotransmitters , cytokines , chemokines , toxic metabolites that condition the final protein / mrna profiles of cells , their activation states and functional outcomes [ 2 , 3 ] . since neuroinflammation accompanies a large variety of neurodegenerative diseases , there is increasing interest in determining how the different cell phenotypes and cellular interconnectivity might contribute to reduce inflammation and reverse neurodegeneration . microglia actively participate to the context - dependent , neuroprotective / neurotoxic molecular network that is triggered during neuroinflammation . among the molecular cues having a key role in this process , extracellular nucleotides are major responsible for intercellular communication and propagation of inflammatory stimuli [ 57 ] . this occurs by specific binding to various receptor subtypes , termed ionotropic p2x and metabotropic p2y , which are simultaneously localized on several different cell phenotypes . among these , the p2y12 receptor subtype belonging to the gi class of g protein - coupled receptors is activated by adp . two transcript variants apparently encoding the same protein isoform have been identified so far for p2ry12 gene , but the determinants for cell specificity of p2y12 protein expression are still unknown . p2y12 is found on the surface mainly , but not exclusively , of blood platelets , where it acts as blood clotting regulator and target for the treatment of thromboembolisms [ 9 , 10 ] . in the nervous system , the tissue- and cellular - selective expression of p2y12 exhibits a pattern throughout white and gray matter consistent with astrocyte expression , although it has not been found colocalization between p2y12 and gfap - positive astrocytes in rat brain cortex and nucleus accumbens , despite the abundant presence of p2y12 mrna . moreover , we previously established in vivo the expression of p2y12 in oligodendrocytes and myelin sheaths of rat cerebral cortex , subcortical areas , and periventricular white matter . this localization is confirmed throughout the corticospinal tract , therefore suggesting high conserved tissue - homogeneity and phenotype - specificity , and a hypothesized role in myelination [ 1215 ] . p2y12 is finally observed in brain and spinal cord resident microglia , where it affects activation , chemotaxis [ 1619 ] and neuropathic pain , but it is not observed , for example , in peripheral macrophages in spleen [ 18 , 20 ] . p2y12 expression in primary microglia is variable with postnatal development and shows sexually dimorphic behavior . through the use of all the available p2y12-immunoreactive antibodies recognizing the c - terminus or the second intracellular loop of the receptor , the aim of the present work is to provide comparative evidence about p2y12 cell specificity in microglia versus oligodendrocytes particularly from the healthy and diseased cns under neuroinflammatory conditions as those sustained during amyotrophic lateral sclerosis ( als ) and multiple sclerosis ( ms ) . adult b6.cg-tg ( sod1-g93a)1gur / j mice expressing high copy number of mutant human sod1 with a gly93ala substitution ( sod1-g93a ) were originally obtained from jackson laboratories ( bar harbor , me , usa ) and housed in our indoor animal facility as described in apolloni and collaborators , . the animals were euthanized , according to the guidelines for preclinical testing and colony management . also neonatal wistar and adult lewis rats ( from charles river laboratories , lc , italy ) were housed in our indoor animal facility . animal procedures were performed according to european guidelines for the use of animals in research ( 86/609/cee ) and the requirements of italian laws ( d.l . ethical procedures were approved by animal welfare office , department of public health and veterinary , nutrition and food safety , general management of animal care and veterinary drugs of the italian ministry of health . all efforts were made to minimize animal suffering and number of animals necessary to produce reliable results . microglia primary cultures prepared from mouse cortex as previously described were about 98% pure , as verified by immunofluorescence with cd11b ( for microglia ) , glial fibrillary acidic protein ( gfap , for astrocytes ) , neuronal nuclei ( neun , for neurons ) , and smi94 ( for oligodendrocytes ) . purified cultures of oligodendrocytes were prepared from forebrain of postnatal day 1 - 2 wistar rats , according to minor modifications from a previously described method . after removing the meninges , cortices were minced , digested , and dissociated by passage through 70 m nylon cell strainer ( bd biosciences , europe ) . cells were plated in dulbecco 's modified eagle 's medium ( dmem ) supplemented with 20% heat - inactivated fetal bovine serum ( fbs ) , 4 mm glutamine , 1 mm sodium pyruvate , 50 u / ml penicillin , 50 g / ml streptomycin , and 100 g / ml gentamicin in t75 poly - d - lysine - coated flasks , at about 10 million cells / flask . the cultures were maintained at 37c in a 5% co2 and 95% air atmosphere for 14 days . mixed glial cultures were then shaken at 200 rpm at 37c for 1 h and 98% pure microglia collected from the supernatant of each flask , as verified by immunofluorescence with gfap , neun , neural / glial antigen 2 ( ng2 , for oligodendroglial precursor cells ) , myelin basic protein ( mbp , for mature oligodendrocytes ) , and cd11b . after further shaking at 200 rpm and 37c for 1518 h , the detached cell suspension was finally incubated for 1 h at 37c for differential adhesion of contaminating cells . the non - adherent cells were filtered through 40 m nylon cell strainer ( bd biosciences ) , spun for 10 min at 100 g , resuspended in oligodendroglial precursor cells medium ( basal chemically defined medium : dmem , 4 mm l - glutamine , 1 mm sodium pyruvate , 0.1% bovine serum albumin ( bsa ) , 50 g ml apo - transferrin , 5 g ml insulin , 30 nm sodium selenite , 10 nm d - biotin , and 10 nm hydrocortisone ) containing 10 ng ml platelet derived growth factor - aa ( pdgf - aa ) and 10 ng ml basic fibroblast growth factor ( bfgf ) , and seeded at the density of 1 10 cells / cm into poly d , l - ornithine - coated plates . the cells were then induced to differentiate into mature oligodendrocytes when the basal chemically defined medium was added with 15 nm triiodothyronine , 10 ng ml ciliary neurotrophic factor ( cntf ) and 0,05 mg 10 ml n - acetyl - l - cysteine ( nac ) , for 47 days . a 98% pure population of oligodendrocytes was thus obtained , as verified by immunofluorescence with ng2 , mbp , gfap , neun , and cd11b antibodies . organotypic cerebellar slice cultures were prepared with modifications from previously published protocols [ 26 , 27 ] . briefly , cerebella were excised from neonatal wistar rat ( 810 days old ) , cut on a mc ilwain tissue chopper ( 400 m ) and parasagittal slices separated into cold hank 's balanced salt solution ( hbss ) . two to three slices were plated on each millicell cm culture inserts ( millipore , italy ) and kept in organotypic maintenance medium ( 50% basal medium eagle - bme , 25% hbss , 25% heat - inactivated horse serum , supplemented with 5 mg / ml glucose , 1 mm glutamine , 50 u / ml penicillin , and 50 g / ml streptomycin ) at 37c in a 5% co2 and 95% air atmosphere . the medium was changed every 2 days and double immunofluorescence was performed in free floating after 710 days in vitro . organotypic cultures were washed three times with pbs , fixed with 4% paraformaldehyde for 1 h , rinsed , and blocked for 1 h in phosphate buffered saline ( pbs)/10% normal donkey serum ( nds)/0.4% triton x-100 . primary antibodies were incubated for 24 h in pbs/2% nds/0.4% triton x-100 ( table 1 ) . the secondary antibodies used for double labelling are cy3-conjugated donkey anti - rabbit igg ( 1 : 100 , jackson immunoresearch , europe ) or alexa fluor 488-affinipure donkey anti - mouse igg ( 1 : 200 , jackson immunoresearch ) . after rinsing , sections were mounted , covered with fluoromount medium ( sigma - aldrich , milan , italy ) and a coverslip , and analyzed by confocal microscopy . microglia and oligodendrocytes were washed three times with pbs , fixed with 4% paraformaldehyde for 10 min ( oligodendrocytes ) or 20 min ( microglia ) , washed , permeabilized with 0.050.1% triton x-100 for 10 min , rinsed , and blocked for 30 min in pbs/1% bsa . microglia were stained for about 3 h at 37c in 1% pbs / bsa with 5 g / ml cy2-phalloidin ( sigma - aldrich ) , in combination with primary antibodies against p2y12 receptor , as reported in table 1 . the secondary antibodies used for double labelling are cy3-conjugated donkey anti - rabbit igg ( 1 : 100 , jackson immunoresearch ) or alexa fluor 488-affinipure donkey anti - mouse igg ( 1 : 200 , jackson immunoresearch ) . cells were extensively washed and stained with the nucleic acid blue dye , hoechst 33342 ( 1 : 1000 ) . after rinsing , cells were covered with fluoromount medium ( sigma - aldrich ) and a coverslip and analyzed by confocal microscopy . animals were anaesthetized by intraperitoneal injection of chloral hydrate ( 500 mg / kg ) and transcardially perfused with pbs followed by 4% paraformaldehyde at ph 7.4 . tissue samples ( mice spinal cord and rat brain ) were then postfixed for 1 - 2 days , and cryoprotected in 30% sucrose in pbs at 4c . mice spinal cords ( l3l5 ) were cut at 30 m thickness with a frozen microtome . sections were mounted on slide glasses and allowed to air - dry ( 1 - 2 h ) . a rectangle was then drawn around the sections with a pap pen ( sigma - aldrich ) . rat brains were cut at 40 m thickness using a cryostat microtome and sections were processed in free - floating . double immunofluorescence analysis was performed after blocking in pbs containing 10% nds and 0.3% triton x-100 for 1 h at room temperature . sections were incubated with different combinations of primary antibodies ( table 1 ) , in pbs , 0.3% triton x-100 and 2% nds , for 2448 h at 4c . finally , sections were washed with pbs and incubated with appropriate fluorescent - conjugated secondary antibodies for 3 h at room temperature . after rinsing , sections were covered with fluoromount medium ( sigma - aldrich ) and a coverslip and analyzed by confocal microscopy . tissues supplied by uk multiple sclerosis tissue bank at imperial college , london , were collected postmortem with fully informed consent from both donors and close relatives . procedures for retrieval , processing , and storage have gained ethical approval from all appropriate committees . the brain tissues analyzed were from 7 neuropathologically confirmed cases of secondary progressive ms ( spms ) . analysis was performed also on samples from patients who died due to nonneurological diseases ( healthy ) . cerebral hemispheres were fixed with 4% paraformaldehyde for about two weeks , coronally sliced , and blocked . individual blocks were cryoprotected in 30% sucrose for one week and frozen by immersion in isopentane precooled on a bed of dry ice . cryostat sections ( 3040 m thick ) were either stained with luxol fast blue and cresyl fast violet ( kluver - barrera staining ) , in order to detect white matter lesions and their cellularity , or subjected to immunohistochemistry for mbp , in order to distinguish grey matter lesions . sections were processed in free - floating for double immunofluorescence studies , as described in amadio and collaborators . immunofluorescence was analyzed by means of a confocal laser scanning microscope ( zeiss , lsm700 , germany ) equipped with four laser lines : 405 , 488 , 561 , and 639 nm . the brightness and contrast of the digital images were adjusted using the zen software ( zeiss ) . human p2y12 complete cdna was obtained by reverse transcription with enhanced avian rt - pcr kit ( sigma - aldrich ) from total human brain rna ( life technologies , paisley , uk ) . the obtained cdna was then cloned into the xhoi and xbai sites of the eukaryotic expression vector cs2 + mt to provide n - terminal 6x c - myc epitope - tagged mammalian expression plasmids , which has been validated by dna sequencing . oligos used for amplification were as follows : forward 5gcctcgagatgcaagccgtcgacaacctc3 and reverse 5gctctagagtttacattggagtctcttc designed on human p2y12 mrna ( nm_022788.3 ) . human embryonic kidney 293 cells ( hek 293 ) and sloan - kettering neuroblastoma sh - sy5y clone ( sh - sy5y ) cells were grown in dmem supplemented with 10% fbs , 100 unit / ml penicillin , and 100 g / ml streptomycin at 37c in atmosphere containing 5% co2 . one day before transfection , hek293 or sh - sy5y cells were plated and transfection of p2y12-cs2 + mt or cs2 + mt empty vector was performed with lipofectamine 2000 ( life technologies ) , according to manufacturer instructions . primary rat microglial and oligodendrocyte cells were lysed with trizol ( life technologies ) and total rna was extracted following the manufacturer 's instructions . after dnase treatment ( qiagen , hilden , germany ) , equal amount of total rna ( 1 g ) was subjected to retrotranscription by high capacity rna - to - cdna kit ( life technologies ) and 50 ng of each cdna were amplified with rat p2y12 specific primers ( f : 5-gattgataaccattgacc-3 ; r : 5-ggtgagaatcatgttagg-3 ) . amplification products ( 10 l of 20 ) were electrophoresed on 2% agarose gel containing ethidium bromide ( 1 g / ml , sigma aldrich ) , photographed under uv light using kodak image station 440cf , with molecular imaging software 4.0.1 . in order to isolate total protein extracts , microglia and oligodendrocytes were harvested with ice - cold ripa buffer ( pbs , 1% nonidet p-40 , 0.5% sodium deoxycholate , and 0.1% sds ) , added with protease inhibitor cocktail ( sigma aldrich ) . lysates were kept for 30 min on ice and then centrifuged for 10 min at 14,000 rpm , at 4c . snap - frozen blocks from cases ms114 , ms125 , and ms163 supplied by uk multiple sclerosis tissue bank at imperial college in london were homogenized in ripa buffer , added with protease inhibitor cocktail , incubated on ice for 1 h , and centrifuged at 14,000 rpm for 10 min at 4c . rat brain and mouse brain and spinal cord were homogenized and sonicated in ripa buffer , added with protease inhibitor cocktail , kept for 1 h on ice , and centrifuged at 4c for 10 min at 13,000 rpm . supernatants were collected and analyzed for protein content by bradford colorimetric assay ( biorad , milan , italy ) . analysis of protein components was performed by polyacrylamide gel ( sds - page ) separation ( biorad ) and transfer onto nitrocellulose membranes ( amersham biosciences , cologno monzese , it ) . after saturation , blots were probed overnight at 4c , with the specified antibody ( table 1 ) , and finally incubated for 1 h with hrp - conjugated secondary antibodies and detected on x - ray film ( aurogene , rome , italy ) , using ecl advance western blotting detection kit ( amersham biosciences ) . p2y12 protein was detected with molecular mass comprised between 40 kda ( as predicted by amino acid sequence ) and 49 - 50 kda ( as predicted by the manufacturer data sheet ) . analysis was performed with the statistical software package medcalc ( medcalc software , mariakerke , belgium ) . in order to provide wide ranging comparative analysis of p2y12 expression particularly in microglia and oligodendrocytes under neuroinflammatory conditions , we performed immunofluorescence and confocal analysis of receptor expression in primary cortical and organotypic cerebellar cultures , in tissue slices from rat striatum and cerebellum , in spinal cord sections from symptomatic and end stage sod1-g93a als mouse model , finally in autoptic cortical tissue from progressive ms donors . p2y12 receptor is formed by two transcript variants that give rise to identical proteins with 342 amino acids , a secondary structure constituted by seven hydrophobic transmembrane domains connected by three extracellular and three intracellular loops , with four extracellular cysteine residues most likely contributing to the nucleotide binding site ( figure 1(a ) ) . the commercially available p2y12 antibodies that we mostly used in our work are raised against the second intracellular loop , and precisely amino acids 125142 ( here named intra1 , intra2 , and intra fl ; see red circle in figure 1(a ) ) , and against the c - terminus , here named c - ter ( see green oval in figure 1(a ) ) ( see also table 1 ) . in order to validate the use of these different antibodies for p2y12 receptor , we compared them on recombinant p2y12 receptor protein obtained by cloning the complete cdna of the human receptor into the eukaryotic expression vector cs2 + mt , to provide the expression of a n - terminal c - myc tagged fusion protein . after transfection into sh - sy5y and hek293 cell lines , we analyzed total protein extracts by sds - page and western blotting using c - ter , intra1 , and intra2 antibodies . although with different intensities , all the antibodies recognize the myc - p2y12 protein band at the predicted molecular mass of 50 kda . these results confirm the specificity of the used antibodies toward denatured recombinant p2y12 receptor ( figure 1(b ) ) . in order to verify the expression of p2y12 mrna in rat primary oligodendrocytes ( ol ) and microglia ( rmg ) , rt - pcr was performed using specific primers designed on receptor sequence . as shown in figure 1(d ) , rt - pcr on both rmg and ol reveals the presence of the predicted p2y12 mrna band . next , we validated the antibodies with protein extracts from dissociated primary cultures or tissues from different species . p2y12 protein is specifically recognized in extracts from human cerebral cortex snap frozen tissue , rat and mouse brain , mouse primary microglia ( mmg ) and ol ( figure 1(c ) ) . no signals are obtained when the immunoreactions are performed in the presence of p2y12 neutralizing peptides , when available from manufacturer ( data not shown ) . in order to establish cell specificity of p2y12 receptor expression , we performed comparative immunofluorescence and confocal analysis in primary dissociated cortical microglia and oligodendrocytes ( figures 2(a ) and 2(b ) ) , as well as organotypic cerebellar cultures ( figure 2(c ) ) . p2y12 is strongly recognized by both c - terminus- and second intracellular loop - recognizing antibodies ( red , c - ter , upper left inset ; intra fl , upper right inset ; intra1 , lower right inset and intra2 , lower left inset ) , specifically distinguishing the very heterogeneous morphological features of mouse primary microglia , as shown by double fluorescence and confocal analysis performed with phalloidin ( green ) , a marker for filamentous actin ( figure 2(a ) ) . fan - like cells ( insets ) , elongated rod - like cell bodies with short and tiny branches , asymmetrical hairy cells with miniature processes or lamellipodia are simultaneously observed ( c - ter phalloidin hoechst , merged ) . likewise , all the p2y12 antibodies ( intra2 , intra1 , c - ter , and intra fl ) recognize the multibranched morphology of rat mature oligodendrocytes ( figure 2(b ) , ol , insets , red ) in primary cultures , as confirmed by double immunofluorescence and confocal analysis with mbp antibody ( ol , intra1-mbp , yellow merged image ) . in addition , both intra2 and c - ter antibodies distinguish the ng2-positive rat oligodendrocyte precursor cells ( opc , merged insets ) . p2y12 immunoreactivity is confirmed in the ex - vivo system of organotypic rat cerebellar cultures ( figure 2(c ) ) . however , differently from mouse microglia and rat oligodendrocyte primary cultures , the second intracellular loop- ( red , intra1 ) and c - terminus - recognizing ( red , c - ter ) antibodies surprisingly immunoreact with different cell phenotypes when the integrity and cytoarchitecture of the tissue is preserved , as in organotypic cultures . while intra1 antibody ( red ) exclusively labels mbp - positive fibers ( green ) and highlights myelin structures , c - ter antibody does not immunoreact with myelinated fibers , being present on cells likely resembling microglia ( insets ) , as also confirmed by colocalization with microglial marker cd11b ( data not shown ) . results similar to those found with intra1 were also obtained with intra2 antibodies , lots an01/02/04 ( data not shown ) . we next verified if the cell type - specific presence of p2y12 receptor observed in organotypic culture , either in myelin structures or in microglia , respectively , by the use of the second intracellular loop-(intra1 ) or the c - terminus - recognizing ( c - ter ) antibody , is also confirmed in rat cerebellar and striatal slice tissues ( figure 3 ) . all the antibodies raised against the second intracellular loop ( red , intra1 , intra2 , intra fl ) identify the abundant presence of p2y12 receptor only on myelinated fibers from both cerebellum ( figures 3(a ) , 3(b ) , and 3(c ) ) and striatum ( figures 3(d ) , 3(e ) , and 3(f ) ) , as confirmed by colocalization of signals obtained with intra fl and mbp antibodies in cerebellum ( + mbp , inset c2 , merged , yellow ) and striatum ( data not shown ) ; colocalization of signals obtained with intra1 and intra2 with mbp antibodies in cerebellum and striatum ( data not shown ) ; immunoreactivity of intra1 , intra2 , and intra fl antibodies for structures identical to those observed in cerebellum ( data not shown ) and striatum with mbp antibody ( see for instance mbp , inset e1 , green ) ; identification by second intracellular loop p2y12 antibodies of structures totally different from both bergmann glia and astrocytes recognized by specific gfap antibody in cerebellum ( inset b1 , green ) , and from microglia identified by specific cd11b antibody in cerebellum ( + cd11b , inset c1 , merged , green ) and striatum ( + cd11b , inset f1 , merged , green ) . of notice , all the 2nd intracellular loop antibodies are able to describe the specific cytoarchitecture of both cerebellum , where radiant and sparse fibers clearly characterize the lobuli , and of striatum , where white matter is instead organized in distinct bundles . as in organotypic cultures , the c - terminus antibody ( red , c - ter ) instead recognizes only microglia in rat cerebellum ( figures 3(g ) , 3(h ) , and 3(i ) , and insets g1 , h1 , i1 ) , striatum ( figures 3(j ) , 3(k ) , and 3(l ) ) and cerebral cortex slices ( data not shown ) , with a signal more uniformly distributed throughout the whole tissue . by comparing the cd11b and c - ter immunolabelling in the striatum , we furthermore observe that the mutual intensity of signals is cell - selective within the microglia population , with some cells exclusively positive for c - ter ( arrow ) , and others instead showing different grades of cd11b - c - ter colocalization ( see for instance orange , yellow and greenish cells in the merged field ) . regrettably , double immunofluorescence with c - ter and iba1 microglia marker is not practicable , since both antibodies are raised in the same species . however , no colocalization of signals is ever shown between mbp ( green ) and c - ter antibodies ( + mbp , inset j1 , merged ) . in order to verify if p2y12 recognized by c - ter antibody exclusively in microglia from rat brain slices could be used as specific microglia marker during neuroinflammation , we validated its use in a typical neuroinflammatory disease such as als and for the first time characterized the presence of p2y12 receptor in sod1-g93a mouse model ( figure 4 ) . by immunofluorescence and confocal analysis on lumbar spinal cord sections ( l3l5 ) of wild - type ( wt ) mice , we first compared the immunoreactive signals obtained with c - ter and specific microglia markers cd11b ( red , recognizing ramified microglia ) and cd68 ( red , recognizing roundish activated microglia ) . in wt mice , c - ter ( green ) is abundantly and strongly immunoreacting with the microglia population and colocalizing with the majority of cd11b - positive cells , in both dorsal ( dh ) and ventral ( vh ) horns of spinal cord ( figure 4(a ) , left panel , merged yellow signal ) . all cd11b - positive cells share immunoreactivity for c - ter , as proved by the absence of red cd11b signal . conversely , not all c - ter - positive cells immunoreact also with cd11b antibody , as proved by the presence of some green c - ter signals . in addition , we never observe colocalization with the rare activated cd68-positive ( red ) microglia cells present in wt healthy tissue ( figure 4(a ) , right panel , merged and inset ) . to test if c - ter antibody can further recognize microglia activation during the progression of als , we next examined sod1-g93a spinal cord sections at two different stages of the disease , that is , 20 weeks , a phase when the disease accelerates , and end stage , that is , 23 weeks , when the animals reach the score of 1 accordingly to a behavioral score system . at both stages , sod1-g93a mice show a significant increase in cd68 immunostaining not only when compared to wt mice ( figure 4(a ) ) , but also in vh with respect to dh , and this is even more evident at end stage with respect to 20 weeks ( red , figure 4(b ) ) . remarkably , the immunoreactive signal of c - ter ( green , figure 4(b ) ) decreases during disease progression and the effect is pronounced especially in vh , where motor neuron loss , tissue damage , and microglia activation are known to be increased . particularly at 20 weeks , c - ter - expressing microglia are still ramified in dh , where few cd68-positive cells are present , but start to disappear in vh , where instead cd68-positive cells are increased . at end stage , c - ter staining decreases in dh with respect to 20 weeks and disappears almost completely in vh , concomitantly with a robust increase in cd68 staining . a parallel decrease in c - ter and increase of cd68 immunoreactivities is also confirmed by western blot analysis performed on sod1-g93a lumbar spinal cord homogenates at end stage ( figure 4(c ) ) . as in rat organotypic cerebellar cultures ( figure 2(b ) ) and rat cerebellar , striatal , cortical , or mouse spinal cord tissue slices ( figures 3 and 4 ) , a similar pattern of p2y12 receptor expression is shown in human frontal cortex autoptic tissue by using c - ter antibody that highlights only microglia uniformly distributed throughout the entire healthy tissue ( red ) , but not mbp - positive myelinated structures ( green ) , as detected by the absence of overlapping immunofluorescent signals ( figures 5(a ) , 5(b ) , and 5(c ) , merged and insets a1 , c1 ) . on the contrary , intra1 ( red , figures 5(d ) , 5(e ) , and 5(f ) ) , intra2 , and intra fl ( data not shown ) antibodies recognize exclusively myelinated fibers in tissue from healthy ( insets d1 , e1 , and f1 ) and progressive ms donors ( figures 5(d ) , 5(e ) , and 5(f ) ) , as it is found with rat tissue ( figures 2 and 3 ) . moreover , we confirm a decrease of p2y12 receptor signal in proximity to the demyelinating lesion , as detected by loss of mbp - positive fibers ( see asterisks ) . no colocalization of signals is shown with mhc ii - positive microglia ( + mhc ii , inset d3 , merged ) . importantly , c - ter , intra1 ( red , insets a - f2 ) and intra2 , and intra fl ( data not shown ) antibodies all recognize the presence of p2y12 receptor in integrin ii/3-positive platelets ( green ) contained in the blood vessels of the analyzed tissues . as observed in als mouse spinal cord where p2y12 receptor detected by c - ter antibody is shown to temporally and regionally decrease in microglia as a function of increasing inflammatory damage ( figure 4 ) , we notice that microglia gradually lose immunoreactivity for c - ter antibody ( figure 6 ) in proximity to the demyelinating active cortical lesions of ms expressing augmented positivity for mhc ii [ 3335 ] . moreover , by comparing mhc ii and c - ter signals , we recognize four areas ( a d ) inside and around the lesion , where microglia express different amount of these proteins . in zone a , at the edge of the lesion , we observe a predominance of c - ter immunoreactivity ( red ) compared to that of mhc ii ( green ) , as depicted in the merged field by a major occurrence of red signal . in zone b , closer to the lesion , we notice a prevalence of active mhc ii - positive green cells , but still the presence of few red and yellow signals . c and d , apparently in the core or outside the lesion , respectively , where microglia express either almost exclusively mhc ii protein ( c ) or p2y12 receptor on ramified microglia ( d ) . the interchange among different cell types of molecular cues that condition the cell specificity and the protein profile of each cell characterizes the morphological and functional heterogeneity in particular of microglia within various cns regions , developmental stages [ 37 , 38 ] and , even more , states of activation during pathological conditions . in the case , for instance , of als , the release of signals from motor neurons apparently denotes one of the earliest phase of the disease , with microglia behaving as an m2 phenotype producing neuroprotective factors to repair motor neurons and preventing them against further injury . as disease rises , motor neurons start releasing alarm signals that in turn convert microglia from beneficial m2 to cytotoxic m1 phenotype , with consequent release of proinflammatory cytokines . for instance , m1 markers are abundantly expressed in normal appearing white matter and throughout active demyelinating ms lesions by activated microglia and macrophages , although in human active ms lesions microglia show an intermediate activation status . in addition , m2 microglia appear fundamental to guide oligodendrocyte remyelination in mice , and a switch from m1- to m2-dominant response occurs in microglia and peripherally derived macrophages when remyelination starts . only therapeutic procedures that both down - regulate the harmful responses and up - regulate the beneficial responses may hopefully slow pathological progression and provide meaningful hope for treatment . at the same time , the identification of clear markers involved in the m2/m1 microglia transition becomes mandatory for presymptomatic diagnosis , monitoring of disease progression , and efficacy of therapies . under this perspective , and consistently with previous findings establishing the role of purinergic receptors in the pathogenesis of both als and ms [ 14 , 43 ] , our present work serves this aim , by highlighting the gradual loss of p2y12 immunoreactivity as an early marker of neuroinflammation and microglia metamorphosis . we have indeed demonstrated here that p2y12 receptor protein identified in primary cultures of both microglia ( figure 2(a ) ) [ 18 , 44 ] and oligodendrocytes ( figure 2(b ) ) by different but p2y12-selective antibodies can be instead recognized in branched microglia exclusively by the use of c - ter antibody ( figures 36 ) . this occurs only when the integrity and cytoarchitecture of the tissue is typically preserved in the presence of the least experimental manipulations , that is , in organotypic cultures ( figure 2(c ) ) and tissues slices for instance from rat striatum and cerebellum ( figure 3 ) , mouse spinal cord ( figure 4 ) , and human cerebral cortex ( figures 5 and 6 ) . a similar difference in primary cultured cell versus tissue distribution of a protein was previously demonstrated with large - conductance calcium - activated potassium channel expression , in vascular endothelium . a first implication emerging from these results is that a reliable evidence about selective p2y12 expression in cells of healthy or neuroinflammatory states is genuine only when cell connectivity and tissue architecture are fully preserved . we have indeed shown this , by proving that the antibodies used for p2y12 , recognizing either the c - terminus or the second intracellular loop of the receptor ( figure 1 and table 1 ) and immunolabelling , respectively , microglia or myelinated fibers in the cns , are all still able to immunoreact for instance with platelets ( figure 5 ) , where the receptor was originally described to be present and to have a role in the processes of activation , aggregation [ 4649 ] , primary hemostasis , and arterial thrombosis [ 5055 ] . a possible explanation for the microglia versus oligodendrocyte selectivity of the p2y12 antibodies might be that gi - coupling , and/or quaternary structure , post - transcriptional modifications , and subcellular localization of p2y12 that remain strictly preserved in platelets , are instead divergent in microglia with respect to oligodendrocytes / myelinated fibers . in this case , a cell - specific network of p2y12 oligomeric interactions and/or a distinct subcellular partitioning might simply mask the recognition sites of the different antibodies on p2y12 protein in different cell types . to support this hypothesis , we know that in platelets p2y12 indeed resides in subcellular lipid raft structures and its partitioning out from rafts causes for instance inactivation and that also the presence of another purinergic receptor , the ionotropic p2x3 , in lipid rafts has cell - specific properties shared in cerebellar granule neurons and total brain tissue but not in neuroblastoma cells and dorsal root ganglia and that the specific antagonist clopidogrel inhibits p2y12 by breaking down the homoligomeric complex to single monomers and finally , that hetero - oligomerization of p2y12 is demonstrated with p2y1 , p2y2 , p2y13 , and with adenosine a1 , a2a receptors in different cellular backgrounds . all these features might very well explain also why it has not been found colocalization between p2y12 and gfap - positive astrocytes in rat brain cortex and nucleus accumbens , despite the abundant presence of p2y12 mrna and , moreover , why p2y12 is specifically observed in brain and spinal cord resident microglia but is not observed , for example , in peripheral macrophages in spleen [ 18 , 20 ] . a second implication that emerges from our results is that the morphological metamorphosis that microglia undergo under neuroinflammatory conditions as those triggered during als and ms , can be remarkably highlighted by the progressive reduction of p2y12 immunostaining obtained with c - ter antibody that reacts , also in this case , exclusively with multibranched microglia still present in the tissue ( figures 4 and 6 ) . this closely reflects the expression of p2y12 that is robust in the resting / surveillant branched state but dramatically decreased after morphological transition and activation of microglia . our observations are also in line with the central role played by p2y12 in branched microglia membrane ruffling and inspection of the environment . on the other hand , they depict a morphological / functional state of microglia that only partially overlaps with cd11b and mhc ii immunoreactivities , which are furthermore known to increase during activation but instead highly contrasts with cd68 immunostaining that is totally absent in ramified microglia . in parallel to our results , these last antibodies actually accentuate the progressive transition of microglia from a lesser ramified shape to a significantly more activated amoeboid phenotype ( figures 4 , 6 , and 7 ) , thus suggesting the dual use of c - ter and cd68 antibodies as markers , respectively , for branched resting / surveillant versus roundish / activated microglia . a reduction of c - ter - p2y12 immunostaining that is concomitant to an increase , for instance , of mhc ii / cd11b / cd68 immunoreactivity , could thus become a feasible approach to detect an increasing neuroinflammatory condition . indeed , p2y12 is considered an essential component and primary site at which nucleotides such as adp act to promote directional microglia movement or chemotaxis at early stages of cns injury . in particular , microglia from mice lacking p2y12 exhibit normal baseline motility but are unable to polarize and to elicit directional branch extension and migration toward nucleotides in vitro , or sites of cortical damage in vivo . these notions are consistent with our results that fail to describe c - ter - p2y12 immunoreactivity on roundish phagocytizing , or polarized migratory microglia . however , we still do not know if reduction / absence of p2y12 c - ter - immunolabelling on activated microglia might be a cause or a consequence of morphological / functional transition or might simply reflect a cell - selective hindrance and lack of access to the immunogenic sites by the antibody . anyhow , we can assert that the distinctive recognition of multibranched microglia renders the c - ter antibody a novel and useful tool to discriminate among microglia morphological states , thus making p2y12 receptor a selective and early marker for the ramified phase . in parallel to this , we have also proven that all the several antibodies raised against the second intracellular loop of p2y12 ( intra1 , intra2 , and intra fl ) can likely be employed as markers for the presence of ms lesions . although with different intensities , they not only recognize the receptor specifically on myelinated fibers of organotypic cultures ( figure 2(c ) ) , tissues slices from rat striatum or cerebellum ( figure 3 ) and human cerebral cortex , but also furthermore highlight the reduction of p2y12 signal that occurs for instance in ms tissue ( figure 5 ) in correlation to the extent of demyelination found in all types of grey matter cortical plaques ( i iii ) and subcortical white matter . in brief , we have shown here that the presence of p2y12 receptor can be simultaneously identified by the c - terminus and the second intracellular loop antibodies . when this occurs , a condition of intact myelinated fibers and branched / surveillant microglia is represented at once that perhaps signifies a for instance in ms , a decrease in the second intracellular loop immunoreactions accompanied by an increase of c - terminus immunoreactivity will possibly depict the loss of myelin and replacement by ramified microglia that often occur in an inactive plaque . on the contrary , a decrease in c - terminus immunolabelling in the abundant presence of second intracellular loop - positive myelinated fibers would indicate an early active plaque where m1/m2 microglia reactivity starts to take place . by comparative analysis of all the available p2y12-immunoreactive antibodies recognizing the c - terminus or the second intracellular loop of the receptor , we have established here that , under experimental conditions of well - preserved cytoarchitecture and tissue integrity , p2y12 receptor expressed by both ramified microglia or oligodendrocytes / myelinated fibers might serve a dual function as specific marker , respectively , of branched / surveillant microglia as well as demyelinating lesions . we believe that p2y12 identification and modulation might potentially acquire an important predictive value under neuroinflammatory conditions , as those found in als and ms . p2y12 is likely to deserve a key role in the verge of a neuroinflammatory breakdown .

in the cns , neuroinflammation occurring during pathologies as amyotrophic lateral sclerosis ( als ) and multiple sclerosis ( ms ) is the consequence of an intricate interplay orchestrated by various cell phenotypes . among the molecular cues having a role in this process , extracellular nucleotides are responsible for intercellular communication and propagation of inflammatory stimuli . this occurs by binding to several receptor subtypes , defined p2x / p2y , which are widespread in different tissues and simultaneously localized on multiple cells . for instance , the metabotropic p2y12 subtype is found in the cns on microglia , affecting activation and chemotaxis , on oligodendrocytes , possessing a hypothesized role in myelination , and on astrocytes . by comparative analysis , we have established here that p2y12 receptor immunolabelled by antibodies against c - terminus or second intracellular loop , is , respectively , distributed and modulated under neuroinflammatory conditions on ramified microglia or myelinated fibers , in primary organotypic cerebellar cultures , tissue slices from rat striatum and cerebellum , spinal cord sections from symptomatic / end stage sod1-g93a als mice , and finally autoptic cortical tissue from progressive ms donors . we suggest that modulation of p2y12 expression might play a dual role as analytic marker of branched / surveillant microglia and demyelinating lesions , thus potentially acquiring a predictive value under neuroinflammatory conditions as those found in als and ms .

acute kidney injury ( aki ) requiring dialysis is a serious complication in critically ill patients , bringing increased morbidity , mortality , and costs of care [ 14 ] . aki requiring dialysis is usually considered the most severe form of kidney injury , and these patients have been conventionally regarded as a relatively homogenous group of patients , either when describing epidemiological information or while conducting clinical trials [ 5 , 6 ] . however , studies examining interventions in dialysis patients ( e.g. , dialysis modality or frequency have not demonstrated unequivocal survival benefits [ 79 ] . it is well recognized that small changes in creatinine ( mild - to - moderate aki ) independently predict mortality [ 10 , 11 ] ; we also recently reported that patients with aki requiring dialysis represent a wider spectrum of severity of kidney injury , contrary to the prevalent notion . thus , it can be hypothesized that the degree of elevation of creatinine prior to initiating dialysis may discriminate risk - adjusted mortality , similar to the observations in nondialysis requiring aki . the acute kidney injury network ( akin ) has issued standard definitions of aki ; currently , in these criteria , aki requiring dialysis is classified as stage iii ( or severe ) aki . the consensus panel also proposed that the examination of natural history of severe aki in icu to be one of the high - priority research areas , with a goal that new information may improve our ability in conducting prospective trials of intervention aki [ 14 , 15 ] . to date , there are limited studies that have examined the course or progression of aki after icu admission until the point of dialysis initiation . whether the degree of severity of kidney injury prior to dialysis requirement independently influences mortality risk in aki is not known . in order to facilitate risk stratification that may be useful for the development of prospective studies , which are geared towards early diagnosis or intervention in severe aki , we characterized the spectrum of severity of kidney injury in a cohort of icu patients with aki who required dialysis . we specifically tested the association of creatinine elevation during icu stay , starting with icu admission and prior to dialysis initiation , with the risk of hospital mortality . we also examined the impact of severity of illness upon icu admission on the mortality risk in these patients . we examined a cohort derived from the veterans affairs ( va ) inpatient evaluation center ( ipec ) ; a national quality improvement program which reports risk - adjusted mortality , length of stay , and adherence to evidence - based practices in all va icus , by collecting data electronically from va computer databases . an analytic dataset was formed from the ipec retrospective cohort including all patients ( n , 617,927 ) admitted to all 191 va icus in the usa between october 2001 and september 2006 . we excluded patients with ( i ) less than three creatinine measurements during the icu stay ( n , 204,963 ) , ( ii ) readmissions to the icu ( n , 50,874 ) , ( iii ) transferred to other hospitals at discharge ( n , 14,219 ) , ( iv ) transplant recipients ( n , 317 ) , and ( v ) those with chronic renal failure defined as prior dialysis ( n , 3,862 patients ) , or international classification of disease-9th clinical modification ( icd-9-cm ) codes for end - stage renal disease ( esrd ; 1,388 patients 585.6 ) , or with a calculated glomerular filtration rate ( gfr ) < 15 ml / min/1.73 m ( 16,571 ) ( gfr estimated by four variable modified diet in renal disease equation ) ; 296 additional patients who required dialysis in icu but had icd-9 codes for advanced ckd / stage v ckd were also excluded ( based on random q / a , these codes identified esrd status in those who required dialysis ) . thus , 324,999 patients were available for analysis . the analyses were reviewed and approved by the university of cincinnati institutional review board and va r&d committees . briefly , from each hospital database , a customized program identifies patients whose hospitalization included an icu stay , and extracts administrative data from the index hospitalization that includes icd-9-cm codes representing the reason for admission to the icu , length of stay , and all procedure codes . measured values of 11 laboratory tests are extracted to estimate severity of illness from a period of time 7 days prior to icu admission through hospital discharge . mortality is defined at death during hospitalization and is validated using the vital status file at the va national database ( austin , tx ) . aki was present if there was an increment of serum creatinine by > 0.3 mg / dl ( or 1.5 times increase ) during the icu stay relative to the creatinine on icu admission . for this analysis , we specifically focused on examining characteristics and outcomes of those aki patients who progressed to new requirement of dialysis ( aki - d ) during icu stay ( part of stage iii aki according to the akin classification system ) . dialysis requirement in icu included both intermittent and continuous dialysis , as prescribed by the clinician , and the modality can not be differentiated based on va electronic records that were available . we categorized aki - d into four groups based on the change in creatinine level after icu admission but prior to initiation of dialysis : group i had an increase in creatinine ranging from 0.3 mg / dl to < 2-fold increase , group ii an increase in creatinine 2 times baseline but <3 times , group iii 3-fold increase in creatinine , and group iv included those patients with new dialysis requirement in icu but who experienced a creatinine elevation of < 0.3 mg / dl . the purpose of this grouping was to determine an equivalent stage of aki that a patient may experience prior to initiating dialysis based on the standard definitions of aki put forth by the akin . some patients may have had creatinine values available after dialysis initiation , but these values were not considered for analysis . the study accounts for differences in patient characteristics using a previously validated logistic regression model that predicts mortality risk for each patient from independent predictors ( age , thirty - one comorbid disease groups ( by icd-9-cm codes ) , eighty - four mutually exclusive admission diagnoses determined from the icd-9-cm codes denoting the reason for admission to the icu , source of admission ( emergency room , outpatient clinics , ward , hospital , operating room , or nursing home ) , and the worst value of each of the 11 laboratory tests measured within 24 hours of icu admission ( sodium , urea , creatinine , bilirubin , albumin , glucose , hematocrit , white blood cell count , pao2 , paco2 , and ph ) . diagnosis and comorbid diseases were included in this model as binary variables , most laboratory values and age were included as cubic splines , and paco2 and ph were treated as a categorical interaction term . the model has been validated across multiple centers with excellent calibration and discrimination ( c - statistic = 0.88 ; brier 's = 0.06 ) , and comparable with traditional methods of severity of illness assessment ( e.g. , apache ) . from the logistic regression model , a standardized mortality rate for groups can be determined ( smr = observed / predicted mortality ; where predicted mortality is estimated by the mortality model described above ) . a more detailed account of development and validation of these variables and risk adjustment methods has been published earlier [ 16 , 19 , 20 ] . univariate comparison of diagnosis categories , comorbid diseases , and their relationship with laboratory variables was tested by chi - square test and kruskal - wallis test . a two - step logistic regression model determined the independent contribution of patient groups ( based on creatinine elevation ) to hospital mortality . the first step predicted each individual patient 's hospital mortality using the validated method described above . independent predictors in the second step included the predicted mortality ( determined from the first step ) and the patient group based on creatinine elevation before dialysis . based on the predicted mortality , as determined by the mortality model described above , we also estimated the standardized mortality rate ( observed / expected mortality ) for each of the patient groups under consideration . risk estimates were expressed as odds ratios ( or ) and 95% confidence intervals ( 95% ci ) . overall , 21.9% of patients developed aki ( 71,090/324,999 ) of whom 2,744 patients experienced severe aki requiring dialysis . 561/2744 ( 20.4% ) patients were admitted to icu from the hospital floors , whereas 2,183/2,744 ( 79.6% ) were either direct admission or admissions from emergency rooms . the median time for aki - d patients to meet the criteria for aki from the time of admission creatinine measurement was 23.4 hours ( interquartile range , 12.2 , 46.6 ) . the median time to initiation of dialysis in icu after icu admission was 96.0 hours ( interquartile range , 33.6 , 231.6 ) ; median time to dialysis initiation from icu admission was 72 hours in group i , 145.1 hours in group ii , 216.0 hours in group iii , and 14.6 hours in group 4 . when examined from the time when patients reached their peak creatinine value to the time of dialysis initiation , the median time was 43.0 , 39.5 , and 67.4 hours in groups i , ii , and iii respectively . figure 1 shows the percentage of patients initiating dialysis by days after icu admission . the proportion of patients in each group is shown in figure 2 ; of the 2,744 patients who required new dialysis during icu stay , only about a third of patients ( 31.5% ) experienced greater than 3 times increase in serum creatinine prior to dialysis initiation , and a small proportion of patients ( 11.2% ) required dialysis with little or no elevation in serum creatinine ( likely for fluid - electrolyte / acid - base disturbances ) . the baseline comorbid and laboratory characteristics of patients who developed aki requiring dialysis , and their univariate comparison across groups i through iv are shown in table 1 . group iii patients ( those with > 3 times increase in creatinine prior to dialysis ) had the lowest levels of creatinine or bun on icu admission . in contrast patients in other groups , who sustained only milder or no elevation in creatinine before dialysis initiation , had significantly higher creatinine and bun levels on icu admission . table 2 shows the frequency of admission diagnosis categories across four subgroups of patients with aki requiring dialysis . the frequencies of patients admitted with primary icu admission diagnoses of kidney disorders or electrolyte and metabolic disorders significantly varied across four groups and were 24% and 21% , respectively , in group iv ( < 0.3 mg / dl increase in creatinine before dialysis ) compared with only 1.9% and 2.1% in group iii ( > 3 times increase in creatinine before dialysis ) . this suggests that majority of patients in group iv may have been dialyzed for indications other than kidney injury ( e.g. , for electrolyte or metabolic disturbances ) . in contrast , cardiothoracic surgery admissions occurred at a higher frequency ( 26% ) among group iii patients ( > 3 times increase in creatinine ) , suggesting that these patients are more likely to have normal renal function on icu admission and hence sustain > 3 times increment in creatinine prior to dialysis requirement . the degree of creatinine elevation prior to dialysis initiation was associated with increase in odds of death . table 3 shows odds ratios of mortality across all four groups , with group i as reference group . dialysis patients in group ii had greater odds of death ( odds ratio ( or ) , 1.76 , 95% confidence intervals ( 95% ci ) , 1.40 , 2.22 ) ) , those in group iii were more than twice likely to die ( or 2.20 , 95% ci , 1.792.71 ) , whereas patients in group iv were less likely to die compared to group i ( or 0.39 , 95% ci , 0.29 , 0.52 ) . the smr ( table 3 ) confirmed a graded association between the degree of creatinine elevation prior to dialysis initiation and mortality risk . the relationship between creatinine elevation and risk of death remained qualitatively similar even after stratifying patients by level of renal function on icu admission ( stratified at creatinine level of 1.2 mg / dl ) , but the sample size was relatively small in these subgroups ( data not shown ) . figure 3 shows smr associated with creatinine elevation stratified by severity of illness on icu admission . dialysis patients in groups ii and iii had a significantly higher smr than those patients in groups i and iv , when predicted mortality was < 10% or 1030% ; smr was not significantly different across all four groups in patients admitted with > 30% predicted mortality on icu admission . the present study advances our understanding of natural history of progression of acute kidney injury during icu stay , in those patients who require dialysis . the study finds that , in patients with aki requiring dialysis , the risk of mortality is independently associated with the degree of severity of kidney injury sustained during icu stay prior to dialysis initiation and that severity of illness further influences this relationship . several epidemiological studies have confirmed that icu patients developing aki requiring dialysis experience a high risk of mortality across different clinical settings , including cardiovascular surgery , or in other medical or surgical icus [ 12 , 2123 ] . it is also observed that the severity of kidney injury , determined by the degree of creatinine elevation , in non - dialysis requiring aki is associated with a graded increase in mortality risk [ 2 , 10 ] . the present study is one of the first reports to show that in patients who developed aki requiring dialysis , the degree of kidney injury prior to dialysis initiation determines mortality risk , after accounting other major predictors of hospital mortality . it does so by examining a large , diverse , multicenter cohort that includes all icu settings , with an ability to perform electronic data extraction starting ( including serial creatinine measurements ) from the time of icu admission until the point of dialysis initiation in aki . the results indicate that majority of the aki - d patients sustain mild - to - moderate degrees of creatinine elevation ( equivalent of stage i or stage ii aki ) prior to dialysis , whereas less than a third of patients require dialysis after sustaining > 3 times increase in serum creatinine during icu stay ( or equivalent of stage iii aki ) . this confirms prior multicenter observations that aki requiring dialysis in icu usually represents a group of patients that sustain acute kidney injury on an underlying renal dysfunction . there were 11% of patients who required dialysis with < 0.3 mg / dl increase relative to icu admission . although the specific indications for dialysis could not be ascertained by electronic data extraction , however , based on the distribution of comorbid conditions and admission diagnoses , it can be suggested that the indications for dialysis in this subgroup may have been for volume overload or electrolyte / metabolic abnormalities rather than the degree of kidney injury . these observations are consistent with our prior experience in cardiac surgery settings , where 1015% of cases were dialyzed for reasons related to volume overload or electrolyte disorders and not azotemia / creatinine elevation . in terms of mortality , however , this group was associated with the lowest risk of death among those who required dialysis consistent with the study hypothesis . prior large multicenter observations provide only a limited insight into the effects of severity kidney injury prior to dialysis initiation on patient outcome . somewhat indirect evidence comes from the studies that have reported the impact of baseline chronic kidney disease ( ckd ) in patients with aki requiring dialysis . for example , a third of patients in a multicenter us cohort ( picard cohort ; 618 aki cases , 60% of whom required dialysis ) had stage iv or worse ckd prior to aki . by multivariate analysis , baseline ckd status conferred 43% lower adjusted odds of in - hospital mortality ( or , 0.57 , 95% ci , 0.390.84 ) in aki . similar observations have been reported in a medicare administrative sample that examined outcomes in aki . unadjusted mortality rate in patients that were coded for aki requiring dialysis on an underlying ckd was 22% compared to 30% among those with aki requiring dialysis but without underlying ckd [ 2426 ] . our study may offer one of the explanations to the seemingly paradoxical observation that ckd status in aki may confer a protective effect : that is , patients with impaired renal function on icu admission are likely to require dialysis after sustaining milder degrees of kidney injury than those admitted with relatively better renal function and thus experience a lower risk of mortality . severity of illness on icu admission further modified the relationship between the degree of creatinine elevation and risk - adjusted mortality in aki require dialysis . the degree of kidney injury sustained prior to dialysis did not discriminate mortality risk in those patients with very high severity of illness ( > 30% predicted mortality ) on icu admission . in contrast , in patients with low - to - moderate severity of illness , it is the degree of injury that determines excess mortality risk rather than dialysis status alone . first , the data indicates that in half of the patients , dialysis was initiated within 96 hours after icu admission , with ~25% of then requiring it in the first 48 hours . thus , in order to target these patients for early interventions , we need to develop better predictive models that are based on information derived within 24 hours of icu admission ; similarly , the development of novel biomarkers that may predict dialysis requirement would need to discriminate acute kidney injury from underlying renal dysfunction at the time of icu admission . second , it indicates that the efficacy of therapeutic measures in aki requiring dialysis can not be equitably compared without accounting for both severity of illness on admission and degree of kidney injury sustained before dialysis initiation . for example , given the same predicted mortality or level of renal function on admission , patients in group i or iv could not be optimally compared with group iii in an intervention trial . finally , it also raises the question that whether early dialysis , as indicated by initiation of therapy after a relatively lower magnitude of rise in creatinine , may offer a survival benefit . the va patient population is predominantly represented by male gender and white race and hence may limit in terms of generalizability of findings to specific demographic subgroups . by way of retrospective design , and lack of standardized criteria to initiate dialysis , the data can not account for differences in dialysis practices across different centers . we , however , examine a multicenter , diverse cohort of icu patients that records patient level information that is available for automated extraction by computerized methods . our cohort derivation excluded patients with advanced renal dysfunction on icu admission , and based on the present findings it can be expected that a significant proportion of these patients may have required new dialysis in icu . we still chose to use this criterion to minimize the likelihood of misclassifying patients with dialysis dependent renal failure prior to icu admission but who were not appropriately coded for it . additionally , we considered the degree of creatinine elevation relative to admission creatinine , which may not reflect a true baseline assessment of renal function ; this still allowed us to assess the relationship between magnitude of creatinine elevation and hospital mortality . another limitation is that we did not have information on modality or frequency of dialysis and hence could account for those variables in the multivariate analyses . the present study relies on the va icu mortality model instead of other traditional methods of measures of severity of illness assessment such as apache criteria . the rationale to do so is because the va icu mortality model is derived from an automated electronic data extraction method , which has been validated across multiple va sites with excellent predictive accuracy and calibration ( c - statistic = 0.88 ) and is comparable with other methods . in summary , within a cohort of patients who develop aki requiring dialysis during icu , the mortality risk is independently associated with the degree of creatinine elevation prior to dialysis initiation . the mortality risk is lowest in those who experience minimal or no elevation in creatinine and may represent less severe acute illness in patients with ckd , or easily reversible fluid - electrolyte or acid - base disturbances . the study also indicates that aki requiring dialysis represents a heterogeneous group of patients , and directly confirms earlier observations that only minority of these cases sustain severe kidney injury prior to dialysis initiation . we interpret the data to suggest that prospective studies aimed at examining therapeutic interventions in aki requiring dialysis ( e.g. , timing of dialysis initiation ) would need to consider both the degree of kidney injury sustained prior to dialysis initiation and overall severity of illness , prior to performing equitable comparisons .

in a multicenter observational cohort of patients - admitted to intensive care units ( icu ) , we assessed whether creatinine elevation prior to dialysis initiation in acute kidney injury ( aki - d ) further discriminates risk - adjusted mortality . aki - d was categorized into four groups ( grp ) based on creatinine elevation after icu admission but before dialysis initiation : grp i > 0.3 mg / dl to < 2-fold increase , grp ii 2 times but <3 times increase , grp iii 3-fold increase in creatinine , and grp iv none or < 0.3 mg / dl increase . standardized mortality rates ( smr ) were calculated by using a validated risk - adjusted mortality model and expressed with 95% confidence intervals ( ci ) . 2,744 patients developed aki - d during icu stay ; 36.7% , 20.9% , 31.2% , and 11.2% belonged to groups i , ii , iii , and iv , respectively . smr showed a graded increase in grp i , ii , and iii ( 1.40 ( 95% ci , 1.291.42 ) , 1.84 ( 1.662.04 ) , and 2.25 ( 2.072.45 ) ) and was 0.98 ( 0.781.20 ) in grp iv . in icu patients with aki - d , degree of creatinine elevation prior to dialysis initiation is independently associated with hospital mortality . it is the lowest in those experiencing minor or no elevations in creatinine and may represent reversible fluid - electrolyte disturbances .

many previous studies have revealed that parents , especially mothers , of children with developmental disabilities such as intellectual disabilities ( ids ) , developmental delay , and physical and sensory handicaps are more likely to show signs of psychological distress or depressive symptoms and to exhibit lower well - being than parents of typically developing children [ 15 ] . it is generally accepted that caring for a child who has a developmental disability may involve significant and prolonged periods of time and energy . since the majority of this increased daily care - giving burden for these children is carried by their parents , they are supposed to have an increased risk for high levels of stress , and thus some cases may be linked to depression [ 7 , 8 ] . recently , the mental health needs of adults with i d have also been taken up for discussion . studies have revealed that psychiatric disorders are more prevalent in people with i d compared within the general population . specialist psychiatric services for people with i d are available in some countries such as the uk and the usa ; however , the provision of high - quality psychiatric services remains a major concern in many countries . . evidently , the current psychiatric services in japan are not adequate to meet the complex mental health needs of people with i d appropriately , sensitively , and effectively . failure to provide services that meet the needs of these individuals may lead to problems such as high - level caregiver stress and inappropriate therapy . the paucity of trained psychiatrists and other allied professionals is a major barrier to the development of this subspecialty in many countries . in japan , high - quality psychiatric services for the parents of children with i d are assumed to be not well established . so far , the major portion of resources has been directed to the treatment of and services connected with the children , and not enough attention has been paid to the actual mental condition and needs of the parents . to our knowledge , there have been no reports on a nationwide scale on the mental health and distress of parents having children with developmental disabilities in japan . since the understanding of the parents ' mental health may be essential for providing more appropriate support , we aimed to obtain a clear perspective on the situation facing the parents through this facility - based nationwide study . the current survey included 1,102 institutions and consultation centers for children with developmental disabilities distributing in all prefectures in japan , which were listed in the national register of agencies , organizations , and institutions related to intellectual disabilities and the directory of support centers for individuals with developmental disabilities . in japan , these institutions and centers play a key role in social support networks . professional workers in these facilities come into contact with children as their clients , and also with the children 's guardians , mainly with the mothers , on a daily basis . the questionnaire was written in japanese and was divided into two sections with a total of 47 multiple choice questions . the first section sought information about the characteristics of the facility , such as staff adequacy , the scale of the facility , the health promotion services provided , the ages of their clients , and the clients ' main disabilities , namely , i d , pervasive developmental disorder ( pdd ) , profound intellectual and multiple disabilities ( pimd ) , physical disabilities , and others . the second section sought information about the clients ' parents , such as signs of mental health distress and their needs , from the professional caregivers ' point of view . also measures taken by the professional caregivers when encountering distressed parents were asked in detail . questionnaires were mailed in october 2008 , with an explanatory letter to the person in charge of each institution or consultation center . when agreement was met to participate in this study , one or more service providers in each facility completed the questionnaire based on all sources of information available . the questionnaire was to be returned unsigned , using the stamped , self - addressed envelope enclosed . for ethical considerations , the information disclosed in the questionnaire was in a form which made it impossible to identify the facility and clients this study was approved and carried out under the supervision of the japan league on developmental disabilities ( jidd ) . the statistical analysis including the chi - squared test was performed with the aid of the statistical package for the social sciences 15.0 ( spss japan , inc . ) . a two - sided p value of 0.05 or less was considered to be statistically significant . answers to open - ended questions a total of 460 out of 1,102 facilities replied within four weeks , the response rate being 41.7% . according to the clients ' main disabilities , each facility was classified into the following categories : i d , pdd , pimd , physical disability group , and others ( table 1 ) . the last group was small in number and diverse in character and also included responders who provided incomplete information ; thus , data from this group were omitted from analysis hereafter . also in table 1 , the number of clients ( mean persons per facility ) as an index of facility scale , their mean age , and proportion of male gender are shown . their average age ( sd ) was 44.7 ( 9.5 ) years old for the i d group , 53.0 ( 7.6 ) for the pdd group , 48.3 ( 8.4 ) for the pimd group , and 55.3 ( 4.9 ) for the physical disability group . 373 out of the 415 facilities ( about 90% ) experienced cases with difficulties when communicating with the clients ' parents . the percentage was high regardless of the children 's disabilities , being concretely highest in the pdd group ( 96.9% ) and lowest in the physical disability group ( 86.7% ) . the proportion of such parents , out of the total , was reported to be almost none ( 5% or less ) or few ( less than one fourth of the clients ) in most of the facilities ( table 2 ) . it should be noted that in the i d and pdd groups , about 15% of the facilities responded that about half or more of the parents were difficult to communicate with . in all groups , the characteristics of the i d and pdd groups were similar to each other , while the pimd and physical disability groups resembled each other . in other words , in the i d and pdd groups , slightly more difficulties were experienced with the fathers compared to in the pimd and physical disability groups , where they experienced more difficulties with other family members such as the grandparents and siblings compared to the i d and pdd groups . the primary reason for these difficulties was thought to be the parent 's personal condition , such as poor mental health ( table 2 ) . this was especially true for the i d and pdd groups [ (df = 9 ) = 41.9 , p < 0.001 ] . in the pimd and physical disability groups , the parent 's poor socio - economic status and heavy care - giving burden , reflecting the child 's level of disability , were considered to be major factors in approximately one - third of the cases . signs of depression or a depressive state were the most common psychiatric disturbances seen in the parents . among the four groups , the pdd group most commonly experienced cases which showed signs of depression or a depressive state in parents . the proportion of such parents was approximately 70% of the total [ (df = 12 ) = 65.1 , p < 0.001 ] . well - treated cases were only 5% out of the total ; thus , most of these conditions were considered to be not medically well treated . for further analysis , we selected 159 cases where symptoms of depression or a depressive state were reported in a descriptive manner . according to these reports , we asked about the time of the onset , that is , whether it was prior to the birth of the child , when the parent first noticed the child 's disability , or after the child had been fully medically diagnosed . apart from the answer unknown which accounted for approximately half of the cases , prior to the birth of the child was relatively common in the pdd group ( 22.1% ) , whereas after the child had been fully medically diagnosed was relatively common in the pimd ( 23.5% ) and physical disability groups ( 28.6% ) . when a parent suffered from signs of psychiatric disturbances , more than half of the children experienced maltreatment . physical neglect was most common in all groups , accounting for about 50% of the cases . in comparison with the other groups , in the pdd group , the child was likely to be emotionally disturbed and confined indoors ( 32.6% ) , in the pimd group , the child was mostly confined indoors ( 33.3% ) , and in the physical disability group , medical care neglect made the utilization of medical and welfare support impossible ( 50% ) [ (df = 12 ) = 27.6 , p = 0.006 ] . the leading measures were gathering and disclosure of necessary information , discussion among service providers within the facility about ways to deal with the parent , protection of the child by providing a safe environment , making approaches to other family members for cooperation , intensive collaboration with other organizations such as institutions and consultation centers for better community support , and medical support for the parent provided by specialists such as physicians and psychologists . many factors were reported to affect the mental health of parents . the presence or absence of support from other family members ( i.e. , a spouse ) and the existence ( or not ) of a medical condition in the parent were major factors ( table 5 ) . practical use ( or not ) of social support networks , whether the child 's level of disability was severe or not , and parent participation in face - to - face family support groups or not were also thought to be influential factors . in all groups , regardless of the child 's disability , more direct welfare support for the child was considered to be helpful and thus requested to be improved , in order to meet the mental health needs of the parent . besides this , in the physical disability group , family support groups , in the pimd group , support ( including financial support ) for other family members , and in the i d and pdd groups , support for other family members ( mainly siblings and the other parent ) and medical treatment of the parent 's psychiatric disturbance were also emphasized . to fulfill the demanded future role of their facility , respondents expressed the importance of more collaboration with other institutions and consultation centers and the necessity for more special training courses targeting support professionals and recognized the urgent demand for more specialists especially psychologists who can support the mental health needs of both the children and their parents . in the present study , we surveyed institutions and consultation centers for children with developmental disabilities in japan , which play a central role in the social support networks . this facility - based nationwide survey revealed the situation facing the parents of children with i d and other developmental disabilities from the professional caregivers ' point of view . almost all of the facilities experienced difficulties in keeping good relations with the clients ' parents , regardless of the children 's disabilities , and the majority of service providers noted that the proportion of such parents , out of the total , was roughly between 5 and 25% , and still increasing . the primary reason for these difficulties was thought to be the parent 's condition , such as their mental health and background . also , signs of depression or a depressive state were assumed to be the most common psychiatric disturbance seen . these results suggested that thorough consideration of the parents ' mental health needs , especially prevention of depressive conditions and support for parents suffering from them , is urgent - demanded task for facilities . i d , attention - deficit / hyperactivity disorder ( ad / hd ) , and pdd are common developmental disorders , and the latter two are noted to have substantial genetic components . it has been reported that a lifetime prevalence of major mood disorders is higher in parents of children with autism and that the onset for the majority was prior to the birth of their child with autism . family history studies of autism consistently uncovered a large subgroup with a high incidence of major mood disorders among family members , suggesting the two entities are related clinically and genetically [ 19 , 20 ] . the findings of the present survey must be interpreted allowing for the limitations of the use of unconfirmed data reported by professional caregivers ' who come into contact with children as their clients , and their parents on a day - to - day basis . evidently , the service providers ' perspective on parental mental health was consistent with previous reports from western countries on major mood disorders and pdd . in other words , facilities where the clients were diagnosed as having pdd experienced significantly more cases of parents with signs of a depressive state compared to other facilities , and the onset of depression was considered to be prior to the birth of the child in many of the cases . although these professional caregivers were well trained and well experienced , only , one - third were physicians , nurses or psychologists . on account of the limitations of these data , we emphasize confirming the diagnoses of psychiatric disturbances through direct contact with the parents by specialists such as psychiatrists is necessary in future studies , for accurate understanding of the overall problem . in japan , there is no official system to check and document the parents ' condition including their mental health status . parents are not accustomed to being asked about their own mental health , so there is a tendency to avoid discussion on matters concerning themselves . this was the major reason why we asked the professional caregivers about the parents ' condition , not the parents directly , in this study . but still we judged it to be worthwhile at this moment to summarize the professional caregivers ' impression on this issue , since the case in japan has never been described in detail despite of its importance . because the majority of burden for daily care of handicapped children is typically carried by mothers , particular concern is raised for their adaptation . in the past studies , the psychosocial outcomes in mothers were said to be better predicted by psychosocial factors such as more active social life and family resources . recent research focusing on parenting stress illustrated the value of the participation of fathers also . greater marital quality predicted lower parenting stress for both mothers and fathers , while greater social support predicted increased parenting efficacy for fathers . although the relationship between child characteristics and parental well - being has not reached an invariable agreement , the severity of a child 's disability , intellectual functioning , and so forth are generally considered to be risk factors . since the burden for daily care is mainly carried by the mother , similarly in japan , service providers tend to be in contact with the mother more frequently than other family members . this may be partially the reason why service providers experienced difficulties in communicating with mostly the mothers in this study . the presence of support from other family members , such as a spouse , is considered to be a major influential factor for the mental health of parents in japan also . practical use of social support networks such as participation in face - to - face family support groups , more financial support for the household , more direct welfare support for the child , and medical treatment of the parent 's psychiatric disturbance were reported to be helpful and thus requested to be improved , in order to meet the mental health needs of the parent . we would like to emphasize the fact that although the importance of medical treatment for the parents ' psychiatric disturbances was pointed out , it was also realized that these conditions were presently not being medically well treated . this shows that current psychiatric services in japan are not adequate to meet the complex mental health needs of the parents appropriately . to resolve this challenging issue , more intensive collaboration between other child - care facilities and organizations , aiming at the mental health needs of the parents , is demanded . the paucity of trained psychiatrists and other allied professionals also needs to be addressed , and in order to fulfill this , sufficient discussion and research on the establishment of an effective training system for specialists is warranted . providing direct advice for the parents on the utilization of social support networks is a reliable measure that can be immediately taken . training more professionals who can properly deal with the parents ' mental health needs is an urgent matter that must be tackled . overall , the present study is the first nationwide survey that examined the professional caregivers ' perspective on the mental health of parents of children with developmental disabilities , and it underscores the importance of understanding and supporting the parents ' mental health needs , which leads to more appropriate support for children with developmental disabilities .

parents of children with intellectual disabilities and/or physical disabilities are supposed to have an increased risk for parenting stress and psychological distress . we as professional caregivers sometimes experience difficulties in keeping good relations or communicating with the parents . professional workers in 460 institutions and consultation centers throughout japan answered a questionnaire on their clinical experiences . about 90% of the facilities experienced distressed parents , and the parents ' condition such as mental health seemed to influence this . signs of a depressive state were the most common psychiatric disturbances detected , and it was notable in the pervasive developmental disorder group . more welfare support , presence of support groups , support from other family members , and medical treatment of the parents ' problems were considered to be helpful and thus requested to be improved . training more professionals who can properly deal with the parents ' mental health needs is an urgent matter that must be tackled .

in may 2001 , 24 cases of s. typhimurium dt160 salmonellosis were reported in the auckland region compared with an average of four sporadic cases each month with this serotype in the previous 7 months . raw and undercooked egg consumption was commonly reported by the first 10 case - patients interviewed . a case - control study and environmental investigation were undertaken to identify the vehicle of infection and source of the outbreak . recognizing the potential for a widely dispersed foodborne outbreak , we expanded the investigation throughout new zealand . we defined a case as diarrhea ( 3 loose stools in a 24-hour period ) or vomiting after april 28 , 2001 , with a stool specimen positive for s. typhimurium dt160 . patients were excluded if they had a history of contact with another person with culture - confirmed s. typhimurium dt160 infection , or if they had a history of recent overseas travel . each case was matched with two controls found from randomly drawn telephone numbers , matching for neighborhood and age ( < 1 , 14 , 514 , > 14 years ) . the questionnaire covered symptoms ( patients only ) and contact with other symptomatic persons , bird or animal contact , and food consumption in the 3-day period before onset of illness ( cases and controls ) . parents or guardians were interviewed on behalf of children ages < 12 years . stepwise conditional logistic regression analyses were performed , also using sas , to identify the combination of variables that best explained the differences between case - participants and controls . samples from the drinking water supply of case - patients with a history of recent consumption of nonreticulated water were collected and tested for coliforms and s. enterica by using standard methods ( 6 ) . brands of eggs eaten raw within the incubation period were sampled at random from retail displays at the case - patients purchase site . eggshell surfaces and contents were tested with standard methods ( 7 ) . broken or cracked eggs were excluded from analysis . salmonella isolates were serotyped by using the kauffman - white scheme ( 8) and s. typhimurium isolates were phage typed by using the laboratory of enteric pathogens method ( 9 ) . from may to august 2001 , a total of 170 case - patients meeting the case definition were identified . of these , 119 ( 70% ) agreed to participate and were enrolled in the study , along with 235 matched controls . the median age of case - patients was 8 years ( range 4 months to 90 years ) , and 57% were female . the most frequently reported symptoms were diarrhea ( 97% ) , abdominal pain ( 77% ) , excessive tiredness ( 67% ) , and fever ( 66% ) . the median duration of illness was 7 days ( range 144 days ) ; 17 ( 15% ) patients were hospitalized , and none died . case - patients and controls did not differ significantly according to age , sex , immunosuppressive therapy , treatment to reduce gastric acidity , or use of antibiotics . all s. typhimurium dt160 isolates were sensitive to ampicillin , cephalothin , chloramphenicol , ciprofloxacin , co - trimoxazole , gentamicin , streptomycin , sulfonamides , tetracycline , and trimethoprim . four represented different levels of contact with other persons with gastrointestinal illness ( i.e. , within 28 days of illness onset ; within 3 days of onset ; within the household ; or outside the household ) . direct handling of dead wild birds , consumption of fast food , and consumption of food at a large gathering , such as at a party or large barbecue , were also significantly associated with illness . after stepwise regression , contact with a person with gastrointestinal illness in the 28 days before onset of illness in the case - patient ( adjusted odds ratio [ or ] 2.8 ; 95% confidence interval [ ci ] 1.4 to 5.4 ) , exposure to dead wild birds ( adjusted or 10.5 ; 95% ci 2.3 to 47.5 ) , and consumption of fast food ( adjusted or 1.7 ; 95% ci 1.0 to 2.9 ) had independent significant associations with illness . dt , definitive type ; d&v , diarrhea and vomiting ; or , odds ratio ; ci , confidence interval . twelve case - patients throughout new zealand indicated that they had drunk water from nonreticulated and untreated water sources . seven patients used roof - collected rainwater , and one used rainwater plus water from a dam . four of the five patients who had eaten raw eggs could identify the retail brand and outlet of purchase . these four patients had purchased six different brands of eggs from seven different retail outlets . samples for two brands were positive for s. thompson , both from shell surface washings . epidemiologic investigation of an outbreak of s. typhimurium dt160 infection in new from may to august 2001 found that contact with dead wild birds , contact with other persons with gastrointestinal illness , and consumption of fast food were all significantly associated with illness . in addition , s. typhimurium dt160 was found in roof - collected rainwater drunk by five patients . s. typhimurium dt160 had been previously identified as the cause of large numbers of sparrow deaths in new zealand in 2000 , and analysis by pulsed - field gel electrophoresis ( using the method described by barrett et al . and restriction enzyme xbai ) demonstrated that bird and human isolates in 2000 were indistinguishable ( 4 ) . in our study , information was not collected on exposure to environments contaminated by wild bird feces , such as parks and play areas , a fact that may have underestimated the avian contribution to human illness . s. typhimurium dt160 has previously been recognized as a bird pathogen in canada ( 11 ) and in england ( 12 ) . before its emergence in new zealand , the human s. typhimurium dt160 infection had only been reported in the context of a 1979 institutional outbreak in the united kingdom , linked to food contamination by sparrow droppings ( 13 ) . consumption of undisinfected water has previously been identified as a risk factor for salmonellosis linked to bird transmission ( 14 ) . this risk factor was not confirmed by our case - control study , despite the finding of s. typhimurium dt160 in roof - collected rainwater . this discrepancy is probably because case - patients and controls were matched by neighborhood , and types of water sources are usually consistent within neighborhoods . the association of illness with contact with another person with gastrointestinal illness is likely underestimated because secondary salmonellosis cases were excluded . consumption of fast food was associated with illness ; however , no single type of food outlet or food was identified . case - patients were equally likely to have eaten food from chain fast - food restaurants as from family - owned fast - food outlets . consumption of fast food may have occurred in environments contaminated by bird feces , or the foods themselves may have been contaminated , either during production or by infected foodhandlers ( 15 ) . asymptomatic salmonella carriers would not have been excluded from selection as controls , potentially reducing the magnitude of observed associations . recall may have been influenced by delays between exposure and interview , although participants were asked to refer to a memory aid ( personal diary or calendar ) . the investigation successfully excluded a single common source exposure for this outbreak and instead suggested that multiple exposures contribute to s. typhimurium dt160 infections in new zealand . strategies for addressing these exposures include routine treatment of roof - collected rainwater , hygienic disposal of dead birds , and promotion of hand - hygiene protocols and sick foodhandler policies in fast - food outlets . the source of this incursion of s. typhimurium dt160 into new zealand remains unknown : bird isolates have been exclusively from nonmigratory birds , s. typhimurium dt160 has not been identified in neighboring countries in the pacific basin , and early case - patients did not have a history of overseas travel .

an outbreak of human salmonella enterica serotype typhimurium dt160 infection in new zealand was investigated from may to august 2001 . handling of dead wild birds , contact with persons with diarrheal illness , and consumption of fast food were associated with infection . contaminated roof - collected rainwater was also detected .

non - crystallographic symmetry ( ncs ) is common in macromolecular crystals , occurring in about 1/3 or more of structures in the protein data bank [ 2 , 3 , 24 ] . though non - crystallographic symmetry increases the complexity of structure determination , in most cases it yields a distinct advantage in this process because it brings with it information on relationships between density in different parts of the crystal and between coordinates in different parts of the structure [ 4 , 1214 ] . these relationships greatly improve density modification and refinement , two key steps in structure determination . one is simple examination of a model that has more than one copy of a chain . it can be used after molecular replacement has been carried out or after a model has been built . another approach is to find symmetry relationships among subsets of atoms in a heavy - atom or anomalously scattering atom substructure [ 11 , 17 ] . these symmetry relationships often reflect non - crystallographic symmetry , and the presence of the ncs can be checked by comparison of the density in the resulting electron density map at ncs - related positions and orientations . this approach can be used near the beginning of a sad , mad , or other structure determination where a substructure is obtained prior to full structure determination . this approach may be difficult to apply if there are only one or two atoms per ncs copy in the substructure and the ncs does not have point - group symmetry . this is because the relative orientations of the ncs - related parts of the substructure are not known in this case . a third approach that can be applied in cases where proper ncs ( e.g. , a two - fold axis ) is present is to search for locations in a map where nearby points related by proper symmetry have similar density . a fourth and very general approach to finding non - crystallographic symmetry is to find patterns of density that are present in more than one part of an electron density map . used a step - wise procedure of first finding possible c locations with pattern - matching techniques , classifying the locations with rotation - invariant classifiers , and then finding pairs of these locations that have similar patterns of density surrounding them . this method can be fast and effective , but has the potential limitations that the map has to be of high enough quality to locate c positions and that for maximal speed the patterns of density need to be initially represented in a rotation - invariant fashion . here we present an approach to finding non - crystallographic symmetry by directly searching for patterns of density that are present in more than one place in a map . this approach uses superposition of a cut - out region of the map with the remainder of the map , and therefore can make use of all the local features in a map , not just rotation - invariant ones . in this approach , an fft - based convolution search [ 5 , 16 ] is used to find orientations and translations that relate parts of a map to other parts of a map . we find that this is a rapid and accurate way to identify ncs , even in a case where the map is poor and the correlation of ncs - related density is low . the approach used here to identify non - crystallographic symmetry from an electron density map has three basic parts . first , a location in the map is found that is likely to be inside the molecule . this is important as it is the local symmetry of the molecule or molecules in the crystal that are of interest , so considering a region inside the molecule is essential for success . the method used to identify such a location in the map is similar to a method commonly used to distinguish macromolecule from solvent in a map . for each grid point in the electron density map , the local rms variation in electron density is calculated . this is done by simply calculating the standard deviation of the density in the map inside a sphere with radius r ( typically 10 ) , centered at the grid point in question . then the grid point in the map with the highest local standard deviation of density is considered the best candidate for a position inside the macromolecule . additional points likely to be inside the macromolecule can optionally be found by choosing additional grid points with high local variation in density that are well separated ( typically at least 15 ) from previously chosen locations . next , a search for ncs relating the density near the candidate point inside the macromolecule with density elsewhere in the asymmetric unit of the crystal is carried out . to make the calculation rapid , the resolution of the map used for this calculation may be chosen to be lower than the resolution of the original map ( typically a resolution of 4 is used ) . a sphere of density ( typically with a radius of 10 ) is cut out of the map , centered at this position within the molecule . this sphere of density is the object that is to be compared with density everywhere in the map . possible rotations of this sphere of density are sampled ( typically each rotation differing by about 20 degrees from all others ) . for each rotation , the density is transformed to the new orientation , and an fft - based convolution search [ 5 , 16 ] is used to identify non - crystallographic translations that yield a high correlation of density . these rotation / translation pairs that yield high correlation ( typically all those at or above 75 % of the highest value found ) represent a possible set of ncs operators relating the various copies of the molecule in the asymmetric unit of the unit cell . additionally , the candidate point used to identify ncs and the ncs - related points in the map may be considered approximate centers of regions where ncs applies . they are used below as the starting point for identification of the region where ncs operators apply . once a set of candidate ncs operators is obtained , the correlation of ncs - related density is examined in the original map , and the ncs operators are refined . this is done using the same approach as has been applied to ncs operators obtained from heavy - atom positions . first the shape and position of the local region that is repeated by ncs ( the asymmetric unit of ncs ) is found . then the ncs operators are refined to maximize the correlation of density among these ncs - related regions . the region where ncs applies is assumed to contain the point identified in the first step of our procedure that is within the macromolecule . a region with arbitrary shape surrounding this point is then chosen by sequential addition of new boundary points such that the local correlation of ncs - related density at each point in the region is above a threshold ( typically 1/4 of the maximum correlation in the region ) . the local correlation of density is calculated using map grid points within a sphere ( normally with the radius used for identification of the solvent boundary , terwilliger 2000b ) , centered at the point in question . if the final correlation of ncs - related density ( averaged over all pairs of ncs - related points ) is above a threshold ( typically 0.4 ) then the ncs operators are considered to represent actual ncs in the crystal and are used in later stages of structure determination . in cases where the starting map is very poor , this threshold may be decreased , increasing the sensitivity of the procedure but also increasing the probability of finding ncs where none is present . the approach used here to identify non - crystallographic symmetry from an electron density map has three basic parts . first , a location in the map is found that is likely to be inside the molecule . this is important as it is the local symmetry of the molecule or molecules in the crystal that are of interest , so considering a region inside the molecule is essential for success . the method used to identify such a location in the map is similar to a method commonly used to distinguish macromolecule from solvent in a map . for each grid point in the electron density map , the local rms variation in electron density is calculated . this is done by simply calculating the standard deviation of the density in the map inside a sphere with radius r ( typically 10 ) , centered at the grid point in question . then the grid point in the map with the highest local standard deviation of density is considered the best candidate for a position inside the macromolecule . additional points likely to be inside the macromolecule can optionally be found by choosing additional grid points with high local variation in density that are well separated ( typically at least 15 ) from previously chosen locations . next , a search for ncs relating the density near the candidate point inside the macromolecule with density elsewhere in the asymmetric unit of the crystal is carried out . to make the calculation rapid , the resolution of the map used for this calculation may be chosen to be lower than the resolution of the original map ( typically a resolution of 4 is used ) . a sphere of density ( typically with a radius of 10 ) is cut out of the map , centered at this position within the molecule . this sphere of density is the object that is to be compared with density everywhere in the map . possible rotations of this sphere of density are sampled ( typically each rotation differing by about 20 degrees from all others ) . for each rotation , the density is transformed to the new orientation , and an fft - based convolution search [ 5 , 16 ] is used to identify non - crystallographic translations that yield a high correlation of density . these rotation / translation pairs that yield high correlation ( typically all those at or above 75 % of the highest value found ) represent a possible set of ncs operators relating the various copies of the molecule in the asymmetric unit of the unit cell . additionally , the candidate point used to identify ncs and the ncs - related points in the map may be considered approximate centers of regions where ncs applies . they are used below as the starting point for identification of the region where ncs operators apply . once a set of candidate ncs operators is obtained , the correlation of ncs - related density is examined in the original map , and the ncs operators are refined . this is done using the same approach as has been applied to ncs operators obtained from heavy - atom positions . first the shape and position of the local region that is repeated by ncs ( the asymmetric unit of ncs ) is found . then the ncs operators are refined to maximize the correlation of density among these ncs - related regions . the region where ncs applies is assumed to contain the point identified in the first step of our procedure that is within the macromolecule . a region with arbitrary shape surrounding this point is then chosen by sequential addition of new boundary points such that the local correlation of ncs - related density at each point in the region is above a threshold ( typically 1/4 of the maximum correlation in the region ) . the local correlation of density is calculated using map grid points within a sphere ( normally with the radius used for identification of the solvent boundary , terwilliger 2000b ) , centered at the point in question . if the final correlation of ncs - related density ( averaged over all pairs of ncs - related points ) is above a threshold ( typically 0.4 ) then the ncs operators are considered to represent actual ncs in the crystal and are used in later stages of structure determination . in cases where the starting map is very poor , this threshold may be decreased , increasing the sensitivity of the procedure but also increasing the probability of finding ncs where none is present . we applied our procedure to finding non - crystallographic symmetry to a structure where an electron density map of moderate quality could be obtained using mad phasing . this structure ( gere , pdb entry 1fse , ) consists of 6 copies of a protein chain with 74 residues . figure 1a shows a schematic of the protein structure . figure 1b , c illustrates the mad - phased electron density map for this structure for corresponding views of chains c and f , respectively , without any density modification or including any information on ncs . it may be seen that this map is of moderate quality in the region of chain c and rather poorer quality in the region of chain f.fig . 1 a ribbon view of 6 chains of gere in crystal structure pdb entry 1fse . b view of experimental mad - phased , non density - modified electron density at a resolution of 2.7 near chain c of gere . the high density - variation location identified by phenix.guess_molecular_centers is marked , and circle with radius approximately 10 centered at this point illustrates the region that is to be cut out and compared to other density in the map . contours for b d are at 1.5. c as in b , except view is near chain f of gere , shown in grey . d view as in b and c , except that the averaged density based on all 6 ncs - related copies is shown . a created with pymol ; b d created with coot a ribbon view of 6 chains of gere in crystal structure pdb entry 1fse . b view of experimental mad - phased , non density - modified electron density at a resolution of 2.7 near chain c of gere . the high density - variation location identified by phenix.guess_molecular_centers is marked , and circle with radius approximately 10 centered at this point illustrates the region that is to be cut out and compared to other density in the map . contours for b d are at 1.5. c as in b , except view is near chain f of gere , shown in grey . d view as in b and c , except that the averaged density based on all 6 ncs - related copies is shown . a created with pymol ; b d created with coot the phenix tool phenix.find_ncs_from-_density was used to carry out the ncs identification . the density map was examined to find locations where the local variation of density was highest using the method phenix.guess_molecular_centers . it can be seen that this point is well within the region of the map with protein - like characteristics ( relatively connected and high density ) . next , after truncating the resolution of the map to 4 , test density corresponding to a sphere of radius 10 of density centered at the point marked in fig . 1b was cut out ( the region inside the circle in fig . 1b , except for the change in resolution of the map ) . the density within all 10 spherical regions in the 4 map was then compared to this test density using an fft - based method , sampling all possible rotations of the test density on a grid . this density correlation procedure correctly identified all 5 regions that had high similarity to the test density . figure 1c shows one of these related regions of density , the one corresponding to chain f of gere , in the same view as fig . it may be seen that within the 10 sphere the density in fig . 1b , c are similar , though the density near chain f ( fig . the shape of the density becomes less similar , as expected because the protein chains surrounding the chain located outside the sphere in fig . 1b have a different arrangement than those outside the sphere in fig . 1c . 1c and the other 4 locations within the asymmetric unit of the density in this crystal were then refined to identify the region over which correlation of density existed and to optimize the correlation of density among all copies of this repeated density . this process yields as well the transformation matrices describing the relationships among all the ncs - related copies in the asymmetric unit of the crystal . the final average pair - wise correlation of density among the 6 copies was 0.53 . 1b ) the average correlation to other ncs - related regions was 0.56 , while for chain f ( fig . figure 1d shows the average density for all the regions of density that were found to be similar from the same viewpoint as shown in fig . it can be seen that this density has features even more like those expected of a density map of a protein than the individual regions of density ( the tubes of density are better - connected ) . the approach described here for finding ncs in a map can be used in a number of situations . in the phenix crystallographic software , the method phenix.find_ncs_from_density is used during structure solution with phenix.autosol if ncs is expected but can not be found from heavy - atom sites , and it is used during iterative model - building with phenix.autobuild any time it is expected and has not been identified by any other means . the ncs relationships identified in this way can be used as a powerful addition to the information available for density modification [ 4 , 1214 ] . these relationships can also be used in model - building , where an incompletely built chain can be built using a more complete ncs - related copy of the same chain as a template [ 9 , 15 , 20 ] . further they could potentially be used to place copies of entire chains in low - resolution maps . the maps to be analyzed for ncs can be crystallographic maps , maps from electron microscopy , or maps from other sources . the approach described here could also be used to find density that is similar in two different electron density maps . this last application could be useful in automatically finding relationships between molecules in different crystals when carrying out cross - crystal averaging .

