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a 25-year - old male was presented with progressive pain of alveolar ridge of maxilla and mandible over 5 years . the pain began 5 years ago and has been aggravated during the last 10 months . the pain was diffuse and compatible with expansile cyst in maxillary and mandibular ridges on panoramic radiography . he was admitted 5 times to azahra hospital for removal of maxillary and mandibular odontogenic cysts during the previous year . he was referred to our institution for taking 3-dimetional face multislice ct - scan . in physical examination non - enhanced computer tomography of face in axial section revealed two expansile maxillary and one mandibular cyst ( figure 1 , 2 , 4 ) . in coronal and sagittal sections of bone window , calcification in entire length of falx and tentorium were detected ( figure 3 , 4 ) . in axial section , abnormal bony excrescence arising from left aspect of upper cervical vertebra was also observed ( figure 5 ) . at surgery , three expansile jaw cysts were removed and the histological examination revealed that they were keratogenic cysts . axial section showes one expansile odontogenic cyst in body of mandible axial section showes two expansile odontogenic cyst in maxillary alveolar ridge coronal section shows calcification of falx cerebri and one expansile maxillary odontogenic cyst sagittal section shows tentorial calcification axial section shows bony excrescence arising from body of upper cervical vertebra . gorlin goltz syndrome or nevoid basal cell carcinoma ( nbccs ) is an autosomal dominant disorder and a rare hereditary disease.1 it was first explained in the 1950s and 1960s by gorlin and goltz.2 it is seen in males and females equally and in whites more than blacks.3 the estimated prevalence varies from 1/57000 to 1/256000.4 this syndrome is due to tumor suppressor gene mutation in the long arm of chromosome 9 ( q22.3-q31).1 approximately , 35 - 50% of cases are due to new mutations.2 patients have different type of signs and symptoms that include basal cell carcinoma ( bcc ) , odontogenic cyst , skeletal anomalies , prognathism , calcification of tentorium and falx.1 because of the mutation of tumor suppressor gene , patients are predisposed to various neoplasm , such as medulloblastoma , meningioma , fibrosarcoma and cardiac fibroma . evans et all5 first described major and minor criteria for diagnosis of this syndrome , then were modified by kimonis et al.6 major criteria : more than two basal cell carcinomas or one appearing in patient < 20 years old ; odontogenic cyst of the jaw that confirmed by histopathology ; three or more palmar or plantar pits ; calcifications of falx cerebri and tentorium ; first - degree relative with gorlin - goltz syndrome . minor : macrocephaly , congenital and skeletal anomalies ( frontal bossing , coarse facies , prognathism ; moderate or severe hypertelorism , cleft lip and palate , vertebral anomalies ) ; other skeletal anomalies ( sprengel deformity , marked pectus deformity , and marked syndactyly of the digits ) ; bridging of the sella turcica , ovarian fibroma or medulloblastoma ; neurological anomalies ( dysgenesis of corpus callosum , mental retardation ) . two major criteria or 1 major and 2 minor criteria are necessary to diagnose gorlin - goltz syndrome.6 in our case , two major criteria ( keratogenic cyst that was approved with histological exam , calcification of falx cerebri and tentorium ) and two minor criteria ( vertebral anomaly and coarse face ) were seen . bccs are seen in 50- 97% of these cases.7 face and neck in first and chest in second are affected . odontogenic cysts are often multiple and presented in about 75% of patients , recurrent operation and removal is necessary.2 fine needle aspiration cytology ( fnac ) is also useful in preoperative diagnosis of odontogenic keratocyst.8 medulloblastoma is detected in 5 - 10% of cases , and is a fatal complication.1 early recognition of this syndrome is necessary for appropriate management . sun ray protection and vitamin a may have protective effect against skin cancer growth.9 repeated surgical excision of odontogenic cysts is necessary . major criteria : more than two basal cell carcinomas or one appearing in patient < 20 years old ; odontogenic cyst of the jaw that confirmed by histopathology ; three or more palmar or plantar pits ; calcifications of falx cerebri and tentorium ; first - degree relative with gorlin - goltz syndrome . minor : macrocephaly , congenital and skeletal anomalies ( frontal bossing , coarse facies , prognathism ; moderate or severe hypertelorism , cleft lip and palate , vertebral anomalies ) ; other skeletal anomalies ( sprengel deformity , marked pectus deformity , and marked syndactyly of the digits ) ; bridging of the sella turcica , ovarian fibroma or medulloblastoma ; neurological anomalies ( dysgenesis of corpus callosum , mental retardation ) . two major criteria or 1 major and 2 minor criteria are necessary to diagnose gorlin - goltz syndrome.6 in our case , two major criteria ( keratogenic cyst that was approved with histological exam , calcification of falx cerebri and tentorium ) and two minor criteria ( vertebral anomaly and coarse face ) were seen . bccs are seen in 50- 97% of these cases.7 face and neck in first and chest in second are affected . odontogenic cysts are often multiple and presented in about 75% of patients , recurrent operation and removal is necessary.2 fine needle aspiration cytology ( fnac ) is also useful in preoperative diagnosis of odontogenic keratocyst.8 medulloblastoma is detected in 5 - 10% of cases , and is a fatal complication.1 early recognition of this syndrome is necessary for appropriate management . sun ray protection and vitamin a may have protective effect against skin cancer growth.9 repeated surgical excision of odontogenic cysts is necessary . major criteria : more than two basal cell carcinomas or one appearing in patient < 20 years old ; odontogenic cyst of the jaw that confirmed by histopathology ; three or more palmar or plantar pits ; calcifications of falx cerebri and tentorium ; first - degree relative with gorlin - goltz syndrome . minor : macrocephaly , congenital and skeletal anomalies ( frontal bossing , coarse facies , prognathism ; moderate or severe hypertelorism , cleft lip and palate , vertebral anomalies ) ; other skeletal anomalies ( sprengel deformity , marked pectus deformity , and marked syndactyly of the digits ) ; bridging of the sella turcica , ovarian fibroma or medulloblastoma ; neurological anomalies ( dysgenesis of corpus callosum , mental retardation ) . two major criteria or 1 major and 2 minor criteria are necessary to diagnose gorlin - goltz syndrome.6 in our case , two major criteria ( keratogenic cyst that was approved with histological exam , calcification of falx cerebri and tentorium ) and two minor criteria ( vertebral anomaly and coarse face ) were seen . bccs are seen in 50- 97% of these cases.7 face and neck in first and chest in second are affected . odontogenic cysts are often multiple and presented in about 75% of patients , recurrent operation and removal is necessary.2 fine needle aspiration cytology ( fnac ) is also useful in preoperative diagnosis of odontogenic keratocyst.8 medulloblastoma is detected in 5 - 10% of cases , and is a fatal complication.1 early recognition of this syndrome is necessary for appropriate management . sun ray protection and vitamin a may have protective effect against skin cancer growth.9 repeated surgical excision of odontogenic cysts is necessary . after spinal anesthesia , the painless period is approximately 2 to 3 hours and then lbp and rlp appear . the use of eat may have a protective effect on late adhesive irritating scar tissue and when impregnated with bupivacaine or methylprednisolone acetate could increase the duration of painless interval . according to our data bupivacaine , was effective on lbp relief and steroid was effective on rlp relief during the first 12 hours after surgery .

gorlin goltz syndrome is an autosomal dominant and a rare hereditary disease . diagnosis of this syndrome is based on major and minor criteria . we report a gorlin goltz syndrome in a 25-year - old male who was presented with progressive pain of maxilla and mandible over 5 years . the pain was diffuse and compatible with expansile cyst in alveolar ridges on panoramic radiography . in physical examination , he had coarse face and prognathism . computer tomography of face revealed two expansile maxillary and one mandibular cyst . calcification of entire length in falx and tentorium were detected in bone window .

cystic fibrosis ( cf ) is an autosomal recessive disease affecting the respiratory , digestive , endocrine , and reproductive systems . although it primarily affects the lungs , disturbances in the gastrointestinal system is also critical for the management of the disease . management of pancreatic insufficiency is needed to extend life expectancy and to maintain a good quality of life ( 1 , 2 ) . the incidence of cf in caucasian populations is about one in 2,500 newborns ( 3 ) . the reported incidence is one in 90,000 asian infants in hawaii ( 4 ) and one in 350,000 live births in japan ( 5 ) . to date , only five patients with cf have been reported in korea and they all presented with respiratory symptoms rather than pancreatic insufficiency ( 6 - 9 ) . cf was confirmed by a sweat chloride test and genetic analysis and the infant was treated successfully with fat - soluble vitamin supplementation and pancreatic enzyme replacement therapy . a 9-month - old female infant was admitted to our hospital because of greasy stools and failure to thrive . she had been well until two months of age , when a productive cough and fever developed . she was hospitalized twice with recurrent pneumonia , which was treated with antibiotics . at three months of age , failure to thrive was noted and methicillin - resistant staphylococcus aureus ( mrsa ) was isolated from sputum cultures obtained by ventilating bronchoscopy . at seven months of age , her parents were aware of her greasy and foul - smelling loose stools that were often pale ; the infant also showed poor appetite . the patient had been born to a nonconsanguineous healthy couple by normal spontaneous vaginal delivery at 40 weeks of gestation . the patient 's birth weight was 2.60 kg and there was no history of delayed passage of meconium . she had a 6-yr - old brother and the family history was unremarkable . on admission , the infant appeared to be malnourished but was not in acute distress . the body weight was 4.5 kg ( < 3rd percentile for age ) , height 54.7 cm ( < 3rd percentile ) , and head circumference 33 cm ( <3 percentile ) . the patient 's vital signs included a temperature of 36.9 , heart rate of 130 beats per minute and respiratory rate of 40 per minute . no digital clubbing was found and a neurologic examination was negative . a complete blood count and serum electrolytes the serum albumin level was 2.6 g / dl and cholesterol was 100 mg / dl . ast and alt levels were 36 u / l and 14 u / l , respectively . plasma carotene level was 22.5 g / dl ( reference range 50 - 250 g / dl ) . immunologic studies of the patient were unremarkable , including normal proportions of polymorphonuclear cells , b cells , cd4 and cd8 t cells , and normal immunoglobulin levels . stool fat was demonstrated microscopically . to confirm the presence of steatorrhea and estimate the quantity of stool fat , an acidified steatocrit test was performed : this gave a value of 33.3% ( reference range ; 0 - 6.5% ) . stool alpha-1 antitrypsin clearance was 0.49 ml over 24 hr , which was within the normal limits . chest radiographs and computed tomography ( ct ) scans revealed segmental atelectasis and patchy infiltration . cf was suspected on the basis of isolation of mrsa in the respiratory tract , recurrent respiratory infection , and fat malabsorption . a quantitative pilocarpine iontophoresis sweat test was performed , showing the average sweat chloride concentration on both thighs to be 102.0 mm / l ( reference limit < 40 mm / l ) . after obtaining informed consent from the parents , direct sequence analysis of the cf transmembrane conductance regulator ( cftr ) gene was performed as described ( 9 ) . ( c.1766 + 2t > c ) and a 1-bp duplication in exon 21 resulting in a frameshift mutation ( c.3908dupa ; asn1303lysfsx6 ) ( fig . pancreatic enzymes ( lipase 25,000 iu ) , fat - soluble vitamins , and long - chain polyunsaturated fatty acids were given to treat the infant 's pancreatic insufficiency . she was fed with breast milk and standard infant formula . with pancreatic enzyme replacement , her weight was at the 3rd percentile for age and her height was at the 10th percentile . the low plasma carotene level indicated malabsorption of fat - soluble vitamin a. the increase in acid steatocrit was indirect evidence of pancreatic insufficiency . this test can be performed accurately on random spot stools and can be used to detect the presence of steatorrhea and to estimate fecal fat quantitatively ( 10 ) . cftr sequencing demonstrated compound heterozygous mutations and the sweat test was positive , so cf was diagnosed in this patient . pancreatic enzyme replacement and fat - soluble vitamin supplementation led to the improvement of the patient 's steatorrhea and catch - up growth by one year after discharge from hospital . patients are described as having pancreatic insufficiency when they have measurable steatorrhea , but this does not occur until only about 1 - 2% of pancreatic enzymatic secretory capacity remains ( 11 ) . pancreatic insufficiency results in the loss of amylase , lipase , and protease activities , as well as bicarbonate secretion . lipases are the primary enzymes used to hydrolyze fat which is important in infants and young children as the primary source of energy . pancreatic function should be tested for the initial diagnosis of cf and monitored subsequently to delineate any changeover from pancreatic sufficiency to insufficiency ( 13 ) . wasting is a significant predictor of survival in patients with cf independent of lung function . enzyme therapy improves nutrient absorption and , with appropriate dietary therapy , normal nutritional status can be expected in most patients with cf ( 1 ) . cf is an autosomal recessive disease caused by mutations of the gene located on chromosome 7 . the gene product is cftr , which regulates the transport of electrolytes across epithelial cell membranes ( 14 ) . cftr mutation patterns in asian populations are different from those observed among caucasians ( 15 , 16 ) . the f508del genotype , which accounts for 66% of cf cases worldwide , is very rare in the korean population ( 16 ) , and four mutations reported in korean patients with cf are not commonly seen in caucasians ( 8 , 9 ) . we found two novel mutations in this patient , which have not been reported in caucasians . previous genotype and phenotype analyses showed that nonsense , frameshift , and splice junction mutations are associated with the pancreatic insufficiency phenotype ( 17 ) . mutations leading to cf have grouped into five classes according to the mechanism by which the mutation affects normal cftr protein function . class i mutations fail to produce cftr proteins because of the presence of nonsense , frameshift , or splice mutations ( 18 ) . our patient harbored a heterozygous splicing mutation and a heterozygous frameshift mutation , which might result in defective production and severe phenotype . previous korean patients with cf , including children and adults , did not show pancreatic insufficiency ( 6 , 8 , 9 ) . in summary , we treated an infant with cf and pancreatic insufficiency .

cystic fibrosis ( cf ) is an autosomal recessive disease that is very rare in asians : only a few cases have been reported in korea . we treated a female infant with cf who had steatorrhea and failure to thrive . her sweat chloride concentration was 102.0 mm / l . genetic analysis identified two novel mutations including a splice site mutation ( c.1766 + 2t > c ) and a frameshift mutation ( c.3908dupa ; asn1303lysfsx6 ) . pancreatic enzyme replacement and fat - soluble vitamin supplementation enabled the patient to get a catch - up growth . this is the first report of a korean patient with cf demonstrating pancreatic insufficiency . cf should therefore be considered in the differential diagnosis of infants with steatorrhea and failure to thrive .

drug conditioned stimuli , which can be discrete ( i.e. , a syringe ) and/or environmental ( i.e. , a room ) , acquire the ability to activate drug - oriented behaviors because they are repeatedly perceived in conjunction with the unconditioned effects of drugs of abuse [ 13 ] . hence , through pavlovian conditioning , drug conditioned stimuli become wanted and preferred , grab attention [ 68 ] , and produce a variety of physiological and psychological responses [ 915 ] . the first was to establish whether drug conditioned stimuli ( i.e. , images of drugs and drug paraphernalia ) can serve as conditioned reinforcers . conditioned reinforcing stimuli , unlike primary reinforcing stimuli , strengthen behavioral responses in virtue of their learned value . therefore , the objective of this study was to determine whether the occurrence of stimuli associated with the effects of drugs can maintain an arbitrary operant response ( i.e. , pressing a computer key ) in the absence of drugs . this is of interest because it is possible that the assessment of the reinforcing value of drug conditioned stimuli could complement other measures of cue reactivity such as self - reported craving [ 17 , 18 ] , and thus help predict clinical outcomes [ 19 , 20 ] . to determine whether drug conditioned stimuli would reinforce arbitrary operant responses , subjects were recruited from stonehenge therapeutic community , a long - term ( 6 months ) residential treatment facility designed for chronic and relapsing substance dependence . therefore , these individuals were likely to have experienced substantial conditioning as a result of excessive exposure to various drugs of abuse . although the selection of this population precluded manipulation of important variables such as availability of the unconditioned stimuli ( i.e. , drugs ) , it allowed the exploration of whether this novel putative index of cue - reactivity could be related to self - reported drug cravings , and predictive of treatment completion , which is typically low in therapeutic communities . the second objective was to study the relationship between explicit behavioral reactivity ( i.e. , operant responding ) and automatic evaluative processes elicited by drug conditioned stimuli . using the implicit association test ( iat ) , for example , it has been established that words such as beer , wine , whisky , and rum generate significant automatic negative responses in heavy drinking individuals who do not try to abstain . that is , subjects are faster at categorizing alcohol - associated words with negative concepts such as bad or disgust , than with positive concepts such as good or pleasant . in light of leading neurobiological theories of addiction predicting dissociations between what people do when they are exposed to drug associated stimuli compared to how they feel [ 23 , 24 ] , the iat was used to assess automatic responses to drug conditioned stimuli within the context of their conditioned reinforcing effect . that is , it can often be questioned whether the results apply to other clinical populations , or if they can help understand basic psychological processes that play a role in the behavior of nonclinical samples . because the third objective of this research was to explore the relationship between basic psychological processes , two additional studies were performed . these studies were specifically implemented in nonmatched groups to ascertain whether significant relationships could be observed between cravings for incentives , behavioral responses to stimuli - associated with these incentives , and automatic evaluative processes elicited by these stimuli . therefore , one study investigated whether drug conditioned stimuli can reinforce operant behavior also in individuals who regularly consume drugs , but are not dependent and not in treatment for excessive use . therefore , volunteers were recruited from the population of undergraduate students at the university of guelph on the basis of self - reported levels of alcohol consumption . the focus on this particular drug was constrained by the selection of the sample : undergraduate students in this university rarely report the use of other drugs , including cannabis . the second study investigated whether only stimuli paired with drugs of abuse can function as conditioned reinforcers . therefore , always in undergraduate students , it was tested whether images of sexy attractive models in swimsuits could support operant responding . sexual stimuli were selected because : ( 1 ) it is fairly intuitive to predict the gender of the reinforcing stimulus in heterosexual individuals ; ( 2 ) it is known that sexual stimuli can act as conditioned reinforcers in animals ; and ( 3 ) it is clear that responses to sexual stimuli can be observed in the absence of sexual addiction . similar methodologies were employed in the three studies to allow for meaningful comparisons across findings . the sample consisted of 28 males and 11 females , aged ( mean standard error of the mean ; sem ) 36.8 1.5 and 37.5 3.9 , respectively , primarily caucasian ( 87% ) , with education below university level ( 98% ) . the power calculation was performed using the effect size estimated using the cohen 's d model , although subsequent analyses required to split the sample in subgroups ( see below ) . the vast majority ( 92% ) of subjects had received previous treatment ; 38% reported one , and 54% reported 2 or more treatment attempts in different programs . all subjects were poly - drug users . excluding tobacco ( because almost all smoked cigarettes ) , the drug most often used ( more than 15 days ) in the 30 days prior to treatment admission was crack / cocaine ( 77% of subjects ) . volunteering participants were eligible only if they had completed at least two weeks of treatment . the average ( sem ) number of days in treatment at the time of study interview was 70 5.5 . the study of responses to alcohol - related stimuli included 49 participants ( 17 males and 32 females , aged 19.1 0.2 and 19.2 0.2 , resp . ) . the study of responses to sexual stimuli included 106 heterosexual participants ( 43 males and 63 females , aged 18.6 0.2 and 18.3 0.1 , resp . ) . all participants were undergraduate students at the university of guelph , recruited by mass - testing questions about drinking and sexual behavior ( see below ) . first , a brief survey assessed drug use in the 30 days prior to arrival at the community , as well as drug of choice and preferred route of administration . this questionnaire included 10 questions about desire for the drug ( i.e. , i have an urge for _ _ _ _ _ _ _ _ _ ) and 10 questions about anticipated drug effects ( i.e. , using _ _ _ _ _ _ _ _ _ _ _ _ right now would make me feel less tired ) . the experimenter completed the blank for each item with the particular participant 's drug of choice . the questions were derived from items common to half of 12 validated questionnaires assessing craving for alcohol , cocaine , speed , heroin , or tobacco [ 2737 ] . this questionnaire was created because participants drug of choice was not known prior to the initial survey , and thus there was a need for questions that would apply regardless of the name of the drug that was used to fill the blanks . this was an adaptation of a conditioned reinforcement procedure [ 3841 ] in which operant behavior is reinforced by stimuli previously associated with the effects of drugs of abuse . in the current study , participants responded to keys generating pictures ( see figure 1 ) of drug look - alike substances ( i.e. , white powder , crystals ) , of actual drugs ( i.e. , bottles of different alcoholic beverages ) , of simulated drug taking behavior ( i.e. , snorting , smoking , injecting , drinking ) , and of drug paraphernalia ( i.e. , syringe , needle , crack pipe ) . six keys were linked to 6 categories of images , and each category included 40 images . four categories were created to represent drugs of choice commonly reported by individuals in treatment at stonehenge : cocaine / crack , heroin , alcohol , and marijuana . two additional categories were created for control purposes and included pictures of buildings , and random colours . these categories were selected because buildings are recognizable visual stimuli with neutral motivational value , and random colors can have motivational value but do not represent identifiable objects . all images were equalized for contrast and luminance . pressing any of the 6 keys activated a single image of a specific category for 1 second according to a progressive ratio schedule of reinforcement . the progressive ratio schedule has been employed in animal and humans [ 43 , 44 ] to measure motivation to self - administer drugs when the response requirement for each subsequent administration progressively increases within the session . of course , in the current study , no drug was provided after completion of each response ratio . participants were not informed about the association between keys and image categories prior to the beginning of testing , no practice trials were given , and there was no time limit to perform the task . the test began after the following instructions were read:pressing the keys d , f , g , h , j , and k will produce pictures on the screen pressing the keys d , f , g , h , j , and k will produce pictures on the screen . some of these will be drug - related and some will not . pressing the same key twice will produce another picture and so on . fourth , after a short break , all participants completed a personalized iat [ 4649 ] to assess automatic responses to drugs generated by exposure to the same drug - associated images employed in the crt task . unlike the traditional iat that includes general attribute categories such as good and bad , the personalized iat requires a categorization of test items into attributes that are specific to the individual being tested : this particular version was selected because it reduces extrapersonal automatic contamination [ 46 , 50 , 51 ] and thus better taps into personal automatic associations with drug ( and nondrug ) stimuli . therefore , participants were asked to categorize 40 drug associated images of their drug of choice , 40 control images ( building images ) , 6 positive words ( i.e. , joy , happy ) , and 6 negative words ( i.e. , rotten , disgust ) into one of four categories : two concept categories ( drugs and buildings ) , and two attribute categories ( i like and i dislike ) . the iat consisted of 5 blocks of trials . for each trial , participants were required to sort a target word or a target picture that appeared in the middle of the screen into a category that appeared at the top left or the top right of the screen using respective computer keys . in the first practice block , participants sorted drug and control images into the concept category buildings on the left or drugs on the right . in the next practice block the third block was a test block : the earlier tasks were combined and now participants sorted both picture and word targets in categories buildings combined with i like that appeared on the left of the screen , or categories drugs combined with i dislike that appeared on the right side of the screen . in the final two test blocks , buildings on the right side of the screen . and , in the final test block , stimuli were sorted into either categories drugs combined with i like that appeared on the left , or categories buildings combined with i dislike that appeared on the right side of the screen . the dependent measure in this task is time ( msec ) required to assign target words and pictures to the matched concept / attribute categories on test two blocks . faster reaction times reflect dominant automatic associations between concept and attribute categories that share a side of the computer screen . the interesting comparison was between reaction times displayed on the test blocks when drugs and i dislike shared a side of the screen versus when the side was shared by drugs and i like . the measures and procedure employed in the two additional studies differed from the main study in three ways . first , in the alcohol study , student participants completed the alcohol dependency scale ( ads ) , in which a score of 9 or greater indicates potential problematic drinking . they also ( 1 ) self - reported drinking in the 30 days prior to study interview ( days of drinking and number of times they drank 04 , 59 , or 10 + standard drinks on each of those occasions ) ; ( 2 ) completed the timeline follow - back measure ( tfm ) ; and ( 3 ) completed the questionnaire about craving with the word alcohol included in each question . in the sex study , participants answered a questionnaire about aspects of sexual behavior in the 30 days prior to study interview ( sexual relationship status , frequency of intercourse , and number of partners ) , and completed the questionnaire about craving employed in the other studies with the spaces for drug names ( i.e. , cocaine , alcohol ) filled by the word sex . second , in the sex study , the images of drugs / drug use / drug paraphernalia employed in the crt task were replaced by pictures of sexy , attractive models ( women and men ) in swimsuits taken from magazines such as maxim , fhm , and gq . previously , the pictures were ranked on sexiness by a focus group , and the top 40 were selected for the study . two additional control categories were included representing stimuli likely to have motivational value in undergraduate students : junk food ( mcdonald 's , pizza ) and snack food ( chocolate , potato chips ) . as in the study at stonehenge therapeutic community finally , in the alcohol study , participants categorized the images of alcoholic beverages and drinking ( from the crt ) , control images ( buildings ) , positive words ( i.e. , joy , happy ) , and negative words ( i.e. , rotten , disgust ) , into one of four categories : two concept categories ( alcohol and buildings ) and two attribute categories ( i like and i dislike ) . similarly , in the sex study , participants categorized the sexy images of opposite sex models ( from the crt ) , control images ( buildings ) , positive words ( i.e. , joy , happy ) , and negative words ( i.e. , rotten , disgust ) into one of four categories : two concept categories ( sex and buildings ) , and two attribute categories ( i like and i dislike ) . for the crt , one- , two- , and three - factor repeated measure anovas were used to compare total responding across the various keys . when data were not normally distributed , the analysis was performed using the friedman repeated measures anova on ranks . in case of significant interactions or significant main effects , multiple comparisons were performed using the student - newman - keuls method to identify individual mean differences ( = 0.05 ) . for iat the data , mean response latencies to categorize stimuli in the critical test blocks were computed and compared using paired t - tests . if they were not normally distributed , the data were analyzed using the wilcoxon signed rank test . furthermore , an iat difference score was calculated for each individual with lower scores reflecting more negative - automatic attitudes toward drugs / alcohol / sex . pearson correlations were employed to explore relationships between iat difference scores and other variables . for all analyses , the specific values of nonsignificant findings are not reported . for analyses presented below , subgroups were created on the basis of drug of choice and preferred route of administration . unfortunately , for the heroin- and alcohol / oral administration - based groupings , the sample sizes were too small for statistical analyses . from the admission survey , it was determined that 31 , 4 , and 4 subjects identified crack / cocaine , heroin , and alcohol as their drug of choice , respectively . as a result , the overall average ( sem ) level of self - reported craving was low ( 47 6.3 ) , with no significant differences between the groups . time spent on the crt task varied between approximately 3 and 5 minutes . from a conditioning perspective , it was predicted that specific images of drugs / drug paraphernalia / drug - taking behavior would serve as reinforcers primarily in those subjects who identified that drug as their drug of choice . in fact , the crack / cocaine group emitted significantly more responses on the keys generating images of crack / cocaine and heroin ( in comparison to control images figure 2(a ) ; [ x(5 ) = 25.04 , p = 0.0001 ] ) , the heroin group emitted more responses on the key generating heroin images ( in comparison to control images figure 2(b ) ) , but the alcohol group showed no apparent response bias ( figure 2(c ) ) . when considering the interpretations of these results , it was noted that many subjects who reported crack / cocaine as drug of choice also reported intravenous use , and that images of needles and injection / injection rituals were included only in the heroin category . therefore , the subjects were re - grouped on the basis of preferred route of administration : nonintravenous ( smoked and snorted ) , n = 19 ; intravenous , n = 16 ; and oral ( drank alcohol ) , n = 4 , and the analysis of responses was repeated . it was found that the non - intravenous group responded significantly more to the key generating images of powder / crack smoking and snorting paraphernalia in comparison to control images ( crack / cocaine category in figure 3(a ) ; [ x(5 ) = 20.79 , p = 0.0008 ] ) . by contrast , the intravenous group responded significantly more to the key generating images of needle paraphernalia and intravenous usage in comparison to control images ( heroin category in figure 3(b ) ; [ x(5 ) = 13.74 , p = 0.017 ] ) . level of operant responding of the third group ( oral ) was already represented in figure 2(c ) ( alcohol ) , and no differences were apparent . on the iat , it was found that reaction times were quicker when the categories drugs and i dislike shared the same side of the computer screen , in comparison to when the same side of the screen was shared by the categories drugs and i like . this effect was equivalent when groups were created by drug of choice ( figure 4(a ) : crack / cocaine group [ t(30 ) = 3.85 , p = 0.0006 ] ; heroin ( figure 4(b ) ) and alcohol ( figure 4(c ) ) groups : trend in the same direction ) or by preferred route of administration ( figure 4(d ) : non - intravenous group [ t(17 ) = 3.12 , p = 0.006 ] ; figure 4(e ) : intravenous group [ w = 74.00 , z = 2.90 , p = 0.001 ] ; ( figure 4(c ) ) oral group : trend in the same direction ) . there were no significant correlations between craving scores , responding on the preferred key in the crt ( regardless of grouping ) , and iat d scores . however , in the non - intravenous group , there was a significant negative correlation between days in treatment and responding on the key generating powder / crack smoking and snorting paraphernalia [ r = 0.59 , p = 0.0068 ; corrected = 0.016 ] . finally , when treatment completers ( 87% ) and noncompleters ( 13% ) were compared , no significant differences were found in responding to the preferred key in the crt ( regardless of grouping ) , craving scores , or iat d scores . from the tfm questionnaire , it was established that the average number of days on which drinking occurred in the 30 days previous to the interview was 5.6 0.5 . in spite of infrequent drinking , more than half of the subjects had an ads score equal or greater than 9 ( see table 1 ) and , overall , there was a significant positive correlation between ads score and self - reported craving for alcohol ( r = 0.40 , p = 0.004 ) . unlike in participants tested at stonehenge , the undergraduates in this study did not display a significant response bias on the crt ( time spent on the crt task varied between approximately 3 and 4 minutes ) , regardless of the ads score ( see table 1 ) . however , as in the study at stonehenge , reaction times on the iat were significantly quicker when the categories alcohol and i dislike shared the same side on the screen ( table 2 ; < 9 group : [ t(41 ) = 2.96 , p = 0.007 ] ; 9 group : [ t(26 ) = 2.67 , p = 0.013 ] ) . although there were no overall significant correlations between the iat d scores and ads scores , craving scores , or responding on the crt , it was noted that 65% of the participants were females , and when ads scores were correlated to iat d scores and craving scores separately in females and males , it was found that , in males , higher ads scores were associated with more positive automatic attitudes ( r = 0.66 , p = 0.005 ) and with higher craving scores ( r = 0.49 , p = 0.051 ) . from the questionnaires about sexual behavior and sexual craving , it was noted that although males and females did not differ on craving ( 97.7 4.1 and 92.7 4.2 ) , there was a significant modulation by relationship status . in fact , craving for sex was significantly higher in both males [ t(41 ) = 2.39 , p = 0.021 ] and females [ t(61 ) = 2.49 , p = 0.015 ] that were actively involved in a relationship at the time of testing ( table 2 ) . this suggested that regular access to a sexual partner could play an important role in modulating performance on the crt and iat . therefore , for analyses of performance on these tests , both males and females were further subdivided in those involved or not involved in a relationship ( table 2 ) . on the crt , both males and females responded significantly more to the key generating images of sexy women or men , compared to all other keys , respectively ( table 2 ; significant main effect of key [ f(5,510 ) = 11.02 , p < 0.0001 ] and significant interaction between gender and key [ f(5,510 ) = 29.53 , p < 0.0001 ] ; statement above based on the results of multiple comparisons ) . time spent on the crt task varied between approximately 3 and 4 minutes . furthermore , within both males and females , those involved in relationships responded significantly more to activate images of models of the opposite sex ( significant main effect of relationship status [ f(1,102 ) = 11.97 , p = 0.0008 ] , significant interaction between relationship status , and key [ f(5,510 ) = 3.61 , p = 0.003 ] , and significant interaction between gender , relationship status , and key [ f(5,510 ) = 4.51 , p = 0.0005 ] ; statement above based on the results of multiple comparisons ) . finally , there was a significant correlation between responses to view images of models of the opposite sex and craving scores ( males and females combined ; r = 0.43 , p = 0.001 ) , but only for those involved in a relationship . the analysis of iat data revealed that both males and females were slower to respond when the categories sex and i dislike shared the same side of the screen , and this effect was not significantly altered by relationship status ( males : main effect of category [ f(1,39 ) = 9.99 , p = 0.003 ] ; females : main effect of category [ f(1,54 ) = 12.74 , p = 0.0008 ] ) . there were no significant correlations between the iat d scores and craving scores or responding to the crt . the principal finding of this study is that individuals in long - term residential treatment for substance dependence emitted a significant number of operant responses ( i.e. , presses on a computer key ) to view images of drugs , drug use , and drug paraphernalia . responding was selective to images of drug of choice and of paraphernalia associated with participants ' preferred route of administration . in fact , those reporting crack cocaine as their drug of choice responded significantly more on the key activating images of crack cocaine and crack cocaine use / pipes . and , when groups were re - established on the basis of typical route of administration , it was found that injectors responded preferentially to the key generating images of needles and associated paraphernalia / use , and smokers / inhalers responded preferentially to the key generating images of white power , crystals , and associated paraphernalia / use . it is widely believed that selective attention to drug related stimuli is critical for the experience of cravings and the maintenance of addictive behaviors , and it is known that users display attention biases for drug related words , scenes , and images [ 5 , 7 , 55 ] . for example , moeller et al . found that the choice of cocaine - related picture correlated with subjects ' concurrent cocaine and other drug use , and predicted cocaine and other drug use over a period of 6 months . the results of the conditioned reinforcement task in abstinent participants corroborate and expand these findings . not only did subjects voluntarily select the key(s ) generating images of drug / route of choice while in treatment , but also responded more to these keys ( versus other keys ) in spite of progressively escalating response requirements . the primary interpretation of this finding is based on classical learning theory , which suggests that drug associated stimuli acquire conditioned reinforcing properties through association with the effects of drugs , and hence gain the ability to reinforce behavior in the absence of drugs . however , there are possible alternative interpretations . for example , subjects may have been bored , and thus willing to respond to any novel image . that said , participants ' responding was significantly greater on keys generating images directly associated with their drug of choice or preferred route of administration . alternatively , the images of drugs and drug paraphernalia might have been very appealing , and thus capable of promoting responding independent of prior learning . however , this seems unlikely given that undergraduate students selected on the basis of alcohol use ( alcohol study ) responded very little to keys generating images of powders , needles , crack pipes , or burning spoons . finally , the conditioning interpretation is further supported by the findings of the sex study . in fact , participants ' magnitude of responding to view pictures of sexy opposite - sex models was significantly modulated by the frequency of sexual behavior . hence , more frequent contact with the unconditioned stimulus ( in this case sexual partner ) increased responses to stimuli predictive of sexual behavior ( sexy models ; conditioned stimuli ) . therefore , although the alcohol and the sex studies were not performed in subjects that were matched to subjects in the stonehenge studies for age , gender , race , and education , they provided important results about basic psychological processes activated by the exposure to learned incentive stimuli . interestingly , a different pattern of responding was observed in abstinent alcoholics in treatment at stonehenge , who did not preferentially respond to the key generating images of beer , wine , spirits , and consumption of these beverages . although it is possible that this was due to a low - sample size ( n = 4 ) , it should be noted that low response to these images was also observed in undergraduate students who scored above 9 on the ads ( see results of alcohol study ) . the discrepancy between findings with alcohol and other drugs / sexual images is difficult to explain . it could be that there is something peculiar about alcohol or alcoholics [ 58 , 59 ] , although it is more likely that the images of alcohol / drinking were not specific enough ( i.e. , preferred drink or brand ) . such possibility could be tested by recruiting a larger sample and by presenting subjects with keys generating images of specific alcoholic beverages and then determine whether key selection is related to beverage of choice . in the study at stonehenge , the lack of correlation between responding to the crt and score on the drug craving questionnaire could imply that the psychological constructs assessed by these tasks are independent . although it is possible that subjects may have not been willing to disclose their craving because of the therapeutic setting in which testing was conducted , it is more likely that the low craving scores may have resulted from perceived nonavailability of drugs [ 6163 ] . such interpretation is supported by the sex study , in which it was found that self - reported levels of craving for sex were significantly higher in those in active relationships , and frequency of sexual behavior was significantly associated with responding to the crt . furthermore , in a separate pilot study ( n = 18 ) also performed at the stonehenge therapeutic community , craving was assessed before and after performance on the crt , and the pre- and post - crt craving scores were virtually identical . therefore , it is likely that within the context of long - term treatment centers , craving may be a psychological dimension of substance dependence that is more difficult to assess using a questionnaire and/or manipulate by exposure to drug - associated stimuli . this is consistent with low levels of spontaneous craving described within inpatient addiction units for alcohol and cocaine dependence ( see for review ) . also , in the study at stonehenge , it was found that the drug images employed in the crt elicited significant negative automatic associations assessed by the personalized iat . it is important to note that the iat does not measure attitudes toward the exemplars ( i.e. , a specific picture of cocaine ) but rather the concepts primed by the exemplars . furthermore , the specific task employed in the current study has been found to assess personal evaluative associations independent from cultural norms . therefore , this pattern of results suggests that conditioned stimuli can reinforce operant responding independently from their automatic valence , and that they retain the ability to generate these responses in abstinent individuals . one study of light drinkers found significant negative implicit attitudes and one study of heavy drinkers found a nonsignificant trend toward positive implicit attitudes . because negative implicit associations were also observed in student drinkers tested in the alcohol study , it is possible that the image - based version of the personalized iat does not explore the same automatic concepts that are generated by words ( i.e. , beer , wine ) . first , students completing the image - based sex iat generated scores reflective of positive automatic attitudes toward sex . second , the personalized iat has never been administered to substance dependent individuals recovering from cocaine and opiate addiction , and it is very likely that implicit attitudes toward these drugs change during the development of dependence . finally , in the alcohol study , ads scores were associated with more positive automatic attitudes ( r = 0.66 , p = 0.005 ) and with higher craving scores ( r = 0.49 , p = 0.051 ) , but in males only . the reason for the discrepancy between males and females in not clear , although it is fairly well established that there are significant sexual differences in psychological and physiological responses to alcohol [ 6769 ] . somewhat disappointing was the lack of significant relationship between performance on the crt and treatment completion , even though duration in treatment was negatively correlated to amount of responding on the crack / cocaine key . clearly , this issue should be addressed more systematically by additional studies that could administer the crt at multiple times during treatment . and , it may be premature to dismiss the predictive clinical value of the crt because it is also possible that self - selection bias played an important confounding role . in fact , approximately 87% of the individuals who volunteered for this study completed the program , and this is at odds with typical retention rates at stonehenge of 40%50% , which are in line with those of other therapeutic communities . in conclusion , although the data should be considered preliminary in light of small group sizes , this paper reports that substance dependent individuals in a long - term residential treatment program who did not report significant cravings for drugs voluntarily responded to view images of preferred drugs / drug use or preferred route of administration . although the predictive clinical utility of the crt is yet to be fully validated , current treatment approaches based on cue - exposure and extinction could profit from assessing behavioral responses to drug conditioned stimuli when self - reports of drug craving are uninformative .