the internal symmetry of a macromolecule is both an important aspect of its function and a useful feature in obtaining a structure by x - ray crystallography and other techniques . a method is presented for finding internal symmetry and other non - crystallographic symmetry in a structure based on patterns of density in a density map for that structure . regions in map that are similar are identified by cutting out a sphere of density from a region that has high local variation and using an fft - based correlation search to find other regions that match . the relationships among correlated regions are then refined to maximize their correlations and are found to accurately represent non - crystallographic symmetry in the map .

putting a halt to the rising trend in overweight prevalence has high priority in public health policy all over the developed world . prevention , preferably starting at an early age , is considered as the key strategy to achieve this goal , and considerable effort is put into the design of preventive measures . whereas we know in general terms that overweight is the result of an unbalance between energy intake and energy expenditure , development of effective preventive measures requires a much more specific understanding . specific dietary habits and ( in)activities associated with overweight risk in specific age groups need to be identified and their quantitative contribution to the overweight prevalence in the population needs to be assessed . in addition , we need to know whether the importance of specific behaviors for the development of overweight differs between sub groups in the population . it is well established that children from families with a low socio - economic status and children with overweight parents are at increased risk to develop overweight [ 39 ] . it is therefore particularly important to assess the role of behavioral risk factors specifically in these children , since they are the priority target groups for interventions to prevent and reduce overweight . the aim of this study was to identify specific behavioral risk factors for overweight in young children and to assess their quantitative contribution to the prevalence of overweight in the general population and in high risk sub groups . in a large population - based birth cohort , we prospectively investigated associations between dietary habits , screen time and physical activity when the children were 5 and 7 years old and overweight at the age of 8 years . we also assessed the role of these behavioral factors separately in high risk sub groups : children of mothers with overweight and children of mothers with low education . we estimated the reduction of overweight prevalence that could be achieved if the prevalence of risk factors would be reduced . the study population consisted of children who participated in the dutch prevention and incidence of asthma and mite allergy ( piama ) birth cohort study . the baseline study population consisted of 4146 pregnant women , recruited from the general population in 1996 - 1997 . postal questionnaires , including questions on the child 's lifestyle and health , were sent to the parents during pregnancy , at the child 's ages of 3 and 12 months , and yearly thereafter up to the age of 8 years . at the age of 8 years , the children were invited for a medical examination which included measurement of weight and height . the study protocol was approved by the medical ethics committees of the participating institutes , and all parents gave written informed consent . of the baseline population of 4146 pregnant women , 183 ( 5% ) were lost to follow - up before any data on the child had been collected . parental completed questionnaires from age 3 months to 8 years were available for 3934 , 3817 , 3694 , 3563 , 3518 , 3473 , 3373 , and 3269 children , respectively . when the medical examination of 8-year - old children was planned , 3668 children ( 93% of 3963 ) were still in the study , 3522 children were invited and 2214 participated ( 63% of those invited ) and had their weight and height measured . 146 of the 3668 children were not invited for the medical examination , although they were still participating in the study . in the majority of cases the reason for this was that their families had moved and were now living too far away from the study centers where the medical examination was conducted . complete data on overweight and risk factors were available for 1871 children ( 47% of the 3963 children in the study at birth ) . during the medical examination of the 8-year - old children , weight was measured to 0.1 kg and height to 0.1 cm by trained research staff using calibrated measuring equipment . from the weight and height measurements bmi ( body mass index : weight ( kg)/height ( m ) ) was calculated . overweight and obesity were defined according to age and gender - specific international standards that use cutoff points equivalent to the 25 kg / m and 30 kg / m cut - offs that are commonly used for adults . the term overweight is used for the total group of children who were overweight , including those who were obese . for physical activity , 3 indicators were selected : time spent walking or cycling to school ( 3 categories ) , membership of a sports club ( yes / no ) , and time spent playing outside ( 3 categories ) . screen time ( 4 categories ) included time spent watching television , video 's , or at the computer . for dietary intake , 3 indicators were used : fast food consumption , snack consumption , and soft drink consumption . data on these exposures were obtained from the questionnaires administered at the ages of 5 and 7 years . these questionnaires contained a food frequency questionnaire with 40 different items and 5 answering categories per item ( ranging from never in the last month to on 6 - 7 days per week ) , which was used to construct the dietary exposure variables . for fast food consumption a score was constructed based on the consumption frequencies of chips / french fries and of foods like hamburgers and hot dogs . snack consumption was based on the reported consumption frequencies of 7 different foods / food groups including pie / cake , muffins , candy bars , sweets , chocolate , biscuits , and crisps . soft drink consumption was based on the reported frequencies of consumption of 4 types of soft drinks . each of these variables was based on a number of different items , which differed with respect to their energy content . to be able to add up the different items , total weekly energy intake from each item was calculated based on the reported frequencies , assuming average portion sizes and average energy content per portion . data on maternal education and maternal weight and height were obtained from the questionnaire completed when the child was 1 year old . maternal bmi was calculated from reported weight and height and overweight and was defined as a bmi 25 kg / m . the associations between the exposure variables and overweight at the age of 8 years were analysed by logistic regression , adjusted for gender and birth weight . exposure variables were constructed using the data collected at both the age of 5 and at the age of 7 years in order to obtain a more stable estimate of exposure during the years preceding the measurement of weight and height . walking / cycling to school , playing outside and screen time were recorded in categories , numbered 1 , 2 , and 3 for walking / cycling to school , 1 , 2 , and 3 for playing outside , and 1 , 2 , 3 , and 4 for screen time ( see table 1 ) . the data collected at the ages of 5 and 7 years , were combined by taking the mean of the numbers of the categories reported at the ages of 5 and 7 years respectively . this resulted in ordinal variables with 5 categories for walking / cycling to school ( ranging from never at ages 5 and 7 to > 1/2 an hour per day at both ages ) , 5 categories for playing outside ( ranging from < 1 x per week at ages 5 and 7 to > 3 x per week at both ages ) and 7 categories for screen time ( ranging from < 1/2 an hour per day at ages 5 and 7 to > 2 hours per day at both ages ) . after having checked for nonlinearity fast food , snack and soft drink consumption at age 57 ( the mean of the consumption scores at age 5 and at age 7 ) , were included in the regression analyses as linear variables , but are shown in categories ( tertiles ) in table 1 . overweight ( including obesity ) was used as the outcome measure in the regression analyses . the number of obese children was too small for analyses with fully adjusted regression models , and obesity was therefore not used as outcome measure in these analyses . in previous studies in the piama cohort , breast feeding was investigated in relation to overweight risk and was found to be associated with reduced overweight risk [ 12 , 13 ] . although breastfeeding is not the subject of this study , results on breast feeding are shown in the tables for completeness . in additional analyses , the associations of physical activity and diet with overweight were also assessed separately for the exposures at age 5 and at age 7 . thirteen percent of all the data potentially in the study ( 3963 participants 20 variables = 79260 ) were missing and 2092 children ( 53% ) had a missing value on at least 1 of the variables . if data are not missing completely at random ( mcar ) , complete case analysis may lead to biased results [ 14 , 15 ] . in our dataset several variables ( including maternal education and breast feeding ) were associated with the probability of a subject having one or more missing values . missing data were multiple times imputed , using the multivariate imputation by chained equations ( mice ) procedure [ 16 , 17 ] , that runs under the statistical program r version 2.5.0 . for the multiple imputation we used all the data that were used in the analyses , as well as data on the children 's weight and height that were measured and reported by the parents in the yearly questionnaires . . maternal education and maternal overweight could be confounders of the associations studied but could also be factors in causal pathways . in addition , the analyses were repeated , stratified for maternal education , maternal overweight and gender . differences between the associations observed in the different sub groups were tested by inclusion of interaction terms in the regression models . for the behavioral risk factors that were found to be statistically significantly associated with overweight , adjusted population attributable risk percentages ( par% ) were calculated , using the mantel - haenszel approach , as described by benichou . table 1 shows characteristics of the study population and the prevalence of overweight and obesity in different sub groups . children with incomplete data on either risk factors or on measured weight and height at 8 years were compared to the children with complete data , with respect to a number of characteristics assessed during the first year of the study ( data not shown ) . children with incomplete data had , compared to children with complete data , a relatively high prevalence of low maternal education ( 27.5% versus 19.4% ) and of no breast feeding ( 19.7% versus 15.9% ) . as expected , in the imputed data the prevalence of these characteristics was somewhat higher ( 23.6% and 17.9% , see table 1 ) than in the children with complete data . the prevalence of maternal overweight was similar in children with incomplete data and in children with complete data ( 24.1% and 26.1% , resp . ) . the prevalence of overweight in the children was almost the same in the imputed data and in the complete cases ( 13.8% and 14.4% , resp . ) and the prevalence of obesity was the same in both datasets ( 2.7% ) . table 2 shows the associations between diet , physical activity , screen time and overweight . in the analyses adjusted for gender and birth weight only ( model 1 ) , fast food consumption and screen time , sports club membership , active transport to school , playing outside , snack consumption , and soft drink consumption were not associated with overweight . when all risk factors ( including breast feeding ) were included in the same regression model ( model 2 ) , the associations with overweight became somewhat weaker for most factors , indicating some association between the individual risk factors . the association of fast food consumption with overweight lost statistical significance in this analysis whereas the association with screen time remained statistically significant . surprisingly , an inverse association between snack consumption and overweight was observed . in additional analyses , the analyses shown in table 2 were repeated with the behavioral risk factors measured at age 5 and ( separately ) with those factors measured at age 7 instead of the variables combining the exposures measured at age 5 and age 7 . for the physical activity indicators and for screen time , results of these analyses were similar to those shown in table 2 , showing associations between screen time and overweight ( aor ( 95% ci ) at age 5 : 1.35 ( 1.191.53 ) and aor ( 95% ci ) at age 7 : 1.22 ( 1.081.39 ) ) but not between the physical activity indicators and overweight . snack consumption at age 5 was not associated with overweight at the age of 8 years ( aor ( 95% ci ) : 0.87 ( 0.661.14 ) ) , but there was a significant inverse association between snack consumption at age 7 and overweight at the age of 8 years ( aor ( 95% ci ) 0.70 ( 0.540.91 ) ) . low maternal education and especially maternal overweight were strong predictors of childhood overweight ( see table 2 , model 1 ) . the association between low maternal education and overweight weakened substantially in the model including the behavioral risk factors ( an almost 25% change in the regression coefficient from 0.50 to 0.38 ) , indicating that these specific factors explain at least part of the excess overweight prevalence in children of mothers with low education ( see table 2 , model 3 ) . the association between maternal overweight and overweight in the child weakened only marginally in the model including behavioral risk factors ( 5% change in regression coefficient from 1.07 to 1.02 ) , indicating that the association between maternal overweight and overweight of the child is only to a limited extent mediated by the behavioral factors studied ( see table 2 , model 4 ) . the analyses were repeated after stratification for low maternal education , maternal overweight and gender , respectively . in the sub groups with low maternal education and with maternal overweight children of mothers with a low level education had a higher prevalence of high fast food and snack intakes , of > 2 hours screen time per day and of not being member of a sport club than children of more highly educated mothers ( see table 3 ) . children of overweight mothers had a higher prevalence of high fast food intake and of > 2 hours screen time per day ( at the age of 5 , but not at 7 years ) than children of normal weight mothers ( see table 3 ) . girls had a slightly higher prevalence of overweight and slightly less screen time than boys ( data not shown ) . the results suggest that the associations between screen time and overweight were weaker in children of mothers with overweight or low education than in children of mothers with a normal weight or higher level of education ( table 4 ) . interactions between the exposure variables and low maternal education and maternal overweight were not statistically significant , however . for boys and girls these associations were very similar ( data not shown ) . of the behavioral risk factors studied , only screen time was statistically significantly associated with overweight . based on the prevalence and adjusted odds ratios for screen time > 1 hr per day ( see table 5 ) , we estimated how much screen time contributed to the total prevalence of overweight . table 5 shows the adjusted population attributable risk percentage ( par% ) for a screen time of more than 1 hour per day at age 5 and/or age 7 as compared to less than 1 hour at both ages . of the total overweight prevalence in the low risk sub groups an estimated 17% could be attributed to screen time of > 1 hour per day as compared to 10% in the high risk sub groups . this means that , if all children would reduce their screen time to less than 1 hour per day , the following reductions in overweight prevalence could be achieved : from 18.3% to 16.4% in children of mothers with low education ; from 12.4% to 10.3% in children of mothers with higher education from 25.0% to 22.4% in children of overweight mothers and from 10.0% to 8.3% in children of normal weight mothers . all analyses were conducted in a data set containing only complete cases as well as in the imputed data . the odds ratios ( 95% ci ) for screen time , for example , were 1.47 ( 1.241.75 ) and 1.41 ( 1.171.70 ) in model 1 and model 2 , respectively , in the complete case analysis , compared to 1.45 ( 1.261.66 ) and 1.39 ( 1.211.61 ) in the analysis in the imputed data set . of 8 different potential behavioral risk factors , only screen time showed consistent and significant associations with overweight . in high risk sub groups of the population , screen time was higher than in low risk sub groups , but the associations between screen time and overweight were weaker ( although not significantly weaker ) in the high risk sub groups . important strengths of the study were the prospective design , the large study population , the repeated measurements ( at ages 5 and 7 ) of different behavioral risk factors and the availability of measured ( rather than parental reported ) data on weight and height.however , a number of limitations have to be considered . thirteen percent of our data were missing and 53% of the study population had a missing value on 1 or more of the variables used in the analyses . we used multiple imputation to deal with missing data in our study in order to avoid bias due to selective missing of data and to make more efficient use of the available data . results of the complete case analysis and the analyses in the imputed data sets were similar . information on the behavioral risk factors was obtained from parental completed questionnaires and we therefore need to address the possibility of misreporting . the questions asked ranged from simple matter - of - fact items ( like sports club membership yes / no ) to items that are more difficult to observe and recall accurately ( like the consumption frequency of a range of different food items ) . we expect that recalling or reporting problems will not have substantially influenced the data on sports club membership or transport to school and that any misreporting on these items will not have been systematically associated with overweight development . the data on foods and drinks consumption may well have been influenced by reporting bias . many of the foods included in the dietary variables have a bad reputation in relation to overweight , and selective underreporting by parents of children who were developing overweight may have occurred . another problem with respect to the diet variables is that data were available on the consumption frequencies of food items , but not on portion sizes . estimates of the intakes of fast food , snacks and soft drinks and the ranking of children according to their intakes were therefore based on reported consumption frequencies only , and the possibility of misclassification of food intake can not be excluded . membership of a sports club and active transportation to school were not associated with overweight in this population . the reason that we did not observe the hypothesized associations might be that in young children the duration and intensity of these activities are usually too low to have a sizeable impact on total activity . if this would be confirmed in further studies , it may have potentially important implications for the allocation of budgets for the prevention of overweight in this age group . in older children and adolescents active transport to school and sports club membership we found some evidence for an inverse relation between playing outside and overweight , but the associations were weak and not statistically significant . children who played outside 1 x per week seemed to have a higher overweight risk , but such children were a small minority in the study population . screen time was consistently and significantly associated with overweight and the association was largely independent of other lifestyle factors . a dose - response relationship was observed when it was included as a categorical variable in the regression analysis . our results thus suggest that , among the indicators of active and sedentary behavior that we studied , screen time is the most important risk factor for overweight in the age group studied . this result is in agreement with the conclusion of a recent study in which the evidence was summarized for six different strategies to prevent or treat pediatric overweight . our findings on screen time and overweight risk are also in line with the results of a comprehensive meta - analysis on the subject , which observed a statistically significant relationship between tv viewing and body fatness among children and youth . we estimated that a reduction in overweight prevalence of up to 2 percentage points could be achieved if all children would reduce their screen time to less than 1 hour per day . maximum achievable reduction of prevalence ( marp ) of 1.5 percentage points that was estimated for watching television > 1 hr / day in a study on 5 - 6 year old german children . consumption of soft drinks and fast food at ages 57 was not associated with overweight whereas an unexpected inverse association between snack consumption and overweight was observed . snack consumption at the age of 5 years was not associated with overweight at age 8 , but snack consumption at age 7 was inversely associated with overweight at age 8 . as indicated previously , these results may have been influenced by selective underreporting of snack consumption by parents of children who were becoming overweight . besides selective underreporting , reverse causation may also have played a role , in the sense that parents of children who were becoming overweight really have reduced their children 's consumption of specific foods and drinks . the results on fast food , snack and soft drink intakes seem to indicate that in particular the items included in our snack , variable are the kind of foods that come to parents ' minds when they think of trying to limit excessive weight gain in their child . the selective underreporting and reverse causation that are likely to be present in our dietary data , make it impossible to judge from our results how diet influences the development of overweight . we hypothesize that these mechanisms might play a role also in other epidemiological studies on diet and overweight . whereas , on theoretical grounds , it is hard to imagine that food intake is unrelated to weight status , observational studies have generally not produced consistent evidence for the expected associations [ 20 , 2327 ] . our results do not provide an answer to the question whether consumption of soft drinks , fast food , and snacks causes overweight but seem to indicate that parents may see reduction of snack consumption as a method to counteract the development of overweight . further studies into the strategies that parents themselves use to influence their children 's weight gain might provide insights that could be useful for the design of preventive strategies . low maternal education and maternal overweight were found to be strongly associated with overweight in the child . the association between low maternal education and overweight weakened substantially when the behavioral risk factors were included in the model , indicating that these factors explain part of the association . the behavioral risk factors had little influence on the association between maternal overweight and overweight in the child , indicating that other factors , possibly genetic factors , play a more important role in this association . we repeated the regression analyses in sub groups stratified by maternal weight status and maternal education . whereas in the high risk sub groups , screen time was higher than in low risk sub groups , the associations between screen time and overweight were weaker ( although not statistically significantly ) in these groups , and the population attributable risk percentage was lower than in the low risk sub groups . as the children of mothers with overweight and/or low education are the children at highest risk to develop overweight , this observation is potentially important for the development of preventive strategies and needs to be clarified in future observational as well as intervention studies . reduction of screen time should be part of interventions to prevent or reduce overweight in young children and could result in a reduction of overweight prevalence in the order of 2 percentage points in both high and low risk sub groups . our results also suggest however that interventions aimed to promote sports club membership , active transport to school , and playing outside may have little impact on the prevalence of overweight in this age group . the possibility that , due to reverse causation , associations between food intake and overweight can not be assessed in observational studies , needs further study .

objective . to prospectively identify behavioral risk factors for childhood overweight and to assess their relevance in high risk sub groups ( children of mothers with overweight or low education ) . methods . in the piama birth cohort ( n = 3963 ) , questionnaire data were obtained at ages 5 and 7 on screen time , walking or cycling to school , playing outside , sports club membership , fast food consumption , snack consumption and soft drink consumption . weight and height were measured at age 8 years . results . screen time , but none of the other hypothesized behavioral factors , was associated with overweight ( aor 1.4 ( ci : 1.21.6 ) ) . the adjusted population attributable risk fraction for screen time > 1 hr / day was 10% in the high risk and 17% in the low risk sub groups . conclusion . reduction of screen time to < 1 hr / day could result in a reduction of overweight prevalence in the order of 2 percentage points in both high and low risks sub groups .

the protection of water supplies and water distribution infrastructure such as reservoirs is of great concern for those defending against bioterrorism . the us military is concerned because there exist multiple biological warfare agents ( bwas ) that could be intentionally introduced into the water supply which could have serious implications for military operations as well as civilians located within a theater of operations . to protect potable water supplies , the department of defense ( dod ) seeks to develop a rapid field test that would determine if field supplies of water are safe to drink by the warfighter during a military operation . the us armed services have fielded biological defense systems that are designed to protect against a large scale attack via bwas dispersed through the air . these defensive systems utilize high - efficiency aerosol collectors that impinge the sampled air into a liquid buffer which is tested for bwas using antibody - based handheld assays ( hhas ) . civilian first responders use similar versions of these antibody - based hhas for testing suspicious powder samples suspected to contain bwas . depending on which manufacturer of hha is being tested , the technology can be low cost , requires no electrical power , and requires minimal to no sample preparation to test a sample . it was an hha panel manufactured by the dod that first identified bacillus anthracis in the powder that spilled across a desk in senator daschle 's office in the hart senate office building in 2001 . three years later , a dod hha was again used to identify ricin toxin in the mail room of the dirksen senate office building . these instances demonstrate that , when hha technologies are used appropriately by an experienced operator trained to understand what the results mean , they can be a useful tool . additionally , hhas have proven fast and effective in the laboratory for the detection of francisella tularensis and biotoxins such as staphylococcal enterotoxin b ( seb ) and microcystins . the dod has initiated an effort called the joint chemical biological and radiological agent water monitor ( jcbrawm ) program to develop a system which will allow rapid field testing of drinking water supplies used by military personnel . the effectiveness of hhas in detecting biological agents in the air and in powders has resulted in a desire to test these assays to gauge their effectiveness to protect drinking water supplies used by us warfighters . in the present study , hhas from multiple manufacturers were evaluated for their effectiveness at detecting two specific targets , seb and ricin , in multiple water matrices . the hhas were not modified in any way to optimize for the detection of toxins within water supplies despite the fact that they were initially developed to test for concentrated agent collected from high - efficiency aerosol collectors and impinged into a phosphate buffered saline ( pbs ) solution rather than to test unbuffered water supplies . the ability of the hhas to detect seb and ricin in four different water matrices , representative of waters used by different branches of the armed forces , was evaluated . the four different water matrices were formulated tap water , formulated tap water with 1 mg / l free available chlorine ( fac ) , water treated by reverse osmosis ( ro ) with 1 mg / l fac , and ro water with 2 mg / l total residual bromine . suggested health effect levels as used by the us military for ricin and seb are 455 ng / ml and 4.55 ng / ml , respectively . these minimal health effect levels were derived using the tri service standards for field water and based on an average consumption of 10 liters per day for seven days for a 70 kg service member . therefore , detection of ricin and seb in water at or below the suggested health effect level is desired . both singleplex ( one - line ) and multiplex ( two - line and two - dot ) hha models produced by two manufacturers were evaluated in the present study for the detection of ricin toxin and seb [ 5 , 6 ] . active seb ( toxin technologies , sarasota , fla , usa ) and active ricin whole toxin ( vector laboratories , burlington , on , canada ) were obtained from the dod 's critical reagents program ( crp ) for use in the present study . briefly , approximately 500 ml of astm type i deionized water was added to a one - liter volumetric flask to which each stock solution was added to achieve the desired final concentration and the total volume was brought to one liter with deionized water . after 1520 minutes , the ph was assessed and adjusted if needed ( with minimal volume change ) to 7.67.8 . once prepared , synthetic tap water was stored at 4c with a shelf life of one week . briefly , one gram of calcium hypochlorite was added to twenty milliliters of astm type i deionized water and shaken vigorously for one to two minutes . briefly , approximately five microliters of the chlorine stock solution ( supernatant , not solids ) was added to 100 ml of the formulated tap water or ro water , which was prepared by running local tap water through a commercial ro unit . the fac was measured using a hach dr 2500 spectrophotometer per manufacturer 's instructions , and the fac level was adjusted as needed to achieve the desired fac level of 1 chlorinated water was considered stable for only one day and therefore made fresh daily just prior to use . briefly , 25 ml of bromine resin was transferred to a 500 ml , 0.20 m filter unit ( corning life science , lowell , mass , usa ) . total residual bromine in the collected water was measured using a hach dr 2500 spectrophotometer per manufacturer 's instructions and adjusted as needed to achieve the desired final concentration of 2 mg / l . each water type was prepared fresh daily as described above and spiked separately with each target at desired concentrations . after spiking , 100 l of sample was loaded onto the sample well of the hha . the hha was allowed to develop for 15 minutes prior to reading the result by eye . each challenge concentration was tested a minimum of two independent times with a minimum of three handheld assays per concentration . limits of detection ( lod ) were determined for each hha type in each water matrix and defined as the minimum concentration of toxin that could be detected by eye . samples for circular dichroism ( cd ) were prepared by spiking ro water containing 1 mg / l fac with seb to a final concentration of 2 g / ml . control samples received no chlorine . after 24 hours , sodium thiosulfate ( 0.005% w / v final concentration ; fisher scientific catalog number s446 ) was added to all samples to quench any remaining chlorine prior to analysis by cd . cd experiments employed a jasco model j-810 spectropolarimeter ( jasco analytical instruments , easton , md , usa ) equipped with a ptc-423s peltier thermoelectric temperature control system . cd spectra were obtained for seb at 25c from 260 to 190 nm with a data pitch of 0.5 nm , a band width of 1 nm , a response time of 1 sec , and a scanning speed of 10 nm / min . the cd was carried out on 2 g / ml samples in 1 cm quartz cuvettes and required long - time signal averaging . the concentration of seb was verified for a representative stock solution by uv absorption at 277 nm using an e for seb of 14 . for each sample , the blank - subtracted cd curve for 100 averaged scans was converted to molar ellipticity using a molecular weight of 28,366 daltons and fit with the secondary structure estimation application in jasco 's spectra manager ( version 1.53.01 ) software to obtain the fraction of helix , beta sheet , turn , and random coil secondary structure . the percentages reported here were obtained using the reed reference set for the secondary structure calculations . samples for sds - page were prepared by spiking ro water containing 1 mg / l fac with seb to a final concentration of 2 g / ml . control samples received no chlorine . after one minute , one hour , and 24 hours , sodium thiosulfate ( 0.005% w / v final concentration ) was added to all samples to quench any remaining chlorine prior to analysis by sds - page . reducing gel eletrophoresis was carried out using the mini - protean 2 cell and 1020% tris - hcl pager gold precast gels from lonza ( rockland , me , usa ) . fifty nanograms of seb samples and 1 : 20 diluted mark12 unstained standard ( invitrogen ) were prepared in sds sample buffer , followed by heating at 95c for 5 minutes prior to loading . running buffer was prepared by dilution from 10x tris / glycine / sds buffer ( biorad ) . following electrophoresis , the gel was fixed with 50% methanol and 7% acetic acid ( both v / v ) for a total of 30 minutes . the rapid staining protocol with sypro ruby protein stain ( invitrogen ) was used for the detection of seb . the gel was washed with a solution of 10% methanol and 7% acetic acid ( both v / v ) for 30 minutes and imaged and documented with uv light using syngene ( frederick , md , usa ) . all hhas were evaluated for their performance in formulated tap water , formulated tap water with chlorine , ro water with chlorine , and ro water with bromine for the detection of ricin and seb . each water type was spiked with multiple concentrations of each toxin and loaded onto hhas for testing until the lod was determined for each hha model . it was determined that ricin could be detected down to approximately 1 ng / ml for most hha models in both formulated tap water and formulated tap with chlorine , while exhibiting only slightly higher lods in ro with chlorine for most models . however , in ro with bromine , the lod for ricin increased to 3,000 ng / ml for all models tested . for seb , lods in formulated tap water were low , ranging from 0.9 ng / ml to 12.5 ng / ml . however , in both chlorinated and brominated water , lods increased to 2,500 ng / ml for singleplex hhas and even higher to 5,000 or 10,000 ng / ml for multiplex models . in all cases detection below the suggested health effect level was desired ; however , seb was not detected below the suggested health effect level in chlorinated and brominated water and ricin was not detected below that level in brominated water . due to the increased detection limits observed in chlorinated and brominated water , cd spectroscopy , which assesses the three - dimensional fold of a protein , was performed on samples of untreated seb and seb exposed to chlorine in an effort to evaluate the basis for increased lods ( figure 1 ) . for each sample , the individual cd scans were compared prior to averaging to ascertain the structural stability of the sample . untreated seb at 2 g / ml was determined to contain 14.3% helix , 34.50% sheet , 17.6% turn , and 33.6% random coil ( rmsd < 0.1% ) . the cd spectrum of seb treated with 1 mg / l fac was stable over the time of experimentation . the curve - fitting calculation for chlorine - treated seb resulted in 25.9% helix , 0.0% sheet , 0.0% turn , and 74.1 random coil ( rmsd < 0.1% ) . sds - page was also performed to ascertain the overall effect of chemical reaction with chlorine on the molecular weight of seb . for the treated and untreated seb , 1.5 g of seb was loaded per well . as shown in figure 2 , for untreated seb , the seb was visible on the gel as a band with a molecular weight of approximately 28 kda for all time points tested . for chlorine - treated seb , no band was visible at the same molecular weight as untreated seb for any of the time points tested . ricin and seb are considered potential biological threat agents as both can cause illness either by inhalation or ingestion . seb could potentially be used to attack low - volume water supplies ; however , due to the fact that ricin is less toxic by oral ingestion , it may be a less likely threat to drinking water . however , detection of both agents in water is still a concern and technologies capable of detection in water must be investigated . performance of hhas for air and surfaces is well known ; however , the use of hhas for detection of agents in water is less recognized . therefore , the application of hhas for detection in water was assessed in this study . in formulated tap water , detection of seb and ricin was achieved at or below the suggested health effect levels for each model of hha in almost every case . however , the presence of disinfectants proved problematic with regard to detecting either seb or ricin using all models of hhas . the increased lods observed when disinfectants were present are believed to be due to the degradation of the target or toxins by chlorine and bromine . therefore , it is likely that protein toxins would also be affected by these strong oxidants . in fact , sodium hypochlorite is known to completely inactivate seb and ricin toxin [ 11 , 12 ] . therefore , when exposed to chlorine or bromine , protein toxins could become damaged or denatured causing antibodies present in an antibody - based assay , such as hhas , to no longer bind to the altered antigen as effectively . this decrease in binding efficiency could yield higher lods for the target , such as those observed in the present evaluation . to address this issue , cd spectroscopy and sds - page the cd results for untreated seb were similar to those obtained by singh et al . for seb at 0.5 - 0.6 mg / ml in 10 mm sodium phosphate . the cd results for chlorine - treated seb indicate a significant change in three - dimensional fold resulting in less well - defined structure for the chlorine - treated seb relative to the untreated seb . based on curve - fitting calculations , the percentage of random coil more than doubled upon treatment with chlorine . the secondary structure percentages obtained from the cd curve - fitting routine may well represent an average of multiple seb - based species in solution , which would be consistent with the sds - page results . the absence of bands for the chlorine - treated seb could occur if the seb was cleaved into smaller peptides that diffuse out of the gel or if multiple higher molecular weight species are formed , with no single species being concentrated enough to be visualized on the gel . in either of these cases , chiral components would be present in the sample and would lead to a measureable cd spectrum . the cd and page results together suggest a major change in seb structure that may result in multiple seb - based antigens , the majority of which are less easily recognized and not bound as effectively as untreated seb by the antibody present on the membrane of the assay . the decreased binding capacity for the seb - based antigen(s ) would account for the increased detection limits seen for seb in chlorinated water . these results support the idea that increased lods in disinfected water are caused by disruption in antigen - antibody binding due to degradation of the antigen . in addition to the detection studies reported herein , currently studies are being performed to address whether both ricin and seb remain active , or toxic , after exposure to chlorine and bromine . however , regardless of whether activity remains , the ability to detect the presence of each toxin in disinfected water supplies is important . presence of these agents could indicate a potential compromise or attack on the water supply , thereby revealing security risks as well as potential health risks . additionally , it was noted in the present study that chlorine did not impact detection levels of ricin as significantly as detection levels of seb under the test conditions employed . additional exposure time may be required in order for ricin detection to be affected in the same manner as that observed for seb in chlorinated water . this could be due to a difference in protein structure or stability in chlorinated water between the two toxins . further tests allowing for prolonged incubation of ricin in chlorinated water prior to detection could address this issue .

development of a rapid field test is needed capable of determining if field supplies of water are safe to drink by the warfighter during a military operation . the present study sought to assess the effectiveness of handheld assays ( hhas ) in detecting ricin and staphylococcal enterotoxin b ( seb ) in water . performance of hhas was evaluated in formulated tap water with and without chlorine , reverse osmosis water ( ro ) with chlorine , and ro with bromine . each matrix was prepared , spiked with ricin or seb at multiple concentrations , and then loaded onto hhas . hhas were allowed to develop and then read visually . limits of detection ( lod ) were determined for all hhas in each water type . both ricin and seb were detected by hhas in formulated tap water at or below the suggested health effect levels of 455 ng / ml and 4.55 ng / ml , respectively . however , in brominated or chlorinated waters , lods for seb increased to approximately 2,500 ng / ml . lods for ricin increased in chlorinated water , but still remained below the suggested health effect level . in brominated water , the lod for ricin increased to approximately 2,500 ng / ml . in conclusion , the hhas tested were less effective at detecting ricin and seb in disinfected water , as currently configured .

osteosarcoma is a common primary malignant bone tumor with complicated pathogenesis and frequent distal metastasis , thus causing higher mortality rates , especially in the teenagers . epidemiological surveys revealed that nearly 40% of osteosarcoma patients died from misdiagnosis and/or early tumor metastasis , especially toward the lung tissue [ 24 ] . therefore , it is of critical importance to find a simple and effective biological marker for osteosarcoma , in order to make early diagnosis . current studies have revealed the role of gene mutation in the proliferation of tumor cells . cell apoptosis - related genes , including vascular epithelial growth factor ( vegf ) and microrna ( mir)-96 , were found to be closely correlated with the occurrence and prognosis of osteosarcoma . p15 , a novel tumor - suppressor gene , has been reported to play an important regulatory role in the progression of various malignant tumors , such as pulmonary cancer , nasopharynx cancer , and kidney cancer . the fragment loss , point mutation , or methylation of p15 gene may all lead to its abnormal tumor - suppressing function , thus facilitating the malignancy and metastasis of tumor cells . previous studies have been performed on the pathogenesis and related mechanisms of pulmonary metastasis of osteosarcoma , but the involvement of p15 gene has not been reported . as it is of critical importance for early diagnosis of osteosarcoma to discover a simple but effective biological tumor marker , this study aimed to describe the mechanism of p15 gene mutation in the occurrence and lung metastasis of osteosarcoma in a rat model , as previously described . this study may provide further evidence for developing methods in clinical diagnosis of malignant osteosarcoma . a total of 60 sd rats ( both males and females , ages 4~5 weeks , body weight 150~170 g ) from the laboratory animal center of harbin medical university , were randomly divided into control and model groups ( n=30 each ) . the osteosarcoma model was prepared as previously reported . in brief , after general anesthesia , 0.5 ml umr-106 cell suspensions ( atcc , us , 110 per ml ) , which were cultivated in dmed containing 10% fetal bovine serum ( gibco , usa ) , were subcutaneously injected into the right forelimb of each rat in the model group . rats were used for all experiments , and all procedures were approved by the animal ethics committee of harbin medical university . after 0 , 1 , 2 , 4 , and 6 weeks of the inoculation , 5 rats from model or control groups were sacrificed to separate osteosarcoma and pulmonary tissues . the tumor volume was approximated as : ( short diameter long diameter)/2 , as reported elsewhere . the isolated pulmonary tissue was fixed in 10% neutral buffered formalin , followed by xylene dewaxing and gradient ethanol ( 100% , 95% , 80% , and 70% ) hydration . we added 20 g / ml protease k without dnase to incubate at 37c for 15 min , and then it was boiled in citric acid buffer ( 0.01 m , ph = 6.0 ) for 20 min after being washed 3 times ( 5 min each time ) with pbs until naturally cooling down to room temperature . pbs containing 5% normal goat sera was used to block at 37 for 10 min , then the sera was decanted and added to primary antibody . after being washed with pbs for 3 times , secondary antibody containing 1% labeled biotin was applied for incubation at 37c for 15 min . after being washed with pbs for 3 times , 3% horseradish peroxidase - labeled avidin working solution was used for incubation at 37c for15 min . dab staining was conducted after being washed with pbs 3 times , and then it was washed with tap water , following hematoxylin re - dyeing , drying , and mounting . sp - ihc staining was used to show the positive expression of tumors cells and metastatic conditions . for a quantitative analysis , 10 randomly selected fields we carefully followed the instructions in the sp - ihc detection kit ( shanghai yanhui bio - tech , china ) . peripheral blood samples ( 0.5 ml ) were collected from all animals at the same time points , as detailed above . after centrifugation for 10 min at 3000 g , lower blood clots were lysed in pbs buffered - lysing reagents . total rna were then extracted from blood cells for further synthesis of cdna , which was then used as the template in pcr assay for the analysis of 2 exon of p15 gene . using specific designed primers ( p15-f , 5-tggct ctgac cactc tgc-3 ; p15-r , 5-agcga attcg ggtgg gaa-3 ) and internal reference gene -actin ( forward , 5-gaaac tacgt tcaag cgatc-3 ; reverse , 5-ctaga agcat ttgcg gtgga-3 ) , pcr was performed in the following mixture : 1 l 10 reaction buffer , 70 m dntps ( 4 ) , 100 ng dna templates , 30 ng primers , 100 ng taq dna polymerase , plus ddh2o ( up to 10 l ) . the reaction parameter was : pre - nature at 95c for 5 min , followed by 40 repeating cycles each containing 95c denature for 30 s , 60c annealing for 30 s , and 72c elongation for 30 s. the reaction was ended by final elongation at 72c for 8 min . agarose gel ( 1% ) electrophoresis was used to visualize pcr fragments lengths , along with -actin - controlled fragments ( 232 bp ) . spss 20.0 software package was used to analyze all collected data , which are presented as meanstandard deviation ( sd ) . between - group comparison a total of 60 sd rats ( both males and females , ages 4~5 weeks , body weight 150~170 g ) from the laboratory animal center of harbin medical university , were randomly divided into control and model groups ( n=30 each ) . the osteosarcoma model was prepared as previously reported . in brief , after general anesthesia , 0.5 ml umr-106 cell suspensions ( atcc , us , 110 per ml ) , which were cultivated in dmed containing 10% fetal bovine serum ( gibco , usa ) , were subcutaneously injected into the right forelimb of each rat in the model group . rats were used for all experiments , and all procedures were approved by the animal ethics committee of harbin medical university . after 0 , 1 , 2 , 4 , and 6 weeks of the inoculation , 5 rats from model or control groups were sacrificed to separate osteosarcoma and pulmonary tissues . morphological changes were observed , along with the measurement of tumor size . the tumor volume was approximated as : ( short diameter long diameter)/2 , as reported elsewhere . the isolated pulmonary tissue was fixed in 10% neutral buffered formalin , followed by xylene dewaxing and gradient ethanol ( 100% , 95% , 80% , and 70% ) hydration . we added 20 g / ml protease k without dnase to incubate at 37c for 15 min , and then it was boiled in citric acid buffer ( 0.01 m , ph = 6.0 ) for 20 min after being washed 3 times ( 5 min each time ) with pbs until naturally cooling down to room temperature . pbs containing 5% normal goat sera was used to block at 37 for 10 min , then the sera was decanted and added to primary antibody . after being washed with pbs for 3 times , secondary antibody containing 1% labeled biotin was applied for incubation at 37c for 15 min . after being washed with pbs for 3 times , 3% horseradish peroxidase - labeled avidin working solution was used for incubation at 37c for15 min . dab staining was conducted after being washed with pbs 3 times , and then it was washed with tap water , following hematoxylin re - dyeing , drying , and mounting . sp - ihc staining was used to show the positive expression of tumors cells and metastatic conditions . for a quantitative analysis , 10 randomly selected fields we carefully followed the instructions in the sp - ihc detection kit ( shanghai yanhui bio - tech , china ) . peripheral blood samples ( 0.5 ml ) were collected from all animals at the same time points , as detailed above . after centrifugation for 10 min at 3000 g , lower blood clots were lysed in pbs buffered - lysing reagents . total rna were then extracted from blood cells for further synthesis of cdna , which was then used as the template in pcr assay for the analysis of 2 exon of p15 gene . using specific designed primers ( p15-f , 5-tggct ctgac cactc tgc-3 ; p15-r , 5-agcga attcg ggtgg gaa-3 ) and internal reference gene -actin ( forward , 5-gaaac tacgt tcaag cgatc-3 ; reverse , 5-ctaga agcat ttgcg gtgga-3 ) , pcr was performed in the following mixture : 1 l 10 reaction buffer , 70 m dntps ( 4 ) , 100 ng dna templates , 30 ng primers , 100 ng taq dna polymerase , plus ddh2o ( up to 10 l ) . the reaction parameter was : pre - nature at 95c for 5 min , followed by 40 repeating cycles each containing 95c denature for 30 s , 60c annealing for 30 s , and 72c elongation for 30 s. the reaction was ended by final elongation at 72c for 8 min . agarose gel ( 1% ) electrophoresis was used to visualize pcr fragments lengths , along with -actin - controlled fragments ( 232 bp ) . spss 20.0 software package was used to analyze all collected data , which are presented as meanstandard deviation ( sd ) . between - group comparison one week after the inoculation of tumor cells , there were small tumor lesions underneath the skin around the injection sites . as shown in table 1 , the average size of these tumors was 4.000.18 ( in mm ) . abnormally enlarged microvessels can be observed on the surface and in the peripheral muscles of the tumor , even by the naked eye . on the second week , the tumor size increased to 4.000.25 ( in mm ) , with severe edema on the surface and in peripheral tissues . on the fourth and sixth weeks after injection , tumor size continued to grow ( 17.000.23 and 21.000.31 , in mm respectively ) but more slowly . a longitudinal dissection of the tumor revealed white and hard tissues with centralized necrosis . before tumor cell inoculation after the injection of osteosarcoma cells , model rats had significantly elevated tumor cell percentage with the elongation of time . on the first week , most of osteosarcoma and pulmonary tissues showed negative staining for tumor cells . from week 2 , positive staining cells began to occur in model rats but not in control rats . on week 6 , the tumor positive rates were as high as 85% and 72% in primary and pulmonary tissues , respectively ( table 2 , figure 1 ) . in a total of 25 peripheral blood samples in osteosarcoma rats , 18 of them ( 72% ) showed mutation in the 2 exon of p15 gene , while control rats had normal gene isoforms ( table 3 , figure 2 ) . in examining rats from different time points further studies also revealed significant correlation between p15 gene mutation and occurrence of osteosarcoma ( r=0.998 , p<0.05 ) . one week after the inoculation of tumor cells , there were small tumor lesions underneath the skin around the injection sites . as shown in table 1 , the average size of these tumors was 4.000.18 ( in mm ) . abnormally enlarged microvessels can be observed on the surface and in the peripheral muscles of the tumor , even by the naked eye . on the second week , the tumor size increased to 4.000.25 ( in mm ) , with severe edema on the surface and in peripheral tissues . on the fourth and sixth weeks after injection , tumor size continued to grow ( 17.000.23 and 21.000.31 , in mm respectively ) but more slowly . after the injection of osteosarcoma cells , model rats had significantly elevated tumor cell percentage with the elongation of time . on the first week , most of osteosarcoma and pulmonary tissues showed negative staining for tumor cells . from week 2 , positive staining cells began to occur in model rats but not in control rats . on week 6 , the tumor positive rates were as high as 85% and 72% in primary and pulmonary tissues , respectively ( table 2 , figure 1 ) . in a total of 25 peripheral blood samples in osteosarcoma rats , 18 of them ( 72% ) showed mutation in the 2 exon of p15 gene , while control rats had normal gene isoforms ( table 3 , figure 2 ) . in examining rats from different time points further studies also revealed significant correlation between p15 gene mutation and occurrence of osteosarcoma ( r=0.998 , p<0.05 ) . due to its unfavorable prognosis , early diagnosis and treatment is of critical importance for preventing metastasis of osteosarcoma lesions . the clinical features of osteosarcoma mainly include bone pain , hypercalcinemia , and osteolysis metastasis . the complication of other organ / tissues , such as the lung , may indicate the metastasis of tumors . the proliferation of primary tumor lesions causes the detachment of tumor cells , which penetrate vessel walls to enter the general lymph or blood circulation system for the invasion of distal tissues , where tumor seeds investigation of the mechanisms underlying the progression and metastasis of osteosarcoma is a popular research topic . recent studies revealed complicated genetic processes regulating the tumor metastasis , as a hot - spot located in chromosome 9p21 - 22 region affects gene fragment loss or transcriptional abnormalities in various malignant tumors [ 1719 ] . p15 gene , as a representative gene in this region , has been shown to have mutants and expression down - regulation in kidney and bladder cancer . in approximately 23% of cancer cells of it has been indicated that single - base mutation occurs in 67 , 119 , and 135 codons of p15 gene . due to the methylation of p15 gene , hematopoietic progenitor cells lose the regulation of tgf--mediated growth inhibition , which in turn increased the incidence of malignant bone tumors . recent evidence suggests the complexity of the abnormal regulation of p15 gene involved in bone tumor metastasis . however , the relationship between p15 gene and lung metastasis of osteosarcoma remains to be determined . this study utilized umr-106 cell line , which is a classical osteosarcoma cell line induced by radioactive phosphorous isotope ( 32-p ) , to generate an osteosarcoma rat model by subcutaneous injection . the observation of tumor size and pulmonary tissue damage , along with p15 gene expression at different time points after the injection , showed normal tissue structures and almost no tumor cells in the control group . after the inoculation , the overall survival rate of rats was 83.3% , in which about 48% of cases had pulmonary metastasis , suggesting the effectiveness and usefulness of this model . the tumor cell expression maintained at minimal level with lower mutation rate of p15 gene at 1 week after the inoculation . at 26 weeks , however , abnormal tumor - like structures began to occur in the pulmonary tissues in the model group , along with elevated p15 gene mutation rates over time . the tumor - positive cell percentage in pulmonary tissues increased from 10% to more than 72% at the final time point ( 6 weeks ) , suggesting the occurrence of lung metastasis , as consistent with previous reports . other studies demonstrated the suppressed incidence of esophagus cancer and pharynx squamous carcinoma by the inhibition of p15 gene methylation . furthermore , p15 expression level was higher in tongue squamous carcinoma without lymph node metastasis , compared to those patients with metastasis , who also had unfavorable prognosis . all these studies point to the importance of p15 gene in inhibiting tumor growth and invasion , both of which are consistent with our results in osteosarcoma . the proliferation of cells is highly dependent on the complete cell cycle , whose disruption or dysfunction may directly affect the tumor growth . p15 gene , as an inhibitory factor in cell - cycle - dependent kinase ( cdk ) , exerts its function mainly on g1 phase of the cell cycle via modulating the n - terminal protein expression of multiple tumor - suppressing factors , thereby inhibiting the abnormal growth of various malignant tumor cells . the correlation between p15 gene mutation in peripheral blood cells and the growth curve of osteosarcoma lesion suggests the invasion of tumor cells via blood circulation into distal sites , in addition to the facilitating role of mutant p15 in the pulmonary metastasis of osteosarcoma . our results indicate the close correlation between p15 gene mutation and growth of osteosarcoma , and the potentially direct involvement of p15 gene in pulmonary metastasis . our results suggest the potency of p15 mutant genes as a novel biological marker in early diagnosis of osteosarcoma in clinics , although its detailed molecular mechanism in progression and pulmonary metastasis require further study . molecular biology research is still required to elucidate the mechanism of mutation location of p15 gene , as well as its role in the metastasis of osteosarcoma to the lung .

backgroundas a type of primary malignant bone tumor , osteosarcoma has high incidence and poor prognosis , and is predisposed for pulmonary metastasis . the abnormal expression of p15 gene directly participates in the invasion of various cancers . therefore , this study investigated the gene mutation of p15 in both primary lesion and pulmonary metastasis lesion of osteosarcoma in a rat model , in an attempt to elucidate the value of p15 gene as a biological marker.material/methodsa total of 60 sd rats were randomly divided into 2 groups . model rats had injection of osteosarcoma umr-106 cells ( 5106 ) inoculated underneath the right forelimb skin , while control rats received saline injection instead . six rats were sacrificed after 0 , 1 , 2 , 4 , and 6 weeks of the inoculation . tissue samples from inoculation sites and lungs were extracted for measuring the tumor size . sp immunohistochemical ( ihc ) staining was used to detect the positive expression rate , while p15 gene mutation was detected by pcr method.resultswith the elongation of inoculation time , tumor size was significantly increased ( p<0.05 ) . the positive expression rates in both primary and pulmonary metastasis lesions were also significantly elevated ( p<0.05 ) . the occurrence rate of p15 gene mutation in model rats was significantly elevated and showed a correlation with the tumor formation ( r=0.998 , p<0.05).conclusionsthe p15 gene mutation was significantly correlated with osteosarcoma formation and metastasis towards the pulmonary tissue , suggesting its potency as a novel biological marker for early diagnosis of osteosarcoma .

the term was used by hallopeau in 1889 and is derived from the greek ; thrix - hair , tillein - pull out and mania - madness . scalp is the most common site for pulling hair although other hair bearing areas may be involved . in less severe forms diagnosis is mainly by history and clinical examination , nevertheless , sometimes it is very difficult to differentiate it from other causes of noncicatricial alopecia . trichoscopy , dermoscopy hair and scalp , is a noninvasive technique for differential diagnosis of various hair and scalp diseases . here authors evaluated trichoscopic patterns in ttm and authors believe that these patterns are specific to ttm , which can aide in early diagnosis of this chronic condition . this study was carried out on 10 patients attending department of dermatology in a tertiary hospital attached to s. nijalingappa medical college at bagalkot , south india between january 2014 and july 2014 . ten patients with clinical features of ttm were subjected for a complete history and dermatological examination . demographic data such as age and gender and clinical variables in terms of site of lesions and disease duration were documented . dermlite 3 dermoscope ( 10 magnifications ) with both polarized and nonpolarized lights was employed in the study . sony camera ( digital , 14 mega pixels ) was attached to save the images . initially , ultrasound gel was applied either on the faceplate of dermoscopy or on the skin lesions and then lesions were observed through the eyepiece of dermoscopy . although polarized dermoscopy was employed , ultrasound gel was applied for clarity of images and to lessen distortions associated with light . this study was carried out on 10 patients attending department of dermatology in a tertiary hospital attached to s. nijalingappa medical college at bagalkot , south india between january 2014 and july 2014 . ten patients with clinical features of ttm were subjected for a complete history and dermatological examination . demographic data such as age and gender and clinical variables in terms of site of lesions and disease duration were documented . dermlite 3 dermoscope ( 10 magnifications ) with both polarized and nonpolarized lights was employed in the study . however , only polarized light version was used in our study . sony camera ( digital , 14 mega pixels ) was attached to save the images . initially , ultrasound gel was applied either on the faceplate of dermoscopy or on the skin lesions and then lesions were observed through the eyepiece of dermoscopy . although polarized dermoscopy was employed , ultrasound gel was applied for clarity of images and to lessen distortions associated with light . mean age of the patients was 34 years ( minimum age 13 years and maximum age 55 years ) . mean duration of disease was 19 months ( minimum 2 months and maximum 36 months ) . most common symptom was patchy loss of hair over the scalp ( 100% ) especially in the frontal area [ figure 1 ] and one patient ( 10% ) had tonsure pattern of hair loss . four patients agreed to knowingly pulling of hair , and one patient ( 10% ) gave a history of eating them ( trichophagia ) . the most common trichoscopic pattern observed in all patients was decreased hair density and hairs broken at different lengths ( 100% ) [ figure 2 ] . excoriations are also seen on the forehead trichoscopy showing decreased hair density , trichoptilosis ( yellow arrows ) , tulip hairs ( red arrows ) and perifollicular whitish areas ( red stars ) short hair with trichoptilosis ( split ends ) [ figures 25 ] , irregular coiled hairs [ figure 3 ] and upright re - growing hairs were demonstrated in 8 patients ( 80% ) each . the novel diagnostic signs like black dots and flame hair [ figure 4 ] , v - sign [ figure 3 ] , follicular hemorrhages [ figure 5 ] were observed in 3 patients ( 30% ) each . tulip hair and hair powder [ figure 6 ] patterns were observed in 1 ( 10% ) patient each . all patients ( 100% ) had the noninflammatory alopecia with follicles having distorted and collapsed inner root sheath [ figure 7 ] in histopathology . trichoscopy showing coiled hairs ( red arrows ) and v - hairs or v - sign ( yellow arrows ) trichoscopy showing flame hairs ( red arrows ) and black dots ( yellow arrows ) trichoscopy showing decreased hair density , follicular hemorrhage ( black arrow ) and trichoptilosis ( red arrows ) trichoscopy showing tulip hairs ( red arrows ) and hair powder ( yellow arrows ) histopathology showing diffuse , noninflammatory alopecia , missing hair shafts ( black stars ) and inward collapse of outer root sheath ( black arrows ) . few unaffected follicles are also seen ( yellow arrows ) ( h and e , 4 ) histopathology showing an irregular , deformed and pigmented shaft characteristic of trichomalacia ( h and e , 40 ) trichotillomania is a chronic impulse control disorder characterized by repetitive hair pulling resulting in alopecia . in young children , it is usually a habit that resolves spontaneously or with minimal treatment . in older age groups , adolescents and adults , seen predominantly in females and evidence of some form of psychological or behavioral stress is often apparent . the favorite site is the easily reached fronto - parietal region of the scalp followed by eyelashes , eyebrows , pubic hair , body hair and facial hair . this results in patchy loss of hair , often in a bizarre or angular pattern , in which the hairs are twisted and broken at various distances from the clinically normal scalp . a hairball , trichobezoar , is a rare accompaniment of ttm in those who also eat the plucked hair ( trichophagia ) . in our study , most of the patients belonged to adolescent , and middle age group and most of them were females . all of them presented with patchy hair loss over the scalp , none of the patients gave a history of hair loss in other areas . only one patient had a history of eating of pulled hairs , but there were no signs and symptoms of trichobezoar in her . the diagnosis of ttm is often difficult as other hair loss diseases appear similar in clinical manifestations . this difficulty is encountered especially with alopecia areata ( aa ) . as ttm often coexists with aa trichoscopy is a novel diagnostic technique , both simple and noninvasive , can be used as a handy bed side tool for diagnosing common hair and scalp disorder . several common trichoscopy features of ttm were identified . decreased hair density , hairs broken at different lengths , short hairs with trichoptilosis ( " split ends " ) are described in the literature as diagnostic of ttm . authors observed these findings in all the patients , and this is due to irregular and repetitive pulling of hairs leading to damage to the cuticle . irregular coiled hairs and upright re - growing hairs are also reported to be seen in ttm . in this study , these findings were demonstrated in 80% of patients . coiled hair results from hair shaft fracture and coiling of the remaining proximal part which is fixed to the scalp . black dots are believed to be remnants of hair shafts arising from tapering hairs , broken hairs , and bent hairs . in a study conducted by shim et al . rakowska et al . observed that black dots tend to be uniform in size and shape in aa , whereas in ttm and tinea capitis they are variable in diameter and round , oval , irregular in shape . it presents as a red dot corresponding to follicular ostia capped or stuffed with the blood clot and suggests a history of traumatic forced plucking . recently , flame hairs , v - sign , tulip hairs , and hair powder are described , and authors claim these signs are specific only for ttm . flame hairs are semi - transparent , wavy and cone - shaped hair residues , which develop as a result of severe mechanical hair pulling and shredding . v - sign is created when two or more hairs emerge from one follicular ostium , which are pulled simultaneously and break at the same length above scalp surface . when hair shafts are almost totally damaged by mechanical manipulation , only a sprinkled hair residue is visible . this finding is referred to as hair powder . in the present study , these trichoscopic patterns were demonstrated in variable frequency . in aa , the common trichoscopic findings are yellow dots , uniform black dots , broken hair , trichoptilosis , upright re - growing hair , and vellus hair . as some of the trichoscopic features are overlapping in ttm and aa , histopathology plays a corroborative role in definitive diagnosis . the histopathological features of ttm include empty follicles , incomplete disrupted follicular anatomy , trichomalacia , and pigment casts without significant inflammation . collapsed inner root sheath and missing hair shaft indicate the extraction of the hair follicle due to forcible hair pulling . partially extracted and distorted root sheath material with fragments of pigment can be identified as the germinative follicular epithelium may produce abnormally formed , diminished and distorted hair shaft with irregular pigmentation as a result of trauma . a new trichoscopic pattern was observed by the authors in one patient , in the form of perifollicular whitish areas [ figure 2 ] . authors believe these whitish areas represent hyperkeratosis that is due to perifollicular damage as a result of repeated hair pulling trichotillomania is often chronic and difficult to treat . patients may attempt to disguise the condition due to its social implications . hence trichoscopy , being a noninvasive and in vivo diagnostic technique , can be utilized in this condition as it plays a vital role in the diagnosis of this condition by demonstrating specific trichoscopic patterns . further studies on trichoscopic patterns correlating with duration of disease and histopathology of ttm are suggested .

introduction : trichotillomania ( ttm ) is characterized by patchy alopecia of hair bearing areas . this is because of compulsive urge to pull the hair . scalp is the most common site for pulling hair . usually , patients may have only small areas of baldness , in severe forms , tonsure pattern of baldness is observed . diagnosis is by history and clinical examination . however , it is difficult to differentiate from other causes of noncicatricial alopecia . here , authors observed trichoscopic patterns and evaluated their importance in the diagnosis of ttm.materials and methods : this study was conducted in s. nijalingappa medical college , bagalkot , from january 2014 to july 2014 . ten patients with clinically suspected ttm were included in the study . informed consent was taken and ethical clearance was obtained . dermlite3 dermoscope was used with sony camera attachment to save the images . histopathological examination was conducted in all the patients to confirm the diagnosis.results:ten patients were included in the study . mean age of the patients was 34 years . most common symptom was patchy loss of hair in the frontal area ( 100% ) . common trichoscopic feature was decreased hair density and broken hairs . trichoptilosis ( split ends ) and irregular coiled hairs were seen in 80% patients . novel diagnostic signs like black dots , flame hair , v - sign , follicular hemorrhages , were seen in 30% each . tulip hair and hair powder were observed in 10% of patients . all patients had the noninflammatory alopecia with distorted and collapsed inner root sheath in histopathology.conclusion:trichotillomania is often chronic and difficult to treat . hence , early diagnosis and treatment is necessary . authors believe that the trichoscopy plays a vital role in the diagnosis of this condition by demonstrating specific trichoscopic patterns .

the prevalence of syphilis in the normal population has decreased significantly since 1977 in korea ( 1 , 2 ) . however , in western countries the incidence of syphilis has risen , mainly in the 20 - 24 age group and its clinical manifestations have been various ( 3 ) . sexual activity has become more acceptable in our culture recently , initiating various forms of sex , and as a result , many different clinical conditions of sexually transmitted disease ( std ) have appeared . it is usually asymptomatic and less frequently presents as proctitis , ulcer and pseudotumors ( 3 , 4 ) . therefore , it is difficult to diagnose and physicians occasionally might prescribe inappropriate treatments ( 5 , 6 ) . in this paper we describe a case of primary rectal syphilis which was suspected to be rectal cancer . he complained of bowel habit change with tenesmus , mucous discharge , anal pain and intermittent presence of blood in stool . he had a 2 cm sized , firm , non - tender and indurated nodule on the left side of the inguinal area and multiple condyloma acuminatum around the anus . sigmoidoscopy in our gastrointestinal endoscopy unit showed two indurated masses of 1.5 to 2 cm on the posterior wall of the middle and lower rectum ( fig . the masses had a coin like appearance , and each of them had a slightly depressed and ulcerated surface . the vdrl quantitative test was 1:64 and the fta - abs igm and igg test was positive . a human immunodeficiency virus ( hiv ) the patient was treated with intramuscular penicillin g benzathine ( 2,400,000 iu im/1 week , thrice ) . follow - up sigmoidoscopy after 1 month showed nearly complete regression of the chancre ( fig . a repeat biopsy was done and the histologic result showed focal lymphoid hyperplasia . a vdrl quantitative test after 3 months was 1:1 . most physicians have a tendency to consider rectal ulceration as a neoplasm ( 7 , 8) . similarly in our case symptoms and signs suggested rectal neoplasia and the clinical history did not reveal homosexuality . however , after the vdrl test the patient confessed that he had been raped by a homosexual male . therefore , when rectal syphilis is suspected ( i.e. , perianal condyloma acuminatum associated with inguinal lymphadenopathy ) the history of sexual intercourse , especially rectal intercourse , has to be taken . a dark - field examination is recommended by many studies ( 3 ) . in our case , however , a dark - field examination was not performed , because rectal chancre was highly suggested by the clinical history , anorectal lesions , pathologic findings and positive serologic test . additionally , like other syphilis cases benzathine penicillin therapy induced a rapid regression of the rectal lesions . we believe when a positive serologic syphilis test is associated with ulcerative lesion , additional laboratory tests are not necessary . inguinal lymphadenopathy need not require an initial biopsy when evidence of regional infection is present . usually after antibiotic therapy of at least 4 weeks , most physicians might decide whether a biopsy is necessary . in this case , however , the physician did not have experience recognizing rectal syphilis , so he immediately obtained a biopsy specimen from the inguinal lesion . . in western countries the incidence of rectal syphilis is rising , especially in groups of active homosexual males . bassi et al . emphasized in his case report that endoscopists should keep in mind the rising incidence of syphilis ( 3 ) . therefore an initial physician should encourage his / her patient to inform him / her of the patient 's sexual history .

a 30-yr - old man was referred for suspicious rectal cancer because of ulcerated lesions in the rectum and a palpable mass in left inguinal area . sigmoidoscopy showed two indurated masses and histologic evaluation of biopsy revealed obliterative endarteritis with heavy plasma cell infiltration . both venereal disease research laboratories ( vdrl ) and fluorescent treponemal antibody absorption ( fta - abs ) tests were positive . after injection of penicillin g benzathine for 3 weeks , the rectal chancre and the palpable mass disappeared .

the protective effects of vagus nerve during ischemia / reperfusion ( i / r ) have been suggested ( 1 ) . vagus nerve stimulation decreases the inflammation and the injuries resulted from i / r through affecting neutrophils while vagotomy increases these processes ( 1 ) . moreover , it has been observed that stimulation of vagus nerve can cause protective effects on gastric injury through releasing prostaglandins , nitric oxide ( no ) and calcitonin gene related peptide ( cgrp ) ( 2 ) . it is probable that vagus nerve exerts its gastric protective effects through releasing the aforementioned mediators . also , it has been reported that cholinergic activity of the efferent vagus nerve can participate in immunity modulation ( 3 ) . another study has shown that nicotine in monocytes not only decreases the production of pre - inflammatory cytokines , but also changes the response to il-10 , an anti - inflammatory cytokine ( 4 , 5 ) . cho et al have shown that alternative stimulation of cervical vagus nerve increases intra - gastric pressure and also causes bleeding ulcers in mucosal glands of stomach , and these effects can be prevented by atropine administration or sub - diaphragmatic vagotomy ( 6 ) . it has been reported that gastric vagotomy is effective in treating gastric and duodenal ulcers ( 7 ) . melatonin as a neurohormone is basically produced in the pineal gland and also in epiphysis , gastrointestinal ( gi ) mucosa and other organs . melatonin has several physiological activities such as controlling circadian rhythm , sleep induction , regulation of seasonal reproduction , and improvement of immunity . the most important effect of melatonin is an anti - oxidant effect that protects living organisms against oxidative stress ( 11 ) . in several studies , the ability of melatonin in decreasing molecular injury has been shown during i / r . melatonin prevents myocardial infarction , necrotic cell death and renal abnormality after i / r ( 8 , 12 ) , reduces brain edema in ischemia ( 12 ) , and prevents acute gastric ulcers resulted from stress . these effects can be done through direct reactive oxygen species / reactive nitrogen species ( ros / rns ) detoxification , increase in anti - oxidative enzymes , and the prevention of electron leakage in mitochondrial inner membrane ( 11 , 13 ) . melatonin has a role in expression and function of nicotinic acetylcholine receptors and increases the efficacy of beta bungarotoxin - sensitive acetylcholine receptors ( 14 ) . it has been recognized that intra - cerebral injection of melatonin is effective in prevention of acid and pepsin secretion through cholinergic activity ( 15 ) . it has been identified that 5ht3 and 5ht2 receptors are involved in stimulatory effects of melatonin in releasing pancreatic enzymes ( 11 ) . protective effects of melatonin are not only due to antioxidant activity , but it also activates capsaicin - sensitive afferent fibers ( 1 , 16 ) . melatonin may have a potential impact on the treatment of peptic ulcer via significant increase in ghrelin expression ( 17 ) . melatonin increases the release of pancreatic amylase and proteins through vagus nerve ( 18 ) . the effects of melatonin on pancreatic enzymes are reversed by vagotomy and administration of capsaicin ( 18 , 19 ) . intra - lumen administration of melatonin increases plasma cholecystokinin ( cck ) and antioxidants ( 11 , 19 , 20 ) . it has been shown that sensory nerve fibers are involved in protective effects of melatonin in the healing of acute gastric injury and peptic ulcer ( 16 ) . it has been reported that vagus nerve increases the secretion of bicarbonate from duodenum mucosa through an increase in melatonin secretion ( 21 ) . vagal pathways are the most important regulatory pathways in the gastrointestinal tract which is one of the most important sources of melatonin production . in previous studies , both protective and harmful effects of the stimulation of vagus nerve in digestive system have been reported . the role of sensory neurons in protective effects of melatonin in the healing of acute gastric injury and peptic ulcer has been reported . there is no study about the combined effects of melatonin administration and intervention of vagus nerve during gastric i / r injury . probably , vagus nerve and melatonin have interactions in their protective effects during gastric i / r events so , the present study was performed to examine this interaction during gastric i / r . this study was performed in 42 male wistar rats weighing 180 - 220 g. animals were kept at room temperature 20 - 22 c and 12 hr-12 hr light - dark cycle . animals had free access to water and food and were randomly divided into 6 groups of 7 rats . rats were fasted for 12 hr prior to the experiment but had access to water . experimental groups were : 1 ) base+i / r+vehicle ; 2 ) base+i / r+melatonin ; 3 ) vagotomy+i / r+vehicle ; 4 ) vagotomy+i / r+melatonin ; 5)vagus nerve stimulation+i / r+vehicle ; 6)vagus nerve stimulation+i / r+ melatonin . base , means condition that neither vagus nerve was stimulated nor vagotomy was performed . animals underwent tracheostomy and were cannulated ( in order to prevent probable airway occultation ) under anesthesia induced by intraperitoneal pentobarbital ( 50 mg / kg ) . after local shaving , a small incision was made in cervical area and branches of cervical vagus nerve were carefully dissected from carotid artery and cut ( 1 ) . after dissection of vagus nerve , distal end was covered with mineral oil and stimulated with bipolar electrode of a stimulator using 10 volt / msec pulses at the frequencies of 0.625 , 1.25 , 2.5 , 5 or 7.5hz for 30 sec . stimulations were performed with 2-min intervals ( 23 ) . in order to ensure the stimulation of vagus nerve , electrocardiogram gastric i / r injury was induced by dissecting celiac artery from the surrounding tissues in rats anesthetized with 50 mg / kg pentobarbital , 30 minutes after dissection of vagus nerve . then , celiac artery was closed with a microvascular clamp and returned to its place and the area was sutured using 4 - 0 silk . occlusion was continued for 30 min ( 18 ) followed by a 3-h circulation ( reperfusion ) . melatonin ( 10 mg / kg ) was injected intraperitoneally before reperfusion in groups 5 , 6 and 7 ( 18 , 25 ) . melatonin was dissolved in 1% ethanol , diluted with 0.9% saline , and injected at a final volume of 0.3 ml ( 18 ) . one part was kept in 10% formalin solution for histopathological assessment and the rest was kept at -70 c for evaluation of oxidant and antioxidant factors ( 18 , 25 ) . mda level was measured according to hiroshi ohkawa method via reaction with thiobarbituric acid and color formation . optical absorbance was determined at 532 nm and mda level was reported as nmol / mg protein ( 26 ) . glutathione peroxides activity was determined by the method described by valentine and paglia method ( 20 ) , and enzyme activity was reported as u / mg protein . superoxide dismutase activity was determined using ransod kit ( randox , uk ) and enzyme activity was reported as u / mg protein ( 19 ) . catalase activity was determined by the method described by mari and reported as u / mg protein ( 20 ) . the h & e stained sections were examined under low power ( 40x ) to identify the areas of neutrophil aggregates within all tissue blocks . the number of neutrophils was calculated in a semiquantitative manner using the mean value of 20 non - overlapping high power fields ( hpf ; magnification of 400x ; 0.08 mm ) by using a 40x objective and a square grid mounted in a 10x microscopic eyepiece . comparisons among different groups were made by one - way and two - way anova followed by post hoc tukey s test . animals underwent tracheostomy and were cannulated ( in order to prevent probable airway occultation ) under anesthesia induced by intraperitoneal pentobarbital ( 50 mg / kg ) . after local shaving , a small incision was made in cervical area and branches of cervical vagus nerve were carefully dissected from carotid artery and cut ( 1 ) . after dissection of vagus nerve , distal end was covered with mineral oil and stimulated with bipolar electrode of a stimulator using 10 volt / msec pulses at the frequencies of 0.625 , 1.25 , 2.5 , 5 or 7.5hz for 30 sec . stimulations were performed with 2-min intervals ( 23 ) . in order to ensure the stimulation of vagus nerve , electrocardiogram gastric i / r injury was induced by dissecting celiac artery from the surrounding tissues in rats anesthetized with 50 mg / kg pentobarbital , 30 minutes after dissection of vagus nerve . then , celiac artery was closed with a microvascular clamp and returned to its place and the area was sutured using 4 - 0 silk . occlusion was continued for 30 min ( 18 ) followed by a 3-h circulation ( reperfusion ) . melatonin ( 10 mg / kg ) was injected intraperitoneally before reperfusion in groups 5 , 6 and 7 ( 18 , 25 ) . melatonin was dissolved in 1% ethanol , diluted with 0.9% saline , and injected at a final volume of 0.3 ml ( 18 ) . one part was kept in 10% formalin solution for histopathological assessment and the rest was kept at -70 c for evaluation of oxidant and antioxidant factors ( 18 , 25 ) . mda level was measured according to hiroshi ohkawa method via reaction with thiobarbituric acid and color formation . optical absorbance was determined at 532 nm and mda level was reported as nmol / mg protein ( 26 ) . glutathione peroxides activity was determined by the method described by valentine and paglia method ( 20 ) , and enzyme activity was reported as u / mg protein . superoxide dismutase activity was determined using ransod kit ( randox , uk ) and enzyme activity was reported as u / mg protein ( 19 ) . catalase activity was determined by the method described by mari and reported as u / mg protein ( 20 ) . the h & e stained sections were examined under low power ( 40x ) to identify the areas of neutrophil aggregates within all tissue blocks . the number of neutrophils was calculated in a semiquantitative manner using the mean value of 20 non - overlapping high power fields ( hpf ; magnification of 400x ; 0.08 mm ) by using a 40x objective and a square grid mounted in a 10x microscopic eyepiece . comparisons among different groups were made by one - way and two - way anova followed by post hoc tukey s test . mean number of neutrophils in base+ i / r+ melatonin group ( 23.80.37 ) was lower than that in base+ i / r+ vehicle group ( 27.20.37 ) ( p<0.01 ) . also , this parameter in vagus stimulation + i / r+ vehicle group ( 450.32 ) was higher than that in base+ i / r+ vehicle and vagotomy+ i / r+ vehicle groups ( 27.20.36 ) ( p<0.001 ) . / r+melatonin group ( 20.6 0.4 ) in comparison to vagus stimulation+i / r+vehicle group ( p<0.001 ) . / r+ melatonin group as compared to base+i / r+ melatonin group ( p<0.01 ) . a significant increase in neutrophils count was shown in vagotomy+i / r+melatonin group ( 260.32 ) as compared to base+i / r+melatonin group ( p<0.05 ) . the number of neutrophils in gastric tissue of the study groups ( n=7 ) . # # # p<0.001 : vagus stimulation+i / r+veh group vs. vagotomy+i / r+veh group . base : condition that neither vagus was stimulated nor vagotomy was performed ; i / r : ischemia / reperfusion ; mel : melatonin ; veh : vehicle mda level in the study groups is presented in figure 2 . as it is seen , mda level in gastric tissue was lower in base+i / r+melatonin group ( 4.090.15 nmol / mg protein ) in comparison with base+i / r+vehicle group ( 5.530.11 nmol / mg protein ) ( p<0.05 ) . moreover , a significant increase in mda level was shown in vagus stimulation+i/-r+vehicle group ( 7.310.25 nmol / mg protein ) in comparison with base+i / r+vehicle and vagotomy+i/- r+vehicle ( 5.570.14 nmol / mg protein ) groups ( p<0.01 ) . / r+vehicle group ( p<0.001 ) . also , mda level was lower in vagus stimulation+i / r+melatonin group ( 2.48 0.06 nmol / mg protein ) in comparison with base+i / r+melatonin group ( p<0.01 ) . mda level was higher in vagotomy+i / r+melatonin group ( 5.75 0.63 nmol / mg protein ) in comparison with base+ i / r+melatonin group ( p<0.05 ) . * p<0.05 : base+i / r+ mel group vs. base + i / r+ veh group . # # p<0.01 : vagus stimulation+i / r+veh group vs. vagotomy+i / r+veh group . a p<0.01 : vagus stimulation+i / r+mel group vs. base + i / r+mel group . base : condition that neither vagus was stimulated nor vagotomy was performed ; i / r : ischemia / reperfusion ; mel : melatonin ; veh : vehicle catalase activity in the study groups are shown in figure 3 . catalase activity was increased in base+i / r+melatonin group ( 0.060.003 u / mg protein ) in comparison with base+i / r+vehicle group ( 0.040.003 u / mg protein ) ( p<0.001 ) . a significant increase in catalase activity was shown in vagus stimulation+ i / r+vehicle group ( 0.07 0.004 u / mg protein ) in comparison with base+i / r+vehicle and vagotomy+i / r+vehicle groups ( 0.0420.002 u / mg protein ) ( p<0.001 ) . / r+melatonin group ( 0.070.004 u / mg protein ) in comparison with base+i / r+melatonin group ( p<0.05 ) . catalase activity was lower in vagotomy+i / r+melatonin group ( 0.040.003 u / mg protein ) in comparison with base+i / r+melatonin group ( p<0.05 ) . catalase activity ( u / mg protein ) in gastric tissue of the study groups ( n=7).***p<0.001 : base+i / r+mel group vs. base+i / r+veh group . # base : condition that neither vagus was stimulated nor vagotomy was performed ; i / r : ischemia / reperfusion ; mel : melatonin ; veh : vehicle figure 4 shows glutathione peroxidase activity in the studied groups . a significant increase in gpx activity was shown in gastric tissue of basal+i / r+melatonin group ( 10.310.37 ) ( p<0.001 ) in comparison with that in basal+i / r+vehicle group ( 4.420.57 ) . / r+vehicle group ( 7.120.31 ) as compared to that observed in basal+i / r+vehicle and vagotomy+i / r+vehicle groups ( 4.430.38 ) ( p<0.001 ) . also , a significant increase in gpx activity was indicated in vagus stimulated+i this parameter was significantly lower in vagotomy+ i / r+melatonin group ( 4.610.4 ) as compared to that in basal+i / r+melatonin group ( p<0.01 ) . level of gpx activity ( u / mg protein ) in gastric tissue of the study groups ( n=7 ) * * * p<0.001 : base+i / r+mel group vs. base+i / r+veh group . # b p<0.001 : vagotomy + i / r+ mel group vs. base+i / r+mel group . base : condition that neither vagus was stimulated nor vagotomy was performed ; i / r : ischemia / reperfusion ; mel : melatonin ; veh : vehicle sod activity ( u / mg protein ) in gastric tissue of studied groups has been shown in figure 5 . as it is seen , this parameter in basal+i / r+melatonin group ( 0.310.004 ) was significantly higher ( p<0.05 ) than that in basal+i / r+vehicle group ( 0.230.016 ) . a significant decrease in sod activity / r+ vehicle group ( 0.310.004 ) in comparison with that in vagotomy+i / r+vehicle group ( 0.360.003 ) ( p<0.05 ) , but it was higher than that in basal+ i / r+vehicle group . this parameter in vagotomy+i / r+melatonin group ( 0.170.04 ) was lower than that in basal+i/-r+melatonin group ( p<0.05 ) . superoxide dismutase activity ( u / mg protein ) in gastric tissue of the study groups ( n=7 ) * p<0.05 : base+i / r+mel group vs. base+i / r+veh group . base : condition that neither vagus was stimulated nor vagotomy was performed ; i / r : ischemia / reperfusion ; mel : melatonin ; veh : vehicle mean number of neutrophils in base+ i / r+ melatonin group ( 23.80.37 ) was lower than that in base+ i / r+ vehicle group ( 27.20.37 ) ( p<0.01 ) . also , this parameter in vagus stimulation + i / r+ vehicle group ( 450.32 ) was higher than that in base+ i / r+ vehicle and vagotomy+ i / r+ vehicle groups ( 27.20.36 ) ( p<0.001 ) . / r+melatonin group ( 20.6 0.4 ) in comparison to vagus stimulation+i / r+vehicle group ( p<0.001 ) . / r+ melatonin group as compared to base+i / r+ melatonin group ( p<0.01 ) . a significant increase in neutrophils count was shown in vagotomy+i / r+melatonin group ( 260.32 ) as compared to base+i / r+melatonin group ( p<0.05 ) . the number of neutrophils in gastric tissue of the study groups ( n=7 ) . # # # p<0.001 : vagus stimulation+i / r+veh group vs. vagotomy+i / r+veh group . base : condition that neither vagus was stimulated nor vagotomy was performed ; i / r : ischemia / reperfusion ; mel : melatonin ; veh : vehicle as it is seen , mda level in gastric tissue was lower in base+i / r+melatonin group ( 4.090.15 nmol / mg protein ) in comparison with base+i / r+vehicle group ( 5.530.11 nmol / mg protein ) ( p<0.05 ) . moreover , a significant increase in mda level was shown in vagus stimulation+i/-r+vehicle group ( 7.310.25 nmol / mg protein ) in comparison with base+i / r+vehicle and vagotomy+i/- r+vehicle ( 5.570.14 nmol / mg protein ) groups ( p<0.01 ) . / r+vehicle group ( p<0.001 ) . also , mda level was lower in vagus stimulation+i / r+melatonin group ( 2.48 0.06 nmol / mg protein ) in comparison with base+i / r+melatonin group ( p<0.01 ) . mda level was higher in vagotomy+i / r+melatonin group ( 5.75 0.63 nmol / mg protein ) in comparison with base+ i / r+melatonin group ( p<0.05 ) . * p<0.05 : base+i / r+ mel group vs. base + i / r+ veh group . # a p<0.01 : vagus stimulation+i / r+mel group vs. base + i / r+mel group . base : condition that neither vagus was stimulated nor vagotomy was performed ; i / r : ischemia / reperfusion ; mel : melatonin ; veh : vehicle catalase activity was increased in base+i / r+melatonin group ( 0.060.003 u / mg protein ) in comparison with base+i / r+vehicle group ( 0.040.003 u / mg protein ) ( p<0.001 ) . a significant increase in catalase activity was shown in vagus stimulation+ i / r+vehicle group ( 0.07 0.004 u / mg protein ) in comparison with base+i / r+vehicle and vagotomy+i / r+vehicle groups ( 0.0420.002 u / mg protein ) ( p<0.001 ) . / r+melatonin group ( 0.070.004 u / mg protein ) in comparison with base+i / r+melatonin group ( p<0.05 ) . catalase activity was lower in vagotomy+i / r+melatonin group ( 0.040.003 u / mg protein ) in comparison with base+i / r+melatonin group ( p<0.05 ) . catalase activity ( u / mg protein ) in gastric tissue of the study groups ( n=7).***p<0.001 : base+i / r+mel group vs. base+i / r+veh group . # # # p<0.001 : vagus stimulation+i / r+veh group vs. vagotomy+i / r+veh group . base : condition that neither vagus was stimulated nor vagotomy was performed ; i / r : ischemia / reperfusion ; mel : melatonin ; veh : vehicle a significant increase in gpx activity was shown in gastric tissue of basal+i / r+melatonin group ( 10.310.37 ) ( p<0.001 ) in comparison with that in basal+i / r+vehicle group ( 4.420.57 ) . / r+vehicle group ( 7.120.31 ) as compared to that observed in basal+i / r+vehicle and vagotomy+i / r+vehicle groups ( 4.430.38 ) ( p<0.001 ) . also , a significant increase in gpx activity was indicated in vagus stimulated+i this parameter was significantly lower in vagotomy+ i / r+melatonin group ( 4.610.4 ) as compared to that in basal+i / r+melatonin group ( p<0.01 ) . level of gpx activity ( u / mg protein ) in gastric tissue of the study groups ( n=7 ) * * * p<0.001 : base+i / r+mel group vs. base+i / r+veh group . # # p<0.01 : vagus stimulation+i / r+veh group vs. vagotomy+i / r+veh group . b p<0.001 : vagotomy + i / r+ mel group vs. base+i / r+mel group . base : condition that neither vagus was stimulated nor vagotomy was performed ; i / r : ischemia / reperfusion ; mel : melatonin ; veh : vehicle sod activity ( u / mg protein ) in gastric tissue of studied groups has been shown in figure 5 . as it is seen , this parameter in basal+i / r+melatonin group ( 0.310.004 ) was significantly higher ( p<0.05 ) than that in basal+i / r+vehicle group ( 0.230.016 ) . a significant decrease in sod activity / r+ vehicle group ( 0.310.004 ) in comparison with that in vagotomy+i / r+vehicle group ( 0.360.003 ) ( p<0.05 ) , but it was higher than that in basal+ i / r+vehicle group . this parameter in vagotomy+i / r+melatonin group ( 0.170.04 ) was lower than that in basal+i/-r+melatonin group ( p<0.05 ) . superoxide dismutase activity ( u / mg protein ) in gastric tissue of the study groups ( n=7 ) * p<0.05 : base+i / r+mel group vs. base+i / r+veh group . # # p<0.01 : vagus stimulation+i / r+veh group vs. vagotomy+i / r+veh group . base : condition that neither vagus was stimulated nor vagotomy was performed ; i / r : ischemia / reperfusion ; mel : melatonin ; veh : vehicle the present study was performed to investigate the protective effect of interaction between melatonin and vagus nerve during gastric i / r . the results of this study showed that melatonin administration decreased neutrophils infiltration and mda level and increased sod , cat and gpx activities in gastric tissue . also , vagus stimulation increased neutrophils infiltration and mda level in gastric tissue while melatonin administration along with vagus stimulation decreased the effect of vagus stimulation in gastric tissue . finally , vagotomy prevented some of the protective effects of melatonin during gastric i / r . according to the results of this study , melatonin can reduce gastritis during i / r , probably through decreasing the activity of oxidant enzymes and increasing the activity of anti - oxidant enzymes such as sod , cat and gpx . it has also been demonstrated that melatonin decreases mad level ( 27 - 29 ) . other probable mechanisms underlying the protective effect of melatonin include an increase in gastric microcirculation ( 1 ) , a reduction in acid and pepsin secretions ( 15 ) , a reduction in production of inflammatory cytokines ( 16 ) and an increase in the expression of anti - oxidant gene ( 17 , 30 - 32 ) . moreover , in our study , a stimulation of vagus nerve increased gastritis which was associated with an increase in mda level while vagotomy after i / r was ineffective . it is probable that the stimulation of vagus nerve following gastric i / r aggravated the condition and increased the activity of oxidant enzymes . since vagotomy in basal condition did not have any effect , it seems that the control of gastritis and the activity of oxidant and anti - oxidant enzymes are not under the control of vagus nerve in basal conditions . it has been demonstrated that the stimulation of vagus nerve increases output of gastric juice and acid ( 33 ) , and decreases the activity of anti - oxidant enzymes like sod ( 33 ) . on the other hand , vagotomy showed healing effects on duodenal ulcers ( 7 ) similar to the effect of atropine ( 34 ) . in a study , diaphragmatic vagotomy did not change the activity of catalase in the intestine ( 35 ) which was similar to the result of the present study . the results of some studies are not consistent with our data . for example , it has been identified that vagal afferent fibers decrease gastrointestinal tract inflammation through nicotinic receptors leading to a decrease in inflammatory cytokines ( 16 ) . cholinergic agonists such as neostigmine decrease superoxide anion and inflammation in i / r ( 36 ) . elevation of acetylcholine transferase activity and decrease in acetylcholine esterase inhibitors increase gpx ( 35 , 37 ) . treatment with acetylcholine esterase inhibitors increases catalase half - life ( 38 ) . in the present study , melatonin administration affected the outcomes in i / r+vagus stimulation group . melatonin decreased gastritis and mda level , and increased sod and gpx activity , but not in vagotomy group . these findings show that protective effects of melatonin are probably mediated through vagus nerve . also , interaction between melatonin and vagus stimulation is suggested since these show a synergic effect in relation with anti - oxidant factors . regarding this probable interaction , it is suggested that melatonin exerts its gastric protective effect through stimulation of gi tract neurons leading to cgrp release ( 1 ) . intra - cerebral injection of melatonin inhibits acid and pepsin secretions through vagus cholinergic activity ( 15 ) . melatonin increases the release of amylase and pancreas proteins through vagus nerve ( 16 ) . effect of melatonin administration accompanied with vagotomy has not suggested in the release of amylase(18 ) . studies have shown that intra - lumen administration of melatonin or its precursor induces the release of pancreatic enzymes , while this effect is not shown in isolated pancreas . moreover , these effects of melatonin are reversed by vagotomy and capsaicin administration ( 11 , 18 , 19 ) . it has been reported that melatonin effect is mediated indirectly through the release of cck followed by vagovagal reflex ( 11 , 19 , 39 ) . it has been shown that intra - lumen administration of melatonin increases plasma cck level ( 11 , 19 ) and cck acts through stimulation of vagal afferent fibers in gi tract ( 40 , 41 ) . it has been reported that vagotomy in combination with melatonin administration inhibits the release of pancreas proteins ( 7 , 11 , 16 ) . it has been reported that melatonin increases the expression and activity of ach receptors , and synergistic effects are induced during vagus stimulation ( 42 ) . according to a study , injection of phenylephrine causes melatonin release from mucosa enterochromaffin cells in the presence of normal vagus nerve and sympathetic paths ( 39 ) . melatonin increases the release of bicarbonate from gastric mucosa ( 39 ) and decreases the release of inflammatory cytokines of gi through increasing vagus activity ( 16 ) . it is suggested that melatonin is neuroprotective in gastric i / r probably by decreasing gastritis and mda and increasing the activities of cat , sod and gpx . these effects of melatonin are probably mediated by vagus nerve . moreover , in gastric i / r , melatonin can reverse harmful effects of vagus stimulation . it is suggested that further studies are required to find the mechanisms involved in this interaction .