although it is well established that drug conditioned stimuli produce a variety of conditioned responses , it is not known whether such stimuli can also reinforce an arbitrary operant response and thus serve as conditioned reinforcers . volunteers ( n = 39 ) recruited from a residential treatment center for substance dependence were tested on a task in which presses on computer keys activated images of drugs / drug paraphernalia on a progressive ratio schedule of reinforcement . they also completed a personalized craving questionnaire and a personalized implicit association test . a significant bias in responding was found for images of preferred drugs / route of drug administration . craving , however , was low and the images generated negative evaluative reactions . two additional studies were performed to ascertain the generalizability of the effects to a different population of drug - using individuals ( i.e. , students who drink ) and to incentive stimuli of a different nature ( i.e. , sexual ) . the additional studies partially replicated and extended the central findings of the main study . therefore , although these data should be considered preliminary in light of small group sizes , it is concluded that cue specificity and availability of the unconditioned stimuli ( drugs and sex ) plays a role in modulating responding maintained by conditioned reinforcers .

worldwide , thyroid cancer is the most common endocrine malignancy making up about 15% of all cancers in both males and females . over the past several decades papillary thyroid carcinoma ( ptc ) is the most common type of thyroid cancer constituting 7585% of all cases and therefore represents a major contributor to the worldwide increase of thyroid cancer . the braf mutation is the most common genetic alteration in ptc , found in 45% of cases . threonine protein kinase that plays a critical role in cell signaling as an activator within the mitogen - activated protein kinase pathway . the most common braf mutation is a valine - to - glutamic acid change in codon 600 , creating the so - called braf v600e mutation , which elevates the serine threonine activity and causes a constitutive kinase signaling pathway in ptc . the braf mutation confers a worse prognosis for ptc such as a higher risk of lymph node metastasis , extrathyroidal extension , advanced stage , vascular invasion , impaired iodine uptake , recurrence , and mortality . in addition , ptcs that are < 1 cm , but harbor braf mutations have been reported to be associated with adverse prognostic features such as extrathyroidal extension and lymph node metastasis , conveying a more ominous prognosis than their size would predict . braf v600e detection has also been a helpful diagnostic marker for ptc , and it has been shown to be a possible follow - up step to evaluate potential malignant thyroid nodules , particularly when the cytomorphological findings of the initial fine - needle aspiration ( fna ) are equivocal . studies have found that at least 20% of fnas of thyroid nodules show indeterminate cytological findings . in these cases , detection of the braf v600e mutation in the fna specimen would favor a diagnosis for ptc and patients may be considered candidates for surgical intervention at higher rates than with fna diagnosis alone . recent studies have indicated that an average of 17% of thyroid cancers , which were initially considered indeterminate on fna biopsy , were able to be diagnosed with braf mutation analysis as malignant . to maximize the diagnostic utility of fna specimens , the goal of the current study is to explore the diagnostic implication of detecting braf mutations in the supernatant fluid from cytocentrifugation of fna specimens of thyroid lesions . the current practice for the detection of braf mutations is primarily done using the cellular dna extracted from fna specimens , while the supernatant is routinely discarded . however , at times , paucicellular or acellular fna specimens may fail to provide adequate material for mutation testing . we hypothesize that enough dna may be present in the fna supernatant to conduct mutation testing and that braf mutation analysis using supernatant may increase the diagnostic yield of the fna . a total of 78 thyroid fna samples from 72 patients ( 59 females , 13 males , mean : 56 years , range : 1391 years ) were used for the current study . samples were submitted to a reference laboratory ( asuragen , austin , tx , usa ) , primarily due to indeterminate cytology results , for genetic mutation profiling ( including braf ) between may 2014 and january 2015 [ table 1 ] . samples were obtained , during fna procedures performed under ultrasound guidance , from thyroid nodules . following the fna , some patients had thyroidectomies and corresponding surgical pathology diagnosis . braf mutation testing results within fine needle aspiration cytology classification groups both the cellular dna and supernatant dna from each individual sample were analyzed for braf v600e mutation . routinely , four passes of fna were performed during each fna procedure . from the first three passes of the fna , the supernatant was removed and a thinprep slide was prepared from the cells remaining in the cytolyt tube . a fourth pass containing cellular dna was collected directly into rnaretain media for molecular testing at the reference laboratory ( asuragen , austin , tx , usa ) using allele - specific polymerase chain reaction ( pcr ) with sensitivity of detecting 2% mutant braf alleles . of note , the asuragen laboratory also tested other mutations such as kras and nras in these samples . however , the results of the other mutations were not the focus of the current study and were not used for analysis . supernatant dna was extracted using the qiagen biorobot ez1 ( qiagen , hilden , germany ) with 200 l of supernatant according to the manufacturer 's instruction . dna then underwent braf mutation testing using an in - house developed pyrosequencing - based method with a limit of 10% detection of mutant alleles . this assay has been used for more than 3 years to diagnose braf mutations in melanoma and colorectal cancers . pcr amplification was performed by applied biosystems 9700 pcr system ( applied biosystems , foster city , ca , usa ) using primers ( braf - f : 5-tct tca tga aga cct cac agt aaa aa-3 ; braf - r : 5-cca caa aat gga tcc aga ca-3 ) . the amplicons were immobilized on streptavidin sepharose high - performance beads ( ge healthcare bio - sciences , uppsala , sweden ) as instructed by the manufacturer . codons 594 - 601 of braf were sequenced with a sequencing primer ( 5-gga ccc act cca tcg aga-3 ) in the reverse direction using pyromark q24 system ( qiagen , venlo , the netherlands ) . in addition , braf mutation data were correlated with clinical , cytology , and/or surgical pathology findings . of the 78 samples evaluated , 63 ( 80% ) samples had amplifiable dna from both the supernatant and cellular components for braf mutation analysis . about 68 ( 87% ) samples had amplifiable dna from the supernatant and 73 ( 94% ) samples had amplifiable dna from the cellular . all samples had dna that was either amplified in the cellular or acellular supernatant [ table 1 ] . cytological fna diagnosis was rendered using the six diagnostic categories of the bethesda system for reporting thyroid cytopathology : nondiagnostic or unsatisfactory , benign , atypia of undetermined significance ( aus ) or follicular lesion of undetermined significance , follicular neoplasm or suspicious for a follicular neoplasm , suspicious for malignancy , and malignant . after cytological evaluation of our samples , 0 ( 0% ) were classified as nondiagnostic or unsatisfactory , 5 ( 6% ) were benign , 50 ( 64% ) were aus or follicular lesion of undetermined significance , 16 ( 21% ) were follicular neoplasm or suspicious for a follicular neoplasm , 6 ( 8% ) were suspicious for malignancy , and 1 ( 1% ) was malignant [ table 1 ] . two ( 3% ) of the 68 supernatant samples with amplifiable dna were positive for braf mutations [ table 1 ] . of importance , the cellular dna counterparts of these two samples showed no braf v600e mutations . the first case was a 29-year - old female who had an fna diagnosis of aus [ figure 1a ] with 27% mutant v600e allele by pyrosequencing [ figure 2b ] . the second case was a 52-year - old female who had an fna showing findings suspicious for ptc [ figure 1b ] which was confirmed in the resected thyroid [ figure 1c ] . this case had 34% mutant v600e allele by pyrosequencing in the supernatant [ figure 2c ] . these samples were checked for validity against a pyrosequenced wild type control [ figure 2a ] and reagent ( negative ) control [ figure 2f ] . in addition , the resected thyroid specimen showed the identical v600e mutation [ figure 2e ] by the same method used for supernatant , confirming that the supernatant result was truly positive . ( a ) fine - needle aspiration showing a cluster of atypical follicular cells characterized by slight nuclear enlargement and nuclear overlapping ( pap , 400 ) . ( b ) fine - needle aspiration shows clusters of atypical follicular cells displaying slight nucleomegaly and nuclear overlapping ( diff - quick , 400 ) . ( c ) surgical resection reveals 1.2 cm focus of papillary thyroid carcinoma ( 200 ) braf mutation site comparison between wt and v600e samples . ( a ) wild type control ; ( b and c ) two samples with braf v600e mutations in the supernatants and wild type in the cellular counterparts ; ( d ) an example of braf v600e mutation in the cellular counterpart but wild - type allele in the supernatant ; ( e ) braf v600e mutation as identified in the thyroidectomy specimen of the case shown in c ; ( f ) reagent ( negative ) control five samples were positive for braf mutations using cellular dna [ table 1 ] . two of the samples had no amplifiable dna in the supernatant , and the remaining three showed no mutation in the supernatant [ figure 2d ] . these five samples showed aus ( n = 3 ) , suspicious for malignancy ( n = 1 ) , and malignant ptc ( n = 1 ) by fna . of note , one of the aus cases had follow - up surgical resection specimens revealing ptc in which the v600e mutation was again confirmed by the in - house pyrosequencing assay ( data not shown ) . among the eleven samples that showed fna and/or surgical pathology findings suspicious for or diagnostic of ptc ( suspicious for malignancy [ n = 6 ] , malignant [ n = 1 ] , and/or surgical pathology diagnostic of ptc [ n = 4 ] ) [ table 1 ] , 6 ( 55% ) samples ( one supernatant and 5 cellular ) were positive for the braf v600e mutation . this suggests that testing the supernatant dna in fna specimens may increase the diagnostic yield by 1/11 ( 9% ) in this setting . however , in the setting of indeterminate cytology , the yield of testing supernatant was less and increased the braf mutation detection by only about 2.4% ( 1/42 supernatant samples with amplifiable dna ) . our results indicate that the vast majority ( 87% of our samples ) of routinely discarded fna supernatants contain amplifiable dna that could be used for molecular analysis . in addition , some samples of the supernatant contained the mutated braf v600e allele which was not present in the cellular counterpart of the same individual patient sample . therefore , this routinely discarded material could be critical in some cases to diagnose and properly treat ptc patients . in the current study , all of the braf v600e mutations detected in the supernatant were paired with wild - type braf results in the cellular component of the same fna sample . of interest , all patients with cellular components showing braf v600e mutations had the corresponding supernatants revealing either wild - type braf or no amplifiable dna . the higher sensitivity ( detecting 2% of mutant alleles ) of the method used to detect braf mutations in the cellular component may have contributed to the increased detection of mutated braf genes in the cellular component as evaluation of the supernatant component used a method with sensitivity of detecting only 10% of alleles . using a more sensitive method to test the supernatant , similar to the sensitivity of the one performed on the cellular component at the reference laboratory , may detect more mutations in these samples . however , this is beyond the scope of this exploratory study where the goal is to preliminarily show the diagnostic utility of the fna supernatant . however , the differences in sensitivity between the two different assays used can not explain the discrepancies within the samples with mutant alleles in the supernatant and wild type alleles in the cellular component . it is conceivable that some tumors may have a higher cellular turnover ( i.e. , increased mitosis , apoptosis , and necrosis ) , resulting in the shedding of dna ( cell - free dna ) in the interstitial fluid of the tumor , which is then caught only in the supernatant . therefore , the lack of braf mutations in the cellular component of these samples could be likely due to sampling issues ( i.e. , cancer cells with braf mutations were missed by the fna needle in the fourth pass , which is the sample submitted to the reference laboratory , resulting in a negative cellular dna analysis while the tumor cells with the braf mutations were obtained from the first three fna passes and therefore were present in the supernatant fluid leading to a positive supernatant dna analysis ) . another possibility , although less likely , is that the supernatant contained a few tumor cells that remained after centrifugation . to the best of our knowledge , the current study represents the first attempt to use fna thyroid specimen supernatants to test for braf mutations in ptc . our findings of detecting braf mutations in fna supernatants of ptc are in agreement with the results of a recent study , which demonstrated that the use of fna supernatant specimens from patients with pancreatic cancers for molecular testing may outperform cellular dna analysis in certain situations . we attempted to quantify the dna amounts in the supernatant ; however , the majority of samples showed the amount of dna was below the linear range of nanodrop 1000 ( thermo scientific , wilmington , de ) even though 87% of our samples had enough amplifiable dna for the detection of braf mutation . of note , we only used 200 l of about 2550 ml supernatant for dna extraction ; therefore the yield of dna could potentially be increased if a greater amount of supernatant was used for dna extraction . the percentage of braf mutations among aus cases in the current study is lower than in the literature ( 8% or 4/50 combining both supernatant and cellular samples vs. the 1520% found in other studies ) . however , this braf mutation rate is compatible to that of a recent study using residual thinprep material from thyroid fnas for braf testing . our results indicate that profiling the mutations of braf and other genes using dna from routinely discarded fna supernatants may provide valuable diagnostic information to assist with the diagnosis of ptc in a substantial percentage of patients , particularly if clinical / morphologic findings are suspicious for malignancies and no mutations are identified in cellular dna samples . in the current study , testing supernatant dna in the fna specimens increases the diagnostic yield by 1/11 ( 9% ) . based on the findings of the current study , we suggest that the fna supernatants should be kept until the cellular molecular testing results are available . if the molecular test results using the cellular dna are inconclusive ( i.e. , negative or no amplifiable dna ) , the supernatants can be then submitted for molecular testing to maximize the diagnostic yield of an fna when clinically indicated . this may prevent patients from being subjected to unnecessary further work - ups such as repeat fna procedures . gc performed the mutation study . at and mh made the diagnosis and provided the photographs . aus : atypia of undetermined significance dna : deoxyribonucleic acid fna : fine - needle aspiration pcr : polymerase chain reaction ptc : papillary thyroid carcinoma wt : wild type genotype . to ensure the integrity and highest quality of cytojournal publications , the review process of this manuscript was conducted under a double - blind model ( authors are blinded for reviewers and vice versa ) through automatic online system .

objective : braf mutations using cellular dna from fine - needle aspiration ( fna ) specimens are commonly used to support the diagnosis of papillary thyroid carcinoma ( ptc ) . the goal of this study was to preliminarily evaluate the diagnostic utility of detecting braf mutations in the routinely discarded fna specimen supernatant fluid.materials and methods : seventy - eight fnas of thyroid lesions were evaluated for braf mutations using both cellular and supernatant dna . braf mutation data were correlated with cytology and surgical pathology.results:of the 78 samples evaluated , 68 ( 87% ) had amplifiable dna in the supernatant with 2 ( 3% ) positive for braf mutations . these two samples showed no mutations in the cellular counterpart . among the 11 samples showing morphologic findings ( fna / surgical pathology ) suspicious / diagnostic of ptc , 6 ( 55% ) samples ( one supernatant and five cellulars ) were positive for braf mutations . this suggests that testing supernatant dna in fna specimens may increase the diagnostic yield by 1/11 ( 9% ) in this setting.conclusions:the vast majority of routinely discarded fna supernatants contain amplifiable dna . in addition , profiling the mutations of braf and other genes using supernatant dna may provide valuable diagnostic information to assist the diagnosis of ptc in patients with clinical / morphologic findings suspicious for malignancies and cellular dna showing no mutations .

amyotrophic lateral sclerosis ( als ) is a neurodegenerative disorder that affects the upper and lower nervous system . clinical features are progressive weakness , muscle atrophy , fasciculation , muscle spasms , dysarthria , dysphagia , and dyspnea . pemphigus is an autoimmune blistering disease that affects skin and mucous membrane and usually occurs in adults with a mean age of 4060 years . the incidence of pemphigus ranges from 0.1 to 0.5/100,000 per year . in iran , the incidence rate is estimated approximately 5/100,000 . the common manifestations of the disease are painful erosions in oral mucosa and flaccid blisters on the normal skin or erythematous base . we report a case of an als in a male patient , followed by pemphigus vulgaris ( pv ) . to the best of our knowledge a 60-year - old male patient was admitted to our clinic with painful mucocutaneous lesions since 1 week before admission . due to als , the patient suffered from dysphagia and odynophagia about 6 months ago . nutritional support through a nasogastric tube was performed andpercutaneous endoscopic gastrostomy ( peg ) was inserted after several days . the patient s functional status and quality of life ( qol ) decreased gradually over time . in general , we observed flaccid blisters in different sizes and multiple erosions on his trunk , lower extremity , and flexural area [ figure 1 ] . flaccid blisters , erosions , and crusted plaques were observed on the patient s trunk and inguinal areas a complete blood count and serum biochemical studies showed normal results . skin biopsy was performed to examine histology which revealed suprabasal acantholysis [ figure 2 ] . an immunofluorescence study demonstrated intercellular deposition of igg and c3 , confirming the diagnosis of pv . 10 ) the patient was treated with systemic prednisone ( 60 mg / day in divided doses , 1 mg / kg ) and azathioprine 50 mg 3 times a day ( 2/5 mg / kg / day ) . after 9-month follow - up , oral feeding was improved and the improvement in qol was achieved . therefore , the peg tube was removed . he was close in follow - up and was doing well [ figure 3 ] . the written consent form was obtained from the patient . healed skin lesions with remained hyperpigmentation after 9 months of treatment studies show that the incidence and prevalence rate of als in iranian populations , in comparison to other countries , are lower . the disease is associated with human leukocyte antigen ( hla ) class ii , in particular hla - dr and hla - dq . in keeping with previous studies , hla - dr , hla - dp , and hla - dq staining in addition to these straightforward descriptions , autoimmunity may justify the association between pv and als . reported that three cases out of 168 patients suffering from als in 2000 and showed clinical and histological features of bullous pemphigoid . as a recent epidemiologic study , the association between bullous pemphigoid and als is more than coincidental . they discussed that the interrelation between bullous pemphigoid antigen 1 ( bp ag1 ) and neurofilaments may lead to cutaneous consequence . hence , it seems that neurological disorders may be associated with bullous dermatitis . in another study , foureur et al . reported an 84-year - old woman with right hemiparesis secondary to ischemic cerebral stroke in 2001 . two hypotheses were proposed for the coexistence of these two diseases : the neuroautoimmunity association with the aging processthe autoantibody reactivity against dystonin , which shares homology with bp ag1 in c - terminal location . the neuroautoimmunity association with the aging process the autoantibody reactivity against dystonin , which shares homology with bp ag1 in c - terminal location . cenk kohen and beril kucumen described that a 53-year - old male als patient in 2011 whose disease had started 10 years ago . it is the only study that reported a case of pv accompanied with als . in 2013 , thongprasom et al . the patient suffered from discoid lupus erythematosus , dermatomyositis , asymmetric polyarthritis , and multiple enthesopathy during a long - term follow - up . in this rare case , pv is an uncommon condition in als patient . according to mentioned studies and the existence of autoantibodies in pathogenesis of both diseases , it seems that the association between pv and als is more than being coincidental . however , further studies are needed to be investigated . if patients present with bullous skin lesions in the course of advanced neurologic disorder , physicians should consider an autoimmune bullous disease such as pv and bp . in summary , the early successful detection in control of both neurologic disease and skin lesion is important . fm contributed to the conception of the work , conducting the study , drafting and revising the draft , approval of the final version of the manuscript , and agreed for all aspects of the work . mm contributed to the conception of the work , conducting the study , drafting and revising the draft , approval of the final version of the manuscript , and agreed for all aspects of the work . fr contributed to the conception of the work , conducting the study , drafting and revising the draft , approval of the final version of the manuscript , and agreed for all aspects of the work .

pemphigus vulgaris ( pv ) is an autoimmune bullous and erosive mucocutaneous disease . rarely , it occurs in patients with other autoimmune disease . the relation between pv and neurological disorders is unclear and needs to be more studied . here , we report a case of amyotrophic lateral sclerosis ( als ) , followed by dermatologic involvement . histopathological evidence and direct immunofluorescence are consistent with pv . systemic corticosteroid and azathioprine were effective in the treatment of mucocutaneous lesions . pv seems to be accidentally associated with als . expression of major histocompatibility complex class ii in autoimmune disease and production of autoantibodies have been proposed to describe the association of pv with als .

approximately one in every five hospitalized patients is readmitted within 30 days . currently , two - thirds of us hospitals have reimbursement penalties for higher than expected 30-day readmission rates from the center for medicaid and medicare services [ 2 , 3 ] . it is expected that similar penalties will be extended to other procedures and diagnoses including cardiac surgery . in preparation for the expansion of the penalty system in the usa and to improve prediction of patients at high risk of postdischarge complications leading to readmissions or premature death , risk factors must be identified early in the hospital course to align the best possible quality and continuity of care . currently , a validated risk model for predicting readmissions after cardiac surgery is not available and few risk factors for readmission are known . recent evidence from california reported an association between infection and higher rates of 30-day readmission after cardiac surgery . however , identification of infection after discharge without routine monitoring of a postcardiac surgical patient is problematic . what is needed is for clinical care teams to identify patients at high risk of infection before cardiac surgery to determine readiness and safety for the patient to undergo surgery . a common marker of inflammation is white blood cell ( wbc ) count , routinely measured prior to cardiac surgery . wbc count provides a broad measure of inflammation status , whether as a result of infection or proinflammatory disease states such as diabetes , copd , or hemodialysis [ 58 ] . elevated wbc count is reported as a component of the systemic inflammatory response syndrome ( sirs ) to sepsis and is endorsed as a marker for reporting the systemic inflammatory response to cardiopulmonary bypass [ 9 , 10 ] . in addition , current evidence has shown that preoperative wbc count is predictive of in - hospital mortality and stroke and major bleeding after coronary artery bypass graft surgery and associated with complications in other endovascular and thoracic procedures [ 13 , 14 ] , suggesting that preoperative wbc count may aid clinical care teams in risk - stratifying patients prior to surgery . however , it is not known if a patient 's baseline inflammatory state measured by crude wbc count could predict 30-day readmission . therefore , we sought to evaluate whether preoperative wbc count was associated with 30-day readmissions after cardiac surgery . patients undergoing coronary artery bypass graft ( cabg ) surgery and/or valve surgery within the northern new england cardiovascular disease study group ( nne ) between july 2008 and december 2010 were enrolled in the cohort . a total of 2,209 consecutive patients were included along with 268 readmissions to the hospital performing the index cardiac surgery . twelve patients were excluded due to missing white blood cell counts and twenty - one for incomplete data , leaving a total of 2,176 patients and 259 readmissions occurring within 30 days of discharge from the index cardiac surgery admission . all institutional review boards for each center reviewed and approved the data collection for the nne registry and supplementary data collection for readmissions . the nne is a voluntary regional consortium of physicians , allied health professionals , research scientists , and hospital administrators from institutions in maine , new hampshire , and vermont that support coronary revascularization and open - heart surgery . the goal of the consortium is to foster continuous improvement in the quality , safety , and effectiveness of care for patients with cardiovascular disease through the analysis of process and outcomes data with timely feedback to the health care professionals providing these services . all the hospitals providing open - heart surgery in this region contribute data on consecutive cases with validation of procedure numbers and mortality performed every two years . the registry collects data on patient characteristics , procedural indication , priority , and process , and in - hospital outcomes ( see http://www.nnecdsg.org/ for the data forms and publically available data ) . wbc count was defined as the last preoperative measurement of wbc taken prior to procedure , was collected by data abstractor at each center . categories of wbc counts were divided into predefined categories ( < 6.0 , 6.07.9 , 8.09.9 , 10.012.0 , and > 12.0 thousands per cubic millimeter , mm ) . baseline , operative , and postoperative outcomes were compared using chi - square tests and continuous data using student 's t - test or wilcoxon rank sum tests where appropriate . we conducted both univariate and backwards stepwise logistic regression removing risk factors that did not reach an alpha < 0.1 among only risk factors with an alpha < 0.1 from univariate comparisons . all risk factors meeting an alpha < 0.1 were included in the final model multivariate logistic regression model . categories of white blood cell counts were then added to the multivariate clinical risk prediction model . we conducted a hosmer - lemeshow goodness of fit test and calculated the area under the receiver operating characteristic ( roc ) curve for the final multivariate model with categories of white blood cell count and reported the roc and 95% confidence intervals for each model . among the 2,176 patients , 259 patients were readmitted within 30 days ( 11.9% ) . patient demographics were similar between patients with a 30 day readmission and those without a readmission . patients readmitted within 30 days were more likely to have chronic obstructive pulmonary disease , history of dialysis , single vessel coronary disease , and white blood cell counts greater than ten thousand prior to surgery ( table 1 ) . procedural factors associated with 30-day readmission included valve or combined cabg / valve procedure , on - pump surgery , nadir hematocrit < 20 on bypass , three or more packed red blood cell transfusions , use of inotropes , and the development mediastinitis , aki , or atrial fibrillation ( table 2 ) . patients with elevated wbc counts at baseline ( 10,00012,000 and > 12,000 mm ) had higher 30-day readmission than those with lower wbc counts prior to surgery ( 15% and 18% compared to 10%12% , p = 0.037 , figure 1 ) . after backwards stepwise regression , wbc count and other risk factors remained significantly associated with 30-day readmission including number of diseased vessels , on - pump surgery , nadir hematocrit < 20 on bypass , receiving three or more packed red blood cells , developing mediastinitis , and acute kidney injury ( table 3 ) . type of surgery ( valve , isolated coronary artery bypass graft , or combined valve / graft ) and duration of bypass were not significantly associated with readmissions in the multivariate model . adjusting odds ratios for preoperative wbc counts were 1.42 ( 0.86 , 2.34 ) for counts 10,00012,000 ( mm ) and 1.81 ( 1.03 , 3.17 ) for counts > 12,000 ( mm ) ( table 2 ) . the calculated c - statistic was 0.66 with a hosmer - lemeshow goodness of fit chi - square of 10.94 and p value of 0.2 . patient and procedural characteristics stratified by white blood cell categories are summarized in table 4 . we explored the predictive ability of wbc counts prior to cardiac surgery on 30-day readmission . with and without adjustment of other risk factors for readmission , patients with preoperative wbc counts > 12,000 ( mm ) were significantly more likely to be readmitted to the hospital within 30 days from discharge . we are the first to demonstrate that a marker of inflammation prior to the start of surgery demonstrates increased risk of 30-day readmission and should be incorporated into risk models to predict readmission prior to discharge from cardiac surgery . wbc count has enjoyed a resurgence in recent years as a valid marker of inflammation and as a strong independent predictor of future coronary heart disease and stroke [ 15 , 16 ] . after an acute event , patient outcomes remain influenced by wbc count at the time of hospital admission . in several studies , peak wbc count or elevated monocyte count has been linked to death or major adverse cardiac events ( maces ) outcomes , including readmission [ 1719 ] . other strong evidence has linked high wbc count at admission with adverse outcomes ( mortality and bleeding ) in patients undergoing coronary revascularization with cardiopulmonary bypass [ 11 , 12 ] . however , in the case of cardiopulmonary bypass it is unclear whether high wbc count contributes to preexisting risk or to development of the systemic inflammatory response postoperatively or both . the systemic inflammatory response is a complication in cardiopulmonary bypass patients that is caused by a combination of surgical stress and contact activation of blood component in the extracorporeal circuit [ 20 , 21 ] . it is poorly defined and the only formal definition is the systemic inflammatory response syndrome ( sirs ) , borrowed from the sepsis field . according to the definition , sirs exists when any two out of four criteria relating to abnormal temperature , heart rate , respiratory rate , or white cell counts exist . an evidence - based review of the inflammatory response indicated that all four sirs criteria were rarely monitored in the setting of cardiopulmonary bypass as they were felt to be too nonspecific and if taken literally would apply to approximately 40% of all patients [ 2426 ] . a more recent update on minimal reporting criteria by the outcomes consensus panel singled out wbc count as the only criterion measured on its own as being relevant to the inflammatory status . this recommendation was supported by other fields in which wbc count is recognized as a valid marker of inflammation [ 5 , 6 , 8 , 27 ] . an alternative theory for the development of the systemic inflammatory response is that this is determined less by the extracorporeal circuit itself but rather by preexisting activation of white cells and endothelium or by preoperative transfusion . consistent with this theory is that high wbc count prior to coronary surgery utilizing cardiopulmonary bypass is linked with adverse outcomes including mortality and bleeding [ 11 , 12 ] . our present findings that high wbc count before - surgery is linked to an increased risk of 30-day readmission after discharge add further weight to this idea . we therefore conclude that wbc count measured prior to cardiac surgery may serve as a measure of the patient 's inflammatory status and could aid in identifying and managing patients at heightened risk of readmission after discharge from cardiac surgery . this becomes especially relevant in an era when higher than expected readmission rates may attract financial penalties to hospitals .

approximately 1 in 5 patients undergoing cardiac surgery are readmitted within 30 days of discharge . among the primary causes of readmission are infection and disease states susceptible to the inflammatory cascade , such as diabetes , chronic obstructive pulmonary disease , and gastrointestinal complications . currently , it is not known if a patient 's baseline inflammatory state measured by crude white blood cell ( wbc ) counts could predict 30-day readmission . we collected data from 2,176 consecutive patients who underwent cardiac surgery at seven hospitals . patient readmission data was abstracted from each hospital . the independent association with preoperative wbc count was determined using logistic regression . there were 259 patients readmitted within 30 days , with a median time of readmission of 9 days ( iqr 416 ) . patients with elevated wbc count at baseline ( 10,00012,000 and > 12,000 mm3 ) had higher 30-day readmission than those with lower levels of wbc count prior to surgery ( 15% and 18% compared to 10%12% , p = 0.037 ) . adjusted odds ratios were 1.42 ( 0.86 , 2.34 ) for wbc counts 10,00012,000 and 1.81 ( 1.03 , 3.17 ) for wbc count > 12,000 . we conclude that wbc count measured prior to cardiac surgery as a measure of the patient 's inflammatory state could aid clinicians and continuity of care management teams in identifying patients at heightened risk of 30-day readmission after discharge from cardiac surgery .