objectives : vagal pathways in gastrointestinal tract are the most important pathways that regulate ischemia / reperfusion ( i / r ) . gastrointestinal tract is one of the important sources of melatonin production . the aim of this study was to investigate probable protective effect of the interaction between vagus nerve and melatonin after i / r.materials and methods : this study was performed in male rats that were divided into six groups . cervical vagus nerve was cut bilaterally after induction of i / r and the right one was stimulated by stimulator . melatonin or vehicle was injected intraperitoneally . the stomach was removed for histopathological and biochemical investigations.results:a significant decrease in infiltration of gastric neutrophils and malondialdehyde ( mda ) level after i / r was induced by melatonin and was disappeared after vagotomy . the stimulation of vagus nerve significantly enhanced these effects of melatonin . however , a stimulation of vagus nerve alone increased neutrophils infiltration and mda level . melatonin significantly increased the activities of catalase , glutathione peroxidase ( gpx ) , superoxide dismutases ( sod ) . unlike stimulation of vagus nerve , vagotomy decreased these effects of melatonin.conclusion:according to these results , it is probable that protective effects of melatonin after i / r may be mediated by vagus nerve . therefore , there is an interaction between melatonin and vagus nerve in their protective effects .

the 2002 cnnhs is a nationally representative cross - sectional study on nutrition and chronic diseases . a stratified , multistage probability cluster sampling design was used in this survey ( 13 ) . based on socioeconomic characteristics , in the first stage of sampling , 22 counties were randomly selected from each of the 6 regions in china . in the second stage , three townships were randomly selected from each of the selected counties . from each of the townships , 2 residential villages were randomly selected ; and 90 households were then randomly sampled from each village for physical examination . one - third of the households were selected to participate in the dietary survey and blood draw . for the present study , we used residents who were living in rural areas and were born between october 1 , 1952 , and september 30 , 1964 , as our analytic population . to minimize misclassification of the exposure periods , subjects who were born between october 1 , 1958 , and september 30 , 1959 , and between october 1 , 1961 , and september 30 , 1962 , were excluded since the exact dates of the start and the end of the chinese famine were not available and not the same across regions . flow chart on the sampling method in each region * of the 2002 china national nutrition and health survey . * the mainland of china is classified into 6 regions defined by the chinese bureau of statistics according to their socioeconomic development . they are metropolis , general city , type i rural site , type ii rural site , type iii rural site , and type iv rural site . subjects were categorized into five exposure cohorts : nonexposed cohort , fetal - exposed cohort , early childhood exposed cohort , mid childhood exposed cohort , and late childhood exposed cohort . subjects who were born between october 1 , 1962 , and september 30 , 1964 , were classified as the nonexposed cohort ; and subjects who were born between october 1 , 1959 , and september 30 , 1961 , were classified as fetal - exposed cohort . subjects who were born between october 1 , 1952 , and september 30 , 1958 , were grouped by every 2 years and were classified into one of the three childhood - exposed cohorts . mean ages for subjects in nonexposed cohort , fetal - exposed cohort , early childhood exposed cohort , mid childhood exposed cohort , and late childhood exposed cohort were 39 , 42 , 45 , 47 , and 49 years , respectively . the chinese famine affected the entire mainland of china , but the severity varied across regions due to different weather conditions , population density , and local policies regarding food shortage ( 7 ) . as previously described , we used the excess death rate of each province to determine the severity of the famine ( 7 ) . the excess death rate was calculated as the percentage change in mortality rate from the mean level in 19561958 to the highest value during the period 19591961 ( 7 ) . an excess death rate of 50% was used as the threshold : regions that had an equal or higher rate than this cutoff were categorized as severely affected famine areas , and otherwise as less severely affected famine areas . we split all five cohorts into severely affected famine areas and less severely affected famine areas . this enabled us to test the hypothesis that the famine effect is stronger in the severely affected famine areas than that in the less severely affected famine areas and to consider both birth cohort effects and regional differences . all subjects were invited for blood collection after an 10 to 14 h overnight fast . the plasma was separated by centrifugation at 3,200 rpm for 1015 min within 1 h of collection , and kept at room temperature without sunshine . fasting plasma glucose ( fpg ) concentration was measured using the glucose oxidize enzymatic method within 3 h of plasma preparation . every tenth sample was measured twice ( the correlation coefficient of duplicate measurements was 0.98 ) . we used criteria proposed by the who expert committee on diabetes mellitus ( 14 ) . mmol / l , including impaired fasting glucose , impaired glucose tolerance , and type 2 diabetes . in addition , subjects who had been previously diagnosed with type 2 diabetes were added as cases of hyperglycemia and type 2 diabetes . dietary patterns , economic status , and bmi measured in 2002 were used as measures of the nutritional environment in adulthood and to examine the mismatch between fetal nutrition and adult nutrition . the method for assessing dietary patterns has been described in detail elsewhere ( 15 ) . briefly , four dietary patterns were derived through cluster analysis , which were labeled as green water , yellow earth , new affluence , and western adopter . green water and yellow earth patterns represent the traditional chinese diets in the south and the north , respectively , whereas the other two represent westernized dietary patterns . in this study , we combined the clusters of green water and yellow earth as the traditional dietary pattern , and we combined the clusters of new affluence and western adopter as the affluent / western pattern . current economic status was assessed by the mean annual income in the year prior to the 2002 cnnhs , which was treated as a dichotomous variable . the mean level of the current sample ( 2,000 chinese yuan per person per year ) was used as a cutoff point for economic status . we used the criteria recommended for chinese adults and classified subjects as overweight if bmi 24 kg / m , or otherwise normal ( 16 ) . the protocol of the 2002 cnnhs was approved by the ethical committee of the national institute for nutrition and food safety , chinese center for disease control and prevention . signed consent forms were obtained from all participants . we performed survey analyses with sas 9.2 for windows ( sas institute , cary , nc ) to estimate statistics for this complex , multistage - designed survey sample . survey weights were derived from the 2000 china national population census and associated administrative data . mean fpg differences between the exposed cohorts and the nonexposed cohort were tested by generalized least squares estimation ( 17 ) . risks of hyperglycemia and type 2 diabetes among fetal and childhood - exposed subjects , compared with nonexposed subject , were examined with the method of maximum likelihood by using the survey logistic regression model . interaction between famine exposure cohort ( fetal- or childhood - exposed vs. nonexposed ) and area ( severely affected and less severely affected ) was tested by adding a multiplicative factor in the survey logistic regression model . analyses were adjusted for sex , family history of diabetes , educational level , current smoking , alcohol use , and physical activity level , all assessed in 2002 . to explore whether the associations between fetal exposure to severe famine and hyperglycemia were affected by an improved nutritional environment in later life , we subsequently stratified the analyses by dietary patterns , economic status and bmi in adulthood . the odds ratio of hyperglycemia in the fetal - exposed cohort compared with the nonexposed cohort was calculated within each category of the stratified factor . to distinguish severely and less severely affected famine areas more appropriately , we performed sensitivity analysis by using a more stringent cutoff point , i.e. , we used an excess death rate 100% to define the severity of famine . in addition , we performed analyses by using the cohort born during october 1 , 1962 , to september 30 , 1968 , as a nonexposed cohort for association analyses , or by excluding participants with a family history of diabetes . subjects were categorized into five exposure cohorts : nonexposed cohort , fetal - exposed cohort , early childhood exposed cohort , mid childhood exposed cohort , and late childhood exposed cohort . subjects who were born between october 1 , 1962 , and september 30 , 1964 , were classified as the nonexposed cohort ; and subjects who were born between october 1 , 1959 , and september 30 , 1961 , were classified as fetal - exposed cohort . subjects who were born between october 1 , 1952 , and september 30 , 1958 , were grouped by every 2 years and were classified into one of the three childhood - exposed cohorts . mean ages for subjects in nonexposed cohort , fetal - exposed cohort , early childhood exposed cohort , mid childhood exposed cohort , and late childhood exposed cohort were 39 , 42 , 45 , 47 , and 49 years , respectively . the chinese famine affected the entire mainland of china , but the severity varied across regions due to different weather conditions , population density , and local policies regarding food shortage ( 7 ) . as previously described , we used the excess death rate of each province to determine the severity of the famine ( 7 ) . the excess death rate was calculated as the percentage change in mortality rate from the mean level in 19561958 to the highest value during the period 19591961 ( 7 ) . an excess death rate of 50% was used as the threshold : regions that had an equal or higher rate than this cutoff were categorized as severely affected famine areas , and otherwise as less severely affected famine areas . we split all five cohorts into severely affected famine areas and less severely affected famine areas . this enabled us to test the hypothesis that the famine effect is stronger in the severely affected famine areas than that in the less severely affected famine areas and to consider both birth cohort effects and regional differences . all subjects were invited for blood collection after an 10 to 14 h overnight fast . the plasma was separated by centrifugation at 3,200 rpm for 1015 min within 1 h of collection , and kept at room temperature without sunshine . fasting plasma glucose ( fpg ) concentration was measured using the glucose oxidize enzymatic method within 3 h of plasma preparation . every tenth sample was measured twice ( the correlation coefficient of duplicate measurements was 0.98 ) . we used criteria proposed by the who expert committee on diabetes mellitus ( 14 ) . mmol / l , including impaired fasting glucose , impaired glucose tolerance , and type 2 diabetes . in addition , subjects who had been previously diagnosed with type 2 diabetes were added as cases of hyperglycemia and type 2 diabetes . dietary patterns , economic status , and bmi measured in 2002 were used as measures of the nutritional environment in adulthood and to examine the mismatch between fetal nutrition and adult nutrition . the method for assessing dietary patterns has been described in detail elsewhere ( 15 ) . briefly , four dietary patterns were derived through cluster analysis , which were labeled as green water , yellow earth , new affluence , and western adopter . green water and yellow earth patterns represent the traditional chinese diets in the south and the north , respectively , whereas the other two represent westernized dietary patterns . in this study , we combined the clusters of green water and yellow earth as the traditional dietary pattern , and we combined the clusters of new affluence and western adopter as the affluent / western pattern . current economic status was assessed by the mean annual income in the year prior to the 2002 cnnhs , which was treated as a dichotomous variable . the mean level of the current sample ( 2,000 chinese yuan per person per year ) was used as a cutoff point for economic status . we used the criteria recommended for chinese adults and classified subjects as overweight if bmi 24 kg / m , or otherwise normal ( 16 ) . the protocol of the 2002 cnnhs was approved by the ethical committee of the national institute for nutrition and food safety , chinese center for disease control and prevention . signed consent forms were obtained from all participants . we performed survey analyses with sas 9.2 for windows ( sas institute , cary , nc ) to estimate statistics for this complex , multistage - designed survey sample . survey weights were derived from the 2000 china national population census and associated administrative data . mean fpg differences between the exposed cohorts and the nonexposed cohort were tested by generalized least squares estimation ( 17 ) . risks of hyperglycemia and type 2 diabetes among fetal and childhood - exposed subjects , compared with nonexposed subject , were examined with the method of maximum likelihood by using the survey logistic regression model . interaction between famine exposure cohort ( fetal- or childhood - exposed vs. nonexposed ) and area ( severely affected and less severely affected ) was tested by adding a multiplicative factor in the survey logistic regression model . analyses were adjusted for sex , family history of diabetes , educational level , current smoking , alcohol use , and physical activity level , all assessed in 2002 . to explore whether the associations between fetal exposure to severe famine and hyperglycemia were affected by an improved nutritional environment in later life , we subsequently stratified the analyses by dietary patterns , economic status and bmi in adulthood . the odds ratio of hyperglycemia in the fetal - exposed cohort compared with the nonexposed cohort was calculated within each category of the stratified factor . to distinguish severely and less severely affected famine areas more appropriately , we performed sensitivity analysis by using a more stringent cutoff point , i.e. , we used an excess death rate 100% to define the severity of famine . in addition , we performed analyses by using the cohort born during october 1 , 1962 , to september 30 , 1968 , as a nonexposed cohort for association analyses , or by excluding participants with a family history of diabetes . basic characteristics of the study population are shown in table 1 . in our main study population ( n = 7,874 ) , 1,005 ( 12.8% ) subjects had been exposed to the chinese famine during fetal life , and 4,915 ( 62.4% ) subjects had been exposed during childhood . as compared with the nonexposed individuals , fetal - exposed subjects were 0.9 cm shorter as adults , and childhood - exposed subjects were 1.5 cm shorter ( table 1 ) . the prevalence of hyperglycemia among adults in the nonexposed , fetal - exposed , early childhood , mid childhood , and late childhood exposed birth cohorts was 2.4% , 5.7% , 3.9% , 3.4% , and 5.9% , respectively . basic characteristics of study population according to chinese famine exposure * * data are adjusted means ( se ) . adjusted factors included sex , educational level , family history of diabetes ( only for glucose ) , current smoking , alcohol use , and physical activity level . compared with the nonexposed cohort , p < 0.05 . in severely affected famine areas , fpg concentration was significantly higher in the fetal - exposed cohort than in the nonexposed cohort with a mean difference of 0.20 no significant difference was observed in the less severely affected famine areas ( p for interaction = 0.001 , table 2 ) . compared with nonexposed subjects , fpg was higher in the late childhood exposed cohort in both the severely affected famine areas and less severely affected famine areas . a significant interaction between the exposed cohort and areas was found only for the fetal - exposed cohort ( table 2 ) . concentrations of fasting plasma glucose and prevalence rates of hyperglycemia and type 2 diabetes by birth cohort and severity of the chinese famine area * data are adjusted means ( se ) for fasting plasma glucose , sex standard prevalence , and odds ratio for hyperglycemia and diabetes . adjusted factors included sex , education level , family history of diabetes , and current smoking , alcohol use , and physical activity level . subjects exposed to famine during fetal life in severely affected famine areas had a higher prevalence of hyperglycemia than the nonexposed cohort . the odds ratios were significantly different between the severe and less severe famine areas ( table 2 ) , suggesting a stronger famine effect in the severely affected famine areas . compared with the nonexposed cohort , subjects in the late childhood exposed cohort had a higher risk of hyperglycemia in both severely and less severely affected famine areas , but the odds ratios were not significantly different between the severe and less severe famine areas ( table 2 ) . a significantly higher prevalence of type 2 diabetes was observed among subjects exposed in late childhood as compared with the nonexposed cohort ( table 1 ) . however , table 2 shows that after stratification of this group by severity of famine exposure , no significant difference of type 2 diabetes risk was observed anymore between different famine cohorts . stratified analyses by dietary pattern , economic status , and bmi for severely affected famine areas are shown in fig . figure 2a1 shows that the prevalence of hyperglycemia was highest ( 18.9% ) in subjects in the fetal - exposed cohort and who consumed an affluent / western diet . as compared with the relatively nonexposed cohort , the odds ratio of hyperglycemia in the fetal - exposed cohort was 7.63 ( 95% ci : 2.4124.1 , p = 0.0005 ) for those who had an affluent / western dietary pattern , and 2.34 ( 95% ci : 0.826.70 , p = 0.112 ) for those with a traditional dietary pattern . prevalence of hyperglycemia among birth cohorts according to early life famine exposure and later life dietary patterns ( a1 and b1 ) , socioeconomic status ( a2 and b2 ) , and bmi ( a3 and b3 ) in severely ( column a ) and less severely affected famine areas ( column b ) . figure 2a2 shows that as compared with nonexposed subjects , the odds ratio of hyperglycemia in the fetal - exposed cohort was 6.20 ( 95% ci : 2.0818.5 , p = 0.001 ) in subjects with a higher adult economic status , and 1.68 ( 95% ci : 0.505.71 , p = 0.404 ) in subjects with a lower adult economic status . figure 2a3 shows that overweight subjects in the fetal - exposed cohort had the highest prevalence of hyperglycemia ( 13.9% ) . however , the risks of hyperglycemia were largely comparable in these two groups ; the odds ratio of hyperglycemia in the fetal - exposed cohort was 3.71 ( 95% ci : 1.1312.2 , p = 0.031 ) in overweight subjects and 4.37 ( 95% ci : 1.1516.5 , p = 0.030 ) in normal weight subjects , respectively , compared with the nonexposed cohort . similar analyses were performed in subjects exposed to less severely affected famine areas during fetal life and childhood ( fig . 2 , right column , graphs b1 , b2 , and b3 ) , but did not show consistent associations . when we defined the severely affected famine areas as those with an excess death rate 100% , the prevalence of hyperglycemia among the fetal - exposed cohort in severely affected famine areas increased to 8.1% , but this did not change the associations between fetal exposure to famine and risk of hyperglycemia in adulthood . in addition , neither using subjects who were born between october 1 , 1962 , and september 30 , 1968 , as a nonexposed cohort nor excluding subjects with a family history of diabetes materially changed the associations ( table 3 ) . prevalence rate of hyperglycemia by birth cohorts and severity of famine areas : sensitivity analyses all odds ratios used the nonexposed cohort as the reference cohort . adjusted factors include sex , educational level , family history of diabetes , and current smoking , alcohol use , and physical activity level in 2002 . in this study of a large sample of chinese adults , we found a significant association between severe famine exposure during the fetal period and an increased risk of hyperglycemia in adulthood . this association was stronger in subjects with a western dietary pattern or higher economic status in adulthood . several mechanisms might explain the associations between fetal famine exposure and risk of diabetes in later life . exposure to extreme starvation in rats led to poor development of pancreatic -cell mass and function and insulin resistance , which might persist in later life ( 18 ) . a poor intrauterine environment may also reduce skeletal muscle development ( 19 ) , which may subsequently lead to insulin resistance in peripheral tissues ( 20 ) . it has also been suggested that stress suffering from fetal famine exposure could change the setpoint of the hypothalamic - pituitary - adrenal ( hpa ) axis , which could result in long - term changes in secretion of neuroendocrine mediators of the stress response , and predispose to cardiovascular and metabolic disease in later life ( 21,22 ) . to our knowledge , thus far three studies have assessed the associations of exposure to famine with measures of glucose intolerance . these studies were performed in the netherlands ( the dutch famine study ) ( 4,5 ) , russia ( the leningrad siege study ) ( 6 ) , and china ( our chinese famine study ) . the dutch famine study reported higher 2-h glucose and insulin levels among subjects who were exposed to famine during fetal life ( 4,5 ) , but this association was not observed in the leningrad siege study ( 6).the inconsistent results might be due to differences in postnatal environmental life exposures . although the dutch population rapidly developed into a wealthy and rich population after the famine , the leningrad people remained relatively poor . in our study , we observed that fetal exposure to the severe chinese famine increases the risk of hyperglycemia in adulthood , which was exacerbated by an unhealthy adult diet and higher economic status . our results support the hypothesis that exposure to a nutritionally rich environment modifies the association between fetal famine exposure and disease in later life ( 1,20,23 ) . the association between fetal famine exposure and hyperglycemia was stronger in participants with an affluent / western dietary pattern . these subjects were , to a large extent , less poor and more highly educated ( 15 ) , and they have benefited most from dramatically enhanced economic opportunities and have broken away from traditional chinese food patterns ( 15 ) . their diet is characterized by a high intake of meat , eggs , dairy , sugary beverages , edible oils , and a low vegetable use ( 15 ) . apparently , this nutrition rich environment did not match the fetal starvation environment that people of fetal exposed cohort experienced , which in turn increased the risk of hyperglycemia in later life ( 1,20,23 ) . our study used annual mean income as the cutoff to categorize economic status ( 2,000 chinese yuan / person / year ) . subjects in the lower economic group might consume mostly traditional plant foods with little meat . therefore , the discrepancy between the nutritional environment in adulthood and fetal undernutrition conditions may be less evident for those with a higher economic status . in other words , there was probably greater mismatch between in utero and adulthood environments in the higher economic group , which triggered an increased prevalence of hyperglycemia in the fetal - exposed cohort . similar results were described in the dutch famine study ( 4 ) , showing that 2-h glucose concentrations were especially high among people exposed to the famine during fetal life and who became obese in later life . however , the relative risk of hyperglycemia in overweight subjects was not different from that in normal weight subjects . this may be partly due to the increased prevalence of hyperglycemia in the nonexposed cohort in overweight subset . these results therefore indicate that both improving fetal nutritional environmental and controlling bmi in later life are important for prevention of a disturbed glucose metabolism . childhood nutritional status , particularly during infancy , is another key factor in influencing the propensity to develop disease in adulthood ( 23 ) . animal studies have shown that postnatal caloric restriction might hamper -cell development ( 24 ) and might disturb glucose metabolism in later life in rats ( 25 ) . our study found significantly increased fpg in the early childhood exposed cohort in the severely affected famine areas , but no significant differences in fpg in the less severely affected famine areas . we also observed a higher risk of hyperglycemia among subjects exposed in late childhood in both severely and less severely affected famine areas . these results suggest that famine exposure during childhood may increase the risk of hyperglycemia in later life . however , we can not exclude a potential cohort effect , such as aging ( 26 ) . similar risks of hyperglycemia among subjects exposed during childhood in both famine - exposed areas and nonfamine - exposed areas suggest rather a cohort ( older age ) effect than a famine effect . however , since almost all rural regions in china were affected by the famine during 19591961 , no valid nonfamine - exposed cohort comprising subjects born in the same time period was available . thus , the association between childhood exposure to famine and risk of hyperglycemia needs to be studied in more detail . first , we assumed that the residents we investigated at the time of the survey were born in the same province and in a similar rural area . however , severe restrictions on migration and relocation in china made our sample quite stable . migration with permanent resident permission still needed to be approved by authorities on a case - by - case basis in china . according to the 2000 china national population census , 2.68% of the rural population lived in provinces other than the provinces of their birthplaces ( 27 ) . our study sample was based on the residence registration system ; only subjects with permanent resident permission in local areas were involved in our study . therefore , we do not expect that intraprovince migration leading to measurement error in the coding of birth place is a major concern in our results ( 12 ) . second , subjects in our fetal - exposed cohort may have actually experienced severe famine during both the fetal period and the infancy period because the famine lasted approximately 3 years . it was therefore difficult to distinguish whether the fetal period or the infancy period was more important . however , the early childhood cohort also included subjects exposed to famine in infancy , which did not have a substantial influence on the risk of hyperglycemia . thus , our results indicate that the fetal period should be considered as the primary critical period . third , our subjects who experienced severe famine in the fetal period were in their early 40s in 2002 , and the cases of type 2 diabetes were few . the small numbers may partly explain why we did not observed significant associations with the risk of type 2 diabetes . we used the excess death rate as an indirect measure of famine exposure . with this method , we could not distinguish death due to famine from death due to unfavorable weather conditions or infections . we also did not have reliable information about individual food availability during the famine period . therefore , from our data , we can not conclude that the higher risk of hyperglycemia among subjects exposed to famine is exclusively due to malnutrition in early life . however , nutrition deficiency was highly prevalent during the chinese famine . china 's grain output declined by 15% in 1959 and in the following 2 years , and its food supply plunged further to 70% of its 1958 level ( 8) . as almost all foods were delivered through communal kitchens at that time , no social groups were spared from the effects of the famine ( 9 ) . however , since the famine effect on glucose intolerance did not depend on birth size in the dutch famine study ( 4 ) , we do not consider the lack of information about individual birth outcomes as a major limitation . in conclusion , we found that exposure to severe famine in fetal life increased the risk of hyperglycemia in adulthood . the mismatched nutrition postnatal environment represented by a western dietary pattern and improved economic status further increased susceptibility to hyperglycemia in those who experienced fetal exposure to famine . together with previous studies , our study emphasizes that early life environment is critical for the risk of hyperglycemia in adult life .

objectiveearly developmental adaptations in response to undernutrition may play an essential role in susceptibility to type 2 diabetes , particularly for those experiencing a mismatched rich nutritional environment in later life . we examined the associations of exposure to the chinese famine ( 19591961 ) during fetal life and childhood with the risk of hyperglycemia and type 2 diabetes in adulthood.research design and methodswe used the data for 7,874 rural adults born between 1954 and 1964 in selected communities from the cross - sectional 2002 china national nutrition and health survey . hyperglycemia was defined as fasting plasma glucose 6.1 mmol / l and/or 2-h plasma glucose 7.8 mmol / l and/or a previous clinical diagnosis of type 2 diabetes.resultsprevalences of hyperglycemia among adults in nonexposed , fetal exposed , early - childhood , mid - childhood , and late - childhood exposed cohorts were 2.4% , 5.7% , 3.9% , 3.4% , and 5.9% , respectively . in severely affected famine areas , fetal - exposed subjects had an increased risk of hyperglycemia compared with nonexposed subjects ( odds ratio = 3.92 ; 95% ci : 1.649.39 ; p = 0.002 ) ; this difference was not observed in less severely affected famine areas ( odds ratio = 0.57 ; 95% ci : 0.251.31 ; p = 0.185 ) . the odds ratios were significantly different between groups from the severe and less severe famine areas ( p for interaction = 0.001 ) . in severely affected famine areas , fetal - exposed subjects who followed an affluent / western dietary pattern ( odds ratios = 7.63 ; 95% ci : 2.4124.1 ; p = 0.0005 ) or who had a higher economic status in later life experienced a substantially elevated risk of hyperglycemia ( odds ratios = 6.20 ; 95% ci : 2.0818.5 ; p = 0.001).conclusionsfetal exposure to the severe chinese famine increases the risk of hyperglycemia in adulthood . this association appears to be exacerbated by a nutritionally rich environment in later life .

with the goal of discovering the genes that contribute to the risk of common diseases , numerous susceptibility loci have been identified using linkage analysis . despite replication of many of these linkages in a second sample , some exceptions include tbc1d1 , identified as the gene responsible for the obesity linkage on chromosome 4p15 - 14 , and hoxb13 , identified as the gene responsible for the prostate cancer linkage on chromosome 17q21 - 22 . however , for many other common disease linkage signals , the underlying causal genes and variants await discovery . the expectation that a single gene accounts for a linkage peak may contribute to the difficulty in identifying causal genes . on the contrary attributed a triglyceride linkage on chromosome 7q36 to variants in five genes and christians et al . found that fine - mapping caused a single quantitative trait locus ( qtl ) for body size in mice to resolve first into three qtls , then one of those to split into two . furthermore , the clustering of causal genes may have stymied the multi - group effort to identify type 2 diabetes ( t2d ) genes on chromosome1q ; two strong associations present in populations of european ancestry failed replication and confirmation in other ethnic groups . these examples suggest that abandoned linkage findings might yet reveal susceptibility genes if reappraised while considering the possibility of multiple causal genes . as for other common diseases , the challenge is even greater in african american ( aa ) populations where both prevalence and genetic diversity are higher , and pathophysiology may differ . genome wide case - control association studies ( gwass ) have identified single nucleotide polymorphisms ( snps ) with small effects on t2d risk , but few of the associated snps , identified primarily in european ancestry populations , are replicated in aas and few snps have been identified in aa populations . in general , the widespread rejection of family studies in favor of gwass has failed to produce the promised prognostic and diagnostic variants for t2d . as a resource for the discovery of genes related to t2d and its complications , the american diabetes association established the genetics of niddm ( gennid ) study . from 1993 to 2003 we have used this resource to increase understanding of the genetics of t2d by applying linkage and family - based association analysis to the african american ( aa ) subset of the gennid sample . genome - wide linkage analysis using genotypes on 5,914 snps identified chromosomal regions that potentially harbor risk genes for t2d and age of t2d diagnosis ( aod ) . the strongest signal for t2d occurred on chromosome 2 at 95121 megabases ( mb ) , with weaker support for aod in the same region and for t2d at 6895 mb . both t2d and aod also showed linkage on chromosome 13 at 1930 mb , but linkage was limited to t2d on chromosome 7 at 5079 mb and to aod on chromosome 18 at 3165 mb . other analyses inferred pleiotropy with triglyceride in the chromosome 2p region and pleiotropy with obesity in the chromosome 13 region . herein we present linkage and association analyses on the aa subset of the gennid sample using genotypes on 9,203 fine - mapping snps added to the genome scan snps used previously . our first goal was to more precisely localize the t2d susceptibility genes in each of the five chromosomal regions identified in the genome scan ( chromosome 2 at 6895 and 95121 mb , chromosome 7 at 5079 mb , chromosome 13 at 1930 mb , and chromosome 18 at 3165 mb ) . our second goal was to test for association of the gene - based fine - mapping snps with t2d and aod in order to identify genes related to t2d in aas . the gennid study ascertained families through sibling pairs each with a t2d diagnosis . during phase 1 , extended family members were also studied ; one site ascertained aas . during phase 2 , data collection beyond the sibling pair was limited to parents , or , if parents were unavailable , unaffected siblings ; five sites ascertained aas . during phase 3 , only affected sibling pairs and trios were studied ; 5 additional sites ascertained aas . in total , 1,496 aa members of 580 pedigrees were studied at 10 sites . data cleaning and re - evaluation of t2d diagnoses by current criteria reduced the analysis sample to 1,344 members of 524 pedigrees ; for this study , we selected an informative subset of 1,077 members of 415 pedigrees . t2d diagnoses , originally following national diabetes data group criteria , were re - evaluated using current criteria prior to all analyses ; 84 affected and 13 unaffected sample members were re - assigned as unknown . body mass index ( bmi ) was computed from height and weight obtained from physical examination . we selected a subset of the sample for fine - mapping by excluding unaffected individuals below age 53 , the age by which onset had occurred in 75% of cases , and then any individual , either affected or unaffected , who consequently had no family members in the sample . this sample subset maintained significant lod scores in each of the linkage regions and produced power over 80% to detect association . power to produce a p - value of 0.00001 was estimated from simulation of 1000 replicates of a snp with minor allele frequency 0.4 and heterozygous effect a 10% increase in penetrance at age 50 for t2d or 3 year increase in aod . for fine - map genotyping , we used snagger ( http://snagger.sourceforge.net/ ) to select tagsnps within genes in the linkage regions on chromosomes 2 , 7 , 13 , and 18 . using hapmap phase 1 and 2 ( http://hapmap.ncbi.nlm.nih.gov ) , variants in the region were tagged at linkage disequilibrium ( ld ) r 0.7 . each snp selected had minor allele frequency > 0.1 and illumina design score > 0.4 ; snp pairs had minimum spacing of 60 base pairs ( bp ) . the genetic map locations of the snps in centimorgans ( cm ) were obtained from the rutgers combined linkage - physical map of the human genome ( http://compgen.rutgers.edu/maps ) ; locations of snps not mapped directly were estimated using physical positions from ncbi dbsnp build 123 in a smoothing calculation ( conway institute bioinformatics service ; http://integrin.ucd.ie ) . genotyping errors were identified using pedcheck and merlin ; we zeroed 4978 genotypes of 710 snps , 74 identified by pedcheck and 4904 by merlin . genotypes for 24 snps , available on less than 95% of the sample following data cleaning , were retained after confirmation of minimal effects on the results . multi - point identity by descent ( ibd ) probabilities were computed at each cm using merlin , treating as haplotypes snp sets with pairwise ld r > 0.7 . we identified 279 , 175 , 133 , and 259 snp sets on chromosomes 2 , 7 , 13 , and 18 , respectively . likelihood analysis , as implemented in jpap , was used for univariate linkage analysis of unadjusted t2d ( ut2d ) , t2d adjusted for bmi ( bt2d ) , and aod and for bivariate association analysis of ut2d and aod . aod was adjusted for gender and modeled as a normal density with mean , standard deviation , and gender effect as parameters . t2d risk was modeled to account for aod in affected pedigree members , while allowing for censored observations with age , gender , and bmi ( for bt2d only ) effects and penetrance as parameters . for each trait , additional parameters included heritability , a quantitative trait locus ( qtl ) effect in linkage analysis , and an additive snp effect in association analysis . we applied variance components linkage analysis using the univariate model for each trait in conjunction with the ibd probabilities . at each cm across the 5 regions , all parameters were estimated for aod while only heritability and qtl effect were estimated for ut2d and bt2d with all other parameters for those traits fixed at estimates obtained upon maximizing the likelihood while correcting the likelihood for the ascertainment of each pedigree through an affected sib pair . the lod score at each cm was computed as the common logarithm of the ratio of the maximized likelihoods with the qtl effect estimated to the maximized likelihood with the qtl effect set to zero . we assessed each fine - mapping snp for association with ut2d and aod by coding the snp genotype as an additive covariate and testing its effect on both traits in a bivariate model . p values were determined using a 2 degree of freedom statistic computed as twice the natural logarithm of the ratio of the maximized likelihood with covariate effects estimated for both t2d and aod to the maximized likelihood with both effects set equal to zero . we controlled for multiple testing separately within each of the 5 linkage regions by specifying a false discovery rate of 0.05 accounting for 1291 , 1780 , 1819 , 1336 , and 2977 fine - mapping snps for chromosomes 2p , 2q , 7 , 13 , and 18 , respectively . we tested for an independent effect of each secondary associated snp , conditional on the snp within the same gene that attained the highest significance , by comparing the maximized likelihood estimating t2d and aod covariate effects for both snps simultaneously to the maximized likelihood estimating t2d and aod covariate effects for the most significant snp alone . p < 0.05 after bonferroni correction for the number of secondary snps in the gene supported the independence of the secondary snp . we genotyped 9,203 snps within five regions encompassing a total of 133 cm and 128 mb ( table 1 ) and merged them with 244 ( 36 duplicates ) genome scan snps . spacing between snps averaged 0.0145 cm ( 13,910 bp ) and ranged from 0.0 to 5.3 cm and from 26 bp to 6 mb . to comply with the sample size limitations of the cidr genotyping platform , we selected a subset of the original linkage sample to be informative for both linkage and association . the genotyped subset included 1077 members of 415 pedigrees that ranged from 2 to 11 members ( 1 to 8 were genotyped ) . in this subset , aod and bmi differed little from the complete sample , but the unaffected sample members , selected for a minimum age of 53 years , were older ( table 2 ) . we previously identified 5 linkage peaks within four chromosomal regions from autosomal scans of t2d and aod ; the chromosome 2 region contained 2 peaks separated by 30 mb and the centromere . using updated genetic map positions for the genome scan snps , we repeated the linkage analyses on the smaller sample : the genome scan lod scores for all 5 peaks ( table 3 ) generally agreed with our published lod scores for bt2d and aod and for ut2d . upon adding the fine - mapping snps , linkage evidence remained in all 5 regions , but strengthened only on chromosome 2 ( table 3 ) . on chromosome 18 , only aod supported linkage , although ut2d provided weak support ( lod = 2.62 ) upon elimination of sample members with bmi > 45 . the most remarkable effect of fine - mapping was the splitting of the lod score curves into multiple peaks in all of the regions except on chromosome 18 . additional evidence that multiple susceptibility genes contribute to the linkage signals derived from the identification of associated snps that reside in multiple genes within each of the 5 regions , including on chromosome 18 ( table 4 ) . the number of associated snps ranged from 4 on chromosome 13 to 17 on chromosome 2q ; the number of associated genes ranged from 2 on chromosome 13 to 9 on chromosome 7 . although 20 of the 27 associated genes were identified through a single snp , 5 genes were identified through 2 or 3 snps and arhgap25 and dpp10 were identified by 8 and 10 , respectively . however , six associated snp pairs exceeded ld r 0.7 : arhgap25 ( rs6714065 and rs7605681 ) , ctnna2 ( rs968820 and rs1368915 ) , dpp10 ( rs843417 and rs1823267 , rs10204212 and rs13432035 , rs4848376 and rs11694256 ) , and hip1 ( rs1179625 and rs1179622 ) . nevertheless , conditional association analysis supported the independence of two or more snps in arhgap25 , polr1b , dpp10 , and mtus2 ( supplementary table s1 ) . the fine - mapping of five chromosomal regions allowed us not only to confirm our genome scan linkages , but also to infer the presence of multiple t2d susceptibility genes in each of the regions . two types of evidence supported the presence of multiple susceptibility genes underlying the linkage peaks . first , except chromosome 18 , each lod score peak split into at least two peaks when fine - mapping snps were added to the linkage analysis . second , in every region including chromosome 18 , two or more potential susceptibility genes were identified through the association with t2d and aod of fine - mapping snps residing in those genes . in fact , martin et al . attributed many failures to identify causal variants to the incorrect assumption that a single or a limited number of variants are responsible for a linkage signal . as evidence , they identified variants in five genes that fully accounted for a linkage peak for plasma triglyceride level . if small effects are typical of common disease susceptibility genes , their detection through linkage analysis may be limited to locations where the genes cluster . however , fine - mapping using next generation sequencing may provide sufficient power necessary to detect even isolated genes . although chromosome 18 failed to show t2d linkage and chromosome 7 failed to show aod linkage , we nevertheless expect that the susceptibility genes residing in all regions both increase t2d risk and decrease aod . variation between regions in the information available undoubtedly affects the relative strength of the corresponding lod scores for t2d or aod . as evidence , the elimination of t2d cases with bmi > 45 kg / m revealed a weak t2d linkage on chromosome 18 and fine - mapping revealed aod linkages on chromosomes 2p and 2q . the lod scores are generally lower for aod than for t2d as expected since aod reflects onset age imprecisely and the familial correlation of aod may partially result from temporal clustering of t2d diagnoses among relatives . nevertheless , aod proved sufficiently accurate to produce linkage evidence in 4 of the chromosomal regions . few of the associated genes have previously been reported to associate with t2d or related traits . the exceptions include grb10 with t2d , nedd4l with diabetic nephropathy , and lipg with lipid metabolism . novel candidates reported herein include a cytokine ( il36b ) and genes involved in lipid metabolism ( acoxl ) and cell - cell and cell - matrix adhesions ( magi2 , cldn4 , ctnna2 ) . interestingly , the candidates also included genes involved in williams - beuren syndrome ( wbscr28 , wbscr17 , cldn4 ) , whose sufferers have a high prevalence of diabetes and pre - diabetes . dpp10 is related to dpp4 , whose inhibitors are oral anti - hyperglycemics used in t2d therapy , and dpp6 , which contributes to a triglyceride linkage , however , unlike dpp4 , ddp10 lacks serine protease / dipeptidyl peptidase activity . support for the functionality of these genes derived from evidence that their expression levels are affected by our associated snps or proxies with high ld . we tested 14 snps in the 5 genes for which expression levels from transformed lymphocytes were measured on a subset of 160 sample members ; nominal significance was obtained for one : p = 0.0148 for an effect of rs7579103 on arhgap2 expression levels . in other populations and various tissues , expression levels of arhgap2 , aff3 , polr1b , hip1 , lipg , and nedd4l showed evidence of an effect of an associated snp or its proxy ( p<1105 ) . for further insight into the nature of the associations , we haplotyped sets of 1530 fine - mapping snps , each encompassing two or more of the associated snps reported in table 4 . for arhgap25 , two haplotypes associated with t2d : the first extended across 4 of the associated snps and contained the risk alleles for all 4 ; the second extended across 6 associated snps and contained the risk alleles for all 6 , but differed from the first haplotype for alleles at other snps . we propose that each haplotype harbors a distinct causal variant , despite sharing associations with the same risk alleles , demonstrating the complexity of disease associations . the advantages include the opportunity to exploit information on local ancestry to localize disease susceptibility genes . as with any such analysis , confirmation of these findings awaits replication in another sample . in summary , linkage and association analysis using genotypes on an additional 9,203 fine - mapping snps added to five chromosomal regions confirmed each linkage and identified potential susceptibility genes in each of the regions .

we previously localized type 2 diabetes ( t2d ) susceptibility genes to five chromosomal regions through a genome - wide linkage scan of t2d and age of diagnosis ( aod ) in the african american subset of the gennid sample . to follow up these findings , we repeated the linkage and association analysis using genotypes on an additional 9,203 fine - mapping snps selected to tag genes under the linkage peaks . in each of the five regions , we confirmed linkage and inferred the presence two or more susceptibility genes . the evidence of multiple susceptibility genes comprised : 1 ) multiple linkage peaks in four of the five regions ; 2 ) association of t2d and aod with snps within 2 or more genes in every region . the associated genes included 3 previously reported to associate with t2d or related traits ( grb10 , nedd4l , lipg ) , and 24 novel candidate genes including genes in lipid metabolism ( acoxl ) and cell - cell and cell - matrix adhesion ( magi2 , cldn4 , ctnna2 ) .

the circadian system organizes the different biological functions in 24 h , such as sleep / activity , temperature , heart rate , glucose level , cortisol production , and oxidative stress . in mammals , this system is organized by a central clock localized in the suprachiasmatic nucleus of the hypothalamus ( scn ) and in a series of peripheral oscillators such as the liver , lung , adrenal gland , fibroblast cells , and others tissues [ 2 , 4 ] . the peripheral oscillators are synchronized every day via nervous or humoral signals , and the most important humoral signal is the melatonin hormone , secreted by the pineal gland during the dark hours , and its impairment is associated with different disorders such as insomnia , cardiovascular disease , and cancer . the molecular clock is organized by transcriptional / translational feedback loop of clock genes named clock , bmal1 , per13 , and cry1 - 2 . at the molecular level , the complex clock - bmal1 stimulates the expression of negative regulators per13 and cry1 - 2 , and their protein inhibits the effect of the heterodimer clock / bmal1 [ 6 , 7 ] . moreover , the molecular clock has different modulators which give fine tuning of output signals such as rev - erb , a negative regulator of bmal-1 expression [ 79 ] , sirt1 , a regulator of clock - mediated acetylase activity [ 10 , 11 ] , and pgc1 , a stimulator of bmal1 expression [ 12 , 13 ] ( see figure 1 ) . this system provides an output signal to genes such as hexokinase , dbp [ 1517 ] , vegf , steroidogenic enzymes star , 3-hsd , and wee-1 , giving a circadian oscillation of physiological functions such as metabolism , angiogenesis , cortisol production [ 2 , 19 ] , and cellular proliferation . the molecular clock can be modified by environmental changes and our modern lifestyle , resulting in physiological alteration and risk of pathologies ; for example , during the monkey 's pregnancy , the light exposition during night hours induces a lower body temperature and absence of circadian rhythm of temperature in the newborn . in humans , different reports showed that the alterations of the circadian system increased the risks of cancer [ 2123 ] , preeclampsia , diabetes [ 3 , 25 ] , and mood disorder . curiously , a large quantity of polymorphisms has been detected in clock genes , which can be influencing the development of diseases through different physiological systems . this review will primarily focus on the hypothesis which states that the variation of genetic components of the circadian system , similar to environmental changes , can affect several physiological systems and produce an elevation of the risk of developing a disease . in recent years , there has been a significant increase in available information from polymorphic variations and epigenetic modification over clock genes and their risk of diseases . the report of health from the united states ( 2014 ) showed about 10% of population have a poor health associated with pathologies such as obesity ( 35.5% ) , hypercholesterolemia ( 30% ) , diabetes ( 32% ) , and cancer ( 6.4% ) . curiously , despite its low frequency in population , cancer is the second cause of death in the united states . the above pathologies add the infertility , which affects about 10.9% of women in the united states and 17% of women in other developed countries . during short - stay in the hospital , mood disorder and psychosis are the principal causer of hospital stay during 2014 and represent the third cause of morbidity worldwide . this present research reports the last single nucleotide polymorphism ( snp ) associated with an elevated risk of developing principal pathologies worldwide such as mood disorder , infertility , cancer , metabolism and diabetes and addictions , and a description of usefulness for a more detailed study in other pathologies . in mammals , the circadian system is a supraphysiological system that regulates biological functions every 24 h such as glucose homeostasis , temperature , blood pressure , tone in the coronary artery , and heart rate . the bmal1/clock complex target genes are related to the circadian expression of metabolic pathways as was observed by hatanaka et al . through high resolution the author detected a circadian expression of ( i ) glucose metabolism - related genes such as glut2 , por , pck1 , and gys2 and ( ii ) cholesterol metabolism - related genes such as cyp2a4 , cyp2a5 , cyp4a14 , cyp7a1 , and cyp2c55 ; and considering the very important role these systems have in metabolism , any alteration of them can have negative health effects . today there are several metabolic problems affecting the population , such as obesity , hyperglycemia , dyslipidemia , and hypertension . the cooccurrence of three or more metabolic disorders , including obesity , is defined as metabolic syndrome , showing a prevalence in the united states during 2009 - 2010 of about 22% of adults ( 21.4626.15 ) . the insulin resistance or type 2 diabetes is one of the most important metabolic diseases currently affecting about 366 million people , causing more than 3.8 million deaths , and showing a rise in the number of affected people as observed in the united states population during 19992010 . several factors could be increasing the risk of death by diabetes , such as dyslipidemia , hypertension , and obesity , all of which are driving to an elevated risk of cardiovascular disease . a variant of this pathology can also develop during the pregnancy ( gestational diabetes mellitus ) and is characterized by glucose intolerance in the mother and adverse consequences for her offspring , such as an increase in blood pressure , body mass index ( bmi ) , and body fat and a decrease in hdl levels . at the molecular level , both diseases have in common the impairment of signal transduction of insulin receptors such as pi3kinase / akt and ras / map kinases [ 42 , 45 , 46 ] , leading to metabolic dysregulations such as inhibitions of glycogen synthesis , impaired translocation of glut4 to plasmatic membrane , or antilipolytic effects of insulin in white adipose tissue [ 39 , 46 ] . meta - analyses from 448 articles reporting shift work and health consequence have detected a strong relationship between shift work , a potent chronodisruptor , and diabetes mellitus type 2 ( odd ratio about 1.42 times higher than day workers ) . however , this is not the only chronodisruption which we are exposed too . in animal models exposed to light / dark patterns similar to shift work , the length of the working day and changes of feeding times induce a modification of the liver weight and plasmatic glucose and a modification of the circadian profile for glucose , insulin , and triglyceride . these findings add to the observation in the circadian disruption in mutant animals , showing alterations of cholesterol metabolism , abolition of the circadian production of glycerol , free fatty acids , and impaired expression of rate - limiting lipolytic enzymes such as lipase , all of which add to increased weight gain , adipocyte hypertrophy , high level of glucose , glucose intolerance , and hypersecretion of insulin [ 4952 ] . polymorphic variations of clock genes can be incrementing the risks of developing a disease similar to chronodisruption by environmental changes as it has been detected in human metabolic disorders ( see table 1 ) . for example , the genotyping of clock genes in 346 greek pregnant women and their risk of diabetes was performed , detecting that the polymorphisms of bmal1 rs7950226 and rs11022775 are associated with gestational diabetes mellitus ( p = 0.025 , or = 1.46 and p = 4.455e 06 , or = 2.64 , resp . ) , while the study of the haplotype analysis of rs7950226/rs11022775 showed a major frequency in women with gestational diabetes mellitus ( p = 0.0069 , or = 6.96 ) . similarly , a study performed in subjects from the united kingdom and pakistan reported that cry1 and cry2 polymorphisms rs2292912 and rs12315175 , respectively , are associated with diabetes ( p = 0.015 and 0.008 , resp . ) . moreover , the variant rs12315175 for cry2 has a tendency to elevate the risk at about 5% compared to the variation of cry1 ( or = 1.05 and 0.95 , resp . ) . also , the authors did not find associations between diabetes and bmal1 polymorphisms rs7950226 and rs11022775 , as occurs in gestational diabetes mellitus . genotyping of 19,000 adults from northern sweden for variants rs8192440 for cry1 and rs11605924 for cry2 is associated with higher level of glucose concentrations at 2 hours ( p = 0.06 and p = 0.005 , resp . ) . another study genotyping 1304 individuals from 424 british families , containing at least one patient with diabetes type 2 ( diabetes in families study collection ) , demonstrates the relation between bmal1 polymorphisms rs7950226 and rs11022775 and diabetes ( p = 0.002 ) , reinforcing what was previously described . similar relations are observed for bmal2 polymorphism rs7958822 in obese men and women ( or 2.2 and 2.7 , resp . ) and the deletion / insertion of 54 base pair sequences of five repeat alleles on per3 gene ( rs57875989 ) [ 57 , 58 ] . in contrast , the per2 polymorphism rs7602358 is associated with a protection from type 2 diabetes in the uk population , which suggests that not all polymorphisms are negative for health . at the level of dyslipidemia , an interesting correlation has been detected between small dense ldl level and polymorphism of clock genes . in individuals carrying the polymorphism associated with the clock gene ( rs1801260 ) , the genotype tt or tc showed a major level of small dense ldl , which leads to increased triglycerides and increased risk of cardiovascular and obesity diseases , similar to the metabolic syndrome . likewise , genotyping in the japanese population identified clock gene polymorphism rs1801260 associated with higher odds ratio ( or ; 1.5 ) of type 2 diabetes ; in contrast , a multicenter study detected the clock polymorphism rs4580704 is associated with prevention of diabetes and cardiovascular disease . the haplotypes of rs10002541 and rs4864546 from clock ( cg and tg variations ) are associated with abdominal obesity in chinese population ( or 0.74 and 1.70 , resp . ) and polymorphism rs4864546 is associated with low level of hdl / apolipoprotein a1 ratio in spain . moreover , clock polymorphisms rs12649507 and rs3749474 are associated with higher intake of polyunsaturated fatty acid and fat intake , which can modify the body mass index ( bmi ) . this is an antecedent which , bearing in mind the ideas above , suggests the genetic components of the circadian system as a critical factor in the development of metabolic diseases . in humans , infertility affects about 9% of couples , and about one - third of infertility cases associated with idiopathic male infertility are multifactorial , with 50% due to genetic abnormalities . the circadian system is important during reproduction and development [ 89 , 90 ] ; for example , it is involved in the timing of the lh surge [ 90 , 91 ] , stimulation of ovulation [ 90 , 92 ] , and regulating the level of steroidogenic acute regulatory protein ( star ) expression and is critical for cholesterol translocation inside mitochondria and sperm count . the genetic factors of clock genes are implicated in the pathogenesis of infertility ( see table 2 ) , as it occurs in male partners of infertile couples . the genotyping of male partners detected a correlation between single nucleotide polymorphism of clock rs11932595 , rs6811520 , and 6850524 , with infertility ( p < 0.05 and or ranged between 1.4 and 1.9 ) . similarly , the analysis of bmal1 polymorphism rs4757144 in slovenian and serbian caucasian men showed a significant correlation with infertility ( p = 0.047 ) , suggesting that clock genes clock and bmal1 contribute to successful fertilization . mood or affective disorders are important causes of morbidity , where we can highlight depressive pathologies and hypomaniac and maniac disorders as major diseases affecting the population [ 29 , 66 ] , and it can be highlighted that depressive pathologies are the third largest source of morbidity in the world . curiously , a potent correlation has been detected between mood disorders , sleep / activity , and the circadian system suggesting that the circadian system can be modulating the neuronal activity . during pregnancy , the prevalence of mental illnesses in women is about 8% , suggesting an imbalance between physiology of sleep and/or the circadian system . this can be observed in the onset of a mental disease as has been observed in the impaired circadian production of melatonin during the pregnancy of depressed women ( approximately 34 weeks of gestation ) , which shows an advance onset time of melatonin production of about 40 minutes and a minor production during the dark hours [ 97 , 98 ] . moreover , workers who do shift work ( circadian disruption ) showed decreased alertness , cognitive functions , mood , social and work activities , and health [ 26 , 99 ] , which are associated with an impaired circadian system via melatonin suppression , all of which leads to the appearance of a mood disorder . for this reason , we can say that the circadian system may contribute to the risk of developing a mood disorder and the genetic component of clock genes can be involved in the development of mood pathologies ( see table 2 ) . at the genetic component level of the circadian system , a study of 744 people who carry polymorphisms rs2291739 and rs11171856 from tim , a member of the clock gene family which interacts with per1 - 2 proteins [ 100 , 101 ] , showed that they have an elevated risk of developing mood disorders ranging between 19 and 23% ( or 1.19 and 1.23 , resp . ) . moreover , gene variants of positive regulator clock have also been associated with mood disorders . people who carry the variants rs1801260 and rs11932595 showed a major risk of developing a bipolar disorder ranging between 45 and 37% , respectively , in comparison to a patients without polymorphism ( or 1.45 and 1.37 , resp . ) . in the same study , it was also observed that the polymorphisms rs2291739 and rs11171856 from tim gene are associated with unipolar disease , with a risk ranging between 37 and 40% in comparison to a patient without polymorphism ( or 1.37 and 1.40 , resp . ) . / rs11022779g / rs1122780 t ( haplotype ) are associated with mood disorders and bipolar disease [ 29 , 66 ] . it has also been seen that the bipolar pathologies are associated with other genetic variations of bmal1 gene ( rs4757144 , rs1982350 , and rs1481892 ) and the negative regulator per3 ( rs2859387 ) . curiously , a study performed an indian families reported the bmal1 polymorphisms rs2279287 are associated with seasonal affective disorder . in addition , the gene variant of cry2 gene ( rs4132063 ) and deletion / insertion of 54 base pair sequences on per3 gene ( rs57875989 ) may also be contributing to mood disorders via increased risk of developing bipolar disease . for example , a study in south india showed the prevalence of five repeat homozygotes from rs57875989 is associated with bipolar disease ( or 1.72 ) but not with schizophrenia . at the level of depressive pathologies , a study performed in china compared 485 subjects ( control ) to 105 patients suffering from a depression disorder . the genotyping for clock genes showed that the variants of cry1 ( rs2287161 ) and cry2 ( rs10838524 ) are correlated to the depression disease with an odds ratio of 1.75 ( p = 0.012 ) , which suggests that a patient with the allele has 1.75 times more risk of developing depression . moreover the authors detected a single nucleotide polymorphism for tef gene , the variant rs738499 , is associated with 2.22 times more risk of development of depression ( odds ratio of 2.22 . finally , in a study performed in young adults , the risk of excessive intake of alcohol is minor when the polymorphism of per2 gene rs56013859 is present , suggesting a possible protector role for allelic variation of clock genes in addictions . in fact , this suggests that some polymorphisms are not negative for our health and it is necessary to study them in greater depth . cellular proliferation is a critical event for the survival and restitution of tissues of all living things and the circadian system is capable of delivering temporary information to the cell cycle [ 102104 ] . however , an uncontrolled cellular proliferation and an excessive tissue growth are observed by an altered cellular cycle during cancer , a pathology that showed a higher incidence in patients exposed to an impaired circadian system such as a shiftwork [ 106 , 107 ] . the cell cycle is a finely regulated process from a cell that is capable of generating multiple cells through a series of cell divisions , including four critical and successive steps named g1 phase ( growth phase 1 ) , s phase ( synthesis ) , g2 ( growth phase 2 ) , and m phase ( mitosis ) . during the cell cycle , cyclin - dependent kinases ( cdk ) are critical for the transition between different stages , for example , cdc2 plus cyclin b protein kinase form the complex cdk1 which regulates g2/m transition [ 108 , 109 ] . during dna replication , there are a number of controls or checkpoints that are critical in the cell cycle when dna damage is detected : the activation of the rad protein , which induces the action of cds 1 proteins , is triggered , as well as chk1 which are involved in cell cycle arrest via action of wee-1 and mik-1 proteins . however , the efficiency of this process changes depending on the time of the day when the lesion occurred . a lesion in the liver , occurring during the last light hours , induced a massive entry to m phase compared to a lesion which occurs early in the morning , which shows that the hour of surgery and the circadian system are critical for liver regeneration . similarly , oral mucosa is a highly proliferative tissue and it showed a circadian expression of clock genes bmal1 , per1 , and cry1 and thymidylate synthase activity , critical for dna synthesis during phase s. curiously , the peak of per1 mrna precedes the peak of thymidylate synthase activity , which suggests that the circadian system modulates the cellular proliferation in mucosa . wee-1 gene regulates cellular proliferation , and its promoter has three conserved sequence cacgtg ( e - box ) critical for the circadian expression of wee-1 . its protein is capable of inactivating the cdc2-cyclin b complex via phosphorylation which inhibits transition between g2/m . curiously , knock - down of bmal1 in carcinoma cells of the colon ( c26 cells ) , fibroblast cells ( l929 cell ) , and intestine epithelial cells ( iecs ) produces cellular proliferation in vitro and increments the size of tumor cells injected subcutaneously , via the inhibition of apoptosis and the reduction of the time transition between g2/m , reduction of p53 and wee-1 expression . moreover , the knock - out for clock genes bmal1 y per2 in mice previously exposed to gamma radiation caused in mice hyperplasic growth and development of lymphoma , hepatic carcinomas , ovary tumors , and osteosarcomas via reduction of p53 expression . a precedent that reinforces the idea that clock genes can be modulators of cellular cycle via wee-1 and p53 proteins . at the genetic component level , different studies reveal the importance of the polymorphic variant of clock genes ( see table 3 ) and how the chrono - type modulates the risk of cancer , such as observed in breast cancer , in which the risk is more elevated in premenopausal women ( or , 2.43 and 2.55 ; resp . ) . similarly , women showed evening or night preference such as shown in the case of a study performed on norwegian nurses working in night shift which revealed two polymorphisms associated with breast cancer . the risk is incremented when women spend more time working during night hours , but this risk is higher when women have the alleles for bmal1 rs2290035 ( or 1.91 ) , rs969485 ( or 1.64 ) , and rs3903529 ( or 2.77 ) or variant rs3750420 ( or 1.6 ) of roreb gene . similarly , an incremented risk is associated with breast cancer in women from france when they have the allele rs11932595 in clock gene ( or = 0.74 ) or in connecticut ( usa ) when they have the polymorphisms rs7698022 ( or 1.34 ) and rs1048004 ( or , 1.43 ) . curiously , when norwegian nurses are exposed to shift work during four nights , they showed three times more risk ( or 2.75 ) of developing breast cancer when they were carrying clock polymorphism rs11133373 , which suggests that the disruption of endogenous circadian rhythm by polymorphism associated with clock genes increases the risk of cancer . moreover , clock gene expression is induced in breast tissue from patients with breast cancer ; this expression is associated with hypomethylation of clock gene . the silencing of clock gene lowered when women carry polymorphisms associated with cancer such as rs10448004 and rs7698022 , which suggests that clock gene is a critical protagonist of cancer development . at level of colorectal cancer , a strong correlation is detected between cancer and the genetic component of the circadian system . for example , a polymorphism in the clock gene ( rs1801260 ) showed a major prevalence in a cancer patient ( p < 0.0001 , or = 1.78 for c - allele ) compared to a control patient . similarly , a screening performed in south carolina , usa , showed that a variation by deletion / insertion of a 54 base pair sequence on per3 gene ( rs57875989 ) is associated with a higher risk of colorectal adenoma formation with odds ratio within 2.15.1 . moreover , a population study performed on the residents of king county , washington ( usa ) , detected six different types of polymorphism , which are significantly associated with the risk and aggressiveness of prostate cancer . these genetics variants are rs1012477 for per3 ( or 1.3 ) , rs7602358 for per2 ( or 1.24 ) , and rs2289591 in per1 ( or 1.7 ) . similarly , a genotyping of a patient with prostate cancer showed a strong correlation between cry1 polymorphisms rs7297614 , rs1921126 , and rs12315175 and fatal prostate cancer ( or mean within 1.52 ) and a study conducted in china showed that the cry2 variant rs1401417 and the deletion / insertion of 54 base pair sequences on per3 gene ( rs57875989 ) were associated with a major risk of developing prostate cancer ( or ; 1.7 and 1.3 , resp . ) . a study performed in brazilian patients with pulmonary cancer showed a strong correlation of per3 polymorphism rs228644 and the risk of cancer ( or 1.99 ) . moreover , the authors reported the ancestral haplotypes for per3 rs228729 , rs228727 , rs707467 , rs228644 , and rs10462020 are associated with a higher cancer frequency ; similarly , a meta - analysis performed by literature search showed that the variant insertion / deletion of per3 rs57875989 is associated with an increase of cancer susceptibility in about 17% or 70% . in a similar way , a soft risk is associated with breast cancer in premenopausal women from india , which suggests there is a relation between per3 and cellular proliferation . the frequency analysis of 1,538 breast cancer cases and 1,605 controls in china for clock gene variants showed the strong associations between three snps in circadian clock genes and the risk of developing breast cancer . the variants for cry1 rs1056560 are correlated to cancer , which elevate the risk in about 11% ( or 1.11 ) ; per2 rs934945 in about 15% ( or 1.15 ) ; and clock rs3805151 in about 35% ( or 1.35 ) . in contrast , tim protection for breast cancer development is detected in patients that carry the c - allele of rs7302060 ( or , 0.54 ) . the g allele of rs2291738 and the c - allele of rs7302060 are associated with reduced risk of breast cancer among estrogen receptor ( ) or progesterone receptor ( ) positive breast cancer cases ( or , 0.46 and 0.36 , resp . ) . the exogenous expression of human clock in cell lines of colorectal carcinoma induces the cellular proliferation in about 28% . in contrast , knock - down of endogenous clock gene expression inhibits the cell proliferation in about 34% . moreover , exogenous clock inhibits the apoptosis ( 42% reduction ) via inhibition on apoptosis associated proteins expression of bax and bid and the increase of phosphorylation of akt . in vivo experiments by xenograft transplant of colorectal carcinoma cell line transduced with clock increase the tumor volume and tumor weight in about 61% and a 91% , respectively . however , the pharmacological inhibition of cry in human breast cancer by treatment with pharmacological agent ks15 inhibits the proliferation and cell viability by stimulation of wee-1 expression and stimulates the activity of heterodimer complex bmal1:clock [ 112 , 113 ] . curiously , two polymorphisms for clock gene cry2 , rs11038689 and rs1401417 , have a protective action over the mammary cancer , which suggests that not all polymorphisms associated with clock genes are negative for our health . non - hodgkin 's lymphoma is characterized by lymphoproliferation and advanced clinical stages , invasion to other tissues , and death . the estimated deaths from non - hodgkin 's lymphoma in the united states amounted to 19,790 during 2015 . analysis of cry2 variants showed the polymorphisms rs11038689 , rs7123390 , and rs1401417 increase the risk of lymphoma ( or , 2.34 ; 2.40 and 2.97 , resp . ) . moreover , a minor association of clock gene variants and glioma is detected for per1 rs2585405 , clock rs11133391 , and cry1 rs12315175 ( or , 1.16 ; 1.08 and 1.02 , resp . ) . genotyping of han chinese patients diagnosed with primary hepatocellular carcinoma showed an association between single snps of per3 rs228669 and cry1 rs3809236 with odds ratio of 1.41 and 1.26 , respectively . these precedents reinforce the idea that clock genes can be modulators of cellular cycle and that modulates the risk of cancer . the circadian system is a supraphysiological system which modulates different physiological systems , and any alteration of this can have a negative impact on human health . different chronodisruptors have been described in literature such as light / dark pattern and inhibition of melatonin production as occurs in shifts work . however , other factors can be contributing on a minor scale , such as mealtimes or a genetic component . the relevance of the genetic variation of clock genes and how it can interact with the environment is unknown . but it has been described that the genetic component in the population predisposes the development of different pathologies such as diabetes , dyslipidemias , obesity , mood disorders , and addiction , all of which suggest the importance of this system to our health . however , it is also necessary to say that the genetic component could be protecting our health such as in the case of polymorphisms associated with per2 and cry2 genes in diabetes , alcohol intake , and cancer . these findings will need to be implemented and evaluated at the genetic interaction level and also the way in which the environment factors trigger the expression of these pathologies will be examined . finally , prospective studies are necessary to assess the predictive potential of these markers and to implement early treatment with consequent cost reduction for the health system .

the circadian system is a supraphysiological system that modulates different biological functions such as metabolism , sleep - wake , cellular proliferation , and body temperature . different chronodisruptors have been identified , such as shift work , feeding time , long days , and stress . the environmental changes and our modern lifestyle can alter the circadian system and increase the risk of developing pathologies such as cancer , preeclampsia , diabetes , and mood disorder . this system is organized by transcriptional / tranductional feedback loops of clock genes clock , bmal1 , per13 , and cry1 - 2 . how molecular components of the clock are able to influence the development of diseases and their risk relation with genetic components of polymorphism of clock genes is unknown . this research describes different genetic variations in the population and how these are associated with risk of cancer , metabolic diseases such as diabetes , obesity , and dyslipidemias , and also mood disorders such as depression , bipolar disease , excessive alcohol intake , and infertility . finally , these findings will need to be implemented and evaluated at the level of genetic interaction and how the environment factors trigger the expression of these pathologies will be examined .

cellular adaptation requires biochemical processes including post - translational mechanisms to modify existing proteins . catalyzed by opposing kinases and phosphatases , reversible phosphorylation of serine , threonine , and tyrosine residues is now appreciated as a fundamental regulatory mechanism with the majority of phosphorylation ( > 99% ) occurring on serine and threonine residues [ 1 , 2 ] . due to their untapped therapeutic potential , protein phosphatases have been identified as promising targets for xenobiotic manipulation through rational drug design ( reviewed in [ 36 ] ) . in particular , the ubiquitously expressed protein phosphatase 2a ( pp2a ) has been proposed as a target for the treatment of a number of pathologies ranging from neurodegenerative diseases such as alzheimer disease to a variety of neoplasias [ 79 ] . compared to other members of the phosphoprotein phosphatase ( ppp ) superfamily of serine / threonine phosphatases , a detailed understanding of the mechanism by which pp2a recognizes substrates and mediates site - specific dephosphorylation remains to be developed . sequence and structural homology of the catalytic subunits of ppp family members has revealed a conserved catalytic mechanism in which a divalent metal cation activates a water molecule to hydrolyze phospho - serine / threonine without the formation of a phosphoenzyme intermediate ( reviewed in [ 1012 ] ) . despite a shared catalytic mechanism , substrate specificity within the ppp family for example , the ppp calcineurin ( also known as protein phosphatase 2b ) has been shown to interact with two consensus sequences , pxixit and lxvp , found on nonsubstrate - interacting proteins and target substrates ( reviewed in [ 11 , 13 ] ) . for protein phosphatase-1 ( pp1 ) , substrate specificity is conferred by incorporation of pp1-interacting proteins via a conserved docking motif with a general consensus sequence of rvxf ( reviewed in [ 11 , 12 ] ) . at present , consensus sequences in pp2a substrates have not been identified . this review will focus on our emerging understanding of pp2a substrate specificity , which appears to involve additive effects of multiple discrete interactions . pp2a is a highly conserved serine / threonine phosphatase which , depending on the tissue of origin and cell type , may account for up to 1% of cellular protein and the majority of serine / threonine phosphatase activity . the physiological functions of pp2a have been implicated in all facets of cellular existence ( reviewed in ) . further , pp2a functions as a critical tumor suppressor whose interruption leads to proliferative diseases . the heterotrimeric holoenzyme is composed of a catalytic subunit ( c ) a scaffold subunit ( a ) and one member of four families of regulatory subunits ( b ) ( figure 1 ) . the diversity of pp2a heterotrimers is achieved through expression of two c subunits , two a subunits and approximately fifteen b subunits in vertebrates . the b subunits are derived from four diverse gene families ( b , b , b , and b ) that have little sequence similarity between families but maintain high sequence similarity within families . the b family ( b55 , pr55 , ppp2r2 ) of regulatory subunits consists of four genes ( , , , ) , the b - family ( b56 , pr61 , ppp2r5 ) is comprised of five isoforms ( , , , , ) , the b family ( pr72 , ppp2r3 ) includes three isoforms ( /pr72/130 , /pr59 , /pr48 ) , and the b family ( pr93/pr110 ) is made up of three proteins ( sg2na , striatin , and mmob1 ) . there is some controversy as to whether the b family members , most notably sg2na , are bona fide pp2a regulatory subunits that always associate with the ac dimer or whether they are merely regulated by association with the pp2a dimer . given the large number of pp2a subunits , it is thought that each cell expresses a dozen or more distinct holoenzyme complexes which act on a diverse array of substrates . pp2a holoenzyme diversity has been the subject of several excellent papers [ 14 , 15 , 18 ] . like pp1 , regulatory subunit incorporation is thought to dictate the substrate specificity of the pp2a complex , however , only recently have molecular studies begun to develop insight into the mechanism by which the regulatory subunit acts . the results from recent studies suggests a multitiered mechanism wherein pp2a substrate specificity arises from ( 1 ) subcellular localization of pp2a defined by the b subunit , ( 2 ) selective holoenzyme assembly by posttranslational modification , ( 3 ) interaction with specific endogenous inhibitors , ( 4 ) interactions between the b subunit and phosphosubstrates at sites distant from the active site , and ( 5 ) b - subunit residues which infiltrate the catalytic cleft of the c subunit . this paper will provide a summary of these studies and how the understanding of the determinants of pp2a substrate specificity has advanced . the heterotrimeric holoenzyme is targeted to discrete subcellular locales dictated in part by which b - regulatory subunit is incorporated . the localization imparted by the b - regulatory subunit dictates the spatial sphere of influence of the holoenzyme complex for potential substrates . this mechanism of targeting pp2a activity is highlighted by extensive studies of the b family of regulatory subunits . for instance , the b family regulatory subunits target the holoenzyme to different cellular compartments in the brain . specifically , b and b are primarily cytosolic where as the b-regulatory subunit associates with a detergent - resistant protein fraction consistent with an interaction at the cytoskeleton . similar diversity has been observed in the b family of regulatory subunits . a c - terminal nuclear export signal common to b , b , and b which , when these regulatory subunits are incorporated into the pp2a holoenzyme , results in cytoplasmic localization of the heterotrimer . b and b isoforms lack a similar sequence and are found primarily in the nucleus . in cardiomyocytes , b interacts with the protein ankyrin - b through its c - terminus which leads to localization at the cardiac m - line . b on the other hand has been shown to target the holoenzyme complex to subnuclear structures in cardiomyocytes where pp2a / b may regulate gene expression . loss of proper subcellular targeting of pp2a has been implicated in the biogenesis and aggressive phenotype of neoplastic growths . specifically , a truncated form of b ( b ) , has been isolated from a melanoma cell line wherein the pp2a/b complex is targeted to the trans - golgi network , blunts p53 responsiveness , contributes to genetic instability and increases metastatic motility [ 2427 ] . this was first observed in a member of the b family , b. two neuron - specific isoforms of b , b1 , and b2 , are generated by the use of an alternative 5 exon which results in the production of a divergent n - terminal extension on b2 [ 28 , 29 ] . the n - terminal extension of b2 directs the holoenzyme complex to the outer mitochondrial membrane ( omm ) by targeting the mitochondrial translocase complex and forming an abortive complex resistant to import into the mitochondrial matrix [ 28 , 30 ] . the omm - directed pp2a / b2 complex promotes fragmentation of the mitochondria reticulum and increases cell susceptibility to proapoptotic insults through an unknown mechanism . alternative splicing of b - regulatory subunits directing subcellular localization of the pp2a holoenzyme has also been observed in both the b and b families . two b isoforms differ by the inclusion or exclusion an n - terminal nuclear localization signal leading to isoform - specific nuclear or cytoplasmic localization . the murine - specific b family member , b , is also subject to alternative splicing leading to isoform - specific nuclear or cytoplasmic localization through an as yet unidentified mechanism . for example , the interaction between pp2a / b and shugoshin during meiosis is crucial for spatial and temporal regulation of sister chromatid disjunction . during meiosis , the cohesin complexes , which link the arms of bivalent chromosomes and the centromeres of sister chromatids , must be released in a stepwise fashion by the protease separase ; the cohesin complex is firstly hydrolyzed along the arms of the bivalent chromosomes for completion of anaphase i and secondly at the centromeres of sister chromatids for completion of anaphase ii . during anaphase i , the centromeric cohesin complex is protected from separase - dependent proteolysis by shugoshin [ 34 , 35 ] . shugoshin recruits pp2a / b to the centromere which likely results in dephosphorylation of the cohesin complex leading to protection of the cohesin complex from separase - dependent proteolysis [ 36 , 37 ] . through cocrystallization , xu and colleagues , have revealed that dimeric human shugoshin 1 interacts with pp2a / b through a coiled - coil region across a broad composite surface of the c and b subunits . incorporation of specific regulatory subunits is influenced by reversible posttranslational modification of the c subunit . many groups have shown that the c - terminus of the c subunit is modified through phosphorylation and methylation on y307 and l309 , respectively [ 3946 ] . phosphorylation of y307 is catalyzed by src kinase and is likely opposed by pp2a - catalyzed autodephosphorylation of this phosphotyrosine . phosphorylation of y307 selectively inhibits recruitment of the b family and some b family members to the dimeric ac complex whereas b recruitment is not effected . methylation of the c subunit at the c - terminal l309 is catalyzed by the protein phosphatase methyltransferase ( ppm1 ) and is opposed by the phosphatase methylesterase ( pme-1 ) [ 4751 ] . reversible methylation of pp2a is absolutely critical as knocking - out pme-1 in mice changes the phosphoproteome and results in early perinatal lethality . further , methylation of the c subunit is a dynamic process which plays a role in cellular response to acute stimuli . the recruitment of the b subunit to the ac dimer has been postulated to require methylation of the c subunit for some of the b - subunit families . however , conflicting results have been reported that may reflect differences in experimental design and will be discussed further . studies wherein pp2a / b is isolated from intact cells have revealed that methylation of the c subunit at l309 is required for incorporation of the b family of regulatory subunits into the holoenzyme complex [ 39 , 40 , 42 , 44 , 54 , 55 ] . conversely , in vitro assembly of the pp2a / b holoenzyme complex does not require methylation of the c subunit for incorporation of the b family of regulatory subunits [ 56 , 57 ] . similarly controversial , the requirement for c subunit l309 methylation was observed to be study - specific in in vitro pp2a / b timer formation [ 58 , 59 ] . methylation was dispensable for isolation of the pp2a / b holoenzyme complex from intact cells . the role of methylation of the c subunit in recruitment of b and b families of regulatory subunits is less controversial with methylation of the c subunit being dispensable [ 44 , 54 ] . for more information , posttranslational modifications which influence formation of the pp2a holoenzyme complex also occur on the b subunit . pp2a / b negatively regulates the erk map kinase signal transduction pathway . through formation of a ternary complex of the early response gene product iex-1 , b1 , and erk , erk mediates its own disinhibition by phosphorylation of b1 on s327 leading to b1 disassociation from the pp2a holoenzyme . since s327 is conserved among b - subunit family members , it is likely that other b subunits are regulated similarly . additionally , b is likely phosphorylated on s167 to disrupt the b subunit from the ac dimer in early mitotic stages . however , pp2a / b activity is necessary to resolve the mitotic spindles and conclude mitosis ; therefore , autodephosphorylation may occur on b to allow efficient pp2a / b heterotrimer formation and cell cycle progression . thus , phosphorylation of the b - regulatory subunits also influences holoenzyme assembly and , therefore , substrate specificity . phosphorylation of the b subunit of heterotrimeric pp2a also potentiates the catalytic activity of the holoenzyme complex . in response to activation of d1 dopamine receptors on striatal neurons , camp - dependent protein kinase a ( pka ) phosphorylates b at s566 increasing activity of the pp2a/ b holoenzyme towards dopamine- and camp - regulated neuronal phosphoprotein ( darpp-32 ) [ 64 , 65 ] . dephosphorylation of t75 on darpp-32 by pp2a / b disinhibits pka - mediated phosphorylation of darpp-32 at t34 which converts darpp-32 into a potent pp1 inhibitor leading to changes in neuronal signaling . this circuit acts to attenuate phospho - t75 inhibition of t34 phosphorylation of darpp-32 . this circuit has been shown to be differentially regulated by psychomotor stimulants and antipsychotics acting on different striatal neuron subpopulations . while assembly of the many trimeric pp2a holoenzymes directs cellular localization and substrate specificity , further regulation is afforded through binding of specific protein inhibitors of pp2a . one such inhibitor set ( i2/taf-1 ) is upregulated during the progression of chronic myelogenous leukemia through bcr / abl activity and results in decreased pp2a activity . an additional pp2a inhibitor is the protein cip2a ( cancerous inhibitor of pp2a ) , overexpression of which is associated with several human malignancies . cip2a associates with c - myc to protect its phosphorylated s62 from pp2a - directed activity stabilizing the c - myc protein and allowing it to promote oncogenesis . recently an interplay between the drosophila serine / threonine kinase greatwall ( gwl ) and pp2a / b was observed during mitotic entry in two separate studies [ 69 , 70 ] . pp2a / b activity prevents mitotic entry by maintaining cdc25 in a dephosphorylated and inactive state . gwl reverses this inhibition through the phosphorylation of s67 of both -endosulfine ( ensa ) and cyclic adenosine monophosphate-(camp- ) regulated phosphoprotein-19 ( arpp-19 ) . phosphorylation converts ensa and arpp-19 into very specific inhibitors of pp2a / b activity and produces activation of cdc25 leading to cell cycle progression . similar cell cycle regulatory activity has been observed with the mammalian ortholog of gwl , mastl ; however , the mastl - pp2a interaction has yet to be characterized . once targeted to specific subcellular locales , the pp2a holoenzyme must recruit and dephosphorylate target substrates . recent structural studies have begun to suggest the mechanism by which the regulatory subunit of pp2a mediates initial binding to target substrates . other -propeller proteins have been shown to bind ligands in the central depression on the top surface of the toroid . crystallization of pp2a / b revealed a cluster of acidic residues in this depression that is available to recruit potential substrates containing a basic motif . in this same study , the acidic central depression of b was experimentally confirmed to bind the microtubule - associated protein tau , an established pp2a / b substrate . several conserved aspartate and glutamate residues in b engage in weak , electrostatic interactions across a large basic portion of tau and support dephosphorylation of tau at multiple sites through cycles of binding and unbinding . structurally divergent b family members may recruit substrates in a similar fashion as the b family . the crystal structure of pp2a / b shows that the b subunit contains 18 stacked -helices which adopt 8 huntingtin elongation a subunit tor- ( heat- ) like repeats [ 58 , 59 ] . a portion of these heat - like repeats interact with the a subunit of the holoenzyme to mediate regulatory subunit incorporation into the holoenzyme complex . like the b subunit , an acidic patch is exposed in the b family of regulatory subunits and may mediate protein - protein interactions and substrate recruitment . structural studies of pp2a have revealed a conserved loop in the b family of regulatory subunits which infiltrates the catalytic core of the holoenzyme [ 58 , 59 ] ( figure 2(a ) ) . at the tip of this loop is a conserved glutamate residue , e153 ( b numbering ) , which contacts through its carbonyl oxygen the catalytic subunit and through its carboxyl group a cocrystallized microcystin molecule in the active site . mutational analysis revealed that e153 is an absolute requirement for efficient dephosphorylation of tyrosine hydroxylase ( th ) , a known pp2a / b substrate , as well as other as yet unidentified cellular substrates of this pp2a holoenzyme . further , it was determined that e153 of b interacts with r37 and r38 of th to mediate dephosphorylation of both s31 and s40 on th . positively charged residues in the vicinity of target phospho - serine / threonine residues could represent a consensus sequence for b - subunit - mediated dephosphorylation . further , the infiltrating loop is likely a conformationally dynamic structure which is not sterically hindered by surrounding portions of the a , c , or b subunits . since r37/38 are important for dephosphorylation of both upstream ( s40 ) and downstream ( s31 ) sites , it appears that the orientation of phosphopeptides relative to the catalytic cleft is not constrained by additional interactions . collectively , the above observations support a model in which the sites of interaction between the substrate and the b regulatory subunit that are distant and near the active site together control substrate specificity . first , the interaction occurring at sites distant from the active site increases the local substrate concentration . following this initial substrate recruitment , although divergent in its sequence , an analogous structure from the -propeller fold of the b family of regulatory subunits extends to the catalytic core of the holoenzyme ( figure 2(b ) ) . this loop places conserved residues of the b - family subunits very near the holoenzyme active site . unpublished observations generated in our lab suggests that of these loop residues k87 of b2 may play a similar role as e153 of b in site - specific dephosphorylation of target substrates ; however , further characterization of this substrate specificity loop is required . pp2a is a ubiquitous protein phosphatase responsible for the dephosphorylation of many different intracellular targets . the diverse repertoire of potential substrates for pp2a is imparted by the incorporation of one of fifteen unique b - regulatory subunits . recent studies have increased our understanding of the mechanisms by which the b subunit imparts specificity to the holoenzyme complex . through selective incorporation of the b - regulatory subunit , the holoenzyme complex is recruited to discrete subcellular locales which define the sphere of influence for the phosphatase . secondly , interactions between endogenous inhibitors and specific pp2a heterotrimers further restrict phosphatase activity . as shown for the b family , regulatory subunits mediate low - affinity interactions with substrates to increase the local concentration of substrates . through a flexible substrate selectivity loop which contacts the catalytic subunit , interactions between the regulatory subunit and phosphosubstrate may mediate multiple nearby dephosphorylation events . with the current structural information available for the pp2a complexes , future high - resolution studies will further define the molecular mechanism of pp2a substrate specificity . as general , inhibitors of pp2a are either clinically irrelevant or toxic , as in the case of the small molecule inhibitor microcystin , novel methods to increase the specificity of pp2a inhibition or activation must be developed . a clearer understanding of the pp2a substrate specificity mechanisms will serve as the foundation for rational drug design of selective inhibitors and activators of specific pp2a holoenzyme complexes .

protein phosphatase 2a- ( pp2a- ) catalyzed dephosphorylation of target substrate proteins is widespread and critical for cellular function . pp2a is predominantly found as a heterotrimeric complex of a catalytic subunit ( c ) , a scaffolding subunit ( a ) , and one member of 4 families of regulatory subunits ( b ) . substrate specificity of the holoenzyme complex is determined by the subcellular locale the complex is confined to , selective incorporation of the b subunit , interactions with endogenous inhibitory proteins , and specific intermolecular interactions between pp2a and target substrates . here , we discuss recent studies that have advanced our understanding of the molecular determinants for pp2a substrate specificity .

connectivity is key to understanding activity in neural systems ( sporns et al . , 2000 ) . network connectivity in science and in engineering fields as diverse as mechanics , communication technology , public health , geography and town planning , is studied mathematically using the concepts of graph theory ( bollobas , 1998 ) . recently , graph theory is being applied to brain connectivity ( sporns and zwi , 2004 ; bullmore and sporns , 2009 ) and its pathologies in alzheimer 's disease ( stam , 2004 ; stam et al . , 2007 ) , brain tumors ( bartolomei et al . , 2006 ) , epilepsy ( ponten et al . , 2007 ) and , in particular , schizophrenia ( bleuler , 1911/1950 ; friston and frith , 1995 ; andreasen , 1999 ; micheloyannis et al . , 2006 ; applying graph - theoretic concepts to the brain sheds new light on the basic principles of integration and segregation underlying adaptive cognitive processes , and on their disruption in maladaptive states . schizophrenia has been understood as a cognitive disorder ( bleuler , 1911/1950 ) based on the breakdown of large - scale cortico - cerebellar - thalamic - cortical ( andreasen , 1999 ) or prefronto - temporal circuits ( friston and frith , 1995 ; goldman - rakic and selemon , 1997 ) , or more generally the inability to integrate neural processes in different brain areas , a syndrome termed dysconnectivity ( stephan et al . this condition may pare down , in particular , the input to pyramidal cells of the dorsolateral prefrontal and temporal cortex ( garey et al . these cells are glutamatergic and receive projections from the thalamus and widespread cortical areas , and hence are likely to be involved in higher - level cognition . reduced connectivity may thus lead to fragmentation , a loss of coherence in cognitive activity . graph theory enables us to model the loss of connectivity in simulated neuronal networks and predict the time course of fragmentation . on the face of it , percolation plays an important role in the evolution , growth , and maintenance of a large variety of natural , technological , and social systems ( ben avraham and havlin , 2000 ) . it refers to the probability of existence of a path between every pair of nodes in a graph , or equivalently , the graph being connected whilst many previous studies have examined cortical network connectivity in schizophrenia and other disorders , none to our knowledge have employed the concept of percolation , an issue that we presently redress . the percolation function is the cumulative density function ( cdf ) of percolation as a function of connectivity . it is possible , in principle , to measure the percolation function in living neural tissue , by using progressive lesioning , for instance through the administration of inhibitory neurotransmitters ( breskin et al . , observations on human brain functional connectivity may be compared to the theoretical percolation function for random networks ( erds and rnyi , 1959 ) . the percolation function cp(n ) of a random graph of n vertices and e edges is given by cp(n ) = e the presence of a critical threshold motivates us to revisit the notion of cortical dysconnectivity as a sudden breakdown of percolation . there are , however , reasons to assume that the percolation threshold is neither the first , nor the most predominant , critical transition in the development of schizophrenia : brains are not random networks . in both the structural ( sporns and zwi , 2004 ) and functional ( salvador et al . 2006 ) domains , the hallmarks of brain organization include local clustering as expressed in high values for the clustering coefficient ( cc ) , high global connectedness as specified by a short characteristic path length ( cpl ) ( watts and strogatz , 1998 ) , and modularity ( murre and sturdy , 1995)a combination characteristic of modular small - world networks ( he et al . , 2009 ) . graph - theoretical studies ( murre and sturdy , 1995 ; watts and strogatz , 1998 ) showed that small - world and modular networks can secure global connectivity with a small number of connections . for brains configured as modular small - worlds , a few connections will suffice to ensure percolation . most likely , therefore , percolation is not the crucial bottleneck for brain pathologies such as schizophrenia . we will propose as an alternative theoretical possibility that , instead , brain pathologies are associated with a breakdown in the local organization . in schizophrenia patients , functional connectivity in scalp eeg channels appears to reflect a loss of clustering after correcting for differences in the density of functional connections ( micheloyannis et al . the question , therefore , arises , whether fragmentation can be understood as a critical breakdown in the ability of the brain to establish and maintain a modular small - world functional architecture . here we show by numerical simulations that in neural activity networks , with loss of connectivity a self - organizing small - world neural network can not sustain its local clustering , well before global connectivity breaks down . the iterated logistic map f(x)= 1 ax is unimodal on [ 1;1 ] [ 1;1 ] and capable of both periodic and chaotic behavior depending on its control parameter a. in this study , we construct networks of coupled logistic maps , all with parameter a = 1.7 such that the dynamics of a single unit are chaotic under iteration of a randomly chosen initial activation value . a unit x is coupled with coupling strength = 0.4 to any number mi of other units in the network such that its activation value xn + 1 at iteration n + 1 depends on the activation value of itself and all adjacent units at iteration n : in this equation , b(i ) is the set of units adjacent to unit i , mi is the number of units adjacent to unit i. the coupling strength is divided by mi , and has a compensation term ( 1 ) to make sure that logistic map of an individual unit retains its mapping [ 1:1 ] [ 1:1 ] , and thus functions properly for any numbers of adjacent units . a network of this type can be used to study the buildup and breakdown of modularity resulting from hebbian adaptive structural self - organization . it implements a simple rewiring rule based on synchronization of chaotic activity and rewires at most one connection per iteration , carefully keeping the network 's total number of connections constant throughout the process . a single iteration of the network consists of four steps , to be repeated several times after an initial random inception : initialize the network . randomly establish e connections between v units to create a ( v , e ) random network , and initialize every unit with a random activation value [ 1:1 ] . though values of v and e are chosen such that a network has a high probability of being connected , this is not required.update units . synchronously update every unit 's activation value from its own and all its adjacent units ' activation values according to equation ( 1).select pivot and candidate . randomly select one unit from the network ( the pivot ) . from all other units , select the one whose activation value is closest to the pivot 's . then , from the units already adjacent to the pivot , select the one whose activation value is farthest from the pivot 's , and cut its connection to keep the number of connections constant . if there is already a connection between the pivot and the candidate , or if the pivot has zero connections , nothing happens and this step is skipped.iteration completed . randomly establish e connections between v units to create a ( v , e ) random network , and initialize every unit with a random activation value [ 1:1 ] . though values of v and e are chosen such that a network has a high probability of being connected , this is not required . synchronously update every unit 's activation value from its own and all its adjacent units ' activation values according to equation ( 1 ) . randomly select one unit from the network ( the pivot ) . from all other units , select the one whose activation value is closest to the pivot 's . then , from the units already adjacent to the pivot , select the one whose activation value is farthest from the pivot 's , and cut its connection to keep the number of connections constant . if there is already a connection between the pivot and the candidate , or if the pivot has zero connections , nothing happens and this step is skipped . networks implementing these iterative steps exhibit development from an initial random configuration to modular small - world configurations ( gong and van leeuwen , 2003 ; van den berg and van leeuwen , 2004 ; rubinov et al . , 2009b ) but as it turns out , both the consistent build - up of connective modularity on one hand , or the loss of structural coherence due to functional fragmentation on the other , are a result of changing dynamic activity depending critically on the number of connections in the network . the influence of these numbers shows a close relationship to the percolation function of random graphs . a common principle for neural network evolution is preferential attachment ( barabsi and albert , 1999 ) . this mechanism leads to networks that are scale - free , but not modular small - worlds . only by combining preferential attachment with adaptive , hebbian rewiring , does a network emerge that is scale - free and also has modular small - world network structure ( gong and van leeuwen , 2003 ) . an adaptive rewiring scenario for evolving networks allows networks with initially random or regular structures to develop into modular small - world structures ( gong and van leeuwen , 2004 ; rubinov et al . , 2009b ) . the scenario requires network units ( edges ) that produce ongoing , non - random , non - periodic oscillatory activity . these could , for instance , be represented by spiking model neurons ( kwok et al . , 2007 ) or by nonlinear maps as an extremely simplified model of neural mass activity ( breakspear et al . , 2003a , b ) . with these simple units as edges , the vertices of the network represents the couplings of a coupled nonlinear map ( kaneko , 1989 ) . adaptive rewiring operates on this activity according to the general hebbian principle of what fires together wires together ( paulsen and sejnowski , 2000 ) . at successive points during the systems ongoing spontaneous activity , connections are added between pairs of synchronously active but hitherto unconnected units , while connections between desynchronized units are removed ( see methods ) . over time the network gradually assumes a modular , small - world structure ( figure 1 ) . adaptive rewiring leads from an initial random network ( left ) , to modular small - world structure ( right ) in small iterative steps . coupled chaotic oscillators intermittently synchronize and desynchronize their activity spontaneously in patterns of great variability . after some time a momentarily synchronized pair of units that are not connected receive a connection , which is removed from a pair that are connected but not synchronized . as this process continues , a modular , small - world structure emerges from an initially random configuration . to obtain a more detailed view of this phase transition , we use the adaptive rewiring scenario with coupled nonlinear maps ( kaneko , 1989 ) with initially randomly structured graphs , for a range of different numbers of vertices v : v = 300 , 400 , 500, ,1000 vertices and numbers of edges e that differ by small steps of 20 . for each combination of v , e , across four million iterations we measured the cc and the cpl every one thousand iterations , resulting in a 4000 point record for each of five runs . the maximum , minimum , and mean values of the last 2000 points in each run were averaged over the five runs as illustrated in figure 3 . meanwhile a mixture of regular and irregular behavior is established in the network activity that is itself optimal for sustaining the small - world structure . crucially , whilst low dimensional , ordered , and synchronized activity dominates within modular communities , high dimensional unsynchronized activity in connector hubs ensures that the system does not fragment ( rubinov et al . attractors in the space of possible systems ( gong and van leeuwen , 2004 ) , which offers a potential explanation for their ubiquity in biological neural networks at different scales , including the entire brain ( barabsi and albert , 1999 ) . in this scenario , connectivity constitutes a critical limit for the evolution to small - world structure ( figure 2 ) . when the number of edges is large enough , adaptive rewiring guarantees a robust evolution from random to small - world connectivity . below this limit , this evolution is frustrated , and fails to reach a stable asymptotic state . with reduced connectivity levels , we first encounter critical fluctuation : intermittently during some episodes , clusterings are formed intermittently , which are annihilated in other episodes . this may reflect the intermittent occurrence of certain symptoms ( e.g. , delusions ) as the brain disease first becomes manifest . for still lower connectivity levels , adaptive rewiring becomes completely ineffective ; this may reflect the advanced state of the disease . self - organization from random to small - world critically in a network of 700 vertices . the self - organization occurs through adaptive rewiring . whether a small - world emerges depends on the number of edges . we compared the critical limit on the evolution to small - world structures to percolation thresholds of random networks with the same numbers of edges and vertices . figure 4 shows that the observed minimum cc can be modeled as a linear function of cp(n ) , with k3 for offset and k4 for amplitude : ccpred = k3 + k4 cp(n ) . parameter k3 was in the range [ 0.107:0.196 ] , parameter k4 in [ 0.392:0.459 ] and parameter k1 in [ 0.001:0.006 ] . the behavior of these parameters across network sizes was not monotonic ( figure 4 ) . parameter k2 however , the horizontal position of the anchor point , showed a universal scaling law to the anchor point in the percolation function of random graphs , namely ( table 1 ) : aswn(n ) = arand(n ) . note : anchor point arand(n ) = n ln(n ) for classic random graphs of n vertices ; this anchor point indicates the percolation threshold , where the percolation function cp(n ) shows the greatest inflection . aswn(n ) : anchor point for the small - world networks fitted according to figure 4 . marquardt algorithm implemented in fityk . scaling power : the value of h in the equation aswn(n ) = arand(n ) . a common principle for neural network evolution is preferential attachment ( barabsi and albert , 1999 ) . this mechanism leads to networks that are scale - free , but not modular small - worlds . only by combining preferential attachment with adaptive , hebbian rewiring , does a network emerge that is scale - free and also has modular small - world network structure ( gong and van leeuwen , 2003 ) . an adaptive rewiring scenario for evolving networks allows networks with initially random or regular structures to develop into modular small - world structures ( gong and van leeuwen , 2004 ; rubinov et al . , 2009b ) . the scenario requires network units ( edges ) that produce ongoing , non - random , non - periodic oscillatory activity . these could , for instance , be represented by spiking model neurons ( kwok et al . , 2007 ) or by nonlinear maps as an extremely simplified model of neural mass activity ( breakspear et al . , 2003a , b ) . with these simple units as edges , the vertices of the network represents the couplings of a coupled nonlinear map ( kaneko , 1989 ) . adaptive rewiring operates on this activity according to the general hebbian principle of what fires together wires together ( paulsen and sejnowski , 2000 ) . at successive points during the systems ongoing spontaneous activity , connections are added between pairs of synchronously active but hitherto unconnected units , while connections between desynchronized units are removed ( see methods ) . over time the network gradually assumes a modular , small - world structure ( figure 1 ) . adaptive rewiring leads from an initial random network ( left ) , to modular small - world structure ( right ) in small iterative steps . coupled chaotic oscillators intermittently synchronize and desynchronize their activity spontaneously in patterns of great variability . after some time a momentarily synchronized pair of units that are not connected receive a connection , which is removed from a pair that are connected but not synchronized . as this process continues , a modular , small - world structure emerges from an initially random configuration . to obtain a more detailed view of this phase transition , we use the adaptive rewiring scenario with coupled nonlinear maps ( kaneko , 1989 ) with initially randomly structured graphs , for a range of different numbers of vertices v : v = 300 , 400 , 500, ,1000 vertices and numbers of edges e that differ by small steps of 20 . for each combination of v , e , across four million iterations we measured the cc and the cpl every one thousand iterations , resulting in a 4000 point record for each of five runs . the maximum , minimum , and mean values of the last 2000 points in each run were averaged over the five runs as illustrated in figure 3 . meanwhile a mixture of regular and irregular behavior is established in the network activity that is itself optimal for sustaining the small - world structure . crucially , whilst low dimensional , ordered , and synchronized activity dominates within modular communities , high dimensional unsynchronized activity in connector hubs ensures that the system does not fragment ( rubinov et al . attractors in the space of possible systems ( gong and van leeuwen , 2004 ) , which offers a potential explanation for their ubiquity in biological neural networks at different scales , including the entire brain ( barabsi and albert , 1999 ) . in this scenario , connectivity constitutes a critical limit for the evolution to small - world structure ( figure 2 ) . when the number of edges is large enough , adaptive rewiring guarantees a robust evolution from random to small - world connectivity . below this limit , this evolution is frustrated , and fails to reach a stable asymptotic state . with reduced connectivity levels , we first encounter critical fluctuation : intermittently during some episodes , clusterings are formed intermittently , which are annihilated in other episodes . this may reflect the intermittent occurrence of certain symptoms ( e.g. , delusions ) as the brain disease first becomes manifest . for still lower connectivity levels , adaptive rewiring becomes completely ineffective ; this may reflect the advanced state of the disease . self - organization from random to small - world critically in a network of 700 vertices . the self - organization occurs through adaptive rewiring . whether a small - world emerges depends on the number of edges . we compared the critical limit on the evolution to small - world structures to percolation thresholds of random networks with the same numbers of edges and vertices . figure 4 shows that the observed minimum cc can be modeled as a linear function of cp(n ) , with k3 for offset and k4 for amplitude : ccpred = k3 + k4 cp(n ) . parameter k3 was in the range [ 0.107:0.196 ] , parameter k4 in [ 0.392:0.459 ] and parameter k1 in [ 0.001:0.006 ] . the behavior of these parameters across network sizes was not monotonic ( figure 4 ) . parameter k2 however , the horizontal position of the anchor point , showed a universal scaling law to the anchor point in the percolation function of random graphs , namely ( table 1 ) : aswn(n ) = arand(n ) . note : anchor point arand(n ) = n ln(n ) for classic random graphs of n vertices ; this anchor point indicates the percolation threshold , where the percolation function cp(n ) shows the greatest inflection . aswn(n ) : anchor point for the small - world networks fitted according to figure 4 . marquardt algorithm implemented in fityk . scaling power : the value of h in the equation aswn(n ) = arand(n ) . we propose that important insights into cortical activity and architecture can be obtained by modeling the activity - dependent rewiring of neural connections during development ( gong and van leeuwen , 2003 ; rubinov et al . , 2009b ) . in our model , network connections evolve in accordance with the principle that the structure rewires in adaptation to spontaneous , on - going activity . this evolution , however , is only guaranteed if there are sufficiently many connections available . if connectivity is reduced below this number , the structure shifts toward randomness ; in particular , local clustering is reduced . andreasen ( 1999 ) and friston and frith ( 1995 ) considered schizophrenia as fragmentation , understood as the breakdown of integration between widely distributed brain areas ( stephan et al . , 2006 , 2009 ) . this breakdown can be associated with the loss of connectivity ( zalesky et al . , 2011 ) , in particular of input to layer 3 pyramidal cells , an effect which is well - documented ( e.g. , garey et al . ( 2011 ) observed widespread impairment in structural connectivity in schizophrenic patients , involving medial frontal , parietal / occipital and left frontal cortex . it should be observed that the loss of connectivity that may lead to the onset of schizophrenia can be relatively subtle . across the population , inputs to layer 3 pyramidal cells are substantially reduced during late adolescence , the typical period for the onset of schizophrenia ( bourgeois et al . , 1994 ) . given that brain connectivity is costly , it may well be that in normals , its density hovers just above the critical level ( the anchor point in figure 4 ) , but in early schizophrenia it may fall just below this point . the graph - theoretical concept of percolation tells us that a small decline in connectivity can lead to a sudden breakdown of global network coherence . based on our results , however , we argue that fragmentation in brain pathologies such as schizophrenia may be considered theoretically as a breakdown in the local connectivity structure , prior to the loss of global coherence . the number minimally needed to secure local modularity , and hence to prevent it from shifting toward randomness in structure , is systematically related to , and greater than , that needed to secure global connectivity , even if the system has fallen into entirely random connectivity . this result is of potential importance for understanding the pathophysiological processes that give rise to this disorder . the loss of local clustering in our model is in accordance with observations in schizophrenic patients by micheloyannis et al . et al . ( 2006 ) , the clinical group also showed longer path lengths than the controls , whereas in rubinov et al . nevertheless , we might attribute the discrepancy to the fact that in both studies comparisons were made , for statistical reasons , between networks that were thresholded to have identical connectivity . whereas the above - mentioned effects of clustering remain relatively unaffected by threshold setting , the differences in path length rapidly disappear for lower thresholds ( figure 1 in micheloyannis et al . , 2006 ) . ( 2003b ) reported that although there were no significant increases in the occurrence of nonlinear interdependence between pairs of electrodes in schizophrenia , there was an increase in the co - occurrence in multiple ( widespread ) instances of nonlinear interdependence . this means that a relatively large number of global connections will have survived thresholding in rubinov 's study , leading to their observation of path length shortening . it can not be concluded from rubinov 's study , therefore , that global connectivity is stronger in schizophrenics than in normals ; it could , however , be concluded that the global connectivity becomes stronger in schizophrenia relatively to their local connectivity . such a conclusion would entirely be in accordance with the modularity breakdown observed in our model . along the lines set out here , a shift in the balance from local to global connectivity is perfectly consistent with an overall loss of connectivity in early schizophrenia . ( 2003 ) introduced the notion of overbindingthe formation of excessive connections that are effectively random and , as such , do not enable distinguishing external from internal sources , thus providing conditions favorable for phenomena such as hallucination . a possible objection to our findings is the specific choice of our rewiring algorithm . note , however , that in the present paper we sought to establish the principled possibility using the simplest possible model , rather than to establish the empirical validity through the most realistic model possible . note that , as a consequence , the model contains only generic dynamical and adaptive principles . we have discussed elsewhere the robustness of this model ( gong and van leeuwen , 2003 , 2004 ; van den berg and van leeuwen , 2004 ; kwok et al . an important limitation is that the model inevitably makes over - simplifying assumptions . in particular inter - modular connections are physically of longer range than intra - modular ones and , therefore , have a higher metabolic cost and a greater vulnerability . preliminary analysis of models with more realistic constraints does not appear , however , to affect our conclusions . clearly , a more differentiated model is needed to address empirical datasets such as ( rubinov et al . , 2009a ) , an important goal of future work . however , it should also be noted that uncovering universal principles such as those reported here has the advantage of being detail invariantthat is , robust across a range of potential constraints , whereas findings arising in detailed models may not be robust to changes in those details . we observed universal scaling behavior in adaptive self - organization of clustered small - world networks : the connectivity needed for these network properties to emerge under hebbian rewiring scales with a universal power = 1.17 to the percolation function in random networks . note , first , that > 1 might have been expected , given that the requirement to observe clustering and small - world structure are constraints additional to percolation . what is surprising is that these requirements are met with alpha very close to unity ; near - linear scaling implies that these additional constraints can be realized with great efficiency . in terms of kolmogorov - complexity , small - worlds are compressible , whereas almost every possible network of n nodes and e edges ( or equivalently a bit string of length l = n(n 1 ) with e ones and l - e zeros ) will be incompressible ( li and vitnyi , 1993 ) . in this perspective , the ubiquity of small - world structure in real - world networks is quite astonishing : within human brains ( sporns and zwi , 2004 ; stam , 2004 ; eguluz et al . bartolomei et al . , 2006 ; micheloyannis et al . , 2006 ; ponten et al . , 2007 ; rubinov et al . , 2009a ; bassett et al . , 2010 ) , as well as between them : networks of scientific co - authorship ( newman , 2001 ) , collaborating movie actors ( watts and strogatz , 1998 ; amaral et al . , 2000 ) , social networks in general ( wasserman and faust , 1994 ) . here we showed how such a network could arise with minimal connectivity close to random network percolation what is the reason for the universality of the scaling exponent ? we may wonder whether the same exponent found in other domains , could help us understand the principle . a study of class graphs in open - source , object - oriented software systems ranging from simple paint programs , peer - to - peer downloaders , racing games , database management software to a complete operating system , showed that the number of links between classes scales to the number of classes with an exact power = 1.17 . the authors found that class graphs are small - world networks at the critical threshold for the breakdown of modularity , which happens when developments to the system are widely dispersed and affect many unrelated classes in apparently distant modules ( valverde and sol , 2007 ) . the similarity of this finding to ours supports the view that the scaling exponent reflects a general feature in the emergence and breakdown of modular network structure . the study of self - organizing modular small - world networks casts a new perspective on psychiatric illnesses characterized by disorganized cognition , such as schizophrenia , of which the expression has been attributed to fragmentation a subtle but pernicious disconnection ( friston , 1996 , p. 644 ) . rather than a breakdown in global connectivity , we propose that fragmentation is to be understood as a failure to organize the functional connectivity of the brain into a modular small - world structure . this is in accordance with the observed random shift in schizophrenic ( micheloyannis et al . , 2006 ; random networks are considered extremely uneconomical ; in terms of cable length , an optimal configuration combines local modules with a limited number of large - scale connections ( murre and sturdy , 1995 ) . even though our model does not consider distance , in terms of network topology it is still the case that information travels efficiently both within locally connected circuits of modular small - world graphs and between their circuits , which makes these networks efficient for transport or communication ( latora and marchiori , 2001 ; bassett et al . , 2010 ) . the scaling observations tell us that fragmentation is a result of a breakdown in local , rather than global structure . with progressive loss of connectivity , ultimately , it may not matter which connections are lost first , the result may be a cascade of changes that lead to the network falling apart . for diagnosis , however , a proper understanding of the early stages of the disease is crucial ; loss of modularity might offer a new perspective on the origins of the disease . one simulation consists of one network of v units and e connections , which is randomly initialized and then iterated exactly 4,000,000 times , simultaneously rearranging its connections and activity patterns , according to the adaptive rewiring scenario . the smallest simulation we adopt has v = 300 units and e = 2400 connections . during iteration , its cpl and its cc are taken every 1000th iteration ( 1000 , 2000 , , 40,00,000 ) resulting in a 4000 point record , with a value for cc and a value for cpl at each point . although the speed of convergence depends on the size of a network , 4 , 000 , 000 iterations prove to be enough to clearly discern asymptotic behavior for all simulations used in this investigation ( figure 3 ) . evolution under adaptive rewiring of maximum , minimum , and average cluster coefficient and characteristic path length . ( a ) the values of minimum , maximum and average cc for networks of 700 vertices and edges ranging from [ 7000 , 7020 , 7040, ,10,000 ] after extensive adaptive rewiring . note that beyond 9000 edges , cc - values tighten to a narrow range , indicating strong and consistent clustering behavior . ( b ) the values of minimum , maximum , and average cpl for networks of 700 vertices and edges ranging from [ 7000 , 7020 , 7040, ,10,000 ] after extensive adaptive rewiring . beyond 9000 edges thus , for 700 vertices , at least 9000 edges are needed for adaptive rewiring to converge to small - world structure . from the 4000 point record , the maximum , minimum , and average values for both cpl and cc are calculated from the last 2000 points . for statistical robustness , we do any single simulation five times , and average the five values over this simulation - quintuple , resulting in a maximum , minimum , and average cc and cpl for the ( v = 300 , e = 2400 ) network . we then start a new quintuple of simulations , increasing the number of connections e by 20 , generating five networks with v = 300 units and e = 2420 connections , and calculate the maximum , minimum , and average cc and cpl values from these five new simulations . we keep starting new quintuples , repeatedly increasing e by 20 , until e = 3300 and the batch of 300-quintuples is complete . from the entire batch , the six cc and cpl values of every ( v , e ) are taken to graph the asymptotic clustering and path - length behavior of networks of 300 units as it depends on the numbers of connections ( figure 4 ) . gray lines represent minimal , maximal and average observed values for clustering coefficient , the dotted line is the predicted clustering coefficient , ccpred , a linear function of the percolation function cp(n ) of a random graph of n vertices : ccpred = k3 + k4 cp(n ) fitted with parameters k3 and k4 to the minimum observed clustering ; the arrow indicates its anchor point aswn(n ) with the corresponding number of edges in parentheses . this process is then repeated for a batch of quintuples of networks with 400 units and numbers of connections 3600 , 3620 , , 5000 ( see figure 4 , top - right box ) . we continue doing this for batches of networks with 500 , 600 , 700 , 800 , 900 , and 1000 units , with connections increasing by 20 , showing asymptotic clustering and path - length behavior depending on connectivity for networks of different sizes ( figure 4 ) . note that although for 300 units , connections ranged from e = 2400 to e = 3300 , these numbers are different for larger networks . for each of the eight batches , a phase transition was witnessed for both the cc and the cpl . to pin down the exact location of the steepest inclination in the phase transition of the cc ( its center , or anchor point ) , the percolation function for classic random graphs was function - matched to the minimum cc of every batch . the minimum cluster coefficient was chosen over the average and the maximum cluster coefficient because it has the steepest inclination , which facilitates the fitting best . macquardt an iterative curve - fitting algorithm which operates by minimizing the summed squares of the residuals , in this case the difference between minimal cc - values of n - edge simulation quintuples on the one hand , and the ( erds and rnyi , 1959 ) percolation function 's value for n edges on the other . macquardt algorithm is sensitive to local minima which makes it inefficient when using completely random initial values . initial parameters were hand - guessed separately for each of the eight subgraphs in figure 4 , after which the algorithm was ran until convergence beyond the program 's six - digit resolution , a procedure that was repeated three times with small differences in the hand - guessed initial parameters . the final values did not differ within the program 's six - digit resolution over the three repetitions , and convergence was very fast ( typically well before 100 iterations ) . the fits show significant deviations from the data curve , due to intrinsic fluctuations in the data . we , therefore , considered reliable the estimates of the scaling power and other model parameters . even more reliable estimates could , in principle , be obtained by scaling up the network size to 2000 , 5000 , and 10,000 vertices , resources permitting , as computation time and data grow nonlinearly with network size . 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 . 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 .

psychiatric illnesses characterized by disorganized cognition , such as schizophrenia , have been described in terms of fragmentation and hence understood as reduction in functional brain connectivity , particularly in prefrontal and parietal areas . however , as graph theory shows , relatively small numbers of nonlocal connections are sufficient to ensure global coherence in the modular small - world network structure of the brain . we reconsider fragmentation in this perspective . computational studies have shown that for a given level of connectivity in a model of coupled nonlinear oscillators , modular small - world networks evolve from an initially random organization . here we demonstrate that with decreasing connectivity , the probability of evolving into a modular small - world network breaks down at a critical point , which scales to the percolation function of random networks with a universal exponent of = 1.17 . thus , according to the model , local modularity systematically breaks down before there is loss of global coherence in network connectivity . we , therefore , propose that fragmentation may involve , at least in its initial stages , the inability of a dynamically evolving network to sustain a modular small - world structure . the result is in a shift in the balance in schizophrenia from local to global functional connectivity .

dysphagia is the subjective awareness of difficulty in the passage of solids or liquids from the oropharynx to the stomach . dysphagia can be classified into an oropharyngeal or an esophageal location , and it is caused by neuromuscular motility disorders and mechanical obstruction.1 ) mechanical dysphagia is associated with intrinsic or extrinsic compression , resulting in progressive intolerance to solids . the term , dysphagia aortica , has been used to describe difficulty in swallowing caused by external compression from an ectatic , tortuous , or aneurysmal aorta as a result of age - related degeneration.2 ) dysphagia aortica is classically seen in elderly women with short stature who have hypertension and kyphosis.3 ) we report herein a patient with dysphagia associated with an aortic aneurysm . an 86-year - old woman presented with worsening nausea and vomiting . because of her progressive dysphagia to solids for the last 6 months , she had ingested only semisolids and liquids . three days before seeking evaluation at our hospital , she had difficulty in swallowing liquids , along with nausea and vomiting . the medical history revealed that she had been diagnosed with primary hypertension , an ascending aortic aneurysm , congestive heart failure , moderate aortic regurgitation , and moderate mitral regurgitation 6 years previously . because of old age she had undergone vertebroplasty due to multiple compression fractures of the thoracic and lumbar vertebrae 5 years ago . on admission to the hospital the blood pressure was 130/90 mmhg , the pulse rate was 64 beats / min , the respiratory rate was 28 breath / min , the body temperature was 36.0 , the height was 1.43 m , and the body weight was 37 kg . the physical examination showed a diastolic murmur at the right upper sternal border and a pansystolic ejection murmur at the left lower sternal border . the laboratory findings were as follows : the white blood cell ( wbc ) count was 5,100/mm , the hemoglobin was 11.8 g / dl , the platelet count was 151,000/mm , the blood urea nitrogen ( bun ) was 39.8 mg / dl , the creatinine was 1.5 mg / dl , the total protein was 5.8 g / dl , the albumin was 3.2 g / dl , the lactate dehydrogenase ( ldh ) was 546 her chest radiograph revealed blunting signs at both costophrenic angles , cardiomegaly with a cardio - thoracic ratio of 0.8 , and an enlarged , tortuous aorta ( fig . 1 ) . computed tomography ( ct ) of the chest demonstrated an enlarged , tortuous aorta ( fig . the diameters of the ascending thoracic aorta , the descending thoracic aorta , and the proximal abdominal aorta were 7 cm , 6 cm , and 5.3 cm , respectively . the upper gastrointestinal barium study revealed marked extrinsic compression of the distal esophagus just above the esophagogastric junction ( fig . we concluded that the symptoms and the results of the imaging studies were consistent with dysphagia aortica . although we recommended surgical correction of the aortic aneurysm or percutaneous endoscopic gastrostomy , the patient declined any invasive procedures and she was transferred to a nursing home on the 12 hospital day . the esophagus normally begins on the right side of the thoracic aorta and then descends . then , the esophagus lies on the left side of the aorta and penetrates the diaphragm through the diaphragmatic histus.4 ) the aging process and the accompanying degenerative changes with the loss of elasticity causes a dilated , elongated , and distorted aorta , which may result in a so - called reverse c- or reverse s - shaped aorta.3 ) as a result , the esophagus is pushed and compressed by the aorta against the cardiac chambers , which are anterior in location.5 ) there is no gold standard diagnostic procedure for dysphagia aortica . the association of suggestive symptoms , such as progressive intolerance to solids with concomitant weight loss along with the results of imaging and other diagnostic studies provide a high index of suspicion.6 ) the diagnostic work - up includes radiologic , endoscopic , and manometric studies . on a standard chest radiography and ct scan , the enlargement of the aortic arch and the tortuous dilated aorta can be observed . a barium swallow test may show partial esophageal obstruction and pulsatile movement of the barium synchronous with aortic pulsation.7 ) endoscopy reveals pulsatile extrinsic compression and stenosis of the lower esophagus with proximal dilatation . esophageal manometry may demonstrate a localized high pressure band with superimposed pounding that is synchronous with the cardiac pulsation.8 ) however , the typical findings of dysphagia aortica can be inconsistent . the radiographic findings are often inconclusive because a dilated , tortuous thoracic aorta is frequently encountered in elderly patients with dysphagia . although a ct scan is useful for evaluating not only the aortic lumen , but also the aortic wall,9 ) occasionally this is of no value in assessing compression of the esophagus by the aorta.2 ) false negative results are common for barium swallow studies . in addition , the classical manometric features suggestive of dysphagia aortica also occur in normal subjects.8 ) the differential diagnosis of dysphagia aortica includes various common structural and neuromuscular abnormalities.1 ) gastroesophageal reflux disease and motility disorders are common causes of dysphagia . the co - existence of these conditions and the lack of sensitivity and specificity of the usual diagnostic tests make it difficult to diagnose dysphagia aortica with certainty . wilkinson et al.2 ) demonstrated that a video solid bolus swallow test could be useful in determining the manometric findings that are suspicious for dysphagia aortica when the standard evaluation fails . mild cases may be treated conservatively , such as avoiding sticky solids and feeding on a liquid diet . the surgical procedures include transposition of the distal esophagus , separation of the distal esophagus from the aorta , esophagomyotomy , division of the right crus of the diaphragm , aortic resection , and repair of an aneurysm . for patients who are not candidates for surgery , insertion of a feeding tube via percutaneous endoscopic gastrostomy ( peg ) although we did not perform esophageal manometry , the patient 's symptoms and imaging studies were consistent with the classic findings of dysphagia aortica . although the patient declined any invasive procedures , a peg for a feeding tube might have been helpful for nutritional support . dysphagia aortica is an uncommon type of dysphagia that is caused by extrinsic mechanical compression . it should be differentiated from other causes of dysphagia , such as gastroesophageal reflux disease or motility disorders , because dysphagia aortica often requires surgical intervention that can significantly reduce the morbidity and these interventions can be curative in some situations .

dysphagia aortica is difficulty in swallowing caused by extrinsic compression of the esophagus due to an ectatic , tortuous , or aneurysmatic atherosclerotic thoracic aorta . this condition is very uncommon , and it is usually associated with old age , women with short stature , hypertension , and kyphosis . we report herein a case involving a patient with dysphagia who had an aortic aneurysm .

from now on , molecular study regarding female sexuality has focused on female sexual hormone , like estrogen receptor ( er ) and er derivatives.12 but flibanserin is an agent of 5-hydroxytryptamin ( 5-ht ) type 1a receptor and an antagonist of 5-ht type 2a and thus a new molecular entity.3 flibanserin was originally developed as antidepressant drug . in clinical stage 2a as antidepressant , flibanserin could not prove its effectiveness , but almost no sexual function disorder was reported in subjects . for this reason , the arizona sexual experiences scale ( asex ) was used for making a comparison of the sexual function effectiveness of flibanserin on antidepressant vs. placebo proven in stage 2b studies among four depression programs.4 stage 2b studies have failed to prove its general effectiveness on depression , but in answering this question " how intense is your sexual desire ? " in the asex , flibanserin turned out to be more excellent in both placebo and active - comparator . since then , drug development has changed its directivity toward being a potential medicine for treating hypoactive sexual desire disorder ( hsdd ) . hsdd is chara or absence of sexual fantasy and sexual desire associated with personal pain in the 4th edition of the diagnostic and statistical manual - text revision ( dsm - iv - tr).5 determining lack or absence is done by clinicians by considering the factors influencing sexual activitie such as age and contexts of personal life . sexual function disorder is not explained well by other axis 1 disorders ( except other sexual function disorders ) and also it is not that it is caused only by direct physiological effects of materials ( i.e. , drug overuse , drug ) or overall medical status . the dsm-5 completed in 2013 addresses new illness called as female sexual interest / arousal disorder ( fsiad ) , which has both properties of hsdd and another illness of dsm - iv known as female sexual arousal disorder . flibanserin underwent new drug application ( nda ) as original drug in 2009 . the two central phase 3 studies ( violet6 , daisy7 ) all used e - diary and did not show big statistical improvement compared to placebo in the pre - specified co - primary efficacy endpoint that assessed daily sexual desire . in both violet6 and daisy7 studies , flibanserin showed an increase in satisfying sexual events ( sse ) and female sexual function index ( fsfi ) and reduction in female sexual distress scale - revised ( fsds - r ) , but did not show a meaningful increase in e - diary scores compared to placebo . in both studies , the most commonly reported adverse effects in women prescribed with flibanserin include drowsiness ( 11.8% ) , vertigo ( 10.5% ) , and fatigue ( 10.3%).67 these studies show a statistically significant improvement in secondary end points that measured sexual desire with other tools known as fsfi compared to placebo . applicants told that the effectiveness of flibanserin against sexual desire is better explained with fsfi , but most advisory committees did not agree to alteration in the e - diary evaluation method set as the one designed to evaluate sexual desire . the safety consciousness expressed by the advisory committees includes adverse effects such as fatigue and drowsiness as well as drug - drug interactions ( ddis ) and flibanserinalcohol interactions . another central stage 3 study is begonia,8 which set sse , fsfi , fsds - r as major evaluation methods rather than using e - diary in the existing viole and daisy studies and reported that flibanserin shows a statistically significant increase in sse and fsfi and reduction in fsds - r compared to placebo . however , many problems with safety were brought up in the second submission , too . vertigo , drowsiness , and vomiting are the most commonly reported adverse effects , and these occurred 2% to 3% , respectively if using placebo during stage 3 placebo - control premenopausal hsdd , whereas occurred close to 11% in subjects who used flibanserin as 100 mg every night at bedtime . events matching central nervous system depression ( fatigue , drowsiness , and vertigo ) occurred close to 21% in subjects who used flibanserin as 100 mg every night at bedtime and this proportion is three times higher than in placebo group . flibanserin is very difficult to endure if taking in combination with fluconazole and strong cytochrome p450 3a4 ( cyp3a4 ) suppressant and this combination may increase the risk of swoon and symptomatic low blood pressure . and ethanol administered in combination with flibanserin greatly increases the risk of drowsiness , fatigue , orthostatic hypotension , and swoon . additionally , these studies were limited to general healthy women who did not take benzodiazepines , sleep aid , narcotics and many other drugs . the effectiveness was not so big in general , and considering these concerns , fda refused to approve it again and recommended that additional safety studies are required . according to the latest fda review , there is no data indicating new efficacy . instead , flibanserin sponsors submitted additional safety data next day including research data proving that there is no obstacle to driving , data comparing the commercialized products and the side effects profile of these products , and analysis clarifying the potential effects of alcohol on side effects.9 despite the trustfulness of highly influential studies , fda product review is not a fundamental comparison , in fact , and so comparison of safety between individual products may be misunderstood . in particular , alcohol interaction study was conducted in 25 healthy volunteers as a sample , among which only two were female . since the refusal from fda in 2013 , a civic group called as even the score was formed to support what was called as " gender equality " in the manner approaching sexual function disorder treatment.10 this group insisted that there is no treatment for women although even 26 items for male sexual function disorder were already approved . this argument was rejected by fda on the ground that there are no approved products for low sexual desire in men and the 26 items for treatment contain various mixtures of testosterone . despite the fact that flibanserin is supported by consumer advocacy groups and consequently not the first product supported by pharmaceutical companies , fda 's argument on gender bias while regulating is worthy of notice in that the range of making efforts against this argument is from campaigns via social media even to letters from the members of the national assembly.11 another noteworthy feature in application for flibanserin to fda is use of the findings on sexual desire reported by patients as primary efficacy variable for approval . sexual desire can be seen by those who experience it and the results reported by patients can be measured without confounding this concept from others . the results reported by patients have become more important in studies and other drugs that have such results as primary end point have been approved ( although none were based on sexual desire ) . among all new molecular entities and biological permit applications approved by fda from 2006 to 2010 , gnanasakthy et al.12 found that among which , 17% ( 20 in 116 ) used the results reported by acknowledged patients as primary outcome in approval labels . the complex element in evaluating flibanserin lies in recalling the frequency of desire and extent of intensity and then measuring with fsfi index four weeks after the daily records of the most intense desires when applying for the primary desire end point first time . fda raised some concerns about optimizing fsfi as desire assessment tool , including the validity of contents and the period of recall , but the recent advisory committees did not focus on changing the primary end points or consulting the validity of fsfi . the final concern with flibanserin is associated with its use in clinical settings and this was an important issue for the committees . despite little reliable estimate of hsdd prevalence , this product will be used obviously in women in a broader sense than had been studied so far like others , many will not satisfy the official diagnostic criteria of hsdd , and many will increase the risk of adverse effects or be administering other drugs . the concerns with its use without labeling were reemphasized again by speakers who argued that they need flibanserin , but at the same time , it was reported that those who were diagnosed with cancer or in menopause should be excluded from treatment with flibanserin by labeling when sold . on june 4 , 2015 , fda called a committee meeting to review the efficacy and safety of flibanserin , and after the meeting , the committee approved flibanserin under the condition that risk management tools should be enforced mandatorily . despite the presence of many problems with flibanserin , fda finally approved it under the condition that risk evaluation and mitigation strategies ( rems ) should be enforced for flibanserin . in the future , flibanserin is expected to gradually increase in amount with changes from off - label to on - label . if prescription with flibanserin is determined as necessary , before using it , it is important to check prescription criterion , possible adverse effects after prescription , and precautions during administration . to get a prescription with flibanserin , the first criteria is premenopausal women aged 18 year or more who met the stages of reproductive aging workshop ( straw ) criteria . the straw criteria are that she has a regular menstrual cycle for 21 to 35 days in the previous twelve months and a normal level of follicle stimulating hormone ( fsh ) . in addition , it should be prescribed if primary diagnosis is hsdd according to dsm - iv - tr criteria or if there is any secondary sexual arousal disorder or female disposition disorder accompanied by hsdd . besides , its prescription is likely to be considered if the fsds - r ( range , 0 - 52 ) score13 used in clinical studies is 15 points or higher . in addition , in case that sexual desire disorder comes from disease , psychiatric problems , and interpersonal problems or in case that it does so from medications or other substances taken in conjunction , these cases are not the diseases for which medicine is efficacious . besides , postmenopausal women and sexual desire decline disorder male patients are not adapted during use and i t is not intended for reinforcing sexuality.1415 the adverse effects most common in prescribing flibanserin are reported as vertigo , fatigue , vomiting , insomnia , and mouth dryness . precautions in prescription include avoiding the use of other substances associated with cyp3a4 and cytochrome p450 2c19 ( cyp2c19 ) which are associated with drug metabolism , for example , administration with ketoconazole as cyp3a4 inhibitor and grapefruit juice and fluconazole as cyp3a4 or cyp2c19 inhibitor , which are likely to increase the risk of orthostatic hypotension side effects . furthermore , selective serotonin reuptake inhibitors and selective norepinephrine reuptake inhibitors ( ssri / snri ) may cause anxiety , drowsiness , fatigue , insomnia , and vertigo , if used . so carefulness is required . alcohol is contraindication because if administered with alcohol , it is highly likely to cause central nervous system depression ( fatigue , drowsiness , and vertigo ) , low blood pressure , and swoon . hormonal contraceptives are known as weak cyp3a4 inhibitors , and likely to cause side effects such as vertigo , drowsiness , and fatigue if coadministered with flibanserin . triptans is used for treating migraine as agonist of 5-ht type 1b and 1d receptor and may cause side effects of drowsiness if administered with flibanserin . in addition , if flibanserin is administered to patients with decline in liver failure , increase in concentration of drug in blood is observed and thus it 's considered that adverse effects are likely to occur . besides , taking it with herb extracts and digesters and gastroprotective drugs requiring no doctor prescription is likely to cause the risk of adverse effects and the use of flibanserin to women who are breast - feeding is contraindication . as we discussed above , flibanserin was refused to approve by fda in 2009 for its adverse effects and discordance of its efficacy from different evaluation methods . most common side effects were central nervous system depression , like fatigue , drowsiness , and vertigo . ddis with fluconazole , ethanol , and many other drugs were mainly discussed and fda refused to approve it again recommending additional safety data . with the additional profile and analysis which can prove their safety compared with other products , on june 4 , 2015 , fda finally approved it on the grounds that rems should be enforced , and soon we can get flibanserin on - label under the strict prescription . flibanserin can be prescribed to premenopausal women aged 18 years or higher who has a regular menstrual cycle , but if liver function fails , if breast - feeding , and if drinking alcohol , the use of flibanserin is contraindication . also , its administration with fluconazole , ketoconazole , ssri / snri , triptans , hormonal contraceptives , herb extracts , and several other drugs requiring no doctor prescription is prohibited because it is likely to increase the risk of adverse effects . we are waiting for patients ' reaction , considering the cost and effectiveness , in countries where flibanserin will be on sale , and more agents like unicenta ( uncnt ) and melsmon , which proved as having a efficacy of menopausal symptoms , have to be studied as treating for female sexual dysfunction.16

there have been several products developed for male sexual dysfunction . however , developing agents for female sexual dysfunction is lagging behind for various reasons . sildenafil citrate ( viagra ) and tadalafil ( cialis ) , which have been prescribed for male sexual function disorders , are known to act on vessels.[1 ] on the other hand , flibanserin is thought to act on brain . flibanserin has been approved by u. s. food and drug administration ( fda ) for treatment of hypoactive sexual desire disorder ( hsdd ) of premenopausal women in 2015 , and is expected to be released in south korea soon . authors wrote this article to acknowledge flibanserin to sexologists for females or physicians for menopausal medicine , so that this agent can be safely used for females who have hsdd .

kcm is itself very rare and distinct type of keratoacanthoma which usually occurs over extremities and scalp is unusual site for development of lesion . treatment is difficult as lesion of kcm present with large raised , rolled borders with peripheral extension . keratoacanthoma ( ka ) is a rapidly evolving tumor , composed of keratinizing squamous cells originating in pilosebaceous follicles and resolving spontaneously if left untreated . it is relatively common , especially in whites occurring in middle age while being uncommon in dark - skinned . . it presents as firm , rounded , flesh - colored or reddish papule ; with a rapid growth phase becoming 10 - 20 mm and then spontaneous healing taking place over three months . there are three rare clinical variants of solitary ka , namely giant ka , keratoacanthoma centrifugum marginatum ( kcm ) and subungual ka . in kcm , lesions are large , reaching upto 20 cms . the most common locations are dorsa of hands and legs , lesions on scalp being rare . we report a rare case of kcm occurring on the scalp which is an unusual site . a 62-year - old male , watchman by occupation presented with asymptomatic raised lesion on scalp since one year . lesion had developed de novo , as a pea sized lesion with gradual increase to cover entire vertex . cutaneous examination revealed a single , irregular , 12 15 cm , yellowish plaque on vertex of scalp with nodular surface and a central crater [ figure 1 ] . on palpation , the plaque was firm , non - tender , non - indurated and not attached to underlying structure . ultrasonography of lesion showed ill defined hypo echoic mass involving epidermis , dermis and subcutaneous tissue . a single , irregular , 12 15 cm , yellowish plaque on vertex of scalp with nodular surface and a central crater . deep punch biopsy revealed exoendophytic , globular , well circumscribed central cup shaped crater , epidermal invagination with marked hyperkeratosis and horn cyst with rich keratin filled crater imparting glassy appearance and well demarcated regular base . . marked epidermal proliferation with crater formation with horn pearls and inflammatory infiltrate at the base of the lesion was suggestive of a fully developed lesion of kcm . complete surgical excision with grafting was done successfully with dramatic improvement with no recurrence at 9 months [ figure 5 ] . exoendophytic , globular , well circumscribed central cup shaped crater , epidermal invagination with marked hyperkeratosis and horn cyst with rich keratin filled crater imparting glassy appearance and well demarcated regular base epidermis forming buttress over the pseudhorn cyst dense nodular lymphocytic infiltrate in the upper dermis surgical excision with grafting the etiology is multifactorial that includes chronic ultraviolet ray exposure , smoking , contact with chemical carcinogens like pitch , mineral oil , tar , trauma and vaccination . the role of human papilloma virus remains inconclusive but in one study , hpv type 6 and 11 were detected within the lesion . it can be localized to any region of the body but is more frequently seen on dorsum of hands and legs . progressive peripheral extension with a raised rolled - out margin and atrophy at the centre is a characteristic feature of kcm . the margin of the lesion showing multiple comedonal orifices giving rise to a cribriform pattern may represent a unique phenomenon of kcm . it is hypothesized that this typical appearance may arise as a result of sequential involvement of multiple adjacent hair follicles in a centrifugal fashion . kcm does not show tendency for spontaneous regression , a feature also seen in giant ka , which grows rapidly reaching a size of 5 cm or more and occurring commonly on nose and eyelids . kcm is differentiated from giant ka by absence of downward vertical spread and destruction of underlying tissue . nevertheless , in giant ka spontaneous involution takes place after several months , often accompanied by detachment of a large keratotic plaque . the other differential diagnoses include squamous cell carcinoma , lupus vulgaris , botryomycosis , blastomycosis - like pyoderma and pseudoepitheliomatous hyperplasia , hypertrophic lupus erythematosus , atypical mycobacterial infections , or deep fungal infections . there are reports that kcm has been treated successfully with oral retinoids ( acitretin , etretinate , or isotretinoin 0.5 - 1 mg / kg / day ) which should be given until complete clearance of lesion . surgical intervention is a preferred mode of therapy keeping in mind that a wide excision is performed to prevent subsequent recurrence . other treatment modalities successfully used include topical 5-fluorouracil , intralesional injections of interferon alpha , methotrexate , or bleomycin , or mohs micrographic surgery . in kcm , lesions are usually too large for excisional biopsy for diagnosis , but certain characteristic features like clinical behavior , appearance of lesions , histological features ( hyperkeratosis and hyperplasia ) , and cytological features ( tumor cells with eosinophilic and glassy cytoplasm , few mitoses ) help clinch the correct diagnosis . first time from india we are reporting case of kcm presenting over scalp which is successfully treated with surgical excision without any recurrence .

keratoacanthoma ( ka ) is a rapidly evolving tumor , composed of keratinizing squamous cells originating in pilosebaceous follicles and resolving spontaneously if left untreated . it is relatively uncommon in dark - skinned and occurs in middle aged individuals . males are three times more affected than females . it presents as firm , rounded , flesh - colored or reddish papule ; with a rapid growth phase followed by spontaneous healing over three months . two types of ka exist i.e. , solitary and multiple . there are three rare clinical variants of solitary ka , namely giant ka , keratoacanthoma centrifugum marginatum ( kcm ) and subungual ka . in kcm , lesions are large , reaching upto 20cms . there is peripheral extension with raised , rolled border and atrophy in the center . there is no tendency toward spontaneous involution . the most common locations are dorsa of hands and legs , lesions on scalp being rare . a rare case of kcm occurring on scalp which is an unusual site is reported .

hydroxyapatite biomaterials are materials that are very widely used in several health purposes , including as a source of calcium for the manufacture of toothpaste and as an important material in the formation / bone repair . the chemical properties of hydroxyapatite are bioactive and compatible with the adjacent bone and teeth . hydroxyapatite is a calcium phosphate ceramic that is totally biocompatible and nontoxic and becomes an integral part of living bone and teeth tissue [ 18 ] . the raw material for the production of hydroxyapatite biomaterial is very easily available and abundant in indonesia . the production process was easy and the cost is also relatively inexpensive if done on a large scale . among the abundant raw materials are the shells of crabs , which are one of indonesia 's main export commodities . export of crabs commodity by indonesia amounted to 604.215625.000 tons / year without the shell form , while domestic consumption is expected to be so much more , as in makassar , carrying 292.5 tons exported in the form of crab without shell with the main export destination being singapore . if the mass of crab shells 2550% of the total mass , it can be estimated that in 2012 produced the shells of crabs around 151,053.75 to 302,107.5 tons in indonesia and 73.125 to 146.25 tons in makassar ( a region of indonesia ) , there was just from crabs production were exported . this value is of course even more if the crab 's consumptions in the country are also taken into account . this suggests the existence of crab shells is abundant in indonesia , including in makassar . as known in indonesia , the shells of crabs have not been used , so it will only be a waste disturbing environment [ 7 , 8 ] . crabs shells containing calcium carbonate ( caco3 ) are very abundant ; amount 4070% , varies according to the species . calcium carbonate can be further processed into calcium hydroxyapatite [ ca10(po4)6(oh)2 ] [ 4 , 5 ] . according to strassler , hydroxyapatite is one of the active ingredient materials that are widely used in toothpaste products for protection against teeth demineralization [ 1113 ] . the crystals structure of hydroxyapatite will be better by using cao as a precursor of calcium . however , the use of these compounds also produces carbonate apatite [ ca10(po4)6co3 ] in a fairly large percentage . this is because the calcination process can not completely eliminate carbon dioxide ( co2 ) in caco3 so that there can be reaction with the precursor phosphate . however , the carbonate apatite heating at a temperature of 700900c for 25 hours , followed by washing using distilled water , the carbonate apatite can be hydroxyapatite [ 1113 ] . the instrument were used in this research were glass apparatus , ohaus analytical balance , petri dish , porcelain cup , buchner flask , buchner funnel , vacuum pump sargent - welch co. model 1400 , magnetic stirrer , magnetic bar , hotplate idealife , ph meter , furnace thermolyse 6000-barnstead , dessicator , thermometers , spnisosfd oven , stopwatch , shimadzu x - ray diffractometer ( xrd ) model 6000 , x - ray fluorescence ( xrf ) shimadzu , fourier transform infra - red ( ftir ) prestige-21 shimadzu , scanning electron microscopy combined with the ability to generate localized chemical information ( sem - edxa ) variant , and uv - vis shimadzu model 6105 . this is because the carbonate ion compounds are able to inhibit the crystallization process ca10(po4)6(oh)2 so that the results will be dominated by an amorphous phase . if the caco3 burned at temperature calcinations , decomposition reaction of caco3 into cao will occur and co2 emissions are dominant and will be issued as a result of the combustion reaction [ 1315 ] . waste of crabs shells portunus pelagicus species was taken from an exporter crabs company in industry areal , makassar city , south sulawesi province , indonesia . wahidin hospital , and ( nh4)2hpo4 was obtained from fluka chemical , ch3cooh glacial 100% , ch3coona3h2o from merck , ( nh4)6mo7o244h2o from aldrich , nh4vo3 from aldrich , hno3 and naf from fluka chem . , ( nh4)2c2o4 , distilled water , aluminum foil , and whatman filter paper numbers 40 and 42 . the instruments were used in this research that glass as commonly used in laboratories , ohaus analytical balance , petri dish , porcelain cup , buchner flask , buchner funnel , vacuum pump sargent - welch co. model 1400 , magnetic stirrer , magnetic bar , hotplate idealife , ph meter , furnace thermolyse 6000 barnstead , dessicator , thermometers , oven spnisosfd , stopwatch , shimadzu x - ray diffractometer ( xrd ) model 6000 , x - ray fluorescence ( xrf ) shimadzu , fourier transform infrared ( ftir ) prestige-21 shimadzu , scanning electron microscopy combined with the ability to generate localized chemical information ( sem - edxa ) variant , and uv - vis shimadzu model 6105 . crabs shells portunus pelagicus waste was cleaned with distilled water and dried at room temperature . furthermore , to transform crabs shells into caco3 and then into cao , calcinations were performed on the samples at 1000c temperature for 5 hours at a rate of temperature rise 5c / minute . calcium oxide ( cao ) obtaining dominated as result of calcinations and then made suspensions in 100 ml of distilled water with a calcium concentration of 0.3 m. the suspensions reacted by dropwise with a 100 ml 0.2 m of ( nh4)2hpo4 solution through the coprecipitation method , at temperatures around 40c while the solution was stirred for 25 hours . the precipitation allowed stand overnight or 24 hours at room temperature , and the precipitate is filtered with a whatman filter paper number 40 and dried at 110c for 5 hours . the pure hydroxyapatite obtained by sintering to the dried precipitate at various temperatures of 500900c for 4 hours [ 16 , 17 ] . the characterization of the compounds was performed by using x - ray diffraction ( xrd ) , ftir , and sem - edxa . the proven in vitro demineralization of teeth was conducted through evaluating the concentration of phosphate in solution by using the uv - vis spectroscopy . the effectiveness of hydroxyapatite to protection against teeth demineralization was tested in acetate buffer ph 5.0 solutions , with 1 m of acetic acid concentration with the addition of hydroxyapatite in varying concentrations and immersion time . each of 5 beakers was filled with 300 ml of acetate buffer ph 5.0 with acetic acid concentration of 1 m. an acetate buffer was left without adding anything as a comparison . an acetate buffer was then added 10 ppm of naf left without addition of ca10(po4)6(oh)2 . other three pieces beaker of acetate buffer containing 10 ppm of naf are adding of ca10(po4)6(oh)2 with variation concentration of 25 ppm , 50 ppm , and 100 ppm . the immersed times of tooth samples in solution are 3 , 6 , 9 , 24 , and 48 hours , respectively . furthermore , the phosphate concentration in each solution was measured by using uv - vis spectrophotometer with the wavelength for phosphate ( maks ) being 432 nm . the calcinations aim to eliminate the organic component in the shells of crabs and convert caco3 compound which is the dominant compound in the shells of crabs into cao through the elimination of co2 in gas form . characterizing the calcinations results the results can be seen from the diffraction pattern of the crab shells before and after calcinations at a temperature of 1000c for 5 hours ( figure 1 ) . in this figure can be observed changes in the diffraction pattern of crab shells , where the change in the diffraction pattern is due to the chemical change from mixture of caco3 with organic matter to pure cao . the appearance of the sharper peaks in figure 1 after calcinations ( b ) is a result of the crystallinity of the cao . identification by ft - ir as shown in figure 2 showed there is a reduction process of -co3 groups and some of ir - spectra were missing after calcinations . the elimination of -co3 groups and organic components can also be seen from the data of mass reduction of sample calcinations . the determinations of calcium contained in sample was conducted by using x - ray fluorescence , where obtained calcium is 66.62% after calcinations . these results are then used to calculate the stoichiometry in determining the number of results calcinations which is needed to react with ( nh4)2hpo4 as the precursor phosphate . the precipitation reactions aiming to produce ca10(po4)6(oh)2 used phosphate , ( nh4)2hpo4 , as the precursor and then reacted with cao as calcinations results . side results ca10(po4)6co3 also occur as a product of reaction of ( nh4)2hpo4 and caco3 presence in the calcinations results . dried precipitate further sintered on temperature variations of 4001000c for 2 hours ; it is intended to determine the optimum temperature where the ca10(po4)6(oh)2 is formed , furthermore characterized by using xrd , ft - ir , and sem - edxa . the diffractograms of each sintering results compounds indicate that the temperature is closely related to the formation of crystals . this is due to the nature of the vibrating atoms moving faster in higher temperatures . the optimum temperature formation of hydroxyapatite was determined by calculation of the probability of the sample phase from the xrd results analysis according to jcpds standard data , which , jcpds ; 24 - 0033 is standard data for ca10(po4)6(oh)2 ; 09 - 0169 for -ca3(po4)2 ; 29 - 0359 for -ca3(po4)2 , 35 - 0180 -ca3(po4)2 ; 35 - 0180 for ca10(po4)6co3(oh)2 and 19 - 0272 standard data for ca10(po4)6co3(oh)2 . figures 3 and 4 showed that the temperature is closely associated with the formation of hydroxyapatite phase . in the both graphs it can be seen that the maximum intensity of the phase formation of hydroxyapatite has been found by sintering at temperature of 800c , it means the optimum temperature of hydroxyapatite formation is 800c , and then this result will be used for another application . the formations of hydroxyapatite phase had been dominated at 800c confirmed by percentage probability sample phase ( figure 5 ) , in which the percentage of hydroxyapatite phase formed around 46.61% , while phases of -ca3(po4)2 and phase -ca3(po4)2 are 17.76% and 19.37% , respectively . however , also there is still presence of a phase ca10(po4)6co3 and ca10(po4)6co3(oh)2 with a range of 11.84% and 4.43% , respectively , which indicates the presence of carbonates . the x - ray diffractograms of ca10(po4)6(oh)2 synthesized at a 800c temperature sintering can be seen in figure 6 , where the peaks of ha are symbolized by the peak of the crystal ca10(po4)6(oh)2 , while peak -tkf is symbolized crystalline peaks of -ca3(po4)2 , the symbol -tkf for crystalline -ca3(po4)2 , aka for ca10(po4)6co3 , and akb for ca10(po4)6co3(oh)2 . the highest intensity peak at 31.2572 deg corresponding to crystalline -ca3(po4)2 is seen , the second highest peak intensity is 31.7783 , and the third highest peak intensity is 28.0565 crystals suitable for ca10(po4)6(oh)2 . ftir results showed that the sintering temperature variation affects the absorption band shapes which generally all sintering results showed absorption band of -oh , absorption band 1 , 2 , 3 , and 4 of po4 , and co3 groups . infrared spectra in figure 7 show the -oh groups at 633 cm which are characteristic of hydroxyapatite appearing on the sintering temperatures of 4001000c . additionally spectrum also showed higher sintering temperature causing the sharper peaks phosphate group ( po4 ) because the nature of the vibrating atoms moves faster at higher temperatures . the ft - ir spectra of sample were sintered at a temperature of 800c ( figure 8) showing that hydroxyapatite is the dominant compound formed . the stretching frequencies of po4 group are indicated by 1120.64 cm , 1091.71 cm , 1043.49 cm ( 3 ) , 993.34 cm , 877.61 cm ( 1 ) , 603.72 cm , 565.14 cm , ( 4 ) , and 370.33 cm ( 2 ) . and a sharp spectrum in the area of 3570.24 cm indicates the presence of free -oh and 3427.51 cm indicating -oh bounded , and this is indicating that the dominant compound is ca10(po4)6(oh)2 . while area of 1654.92 cm , 1458.18 cm , and 1421.54 cm indicates the presence of carbonate groups ( co3 ) it can be identified as ca10(po4)6co3 and ca10(po4)6co3(oh)2 which has not been transformed into ca10(po4)6(oh)2 during the sintering process . the results of characterization by sem - edxa shown in figure 9(a ) show that the size of hydroxyapatite formed from the synthesis tends to be small and only a few are large . while figure 9(b ) shows that the surface of hydroxyapatite is smooth and nonporous , this shows that hydroxyapatite which has synthesis from crab shells can function well as inhibiting tooth demineralization . while in figure 10 the edxa spectrum shows the composition of the synthesis yield was dominated by oxygen ( o ) up to 59.52% , calcium ( ca ) up to 23.76% , and phosphorus ( p ) up to 13.32% . demineralization of teeth is a process of decomposition of the crystal of ca10(po4)6(oh)2 due to the acidic conditions by releasing ca and po4 ions . demineralization of tooth causing increased levels of ca and po4 in saliva in direct contact with the tooth . in vitro , the rate of tooth demineralization can be observed through the concentrations of ca and po4 ions in solutions where the tooth was soaked each unit of time . therefore , increase of po4 ions concentrations in solution a soaked gear can be one of indicators to measure the rate of tooth demineralization . figure 11 shows the relationship between the soaking time of teeth versus the increase of the ion po4 levels in solution where the tooth was soaked as well ; it appears that with the increasing addition of ca10(po4)6(oh)2 into the acetate buffer equals to the rate of demineralization decrease . it is can be altered by the amount of po4 ions in solutions ; however the addition of ca10(po4)6(oh)2 ions showed lower amount of po4 ions compared to solutions without the addition of ca10(po4)6(oh)2 . this proves that the ca10(po4)6(oh)2 were synthesized from the crab shell effective for protection against tooth demineralization . the decrease in the rate of tooth demineralization with the addition of ca10(po4)6(oh)2 can also be observed through analyzing the tooth mass reduction in the fifth variation of acetate buffer solution as shown in table 1 . the greater concentrations of ca10(po4)6(oh)2 in the acetate buffer solution were teeth immersed exhibit the smaller mass of teeth in the solution . based on these results it is concluded as follows.waste of shells crabs ( portunus pelagicus ) proved to be used as raw material for the synthesis of ca10(po4)6(oh)2 due to high calcium levels which amounted to 66.62% in addition to the abundant existence as waste.the optimum temperature formation of ca10(po4)6(oh)2 is at 800c.ca10(po4)6(oh)2 were synthesized from waste of shell crabs ( portunus pelagicus ) in vitro effectively inhibiting the rate of demineralization of the tooth where the greater the addition of ca10(po4)6(oh)2 in the solution , the more the inhibiting demineralization of the tooth or the smaller the rate of tooth demineralization in solution . waste of shells crabs ( portunus pelagicus ) proved to be used as raw material for the synthesis of ca10(po4)6(oh)2 due to high calcium levels which amounted to 66.62% in addition to the abundant existence as waste . ca10(po4)6(oh)2 were synthesized from waste of shell crabs ( portunus pelagicus ) in vitro effectively inhibiting the rate of demineralization of the tooth where the greater the addition of ca10(po4)6(oh)2 in the solution , the more the inhibiting demineralization of the tooth or the smaller the rate of tooth demineralization in solution .

crab 's shells of portunus pelagicus species were used as raw materials for synthesis of hydroxyapatite were used for protection against demineralization of teeth . calcination was conducted to crab 's shells of portunus pelagicus at temperature of 1000c for 5 hours . the results of calcination was reacted with ( nh4)2hpo4 , then dried at 110c for 5 hours . sintering was conducted to results of precipitated dried with temperature variations 4001000c for a hour each variation of temperature then characterized by x - ray diffractometer and ftir in order to obtain the optimum formation temperature of hydroxyapatite is 800c . the hydroxyapatite is then tested its effectiveness in protection against tooth demineralization using acetate buffer ph 5.0 with 1 m acetic acid concentration with the addition of hydroxyapatite and time variation of immersion . the results showed that the rate of tooth demineralization in acetate buffer decreased significantly with the provision of hydroxyapatite into a solution where the addition of the magnitude of hydroxyapatite is greater decrease in the rate of tooth demineralization .

brain injury is caused by intrinsic or extrinsic factors and it can result in various disabilities such as motor , sensory , behavioral , or cognitive dysfunction depending on the area of the brain lesion1 . cognitive impairment due to brain injury is an important factor affecting patients independent functions and participation in activities2 , interfering with their return to daily living and work3 . it can also influence motivation and the ability to participate in rehabilitation programs and interfere with a return to the community . therefore , for successful rehabilitation , accurate and comprehensive cognitive assessment and treatment are required4 . for cognitive rehabilitation of patients with brain injury , traditional treatment and computer - based cognitive therapy ( vr ) technology is gaining recognition as a useful tool for cognitive research , evaluation , and rehabilitation5 . vr systems allow users to interact in various sensory environments and to obtain real - time feedback on their performance using computer technology6 . the virtual environment offered via vr technology makes it possible for patients to participate in activities in settings and environments similar to those encountered in real life7 , 8 . in addition , vr tools can be used to record accurate measurements of the subject s performance9 and to deliver greater therapeutic stimulation to users5 . vr has been used as a tool to diagnose cognitive impairment and as a vehicle to provide new treatments5 . although the use of vr in cognitive rehabilitation has been increasing , few systematic reviews have investigated the use of vr programs in cognitive rehabilitation and the overall effect of these programs on cognition . therefore , this systematic review investigated the different types of vr programs used for cognitive evaluation and interventions for patients with brain injury . studies using vr programs for cognitive intervention were reviewed according to pico ( patient , intervention , comparison , and outcome ) methods . ( virtual reality or virtual or game based virtual reality or computer based virtual reality ) and ( stroke or cerebral vascular accident or hemiplegia or brain injury or traumatic brain injury ) and ( cognition or cognitive or memory or attention or executive function ) . inclusion criteria were : ( 1 ) subjects over the age of 19 years with brain injury ; ( 2 ) articles written in english ; and ( 3 ) studies that used vr in cognitive rehabilitation . exclusion criteria were : ( 1 ) subjects who were animals or children ; ( 2 ) review articles ; and ( 3 ) 2d computer - based cognitive rehabilitation . randomized controlled trials ( rcts ) and crossover studies were scored on the physiotherapy evidence database ( pedro ) scale12 . two authors independently assessed the methodological quality of the included studies , and disagreements were resolved by reaching consensus . memory assessment was the most common study topic , followed by assessments of executive function and attention . a variety of vr programs were used ( table 1table 1.analysis of studies of cognitive assessment using vrauthor , yearassessment areatype of vrbrooks et al . three were rcts24,25,26 , one was a crossover study27 , and one was a case report28 . excepting the case report , four studies were assessed using the pedro scale . two studies24 , 25 scored 4 , and the remaining two studies scored 326 and 127 , respectively . table 2table 2.analysis of studies with cognitive interventions using vrstudypatientinterventioncomparisonoutcomenmean agetype of vrintensityoutcome measure(s)findingsakinwuntan et al . , 20102469eg 55 cg 54stisim drive system60 min / day , 3 times / week , total 5 weeksnon - computer based cognitive therapyufov testsignificant within group improvements but no significant difference between two groupscaglio et al . , 201228124midtown madness 290 min / day , 3 times / week , total 5 weeks - fdst , bdst , ravlt - ir , dr , rbmt - ir , dr , corsi s block tapping test , corsi s supraspan test - ir , dr , tmt - a , b , phonemic fluency mmse , adassignificant improvements in ravlt - ir and corsis supraspan - ir , drgrealy et al . , 1999271332.38nonimm - ersive vr exercise bicycle25 min / day , 3 times / week , total 4 weeksno treatmentfdst , bdst , digit symbol test , tmt - a & b , auditory learning test , velt , vilt , logical memory , complex figure testeg showed significant improvements in digit symbol test , velt , vilt compared to cgjacoby et al . , 20132512eg 27.83 cg 30.67irex45 min / day , 34 times / week , total 3 weeksconventional otmet - sv , efpteg showed significant improvements in all outcome measures compared to the cgkim et al . , 20112628eg 66.5 cg 62.0irex30 min / day , 5 times / week , total 4 weekscomputer based cognitive therapyk - mmse , tol , vcpt , acpt , word - color test , fdst , bdst , fvst , bvst , vilt , velt , tmt - asignificant difference between experimental group and control group in vcpt and bvsteg : experimental group , cg : control group , met - sv : multiple errands test - simplified version , efpt : executive function performance test , fdst : forward digit span test , bdst : backward digit span test , tmt - a : trail making test - type a , tmt - b : trail making test - type b , velt : verbal learning test , vilt : visual learning test , ufov : useful field of view test , k - mmse : korean version of the mini - mental status examination , tol : tower of london test , vcpt : visual continuous performance test , acpt : auditory continuous performance test , fvst : forward visual span test , bvst : backward visual span test , ravlt : rey auditory verbal learning test , ir : immediate recall , dr : delayed recall , rbmt - the rivermead behavioural memory test , adas : alzheimer s disease assessment scale presents the characteristics of the five studies . flowchart of the article search and study selection eg : experimental group , cg : control group , met - sv : multiple errands test - simplified version , efpt : executive function performance test , fdst : forward digit span test , bdst : backward digit span test , tmt - a : trail making test - type a , tmt - b : trail making test - type b , velt : verbal learning test , vilt : visual learning test , ufov : useful field of view test , k - mmse : korean version of the mini - mental status examination , tol : tower of london test , vcpt : visual continuous performance test , acpt : auditory continuous performance test , fvst : forward visual span test , bvst : backward visual span test , ravlt : rey auditory verbal learning test , ir : immediate recall , dr : delayed recall , rbmt - the rivermead behavioural memory test , adas : alzheimer s disease assessment scale in this review , the types of vr programs that have been used in cognitive evaluations of patients with brain injury were identified and studies of cognitive interventions were reviewed according to pico methods . in the included studies , the vr programs could distinguish the cognitive disability of patients in comparisons with healthy subjects . thus , vr could be used as a new assessment method of the cognitive function of patients with brain injury . therefore , in contrast to conventional cognitive assessments , vr programs can provide consistently accurate measurements of cognitive function . however , some methodological problems were found in the reviewed articles . in most of the studies , the vr tool used was not compared with a standardized assessment tool , and the inter - rater reliability was not measured . the five studies of cognitive therapy using vr all reported positive effects . in the assessment of cognitive function , the vr interventions resulted in improvements in the areas of memory and attention but not executive function . ben - yishay et al.29 stated that to effectively raise cognitive function , normal attention is needed . if the ability to concentrate on external information is impaired , memory , problem - solving skills , and appropriate behavior may be difficult . thus , they suggested that the impairment of attention due to brain injury may interfere with the recovery of other cognitive functions , such as memory , executive function , and planning . the results of this systematic review indicate that the cognitive improvement of attention using vr programs will have a positive impact on the recovery of general cognitive function . the advantage of cognitive rehabilitation using vr is that it provides a variety of environments similar to those encountered in real life30 . the results of this review suggest that patients are more motivated in virtual environments than they are in conventional settings . therefore , vr programs can be expected to lead to an improvement in cognitive function . in vr interventions , patients can be treated in a safe environment compared to real settings . in addition , vr programs can be tailored to the type of injury and easily adjusted to the level of cognitive disability , the complexity of a task , the reaction conditions , and the characteristics and patterns of feedback30 . as vr systems are constantly evolving and becoming smaller and more easily adjustable , they can be expected to provide specialized therapy in new settings , such as patients homes or clinics . these advantages of vr systems can benefit patients who find it difficult to visit health care organizations5 . the results of this systematic review suggest that vr is an effective cognitive therapy for patients with brain injury compared to control therapy . , there was a significant risk of bias with regard to allocation concealment and blinding . given the heterogeneity of the included studies , the ability to draw conclusions is limited . well - designed rcts and blind studies will be needed to provide evidence of the benefits of vr on cognitive function .