1,2,3-trichloropropane ( tcp , cas no . 96 - 18 - 4 ) is a non - natural , biodegradation - recalcitrant and toxic compound that occurs in groundwater and soil , mainly as a result of improper disposal of tcp - contaminated chemical waste ( agency for toxic substances and disease registry [ atsdr ] 1992 , 2011 ) . tcp is formed as a by - product during the synthesis of various chemicals , most notably in the classical synthetic route to epichlorohydrin , and was present in commercial preparations of the soil fumigant 1,3-dichloropropene ( also known under the trade name d - d ) , which is now abandoned ( tesoriero et al . 2001 ) . for example , fluorination of tcp is used to produce the cross - linking agent hexafluoropropylene , which is applied for making elastomers . tcp is also used in the chemical industry as a solvent for oils and fats , waxes , and resins . other past uses of this compound include in paint thinner and varnish remover , and as a degreasing agent . the toxic substances control act inventory of the us environmental protection agency ( us epa ) estimated the usage of tcp in 2002 as 15 10 kg ( us epa 2004 ) . the federal facilities restoration and reuse office ( ffrro ) of the us epa has listed tcp as an emerging contaminant in december 2010 for which physical , toxicological and environmental data were summarized in a fact sheet ( ffrro 2011 ) and a review of the toxicology of tcp was written ( us epa 2009 ) . the european union chemicals agency ( echa ) has listed tcp as a chemical of very high concern because of its carcinogenic , mutagenic and reproductive effects ( echa 2012a ) . contamination of soil and groundwater by tcp has occurred both as point source pollution , due to improper disposal of wastes or accidental spillage , and as diffuse contamination , due to its presence in the nematicide 1,3-dichloropropene . tcp has been detected in hundreds of surface water and drinking water sources , e.g. , in the united states , at levels of 0.1100 g / l ( oki and giambelluca 1989 ; atsdr 1992 , 2011 ; california environmental protection agency 2009 ) . in a dutch monitoring program , tcp was detected in surface water of the rhine , meuse , westerscheldt , and in the northern delta area ( miermans et al . groundwater samples from the netherlands were found to contain tcp as well as 1,2-dichloropropane due to the application of impure nematicides , especially in potato fields ( lagas et al . 1989 ) . tcp was also detected in the river nitra , slovakia ( liska et al . 1996 ) and along with a range of other volatile organohalogens in water at an industrial site in the osaka area , japan ( yamamoto et al . tcp is a suspected human carcinogen based on evidence of tumor formation in studies with rats and mice ( irwin et al . because of its toxicity , the presence of tcp in groundwater can pose a serious risk to human health and ecosystem quality , and the national toxicology program ( 1993 , 2005 ) of the us department of health has listed tcp as reasonably anticipated to be a human carcinogen ( us national library of medicine 2011 ) . toxicological properties of tcp are included in the echa registered substances database ( echa 2012b ) and the nih hazardous substances data bank ( hsdb 2000 ) . remediation of tcp - contaminated sites is difficult due to its persistent nature and its physiochemical properties , which cause spreading with flowing groundwater ( salter et al . biodegradation has been observed both under anaerobic and aerobic conditions , but appears to be slow and mostly due to cometabolism , with little evidence for tcp supporting growth or adaptation of bacteria . based on laboratory studies there are indications that tcp may serve as an electron acceptor under anaerobic conditions . in this review , we discuss abiotic and biotic transformations of tcp , as well as the possibilities of enzymatic dehalogenation of tcp and genetic construction of tcp - degrading bacteria . tcp , also known as allyl trichloride , trichlorohydrin or glycerol trichlorohydrin , is a clear and colorless liquid , with a strong odor similar to that of chloroform or trichloroethylene . it is soluble in ethanol , ether , and chloroform , and only slightly soluble in water . like other chlorinated hydrocarbons tcp is flammable , and when heated to decomposition , it yields toxic fumes of hydrogen chloride gas ( us national library of medicine 2011 ) . the toxicological properties of tcp were recently reviewed by the us epa ( 2009 ) and earlier by the who ( 2003).the major pathways for metabolism of tcp in higher organism starts with oxidative transformation by microsomal cytochrome p-450 or substitution by glutathione transferase ( mahmood et al . p450-monooxygenase - mediated conversion leads to formation of 2,3-dichloropropanal and 1,3-dichloroacetone , which can be reduced to 2,3-dichloropropanol and 1,3-dichloropropanol , presumably by dehydrogenase activity ( fig . 1 ) . glutathione conjugation of tcp may produce glutathione adducts that can undergo intermolecular substitution to form a highly reactive episulfonium ion , but glutathione can also prevent alkylation of proteins by the electrophilic monooxygenase products 2,3-dichloropropanal and 1,3-dichloroacetone ( weber and sipes 1992 ) . metabolism of glutathione conjugates formed from tcp and its oxidation products also gives rise to various glutathione , cysteine and n - acetylcysteine conjugates . thus , tcp itself is not mutagenic , but the products ( such as 2-chloroacrolein and episulfonium ions ) are strong alkylating agents , explaining the toxicity and mutagenicity of tcp . using radiolabeled tcp , formation of dna adducts after exposure to tcp has indeed been demonstrated in mice ( la et al . 1metabolic pathways of 1,2,3-trichloropropane ( tcp ) ( mahmood et al . 1991 ; la et al . 1995 ; who 2003 ; us epa 2009 ) . reactions : a initial monooxygenase ( p450)-mediated conversion and dehydrogenation leading to dichloro - metabolites ; b glutathione conjugation can occur on tcp and oxidized derivatives ; c glutathione adducts can be converted to highly toxic species , such as episulfonium ions ; d the final products include dna adducts , as well as glutathione derivatives that are secreted metabolic pathways of 1,2,3-trichloropropane ( tcp ) ( mahmood et al . 1991 ; la et al . 1995 ; who 2003 ; us epa 2009 ) . reactions : a initial monooxygenase ( p450)-mediated conversion and dehydrogenation leading to dichloro - metabolites ; b glutathione conjugation can occur on tcp and oxidized derivatives ; c glutathione adducts can be converted to highly toxic species , such as episulfonium ions ; d the final products include dna adducts , as well as glutathione derivatives that are secreted animal studies have shown that long - term exposure to tcp may cause kidney failure , reduced body weight and tumors ( irwin et al . 1995 ; atsdr 1992 ; us epa 2009 ; echa 2012b ) . the guidelines for carcinogen risk assessment and irac monographs reported that tcp is expected to be carcinogenic to humans , based on animal studies , as well as resemblance of metabolism between human and rodent microsomes , in vitro mutagenicity , and the ability to form dna adducts ( irwin et al . properties that determine the behavior of tcp in the environment include : its high density ( 1.39 g / ml ) ; the modest water solubility ( 1.75 water partitioning coefficient ( log kow = 2.02.5 , quite similar to the value for trichloroethylene ) ; and the low henry coefficient ( h = 3.23.4 10 atm m / mol at 25 c ) . the high density causes tcp , when dumped as a liquid on soil or in ponds , to sink to lower levels , e.g. , into groundwater or into accessible subsurface structures like rock fissures . water partitioning coefficient implies substantial distribution via groundwater flows , even when organic carbon is present . furthermore , tcp has a tendency to evaporate from surface water to air ( but less than trichloroethylene , h = 10 10 atm m / mol ) , where , on exposure to sunlight , it is subjected to photo - degradation by reaction with hydroxyl radicals with a half - life of about 15 days . the volatility of tcp , the possibility of washout by precipitation , and its resistance to degradation in water , may result in cycling of tcp between environmental compartments ( atsdr 1992 ; us epa 2009 ) . bioaccumulation and biomagnification are expected to be of minor importance in view of the modest lipophilicity of tcp . tcp can stay in groundwater for a prolonged period , in part due to its low organic carbon partitioning coefficient , but especially because rates of abiotic and biotic degradation in groundwater are low . abiotic hydrolysis of tcp under basic and neutral abiotic conditions has been studied at different temperatures and ph values and in the presence of different ions such as sulfide and carbonate ( sarathy et al . tcp appears to be highly stable under a variety of conditions , with an expected half - life of hydrolysis under environmental conditions ( 25 c , ph = 7 ) in the order of hundreds of years ( pagan et al . 2,3-dichloro-1-propene was detected as a product that could be converted to 2-chloro-2-propen-1-ol ( fig . 2 ) . other non - stimulated abiotic reactions in water under environmental conditions have not been characterized.fig . 2abiotic transformations of tcp under non - stimulated conditions ( a ) , photochemical conversion or catalytic conversion in the presence of radical - generating oxidants ( b ) , conversion in the presence of fenton reagent ( c , d ) , and conversion under stimulated anaerobic ( reductive ) conditions ( e ) . see text for details abiotic transformations of tcp under non - stimulated conditions ( a ) , photochemical conversion or catalytic conversion in the presence of radical - generating oxidants ( b ) , conversion in the presence of fenton reagent ( c , d ) , and conversion under stimulated anaerobic ( reductive ) conditions ( e ) . see text for details the physical and chemical properties of tcp obviously strongly influence possibilities for remediation of soil and groundwater ( salter - blanc and tratnyek 2011 ) . on - site methods for treating tcp - polluted groundwater include pump and treat and vacuum extraction . the latter is used for different volatile organohalogens , but it is not a very favorable approach because of the low henry s law constant of tcp ( ch2 m hill 2005 ) . extracted water can be treated by absorption , using activated carbon , by chemical oxidation or by vacuum extraction . for example , full - scale remediation of tcp - contaminated groundwater is under investigation at the tyson superfund site near philadelphia , pa , using vacuum extraction of soil , extraction of groundwater , and treatment of extracted water and vapor with activated carbon for the removal of tcp ( pezullo et al . 2005 ) . chemical oxidation is performed with oxidizing agents such as ozone , permanganate or hydrogen peroxide . they cause decomposition of tcp into carbon dioxide , water and chloride ions ( dombeck and brog 2005 ) ( fig . 2 ) . with a mild oxidant such as permanganate , transformation of tcp is slow whereas in the presence of strong oxidants such as hydroxyl radicals , generated photochemically , and sulfate radicals , catalytically generated from persulfate , transformation is much faster ( ch2 m hill 2005 ) . chemical tcp oxidation may also be initiated by the fenton reagent ( h2o2 with an iron catalyst ) . khan et al . ( 2009 ) studied the effect of iron type ( fe , fe and fe ) on the removal of tcp by h2o2 , and found that fe was most effective in reducing tcp and increasing its biodegradability . degradation products were 1,3-dichloroacetone and 2,3-dichloro-1-propene if fe and fe were used , and isopropanol and propionaldehyde if fe was used , confirming extensive oxidative conversion . chemical oxidation can also be used for in situ treatment of tcp contamination . in case tcp is present in the form of a dense non - aqueous - phase liquid ( dnapl ) , oxidants can be introduced into the subsurface to achieve contaminant oxidation . chlorinated solvents such as trichloroethene , carbon tetrachloride and tcp may also be removed by abiotic reduction with zero - valent iron . thus , distribution of tcp via flowing groundwater can be prevented by using a zero - valent iron ( fe ) barrier . ( 2003 ) investigated the effects of carbonate , silica , chloride and organic matter on the removal of various organohalides by granular iron using column studies . the results indicated that differences in groundwater chemistry have a strong effect on the activity and longevity of the granular iron , which will influence the design of reactive barriers . compounds enhancing metal corrosion ( carbonate , chloride ) may improve reactivity , whereas compounds such as feco3 and na2sio3 can reduce the activity , especially upon prolonged treatment , through deactivation of the metal surface . propane , propylene , and trace amounts of 1-chloro-2-propene were detected as tcp transformation products ( fig . 2 ) , indicating a role for reductive dechlorination and elimination of hcl in the removal of tcp . reductive transformation of tcp was also found with zero - valent zinc , which exhibited a reactivity that was more than an order of magnitude higher than that of iron ( sarathy et al . groundwater components that influenced zinc surface properties through corrosion or formation of an inactive layer of zno or zn(oh)2 had a large influence on the removal kinetics . the toxicological properties of tcp were recently reviewed by the us epa ( 2009 ) and earlier by the who ( 2003).the major pathways for metabolism of tcp in higher organism starts with oxidative transformation by microsomal cytochrome p-450 or substitution by glutathione transferase ( mahmood et al . p450-monooxygenase - mediated conversion leads to formation of 2,3-dichloropropanal and 1,3-dichloroacetone , which can be reduced to 2,3-dichloropropanol and 1,3-dichloropropanol , presumably by dehydrogenase activity ( fig . 1 ) . glutathione conjugation of tcp may produce glutathione adducts that can undergo intermolecular substitution to form a highly reactive episulfonium ion , but glutathione can also prevent alkylation of proteins by the electrophilic monooxygenase products 2,3-dichloropropanal and 1,3-dichloroacetone ( weber and sipes 1992 ) . metabolism of glutathione conjugates formed from tcp and its oxidation products also gives rise to various glutathione , cysteine and n - acetylcysteine conjugates . thus , tcp itself is not mutagenic , but the products ( such as 2-chloroacrolein and episulfonium ions ) are strong alkylating agents , explaining the toxicity and mutagenicity of tcp . using radiolabeled tcp , formation of dna adducts after exposure to tcp has indeed been demonstrated in mice ( la et al . 1metabolic pathways of 1,2,3-trichloropropane ( tcp ) ( mahmood et al . 1991 ; la et al . 1995 ; who 2003 ; us epa 2009 ) . reactions : a initial monooxygenase ( p450)-mediated conversion and dehydrogenation leading to dichloro - metabolites ; b glutathione conjugation can occur on tcp and oxidized derivatives ; c glutathione adducts can be converted to highly toxic species , such as episulfonium ions ; d the final products include dna adducts , as well as glutathione derivatives that are secreted metabolic pathways of 1,2,3-trichloropropane ( tcp ) ( mahmood et al . 1991 ; la et al . 1995 ; who 2003 ; us epa 2009 ) . reactions : a initial monooxygenase ( p450)-mediated conversion and dehydrogenation leading to dichloro - metabolites ; b glutathione conjugation can occur on tcp and oxidized derivatives ; c glutathione adducts can be converted to highly toxic species , such as episulfonium ions ; d the final products include dna adducts , as well as glutathione derivatives that are secreted animal studies have shown that long - term exposure to tcp may cause kidney failure , reduced body weight and tumors ( irwin et al . 1995 ; atsdr 1992 ; us epa 2009 ; echa 2012b ) . the guidelines for carcinogen risk assessment and irac monographs reported that tcp is expected to be carcinogenic to humans , based on animal studies , as well as resemblance of metabolism between human and rodent microsomes , in vitro mutagenicity , and the ability to form dna adducts ( irwin et al . properties that determine the behavior of tcp in the environment include : its high density ( 1.39 g / ml ) ; the modest water solubility ( 1.75 water partitioning coefficient ( log kow = 2.02.5 , quite similar to the value for trichloroethylene ) ; and the low henry coefficient ( h = 3.23.4 10 atm m / mol at 25 c ) . the high density causes tcp , when dumped as a liquid on soil or in ponds , to sink to lower levels , e.g. , into groundwater or into accessible subsurface structures like rock fissures . water partitioning coefficient implies substantial distribution via groundwater flows , even when organic carbon is present . furthermore , tcp has a tendency to evaporate from surface water to air ( but less than trichloroethylene , h = 10 10 atm m / mol ) , where , on exposure to sunlight , it is subjected to photo - degradation by reaction with hydroxyl radicals with a half - life of about 15 days . the volatility of tcp , the possibility of washout by precipitation , and its resistance to degradation in water , may result in cycling of tcp between environmental compartments ( atsdr 1992 ; us epa 2009 ) . bioaccumulation and biomagnification are expected to be of minor importance in view of the modest lipophilicity of tcp . tcp can stay in groundwater for a prolonged period , in part due to its low organic carbon partitioning coefficient , but especially because rates of abiotic and biotic degradation in groundwater are low . abiotic hydrolysis of tcp under basic and neutral abiotic conditions has been studied at different temperatures and ph values and in the presence of different ions such as sulfide and carbonate ( sarathy et al . tcp appears to be highly stable under a variety of conditions , with an expected half - life of hydrolysis under environmental conditions ( 25 c , ph = 7 ) in the order of hundreds of years ( pagan et al . 2,3-dichloro-1-propene was detected as a product that could be converted to 2-chloro-2-propen-1-ol ( fig . 2 ) . other non - stimulated abiotic reactions in water under environmental conditions have not been characterized.fig . 2abiotic transformations of tcp under non - stimulated conditions ( a ) , photochemical conversion or catalytic conversion in the presence of radical - generating oxidants ( b ) , conversion in the presence of fenton reagent ( c , d ) , and conversion under stimulated anaerobic ( reductive ) conditions ( e ) . see text for details abiotic transformations of tcp under non - stimulated conditions ( a ) , photochemical conversion or catalytic conversion in the presence of radical - generating oxidants ( b ) , conversion in the presence of fenton reagent ( c , d ) , and conversion under stimulated anaerobic ( reductive ) conditions ( e ) . the physical and chemical properties of tcp obviously strongly influence possibilities for remediation of soil and groundwater ( salter - blanc and tratnyek 2011 ) . on - site methods for treating tcp - polluted groundwater include pump and treat and vacuum extraction . the latter is used for different volatile organohalogens , but it is not a very favorable approach because of the low henry s law constant of tcp ( ch2 m hill 2005 ) . extracted water can be treated by absorption , using activated carbon , by chemical oxidation or by vacuum extraction . for example , full - scale remediation of tcp - contaminated groundwater is under investigation at the tyson superfund site near philadelphia , pa , using vacuum extraction of soil , extraction of groundwater , and treatment of extracted water and vapor with activated carbon for the removal of tcp ( pezullo et al . 2005 ) . chemical oxidation is performed with oxidizing agents such as ozone , permanganate or hydrogen peroxide . they cause decomposition of tcp into carbon dioxide , water and chloride ions ( dombeck and brog 2005 ) ( fig . 2 ) . with a mild oxidant such as permanganate , transformation of tcp is slow whereas in the presence of strong oxidants such as hydroxyl radicals , generated photochemically , and sulfate radicals , catalytically generated from persulfate , transformation is much faster ( ch2 m hill 2005 ) . chemical tcp oxidation may also be initiated by the fenton reagent ( h2o2 with an iron catalyst ) . khan et al . ( 2009 ) studied the effect of iron type ( fe , fe and fe ) on the removal of tcp by h2o2 , and found that fe was most effective in reducing tcp and increasing its biodegradability . degradation products were 1,3-dichloroacetone and 2,3-dichloro-1-propene if fe and fe were used , and isopropanol and propionaldehyde if fe was used , confirming extensive oxidative conversion . chemical oxidation can also be used for in situ treatment of tcp contamination . in case tcp is present in the form of a dense non - aqueous - phase liquid ( dnapl ) , oxidants can be introduced into the subsurface to achieve contaminant oxidation . chlorinated solvents such as trichloroethene , carbon tetrachloride and tcp may also be removed by abiotic reduction with zero - valent iron . thus , distribution of tcp via flowing groundwater can be prevented by using a zero - valent iron ( fe ) barrier . ( 2003 ) investigated the effects of carbonate , silica , chloride and organic matter on the removal of various organohalides by granular iron using column studies . the results indicated that differences in groundwater chemistry have a strong effect on the activity and longevity of the granular iron , which will influence the design of reactive barriers . compounds enhancing metal corrosion ( carbonate , chloride ) may improve reactivity , whereas compounds such as feco3 and na2sio3 can reduce the activity , especially upon prolonged treatment , through deactivation of the metal surface . propane , propylene , and trace amounts of 1-chloro-2-propene were detected as tcp transformation products ( fig . 2 ) , indicating a role for reductive dechlorination and elimination of hcl in the removal of tcp . reductive transformation of tcp was also found with zero - valent zinc , which exhibited a reactivity that was more than an order of magnitude higher than that of iron ( sarathy et al . groundwater components that influenced zinc surface properties through corrosion or formation of an inactive layer of zno or zn(oh)2 had a large influence on the removal kinetics . few studies have been done aimed at establishing the possibilities for biodegradation and bioremediation of tcp . growth - supporting biodegradation of halogenated compounds is generally based on one of the following processes : ( 1 ) chemotrophy with an oxidizable electron donor ( hydrogen , lactate ) and use of the halogenated compounds as a physiological electron acceptor ( anaerobic conditions ) ; ( 2 ) use of the halogenated compound as a carbon and energy source with an external electron acceptor ( oxygen , nitrate ) ; ( 3 ) fermentative metabolism , in which the halogenated compound serves both as electron donor and ( indirectly ) as electron acceptor . such cometabolic transformations are due to the broad substrate spectrum of many microbial enzymes , the general reactivity of cofactors , or the formation of reactive intermediates in the catalytic cycle of some enzymes . examples are reductive dechlorination by cobalamin cofactors of anaerobic bacteria , and oxidative transformation by broad - specificity metal - containing monooxygenases of aerobic bacteria . obviously , a biodegradation process that stimulates growth of the active organisms is preferable in a bioremediation situation since it allows adaptation at the population level , leading to an increase of the amount of active biomass during the treatment process . reductive biotic transformation of tcp has been demonstrated under anaerobic conditions ( lffler et al . 3 ) and sequential aerobic anaerobic conditions ( long et al . 1993 ) . reductive dechlorination of both tcp and chloroethanes was observed with an enrichment culture that dechlorinated 1,2-dichloropropane , and propene and 1,2-dichloropropane were detected as products ( lffler et al . 1997 ) . experiments in which various halogenated aliphatic compounds were incubated with anaerobic sediments indicated zero - order conversion kinetics for tcp and dichloromethane , whereas most other organohalogens were transformed according to first order kinetics ( peijnenburg et al 2009 ) or possibly via 1,3-dichloropropene ( broken arrows ) anaerobic biotransformations of tcp . both reductive dehalogenation ( rd ) and dihaloelimination reactions ( dhe ) are observed . formation of allylchloride may occur by dihaloelimination ( yan et al . 2009 ) or possibly via 1,3-dichloropropene ( broken arrows ) recently , two strains ( bl - dc-8 and bl - dc-9 ) of an anaerobic gram - negative bacterium were isolated from contaminated groundwater at a superfund site located near baton rouge , and characterized as belonging to the new species dehalogenimonas lykanthroporepellens ( moe et al . these bacteria utilize tcp as an electron acceptor under anaerobic conditions but not chlorinated alkenes . for both strains , allyl chloride was detected as the main initial dechlorination product ( fig . however , allyl chloride is unstable and is hydrolyzed abiotically to allyl alcohol , whereas in the presence of cysteine or sulfide , allyl chloride was transformed to allyl mercaptan , s - allyl mercaptocysteine and allyl sulfides . the mechanism of the reductive dechlorination reaction is not completely clear , as the enzymes responsible for tcp dechlorination have not yet been isolated and characterized . in freshwater environments , transformation of tcp into allyl chloride followed by the formation of allyl alcohol could be toxic to fish and aquatic life ( ewell et al . 1986 ) . bioremediation of contaminated groundwater through in situ reductive dechlorination can be performed by injecting a compound such as hydrogen , lactic acid or another oxidizable organic substrate that is used by microorganisms to produce hydrogen , which induces reductive dechlorination and serves as electron donor ( tratnyek 2008 ) . at a site in california , 99.9 % reduction of tcp contamination has been found over a period of 1,000 days . however , biotic dechlorination through hydrogen - releasing compounds may be applicable only at low concentrations , such as less than 1 mg various halogenated aliphatic hydrocarbons can be transformed in a cometabolic manner by broad - specificity monooxygenase involved in hydrocarbon degradation , such as methane monooxygenase ( hanson et al . the soluble methane monooxygenase produced by cells of the methanotrophic bacterium methylosinus trichosporium ob3b can convert tcp , giving rise to dichloropropanols after subsequent reduction ( bosma and janssen 1998 ; fig . , tcp is a poor substrate for the enzyme as compared to other pollutants such as trichloroethylene . the conversions are analogous to those catalyzed by cytochrome p450 in mammalian systems ( fig . 1 ) . the major drawback of such cometabolic conversions is product toxicity . in case of tcp conversion by methane monooxygenase , the insertion of oxygen preferentially occurs on the terminal carbon atom , which yields chlorinated carbonyl compounds that may undergo elimination to produce 2-chloroacrolein , a very reactive compound.fig . 4conversions of 1,2,3-trichloropropane initiated by methane monooxygenase ( mmo ) produced by m. trichosporium ob3b cells . reduction to alcohols is caused by alcohol dehydrogenase activity ( dh ) conversions of 1,2,3-trichloropropane initiated by methane monooxygenase ( mmo ) produced by m. trichosporium ob3b cells . reduction to alcohols is caused by alcohol dehydrogenase activity ( dh ) the aerobic conversion of tcp reported by leahy et al . ( 2003 ) using a mixture of hydrocarbon - degrading bacteria is probably based on similar reactions , but the products were not identified . aromatic hydrocarbon - degrading bacteria produce monooxygenases that are capable of chlorinated hydrocarbon degradation through similar oxidative reactions as the methane monooxygenase of methanotrophs . for example , the toluene monooxygenase of a pseudomonad was described to convert chlorinated hydrocarbons ( newman and wackett 1997 ) . microbial transformation of tcp to co2 , h2o and hcl by oxidative metabolism with oxygen as an electron acceptor and by reduction to lesser chlorinated propanes and hcl is thermodynamically possible ( dolfing and janssen 1994 ) . however , no aerobic organisms , enrichment cultures , or bioreactors have been described that demonstrate the use of tcp as a growth - supporting oxidizable substrate . various attempts to enrich tcp - degrading microorganisms from environmental samples , including from sites with a long history of tcp or epichlorohydrin pollution , or to obtain tcp degradation in continuous - flow columns inoculated with samples from contaminated sites , have failed ( bosma et al . 2002 ) . this indicated that tcp is indeed a very recalcitrant compound and nature has not yet evolved aerobic organism that are adapted to it . the fact that the thermodynamic calculations indicate that aerobic oxidation of tcp is energetically favorable suggests biochemical hurdles instead of another fundamental reason as the cause of the apparent recalcitrance of tcp . an example of such a biochemical hurdle is toxicity of intermediates . in case of halogenated aliphatic compounds , several reactive intermediates occur along the metabolic pathways , requiring optimization of fluxes to prevent accumulation of such reactive intermediates to toxic levels ( van hylckama vlieg and janssen 2001 ) . it also may be due to formation of dead - end side products that are toxic . formation of such reactive intermediates will act against evolutionary selection of more efficient initial enzymes for tcp metabolism . the recalcitrant nature of a non - natural compound might also be due to presence of structural elements that can not be recognized and converted by microbial enzymes , which evolved for the conversion of natural compounds ( rieger et al . 2002 ) . when inspecting the possible pathways for productive aerobic metabolism of tcp , hydrolysis of a carbon halogen bond as the first step seems the most attractive reaction , because it does not involve reactive intermediates and leads to 1,3-dichloro-2-propanol 2,3-dichloro-1-propanol . these compounds are known to be biodegradable and pure cultures capable of using dichloropropanols for growth under aerobic conditions are known ( effendi et al . 2000 ; higgins et al . 2005 ; van den wijngaard et al . 1989 ; halogen bonds in chlorinated compounds is carried out by a diversity of microbial enzymes called dehalogenases . these belong to different phylogenetic classes , of which the haloalkane dehalogenases that are members of the /-hydrolase fold superfamily of proteins are the best characterized ( janssen 2004 ; koudelakova et al . another prominent class is the had - superfamily of haloacid dehalogenases and phosphatases , with dehalogenases that act on 2-chloroacetate and 2-chloropropionate . haloalkane dehalogenases are known to convert compounds such as 1,2-dichloroethane , 1,2-dibromoethane , 1,3-dichloropropane , 1,2-dichloropropene , and ( slowly ) hexachlorocyclohexane ( janssen et al . 2005 ; poelarends et al . the conversion of tcp by a haloalkane dehalogenase was first described by yokota et al . ( 1987 ) using an enzyme from corynebacterium strain m15 - 3 , but the activity was very low ( kcat / km = 36 s m ) ( bosma et al . 1999 ) . sequence analysis and structural studies identified the protein ( which is commonly called dhaa ) as a member of the /-hydrolase fold family . another dehalogenase that has a low activity with tcp is linb , and enzyme originally discovered in bacteria that degrade hexachlorocyclohexane ( monincov et al . ( 2000 ) found that the same gene is geographically widely distributed by using pcr analysis and dehalogenase gene sequencing of different bacteria enriched with other haloalkanes , including 1,3-dichloropropene . comparison of the genetic organization in different organisms revealed that the haloalkane dehalogenase gene likely originates from rhodococcus strains , where it is present in an operon together with an alcohol dehydrogenase and an aldehyde dehydrogenase gene , as well as a regulatory gene that influences gene expression . the latter may act as a repressor in the absence of a halocarbon substrate ( like 1-chlorobutane ) . when the dehalogenase gene regions from a 1,2-dibromoethane degrading mycobacterium and a 1,3-dichloropropene dehalogenating pseudomonas were examined , it appeared that the repressor gene was absent or inactivated by mutations to allow production of the enzyme in the presence of these new , non - inducing substrates ( poelarends et al . 1999 , 2000 ) . in the absence of a functional regulatory gene , inactivation of the repressor causing constitutive expression of a dehalogenase lack of microbial growth on tcp and lack of adaptation in column or enrichment experiments is most likely due to the very rare occurrence of a haloalkane dehalogenase gene with a suitable activity in a host organism that is capable of dichloropropanol conversion . mutations in the haloalkane dehalogenase that would lead to an enhanced substrate range that includes tcp would be unlikely to propagate in an organism that does not grow on the hydrolysis product and thereby provide a selective growth advantage . when dna sequence databases , both of completed bacterial genomes and environmental sequences , are searched for genes that encode the dhaa - type haloalkane dehalogenase , or the haloalcohol dehalogenases known to be involved in 2,3-dichloro-1-propanol metabolism ( except in organisms isolated on these compounds ) , no hits are found . these genes seem very rare and can only be recovered by appropriate enrichment culture techniques starting with polluted environmental samples . the evolution of bacteria that have the capacity to degrade tcp aerobically is thus restricted by the selectivity of haloalkane dehalogenases , and the rare occurrence of bacteria growing on dichloropropanols ( fig . 5 ) . consequently , attempts were made to obtain organisms capable of tcp detoxification by a combination of protein engineering and heterologous gene expression ( bosma et al . dce bioremediation has been established at full scale , using bacterial cultures that use dce as carbon source for growth according to the pathway that is shown ( a ) . tcp is much more recalcitrant , but productive catabolic pathways can be envisaged ( b ) . the upper routes could proceed from 2-chloroacrylic acid either via dehydrogenation ( dh ) ( kurata et al . 2005 ) or dechlorination ( dhl ) ( mowafy et al . 2010 ) . the lower route is thought to proceed in the strain constructed by bosma et al . ( 2002 ) in a. radiobacter ad1 expressing a mutants haloalkane dehalogenase ( dhaam2 ) and involves dehalogenases ( hhe ) and epoxide hydrolase ( eh ) comparison of catabolic pathways for 1,2-dichloroethane ( dce ) and tcp . dce bioremediation has been established at full scale , using bacterial cultures that use dce as carbon source for growth according to the pathway that is shown ( a ) . tcp is much more recalcitrant , but productive catabolic pathways can be envisaged ( b ) . the upper routes could proceed from 2-chloroacrylic acid either via dehydrogenation ( dh ) ( kurata et al . 2005 ) or dechlorination ( dhl ) ( mowafy et al . 2010 ) . the lower route is thought to proceed in the strain constructed by bosma et al . ( 2002 ) in a. radiobacter ad1 expressing a mutants haloalkane dehalogenase ( dhaam2 ) and involves dehalogenases ( hhe ) and epoxide hydrolase ( eh ) different reports on the engineering of haloalkane dehalogenase variants with enhanced activity towards tcp have been published . by using error prone pcr and dna shuffling , bosma et al . ( 2002 ) generated a dhaa mutant ( e.g. , a variant called dhaam2 with the mutations c176y and y273f ) that had three times higher catalytic efficiency ( kcat / km = 280 s m ) than wild - type enzyme . similarly , gray et al . ( 2001 ) performed in vitro evolution studies which also yielded a mutant with a substitution at position 176 and a mutation close to the n terminus that showed higher activity with tcp as compared to wild - type , and further mutations enhanced the stability of the enzymes . the strategy to construct a recombinant tcp - degrading strain was based on the use of an improved haloalkane dehalogenase into an organism that grows on the product of hydrolytic dehalogenation , which is 2,3-dichloro-1-propanol . for this , a host was used that could degrade both 2,3-dichloropropanol and 1,3-dichloropropanol : agrobacterium radiobacter ad1 ( van den wijngaard et al . 1989 ) . first , the wild - type haloalkane dehalogenase gene for dhaa from rhodococcus was placed under control of a strong constitutive promoter and cloned on a broad host range plasmid ( plafr3 ) that was introduced into strain ad1 ( bosma et al . growth of the resulting strain was not significant , but after incubation of 25 days 0.7 mm of tcp was converted and a small increase of biomass was observed . the strain did utilize 1,2,3-tribromopropane and 1,2-dibromo-3-chloropropane as sole carbon source , showing for the first time growth on a trihalopropane . growth on tcp could be obtained when a dhaa - type dehalogenase with improved activity for tcp was used . the resulting strain , a. radiobacter ad1(ptb3-m2 ) , was able to utilize tcp as carbon and energy source under aerobic conditions . after 10 days , 3.6 mm tcp was converted by a culture initially inoculated to an od450 of 0.14 . due to production of hydrochloric acid , the ph dropped to 6.0 ( bosma et al . the construction of a recombinant strain using an improved haloalkane dehalogenase that was expressed under a strong constitutive promoter in a host that degrades a dichloropropanol , is an important step towards obtaining an organism that is suitable for tcp bioremediation under aerobic conditions . 2002 ) : ( 1 ) although the initial dehalogenase is significantly improved for tcp conversion ( ca . 5-fold as compared to wild - type ) , the activity of dhaam2 is still too low to rapidly transform tcp . consequently , the estimated doubling time of the constructed strain was 90 h , which , for comparison , is much slower than the ca . 10 h measured for the 1,2-dichloroethane - degrader xanthobacter autotrophicus strain that is used for full - scale groundwater bioremediation . ( 2 ) degradation of tcp was incomplete due to the enantioselective conversion of only the ( r)-2,3-dichloropropanol by the host a. radiobacter ad1 . the dhaam2 dehalogenase produced a racemic mixture of ( r)- and ( s)-2,3-dichloropropanol from tcp . ( 3 ) the modified dehalogenase gene for dhaam2 was introduced into strain ad1 using the cloning vector plafr3 , which is a transmissible plasmid . such a plasmid may be modified or lost under stress conditions , or it may be transferred to other bacteria . ( 4 ) application of specialized bacteria in bioremediation operations will likely make use of open systems , such as an immobilized - cells bioreactor from which organisms may detach and end up in effluent water . this may lead to spread of resistance genes ( in this case tetracycline ) if the engineered organism contains additional antibiotic resistance markers . to remedy these limitations , the catabolic potential of naturally occurring organisms towards organic compounds is the result of long evolution processes , whereas the time in which organisms have been tempted to evolve new enzymes , pathways and regulatory mechanisms that allow conversion of xenobiotic industrial chemicals is quite short . the industrial synthesis of compounds such as trichloropropane only started in the first half of the 20th century . nevertheless , the presence of these synthetic compounds in the biosphere has already triggered the evolution of new metabolic activities , as illustrated by various examples ( janssen 2004 ; janssen et al . 2005 ; paul et al . 2005 ) . an important example of bacteria capable of tcp degradation are the strictly anaerobic strains bl - dc-8 and bl - dc-9 of d. lykanthropropepellens , isolated from contaminated groundwater in the usa ( yan et al the net dihaloelimination reaction catalyzed by these organisms implies transfer of electrons to tcp , with chloride release . this suggests the possibility of reductive dehalogenation coupled to electron transfer from hydrogen or another electron donor to tcp ( dehalorespiration ; smidt and de vos 2004 ) . since this process could possibly stimulate growth , as indicated by an increase in cell numbers ( yan et al . 2009 ) , genetic- or population - level adaptation of cultures to tcp under anaerobic conditions can be envisaged . this may yield faster growing cultures than those currently described ( maximum specific growth rate 0.150.17 day ) . it would also be highly interesting to identify the genes , proteins and cofactors involved in anaerobic conversion of tcp to allyl chloride and to establish their possible association with energy metabolism . the biochemical basis of dihaloelimination reactions is currently not well understood , although they may be important for different chlorinated substrates ( de wildeman et al . 2003 ; smidt and de vos 2004 ) . for in situ bioremediation , anaerobic transformation may be more attractive than aerobic processes , due to the difficulty of homogeneous oxygen supply and its preferred use for other oxidative processes if tcp is a low - level contaminant . anaerobic degradation of tcp was described to produce next to allyl chloride also small amounts of further conjugation products ( diallyl sulfide , allyl mercaptan ) , probably due to abiotic reactions with sulfide ( yan et al . the chemically labile carbon halogen bond in allyl chloride , as well as its sensitivity to cleavage by hydrolytic dehalogenases , suggest that more rapid biodegradation of allyl chloride with reduced formation of sulfur conjugates can be achieved when adapted mixed cultures are used . thus , further studies on the anaerobic metabolism of tcp and allyl chloride , in combination with appropriate enrichment and adaptation strategies , may well lead to more rapid anaerobic degradation as compared to what is currently possible . regarding aerobic degradation of tcp , genetic engineering can contribute to the acquisition of new bioremediation organisms , as illustrated by bosma et al . the biodegradation of tcp , use of a better haloalkane dehalogenase is desirable . by using rational design and directed evolution , the activity of dhaa against tcp was recently improved by pavlova et al . tunnel residues leading to the active site of dhaa were selected as target spots for mutagenesis , based on the notion that substrate binding and/or product release may limit the rate of catalysis . the best variants that were obtained carried three new mutations as compared to variant dhaam2 , and had 36 times higher activity ( kcat ) than the natural enzyme towards tcp ( table 1 ) . in the degradation pathway of 1,2-dichloroethane ( dce ) by x. autotrophicus gj10 , the first step is catalyzed by dhla , which is a phylogenetically related haloalkane dehalogenase . since this organism was successfully used for groundwater cleanup at full scale ( stucki and ther 1995 ) , it is interesting to compare the catalytic rates of the initial haloalkane dehalogenases ( table 1 ) . the differences in table 1 are important since kinetic properties and expression levels of the dehalogenases have a major impact on the kinetic properties of chloroalkane degradation ( substrate affinity , growth rate ) by the host organism ( van den wijngaard et al . 1993 ) . even though the activity of dhaa31 is significantly improved by directed evolution , the kcat and kcat / km values of dhaa31 for tcp are still lower than the corresponding values of dhla for dce ( table 1 ) . thus , an engineered organism expressing the evolved dhaa31 will still have a lower affinity for tcp than strain gj10 for dce . it is well possible that further variants of haloalkane dehalogenases that convert tcp even better can be obtained . strategies for laboratory evolution of new enzyme activities are still improving , and recently we were able to obtain complementary tcp dehalogenating mutants that produce almost enantiopure ( r)- or ( s)-2,3-dichloro-1-propanol . although dehalogenase enantioselectivity may be unimportant for groundwater and soil bioremediation , it holds great promise for converting tcp waste to economically valuable chiral building blocks for use in the fine chemicals and pharmaceutical industries ( van leeuwen et al . 2012).table 1kinetic parameters of haloalkane dehalogenase variants with tcp and 1,2-dichloroethane ( dce)variantsubstratekcat ( s)km ( mm)kcat / km ( m s)referencedhaa wild - typetcp0.035 0.0020.98 0.1736bosma et al . ( 2002)dhaa27tcp1.02 0.0311.09 0.10930pavlova et al . ( 2009)dhaa31tcp1.26 0.0311.2 0.151060pavlova et al . ( 2009)dhla wild - typedce3.3 0.50.53 0.26200krooshof et al . ( 1998)dhaa and variants thereof indicate the rhodococcus enzyme that was subjected to directed evolution for enhanced tcp conversion . dhla indicated the x. autotrophicus dehalogenase that was applied in a whole - cell cleanup process for 1,2-dichloroethane removalmargin of error not given kinetic parameters of haloalkane dehalogenase variants with tcp and 1,2-dichloroethane ( dce ) dhaa and variants thereof indicate the rhodococcus enzyme that was subjected to directed evolution for enhanced tcp conversion . dhla indicated the x. autotrophicus dehalogenase that was applied in a whole - cell cleanup process for 1,2-dichloroethane removal margin of error not given improved conversion of 2,3-dichloropropanol by a better host is under investigation with new isolates that were obtained from a site contaminated with epichlorohydrin and chloropropanols due to leakage of waste from epichlorohydrin manufacture . this organism , a strain of pseudomonas putida , uses a pathway for 2,3-dichloropropanol degradation that is different from the route detected in agrobacterium strains ( higgins et al . 2005 ; van den wijngaard et al . however , none of the current dichloropropanol degraders has been selected on the basis of its potential to form a biofilm on a solid support under groundwater flow conditions , and in competition with other bacteria . furthermore , substrate supply will likely be low , which also may impose physiological requirements on the host organism . the use of plasmid - based systems , as in the a. radiobacter ad1(ptb3-m2 ) recombinant ( bosma et al . 2002 ) is undesirable for the construction of bioremediation organisms , especially when in situ remediation is targeted ( de lorenzo and timmis 1994 ; timmis and pieper 1999 ) . a recombinant organism applied in situ should be capable of establishing itself an environment where the conditions can not be controlled ( de lorenzo 2009 ) . this may cause stress , leading to plasmid loss or lysis , as well as to spread of recombinant dna . the presence of antibiotic resistance - based selection markers and the use of transmissible plasmids can be avoided by employing chromosomal integration , for which efficient transposon - based systems were developed . for example , a modified tn5 transposon system can be used to integrate a foreign gene into the chromosome , leading to stable integration ( de lorenzo and timmis 1994 ) . such cloning vectors have been used successfully to construct strains for environmental applications ( panke et al . 1998 ) . if an efficient pathway can be assembled or evolved in the laboratory , in a robust host organism that can maintain itself under practical conditions , the prospects of successful application of such a genetically engineered organism for bioremediation are good . the limited success that has been achieved so far in this area , is mainly due to the fact that few recombinant organism have been engineered to degrade compounds which are really recalcitrant and where the poor degradability is due to biochemical factors instead of low solubility , limiting oxygen supply , poor bioavailability , etc . on the other hand , evolution of dehalogenases also occurs in natural environments ( janssen 2004 ) , and it is well possible that at some day , due to continued evolutionary pressure , tcp becomes a degradable compound and that tcp - degrading organisms can be obtained by classical enrichment . few studies have been done aimed at establishing the possibilities for biodegradation and bioremediation of tcp . growth - supporting biodegradation of halogenated compounds is generally based on one of the following processes : ( 1 ) chemotrophy with an oxidizable electron donor ( hydrogen , lactate ) and use of the halogenated compounds as a physiological electron acceptor ( anaerobic conditions ) ; ( 2 ) use of the halogenated compound as a carbon and energy source with an external electron acceptor ( oxygen , nitrate ) ; ( 3 ) fermentative metabolism , in which the halogenated compound serves both as electron donor and ( indirectly ) as electron acceptor . such cometabolic transformations are due to the broad substrate spectrum of many microbial enzymes , the general reactivity of cofactors , or the formation of reactive intermediates in the catalytic cycle of some enzymes . examples are reductive dechlorination by cobalamin cofactors of anaerobic bacteria , and oxidative transformation by broad - specificity metal - containing monooxygenases of aerobic bacteria . obviously , a biodegradation process that stimulates growth of the active organisms is preferable in a bioremediation situation since it allows adaptation at the population level , leading to an increase of the amount of active biomass during the treatment process . reductive biotic transformation of tcp has been demonstrated under anaerobic conditions ( lffler et al . 3 ) and sequential aerobic anaerobic conditions ( long et al . 1993 ) . reductive dechlorination of both tcp and chloroethanes was observed with an enrichment culture that dechlorinated 1,2-dichloropropane , and propene and 1,2-dichloropropane were detected as products ( lffler et al . 1997 ) . experiments in which various halogenated aliphatic compounds were incubated with anaerobic sediments indicated zero - order conversion kinetics for tcp and dichloromethane , whereas most other organohalogens were transformed according to first order kinetics ( peijnenburg et al 2009 ) or possibly via 1,3-dichloropropene ( broken arrows ) anaerobic biotransformations of tcp . both reductive dehalogenation ( rd ) and dihaloelimination reactions ( dhe ) are observed . formation of allylchloride may occur by dihaloelimination ( yan et al . 2009 ) or possibly via 1,3-dichloropropene ( broken arrows ) recently , two strains ( bl - dc-8 and bl - dc-9 ) of an anaerobic gram - negative bacterium were isolated from contaminated groundwater at a superfund site located near baton rouge , and characterized as belonging to the new species dehalogenimonas lykanthroporepellens ( moe et al . these bacteria utilize tcp as an electron acceptor under anaerobic conditions but not chlorinated alkenes . for both strains , allyl chloride was detected as the main initial dechlorination product ( fig . however , allyl chloride is unstable and is hydrolyzed abiotically to allyl alcohol , whereas in the presence of cysteine or sulfide , allyl chloride was transformed to allyl mercaptan , s - allyl mercaptocysteine and allyl sulfides . the mechanism of the reductive dechlorination reaction is not completely clear , as the enzymes responsible for tcp dechlorination have not yet been isolated and characterized . in freshwater environments , transformation of tcp into allyl chloride followed by the formation of allyl alcohol could be toxic to fish and aquatic life ( ewell et al . 1986 ) . bioremediation of contaminated groundwater through in situ reductive dechlorination can be performed by injecting a compound such as hydrogen , lactic acid or another oxidizable organic substrate that is used by microorganisms to produce hydrogen , which induces reductive dechlorination and serves as electron donor ( tratnyek 2008 ) . at a site in california , 99.9 % reduction of tcp contamination has been found over a period of 1,000 days . however , biotic dechlorination through hydrogen - releasing compounds may be applicable only at low concentrations , such as less than 1 mg various halogenated aliphatic hydrocarbons can be transformed in a cometabolic manner by broad - specificity monooxygenase involved in hydrocarbon degradation , such as methane monooxygenase ( hanson et al . the soluble methane monooxygenase produced by cells of the methanotrophic bacterium methylosinus trichosporium ob3b can convert tcp , giving rise to dichloropropanols after subsequent reduction ( bosma and janssen 1998 ; fig . , tcp is a poor substrate for the enzyme as compared to other pollutants such as trichloroethylene . the conversions are analogous to those catalyzed by cytochrome p450 in mammalian systems ( fig . 1 ) . the major drawback of such cometabolic conversions is product toxicity . in case of tcp conversion by methane monooxygenase , the insertion of oxygen preferentially occurs on the terminal carbon atom , which yields chlorinated carbonyl compounds that may undergo elimination to produce 2-chloroacrolein , a very reactive compound.fig . 4conversions of 1,2,3-trichloropropane initiated by methane monooxygenase ( mmo ) produced by m. trichosporium ob3b cells . reduction to alcohols is caused by alcohol dehydrogenase activity ( dh ) conversions of 1,2,3-trichloropropane initiated by methane monooxygenase ( mmo ) produced by m. trichosporium ob3b cells . reduction to alcohols is caused by alcohol dehydrogenase activity ( dh ) the aerobic conversion of tcp reported by leahy et al . ( 2003 ) using a mixture of hydrocarbon - degrading bacteria is probably based on similar reactions , but the products were not identified . aromatic hydrocarbon - degrading bacteria produce monooxygenases that are capable of chlorinated hydrocarbon degradation through similar oxidative reactions as the methane monooxygenase of methanotrophs . for example , the toluene monooxygenase of a pseudomonad was described to convert chlorinated hydrocarbons ( newman and wackett 1997 ) . microbial transformation of tcp to co2 , h2o and hcl by oxidative metabolism with oxygen as an electron acceptor and by reduction to lesser chlorinated propanes and hcl is thermodynamically possible ( dolfing and janssen 1994 ) . however , no aerobic organisms , enrichment cultures , or bioreactors have been described that demonstrate the use of tcp as a growth - supporting oxidizable substrate . various attempts to enrich tcp - degrading microorganisms from environmental samples , including from sites with a long history of tcp or epichlorohydrin pollution , or to obtain tcp degradation in continuous - flow columns inoculated with samples from contaminated sites , have failed ( bosma et al . 2002 ) . this indicated that tcp is indeed a very recalcitrant compound and nature has not yet evolved aerobic organism that are adapted to it . the fact that the thermodynamic calculations indicate that aerobic oxidation of tcp is energetically favorable suggests biochemical hurdles instead of another fundamental reason as the cause of the apparent recalcitrance of tcp . an example of such a biochemical hurdle is toxicity of intermediates . in case of halogenated aliphatic compounds , several reactive intermediates occur along the metabolic pathways , requiring optimization of fluxes to prevent accumulation of such reactive intermediates to toxic levels ( van hylckama vlieg and janssen 2001 ) . it also may be due to formation of dead - end side products that are toxic . formation of such reactive intermediates will act against evolutionary selection of more efficient initial enzymes for tcp metabolism . the recalcitrant nature of a non - natural compound might also be due to presence of structural elements that can not be recognized and converted by microbial enzymes , which evolved for the conversion of natural compounds ( rieger et al . 2002 ) . when inspecting the possible pathways for productive aerobic metabolism of tcp , hydrolysis of a carbon halogen bond as the first step seems the most attractive reaction , because it does not involve reactive intermediates and leads to 1,3-dichloro-2-propanol 2,3-dichloro-1-propanol . these compounds are known to be biodegradable and pure cultures capable of using dichloropropanols for growth under aerobic conditions are known ( effendi et al . halogen bonds in chlorinated compounds is carried out by a diversity of microbial enzymes called dehalogenases . these belong to different phylogenetic classes , of which the haloalkane dehalogenases that are members of the /-hydrolase fold superfamily of proteins are the best characterized ( janssen 2004 ; koudelakova et al . another prominent class is the had - superfamily of haloacid dehalogenases and phosphatases , with dehalogenases that act on 2-chloroacetate and 2-chloropropionate . haloalkane dehalogenases are known to convert compounds such as 1,2-dichloroethane , 1,2-dibromoethane , 1,3-dichloropropane , 1,2-dichloropropene , and ( slowly ) hexachlorocyclohexane ( janssen et al . the conversion of tcp by a haloalkane dehalogenase was first described by yokota et al . ( 1987 ) using an enzyme from corynebacterium strain m15 - 3 , but the activity was very low ( kcat / km = 36 s m ) ( bosma et al . sequence analysis and structural studies identified the protein ( which is commonly called dhaa ) as a member of the /-hydrolase fold family . another dehalogenase that has a low activity with tcp is linb , and enzyme originally discovered in bacteria that degrade hexachlorocyclohexane ( monincov et al . ( 2000 ) found that the same gene is geographically widely distributed by using pcr analysis and dehalogenase gene sequencing of different bacteria enriched with other haloalkanes , including 1,3-dichloropropene . comparison of the genetic organization in different organisms revealed that the haloalkane dehalogenase gene likely originates from rhodococcus strains , where it is present in an operon together with an alcohol dehydrogenase and an aldehyde dehydrogenase gene , as well as a regulatory gene that influences gene expression . the latter may act as a repressor in the absence of a halocarbon substrate ( like 1-chlorobutane ) . when the dehalogenase gene regions from a 1,2-dibromoethane degrading mycobacterium and a 1,3-dichloropropene dehalogenating pseudomonas were examined , it appeared that the repressor gene was absent or inactivated by mutations to allow production of the enzyme in the presence of these new , non - inducing substrates ( poelarends et al . 1999 , 2000 ) . in the absence of a functional regulatory gene , inactivation of the repressor causing constitutive expression of a dehalogenase appears a way to allow genetic adaptation and biodegradation . lack of microbial growth on tcp and lack of adaptation in column or enrichment experiments is most likely due to the very rare occurrence of a haloalkane dehalogenase gene with a suitable activity in a host organism that is capable of dichloropropanol conversion . mutations in the haloalkane dehalogenase that would lead to an enhanced substrate range that includes tcp would be unlikely to propagate in an organism that does not grow on the hydrolysis product and thereby provide a selective growth advantage . when dna sequence databases , both of completed bacterial genomes and environmental sequences , are searched for genes that encode the dhaa - type haloalkane dehalogenase , or the haloalcohol dehalogenases known to be involved in 2,3-dichloro-1-propanol metabolism ( except in organisms isolated on these compounds ) , no hits are found . these genes seem very rare and can only be recovered by appropriate enrichment culture techniques starting with polluted environmental samples . the evolution of bacteria that have the capacity to degrade tcp aerobically is thus restricted by the selectivity of haloalkane dehalogenases , and the rare occurrence of bacteria growing on dichloropropanols ( fig . consequently , attempts were made to obtain organisms capable of tcp detoxification by a combination of protein engineering and heterologous gene expression ( bosma et al . dce bioremediation has been established at full scale , using bacterial cultures that use dce as carbon source for growth according to the pathway that is shown ( a ) . tcp is much more recalcitrant , but productive catabolic pathways can be envisaged ( b ) . the upper routes could proceed from 2-chloroacrylic acid either via dehydrogenation ( dh ) ( kurata et al . 2005 ) or dechlorination ( dhl ) ( mowafy et al . 2010 ) . the lower route is thought to proceed in the strain constructed by bosma et al . ( 2002 ) in a. radiobacter ad1 expressing a mutants haloalkane dehalogenase ( dhaam2 ) and involves dehalogenases ( hhe ) and epoxide hydrolase ( eh ) comparison of catabolic pathways for 1,2-dichloroethane ( dce ) and tcp . dce bioremediation has been established at full scale , using bacterial cultures that use dce as carbon source for growth according to the pathway that is shown ( a ) . tcp is much more recalcitrant , but productive catabolic pathways can be envisaged ( b ) . the upper routes could proceed from 2-chloroacrylic acid either via dehydrogenation ( dh ) ( kurata et al . 2005 ) or dechlorination ( dhl ) ( mowafy et al . the lower route is thought to proceed in the strain constructed by bosma et al . ( 2002 ) in a. radiobacter ad1 expressing a mutants haloalkane dehalogenase ( dhaam2 ) and involves dehalogenases ( hhe ) and epoxide hydrolase ( eh ) different reports on the engineering of haloalkane dehalogenase variants with enhanced activity towards tcp have been published . by using error prone pcr and dna shuffling , bosma et al . ( 2002 ) generated a dhaa mutant ( e.g. , a variant called dhaam2 with the mutations c176y and y273f ) that had three times higher catalytic efficiency ( kcat / km = 280 s m ) than wild - type enzyme . similarly , gray et al . ( 2001 ) performed in vitro evolution studies which also yielded a mutant with a substitution at position 176 and a mutation close to the n terminus that showed higher activity with tcp as compared to wild - type , and further mutations enhanced the stability of the enzymes . the strategy to construct a recombinant tcp - degrading strain was based on the use of an improved haloalkane dehalogenase into an organism that grows on the product of hydrolytic dehalogenation , which is 2,3-dichloro-1-propanol . for this , a host was used that could degrade both 2,3-dichloropropanol and 1,3-dichloropropanol : agrobacterium radiobacter ad1 ( van den wijngaard et al . 1989 ) . first , the wild - type haloalkane dehalogenase gene for dhaa from rhodococcus was placed under control of a strong constitutive promoter and cloned on a broad host range plasmid ( plafr3 ) that was introduced into strain ad1 ( bosma et al . growth of the resulting strain was not significant , but after incubation of 25 days 0.7 mm of tcp was converted and a small increase of biomass was observed . the strain did utilize 1,2,3-tribromopropane and 1,2-dibromo-3-chloropropane as sole carbon source , showing for the first time growth on a trihalopropane . growth on tcp could be obtained when a dhaa - type dehalogenase with improved activity for tcp was used . the resulting strain , a. radiobacter ad1(ptb3-m2 ) , was able to utilize tcp as carbon and energy source under aerobic conditions . after 10 days , 3.6 mm tcp was converted by a culture initially inoculated to an od450 of 0.14 . due to production of hydrochloric acid , the ph dropped to 6.0 ( bosma et al . the construction of a recombinant strain using an improved haloalkane dehalogenase that was expressed under a strong constitutive promoter in a host that degrades a dichloropropanol , is an important step towards obtaining an organism that is suitable for tcp bioremediation under aerobic conditions . 2002 ) : ( 1 ) although the initial dehalogenase is significantly improved for tcp conversion ( ca . 5-fold as compared to wild - type ) , the activity of dhaam2 is still too low to rapidly transform tcp . consequently , the estimated doubling time of the constructed strain was 90 h , which , for comparison , is much slower than the ca . 10 h measured for the 1,2-dichloroethane - degrader xanthobacter autotrophicus strain that is used for full - scale groundwater bioremediation . ( 2 ) degradation of tcp was incomplete due to the enantioselective conversion of only the ( r)-2,3-dichloropropanol by the host a. radiobacter ad1 . the dhaam2 dehalogenase produced a racemic mixture of ( r)- and ( s)-2,3-dichloropropanol from tcp . ( 3 ) the modified dehalogenase gene for dhaam2 was introduced into strain ad1 using the cloning vector plafr3 , which is a transmissible plasmid . such a plasmid may be modified or lost under stress conditions , or it may be transferred to other bacteria . ( 4 ) application of specialized bacteria in bioremediation operations will likely make use of open systems , such as an immobilized - cells bioreactor from which organisms may detach and end up in effluent water . this may lead to spread of resistance genes ( in this case tetracycline ) if the engineered organism contains additional antibiotic resistance markers . to remedy these limitations , the catabolic potential of naturally occurring organisms towards organic compounds is the result of long evolution processes , whereas the time in which organisms have been tempted to evolve new enzymes , pathways and regulatory mechanisms that allow conversion of xenobiotic industrial chemicals is quite short . the industrial synthesis of compounds such as trichloropropane only started in the first half of the 20th century . nevertheless , the presence of these synthetic compounds in the biosphere has already triggered the evolution of new metabolic activities , as illustrated by various examples ( janssen 2004 ; janssen et al . 2005 ; paul et al . 2005 ) . an important example of bacteria capable of tcp degradation are the strictly anaerobic strains bl - dc-8 and bl - dc-9 of d. lykanthropropepellens , isolated from contaminated groundwater in the usa ( yan et al the net dihaloelimination reaction catalyzed by these organisms implies transfer of electrons to tcp , with chloride release . this suggests the possibility of reductive dehalogenation coupled to electron transfer from hydrogen or another electron donor to tcp ( dehalorespiration ; smidt and de vos 2004 ) . since this process could possibly stimulate growth , as indicated by an increase in cell numbers ( yan et al . 2009 ) , genetic- or population - level adaptation of cultures to tcp under anaerobic conditions can be envisaged . this may yield faster growing cultures than those currently described ( maximum specific growth rate 0.150.17 day ) . it would also be highly interesting to identify the genes , proteins and cofactors involved in anaerobic conversion of tcp to allyl chloride and to establish their possible association with energy metabolism . the biochemical basis of dihaloelimination reactions is currently not well understood , although they may be important for different chlorinated substrates ( de wildeman et al . 2003 ; smidt and de vos 2004 ) . for in situ bioremediation , anaerobic transformation may be more attractive than aerobic processes , due to the difficulty of homogeneous oxygen supply and its preferred use for other oxidative processes if tcp is a low - level contaminant . anaerobic degradation of tcp was described to produce next to allyl chloride also small amounts of further conjugation products ( diallyl sulfide , allyl mercaptan ) , probably due to abiotic reactions with sulfide ( yan et al . the chemically labile carbon halogen bond in allyl chloride , as well as its sensitivity to cleavage by hydrolytic dehalogenases , suggest that more rapid biodegradation of allyl chloride with reduced formation of sulfur conjugates can be achieved when adapted mixed cultures are used . thus , further studies on the anaerobic metabolism of tcp and allyl chloride , in combination with appropriate enrichment and adaptation strategies , may well lead to more rapid anaerobic degradation as compared to what is currently possible . regarding aerobic degradation of tcp , genetic engineering can contribute to the acquisition of new bioremediation organisms , as illustrated by bosma et al . the biodegradation of tcp , use of a better haloalkane dehalogenase is desirable . by using rational design and directed evolution , the activity of dhaa against tcp was recently improved by pavlova et al . tunnel residues leading to the active site of dhaa were selected as target spots for mutagenesis , based on the notion that substrate binding and/or product release may limit the rate of catalysis . the best variants that were obtained carried three new mutations as compared to variant dhaam2 , and had 36 times higher activity ( kcat ) than the natural enzyme towards tcp ( table 1 ) . in the degradation pathway of 1,2-dichloroethane ( dce ) by x. autotrophicus gj10 , the first step is catalyzed by dhla , which is a phylogenetically related haloalkane dehalogenase . since this organism was successfully used for groundwater cleanup at full scale ( stucki and ther 1995 ) , it is interesting to compare the catalytic rates of the initial haloalkane dehalogenases ( table 1 ) . the differences in table 1 are important since kinetic properties and expression levels of the dehalogenases have a major impact on the kinetic properties of chloroalkane degradation ( substrate affinity , growth rate ) by the host organism ( van den wijngaard et al . 1993 ) . even though the activity of dhaa31 is significantly improved by directed evolution , the kcat and kcat / km values of dhaa31 for tcp are still lower than the corresponding values of dhla for dce ( table 1 ) . thus , an engineered organism expressing the evolved dhaa31 will still have a lower affinity for tcp than strain gj10 for dce . it is well possible that further variants of haloalkane dehalogenases that convert tcp even better can be obtained . strategies for laboratory evolution of new enzyme activities are still improving , and recently we were able to obtain complementary tcp dehalogenating mutants that produce almost enantiopure ( r)- or ( s)-2,3-dichloro-1-propanol . although dehalogenase enantioselectivity may be unimportant for groundwater and soil bioremediation , it holds great promise for converting tcp waste to economically valuable chiral building blocks for use in the fine chemicals and pharmaceutical industries ( van leeuwen et al . 2012).table 1kinetic parameters of haloalkane dehalogenase variants with tcp and 1,2-dichloroethane ( dce)variantsubstratekcat ( s)km ( mm)kcat / km ( m s)referencedhaa wild - typetcp0.035 0.0020.98 0.1736bosma et al . et al . ( 1998)dhaa and variants thereof indicate the rhodococcus enzyme that was subjected to directed evolution for enhanced tcp conversion . dhla indicated the x. autotrophicus dehalogenase that was applied in a whole - cell cleanup process for 1,2-dichloroethane removalmargin of error not given kinetic parameters of haloalkane dehalogenase variants with tcp and 1,2-dichloroethane ( dce ) dhaa and variants thereof indicate the rhodococcus enzyme that was subjected to directed evolution for enhanced tcp conversion . dhla indicated the x. autotrophicus dehalogenase that was applied in a whole - cell cleanup process for 1,2-dichloroethane removal margin of error not given improved conversion of 2,3-dichloropropanol by a better host is under investigation with new isolates that were obtained from a site contaminated with epichlorohydrin and chloropropanols due to leakage of waste from epichlorohydrin manufacture . this organism , a strain of pseudomonas putida , uses a pathway for 2,3-dichloropropanol degradation that is different from the route detected in agrobacterium strains ( higgins et al . 2005 ; van den wijngaard et al . however , none of the current dichloropropanol degraders has been selected on the basis of its potential to form a biofilm on a solid support under groundwater flow conditions , and in competition with other bacteria . furthermore , substrate supply will likely be low , which also may impose physiological requirements on the host organism . the use of plasmid - based systems , as in the a. radiobacter ad1(ptb3-m2 ) recombinant ( bosma et al . 2002 ) is undesirable for the construction of bioremediation organisms , especially when in situ remediation is targeted ( de lorenzo and timmis 1994 ; timmis and pieper 1999 ) . a recombinant organism applied in situ should be capable of establishing itself an environment where the conditions can not be controlled ( de lorenzo 2009 ) . this may cause stress , leading to plasmid loss or lysis , as well as to spread of recombinant dna . the presence of antibiotic resistance - based selection markers and the use of transmissible plasmids can be avoided by employing chromosomal integration , for which efficient transposon - based systems were developed . for example , a modified tn5 transposon system can be used to integrate a foreign gene into the chromosome , leading to stable integration ( de lorenzo and timmis 1994 ) . such cloning vectors have been used successfully to construct strains for environmental applications ( panke et al . 1998 ) . if an efficient pathway can be assembled or evolved in the laboratory , in a robust host organism that can maintain itself under practical conditions , the prospects of successful application of such a genetically engineered organism for bioremediation are good . the limited success that has been achieved so far in this area , is mainly due to the fact that few recombinant organism have been engineered to degrade compounds which are really recalcitrant and where the poor degradability is due to biochemical factors instead of low solubility , limiting oxygen supply , poor bioavailability , etc . on the other hand , evolution of dehalogenases also occurs in natural environments ( janssen 2004 ) , and it is well possible that at some day , due to continued evolutionary pressure , tcp becomes a degradable compound and that tcp - degrading organisms can be obtained by classical enrichment . the toxicity and environmental behavior of tcp has stimulated research into techniques for removal of tcp from polluted sites . however , cleanup of tcp - contaminated water and soil is difficult due to its physiochemical properties and persistent nature . both aerobic and anaerobic processes have been investigated , but further work is needed to obtain cultures and processes with sufficient activity for testing under practical conditions and scale - up . until then , water treatment can be done by chemical methods such as oxidative degradation using a strong oxidant and a catalyst or uv light to generate radicals . reductive dechlorination by zero - valent iron and especially zinc are also suitable options , also for in situ application as barriers to prevent spreading via groundwater flow . soil venting , stripping and activated carbon absorption may be used for removing tcp contaminants from soil and water . for in situ treatment , reductive dechlorination may be the best option , especially if it can be coupled to growth - supporting dehaloelimination . recent developments in molecular biology and protein engineering have led to the construction of genetically engineered strains that allow slow but complete biodegradation of tcp under aerobic conditions . if these strains can be further evolved to exhibit degradation rates that compare favorably or are similar to those of 1,2-dichloroethane degradation by x. autotrophicus gj10 , which is used at full scale for groundwater cleanup ( stucki and ther 1995 ) , it is likely that a full - scale tcp bioremediation is feasible . the construction of such strains is dependent on dehalogenase with high activity , robust host strains that resist uncontrollable conditions , and the possibility to obtain growth - supporting metabolic pathway that completely mineralizes tcp . 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 .