[ purpose ] the purpose of this review was to investigate various types of vr programs and their use in cognitive evaluations and interventions for patients with brain injury . [ subjects and methods ] pubmed , cochrane , and otseeker electronic databases were searched with the search terms . at of 350 titles and abstracts were retrieved , and 17 articles were selected for this review . selected articles were assessed on the level of evidence using the physiotherapy evidence database ( pedro ) scale . [ results ] articles assessing the impact of cognitive impairments in memory were most commonly found , and vr interventions elicited positive effects in patients with brain injury . [ conclusion ] vr can be considered a new tool for cognitive rehabilitation after brain injury . vr interventions also have a number of advantages , e.g. cost - effectiveness , compared to other interventions .

despite a considerable progress in malaria control in iran over the past few years that led to significant reduction of cases , the disease still remains a major health problem in south and southeastern parts of the country . 2008 , afsharpad et al . 2012 ) and insecticide resistance of anopheles vectors ( enayati et al . 2012 ) are aggravated by continuous influx of imported cases , mostly with plasmodium falciparum , from neighboring countries of afghanistan and pakistan ( zakeri et al . the latest checklists of iranian mosquitoes include 28 anopheles species , identified mostly on the basis of morphological features , and a few by dna - based approaches ( azari - hamidian 2007 ) . maculipennis are known to be responsible for transmission of malaria in the country ( manouchehri et al . some important malaria vectors in iran are assumed to be members of species complexes or species groups , which are often difficult to distinguish morphologically . application of dna - based approaches has resolved some cryptic species in iranian complex species including an . fluviatilis , however , the identity of some members are still doubtful or were refuted later ( azari - hamidian 2007 , naddaf et al . this , to great extent , is due to introduction of molecular markers such as its2 and 28s - d3 genes for discriminating the members of this complex species in india ( manonmani et al . biology , variation in behaviors , and role of this species in malaria transmission in different geographical areas of iran has been extensively reviewed by others ( eshghi et al . 1976 , edalat 19971998 , hanafi - bojd et al . 2012 ) . in an early study comparison of its2 sequence of iranian specimens from various localities in south and southeastern iran revealed only species y , which is presumably species t ( naddaf et al . 2003 ) , however , rapd - pcr analysis of same specimens revealed two distinct patterns , separating representatives of fars province from other areas ( naddaf et al . analysis of 28s - d3 gene from same populations corroborated rapd results , fars province specimens showed to be identical to species u in india , while individuals from other areas exhibited heterozygocity at the only base pair position that identifies species u and t ( naddaf et al . 2010 ) . in addition , in a separate study based on 28s - d3 analysis , species t and species u were reported from jiroft of fars province and chabahar of sistan va baluchestan province , respectively ( mehravaran et al . 2011 ) . the aim of this study was to evaluate cytochrome oxidase i ( coi ) gene alongside 28s - d3 as a diagnostic tool for identification of an . coi gene sequences have been extensively used for population studies and resolving evolutionary relationship among closely related species groups of insects ( lunt et al . 1996 ) and anopheline mosquitoes ( krzywinski and besansky 2003 ) . variations in this fragment have been exploited as dna barcodes for identifications of culicidae mosquitoes including an . fluviatilis mosquitoes originated from different localities in south and southeastern areas of iran including fars , hormozgan , kerman , and sistan va baluchestan provinces . the extraction method and identity of some mosquitoes based on its2 and/or 28-d3 genes were described previously ( naddaf et al . all the dna samples were initially subjected to allele specific ( as)-pcr based on 28s - d3 gene as described by singh et al . the coi gene was amplified using universal primers , ubc6 ( 5- gga gga ttt gga aat tga tta gtt cc -3 ) and ubc9 ( 5-ccc ggt aaa att aaa ata taa act tc-3 ) , designed by simon et al . each 25l reaction contained 20 pmol of each primer , 2 mm mg cl2 , 10 mm tris - hcl , 50 mm kcl , 150m of dntps , 1u of taq , and 2l of dna . pcr products were purified using a gel band purification kit ( pharmacia , piscataway , nj , usa ) according to manufacturer s recommendations and later sequenced using the same primers as used for amplification at seqlab laboratory in germany . the sequences were manually edited and corrected using bioedit software , version 7.1.3.0 ( hall 1999 ) and fragments of 474 bp length were selected for analysis . the distances between groups and between individual sequences were calculated , and phylogenetic tree for iranian sequences was generated using the kimura two parameter ( k2p ) model of neighbor - joining method in a complete deletion procedure using mega 4 software ( tamura et al . the sequence data for the coi gene sequences were submitted to genbank with the accession numbers jx020706-jx020729 . fluviatilis mosquitoes originated from different localities in south and southeastern areas of iran including fars , hormozgan , kerman , and sistan va baluchestan provinces . the extraction method and identity of some mosquitoes based on its2 and/or 28-d3 genes were described previously ( naddaf et al . all the dna samples were initially subjected to allele specific ( as)-pcr based on 28s - d3 gene as described by singh et al . the coi gene was amplified using universal primers , ubc6 ( 5- gga gga ttt gga aat tga tta gtt cc -3 ) and ubc9 ( 5-ccc ggt aaa att aaa ata taa act tc-3 ) , designed by simon et al . ( each 25l reaction contained 20 pmol of each primer , 2 mm mg cl2 , 10 mm tris - hcl , 50 mm kcl , 150m of dntps , 1u of taq , and 2l of dna . pcr products were purified using a gel band purification kit ( pharmacia , piscataway , nj , usa ) according to manufacturer s recommendations and later sequenced using the same primers as used for amplification at seqlab laboratory in germany . the sequences were manually edited and corrected using bioedit software , version 7.1.3.0 ( hall 1999 ) and fragments of 474 bp length were selected for analysis . the distances between groups and between individual sequences were calculated , and phylogenetic tree for iranian sequences was generated using the kimura two parameter ( k2p ) model of neighbor - joining method in a complete deletion procedure using mega 4 software ( tamura et al . the sequence data for the coi gene sequences were submitted to genbank with the accession numbers jx020706-jx020729 . all the dna specimens from fars province yield only a product of approximately 375 bp length indicative of species u , whereas specimens from hormozgan , kerman and sistan va baluchestan provinces amplified two bands of 375 bp and 128 bp length . phylogenetic analysis using coi gene grouped individuals from fars province in two distinct clades separate from other iranian individuals representing populations of hormozgan , kerman , and sistan va baluchestan ( fig . the mean distance between iranian and indian groups was 1.66% , while the value between fars group and the group comprising other iranian members was 2.06% . the indian group exhibited the same distance ( 2.06% ) with fars group . the highest distance ( 3.09% ) among iranian individuals was between specimens 930 from fars province and 398 from sistan va baluchestan province . six individuals belonging to two clades ( 87 , 97 , 655 , and 928 ) and ( 92 ) from fars province showed 100% identity . in addition , ten individuals from other geographical areas including four from koveh ( 170 , 172 , 173 , and 186 ) and one from minab ( 121 ) in hormozgan province , two from abchekan ( 392393 ) in sistan va baluchestan province , and three from kahnouj in kerman province ( 815 , 836 , and 839 ) were 100% identical . accurate identification of malaria vectors is not only one of the most basic requisite for success of malaria control programs , but also has become an intriguing issue for understanding speciation process and evolution of anopheles mosquitoes . in absence of cytotaxonomic evidence , rapd - pcr methodology and variation in 28s - d3 gene have resolved two potential sibling species in an . we report further evidence for the occurrence of these two sibling species by coi analysis of mitochondrial dna from the same specimens . anopheles fluviatilis james is a complex of cryptic species ; cytotaxonomic studies of polythene chromosomes has revealed three reproductively isolated species in india known as s , t , and u ( subbarao et al . 1994 ) . application of the first dna - based method using its2 gene identified two putative species of x and y that are presumably equivalent to species s and t , respectively ( manonmani et al . later , a complete as - pcr assay based on variations of 28s - d3 gene against chromosomally examined specimens identified all the members of the complex ( singh et al . fluvialitis mosquitoes of iran . however , the same specimens displayed variations in 28s - d3 gene ; the individuals from fars exhibited similarity with species u in india whereas individuals from others areas showed heterozygocity at the single nucleotide position that identifies species u and t. the identity of an . fluviatilis complex in iran became complicated as the heterozygocity in 28-d3 was not reflected in its2 fragment and individuals from fars province exhibited dual identity of t and u based on its2 and 28s genes , respectively ( chen et al . fluviatilis sibling species were not addressed in their study , however , k2p genetic distances between different species of culicidae were reported to be > 2% . in our study , the mean distance between iranian and indian populations was 1.66% , whereas the value between fars group and the group comprising other iranian individuals was 2.06% . the distance between most individuals from fars province and individuals from other areas was > 2% . two individuals ( 9192 ) from fars province exhibited almost equivalent distance with all other members including those ( 930 , 926 , 928 , 655 , 87 , and 97 ) from the same province . jf966741 , unpublished ) that showed a high divergence from other sequences and appeared as an out group beyond an . the results of present study were almost concordant with earlier results obtained by rapd - pcr methodology and 28s - d3 analysis ( naddaf et al . this study shows that coi gene can be used as a useful tool along other dna markers like 28-d3 gene for dissolving closely related taxa of an . fluviatilis mosquitoes ( by its2 and 28s - d3 genes ) from india , iran , and other geographical areas by this genetic marker can bring more insight into taxonomy of this sibling species . this study shows that coi gene can be used as a useful tool along other dna markers like 28-d3 gene for dissolving closely related taxa of an . fluviatilis mosquitoes ( by its2 and 28s - d3 genes ) from india , iran , and other geographical areas by this genetic marker can bring more insight into taxonomy of this sibling species .

background : anopheles fluviatilis , one of the major malaria vectors in iran , is assumed to be a complex of sibling species . the aim of this study was to evaluate cytochrome oxidase i ( coi ) gene alongside 28s - d3 as a diagnostic tool for identification of an . fluviatilis sibling species in iran.methods:dna sample belonging to 24 an . fluviatilis mosquitoes from different geographical areas in south and southeastern iran were used for amplification of coi gene followed by sequencing . the 474475 bp coi sequences obtained in this study were aligned with 59 similar sequences of an . fluviatilis and a sequence of anopheles minimus , as out group , from genbank database . the distances between group and individual sequences were calculated and phylogenetic tree for obtained sequences was generated by using kimura two parameter ( k2p ) model of neighbor - joining method.results:phylogenetic analysis using coi gene grouped members of fars province ( central iran ) in two distinct clades separate from other iranian members representing hormozgan , kerman , and sistan va baluchestan provinces . the mean distance between iranian and indian individuals was 1.66% , whereas the value between fars province individuals and the group comprising individuals from other areas of iran was 2.06%.conclusion : presence of 2.06% mean distance between individuals from fars province and those from other areas of iran is indicative of at least two sibling species in an . fluviatilis mosquitoes of iran . this finding confirms earlier results based on rapd - pcr and 28s - d3 analysis .

they are found most commonly in the cranial and peripheral nerves , and occurrence in the omentum is very rare . however , there have been some cases reported to develop serious complications and , if there was malignancy , to cause metastasis or recurrence . we presented a case of schwannoma originating from the great omentum , including histological and immunohistological studies . a 55-year - old man was referred to our department for the treatment of a tumor detected close to the stomach by ultrasound . medical examination including ultrasound had been regularly performed to follow up his gallbladder stone for the past 6 years . although he had no complaints and symptoms , the tumor had been increasing in size over one year . abdominal and endoscopic ultrasound showed a 2.0 1.3 cm cystic mass lateral to the wall of the stomach . its component included an 11.8 5.7 mm elevated lesion ( fig . 1 , fig computed tomography scan of the abdomen and pelvis showed a 2.6 1.9 cm cystic mass , which was slightly enhanced , and a gallbladder stone ( fig . 3 ) . magnetic resonance imaging demonstrated a hyperintense mass on t2-weighted image . a distance from the stomach wall laboratory tests , including tumor makers , were normal . under a perioperative diagnosis of cystic tumor in the abdomen and cholelithiasis , we performed laparoscopic resection with vessel sealing system , which revealed a 2.0 cm mass arising from the great omentum , not adherent to other organs . grossly , the tumor was configured by a well - encapsulated round mass measuring 30 18 15 mm in diameter ( fig . histologically , the spindle - shaped cells were arranged in interlacing bundles and fascicles , together with varying numbers of tumor cells containing various amounts of light brown or grayish pigment ( fig . these alternate with looser antoni b tissue , which is comprised of cells showing clear , vacuolated cytoplasm due to lipid accumulation . immunohistochemically , most of the neoplastic cells reacted moderately to nse , ncam and s-100 protein ( fig . schwannomas are classified as one kind of the peripheral nerve sheath tumors , of which schwannnoma and neurofibroma are the most frequent . currently , the most precise determination of the tumor 's cell type is established by its immunohistochemical profile , ultrastructural features , or both . a tumor composed of cells with distinctly schwannian characteristics schwannomas are known to mainly arise from the peripheral and caranial nerves , the extremities and the retroperitoneum . they are seldom found in the abdomen , especially the extragastrointestinal tract , of which solitaly schwannoma of the great omentum is an extremely rare tumor . in 303 schwannomas reported by das gupta , one case originated from the retroperitoneum , others from the central nervous system and peripheral nerve . in another review of the literature , stout and of these , 37 were in the stomach , 3 in the small bowel , and none in the omentum and the abdominal cavity . previously published cases of schwannoma in the omentum were collected from a computerized medical literature search ( pubmed ) . only 6 cases of schwannoma from the great omentum have been observed ( table 1 ) . we can see that more cases of schwannoma from the lesser omentum have been recorded than from the great omentum . it is mentioned that the lesser omentum contains a small amount of nerves in almost equal distribution , the great omentum has a paucity of nervous tissue and anatomically consists of fat and lymphatic tissue . we found a case of malignant schwannoma arising from the omentum which demonstrated peritoneal metastasis , and another reported case of the small intestine emphasized poor prognosis since only 2 of 24 patients survived for more than 5 years . benign schwannomas are also reported to increase in size and to eventually cause complications by compressing other organs or by causing bleeding in or outside the gastrointestinal tract . pigmented schwannoma is another type of schwannoma , usually arising from the sympathetic nervous system . additionally abdominal schwannomas cause diagnostic problems because clinical symptoms are uncharacteristic or misleading even if the tumor is large . ct imaging typically showed a low attenuation mass , peripheral enhancement and cystic degeneration pattern . mri disclosed schwannoma of low signal intensity on t1-weighted image and high signal intensity on t2-weighted image . in spite of these characteristic , it is difficult to exclude other abdominal tumors , leiomyomas , lymphomas and unspecified sarcomas etc . while previous cases confirm that the tumor may attain a considerable size , bigger than 5 cm or producing symptoms , our case of a schwannoma which was small and asymptomatic is hardly diagnosed . however this tumor showing progression in size is thought to have potential to behave very aggressively despite benign histological features , which stresses the need of more information on this type of tumor and diagnosis . consequently , histological analysis of the surgical specimen is necessary for a correct diagnosis , and common treatment for schwannoma is surgery . therefore laparoscopic resection seemed to be the most adequate method to diagnose and rule out malignant tumor as a minimally invasive surgery . the majority of tumors can be safely resected , the surgeon being careful of the dividing feeder vessels because schwannomas are well known as hypervascular tumors . to our knowledge there are no case reports documenting other treatments , including chemotherapy for schwannomas in the abdominal cavity .

schwannoma in the abdomen is an uncommon neoplasm that occurs most frequently in the cranial and peripheral nerves ; it is extremely rare in the great omentum and only 6 cases of schwannoma of the great omentum have been observed previously . we report the case of a schwannoma found in the great omentum of a 55-year - old man who was treated with laparoscopic surgery . though it was difficult to diagnose preoperatively , the tumor showed malignant potential by rapidly increasing in size . histologically it was configured by a well - encapsulated round mass measuring 30 18 15 mm in diameter . immunohistochemically most of the neoplastic cells reacted moderately to nse , ncam and s-100 protein . we document the clinicopathological study of a schwannoma of the great omentum , followed by a review of the literature .

coronary artery disease ( cad ) is a major public health problem worldwide and the single largest cause of mortality in the united states , responsible for one of every six deaths ( aha heart disease and stroke statistics , 2010 ) . cad is caused by atherosclerosis , which is an inflammatory disease that involves multiple cell types , including circulating cells and cells in the vessel wall . despite advances in risk factor management on an epidemiological level , various blood markers associated with increased risk for death and cardiovascular endpoints have been identified , but currently very few have been shown to have a diagnostic impact or important clinical implications that would affect patient management . therefore , there is a great need for innovative biomarkers that can assess risk for cad , assess activity of the atherosclerotic process , and guide evaluation of therapy . several recent studies have suggested that circulating micrornas could be useful as biomarkers for various human disease states , including cancer , acute myocardial infarction [ 47 ] , heart failure , and chronic vascular disease [ 8 , 9 ] . micrornas ( mirnas ) are a recently recognized class of short ( 1925 nt ) , single - stranded , noncoding rnas that regulate an array of cellular functions through the degradation and translational repression of mrnas that contain complementary sequences . more than 1000 human mirnas have been identified , and , in tissues , mirnas regulate the expression of genes involved in critical cellular processes , including differentiation , growth , proliferation , and apoptosis . importantly , mirnas are now regarded as rheostats that fine - tune expression of proteins involved in just about every process in human cells . mirnas have been found in tissues , whole blood , serum , plasma , and other body fluids in a stable form that is protected from endogenous rnase activity [ 3 , 12 ] . although the biological function of mirna is yet to be fully understood , tissue levels of specific mirnas have been shown to correlate with pathological development of different diseases . mirnas function as managers in gene regulatory networks , and they are distinct from other biomarkers because they have a pathogenic role in the disease process and are not merely byproducts of the disease state . thus , mirna expression signatures in tissues and blood have a potential role in the diagnosis , prognosis , and assessment of therapy . in this study , we sought to compare mirna expression in whole blood of patients with angiographically significant cad to that of healthy aged - matched controls . we performed an initial exploratory microarray analysis in 5 cases and controls and then further examined the most highly expressed mirnas in an additional 15 cases and controls . study participants were recruited as part of the emory cardiology biobank , consisting of 3492 consecutive patients enrolled prior to undergoing elective or emergent cardiac catheterization across three emory healthcare sites , between 2003 and 2008 . patients aged 2090 years were interviewed to collect demographic characteristics , medical history , and lifestyle habits . risk factor prevalence was determined by physician diagnosis and/or treatment for hypertension , hyperlipidemia , and diabetes . coronary angiograms were evaluated independently by two operators , who made visual estimation of luminal narrowing in multiple segments based on a modified form of the aha / acc classification of the coronary tree . using these data , significant cad was defined as at least one major epicardial vessel with > 50% stenosis , assessed by quantitative coronary angiography . additionally , we collected whole blood samples from patients with 2 risk factors for cad , but did not have angiographically significant cad . the study was approved by the institutional review board at emory university , atlanta , ga , usa . all subjects provided written informed consent at the time of enrollment . the healthy subject cohort was a random sample of employees from emory university who were fully employed , productive people . these subjects were aged 18 and older , not taking medications , and had not been hospitalized for at least one year prior to participation . venous blood samples were drawn via antecubial venipuncture into paxgene blood rna tubes and stored at 20c within 24 hours before rna extraction . mirna was isolated using the preanalytix paxgene mirna isolation kit ( qiagen ) according to the manufacturer 's protocol . mirna microarray analysis was performed by asuragen , inc . , which uses the affymetrix manufactured genechip mirna arrays . once labeled , the mirna targets were hybridized overnight onto the microarrays following which the arrays were washed and stained using streptavidin - phycoerythrin ( sape ) . expression analysis was performed for all small rnas for homo sapiens ( e.g. , mirna , small nuclear rnas like snorna and scarna ) and separately for sanger mirna registry content ( mirbase 11.0 , http://microrna.sanger.ac.uk/ ) for homo sapiens . mirna reverse transcription was performed using the taqman microrna reverse transcription kit ( applied biosystems ) at 16c for 30 min , 42c for 30 min , and denaturation of the enzyme at 85c for 5 min . the rt reaction was performed at 37c for 1 hour followed by 5 min at 95c . taqman microrna assays ( applied biosystems ) were performed using the 7500 fast real - time pcr system at the 9600 emulation run mode . ct values were converted into copy numbers ( copy no . = 2 ) and normalized to rnu44 . unpaired student 's t - tests were used to compare data . a p value < .05 ( two sided ) was considered significant . for analysis of microarray expression data , a two- sample t - test was carried out for every gene , followed by multiplicity correction to control the false discovery rate ( fdr ) at .05 . we initially performed expression profiling of all small rnas ( 1770 genes , figure 1 ) in the whole blood samples . three mirnas passed the fdr cutoff of 0.05 : mir-584 ( 7.9-fold higher in healthy versus cad patients , p = .000103 , t - test ) , mir-542 - 5p ( 3.9-fold higher in healthy versus cad pts , p = .000168 , t - test ) , and mir-187 * ( 2.77-fold higher in healthy versus cad pts , p = 8.1 10 , t - test ) . however , signal intensities for mir-542 - 5p and mir-187 * were very low and not detected in most samples . separately , we performed an analysis of sanger registry mirnas ( 848 for homo sapiens ) in the whole blood samples , but none of the mirnas passed the fdr cutoff of 0.05 . ten mirnas passed a fdr cutoff of 0.10 , but only one ( mir-129 - 5p , 1.57 fold higher in healthy patients , p = .000044 ) of these mirnas had consistently detectable signal across the 10 blood samples . although we were able to detect some differences in whole blood mirna levels between healthy subjects and cad patients ( mir-584 , in particular ) , our microarray data suggest that , similar to other reports , levels of mirnas in the blood are low and microarrays may lack the sensitivity to adequately identify mirnas that might serve as vascular disease biomarkers . interestingly , among the mirnas that tended to show consistent differences between healthy and cad blood , mirna expression was generally higher in the blood of healthy subjects , a finding previously observed by others . next , we performed qrt - pcr on rna isolated from whole blood of another 10 patients with angiographically significant cad and 15 healthy subjects . we evaluated the levels of mirnas that , based on our microarray data , were highly expressed in blood and tended to have different levels of expression between the two groups . this analysis included mir-150 , mir-584 , mir-21 , mir-24 , mir-126 , mir-92a , mir-34a , mir-19a , mir-145 , mir-155 , mir-222 , mir-378 , mir-29a , mir-30e-5p , mir-342 , and mir-181d . among these we found that mir-19a , mir-584 , mir-155 , mir-222 , mir-145 , mir-29a , mir-378 , mir-342 , mir-181d , mir-150 , and mir-30e-5p were significantly downregulated in the blood of patients with cad compared to healthy subjects ( figure 2 ) . furthermore , we also assessed expression of these 11 mirnas in a cohort of patients who had 2 risk factors for cad , but did not have angiographically significant cad . we found that there was no difference in whole blood mirna expression of this latter group compared to that in patients with significant cad ( not shown ) , suggesting that these mirnas are markers for vascular inflammation rather than markers specific for significant cad . there has been one report that mirna expression in blood may be influenced by medications . indeed , one of the mirnas that we found to be downregulated in the blood of patients with cad is mir-155 , which is known to target the at1 receptor . therefore , we assessed the impact of medications on expression of mir-155 , mir-145 , mir-181d , mir-222 , mir-19a , mir-342 , mir-29a , mir-30e-5p , and mir-378 . we compared the mirna expression levels in the blood of patients with angiographically significant cad or 2 risk factors for cad but not on acei or arb to that in blood of similar patients who were taking at least one of medications . interestingly , levels of mir-155 , mir-19a , mir-145 , mir-222 , mir-342 , mir-30e-5p , and mir-378 ( figure 3 ) were significantly decreased in patients taking acei or arb compared to those who were not , suggesting that these medications may directly modulate expression of these mirnas , or they may influence inflammatory factors that otherwise regulate their expression . importantly , we did not find that statin use had a significant effect on the levels of mirnas identified in this study ( not shown ) . several recent studies have indicated that there is a potential role for circulating mirna levels as valuable biomarkers for different disease processes , including cancer , cardiomyopathy , and acute myocardial infarction . in this study , we wanted to address the hypothesis that mirna expression levels in blood could predict the presence of significant coronary artery disease in human subjects . we identified 11 mirnas whose expression was significantly downregulated in patients with angiographic evidence of significant atherosclerosis compared to healthy subjects that were matched for age and gender . in addition , our data suggest that inhibition of the renin angiotensin system by acei or arb use further influences mirna expression in blood . this study confirms previous reports showing differences in circulating mirna levels of patients with cad compared to those of healthy subjects [ 8 , 16 ] . our study differs from the other studies in the content of the group of mirnas that were downregulated in patients with cad . we performed this analysis using the paxgene blood rna system because it provides a way to stabilize rna immediately after sample collection and facilitates storage of the samples for a relatively long period of time without compromising rna integrity [ 17 , 18 ] . importantly , mrna profiling of whole blood or peripheral mononuclear cells has been previously applied to cardiovascular disease [ 19 , 20 ] , and a relationship has been identified between mrna levels in whole blood and extent of coronary artery disease . our study extends this work by providing insight into the whole blood expression of mirnas that are potentially involved in regulating these cad genes . in our qrt - pcr analysis of whole blood samples from cad patients , we found similar changes in expression of mir-155 and mir-145 as what has been reported previously . however , we failed to detect changes in other mirnas that were described in this previous report , namely mir-126 and mir-92a . first , we studied whole blood samples , whereas plasma was studied previously . thus our mirna profile likely reflects intracellular and extracellular mirnas levels in contrast to exclusively extracellular mirnas that would be detected in plasma . another difference in our study is that we normalized mirna expression to expression of rnu44 , a small nucleolar rna that , based on our microarray analysis , we found to be highly and consistently expressed across blood samples from cad patients and controls . this study confirmed the observation made by others regarding the difficulty of using microarray analysis to profile mirna expression in blood samples . undoubtedly , this is in part due to the limitation of a relatively small sample size for our microarray analysis , as well as reduced sensitivity of the microarray method compared to qrt - pcr . in addition , principal component analysis of our data suggested that two of the mirna profiles for healthy subjects were actually similar to that of the cad patients , suggesting that these individuals may in fact have subclinical vascular inflammation . , we believe that careful selection of patients and well - matched control subjects from a larger group of well - characterized individuals reduced some of the variability in our qrt - pcr analysis . we can only speculate as to why expression of circulated mirnas is generally decreased in patients with cad . it has been hypothesized that levels of circulating mirnas are decreased in vascular disease because they have been taken up into atherosclerotic lesions . the levels of circulating mirnas may be affected by multiple factors , including transcription , processing and stability of the mirnas within circulating cells , as well as the ability of these cells to release mirnas into the plasma . circulating mirnas may be delivered to cells in the heart or blood vessels through microvesicles , exosomes , or apoptotic bodies . because our study assessed mirna expression in whole blood , our findings are likely more reflective of changes in mirna transcription or processing rather than release from the circulating cells . it remains to be determined whether downregulation of mirnas in cad patients is directly involved in inflammation or a compensatory response to this process . based on our observed changes in mirna expression in response to acei / arb therapy , we believe that circulating mirna levels reflect a compensatory response to inflammation . further experimental studies are necessary to explore the mechanisms by which cad and therapies affect tissue versus circulating mirna levels . in summary , the present study provides insight into whole blood levels of mirnas in patients with cad compared to healthy subjects and demonstrates their potential utility as biomarkers for vascular disease . validation of the changes in mirna expression observed here in larger studies will be a necessary step to confirm their candidacy as biomarkers and therapeutic targets . we believe that further elucidation of the role that these mirnas play in the pathogenesis and progression of chronic cad will contribute to our understanding of the disease process and lead to new therapeutic and preventative strategies .

coronary artery disease ( cad ) is the largest killer of males and females in the united states . there is a need to develop innovative diagnostic markers for this disease . micrornas ( mirnas ) are a class of noncoding rnas that posttranscriptionally regulate the expression of genes involved in important cellular processes , and we hypothesized that the mirna expression profile would be altered in whole blood samples of patients with cad . we performed a microarray analysis on rna from the blood of 5 male subjects with cad and 5 healthy subjects ( mean age 53 years ) . subsequently , we performed qrt - pcr analysis of mirna expression in whole blood of another 10 patients with cad and 15 healthy subjects . we identified 11 mirnas that were significantly downregulated in cad subjects ( p < .05 ) . furthermore , we found an association between acei / arb use and downregulation of several mirnas that was independent of the presence of significant cad . in conclusion , we have identified a distinct mirna signature in whole blood that discriminates cad patients from healthy subjects . importantly , medication use may significantly alter mirna expression . these findings may have significant implications for identifying and managing individuals that either have cad or are at risk of developing the disease .

among eusocial insects , paper wasps ( hymenoptera : vespidae : polistinae ) are notable for the diversity of their nest architecture ( jeanne , 1975 ; wenzel , 1991 ) . many swarm - founding polistine wasps , including most species of the neotropical tribe epiponini , construct envelopes around their brood combs . comparative and experimental studies suggest that , among other functions , nest envelopes reduce rates of predation and parasitism on wasp brood ( london and jeanne , 1998 ; smith et al . , 2001 ) . however , little is known about the adaptive significance of variation in the materials that wasps use to construct nest envelopes ( hansell 1984 ; wenzel , 1991 ; cole et al . , 2001 ) . polybia emaciata is unusual among the polistine wasps because it uses mud , rather than wood pulp or plant fibers , as the main raw material for nest construction ( schremmer , 1984 ) . the genus polybia includes 56 described species ; of these , only the other three species in the subgenus pedothoeca , and one species in the subgenus furnariana , share the derived trait of mud nest construction ( richards , 1978 ; jeanne , 1991 ; cooper , 1993 ) . no other swarm - founding polistine wasps are known to build primarily mud nests , although small amounts of inorganic material are sometimes incorporated into the nest paper of other species ( wenzel , 1991 ) . the nest of p. emaciata is otherwise typical of the genus ( jeanne , 1975 ; wenzel , 1991 ) , being phragmocyttarus ( comprising horizontal layers of comb that are conjoined and enclosed by a continuous envelope ) with a single small entrance hole near the bottom of the nest . numbers of adults in mature colonies are relatively small for the genus , ranging from fewer than 100 up to about 500 adults ( richards , 1978 ; chadab , 1979 ; strassmann et al . , 1992 ) . polybia emaciata nests are hard and much more durable than most paper nests of similar size and shape ( rau , 1933 ; richards , 1978 ) . for example , skutch ( 1971 ) noted that nests of this species in costa rica were unlike paper nests in that they persisted for several years . the high durability of p. emaciata construction material suggests that the nest itself may provide greater brood protection than paper nests . if so , we hypothesized that the wasps ' defensive responses to vertebrate attacks have changed to take advantage of nest durability . we predicted that the nests ' protective properties reduced the need for behavioral defenses by p. emaciata workers , relative to paper - nesting polybia species with similar colony and nest sizes . we tested this prediction by disturbing p. emaciata colonies , and comparing the workers ' responses to those of simultaneously observed paper - nesting p. occidentalis colonies in the same location . chadab ( 1979 ) surveyed the defensive responses of swarm - founding paper wasps to vertebrate - like disturbances , including tapping on the nest and/or on the supporting vegetation . the most frequently observed response was for dozens to hundreds of workers to rush out onto the nest envelope , sometimes adopting aposematic postures ( see also o'donnell et al . , response was seen in the genera angiopolybia , charterginus , leipomeles , parachartergus , polybia , pseudopolybia , and synoeca , and accords with our observations of what appears to be typical epiponine defensive behavior . weaker exit - nest responses ( either requiring several taps , or involving fewer wasps ) were seen in species of leipomeles , metapolybia , and protopolybia . interspecific variation in mature colony size was not a good predictor of the type of defensive response , as species with colony sizes smaller and larger than p. emaciata performed nest exiting defensive behavior . an attack response often coincides with , or quickly follows , the nest exit response and involves workers flying from the nest , striking the intruder , and attempting to sting ( hermann and blum , 1981 ) . when paper - using polybia nests are tapped , some of the workers on the nest surface may immediately perform attacks , while large numbers of workers rush out of the nest entrance and stand in alert posture on the envelope ( jeanne , 1981 ; jeanne et al . , 1992 ) . further tapping elicits additional attacks . breathing on the nest surface or into the nest results in immediate attack by several to dozens of workers . we discuss the possible adaptive significance of deviations from the typical nest exit that have been documented in several paper wasp species , including p. emaciata . this study was conducted in and near the town of gamboa , panama province , republic of panama ( 0907n 7942w ; 50 m elevation ) . observations and manipulations were conducted during the wet season from 1 august to 15 october 1990 and from 3 to 5 july 1998 . surveys of eusocial wasp colonies during these periods indicated that p. emaciata was among the most abundant swarm - founding wasps in terms of nest density ( s.o'd . , unpublished data ) . most of the data presented were collected from four colonies of p. emaciata and five colonies of p. occidentalis that had been moved approximately 0.5 km to 1 km from their natural nesting sites to artificial supports to facilitate observation . all subject colonies were moved into a small area ( < 0.25 ha ) , with similar amounts of tree cover ( one p. occidentalis colony was additionally sheltered by building eaves ) . all colonies were in place at least 2 weeks before observations on defense behavior were initiated , and all continued to forage and to raise brood ( visible through the nest entrance in the lower combs ) throughout the observation period . the p. emaciata observation colonies had nests ranging in size from 9 cm long to 22 cm long . the numbers of adult wasps and colony age probably varied positively with nest size , though we did not measure these variables . we also collected data opportunistically from p. emaciata colonies in situ in 1990 and again in 1998 . these nests were located in the gamboa area on thin tree branches from 1.5 m to 5 m above the ground . we disturbed p. emaciata and p. occidentalis colonies in an effort to evoke defensive behavior , using stimuli that elicited nest exit and attack responses in costa rican p. occidentalis ( s.o'd . and we disturbed the polybia nests by first tapping on the side of the outer envelope near the middle of the nest for 30 sec while wearing a full bee suit , and we noted the responses of on - nest and in - nest workers . if there was no attack response to tapping , we blew into the nest entrance for 30 sec . each polybia observation colony was disturbed 10 times over the course of the study period in 1990 . the in situ nests of p. emaciata were tested once each in a similar fashion . we used a non - parametric wilcoxon test for species differences in the probability of immediate attack responses using the four p. emaciata and five p. occidentalis observation colonies . trials were pooled within colonies and each colony was used as a single data point in the analysis . all observation and in situ polybia colonies contained brood ( larvae and/or pupae were visible in the lower combs ) when they were tested . based on forager traffic and on the numbers of wasps exiting the nests during attacks polybia emaciata only rarely ( 5 occasions out of 40 trials on the observation nests and 2 occasions out of > 15 trials on in situ nests ) exhibited a typical polybia attack response within 30 sec when the nest was tapped . polybia emaciata workers never rushed out of the nest and onto the nest envelope . in all trials where the wasps did not attack in response to the initial mechanical disturbance ( within the first 30 sec ) , some of the workers on the nest surface flew off the nest and departed ; the remainder rapidly entered the nest . after the initial disturbance , the nest surface and the lowest layer of comb ( partially visible through the nest entrance , fig . breathing into the nest entrance did not elicit rapid ( within 10 sec ) attack responses . after continued disturbance within a trial ( breath and tapping ) , p. emaciata workers often attacked violently . in approximately 75% of trials , several dozen individuals rapidly exited the nest , took flight , and struck and attempted to sting the observer 's bee veil . the p. emaciata nests were not abandoned following these attacks , and workers later returned to the nests . in contrast , all 50 nest - tapping trials with p. occidentalis resulted in immediate ( within 1 sec ) exit - nest and attack responses . the species difference in the per - colony probability of nest exit and attack within 30 sec of disturbance was significant ( fig . 2 ; wilcoxon 2-tailed test , z=2.60 , p<0.01 ) . breathing in or on p. occidentalis nests always elicited immediate attack responses , therefore , habituation to tapping and breathing stimuli was not evident over the course of the study in p. occidentalis . the four p. emaciata observation nests suffered no damage while exposed to nearly daily heavy rains for 3 weeks ; four of the p. occidentalis observation nests in the same location were visibly damaged by rain during the same period . even during heavy downpours , the mud nests repelled water much longer than nearby p. occidentalis nests . during one rain shower , the mud nests did not appear waterlogged until 15 min of rainfall had elapsed , while p. occidentalis nests stopped shedding water and became waterlogged after < 5 min . the nest fell 2 m to the ground , with only minor damage to its envelope , and the nest was still occupied by adult wasps when it was collected within 12 h of falling . rather than exiting and attacking within a few sec of vibrational disturbance , p. emaciata workers usually either departed or fled into the nest interior . unlike many other paper wasps ( s.o'd . and r.l.j . , personal observation ) , p. emaciata failed to respond aggressively to human breath , even when we blew directly into the nest entrance . polybia emaciata behavioral responses to nest disturbance were unusual among their congeners , and among paper - nesting epiponini with similar colony sizes ( s.o'd and r.l.j . personal observation ; chadab , 1979 ) . however , a few species of paper wasps in other genera exhibit similar responses to human disturbance , which chadab ( 1979 ) labeled richards ( 1978 ) collected a p. emaciata colony in brazil , and the wasps did not exit the nest until 1.5 h after it was collected . residents of costa rica from several rural communities reported collecting and moving active p. emaciata colonies without being stung , by placing a finger over the nest entrance and plucking the nest from its attachment point . chadab ( 1979 ) noted that one colony of p. emaciata in ecuador had durable nest paper and exhibited defensive retreat responses like those that we recorded ; two other p. emaciata colonies had fragile nests and performed exit - nest and attack behavior when jarred . we did not observe qualitative variation in p. emaciata nest durability in panama ( > 50 colonies observed ; not all were tested for defensive responses ) . predation on swarm - founding wasp nests by bats ( jeanne , 1970a ) , birds ( skutch , 1971 ; windsor , 1976 ) , and primates ( vecht , 1967 ) has been documented . we hypothesize that the unusual defensive response of p. emaciata is a behavioral adaptation to its use of mud in nest construction , which may make the nest more resistant than paper to entry by vertebrate predators . it appears that the wasps initially rely on the nest itself , rather than on exit and attack behavior , to thwart vertebrate predators . we suggest that this represents a special type of architectural defense ( hermann and blum , 1981 ) , where the defensive behavior of a species has been modified to exploit the properties of its nest material skutch ( 1959 ) observed a red throated caracara ( daptrius americanus ) removing combs from a p. emaciata nest and feeding on the brood in costa rica . our subject nests often responded with attack behavior after extended disturbances , suggesting that attack can be effective for this species . when attack did come it was sudden and massive , suggesting that it was coordinated by an alarm pheromone , as has been demonstrated for p. occidentalis ( jeanne , 1981 ) . although army ants are among swarm - founding wasps ' most frequent predators in wet tropical habitats , preliminary tests on the reaction to eciton army ants did not suggest that p. emaciata 's mud nest is effective in resisting these predators ( chadab , 1979 ; s.o'd . personal observation ) . the use of mud as a nesting material may have evolved in response to predation pressure , particularly by vertebrate enemies . similar advantages against hornet ( vespa ) predation may have favored the evolution of mud construction in hover wasps of the genus liostenogaster ( hansell 1984 ; turillazzi 1991 ) . however , mud construction appears to provide other benefits . our observations suggest that p. emaciata mud nests were more water repellent , and more resistant to mechanical damage , than similarly sized paper nests . even if increases in general durability were the original selective advantage driving the evolution of mud construction , the workers ' defensive behavior has apparently been secondarily modified . there is variability in average colony size and in the details of nest architecture among species within the mud - nesting polybia subgenus pedothoeca , and in the independently evolved mud - nester p. furnaria ( richards , 1978 ; carpenter et al . , 2000 ) . of special value to our understanding of the evolutionary significance of the mud - nesting habit will be field studies on the defensive behaviors of the other mud - nesting polybia species . in particular , such studies could establish whether retreat behavior in response to vertebrate disturbance always accompanies the use of mud in nest building . useful comparisons of defensive behavior could also be made to epiponine paper - nesters with relatively strong nest paper , such as epipona and chartergus . retreat behavior in response to vertebrate disturbance is not restricted to mud - nesting species of epiponini . similar responses to human approach or nest vibration have been observed in paper- and secretion - nesting species ( protopolybia emortualis , protopolybia exigua , and protopolybia ( formerly pseudochartergus ) fuscatus ) and in cavity - nesting agelaia cajennensis ( jeanne , 1970b ; chadab , 1979 ; carpenter et al . , 2001 ) . paper - nesting polybia jurenei colonies retreated when their nest tree was rubbed , but exited and attacked when the substrate was jarred ( s. o'd . these patterns suggest that retreat behavior has evolved convergently in several lineages of epiponini , perhaps in response to particular types of predators . photograph of a mud nest of polybia emaciata collected july 1998 in gamboa , panama . box plots showing the range of probabilities of response to nest disturbance with attack within 30 sec by polybia emaciata and p. occidentalis colonies .

the swarm - founding wasp polybia emaciata is unusual among eusocial vespidae because it uses mud , rather than wood pulp , as its primary nest construction material . polybia emaciata nests are more durable than similarly sized paper nests . we tested the hypothesis that the defensive behavior of this wasp may have been modified to take advantage of their strong nests in defense against vertebrate attacks . we simulated vertebrate disturbances by tapping on , and breathing in , p. emaciata nests and similarly sized p. occidentalis paper nests in the same location at the same time . polybia emaciata responses to disturbance were qualitatively different from those of p. occidentalis . the latter exit the nest and attack , while p. emaciata workers typically fled or entered the nest , attacking only after repeated and extended disturbances . we conclude that durable nest material may permit predator avoidance behavior in p. emaciata . we compare the defensive responses of p. emaciata workers with those of other swarm - founding vespidae , and discuss several selective forces that could cause the evolution of species variation in nest defense behavior .

human gliomas represent 50% to 60% of all intracranial tumors . according to the world health organization ( who ) guidelines , gliomas are histologically classified into four grades : pilocytic astrocytoma ( grade i ) , low - grade diffuse astrocytoma ( grade ii ) , anaplastic astrocytoma ( grade iii ) , and glioblastoma multiforme ( gbm , grade iv ) . both diagnostic technologies and therapeutic strategies the 5-year survival rates of low - grade ( grade i~ii ) and high - grade ( grade iii~iv ) glioma patients in china are 75.4% and 18.2% , respectively . especially , the median survival time for patients with gbm is still only 12 months . indeed , early diagnosis and prolonging survival in glioma patients remains a great challenge for clinicians in the field of neurooncology . there have been several prognostic factors for glioma patients , such as age , preoperative duration of symptoms , karnofsky performance status ( kps ) score , histologic grade , tumor necrosis , surgical resection extent , use of postoperative radiation therapy , and , probably , adjuvant chemotherapy . however , these clinical parameters can not completely account for the observed variation in survival because of the heterogeneity of glioma patients . thus , there is an urgent need to further investigate the molecular mechanisms of glioma and to identify the effective prognostic indicators for survival prediction.the dna - base excision repair ( ber ) pathway is responsible for the repair of exogenous and endogenous alkylating and oxidative dna damage , which may lead to carcinogenesis , cell death , and aging if left unrepaired this pathway involves the recognition and removal of damaged bases by a dna glycosylase , followed by incision of the resulting abasic ( ap ) site by ap endonuclease , dna synthesis by polymerase , and strand ligation by dna ligase . thus , the ber pathway is an important candidate for intervention into the cellular responses to dna change . n - methylpurine - dna glycosylase ( mpg ) as a dna repair enzyme is a main component in the ber pathway . in previous study aimed at understanding the significance of initiating lesions removed by the ber pathway , kaina et al . identified the over - expression of the human mpg in chinese hamster ovary cells . in the n - alkylpurine repair process , mpg is responsible for the glycolytic removal of the modified base , which leads to the formation of apurinic sites . although n - alkylpurines have not been found to be directly mutagenic , apurinic sites left by this repair process can block replication and lead to mutation . mpg also participates in the repair of 8-hydroxyguanine and hypoxanthine . because of the potential role of dna base lesions in mutagenesis and carcinogenesis , a number of studies have been performed to investigate the association of mpg with various human cancers . ( 1998 ) detected the increased mpg gene and protein expression in the breast cancer cells versus normal breast epithelial cells by northern analysis , southern blots , immunofluorescence , immunohistochemistry , and western blot analysis . in 2001 , sohn et al . reported that the expression of mpg was increased in high - risk hpv - infected cervical neoplasias and the intracellular distribution of mpg protein was altered , suggesting a role of mpg in carcinogenesis . in an effort to improve the efficacy of cancer chemotherapy by intervening into the cellular responses to chemotherapeutic change , many researchers have been interested in the effects of mpg in tumor sensitivity to the clinical chemotherapeutic agents . as their results , mpg - overexpressing ovarian cancer , osteosarcoma , and breast cancer cells are significantly more sensitive to the clinical chemotherapeutic agents , suggesting that the overexpression of mpg may be a possible gene therapy approach to sensitize tumor cells to the cell - killing effects of chemotherapeutic alkylating agents . the biological mechanism behind the increase of sensitivity to the chemotherapeutic agents in mpg overexpressing cell lines may be that the balance between glycosylase activity , leading to apurinic sites and formation of strand breaks , and subsequent excision repair processes may play an important role in determining cellular cytotoxicity and resistance to alkylating agents . of our interests , we focus on the involvement of mpg in human gliomas . recent study has demonstrated that mpg mrna expression was higher in astrocytic tumor tissues than in brain tissues adjacent to tumor , and mpg protein localization in immunohistochemical study was detected only in the nucleus of all tumor tissues , suggesting an mpg 's role in human astrocytic tumors and raise the possibility that the altered mpg expression and intracellular localization could be associated with astrocytic tumorigenesis . tang et al . further demonstrated that mpg overexpression , together with the inhibition of ber , could sensitize glioma cells to the alkylating agent . however , little is known about the expression level of mpg in human gliomas with different clinical grades and its prognostic significance . to address this problem , we used quantitative real - time pcr , immunohistochemistry assay , and western blot analysis to investigate the expression pattern of mpg gene and protein in glioma specimens and normal control brain tissues . next , we analyzed the relationship between mpg expression and the glioma stage as well as the survival of patients . this study was approved by the research ethics committee of the institute for functional neurosurgery p.l.a , tangdu hospital , fourth military medical university , xi'an , p.r . china . written informed consent a total of 128 formalin - fixed , paraffin - embedded specimens of gliomas resected between 2000 and 2010 were retrieved from the archives of the pathology department of tangdu hospital , fourth military medical university , p.r . all the slides were reevaluated according to who classifications by two pathologists , with differences resolved by careful discussion . a total of 76 males and 52 females ( 1.46 : 1 ) were enrolled in this study , and the median age was 42 years ( range , 1271 ) . thirty - two of the 128 gliomas were classified as low - grade [ 18 pilocytic astrocytomas ( who i ) and 14 diffuse astrocytomas ( who ii ) ] , and 96 were classified as high - grade gliomas [ 38 anaplasia astrocytomas ( who iii ) , and 58 primary glioblastomas ( who iv ) ] . the clinicopathological features , and the treatment strategies of all the patients were indicated in table 1 . paraffin and snap - frozen sections of nonneoplastic brain tissues from 10 patients with intractable epilepsy were also included as controls . five - year followup was performed , and all patients had complete followup until death . overall survival time was calculated from the date of the initial surgical operation to death . patients , who died of diseases not directly related to their gliomas or due to unexpected events , were excluded from this study . in addition , 20 glioma specimens [ 5 pilocytic astrocytomas ( who i ) , 3 diffuse astrocytomas ( who ii ) , 3 anaplasia astrocytomas ( who iii ) , and 9 primary glioblastomas ( who iv ) ] were snap - frozen in liquid nitrogen and stored at 80c following surgery for mrna and protein isolation . total rna purified from all 20 glioma tissues and 10 control brain tissues real - time monitoring of polymerase chain reactions ( pcrs ) was performed using the abi 7900ht ( idaho technology , idaho falls , i d , usa ) . cdna was synthesized using random primers and oligo 18dt and superscript ii reverse transcriptase ( invitrogen ) . gene expression was determined using the sybr green i dye ( biogene ) , which binds preferentially to double - stranded dna , and 10 ng of template . fluorescence signals , which are proportional to the concentration of the pcr product , are measured at the end of each cycle and immediately displayed on a computer screen , permitting real - time monitoring of the pcr . the reaction is characterized by the point during cycling when amplification of pcr products is first detected , rather than the amount of pcr product accumulated after a fixed number of cycles . the higher the starting quantity of the template , the earlier a significant increase in fluorescence is observed . the threshold cycle is defined as the fractional cycle number at which fluorescence passes a fixed threshold above the baseline . the primers 5-gtc cta gtc cgg cga ctt cc-3 and 5-ctt gtc tgg gca ggc cct ttg c-3 were used to amplify 603-bp transcripts of mpg , and the primers 5-ggt ggc ttt tag gat ggc aag3 and 5-act gga acg gtg aag gtg aca g3 were used to amplify 161-bp transcripts of -actin . the pcr profile consisted of an initial melting step of 1 min at 94c , followed by 38 cycles of 15 s at 94c , 15 s at 56c , and 45 s at 72c , and a final elongation step of 10 min at 72c . fluorescence data were converted into cycle threshold measurements using the sds system software and exported to microsoft excel . thermal dissociation plots were examined for biphasic melting curves , indicative of whether primer dimers or other nonspecific products could be contributing to the amplification signal . twenty glioma tissues and 10 control brain tissues were homogenized in lysis buffer [ pbs , 1% nonidet p-40 ( np-40 ) , 0.5% sodium deoxycholate , 0.1% sodium dodecyl sulfate ( sds ) , 100 ug / ml aprotinin , 100 g / ml phenylmethylsulfonyl fluoride ( pmsf ) , sodium orthovanadate ] at 4c throughout all procedures , and sonicated for 70 s , then add 300 g pmsf per gram of tissue and incubate on ice for 30 min , followed by centrifugation at 15,000 rpm for 20 min at 4c . the protein content was determined according to bradford 's method , with bovine serum albumin used as a standard . protein samples ( 30 g ) were boiled with 2 sample buffer containing 5% -mercaptoethanol for 5 min , separated by size on 15% polyacrylamide gel under sds denaturing conditions , and transferred to a nitrocellulose membrane at 90 v for 2 h. the nitrocellulose membranes were stained with ponceau s to assess the efficiency of transfer . nonspecific binding was blocked by incubation in block buffer ( 5% nonfat dry milk , 0.05% tween-20 , 1 tris - cl - buffered saline ) overnight at 4c , the membranes were hybridized with a mouse monoclonal antibody to mpg ( ab55461 ; abcam ) , then incubated with a horseradish peroxidase - labeled goat anti - mouse igg ( 1 : 500 ) . the bound secondary antibody was detected by enhanced chemiluminescence ( amersham life science , little chalfont , uk ) . positive immunoreactive bands were quantified densitometrically ( leica q500iw image analysis system ) and expressed as ratio of mpg to -actin in optical density units . immunohistochemical assay was performed using the conventional immunoperoxidase technique according to the protocol of the department of neurosurgery , institute for functional neurosurgery p.l.a , tangdu hospital , fourth military medical university , xi'an , p.r . briefly , following peroxidase blocking with 0.3% h2o2/methanol for 30 min , specimens were blocked with phosphate - buffered saline ( pbs ) containing 5% normal horse serum ( vector laboratories inc . , all incubations with a mouse monoclonal antibody to mpg ( ab55461 ; abcam ) at 1 : 500 dilution were carried out overnight at 4c . then the specimens were briefly washed in pbs and incubated at room temperature with the anti - mouse antibody and avidin - biotin peroxidase ( vector laboratories inc . , the specimens were then washed in pbs and color developed by diaminobenzidine solution ( dako corporation , carpinteria , ca , usa ) . after washing with water , specimens were counterstained with meyer 's hematoxylin ( sigma chemical co. , st louis , mo , usa ) . nonneoplastic brain tissues were used as control tissues , and nonimmune igg was also used as negative control antibody for immunohistochemical staining . immunostaining was scored by two independent experienced pathologists , who were blinded to the clinicopathologic parameters and clinical outcomes of the patients . 5% scored 0 , 625% scored 1 , 2650% scored 2 , 5175% scored 3 , > 75% scored 4 ; ( 2 ) intensity of stain : colorless scored 0 , pallide - flavens scored 1 , yellow scored 2 , brown scored 3 . the staining score was stratified as ( 0 score , absent ) , + ( 14 score , weak ) , + + ( 58 score , moderate ) , and + + + ( 912 score , strong ) according to the proportion and intensity of positively stained cancer cells . all computations were carried out using the software of spss version13.0 for windows ( spss inc , il , usa ) . randomized block design anova was used to analyze the statistical difference among different tissue types . in the analysis of glioma morbidity for all patients , we used the kaplan - meier estimator and univariate cox regression analysis to assess the marginal effect of each factor . a spearman 's analysis was carried out to analyze the correlation between mpg mrna and protein expression levels . the gene expression of mpg normalized to -actin in 20 glioma and 10 control brain tissues was detected by real - time pcr . as shown in figure 2 , the expression levels of mpg gene in human glioma tissues were conspicuously higher than those in control brain tissues ( p < 0.001 ) . interestingly , mpg gene expression increased with the advancement of who grades from i to iv ( p < 0.001 ) . the protein expression of mpg normalized to -actin in 20 glioma and 10 control brain tissues was also detected by western blot analysis . as the results , the expression levels of mpg protein tended to increase from the glioma to the normal tissue ( figures 3(a ) and 3(c ) ) . mpg expression was highest in grade iv and lowest in grade i ( figures 3(b ) and 3(c ) ) , indicating a close correlation of mpg protein expression with who grade . there was a significant positive correlation between the expression of mpg gene and protein expression levels from the same glioma tissues ( rs = 0.82 , p < 0.001 ) . the expression and the subcellular localization of mpg protein in 10 nonneoplastic brain and 128 glioma tissues were detected by immunohistochemistry assay . as shown in figure 4 , the positive staining for mpg was mainly observed in the nuclei of tumor cells in glioma tissues , which was consistent with the previous study of wang et al . the representative photographs were shown in figures 4(a ) and 4(b ) . among the glioma specimens , 102 ( 79.7% ) glioma specimens were positively stained for mpg , whereas its immunoreactivities in nonneoplastic brain sections ranged from undetectable to low ( figure 4(c ) ) . additionally , the immunostaining of mpg in nonneoplastic brain tissues is below detection levels of both real - time pcr and western blot . according to the statistical analysis , the expression level of mpg protein in glioma tissues was significantly higher ( p < 0.001 ) than that in nonneoplastic brain tissues , which was consistent with the results of western blot analysis . in addition , mpg expression was not significantly affected by the gender and age ( both p > 0.05 ) of the patients . in contrast , the mpg expression was the closely correlated with who grade , as well as kps . the expression levels of mpg protein in glioma tissues with higher who grade ( table 1 ; p = 0.002 ) and lower kps ( table 1 ; p = 0.01 ) were significantly higher than those with lower who grade and higher kps . moreover , we reviewed clinical information of these mpg - positive or -negative glioma patients . during the follow - up period , 100 of the 128 glioma patients ( 78.1% ) had died ( 24 from the mpg - negative group and 76 from the mpg - positive group ) . as determined by the log - rank test , the survival rate of patients with positive mpg staining was lower than those without mpg staining ( p < 0.001 ; figure 5(a ) ) . the median survival time of patients with negative expression of mpg could not be estimated by statistical analysis because all patients survived better than the overall median level , and those patients with strong positive ( + + + ) , moderate positive ( + + ) , and weak positive ( + ) of mpg were 9.1 1.5 months , 12.3 1.1 months , and 22.6 2.2 months ( log - rank test : p < 0.001 ) . furthermore , figure 5(b ) shows the postoperative survival curve of patients with glioma and mpg expression after adjusting for age , gender , who grade , and kps . by multivariate analysis ( table 2 ) , the loss of mpg expression was a significant ( p < 0.001 ) and independent prognostic indicator for patients with glioma besides age , who grade and kps . the cox - proportional hazards model showed that mpg over - expression was associated with poor overall survival . because of the dismal survival in the patients with glioma , especially in gbm patients , there is an urgent need to find novel specific and effective prognostic markers in order to apply individualized treatments to specifically target the pathophysiologic and molecular properties of glioma patients . in the current study , we investigated the expression of mpg in 128 cases of human glioma and compared the expression with tumor grade and survival rates of patients . our data demonstrated that mpg gene and protein were both increased in glioma compared to nonneoplastic brain tissue . we found an increased trend of both mpg protein level and gene level from who grade i to who grade iv glioma . these results suggest that the transcriptional and translational activation of human mpg might participate in the tumorigenesis and progression of glioma . dna in all somatic and germ cells in the body are continuously exposed to exogenous and endogenous alkylating and oxidative agents that result in damage to dna . if dna damage was not repaired , it may lead to genetic mutation , chromosome aberration , aging , and carcinogenesis . the lack of precise dna repair activities may lead to defective embryogenesis , tissue - specific dysfunction , hypersensitivity to dna - damaging agents , premature senescence , genetic instability , and elevated cancer rates . the repair of dna bases damaged by alkylation is initiated in mammalian cells by mpg , as an alkyladenine dna glycosylase . the majority of repair that is initiated by mpg occurs via short - patch ber , a mechanism whereby only one nucleotide is replaced . the majority of base damage induced by methylation is removed by mpg , including the principal adducts 3-mea and 7-meg . it has been reported that the expression of mpg could be induced by viral damage and a variety of dna - damaging agents . for example , the increased expression of mpg gene was studied in breast cancer , cervical neoplasia , and thymic carcinoma in sv 40 t - antigen - expressing transgenic mice . as previous investigation , mammalian cells actively regulated dna repair enzymes and genes during cell proliferation . dna repair enzymes are expressed in the cell cycle in a defined temporal pattern relative to the induction of dna replication . in brain cells that are not undergoing dna replication , kim et al . indicated that mpg expression in the brain was relative high in 1 week after birth , and the level remained low in day 400 mature adults , suggesting that brain tissue is terminally differentiated and nonproliferating tissues . it has been demonstrated that dna repair enzymes play an important role in the carcinogenesis of several cancers . of our interests , mpg was overexpressed in breast cancer up to 24-fold as compared to normal primary breast epithelium , suggesting the role of mpg in breast carcinogenesis . in astrocytomas , kim et al . in 2003 reported that mpg mrna level was significantly higher than that in tumor - adjacent brain tissues . with the similar results , we in this study also found the upregulation of mpg protein and gene in glioma tissues . additionally , we provide evidence that mpg protein is found in nuclear localization of the glioma tissues , which was consistent with the results of kim et al . . the nuclear localization of mpg in tumor cells of glioma tissues may be suggestive of a role of mpg in the regulation of cell division and cell cycle . furthermore , the most important finding of this study was the correlation of mpg expression and survival rates of patients . kaplan - meier and multivariate analysis both showed a significantly worse overall survival for patients whose tumors had high mpg levels , indicating that high mpg protein level is a marker of poor prognosis for patients with gliomas . this is the first investigation to demonstrate the prognostic value of mpg in human cancers . our data showed the over - expression of mpg gene and protein in human gliomas and also suggested for the first time that mpg be an unfavorable independent prognostic indicator for glioma patients .

aim . to examine the expression of n - methylpurine - dna glycosylase ( mpg ) gene and protein in glioma samples with different who grades and its association with patients ' survival . methods . immunohistochemistry assay , quantitative real - time pcr and western blot analysis were carried out to investigate the expression of mpg gene and protein in 128 glioma and 10 non - neoplastic brain tissues . results . mpg gene expression level in glioma tissues was significantly higher than that in non - neoplastic brain tissues ( p < 0.001 ) . immunohistochemistry also showed that mpg protein was over - expressed in glioma tissues , which was consistent with the resutls of western blot analysis . additionally , the expression levels of mpg gene and protein both increase from grade i to grade iv glioma according to the results of real - time pcr , immunohistochemistry and western blot analysis . moreover , the survival rate of mpg - positive patients was significantly lower than that of mpg - negative patients ( p < 0.001 ) . we further confirmed that the over - expression of mpg was a significant and independent prognostic indicator in glioma by multivariate analysis ( p < 0.001 ) . conclusions . our data showed the over - expression of mpg gene and protein in human gliomas , and also suggested for the first time that mpg be an unfavorable independent prognostic indicator for glioma patients .

the prevalence of overweight and obesity among children and adolescents has widely increased worldwide [ 1 , 2 ] , making it one of the most common chronic disorders in this age group and in adulthood . the use of body mass index ( bmi ) for age to define being overweight and obese in children and adolescents is well established for both clinical and public health applications , because of their feasibility under clinical settings and in epidemiological studies [ 3 , 4 ] . in children and adolescents , the natural increases in bmi that occur with age necessitate the use of age - sex - specific thresholds . the most widely used growth charts are the centers for disease control and prevention ( cdc-2000 ) , the international task force ( iotf ) , and the 2007 growth references for 5 to 19 year olds produced by the world health organization ( who-2007 ) . the cdc-2000 growth charts were developed to evaluate the nutritional status of us children and were originated from five cross - sectional representative surveys carried out in the us between 1963 and 1994 . these growth charts are routinely applied to identify children and adolescents with a bmi greater than the 85th or 95th percentiles following the advice of the us expert committee on childhood obesity . however , the appropriateness of an american dataset for defining overweight in young people from other countries is questionable . the iotf reference also uses age - sex - specific bmi percentiles , and overweight and obesity definition corresponds to an adult bmi of 25 and 30 kg / m , respectively , and reflects values in children tracking to overweight and obesity in adults . this reference is based on six large international cross - sectional representative datasets , identifying the bmi values that extrapolate to childhood . the who-2007 growth references were created to replace the national center for health statistics ( nchs ) references [ 10 , 11 ] . this reference was constructed using data from the 1977 nchs / who growth reference ( 1 to 24 years old ) merged with data from the 2006 who child growth standards for preschool children ( under 5 years of age ) using state - of - the - art statistical methods . although valuable information has been appearing in the literature or online , such as works from the health behaviour of school - aged children study which is mainly related to social determinants of health and well - being among young people , no systematic review has been conducted to understand the worldwide magnitude of the overweight and obesity problem among the adolescent population . thus , the objective of this study was to systematically review the literature regarding the prevalence of overweight and obesity in adolescents ( 1019 years old ) of both sexes published in the past 12 academic years ( 19992011 ) . a systematic literature search was performed which ended on june 7 , 2012 ( see figure 1 ) . the literature search was conducted in medline and scopus using the following mesh terms : overweight ; obesity ; prevalence ; adolescent . in total , 2537 articles were selected . we also reviewed the data on the prevalence of childhood overweight and obesity on the international obesity task force website at http://www.iaso.org / iotf/. to find the articles included in this review , the following inclusion criteria were used : ( 1 ) cross - sectional studies conducted in the last 12 years ( 19992011)when the original study did not report the survey year , it was not included ; ( 2 ) national and regional representative samples , but articles published on the prevalence of overweight in towns , urban , or rural areas in a country were excluded ; ( 3 ) weight and height objectively measured ; ( 4 ) results presented by sex ; ( 5 ) and for both overweight and obesity prevalence ; ( 6 ) the definition of overweight and obesity using the ( i ) cdc-2000 , ( ii ) iotf , and ( iii ) who-2007 growth references ; and ( 7 ) studies written in english , spanish , italian , or portuguese . moreover , if there were more than one national or regional study in the same country , the most recent one was included in the prevalence tables ( except for usa and canada , countries in which the most recent data was not included in the tables due to differences in the representativeness of the samples and the impossibility to calculate a single prevalence of overweight and obesity for adolescents ' boys and girls ; however , no differences in prevalence were observed between studies as it has been indicated in the discussion ) . the final number of articles included in this review was 39 articles related to overweight and obesity and also a study on the latest statistics on the prevalence of overweight and obesity in south africa . potentially relevant papers were selected by ( 1 ) screening the titles ; ( 2 ) screening the abstracts , and ( 3 ) if abstracts were not available or did not provide sufficient data , the entire article was retrieved and screened to determine whether it met the inclusion criteria . full - text articles were assessed by 2 authors ( m. m. bibiloni and j. a. tur ) . any matter of doubt was discussed by at least two of the reviewers ( m. m. bibiloni , a. pons , and j. a. tur ) . a total of thirty - nine articles and a national health report were eligible according to the inclusion criteria established for this review . table 1 presents a description of the forty studies selected for this review including the continent and the country where it was performed ( and region for not national studies ) , year of publishing , total number of participants in the study , number of adolescents , age range , proportion of girls , and number and definition for overweight and obesity classification used . nationally representative data were obtained in twenty - five countries ( including northern ireland ) [ 1539 ] , and ten countries were represented only by regional data [ 40 , 42 , 44 , 45 , 47 , 5054 ] . table 2 shows overweight and obesity prevalence from the twenty - five national studies ( one of them including data from northern ireland ) that were included in this review according to the continent and the country where it was performed , year of survey , study population , age range , criteria used for classifying overweight and obesity used , and along with total data by sex . there were thirty - two different prevalence levels described in the included articles , because five countries presented data using at least two different criteria for overweight and obesity classification [ 18 , 23 , 27 , 36 , 39 ] . the iotf cut - off was used to classify overweight and obesity in twenty - three of the twenty - five national studies considered in the present review . in general , the prevalence of overweight plus obesity was higher in america [ 1820 ] , oceania [ 38 , 39 ] and europe [ 3037 ] and lower in africa [ 1517 ] and certain parts of asia [ 2129 ] ( in china and iran the total prevalence was less than 10% by the iotf cut - offs ) . overall , about 30% of american adolescents and 22%25% of european adolescents ( excepting the czech republic and italian adolescents ' which showed a prevalence of 13.7% and 17.9% , resp . ) were overweight or obese . among oceanian adolescents the prevalence ranged from 23.2% in australia in 2004 to 34.2% in new zealand in 2007 . in africa , the overall prevalence of overweight and obesity was lower than 20% . among asian adolescents there was a broad range of overweight plus obesity . using iotf cut - off , the prevalence of being overweight or obese for asian boys and girls ranged from 5.2% in china in 2002 to 36.4% in bahrain in 2000 . specific prevalence from all the geographic regions was included in this review from three countries : south africa ( nine provinces ) , usa ( fifty two states ) , and italy ( five regions ) . in europe , data from islands of greece ( crete ) and italy ( sicily and sardinia ) [ 48 , 49 ] and spain ( balearic islands ' archipelago ; and the grand canary island ) were also included . on the other hand , regional but not national data was found for eleven countries ( italy , brazil , india , jordan , denmark , france , hungary , poland , spain [ 51 , 52 ] , switzerland , and turkey ) . the iotf cut - off was used to classify overweight and obesity in fourteen of the eighteen selected studies that included regional data . in one study , data was presented using only the who-2007 growth charts and in two studies using only the cdc-2000 growth reference [ 20 , 43 ] . in south africa and usa , substantial geographic variations in adolescent overweight and obesity prevalence varied in south africa from 13.5% in limpopo to 25.5% in kwazulu - natal . in 2007 , overweight and obesity varied in usa from 23.1% in utah and minnesota to 44.5% in mississippi . in 2002 , the prevalence of overweight and obesity in southern italy and italian islands was higher among boys . in southern italy , the overweight prevalence among girls also was higher than in the other geographic regions . comparison between the islands from greece ( crete ) , italy ( sicily and sardinia ) , and spain ( balearic islands and grand canary island ) which were included in this review showed that crete had the highest prevalence of overweight and obesity despite data were presented using different definition . in spain , using the iotf cut - off ( data not shown for balearic islands but given by authors ) , the prevalence of overweight plus obesity was higher in the grand canary island ( 29.1% ) than in the balearic islands ( 24.7% ) . according to national data , the prevalence of overweight among boys was 10% higher than girls in nine countries ( canada , qatar , taiwan , cyprus , czech republic , germany , greece , italy , australia , denmark , and hungary ) and among girls 10% higher than boys in seven of the twenty - five countries ( south africa , seychelles , tunisia , mexico , bahrain , saudi arabia , and sweden ) . the obesity prevalence was 10% higher among boys in seventeen countries ( canada , usa , china , iran , israel , qatar , saudi arabia , taiwan , cyprus , czech republic , germany , greece , italy , portugal , sweden , australia , new zealand , denmark , and hungary ) and 10% higher among girls in four of the twenty - five countries ( south africa , seychelles , tunisia , and bahrain ) . the aim of this study was to review systematically the literature on overweight and obesity prevalence among adolescents worldwide . thirty - nine articles and one national health report that met the inclusion criteria were considered . the overweight and obesity prevalence in the included studies ranged widely . in sixteen of the twenty - three countries with national representative data using the iotf cut - off , overweight and obesity prevalence higher than 20% were found , five countries showed prevalence above 30% , and just in two countries prevalence was lower than 10% . regarding national data , when prevalence was analysed according to sex , it was observed that boys showed a higher prevalence of overweight in almost half of the countries and a higher prevalence of obesity in almost all countries . these results are consistent with previous studies that pointed out a high prevalence of abdominal obesity among boys . differences of prevalence of overweight and obesity between genders have been related to geopolitical and cultural conditions . eight articles compared data between 1980s and/or 1990s with 2000s [ 16 , 19 , 20 , 22 , 28 , 32 , 37 , 50 ] and pointed out an increased prevalence of overweight and obesity in both sexes over this period . however , among australian adolescents the overweight and obesity prevalence increased significantly among boys but not among girls over the period 19972004 . in the australian national children 's nutrition and physical activity survey 2007 ( ncnpas07 ) , 25% of boys and 30% of girls aged 9- to 13-year - olds and 25% of boys and 23% of girls aged 14- to 16-year - olds were overweight or obese using the iotf criteria . a comparison of the 1985 , 1995 , and 2007 australian national surveys of 7- to 15-year - olds indicated that australian children are changing body shape to a more central fat distribution . in usa , overweight and obesity prevalence increased by 3% and 18% among usa girls over this period . however , a cross - sectional analyses of a representative sample ( n = 4111 ) of the usa child and adolescent population ( birth through 19 years of age ) with data from the national health and nutrition examination survey 2009 - 10 ( nhanes ) indicated a prevalence of overweight and obesity among adolescents aged 12 through 19 years of 15.2% and 18.4% , respectively . analyses of trends in obesity prevalence for the last two nhanes surveys ( 2007 - 08 and 2009 - 10 ) indicated that the prevalence of obesity in children and adolescents has not changed in 2009 - 10 compared with 2007 - 08 . on the other hand , since 2004 the overweight and obesity trends were stabilized or decreased among german adolescents . in usa , substantial geographic disparities in adolescent overweight and obesity were found , with an apparent shift toward higher prevalence in 2007 for several states . reported that children in northern europe countries generally tended to have lower overweight and obesity prevalence ( 1020% ) than in southern europe ( 2035% ) . also within the same country , the prevalence and trends of overweight and obesity may not be homogeneous according to different geographic regions . in italy , a north - south gradient in overweight and obesity prevalence among boys but also in overweight prevalence among girls was also reported . a higher prevalence of overweight and obesity has been reported in southern spain in both children and adults . it is important to note that the choice of a reference and a cut - off point will determine the absolute prevalence of overweight and obesity and its trends , and hence different information will be obtained from the papers . argued that the reference they published , supported by the iotf , is less arbitrary and more international than others and recommended its use in international comparisons . lately , monasta et al . suggested that the iotf reference and cut - offs could be preferable to identify overweight and obesity both at individual and population levels because they are at least based on a crude association with ill and health later in life , namely , the definition of overweight and obesity at age 18 years . however , the iotf cut - offs have been not recommended for clinical use when assessing an individual child 's growth [ 9 , 6264 ] . furthermore , recent findings suggested that a universal bmi classification system for childhood and adolescent overweight and obesity may not correspond to a comparable level of body fatness in all populations . the prevalence estimates may not accurately characterize the population groups most at risk of health disadvantages because the correlation of bmi with adiposity is highly variable and dependent on ethnic group [ 9 , 60 , 65 , 66 ] . the comparisons of overweight and obesity prevalence need interpretation with caution due to the difference in survey sampling methods , sample sizes , age range of subjects , quality of data in terms of height and weight measurement , and whether national programmes or strategies to tackle overweight and obesity are in place . even within the same country , the prevalence and trends of overweight and obesity may not be homogenous in view of different ethnicities , geographic regions , and socioeconomic status . only articles in english , spanish , italian , and portuguese were included in this review . the results of this review allow the following conclusions : ( 1 ) overweight and obesity prevalence is high ; ( 2 ) obesity is higher among boys , although it is not clear which sex has a higher proportion of adolescents with overweight ; ( 3 ) despite that there is no consensus about criteria to be used to classify adolescents as overweighed or obese , the most frequently used was the iotf reference . however , the international reference charts for monitoring the secular trends in childhood obesity need to be continually refined and evaluated . the results of this study would contribute to guiding health planners and administrators to develop proper tools for adolescent obesity management .

objective . to review the extant literature on the prevalence of overweight and obesity in adolescents ( 1019 years old ) of both sexes . design . the search was carried out using medline and scopus considering articles published from the establishment of the databanks until june 7 , 2012 . data on the prevalence of children being overweight and obese from the international obesity task force ( iotf ) website was also reviewed . only original articles and one national health report were considered . forty studies met the inclusion criteria . results . twenty - five of these studies were nationally representative , and ten countries were represented only by regional data . conclusions . the prevalence of overweight and obesity among adolescents worldwide is high , and obesity is higher among boys . the iotf criterion is the most frequently used method to classify adolescents as overweighed or obese in public health research .

sacubitril / valsartan is a combination of a neprilysin inhibitor and an angiotensin ii receptor blocker , indicated to decrease the risk of cardiovascular death and hospitalization for heart failure in patients with chronic heart failure.a patient previously stable on atorvastatin developed severe rhabdomyolysis and an elevation of transaminases within 26 days of initiation of sacubitril / valsartan . rhabdomyolysis is a syndrome characterized by skeletal muscle cell damage , leading to elevated creatine kinase ( ck ) , lactate dehydrogenase ( ldh ) , and aspartate aminotranferase ( ast ) , which can result in severe sequelae , including renal failure , cardiac arrhythmia , hyperthermia and death . associated elevations in transaminases may be seen , even in the absence of significant liver injury . clinically , rhabdomyolysis classically presents with muscle pain , weakness and dark , red or tea - colored urine ; however , less than 10% of patients will demonstrate all three components of this triad . rhabdomyolysis may be triggered by hereditary and/or acquired mechanisms , with approximately 75% of initial episodes being a result of acquired causes . in particular , a large number of prescription drugs and drugs of abuse can cause rhabdomyolysis , with statins being one of the top three prescription drugs most commonly responsible for rhabdomyolysis for this syndrome . myopathy and myositis have been reported in conjunction with all of the currently available statins and are considered to be a class effect of these medications . in addition , it has been recognized that statin - associated muscle complaints are dose - related and drug drug interactions that cause an elevation of statin levels have been identified . subgroups of patients at greater risk of statin - associated muscle pathology have been identified and include age > 70 years , impaired renal function , and impaired hepatic function . entresto is a combination of sacubitril , a neprilysin inhibitor , and valsartan , an angiotensin ii receptor blocker ( arb ) , indicated to decrease the risk of cardiovascular death and hospitalization for heart failure in patients with chronic heart failure ( new york heart association [ nyha ] class ii sacubitril / valsartan is usually administered in conjunction with other heart failure drug regimens instead of an angiotensin - converting enzyme inhibitor ( acei ) or another arb . the commonly occurring ( 5% ) adverse reactions reported are hypotension , hyperkalemia , cough , dizziness , and renal failure [ 6 , 7 ] . sacubitril / valsartan has not been associated with rhabdomyolysis and is commonly used in patients who are also receiving statin therapy . a 63-year - old guyanese woman with hypertension , hyperlipidemia , congestive heart failure ( chf ) , atrial fibrillation , and peripheral vascular disease presented to her primary care provider s office with a complaint of an episode of weakness at home associated with general malaise . she was receiving atorvastatin 40 mg daily , carvedilol 25 mg twice daily , digoxin 0.125 mg daily , furosemide 40 mg daily , spironolactone 25 mg daily , rivaroxaban 15 mg daily , and sacubitril / valsartan 24/26 mg twice daily . she was initiated on sacubitril / valsartan 22 days before her clinical presentation and had been on all of her other concurrent medications for more than 1 year . on the review of systems , the patient reported generalized weakness , pain in her knees , and an ill - defined stinging pain in the skin of her arms and legs . her physical examination was non - focal , with no muscle weakness or tenderness of either the skin or muscles noted . laboratory tests performed on presentation were significant for elevated ast / alanine aminotransferase ( alt ) of 351 u / l/131 be having a reaction to her statin therapy prescribed for hyperlipidemia and therefore the atorvastatin was withheld . she was scheduled to return in 4 days for repeat laboratory testing and re - evaluation . when the patient returned in 4 days , she complained of dark urine , generalized weakness , and increased muscle and skin pain throughout her body . she was then found to have rhabdomyolysis , with ck at 58,349 u / l ( > 50 the upper limit of normal ) . sacubitril / valsartan was stopped at this time as it was the only new medication in her regimen . the ck level decreased steadily and her symptoms resolved over the course of a 5-day hospitalization ( fig . 1 ) . her renal function was preserved throughout and her bilirubin and transaminases briefly increased and also subsequently steadily decreased ( figs . 2 , 3 ) . barr virus , hepatitis a , herpes simplex virus , hiv , and cytomegalovirus ( cmv ) . barr virus , and herpes simplex virus , and had negative hepatitis a and b , hiv , hsv . and cmv tests.fig . ast aspartate aminotransferase , alt alanine aminotransferase creatine kinase values during and post hospitalization . ast aspartate aminotransferase , alt alanine aminotransferase by 23 days after her initial presentation , the patient no longer had any muscle or skin pain and her ck and liver function test ( lft ) levels were completely normalized . the patient has not had any reoccurrence of rhabdomyolysis and elevated transaminases for 46 days , and her bilirubin and transaminase levels also returned to normal ( figs . 2 , 3 ) . one week later , the patient was started on an alternate statin at a low dose ( rosuvastatin 5 mg ) and the ck levels and lfts have remained stable . this patient had been on a variety of statins for more than 7 years , including atorvastatin , simvastatin , and rosuvastatin . her baseline ck and lfts were normal prior to the initiation of sacubitril / valsartan . she had previously experienced two transient elevations in ck , to a maximum of 1608 u / l , while hospitalized for an implantable defibrillator infection and explanation complicated by an acute chf exacerbation and a recurrent chf exacerbation in the 3 years prior to the current admission . her ck levels returned to normal after her acute illnesses , without change in her lfts . in this case , the immediate cause of her rhabdomyolysis was unknown , but the new medication in her profile seemed to be the likely trigger . due to the severity of her rhabdomyolysis the naranjo adverse drug reaction probability scale score was 5 , making it probable that the adverse drug reaction was precipitated by sacubitril / valsartan . the drug interaction probability scale score was 3 , consistent with a possible interaction as a cause for the reaction , with sacubitril / valsartan as the precipitant drug and atorvastatin as the object drug . of note , one of the concurrent medications , carvedilol , is a p - glycoprotein inhibitor that may increase the serum concentration of atorvastatin , a p - glycoprotein substrate ; however , the patient was stable on this combination for 7 years before sacubitril / valsartan was initiated . none of the other medications taken by this patient are known to cause rhabdomyolysis or an elevation in transaminases . a literature search regarding a drug interaction between sacubitril and statins yielded no reports of rhabdomyolysis with statins . as per a recent study , coadministration of sacubitril / valsartan with atorvastatin led to a twofold increase in the maximum concentration ( c max ) of atorvastatin and its metabolites . in addition , the us and canadian package inserts of sacubitril / valsartan cite in vitro data showing sacubitril inhibiting organic anion transporting polypeptides ( oatp ) 1b1 and 1b3 , resulting in increased c max and area under the curve ( auc ) of atorvastatin with coadministration of sacubitril / valsartan [ 6 , 7 ] . the canadian version of the product insert further states that sacubitril / valsartan may increase the systemic exposure of oatp1b1 and oatp1b3 substrates , such as statins . sacubitril / valsartan increases the c max of atorvastatin and its metabolites by up to twofold , and auc by up to 1.3-fold . the canadian insert recommends that sacubitril / valsartan must be coadministered cautiously with statins , especially simvastatin , a sensitive oatp1b1/1b3 substrate , and a decrease in dose of simvastatin and atorvastatin may be considered when coadministered with sacubitril / valsartan . the interaction between atorvastatin and sacubitril is mentioned in the us product package insert as a theoretical possibility . there is currently no recommendation to increase monitoring or empirically decrease the dose of any statin on initiation of sacubitril / valsartan . a number of medications are known to inhibit oatps and have been shown to increase statin plasma concentrations . many single nucleotide polymorphisms ( snps ) have been found within the slco1b1 and/or slco1b3 genes encoding oatp1b1 and oatp1b3 proteins , respectively , and were shown to effect the pharmacokinetics and/or pharmacodynamics of statins [ 12 , 13 ] . the clinical consequences of the drug interactions may vary based on the genetic variation in oatp - encoding genes and inhibition of oatp function . the slco1b1 * 1b haplotype appears to be associated with enhanced hepatic uptake and reduced plasma concentrations of oatp1b1 substrates . discontinuation of the statin alone did not result in a decrease in the patient s lfts . it was not until sacubitril / valsartan was also discontinued , and treatment for rhabdomyolysis was initiated , that the patient s symptoms began to improve and her ck and lfts returned to baseline . this could be due to either a direct effect of sacubitril / valsartan or the increased serum concentration of atorvastatin , as described in the literature . because of the above - stated pharmacodynamics , we believe that the more likely mechanism is the increased serum concentration of atorvastatin . currently , there is no recommendation regarding the initiation or continuation of statins in patients with nyha class ii iv heart failure as there is insufficient information on which to base recommendations for or against statin treatment . in these patients , the choice of statin dose , and using a statin for cardiovascular risk reduction benefit , must be weighed against adverse effects , drug drug interactions , precautions and contraindications to statin therapy . we would have liked to have arranged slcob testing for the patient but were unable to . further research will be useful in identifying patients at risk for this adverse reaction , and will likely include genetic testing . this case of severe rhabdomyolysis should stimulate further investigation of the potential negative effects of the initiation of sacubitril / valsartan in patients receiving statin therapy . since sacubitril / valsartan is targeted for patients with heart failure , we predict that this would comprise a large number of the candidates for treatment . we recommend consideration of obtaining baseline ck levels and lfts and close observation of patients . in this case , rhabdomyolysis developed within 3 weeks of initiation of sacubitril / valsartan and evaluation within a shorter time frame may be appropriate . future reports of this reaction may help to define high - risk patients , but it should be noted that our patient had no known high - risk characteristics for the development of rhabdomyolysis , except for prior mild elevations of ck during hospitalizations for chf exacerbations . we encourage the reporting of any similar reactions to the us fda , the manufacturer , and the medical community . eve s. faber , madhavi gavini , ronald ramirez and richard sadovsky declare that they have no conflicts of interest that are directly relevant to the content of this report . a copy of the written consent may be requested for review from the corresponding author .

a 63-year - old woman previously stable on a regimen of atorvastatin 40 mg daily , carvedilol 25 mg twice daily , digoxin 0.125 mg daily , furosemide 40 mg daily , spironolactone 25 mg daily , rivaroxaban 15 mg daily , and enalapril 20 mg twice daily for heart failure developed rhabdomyolysis 26 days after enalapril was stopped and sacubitril / valsartan ( entresto ) started . the patient received sacubitril / valsartan at 24/26 mg twice daily for heart failure ; however , after 26 days she developed muscle and skin pain . investigations revealed elevated creatine kinase and liver function tests , and rhabdomyolysis with raised transaminases was diagnosed . sacubitril / valsartan and atorvastatin were discontinued and the patient was hydrated . she returned to baseline in 23 days and has not had any reoccurrence of rhabdomyolysis and elevated transaminases for 46 days . a naranjo assessment score of 5 was obtained , indicating a probable relationship between the patient s rhabdomyolysis and her use of sacubitril / valsartan . the drug interaction probability scale score was 3 , consistent with a possible interaction as a cause for the reaction , with sacubitril / valsartan as the precipitant drug and atorvastatin as the object drug .

the nlrp3 inflammasome , a member of the nlr family , is a key player in the production of uric acid - mediated il-1 and is an important cytoplasmic protein complex involved in gouty inflammation ( 12 ) . although the precise pathogenic mechanism of gout has not been clearly determined , several crucial proteins such as the purinergic receptor p2x ligand - gated ion channel 7 ( p2x7r ) ( 345 ) and caspase activation and recruitment domain 8 ( card8 ) ( 67 ) proteins are known to be responsible for the pathogenesis of gout . recent single - nucleotide polymorphism ( snp ) studies suggested that genetic alternations of several target molecules such as card8 and p2x7r contribute to the process of nlrp3 inflammasome activation . genetic variants of card8 were identified to play a role in the pathogenesis of a variety of inflammatory diseases , such as rheumatoid arthritis ( ra ) ( 8) and inflammatory bowel disease ( ibd ) ( 9 ) . chen et al . ( 6 ) demonstrated a significantly different genotypic distribution of the card8 rs2043211 polymorphism between gout and control patients in the chinese population . another candidate gene involved in gouty inflammation might be p2x7r , whose protein product binds with atp and induces the efflux of k ions through the p2x7r channel from cells , finally triggering activation of the nlrp3 inflammasome ( 34 ) . a loss - of - function ( 1513 a > c ) snp of the p2x7r gene affects atp - induced cellular functions such as apoptotic cell death ( 10 ) and il-1 release ( 11 ) . more evidence to support the intimate relationship of p2x7r gene polymorphisms with autoimmune diseases are the demonstrated links between p2x7r polymorphisms and ra and systemic lupus erythematosus ( sle ) ( 1213 ) . however , there are no available data linking p2x7r snps with gout . here , we investigated the association of p2x7r rs3751142 and card8 rs2043211 polymorphisms with the susceptibility and clinical manifestations of gout in the male korean population . a total of 242 male gout patients fulfilled the preliminary criteria for classification of primary gout proposed by the american college of rheumatology ( 14 ) and a total of 280 healthy male controls were consecutively enrolled in this study . clinical and laboratory variables , including age at the time of study , disease onset age , body mass index ( bmi ) , disease duration , and serum uric acid level were identified by individual interviews with each patient and medical record review . medications including corticosteroids , non - steroidal anti - inflammatory drugs ( nsaids ) , colchicine , allopurinol , and benzbromarone that were used for gout treatment within one month of the study onset were evaluated through medical record reviews . the study protocol was reviewed and approved by the institutional review board / ethics committee at each medical center that participated in this study . assay reagents for rs3751142(c > a ) in the p2x7r gene and rs2043211(a > t ) in the card8 gene were designed by applied biosystems ( applied biosystems , foster city , ca , usa ) . the reagents consisted of taqman mgb probes ( fam and vic dye - labeled ) . the reaction in a 10 l total volume was optimized at 0.125 l 40x reagents , 5 l 2x taqman genotyping master mix ( applied biosystems ) and 2 l 50 ng genomic dna . pcr conditions included one cycle at 95c for 10 minutes followed by 40 cycles at 95c for 15 seconds and 60c for 1 minute . the pcr was performed using the abi plus ( applied biosystems ) system and the samples were read and analyzed using abi plus ( applied biosystems ) software . the reference sequence was based on the sequence of human chromosome 12 , 12q24 for the p2rx7 gene , and human chromosome 19 , 19q13.33 for the card8 gene . snp , single nucleotide polymorphism ; p2x7r , purinergic receptor p2x ligand - gated ion channel 7 ; card8 , caspase activation and recruitment domain 8 . clinical parameters are described as the median with interquartile range ( iqr ) or the number with percent ( % ) . the hardy - weinberg equilibrium for gout patients and controls was assessed using a web - based calculator ( http://www.had2know.com/academics/hardy-weinberg-equilibrium-calculator-2-alleles.html ) ( 15 ) . the genotypic frequency of each gene was calculated and assessed using the chi - square and fisher s exact tests , if appropriate . both kolmogorov - smirnov and shapiro - wilk analyses were tested to identify normality of data distribution , which did not show a normal distribution . the kruskal - wallis test was used to compare parametric parameters such as age , onset age , disease duration , bmi , and serum uric acid among the three genotypes of each gene . logistic regression analysis adjusted with age was applied to test whether the combined p2x7r rs3751142 and card8 rs2043211 genotype influenced the risk of gout development compared to those in controls . the results are described as the odds ratio ( or ) with a 95% confidence interval ( ci ) . all patients provided written informed consent , and the protocol of this study was approved by the institutional review board ( irb ) of daegu catholic university medical center ( irb no . the authors assert that all procedures contributing to this work comply with the helsinki declaration of 1975 and its later amendments . a total of 242 male gout patients fulfilled the preliminary criteria for classification of primary gout proposed by the american college of rheumatology ( 14 ) and a total of 280 healthy male controls were consecutively enrolled in this study . clinical and laboratory variables , including age at the time of study , disease onset age , body mass index ( bmi ) , disease duration , and serum uric acid level were identified by individual interviews with each patient and medical record review . medications including corticosteroids , non - steroidal anti - inflammatory drugs ( nsaids ) , colchicine , allopurinol , and benzbromarone that were used for gout treatment within one month of the study onset were evaluated through medical record reviews . the study protocol was reviewed and approved by the institutional review board / ethics committee at each medical center that participated in this study . assay reagents for rs3751142(c > a ) in the p2x7r gene and rs2043211(a > t ) in the card8 gene were designed by applied biosystems ( applied biosystems , foster city , ca , usa ) . the reagents consisted of taqman mgb probes ( fam and vic dye - labeled ) . the reaction in a 10 l total volume was optimized at 0.125 l 40x reagents , 5 l 2x taqman genotyping master mix ( applied biosystems ) and 2 l 50 ng genomic dna . pcr conditions included one cycle at 95c for 10 minutes followed by 40 cycles at 95c for 15 seconds and 60c for 1 minute . the pcr was performed using the abi plus ( applied biosystems ) system and the samples were read and analyzed using abi plus ( applied biosystems ) software . the reference sequence was based on the sequence of human chromosome 12 , 12q24 for the p2rx7 gene , and human chromosome 19 , 19q13.33 for the card8 gene . snp , single nucleotide polymorphism ; p2x7r , purinergic receptor p2x ligand - gated ion channel 7 ; card8 , caspase activation and recruitment domain 8 . clinical parameters are described as the median with interquartile range ( iqr ) or the number with percent ( % ) . the hardy - weinberg equilibrium for gout patients and controls the genotypic frequency of each gene was calculated and assessed using the chi - square and fisher s exact tests , if appropriate . both kolmogorov - smirnov and shapiro - wilk analyses were tested to identify normality of data distribution , which did not show a normal distribution . the kruskal - wallis test was used to compare parametric parameters such as age , onset age , disease duration , bmi , and serum uric acid among the three genotypes of each gene . logistic regression analysis adjusted with age was applied to test whether the combined p2x7r rs3751142 and card8 rs2043211 genotype influenced the risk of gout development compared to those in controls . the results are described as the odds ratio ( or ) with a 95% confidence interval ( ci ) . statistical analyses were performed using ibm spss statistics 19.0 software ( ibm corp . , armonk , ny , usa ) . all patients provided written informed consent , and the protocol of this study was approved by the institutional review board ( irb ) of daegu catholic university medical center ( irb no . the authors assert that all procedures contributing to this work comply with the helsinki declaration of 1975 and its later amendments . the mean age was 55.0 ( iqr 45.063.0 ) years for gout patients and 45.0 ( iqr 41.053.0 ) for controls , which was significantly different between two groups ( p < 0.001 ) . data were expressed as with interquartile range ( iqr ) for continuous variables and number ( % ) for categorical variables . no genotypic deviations of rs3751142(c > a ) in the p2x7r gene and rs2043211(a > t ) in card8 based on the hardy - weinberg equilibrium were noted for gout or control patients ( p > 0.05 ) . table 3 shows that a frequency difference of alleles and genotypes for rs3751142(c > a ) and rs2043211(a > t ) was not found between gout or control patients . under the recessive comparison model ( cc vs. ca / aa in p2x7r and aa vs. at / tt in card8 ) , genotypic frequency differences between the two groups were also not detected at each snp ( p > 0.05 of both snps ) . there were no differences of clinical variables according to genotypic distribution of p2x7r rs3751142 and card8 rs2043211 . in a comparison model sub - analysis for onset age ( 30 vs. > 30 years ) and serum uric acid level ( 7.0 vs. > 7.0 mg / l ) , genotypic differences of each clinical parameter were not observed at the two snps ( data not shown ) . rs3751142 ( c > a ) in p2x7r gene and rs2043211(a > t ) in card8 gene . a pair - wise comparison of p2x7r rs3751142 and card8 rs2043211 genotype combinations showed that a trend toward higher risk of gout development was identified in subjects who were heterozygous for the ca p2x7r rs3751142 genotype and recessive homozygous for the tt card8 rs2043211 genotype compared to the cc / aa combination ( p = 0.056 , or = 2.618 , 95% ci 0.975 - 7.031 ) ( table 5 ) . the statistical analysis was performed using logistic regression analysis after adjustment with age . or , odds ratio ; ci , confidence interval . the main objective of this study was to investigate whether genetic variability in the p2x7r rs3751142 and card8 rs2043211 genes was involved in the process of nlrp3 inflammasome activation , which might influence susceptibility for the development of gout . this study identified that card8 rs2043211 was not associated with increased gout risk , which is in contrast to the known difference of genetic distribution of this snp in the chinese and european gout population ( 67 ) . as far as we know , this study is the first to determine genetic association between the p2x7r snp and gout patients , revealing that p2x7r polymorphism did not increase the risk of gout in our study population . the nlrp3 inflammasome is a cytoplasmic protein complex that activates caspase-1 , inducing the maturation and secretion of il-1 , which is associated with gout pathogenesis ( 12 ) . the mechanisms by which signaling pathways activate the nlrp3 inflammasome are not clearly determined ; however , it is known that cytoplasmic k efflux from the cell through the p2x7r channel in response to danger signals allows for nlrp3 inflammasome activation ( 34 ) . similarly , msu crystals , atp , and asbestos are known to induce inflammasome activation by decreasing intracellular k levels ( 16 ) . the p2x7r gene is markedly polymorphic and includes at least 12 non - synonymous snps in the coding region ( 17 ) . p2x7r snps that encode glu496ala or ala348thr substitutions are most commonly found to be associated with il-1 secretion capacity ( 1117 ) . the loss - of - function ( 1513 a > c ) snp of p2x7r gene induced lower atp - mediated k ion efflux , leading to reduced il-1 levels in monocytes ( 11 ) . ( 18 ) recently suggested the possibility that p2x7r is a key regulator for the production of il-1 by msu crystals during acute gouty arthritis . human myeloid leukemic kg-1 cells contain several snps of the p2x7r gene ( 19 ) . among them , kg-1 cells with rs3751142 a revealed neutral effect on p2x7r function . however , there is no evidence as to whether the p2x7r polymorphism has a potent influence on the development of gout . in our data , a minor allele frequency ( maf , a allele ) of p2x7r rs3751142 in gout and control patients was 0.132 and 0.161 , respectively , which are more compatible to those in the dbsnp rs3751142 ( http://www.ncbi.nlm.nih.gov/snp/snp_ref.cgi?locusid05027 ) . this study found that p2x7r rs3751142 was not a genetic risk factor associated with gout susceptibility . the activation of the nlrp3 inflammasome , which is comprised of multiple cytoplasmic proteins including nlrp3 , an asc protein , and caspase-1 , is completed through the binding of the pyd of nlrp3 with the pyd of asc , which is followed by the recruitment of procaspase-1 by linkages of each card within the two proteins . card8 was shown to be a negative regulator of nuclear factor-b ( nf-b ) and caspase-1 activation ( 2021 ) , which implicates a role in the suppression of nlrp3 inflammasome activation . the rs2043211 snp in exon 5 of the card8 gene is a nonsense mutation resulting in the production of a truncated protein . although the functional role of polymorphic card8 within the nlrp3 inflammasome is complicated , genetic variants of card8 rs2043211 contribute to an enhanced inflammatory response in inflammatory diseases such as ra and ibd through increased expression of il-1 ( 8922 ) . mckinney et al . ( 7 ) demonstrated that the prevalence of card8 rs2043211 was different between gout and control patients in the european and polynesian populations ( or 1.11 , p = 0.023 and or 1.15 , p = 0.078 , respectively ) . another case - control study showed that the maf ( a allele ) card8 gene was shown to have an increasing trend relative to the t allele in the chinese male population ( or = 0.084 , p = 0.08 ) ( 6 ) . however , our study revealed no association of card8 rs2043211 with the presence of gout in korean study population . the frequency of maf ( t allele ) is at 0.457 in gout and 0.452 in control patients , which is similar to the frequency in the new zealand polynesian ( 0.499 in gout and 0.439 in control ) and chinese ( 0.498 in gout and 0.463 in control ) patients , whereas a lower frequency of maf was noted in the european population ( 7 ) . based on conflicting data for card8 rs2043211 between these three studies , the functional role of the card8 gene in gout susceptibility has not been clearly determined . therefore , the distinct role of the card8 gene in the pathogenesis of gout should be considered in a diverse ethnic study population . first , the lack of completeness for clinical information of control subjects is an important weakness in our study . this study used genomic dnas provided by the biobank of wonkwang university hospital , korea as control group samples . we were just able to identify a part of demographic data including gender and age . second , this study did not perform functional analysis for genotypes in each target gene . as shown disparity of genetic susceptibility of gout in several ethnic groups ( 67 ) , it is important to identify functional performance of each gene to understand the role of p2x7r and card8 snps in our study population . it is noted that the p2x7r and card8 proteins are closely involved in activation of the nlrp3 inflammasome and il-1 expression , which could explain their role in the pathogenesis of gout ( 12 ) . our study demonstrated that the p2x7r rs3751142 and card8 rs2043211 genetic variants were not implicated in the development of gout in the male korean population . however , we found that in a pair - wise comparison of the ca / tt p2x7r and card8 genotype combination was shown to have an increased trend for the risk of gout ( or = 2.618 , p = 0.056 ) . in conclusion , this study provides evidence that the interaction of msu crystal - mediated p2x7r with card8 may be at least in part responsible for the pathogenesis of gout .

the aim of this study was to determine the association between p2x7r rs3751142 and card8 rs2043211 polymorphisms and gout susceptibility in male korean subjects . this study enrolled a total of 242 male patients with gout and 280 healthy controls . the polymorphisms of two individual genes including rs3751142(c > a ) in the p2x7r gene and rs2043211(a > t ) in the card8 gene were assessed using taq - man analysis . statistical analyses were performed using the chi - square test , kruskal - wallis test , and logistic regression analyses . a difference in genotypic frequency of the p2x7r rs3751142 and card8 rs2043211 genes was not detected between gout and control patients . clinical parameters including age , onset age , disease duration , body mass index , and serum uric acid levels were not different among the three genotypes for either p2x7r or card8 ( p > 0.05 for all ) . a pair - wise comparison of p2x7r rs3751142 and card8 rs2043211 genotype combinations revealed that subjects with the ca p2x7r rs3751142 genotype and the tt card8 rs2043211 genotype had a trend toward a higher risk of gout compared to the cc / aa combination ( p = 0.056 , or = 2.618 , 95% ci 0.975 - 7.031 ) . in conclusion , this study revealed that genetic variability of the p2x7r rs3751142 and card8 rs2043211 genes might , in part , be associated with susceptibility for gout .

isolated involvement of the appendix in crohn 's disease is reported to be 0.2% to 1.8% , and is usually associated with ileocaecal crohn 's disease in 25% of ileal and 50% of caecal disease . while appendicitis in a patient who was previously diagnosed to have ileocaecal crohn 's may be managed with appendicectomy and ileocaecal resection , appendicectomy alone when performed for appendicitis in a patient with unsuspected ileocaecal crohn 's disease could lead to postoperative complications including enterocutaneous fistula . a young female patient who underwent appendicectomy elsewhere for acute appendicitis presented to us with a persistent enterocutaneous fistula of 6 weeks duration . she had complained of general ill health and occasional altered bowel habits for 6 months prior to the acute appendicitis presentation . our investigations , including a ct scan , suggested the possibility of ileocaecal crohn 's disease . she underwent excision of the enterocutaneous fistula and ileocaecal resection , and histopathology of the resected specimen confirmed crohn 's disease . in the postoperative period she received mesasalazine . when last seen 2 years later during her regular follow - up , she was found to be in good health . the possibility of ileocaecal crohn 's disease should be considered in patients presenting with unexplained postoperative enterocutaneous fistula following appendicectomy . a high index of clinical suspicion is required to make a prompt diagnosis and institute appropriate further treatment in form of ileocaecal resection . crohn 's disease is a chronic inflammatory condition that may involve the entire gi tract . histological features include granuloma , lymphoid aggregates , fissures and ulcers extending into muscularis propria , and transmural inflammation . crohn 's disease has been reported to be the cause of acute appendicitis in 0.2% to 1.8% of all the appendicectomies in some series . operations described in the literature for crohn 's disease of the appendix include appendicectomy with caecectomy and ileocaecal resection . however , patients who undergo appendectomy alone are at the risk of complications including enterocutaneous fistula in 34% to 58% of the cases a 24-year - old unmarried female patient presented to us with a persistent fistula in the right iliac fossa following an appendicectomy done in another hospital 6 weeks previously . on further questioning , she informed us that she had presented to this hospital with a 2-week history of abdominal pain , confined to the right lower abdomen . she complained of general ill health and occasional altered bowel habits for 6 months prior to this acute episode . an enquiry with the doctors who had initially seen her revealed that at the time of presentation for acute appendicitis she was quite unwell with fever , tachycardia , and dehydration . her abdomen was tender all over with guarding , more pronounced over the right lower half . an x - ray of her abdomen showed a few fluid levels and an ultrasound scan revealed free fluid in the abdomen with an associated right iliac fossa mass . a diagnosis of pelvic peritonitis secondary to acute appendicitis was made by the physicians , and she was taken up for surgery after adequate resuscitation . there was pus in the abdomen with an appendicular mass , and the appendix appeared oedematous , thickened , and congested . the abdomen was closed after placing a drain , which was removed on the 3 post - operative day . in spite of antibiotic administration she was managed conservatively with antibiotics and anti - inflammatory medication and eventually improved , except for a persistent purulent discharge from the wound . she then decided to seek our opinion and was admitted under our care for further investigation and management . the appendicular histopathology was retrieved from the previous hospital and revealed transmural inflammation with granulomas suggestive of crohn 's disease . a computerized tomography ( ct ) scan of the abdomen carried out in our hospital showed pericaecal collection with an inflammatory mass in front of the caecum ( fig . the abdomen was surgically explored through the previous incision after excising the fistula leading into the caecum . an inflammatory mass associated with the caecum was noted . the appendicular stump had not healed , and was draining into a cavity which was communicating with the wound , indicating a complex enterocutaneous fistula . a limited right hemicolectomy was carried out and continuity established with a primary anastomosis of macroscopically - appearing healthy bowel of the ascending colon and terminal ileum . a specimen of the resected caecum revealed the cobblestone appearance of the mucosa , strongly suggesting the possibility of underlying caecal crohn 's disease ( fig . the histology was reported as inflammatory bowel disease ( ibd ) consistent with crohn 's disease ( fig . she was referred to a gastroenterologist and was being treated with mesasalazine and a regular annual colonoscopy . when last seen at two years post - surgery she continued to remain in good health . limited right hemicolectomy specimen showing the cobble stone appearance of the mucosa typical of crohn 's disease . crohn 's disease may involve the appendix by extension from the terminal ileum or the caecum and present as an acute or sub - acute appendicitis[15 ] . crohn 's disease limited to the appendix usually affects young adults in their 20s and 30s , although it is not limited to this age group and can occur at any age . the clinical presentation is variable , with acute right iliac fossa pain suggestive of acute appendicitis in about 85% of patients , and chronic pain and a palpable mass in the right iliac fossa in about 25% of patients . when the preoperative diagnosis is acute appendicitis , crohn 's disease should be suspected when an atypical or protracted clinical course is present in a patient prior to the appendicitis , particularly in areas where crohn 's disease is prevalent . crohn 's disease is diagnosed by a combination of clinical manifestations and radiological findings including barium studies , showing string strictures , fissures , and fistula in the ileum and caecum . recently , gray scale sonography and color doppler - flow features have been used in diagnosing crohn 's disease . when the disease is located atypically in the appendix , macroscopically , the appendix will be seen to be enlarged , swollen and adherent to the surrounding structures , these findings being secondary to chronic inflammation . histologically , the disease is characterized by transmural inflammation with thickening of the appendiceal wall , epitheloid granulomas , lymphoid aggregates , and mucosal ulceration . other histological features are multinucleated giant langherhan 's cells , crypt abscess , neural hyperplasia and lymphangiectasia . differential diagnosis includes the presence of foreign bodies and diverticulitis of the appendix , which also give rise to chronic granulomatous inflammation with induration and fibrosis of the appendiceal wall , and granulomatous disease of unknown aetiology such as appendiceal sarcoidosis , which is rare and often accompanies systemic disease . differential diagnosis should also include infectious diseases such as tuberculosis , actinomycosis and yersenia infection . a rare fungal infection such as histoplasmosis or blastomycosis , or a parasitic infestation such as schistosomiasis or enterobius vermicularis infestation can elicit an appendiceal granulomatous reaction . the diagnostic difficulty arises when a patient undergoes an appendicectomy for suspected acute appendicitis and the surgeon unexpectedly encounters ileocaecal pathology whose nature is difficult to ascertain in an emergency situation . in view of this , a definitive treatment in the form of ileocaecal resection for conditions like crohn 's disease may be difficult to carry out . while a frozen section may be helpful to differentiate some of these conditions it may not always be feasible in an emergency situation . if crohn 's disease of the appendix is suspected and is limited to the appendix then appendicectomy alone is a routine surgical procedure with very low intraoperative or postoperative mortality and a low rate of fistula formation.the postoperative enterocutaneous fistula incidence rate in crohn 's disease restricted to appendix alone has been reported to be 3.5% , whereas in patients with crohn 's disease of the ileocaecal segment , as in our patient , the postoperative enterocutaneous rate rises to 34% to 58% . weston et al reported that the majority of the patients whose first presentation of crohn 's disease simulates appendicitis and who undergo appendicectomy alone leaving the ileocaecal segment in place to be treated medically , returned postoperatively within 3 years with symptoms . a significant percentage ( 38% ) of these patients returned within one year and most of them ( 77% ) were frequently ill with their disease in the interim period . patients who had an ileocolic resection at the time of the initial operation did not require early reoperation , did not develop short bowel syndrome , and did not have significant postoperative complications . in the postoperative period these patients receive medical therapy to keep the disease quiescent such as salazopyrine , 5-asa , prednisilone , and azothioprine.crohn's disease - related fistula have also been treated with infliximab with some success . despite improvements in medical therapy , between 70% to 90% of the patients with crohn 's disease will eventually require surgical intervention , and approximately half of these will require additional operations . because of the high risk of reoperations and a relatively young age at the time of first operation , a minimally invasive approach has recently been used to carry out the ileocolic resections . a recent meta - analysis of laparoscopic ileocolic resection has revealed a shorter hospital stay compared to open resection , while the morbidity rates were equal and conversion rates were acceptable . the average interval between surgery and recurrence after appendicectomy for crohn 's disease of the appendix is 4 years , after which some claim the recurrence rate to be almost nil . however , others have reported a recurrence rate of 10% over an 8-year follow - up and is much higher in patients with crohn 's disease of the appendix associated with ileocaecal crohn 's disease . most authors recommend periodic surveillance visits with radiological and endoscopic examination of the small intestinal and colon for a period of at least 3 years to promptly detect recurrence . however , others claim that patients with crohn 's disease should be followed for 10 years , and if disease free by then are considered cured . crohn 's disease of the appendix is usually associated with crohn 's disease of the ileum and caecum . appendicectomy will suffice in those who present subacutely , are diagnosed preoperatively by clinical signs , and radiographic evidence shows that the disease is restricted to the appendix alone . however , in the presence of ileocaecal involvement , resection of the ileocaecal segment may be required to prevent postoperative complications of enterocutaneous fistula . while patients who develop enterocutaneous fistula post - appendicectomy in an unsuspected crohn 's disease case have been treated with infliximab with some success , the majority of the patients required ileocaecal resection . moreover ,

context : isolated involvement of the appendix in crohn 's disease is reported to be 0.2% to 1.8% , and is usually associated with ileocaecal crohn 's disease in 25% of ileal and 50% of caecal disease . while appendicitis in a patient who was previously diagnosed to have ileocaecal crohn 's may be managed with appendicectomy and ileocaecal resection , appendicectomy alone when performed for appendicitis in a patient with unsuspected ileocaecal crohn 's disease could lead to postoperative complications including enterocutaneous fistula.case report : a young female patient who underwent appendicectomy elsewhere for acute appendicitis presented to us with a persistent enterocutaneous fistula of 6 weeks duration . she had complained of general ill health and occasional altered bowel habits for 6 months prior to the acute appendicitis presentation . our investigations , including a ct scan , suggested the possibility of ileocaecal crohn 's disease . she underwent excision of the enterocutaneous fistula and ileocaecal resection , and histopathology of the resected specimen confirmed crohn 's disease . in the postoperative period she received mesasalazine . when last seen 2 years later during her regular follow - up , she was found to be in good health.conclusion:the possibility of ileocaecal crohn 's disease should be considered in patients presenting with unexplained postoperative enterocutaneous fistula following appendicectomy . a high index of clinical suspicion is required to make a prompt diagnosis and institute appropriate further treatment in form of ileocaecal resection .

the random sample comprises 267 caucasian preadolescents ( 121 girls , 146 boys ) and 330 caucasian adolescents ( 164 girls , 166 boys ) of a population belonging to the swedish part of the european youth heart study ( eyhs ) . the eyhs is a cross - sectional school - based study of risk factors for future cardiovascular disease among preadolescents ( 910 years old ) and adolescents ( 1516 years old ) . the mean ages in the swedish sample for preadolescents and adolescents were 9.6 years and 15.6 years , respectively . height , weight , and birth weight were measured by internationally accepted standardised procedures ( 22 ) . body mass index ( bmi ) was calculated as weight / height2 ( kg / m ) . identification of sexual maturity was assessed according to tanner and whitehouse 1976 . a researcher of the same gender as the child recorded the pubertal stage after brief observation . on account of ethical reasons the direction of one school preferred not to take part in the assessment of sexual maturity . fifty subjects did therefore not participate in this assessment ( 3 preadolescent girls , 7 preadolescent boys , 25 adolescent girls , and 15 adolescent boys ) . the consumption of milk was assessed by an interviewer - mediated 24-hour recall . in preadolescents , a qualitative food record completed the day before the interview with the help of parents served as a checklist for the data obtained during the recall . dietary data were processed by stormats ( version 4.02 , rudans lttdata , sweden ) and analysed using the swedish national food database ( version 99.1 ) . as part of a broad - ranging questionnaire , parents of the participants were asked if their child had a chronic illness or adhered to a special diet . since under - nourishment is practically non - existent in a nutritionally replete population such as sweden and milk intake accounts in part for daily energy intake , the bmi was taken as proxy for energy intake . furthermore , bmi is traditionally used to validate energy intake data ( 23 ) . for the genetic analysis of lp and lnp , genomic dna was isolated from edta whole blood samples from the individuals with the qiaamp dna blood mini kit spin procedure . the dna fragment spanning the -13910-c / t polymorphic site was amplified using a biotinylated forward - primer ( 5 gggctggcaatacagataagata-3 ) and an unbiotinylated reverse - primer ( 5 agcagggctcaaagaacaatcta-3 ) . the applied sequencing primer was : 5-ctttgaggccaggg-3. sequencing was performed using a psq96 snp reagent kit and a psq 96ma system ( pyrosequencing ab ) psq 96ma 2.0.1 software . the procedure has been previously described in detail ( 24 , 25 ) . for the determination of socio - economic status ( ses ) we used the dichotomous variable , below versus above the mean income level in the catchment areas of the sample ( below or above the mean in their municipality ) on account of the relatively equal distribution of income in sweden ( 26 ) . for four subjects , data for ses student 's t - test was used to determine differences in milk intake ( g / d ) between lp and lnp subjects . milk intake ( g / d ) was somewhat skewed to the right and therefore split in quintiles of milk intake . stepwise backward multiple linear regression analysis was performed in order to study the relationship between milk intake in quintiles and body height ( cm ) after adjustments for sex , birth weight ( g ) , father 's and mother 's height ( cm ) , bmi , tanner stage ( 15 ) , and ses . the study was approved by the research ethics committees of rebro county council and huddinge university hospital . parents and 15-year - olds gave specific written informed consent to participate in the study . the variables included in the backward multivariate regression model were : milk intake , lct c > t-13910 polymorphism ( lp = ct , tt vs. lnp = cc ) , sex , birth weight , father 's and mother 's height , bmi , tanner stage , and ses . characteristics of preadolescents and adolescents by quintile of milk intake are given in table 2 . basic characteristics of covariates to body height in the study population of swedish preadolescents and adolescents variables associated to body height by quintiles of milk intake fifty - six ( 9% ) of the subjects of the whole sample were lnp . where lnp may restrict the intake of milk in these subjects and can thereby have an effect on the dependent variable , body height as well as the exposure variable , i.e. milk intake . milk intake tended to be lower in lnp compared to lp but the difference did not reach statistical significance ( p=0.067 ) . the studied exposure variables in this model were milk intake in quintiles and the lct c > t-13910 polymorphism , and the outcome variable was body height . the model explained 90% of the observed variance of body height in preadolescents and adolescents ( adjusted r = 0.89 ) . non - significant variables were removed in a stepwise manner by elimination when p(f ) 0.10 . model 1 included the variables : milk intake in quintiles , lct-13910 c > t polymorphism , sex , birth weight , parents height , tanner stage , bmi , and ses . in a first step the variable bmi was eliminated and in a second step ses ( table 3 ) . milk intake ( g / d ) in quintiles remained significantly different ( =0.46 ; 95% ci : 0.040 , 0.87 and p=0.032 ) in the final model . in addition , the lct c > t-13910 polymorphism ( lp vs. lnp ) , remained significant in the final model ( =2.05 ; 95% ci : 0.18 , 3.92 and p=0.032 ) , showing a positive association of lp with height . main effect by quintiles of milk intake and lct-13910 c > t ( exposure variables ) on body height in swedish preadolescents and adolescents ( n=542 ) ; stepwise backward multiple linear regression models were used included variables in model 1 : birth weight , sex , mother 's and father 's height . bmi , tanner stage , ses excluded variable : bmi ( p[f ] 0.10 ) . excluded variables : ses ( p[f ] 0.10 ) . we found that milk intake and lct c > t-13910 polymorphism , known to modulate milk consumption , were significant contributors to the observed variance of body height in the studied population . we primarily did not expect to find our hypothesis confirmed in a nutritionally replete country with already one of the highest intakes of milk and dairy products per capita in the world . the lct c > t-13910 polymorphism might contribute with a small effect to phenotypic variation in height . as far as we know no genome wide association studies or meta - analysis have been performed previously addressing the question whether lp or lnp affects body height . increasing evidence suggests that cow 's milk consumption exerts a positive effect on longitudinal growth in preadolescents and adolescents , for instance , by affecting the igf - i - axis ( 8 , 11 , 2730 ) . but not all studies show a positive effect of milk on height , especially in nordic countries , where average milk intake per capita traditionally is high ( 31 ) . the lct c > t-13910 polymorphism , known to modulate milk intake ( 32 , 33 ) , remained significant in the final model . by including the lct c > t-13910 polymorphism in our model , we subtract for hidden heritability in an already predominantly lp population ( 18 ) . in a recent prospective study , evidence of an association between increased infant size at birth and cow milk consumption during pregnancy it is known that lp subjects consume on average more milk than lnp subjects . where lp might thus lead to an increased susceptibility towards already prenatally programmed increased body sizes , mediated by the higher capacity of lp mothers to consume milk and dairy products . our study accounts for parental height as regards milk intake and its potential effects on body height and growth in preadolescents and adolescents . despite the circumstance that body height is a highly heritable complex trait ; corrections for parental height continue to be an exception , also shown in recent studies ( 31 ) . even twin growth studies have been performed without consideration of parental height ( 35 ) . it has been demonstrated that at the current stage a simple prediction based on phenotype of relatives obviously outperforms sophisticated genomic predictions as regards body height ( 36 ) . limitations of this study are the sample size , and the limits inherent to cross - sectional studies as regards causal inference . cow 's milk is an evolutionary food constructed to promote growth and development in calves and appears to affect growth in human beings also ( 13 , 14 , 28 ) . milk has become a normative food for preadolescents and adolescents beyond weaning age , even in asian populations and developing countries , which traditionally did not have home fare based on domesticated dairy animals ( 8 , 37 ) . the long - term effects of this changed nutritional normative are yet poorly understood , even in western countries . we conclude that actual milk intake and the genetic lp trait is positively associated with body height in preadolescents and adolescents . nevertheless , higher milk consumption in childhood might exert both negative and positive effects , since greater height has been associated with higher risk of some cancers ( 29 , 3841 ) . this study was supported by grants from rebro lns landstings forskningskommitt and by nyckelfonden , rebro , sweden .

backgroundbody height is a classic polygenic trait . about 80%90% of height is inherited and 10%20% owed to environmental factors , of which the most important ones are nutrition and diseases in preadolescents and adolescents.objectivethe aim of this study was to explore potential relations between the lct ( lactase ) c > t-13910 polymorphism , milk consumption , and body height in a sample of swedish preadolescents and adolescents.designin a cross - sectional study , using a random sample of preadolescents and adolescents ( n = 597 ) , dietary intakes were determined . anthropometric measurements including sexual maturity ( tanner stage ) and birth weight were assessed . parental body height and socio - economic status ( ses ) were obtained by questionnaires . genotyping for the lct c > t-13910 polymorphism that renders individuals lactase persistent ( lp ) or lactase non - persistent ( lnp ) was performed by dna sequencing . stepwise backward multivariate linear regression was used.resultsmilk consumption was significantly and positively associated with body height ( = 0.45 ; 95% ci : 0.040 , 0.87 , p = 0.032 ) . adjustments were performed for sex , parental height , birth weight , body mass index ( bmi ) , ses , and tanner stage . this model explains 90% of the observed variance of body height ( adjusted r2 = 0.89 ) . the presence of the -13910 t allele was positively associated with body height ( = 2.05 ; 95% ci : 0.18 , 3.92 , p = 0.032).conclusionsmilk consumption is positively associated with body height in preadolescents and adolescents . we show for the first time that a nutrigenetic variant might be able to explain in part phenotypic variation of body height in preadolescents and adolescents . due to the small sample size further studies are needed .

primary hyperparathyroidism ( php ) is a hypercalcemic disease stemming from an abnormal increase in parathyroid hormone ( pth ) secretion by one or more parathyroid glands . the hallmark of this condition is the presence of high levels of calcium and high or inappropriate levels of pth . primary hyperparathyroidism is more common in women than in men and increases with aging in both genders . the goal of parathyroidectomy is the excision of the abnormal parathyroid gland(s ) , preserving the normal ones in order to achieve and maintain a postoperative normocalcemic state . success rates for surgical treatment depend on the skill and experience of the surgeon in finding and recognizing the pathologic changes and excising the correct amount of hyperfunctioning parathyroid tissue . the surgical treatment of php has undergone substantive changes since the first successful parathyroidectomy was performed by felix mandl in 1925 . it is now expected that the vast majority of patients will be cured during initial surgical exploration at a low probability of morbidity . the conventional time - honored operation employing general endotracheal anesthesia and bilateral cervical exploration is safe and effective when performed by experienced surgeons . however , recent technical innovations , including improved preoperative localization and availability of rapid intraoperative pth assays ( iopth ) , have yielded focused approaches with excellent outcomes . sporadic primary hyperparathyroidism is caused by a single enlarged parathyroid gland ( parathyroid adenoma ) in approximately 85% of the cases , whereas multigland hyperplasia occurs in 15% and parathyroid carcinoma is found in less than 1% of patients . unlike the previous dogma that required surgical identification of both enlarged and normal parathyroid glands , the current paradigm in many centers is to identify and excise the incident enlarged gland and to confirm operative cure , employing a rapid intraoperative pth assay . due to the relatively short half - life of pth ( 4 - 5 min ) , a dramatic drop in circulating hormone can be detected once the abnormally secreting gland or glands have been removed . a curative drop in pth allows the surgeon to terminate the operation and obviate additional exploration , whereas failure of the pth levels to demonstrate an adequate decrement asks for additional exploration because of the presence of presumed additional hypersecreting gland(s ) . the aim of the present study was to evaluate our 10-year experience in employing a rapid intraoperative pth assay for php . this is a prospective study on a cohort of operated patients treated at a university referral center . this investigation was approved by the unifesp / epm ethics committee . from june 2000 to april 2011 , 96 surgeries for php were performed at hospital so paulo unifesp / epm , so paulo , brazil . these 96 procedures were performed in 91 previous unexplored patients who had at least 6 months of postoperative follow - up with enough reported data to be eligible for the study . the php diagnoses were established in the presence of high levels of calcium and high or inappropriate levels of pth . all patients had their age , gender , symptoms ( bone and kidney ) , preoperative localization tests , serum ionized calcium ( ica ) , serum total calcium and intact pth recorded before parathyroidectomy , as well as iopth dosages . the laboratorial tests were repeated 1 month , 6 months , and in 1-year increments after surgery . a series of 91 consecutive patients with primary hyperparathyroidism underwent parathyroidectomy guided by intraoperative pth at federal university of so paulo , brazil , from june 2000 to april 2011 . focused parathyroidectomy guided by intraoperative pth was the initial procedure when preoperative localization tests were positive and when there were no suspicion of malignant disease . a bilateral cervical exploration guided by intraoperative pth was performed when preoperative localization tests were negative . a baseline peripheral venous blood sample was obtained just after anesthesia induction as well as 10 minutes after the abnormal parathyroid tissue removal . the intraoperative criterion used to predict successful parathyroidectomy was a decrease in the intact pth levels exceeding 50% from the preincision hormone level . if this criterion was met , surgical exploration of the neck would be completed and the incision closed . otherwise , further surgical exploration of the neck would have to be carried on . intraoperative pth was measured using elecsys pth immunoassay ( elecsys 1010 system , roche , mannheim , germany ) . the test is an immunometric assay based on monoclonal antibodies , magnetic particles as solid phase , and ruthenium complex as chemiluminescent label . total time to perform the assay is 9 minutes ; reference values are 1065 pg / ml . to validate the rapid pth assay , 170 samples from the study the iopth accuracy calculation was based on the following definitions : a true - positive ( tp ) result of iopth was defined as the correct prediction of postoperative normal calcium levels for at least 6 months ; true negative ( tn ) was the correct prediction of incomplete excision by either resection of an additional gland(s ) or operative failure ; false positive ( fp ) was the incorrect prediction of normocalcemia with subsequent postoperative persistent hypercalcemia and high pth levels ; false negative ( fn ) was the incorrect prediction of incomplete excision followed by postoperative normocalcemia . persistent disease was considered when serum calcium and pth levels remained above normal range just after surgery . recurrence was defined when , after reaching normocalcemic levels , serum calcium and intact pth measurements start to rise to abnormal values at least 6 months after surgery . serum ionized calcium ( 1.151.32 mmol / l ) , serum total calcium ( 8.510.5 mg / dl ) and intact pth ( 1167 pg / ml ) , were measured using standard automatic assays . the demographic , clinical , and biochemical aspects of 91 patients with php are shown in table 1 . among all cohort of 91 patients , 69 ( 75.8% ) had solitary adenoma , 10 ( 11.0% ) had multiple endocrine neoplasia type 1 ( men1 ) , 6 ( 6.6% ) had double adenomas , 4 ( 4.4% ) had carcinomas , and 2 ( 2.2% ) patients are still waiting for remedial surgery for multiglandular disease ( mgd ) , 1 of whom had recurrence disease after 10 years of follow - up and the other persistent disease after a false positive iopth ( table 2 ) . we had 85 ( 93.4% ) successful parathyroidectomies 6 ( 6.6% ) failed parathyroidectomies in 91 previous unexplored patients , and 5 ( 100% ) successful remedial surgeries . among the 85 successful patients , 69 ( 81.2% ) had solitary adenoma , 3 ( 3.5% ) had carcinoma , 4 ( 4.7% ) had double adenomas , 8 ( 9.4% ) had men1 , and 1 ( 1.2% ) still has mgd ( table 2 ) . the mean decay of iopth in the successful group of patients was 80.5% ( 34.3% to 96.0% ) . in these 85 patients , we had 80 tp results ( 67 adenomas , 3 carcinomas , 8 men1 and 2 recurrences we also had 3 cases of double adenoma in which the iopth resulted in a tn value , requiring additional exploration to prevent persistent disease ( table 2 ) . there were only 2 fn results among the successful group patients ( table 4 ) . the mean decay of iopth in the 80 patients with tp results was 81.7% , with a minimum drop of 55.0% ( 55.0% to 96.0% ) . operative failure of the initial surgery occurred in 6 patients : 2 double adenoma , 2 men1 , 1 carcinoma , and 1 mgd who is still waiting for remedial surgery ( table 5 ) . both cases of double adenoma had a tn decay of iopth , but the second adenoma wasnot found during the initial operation . with the aid of new localization exams , successful remedial operations were performed with an iopth tp result . the patient with parathyroid carcinoma developed hypercalcemia and high levels of pth two months after surgery . in search of distant metastasis , this patient underwent a pulmonary computerized tomography and multiple pulmonary nodules were found . for this reason , she was classified as iopth false - positive . in both men1 patients , one patient already had men1 diagnosis at the time of initial surgery and we failed to find the fourth gland . the other patient was first operated as a solitary adenoma and men1 diagnosis surfaced just in the follow - up . there was only one case in the operative failure patients ; the iopth had an fp result ( table 4 ) . the surgical treatment for primary php has undergone some changes in recent years , evolving from the standard bilateral neck exploration technique to a less time - consuming procedure of unilateral neck exploration . taking into account the increasing number of asymptomatic and/or oligosymptomatic primary hyperparathyroidism diagnosis in the recent years , the need for a safe and less time - consuming procedure with low perioperative morbidity is clear . preoperative parathyroid localization imaging study and iopth are essential components of the focused parathyroidectomy that allow the excision of all abnormal parathyroid glands without the examination of the normally secreting glands . the clinical utility of rapid iopth measurements in parathyroidectomy was first reported in 1988 using a modified intact pth irma assay . since then , rapid assays have been developed by means of radioactive [ 4 , 11 , 12 ] as well as nonradioactive formats [ 12 , 13 ] . the predominance of a solitary adenoma disease in 85% to 96% of cases of php and the short half - life of intact pth ( 184 ) of only 1.4 to 4 minutes [ 1420 ] combined with the remaining suppressed normal parathyroid glands after removal of all hyperfunctioning tissue allows the measurement of iopth to evaluate its decline rates . several studies have demonstrated the utility of iopth monitoring in the treatment of single - gland primary hyperparathyroidism [ 3 , 8 , 2127 ] . most experts agree that iopth assay is the most useful intraoperative adjunct to assist the surgeon in php surgical treatment [ 28 , 29 ] . it is worth highlighting the useful employment of preoperative localization imaging study in conjunction with iopth : the former points out where the surgeon should start exploration from , and the latter assures that hypersecretory parathyroid tissue removal was accomplished . the current usual criteria for iopth measurement describe a decrease of 50% or over from either the baseline ( preincision ) or the highest preincision or preexcision value within 10 minutes following hyperfunctioning parathyroid resection , pointing out surgical cure and predicting normocalcemia . by resorting to those criteria , high accuracy in intraoperative prediction of cure is achieved . mostly for practice and cost reasons , we have used just two samples as our criteria since we started making use of the iopth : the preincision and 10 minutes postexcision of hyperfunctioning parathyroid . our series of 96 php consecutive surgeries over 10 years reflects a complex tertiary referral center and has its limitations . most of these patients were referred specifically due to their severe signs / symptoms and comorbid medical conditions , which are related to their high bone and kidney disease and the 4.4% incidence of carcinoma . furthermore , all patients were first evaluated by an endocrinologist group specialized in osteometabolic disease who elected the patients for surgical treatment , reflecting the 11% incidence of men1 and the absence of familial hypocalciuric hypercalcemia case in our surgical series . the php diagnose has not been made in a routine practice in most brazilian centers , and many patients have their diagnoses made just after severe signs / symptoms . we have noticed an increase in the percentage of asymptomatic patients over the years in the number of php cases , but probably such increase has not been enough to raise the number of our small solitary adenomas ( 78.3% ) . we get an inferior number of successful parathyroidectomy ( 93.4% ) in previous unexplored patients compared to some large series [ 9 , 32 ] , but it can be considered a satisfactory result , taking into account the high number of patients with carcinoma and men1 in our series . on the other hand , we have obtained good results ( 100% ) in 5 successful second surgical explorations so far : first , we removed a second adenoma in three patients ( two persistent and one recurrent disease ) ; second , we were able to remove a not found fourth gland in a men1 patient , and finally , we removed three glands in a men1 patient previous operated as an sporadic php . in our series of 91 patients , iopth had true results in 87 patients ( 95.6% ) . however , considering our 96 surgical procedures ( 5 remedial surgeries ) , iopth was able to obviate or to ask for additional exploration because of the presence of presumed additional hypersecreting gland(s ) in 92 ( 95.8% ) procedures . the iopth had a mean decay of 81.7% in the tp patients , and the minimum drop in our series that results in patient cure ( operative success ) was 55.0% . one of these cases occurred in a ruptured parathyroid cystic adenoma that may have resulted in a substantial elevation of the hormone levels after the preincision sample , and it should be related to an inadequate iopth decay . the second patient had the others three parathyroid glands identified in normal conditions . based on such scenario , the iopth decay was 50.9% , dropping from 216 pg / dl to 106 pg / dl . as observed by other authors , the fp cases are usually represented by a marginal pth level decrease with a final pth level above the normal range . for these reasons , stricter criteria for the iopth dynamics have been suggested , such as the return of the 10-minute pth to within normal range . however , these stricter criteria were estimated to increase the operative success only by 0.3% , with significant increase in the false negative results , bringing on more unnecessary bilateral neck dissection [ 9 , 33 ] . using these stricter criteria in our patients would have brought about 21 ( 24.7% ) unnecessary bilateral dissections , and just one additional diagnose . we classified a patient with parathyroid carcinoma that revealed metastatic pulmonary disease two months after surgery as an fp result because she certainly had metastatic disease at the surgery and iopth ( 84.1% decay ) failed to predict the presence of presumed additional hypersecreting tissue . however , it can be controversial to suppose that iopth has failed in this patient , once iopth was not defined to predict distant disease . all 4 patients with parathyroid carcinoma had very suggestive signals of malignant disease at presentation ( very high pth levels , severe hypercalcemia and palpable neck mass ) and were not elected to the focused approach . they were submitted to an en bloc tumor resection , removing the parathyroid tumor , the ipsilateral thyroid lobe , and the lymph nodes related to a central neck dissection . during the follow - up period , 2 ( 2.1% ) one patient removed a second adenoma and had a successful remedial surgery with iopth decay of 78.4% . the second patient recurrence occurred almost 10 years after initial surgery and is still waiting for remedial surgery . published a large series of 1,650 php patients and found 5 ( 0.3% ) recurrent cases . one of our series limitations is the absence of vitamin d dosages , one important factor that should be related to the genesis of php recurrence . the iopth revealed to be an important technological adjunct in the current parathyroid surgery for php .

introduction . primary hyperparathyroidism ( php ) is characteristically determined by high levels of calcium and high or inappropriate levels of parathyroid hormone ( pth ) . technological advances have dramatically changed the surgical technique over the years once intraoperative parathyroid hormone ( iopth ) assay had allowed for focused approaches . objective . to evaluate our 10-year experience in employing a rapid intraoperative pth assay for php . methods . a prospective cohort of 91 php - operated patients in a tertiary institution in so paulo , brazil , from june 2000 to april 2011 . results . we had 85 ( 93.4% ) successful parathyroidectomies , 6 ( 6.6% ) failed parathyroidectomies in 91 previous unexplored patients , and 5 ( 100% ) successful remedial surgeries . the iopth was true - positive in 88.5% , true - negative in 7.3% , false - positive in 2.1% , and false - negative in 2.1% of the procedures . iopth was able to obviate additional exploration or to ask for additional exploration in 92 ( 95.8% ) procedures . conclusion . the iopth revealed to be an important technological adjunct in the current parathyroid surgery for php .

the prevalence of diabetes in the u.s . has increased ( 1 ) , and cardiovascular ( cv ) complications remain the major cause of morbidity and mortality in persons with type 2 diabetes , contributing substantially to increased health care costs ( 2 ) . as individuals attempt to manage their diabetes , there is increasing recognition that comorbid behavioral problems , including both depressive symptoms and stress , are associated with poor glycemic control , poor lifestyle behaviors , and increased health services utilization ( 310 ) . we ( 11 ) and others ( 3,4 ) have shown that these behavioral challenges are associated with inadequate medication adherence , which may be associated with adverse outcomes . similarly , poor meta - bolic control may worsen depressive symptoms ( 5 ) , and the relationship appears to be bidirectional ( 8) . the prevalence of these comorbidities and their potential to worsen disease management have led some to develop interventions designed to address comorbid depressive symptoms or stress in persons with diabetes ( 1214 ) . while many studies have documented the cross - sectional presence of these comorbidities and the effect on glycemic control in subjects with diabetes , only a limited number of studies have examined the potential impact on cv outcomes . most of these studies have been limited to patients with depression recruited from established health care settings , and the true population - level impact is unknown . likewise , there has not been adequate comparison of individuals with and without diabetes who have concurrent depressive symptoms and/or elevated levels of perceived stress to examine the unique consequences of behavioral and medical comorbidity . the objective of the present report was to examine the association between the presence of depressive symptoms and/or increased levels of perceived stress , determined at baseline , and risk of incident cv events over 5 years of follow - up in a major national cohort study that used population - based sampling methods , collecting data in the home both in subjects with and without diabetes . the description of the population for this analysis has previously been reported ( 15 ) . in short , the reasons for geographic and racial differences in stroke ( regards ) study is a population - based , prospective , longitudinal cohort study of 30,239 subjects aged > 45 years , 45% of whom are male , 55% female , 41% black , 59% white , 55% from the stroke belt it was designed to examine factors associated with the geographic and racial differences in stroke incidence as well as excess stroke mortality in the southeastern u.s . ( i.e. , stroke belt ) relative to the rest of the nation and among blacks relative to whites . the regards study included relevant measures for perceived stress and depressive symptoms and provides a unique opportunity to examine the impact of these behavioral comorbidities in individuals with and without diabetes and the incidence of adverse cv outcomes in a national sample of black and white adults . the regards cohort was recruited between january 2003 and october 2007 and is evaluated by computer - assisted telephone interview every 6 months . the initial recruitment call was used to obtain verbal consent and collect cv history , risk factors , and demographic characteristics , including age , sex , and race ( each subject self - reported race , and by design , the study compared only non - hispanic black and white subjects , excluding other racial / ethnic groups ) . the study also included an in - home visit by a trained health professional for data collection . participants were asked to provide all prescription and nonprescription medications they had taken in the past 2 weeks , and medication names were recorded during the in - home visit and subsequently coded into drug classes . written informed consent was obtained during the in - person evaluation . the present analysis included all members of the cohort who had complete follow - up data . a brief physical exam including blood pressure ( assessed as the average of two measurements obtained with an aneroid sphygmomanometer after the subject was in the seated position for at least 3 min with both feet on the floor ) and blood and urine samples , and an electrocardiogram ( ecg ) was conducted during an in - home visit by a trained health professional 34 weeks after the telephone interview . height and weight was measured via standard procedures , and bmi was computed as the body weight in kilograms divided by the square of height in meters ( 2 ) . all ecgs were read by a trained cardiologist in a centralized ecg reading laboratory at the wake forest university / baptist medical center in winston - salem , nc , using a predefined interpretation protocol ( 16 ) . atrial fibrillation was determined to be present if there was a self - reported history ( asked as has a doctor or other health professional ever told you that you had atrial fibrillation ? ) or if characteristic findings were present on the ecg as defined in the protocol . left ventricular hypertrophy was defined to be present or absent based on ecg findings using the sokolow - lyon limb lead criteria ( 17 ) as specified in the protocol . hs - crp was determined by particle - enhanced immunonephelometry using the bnii nephelometer ( n high sensitivity crp ; dade behring , deerfield , il ) , while total and hdl cholesterol and glucose were measured by colorimetric reflectance spectrophotometry using the ortho clinical vitros 950/irc chemistry system ( johnson & johnson clinical diagnostics , new brunswick , nj ) in accordance with the national cholesterol education program guidelines ( 18 ) . diabetes was defined as self - reported diabetes obtained during the telephone interview or a fasting glucose 126 mg / dl , a nonfasting glucose 200 mg / dl , or the presence of oral hypoglycemic medication or insulin each obtained during the home visit . the primary exposure of interest was the presence , at baseline , of depressive symptoms and/or elevated levels of perceived stress among individuals with diabetes at baseline compared with those without diabetes . the presence of depressive symptoms was assessed using the previously validated four - item center for epidemiologic studies depression ( cesd ) questionnaire ( 19 ) . the cesd-4 is derived from the original 20-item cesd ( 20 ) and has been found to be highly correlated at 0.87 ( 19 ) . these questions included responses that ranged from 0 to 12 based on the number of days the subject reported experiencing those feelings in the prior week , with higher scores indicating more depressive symptoms . subjects with a cesd score 4 were determined to have an elevated level of depressive symptoms . perceived level of stress was measured using a four - item version of the cohen perceived stress scale ( 21 ) , a validated and previously used ( 22 ) instrument for measuring the perception of personal stress . it measures the extent to which respondents perceive their lives as unpredictable , uncontrollable , and/or overloaded . subjects with cohen perceived stress scale scores > 4 were determined to have high levels of psychological stress , corresponding to the highest quartile of scores . the primary outcomes of interest were physician - adjudicated cv events including stroke , myocardial infarction ( mi)/acute coronary heart disease ( chd ) , and cv death . living participants or their proxies were contacted every 6 months via telephone to assess new - onset stroke , chd events , and cv mortality . a trained interviewer administered a standardized questionnaire that specifically asks whether , since the last follow - up , they had been hospitalized for stroke or heart disease . medical records were retrieved for all potential stroke- and chd - related hospitalizations and deaths . for suspected stroke , each event was adjudicated via medical record review by a neurologist - led medical review team . stroke events were defined following the world health organization definition ( 23 ) but also included events with symptoms lasting < 24 h with neuroimaging consistent with acute ischemia or hemorrhage and cases where adjudicators agreed that the event was likely a stroke or death related to stroke but information was incomplete for world health organization or clinical classification . after a report of a hospitalization or death that potentially could be related to cvd , medical records were retrieved , and the event was adjudicated by a physician - led medical review team , following published guidelines ( 24,25 ) . specifically , medical records were examined for the presence of signs or symptoms suggestive of ischemia ; a rising and/or falling pattern in cardiac troponin or creatine phosphokinase - mb over 6 or more hours with a peak value greater than or equal to twice the upper limit of normal ( diagnostic cardiac enzymes ) ; and ecg changes consistent with ischemia or mi , guided by the minnesota code and classified as evolving diagnostic , positive , nonspecific , or not consistent with ischemia ( 26,27 ) . participant deaths were detected by report of next of kin , online sources ( e.g. , the social security death index ) , and the national death index . for information surrounding the circumstances of participant death , proxies or next of kin were interviewed . additionally , following published guidelines , medical records in the last year of life , death certificates , and autopsy reports were collected and reviewed by the physician - led adjudicators to determine whether the death was a cvd death ( 24,25 ) . data on traditional cv risk factors and socioeconomic factors were also collected in order to adjust for potential confounding in our analyses of the relationship between the presence of comorbid behavioral problems , diabetes , and adverse cv outcomes . additional data collected included the following : annual household income ( < $ 20,000/year , $ 20,000$35,000/year , $ 35,000$75,000/year , and > $ 75,000/year ) , education level ( less than high school education , high school graduate , some college , and college graduate or higher ) , and health insurance ( yes or no ) . also collected was a health history including a history of stroke or heart disease ( self - reported mi , coronary artery bypass grafting , bypass , angioplasty , stenting , or evidence of myocardial infarction on the study ecg ) . smoking behavior was collected and categorized as nonsmoker , past smoker , or current smoker . the current study examined the relationship between comorbid depressive symptoms and/or elevated levels of perceived stress , both measured at baseline , in subjects with and without diabetes and the adjudicated incidence of stroke , acute chd , and cvd death for events occurring through 30 december 2010 . follow - up time for each participant was calculated from the date of the in - home visit to date of first stroke , acute chd , death , or last telephone follow - up . the analysis cohort for this report was 22,003 after exclusion of participants with a history of stroke or heart disease at baseline ( n = 7,164 ) , missing diabetes status at baseline ( n = 845 ) , or missing follow - up data ( n = 171 ) . the initial analysis compared the demographic , socioeconomic , and cv risk characteristics of subjects with and without diabetes in three groups : 1 ) those reporting no comorbid depressive symptoms and without elevated levels of perceived stress , 2 ) those reporting either depressive symptoms or elevated levels of perceived stress , and 3 ) those reporting both depressive symptoms and elevated levels of perceived stress . age - adjusted incidence rates were then compared for each type of cv event in individuals with and without diabetes in the same three comparison groups . the longitudinal relationship between baseline behavioral morbidity ( i.e. , the three comparison groups ) , in both those with and without diabetes , and subsequent adjudicated cv events during follow - up was then examined in a series of cox proportional hazards models . these models examined the relationships first in an unadjusted ( crude ) model and then in additional models that incrementally added the following groups of variables : 1 ) demographic characteristics ( race , age , sex , and region [ stroke belt vs. non stroke belt ] ) , 2 ) socioeconomic factors ( income , education , and health insurance ) , and , finally , 3 ) cv risk factors ( current smoking , history of heart disease , left ventricular hypertrophy , atrial fibrillation , bmi , systolic blood pressure , total cholesterol , hs - crp , and statin use ) . the description of the population for this analysis has previously been reported ( 15 ) . in short , the reasons for geographic and racial differences in stroke ( regards ) study is a population - based , prospective , longitudinal cohort study of 30,239 subjects aged > 45 years , 45% of whom are male , 55% female , 41% black , 59% white , 55% from the stroke belt it was designed to examine factors associated with the geographic and racial differences in stroke incidence as well as excess stroke mortality in the southeastern u.s . ( i.e. , stroke belt ) relative to the rest of the nation and among blacks relative to whites . the regards study included relevant measures for perceived stress and depressive symptoms and provides a unique opportunity to examine the impact of these behavioral comorbidities in individuals with and without diabetes and the incidence of adverse cv outcomes in a national sample of black and white adults . the regards cohort was recruited between january 2003 and october 2007 and is evaluated by computer - assisted telephone interview every 6 months . the initial recruitment call was used to obtain verbal consent and collect cv history , risk factors , and demographic characteristics , including age , sex , and race ( each subject self - reported race , and by design , the study compared only non - hispanic black and white subjects , excluding other racial / ethnic groups ) . the study also included an in - home visit by a trained health professional for data collection . participants were asked to provide all prescription and nonprescription medications they had taken in the past 2 weeks , and medication names were recorded during the in - home visit and subsequently coded into drug classes . written informed consent was obtained during the in - person evaluation . the present analysis included all members of the cohort who had complete follow - up data . a brief physical exam including blood pressure ( assessed as the average of two measurements obtained with an aneroid sphygmomanometer after the subject was in the seated position for at least 3 min with both feet on the floor ) and blood and urine samples , and an electrocardiogram ( ecg ) was conducted during an in - home visit by a trained health professional 34 weeks after the telephone interview . height and weight was measured via standard procedures , and bmi was computed as the body weight in kilograms divided by the square of height in meters ( 2 ) . all ecgs were read by a trained cardiologist in a centralized ecg reading laboratory at the wake forest university / baptist medical center in winston - salem , nc , using a predefined interpretation protocol ( 16 ) . atrial fibrillation was determined to be present if there was a self - reported history ( asked as has a doctor or other health professional ever told you that you had atrial fibrillation ? ) or if characteristic findings were present on the ecg as defined in the protocol . left ventricular hypertrophy was defined to be present or absent based on ecg findings using the sokolow - lyon limb lead criteria ( 17 ) as specified in the protocol . hs - crp was determined by particle - enhanced immunonephelometry using the bnii nephelometer ( n high sensitivity crp ; dade behring , deerfield , il ) , while total and hdl cholesterol and glucose were measured by colorimetric reflectance spectrophotometry using the ortho clinical vitros 950/irc chemistry system ( johnson & johnson clinical diagnostics , new brunswick , nj ) in accordance with the national cholesterol education program guidelines ( 18 ) . diabetes was defined as self - reported diabetes obtained during the telephone interview or a fasting glucose 126 mg / dl , a nonfasting glucose 200 mg / dl , or the presence of oral hypoglycemic medication or insulin each obtained during the home visit . the primary exposure of interest was the presence , at baseline , of depressive symptoms and/or elevated levels of perceived stress among individuals with diabetes at baseline compared with those without diabetes . the presence of depressive symptoms was assessed using the previously validated four - item center for epidemiologic studies depression ( cesd ) questionnaire ( 19 ) . the cesd-4 is derived from the original 20-item cesd ( 20 ) and has been found to be highly correlated at 0.87 ( 19 ) . these questions included responses that ranged from 0 to 12 based on the number of days the subject reported experiencing those feelings in the prior week , with higher scores indicating more depressive symptoms . subjects with a cesd score 4 were determined to have an elevated level of depressive symptoms . perceived level of stress was measured using a four - item version of the cohen perceived stress scale ( 21 ) , a validated and previously used ( 22 ) instrument for measuring the perception of personal stress . it measures the extent to which respondents perceive their lives as unpredictable , uncontrollable , and/or overloaded . subjects with cohen perceived stress scale scores > 4 were determined to have high levels of psychological stress , corresponding to the highest quartile of scores . the primary outcomes of interest were physician - adjudicated cv events including stroke , myocardial infarction ( mi)/acute coronary heart disease ( chd ) , and cv death . living participants or their proxies were contacted every 6 months via telephone to assess new - onset stroke , chd events , and cv mortality . a trained interviewer administered a standardized questionnaire that specifically asks whether , since the last follow - up , they had been hospitalized for stroke or heart disease . for each positive response , the date and time of each event were recorded . medical records were retrieved for all potential stroke- and chd - related hospitalizations and deaths . for suspected stroke , each event was adjudicated via medical record review by a neurologist - led medical review team . stroke events were defined following the world health organization definition ( 23 ) but also included events with symptoms lasting < 24 h with neuroimaging consistent with acute ischemia or hemorrhage and cases where adjudicators agreed that the event was likely a stroke or death related to stroke but information was incomplete for world health organization or clinical classification . after a report of a hospitalization or death that potentially could be related to cvd , medical records were retrieved , and the event was adjudicated by a physician - led medical review team , following published guidelines ( 24,25 ) . specifically , medical records were examined for the presence of signs or symptoms suggestive of ischemia ; a rising and/or falling pattern in cardiac troponin or creatine phosphokinase - mb over 6 or more hours with a peak value greater than or equal to twice the upper limit of normal ( diagnostic cardiac enzymes ) ; and ecg changes consistent with ischemia or mi , guided by the minnesota code and classified as evolving diagnostic , positive , nonspecific , or not consistent with ischemia ( 26,27 ) . participant deaths were detected by report of next of kin , online sources ( e.g. , the social security death index ) , and the national death index . for information surrounding the circumstances of participant death , proxies or next of kin additionally , following published guidelines , medical records in the last year of life , death certificates , and autopsy reports were collected and reviewed by the physician - led adjudicators to determine whether the death was a cvd death ( 24,25 ) . data on traditional cv risk factors and socioeconomic factors were also collected in order to adjust for potential confounding in our analyses of the relationship between the presence of comorbid behavioral problems , diabetes , and adverse cv outcomes . additional data collected included the following : annual household income ( < $ 20,000/year , $ 20,000$35,000/year , $ 35,000$75,000/year , and > $ 75,000/year ) , education level ( less than high school education , high school graduate , some college , and college graduate or higher ) , and health insurance ( yes or no ) . also collected was a health history including a history of stroke or heart disease ( self - reported mi , coronary artery bypass grafting , bypass , angioplasty , stenting , or evidence of myocardial infarction on the study ecg ) . smoking behavior was collected and categorized as nonsmoker , past smoker , or current smoker . the current study examined the relationship between comorbid depressive symptoms and/or elevated levels of perceived stress , both measured at baseline , in subjects with and without diabetes and the adjudicated incidence of stroke , acute chd , and cvd death for events occurring through 30 december 2010 . follow - up time for each participant was calculated from the date of the in - home visit to date of first stroke , acute chd , death , or last telephone follow - up . the analysis cohort for this report was 22,003 after exclusion of participants with a history of stroke or heart disease at baseline ( n = 7,164 ) , missing diabetes status at baseline ( n = 845 ) , or missing follow - up data ( n = 171 ) . the initial analysis compared the demographic , socioeconomic , and cv risk characteristics of subjects with and without diabetes in three groups : 1 ) those reporting no comorbid depressive symptoms and without elevated levels of perceived stress , 2 ) those reporting either depressive symptoms or elevated levels of perceived stress , and 3 ) those reporting both depressive symptoms and elevated levels of perceived stress . age - adjusted incidence rates were then compared for each type of cv event in individuals with and without diabetes in the same three comparison groups . the longitudinal relationship between baseline behavioral morbidity ( i.e. , the three comparison groups ) , in both those with and without diabetes , and subsequent adjudicated cv events during follow - up was then examined in a series of cox proportional hazards models . these models examined the relationships first in an unadjusted ( crude ) model and then in additional models that incrementally added the following groups of variables : 1 ) demographic characteristics ( race , age , sex , and region [ stroke belt vs. non stroke belt ] ) , 2 ) socioeconomic factors ( income , education , and health insurance ) , and , finally , 3 ) cv risk factors ( current smoking , history of heart disease , left ventricular hypertrophy , atrial fibrillation , bmi , systolic blood pressure , total cholesterol , hs - crp , and statin use ) . the current study included a total of 22,003 subjects , of whom 42% were black , 58% female , and 56% living in the southeastern u.s . stroke belt . among all subjects at baseline , 18.6% subjects ( n = 4,090 ) had diabetes , 10% ( n = 2,202 ) reported increased depressive symptoms , and 28% ( n = 6,132 ) reported elevated levels of stress . subjects with diabetes were more likely to report either elevated depressive symptoms or stress or both than were subjects without diabetes ( 36.8% vs. 29.5% ; p < 0.001 ) . detailed demographic characteristics for the study group are given in table 1 , separated into those with and without diabetes and stratified by the presence of behavioral comorbidities . among subjects with diabetes , those reporting either increased depressive symptoms or stress or the combination were more likely to be women , black , live in the stroke belt , and have limited income . subjects reporting either elevated depressive symptoms or stress or both had a higher prevalence of elevated baseline hs - crp values . across each category of behavioral comorbidity ( i.e. , none , either elevated depressive symptoms or stress , both elevated ) , subjects with diabetes had a higher prevalence of elevated baseline hs - crp values , higher mean systolic bp , and greater prevalence of statin use than those without diabetes . baseline characteristics of regards participants with or without diabetes , by depressive symptom / stress category * p value for comparison of subjects with diabetes vs. subjects with no diabetes . table 2 provides age - adjusted incidence rates per 1,000 person - years of follow - up for each of the three independent cv outcomes , broken out by the presence of behavioral comorbidities in those with and without diabetes . with respect to individual cv outcomes , the pattern of age - adjusted incidence rates was approximately twofold higher in those with diabetes than in those without diabetes across the range of comorbid behavioral illness . among individuals with diabetes and one or both behavioral comorbidities , age - adjusted incidence rates were highest for acute chd , followed by cv death , and stroke . among those without diabetes but with one or more behavioral comorbidities , incidence rates were again highest for acute chd but were followed by stroke and cv death . event numbers , age - adjusted incidence rates per 1,000 person - years , and crude and adjusted hrs for stroke , acute chd , and cv disease death for individuals with stress or depressive symptoms and with stress and depressive symptoms compared with those with neither , for individuals with and without diabetes crude model adjusted for depressive symptom and stress category . model 1 adjusted for crude model plus demographic factors ( race , sex , age , region ) . model 2 adjusted for crude model plus demographic factors plus social and economic factors ( income , education , health insurance ) . full model adjusted for crude model plus demographic factors , social and economic factors , and risk factors ( bmi , total cholesterol , hs - crp , atrial fibrillation , left ventricular hypertrophy , systolic blood pressure , statin use , current cigarette smoking ) . table 2 also provides the results of a series of crude and progressively adjusted hazard ratio ( hr ) models for each of the three independent cv outcomes , demonstrating the progressive impact of comorbidity on acute chd and cv death among subjects with diabetes , relative to those without diabetes , even in fully adjusted models that accounted for demographic , socioeconomic , and cv risk factors . unlike the pattern for acute chd and cv death , the magnitude of the hrs for stroke was very similar in subjects with one versus both behavioral comorbidities and diabetes . figure 1 illustrates this significant and progressive pattern of worsening fully adjusted hrs for cv death among those with behavioral comorbidities , relative to those with no behavioral comorbidities , in subjects with diabetes and in comparison with those who do not have diabetes . among individuals with diabetes , the presence of a single behavioral comorbidity either increased depressive symptoms or stress increased the risk of cv death by 53% relative to individuals with diabetes but without either behavioral comorbidity . among individuals with diabetes , the presence of both behavioral comorbidities increased depressive symptoms and stress increased the risk of cv death by 115% relative to individuals with neither behavioral comorbidity , even after adjustment for a wide range of demographic and cv risk factors . while there was a pattern of increasing risk of cv death among those with one or both behavioral comorbidities ( vs. neither ) in subjects without diabetes , the differences were substantially smaller ( 12% and 27% increases , respectively ) and were not statistically significant . consequences of comorbid diabetes and elevated depressive symptoms and/or stress on cv death during follow - up among participants in the regards study . this article joins a growing body of literature that demonstrates a compelling pattern of augmented risk for cv events or cv death associated with comorbid behavioral illness among individuals with diabetes that far exceeds that observed in individuals with these same illnesses but without diabetes . while several studies have shown that formally diagnosed depression and/or depressive symptoms in subjects who are patients in an existing health care system are associated with incident chd , this is among the first studies to compare the impact of these behavioral comorbidities on cv outcomes and mortality in a population - based sample of subjects with versus without diabetes in the same study . it is the first study to compare the effects of both depressive symptoms and stress together and separately in subjects with versus without diabetes , obtained by population - based sampling rather than recruitment in established health care settings . further , the regards study has a large sample size ; includes oversampling of african americans in the stroke belt region , allowing important racial comparisons ; and also includes rich data obtained in the subject s home environment . the study is novel in that , as a community - sampled study , it provides more precise estimates of the population burden and racial disparities associated with these comorbidities and their cv consequences than other studies in which patients were recruited in health care settings . the study provides a much larger sample of african americans and illustrates the greater prevalence of comorbidities among african american women in particular , suggesting the need for more careful screening . further , cv outcomes and cv death were carefully adjudicated events in the current study ; by contrast , no adjudication process occurred in many other studies . our findings suggest that the adverse consequences observed in individuals with behavioral comorbidities and diabetes are best understood in a larger context or spectrum of linkages between physical and behavioral illness and subsequent outcomes . elevated levels of stress and depressive symptoms , alone and together , were associated with a pattern of increased hrs for acute coronary disease and cv death in adjusted models both in subjects with and in subjects without diabetes , achieving statistical significance in those with diabetes . together this pattern suggests the potential for progressive impact of single and multiple behavioral comorbidities on adverse cv outcomes and mortality that is of greatest concern in subjects with comorbid diabetes . there was a significant pattern of increasing hrs for acute chd and cv death in those with diabetes and with one or both behavioral comorbidities ( none , stress or depressive symptoms alone , or both together ) . while diabetes has long been known to increase cv outcomes , our findings suggest that , relative to those individuals with diabetes who have no behavioral comorbidity , those with either depressive symptoms or elevated levels of stress as well as those with both together have progressively increased risks for acute chd events and cv mortality during 5 years of follow - up , even when other risk factors are controlled for . in particular , behavioral comorbidities such as stress or depressive symptoms are not usually screened for in busy primary care practices , and many patients are reluctant to share these symptoms with busy primary care providers . further , many individuals are reluctant to seek care from mental health providers . among the demographic group in this study shown to have the highest prevalence of comorbid behavioral disease and diabetes ( i.e. , black females with limited income in the southeastern u.s . ) , there may also be distrust of the traditional health care delivery system . as a result , these comorbidities may go unrecognized and unmanaged . this study therefore provides strong evidence for adverse consequences associated with these comorbidities and suggests the need for more careful research to identify optimal screening and treatment strategies that work in busy primary care practices or other community settings . interestingly , while the presence of any behavioral comorbidity resulted in important increases in the hr for stroke , the increased hrs for one versus both behavioral comorbidities in subjects with diabetes were the same in fully adjusted models . this may suggest that a different mechanism is operative in the increased relationship of behavioral comorbidities and stroke . these findings build on and expand an established literature that links the cross - sectional and longitudinal presence of stress , depressive symptoms , and/or established depression with cv outcomes and/or mortality including in individuals with comorbid diabetes ( 10,2832 ) . because our study measured these symptoms at baseline and then followed patients longitudinally , it suggests the potential for the chronic influence of these behavioral comorbidities in subjects with diabetes to across time lead to adverse cv outcomes . indeed , the chronic nature of depressive symptoms and stress in individuals with type 2 diabetes has been described by fisher et al . ( 10,33 ) , who called for a more careful understanding of the conjoint physical and emotional burden in these patients . further , these authors join our findings in calling for integrating the screening and management of the emotional side of diabetes into regular diabetes care . in addition , hamer et al . ( 34 ) demonstrated an association between stress and cv events with hr of 1.5 and with the suggestion that behavioral processes explained the largest proportion of the variance in the hazards model . this raises an important question regarding the mechanism(s ) through which behavioral comorbidities result in increased cv events and/or cv death . an early review by plante ( 35 ) suggested that stress may lead to depressive symptoms that are subsequently associated with accelerated atherosclerosis , endothelial dysfunction , inflammatory reactions , and interstitial disturbances that may predispose the subject to premature cv disease or death . in the current study , hs - crp levels were elevated in subjects with diabetes relative to those without diabetes , but among subjects with diabetes , there was a clearly progressive pattern of higher hs - crp levels among those with either versus both behavioral comorbidities . these elevated hs - crp levels may suggest a pattern of worsening atherosclerotic disease in subjects with diabetes with one or both behavioral comorbidities relative to those with no comorbidity . a recent article by pizzi et al . ( 36 ) supports this and shows that depressive symptoms are associated with progressive longitudinal increases in carotid intima - media thickness relative to those who did not report these symptoms . this progressive pattern of atherosclerosis associated with these symptoms or potentially other behavioral problems might be expected to be more common in subjects with diabetes and therefore contribute to adverse cv outcomes . behavioral comorbidities may also interfere with self - care behaviors . among individuals with diabetes , a study by bonnet et al . ( 37 ) showed that those with anxiety or depressive symptoms were less likely to engage in healthy behaviors including physical activity , proper eating behaviors , and avoidance of smoking than were those without anxiety or depressive symptoms . similarly , a meta - analysis by gonzalez et al . ( 3 ) that included studies of individuals with depressive symptoms showed that those with depressive symptoms or depression were more likely to be nonadherent with medications as well as with the larger treatment regimen . ( 38 ) suggest a stronger relationship between diabetes - related distress and glycemic control than between depressive symptoms and glycemic control , and recent data by this group ( 39 ) show that specific reductions in regimen - related distress can result in improvements in medication adherence , physical activity , and glycemic control , all of which may influence cv risk . the scope of the implications from these important associations with cv outcomes should be considered in the context of the recent second diabetes attitudes , wishes and needs ( dawn2 ) study , which shows that these behavioral comorbidities know no geographic or ethnic boundaries and are highly prevalent in subjects with diabetes in multiple countries around the world ( 6 ) . this suggests the need for additional research to understand the mechanisms by which these behavioral comorbidities are associated with cv outcomes in subjects with diabetes as well as to understand innovative intervention strategies that will not only improve the behavioral comorbidities but will also decrease the risk for cv outcomes . clearly , more research is needed to understand whether the effective treatment of depressive symptoms and stress can lower cvd events in individuals with diabetes . and , as noted above , more work needs to be done to establish appropriate screening strategies for behavioral comorbidities that can be effectively disseminated in busy primary care settings where most individuals with diabetes are managed . this study is a prospective cohort study and not a randomized clinical trial , and the incident outcomes should be understood as associations with baseline characteristics without clear evidence of causality . by design , included only non - hispanic white and black subjects ; extrapolation to other ethnic groups or to other nations should not be undertaken . the current study did not include a measure of diabetes duration , which may have influenced the risk of cv events . the study included data on a large number of cv risk factors that were used to help minimize the influence of confounders ; however , there may have been other unmeasured risk factors that were associated with adverse cv outcomes . the presence of elevated stress or depressive symptoms in community - dwelling subjects with diabetes was associated with an increased risk for acute chd or cv death . moreover , subjects with diabetes who reported both stress and depressive symptoms together had the greatest risk for acute chd and cv death . the presence of either or both behavioral comorbidities in subjects with diabetes was also associated with increased hrs for stroke . elevated stress and/or depressive symptoms in subjects without diabetes resulted in increased hrs in fully adjusted models that were much smaller in magnitude and not statistically significant . these findings demonstrate the persistent disparities and negative cv impact of these comorbidities at the population level and suggest the need for more careful integration of behavioral screening and management in primary care settings , where most patients with type 2 diabetes are managed .

objectiveto evaluate the impact of comorbid depressive symptoms and/or stress on adverse cardiovascular ( cv ) outcomes in individuals with diabetes compared with those without diabetes.research design and methodsinvestigators examined the relationship between baseline depressive symptoms and/or stress in adults with and without diabetes and physician - adjudicated incident cv outcomes including stroke , myocardial infarction / acute coronary heart disease , and cv death over a median follow - up of 5.95 years in the national regards cohort study.resultssubjects included 22,003 adults ( 4,090 with diabetes ) ( mean age 64 years , 58% female , 42% black , and 56% living in the southeastern stroke belt ) . elevated stress and/or depressive symptoms were more common in subjects with diabetes ( 36.8% vs. 29.5% ; p < 0.001 ) . in fully adjusted models , reporting either elevated stress or depressive symptoms was associated with a significantly increased incidence of stroke ( hr 1.57 [ 95% ci 1.05 , 2.33 ] vs. 1.01 [ 0.79 , 1.30 ] ) and cv death ( 1.53 [ 1.08 , 2.17 ] vs. 1.12 [ 0.90 , 1.38 ] ) in subjects with diabetes but not in those without diabetes . the combination of both elevated stress and depressive symptoms in subjects with diabetes was associated with a higher incidence of cv death ( 2.15 [ 1.33 , 3.47 ] ) than either behavioral comorbidity alone ( 1.53 [ 1.08 , 2.17 ] ) and higher than in those with both elevated stress and depressive symptoms but without diabetes ( 1.27 [ 0.86 , 1.88]).conclusionscomorbid stress and/or depressive symptoms are common in individuals with diabetes and together are associated with progressively increased risks for adverse cv outcomes .

antineutrophil cytoplasmic antibodies ( anca)-associated glomerulonephritis ( gn ) is characterized by necrotizing and crescentic gn with paucity of immunoglobulin ( ig ) and complement deposition , which is also known as pauci - immune crescentic gn . membranous nephropathy ( mn ) is characterized by the formation of subepithelial immune deposit with resultant changes in glomerular basement membrane ( gbm ) , most notably spike formation . a 48-year - old man presented with marked proteinuria , hypoalbuminemia , and renal dysfunction with positive results for myeloperoxidase ( mpo ) and anca . renal biopsy revealed crescents and thick gbm with subepithelial spikes along with igg deposition on immunofluorescent staining . after one - month follow - up , antibody level and renal function did not improve . coexistence of mn with mpo - anca crescentic gn is very rare and should be managed aggressively . membranous nephropathy ( mn ) is characterized by the formation of subepithelial immune deposit with resultant changes in glomerular basement membrane ( gbm ) , most notably spike formation . approximately 75% of mn represent as primary disease and the rest results from secondary causes , most commonly systemic lupus nephritis ( sle ) , infections such as hepatitis b or c viruses , malignancy , or drugs . antineutrophil cytoplasmic antibodies ( anca)-associated glomerulonephritis ( gn ) is characterized by necrotizing and crescentic gn with paucity of immunoglobulin ( ig ) and complement deposition , which is also known as pauci - immune crescentic gn ( 1 - 3 ) . we report a rare case of mn with myeloperoxidase ( mpo)-anca - associated crescentic gn in a 48 year - old - man who was admitted to our institute . a 48-year - old man presented with intermittent puffiness of face and edema of the feet for two months . he did not have fever , hematuria , or breathlessness . on examination , he had bilateral pitting pedal edema ( + + ) , pulse rate of 98 per minute , and blood pressure of 140/96 mm hg . cardiovascular and respiratory examinations were unremarkable . on investigation the following laboratory results were reported : hemoglobin , 6.1 gm / dl ; white blood cell ( wbc ) count , 5.6 10/l ; platelet count , 2.11 10/l ; blood urea nitrogen , 35 mmol / l ; serum creatinine , 807 mol / l ; random blood sugar , 5.33 mmol / l ; total serum protein , 500 g / l ; serum albumin , 31 g / l ; serum sodium , 132 mmol / l ; serum potassium , 4.54 mmol / l ; and serum cholesterol , 5.28 mmol / l . urine analysis showed 3 + albumin with 35 to 40/hpf of red blood cells and 8 to10/hpf of wbc . results of viral screening for human immunodeficiency virus , hepatitis b and hepatitis c viruses were negative . serum mpo - anca level was 220 u / ml ( normal range , 1 - 5 ) . serum anti - nuclear antibody ( ana ) , the levels of serum complements c3 and c4 were in normal limits . chest radiograph revealed normal findings and renal ultrasonography showed right kidney dimension of 8.6 3.4 cm and left kidney dimension of 9.0 4.5 cm , with increased echogenicity and maintained corticomedullary differentiation . renal biopsy was performed and after paraffin embedding , 3-m - thick sections were prepared and stained by hematoxylin and eosin ( h and e ) , periodic acid schiff , jone s silver methenamine , and gomori s trichrome stains . histopathologic examination ( figures 1 and 2 ) showed a single core of renal tissue containing 14 glomeruli with surrounding tubules and vessels . immunofluorescence ( if ) studies ( figure 3 ) showed fine granular fluorescence ( + 3/4 ) across 80% to 90% of glomerular capillary walls on staining with anti - human igg . no fluorescence was revealed on staining with anti - human iga , c3 , c1q , fibrinogen , and igm antisera . he was diagnosed as a case of mpo - anca - associated crescentic gn with mn . he was treated with intravenous methylprednisolone ( 500 mg / d ) for three days , followed by intravenous cyclophosphamide ( 500 mg ) and oral prednisolone ( 0.5 mg / kg / d ) with antihypertensive drugs . after two - month follow - up , his serum creatinine was 389 mol / l , urine albumin was 3 + with 5 to 7/hpf of rbcs . his renal biopsy revealed crescents and thick gbm with subepithelial spikes along with igg deposition on if staining . coexistence of anca - gn with mn is very rare ( 1 , 4 - 7 ) . surindran et al . detected coexistence of anti - phospholipase a2 receptor antibody ( pla2r)-positive mn and anca - gn ( 9 ) . ram et al . reported mn superimposed on churg - strauss syndrome ( 10 ) . the combination of anti gbm - gn and mn are also reported ( 11 , 12 ) . nasr et al . and nayak et al . reported that mn preceded the development of anti gbm in 50% of cases ( 1 , 13 ) . morizane et al . have reported a case of mpo - anca - associated gn , which was superimposed on type-3 mpgn . mn can be classified into primary and secondary forms according to igg subclass . in primary mn , igg4 is positive and in secondary mn , igg1 and igg4 . speculated that mpo - anca - associated gn might cause secondary mn as both igg1 and igg4 were present in their case ( 4 ) . the membranous lupus nephritis ( ln ) with focal or diffuse ln may have crescents with mn . such mixed patterns of ln have endocapillary proliferation and concomitant subendothelial immune deposits with full - house staining by if . nasr et al . reviewed kidney biopsies from january 2000 to february 2008 and concluded that coexistence of mn and anca - associated gn would be coincidental ( 1 ) . the primary mn is associated with hla - dq1 and pla2r , suggesting genetic predisposition ( 9 , 15 ) . anca - associated gn is also associated with environmental triggers such as drugs and infections ( 9 ) . when patient presents as rapidly progressive glomerulonephritis with marked proteinuria , further investigations to rule out possibility of mn with anca associated crescentic gn should be performed and the patient should be managed aggressively .

introduction : antineutrophil cytoplasmic antibodies ( anca)-associated glomerulonephritis ( gn ) is characterized by necrotizing and crescentic gn with paucity of immunoglobulin ( ig ) and complement deposition , which is also known as pauci - immune crescentic gn . membranous nephropathy ( mn ) is characterized by the formation of subepithelial immune deposit with resultant changes in glomerular basement membrane ( gbm ) , most notably spike formation.case presentation : a 48-year - old man presented with marked proteinuria , hypoalbuminemia , and renal dysfunction with positive results for myeloperoxidase ( mpo ) and anca . renal biopsy revealed crescents and thick gbm with subepithelial spikes along with igg deposition on immunofluorescent staining . the condition was diagnosed as mn with mpo - anca - associated crescentic gn . he was treated with intravenous methylprednisolone and cyclophosphamide . after one - month follow - up , antibody level and renal function did not improve.conclusions:coexistence of mn with mpo - anca crescentic gn is very rare and should be managed aggressively .