purpose1,2,3-trichloropropane ( tcp ) is a persistent groundwater pollutant and a suspected human carcinogen . it is also is an industrial chemical waste that has been formed in large amounts during epichlorohydrin manufacture . in view of the spread of tcp via groundwater and its toxicity , there is a need for cheap and efficient technologies for the cleanup of tcp - contaminated sites . in situ or on - site bioremediation of tcp is an option if biodegradation can be achieved and stimulated . this paper presents an overview of methods for the remediation of tcp - contaminated water with an emphasis on the possibilities of biodegradation.conclusionsalthough tcp is a xenobiotic chlorinated compound of high chemical stability , a number of abiotic and biotic conversions have been demonstrated , including abiotic oxidative conversion in the presence of a strong oxidant and reductive conversion by zero - valent zinc . biotransformations that have been observed include reductive dechlorination , monooxygenase - mediated cometabolism , and enzymatic hydrolysis . no natural organisms are known that can use tcp as a carbon source for growth under aerobic conditions , but anaerobically tcp may serve as electron acceptor . the application of biodegradation is hindered by low degradation rates and incomplete mineralization . protein engineering and genetic modification can be used to obtain microorganisms with enhanced tcp degradation potential .

the data from national cancer registry department in ministry of health and medical education ( moh&me ) was used as the main source of incident for stomach cancer in iran from 2001 to 2005 which was based on pathology reports ( lab based registry ) . the data in the maximum capability consists 81% of incident cases , and in the first year of registration less than 40%.28 totally 19578 cases were identified as stomach cancer between years 2001 to 2005 . telephone numbers were available for 8733 families ( 44.6% ) . to obtain information about patients , a trained nurse called and interviewed all patients or their families . with this method , we collected data from 3439 cases as the study population ( 40% ) . to find whether the study cases are the representative of target population , their demographic features of age group and gender and also the anatomical site were compared with those of the whole 19578 cases across the country as the target population ( table 1 ) . comparing the basic characteristics of target population and study subjects the demographic features were precise and complete , items such as age , sex , name and father name , address , telephone number , cancer site and its pathology report with the name and address of responsible doctor and hospital were well recorded . all registered cases with tumor in cardia , fundus , corpus ( lesser curvature , greater curvature ) and antrum ( including prepyloric region ) were considered as stomach cancer . international classification of disease for oncology ( icd - o-3 ) was used for coding morphology and topography of tumors . for statistical analyses , the continuous quantitative variable of age was categorized into four age groups : less than 50 year , 50 - 59 , 60 - 69 and 70 or more . the anatomical sites of cancer were divided into four main categories of cardia and fundus , corpus or body , antrum or pre pyloric and unknown region . according to the number of patients in each pathologic type , the patients were categorized into three main groups : adenocarcinoma , lymphoma and others ( table 2 ) . pathologic type categorization of 3439 study cases the period between diagnosis and date of death or end time of the study ( march 2005 ) has been considered as follow up time ( days ) . by definition , survival means the time extend from diagnosis to the time of death or last visit . survival rates were calculated using the kaplan - meier method , and the survival probability was estimated for overall cohorts and also for gender , anatomical sites and pathologic type . the significant differences in survival rates among gender and age group classifications were tested by the log rank test for trends . relative ratios ( rrs ) according to demographic and risk variables were calculated by cox 's proportional hazard model , with 95% confidence interval ( ci ) up to the longest follow - up time of 2495 days . four variables of gender , anatomical site , histopathologic type and age groups were entered in cox 's model for multivariate analysis . the log minus log plotted against survival time for each covariate did not show any deviation from the proportionality assumption . of 3439 , 250 cases had a date of death before the time of diagnosis . survival rates were calculated using the kaplan - meier method , and the survival probability was estimated for overall cohorts and also for gender , anatomical sites and pathologic type . the significant differences in survival rates among gender and age group classifications were tested by the log rank test for trends . relative ratios ( rrs ) according to demographic and risk variables were calculated by cox 's proportional hazard model , with 95% confidence interval ( ci ) up to the longest follow - up time of 2495 days . four variables of gender , anatomical site , histopathologic type and age groups were entered in cox 's model for multivariate analysis . the log minus log plotted against survival time for each covariate did not show any deviation from the proportionality assumption . of 3439 , 250 cases had a date of death before the time of diagnosis . survival rates were calculated using the kaplan - meier method , and the survival probability was estimated for overall cohorts and also for gender , anatomical sites and pathologic type . the significant differences in survival rates among gender and age group classifications were tested by the log rank test for trends . relative ratios ( rrs ) according to demographic and risk variables were calculated by cox 's proportional hazard model , with 95% confidence interval ( ci ) up to the longest follow - up time of 2495 days . four variables of gender , anatomical site , histopathologic type and age groups were entered in cox 's model for multivariate analysis . the log minus log plotted against survival time for each covariate did not show any deviation from the proportionality assumption . of 3439 , 250 cases had a date of death before the time of diagnosis . the number of patients with stomach cancer recorded in data set was 3189 between years 2001 and 2005 of whom , 599 ( 18.8 % ) cases survived during the follow - up time . table 1 shows the baseline characteristics of the patients . of all , 833 ( 26.1% ) cases were women and 2273 ( 71.3% ) cases were more than 60 years old . the mean age was 64.72 12.72 years old . the crude one - year , 2 years , 3 years , 4 years and 5 years survival rate for the whole cohorts was 47.6% , 27.2% , 19.3% , 15.7% and 12.8 % respectively . the median for survival time were 396 and 329 days for women and men respectively . the difference of the median for survival time by age group was also statistically significant ( p < 0.001 ; 494 and 281 days for less than 50 years and more than 70 years respectively ) . the 5-year survival rate for men and women was 11.5% and 16.4% respectively with overall survival rate of 12.8% . ( figure 1 ) in terms of anatomical site , the 5-year survival rate for tumors located in antrum was significantly higher than corpus and cardia ( 15.3% , 13.2% , 7% respectively ; p = 0.009 ) . the univariate and multivariate analysis for gender , age group , anatomical site and histopathologic types are shown in table 3 . the likelihood of death was higher in men compared to women ( rr = 1.21 ; 95% ci : 1.11 - 1.33 ) . patients in age group more than 70 years old had a worse prognosis rather than those of fifty years old ( rr = 1.67 ; 95% ci : 1.46 - 1.91 ) . the kaplan - mayer overall survival curve by gender ( p < 0.001 ) relative ratios ( rr ) ( 95 + ci ) estimated from univariate and multivariate survival analysis of stomach cancer the likelihood of death was lower in patients with tumor located in antrum compared to those had tumor in corpus site ( rr = 0.87 ; 95% ci : 0.76 - 1.00 ; p = 0.05 ) . in terms of histopathologic type of tumor , patients with lymphoma type had a significantly higher 5-year survival rate compared to those with adenocarcinoma ( rr = 0.46 ; 95% ci : 0.31 - 0.66 ; p < 0.001 ) . among patients with lymphoma type , the mean ( median ) survival rate of those with large cell type was 1280 ( median = 427 ) days and for non - large cell type was 1195 ( median = 1836 ) days . only five and ten cases were diagnosed histopathologically as sarcoma and neuroendocrine tumors and the mean survival time for them was 545 ( median = 501 ) and 674 ( median = 598 ) days , respectively . the number of cases diagnosed histopathologically as signet cell ring , mucinous adenocarcioma and papillary adenocarcinoma was 360 , 142 and 15 cases respectively . the mean ( median ) survival time for them was 696 ( median = 324 ) , 640 ( median = 334 ) and 514 ( median = 349 ) days , respectively . gastric cancer is one of the most common malignancies in iran and its incidence is particularly high in the northwest of the country.810121516 patterns of gastric cancer in our country are similar to those reported from high - risk regions worldwide.17 in this national level study , the male to female ratio was 2.8:1 and the peak incidence was in the age group more than 60 years old , followed by the age group 50 - 59 years old . , there has been a dramatic decrease in gastric cancer incidence in western countries18 which has been mostly in intestinal type rather than ( table 2 ) intestinal type is more related to preventable environmental factors rather than diffuse type . lymphoma was seen in 2% of whole cancers in this study and in 3% in developed countries . sarcoma in the western countries accounts for 1.5 - 1.7% but in present study it was less than 10% . the most frequent primary site of tumor in present study cases was corpus followed by cardia and then pre - pyloric ( antrum ) area with better survival rate and some manageable predicting factors . a shift in distribution of anatomical site of tumor within the stomach is a considerable issue in the epidemiology of gastric cancer . in the first quarter of 20th century two third of gastric cancers were located in the antrum and prepyloric area and only 10% arose in the cardia or the esophagogastric junction . since 1970s , however , adenocarcinoma of the proximal stomach has become increasingly common worldwide ; such anatomical trend has been followed by iranian patients . over 36 years , the prevalence of cancers in the upper and middle third of the stomach has increased and that of the lower third has decreased . these changes were seen in both genders and age groups under and over 50 years old and it was more significant in younger people.19 understanding survival rate of the disease is important for health care planning and may also suggest opportunities for research in cancer prevention . although gastric cancer is the most common cancer among men in china , japan and many other asian countries , the mortality rate is lower in these countries and in europe rather than iran.20 the findings of present study showed that the median survival time of all patients was 346 days . overall survival rate for both sexes was 12.8% in this study which is approximately half of the rate of western countries ( 23% ) . the survival rate was relatively low particularly among men and age group of 70 + years . were registered with gastric cancer referred to the medical oncology department of the cancer institute from 1998 to 2003 found that the median survival time of all patients ( with or without treatment ) was 10 months overall.14 only 3189 of 19578 ( 16.3% response rate ) were accessible to follow their survival status which make it difficult to generalize easily the results of study to target population . however , there was no statistically significant difference between study population and target group , so the subjects might be considered as a representative of the whole target population of patients with stomach cancer across the country . ( table 1 ) on the other hand data were obtained from cancer office in ministry of health which is pathologic - based . thus there is some possibility of existence of selection bias for this study , but due to sample size it will not affect the results . in terms of histopathologic types of gastric cancer , since they have been reported by various pathologists across the country , inter - observation variation is another possible limitation for this study which might affect the results . in conclusion , this study highlights the relatively low survival rate of gastric cancer and changing in anatomical site pattern for iranian patients at national level . the status of stomach cancer in iran indicates the extremely urgent needs for health authorities to adopt measures of cancer prevention that proved effective in other countries .