although enhanced cardiovascular reactivity is generally associated with future development of hypertension and other cardiovascular events [ 15 ] , there are studies that have failed to show any relationship between reactivity to stress and future elevation of blood pressure [ 1 , 612 ] . emerging evidence suggest that some stress tests may be better predictors of future cardiovascular events than other stressors [ 4 , 5 , 10 ] . for example , blood pressure response to arithmetic and star tracing stress tests predicted high blood pressure while reactivity to cold pressor stress test did not [ 13 , 14 ] . furthermore , metanalysis of studies that assessed mental stress tests and hypertension development revealed variable success of mental tests in predicting hypertension . among the different types of mental stressors , cognitive mental stressors were more consistent in predicting hypertension compared to emotion evoking , interview , and public speaking stressors . the inconsistencies in prediction do not appear to be explained by differences in the type ( mental , physical , or psychophysical ) of stress tests for there is inconsistent predictability even among the types of stressors . another possible explanation for the inconsistencies in reactivity prediction of adverse cardiovascular outcomes is the interaction of psychosocial factors with cardiovascular responses to acute laboratory stressors . metanalysis of over 700 studies revealed that chronic ( trait ) anxiety is associated with decreased cardiovascular reactivity . in contrast , a study of young european population revealed that acute ( state ) anxiety was associated with significantly increased reactivity to cold pressor test but not mental stress test . these observations , when taken together , suggest that individuals may be inaccurately identified as hyperresponsive if anxiety is not considered as a confounder in the reactivity response to acute laboratory stress tests . consequently , inaccurate assessment of increased reactivity due to the interaction of anxiety with acute stressors may explain the inconsistent reports of increased risk of hypertension with increased reactivity . this study sought to investigate whether ( 1 ) anxiety determined the blood pressure response to stress tests and ( 2 ) anxiety differentially influenced blood pressure response to anger recall and cold pressor stress tests in african americans . we chose to study african americans for several reasons : ( 1 ) this group is characterized as hyperresponsive to stress [ 7 , 1720 ] , ( 2 ) several reports have failed to find increased reactivity in this population [ 4 , 8 , 2123 ] , and ( 3 ) psychosocial factors , including anxiety , are significantly associated with blood pressure in this population [ 2429 ] . we report that state ( in the moment ) anxiety was significantly associated with blood pressure response to both stressors ( anger recall and cold pressor stress tests ) in this population . these results support the idea that identification of hyperresponders to acute stress tests among african americans must take into account anxiety levels before determining whether an individual has increased reactivity to acute stress and/or that anxiety may play an important role along with reactivity response in hypertension development . however , our results do not support the idea that anxiety differentially impacts reactivity response to psychological , psychophysical , and physical stressors . a sample of 179 ( 116 males , 63 females ) participants of african descent were recruited to the study . all study procedures and materials were approved by and in compliance with the north carolina central university institutional review board . eligibility criteria for entry were ( 1 ) be 18 to 65 years old ( 2 ) being a student or employee at north carolina central university or living in the surrounding regions of durham , orange and wake counties , ( 3 ) having no diagnosed cardiovascular disease ( self - reported ) , and ( 4 ) not taking any hypertensive medication . these regions of durham , orange , and wake counties make up the north carolina triangle region that is in the stroke belt ( e.g. , a geographic region with a higher occurrence of stroke ) . of these 179 , only 50 are reported in the current report ; these were selected based on the type of mental stress used . the 50 participants reported here met the following criteria : ( 1 ) completed both the trait anxiety scale and the state anxiety scale , and ( 2 ) were between the ages of 18 and 40 years old . study participants were scheduled at either 9 am or 1 pm for the three - hour study protocol . after receiving informed consent , trained staff measured blood pressure by sphygmomanometer method with a ge dinamap pro 100 automatic model and a cuff size appropriate for the body size . each participant was allowed five minutes to sit quietly before taking the first resting parameters . the dinamap was set to assess systolic blood pressure ( sbp ) and diastolic blood pressure ( dbp ) at one - minute intervals for the resting measurements as well as during the two acute stressor tasks . following resting blood pressure and heart rate measurements , participants were administered the cold pressor test , consisting of submersion of the hand in ice cold water for three minutes followed by a five - minute recovery period . a psychological stressor , anger recall , was given only after blood pressures and heart rate returned to baseline resting values . anger recall stress consisted of 5 minutes of contemplating an event that evoked anger , 5-minute discussion about the event , and 5-minute recovery period . cardiovascular reactivity was calculated as the difference between the average baseline prestressor blood pressure and the average change in blood pressure over the 5-minute stress period . the study protocol consisted of state anxiety assessment , resting bp measurement , second resting bp measurement , cold pressor stressor , third resting bp measurement , anger recall stressor , trait anxiety assessment , recording medical history , body mass index measurement , and completing a demographics questionnaire . state anxiety is defined as an acute response to a threatening or challenging situation , while trait anxiety is defined as a stable and enduring tendency to be anxious . each item is rated on a four - point scale ( 1 = almost never , 4 = almost always ) . items from each subscale are summed to create a total state anxiety score and a total trait anxiety score . higher scores on the state anxiety subscale indicate greater anxiety at the present time ; higher scores on the trait anxiety subscale indicate greater anxiety , in general . the state anxiety subscale has an alpha coefficient of .87 , and the trait anxiety has an alpha coefficient of .88 , indicating good ( since .80 or greater ) internal consistency in this sample . scoring of the psychosocial scales spielberger state trait anxiety inventory utilized scoring protocols documented in prior research as indicated above and were confirmed with factor analysis . mean , standard deviation , standard error , median , and quartile calculations provide data reductions for sbp , dbp , and other clinical measures with multiple measurements . regression models , goodness of fit , multivariate parameter estimates , and confidence intervals were evaluated for each stressors impact on sbp , and dbp . two participants did not complete the cold pressor stressor ; thus , the sample size is 48 for the cold pressor cardiovascular reactivity and 50 for the anger recall cardiovascular reactivity . the african american study samples are relatively young ( median age of twenty - one years ) with normal bmi ( median bmi was 26.2 kg / m ; normal bmi is 2530 kg / m ) and waist circumference normal values of less than 102 for males and 88 cm for females [ 3335 ] . this group also had normal cholesterol ( less than 200 mg / dl ) , triglycerides ( less than 150 mg / dl ) , glucose ( less than 126 mg / dl ) , and insulin ( < 10 miu ) levels . this group was normotensive with median systolic ( sbp ) and diastolic ( dbp ) blood pressures of 114 and 70 mmhg , respectively . a summary of the cardiovascular reactivity responses to ar and cp is shown in table 2 . cardiovascular reactivity was defined as the change in cardiovascular parameters ( sbp , dbp , mean arterial pressure ( map ) , and heart rate ( hr ) ) following the induction of a stress stimulus compared to baseline cardiovascular parameters . both the cp stressor and the ar stressor produced significant rises in sbp , dbp , and hr . all of the values returned to baseline during the recovery period except for sbp during the cp recovery period . repeated measures anova and the multiple comparison test , student - newman - keuls test , verified that the two stressor tasks ( cp and ar ) produced statistically significant increases ( p < 0.0001 ) in the cardiovascular parameters in comparison to the resting measurements . we examined pearson 's correlations of state anxiety , trait anxiety , age , bmi , and resting cardiovascular measures with the cardiovascular reactivity parameters . trait anxiety had a statistically significant , positive correlation with state anxiety ( n = 50 ; pearson 's r = 0.47 ; p < 0.001 , two - tailed pearson 's correlation ) . state anxiety had statistically significant , positive correlations with cp reactivity response for sbp ( average change ; n = 48 ; pearson 's r = 0.37 ; p = 0.01 ) , dbp ( average change ; n = 48 ; pearson 's r = 0.40 ; p = 0.005 ) . similarly , state anxiety was highly correlated with the ar reactivity response for sbp ( n = 50 ; pearson 's r = 0.34 ; p = 0.015 ) and dbp ( n = 50 ; pearson 's r = 0.35 ; p = 0.013 ) . specifically , state anxiety was significantly associated with hr change to cp ( average change ; n = 48 ; pearson 's r = 0.37 ; p = 0.009 ) but was not related to hr changes to ar ( n = 50 ; pearson 's r = 0.14 ; p = 0.30 ) . trait anxiety had a nonsignificant correlation with sbp ( n = 48 ; pearson 's r = 0.23 ; p = 0.12 ) but a positive , significant correlation for dbp ( n = 48 ; pearson 's r = 0.34 ; p = 0.02 ) reactivity response to cp . as for ar , trait anxiety was positively and significantly correlated with sbp ( n = 50 ; pearson 's r = 0.35 ; p = 0.012 ) but was not significantly correlated with dbp ( n = 50 ; pearson 's r = 0.23 ; p = 0.11 ) . age , bmi , and resting cardiovascular measures had nonsignificant correlations with both cp reactivity and ar reactivity as measured by sbp , dbp , and hr changes . resting sbp was only associated with map changes with cp and ar stress tests ( pearson 's r = 0.33 ; p < 0.02 and r = 0.38 ; p < 0.01 , resp . ) . the following variables were evaluated in the stepwise procedure : state anxiety , trait anxiety , resting cardiovascular measures , body mass index , and age . the variable selection results of the stepwise algorithm suggested the use of state anxiety for a parsimonious model of both cold pressor cardiovascular reactivity as well as anger recall cardiovascular reactivity . our next step was to evaluate regression models to predict cardiovascular reactivity with state anxiety as the independent variable . the model for predicting the cp increase in sbp ( see table 3 ) had an r of 0.14 and state anxiety as a significant parameter ( p = 0.009 ) . state anxiety was also a significant independent variable ( p = 0.005 ) in the model of the cp change in dbp ( r = 0.16 ) . similar results were found with the model for predicting the change in cardiovascular reactivity for the ar stressor as shown in table 4 . state anxiety was a significant parameter for the change in sbp ( p = 0.015 , r = 0.12 ) and dbp ( p = 0.013 , r = 0.12 ) . each model was scrutinized to verify adherence to the assumptions of regression modeling ( linear relationship between independent variables and dependent variables , homoscedasticity of the errors , and errors are independent and normally distributed ) . shapiro - wilke statistics did not reject the null hypothesis of normal distribution of the residuals . this study investigated whether anxiety differentially affects cardiovascular reactivity to cold pressor and anger recall stress tests in a sample of young , healthy , community dwelling african american adults , a population prone to develop cardiovascular disease . importantly , we show that the state ( at the moment ) anxiety was significantly associated sbp and dbp responses to both cold pressor and anger recall laboratory stress tests in this population . in contrast to blood pressure , state anxiety differentially predicted hr response to cp but not ar . on the other hand , chronic ( trait ) anxiety was not a significant predictor of reactivity in our statistical models . we interpret these results to mean that the state of anxiety at the time of the stressor must be considered when assessing cardiovascular reactivity to laboratory stress tests . failure to consider state anxiety as a confounder of reactivity responses may lead to misidentifying some individuals as hyperresponders when compared to others . misidentification of individuals may contribute in part to the inconsistent findings of increased reactivity in african americans and to the inconsistent prediction of hypertension in those with increased vascular reactivity . alternatively , these results can be interpreted to mean that the interaction of state anxiety and cardiovascular reactivity may be important determinants of hypertension development in african americans . anxiety , chronic anxiety in particular , has been linked to the development of disease . contrary to what would be expected , chronic anxiety has been shown to be negatively associated with cardiovascular reactivity . although chronic anxiety and cardiovascular reactivity associations have been studied , few studies have investigated the role of state anxiety in determining blood pressure response to stress . white coat response and is effective in predicting ambulatory evening systolic blood pressure in young black males . studies that have investigated the effect of state anxiety on reactivity did not include african americans [ 16 , 37 ] . our study provides evidence that young healthy african americans who are anxious prior to the stress tests are likely to have higher blood pressure responses to the stress . thus , variability in hyperresponsiveness response to laboratory stress tests in african americans may be due in part to a failure to consider state anxiety as a confounder . although many studies have shown that enhanced cardiovascular reactivity predicts hypertension development and other cardiovascular events [ 15 ] , there are several studies that failed to show any relationship between reactivity to stress and hypertension development [ 1 , 612 ] . the inconsistency may be a consequence of the type of stressors used [ 4 , 5 , 38 , 39 ] and the interaction of psychosocial factors with blood pressure response to the stressor [ 4042 ] . psychological stress tests may be better than cold pressor stress tests at predicting future cardiovascular events [ 4 , 5 , 22 , 43 , 44 ] . this differential effect of stressor type and hypertension development may be a consequence of differential psychosocial factor interaction with stressors . in a study of a european population , psychosocial factors appear to have a greater impact on reactivity to cold pressor than reactivity to mental stress . our study compares the impact of anxiety on reactivity to cold pressor and anger recall in african americans . another explanation for the inconsistent findings of increased risk of hypertension development with increased reactivity is the interaction of psychosocial factors with cardiovascular reactivity to promote hypertension development . for example , studies show that hostility , depression , and anger contribute to increased reactivity [ 40 , 45 , 46 ] . psychosocial factors also are associated with increased incidence of cardiovascular events [ 4750 ] as well as the development of cardiovascular disease [ 51 , 52 ] . we show that the psychosocial factor , anxiety , can influence the reactivity response to acute stress . however , because the study design was cross - sectional , it could not be determined whether state anxiety interaction with cardiovascular response to acute stress predicts hypertension development . this study shows that the current state of anxiety was significantly associated with blood pressure response to laboratory stress tests . consequently , anxiety levels should be assessed when using acute laboratory stress tests for identification of those with increased cardiovascular reactivity . further studies are needed to determine whether reactivity normalized to anxiety increases the accuracy in identifying hyperresponders and , subsequently , predicting future hypertension development . these findings need to be validated in a larger cross sectional population of african americans . additionally , our study design did not allow us to determine whether the impact of anxiety on blood pressure response to acute stressor is unique to anxiety or whether other psychosocial factors similarly influence blood pressure response to acute stress in our cohort . another limitation of the study is that adrenergic system activation was not measured ; consequently , it could not be determined if the two stressors differentially activated the beta or alpha - adrenergic receptor pathways . this information would be helpful in future longitudinal studies that will address how activation of the alpha and beta adrenergic receptors pathways ultimately leads to hypertension development and the attending cardiovascular disease . a longitudinal study design will also help to address the question of whether inclusion of anxiety enhances the ability of increased reactivity to predict future elevations of blood pressure .

although several studies have shown that enhanced cardiovascular reactivity can predict hypertension development in african americans , these findings have not been consistent among all studies examining reactivity and hypertension susceptibility . this inconsistency may be explained by the influence of anxiety ( state and trait ) on the blood pressure response to stress . therefore , this study sought to determine whether anxiety is associated with blood pressure response to cold pressor ( cp ) and anger recall ( ar ) stress tests in young healthy african americans . modeling using state and trait anxiety revealed that state anxiety predicts systolic ( sbp ) and diastolic blood pressure dbp response to cp and ar ( p 0.02 ) . interestingly , state anxiety predicted heart rate changes only to cp ( p < 0.01 ; p = 0.3 for ar ) . although trait anxiety was associated with sbp response to ar and not cp , it was not a significant predictor of reactivity in our models . we conclude that anxiety levels may contribute to the variable blood pressure response to acute stressors and , therefore , should be assessed when performing cardiovascular reactivity measures .

sweet 's syndrome ( ss ) presenting in a localized and symmetric pattern is a very rare phenomenon . there have , however , been , a few case reports for the same . ss per se was first reported in 1964 by robert douglas sweet . though classically presenting as tender pseudovesicles , ss may demonstrate numerous topographic variations , which could be quite confusing for the diagnosing dermatologist . a 45-year - old farmer from nepal presented to the department of dermatology with complaints of an abrupt onset of an asymptomatic cutaneous eruption involving the neck and both the forearms since the past 5 days . the lesions were smaller earlier which had progressively increased in size over the past 5 days to reach the current status . there were no associated constitutional symptoms and no history of contact with cattle or sheep . , papules and plaques of varying sizes with a striking central umbilication were identified involving the forearms and neck that gave an illusion of vesiculation [ figures 13 ] . some of the lesions over the neck had a crust overlying the depressed center [ figures 4 and 5 ] . gram staining of the tissue obtained from one of the papules after needle extirpation was inconclusive . a skin biopsy from these lesions showed absence of epidermal changes with a dense inflammatory neutrophilic dermal infiltrate along with dermal edema as the hallmark finding , without evidence of vasculitis [ figures 68 ] . laboratory parameters revealed a hemoglobin level of 14 g / dl , a total white blood cell count of 13,000 cells / mm with 89% neutrophils , and a normal peripheral smear . his renal and hepatic profiles were normal , and imaging studies of the abdomen and pelvis were insignificant . the patient was diagnosed as ss and started on 20 mg of prednisolone once daily for 2 weeks and 100 mg of dapsone once at bedtime for 2 weeks . fleshy pseudovesicles with central umbilication seen on the right forearm of our patient over the left forearm , a plaque with the characteristic central umbilication seen , closely mimicking the lesions encountered in molluscum contagiosum . furthermore , to note here is the symmetric pattern of lesion arrangement molluscoid pseudovesicles over the nape of the neck juicy plaques with a central depression and an overlying crust seen on the right side of the neck similar lesions on the left side of the neck as seen in figure 4 . to note here again , is the striking symmetric distribution of the nuchal lesions section of the plaque on the right forearm demonstrating dermal edema and a dense dermal inflammatory infiltrate ( h and e , 10 ) same section showing a dense inflammatory infiltrate composed of neutrophils around the blood vessels and also diffusely distributed ( h and e , 20 ) absence of vasculitis on microscopy ( h and e , 40 ) sweet 's syndrome is the prototypical neutrophilic dermatosis , generally characterized by fever , an increase in the neutrophilic count along with tender pseudovesicles distributed over the body in an asymmetric manner . etiologically , three types of ss have been described , namely the idiopathic , paraneoplastic , and the drug - induced type of ss . as mentioned earlier , there have , however , been reports of the occurrence of ss localized to specific sites of the body though , some of which have presented with very unusual morphologic patterns , as shown by sommer et al . who described palmoplantar pustulosis as the sole finding in a ss patient , verma et al . demonstrating the occurrence of ss confined to the photo exposed sites alone , verma who elucidated a rare recurrent bullous eruption localized to the flexural aspect of both the forearms in a ss patient and brechtel et al . who reported a localized facial presentation of ss manifesting as an inflammatory lesion with a central depression superimposed with several pustules on the surface of the plaque . a variant of ss referred to as neutrophilic dermatosis of the dorsal hands ( nddh ) is a specific entity confined only to the dorsa of the hands and has been extensively reviewed in literature by galaria et al . , takahama and kanbe , cook et al . nddh usually presents as edematous and violaceous plaques and papulonodules on the radial aspect of the dorsal surface of both hands . our patient presented with molluscoid pseudovesicles distributed over the neck and forearms only , which is a very rare morphologic pattern witnessed in ss and to the best of our knowledge has not been previously described . second , our patient lacked the usual constitutional symptoms that herald ss , which made the diagnosis a challenge . third , as no systemic association with ss could be identified in our patient , he was further sub - classified as a case of idiopathic ss , which again is a rarity in male patients , who usually present with the paraneoplastic or the drug - induced variant of ss . the hallmark finding in our patient was the central depression in all the pseudovesicles examined . the other possibilities , we kept in mind after clinically examining our patient , were , atypical erythema multiforme , histioid leprosy , milker 's nodule , cowpox , and ecthyma contagiosum , but these were eventually ruled out . hence , to conclude , we see that ss is a dermatosis that could be a great mimicker just like leprosy and syphilis and therefore when faced with diagnostic dilemmas in these kinds of atypical presentations , it would be worthwhile considering ss as a differential diagnosis . idiopathic sweet 's syndrome ( ss ) may even occur in males though highly uncommonthe absence of constitutional symptoms does not necessarily rule out the diagnosis of ssthe plaques encountered in ss could even be asymptomatic.molluscoid lesions seen in ss is a very rare morphologic pattern encountered and to the best of our knowledge is being reported for the 1 time in literaturelocalized patterns of ss are not so rare as previously thought . idiopathic sweet 's syndrome ( ss ) may even occur in males though highly uncommon the absence of constitutional symptoms does not necessarily rule out the diagnosis of ss the plaques encountered in ss could even be asymptomatic . molluscoid lesions seen in ss is a very rare morphologic pattern encountered and to the best of our knowledge is being reported for the 1 time in literature localized patterns of ss are not so rare as previously thought .

sweet 's syndrome is a well - known entity in the field of dermatology . it has presented itself in myriad forms , well - recorded in literature . our patient presented with a sudden eruption of nontender molluscoid pseudovesicles distributed over the neck and forearms alone . a complete diagnostic work up for the same did not give any clue regarding an underlying systemic ailment , responsible for the dermatosis . this case is being presented because of this extremely rare morphology of gomm - button disease .

in order to prevent and control microbial proliferation in industrial settings , cleaning and disinfection plans are applied on a regular basis [ 1 , 2 ] . in food processing plants , the control of microbial contamination generally involves clean - in - place ( cip ) procedures which consist of running alternated cycles of detergent and disinfectant solutions with water rinses in high turbulence regimes through the plant and pipeline circuits without dismantling or opening the equipment [ 25 ] . biocides are currently used in industrial processes as the most significant countermeasure to control microbial growth and proliferation . industry moved progressively towards the use of surfactants that are less toxic and more biodegradable . surfactants are classified according to the ionic physiognomies of their hydrophilic group as anionic , cationic , nonionic , and zwitterionic [ 6 , 8 ] . quaternary ammonium compounds ( qacs ) are cationic surfactants that are commonly used because of their hard - surface cleaning , odor removal and antimicrobial properties . besides killing bacteria , the chemical nature of qacs can cause modifications on the properties of abiotic surfaces , decreasing their tension and therefore preventing attachment of microorganisms . the antimicrobial mode of action of cationic surfactants is proposed by some authors as a sequence of events : attraction by the negatively charged cell surface ; adsorption to the cell wall through the hydrophobic headgroup ; reaction with the lipids and proteins that compose the cytoplasmic membrane ; and cell penetration and interaction with intracellular constituents [ 10 , 11 ] . thus , qacs damage the outer layers of bacteria , thereby promoting the release of intracellular constituents . antimicrobial efficacy tests require planning of an adequate strategy and should include all the parameters found in real settings . aspects such as the proper contact time under known water hardness and conditions of high or low soil content should be considered . for an effective cleaning and disinfection plan , the choice of the disinfectant must follow specific criteria such as compatibility with the surfaces to be disinfected , economic constraints , safety in the workplace , toxicological safety , and biological degradability . it should , most of all , target the type of bacteria and the type of soiling . in fact , disinfectants can be seriously affected by the presence of organic matter . interfering substances have been studied in the last years and included in cleaning and disinfection plans regulated by the authorities such as the european standard en-1276 . however , most of these studies only address the effects of bovine serum albumin ( bsa ) and water hardness [ 9 , 14 , 15 , 1921 ] . evaluated the bactericidal activity of disinfectants referred in the german veterinary society guidelines as references for testing disinfectants used in dairy and food industries . in order to simulate the conditions found in practice , they used low fat milk as an organic load and reported the significance in choosing an appropriate disinfectant since the inclusion of a challenging substance ( organic material ) is important to access the proper bactericidal activity . bessems demonstrated that a qac tested on three microorganisms ( pseudomonas aeruginosa , staphylococcus aureus , and candida albicans ) had a similar killing rate in the absence of interfering substances and after the inclusion of 17 dh water hardness , a strong reduction of the killing activity was found for the gram - negative bacteria assessed the effect of dried yeast and human serum on the activity of benzalkonium chloride and concluded that the bactericidal activity of the qac was inhibited by solutions of both interfering substances . this work provides information on the influence of potential interfering substances ( bovine serum albumin ha ) on the antimicrobial activity of two qacs ( benzalkonium chloride and cetyltrimethyl ammonium bromide ) against bacillus cereus and pseudomonas fluorescens , as they are two major contaminants in the food industry , particularly the dairy industry , and are a known cause of produce spoilage and foodborne illnesses [ 2 , 2226 ] . some of the interfering substances used throughout the experiments are proposed in the european standard en-1276 as potential interfering agents in disinfection while the others are extracellular polymeric substances ( eps ) from the biofilm matrix that have an important role in antimicrobial resistance . the bacteria used in this work were pseudomonas fluorescens atcc 13525 and a bacillus cereus strain , isolated from a disinfectant solution and identified by 16s rrna gene sequencing . bacterial strains were grown at a temperature of 30 2c and ph 7 , with glucose as the main carbon source . culture medium consisted of 5 gl glucose , 2.5 gl peptone , and 1.25 gl yeast extract in phosphate buffer ( pb ) ( ph 7 , 0.025 m ) . a bacterial suspension was prepared by inoculation of a single colony grown on solid medium into a 1 l flask containing 250 ml of sterile nutrient medium . this bacterial suspension was incubated overnight at the given temperature with agitation ( 120 rpm ) . the qacs used throughout the experiments were benzalkonium chloride ( bac ) and cetyltrimethyl ammonium bromide ( ctab ) ( sigma , portugal ) . preliminary studies with a concentration range between 0 and 5000 mgl were initially made . in order to ascertain the behaviour of bacteria to the qac , the selected concentrations for further studies were 3 , 5 , 10 , 20 , and 35 mgl . the qacs were used individually and in combination ( both chemicals were combined in equal volumes and concentrations ) . ha ( acros organics , fisher chemical , portugal ) , and yeast extract ye ( merck , portugal ) . after the growth period , the suspensions were centrifuged ( 3999 g , 5 minutes ) , washed two times , and resuspended in pb to a final cell density of approximately 1 10 cellsml . in the case of the consortium , both bacterial suspensions were washed two times resuspended in pb to a final cell density of approximately 1 10 cellsml , and combined in equal volumes to obtain the same cell concentrations of the single species tests . afterwards , all bacterial suspensions were exposed to several concentrations of qac for a period of 30 minutes . the effects of the chemicals were evaluated by the assessment of the oxygen uptake rate due to glucose oxidation , according to simes et al . . to investigate the influence of interfering substances on the antimicrobial efficacy , the same procedure was followed with the addition of 300 mgl of bsa , alg , ye , or ha to the bacterial suspension , simulating low concentrations of interfering substances according to the european standard en-1276 . three independent experiments , each with duplicate samples , were performed for each condition tested . the methodology was performed according to johnston et al . for a period of 10 minutes . bac and ctab were chemically neutralized by a sterile solution of ( w / v ) 0.1% peptone , 0.5% tween 80 , 0.1% sodium thiosulphate , and 0.07% lecithin dissolved in pb . control experiments were performed to ascertain the effects of the 10-minute exposure to the neutralization solution , and no effects were detected on the respiratory activity of b. cereus and p. fluorescens ( data not shown ) . after the neutralization step , the bacterial suspensions were centrifuged ( 3999 g , 5 min ) and resuspended in the same volume of pb . the respiratory activity was ascertained by measuring oxygen uptake rates in a biological oxygen monitor ( yellow springs instruments 5300a ) . demonstrated that this procedure is more adequate and rapid than the assessment of colony forming units to characterize the antimicrobial activity of biocides against heterotrophic aerobic bacteria . samples were placed in the temperature - controlled vessel of the biological oxygen monitor ( t = 25 1c ) each containing a dissolved oxygen probe connected to a dissolved oxygen meter . before measuring , the samples were aerated for 10 minutes to ensure oxygen saturation ( [ o2 ] = 8.6 mgl ) . the vessel was closed , and the decrease of oxygen concentration was monitored over time . the initial linear decrease corresponds to the endogenous respiration rate . to determine the oxygen uptake due to substrate oxidation , 12.5 l of a 5 gl glucose solution was added to each vessel . the slope of the initial linear decrease in dissolved oxygen , after glucose injection , corresponds to the total respiration rate . the difference between these two rates is the oxygen uptake rate due to glucose oxidation . the inactivation was calculated using metabolic activities according to the following equation : ( 1)% inactivation=(mcmt)mc100 , where mc is the metabolic activity of the control experiments ( without antimicrobial exposure ) and mt is the metabolic activity of the bacterial solutions exposed to the antimicrobial . if % inactivation > 0 there was inactivation of the microorganisms whereas if % inactivation < 0 there was metabolic potentiation . the mbc for each situation was determined as the lowest concentration of qac or qac combination where no respiratory activity was detected . for each parameter the statistical significance of the results was evaluated using the wilcoxon test ( confidence level 95% ) to investigate whether the differences between the resulting experimental values could be considered significant . the antibacterial activity of bac , ctab , and their combination was investigated in the absence and in the presence of four selected interfering substances . in the absence of interfering substances bac caused the inactivation of b. cereus at 10 mgl , p. fluorescens at 35 mgl , and the consortium at 20 mgl . ctab at 20 mgl completely inactivated b. cereus and at 35 mgl inactivated the total population of p. fluorescens and the consortium . the combination of both qacs was synergistic in the inactivation of b. cereus ( total inactivation with 3 mgl ) and indifferent for p. fluorescens ( 35 mgl ) and the bacterial consortium ( 35 mgl ) . the inclusion of the selected interfering substances influenced the antimicrobial activity of the qacs to some extent ( figures 13 ) . the inactivation of b. cereus ( figure 1 ) was not affected by the presence of any interfering substances ( p > 0.05 ) , except with ha . the antimicrobial action of the qacs against p. fluorescens ( figure 2 ) was not significantly influenced by the presence of most potential interfering substances ( p > 0.05 ) , except for ha where interference was observed ( p < 0.05 ) . the antimicrobial activity of the qacs against the bacterial consortium ( figure 3 ) was affected by the presence of interfering substances . ha reduced significantly the activity of ctab at higher concentrations ( p < 0.05 ) . bsa and ye resulted in a significant reduction of the activity of the combination of qacs ( p < 0.05 ) . linear correlations were determined to assess the relationship between qac concentrations and the inactivation data . the effect of increasing qac concentration on bacterial inactivation shows that there are strong linear correlations ( r > 0.850 ) for the control assays , with the exception of b. cereus ( this bacterium was inactivated with low qac concentrations ) . the most extreme cases are the treatments with ctab to p. fluorescens with alg as an interfering substance ( r = 0.771 ) and the bacterial consortium in the presence of ye ( r = 0.738 ) . likewise , this decrease of linear correlation factors was found for p. fluorescens and for the consortium exposed to ha where the lowest correlation factor was 0.153 , which was obtained for p. fluorescens treated with ctab . this phenomenon only happened when the qacs were used on p. fluorescens and the bacterial consortium in the presence of ye and ha . the most significant cases of oxygen uptake rate increase were verified for p. fluorescens exposed to bac ( 5 to 35 mgl ) and ctab ( 3 to 35 mgl ) in the presence of ha and combination of qacs ( 3 to 10 mgl ) in the presence of ye . a similar metabolic behaviour was found for the bacterial consortium exposed to bac ( 3 to 35 mgl ) and ctab ( 5 and 10 mgl ) for ha and qac combination ( 3 to 20 mgl ) with ye . the mbc values for the different conditions tested ( single and combined qacs , in the absence and presence of potential disinfection interfering substances ) are shown in table 1 . the presence of bsa increased the mbc of the combination of qacs for b. cereus ( 3 to 5 mgl ) and the consortium . alg increased the mbc of bac for the consortium ( 20 to over 35 mgl ) and qacs combination ( 3 to 5 mgl ) for b. cereus . ye increased the mbc of bac for b. cereus ( 10 to 20 mgl ) and qac combination ( 3 to 5 mgl ) . the mbc values for the consortium of cells increased in the presence of ye ( bac20 to 35 mgl , ctab35 to over 35 mgl , and qac combination35 to over 35 mgl ) . ha increased the mbc for all the scenarios , except of ctab when applied to b. cereus ( in this situation the mbc was reduced ) . the mbc was reduced in other situations such as , for b. cereus , in the presence of alg when using bac and ctab ( 10 to 5 mgl and 20 to 5 mgl , resp . ) and in the presence of ye when using ctab ( 20 to 3 mgl ) . fluorescens inactivation by ctab was reduced by bsa ( 35 to 20 mgl ) . alg also reduced the antimicrobial activity of the combination of qacs against the bacterial consortium ( 35 to 20 mgl ) . it is assumed that the organic material can potentially interfere with the antimicrobial agents by chemical and/or ionic interactions [ 15 , 33 ] . therefore , it is necessary to know the role of each potential interfering substance in the antimicrobial activity in order to develop effective disinfection strategies . the interfering substances tested are commonly found as residuals in the food industry ( from food products and from microbial contaminants , biofilms ) [ 18 , 27 ] . in this study , higher inactivation rates were verified for b. cereus in comparison to p. fluorescens at the same qac concentration . in fact , when b. cereus and p. fluorescens are combined in a 1 : 1 bacterial suspension , it is expected that the first is more affected than the second . b. cereus is more susceptible due to the fact that it is a gram - positive bacterium that lacks an outer membrane , which typically provides increased protection to gram - negative bacteria . this fact is corroborated by previous reports which stated that gram - positive bacteria are more susceptible to cationic surfactants than gram - negative bacteria [ 34 , 35 ] . bsa was already studied as an interfering substance in disinfection practices [ 9 , 14 , 1921 , 36 ] . the negative effect of bsa on the action of biocides against p. fluorescens was demonstrated by simes et al . p. fluorescens treatment with ctab with the addition of 3 gl of bsa resulted in a 10-fold increase on the mbc of this qac [ 9 , 21 ] . in the present study , the efficacy of the combination of qacs against b. cereus and the cell consortium was also reduced . this effect of bsa as an antimicrobial quencher is apparently due to the strong ability of qacs to react with proteins . proteins can precipitate in the form of their anions , in this way , the negative - charged protein ions will cling to the positively charged molecules of the cationic compounds . ctab is a biocide that targets the membrane and has a strong affinity for proteins . bac is composed of a positively charged hydrophobic headgroup which clings to opposite charged surfaces [ 8 , 37 ] . studied the effect of the alkyl chain of bac binding to bsa and dried yeast . their conclusions were that bac is often inactivated by organic matter , either by adsorption to the bacterial surface or by adsorption to the organic matter in general . these authors also suggested that the reduction in the activity of bac was probably related to more than one physical property of the compounds like the chain length ( longer chains result in more adsorption to the bacterial surface ) . alg is a common constituent of the extracellular polymeric substances of the biofilm matrix [ 3840 ] . a function frequently attributed to eps is their general protective effect on biofilm microorganisms against adverse conditions . the eps matrix delays or prevents antimicrobials from reaching target microorganisms within the biofilm by diffusion limitation and/or chemical interaction with the extracellular proteins and polysaccharides [ 32 , 41 ] . in this study , alg either potentiated or hindered the antimicrobial activity of the selected qacs . the presence of this interfering substance was not obvious on the inactivation of p. fluorescens . on the other hand , the inactivation of b. cereus by bac and ctab and the consortium by the combination of qacs was easier in the presence of this interfering substance . the bacterial consortium treatments with bac and b. cereus with the combination of qacs were hampered by the presence of alg . davies et al . found that the production of alg was triggered by membrane perturbation induced by ethanol stress , nitrogen limitation , attachment to surfaces , or even high oxygen tension [ 42 , 43 ] . this substance is suggested as one of the main biofilm resistance vectors either by reacting with the antimicrobials or by hindering antimicrobials diffusion to the cells . the antimicrobial interference caused by alg is apparently due to electrostatic interactions between the anionic alg and the cationic - selected qacs . the presence of ye as interfering substance resulted in three different outcomes on the antimicrobial activity of the qacs : ( 1 ) no effect / indifference , ( 2 ) the respiratory activity reduced , and ( 3 ) the respiratory activity potentiated . this interfering substance worked mainly as a hinderer of the antimicrobial activity by increasing the mbc of b. cereus in all cases except for ctab , of p. fluorescens with the combination of qacs , and of the consortium of cells with ctab and the combination of qacs . ye is listed in the european standard en-1276 as an interfering substance native to the brewery industry . the constituents of ye are very similar to the components of the bacterial cells , thus , it is expected that the antimicrobial agents that target the bacterial cells are also drawn to ye . in a similar study by jon et al . it was shown that the presence of dried yeast decreased the biocidal effectiveness of bac . humic substances are found ubiquitously in the environment and can be found in the biofilm matrix [ 2 , 46 ] . ha reduced the antimicrobial activity of the qacs in most of the cases , although in some cases it promoted the respiratory activity ( potentiation ) . the presence of these compounds had the strongest effect compared to the remaining interfering substances . studied the sorption mechanisms of anionic and cationic surfactants to natural soils concluding that the dominant sorption mechanism of surfactants to clay is cation exchange . koopal et al . also verified the formation of complexes ha - cationic surfactant . respiratory activity potentiation was verified with the addition of ha to p. fluorescens and ye to the bacterial consortium . it is known that ha participates in cellular metabolism processes such as growth , respiration , photosynthesis , and nitrogen fixation . on the other hand , ha were proposed to replace synthetic surfactants such as sds , tween 80 , and triton x-100 in industrial applications such as textile dying or washing . it is therefore possible that the inclusion of humic substances in a solution of qacs may interfere with the chemical characteristics of the solution . the resultant mixture , with an apparent reduced antimicrobial efficacy , seems to potentiate the respiratory activity of the bacteria , particularly of p. fluorescens . as qacs are membrane active agents , their use at sublethal concentrations could improve membrane permeability and consequently the nutrient influx , without compromising the bacterial viability . also , there is the hypothesis that the potentially interfering agents could be used as nutrients . in fact , it was found that the growth rates of anaerobic and aerobic microorganisms increased when humic substances were added , which stimulated enzyme activity [ 52 , 53 ] . in a similar way , ha are likely to be used for growth in the same way as ye ; these might be broken down to smaller molecules that can be used by cells as a carbon or nitrogen sources . the antimicrobial activity of the tested qacs was enhanced in some cases , where the interfering substances were present . this is an unexpected result due to the recognized potential of alg , bsa , ha , and ye to interfere with disinfection . this effect is probably due to the low concentration of interfering substances tested that caused both respiratory activity reduction and potentiation . ethylenediamine tetraacetate ( edta ) was reported as early as 1965 to increase the biocidal effects of bac and chlorhexidine diacetate on pseudomonas aeruginosa . reported that chitosan ( a polysaccharide ) potentiated the antimicrobial action of sodium benzoate on spoilage yeasts . in dairy plants , disinfection is potentiated by pre - washes with alkali or enzyme - based cleaning agents . most of these cases were observed for b. cereus ( four occurrences ) , one was observed for p. fluorescens , and another one was observed for the consortium of cells . the mbc was improved by more than 50% in the cases of b. cereus and less than 30% for p. fluorescens and the consortium of cells . to our knowledge there are no reported cases of antimicrobial agents potentiation by bsa , ye , or alg . although the exact chemical structure of ha has not yet been determined , ha could be chemically similar to the tested qacs , presenting a positive hydrophilic head and a hydrophobic tail . with this structure ha could act as detergents in conditions such as those observed in the treatment of b. cereus with ctab . the present work shows that increasing qacs concentrations lead to an increase in antimicrobial effectiveness . this is valid mainly when the qacs were applied in the absence of interfering substances . this means that disinfection was concentration dependent , as found for most of the antimicrobial chemicals . however , the linear dependency of inactivation versus concentration is not verified for most of the tests where interfering substances were added . this result evidences that the mathematical modelling of disinfection strategies requires a case - to - case analysis when interfering substances are present . the overall results demonstrate that a disinfection process in the presence of the selected interfering substances can reduce the effectiveness of bac , ctab , and their combination . the bacteria were inactivated equally by all qacs , although in the absence of interfering substances ctab was the most efficient solution . p. fluorescens was the bacterium with the highest resistance to inactivation , followed by the bacterial consortium . the tested interfering substances , referred in the european standard 1276 ( bsa and ye ) , and known eps constituents related with biofilm resistance ( alg ) resulted in mild interferences on the activity of the qacs . ha were the interfering substance that resulted in the most severe effect by reducing the activity of qacs , causing , in some circumstances , significant respiratory activity potentiation .

standard cleaning processes may not remove all the soiling typically found in food industry , such as carbohydrates , fats , or proteins . contaminants have a high impact in disinfection as their presence may reduce the activity of disinfectants . the influence of alginic acid , bovine serum albumin , yeast extract , and humic acids was assessed on the antimicrobial activities of benzalkonium chloride and cetyltrimethyl ammonium bromide against bacillus cereus vegetative cells and pseudomonas fluorescens . the bacteria ( single and consortium ) were exposed to surfactants ( single and combined ) in the absence and presence of potential disinfection interfering substances . the antimicrobial effects of the surfactants were assessed based on the bacterial respiratory activity measured by oxygen uptake rate due to glucose oxidation . the tested surfactants were efficient against both bacteria ( single and consortium ) with minimum bactericidal concentrations ranging from 3 to 35 mgl1 . the strongest effect was caused by humic acids that severely quenched antimicrobial action , increasing the minimum bactericidal concentration of the surfactants on p. fluorescens and the consortium . the inclusion of the other interfering substances resulted in mild interferences in the antibacterial activity . this study clearly demonstrates that humic acids should be considered as an antimicrobial interfering substance in the development of disinfection strategies .

social scientists who explore factors mediating and moderating the relationships between social stressors and mental health , including drinking outcomes , have highlighted modes of coping [ 1 , 2 ] . these studies have explored behaviors which protect people from being psychologically harmed and cognitive appraisals which influence behaviors such as problem - focused coping or using alcohol to self - medicate distress . however , studies have not considered the characteristics of the stressful situation itself that may make certain coping strategies more or less effective . in particular , psychiatric epidemiologic studies have tended to emphasize microlevel stressors ( e.g. , stressors in individuals ' role domains ) and , until recently , have ignored the linkages between macrolevel social forces and the daily stressors in people 's lives [ 79 ] . however , macrolevel social conditions can affect the magnitude of stressors experienced in people 's lives and the extent to which they experience cumulative adversity . this paper focuses on coping with the fallout from one type of macrolevel social stressor : the recent great recession . this economic downturn constituted the most severe economic crisis in the united states since the great depression and had persisting economic effects ( e.g. , job loss , less desirable working conditions , loss of home , loss of retirement savings , lack of health care access , and social isolation ) which have been linked with deleterious drinking outcomes . a key issue involving the effectiveness of alternative modes of coping with stressors derived from macrolevel social forces and protecting against deleterious drinking outcomes is the question of whether individual modes of coping outside of the political realm emphasized in the overall coping literature are most efficacious . or , alternatively , do stressors engendered by macrolevel social forces require unique forms of coping , that encompass the political realm ? with respect to coping strategies employed in the wake of the great recession , politically oriented coping strategies might be of particular relevance due to the impact of governmental decision - making on the state of the economy . politically oriented coping strategies might include political activism oriented to altering economic policies or support for campaigns by politicians offering solutions to economically based hardships . scholarly work on the occupy wall street movement , for example , documents involvement by individuals sharing their own economic struggles and collectively gathering to protest their precarious economic situation and uncertain economic future . earlier work also suggests the salience of activities oriented toward changing politically based social realities such as through the act of voting or by collectively challenging community - level decisions such as school closings . by contrast , the traditional coping literature has emphasized individual , nonpolitical modes of coping , such as emotional acceptance of the stressful situation , blaming one 's self for the situation , or taking individual actions such as looking for a job if unemployed . while sociologists have recently accorded greater attention to the prevalence and alcohol - related consequences of macrolevel stressors , they have yet to address the extent to which politically oriented modes of coping may be the most efficacious ways to address problems stemming from macrolevel social forces or events . prior to the more recent focus on macrolevel stressors , kaplan and liu embraced the idea of collective coping as one means for individuals who maintained stigmatized personal identities to challenge and transform conventional socionormative systems through participation in social movements . subsequently , thoits more explicitly addressed collective coping within the context of the stress paradigm involving acute and chronic stressors not limited to the specific area of stigmatized statuses . she argued that individuals who find themselves in problematic situations can deliberately work to transform the meaning of their experiences and they can additionally use these experiences as a basis for helping or effecting changes in the lives of others she proposed the concept of transformatory coping to include engagement in collective activist activities with others who share similar problems . for example , parents of autistic children have lobbied governments for social services perceived to aid in their children 's development . in sum , ( 1 ) collective coping tactics ( such as politically oriented coping ) represent an unmeasured dimension of coping behaviors beyond that represented in the coping literature to date and ( 2 ) collective coping tactics may demonstrate a stronger association between stressors , particularly those stemming from macrolevel social forces , and deleterious drinking outcomes compared to the use of modes of coping previously emphasized in the literature on coping . the present study extends previous work by empirically addressing the extent to which politically oriented coping activities engaged in as a response to a macrolevel social stressor , the great recession , are protective against alcohol - related outcomes compared with coping strategies focused outside of the political realm . we hypothesize that politically oriented coping will be more protective against economic stressors linked with the great recession than nonpolitical modes of coping and will uniquely account for some portion of the associations between economy - related stressors and drinking outcomes . further , we also examine whether politically oriented coping and coping outside of the political realm are more protective for men versus women in the face of macrolevel engendered stressors such as those involving the economic fallout from the great recession . there is consistent evidence that women are more likely to use support - based coping strategies ( e.g. , seeking support from others such as partner family and friends ) in response to stress in contrast to men and some indication that avoidant coping techniques are associated with greater alcohol consumption among men but not women [ 1 , 2022 ] . ( in contrast , parity by gender in the use of individual active coping strategies and their significance for mental health outcomes is generally reported [ 1 , 21 ] . ) however , whether there is a corresponding propensity for men and women to differ in the use of politically oriented coping to offset the alcohol - related effects of economy - related stressors is less certain . earlier research tended to argue that women were less politically interested , informed , and efficacious compared to men . more recent work has shown that women and men differ in particular modes of participation ; women are more likely to vote and engage in individual political actions such as signing petitions or donating money , whereas men are more likely to be engaged in collective forms of action such as group protest activities . thus , we hypothesize that there will be no overall differences in the extent to which women and men manifest politically oriented coping or in the effect that politically oriented coping has on drinking outcomes . following a transactional model of stress [ 4 , 25 ] , we model coping as a mediator of the relationship between the stressor ( i.e. , stressful consequences of the great recession ) and the stress response ( i.e. , drinking outcomes ) . data were derived from a study conducted in the united states between june , 2010 , and january , 2011 , that was undertaken in order to understand life change consequences of the major downturn in the economy known as the great recession . respondents were selected by a random digit dial ( rdd ) phone survey of the continental united states , and those who consented to participate in the study were mailed questionnaires . eligible respondents were selected from the households using the troldahl - carter - bryant method of respondent selection which involves the means to randomly select a respondent from all eligible household members . respondents were told during the phone screen that a $ 50 american express gift card would be sent to the eligible respondent if he or she completed the questionnaire . respondents were mailed an initial survey , a postcard reminder to nonresponders , and a second questionnaire if they still had not responded . figure 1 encompasses a flow chart characterizing each stage in the data collection process through the completion of the questionnaires . 65.9% ( n = 663 ) of the respondents completing the screening calls returned the questionnaire . the telephone screening cooperation rate and the mail survey response rate were each calculated using the conservative aapor response rate formula 3 . the overall survey response rate is the product of the phone screening cooperation rate ( 35.5% ) and the mail questionnaire return response rate ( 65.9% ) or 16.8% . we acknowledge that this response rate is less than ideal and further address this issue in the discussion of study limitations and note other indicators of the representativeness of the final sample . selection weights were calculated for each of the cases to weight for the different probability of selection for each case . poststratification weights were calculated for the dataset to ensure that the distribution of sample cases on important demographic variables ( age , race / ethnicity , and gender ) conformed to the distribution of these variables in census bureau 's 2008 united states population estimates . it should be noted that estimates of alcohol consumption for the present sample did appear to conform to national estimates preweighting . for example , the average number of drinks consumed in the past month on days when one drank for the present sample is 2.16 , versus the estimated average of 2.10 reported by the centers for disease control and prevention 's behavioral risk factor surveillance system for 2010 ( by gender , the averages are 2.35 for men and 1.89 for women in the present sample versus 2.43 for men and 1.81 for women in the cdc estimates ; by race / ethnicity , the averages are 2.16 for non - hispanic whites , 2.08 for asians , 2.12 for african americans , and 2.72 for hispanics in the present sample versus 2.26 for non - hispanic whites , 2.41 for asians , 2.19 for african americans , and 2.68 for hispanics in the cdc data ) . it should additionally be noted that the respondents included in this sample reported an overall higher level of education than the general population based on 2008 census estimates . analysis of variance revealed no significant variation by education in either the outcomes or support coping , avoidant coping , or politically oriented coping . respondents with less than a high school degree are found to be marginally less likely ( p < 0.10 ) to use active coping strategies compared to those with a college degree or postcollege training . given this discrepancy as well as the fact that education is generally protective against problem drinking predictor variables are economy - related stressors , coping strategies enacted outside of the political realm ( i.e. , active coping , support coping , and avoidant coping ) , politically oriented coping , and gender . the sociodemographic characteristics of age , education , and race / ethnicity are controlled in all analyses . to assess past - month drinking patterns , we use the quantity - frequency - variability index ( qfv ) developed by cahalan et al . . frequency of drinking is measured as a count of the days on which alcohol was consumed in the past 30 days , and quantity of drinking is measured as the average number of drinks consumed on those days . variability is calculated by the greatest number of drinks consumed on any one day in the past 30 days . scores are calculated by multiplying responses to the quantity , frequency , and variability questions ( = 0.87 ) . as with the current data , this index tends to approximate a continuous scale , and ample evidence supports its use as such ( see fitzgerald and mulford for a review ) . our measure of problematic drinking is the 10-item bmast ( = 0.74 ) , which is a count measure of difficulties related to alcohol use over the past year . respondents were asked to indicate yes or no in response to 10 items such as having an accident , losing a close friend , spouse , or loved one , being hospitalized , having trouble at work , and soliciting professional help because of one 's drinking . the bmast is one of the most widely used tools for assessing alcohol dependence and problems . it correlates strongly with the full - length mast and evidence of its reliability and validity is widely available [ 31 , 32 ] . moreover , we are not using the bmast as a diagnostic tool for depicting problem versus nonproblem drinking , but as it is intended , as suggestive of different degrees of problematic drinking . the measure of economy - related stressors is the life change consequences of the great recession ( lccgr ) instrument . this construct was developed on the basis of qualitative analyses of transcripts derived from focus groups involving both genders and diverse racial / ethnic groups . the items fall into seven categories : home ownership problems , such as difficulties in mortgage payments , difficulties in paying property taxes , or a drop in credit rating ; undesirable living situation , including having to live in a less desired location to save money or having gas and electricity or heat shut off due to an inability to pay bills ; problematic employment situation , including a pay - cut , furlough days , and increased feelings of competition with fellow employees ; unemployment or underemployment ; inadequate health insurance , including lack of medical or dental coverage , decreased quality of coverage , and inability to obtain coverage ; social role constraints , such as dissolution of spouse / partner relationship , decreased social life , and increased social isolation due to finances ; and inadequate sick time , including inadequate sick days and having to work despite poor health . consistent with common practice , each score for this measure is a straight count of the number of stressors reported . three dimensions of nonpolitical modes of coping are assessed : active coping , support coping , and avoidant coping . these measures of coping are derived from subscales of the brief cope instrument and have previously been validated as stand - alone indices in community samples [ 3436 ] . participants in the present study were asked whether they have used these coping strategies in response to the economic recession . confirmatory factor analysis of the present data supports the inclusion of these items as three separate coping indices , consistent with prior research . active coping ( = 0.84 ) includes eight items measuring acceptance , positive reframing , and planning and taking action in response to the economic recession . support coping ( = 0.81 ) includes four items measuring use of emotional and instrumental support ( i.e. , receiving emotional support and getting advice and help from other people ) . avoidant coping ( = 0.75 ) includes 10 items measuring self - distraction , behavioral disengagement , self - denial , blame , and a tendency to vent about or make fun of the situation . all items were rated on a four - point likert - type scale ranging from 0 ( i did not do this at all ) to 3 ( i did this a lot ) . the measure of politically oriented coping is assessed by a four - item instrument ( = 0.79 ) drawn from the summed responses ( i.e. , not at all , a little , some , quite a bit , and a lot ) to four statements asking how often respondents , in response to the economic recession , have been ( 1 ) engaging in political activities such as signing petitions , leading or participating in rallies or marches , or writing to political representatives ; ( 2 ) organizing with others to challenge politicians currently in office ; ( 3 ) voting in elections to support politicians who share your political beliefs ; and ( 4 ) participating in groups trying to influence the policies of the government at the local , state , or national level . the questions used to construct this index were derived from analyses of focus group transcripts ( see for details about these focus groups ) . confirmatory factor analysis reveals that these items load on a single factor , supporting their inclusion as one index . education is a categorical variable based on the educational attainment categories of ( 1 ) less than high school ( n = 45 ) ; ( 2 ) high school graduate ( n = 350 ) ; ( 3 ) college graduate ( n = 110 ) ; and ( 4 ) postcollege training ( n = 150 ) . race / ethnicity is a dummy variable including non - hispanic whites ( n = 436 ) , african americans ( n = 80 ) , hispanics ( n = 91 ) , asians ( n = 29 ) , and individuals who identify as an other race / ethnicity ( n = 17 ) . in all analyses , non - hispanic whites serve as the reference category . after examining bivariate correlations in order to assess the basic patterns of association among key study variables , we performed structural equation modeling ( sem ) using mplus software to examine the predictive significance of economy - related stressors for coping outside of the political realm and politically oriented coping tactics and the two drinking outcomes considered ( i.e. , past - month drinking and problematic drinking ) , net of the sociodemographic control variables . we considered the potential for nonpolitical coping and politically oriented coping tactics to mediate the associations between economy - related stressors and each of the outcomes assessed in two models . the first model tested for associations between economic stressors and the drinking - related outcomes . the second model adds nonpolitical and politically oriented coping tactics to test the full mediation model . this latter model assesses all of the direct and indirect paths between economic stressors and the drinking outcomes considered through the nonpolitical and politically oriented coping tactics investigated . we formally tested for mediation using the procedures described by muthen and muthen for mplus software , which apply the tests described by mackinnon et al . . finally , because the associations between social stress and drinking are found to vary by gender [ 33 , 39 ] , we examined whether any observed mediating effects of the coping strategies investigated vary by gender . for these tests , separate equations include the interaction term for gender by each coping strategy in the path models linking coping with drinking outcomes . it is noteworthy that stressors related to the economy are associated with each of the alcohol - related outcomes and all of the coping resources considered : economy - related stressors are associated with more alcohol consumption and problematic drinking , as well as higher levels of active coping , support coping , avoidant coping , and politically oriented coping . it is also noteworthy that only two of the coping strategies assessed are associated with the drinking outcomes . avoidant coping and politically oriented coping are associated with both alcohol consumption and problematic drinking , but in opposite directions . that is , greater avoidant coping is associated with greater alcohol consumption and problematic drinking , whereas greater politically oriented coping is associated with less alcohol consumption and problematic drinking . the lack of correlation between active coping and support coping and each of the drinking outcomes , respectively , provides some indication that not all of the coping strategies may be useful in understanding the associations between economic stressors and drinking - related outcomes . additionally , the possibility that coping resources may vary by gender is not strongly supported by the pattern of correlations reported , with the exception that women reported significantly more emotional support than men . however , and consistent with previous research , women are found to drink less and less problematically . the hypothesized associations between economy - related stressors , coping strategies , and the alcohol - related outcomes are further elaborated upon in the structural equation model . estimation of the first model ( figure 2 ) , including only economic stressors , the drinking - related outcomes , and sociodemographic controls , produces a just identified model and , as such , meaningful fit statistics are not provided . the standardized path coefficients demonstrate that economic stressors and each of the drinking - related outcomes considered are significantly and positively related . net of the sociodemographic controls , greater economic strain is associated with greater alcohol consumption over the past month ( = 0.094 , s.e . = 0.004 , and p < 0.01 ) and a higher incidence of problematic drinking over the past year ( = 0.181 , s.e . sem analysis testing the second model , of the hypothesized associations between economy - related stressors , coping strategies , and the alcohol - related outcomes considered , is presented as figure 3 . in figure 3 , solid lines indicate effects that are statistically significant , and dashed lines indicate effects that are not statistically significant . the model fit criteria provided by hu and bentler ( cfi > 0.95 ; rmsea < 0.06 ; srmr < 0.08 ) are used to assess the measurement model . based on these criteria , there is consistent evidence of good fit for this model ( cfi = 0.982 ; rmsea = 0.046 ; this model demonstrates that economic stress is associated with higher levels of each of the coping resources considered . that is , greater economic strain appears to predict a greater propensity to use both maladaptive coping tactics ( i.e. , avoidant coping ) and adaptive coping tactics ( i.e. , active coping , support coping , and politically oriented coping ) . avoidant coping is associated with higher levels of past - month drinking and problematic drinking , whereas politically oriented coping is associated with less alcohol consumption and problematic drinking . formal mediation tests next reveal that the effects of economy - related stressors on the alcohol - related outcomes assessed are partly explained by variation in avoidant coping and politically oriented coping . significant indirect effects are found for the pathways from economy - related stress to both past - month drinking and problematic drinking . of the total effect of economy - related stress on past - month drinking ( 0.071 ) , 0.047 is accounted for by the indirect effect of economy - related stress through avoidant coping and 0.024 is explained by the indirect influence of politically oriented coping . a similar pattern emerges with respect to the relationship between economy - related stressors and problematic drinking . the indirect effects of avoidant coping account for 0.046 of the total effect of economic stressors on problematic drinking ( 0.098 ) and politically oriented coping explains 0.032 . taken together , these findings indicate that both adaptive and maladaptive coping strategies come to bear on drinking patterns associated with economic strain . on the one hand , economic strain appears to be associated with drinking more and more problematically , in the extent to which it is associated with a tendency to engage in avoidant coping strategies . but , then , again , economic strain is also associated with greater politically oriented coping , which is protective against alcohol use and misuse . moderation tests next determine whether the mediating effects of the coping strategies investigated vary by gender . however , the mediating effect of avoidant coping for the economy - related stressor problematic drinking association differs for men and women in this sample . this effect is displayed in figure 4 , which presents the predicted pattern of gender contrasts in the effects of avoidant coping on problematic drinking based on the mean , plus and minus two standard deviation values of avoidant coping ( as displayed in table 1 ) . as shown in figure 4 , the mediating effects of avoidant coping differ for men and women because the relationship between avoidant coping and problematic drinking is significantly less strong for women compared to men ( = 0.319 , s.e . thus , the observation that economic strain is associated with greater problematic drinking in the extent to which it is associated with avoidant coping strategies appears to be more pronounced among men than among women . the findings from this study support a broadened conceptualization of modes of coping in stress paradigm - oriented research to encompass politically oriented coping , in addition to modes of coping outside of the political realm which have predominated in studies of the coping - related moderators or mediators of the associations between social stressors and drinking outcomes . moreover , given our focus on economic stressors deriving , at least in part , from the macrolevel social forces producing the great recession , we suggest that politically oriented coping is particularly salient as a mode of behavioral adaptation in relation to societally engendered stressors as opposed to stressors that are less affected by macrolevel social forces . while this study specifically addressed deleterious drinking consequences of the recent great recession , social scientists have also delineated broader social - structural and political forces occurring over the last three decades which have led to pervasive job insecurity across all sectors of the us workforce and the erosion in the standard of living for most of the population [ 4143 ] . these phenomena include globalization and the outsourcing of work , the downsizing of corporate entities , the shift from secure semiskilled industrial jobs which paid a living wage to low wage service sector jobs , an increase in contingent workers with lower pay , and lack of job security and fringe benefits . thus , politically oriented coping encompassing the goal of changing governmental policies ( e.g. , rallying for changes in the tax structure influencing the distribution of wealth throughout society , fighting for government stimulus policies oriented toward job creation , or rallying support for raising the government - mandated minimum wage ) may prove to be a more efficacious mode of coping with economic stressors and more protective against deleterious drinking outcomes compared to nonpolitically oriented active coping activities such as looking for a job when unemployed , especially if adequate numbers of jobs relative to demand do not exist and a large proportion of jobs that do exist pay less than a living wage . consistent with our gender - linked hypothesis , our data showed that males and females did not differ either in the use of politically oriented coping or in the extent to which politically oriented coping was protective in relation to drinking outcomes . however , our data showed that male but not female avoidant coping significantly predicted problem drinking . thus , with regard to nonpolitically oriented coping with economic stressors , males clearly utilized a coping mode that was maladaptive . this finding might be seen as congruent with early male socialization patterns which have been viewed as fostering men 's sense of self - importance rather than connectedness in social relationships insofar as men 's avoidance in dealing with economic problems may affect both themselves and others close to them . alternatively , avoidant coping may predict problem drinking in men but not women to the extent to which males are socialized to drink more heavily than women . our findings should be viewed within the context of the methodological limitations of this study . the politically oriented coping measure which we developed for this study represents an initial attempt to operationalize this concept . however , it is limited to 4 items in contrast to the much longer nonpolitically oriented coping instrument and does not differentiate politically oriented coping tactics into discrete modes , similar to the measurement of nonpolitically oriented coping . although factor analysis supported the inclusion of the indicators of politically oriented coping as a single measure , additional work on the concept of politically oriented coping would be useful , with a differentiation between alternative types of politically oriented coping . first , the differentiation between individual modes of political action ( e.g. , voting , signing petitions , and donating money ) and collective political activities ( participation and leadership roles in different types of political action groups ) would be useful . secondly , some types of collective coping could be viewed as relatively more adaptive versus maladaptive . for example , the tea party social movement was motivated by both economic and cultural concerns [ 45 , 46 ] . however , one might differentiate between the tea party articulation of problem - focused social policies such as its belief in the need to diminish the size of government and racially oriented collective venting , such as screams of kill him by audience members in response to sarah palin 's critique of barak obama at rallies during the 2008 presidential election . second , this study utilized cross - sectional data and , thus , likely provides only a snapshot of the complex processes linking economy - related stressors , coping strategies , and drinking outcomes . while our theoretical framework postulated that exposure to economic stressors coupled with particular modes of coping leads to problematic drinking , it is possible that problem drinking coupled with maladaptive coping might make one prone to experiencing economic stressors such as losing one 's job . moreover , we lack data regarding alcohol consumption prior to exposure to the stressors focused on in this study and the extent to which alcohol may have been used as a coping mechanism . thus , further longitudinal studies are necessary to more clearly delineate the causal directions of the relationships between economic stressors , modes of coping , and drinking outcomes . third , the study methodology encompassed random - digit - dialing for recruiting the sample , thus only reaching individuals with landline telephone numbers . consequently , individuals relying on cell phones only , along with households without access to any telephone , were not included in this study . this potential noncoverage error is a source of concern because comparisons of our data with the us population revealed that the sample underrepresented african americans , latinos , and younger ( < age 40 ) and less - educated ( high school or less ) persons . however , our data were weighted to reflect the demographics of the overall population , and we compared weighted and unweighted estimates of each of our dependent variables to determine if nonresponse and/or noncoverage may have introduced serious bias into one or more of them . in each instance , we found that the weighted values of each measure fell well within 1 sd of the unweighted values , suggesting that the distributions of our key measures were not appreciably influenced by the underrepresentation of particular demographic groups . despite the noted limitations , this study extends prior research on the moderators and mediators of the social stressor - drinking outcome relationships to broaden notions of coping to include politically oriented coping . future studies incorporating this mode of coping may more clearly elucidate the political dynamics involved in both the macrolevel production of social stressors and the deleterious alcohol - related consequences of these stressors . this line of research would also have implications for the treatment of alcohol - related problems . in particular , considerations of more adaptive modes of coping might go beyond recommending individual behaviors such as job seeking by unemployed individuals to also suggest politically oriented coping to collectively try to influence the social conditions such as unemployment levels that may give rise to the propensity to self - medicate distress through the use of alcohol .

research derived from the stress paradigm suggests that certain types of coping ( e.g. , problem - focused coping instead of behavioral disengagement ) are protective against problem - related drinking to deal with social stressors . going beyond the typical focus in the coping literature , we hypothesize that stressors engendered by macrolevel social forces may require coping actions within the political realm in contrast to modes of coping focused outside of the political realm . a united states sample of 663 respondents completed a mail survey in 2010 , including measures of stressful consequences of the great recession , drinking patterns and problems , modes of coping encompassed in the brief cope instrument , and politically oriented coping . structural equation modeling examined whether modes of coping mediated the links between stressors and drinking outcomes . a substantial portion of the associations between stressors and drinking was explained by modes of coping . politically oriented coping was protective against problem drinking for both genders . future studies should further explore politically oriented coping in addition to modes of coping outside of the political realm when studying the relationships between macrolevel social stressors and deleterious drinking outcomes .

the development of titanium fixtures has brought several benefits for the rehabilitation of edentulous patients . when biological and mechanical principles are respected , this treatment modality may successfully restore the functional and esthetic impairments caused by tooth loss3 . in spite of the significant evolution of a number of implant systems , implant design and features , such as those related to the mechanical behavior of implant - supported prostheses , dental prostheses do fail during function mainly due to abutment and prosthesis screw loosening and/or fracture . in addition , it has been reported that abutment screw loosening is only surpassed by loss of osseointegration as the main cause of failure on implant - supported restorations , as shown in longitudinal follow - up studies5 . when two metal surfaces are in contact , adhesion and friction forces do limit the movement between them . an applied method aimed to reduce this friction and improve adhesion consists of interposing a lubricating film between these surfaces . a metal with low shear strength , such as pure gold , may act as a dry lubricant . when compared to screws without gold coating , it has been found that gold - coated abutment screws subjected to torques of 12 , 20 , and 32 ncm aiming a 0.0064 mm opening of the implant - abutment interface presented 26 , 24 , and 24% of preload increase , respectively7 . in another study , it was found that when a torque force is applied to an abutment screw , a significant part of this force is lost due to friction between the contact points of metal surfaces , inhibiting the rotation of the screw . thus , decreasing of friction between metallic surfaces may increase the screw rotation and , consequently , the preload . the rotation of gold - coated abutment screws placed with torque forces of 12 , 20 , and 32 ncm increased 73 , 76 , and 62% , respectively , when compared to titanium abutment screws2 . the purpose of this study was to evaluate by strain gauges the preload and torque removal values on three abutment screws ( gold , titanium , and titanium with surface treatment screws ) applying an equivalent torque force ( 30.070.28 ncm ) . three machined self - tapping external hex titanium screws with 4 mm in diameter and 15 mm in length were used in this investigation ( master screw , conexo sistemas de prtese ltda . three transmucosal cera one type abutments ( 2.0 mm height , cera one , conexo sistemas de prtese ltda . , reference 045022 , batch # 5080915121 ) , designed for cemented single standing implant - supported restorations were also used . the transmucosal abutments were attached to the fixtures using 10 gold screws ( conexo , sistemas de prtese ltda . , reference 121022 , batch # 5073147 ) ( figure 1 a a ) , 10 titanium screws ( conexo sistemas de prtese ltda . , reference 121024 , batch # 5063223 ) ( figure 1a b ) , and 10 surface treated titanium screws ( ti - tite , conexo sistemas de prtese ltda . , reference 121026 , batch # 5063035 ) ( figure 1a c ) . a load cell adapted from a model described in the literature this load cell presented a central void for fixture fixation in its upper portion and a horizontal plate for fixation of four strain gauges on its lower portion ( figure 1 b ) . the abutments were placed on the upper plate of the cell ( figure 1 c ) in such way that the contact between abutments and fixtures was free of any interference . therefore , when the abutment was fixed to the fixture by the abutment screw , tensions generated by this fixation pulled the unit against the abutment , producing a deformation in the lower plate connected to the strain gauges . next , the force ( preload ) generated on the abutment screws was captured by the strain gauges in volts ( v ) and later converted to newton ( n ) . the aforementioned sample was arranged into 3 different groups according to the characteristics of each abutment screw : group a was formed by gold screws , group b was formed by titanium screws , and group c was formed by surface - treated titanium screws . to determine fixation and removal torques , a digital torquemeter was used ( torqueleader , model tsd150 , type i , class e , part # 117317 ) , previously calibrated according with iso 6789:2003 ( e ) standard . a square hand wrench ( 1.27 mm diameter , conexo , , reference 062300 , batch # 5091632 ) was attached to the torquemeter to allow proper connection between torquemeter and screws ( figure 1 d ) . following the proper assembly of fixture and abutment on group a , the gold screw was then attached to the fixture with an applied torque force of 30.070.28 ncm , being repeated afterwards for groups b and c. then , the initial preload value for the abutment screws was determined ( in volts ) and all screws were kept in their positions for 5 minutes for preload stabilization , according to the literature9 . during this the preload stabilization period , preload values were obtained after 1 , 2 , 3 , 4 , and 5 minutes after fixation , and thereafter a mean value was calculated . after this evaluation , the abutment screw was removed using a digital torquemeter and the maximum value of reverse torque force required for screw removal was recorded in ncm . next , the same screw was fixed again another 4 times , obtaining the preload and torque removal mean values . nine other screws from group a as well as 10 other screws from b and c groups were subjected to the same procedure described above . the values of fixation torque forces of the 3 groups were analyzed statistically by to one - way analysis of variance ( anova ) . comparison of preload values within groups was possible using the mean of 6 measurements ( 0 to 5 minutes ) obtained at the first abutment fixation , using two - way anova . one - way anova was used to compare the mean preload values among the groups . tukey 's test was used for multiple comparisons among the groups . for all statistical tests , all fixations for each screw ( n=5 ) were considered for evaluation of the torque removal to obtain an average value , and the statistical analysis was performed similarly to the preload data . two - way anova was used to evaluate the mean torque removal among the groups , and one - way anova to evaluate the differences between screws . tukey 's multiple comparison test was used for individual comparisons among the groups . for all statistical tests , the mean fixation torque force when all screws were considered was 30.070.28 ncm ( c.i . there were no statistically significant differences ( p=0.1244 ) among the groups regarding the fixation torque . the gold screws presented the highest preload values and the titanium screws the lowest values ( figure 2 ) . the mean preload values of the gold screws was 131.728.98 n , and presented statistically significant differences ( p<0.01 ) compared to groups b and c. screw # 3 and screw # 1 presented the highest ( 140.48 n ) and the lowest ( 117.73 n ) preload values , respectively . the titanium screws presented statistically significant differences ( p<0.01 ) ( mean 37.035.69 n ) . the highest preload value was obtained for screw # 7 ( 49.68 n ) , while screw # 3 presented the lowest value ( 25.30 n ) . in the group of surface - treated titanium screws , the mean preload value was 97.784.68 n. statistically significant differences ( p<0.01 ) were also observed in this group , ( maximum : 104.09 n for screw # 4 ; minimum : 90.28 n for screw # 2 ) . the removal torque presented statistically significant differences among the analyzed materials ( p<0.001 ) ( figure 3 ) . the gold screws presented a mean removal torque of 17.641.12 ncm , i.e. they did not shown statistically significant differences ( p=0.3713 ) . titanium screws presented a mean removal torque of 18.751.89 ncm , and showed statistically significant differences ( p<0.001 ) . the group of surface - treated titanium screws present statistically significant difference ( p=0.004 ) ( mean : 16.431.33 ncm ) . the results of this study are similar to those of previous investigations4,8 , which found statistically higher preload values for gold fixation screws in comparison to other tested materials , in spite of using different methodologies and materials . a maximum preload value of 666.4 n for gold screws and 458.2 n for titanium screws was reported8 . in addition , the finds of the present study suggest that the evaluated surface treatment of titanium screws was effective , since these screws presented higher preload values than conventional titanium screws . most previously published reports in the dental literature do not specify the removal torque of abutment screws . the effect of different cyclic loads has been evaluated using a 32 ncm torque for fixation of the screws , which was repeated after 10 minutes to avoid contact relaxation6 . several manufacturers have suggested this clinical approach to decrease screw loosening . in the present study , to allow a direct comparison between results of preload and torque removal for the studied groups , all screws received a similar fixation torque of30.070.28 ncm , despite the manufacturer 's recommendations of using 35 ncm torque for the ti - tite screws . this was performed to eliminate a possible bias caused by elastic deformation that might occur in titanium screws during fixation , which might not only quantitatively but qualitatively influence the study outcomes and impair comparison among groups . this approach allowed that , in this investigation , only the screw was left as a study variable . another relevant issue is that up to 10% of the initial preload may be lost to smooth contact surfaces ( embedment relaxation ) , rather than elongation stresses . however , it has been previously observed that when the same screw is fixed several times , its preload values increased4 . comparison to different results obtained in other studies might be a difficult task due to variations of tested products and variables that may influence the produced preload value , such as the elastic modulus of the screws , opposing joint surfaces , abutment design , friction coefficient , lubrication , rate of the applied torque and the adaptation between the fixture hexagon and the abutment2 . the findings of the present study suggest a trend for greater preload values verified for gold screws , followed by surface - treated titanium screws and titanium screws , confirming the outcomes of previous investigations4,8 . finite element analysis has been used to evaluate preloading through elongation of the abutment screw . a study evaluating the preload of two different systems of the same implant manufacturer in function , found that the optimal preload produced should be 75% of the screw yield stress , and also that the preload value increases dramatically as the friction coefficient between implant and screw decreases7 . as loosening of implant / abutment joint causes clinical problems , another question that may be raised is whether the torque values recommended by the manufacturers should be increased in order to obtain greater longevity of screw tightening . the preload produced by gold or titanium fixation screws can be evaluated through the measurement of elongation stresses . it has been found that the stresses were 60% lower than the strengths against the torques applied according to the manufacturer 's instructions4 . on the other hand , it has been reported that gold or titanium screws could support higher torques than those indicated by the manufacturers without presenting plastic deformations , although it has been recommended elsewhere that the stresses should not exceed 65% of the screw 's fracture strength1 . although the results of this study showed that gold screws have a clear superiority of the produced preload , application of cyclic loads would be required for a closer simulation of the masticatory function on implanted - supported restorations . it may be concluded : 1 . gold should be the material of choice for abutment fixation screws , since it produced the highest preload values , followed by surface - treated titanium screws and conventional titanium screws ; 2 . titanium screws presented the highest torque removal values , followed by gold screws , and surface - treated titanium screws .

several authors still consider the mechanical problems of fracture and component loosening as the main causes of failure of implant - supported restorations . the purpose of this in vitro study was to compare the preload of three types of screw for transmucosal abutment attachment used in single implant - supported prosthesis through strain gauge and removal torque measurements . three external hex fixtures were used , and each received a transmucosal abutment ( cera one ) , which was fixed to the implant with its respective screw : group a- gold screw , group b- titanium screw and group c- surface - treated titanium screw ( ti - tite ) . ten screws of each type were attached applying a 30.070.28 ncm torque force and maintained in position for 5 minutes . after this , the preload values were measured using strain gauges and a measurement cell . gold screws presented higher preload values ( 131.728.98 n ) , followed by surface - treated titanium screws ( 97.784.68 n ) and titanium screws ( 37.035.69 n ) . anova ( p<0.05 ) and tukey 's test ( p<0.05 ) were applied . statistically significant differences were found among the groups for both preload and removal torque values . in conclusion , gold screws may be indicated to achieve superior longevity of the abutment - implant connection and , consequently , prosthetic restoration due to greater preload values yielded .

the central nervous system ( cns ) represents an important target for hiv infection during multiple stages of the disease : early , after invasion of the host , since the virus rapidly enters the cns , which then constantly acts as a viral reservoir ; lately , subverting its function and causing peripheral neuropathies and neurocognitive disorders ; and lastly , during the final stage of neuroaids , triggering opportunistic cns infections , cancers , and dementia ( tardieu and boutet , 2002 ) . highly active antiretroviral therapy ( haart ) , a combination of drugs that inhibits enzymes essential for hiv replication , can reduce the viremia and the onset of opportunistic infections in most patients , and prolong the survival ( see table 1 ) . although haart has reduced the incidence of clinical signs of neurological disease in hiv - infected individuals , autopsy studies suggest that there has been no corresponding decline in the incidence of inflammatory lesions in the cns . haart currently in use in the clinical practice . among the limits of the current treatments the most noticeable is the inability to eradicate hiv - infected cells , both , limiting the time frame in which haart initiated after exposure to hiv can prevent infection , and allowing replication - competent virus that persists in infected cells to emerge rapidly after the cessation of haart . according to those considerations , there is evidence that haart is less effective in lowering virus replication in the cns than in the blood ( gisolf et al . , 2000 ) ; and haart resistant viruses are more often found in the cerebrospinal fluid ( csf ) than in biological samples ( cinque et al . , the principal challenges in drug development for cns hiv infections are : ( 1 ) understanding the pathways for viral entry , replication , reservoirs , and glial activation in order to identify new targets for treatment , ( 2 ) developing selective therapeutics agents for those targets , or shaping effective strategies to deliver them within the cns , and finally but not to underestimate ( 3 ) collaborating with pharmaceutical companies to test promising drugs in clinical trials , and eventually exploit them in clinical practice . hiv is known to productively replicate in macrophages and microglia of the cns , but it also infects , via a cd4-independent pathway , macroglial cells such as astrocytes and most - importantly brain microvascular endothelial cells , which represent the major component of the blood brain barrier ( bbb ; bissel and wiley , 2004 ) . although it is generally accepted that neurons do not become infected by hiv , recent reports have shown that neuronal progenitor brain cells can be infected by various viral strains ( i.e. , hiv-1 ) , eventually playing a role as viral reservoirs ( lawrence et al . , 2004 ) . moreover , one of the important features of cns infection is that its viral populations can diverge from those of plasma : indeed , genetic compartmentalization of csf hiv-1 infection has been nicely demonstrated in a series of studies using the heteroduplex tracking essay to compare blood and csf viral populations cross - sectionally and longitudinally ( ritola et al . , 2005 ) ; therefore even if during acute infection the two population are identical , during chronic infection and especially in cases of hiv encephalitis they genetically and functionally diverge , and may justify the failure of antiretroviral therapy ( strain et al . , 2005 ) . identification of viral and cellular mechanisms in the cns that control virus replication after acute infection , maintain latency during the asymptomatic stage , and mediate resurgence of virus replication and development of hiv - associated encephalitis during late infection , is critical . the dynamic signaling pathways that are activated by virus replication and active / infiltrating macrophages and lymphocytes and the cytokines , chemokines , and neurotoxic products they elaborate may interfere with the homeostatic signaling pathways that maintain normal quiescent brain function . to understand the correlates in terms of neuroprotection , neuroinflammation , and neurodegeneration processes many preclinical models have been developed , such as : hiv transgenic rat ( i.e. , tg rat from reid et al . , 2001 ) , or mice ( i.e. , gp 120 tg mice from toggas et al . , 1994 ) , but also those obtained by injection of hiv proteins ( i.e. , gp 120 from sundar et al . , 1991 ; or tat and bansal et al . , 2000 ) , or hiv - infected macrophages into the brain of mice ( anderson et al . , 2003 ) the most studied signaling pathways include , among others , the gsk3 , cdk5 , erk , notch , p38 , and jnk cascades . for instance , it is noteworthy that many mitogen - activated protein kinases are pivotal during acute , asymptomatic , and terminal infection : some of them seems to promote cell growth and differentiation in response to initial infection ( i.e. , erk ) , whereas others are associated to growth arrest , apoptosis , and oncogenic transformation during aids . cns infection : in contrast to hiv - infected cd4 + t cells , infection in brain macrophages and microglial cell lines leads to an extended life span and elevated survival against apoptotic stresses ( chugh et al . , 2007 ) , and only during terminal infection , when there is resurgent virus replication , the balance favors neuronal death ( i.e. , via activation of jnk and p38 ) reflecting a failure to maintain homeostasis in the cns ( barber et al . , 2004 ) with the consequent motor and cognitive impairments . many strategies are currently under evaluation to improve hiv treatment , but a relatively small number are primarily dedicated to the alleviation of neurological complaints or the control of neuroaids , and unfortunately more than 98% of drug candidates for cns disorders never make it to the clinic ( pardridge , 2003 ) . herein are a couple of examples picturing how researches , initially conducted for neoplastic , neurodegenerative , or epileptic disorders , on specific pathways and delivery systems , might be adapted and applied to the field of cns hiv infection ( see figure 1 ) . neutralization of vif on microvascular endothelial cells ( ifn and ifn ) and subsequent inhibition of viral entry into the cns ; 3 . inhibition of hiv replication in macrophages and viral spread into the cns ( nk-1-agonists ) ; 4 . inhibition of cytoprotective effect exerted by tat proteins over microglial cells which act as cns viral reservoirs ( pi3k and akt inhibitors ) ; 5 . protection of neural integrity and plasticity along with prevention of neurotoxicity and neuronal death ( glutamate receptors- , gsk3- , jnk- , and p38-inhibitors ) . discovered in 1898 , mitoguazone ( mgbm ) had been investigated but abandoned as an anti - cancer drug , recently it was found to cause selective loss of cd16 + hiv - infected cells , specifically by blocking osteopontin needed to transform a circulating macrophage into a cns / resident one . mgbg although ineffective in reducing hiv viremia , does appear to shut down viral evolution by selectively killing recent migrants macrophages rather than resident cells , and to be safe over the long - term treatment in aids patients ( jeymohan et al . , 2009 ) . the manipulation of ifn and ifn apobec pathways could prevent hiv invasion of cns because of the potent intrinsic immunity exerted by the expression of apobec3 g gene in microvascular endothelial cells ( argyris et al . , 2007 ) . this gene code for proteins possessing cytidine deaminase activity and allow the bbb to actively neutralize the effects of viral infectivity factors ( vif ) , which are essentials for viral entry and spread into the cns ( jeong et al . , 2008 ) . nk-1 , also known as substance p , was the first neuropeptide discovered in 1931 , it is widely distributed in the central and peripheral nervous system where it functions as a potent mediator of immunoregulation . agonists to nk-1 receptor potently inhibit both in vitro and in vivo hiv replication in human macrophages , apparently down regulating ccr5 ( jeymohan et al . , 2009 ) . initially recognized , and extensively studied as a promising target for anti - cancer therapies because of its role during tumorigenesis , the pi3k / akt cell survival pathway became a target for anti - hiv treatment after the observation that pi3k inhibitors ( such as wortmannin and ly294002 ) or akt inhibitors ( such as miltefosine ) , both able to cross the bbb , contrast the cytoprotective effect exerted by tat proteins over macrophage / microglial cells , and make them again susceptible to cell death following an apoptotic stimulus ( chugh et al . most interestingly pi3k / akt inhibitors proved to be effective without harming uninfected cells and therefore experimental data as far available support their possible use for hiv - therapy and targeting of long - lived viral reservoirs . nanoformulated haart essentially manipulates the immune system for therapeutic purposes by using synthetic or biological nanoengineered drug carriers . those nanoformulated drugs have been proven to be efficient delivery vehicles for a wide range of therapeutic agents , and can be easily injected in the blood circulation or virtually into any site of the brain with minimal invasiveness . major concerns to this therapeutic approach are the choice of drug administration ( systemic or local ) and consequently the residence in the systemic blood circulation , or in the brain tissue adjacent to topic injection , the drug loading efficiency , and the burst release effect . furthermore , many other question marks are still to be answered , or even formulated . concerning synthetic carriers , hyaluronic acid , a linear polysaccharide composed of alternating d - glucuronic acid and n - acetyl - d - glucosamine units , because of its immunoneutrality , has been proposed as a biocompatible and biodegradable biomaterial for tissue engineering , and in drug delivery systems into the cns : particularly appealing would seem the opportunity to produce drug - incorporated hyaluronic acid nanoparticles ( jeong et al . , 2008 ) . as well monocytes , because of their abilities to move drugs inside the cells have advantages as a depot for haart , but also they might improve compliance as drugs can be released from cells for periods up to weeks or longer . nevertheless , further application of biological nanoengineered drug carriers will require optimization of nanoparticles uptake , deeper knowledge of their kinetics across the bbbs , and more sophisticated imaging systems to track migration of monocyte macrophages to the brain and target the delivery of specific therapeutics ( jeymohan et al . , 2009 ) . beside compounds aimed at control the virus , others try to treat the effects of the underlying neuroinflammation and neurodegeneration mechanisms , and might provide a beneficial supplement to haart . neurotoxicity is mainly mediated through glutamate production , which is triggered by nmda subtypes of glutamate receptors , and correlated to caspase activation and subsequent neuronal apoptosis ( erdmann et al . , 2006 ) ; accordingly , pharmacologic inhibition of glutaminase directly correlate to neuronal survival ( tian et al . , 2008 ) . those insights support the hypothesis that inhibition of glutamate production by blocking mitochondrial glutaminase activity may clinically prevent neurotoxicity during hiv-1 infection . taken together the gsk3 , cdk5 , jnk , and p38 signaling pathways are important regulators of neurotoxicity , being responsible for synaptic and dendritic damage to pyramidal neurons , loss of immunoreactive neurons , and demyelinization . gsk3 inhibitors ( i.e. , lithium , valproic acid ) as well as cdk5 inhibitors ( i.e. , roscovitine ) seems to protect neuronal integrity and plasticity ( crews et al . , 2009 ) , while jnk- and p38-inhibitors ( i.e. , minocycline ) may prevent neuronal death ( lin et al . , 2001 ; bozyczko - coyne et al . , 2002 ) each of those target holds promise for the development of treatment strategies to ameliorated the neuropathological effects exerted by hiv proteins ( tat , vif , etc . ) . discovered in 1898 , mitoguazone ( mgbm ) had been investigated but abandoned as an anti - cancer drug , recently it was found to cause selective loss of cd16 + hiv - infected cells , specifically by blocking osteopontin needed to transform a circulating macrophage into a cns / resident one . mgbg although ineffective in reducing hiv viremia , does appear to shut down viral evolution by selectively killing recent migrants macrophages rather than resident cells , and to be safe over the long - term treatment in aids patients ( jeymohan et al . , 2009 ) . the manipulation of ifn and ifn apobec pathways could prevent hiv invasion of cns because of the potent intrinsic immunity exerted by the expression of apobec3 g gene in microvascular endothelial cells ( argyris et al . , 2007 ) . this gene code for proteins possessing cytidine deaminase activity and allow the bbb to actively neutralize the effects of viral infectivity factors ( vif ) , which are essentials for viral entry and spread into the cns ( jeong et al . nk-1 , also known as substance p , was the first neuropeptide discovered in 1931 , it is widely distributed in the central and peripheral nervous system where it functions as a potent mediator of immunoregulation . agonists to nk-1 receptor potently inhibit both in vitro and in vivo hiv replication in human macrophages , apparently down regulating ccr5 ( jeymohan et al . , 2009 ) . initially recognized , and extensively studied as a promising target for anti - cancer therapies because of its role during tumorigenesis , the pi3k / akt cell survival pathway became a target for anti - hiv treatment after the observation that pi3k inhibitors ( such as wortmannin and ly294002 ) or akt inhibitors ( such as miltefosine ) , both able to cross the bbb , contrast the cytoprotective effect exerted by tat proteins over macrophage / microglial cells , and make them again susceptible to cell death following an apoptotic stimulus ( chugh et al . most interestingly pi3k / akt inhibitors proved to be effective without harming uninfected cells and therefore experimental data as far available support their possible use for hiv - therapy and targeting of long - lived viral reservoirs . nanoformulated haart essentially manipulates the immune system for therapeutic purposes by using synthetic or biological nanoengineered drug carriers . those nanoformulated drugs have been proven to be efficient delivery vehicles for a wide range of therapeutic agents , and can be easily injected in the blood circulation or virtually into any site of the brain with minimal invasiveness . major concerns to this therapeutic approach are the choice of drug administration ( systemic or local ) and consequently the residence in the systemic blood circulation , or in the brain tissue adjacent to topic injection , the drug loading efficiency , and the burst release effect . furthermore , many other question marks are still to be answered , or even formulated . concerning synthetic carriers , hyaluronic acid , a linear polysaccharide composed of alternating d - glucuronic acid and n - acetyl - d - glucosamine units , because of its immunoneutrality , has been proposed as a biocompatible and biodegradable biomaterial for tissue engineering , and in drug delivery systems into the cns : particularly appealing would seem the opportunity to produce drug - incorporated hyaluronic acid nanoparticles ( jeong et al . , 2008 ) . as well monocytes , because of their abilities to move drugs inside the cells have advantages as a depot for haart , but also they might improve compliance as drugs can be released from cells for periods up to weeks or longer . nevertheless , further application of biological nanoengineered drug carriers will require optimization of nanoparticles uptake , deeper knowledge of their kinetics across the bbbs , and more sophisticated imaging systems to track migration of monocyte macrophages to the brain and target the delivery of specific therapeutics ( jeymohan et al . , 2009 ) . beside compounds aimed at control the virus , others try to treat the effects of the underlying neuroinflammation and neurodegeneration mechanisms , and might provide a beneficial supplement to haart . neurotoxicity is mainly mediated through glutamate production , which is triggered by nmda subtypes of glutamate receptors , and correlated to caspase activation and subsequent neuronal apoptosis ( erdmann et al . , 2006 ) ; accordingly , pharmacologic inhibition of glutaminase directly correlate to neuronal survival ( tian et al . , 2008 ) . those insights support the hypothesis that inhibition of glutamate production by blocking mitochondrial glutaminase activity may clinically prevent neurotoxicity during hiv-1 infection . taken together the gsk3 , cdk5 , jnk , and p38 signaling pathways are important regulators of neurotoxicity , being responsible for synaptic and dendritic damage to pyramidal neurons , loss of immunoreactive neurons , and demyelinization . gsk3 inhibitors ( i.e. , lithium , valproic acid ) as well as cdk5 inhibitors ( i.e. , roscovitine ) seems to protect neuronal integrity and plasticity ( crews et al . , 2009 ) , while jnk- and p38-inhibitors ( i.e. , minocycline ) may prevent neuronal death ( lin et al . , 2001 ; bozyczko - coyne et al . , 2002 ) each of those target holds promise for the development of treatment strategies to ameliorated the neuropathological effects exerted by hiv proteins ( tat , vif , etc . ) combinations of drugs work better and longer than seen previously , patients benefit demonstrably from the magnitude and duration of viral suppression , and require more and more symptomatic treatment of neuroaids cognitive impairment . however haart is very demanding for patients , non - adherence can lead to therapeutic failure , and in addition drug resistance is a continuing problem ; therefore , the need for improved drug and delivery systems , but also an interest for adaptive treatment strategies and mathematical modeling to manage structured treatment interruptions ( rosenberg et al . , 2007 ) , have been outlined by many researchers . to this regard a thigh collaboration with industry is strictly required and introduces a new set of challenges such as : ( 1 ) circulation of promising ideas , often just developed with university grants , ( 2 ) protection of intellectual property and patent rights , ( 3 ) fund - raising opportunities for start - ups during preclinical researches on novel compounds , until ( 4 ) identification of potential blockbusters appealing to big pharma companies for introduction in the market and their clinical exploitation . finally , current criteria for initiation of haart do not consider cns outcomes as a benchmark of efficacy , thus establishing cns - based criteria for initiation of protocols should be the ultimate goal not only for therapeutic but also for neuroprotection strategies ; as recently stated by jeymohan et al . ( 2009 ) on behalf of the neuroaids research participants these priorities should be seen as a template for future growth of the field and its long - term impact . 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 .