background : stomach cancer is one of the most common malignancies worldwide . in iran , the mortality of stomach cancer is the first cause of death due to cancer in both sexes . this study was designed to estimate survival rate of stomach cancer at national level.methods:from the national cancer registry file , 3439 cases of stomach cancer that had telephone number and were diagnosed between years 2001 and 2005 were called to obtain information about their life status . survival estimates were calculated using the kaplan - meier method , and the survival probability was calculated for the overall cohorts and also for gender and anatomical sites of tumor . relative ratios ( rr ) according to demographic and risk variables were calculated by cox 's proportional hazard model.results:the overall 5-year survival rate was 12.8% . the likelihood of death was higher in men ( rr = 1.21 ; 95% ci : 1.11 - 1.33 ) and patients more than 70 years old had worse prognosis rather than those below fifty years old ( rr = 1.67 ; 95% ci : 1.46 - 1.91 ) . the 5-year survival rate for tumors located in antrum was significantly higher than corpus and cardia ( p = 0.009 ) . patients with lymphoma had a significantly higher 5-year survival rate compared to those with adenocarcinoma ( rr = 0.46 ; 95% ci : 0.31 - 0.66).conclusions : the status of stomach cancer , including relative low survival rate in iran , indicates the extremely urgent needs for health authorities to adopt measures of cancer prevention that proved effective in other countries .

the high variability of disease activity among patients newly presenting with rheumatoid arthritis ( ra ) or undifferentiated arthritis ( ua ) makes it necessary to know which patients will develop persistent disease , regardless of diagnosis , so that they can be treated more aggressively from the outset and to avoid inappropriate treatment of patients more prone to remission . several methodological issues must be considered when studying predictors of persistent activity in patients with recent - onset ra . first , when the disease is in its early stages , patients seldom fulfill the 1987 american rheumatism association ( ara ) revised criteria for ra . patients who do not fulfill criteria for definite ra at first presentation might be classified as having definite ra at a subsequent time point , but many cases remain unclassifiable ( ua ) [ 25 ] . there is an important proportion of newly presenting patients who do not satisfy these criteria , but for whom there is a compelling reason to treat with disease - modifying antirheumatic drugs ( dmard ) , or who on followup develop persistent disease even if there is no change in their classification status . recently , new classification criteria for ra have been developed in an attempt to increase sensitivity in recent - onset cases . whether the fulfillment of ara criteria is useful to predict activity is unknown . second , since treatments are not randomly assigned in nonexperimental studies , disease activity may be influenced by the type of treatment patients receive . third , factors selected by different authors as potentially predictive of a poor outcome are very heterogeneous and highly variable . the combined role of genetic and immunologic factors in the development of severe ra has been the subject of recent investigations . recent data support the hypothesis that the presence of hla drb1 shared epitope ( se ) alleles can trigger immune reactions such as the production of anticyclic citrullinated peptide antibodies ( anti - ccp ) . ra patients showing these antibodies in the early stages of the disease could develop more severe disease than those who lack them . rf positivity seems to be related to active disease , but no definite conclusions have been reached regarding its value as a predictor of disease activity in ra . tumor necrosis factor alpha ( tnf ) plays a pivotal role in regulating the inflammatory response in ra . however , there are few reports on the role of the g - to - a polymorphism at position 308 of the tnf gene promoter ( 308 tnf ) as an independent marker of disease activity in recent - onset ra [ 12 , 13 ] ; no association has been seen in rf - positive patients in particular . although 308 tnf [ 1214 ] , se alleles , [ 1522 ] rf , [ 2329 ] , and anti - ccp [ 3038 ] have all been studied as potential predictors for persistent activity in cohort studies of recent - onset ra , so far no study has investigated the combined effect of this particular set of factors . the combination of several markers could increase the capacity to predict persistent disease in patients with recent - onset ra and the identification of markers associated with a poor outcome would facilitate the development of new drug targets . finally , since there is no consensus definition of disease activity in recent - onset ra , the use of different definitions may generate substantial variation among studies . as no gold standard exists , a disease activity score based on a reduced joint count ( das28 ) , or other disease activity indexes can be used . a das28 2.6 is considered indicative of active disease , while a das28 < 2.6 corresponds to fulfillment of the preliminary ara criteria for clinical remission in ra . in this study , multivariate logistic and lineal regression was used to find a model based in immunogenetic markers that predicts persistent activity in patients with recent - onset ra or ua . the study is based in a recent - onset inflammatory polyarthritis ( ip ) register established in seville , spain , in january 2002 to look into various diagnostic , prognostic and therapeutic issues [ 4547 ] . we studied a prospective cohort of 201 consecutive patients with recent - onset ra or ua ( disease duration 1 year ) who were referred to our recent - onset ip unit from january 2002 through december 2006 . patients were referred from primary health care centers , emergency services , and outpatient rheumatology clinics of the virgen del roco university hospital health district in seville , spain ( population 774 619 according to the 2002 census ) . details of the case - ascertainment and follow - up procedure have been previously described . to be included in the recent - onset ip register , patients referred to the unit had to reside in the hospital health district catchment area , be at least 16 years old , and have at least two swollen joints lasting for a minimum of 4 weeks and a maximum of 12 months . the 1987 ara criteria for ra and international classification criteria for other rheumatic diseases were used at baseline and in all follow - up assessments and cumulatively applied . patients were classified as having ra if they fulfilled at least four of the seven 1987 ara criteria for ra ; those who did not fulfill at least four of these seven criteria and did not fulfill the classification or diagnostic criteria of any other particular rheumatic disease were classified as having ua . cases classified as ra during any visit ( at 0 , 1 , 3 , 6 , 9 , and 12 months ) and cases still classified as having ua at the end of followup were included in this study ; patients with alternative diagnoses were excluded . even if the new acr / eular classification criteria for ra have been published after our statistical analysis was completed , we have calculated the proportion of patients who fulfill them for informative purposes . from january 2002 through december 2006 of such patients , 469 ( 47.0% ) fulfilled the criteria for inclusion in the register , but 33 ( 7.0% ) were lost to follow - up . this left a total of 436 registered patients , of whom 201 ( 46.1% ) had completed the first year of followup by the time of this analysis . hla - drb1 se alleles were genotyped using a reverse dot - blot kit with sequence - specific oligonucleotide probes ( dynal reli sso hla - drb1 typing kits ; dynal biotech , bromborough , uk ) . when necessary , high - resolution typing of hla - drb1 * 01 , drb1 * 04 and drb1 * 14 samples was performed using dynal allset ssp drb1 * 01 , drb1 * 04 and drb1 * 14 , respectively . the frequency of se in a healthy control group from our district catchment area was 30% ( 28% heterozygous and 2% samples were genotyped for 308 tnf using a taqman 5 allelic discrimination assay ( custom taqman snp genotyping assays method , applied biosystems , foster city , calif , usa ) . allele - specific probes were labeled with vic and fam fluorescent dyes . polymerase chain reaction ( pcr ) was carried out in a total reaction volume of 8 l with the following amplification protocol : denaturation at 95c for 10 min , 40 cycles of denaturation at 93c for 15 sec and annealing and extension at 60c for 1 min . after pcr , the genotype of each sample was automatically attributed using the sds 1.3 software for allelic discrimination . the frequencies of 308 tnf genotypes in a healthy control group from our district catchment area were 80% for gg , 17% for ga and 3% for aa . anti - ccp antibodies were tested by second - generation elisa ( quanta lite ccp igg elisa . , san diego , calif , usa ; positive : > 20 iu / ml ) , and rf by nephelometry on a bn ii instrument ( dade behring , marburg , germany ) using the n latex rf method ( dade behring ) [ 46 , 47 ] ; levels > 50 iu / ml were considered positive using the optimal cutoff value reported by other authors . in a healthy control group from our district catchment area , the rf level at percentile 95 was 15 iu / ml , and the highest anti - ccp level was 10 ui / ml . treatment with corticosteroids and dmard ( methotrexate , sulphasalazine , chloroquine , leflunomide , cyclosporine , azathioprine , or combinations thereof ) during the entire followup period was assessed . a das28 < 2.6 was considered indicative of no disease activity or remission , and a das28 2.6 was considered indicative of active disease . the independent variables were the se status , anti - ccp and rf ( either status or levels ) , and 308 tnf genotype ( gg or ga / aa ; as there were few ga and aa cases , these two categories were collapsed ) obtained at baseline . as the probability of persistent activity may be influenced by the treatment the patients received , this confounding factor was entered as an additional independent variable . the treatment given throughout the 12 months of followup was corticosteroids , categorized dichotomously ( yes / no ) , and/or dmard , categorized either dichotomously ( yes / no ) or as the number of drugs given ( from 0 to 3 ) . multinomial regression models were also used to adjust the possible differences between disease classification ( ra or ua ) throughout followup . all data were recorded in an access 2000 database and then exported to the statistical package for the social sciences ( spss ) v. 15.0 for statistical analysis . for an alpha level of 0.05 , an anticipated medium effect size of 0.15 ( according to cohen 's convention for multiple regression ) and an assumed 10% rate of attrition , the minimum sample size required to reach a statistical power of 0.80 in a multiple regression model with eight predictor variables would be 108 . we calculated absolute frequencies and percentages for qualitative variables , and means and standard deviations for quantitative variables . variables that are predictive for disease activity at one year were identified by univariate and multivariate logistic and linear regression models . for univariate analyses we used student 's t - test , or fisher 's exact test , as appropriate . normality and homoscedasticity contrasts ( kolmogorov - smirnov and levene tests , resp . ) were undertaken for parametric tests . for multivariate analysis , wald 's statistic ( logistic regression ) or student 's t - test ( linear regression ) were used for stepwise exclusion of variables weakly associated with the dependent variable , as indicated by a p value 0.15 . since the se variable is polytomic , it was analyzed by creating a dummy variable with the first category ( / ) used as the reference . full and reduced models were compared with the g statistic ( logistic regression ) or partial multiple f - test ( linear regression ) . variables with a p value > 0.05 were analyzed as potential confounders , and they were considered as such whenever their coefficients changed by > 20% . multicollinearity among independent variables was assessed by the variance inflation factor , independence by the durbin - watson test , normality by the shapiro - wilk test , and homoscedasticity of the residues by the dispersion diagram among residues and the estimated values . goodness of fit was assessed with the hosmer lemeshow goodness - of - fit analysis , and discrimination was reported as the area under the receiver operating characteristic ( roc ) curve . in the linear analysis , all contrasts were two - tailed , and the significance level was set at < 0.05 . the 308 tnf could not be genotyped in 7 patients . of the remaining 194 patients , 39 ( 20.1% ) sixty - nine patients ( 34.3% ) were heterozygous ( /+ ) , and 16 ( 8.0% ) were homozygous ( + /+ ) for the se allele ; 85 ( 42.3% ) were rf - positive , and 88 ( 43.8% ) were anti - ccp - positive . the number of patients fulfilling 4 ara criteria for ra increased from 108 ( 53.7% ) at baseline to 142 ( 70.6% ) after 1 year ( i.e. , 34 of the 93 patients having ua at baseline fulfilled 4 ara criteria for ra after 1 year ) . the number of patients fulfilling the new acr / eular classification criteria for ra increased from 145 ( 72.1% ) at baseline to 154 ( 76.6% ) after 1 year . sometime between the baseline visit and the end of the follow - up period , 190 patients ( 94.5% ) mean ( sd ) values for das28 were 6.3 ( 1.4 ) at study entry and 3.5 ( 1.3 ) at 1 year . in univariate analyses , qualitative variables significantly associated with a das28 2.6 at one year were positive se ( p < 0.001 ) , fulfillment of the 1987 ara criteria ( p = 0.002 ) , and treatment with dmard ( p = 0.003 ) . as for quantitative variables , only anti - ccp levels ( p = 0.030 ) and the number of dmard ( p < 0.001 ) were significantly associated with a das28 2.6 ( table 2 ) . table 3 shows the results of univariate and multivariate logistic regression for das28 at one year . in univariate regression analyses , only positive se ( p < 0.001 ) and the number of dmard given during followup ( p < multivariate logistic regression analysis resulted in a model in which a das28 2.6 at one year of followup was significantly predicted by the presence of se ( or : 4.72 [ 95% ci : 2.17 , 10.25 ] ; p < 0.001 ) and the number of dmard ( or : 2.54 [ 95% ci : 1.64 , 3.95 ] ; p that means that for any two patients administered the same number of dmard , the probability of persistent activity at 1 year is almost 5 times greater in a patient with se than in a patient without se . for a cutoff value of 0.05 , the model had a sensitivity of 81.9% and a specificity of 56.1% , with an auc of 76.4% ( 95% ci : 68.9% , 83.8% ) , that is , significantly higher than 50% ( p < 0.001 ) , indicating that the model showed fair discriminatory power . the model had also fair accuracy ( i.e. , it correctly predicted 74.6% of the cases ) . table 4 shows the results of univariate and multivariate linear regression for das28 at one year . in univariate regression analyses , anti - ccp status ( p = 0.003 ) , rf status ( p = 0.004 ) , se heterozygosity ( p < 0.001 ) , se homozygosity ( p = 0.017 ) and the number of dmard ( p < 0.001 ) were associated with higher das28 at one year . in the linear regression analysis , a higher das28 was significantly predicted by se heterozygosity ( coefficient : 0.67 [ 95% ci : 0.32 , 1.01 ] ; p < 0.001 ) , se homozygosity ( coefficient : 0.73 [ 95% ci : 0.11 , 1.35 ] ; p = 0.021 ) and the number of dmard ( coefficient : 0.63 [ 95% ci : 0.43 , 0.82 ] ; p < 0.001 ) , but not by any other variable ( partial f tests = 0.115 ; p = 0.995 ; df = 6.195 ) . that means that for any two patients administered the same number of dmard , the das28 score at 1 year will be 0.73 points greater in a patient homozygous for se than in a patient without se . this model explained only 22.5% of the variability of the dependent variable ( r = 0.225 ) . in these models , no significant interactions among variables all criteria for the use of multivariate linear regression were fulfilled : independence , normality and linearity of the independent variables , absence of multicollinearity among them , and homoscedasticity of the residues . several cohort studies of populations similar to ours have investigated the value of different combinations of variables , including hla - drb1 se alleles , 308 tnf , rf , and anti - ccp for predicting disease activity among patients with recent - onset ra [ 1237 ] . these studies differed methodologically in terms of referral and recruitment procedures , inclusion criteria , disease duration , variables assessed at presentation , followup until assessment of outcome , and disease activity scoring methods . our study is the first to investigate this particular set of four immunogenetic markers using multivariate regression . moreover , the potentially confounding effects of the classification criteria ( ra versus ua ) and the type of treatment given were controlled for by including these variables in the regression analyses . some studies have found a significant association between se alleles and disease activity in recent - onset ra [ 15 , 17 , 19 ] , and some have not . [ 16 , 18 , 2022 ] several have not used multivariate statistical methods [ 15 , 17 , 21 ] . our results show that persistent activity at one year , assessed with the das28 , is significantly influenced by the presence of se in patients with recent - onset ra or ua . this finding is consistent across univariate and multivariate logistic and linear analyses ( tables 2 , 3 , and 4 ) . however , since ra is a multigenic inflammatory disorder , it is likely that other factors are involved in its outcome . the possibility that the 308 tnf may have prognostic implications is currently being debated . in a seropositive ra inception cohort , no statistically significant differences were seen in das between patients with ga or aa genotypes and those with the gg genotype . other studies that , like ours , were not confined to seropositive ra patients have also suggested that the 308 tnf is not a genetic marker for disease activity in recent - onset ra [ 12 , 13 ] . in this study , the ga / aa genotypes were not retained in any model , either logistic or linear , and not even in the univariate analyses ( tables 2 , 3 , and 4 ) . [ 9 , 18 , 28 ] besides the fact that our patients had recent - onset ra or ua rather than long - term ra , another possible explanation for the low frequency of rf positivity may be that , as recommended by some to predict outcome , we used high cutoff values for rf ( > 50 iu / ml , instead of > 40 iu / ml , > 20 iu / ml , or even > 10 iu / ml in other studies ) . had we used a cutoff value of 40 iu / ml , the frequency of rf positivity would have been 58.7% , instead of 42.3% . several studies have reported that rf is a good predictor of disease activity [ 23 , 24 , 2631 ] . however , in our univariate analyses rf , treated either as a qualitative or a quantitative variable , was not significantly associated with das28 ( table 2 ) . additionally , in the multivariate analyses , rf was not a prognostic factor for disease activity ( tables 3 and 4 ) . similar results have been found in other cohorts of recent - onset ra patients , both in spain and elsewhere [ 21 , 25 , 30 , 32 ] . in this community - based cohort , only 88 ( 43.8% ) of the 201 patients with recent - onset ra or ua were positive for anti - ccp at baseline . a low frequency of positivity at presentation has been recorded in other recent - onset ra cohorts [ 30 , 32 , 3436 ] , and it may be indicative of early - stage disease . the usefulness of anti - ccp for predicting disease activity in patients with recent - onset ra has been evaluated in several cohort studies . some have suggested it is a marker for active disease , as measured with either the sjc [ 30 , 3336 , 38 ] or the das28 , [ 21 , 30 , 36 ] , but others have not confirmed an association . [ 22 , 31 , 32 , 37 ] only a few of these studies have used multivariate statistical methods [ 22 , 30 , 32 , 38 ] . predictive value may depend on whether anti - ccp status or titers are considered . in our univariate analyses , patients who were positive for anti - ccp at presentation had not more disease activity at 1 year than patients who were negative ( table 2 ) . when quantitative values were used , anti - ccp antibodies were significantly associated with das28 ( table 2 ) . however , this marker was not a predictor of this outcome in regression models ( tables 3 and 4 ) . similar results have been found in other studies in which multivariate analyses have been performed [ 22 , 32 , 38 ] . the number of patients who fulfilled 4 ara criteria for ra increased with length of followup . thus , it is advisable to use a cumulative approach to the classification of disease . in the community - based norfolk arthritis register ( noar ) , the percentage of patients classified as having ra using the above criteria increased from 38% at baseline to 66% at 5 years . in our cohort of 463 patients with recent - onset ip , 108 ( 23.3% ) fulfilled 4 ara criteria for ra when first seen , and 142 ( 30.7% ) at 1 year . the number of patients fulfilling the new ra classification criteria increased from 145 ( 72.1% ) at baseline to 154 ( 76.6% ) after 1 year . the 1987 ara classification criteria for ra , derived from patients with long - standing established ra , were not designed to identify patients with recent - onset disease , and the current management of ra is intended to prevent patients reaching a stage when they satisfy these criteria . in this cohort , we included patients fulfilling 4 of the 7 ara criteria for ra and ua patients , since , regardless of diagnosis , dmard therapy was used as an indicator of the physician 's opinion that the patient was at risk of developing persistent disease in 94.5% of patients . the value of these criteria to predict active disease in patients with recent - onset disease has been questioned . in this cohort , the fulfillment of ara criteria for ra was not predictive for disease activity at 1 year ( tables 3 and 4 ) . in our study , based on routine care , the treatment given over the 12 months of followup was included in the univariate and multivariate analyses and was significantly and negatively related to disease activity in every analysis ( tables 2 , 3 , and 4 ) . since treatment was not a confounder in multivariate analyses and dmard have limited efficacy , this could indicate that , at least in a subgroup of patients , persistent disease activity might be related not to insufficient treatment with dmard but to a failure to respond to conventional dmard . a post hoc analysis of data from the best study has shown that patients who failed to respond to methotrexate were unlikely to respond to other conventional dmard , and a recent study from the community - based noar has identified se positivity as the strongest predictor of methotrexate monotherapy inefficacy in patients with early inflammatory polyarthtitis . the ability of dmard to prevent radiological damage has also been questioned . in a previous study we have found that erosive damage at 1 year in patients with recent - onset ra is significantly influenced by se homozygosity and the presence of baseline erosions , but not by rf status , anti - ccp status , 308 tnf genotype or treatment with conventional dmard . in conclusion , for patients with recent - onset ra or ua treated with the same number of dmard , the probability of persistent activity is significantly influenced by se presence . positive rf and anti - ccp at baseline , as well as the presence of the aa or ga genotypes of 308 tnf or the fulfillment of criteria for ra , as opposed to ua classification , were not good predictors of disease activity . immunogenetic tests were partially funded by grant fis-07/0061 from the fondo de investigaciones sanitarias ( spain ) .

we assessed the contribution of four baseline markers hla - drb1 shared epitope ( se ) , 308 tumor necrosis factor gene promoter polymorphism , rheumatoid factor , and anticitrullinated peptide antibodies for predicting persistent activity ( das28 score 2.6 ) after one year of followup in a cohort of 201 patients with recent - onset rheumatoid arthritis ( ra ) or undifferentiated arthritis ( ua ) aged 16 years or older who had a 4-week to 12-month history of swelling of at least two joints . patients had not been previously treated with corticosteroids or disease - modifying antirheumatic drugs ( dmard ) . in the best logistic regression model , only two variables were retained : se positivity and number of dmard administered ( area under the curve = 76.4% ; 95% ci : 69.2% , 84.4% ; p < 0.001 ) . the best linear regression model also included these two variables , explaining only 22.5% of the variability of das28 score . in this study , given an equal number of dmard administered , the probability of persistent activity in patients with recent - onset ra or ua was significantly influenced by se presence .

aging and weight loss have visible effects on the skin . structural changes to the skin associated with aging , such as changes in fat distribution and loss of collagen and elastic fibers , cumulate in a loss of tone and elasticity and manifest as wrinkles.1,2 substantial weight loss has also been shown to be associated with decreased levels of skin collagen and elastin that might result in undesirable flaccidity.3 soft tissue facial augmentation with dermal fillers is a minimally invasive esthetic technique that is growing in popularity.46 several injectable , cross - linked hyaluronic acid - based gel fillers are available that are intended for use as an implant for restoring facial volume loss . the calcium hydroxylapatite - based volumetric filler , radiesse ( merz pharmaceuticals gmbh , frankfurt , germany ) , is formulated to augment volume immediately , and subsequently to stimulate collagen production and thus restore volume in a process that mimics physiological remodeling of the extracellular matrix.79 the ability of radiesse to evoke physiological collagenogenesis is associated with improvements in skin quality , such as skin tightening , and is evidenced by its efficacy for the correction of moderate - to - severe facial wrinkles and folds , particularly nasolabial folds.10,11 the aim of the present study was to investigate the efficacy , safety , and subject satisfaction of the volumetric effect of radiesse in a novel body vectoring technique for correction of skin flaccidity in the thighs , abdomen , and brachial zones . this was a single - arm , prospective clinical study performed at a single center in spain . healthy female subjects aged 1869 years were eligible for inclusion if they had a self - evaluated skin flaccidity rating of 3 or more in the treatment area of interest ( thigh , abdomen , or brachial zone ) on a 6-point scale from 0 ( no flaccidity ) to 5 ( very severe flaccidity ) . exclusion criteria comprised pregnancy , breast feeding , chronic use of nonsteroidal anti - inflammatory drugs , autoimmune conditions , obesity , and a known allergy to lidocaine . written informed consent was obtained from each subject and the study was performed in line with local guidelines and those that have their origins in the 1975 declaration of helsinki . twenty female subjects aged 2867 years participated in the study , 25% of whom were current smokers . digital photographs of the three treatment zones were taken before and 5 weeks after treatment . a cutometer mpa 580 ( courage + khazaka electronic gmbh , cologne , germany ) and an ultrascan uc22c 22mhz ( taberna pro medicum gmbh , luneburg , germany ) were used to locate and record skin density , thickness , and flaccidity measurements from an exact point , which was used before and 5 weeks after treatment . cutometer and ultrascan measurements were conducted at the following approximate positions : for the thigh , 20 cm below the iliac crest bone , 10 cm from the internal edge ; for the abdomen , 3 cm from the navel ( left or right ) ; and for the brachial zone , 20 cm from the acromion in a coronal cut of the arm , 12 cm internal . subjects were also asked to rate the flaccidity of their skin before treatment using the 6-point scale , and to perform a skin pinch test before treatment to assess skin thickness/ density at baseline . the right hand side of the body was treated first ( visit 1 ) ; the left hand side was treated following completion of posttreatment assessments of the right hand side ( 5 weeks after treatment ) . follow - up assessments of left hand side treatment occurred a further 5 weeks later . prior to treatment , vector maps for the right hand side of the body were designed to inform positioning of needles during administration ( figure 1 ) . for the thigh , an anchor point on the border between the different skin types of the inner and outer thigh was located and two lines drawn . one followed the transition line of the two skin types and the other extended to the middle of the fat of the inner thigh . several lines were then drawn protruding from these , with one line prolonged to ensure coverage of the entire inner thigh . this positioning approach aimed to lift the fat tissue of the internal side of the leg and improve skin quality . for the abdomen , a fix point to anchor the vectors was found under the ribs and two lines were drawn from this point , one line downward passing next to the navel ; the second was drawn at a 45 degree angle to the first . several protruding lines were drawn to cover almost the entire zone with the aim of correcting navel shape and improving skin quality . for the brachial zone , the aim was to lift the internal side of the arm and improve skin quality . a fix point was found at the deltoid muscle , then one line was drawn 3 cm into the axillary zone and a second was drawn to protrude two thirds into the arm . five weeks after the right hand side of the body was treated , the same approach was used to design vectors for the left hand side ahead of treatment . radiesse ( 3 ml ) was diluted , in line with the us food and drug administration approval , with 0.6 ml lidocaine ( 2% without epinephrine ) . in all , 3 ml radiesse solution was administered per thigh , 1.5 ml per hemiabdomen , and 1.5 ml per brachial zone . injections were administered into the deep dermis using a 1 ml luer - lok syringe and a 27 g 40 mm needle . subjects rated the pain experienced during treatment on a 10-point scale from 1 ( no pain ) to 10 ( very severe pain ) . digital photographs of the three treatment zones were taken before and 5 weeks after treatment . a cutometer mpa 580 ( courage + khazaka electronic gmbh , cologne , germany ) and an ultrascan uc22c 22mhz ( taberna pro medicum gmbh , luneburg , germany ) were used to locate and record skin density , thickness , and flaccidity measurements from an exact point , which was used before and 5 weeks after treatment . cutometer and ultrascan measurements were conducted at the following approximate positions : for the thigh , 20 cm below the iliac crest bone , 10 cm from the internal edge ; for the abdomen , 3 cm from the navel ( left or right ) ; and for the brachial zone , 20 cm from the acromion in a coronal cut of the arm , 12 cm internal . subjects were also asked to rate the flaccidity of their skin before treatment using the 6-point scale , and to perform a skin pinch test before treatment to assess skin thickness/ density at baseline . the right hand side of the body was treated first ( visit 1 ) ; the left hand side was treated following completion of posttreatment assessments of the right hand side ( 5 weeks after treatment ) . follow - up assessments of left hand side treatment occurred a further 5 weeks later . prior to treatment , vector maps for the right hand side of the body were designed to inform positioning of needles during administration ( figure 1 ) . for the thigh , an anchor point on the border between the different skin types of the inner and outer thigh was located and two lines drawn . one followed the transition line of the two skin types and the other extended to the middle of the fat of the inner thigh . several lines were then drawn protruding from these , with one line prolonged to ensure coverage of the entire inner thigh . this positioning approach aimed to lift the fat tissue of the internal side of the leg and improve skin quality . for the abdomen , a fix point to anchor the vectors was found under the ribs and two lines were drawn from this point , one line downward passing next to the navel ; the second was drawn at a 45 degree angle to the first . several protruding lines were drawn to cover almost the entire zone with the aim of correcting navel shape and improving skin quality . for the brachial zone , the aim was to lift the internal side of the arm and improve skin quality . a fix point was found at the deltoid muscle , then one line was drawn 3 cm into the axillary zone and a second was drawn to protrude two thirds into the arm . five weeks after the right hand side of the body was treated , the same approach was used to design vectors for the left hand side ahead of treatment . radiesse ( 3 ml ) was diluted , in line with the us food and drug administration approval , with 0.6 ml lidocaine ( 2% without epinephrine ) . in all , 3 ml radiesse solution was administered per thigh , 1.5 ml per hemiabdomen , and 1.5 ml per brachial zone . injections were administered into the deep dermis using a 1 ml luer - lok syringe and a 27 g 40 mm needle . for each line of the vector map , subjects rated the pain experienced during treatment on a 10-point scale from 1 ( no pain ) to 10 ( very severe pain ) . across all treatment zones , cutometer data showed improvements in skin flaccidity versus baseline in 78% of cases at 5 weeks after treatment ( figure 2 ) , most commonly in the thighs ( 82% of cases ) . the mean reduction in flaccidity from baseline to 5 weeks after treatment was also calculated , and was 0.0924 mm , 0.0117 mm , and 0.0814 mm for the brachial zone , thighs , and abdomen , respectively . the overall mean reduction in flaccidity from baseline to 5 weeks after treatment was 0.093 mm . at 5 weeks after treatment improvements in skin density relative to baseline were recorded in the majority of measurements across all treatment zones ( figure 3 ) . the abdomen showed the best treatment response , with improvements versus baseline in 88% of cases . skin thickness also showed improvement versus baseline in the majority of cases ( figure 3 ) , but most frequently in the thighs ( 88% of cases ) . representative clinical photographs before and after treatment are provided in figure 4 . at baseline , mean subject - assessed flaccidity scores for the thighs , abdomen , and brachial zones were 3.6 , 3.7 , and 3.8 , respectively ( figure 5 ) . following treatment , subject - assessed flaccidity scores showed an improvement versus baseline in 27 of the 36 treated zones ( 75% ) . the mean self - assessed flaccidity score following treatment was 2.6 for the thighs , 2.7 for the abdomen , and 3.0 for the brachial zones . all subjects ( 100% ) reported a positive pinch test , indicating an improvement in skin thickness . bruising was reported for 16 of the 36 treated zones ( 44.4% ) following treatment . seventeen subjects ( 85% ) rated the pain they experienced during treatment as 4 on a 10-point scale from 1 ( no pain ) to 10 ( very severe pain ) . across all treatment zones , cutometer data showed improvements in skin flaccidity versus baseline in 78% of cases at 5 weeks after treatment ( figure 2 ) , most commonly in the thighs ( 82% of cases ) . the mean reduction in flaccidity from baseline to 5 weeks after treatment was also calculated , and was 0.0924 mm , 0.0117 mm , and 0.0814 mm for the brachial zone , thighs , and abdomen , respectively . the overall mean reduction in flaccidity from baseline to 5 weeks after treatment was 0.093 mm . at 5 weeks after treatment improvements in skin density relative to baseline were recorded in the majority of measurements across all treatment zones ( figure 3 ) . the abdomen showed the best treatment response , with improvements versus baseline in 88% of cases . skin thickness also showed improvement versus baseline in the majority of cases ( figure 3 ) , but most frequently in the thighs ( 88% of cases ) . at baseline , mean subject - assessed flaccidity scores for the thighs , abdomen , and brachial zones were 3.6 , 3.7 , and 3.8 , respectively ( figure 5 ) . following treatment , subject - assessed flaccidity scores showed an improvement versus baseline in 27 of the 36 treated zones ( 75% ) . the mean self - assessed flaccidity score following treatment was 2.6 for the thighs , 2.7 for the abdomen , and 3.0 for the brachial zones . all subjects ( 100% ) reported a positive pinch test , indicating an improvement in skin thickness . bruising was reported for 16 of the 36 treated zones ( 44.4% ) following treatment . seventeen subjects ( 85% ) rated the pain they experienced during treatment as 4 on a 10-point scale from 1 ( no pain ) to 10 ( very severe pain ) . this single - arm , prospective clinical study demonstrated that the body vectoring technique using radiesse induced notable reductions in skin flaccidity , increased skin density , and increased skin thickness in three body zones . subjects were satisfied with their treatment , with the majority recording an improvement in skin flaccidity via self - evaluation and all subjects recording a positive pinch test . all three treatment zones showed a good response in terms of efficacy and subject satisfaction , particularly at the abdomen and thighs . the treatment was well tolerated , with any treatment - related bruising resolving within a week . while body vectoring is a relatively novel application of radiesse , its use in facial soft tissue augmentation is established4 and it has been demonstrated to have a good safety and efficacy profile.10,1214 a vectoring approach has also been documented as an effective method for restoring facial proportions.15 the simple application technique used in the present study and the high reproducibility of positive responses suggest that the potential applications of radiesse could expand beyond that of facial soft tissue augmentation to include correction of flaccid skin in the abdomen , thigh , and brachial zones . such a procedure may be desirable to counteract the structural changes in the skin that are associated with aging and with substantial weight loss.1,3 in addition to an immediate volumetric effect , radiesse has the ability to stimulate long - term physiological remodeling of the extracellular matrix.7,8 specifically , radiesse has demonstrated an effect on collagen , elastin , and fibroblasts,79 which may give radiesse the potential to have long - lasting esthetic effects in skin rejuvenation . on the basis of the positive results in the present study , it is envisaged that this body vectoring technique could be used as a preventative measure ; treatments would probably not be needed more than once per year , due to the potential of radiesse to have sustained efficacy.11 in this study , radiesse was reconstituted with lidocaine , which has previously not demonstrated any notable effect on the physical properties of the filler ; however , any impact on subject comfort , durability , and efficacy remains to be determined.16 the use of lidocaine injection followed by a massaging technique has been found to facilitate the dispersion of radiesse.17 the long - term maintenance of the esthetic effect was not explored in this preliminary study , but long - term durability of the effect of radiesse in facial augmentation has previously been demonstrated.11,18 future studies should monitor subjects in the longer term to explore the sustained efficacy of the treatment . we recorded improvements in self - evaluation scores following treatment , indicative of a good level of patient satisfaction . it is important to note the subjective nature of these data , and the impact that patient expectations of the treatment might have had on their assessment of the results . this novel body vectoring technique with radiesse had notable positive results on skin flaccidity in the thigh , abdominal , and brachial zones , was well tolerated , and had good subject satisfaction .