the central nervous system ( cns ) represents an important target for hiv infection during multiple stages of the disease : early , after invasion of the host , acting as a viral reservoir ; lately , subverting its function and causing peripheral neuropathies and neurocognitive disorders ; and lastly , during the final stage of neuroaids , triggering opportunistic infections , cancers , and dementia . highly active antiretroviral therapy , a combination of drugs that inhibits enzymes essential for hiv replication , can reduce the viremia and the onset of opportunistic infections in most patients , and prolong the survival . among the limits of the current treatments the most noticeable is the inability to eradicate hiv - infected cells , both , limiting the time frame in which antiretroviral therapies initiated after exposure to hiv can prevent infection , and allowing replication - competent virus that persists in infected cells to emerge rapidly after the cessation of treatments . many strategies are currently under evaluation to improve hiv treatment , unfortunately more than 98% of drug candidates for cns disorders never make it to the clinic ; here in we report how nanoformulated strategies might be adapted and applied to the field of cns hiv infection .

colorectal adenocarcinoma is the fourth most frequently diagnosed malignancy in korea , accounting for 12% of newly diagnosed cancer cases . according to the ministry of health and welfare , the incidence of colorectal cancer has increased in both males and females during the past 2 decades . although surgery is potentially curative , approximately one third of all newly diagnosed patients present with inoperable metastatic disease . palliative chemotherapy is more effective than the best supportive care for improving overall survival ( os ) as well as quality of life in advanced colorectal cancer . while significant advances have been made in recent years , cure is rarely possible in advanced colorectal cancer ( acc ) , making further improvements in therapy imperative . for more than three decades , the treatment options for acc have been almost exclusively based on 5-fluorouracil ( 5-fu ) and folinic acid ( fa ) . until the early 1990s , 5-fu , often modulated by fa , was the single effective chemotherapy available , but only led to meaningful responses in a small minority of treated patients . the recent integration of oxaliplatin and irinotecan for the management of acc patients has extended median os in a meaningful way . both drugs have been shown to have synergistic effects with 5-fu and fa in colorectal cancer cell lines . since irinotecan and oxaliplatin have been used for acc treatment , the efficacy of palliative chemotherapy for acc has considerably improved . first - line treatment with irinotecan and either bolus or infusional 5-fu / fa , namely irinotecan / bolus fluorouracil / lv or 5-fluorouracil , folinic acid plus irinotecan ( folfiri ) , significantly improved outcomes as compared with 5-fu / fa . similarly , a combination of oxaliplatin and infusional 5-fu / fa , known as 5-fluorouracil , folinic acid plus oxaliplatin ( folfox ) , significantly increased the response rate and the time to progression compared with 5-fu / fa . these results established 5-fu - based chemotherapy , in combination with either irinotecan or oxaliplatin , as standard first - line chemotherapy regimens for patients with acc . furthermore , capecitabine has been shown to exhibit antitumor activity against acc both as a single agent and in combination with oxaliplatin [ 12 - 14 ] , as well as with irinotecan . in korea , capecitabine was approved in january 2006 as a partner with oxaliplatin or irinotecan for the treatment of acc . a decision regarding chemotherapy regimens for factors considered include the extent of disease , potential toxicities , especially for those with impaired oral intake or with decreased performance status , activity of chemotherapy , and the patient preference . we therefore decided to evaluate various combination chemotherapy regimens as first - line chemotherapy for acc . this report describes a single - center , retrospective study . between january 2006 and december 2007 , a total of 537 acc patients were treated with first - line chemotherapy for advanced disease at samsung medical center ( seoul , korea ) . fifty - nine patients were excluded because they were treated with single - agent chemotherapy . the criteria for case inclusion were as follows ; 1 ) histologically confirmed diagnosis of adenocarcinoma arising from colon or rectum , 2 ) no prior chemotherapy or radiotherapy except for adjuvant use , 3 ) advanced ( metastatic and/or recurrent ) disease , 4 ) availability of clinical data at the beginning of therapy and follow - up . we attempted to exclude patients who were enrolled in clinical trials to ensure the choice of chemotherapy regimen was solely at the discretion of the treating physician . all the data were prospectively recorded and only the survival data was updated at the time of analysis . written informed consent was given by all patients prior to receiving chemotherapy , according to institutional guidelines . the patients were required to have a life expectancy of 12 weeks of more ; age of at least 18 years or older ; eastern cooperative oncology group ( ecog ) performance status of 2 or lower ; adequate hematologic ( neutrophil count>1,500/mm and platelet count>100,000/mm ) , hepatic ( serum total bilirubin level<1.5 mg / dl and aspartate aminotransferase / alanine aminotransferase<3upper limit normal ) and renal ( serum creatinine level<1.5 mg / dl ) functions . our department guidelines define combination chemotherapy regimens as follows : in folfox or folfiri , fa is given at the dose of 200 mg / m followed by bolus 5-fu 400 mg / m on day 1 , and a 46-hour continuous infusion of 5-fu 2,400 mg / m on days 1 and 2 . oxaliplatin 85 mg / m or irinotecan 180 mg / m was administered on day 1 as a 2-hour infusion , concurrent with fa . in capecitabine plus oxaliplatin ( xelox ) and capecitabine plus irinotecan ( xeliri ) , oxaliplatin 130 mg / m or irinotecan 240 mg / m was given on day 1 in combination with oral capecitabine 1,000 mg / m twice daily on days 1 to 14 . folfox and folfiri were repeated every 2 weeks , whereas xelox and xeliri were repeated every 3 weeks until disease progression or unacceptable toxicity occurred , or until a patient chose to discontinue treatment . the dosages for subsequent cycles were adjusted according to the toxic effects that developed during the preceding cycle . the prophylactic use of hematopoietic growth factors was not allowed during treatment , except for patients with febrile neutropenia or grade 4 myelosuppression at the treating physician 's discretion . after this combination chemotherapy had failed , second - line chemotherapy was recommended to all the patients if their performance status was preserved . according to department policies , all tumor measurements were assessed every 2 or 3 cycles of chemotherapy by using an abdominopelvic computed tomography ( ct ) scan and other tests that were initially used to stage the tumor . tumor response and progression were evaluated according to the response evaluation criteria for solid tumors ( recist ) . the primary endpoint of this retrospective study was progression - free survival ( pfs ) . the date of disease progression or death from causes other than acc was used in calculating pfs . pfs and os were estimated according to the kaplan - meier method and the statistical significance of differences in survival curves between groups was tested with a log - rank test . multivariate models were used for exploratory purposes to examine the impact of each regimen on the outcomes of chemotherapy . covariates included were age ( below vs.median ) , gender , previous adjuvant therapy , an ecog performance status ( 0 - 1 vs.2 ) , number of involved sites ( 1 vs.2 ) , metastases ( liver , peritoneum , and lung ) , baseline chemistry profiles ( serum albumin , alkaline phosphatase , and bilirubin ) , combination with bevacizumab , and chemotherapy regimens . laboratory parameters were initially recorded as continuous variables and later dichotomized according to the median value of each variable ( below vs.median ) . between january 2006 and december 2007 , a total of 478 acc patients were treated with combination chemotherapy in a first - line setting : folfox ( n=172 ) , folfiri ( n=95 ) , xelox ( n=155 ) , and xeliri ( n=56 ) . in 29 patients , chemotherapy involved bevacizumab . . sixty percent of patients were male , and 33 patients ( 7% ) had an ecog performance status of 2 or more . more than half of the patients ( n=263 ) had received surgery for curative intent , and 206 patients received adjuvant chemotherapy or chemoradiotherapy . approximately one - third of the patients had two or more metastatic disease sites , mostly involving liver and abdominal lymph nodes . we noted that more folfiri or xeliri patients had received adjuvant therapy involving oxaliplatin than folfox or xelox patients . after first - line failure , second - line chemotherapy was administered for more than half of the patients ( n=290 ) . median follow - up duration was 40.6 months ( 95% confidence interval [ ci ] , 39.3 to 41.8 months ) . at the time of data collection , 449 ( 89% ) median chemotherapy durations for folfox , folfiri , xelox , and xeliri were 4.9 , 4.5 , 5.7 and 5.4 months , respectively . we found no statistically significant difference among chemotherapy durations for these regimens ( log - rank p=0.19 ) . the most common reason for discontinuation of treatment was disease progression . in 172 patients receiving folfox , two - thirds of patients discontinued therapy due to disease progression ( n=118 ) , physician 's recommendation or patient withdrawal ( n=33 ) , or toxicity ( n=21 ) . folfiri patients discontinued therapy due to progression ( n=60 ) , physician 's recommendation or patient withdrawal ( n=13 ) , toxicity ( n=20 ) , or unknown causes ( n=2 ) . similarly , disease progression was the most common cause of therapy discontinuation in 101 xelox patients and 34 xeliri patients . we found no relevant difference in the occurrence of overall grade 3 or 4 toxicities among regimens ( table 2 ) . in brief , the main difference in grade 3 or 4 toxicities with folfiri or xeliri was diarrhea ( 21% for folfiri and 27% for xeliri vs. 11% for folfox and 9% for xelox ; p=0.02 ) , and with folfox or xelox the difference involved peripheral neuropathy ( 9% for folfox and 7% for xelox vs. 2% for folfiri and xeliri ; p<0.01 ) . although patients who were treated with irinotecan - containing chemotherapy more frequently experienced grade 3 or 4 leukopenia ( 13% vs. 10% ; p=0.09 ) than those treated with oxaliplatin - containing regimens , we found no significant difference in the incidence of febrile neutropenia ( 5% in each arm ) . in addition , grade 2 or more hand - foot syndrome was more frequently observed in xelox and xeliri ( 14% and 12% , respectively ) than in folfox and folfiri ( 6% and 8% , respectively ) . in the current retrospective analysis , one death occurred in the midst of treatment with folfox , with no clinical evidence of progression having been demonstrated . another patient died of respiratory failure shortly after completion of the second folfox cycle , in which the possibility of disease progression could not be completely excluded . the patient , initially presenting with multiple pulmonary nodules and bilateral pleural effusion , complained about increasing dyspnea and suffered a sudden respiratory arrest . five other deaths occurred in patients while receiving folfiri or xelox , and were attributed to neutropenic sepsis . of a total of 478 patients , 61 could not be evaluated for responses because of the absence of any measurable lesions or early discontinuation of therapy . objective responses to chemotherapy were noted in 200 patients ( response rate , 48% ; 95% ci , 43 to 53% ) ; 42% for folfox , 49% for folfiri , 55% for xelox , and 51% for xeliri . the difference in the response rates for each regimen was not statistically significant ( p=0.09 ) . patients who had a poor performance status ( 2 in ecog scale ) were significantly less likely to respond to first - line chemotherapy ( 24% vs. 50% ; p<0.01 ) compared to those with an ecog performance status of 0 or 1 . response rate was not significantly influenced by age , gender , prior treatments , baseline laboratory parameters , metastatic sites , or chemotherapy regimen . the estimated median pfs for all patients was 6.8 months ( 95% ci , 6.3 to 7.3 months ) , and the median os was not yet reached . pfs was shorter , although statistically insignificant ( p=0.12 ) , in patients receiving folfox or folfiri ( 6.2 months and 6.0 months , respectively ) than those receiving xelox ( 7.9 months ) or xeliri ( 7.1 months ) . one - year pfs rates for folfox , folfiri , xelox , and xeliri were 24% , 21% , 32% , and 32% , respectively ( fig . 1 ) . a multivariate regression model revealed that pfs was significantly affected by performance status ( hazard ratio , 0.39 ; 95% ci , 0.28 to 0.57 ; p<0.01 ) . we also tested whether the pfs was modified by interaction between the effect of baseline clinical parameters and the chemotherapy regimens ; the first - level interaction term between these variables was entered into a separate multivariate model . however , we found no interaction between any first - line chemotherapy regimens for each clinical parameter . despite recent advances in the treatment of acc , patients treated with first - line chemotherapy have a median os rarely exceeding 24 months . in the current analysis , our observation that pfs was slightly shorter with folfox or folfiri than with xelox or xeliri is possibly related to negative prognostic factors influencing the choice of intravenous chemotherapeutic agents instead of oral agents . similarly , the choice of a chemotherapeutic agent depends on the ones previously used in the adjuvant setting . although this study is retrospective in nature , the results provide a piece of evidence that patients with acc may derive an indisputable benefit from fluoropyrimidine - based combination chemotherapy . the decision to use specific chemotherapeutic agent(s ) in patients with acc should be determined by their relative merits on a case - by - case basis . the evolution of treatment using irinotecan and oxaliplatin in combination with 5-fu regimens has resulted in a significant prolongation of life in patients with acc . since both drugs have demonstrated similar activity in first- and second - line settings , the optimal chemotherapy regimen and dosing schedule in patients with acc remains undecided . randomized trials comparing first - line folfox with folfiri indicated equivalent activity and moderate toxicity differences between these two regimens , suggesting that use of either oxaliplatin or irinotecan is reasonable . in another phase iii study in which patients crossed over to the alternative regimen upon progression while on their first - line regimen , the overall pfs on first - line therapy was 8.1 months for folfox and 8.5 months for folfiri . in a combined analysis of seven phase iii trials in acc , grothey et al . reported that the strategy of making these three active drugs ( fluoropyrimidine , oxaliplatin , and irinotecan ) available to patients with acc could maximize os . in addition to efficacy data , patient convenience and satisfaction are assuming increased importance in cancer management . oral administration has the advantage of permitting convenient , patient - orientated therapy , providing the patient with a degree of independence and improved quality of life , while avoiding complications associated with intravenous drug administration . chemotherapy regimens involving oral capecitabine showed similar safety and efficacy profiles compared with 5-fu - based regimens . oral capecitabine was proved to have a similar quality of life profile to that of 5-fu , but seemed to be more convenient in terms of administration . in korea , a capecitabine - containing regimen was approved in january 2006 as the first - line treatment of acc . data from the current retrospective study suggest that patients are highly compliant with capecitabine , when given in combination with either oxaliplatin or irinotecan . patients treated with capecitabine experienced a higher incidence of clinically relevant hand - foot syndrome , but this toxic effect was predictable and manageable . an improved understanding of the molecular pathogenesis of cancer has lead to the development of novel agents designed to target critical cellular pathways . one of the most successful novel approaches in the treatment of acc involves therapeutic agents that inhibit the neovascularization process of growing tumors . bevacizumab is a recombinant humanized anti - vascular endothelial growth factor monoclonal antibody that is being clinically evaluated in many tumor types , including acc . because bevacizumab was only recently made available in korea as first - line chemotherapy , only 29 patients received bevacizumab . this study confirms that both oxaliplatin- and irinotecan - containing combination chemotherapy are effective for the first - line treatment of acc . capecitabine clearly represents an effective and well - tolerated oral alternative to 5-fu . while the current study was primarily focused on cytotoxic therapies , the integration of targeted agents , including bevacizumab , is a novel standard option to further improve outcomes for patients with acc .

purposefluoropyrimidine - based combination chemotherapy , in combination with either oxaliplatin or irinotecan , has demonstrated efficacy and tolerability in treatment of advanced colorectal cancer ( acc).materials and methodsbetween january 2006 and december 2007 , a total of 478 acc patients were treated with combination chemotherapy in first - line settings . combination therapies included : 5-fluorouracil , folinic acid plus oxaliplatin ( folfox , n=172 ) , 5-fluorouracil , folinic acid plus irinotecan ( folfiri , n=95 ) , capecitabine plus oxaliplatin ( xelox , n=155 ) , and capecitabine plus irinotecan ( xeliri , n=56 ) . folfox and folfiri were repeated every 2 weeks , whereas xelox and xeliri were repeated every 3 weeks until occurrence of disease progression or unacceptable toxicity , or until a patient chose to discontinue treatment.resultsthe median age was 58 years ( range , 19 to 84 years ) and the median chemotherapy durations for folfox , folfiri , xelox , and xeliri were 4.9 , 4.5 , 5.7 , and 5.4 months , respectively . combination chemotherapy regimens were generally well tolerated . the estimated median progression - free - survival ( pfs ) for all patients was 6.8 months ( 95% confidence interval , 6.3 to 7.3 months ) . no statistically significant difference in pfs was found among regimens used as first - line chemotherapy . sixty percent ( n=290 ) of patients received second or further lines of therapy after failure.conclusionfluoropyrimidine-based combination chemotherapy regimens appear to be equally active and tolerable as first - line therapy for acc .

lymphomatoid granulomatosis ( lyg ) is a rare angiocentric and angiodestructive lymphoproliferative disease with granulomatous reaction involving the lungs most frequently , however , it may also involve the kidneys , skin and especially the central nervous system4,6,9,11 ) . lyg is characterized by an infiltration of atypical lymphocytoid and plasmocytoid cells , with granulomatous inflammation in an angiocentric and angiodestructive polymorphic cellular infiltrate6,8 ) . therefore the purpose of this study is to submit the first report about the case with thoracic spinal lyg after diagnosis with b - precursor childhood acute lymphoblastic leukemia ( all ) . after surgical decompression with biopsy on the spinal lesion , diagnosis of grade ii lyg was made and rituximab was administered . the patient is in good neurological condition without recurrence at the 6-year follow - up . a 4-year - old girl was diagnosed with b - precursor all with the involvement of kidneys and pancreas in november , 2004 . treatment was initiated according to the pediatric oncology group ( pog ) protocol 900612 ) . on april 12 , 2006 , the patient was admitted to pediatric clinic with a complaint of intermittent fever for 3 days . on april 19 , 2006 , 7 days after the admission , the patient developed a gait disturbance . neurological examination revealed paraparesis of grade 3 on medical research council ( mrc ) muscle strength grading scale1 ) with increased deep tendon reflex in both ankles , knee jerks , and bilateral positive babinski sign , but her senses were intact . cerebrospinal fluid ( csf ) findings were as follows : elevation of protein ( 243.3mg / dl ) , and decrease of glucose ( 44mg / dl ) . the condition was suspected to be an acute inflammatory demyelinating polyneuropathy and was treated with intravenous immunoglobulin ( 400 mg / kg / day ) for 5 days . however , her paraparesis was aggravated to grade 1 on mrc muscle strength grading scale from april 26 , 2006 , 14 days after the admission , and spinal mri was performed . t2 weighted mri demonstrated heterogeneous high signal intensity infiltration on t4 vertebral body and subligamental spreading soft tissue mass on t3 - 5 epidural space from t4 posterior vertebral body , causing spinal cord compression ( fig . 1 ) . on april 27 , 2006 , laminectomy of t3 , 4 with open biopsy were performed . the epidural mass was encircling the spinal cord mainly on ventral surface and was slightly grey to yellow , solid and compact nature . a histopathologic examination showed an angiocentric distribution of polymorphous lymphoid infiltrate , with extensive necrosis . immunohistoche mical staining for cd3 and cd20 showed the predominance of mature t cells and atypical b cells ( fig . hybridization revealed positivity for epstein - barr virus ( ebv ) encoded rna in a few lymphocytes , confirming the diagnosis of grade ii lyg . one - week post - operative neurologic examination showed paraparesis grade 3 on mrc scale , and bare independent walking but as she still had gait impairment , she received rehabilitation . after four weeks of the surgery , she restored her normal state and could walk quite well . at that time , there were no pulmonary lesions , so we suspected the spinal lesion as the primary involvement site . two weeks after , chest and abdomen ct revealed multiple low density nodular mass on the liver and the lower lobe of the left lung . histopathologically , lesions were not blastic in appearance and in immunophenotype , and the lesions were negative for cd10 , tdt , and cd22 , so we ruled out the relapse of all . she was treated with rituximab ( 275mg / m / day , 4 times per week ) for 4 weeks and intravenous high dose methylprednisolone ( 30mg / kg per day for 3 days , 20mg / kg per day for 2 days and 10mg / kg per day for 2 days ) . chest and abdomen ct showed marked improvement of the lesions 3 months after the treatment compared with the lesions in the images obtained before the treatment . a follow - up mri study performed 4 months after the operation documented no evidence of residual mass or recurrence ( fig . 3 ) . chest and abdomen ct performed 1 year after the operation showed no demonstrable focal lesion in lung field and mediastinum and unremarkable abdominal organs . her weakness improved and she was able to walk independently and had no gait impairment . but unfortunately we failed to take additional follow - up of mri for the patient , as she was terrified to take mri and full sedation was not possible . moreover the parents of the patient did not agree with the idea of taking mri one more time because she was in good state at that time . to date , lyg is a rare lymphoproliferative disease characterized by angiocentric and angiodestructive ebv - positive b - cell proliferation of uncertain malignant potential , which might be associated with exuberant reactive t cell infiltration2,9 ) . lyg affects men more than women with ratio 2 to 1 and presents mainly in adults between the fourth and sixth decades of life , although patients of all ages can be affected . its occurrence in children is quite rare4 ) . there were three cases of lymphomatoid granulomatosis with onset after all . two of these patients died of the disease , only one obtained benefit from acyclovir therapy7 ) . lyg usually affects lungs primarily , and it may also present several significant extrapulmonary manifestations , affecting skin , kidney , spleen , and the central nervous system . involvement of the spine has been rarely reported . to my best knowledge , this is the first report of thoracic spinal lyg presenting with unique progressive myelopathy . however , numerous granulomatous lesions in the central nervous system and lungs are highly suggestive of lyg . lyg with central nervous system involvement , observed in approximately 30% of the affected patients , indicates poor prognosis4,9,11 ) . there was only one case report about lyg with spinal involvement . in that case , lyg involve cervical spinal cord , and the patient submitted four cycles of adjuvant cyclophosphamide , doxorubicin , vincristine and prednisone ( chop ) . but the patient died 5 months after the treatment due to systemic progression of the disease7 ) . a grading system of lyg is based on the number of atypical lymphocytes , ebv - positive b - cells and amount of necrosis5 ) . pathophysiologically , grade i lesions consist of a polymorphous angiocentric and angiodestructive infiltrate of lymphocytes , plasma cells , and histocytes , with or without eosinophils . large lymphoid cells or immunoblasts were less than five in high power field ( 400 ) or absent . if large lymphoid cells or immunoblasts were present , there would be no cellular atypia and minimal - to - absent necrosis . grade ii lesions consisted of large lymphoid cells with less than twenty ebv - positive cells in high power field with some cellular atypia in small lymphoid cells , and necrosis was more common . grade iii lesions , angiocentric lymphomas , consisted of large atypical cells with more than twenty ebv positive cells in high power field with the marked cytologic atypia in both small and large lymphoid cells4,13 ) . to date , no standard treatment has been established for lyg and treatments approach to systemic lyg include drugs acting on the immune system(corticosteroids , interferon alfa-2b , cyclosporine a , anti - cd20 monoclonal antibody - rituximab ) , mono- or combined chemotherapy , radiotherapy , and autologous stem cell transplantation . several treatments with rituximab have been reported , with or without chemotherapy or radiotherapy3,9,10,15 ) . rituximab was approved for the treatment of recurrent , poorly differentiated refractory or follicular cd20 positive b - cell non - hodgkin 's lymphoma14 ) . in this case , immunohistochemical staining for cd3 and cd20 showed the predominance of mature t cells and atypical b cells . in situ hybridization revealed positive ebv encoded rna in a few lymphocytes , confirming the diagnosis of grade ii lyg . the patient experienced rapid worsening of neurological symptoms and absence of systemic involvement on pre - operative laboratory findings and radiological imaging , and she therefore was treated with surgical decompression and biopsy on the t3 - 5 epidural lesion and improved neurologic symptoms . surgical decompression can improve neurological morbidity of the patient . however , appropriate chemotherapy is highly needed as systemic progression of lyg make poor prognosis . the patient responded to rituximab therapy well without adverse effects , and her condition remained stable for 6 years . therefore it was suggestive that decompression and rituximab is suitable as the initial therapy for symptomatic spinal lyg . this case described an unusual occurrence of thoracic spinal lyg in a 4-year - old girl . childhood lyg is a rare disease but should be considered in the nodular pulmonary infiltrates and central nervous system manifestations , especially in the past diagnosis of childhood acute lymphoblastic leukemia . awareness of the features of lyg can lead to earlier diagnosis and prompt appropriate therapy .

lymphomatoid granulomatosis ( lyg ) is a lymphoproliferative disease involving the lungs most frequently ; however , it may also involve the kidneys , skin and especially the central nervous system . unique initial presentation of spinal involvement is extremely rare and epidural lesion of thoracic spine has not been reported . the prognosis for lyg has been reported to be poor , and there currently exists no satisfactory established treatment protocol . the purpose of this study is to report a case of successful treatment with surgery and rituximab combination therapy in thoracic spinal lyg .

kidney transplant recipients not only have numerous comorbidities associated with renal failure , but also often have additional problems due to chronic immunosuppression [ 13 ] . the surgical management of colorectal cancer is risky to the patient in terms of the surgical procedure and the interruption of immunosuppression . in general , these patients are more susceptible to perioperative complications . although open colorectal resection has been practiced for many years in kidney recipient patients , the minimally invasive procedure has been developed and is gaining popularity . multiple randomized controlled trials have proven that minimally invasive surgery yields better short - term outcomes than open colorectal surgery in terms of reduced intraoperative bleeding , postoperative pain , and hospital stay , as well as a lower incidence of infection and respiratory complications , with equivalent long - term outcomes [ 4 , 5 ] . however , selected high - risk patients may benefit from minimally invasive colorectal resection due to reduced morbidity . the aim of this study was to prove the safety and feasibility of laparoscopic and robotic colorectal resection in kidney transplant recipients by evaluating the technical protocol and the short- and long - term outcomes . this retrospective review of all kidney transplant recipients diagnosed with colon or rectal cancer who received laparoscopic or robotic resection was conducted between may 2007 and august 2012 at yonsei university college of medicine , south korea . data were analyzed using the spss statistical program ( version 18 for windows ; spss inc . , all patients received 4 liters of golytely ( peg-3350 and electrolytes for oral solution , braintree laboratories , inc . , antibiotics and a stress dose of steroids of 100 mg were given on induction of anesthesia and continued postoperatively with tapering dose of steroid . a main concern in laparoscopic and robotic colorectal resection is accurate and safe port placement to avoid injury to the transplanted kidney . after periumbilical port insertion using an open or closed technique , the abdomen was insufflated with medical co2 to 1214 mmhg pressure . the kidney was shifted posterolaterally to provide adequate space for the working ports ( figure 1 ) . next , depending on the type of procedure , two right- or left - sided trocars under direct visualization were easily placed . for robotic trocar positioning , we used a hybrid technique by which the colon was mobilized laparoscopically and the robot was docked between the legs of the patients and used mainly for pelvic dissection . robotic port insertion depended on the location of the kidney : for the right kidney , arms 2 and 3 were on the left and arm 1 was on the right ; for the left kidney , arms 1 and 3 were on the right and arm 2 was on the left . the procedure used colonic mobilization and followed total mesorectal excision principles as required for pelvic dissection . we started medial to lateral mobilization of the colon 2 cm distal to the sacral promontory going upward toward the inferior mesenteric artery where most of it is ligated at its origin . then , lateral dissection followed including splenic flexure mobilization . finally , we dissect the rectum starting posteriorly going deep to the levator ani muscles carefully to avoid hypogastric nerve injury . then , we dissect laterally and anteriorly where we should avoid injury to prostrate and neurovascular puddle . usually , we perform a rectal irrigation before transection . the specimen extracted from the left iliac fossa assistant port and duple stabling technique was used for bowel anastomosis . regarding the right colectomy cases , medial to lateral approach our preferred method is applying complete mesocolic principles where vessel ligation is performed at its origin . ten of these recipients were diagnosed with colon or rectal cancer and underwent minimally invasive surgical resection . five ( 50% ) patients were males ; the mean patient age was 56.8 ( range , 4772 ) years , and the mean body mass index was 22.4 ( range 20.526.4 ) kg / m . nine ( 90% ) patients had hypertension , three ( 30% ) had diabetes mellitus , one patient had a history of hepatitis , and one had a history of chronic pulmonary tuberculosis . the american society of anesthesiologists scores were ii in 60% of patients and i and iii in 20% of patients each . all rectal patients were evaluated by computed tomography ( ct ) scan and a colonoscopy . in addition , magnetic resonance imaging ( mri ) was used for further evaluation of the patients with rectal cancer . preoperative stages were i and ii in 40% of the patients and iii in 10% of the patients . no tumor invasion or extension beyond the mesorectal fat with a maximum stage preoperative chemoradiation therapy was administered to one patient with low - rectal cancer with a preoperative stage iii ( t3n1 m0 ) . four ( 40% ) patients underwent an anterior and a low anterior resection , two ( 20% ) underwent a covering ileostomy and a right hemicolectomy , and one patient ( 10% ) underwent a left hemicolectomy . most ( 90% ) procedures were performed laparoscopically and 10% were conducted robotically ( table 1 ) . pre- and postoperative laboratory blood findings were evaluated every other day to minimize intra- and postoperative complications ( table 2 ) . the mean carcinoembryonic antigen level was 3.66 2.53 ( range 0.729.18 ) ng / ml and the mean white blood cell count was 7 , 315 3,842 ( range 2,99016,520 ) mm . three ( 30% ) patients were anemic , with a mean hematocrit of 34.65 7.72% ( range 26.753.4% ) and a mean hemoglobin concentration of 11.38 2.54 ( range 8.917.4 ) g / dl . mean preoperative urea and creatinine levels were 27.7 23.62 ( range 11.489.4 ) mg / dl and 1.43 0.98 ( range 0.593.94 ) mg / dl , respectively . most of the immunosuppressive medications used pre- and postoperatively were cyclosporine or tacrolimus combined with low dose of steroid ranging between 5 mg to 12 mg . no change in immunosuppressive the mean postoperative hemoglobin concentration was 10.63 2.45 ( range 7.616.1 ) g / dl and no patient required a blood transfusion . the mean postoperative leukocyte count ( 10,655 2,219 ( range 7,53014,220 ) mm ) was slightly but significantly higher than the preoperative value ( p = 0.032 ) , and no severe leukocytosis or leukopenia was seen . mean postoperative urea and creatinine levels were 18.35 17.8 ( range 11.460.7 ) mg / dl and 1.40 0.76 ( range 0.723.09 ) mg / dl , respectively . the mean interval between kidney transplantation and surgery was 11.7 ( range 1.322 ) years . the mean estimated blood loss was 30 53.74 ( range 0150 ) ml . we rarely found adhesions ; most observed were minimal and were noted when the peritoneum was opened accidentally during the kidney transplant or another procedure . no significant difference was observed between pre- and postoperative kidney function parameters , and no organ rejection was reported . the mean timing of the first oral intake was 3.8 5.81 ( range 27 ) days postoperatively . postoperative stages were i in 20% of the patients , ii in 50% of the patients , and iii in 30% of the patients ( table 3 ) . one patient underwent adjuvant chemoradiation ( xeloda ( capecitabine ) + radiation ) and four patients underwent adjuvant chemotherapy alone ( fluorouracil , leucovorin , folic acid , oxaliplatin ( folfox ) in three cases , and 5-fluorouracil + leucovorin in one case ) . over a median follow - up period of 31.4 21.57 ( range 263 ) months , no mortality occurred . one patient showed liver metastasis 1 year postoperatively , which was treated by radiofrequency ablation and no further recurrence till the present date of our study . in six patients who were followed for a mean of 43.5 9.84 ( range 3263 ) months , the 3-year disease - free rate was 83.3% and the 3-year overall survival rate was 100% ( table 3 ) . the risk of colorectal neoplasia is high among renal transplant recipients and is associated with the duration of immunosuppression , regardless of age . the reported incidence of colorectal cancer in kidney transplant recipients has ranged from 0 to 0.9% . kim et al . found that the rate of right colon cancer was significantly higher in transplant recipients than in the general cancer population ( 35.2% versus 15.4% ) , whereas the rate of rectal cancer was significantly lower in transplant recipients ( 33.5% versus 46.5% ; p = 0.031 ) . also reported that the risk of colorectal cancer was twofold higher in the first posttransplantation year than in the general population , and an additional 2.2-fold higher after the third posttransplantation year . in contrast , saidi et al . found no increased risk of colorectal cancer among transplant recipients compared with the general population and noted the need for further evaluation of this issue . parnaby et al . reported that three population - based studies showed increases of up to twofold in the incidence of colonic but not rectal cancer in renal transplant recipients and that two population - based studies showed threefold and 10-fold increases in the incidence of anal cancer in these patients . patients on immunosuppressive medications or who have undergone organ transplantation with comorbidities have increased morbidity ( stress - related complications ) and mortality and a prolonged postoperative recovery period , after major open surgery , including colorectal surgery [ 1215 ] . he concluded that the nonspecific immune response appeared to be less affected by laparoscopic surgery compared with open surgery , while specific cell - mediated immunity was equally affected . laparoscopic colorectal resection is less physically stressful and offers short - term advantages compared with open resection . technically , selection of the port insertion site is the most important step in a laparoscopic or robotic procedure . after the establishment of pneumoperitoneum , the kidney is moved inferolaterally to allow port placement . our hospital is one of the leading hospitals in robotic surgery in korea . as we reported in our results , that one case was performed by robotic method . in that case , we used a hybrid technique where the colonic mobilization was completed laparoscopically and the da vinci robot was used only for pelvic dissection . we found that the robotic port position of the 3rd arm should be positioned on the opposite side of the transplanted kidney because of its close proximity with the anterior superior iliac spine where the kidney is located even after pneumoperitoneum . however , any port position even in totally robotic technique could be safe as long as the ports are placed away from transplanted kidney . we found no or minimal adhesion during the procedures , which may have been related to peritoneum opening during retroperitoneal dissection in the kidney transplant procedure . adhesions from previous surgeries , possibly related to the use of steroids , were not an issue . only one case series of laparoscopic - assisted colectomy in three kidney transplant recipients has been published . in that series , the average time since transplantation was 8 ( range 610 ) years and no patient experienced organ rejection . all patients had colon cancer , and all allografts were contralateral to colon resection . the mean operative time was 103 ( range 100105 ) min and no complications occurred . the average hospital stay was 5 ( range 47 ) days and the mean follow - up time was 17 ( range 340 ) months . our series was larger than that described above , and it included patients with colonic and rectal cancer . moreover , we included patients who underwent laparoscopic and robotic surgery . in our series , the mean interval between kidney transplantation and diagnosis of colorectal cancer ( 11.7 years ) was slightly longer . our mean operative time was 192.5 53.87 ( range 77263 ) min , due to the longer operative time ( 263 min ) of the robotic procedure ; the mean operative time for laparoscopic procedures was 184.6 50.74 min . we observed similarly intact renal function and no rejection during the follow - up period . two patients in our series had minor complications ( wound seroma and chyloperitoneum ) that were managed conservatively . the leukocyte count is normally raised after any major surgical procedure , but no severe leukocytosis or leukopenia occurred in our patients indicating infection or rejection . no change in the form or dosage of immunosuppressive medications was required before surgery , and medications were resumed postoperatively under monitoring by the transplant team . in general , these patients are more susceptible to perioperative complications . immunosuppression can lead to a higher infection rate and surgical stress can increase immunosuppression , a phenomenon that is more pronounced following open ( versus laparoscopic ) surgical procedures [ 12 , 14 , 15 ] . our follow - up period ( mean 31.4 21.57 months ) was longer than that previously reported . in addition , the mean follow - up period was 43.5 9.84 months for the six patients in whom long - term outcomes were assessed . no mortality occurred ; the 3-year disease - free rate was 83.3% , and the 3-year overall survival rate was 100% . in conclusion , minimally invasive surgery is feasible in kidney transplant recipients , who can benefit from fewer wound - related problems . technical proficiency , experience , and the use of a multidisciplinary team approach including an oncologist and transplant team can result in a successful procedure and ensure the safety of the transplanted kidney . therefore , minimally invasive colorectal procedures could be considered safe alternatives to open colorectal resection in selected kidney transplant patients .

aim . to prove the safety and feasibility of minimally invasive ( laparoscopic and robotic ) colorectal resection in kidney recipients by evaluating the technical protocol and reviewing short- and long - term outcomes . methods . between may 2007 and august 2012 , a retrospective review of ten kidney transplant patients diagnosed with colorectal cancer was evaluated for technical tips , short- and long - term outcomes . results . the mean patients ' age was 56.8 9.91 years and 50% of them were male . anterior and low anterior resections were performed in 40% of the patients each ; 20% and 10% of the patients underwent right and left hemicolectomy , respectively . most ( 90% ) procedures were performed laparoscopically and 10% were performed robotically . no open conversions . mean operating time was 192.5 15 min , blood loss was 30 50 ml , and mean hospital stay was 9.7 5.5 days . two ( 20% ) patients had postoperative complications : wound seroma and chyloperitoneum . over a mean follow - up period of 31.4 21.57 months , no mortality or kidney rejection occurred . among the six patients followed up for a mean of 43.5 9.84 months , 83.3% were 3-year disease - free and the overall survival rate was 100% . conclusion . minimally invasive colorectal resection is likely to be safe and feasible , with fewer complications and acceptable short- and long - term outcomes , in kidney transplant recipients .

acute occlusion of the superior mesenteric artery ( sma ) causes extensive intestinal necrosis due to the difficulty of early diagnosis , resulting in poor prognosis , with a high postoperative mortality rate of 65.2% . recent reports indicate that selective thrombolytic therapy with intraarterial infusion of urokinase is effective for acute sma occlusion diagnosed early after onset . early thrombolytic therapy can not always induce complete thrombolysis , and even if intestinal necrosis is avoided by administration of initial thrombolytic therapy , indications for additional thrombolytic therapy or the method for monitoring intestinal viability during subsequent follow - up have not been established . in this report , we present a case of acute sma occlusion diagnosed early after onset that was successfully treated by sequential and intermittent thrombolytic therapy by intraarterial urokinase infusion with angiographic evaluation of blood flow , thereby avoiding intestinal resection . an 82-year - old woman with a past history of atrial fibrillation was admitted to our hospital , complaining of acute epigastric pain and stool with fresh blood . physical examination was as follows : blood pressure 140/80 , heart rate 70/min , o2 saturation 97% ( room air ) , body temperature 37.2c . the abdomen was flat and soft , with no apparent tenderness or sign of peritoneal irritation . laboratory data on admission revealed an elevated wbc count and mildly elevated hepatic enzyme values ( wbc 15,400/mm , crp 0.05 mg / dl , ldh 502 iu / l , ast 57 iu / l , alt 21 iu / l , cpk 113 colonoscopy revealed mildly erosive , edematous mucosa , but no obvious bleeding from the ascending colon to the terminal ileum . abdominal enhanced ct showed a filling defect in the proximal portion of the sma main trunk , which led to the diagnosis of acute sma occlusion ( fig . , selective sma angiography was performed , showing complete occlusion of the sma around the first jejunal artery branch . subsequently , intraarterial bolus infusion of urokinase ( 600,000 iu ) with thrombus suction was performed . as a result , however , the thrombus remained , and intramural blood flow of the affected intestine was not visualized , suggesting possible necrosis of the affected intestine ( fig . a mild ischemic change was observed at the intestinal wall from the jejunum 40 cm distal from the treitz ligament to the ascending colon , but no apparent necrosis . blood flow in the mesentery was well palpable at the central portion of the sma , while peripheral blood flow was not palpable . the operation was completed without intestinal resection or direct removal of the thrombus from the sma . 24 h after the laparotomy , a second angiography was performed via the catheter that remained in place after first angiography , because clinical signs such as abdominal pain suggested the progression of intestinal ischemia . peripheral blood flow was well visualized , and no signs of intestinal necrosis were observed . thereafter , thrombolysis with urokinase infusion ( 240,000 iu ) and suction of the residual thrombus were performed . in addition , 36 h after the laparotomy , a third angiography and thrombolysis ( urokinase 240,000 iu ) were performed , showing improvement in peripheral blood flow via the gradual development of collateral blood flow . follow - up carefully monitored the possible onset of shower embolism by the residual thrombus . intestinal blood flow was well visualized on ct angiography performed 48 h after laparotomy ( 60 h after the first thrombolytic therapy ) . the patient began to take food by mouth on the 6th day after laparotomy and was discharged from our hospital 35 days after laparotomy . early diagnosis and treatment contribute to the improvement of therapeutic results for acute sma occlusion . a delay in diagnosis results in an extremely high mortality rate due to extensive intestinal necrosis ; however , successful resection of necrotic intestine causes short bowel syndrome , severely impairing quality of life . recent reports indicate that enhanced ct is feasible for early diagnosis of acute sma occlusion . when acute sma occlusion is suspected from symptom , clinical history and physical findings , enhanced ct plays a role not only in identifying the causal portion , but also in determining the subsequent treatment plan . early thrombolytic therapy is effective for acute sma occlusion in avoiding extensive intestinal resection . since the first report by jamieson et al . , reports that support the usefulness of selective thrombolytic therapy via a catheter for angiography have increased . the golden hour , the time during which the viability of the ischemic intestine can be preserved , varies , dependent on the portion and extent of occlusion . muneoka et al . studied 23 cases of acute sma occlusion treated by selective thrombolytic therapy in japan , and concluded that the golden hour is 5 h for occlusion of the sma main trunk and 12 h for the occlusion of the distal sma . the goal of thrombolytic therapy is complete thrombolysis , but administration over 48 h increases the risk of complication . easy bleeding due to fibrinolysis promoted by urokinase and shower emboli induced by the release of residual thrombi are known to be the major complications associated with thrombolytic therapy with urokinase . careful monitoring during 24 h after the completion of thrombolytic therapy is essential , and emergency laparotomy is mandatory if intestinal necrosis is suspected . urokinase has no influence during surgery as its half - life in blood is only 16 min . in the present case , the thrombus was detected in the sma main trunk by angiography performed 5 h from onset . therefore , judging that we were within the golden hour , we added urokinase infusion and thrombus suction via the angiography catheter . however , the thrombus remained left , and visualization of intramural blood flow was poor , therefore , we selected exploratory laparotomy . on the other hand , when no apparent intestinal necrosis is observed during exploratory laparotomy , a clinical consensus concerning the indication of additional thrombolytic therapy or optimal methods for evaluation of intestinal blood flow or viability in the subsequent follow - up is not yet established . even if intestinal necrosis can be avoided , intestinal ischemia would cause perforation or stricture of the affected intestine . from 1985 to 2007 , 45 cases of thrombolytic therapy for acute sma occlusion were reported in japan . complete thrombolysis was successful in 28 cases ( 63.6% ) , and 17 cases ( 36.4% ) received laparotomy following thrombolytic therapy . among those 17 patients , intestinal resection was performed in 5 cases ( 11.1% ) due to intestinal necrosis ; the remaining 12 cases ( 26.7% ) without intestinal necrosis at laparotomy were thereafter free from intestinal necrosis . of those 12 patients , 2 received angiographic reevaluation of blood flow and subsequent second - look operation , 3 received embolectomy , and 6 were carefully observed by laboratory examination and physical findings without any invasive treatments . in the present case , we performed thrombolytic therapy with angiographic evaluation of blood flow , sequentially and intermittently , after exploratory laparotomy , which enabled us not only to accurately evaluate intestinal blood flow or viability , but also to avoid a second - look operation . in conclusion , sequential and intermittent thrombolytic therapy with meticulous angiographic evaluation of blood flow is effective in the early stage of acute sma . in particular , it is much less invasive than strategies described in previous reports and enables accurate evaluation of intestinal blood flow or viability over the time course .

acute occlusion of the superior mesenteric artery ( sma ) causes extensive bowel necrosis , resulting in a poor prognosis with an extremely high mortality rate . an 82-year - old woman was admitted to our hospital with the complaint of abdominal pain . she was diagnosed as having acute sma occlusion by enhanced ct . five hours from onset , the first thrombolytic therapy with urokinase was performed , but failed to complete thrombolysis and recanalization of peripheral blood flow . an exploratory laparotomy following the first thrombolytic therapy showed a mild ischemic change in the affected intestine and mesentery , but no sign of necrosis . after the laparotomy , local thrombolytic therapy with angiographic evaluation of blood flow at 24 , 36 and 48 h from the first thrombolysis was performed . as a result , the residual thrombus disappeared and all branches of the sma became well visualized . the patient was discharged well without a second - look operation or major bowel resection . sequential intermittent thrombolytic therapy with meticulous angiographic evaluation of blood flow is effective for early - stage acute sma occlusion .

cervical sagittal alignment is an important component of global spinal alignment , allowing optimized energy expenditure during upright human posture by positioning the head over the pelvis and maintaining appropriate horizontal gaze.1 cervical radiographs ( xrs ) are most commonly performed in the standing position with the arms relaxed on either side of the body , which is considered the most physiologic position during standing . advances in imaging technology have continued , and recently there has been increasing interest in the use of standing whole - body stereoradiography ( sr ) when evaluating spinal deformity . the purported advantages of sr include lack of magnification , lack of parallax distortion , simultaneous two - dimensional ( anteroposterior and lateral ) high - quality imaging of the entire skeleton with lower radiation exposure , as well as capabilities for three - dimensional image reconstruction.2 however , both arms must be forward flexed at the shoulders during sr imaging to reduce obstruction by arm overlapping the thoracic and lumbar regions , and a previous study has found arm positioning , particularly arms flexed with hands touching the clavicle , may change cervical sagittal alignment during xr imaging . previous studies have demonstrated that arm positioning is an important factor during xr imaging , changing sagittal alignment parameters of the thoracic and lumbar spine.3 4 5 6 7 8 in fact , park et al found that lateral whole - spine xrs with hands positioned touching the clavicle were associated with a decrease in t1 slope ( t1-s ) , posterior translation of the head , hypolordotic cervical spine , and downward gazing when compared with standing lateral cervical xrs with arms relaxed on either side of the body.9 to our knowledge , no study to date has compared cervical sagittal alignment parameters obtained from standing lateral cervical xrs versus lateral whole - body srs . the purpose of this study is to evaluate the correlation and reliability of cervical sagittal alignment parameters measured from lateral cervical xrs compared with lateral whole - body eos srs ( eos imaging , paris , france ) . we hypothesized that the difference in arm position and in the trajectory of the radiation beam between standard cervical xr and sr would result in a difference for all cervical sagittal alignment parameters . we retrospectively evaluated adult patients with a primary diagnosis of cervical deformity treated by a single surgeon between january 2010 and december 2014 with both lateral cervical xrs and lateral srs obtained within 1 week of each other . at our institution , lateral cervical xrs are routinely obtained with the patient standing , with the arms in a relaxed neutral position , hanging at the side of the body ; this image is compared with srs obtained with eos imaging , which are acquired with patients standing and arms forward flexed at the shoulders . cervical sagittal alignment measurements were performed using a picture archiving and communications system , with each xr measured side by side on the same computer monitor to allow equal magnification / resolution of the cervical region from the lateral whole - body sr compared with the lateral cervical xr ( fig . were measured by two independent spine surgeon reviewers on two separate occasions , using the following cervical sagittal alignment parameters : c2c7 sagittal cobb angle ( sca ) , c2c7 sagittal vertical axis ( sva ) , c1c7 translational distance ( c1c7 ) , t1-s , neck tilt ( nt ) , and thoracic inlet angle ( tia ) ( fig . cobb angle was measured by drawing the angle between a line parallel to the inferior end plate of c2 and a line parallel to the inferior end plate of c7.10 positive values demonstrate kyphotic alignment and negative values , lordotic alignment . the distance between the vertical plumb line from the center of c2 and the vertical line from the posterosuperior corner of c7 was measured for c2c7 sva . translational distance was defined by the distance from anterior tubercle of c1 to the vertical line from the posteroinferior corner of c7 to evaluate the horizontal translation of the head position.10 t1-s was defined by the angle between the line parallel to the upper end plate of t1 and the horizontal line.11 nt is the angle formed by the line from the mid - t1 upper end plate to the cranial - most aspect of the midsternum and the vertical line from the cranial - most aspect of the midsternum . tia was then calculated by the formula tia = t1 slope + nt.12 the measuring parameters from cervical xr and sr were analyzed using r package irr . the intrarater reliability was quantified by intraclass correlation coefficient ( icc ) ( 3 , 1 ) , termed by shrout and fleiss.13 this type of icc is appropriate when study patients are a random sample of the underlying patient population , and a test and retest are only two occasions of interest . only two variance components are involved : the variance of study subjects and the variance of random errors . this type of icc is appropriate when both study patients and raters are a random sample from their respectively underlying populations . three variance components are involved : the variance of the study subjects , the variance of the raters , and the variance of random errors . icc values of more than 0.75 represent excellent reliability , values between 0.4 and 0.75 represent fair to good reliability , and value less than 0.4 represent poor reliability.14 the relationships between the parameters obtained from xrs and those from srs were compared by correlation analysis ( pearson correlation coefficient ) and paired t test . a total of 35 ( 12 male , 23 female ) patients , with mean age of 59 years old , were included in the study . intrarater reliability was excellent for all cervical sagittal alignment parameters in both the xr and sr groups , with icc ranging from 0.799 to 0.994 for xr and 0.791 to 0.995 for sr . c1c7 demonstrated the highest intrarater icc in both groups ( 0.994 xr , 0.995 sr ) , with nt having the lowest intrarater icc in both groups ( 0.799 xr , 0.791 sr ; table 1 ) . abbreviations : ci , confidence interval ; icc , intraclass correlation coefficient ; nt , neck tilt ; sva , sagittal vertical axis ; t1-s , t1 slope ; tia , thoracic inlet angle . note : 95% ci is the 95% upper and lower boundaries for the confidence interval of icc . icc ( 3 , 1 ) : two random sample components model , two tails . interrater reliability was excellent for all cervical sagittal alignment parameters in both the xr and sr groups , except nt and tia parameters , which had fair reliability . interrater icc was highest for sva ( 0.990 ) in the xr group and c1c7 ( 0.978 ) in the sr group , with nt having the lowest interrater icc in both groups ( 0.465 xr , 0.414 sr ; table 2 ) . abbreviations : ci , confidence interval ; icc , intraclass correlation coefficient ; nt , neck tilt ; sva , sagittal vertical axis ; t1-s , t1 slope ; tia , thoracic inlet angle . note : 95% ci is 95% upper and lower boundaries for the confidence interval of icc . icc ( 2 , 1 ) : three random sample components model , two tails . we found excellent correlations between xr and sr measurements for most cervical sagittal alignment parameters , with sca , sva , and c1c7 having r > 0.90 . t1-s and tia were between 0.75 and 0.90 , and only nt had an r < 0.70 . we found a significant difference between the groups , with sr having lower measurements compared with xr for both sva ( 0.68 cm lower , p < 0.001 ) and c1c7 ( 1.02 cm lower , p < 0.001 ) . there were no differences between the groups for the other cervical sagittal alignment parameters ( sca , t1-s , nt , and tia ; tables 3 and 4 , fig . icc values of cervical alignment parameters between lateral cervical radiograph and lateral whole - body stereoradiography . abbreviations : icc , intraclass correlation coefficient ; nt , neck tilt ; sca , sagittal cobb angle ; sva , sagittal vertical axis ; td , translational distance ; tia , thoracic inlet angle . abbreviations : nt , neck tilt ; sd , standard deviation ; sva , sagittal vertical axis ; t1-s , t1 slope ; tia , thoracic inlet angle . abbreviations : nt , neck tilt ; sd , standard deviation ; sva , sagittal vertical axis ; t1-s , t1 slope ; tia , thoracic inlet angle . intrarater reliability was excellent for all cervical sagittal alignment parameters in both the xr and sr groups , with icc ranging from 0.799 to 0.994 for xr and 0.791 to 0.995 for sr . c1c7 demonstrated the highest intrarater icc in both groups ( 0.994 xr , 0.995 sr ) , with nt having the lowest intrarater icc in both groups ( 0.799 xr , 0.791 sr ; table 1 ) . abbreviations : ci , confidence interval ; icc , intraclass correlation coefficient ; nt , neck tilt ; sva , sagittal vertical axis ; t1-s , t1 slope ; tia , thoracic inlet angle . note : 95% ci is the 95% upper and lower boundaries for the confidence interval of icc . icc ( 3 , 1 ) : two random sample components model , two tails . interrater reliability was excellent for all cervical sagittal alignment parameters in both the xr and sr groups , except nt and tia parameters , which had fair reliability . interrater icc was highest for sva ( 0.990 ) in the xr group and c1c7 ( 0.978 ) in the sr group , with nt having the lowest interrater icc in both groups ( 0.465 xr , 0.414 sr ; table 2 ) . abbreviations : ci , confidence interval ; icc , intraclass correlation coefficient ; nt , neck tilt ; sva , sagittal vertical axis ; t1-s , t1 slope ; tia , thoracic inlet angle . note : 95% ci is 95% upper and lower boundaries for the confidence interval of icc . icc ( 2 , 1 ) : three random sample components model , two tails . we found excellent correlations between xr and sr measurements for most cervical sagittal alignment parameters , with sca , sva , and c1c7 having r > 0.90 . t1-s and tia were between 0.75 and 0.90 , and only nt had an r < 0.70 . we found a significant difference between the groups , with sr having lower measurements compared with xr for both sva ( 0.68 cm lower , p < 0.001 ) and c1c7 ( 1.02 cm lower , p < 0.001 ) . there were no differences between the groups for the other cervical sagittal alignment parameters ( sca , t1-s , nt , and tia ; tables 3 and 4 , fig . icc values of cervical alignment parameters between lateral cervical radiograph and lateral whole - body stereoradiography . abbreviations : icc , intraclass correlation coefficient ; nt , neck tilt ; sca , sagittal cobb angle ; sva , sagittal vertical axis ; td , translational distance ; tia , thoracic inlet angle . abbreviations : nt , neck tilt ; sd , standard deviation ; sva , sagittal vertical axis ; t1-s , t1 slope ; tia , thoracic inlet angle . abbreviations : nt , neck tilt ; sd , standard deviation ; sva , sagittal vertical axis ; t1-s , t1 slope ; tia , thoracic inlet angle . cervical sagittal imbalance remains a complex problem,9 15 and optimal imaging to assess cervical sagittal alignment parameters is important during patient evaluation and particularly preoperative planning . when cervical sagittal imbalance or deformity is identified , additional standing full - length spine xrs are recommended to assess whether other regions of the spine may also have deformity and contribute to global sagittal imbalance . however , obtaining several separate xrs is often time - consuming with repeated radiation exposure . recent advances in imaging techniques include full - body sr , which allows low - dose , high - quality imaging of the entire spinal column and pelvis , as well as assessing for concomitant length discrepancy / deformity of the lower extremities and joints ( hips , knees , ankles).16 despite the purported advantages of sr , the difference in arm positioning compared with the standing lateral cervical xr and the effect on cervical sagittal alignment parameters is not completely understood . our study found most cervical sagittal alignment parameters ( sca , sva , and c1c7 ) measured from lateral cervical xrs and lateral whole - body eos srs had excellent correlation , with excellent intra- and interrater reliability in both groups . our results regarding sca are similar to a previous report by vidal et al that also found excellent intra- ( icc = 0.847 to 0.955 ) and interrater ( icc = 0.846 to 0.954 ) reliability for sca measured on sr imaging , both in the upper cervical spine ( c1c3 ) and lower cervical spine ( c3c7).14 however , this study was performed in patients with adolescent idiopathic scoliosis , and although global sagittal alignment using the c7 plumb line was also found to have excellent reliability , other cervical sagittal alignment parameters were not evaluated . intra- and interrater reliability were lowest for nt and tia , which had fair reliability . we postulate the lower reliability for nt and tia was due to poor visualization of the sternum on both imaging techniques , and they may be suboptimal cervical sagittal alignment parameters in the setting of spinal deformity . similarly , there was only fair correlation between the xr and sr groups for nt , which further emphasizes that nt may not be an ideal cervical sagittal alignment parameter . the most important finding from our study was the significant difference in mean values for both sva and c1c7 between the xr and sr groups . we found the sr group had a mean sva that was 0.68 cm lower and mean c1c7 that was 1.02 cm lower than the xr group , although there was excellent correlation with r > 0.900 for both measurements . the most plausible explanation for our findings is the difference in arm positioning during xr and sr imaging , with forward arm flexion during sr causing the head position to shift posteriorly . our findings are similar to a previous study by park et al , who evaluated cervical sagittal alignment parameters in 101 asymptomatic adults and found that lateral whole - spine xrs with arms flexed in the hand - touching - clavicle position also resulted in posterior head translation compared with lateral cervical xrs.9 however , the study also found the arm - flexed position in the whole - spine xrs resulted in significantly lower t1-s , cervical hypolordosis , and downward gaze . our study did not find differences in t1-s or sca ; however , this discrepancy may be explained by the smaller sample size of our study , as well as the difference in studied population between symptomatic adults with cervical deformity versus asymptomatic adults . we postulate that patients with symptomatic cervical deformity have degenerative changes with less flexibility of the subaxial spine . also , the forward flexion arm position for sr is not as exaggerated as the whole - spine lateral xr , which requires additional muscle activation / strain with elbow and wrist flexion to touch the clavicles . weaknesses of our study include the retrospective design and the potential for selection bias based on nonconsecutive sampling of patients . also , changes in pain or worsening deformity over time may have resulted in differences in cervical sagittal alignment parameters ; however , we attempted to reduce this confounding variable by including only patients with xr and sr images obtained less than 1 week apart . also , our study may have had insufficient sample size to avoid type ii error for various cervical sagittal alignment parameters . based on the findings of our study , we believe sr is a reliable method for determining most cervical sagittal alignment parameters and a viable alternative to lateral cervical xr despite differences in arm position during imaging . however , measurements requiring visualization of the sternum such as nt and tia may not be reliable and do not appear to be optimal cervical sagittal alignment parameters . also , sr results in slight posterior head translation and subsequently lower sva and c17 translational distance compared with lateral cervical xr and may underestimate regional cervical sagittal alignment parameters in patients with cervical deformity or sagittal imbalance . the lower radiation exposure using single sr has to be weighed against its higher cost compared with xr .

study design retrospective analysis.objective to evaluate the correlation and reliability of cervical sagittal alignment parameters obtained from lateral cervical radiographs ( xrs ) compared with lateral whole - body stereoradiographs ( srs).methods we evaluated adults with cervical deformity using both lateral xrs and lateral srs obtained within 1 week of each other between 2010 and 2014 . xr and sr images were measured by two independent spine surgeons using the following sagittal alignment parameters : c2c7 sagittal cobb angle ( sca ) , c2c7 sagittal vertical axis ( sva ) , c1c7 translational distance ( c17 ) , t1 slope ( t1-s ) , neck tilt ( nt ) , and thoracic inlet angle ( tia ) . pearson correlation and paired t test were used for statistical analysis , with intra- and interrater reliability analyzed using intraclass correlation coefficient ( icc).results a total of 35 patients were included in the study . we found excellent intrarater reliability for all sagittal alignment parameters in both the xr and sr groups with icc ranging from 0.799 to 0.994 for xr and 0.791 to 0.995 for sr . interrater reliability was also excellent for all parameters except nt and tia , which had fair reliability . we also found excellent correlations between xr and sr measurements for most sagittal alignment parameters ; sca , sva , and c1c7 had r > 0.90 , and only nt had r < 0.70 . there was a significant difference between groups , with sr having lower measurements compared with xr for both sva ( 0.68 cm lower , p < 0.001 ) and c1c7 ( 1.02 cm lower , p < 0.001 ) . there were no differences between groups for sca , t1-s , nt , and tia.conclusion whole - body stereoradiography appears to be a viable alternative for measuring cervical sagittal alignment parameters compared with standard radiography . xr and sr demonstrated excellent correlation for most sagittal alignment parameters except nt . however , sr had significantly lower average sva and c1c7 measurements than xr . the lower radiation exposure using single sr has to be weighed against its higher cost compared with xr .

nucleotide excision repair ( ner ) plays a central role in preserving the genome of prokaryotes and eukaryotes . this versatile repair system removes structurally and chemically diverse bulky dna lesions , including those induced by exposure to uv light and environmental chemical carcinogens [ 1 , 2 ] . the vital importance of this mechanism is demonstrated by several human ner - deficiency syndromes including xeroderma pigmentosum ( xp ) , cockayne syndrome ( cs ) , and trichothiodystrophy ( ttd ) . xp , for example , is characterized by high photosensitivity , hyperpigmentation , premature skin ageing , and proneness to developing skin cancer . furthermore , the capacity of the ner pathway is important in cancer chemotherapy : ner diminishes the efficacy of chemotherapeutic agents such as cisplatin , which act via the formation of bulky dna adducts . a better understanding of the mechanisms of recognition of dna lesions by the ner system may lead to the design of improved chemotherapeutic drugs that can modulate the repair response . recent findings reveal that polymorphisms in human ner repair genes have an impact on the repair of dna lesions and cancer susceptibility [ 6 , 7 ] , as well as on chemotherapeutic efficacy . the eukaryotic ner pathway is a biologically complicated process and consists of two sub - pathways with different substrate specificity : global genome ner ( gg - ner ) [ 9 , 10 ] and transcription - coupled repair ( tcr ) [ 1114 ] . both sub - pathways consist of ordered multistep processes , which differ in the early steps , when the dna lesions are recognized , but converge in the later steps . in gg - ner , the focus of our present interest , the whole genome is scanned for bulky lesions to initiate the repair process . two independent complexes , one involving the xpc / hr23b / centrin 2 proteins [ 1517 ] and the other involving the ddb1/ddb2 heterodimer [ 1821 ] , have been implicated in the early steps of base - damage recognition during ner . by contrast , the tcr sub - pathway is activated by a stalled rna polymerase during transcription . once the lesion is detected , the two sub - pathways proceed in an essentially identical manner to excise it : the multisubunit transcription factor . tfiih , containing helicases xpb , and xpd , is recruited to the lesion site , followed by xpa , the single - strand dna binding protein rpa , and the two nucleases xpg and xpf - ercc1 . once assembled , a 2432 oligonucleotide stretch containing the lesion is excised from the damaged strand . finally , gap resynthesis by dna polymerases , , and and ligation by dna ligase i complete the ner process . one remarkable characteristic of the ner pathway is its ability to excise an astounding variety of chemically and structurally diverse lesions , and the rates of repair can vary over several orders of magnitude . however , the differences in the structural and thermodynamic properties of the lesions that control the diverse ner efficiencies have remained elusive . it has been suggested that the ner factors do not recognize the lesion itself , but rather the local distortions and destabilizations in the dna that are associated with it [ 2430 ] . a number of different properties of damaged dna that elicit the ner response have been proposed . these include disruption of watson - crick hydrogen bonding [ 24 , 31 ] , kinks in the damaged dna , thermodynamic destabilization [ 24 , 29 , 33 ] , diminished base stacking [ 34 , 35 ] , local conformational flexibility , and flipped - out bases in the unmodified complementary strand [ 3740 ] . a crystal structure of yeast rad4/rad23 , the homolog of the human ner recognition factor xpc / hr23b , bound to dna containing a cyclobutane pyrimidine dimer , shows that rad4/rad23 inserts a -hairpin through the dna duplex and expels two mismatched thymines in the undamaged strand out of the duplex to bind with the enzyme ( pdb i d : 2qsg ) . this structure suggests that lesions which thermodynamically destabilize the dna duplex and facilitate the flipping of base pairs and the intrusion of the beta - hairpin are good substrates to the ner machinery : the more locally destabilized the lesion , the better it is repaired . the modulation of ner susceptibility for the same lesion by neighboring base sequence context , is however , a relatively unexplored area . if a lesion is better repaired in one sequence context than the other , a lesion - induced mutational hotspot could result . in order to elucidate the relationship between ner efficiency and base sequence - governed dna distortion and destabilization induced by a bulky dna adduct , we have employed as a model system the major lesion derived from the cancer - causing compound benzo[a]pyrene ( b[a]p ) . b[a]p is the most well - studied member in a family of ubiquitous environmental pollutants known as polycyclic aromatic hydrocarbons . the tumorigenic metabolite of b[a]p is the diol epoxide r7 , t8-dihydroxy - t9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene ( b[a]pde ) . this intermediate reacts with dna and rna ; the most abundantly stable adduct produced in mammalian cells [ 4446 ] is the 10s ( + ) -trans - anti - b[a]p - n - dg adduct ( [ g * ] ) ( figure 1(a ) ) , the focus of our work . this adduct , unless removed by dna repair mechanisms , is highly mutagenic [ 48 , 49 ] . we have investigated the identical 10s ( + ) -trans - anti - b[a]p - n - dg adduct in the six sequence contexts shown in figure 1(b ) , utilizing an array of approaches : ner in human hela cell extracts , ligation and polyacrylamide gel electrophoresis techniques to assess bending properties of the modified duplexes , and structural studies utilizing high resolution nmr methods as well as unrestrained molecular dynamics ( md ) simulations . the position of the b[a]p ring system in the b - dna minor groove , directed 5 along the modified strand , was first determined by nmr in the 5- c[g*]c - i sequence in 1992 , but sequence - governed structural details as well as dynamic properties remained to be elucidated . one important motivation for our work was to explore the role of nearby guanine amino groups on the structural properties and ner susceptibilities of these duplexes . the key difference in these duplexes is the presence and positioning of guanines flanking the [ g * ] , either immediately adjacent to the lesion or beyond : the b[a]p rings compete for space with the bulky amino group of guanine on the minor groove side of b - dna , which we anticipated would differentially impact the structures of the damaged duplexes in a sequence context - dependent manner . a further motivation was to explore the role of differing sequence contexts beyond the lesion that vary in intrinsic flexibility . we hypothesized that subtle but critical structural effects governed by sequence context would manifest themselves by impacting ner efficiencies . our results determined that sequence context could cause an up to four - fold difference in relative ner susceptibility , with even distant neighbors influencing ner . locally disturbed watson - crick hydrogen bonding and flexible bending are two key sequence - governed structural distortions caused by this lesion that the ner machinery appears to recognize with different efficiencies . more generally , different lesions in varied sequence contexts will cause different kinds of distortions ; thus , the extent of the local thermodynamic destabilization will also vary ; we hypothesize that it is the extent and type of destabilization that determines the relative ner efficiency . the 5- c[g * ] , 5- g[g * ] c , and 5- i[g * ] sequences . high resolution nmr solution studies have shown that the bulky aromatic b[a]p residue is positioned in the minor groove on the 5-side of [ g * ] in the 5- c[g*]g and 5- g[g*]c duplexes ( figure 2 ) . however , there are sequence - governed differences in some of the structural features . duplex , nmr studies revealed that the c : g base pair on the 5-side of [ g * ] is severely disturbed . in the case of the sequence - isomer 5- g[g*]c duplex , this perturbance is not observed . on the other hand , analyses of md simulations [ 51 , 52 ] based on the nmr data revealed significant unwinding near the lesion site combined with an anomalously enlarged roll ( figure 3 ) , not observed in the 5- c[g*]g duplex . duplex , which is a manifestation of a kink that is highly flexible . this flexible bend is caused on a molecular level by the severe untwisting and enlarged roll determined by md from the nmr data : dna bending is largely caused by increased roll , which is correlated with untwisting [ 5557 ] . the underlying structural reasons for the disturbed watson - crick hydrogen bond in the 5- , the bulky amino group on g20 ( figure 3 ) , which is partner to the c on the 5 side of [ g * ] , is sterically crowded by the b[a]p ring system since both are on the minor groove side , and hence this c5 : g20 base pair is episodically denatured ( figure 3(a ) ) ; for the 5- g[g*]c case , the b[a]p rings crowd the g6 amino group , and in this case the crowding is relieved by the severe untwisting accompanied by the increased roll , which produces the flexible bend observed by gel electrophoresis . investigations with the 5- i[g*]c sequence context substantiated the critical role of the guanine amino group since i ( figure 1(b ) ) lacks this group : the gel electrophoretic manifestation of a flexible bend was abolished . the nmr data showed conformational heterogeneity in minor groove conformations , and the md simulations showed episodic denaturation of one of the two hydrogen bonds at the i : c base pair , explaining the heterogeneity . the repair efficiency relative to 5- c[g*]c - i , the standard sequence utilized in many nmr and ner studies [ 53 , 58 ] , is 4.1 0.2 , 1.7 0.2 and 1.3 0.2 for the 5- c[g*]g , 5- g[g*]c and 5- i[g*]c duplexes , respectively ( figure 4 ) . duplex , dynamic episodic denaturation of watson crick base pairing flanking the lesion on the 5-side correlates with the greatest ner susceptibility while the flexible bend in 5- g[g*]c is a less pronounced ner recognition signal , and the disturbance to one hydrogen bond in the 5- i[g*]c case provides a still lesser signal [ 52 , 53 ] in this series . the 5- c[g * ] c - i and 5- t[g * ] t - ii sequence contexts . the 5- c[g*]c - ii and 5- t[g*]t - ii while a single , well - defined minor groove adduct conformation is observed in 5- c[g*]c duplexes , in the 5- t[g*]t - ii sequence context , the minor groove - aligned adduct conformation is heterogeneous . furthermore , polyacrylamide gel electrophoresis studies showed that the adduct induces a rigid bend in the 5-c[g*]c - ii sequence context , the lesion induces a highly flexible bend [ 59 , 60 ] . also , the 5- t[g*]t - ii 11-mer duplex has a lower thermal melting point than the 11-mer 5- duplex ( the exact difference depends on sequence length ) as expected from the thermodynamic properties of t : a and c : g watson - crick base pairs [ 62 , 63 ] . molecular insights on these experimental observations were provided by md simulations for the 5- t[g*]t - ii and 5- c[g*]c - ii duplexes . consistent with the conformational heterogeneity observed in the nmr studies , it was found that the 5- t[g*]t - ii duplex is much more dynamic than the 5- c[g*]c - ii duplex : the highly dynamic base pair on the 5-side of the lesion exhibits episodic denaturation of one of the two watson - crick hydrogen bonds , in agreement with the partial rupturing of this base pair observed by the nmr methods ; also , the 5- t[g*]t - ii duplex shows somewhat increased and more dynamic roll and untwisting compared to the 5- c[g*]c - ii duplex , consistent with the flexible bend observed only for the 5- t[g*]t - ii case ; in addition , the b[a]p ring system exhibits greater mobility and the duplex groove dimensions are more variable . the differences are accounted for by a coupled series of properties : the intrinsically weaker stacking of t - g compared to c - g steps allows for greater flexibility in the 5- t[g*]t - ii duplex ; the weaker t : a pair , with only two hydrogen bonds , compared to the c : g pair , with three bonds , provides enhanced flexibility ; moreover , the absence of guanine amino groups adjacent to the [ g * ] in the 5- t[g*]t - ii case allows for greater mobility for the b[a]p ring system . overall , the greater flexibility of the 5- t[g*]t - ii sequence is attributable to the absence of the guanine amino group . the rates of incision in the human hela cell assay relative to 5- c[g*]c - i is 2.4 0.2 and 1.6 0.2 for the 5- t[g*]t - ii and the 5- c[g*]c - ii duplexes , respectively , corresponding to a 1.5 0.2-fold higher - repair efficiency for the 5- t[g*]t - ii case is consistent with the overall enhanced dynamics manifested in various structural properties , notably watson - crick hydrogen bonding and bending . the 5- c[g*]c - i and 5- c[g*]c - ii sequences ( figure 1(b ) ) differ in the sequences beyond the nearest neighbors to [ g * ] . since different sequence steps are known to be differentially flexible [ 57 , 65 ] , we hypothesized that the same minor groove lesion [ 50 , 64 ] with different distant neighbors would be differentially repaired . polyacrylamide gel electrophoresis and self - ligation circularization experiments revealed that the 5- c[g*]c - ii duplex is more bent and suggested that it has more torsional flexibility than the 5- c[g*]c - i duplex . the key role is played by the unique -c3-a4-c5- segment in the 5- c[g*]c - ii duplex . duplex originates from the guanine amino group at the c3 : g20 pair ( figure 5 ) . this amino group acts as a wedge to open the minor groove ; facilitated by the highly deformable local -c3-a4- base step , the amino group allows the b[a]p ring system to better bury its hydrophobic surface within the groove walls . this produces a yet more enlarged minor groove which is coupled with more local untwisting and more enlarged and flexible roll , causing the greater bend in 5- c[g*]c - ii ( figure 5 ) . the ner efficiencies are 1.6 0.2 times greater in the 5- c[g*]c - ii than in the 5- c[g*]c - i sequence context showing that distant neighbors to [ g * ] modulate the ner susceptibility . the greater ner susceptibility for the 5- c[g*]c - ii duplex is explained by its greater bending with enhanced flexibility : the intrinsic minor groove enlargement caused by both the guanine amino groups [ 55 , 68 ] and the great flexibility of pyrimidine - purine steps , including the c - a step [ 57 , 6972 ] allow the b[a]p moiety ( figure 5 ) to more favorably position itself , but at the expense of the greater bend that makes it more repair - susceptible . we have carried out a series of studies with the same 10s ( + ) -trans - anti - b[a]p - n - dg lesion in a number of sequence contexts that differ in how the lesion is positioned in relation to nearby guanine amino groups . additionally , we have considered differences in intrinsic flexibility of sequences flanking the lesion . these are model systems for gaining understanding of ner lesion recognition factors . we have obtained molecular structural data by nmr and md simulations , bending properties from gel electrophoresis studies , and ner data from human hela cell extracts for all of our investigated sequence contexts ( figure 1(b ) ) . figure 4 summarizes our key findings and enables us to infer a hierarchy of ner recognition signals for the series of sequences and the single lesion we explored . we point out here that a variety of structural disturbances are found in each case , which are correlated . examples include impaired watson - crick pairing that is accompanied by diminished base stacking , and dna bending towards the major groove , that is induced by a minor groove lesion and is accompanied by minor groove enlargement . our present model system suggests that disturbed watson - crick base pairing is a better recognition signal than a flexible bend , and that these can act in concert to provide an enhanced signal : for example , for 5- t[g*]t - ii one episodically ruptured watson - crick hydrogen bond combined with the flexible bend results in better repair than just one disturbed hydrogen bond as in 5- i[g*]c , or the flexible bend alone in 5- g[g*]c ( figure 4 ) . for our system , steric hindrance between the minor groove - aligned lesion and nearby guanine amino groups , if present , determines the exact nature of the disturbances , depending on exactly where the guanine amino groups are situated . the intrinsic flexibility of the specific base steps also plays an important role in causing the differential disturbances . both the nearest neighbor and the more distant neighbor sequence contexts have an impact . more globally , different lesions may cause different types of distortions depending on the specific nature of the lesion and its sequence context . however , regardless of exactly what these distortions are , we hypothesize that they must provide a local thermodynamic destabilization signal for repair to ensue , and the greater the extent of destabilization , the better the repair . the destabilization would facilitate the strand separation , base - flipping , and -hairpin insertion by the xpc / hr23b recognition factor [ 41 , 73 ] needed to initiate ner . in this way , the ner machinery would excise a large variety of lesions with different efficiencies , by recognizing the thermodynamic impact of the lesions rather than the lesions themselves [ 24 , 29 , 41 , 73 ] . lesions that resist ner present a great hazard , as they survive to the replication step and produce a mutagenic outcome ; such ner - resistant lesions provide an important opportunity for gaining further understanding of the mechanism utilized by the ner apparatus to recognize different lesions .

nucleotide excision repair ( ner ) plays a critical role in maintaining the integrity of the genome when damaged by bulky dna lesions , since inefficient repair can cause mutations and human diseases notably cancer . the structural properties of dna lesions that determine their relative susceptibilities to ner are therefore of great interest . as a model system , we have investigated the major mutagenic lesion derived from the environmental carcinogen benzo[a]pyrene ( b[a]p ) , 10s ( + ) -trans - anti - b[a]p - n2-dg in six different sequence contexts that differ in how the lesion is positioned in relation to nearby guanine amino groups . we have obtained molecular structural data by nmr and md simulations , bending properties from gel electrophoresis studies , and ner data obtained from human hela cell extracts for our six investigated sequence contexts . this model system suggests that disturbed watson - crick base pairing is a better recognition signal than a flexible bend , and that these can act in concert to provide an enhanced signal . steric hinderance between the minor groove - aligned lesion and nearby guanine amino groups determines the exact nature of the disturbances . both nearest neighbor and more distant neighbor sequence contexts have an impact . regardless of the exact distortions , we hypothesize that they provide a local thermodynamic destabilization signal for repair .