backgroundthe objective of this study was to investigate the efficacy , safety , and subject satisfaction of the calcium hydroxylapatite - based dermal filler radiesse in a novel body vectoring technique to correct skin flaccidity in the thighs , abdomen , and brachial zones.methodsfemale subjects with self - evaluated flaccidity scores 3 on a 6-point scale ( 0 , no flaccidity ; 5 , very severe flaccidity ) in the zones of interest were included . radiesse was injected according to predesigned vector maps ( 3 ml per thigh , 1.5 ml per hemiabdomen or brachial zone ) . clinical assessments ( skin density and thickness ) were made by an independent reviewer at an exact position before and 5 weeks after treatment using a cutometer and an ultrascan . subjects rated skin flaccidity before and 5 weeks after treatment on the 6-point scale and performed a pinch test to self - assess changes in skin thickness . all adverse events were recorded.resultstwenty females ( aged 2867 years ) were enrolled , contributing 36 treatment zones . across all zones , 78% of flaccidity measurements improved after treatment . improvements in skin flaccidity were most common in the thighs ( 82% of cases ) . an improvement in skin density versus baseline was observed in the majority across all zones , most frequently in the abdomen ( 88% of cases ) . skin thickness in each zone also improved versus baseline for the majority , most frequently in the thighs ( 88% of cases ) . mean self - assessed flaccidity scores at baseline were 3.6 ( thighs ) , 3.7 ( abdomen ) , and 3.8 ( brachial zone ) , and 2.6 , 2.7 , and 3.0 , respectively , posttreatment . all subjects reported a positive pinch test . in total , 47.0% of subjects had bruising after treatment , which resolved within a week . no serious adverse events were reported.conclusionusing this novel technique , radiesse had notable results on skin flaccidity , density and thickness in the thighs , abdomen , and brachial zones , and was well tolerated .

a reproducible and valid method for quantification of the neurological deficit that occurs after stroke is essential for monitoring patients ; many stroke scales have been proposed for this purpose . stroke scales represent a useful tool for estimating the severity of stroke at onset and for assessing prognostic information in hospital . in general , a stroke scale consists of several variables for observing the signs and symptoms and each variable is categorized for scoring . in developing an ideal stroke scale , issues of simplicity , reliability , validity and popularity of use must be pursued , especially if a scale is to be used by a broad array of practitioners . simplicity and time taking is important in any outcome measure , especially for use in stroke patients with cognitive problems and feelings of tiredness . stroke scales can be classified as parametric or clinicometric scales on the basis of physical deficit and functional impairment , handicap scales . evaluating the impact of new treatments requires the use of reliable and valid outcome measures . development of stroke outcome classification systems is necessary because neurological deficits often lead to permanent impairments , disabilities and compromised quality - of - life . medline , ovid , pubmed , google , proquest , scopus , cochrane library , elsevier , thompson , isi , index medicus , index copernicus and science direct was used as search engines . the following string of keywords was selected ( stroke ) and ( scale ) and ( score ) and ( disability ) and ( grade ) with the final search performed on january , 1 , 2013 . at the other side , library archives of mashhad university of medical sciences the national institute of health stroke scale ( nihss ) is the most frequently used stroke deficit scale in routine clinical practice and clinical trials . in spite of its great success , there are problems with the nihss . it contains items with poor reliability and has been criticized for its redundancy and complexity . the nihss overall reliability is clear , however assessments have consistently shown specific items that yield low inter - rater reliability . these items with poorer nihss reliability included facial palsy , ataxia , dysarthria and level of consciousness . among over 15,000 individuals who have taken online nihss certification , the nihss items with poorer inter - rater reliability included facial palsy ( k = 0.25 ) , ataxia ( k = 0.15 ) , level of consciousness ( k = 0.43 ) , dysarthria ( k = 0.46 ) and gaze ( k = 0.44 ) . these nihss items with poor inter - rater reliability have also been identified in spanish , italian and chinese versions of nihss . these elements may contribute to difficulties in practitioner communication , incorrect hospital care patterns that are based on the nihss ; e.g. , decisions to give thrombolytics , variable trial enrollments and even possible difficulties with assessing patient outcome in clinical trials . given the unreliability of some of the nihss items , patients may score high on the nihss when they actually have mild strokes but questionable other findings . alternatively , patients may score as mild even if they have more sever deficits , because unreliability may result in certain items being unscored . patient with sever stroke may not be able to receive nihss scores for ataxia or dysarthria because their arousal state may preclude testing these items . because these items are not scored abnormal unless patients produce testable behaviors , these patients may be too sick to score on these items . though the patients may clinically improve , their nihss scores may artificially worsen since now items such as ataxia and dysarthria can receive the scores that were previously unscored . since these items have been removed from the modified nihss , this difficulty can be avoided or at least lessened . facial palsy and dysarthria showed poor reliability and were redundant , so they were eliminated . the sensory item was simplified due to poor reliability . with fewer items and simpler grading , the resulting modified nihss has shown significantly higher reliability and validity than nihss . in the nihss , 7 of 42 points are related to language function , while only 2 of 42 points are attributed to neglect functions . redundant items are noted in the nihss have been deleted from the modified nihss , resulting in a more balanced clinical scale . the author suggests scoring 0 - 3 to language function and including mute or global aphasia in score 3 as severe aphasia . this scoring strategy improves hemisphere balance between language and neglect items in modified nihss . both nihss and modified nihss failed to accurately or reliably detect stroke severity in patients with posterior circulation findings . with the removal of the ataxia item , there may be a concern that the modified nihss would be even less able to assess brainstem strokes . however , since ataxia is a poorly reliable nihss item , the benefit of using a scale that inconsistently assesses the posterior circulation , may not out weight the consistency of modified nihss . many clinical trials routinely include only anterior circulation strokes , so that there is less need to measure posterior circulation deficit for this purpose . however , stroke severity scale specialized for posterior circulation strokes has been developed and validated in israel . the scandinavian stroke scale ( sss ) is easier than nihss for clinical practice in acute stroke patients and has been used in many clinical trials . the nihss , canadian neurological scale , european stroke scale ( designed for patients with middle cerebral artery stroke ) , sss , japan stroke scale , orpington prognostic scale , orgogozo scale and numerous other scales developed for clinicometric assessment of acute stroke patients . the orpington prognostic scale is easier than nihss in clinical practice and additionally evaluates the cognitive function . despite advantages of modified nihss and sss comparing to the nihss ( including their simplification and less inter - rater variability ) , most of the stroke neurologists around the world continue using the nihss because they have used to work with it for more than two decades , although it could not be an acceptable reason . at the other side , results of previous stroke trials , which are the basis of stroke management guidelines are driven using the initial nihss . the stroke outcome classification of the american heart association is too comprehensive and time consuming to be used in the routine clinical practice and did not enjoy the widespread acceptance around the world . for quality - of - life and outcome measures after stroke , duncan et al . in the us found that eight key areas ( strength , hand function , activities of daily living , mobility , communication , memory , emotion and social participation ) emerged as key areas from the patients perspective . similarly , williams et al . reported that patients identified 12 key domains ( mobility , energy , upper extremity function , work / productivity , mood , self - care , social roles , family roles , vision , language , thinking and personality ) . the basic self - care tasks are feeding , grooming , dressing , bathing , toileting , including sphincter control and mobility , including transferring from place to place . could enable the stroke patient to live at home with the help from family or community providers for meals and other household tasks as needed . these tasks are performed to maintain independence in the home and community and include shopping , using transportation , telephoning , preparing meals , handling finances and maintaining a household . other instrumental activities of daily living that affect quality - of - life are work skills , religious activities and leisure time and recreational activities . the modified rankin scale ( mrs ) and barthel index ( bi ) are widely used functional impairment , disability scales , which have been proven to be a valid and reliable for defining outcome in stroke patients . despite bi inter - rater variability introduces noise into trial outcome assessments and reduces the power of clinical trials to detect treatment outcome . a variety of approaches to minimize inter - rater variation of mrs have been described or proposed , including : ( 1 ) use of a formal structured interview , ( 2 ) training and certification programs using written and video case vignettes and ( 3 ) central panel adjudication of local site - recorded video assessments . however , the instruments and approaches developed to date have not consistently been shown to reduce inter - rater variability for mrs . however , there is little consensus on the optimal implementation of the bi and mrs as an outcome measure in acute stroke trials and it is unclear which outcome scale is preferable . the japan stroke scale and kurashiki pre - hospital stroke scale are clinicometric stroke scales which are designed in asia . the asian stroke disability scale ( asds ) was provided as a simplified functional impairment , handicap scale and inter - rater reliability of asds compared with mrs and bi . development procedure for the procedure is summarized as following steps : ( 1 ) select the variables , ( 2 ) categorize the variables , ( 3 ) evaluate the categorization for their distribution and sensitivity , ( 4 ) modify and re - evaluate the categorization , ( 5 ) repeat procedures 1 through 4 until the appropriate categorizations are obtained . three items including ; self - care , mobility and daily activities were selected as variables for development of the asds based on the contribution of each item to the prognosis and a review of currently available stroke scales . the variables were provisionally graded on a 2- to 4-point scale based on the importance of each item . the total score for a patient could be calculated from the sum of the scores for each of the variables ranging from 0 to 8 . the asds is simple , requires less than 1 min to perform the test and is as valid as mrs and bi in assessment of functional impairment of stroke patients . the quantitative and qualitative inter - rater variability of asds is similar to the mrs and bi . the paired inter - rater variability of mrs , bi and asds scores based on qualitative categorization was not significant for the three methods , p > 0.05 . inter - rater reliability of mrs was poor ( k = 0.16 ) in the study conducted by quinn et al . comparing estimated scores between the paired assessors , there was again poor agreement in 30% and significant variability ( k = 0.38 ) of mrs score . in the evaluation of rankin focused assessment tool , rater 's scores concurred fully in 47 of 50 patients and in the remaining three patients , scores differed by one level . a review of literature about inter - rate reliability of mrs revealed moderate inter - rater reliability , which improved with structured interviews . the difference of disability scores based on the mrs , bi and sss are small and these scores have excellent agreement with each other , whereas modified nihss has substantial agreement with mrs and bi in a uk study . when the mrs and bi scores were dichotomized at 95 and 1 respectively , the nihss appeared more sensitive than the bi or mrs . diagnostic accuracy of bi in serial assessments of ischemic stroke patients was performed in the netherland . assessment of the bi in acute stroke showed good discriminative properties for the final outcome of bi at 6 months . another study in the netherland compared with five stroke scales ; the orgogozo scale , the nihss , the canadian neurological scale and the sss with measures of disability and handicap and quality - of - life according to the mrs and bi . the five stroke scales were highly related to one another but the correlation between stroke scales and functional scales was less than 0.70 and decreased from bi ( 47.5% ) to mrs ( 36.5% ) . therefore , clinicometric stroke scales only partly explain functional health and impact of impairments on functional outcomes seems to be under estimated by the stroke scale weights . the frenchay stroke scale , canadian occupational performance measure , stroke impact scale and numerous other functional impairment scale have been developed for use in stroke patients by stroke specialists and occupational therapists . despite the development of better functional impairment scales , stroke neurologists around the world continue using the mrs and bi , because they have used to work with these scales for decades , although it could not be an acceptable reason . at the other side , results of the previous stroke trials , which are the basis of stroke management guidelines are driven using the initial mrs and bi . the national institute of health stroke scale ( nihss ) is the most frequently used stroke deficit scale in routine clinical practice and clinical trials . in spite of its great success , there are problems with the nihss . it contains items with poor reliability and has been criticized for its redundancy and complexity . the nihss overall reliability is clear , however assessments have consistently shown specific items that yield low inter - rater reliability . these items with poorer nihss reliability included facial palsy , ataxia , dysarthria and level of consciousness . among over 15,000 individuals who have taken online nihss certification , the nihss items with poorer inter - rater reliability included facial palsy ( k = 0.25 ) , ataxia ( k = 0.15 ) , level of consciousness ( k = 0.43 ) , dysarthria ( k = 0.46 ) and gaze ( k = 0.44 ) . these nihss items with poor inter - rater reliability have also been identified in spanish , italian and chinese versions of nihss . these elements may contribute to difficulties in practitioner communication , incorrect hospital care patterns that are based on the nihss ; e.g. , decisions to give thrombolytics , variable trial enrollments and even possible difficulties with assessing patient outcome in clinical trials . given the unreliability of some of the nihss items , patients may score high on the nihss when they actually have mild strokes but questionable other findings . alternatively , patients may score as mild even if they have more sever deficits , because unreliability may result in certain items being unscored . patient with sever stroke may not be able to receive nihss scores for ataxia or dysarthria because their arousal state may preclude testing these items . because these items are not scored abnormal unless patients produce testable behaviors , these patients may be too sick to score on these items . though the patients may clinically improve , their nihss scores may artificially worsen since now items such as ataxia and dysarthria can receive the scores that were previously unscored . since these items have been removed from the modified nihss , this difficulty can be avoided or at least lessened . facial palsy and dysarthria showed poor reliability and were redundant , so they were eliminated . the sensory item was simplified due to poor reliability . with fewer items and simpler grading , the resulting modified nihss has shown significantly higher reliability and validity than nihss . in the nihss , 7 of 42 points are related to language function , while only 2 of 42 points are attributed to neglect functions . redundant items are noted in the nihss have been deleted from the modified nihss , resulting in a more balanced clinical scale . the author suggests scoring 0 - 3 to language function and including mute or global aphasia in score 3 as severe aphasia . this scoring strategy improves hemisphere balance between language and neglect items in modified nihss . both nihss and modified nihss failed to accurately or reliably detect stroke severity in patients with posterior circulation findings . with the removal of the ataxia item , there may be a concern that the modified nihss would be even less able to assess brainstem strokes . however , since ataxia is a poorly reliable nihss item , the benefit of using a scale that inconsistently assesses the posterior circulation , may not out weight the consistency of modified nihss . many clinical trials routinely include only anterior circulation strokes , so that there is less need to measure posterior circulation deficit for this purpose . however , stroke severity scale specialized for posterior circulation strokes has been developed and validated in israel . the scandinavian stroke scale ( sss ) is easier than nihss for clinical practice in acute stroke patients and has been used in many clinical trials . the nihss , canadian neurological scale , european stroke scale ( designed for patients with middle cerebral artery stroke ) , sss , japan stroke scale , orpington prognostic scale , orgogozo scale and numerous other scales developed for clinicometric assessment of acute stroke patients . the orpington prognostic scale is easier than nihss in clinical practice and additionally evaluates the cognitive function . despite advantages of modified nihss and sss comparing to the nihss ( including their simplification and less inter - rater variability ) , most of the stroke neurologists around the world continue using the nihss because they have used to work with it for more than two decades , although it could not be an acceptable reason . at the other side , results of previous stroke trials , which are the basis of stroke management guidelines are driven using the initial nihss . the stroke outcome classification of the american heart association is too comprehensive and time consuming to be used in the routine clinical practice and did not enjoy the widespread acceptance around the world . for quality - of - life and outcome measures after stroke , duncan et al . in the us found that eight key areas ( strength , hand function , activities of daily living , mobility , communication , memory , emotion and social participation ) emerged as key areas from the patients perspective . similarly , williams et al . reported that patients identified 12 key domains ( mobility , energy , upper extremity function , work / productivity , mood , self - care , social roles , family roles , vision , language , thinking and personality ) . the basic self - care tasks are feeding , grooming , dressing , bathing , toileting , including sphincter control and mobility , including transferring from place to place . independence in these activities could enable the stroke patient to live at home with the help from family or community providers for meals and other household tasks as needed . these tasks are performed to maintain independence in the home and community and include shopping , using transportation , telephoning , preparing meals , handling finances and maintaining a household . other instrumental activities of daily living that affect quality - of - life are work skills , religious activities and leisure time and recreational activities . leisure activities are demonstrated as the strongest association to subject well - being . the modified rankin scale ( mrs ) and barthel index ( bi ) are widely used functional impairment , disability scales , which have been proven to be a valid and reliable for defining outcome in stroke patients . despite bi , inter - rater variability introduces noise into trial outcome assessments and reduces the power of clinical trials to detect treatment outcome . a variety of approaches to minimize inter - rater variation of mrs have been described or proposed , including : ( 1 ) use of a formal structured interview , ( 2 ) training and certification programs using written and video case vignettes and ( 3 ) central panel adjudication of local site - recorded video assessments . however , the instruments and approaches developed to date have not consistently been shown to reduce inter - rater variability for mrs . however , there is little consensus on the optimal implementation of the bi and mrs as an outcome measure in acute stroke trials and it is unclear which outcome scale is preferable . the japan stroke scale and kurashiki pre - hospital stroke scale are clinicometric stroke scales which are designed in asia . the asian stroke disability scale ( asds ) was provided as a simplified functional impairment , handicap scale and inter - rater reliability of asds compared with mrs and bi . the procedure is summarized as following steps : ( 1 ) select the variables , ( 2 ) categorize the variables , ( 3 ) evaluate the categorization for their distribution and sensitivity , ( 4 ) modify and re - evaluate the categorization , ( 5 ) repeat procedures 1 through 4 until the appropriate categorizations are obtained . three items including ; self - care , mobility and daily activities were selected as variables for development of the asds based on the contribution of each item to the prognosis and a review of currently available stroke scales . the variables were provisionally graded on a 2- to 4-point scale based on the importance of each item . the total score for a patient could be calculated from the sum of the scores for each of the variables ranging from 0 to 8 . the asds is simple , requires less than 1 min to perform the test and is as valid as mrs and bi in assessment of functional impairment of stroke patients . the quantitative and qualitative inter - rater variability of asds is similar to the mrs and bi . the paired inter - rater variability of mrs , bi and asds scores based on qualitative categorization was not significant for the three methods , p > 0.05 . inter - rater reliability of mrs was poor ( k = 0.16 ) in the study conducted by quinn et al . comparing estimated scores between the paired assessors , there was again poor agreement in 30% and significant variability ( k = 0.38 ) of mrs score . in the evaluation of rankin focused assessment tool , rater 's scores concurred fully in 47 of 50 patients and in the remaining three patients , scores differed by one level . a review of literature about inter - rate reliability of mrs revealed moderate inter - rater reliability , which improved with structured interviews . the difference of disability scores based on the mrs , bi and sss are small and these scores have excellent agreement with each other , whereas modified nihss has substantial agreement with mrs and bi in a uk study . when the mrs and bi scores were dichotomized at 95 and 1 respectively , the nihss appeared more sensitive than the bi or mrs . diagnostic accuracy of bi in serial assessments of ischemic stroke patients was performed in the netherland . assessment of the bi in acute stroke showed good discriminative properties for the final outcome of bi at 6 months . another study in the netherland compared with five stroke scales ; the orgogozo scale , the nihss , the canadian neurological scale and the sss with measures of disability and handicap and quality - of - life according to the mrs and bi . the five stroke scales were highly related to one another but the correlation between stroke scales and functional scales was less than 0.70 and decreased from bi ( 47.5% ) to mrs ( 36.5% ) . therefore , clinicometric stroke scales only partly explain functional health and impact of impairments on functional outcomes seems to be under estimated by the stroke scale weights . the frenchay stroke scale , canadian occupational performance measure , stroke impact scale and numerous other functional impairment scale have been developed for use in stroke patients by stroke specialists and occupational therapists . despite the development of better functional impairment scales , stroke neurologists around the world continue using the mrs and bi , because they have used to work with these scales for decades , although it could not be an acceptable reason . at the other side , results of the previous stroke trials , which are the basis of stroke management guidelines are driven using the initial mrs and bi . the asian stroke disability scale * despite advantages of modified nihss and sss comparing to the nihss , most of the stroke neurologists around the world continue using the nihss . the mrs and bi are widely used functional impairment , disability scales and it is unclear , which outcome scale is preferable . the asds is a simplified functional impairment and disability scale , which is as valid as mrs and bi .

stroke scales can be classified as clinicometric scales and functional impairment , handicap scales . all studies describing stroke scales were reviewed by internet searching engines with the final search performed on january 1 , 2013 . the following string of keywords was entered into search engines ; stroke , scale , score and disability . despite advantages of modified national institute of health stroke scale and scandinavian stroke scale comparing to the nihss , including their simplification and less inter - rater variability ; most of the stroke neurologists around the world continue using the nihss . the modified rankin scale ( mrs ) and barthel index ( bi ) are widely used functional impairment and disability scales . distinction between grades of mrs is poorly defined . the asian stroke disability scale is a simplified functional impairment , handicap scale which is as valid as mrs and bi . at the present time , the nihss , mrs and bi are routine stroke scales because physicians have used to work with these scales for more than two decades , although it could not be an acceptable reason . on the other side , results of previous stroke trials , which are the basis of stroke management guidelines are driven using these scales .

vascular injury is an uncommon but potentially devastating complication of spine surgery . throughout its course , the aorta remains in close proximity to the thoracic and lumbar spine and is at risk from malpositioned pedicle screws or other hardware.1 despite this risk , the reported rate of vascular injury from spine surgery is low . in one systematic review , papadoulas et al found the incidence of vascular injury after excision of a herniated lumbar disc via a posterior approach to be less than 0.05%.2 in addition , hicks et al systematically reviewed 14,570 pedicle screws placed in the thoracic and lumbar spine of 1,666 patients with adolescent idiopathic scoliosis ( ais ) ; they found a 4.2% overall rate of screw malposition with 6 of 8,147 screws ( 0.07% ) abutting the aorta in the subgroup of studies that reported this finding . there were no reports of major vascular complications.3 despite the low rates of vascular injury identified in these reviews , the literature contains multiple case reports in which pedicle screw instrumentation was associated with early or late aortic injuries.4 5 6 7 8 9 10 in the majority of these reports , treatment consisted of removing of the offending hardware with simultaneous open or endovascular aortic repair ( evar ) . here , we describe the case of an adolescent patient with a malpositioned pedicle screw abutting the thoracic aorta and new complaints of deep - seated thoracic pain . intraoperative aortography demonstrated normal flow in the thoracic aorta , so no graft was placed to avoid the known morbidity of evar . the patient was a 20-year - old man who presented to clinic with 4 months of back pain . at the age of 15 years , he had undergone a t6t8 posterior instrumented fusion with excision of an osteoblastoma from the right t7 pedicle and lamina . at that time , his primary symptom was midthoracic pain ; this pain resolved within 3 months of his index surgery . when he returned to clinic 5 years later , he complained of deep - seated substernal and midthoracic pain . , the patient had a well - healed midline incision with no signs of infection . he had normal strength , sensation , and reflexes in his bilateral upper and lower extremities . posteroanterior and lateral radiographs ( fig . 1 ) and a computed tomographic ( ct ) scan of the thoracic spine ( fig . 2 ) demonstrated that the left t6 screw penetrated the lateral cortex of the t6 pedicle and abutted the posteromedial aspect of the aortic wall . there was no pseudoaneurysm or periaortic inflammation identified to suggest frank penetration into the aortic lumen . however , it was felt that because of ongoing mechanical irritation by the abutting screw and the patient 's new pain symptoms , there was an increased risk of aortic perforation , dissection , or thrombotic complication if the screw were left in place , and that therefore , it was advisable to remove it . posteroanterior and lateral radiographs of the thoracic spine show lateral positioning of the left t6 pedicle screw . an axial computed tomographic scan slice through the t6 pedicle and sagittal reconstruction show the left t6 pedicle screw breaches the lateral cortex and impinges against the posteromedial aorta . after a thorough discussion of the risks and benefits with the patient , his family , and the vascular surgery service , the patient elected to go to the operative theater with plans to obtain endovascular control of the aorta , remove the offending hardware , and stent the aorta if required . in the theater , general anesthesia was induced and a double - lumen endotracheal tube was placed in case open thoracotomy were required . the patient was positioned supine and 7-french sheath was introduced into the right common femoral artery . a catheter was passed via this sheath into the thoracic aorta and an aortogram was performed to confirm appropriate position of the catheter and adequate visualization of the aorta . no evidence of stenosis , contrast extravasation , or intraluminal material was noted . with the right groin catheter in place , the previous midline thoracic incision was reopened and dissection was carried down to the level of the posterior instrumentation . the soft tissues were cleared from around the screws and the end caps removed . once the vascular surgery team indicated their readiness to proceed , the left t6 screw was backed out . the vascular team then performed a second aortogram , which again demonstrated no extravasation of contrast or other abnormal finding . a final aortogram after removal of all hardware showed no evidence of injury or abnormal flow within the thoracic aorta ( fig . 3 ) . given the normal aortogram , it was decided no graft placement or further endovascular intervention was necessary . intraoperative aortography demonstrates no extravasation of contrast and normal filling of the descending thoracic aorta . the posterior wound was thoroughly irrigated and closed in layered fashion and the patient was returned to the supine position . estimated blood loss for the procedure was 200 ml . the patient was awakened and found to be neurologically and vascularly intact , unchanged from his preoperative examination . he was admitted to the surgical floor where he progressed appropriately and was discharged home on postoperative day 2 . vascular injury has been recognized as a rare potential complication during anterior spine procedures , with a rate of less than 5% reported in a recent systematic review.11 after posteriorly based procedures , reported rates of acute vessel injury in systematic reviews range from 0 to 0.05%.2 3 however , because of the intimate association of the aorta with the thoracic and lumbar spine , it remains at risk of acute or delayed injury from misplaced posterior instrumentation.1 a growing number of reports suggest such injuries are likely an underappreciated complication.4 5 6 7 8 9 10 in cases of frank perforation of the aorta or another major vessel , immediate operative or endovascular intervention is indicated to repair the injury . potential risks of impinging instrumentation include late catastrophic hemorrhage , pseudoaneurysm formation , or thromboembolic complications . the best evidence in favor of observing impinging hardware without intervening comes from foxx et al who retrospectively reviewed the position of 680 pedicle screws in 107 patients on routine postoperative imaging . of the 680 screws , 33 screws ( 4.9% ) were found to contact a major vessel on imaging , including the aorta in four cases . eight of the nine affected patients were living and asymptomatic at average follow - up of 25 months , with one patient deceased from amyotrophic lateral sclerosis at 16 months after surgery.12 from these data , the authors concluded that asymptomatic , impinging screws may be safely observed with serial imaging . evidence in favor of hardware removal comes primarily from case reports in which pedicle screws impinging against the aorta were diagnosed on a delayed basis and successfully managed with hardware removal and evar.4 5 6 7 8 10 exact details of the procedures varied , but in all cases patients had endovascular stent grafts placed that bridged the affected region of the aorta before or after removal of their hardware . no complications were reported.4 5 6 7 8 10 of note , in several of these reports , patients experienced a symptom - free period after their initial spine surgery and were diagnosed with impinging hardware after returning 4 months to 6 years later with new complaints of pain in the instrumented region ; these complaints resolved after screw removal.6 8 10 we hypothesize that in the setting of impinging hardware , new - onset pain after a symptom - free period may potentially represent a red - flag for surgical intervention . in an in vivo bovine model in which thoracic pedicle screws were left abutting the aorta , faro et al found histopathologic changes in 96% of aortic specimens at 3 , 6 , and 12 months . more than half of the specimens showed significant wall thinning , and in aggregate , the impinged aortas demonstrated decreased stress to failure compared with controls . the severity of tissue damage on histology did not correlate with the depth of impingement on ct scans.13 in the context of the limited clinical data , this study raises concerns that screw impingement may compromise of vessel wall integrity even in the absence of frank perforation . the case considered here is unique in that our patient was much younger than those in previous reports ( 20 years old vs. older than 60 years ) and because he was successfully managed without endovascular grafting given his benign findings on intraoperative aortography . as the incidence of pedicle screw instrumentation for ais continues to rise , one expects to see younger patients facing decisions about how to manage impinging hardware.3 on the basis of multiple case reports , placement of an endovascular aortic graft appears to represent an effective , low - morbidity treatment for impinging or perforating pedicle screws in older population . however , available clinical data after evar in the vascular literature suggest a significant incidence of complication and reoperation at intermediate to long - term follow - up that would be more relevant to a younger patient . in one series of 1,066 patients treated with evar for abdominal aortic aneurysm and followed up between 2 and 16 years , 233 patients ( 22% ) required repeat operation , with 131 operations attributed to graft - related complications ( 12% ) , and the remainder to progression of vascular disease.14 in another study using a modern endovascular aortic graft exclusively ( endurant ; medtronic , minneapolis , minnesota , united states ) , 16 of 100 grafts showed leakage on ct angiography and 5 of 100 patients required endovascular or operative reintervention within the first year.15 the high rate of complications and reoperation associated with evar is sobering , particularly when considering application in an adolescent patient . here , we describe the safe and successful removal of a pedicle screw impinging on the aorta in a symptomatic 20-year - old patient . on the basis of normal intraoperative aortography , we elected against placement of endovascular graft given the high incidence of associated complications and the expected longevity requirement in a young patient . at more than 2 years after the procedure we would recommend considering similar treatment of other young patients when intraoperative aortography shows no abnormality . neuroscience clinical institute , epworth hospital , melbourne , victoria , australia potter et al are to be commended for presenting an important yet very uncommon surgical complication of a misplaced thoracic screw requiring revision for aortic impingement.1 the index procedure was an excision of a t7 osteoblastoma 5 years previously . the presentation of the now 20-year - old male patient was midthoracic and retrosternal pain without any evidence of tumor recurrence . a computed tomographic ( ct ) thoracic scan performed to exclude change in instrumentation or pathology demonstrated a laterally misplaced left t6 screw . interestingly , the ct demonstrated impingement on the thoracic aorta by the screw tip , which was previously unknown . if pain is present , 12% of the patients will report improvement with removal of instrumentation2 ; this also facilitates future imaging for tumor surveillance . the t6 pedicles are among the most technically challenging to cannulate given their narrow width . postoperative ct scans are equally used in many practices , including my own , to exclude neural and/or vascular compromise . delayed aortic rupture is extremely rare.3 however , removal of the t6 screw in this case for vascular impingement is reasonable , especially given the bovine in vivo studies of aortic wall thinning illustrated by faro et al.4 this is especially true in a young adult . in the setting of luminal or gross mural abnormality , the aorta undergoes constant cyclical movement with cardiac pulsatility being the primary dynamic elastic artery . with each cycle , it undergoes radial and longitudinal distortion . if the point of the screw was in contact with the aorta , it is unlikely that it would be in contact with a single adventitial point promoting erosion over time.4 the aorta is tethered to the vertebral column by multiple radicular branches ; one is the artery of the adamkiewicz , classically thought to arise on the left near the t9 level . although the absence of erosion , thickening , or false aneurysm in the referable area is comforting , having an expedited means for brisk vascular control is vital . combined management with the vascular surgery team involves preparing and positioning the patient for an immediate posterolateral thoracotomy ( including a double lung intubation ) as detailed by the authors . endoluminal techniques permit control via a compliant balloon that can be prophylactically placed and repair subsequently effected with an appropriately sized stent graft . ideally , imaging the descending thoracic aorta to position a wire and balloon opposite the screw and fix it relative to the sheath allows immediate control to be achieved . a limitation of this case report is extrapolating results and complication rates from abdominal aortic aneurysm endovascular aortic repair ( evar ) to thoracic endovascular aortic repair ( tevar ) for trauma . even for aneurysm repair , tevar has lower complication rates5 than the more complicated abdominal evar procedures . furthermore , in this report , the injury is small and the thoracic aortic wall probably normal , and so seal is much more assured , provided proper planning has been performed , making an endoleak very unlikely . there is no aneurysm sac to pressurize in this setting that is the main source of reintervention in evar . evar results are not a surrogate for posttraumatic tevar results . not placing the stent graft in the absence of an aortic abnormality the main concerns with placement of a stent graft in this report is the risk of paraplegia , the potential for devastating ( but rare ) graft infection , and the uncertainty of the long - term fate of the stent graft in young patients . the post - discovery decision - making described here by the authors and the commenter is very helpful for all of us clinicians . this is a classic scenario for which there is no evidence base as we have no control group ; we have to use our judgment , experience , and the few descriptions offered in the peer - reviewed literature , even if its level of evidence is 4 or 5 . this case raises the bigger question : should all patients have some form of routine post - instrumentation advanced imaging to check on segmental hardware placement even in asymptomatic patients ? what do you think ?

study design case report and review of the literature . objective the objective of the article is to report the case of a 20-year - old patient with a threatened aortic injury from pedicle screw instrumentation successfully managed without aortic grafting . methods the patient 's clinical course is retrospectively reviewed . the offending hardware was removed after gaining endovascular control of the aorta . results intraoperative aortography was normal and no graft was placed . the patient remains asymptomatic at 2 years after surgery . conclusions hardware impinging on the aorta can safely be removed by gaining endovascular control of the aorta . in the setting of normal intraoperative aortography in a young patient , we recommend against further intervention to avoid the known morbidity of aortic grafting .

during 20052007 ( figure 1 ) , a total of 105 human cases of brucellosis were diagnosed among 2,054 persons who were tested on the basis of clinical suspicion or risky exposure . a human case of brucellosis was considered confirmed if results of serologic tests , such as elisa or complement fixation test , were positive , in accordance with the world health organization case definition ( 5 ) . geographic distribution of human and animal brucellosis in bulgaria during a ) 2005 , b ) 2006 , and c ) 2007 . the alert started in 2005 ( figure 1 , panel a ) , when a case of brucellosis occurred in a bulgarian migrant animal keeper working in greece . active surveillance of persons at risk was implemented , enabling detection of a total of 34 human cases of brucellosis . all cases were classified as imported cases ; therefore , no supplemental active surveillance on animals was implemented . additionally , during routine screening for at - risk workers , 3 other persons employed in a dairy factory were found to be seropositive . due to the lack of traceability of the raw material used in the factory , it was not possible to trace the origin of the infection . at that time ( figure 1 , panel b ) , a total of 10 cases of human brucellosis were reported from different regions of the country . according to anamnestic information , these case - patients had different sources of infection : 3 of the 10 were considered imported infections ; 1 case - patient was diagnosed during hospitalization in sicily ( italy ) , where the patient reported having eaten ricotta cheese , and 2 occurred in bulgarian migrant animal keepers working in greece . concerning the origin of infection , epidemiologic data suggest that 5 of the 10 cases were related to occupational risk and the remaining to consumption of raw milk and milk derivates . surveillance activities enabled detection of 10 animals ( 7 small ruminants and 3 cows ) with positive serologic results ; these animals were then killed and destroyed . during 2007 ( figure 1 , panel c ) , a total of 58 human cases were identified . of 58 cases , 54 were classified as autochthonous ( i.e. , acquired by imported animals found to be infected during regular veterinary surveillance ) . these cases were identified in a bulgarian region bordering greece and turkey ( haskovo region ) . two other cases , which were also classified as autochthonous , were diagnosed in patients who stated they had consumed a risky product ( i.e. , raw milk handled without adherence to hygienic standards ) . the remaining 2 cases were classified as imported because they involved bulgarian migrant animal keepers working in greece . active surveillance in place for animals found a total of 625 heads ( 618 small ruminants , 7 cows ) with positive serologic results ; all were killed and destroyed . analagous with what we observed in humans , most of the infected animals were found in the haskovo region . all animals found to be infected during surveillance activity were bred at the family farm , and their milk and dairy products were prepared and eaten without adherence to proper hygienic standards . our data show that brucellosis is reemerging in bulgaria ( figure 2 ) . on the basis of information provided in this report , we can make several hypotheses regarding the causes of the resurgence of a previously controlled infection in a transitional , rapidly changing country . human and animal cases of brucellosis in bulgaria , 19922007 . in bulgaria , during 19922004 , a total of 22 human cases and 0 animal cases of brucellosis were recorded ; during 20052007 , a total of 105 human cases and 635 animal cases of brucellosis were recorded . overall , 105 human cases of human brucellosis were identified over a 3-year period . of them , this finding suggests that when brucellosis is introduced into naive territories ( i.e. , those territories that were considered officially free of brucellosis ) , the primary source of infection for humans is direct contact with infected animals ( i.e. , exposure to abortion / delivery products ) or domestic consumption of products produced on family farms ( milk , raw cheese ) . however , environmental exposure can also occur , especially in infants and children , who are considered at lower risk for direct contact with potentially infected animals , as recently observed ( 6 ) . this hypothesis appears to be consistent with the context of a naive setting , where preventive measures are not routinely implemented . continuous health education and other strategies may contribute to reduce the circulation of human brucellosis in endemic areas ( 7 ) . the reemergence of brucellosis is not limited to bulgaria but involves several countries in the balkan region and even in the caucasian region ( p. pasquali , unpub . first , due to socioeconomic changes , many countries in these regions are experiencing a dramatic increase of animal trade , animal movement , and occupational migration , which in turn may increase the risk for introduction and spread of infectious diseases , such as brucellosis , from other disease - endemic countries like greece or turkey ( 2 ) . second , the process that has characterized the change of the social and administrative organization since the collapse of the soviet union is far from being completed ; the public health systems are still flawed in many countries . finally , part of the increase may simply be that brucellosis is a complex disease , which has different cycles of expansion and regression . before drawing conclusions first , samples from patients with positive serologic results were used for bacterial culture for brucellosis only if sample collection was properly timed ; no culture positive case is available . second , we can not exclude the possibility that part of the increase in cases of brucellosis could be due to improved surveillance ; in particular , temporal trends and geographic comparison might be , to some extent , affected by the intensity of screening activities . however , this increased surveillance is unlikely to bias the observed shift from imported to locally acquired cases . in conclusion , this report shows how a disease such as brucellosis may increase its public health impact , particularly in transitional countries such as bulgaria . our findings emphasize the importance of the combination of health education and active surveillance systems for controlling infectious diseases and highlight the need for cooperation between public health officials and veterinary officers . creating and improving capacity building are necessary to properly address issues that pose public health hazards .

bulgaria had been free from brucellosis since 1958 , but during 20052007 , a reemergence of human and animal disease was recorded . the reemergence of this zoonosis in the country highlights the importance of maintaining an active surveillance system for infectious diseases that will require full cooperation between public health and veterinary authorities .

breast cancer is the most common cancer and second leading cause of cancer mortality in the united states and texas . it is estimated that 207,090 new cases of invasive breast cancer and 39,840 breast cancer deaths occurred among women in the usa during 2010 . there is considerable variation in the rates of breast cancer mortality at the county level . the reported highest and lowest age - adjusted mortality rates for breast cancer from 2000 to 2008 for texas at the county level were 41.9 and 16.1 per 100,000 , respectively . apart from geographical variation in breast cancer mortality , there is a significant disparity in breast cancer mortality in texas based on racial and ethnic status . mortality rates for female breast cancer were approximately 30% higher during 19922001 and 50% higher during 20012005 among black women compared to white women . both black and hispanic women were found to have significantly increased relative risk of breast cancer mortality compared to non - hispanic white women during 19922000 [ 3 , 5 ] . relatively few studies have been conducted to examine how racial and ethnic status affects the geographic distribution of breast cancer mortality , and no study has been conducted to investigate this at the county level in the state of texas . identification of geographic patterns of breast cancer mortality based on racial and ethnic status could provide impetus to conduct further investigations and target health resources for prevention and treatment in specific geographic areas . the spatial scan statistic method developed by kulldorff , kulldorff and nagarwalla has been shown to be an effective method for investigating geographical patterns of cancer and detecting spatial cancer clusters . a cancer cluster is defined as a greater - than - expected number of cancer cases that occurs within a group of people in a geographic area over a specified period of time . a previous study published by zhan and lin in 2003 detected two statistically significant clusters of female breast cancer mortality at the county level using data provided by the bureau of vital statistics of the texas department of health for the period from 1990 to 1997 . the most likely cluster identified ( p value 0.0004 ) aransas , atascosa , austin , bastrop , bee , bexar , brazoria , brooks , caldwell , calhoun , colorado , de witt , duval , fayette , fort bend , galveston , goliad , gonzales , guadalupe , harris , jackson , jim hogg , jim wells , karnes , kenedy , kleberg , la salle , lavaca , live oak , matagorda , mcmullen , nueces , refugio , san patricio , victoria , waller , wharton , willacy , and wilson . a secondary cluster ( p value 0.0077 ) included only one county : el paso . in this paper , we present results of spatial analyses using the spatial scan statistic method on female breast cancer in the state of texas using county - level female breast cancer mortality data for the years 2000 to 2008 . the objectives of the study were to detect high - risk areas of breast cancer mortality through the discrete poisson model , investigate whether there are high - risk areas where the distribution of breast cancer mortality differs based on the racial / ethnic status of the population using the multinomial model , and examine the spatial distribution of breast cancer mortality cases for non - hispanic blacks ( nhbs ) , hispanics and other races compared to non - hispanic whites ( nhws ) using the bernoulli model . the case definition for this study was a death due to malignant neoplasm of breast cancer ( c50 ) as listed by the international classification of diseases , 10th revision ( icd-10 ) in the female population of texas for the years 2000 to 2008 . the breast cancer mortality data at the county level for the years 2000 to 2008 were obtained from the texas cancer registry , cancer epidemiology and surveillance branch , texas department of state health services . the texas cancer registry classifies cancer mortality data according to the surveillance , epidemiology , and end results ( seer ) cause of death recode , as given by the seer cause of death recode 1969 + ( 9/17/2004 ) ( http://seer.cancer.gov/codrecode/1969+_d09172004/index.html ) , and the seer program has defined major site groups based on the icd-10 . the texas cancer registry is a statewide , population - based registry that collects high - quality , population - based data reported from various sources , including hospitals , cancer treatment centers , ambulatory surgery centers , pathology laboratories , and physician 's offices through active and passive surveillance . it currently meets standards set by the national program of central cancer registries , centers for disease control and prevention for high - quality data , and is gold - certified by the north american association of central cancer registries . in all , 22,820 breast cancer deaths occurred in texas between the years 2000 to 2008 , of which 15,234 ( 66.8% ) were attributed to nhw , 3,503 ( 15.4% ) to nhb , and 3,770 ( 16.5% ) to hispanics . at - risk population data at the county level for texas covering the same time period were obtained from the us census bureau . geographic coordinate data ( i.e. , county centroids ) that represent the locations of the 254 texas county polygons were specified by the 2000 us bureau of the census . we ran purely spatial analyses using 4 different models ( discrete poisson with and without covariates , bernoulli and multinomial models ) . for the discrete poisson model , we assumed that the number of deaths in each county was poisson distributed and ran the model with and without adjustment of racial / ethnic status of the population . for the bernoulli model , we classified breast cancer deaths among nhw as controls and breast cancer deaths among other racial / ethnic groups as cases , thereby creating 3 comparisons ( nhb versus nhw , hispanics versus nhw and other races versus nhw ) . for the multinomial model , we classified breast cancer deaths into 4 racial / ethnic categories ( nhw , nhb , hispanics , and other races ) . we used the satscan v9.1.1 released on march 9 , 2011 to run the spatial scan statistics . the spatial scan statistics detect high - risk areas of cases by gradually scanning a window across space and noting the number of observed and expected observations inside the window . the window sizes are varied continuously up to a prespecified maximum size . the most likely cluster ( the cluster least likely to be due to chance ) is assigned to a window with the maximum likelihood . we used the scanning window in the shape of a circle and specified the maximum window size as one that included 50% of the at - risk population throughout our analyses . we also report secondary clusters that cause a rejection of the null hypothesis ( i.e. , the log likelihood ratio of secondary clusters in the real data is higher than that of the most likely cluster in the simulated data sets ) and do not overlap the most likely cluster . mapinfo professional version 8.0 was used to create the thematic map based on detected counties by spatial scan statistics . this study did not need to be approved by the committee for the protection of human subjects because it used aggregated data on county - level breast cancer mortality in texas . results of spatial analysis using the discrete poisson model without any covariate adjustment suggests that there were five statistically significant clusters of counties with a high rate of female breast cancer mortality in texas for the years 2000 to 2008 ( table 1(a ) , figure 1(a ) ) . the most likely cluster was located in the northeast corner of texas , including 51 counties with relative risk of 1.27 compared to the rest of texas . secondary clusters were located in the central region of texas ; the southeast central region ; the northern part of the state ; the region inclusive of bosque , hood and somervell counties . corresponding relative risks were 1.57 , 1.38 , 1.13 , and 1.66 , respectively , compared to the rest of texas . after adjusting for racial / ethnic status of the population , spatial analysis using the discrete poisson model detected two statistically significant clusters ( table 1(b ) , figure 1(b ) ) . the most likely cluster was located in the western and central region of texas , including 106 counties with relative risk of 1.23 compared to the rest of the state . a secondary cluster was located in southeastern texas with relative risk of 1.32 compared to the rest of texas . results of spatial analysis using the multinomial model suggest that there were three statistically significant clusters of counties where the distribution of risk for female breast cancer mortality based on race / ethnicity is statistically significantly different from the remaining regions of texas ( table 2 , figure 1(c ) ) . the most likely cluster was located in south texas where hispanics had the highest risk and nhb had the lowest risk . the risk of female breast cancer mortality was 4.77 times higher among hispanics and 0.29 times lower among nhb in the cluster compared to the rest of texas . the first secondary cluster was located in northeast texas where nhb had the highest risk and hispanics had the lowest . the risk of female breast cancer mortality was 3.42 times higher among nhb and 0.31 times lower among hispanics in this cluster compared to the rest of texas . the other secondary cluster was located in the northern and western region of texas , where nhw had the highest risk and hispanics had the lowest , although the risk of female breast cancer mortality for one group is not substantially different from the other groups . table 3 shows results of spatial analysis using the bernoulli model for nhb , hispanics , and other races compared to nhw . for nhb versus nhw , we detected one significant cluster located in the northeastern region of texas , with relative risk of 2.63 compared to the rest of the state ( figure 1(d ) ) . for hispanics versus nhw , we detected two significant clusters ( figure 1(e ) ) . the most likely cluster was located in the extreme southern region of texas with relative risk of 4.13 compared to the rest of texas , and the secondary cluster was located in the western region of texas with relative risk of 3.83 compared to the rest of the state . for other races versus nhw , we detected two significant clusters ( figure 1(e ) ) . the most likely cluster included four counties ( brazoria , fort bend , harris , and waller ) in southeast texas , with relative risk of 2.77 , and the secondary cluster included four counties ( collin , dallas , denton , and tarrant ) in northeast texas , with relative risk of 1.70 compared to the rest of the state . we found several statistically significant clusters for female breast cancer mortality at the county level in texas through purely spatial analyses . five significant clusters were found through the discrete poisson model without any covariate adjustment , while the same model after adjusting for racial / ethnic status detected two significant clusters with different geographic distributions . the multinomial model detected three significant clusters with different distributions of risk based on racial / ethnic status . the bernoulli model found one significant cluster for nhb versus nhw , while two significant clusters were detected for hispanics versus nhw and another two for other races versus nhw . zhan and lin ( 2003 ) conducted spatial cluster analysis using the discrete poisson model without any covariate adjustment on female breast cancer mortality data for the years 1990 to 1997 and found two significant clusters . the most likely cluster was located in the southeast region of texas and the secondary cluster included only one county : el paso . we found five significant clusters using the same analysis for the years 2000 to 2008 . the most likely cluster in our findings was located in the northeast region of texas , and three secondary clusters were located in the northern and central regions of the state . only one secondary cluster located in southeast texas had overlapped with the most likely cluster found by zhan and lin ( 2003 ) . this indicates that the geographic distribution of female breast cancer mortality at the county level has shifted over time from the southeast towards northern and eastern areas in texas . one explanation for this change could be rapid growth of urban areas and slower growth of rural areas in texas during the same period . in fact , texas is one of the top five fastest growing states with a 20.6% and 4.3 million ( highest numeric increase ) population increase between 2000 and 2010 . about 85% of the total texas population resided in urban areas in 2005 , with most concentrated in the six largest metropolitan statistical areas austin - san marcos , dallas , fort worth , el paso , houston , and san antonio . houston and dallas - fort worth were among the top 3 fastest growing metro areas of the ten most populous metro areas in the unites states . together , they accounted for almost one - half of the state population and its population growth between 2000 and 2010 . hispanics are the fastest growing population in texas and are expected to outnumber whites by 2020 . this shift in population demographics presents challenges for effective distribution of health care resources to reduce the disparities for breast cancer mortality . after adjusting for racial and ethnic status , the location of the most likely cluster changed from the northeast to the western area of texas . this indicates that the existence of clusters in northeast texas could be explained by racial and ethnic disparities in the region , while other etiological factors might explain mortality clusters in west texas . we found three significant clusters using multinomial analysis where risk for breast cancer mortality differed based on racial / ethnic status . the cluster in the northeast area of texas had significantly highest risk for nhb and lowest risk of hispanics , while the cluster in the southern area of texas had significantly highest risk for hispanics and lowest risk for nhb . it is important to note that risk is not substantially different among racial / ethnic groups in the northern texas cluster . findings from the bernoulli model analysis support the results of the multinomial analysis , indicating that breast cancer mortality is heterogeneously distributed based on racial / ethnic status . nhb and hispanics are more likely to be diagnosed with later stages of breast cancer [ 1113 ] and have relatively higher risk of breast cancer mortality compared to nhw [ 3 , 5 ] . socioeconomic status ( ses ) of the population is an important predictor of stage of breast cancer at diagnosis . less utilization of mammography facilities , cancer radiotherapy , and cancer surgery among minorities is the likely reason that they tend to be diagnosed at later stages and have higher mortality from breast cancer [ 1517 ] . hispanics were the majority in 34 texas counties in the usa - mexico border region , and health care , including preventive and therapeutic resources , are more likely to be scarce or lacking in this underserved population [ 19 , 20 ] . all of these factors could help explain why the highest risks for nhb were in the northeast , and highest risks for hispanics were in the southern areas of texas . the lower risks for other racial / ethnic groups are the result of unknown factors . we identified one cluster in northeast texas that had higher risk of breast cancer mortality for nhb compared to nhw though the bernoulli model analysis . the location of this cluster was the same as that found by the multinomial analysis ; however , the risk was slightly lower . this could be due to fact that we included other races in the multinomial model analysis and breast cancer incidence and mortality have been observed to be lower among this group compared to nhw , nhb , and hispanics . for example , cancer incidence and mortality per 100,000 were 51.7 and 8.6 , respectively , among asian / pacific islanders for the years 2001 to 2005 , while these same rates were 125.4 and 24.3 among nhw , 116.2 and 35.6 among nhb , and 84.6 and 17.2 among hispanics . using bernouli model analysis , we identified two clusters , one in the western part of the state and another along the border with mexico , where hispanics had significantly higher risk of breast cancer mortality compared to nhw . the most likely cluster was the same as that found by the multinomial analysis ; however , the risk was slightly lower . for other races , two significant clusters were detected , with each covering four counties around the houston and dallas metropolitan areas , respectively . this might be related to the fact that the proportion of total population for other races in houston was higher compared to dallas . this type of cluster analysis at the county level can provide useful information to policy makers for the following reasons . the department of state health services divides all texas counties into health service regions ( hsrs ) , identified numerically from 1 to 11 , to provide comprehensive public health services to the citizens of texas through 8 regional public health offices ( figure 2 ) . in addition , public health services in texas are also provided through local health departments . but only one - fourth of counties in texas have local health departments and most of them are located in urban areas . results of our study could be important to regional public health offices and local health departments for establishing priorities and making strategic decisions about distribution of services for breast cancer . for example , secondary cancer cluster 2 identified through the multinomial model ( figure 1(c ) ) covers hsrs 1 , 2/3 and 9/10 and very few counties in these regions are covered through local health departments ( figure 2 ) . regional offices could utilize their limited resources to provide screening services for breast cancer in areas not covered by local health departments . moreover , our results also indicate racial disparities in cluster identification that might be useful to regional and local health departments . for example , the most likely cluster identified through the multinomial model ( figure 1(c ) ) had highest risk for hispanics . regional and local health departments could utilize this information to direct their resources for screening and education to this population . approximately half of texas counties did not have accredited permanent mammography facilities in 2008 as reported in the texas cancer facts & figures , 2008 . moreover , most of counties with more than 1 accredited facility are located around the largest metro areas ( figure 3 ) . though the majority of the texas population is concentrated in these areas , our findings for example , on - site or mobile mammography facilities with more multilingual and follow - up supports could be introduced in the southern areas of texas along the border with mexico , where we identified a cancer cluster with significantly higher risk of breast cancer mortality among hispanics compared to nhw ( figure 1(e ) ) . results of our spatial analyses at the county level provide useful information to guide future spatial analyses at finer scales in texas . moreover , they are useful jumping - off points to conduct subcluster analyses at the county level or finer scales for a particular population group . for example , the census block or tract level analysis of female breast cancer mortality among nhb could be conducted in the hsrs 4 , 5 , 6 , and 7 covering secondary cluster 2 identified through the multinomial model ( figure 1(c ) ) to identify high risk areas for that particular population . we used breast cancer mortality data aggregated at the county level , which could affect the sensitivity of cluster detection . the geographic distribution of total population and number of breast cancer mortality cases at the county level in texas is heterogeneous , with some counties having much lower mortality than other counties . lack of significance for some secondary clusters might be due to this reason or it might be due to the fact that the test is conservative , that is , we compared secondary clusters with the most likely cluster from the simulated datasets . results may not be comparative to other studies using data aggregated at different geographic scales . also , we included only race / ethnicity as a confounder in our analyses , yet there are many other known or hypothesized risk factors for breast cancer that we did not analyze , such as age at diagnosis , family history , alcohol consumption , access to mammography and other health care facilities [ 27 , 28 ] and various environmental [ 29 , 30 ] and reproductive factors [ 31 , 32 ] . results of our analyses indicate that breast cancer mortality at the county level in texas is distributed heterogeneously based on racial / ethnic status . the evidence suggests that highest rates of female breast cancer mortality have shifted over time from southeastern areas towards northern and eastern areas of the state . in texas , nhb had highest risk for breast cancer mortality in the northeastern region and lowest risk in the southern region , while hispanics had highest risk in the southern region along the border with mexico and lowest risk in the northeastern region . these findings , along with continuing trends toward urbanization , growing numbers of hispanic residents , and increasing levels of poverty for many minorities , provide challenges and opportunities for texas policy makers and health advocates . more research is needed to make informed decisions about effective and efficient distribution of health care resources to reduce breast cancer disparities for texas residents .

objective . the objectives of the study were to detect high - risk areas and to examine how racial and ethnic status affect the geographic distribution of female breast cancer mortality in texas . analyses were based on county - level data for the years from 2000 to 2008 . materials and methods . breast cancer mortality data were obtained from the texas cancer registry , and the spatial scan statistics method was used to run purely spatial analyses using the discrete poisson , bernoulli , and multinomial models . results and conclusions . highest rates of female breast cancer mortality in texas have shifted over time from southeastern areas towards northern and eastern areas , and breast cancer mortality at the county level is distributed heterogeneously based on racial / ethnic status . non - hispanic blacks were at highest risk in the northeastern region and lowest risk in the southern region , while hispanics were at highest risk in the southern region along the border with mexico and lowest risk in the northeastern region .

we report a case illustrating how the hazardous cognitive effects of chronic phenytoin administration ( in the form of scholastic deterioration ) transformed into mood and behavioral changes and even suicidality . a 28 year unmarried hindu male of middle socioeconomic status presented with complaints of initial scholastic deterioration , social withdrawal , aggressive and violent behavior , slowness , forgetfulness , anxiety , decreased confidence , sadness , and occasional suspiciousness for the last 5 years and suicidal gestures since 18 months . there was history of epilepsy since last 13 years for which he was receiving 300 mg / d of phenytoin . a provisional diagnosis of mood disorder with epilepsy was kept . however , on investigating , serum phenytoin level was found to be highly elevated at 32.8 mcg / ml ( normal range=10 - 20 mcg / ml ) , while serum folic acid level was below the normal range 5.23 ng / ml ( normal > 5.38 ng / ml ) . thus , diagnosis was revised and the patient was classified as a case of phenytoin toxicity . keeping in mind that cognitive impairment due to phenytoin resolves on drug withdrawal , phenytoin was tapered off ( 50 mg/ every sixth day ) from 300 mg / day and folic acid 5 mg / d was supplemented . the symptom course was closely monitored and supportive psychotherapy instituted during hospitalization ; the patient received no other psychopharmacological agents . within the 2 weeks hospitalization phase , subject showed global improvement of 20% in behavioral symptoms and mood but there was little improvement in his cognitive symptoms . by third follow - up at 11 weeks , cognitive symptoms showed improvement as well and global improvement of 60 - 70% was reported- in line with another study . however , an episode of tonic - clonic seizure occurred at this time , and considering the phenytoin toxicity , the patient was put on divalproex 500 mg / d . no seizures occurred till the 15 week during fourth follow up and the family reported near complete resolution also of his neuropsychiatric symptoms . while their resolution consequent to tapering off phenytoin and folic acid supplementation implicates phenytoin induced decreased folic acid levels and its subsequent correction , it could also be visualized as a secondary reaction to the scholastic handicap due to cognitive deficit . this subject had obtained 53% and 64% in high - school and intermediate examinations , respectively , but thereon could not qualify in competitive examinations for next 4 years and finally gave up switching to take up a bsc ( bachelor of science ) course . in this , feeling unable to cope with the studies , he did not appear in examinations at the end of the session and switched to undertake the less rigorous ba ( bachelor of arts ) course as a desperate last resort . however , he could not pass that also in the first attempt , only clearing in the second but with less than 50% marks . at the time of his presenting to us , he had further switched his career stream , being enrolled in diploma in tourism and was brought to us for very irregular attendance , not appearing for his examinations and not showing interest in current activities and studies . the scholastic deterioration was manifest in his performance decreasing , which invoked certain psychological sequelae which affected not only his vocational domains , but interpersonal and social domains as well . however , when the subject felt that even his enhanced efforts over a considerable period of time did not produce improved cognitive performances , this mismatch led to irritation , depression , worthlessness , hopelessness , and a sense of doom which even led to his showing or attempting suicidal behaviour at times . this mismatch conflict also reflected as changes in his interpersonal behavior . due to the scholastic deterioration , the subject , not only had to compromise much in life , but also faced critical comments and ridicule from family and others which consequently led to violent outbursts towards the family and hostility towards others at times leading to a vicious cycle . such a hypothesis may be in place as two bouts of depression reported during last one and half years lasted for one and one and half months , respectively , and resolved spontaneously without any intervention suggesting it to be arising from the vicious cycle . the diagnosis of phenytoin intoxication was not an easy one to make in our patient as nystagmus and ataxia were absent among the mood , behavior , and cognitive alterations . phenytoin intoxication should be considered when any unusual neurological or behavioral disturbance occurs in a patient receiving the drug . the estimation of the serum phenytoin concentration is invaluable in such a situation.[46 ] it has a long mean half life of 40 h and is very cheap compared to its competitors making it highly acceptable . however , the fact that phenytoin might be more deleterious to higher cognitive functions than carbamazepine or sodium valproate is still commonly ignored and the drug widely used without exercising the necessary precautions entailed in its administration as this case depicts with serum phenytoin levels never having been estimated before hospitalization . importantly , these harmful cognitive effects , if undetected , may roll on to pronounced psychological sequelae like depression , hopelessness , and even suicide . the case illustrates how scholastic performance in students / scholars is extremely vital for them and any scholastic deterioration runs the risk of hazardous ramifications and implications . thus , short - term or long - term use of drugs affecting cognition must be peppered with caution especially in select groups like scholars , students , executive , and the like .

phenytoin is a commonly used antiepileptic medication because of its easy accessibility as well as affordability . however , scientific literature shows various types of side effects of phenytoin . we report a patient who was showing toxicity symptoms in the form of mood , behavior and cognitive symptoms along with scholastic problems and personality change on long term treatment with phenytoin . the patient 's serum phenytoin was found to be quite high ( > 32.8 ng / ml).the symptoms were attributed to phenytoin toxicity which responded within twelve weeks by reducing the dose of phenytoin ( with resultant fall in levels of serum phenytoin ) and the addition of folic acid . while the mood and behavior symptoms recovered early , the cognitive symptoms responded slowly showing 80% -90 % improvement over a period of fifteen weeks .

mechanical dyssynchrony describes the differences in the timing of contraction or relaxation between the left ventricular ( lv ) and right ventricular ( rv ) ( interventricular dyssynchrony ) , or between different myocardial segments of the lv ( intraventricular dyssynchrony ) . it is commonly observed in patients with congestive heart failure ( chf ) , which is caused by electromechanical delay in some regions of the failing heart and will result in further reduction of cardiac function . its presence varies not only with the methodology of assessment , but also the characteristics of patients including the qrs duration , ejection fraction , loading condition , severity of coronary artery disease , and degree of lv hypertrophy or remodeling . mechanical dyssynchrony has been suggested useful for exploration of disease mechanism , stratification of patient risk , selection of therapeutic modality , and prediction of prognosis in chf population , in particular in those who are candidates of cardiac resynchronization therapy ( crt ) . the analysis of mechanical dyssynchrony by echocardiography has been widely adopted due to its advantages of being easily available , non - invasive , radiation free and rapid technological development . the techniques range from conventional m - mode and doppler echocardiography to more advanced tissue doppler imaging ( tdi ) , three - dimensional ( 3d ) echocardiography and two - dimensional speckle tracking imaging , and most recently , 3d speckle tracking imaging . the parameters of systolic dyssynchrony include those signify the dispersion of the time to peak ventricular contraction , as represented by the standard deviation or maximal delay among a certain amount of lv segments , and the difference between the lv and rv . the majority of them were derived from crt trials with cutoff values to define dyssynchrony and therefore suggested by the american society of echocardiography in an expert consensus statement ( table 1).1 - 10 ) the assessment of diastolic dyssynchrony is similar to that of systolic dyssynchrony , in which the parameters calculate the dispersion of the time to ventricular relaxation , as referred to the standard deviation or maximal delay among a certain amount of lv segments.11 - 14 ) of note , tdi velocity is almost the exclusive modality adopted for measurement , because of the discernable and consistent signal of early diastole ( table 1 ) . the compelling evidence from multicenter clinical trials has established crt as the most promising therapeutic modality in heart failure management over the past decade.15 ) crt not only improves symptoms and cardiac function , but also reduces heart failure hospitalization and all - cause / cardiovascular mortality in patients with advanced chf . however , it remains a major problem that non - responders of therapy are constantly observed in about one - third of patients receiving crt , based on the current guidelines for patient selection in which the qrs duration 120 ms is regarded as the only marker of ventricular electromechanical delay.16)17 ) the presence of a prolonged qrs duration in chf is associated with more advanced myocardial disease , more severe lv dysfunction , worse prognosis and higher all - cause mortality.18 ) however , a prolonged qrs duration may not necessarily be equivalent to significant mechanical dyssynchrony , even in the form of left bundle branch block ( lbbb ) . fung et al.19 ) observed that regional electrical conduction delay could be absent in chf patients with lbbb . using the 3d non - contact mapping electrograms , homogenous delay in lv the propagation of endocardial activation similar to that of normal subjects was found in some patients with typical lbbb , while typical delay in depolarization over the lv lateral or posterior wall was observed in others . in fact , the qrs duration on surface ecg is a rather inaccurate estimation of myocardial electrical activation which correlates poorly with the occurrence of systolic mechanical dyssynchrony.20 - 22 ) the correction of cardiac electromechanical delay is suggested to be one of the major mechanisms for crt benefits . therefore , direct measurement of mechanical dyssynchrony by echocardiography has become clinically relevant in estimating the likelihood of response when ecg as a surrogate marker may fail . over the last decade , a number of single - center studies have demonstrated that the lack of mechanical dyssynchrony assessed by noninvasive echocardiographic techniques is closely related to the lack of response in patients who received crt . nevertheless , those results were challenged by the predictors of response to crt ( prospect ) trial , the first multicenter trial in which no single echocardiographic measure of mechanical dyssynchrony could predict crt responses with a good sensitivity and specificity.23 ) however , there were a number of major limitations in the design and execution of the prospect trial , which challenged the value of conducting a highly flawed study that is unable to address the crucial question of role of echocardiography in predicting crt response , and the implication of reducing the amount of unnecessary device implantation.24 - 27 ) the inadequacies in patient selection , study site training , echocardiographic standard for data acquisition and analysis , the lack of training and experience in dyssynchrony assessment by the three echocardiographic core - laboratories , and consideration of other contributing factors may explain the unexpected findings of the trial . they include extensive myocardial scar , absence of contractile reserve , severe mitral regurgitation , high pulmonary pressure , poor lv lead position and suboptimal device programming.16)28)29 ) therefore , the prospect trial shall not be regarded as the final conclusion about the inability of dyssynchrony assessment for predicting crt response , but rather , dedicated training for knowledge and skill transfer can not be overemphasized . our recent study has confirmed the importance of systematic training to ensure the reproducibility of dyssynchrony analysis using tdi when comparing the reading between the " beginners " or the " graduates " of dyssynchrony training with the reference standard of the " experts".30 ) a number of studies are conducted in the " post - prospect era " to examine the ability of mechanical dyssynchrony in predicting crt responses.31 - 46 ) among them , a couple of studies were designed and conducted by the experienced centers in which shared protocol and standardized technique of dyssynchrony analysis could be ensured , as well as having a larger sample size with diversity of patients . furthermore , hard endpoints over long - term follow up were selected , such as all - cause mortality and cardiovascular event . mid - term lv reverse remodeling also frequently occurred as a primary endpoint in these trials , as its presence after crt has been proved to correlate with improvement in clinical status and favorable long - term prognosis.47)48 ) more importantly , multivariate regression models were built up by including other factors at baseline such as age , gender , etiology of heart failure , severity of mitral regurgitation , presence of atrial fibrillation , and lv lead position with mechanical dyssynchrony , to demonstrate its independent or incremental predictive value in predicting crt response.49 - 53 ) functional mitral regurgitation ( fmr ) as a result of the dilation of lv cavity and/or alteration of lv chamber geometry is frequently observed in patients with chf , in particular those with lv systolic dysfunction . in a large study including more than 2000 patients with symptomatic lv systolic dysfunction and ejection fraction < 40% , fmr of any grade assessed by angiography was present in about 60% of the patients.54 ) another study investigated 1421 patients with lv ejection fraction 35% , using color doppler echocardiography , there were moderate fmr in 30% of the patients and severe in 19%.55 ) although mitral valve leaflets , papillary muscles and chordae tendineae appear normal in structure by surgical inspection or echocardiographic examination in fmr , the leaflets fail to coapt properly . fmr , as a complication of lv dilation and systolic dysfunction , can further aggravate lv volumetric overload and exacerbate left atrial ( la ) pressure and volume overload , which will set up a vicious cycle of lv remodeling . several studies revealed that the presence of fmr in heart failure was an independent predictor of worse survival.54)55 ) in addition , a dose - response relationship was observed that a 23% increased risk of death associated with the change from no fmr to mild fmr as well as the change from mild to moderate or severe fmr.54 ) the basic mechanism of fmr is believed to be the mismatch between increased mitral leaflet tethering due to the outward displacement of papillary muscles and reduced closing force caused by lv systolic dysfunction.56 ) furthermore , multiple factors are suggested to be involved in the pathogenesis of this force imbalance , including lv remodeling , leaflet tenting , annular dilation and dysfunction , as well as mechanical dyssynchrony . consequently , these changes lead to the deformation of the mitral valve apparatus and reduction in the coaptation area of the leaflets . in echocardiographic studies , first of all , lv systolic dyssynchrony reduces the efficiency of contraction , resulting in decreased closing forces which worsened reduced leaflet coaptation and increased valve tenting . secondly , uncoordinated contraction of the lv segments adjacent to the papillary muscles may increase mitral leaflet tethering and cause mal - alignment of the leaflet scallops leading to incomplete closure.57 ) soyama et al.58 ) showed in 32 patients with dilated cardiomyopathy that the presence of fmr correlated with a significant delay in mechanical activity between the lv segments supporting the lateral and medial papillary muscle , as assessed by the difference in the time to peak systolic myocardial strain . thirdly , lv mechanical dyssynchrony leads to changes in the mitral valve geometry and kinematics that may induce fmr . in animal models , a more widely opened mitral valve at end - diastole with delayed and dyssynchronous mitral valve closure was created by the rv apical pacing.59 ) the dyssynchronous contraction of the lv basal segments , attributable to the loss of mitral annular contraction , increase in systolic annular area and presence of mitral leaflet tethering , may worsen mitral regurgitation.60 ) therefore , in a cross - sectional study which prospectively enrolled 136 chf patients with lv ejection fraction < 50% and more than mild fmr , liang et al.61 ) included variables of mitral valve deformation , lv global and regional remodeling , lv contractility , mitral annular size and function , and lv mechanical dyssynchrony for multivariate logistic regression analysis . as a result , mitral valve tenting area and lv global dyssynchrony , measured by the standard deviation of the time to peak systolic velocity among the 12 lv segments ( ts - sd ) by tdi , data from multicenter crt trials revealed 13 - 50% reduction in fmr during 6- to 12-month follow up after the device therapy.56 ) intriguingly , pre - pacing mechanical dyssynchrony was found to be one major determinant of fmr reduction after crt.62)63 ) the improvement is suggested to be associated with decreased mechanical dyssynchrony,57)63 ) increased closing force,64 ) improved mitral valve deformation,62 ) and lv reverse remodeling.65 ) mechanical dyssynchrony corrected by crt would have direct impact on fmr and contribute to its improvement by interacting with several other aforementioned factors . therefore , crt would be a potential therapeutic option for selected chf patients with significant fmr when valvular surgery as a current standard treatment carries high risk.61)66 ) diastolic heart failure ( dhf ) , or called heart failure with preserved ejection fraction , is a common condition among chf population.67)68 ) in this condition , echocardiography with doppler studies currently serves as a major diagnostic tool for the differentiation between dhf and systolic heart failure ( shf).69)70 ) although it carries a significant risk of hospitalization and mortality similar to shf , our knowledge of dhf is still limited with regard to its pathogenesis , diagnosis and evidence - based management . hypertension , lv hypertrophy , diabetes and coronary artery disease have been recognized as main risk factors for developing clinically overt dhf , in which lv concentric remodeling , lv segmental wall motion abnormality , lv diastolic dysfunction and la dilatation are commonly observed indices . recently , the concept of lv mechanical dyssynchrony has also been extended to the investigation of patients with dhf as an additional factor involved in the pathogenesis . our early publication demonstrated by tdi that isolated systolic , isolated diastolic , and combined dyssynchrony were observed in 25.0% , 21.7% , and 14.1% of dhf patients , though it was less prevalent than patients with shf.13 ) the study by wang et al.12 ) in their dhf population reported a similar prevalence of systolic dyssynchrony ( 33% ) but a higher prevalence of diastolic dyssynchrony ( 58% ) . in patients with acute coronary syndrome accompanied by dhf , diastolic dyssynchrony was evident in 35% of patients and systolic dyssynchrony in 47% , while the prevalence of diastolic dyssynchrony was much higher than those without dhf.11 ) interestingly , the presence of mechanical dyssynchrony also showed a dynamic change in hypertensive dhf patients , that the prevalence of systolic dyssynchrony increased dramatically during pharmacological stress test from 36% to 85% and diastolic dyssynchrony from 38% to 87%.71 ) although mechanical dyssynchrony is frequently observed in patients with dhf , a number of questions remain unanswered with regard to its contribution to the impairment of cardiac function and clinical manifestation of heart failure , in particular the differences from shf . a wide qrs complex is very uncommon in dhf patients , therefore , the qrs duration is not a major determinant for the presence of systolic and diastolic dyssynchrony . unlike patients with shf , mechanical dyssynchrony in dhf may occur as a result of myocardial disease rather than electromechanical coupling delay . coexistence but not cause - effect relationship of cardiac dysfunction and mechanical dyssynchrony was described in previous studies , while the correlation between the two facets of lv performance differed among studies.11 - 13)71 ) therefore , apart from the severity of myocardial dysfunction , dyssynchronous lv relaxation and impairment of ventricular restoring forces may also interfere the lv filling and lead to a diastolic dyssynchrony,72 ) or vice versa . interestingly , medical therapy for dhf , including diuretics , beta - blockers , calcium - channel blockers , angiotensin - converting enzyme inhibitors and/or angiotensin - receptor blockers , was associated with shortening of diastolic intraventricular delay , which in turn correlated with improvement of lv stiffness and reduction of filling pressure.12 ) however , it remains to define what extent lv dyssynchrony is involved in the pathophysiologic mechanism of dhf . the prognostic implication of mechanical dyssynchrony was initially reported by bader et al.73 ) where 104 chf patients with ejection fraction 45% , over half of them had wide qrs complexes , were examined by the use of pulsed tdi and followed up for one year . although no mortality occurred at the end of follow up , 86 patients ( 83% ) were admitted for decompensated chf . as a result , intraventricular dyssynchrony was found to be most important independent predictor of heart failure hospitalization , and the other two independent predictors included lv ejection fraction and qrs width . in another early study of 106 chf patients with lv ejection fraction < 35% and qrs duration 120 ms who were followed up for a mean of 17 11 months , intraventricular dyssynchrony was measured by tdi as the ts - sd from both basal and middle lv segments in apical 4- and 2-chamber views . a ts - sd cutoff value of > 37 ms was associated with a significant increase in clinical event of including heart failure hospitalization or cardiac transplantation.74 ) the same group recently published their study on 167 chf patients with a mean follow up of 33 months . electrical dyssynchrony defined as the qrs duration 120 ms and mechanical delay as the septal - to - lateral wall delay 65 ms were investigated for their association with adverse events.75 ) in multivariate cox regression analysis , the septal - to - lateral wall delay [ hazard ratio ( hr ) , 2.37 ; p = 0.002 ] showed a better predictive value than qrs duration ( hr , 1.88 ; p = 0.028 ) for cardiac events . moreover , patients with both electrical and mechanical dyssynchrony had a hr of 3.98 ( p < 0.001 ) when compared with those with normal qrs duration and absence of mechanical dyssynchrony.75 ) recently , the impact of mechanical dyssynchrony on prognosis was explored in a subgroup of chf patients who had ischemic cardiomyopathy.76)77 ) in the valsartan in acute myocardial infarction ( valiant ) echocardiography study , mechanical dyssynchrony was assessed in 381 patients with ventricular dysfunction or heart failure after myocardial infarction , who were followed up for a median period of 611 days.76 ) consequently , lv dyssynchrony was independently associated with increased risk of death or heart failure hospitalization , while qrs width 120 ms which occurred in about 5% of patients failed to do so . another study consisted of 215 patients with moderate systolic heart failure undergoing coronary artery bypass graft ( cabg ) surgery , in which mechanical dyssynchrony was calculated by tdi and myocardial viability by single photon emission computed tomography.77 ) post - cabg dyssynchrony 72 ms and 5 viable segments were used to categorize patients into different groups . patients without post - cabg dyssynchrony and with viable myocardium had the least clinical events compared to those with severe post - cabg dyssynchrony and nonviable myocardium ( 3% vs. 64% ; p < 0.001 ) . in addition , qrs duration did not predict cardiac events during the median follow up period of 359 days . importantly , qrs duration was not an independent prognosticator in chf patients who did not exhibit wide qrs complexes . therefore , all of these studies have suggested that assessment of mechanical dyssynchrony is helpful to provide important prognostic value on disease outcome on top of qrs duration . mechanical dyssynchrony is common in chf patients , in particular in those with reduced ejection fraction and prolonged qrs complex . with cumulated knowledge in the advanced imaging techniques and expanded clinical applications of mechanical dyssynchrony , it appears that the assessment of mechanical dyssynchrony has a unique role in heart failure population . not only being useful in crt candidates , it can also be used to predict the development and progression of cardiac diseases , and as prognosticators . however , before the measurement of dyssynchrony is contemplated , it is imperative to receive systematic training in order to achieve high quality online image acquisition and knowledge of offline analysis . furthermore , mechanical dyssynchrony varies with many conditions . therefore , it is important to understand the right clinical context while applying knowledge of dyssynchrony : wide vs. narrow qrs complex , systolic vs. diastolic heart failure , resting vs. stress echocardiography , cause vs. effect , single vs. multiple contributors , and short- vs. long - term outcome .

mechanical dyssynchrony is a common phenomenon in patients with congestive heart failure , which usually identified by noninvasive cardiac imaging tools such as echocardiography . it demonstrates electromechanical delay in some regions of the failing heart which in turn contributes to further impairment of cardiac function . the diagnostic , therapeutic and prognostic values of mechanical dyssynchrony have been reported in a number of studies . therefore , this review describes briefly the methods of measurement , but more importantly , explains the clinical implication of its assessment in heart failure related aspects including cardiac resynchronization therapy , functional mitral regurgitation , diastolic heart failure and mortality .

the corneal scar is responsible for 8% of the 1.3 million blind persons in nigeria . most of these are end - stage scars of persisting , non - clearing , vascularized corneal edema from various forms of keratitis , including , viruses and bacteria like chlamydia , fungus , especially in rural uneducated farming population , and parasites like onchocerca volvulus that cause onchocerciasis , and physical as well as chemical agents . beyond the physical disability , pain , discomfort , and economic burden ; the disfiguring appearance of the eye causes a lot of psychological challenges , which in the traditional african setting may reduce the social values of the affected persons , especially when in the female gender . keratitis of viral nature often tends to be recurrent and persistent because of the difficulty of total eradication of the viral gene from the host - infected cells . these recurrent inflammations break down the corneal anti - neovasculogenesis mechanism with a resulting in - growth of capillaries , and in some cases , larger arterioles and feeder vessels . the normal cornea is an avascular , alymphatic , and immunologically privileged tissue in at least its very center . vascular invasion bridges this immune privilege , allowing the diffusion of immune and allergic mediators , including cytokines , immunoglobulins , vasoactive proteins , and lipids , as well as , cellular infiltrates into the cornea . these mediators are capable of inducing increased vascular permeability in the normal vessels and inducing the growth of abnormal leaky vessels , with resulting edema in the surrounding corneal tissues . the resultant effect of all of these changes is loss of corneal clarity , pain and redness in the short run , as well as lipid and other forms of corneal degeneration , scarring , calcification , and finally functional visual loss in the long run . modalities that can rapidly stop the process of corneal edema will therefore be invaluable in preventing visual morbidity and blindness , with its consequent social and economic impact . a 26-year - old male , unemployed person , presented to the ophthalmology clinic on 14 august , 2012 with a three - week history of blurring , soreness , lacrimation , redness , and pain in the left eye . he had three similar attacks in the last three months , for which he was hospitalized . he had lid edema , cilliary injection , a severely hazy central disciform corneal stromal edema on an old corneal scar , with lipid corneal degeneration , and multiple nummular keratitis at around the 1 oclock position of the left cornea . the anterior chamber of the left eye did not show any cell or flare reaction . the red reflex was poor due to the corneal haze , with no visible details of the fundus . a diagnosis of secondary viral disciform and nummular keratitis was made and patient was put on oral and topical ointment of acyclovir together with a stat dose of cyclopentolate and antibiotics . he was to be followed up in a week by which time a steroid was to be included in his therapy . the patient did not show up until six weeks later when his visual acuity had deteriorated to counting finger in the left eye . he complied with his last medical prescription for only the last two weeks to the date of his presentation . a topical steroid was added to his other medications and he was to be reviewed in another one week . at the scheduled appointment , he was subjectively fine and comfortable , his vision had improved to 6/24 in the left eye and the conjunctiva injection had subsided ; stromal edema had subsided in the superior cornea , and the nummular keratitis had disappeared . he had a prominent feeder vessel into an area of the thick corneal scar in the inferior aspect of the left cornea [ figure 1 ] , with surrounding non - clearing , corneal edema and lipid keratopathy in the inferior - nasal quadrant bordering on the visual axis [ figure 2 ] . he had a deep anterior chamber , but the inferior iris and fundal details were not visible . image of edematous scarred vascularized left cornea image of edematous scarred left cornea the relative quietness of the non - vascularized superior portion of the left cornea contrasted with the active edematous cornea area bordering on the feeder vessel in the inferior portion of the left cornea . it was , therefore , hypothesized that this could be explained by vascular leakage and the inflammatory mediators from the supplying feeder vessel into the adjacent cornea area . after the patient gave informed consent , he was put under a slit lamp microscope . the upper eyelid was manually retracted by the examiners gloved fingers after topical anesthesia with amethocaine . the cautery ballpoint was heated in a spirit lamp and the tip applied to about a 4 mm length of the sclera portion of the feeder vessel , to cauterize and separate the feeder vessel from the scarred and edematous portion of the inferonasal left cornea . the procedure lasted for about four minutes without any complication and was well - tolerated by the patient . the procedure was imaged pre- and post - intervention and the patient was given a stat dose of oral ibuprofen image of the left eye , immediately post intervention five days post intervention , the patient felt well with no more pain and his vision had improved . on examination , visual acuity od remained 6/9 and os there were conjunctival petechial hemorrhages in the area of the cautery and the intra - cornea feeder vessel had regressed [ figure 4 ] . on slit lamp examination , the corneal clearity had improved enough to allow easy visualization of the details of the inferior iris and a normal fundus , with c : d = 0.3 , a pink disc , normal macula , and a peripheral normal pink flat retina . image five days post intervention this patient had achieved rapid corneal edema reversal with significant functional improvement and has remained so , via phone conversation , a month later . this patient had progressive worsening of visual acuity from 6/24 to cf in the left eye despite two weeks usage of antiviral ointment of acyclovir , but had a reversal of visual deterioration from cf to 6/24 in the left eye upon one week 's institution of steroid . this evidence supports the dependency of the pathology and its functional performance on immune mechanism , and therefore , the vessel that brings the mediators as a prospective target for immune modulation . the concept of cauterization , embolization , and new vessel regression in control of abnormal blood flow , infective keratitis , enhanced corneal graft tolerance , control of a lipid keratopathy , and even metaplasia , is not new , and numerous agents have been produced to achieve these purposes . some of these include fine needle diathermy , angiosclerotic agents , obliterative radiotherapy , laser therapy , thrombotic sponge , particles and microspheres , and in the eye , anti - vascular endothelial growth factor and steroids . these vessels are usually microvessels that supply inaccessible parts of the body as opposed to prominent macrovessels ( pre - capillary arterioles ) seen in an accessible part of the cornea in this patient . the size and location of the feeder vessel offered an opportunity for a cost - effective and feasible direct localized surgical cauterizing intervention , with wider diffuse corneal impact well away from the site of the burn . following medical intervention with antiviral , antibiotic , and steroid , this patient showed a differential regression of tissue pathology , edema and functional improvement in the vascularized and non - vascularized portion of the cornea , with the vascularized portion lagging behind in the regression of cornea edema and transparency . the rapid thinning out of the vascularized edematous cornea , assessed by the slit lamp beam , was well and beyond the change seen in the non - vascularized portion , coupled with a complete reversal of the relative thickness of the cornea in the vascularized and non - vascularized portions of the cornea following intervention gives a clue to the cause although the patient had his first post intervention review in five days , the effect of edema regression was probably earlier , but was still adjudged rapid . macrovascular cauterization is affordable , requires no expensive technology , and is feasible even in remote economically poor - resourced regions , as only magnification , a spirit lamp , and a cautery point were required . episcleral and limbal cauterization is a routine procedure in cataract surgery and has not been associated with any significant adverse effect , when correctly applied . this method of cauterization will , however , be more difficult to apply where the panni are multiple , microvascular , and more diffuse . cornea vascularization with edema is an important theme in many important corneal diseases ; therapy can , therefore , achieve a wider application . this case study has demonstrated the accelerated regression of corneal edema and functional corneal performance following cauterization of a cornea feeder vessel . it is a safe , feasible , affordable , and accessible intervention to consider in difficult non - clearing corneal edema of selected patients . we encourage further research on this , to confirm the efficacy of this intervention , as well as researches to develop the applicable intervention of a similar nature for diffuse micropannus diseases of the cornea .

this is a case report of a symptomatic non - clearing , vascularized , disciform , corneal stromal edema with a feeder vessel that has remained refractory to medical therapy of antiviral , steroid , and antibiotics , for a period of three weeks , but showed a rapid improvement in visual acuity of 0.1 log mar within five days of feeder vessel cauterization , together with improvement of two psychometric scales in corneal cloudiness on a scale range of 0 to 3 , clinical evidence of resolution of corneal edema , and subjective resolution of the patient 's symptoms . cauterization was done under magnification with a ball cautery point warmed in a spirit lamp following topical anesthesia . this intervention may become handy in difficult non - clearing corneal edema and prevent blinding consequences , in a low - resource facility , in selected applicable cases .

traditional approaches to drug development and clinical trials based on the premise of one size fits all are becoming less cost - effective and suboptimal clinically.1 this is due mostly to the escalating costs required to develop a new drug , diminishing returns on drug investments , and a high rate of failure for phase iii clinical trials , particularly in oncology drug development.2 as a result , the number of new drugs approved by the us food and drug administration ( fda ) has remained relatively flat and the number of new drug submissions has decreased significantly over the last decade.3 the commitment of the fda to drive innovation in drug development through its critical path initiative appears to have contributed to a modest uptick in new drug approvals in the last several years . in line with the fda initiative to improve the efficiency of drug development and the success rates of late - phase studies , drug sponsors require methodologies that can shorten the length of clinical trial cycles , reduce the number of human subjects required , and provide more reliable early clinical phase assessments for go / no go trial decisions . many sponsor companies are using companion diagnostic assays and diagnostic imaging studies to help streamline the clinical trial process . companion diagnostic assays ( also referred to in the literature as pharmacodiagnostics or theranostics ) provide a test that can identify the presence or absence of a biomarker that is predictive of a patient s phenotype . this approach relies on a detailed understanding of the molecular basis of disease in an individual patient that can subsequently be used to follow - up with a tailored course of treatment based on the presence of specific disease biomarkers . different classes of biomarkers include somatic mutations , polymorphisms , and gene / protein expression profiles that are associated with a particular disease state . in addition to identifying patients likely to respond to a personalized treatment approach , the incorporation of a diagnostic imaging technique or a diagnostic imaging study in clinical trials allows clinicians and scientists to non - invasively assess the presence , location , and extent of disease for objective , quantitative monitoring of disease progression and response to treatments . throughout the clinical trial process , the ability to detect and visualize patient biomarkers using companion diagnostic assays and diagnostic imaging tools provides clinicians and drug developers with tools that facilitate faster , safer , and more efficient clinical trials ( figure 1 ) . early on , they can be used to determine and optimize trial eligibility and enrollment by confirming the presence and quantity of a drug target in an individual patient . during a clinical trial , companion diagnostic assays and diagnostic imaging can be used to monitor and improve treatment responses and patient outcomes by identifying and predicting patient sub - populations that are most likely to respond to a given treatment . diagnostic approaches not only indicate the presence of a molecular target , but can also inform the off - target effects of a therapeutic , providing increased predictive power for toxicity and adverse effects associated with a drug . finally , companion diagnostics and diagnostic imaging can inform whether a treatment is reaching its target , providing drug sponsors with an alternative to strict titration studies for determining optimal dosing . taken together , these approaches are providing new avenues for identifying appropriate patient cohorts for inclusion in a study , monitoring disease , and assessing drug efficacy in individual patients , all of which contribute to potential economic benefits for drug sponsors . as an example , comparative data from drugs approved for the treatment of non - small cell lung cancer ( xalkori , pfizer , inc . , new york , ny , usa ; and tarceva , osi pharmaceuticals , inc . , melville , new york , usa and genentech , inc . , south san francisco , ca , usa ) illustrate the financial benefit of incorporating a companion diagnostic early in the course of drug development . xalkori , which was codeveloped with a companion diagnostic ( vysis alk break apart fluorescence in situ hybridization probe , abbott laboratories , abbott park , il , usa ) required approximately threefold fewer patients in clinical trials ( 960 compared with 3,110 ) , showed an approximately threefold reduction in time from phase i to approval ( 1.8 years compared with 5.3 years ) , and had an overall reduced relative development cost per patient ( 100% compared with 154%).4 furthermore , the addition of epidermal growth factor receptor ( egfr ) testing ( hoffman - la roche ltd . , basel , switzerland ) as a companion diagnostic for tarceva in 2013 helped advance the drug to a first - line treatment in a select population and resulted in positive growth forecasts for a drug already on the market for over 8 years.4 the importance of diagnostic approaches in drug development is highlighted by the growing global cancer diagnostics market which is expected to reach an estimated value of usd 168.6 billion by the year 2020.5 many newer drugs are being codeveloped with a companion diagnostic assay or imaging diagnostic , sparking the growth of more than 125 companies that provide companion diagnostic products and services.6 additionally , nearly two - thirds of breakthrough therapy designations recently granted by the fda include a companion diagnostic.7 at present , much of the activity in companion diagnostics development is focused in the area of oncology . there are currently 23 companion diagnostics approved by fda , 22 of which are approved in oncology.8 the first companion diagnostic , a her2 immunohistochemistry assay , was developed in the late 1990s for use with trastuzumab ( herceptin ) , a monoclonal antibody approved for the treatment of breast cancer . since then , companion diagnostics have been developed for many targeted oncology therapeutics , including tarceva , iressa , erbitux , and vectibix ( table 1).9 although companion diagnostic assays continue to improve personalized medicine , there are a number of significant limitations in current diagnostic assay approaches . specifically , a positive signal generally informs the treating clinician or investigator that a target biomarker is present and , with quantitative assays , to what extent it is present in individual patients . however , the majority of approved diagnostic assays supply very little , if any , information regarding the location and distribution of a target biomarker . in oncology clinical trials , specific knowledge of a target lesion location can be essential , providing accurate biopsy localization and helping to design a treatment plan for tumors involving critical organs ( eg , liver , lung , or bone marrow ) . another limitation of using companion diagnostics is assay sensitivity ( ie , the ability to detect true positives ) . yet another limitation of companion diagnostic assays is the relatively narrow scope of biomarker evaluation . research in the last several years has demonstrated that detection of a therapeutic target is not sufficient to predict drug efficacy and needs to be supplemented by additional data to assess for potential resistance . for example , the presence of kras mutations in colorectal cancers expressing egfr often leads to resistance to anti - egfr therapy.10 lastly , companion diagnostic assays may require large amounts of tissue samples for the evaluation of multiple biomarkers . this is especially challenging for certain solid tumors where tissue samples may be limited . in such instances , objective assessment by other diagnostic methods is essential for effective use of a companion diagnostic assay . in clinical oncology studies , diagnostic imaging helps overcome these limitations by providing a reliable methodology to assess the presence , location , and extent of disease in response to treatment . for many years , computed tomography ( ct ) and magnetic resonance imaging ( mri ) have been primary diagnostic imaging tools used for oncology disease assessments . as the use of diagnostic imaging techniques became widespread in clinical trials , a set of standardized imaging assessment criteria from the world health organization were established.11 in the year 2000 , a modified set of criteria called the response evaluation criteria in solid tumors ( recist 1.0 ) was introduced as part of collaborative efforts between the european organization for research and treatment of cancer , the national cancer institute in the usa , and the national cancer institute of canada clinical trials group.12 recist refined an objective set of criteria that defined when tumor lesions in cancer patients improve ( partial or complete response ) , remain unchanged ( stable disease ) , or worsen ( progressive disease ) during treatment . since its introduction , recist has been updated ( recist 1.1 ) to introduce standards for the assessment of lymph nodes , redefine measurable lesions and assessment of disease progression , as well as establish recommendations for standardized image acquisition.13 today , a large number of oncology clinical trials employ recist to objectively assess cancer treatment response in solid tumors . advances in imaging technologies and our understanding of disease have resulted in additional consortia guidelines for standardizing diagnostic imaging in oncology clinical trials . most notably , the cheson criteria ( 1999 , 2007 , and 2014 ) have established guidelines for the use of diagnostic imaging using ct , mri , and fluorodeoxyglucose ( fdg)-positron emission tomography ( pet ) as well as clinical findings for the assessment of lymphoma patients . in addition , the rano criteria have been established for gliomas , and a number of other criteria have been introduced to specifically assess hepatocellular carcinoma , acute myeloid leukemia , prostate cancer , and the effects of immunotherapies on tumor responses.1419 as these criteria have evolved , it has become clear that conventional anatomical imaging techniques , although very useful , have not supplied all of the objective assessments needed to make accurate early phase go / no go decisions . initiatives by the radiological society of north america , including the quantitative imaging biomarker alliance , to advance volumetric assessments of tumor lesions continue to gain momentum , and researchers are showing increased interest in developing tools for the evaluation of metrics derived from ct and mri studies . as an example , techniques such as dual energy ct and spectral ct imaging are being used to better differentiate and characterize certain cancers . these types of image analysis in conjunction with efforts to assess the relationship of ct and mri to the molecular biology of various tumors , is helping to shape the new fields of radiomics and radiogenomics . although these approaches hold great potential for oncology clinical trials , it is likely to be several years or more before they can be implemented in a clinical environment . the field of molecular imaging is rapidly evolving with many different technologies in various stages of development . at present , nuclear imaging techniques , including planar , single photon emission computed tomography ( spect ) , and pet remain the dominant approach for the diagnosis and treatment of cancers . pet and spect imaging requires the use of a radiotracer that is injected into a patient prior to interrogating its spatial distribution . pet relies on the detection of gamma photon pairs resulting from the annihilation of positrons ( annihilation radiation ) originating from a biologically active radiotracer . using specialized detectors that encircle a patient , ( ie , ring scanners ) two - dimensional or three - dimensional images of radioactivity distribution within the body similarly , spect requires a radiotracer , typically a heavy isotope , and relies on the detection of single gamma photons emitted directly from the radiotracer . since spect tracers can be imaged at the time of injection , they can be used to detect changes in blood flow to various organs in a variety of disease states . spect tracers can also be linked to different biochemical analogs and antibodies to detect tissue specific distribution of cellular targets . one of the first imaging diagnostic agents used in endocrinology and oncology studies was radiolabeled sodium iodide ( nai ) . this compound has been used effectively to identify individuals with hyperthyroidism , monitor residual thyroid tissue post - surgery , and as a follow - up in treatment for thyroid cancer metastesis . clinicians routinely rely on other nuclear medicine techniques to identify appropriate patient cohorts likely to respond to treatment and to monitor treatment responses for various cancers . for example , the use of tc - labeled methylene diphosphonate and f - labeled naf in bone scans to assay for bone metastasis in breast and prostate cancer patients , the use of in - labeled anti - cd20 antibodies for imaging lymphoid malignancies , and the investigational use of i , tc , and f - labeled prostate - specific membrane antigen ( psma ) for monitoring prostate cancer patients ( table 2 ) . the ability to probe for molecular targets in cancer patients has opened the door to better , more accurate assessment of disease . molecular imaging using various pet tracers provides enhanced visualization of tumors , their metabolic activity , and other biological phenotypes ( eg , proliferation , hypoxia , expression of target receptors ) . furthermore , widespread application of non - invasive imaging like pet / spect and gamma scintigraphy , enables the use of many additional tracers in oncology clinical trials ( table 2 ) . f - fdg and f - flt are used to monitor glycolytic activity and proliferation of tumors , respectively , and technetium tc - labeled antibody and peptide compounds are routinely used to label tumors and diagnose sites of cancer . the elucidation and validation of novel oncology targets using high throughput screens is opening the door to development of potent and selective antibodies and other molecules capable of targeting tumor - specific or tumor - enriched receptors.20 these agents can be linked to radionuclides , fluorophores , or other imaging probes ( eg , in , tc ) to confirm the presence and anatomical location of cellular targets or developed as cytotoxic therapeutics ( eg , y , i ) . several well studied proteins serve as oncology targets for imaging diagnostics including psma , the estrogen receptor ( er ) , and the folate receptor ( table 3 ) . psma is a protein amplified on the surface of nearly all prostate cancer cells and is a validated target for the detection of primary and metastatic prostate cancer . radiolabeled small molecules targeting psma are well tolerated tools for the detection of metastatic prostate cancer . a number of academic centers and pharmaceutical companies are developing and testing molecules labeled with f , tc , and i that specifically target psma . molecules capable of targeting er are proving to be extremely valuable for improving breast cancer treatment . several studies have shown that 16-f - fluoro-17-estradiol ( f - fes ) , an er - specific pet tracer , can reliably detect er - positive tumor lesions and that its uptake correlates well with immunohistochemical scoring for the presence of er.2123 currently , f - fes is being evaluated in breast cancer patients to determine patient er status , to help differentiate between benign and malignant lesions , and to differentiate between metastases originating from different tumor types.24 similarly , the folate receptor is overexpressed in many cancer tissues and represents a target for selectively imaging and delivering therapeutics to cancer cells . a companion imaging diagnostic ( tc - labeled folate - targeted molecule ) has already been developed to identify tumors that overexpress the folate receptor , and clinical data have shown that patients with metastases that are positive for the folate receptor benefit from treatment with the corresponding folate - targeted small molecule drug conjugate.25,26 in addition to showcasing the importance of targeted imaging agents for oncology treatment , this approach provides a paradigm for the codevelopment of an imaging diagnostic and a therapeutic agent and may contribute to the design of more efficient drug development workflows in the future . the emergence and development of new spect and pet radiopharmaceuticals , and the use of hybrid imaging modalities such as spect / ct , pet / ct , or pet / mri ( which combine anatomic imaging with physiological imaging in a single device and enable the acquisition of coregistered anatomical and physiological scans ) provide a more complete picture of a patient s disease state . in this respect , the use of molecular imaging is helping to modernize recommendations for the evaluation , staging , and response assessments of cancer patients . as an example , the cheson criteria was recently revised to require f - fdg assessment as the dominant imaging technique for evaluation of fdg - avid lymphomas.27 significant improvements in hardware for pet and spect imaging , primarily driven by the oncology market , continue to advance widespread application of molecular imaging . modern day pet and spect scanners increasingly are using newer crystal detector materials and solid state photon detectors that are smaller in size , provide increased sensitivity , and have better spatial resolution . new collimator designs and specialized gantries additionally , newer image reconstruction techniques and software incorporate iterative reconstruction , time - of - flight data , and resolution recovery , which results in improved image contrast , image resolution , and reduce image noise.28 the value of molecular imaging approaches for drug development has also led to the design and widespread integration of pet and spect techniques in preclinical animal models of disease . small animal micro - pet and micro - spect imaging systems ( as well as small - scale anatomical and hybrid imaging systems ) are commercially available and are being used in the early drug discovery process to monitor drug toxicity and efficacy in efforts to advance the most promising oncology candidate drugs to human clinical trials.29 quantitative approaches using molecular imaging are extremely valuable as they provide insight as to what is occurring at the cellular level and are often predictive of a tumor response before anatomical changes can be observed . the introduction , validation , and use of quantitative molecular imaging continues to drive and optimize the field of imaging diagnostics . in addition to identifying the presence , location , and distribution of a specific tumor biomarker , radiopharmaceuticals can be used to objectively obtain quantitative measurements , including region of interest assessments of single or multiple areas . most clinical trials that use molecular imaging rely on relative or semiquantitative approaches , since absolute quantitation methods using radionuclides are very complex and impractical for routine clinical studies . a common measurement used in molecular imaging for assessing treatment responses is the standardized uptake value ( suv ) . the suv represents the ratio of the concentration of radioactivity in a selected region to the total injected dose of radioactivity distributed evenly throughout a patient s body . suv measurements can be calculated as a mean value ( suvmean ) or as a maximum value ( suvmax ) , and can be further normalized to a patient s lean body mass or whole body mass . additional treatment response information can be gained by quantitative assessment of the changing pattern of uptake at multiple different time points ( figure 2 ) . other quantitative measurements used in clinical trials include glycolytic index determination , which is a measure of the total metabolic activity of a specific targeted area ( eg , target tumor lesion ) and the standardized uptake peak value ( suvpeak ) , currently used with the positron emission tomography response criteria in solid tumors ( percist).30 for longitudinal studies , dynamic measurements acquired over time can be used to generate time / activity curves . in particular , dynamic time / activity curve analysis is useful for assessing diagnostic probes , including their ability to localize in a specific tumor site and duration or residence time in a target site . this type of data can be very useful for determining optimal dosing using the therapeutic equivalent of an imaging companion diagnostic . although molecular imaging offers a wide spectrum of applications in drug development and clinical trials , there is only a single imaging companion diagnostic approved by the fda , called ferriscan . ferriscan uses mri to select patients and manage therapy for non - transfusion - dependent thalassemia . the lack of additional fda - approved imaging companion diagnostics highlights the opportunity and need for additional agents to be adapted , tested , and validated as diagnostic assays . in order for a molecular imaging test to become an integral part of any clinical trial investigation , the specific molecular imaging study has to be validated in prior investigations as an integrated component of a prospective analysis where it is not utilized to direct treatment decisions . upon validation , a molecular imaging test can be used as an integral component of clinical trials and may even be required by regulatory agencies in clinical cases prior to and/or following drug administration.31 as an example , fdg - pet imaging is being used in lymphoma clinical trials in conjunction with the lugano criteria.26 given their pivotal role in clinical trials , it is likely that we will see an increase in the number of imaging companion diagnostics and integrated molecular imaging studies for oncology clinical trials . even in cases where an imaging companion diagnostic is not incorporated into a clinical trial paradigm , it is important to recognize that the combination of a companion diagnostic assay with the appropriate imaging diagnostic can supply complementary information that can not be ascertained from either methodology alone . the importance of companion diagnostics for current and future pharmacotherapy has attracted the attention of global regulatory agencies . for new therapies requiring the use of a diagnostic to qualify patient populations , companion diagnostics must meet typical design control and submission requirements to ensure safety and efficacy . as a result , regulatory agencies are increasing their visibility and offering more structured platforms for diagnostic companies to interact with them . the fda has taken significant steps in the last decade to define the companion diagnostic pathway . the fda has published a drug - diagnostic codevelopment concept paper and created a personalized medicine group within the office of in vitro diagnostics and radiological health . last year , the fda released a formal guidance called in vitro companion diagnostic devices , indicating regulatory pathways and requirements for companion diagnostic devices and therapeutic products.32 the guidance defines in vitro companion diagnostic devices ( ivds ) , informs industry and fda staff on premarket regulatory pathways and enforcement policies , and describes regulatory approval requirements relevant to therapeutic product labeling . in the case of clinical trials , where companion diagnostic assays are used to inform treatments , there are stringent requirements for submission of an investigational device exemption , usually as part of an investigational new drug application ( ind ) . given that the majority of companion diagnostic assays are considered high - risk devices ( class iii ) , there is also a requirement for a premarket approval application . although there are a number of therapeutic drugs that require companion diagnostic testing in the eu , the european medicines agency has been less transparent regarding companion diagnostics . the ivd directive 98/79/ec regulates in vitro diagnostic medical devices in the eu and ivd devices require a ce mark to indicate compliance . currently , any companion diagnostic entering the eu market is classified as a low - risk device based on ce marking by the manufacturer ( self - certification ) . this results in a major divergence in the approval process between the usa and the eu . there are major changes underway in ivd legislation , including regulation that will provide a single regulatory framework for all eu member states . under a new draft guidance which entered parliament last year , companion diagnostics will be assigned as class c devices , requiring design examination certification by a notified body . the process of achieving regulatory approval for new diagnostic imaging agents also remains extremely challenging , highlighted by the fact that only a handful of new radiotracers have received fda approval in the last decade.33 although radiotracers are typically administered at doses that are orders of magnitude lower than therapeutics and are designed to measure molecular processes rather than modify them , they are regulated as though they carry the equivalent risk of a therapeutic . in fact , the commercial development of a new imaging agent shares many of the same challenges as therapeutic drug development , including target validation , lead selection , establishing high affinity and uptake , achieving adequate clearance , and demonstrating low toxicity.2 the fda has issued a three - part guidance in 2004 surrounding the regulatory pathway to the commercialization of new imaging agents , which covers safety assessments , clinical indications , and the design , analysis , and interpretation of clinical studies.3436 in an effort to facilitate the regulatory process for imaging diagnostics , the fda has established an exploratory ind for therapeutics and diagnostics , which provides an early look at the distribution and metabolism of new tracers in a small number of patients using early human screening and microdosing experiments . imaging tracers that show promising results can proceed through traditional clinical trial phases and the filing of a formal ind . the exploratory ind process covers safety and efficacy for measuring a molecular process , but falls short in providing approval for larger clinical trials . to help overcome this , the society of nuclear medicine has put forth a two - step approval process ( safety and efficacy in measuring a molecular process and clinical utility and efficacy ) specifically for diagnostic imaging agents.37 more recently , the society of nuclear medicine created the molecular imaging clinical trials network with the use of centralized inds for non - proprietary radiolabeled tracers to facilitate access to investigational molecular imaging radio - pharmaceuticals for clinical trials . the european medicines agency has also issued a formal guidance document that outlines the qualification process for biomarker development ( emea / chmp / sawp/72894/2008).38 this guidance , updated in 2014 , outlines the scientific pathway leading to either a committee for medicinal products for human use qualification opinion or qualification advice on innovative methods or drug development tools . in vitro companion diagnostic assays and in vivo molecular diagnostic imaging continue to advance the field of personalized medicine and are changing the way in which clinicians are treating cancer and other human diseases . assays and imaging agents are being developed alongside therapeutics to stratify patients and maximize the potential treatment benefit of new oncology therapeutics . these approaches are not only changing the landscape of clinical trials , but are also contributing to important changes in drug development and treatment . with the discovery of new oncology targets and imaging tracers comes increased capabilities to probe , monitor , and evaluate cancer on a molecular level . it is clear that more widespread implementation of imaging diagnostic tools will advance oncology clinical trials and help support new drug approvals in this rapidly expanding therapeutic area .

in the era of personalized medicine , diagnostic approaches are helping pharmaceutical and biotechnology sponsors streamline the clinical trial process . molecular assays and diagnostic imaging are routinely being used to stratify patients for treatment , monitor disease , and provide reliable early clinical phase assessments . the importance of diagnostic approaches in drug development is highlighted by the rapidly expanding global cancer diagnostics market and the emergent attention of regulatory agencies worldwide , who are beginning to offer more structured platforms and guidance for this area . in this paper , we highlight the key benefits of using companion diagnostics and diagnostic imaging with a focus on oncology clinical trials . nuclear imaging using widely available radiopharmaceuticals in conjunction with molecular imaging of oncology targets has opened the door to more accurate disease assessment and the modernization of standard criteria for the evaluation , staging , and treatment responses of cancer patients . furthermore , the introduction and validation of quantitative molecular imaging continues to drive and optimize the field of oncology diagnostics . given their pivotal role in disease assessment and treatment , the validation and commercialization of diagnostic tools will continue to advance oncology clinical trials , support new oncology drugs , and promote better patient outcomes .

abdominal aortic aneurysms ( aaas ) usually occur naturally in the infrarenal part in the human abdominal aorta . in men aged 6580 years , the prevalence of aaas is between 4% and 8% and approximately six times greater in men than women [ 1 , 2 ] . an aaa is a permanent localized dilatation of the abdominal aorta ( beginning at the level of the diaphragm and extending to its bifurcation into the left and right common iliac arteries in human ) that exceeds the normal diameter by 50% , or > 3 cm . the primary risk factors of aaas include family history , smoking , increasing age , male gender , central obesity , and low hdl - cholesterol levels [ 2 , 4 ] . hypertension ( systolic bp > 160 mmhg , diastolic bp > 95 mmhg ) is associated with the aaa risk , but only in women . diabetes , a well - defined risk factor for atherosclerosis , has been shown to be protective against the aaas [ 68 ] . historically , the aaas have been considered as a focal manifestation of the advanced atherosclerosis . however , this conventional theory has been challenged by recent evidences : an aaa was a local representation of a systemic disease of the vasculature . there was a lower incidence of aaas in the individuals suffering from diabetes mellitus that ordinarily considered as the risk equivalent of atherosclerosis [ 68 ] . the inflammatory cells were recruited into the different sites : the outer media and adventitia of aneurysma , and the intima and subendothelium of atheroma [ 1113 ] . three key processes contribute to the aaa phenotype : inflammation , proteolysis , and smooth muscle cell ( smc ) apoptosis . on the basis of the loss of extracellular matrix especially elastin and accumulation of proteolytic enzymes in the aneurysmal tissues , proteolysis has been regarded as the critical pathogenesis of aaas . the extracellular matrix degradation by both predominant proteolytic enzymes mmp-2 and -9 , which synthesized and released mainly by the vascular smcs and infiltrating inflammatory cells such as macrophages , contribute to the anoikis of vascular smcs . the vascular smc apoptosis is another critical pathogenesis of aaas . it has been demonstrated that the decreasing number of the medial vascular smcs in the vascular wall from the aaas patients was relevant to apoptosis [ 1521 ] . degradation of elastin and apoptotic cell death of the medial vascular smcs destroys the aortic wall integrality , weaken the wall tensile strength , consequently facilitate the development of aaas . the inflammatory responses in vascular wall play a pivotal role in the mmps expression and vascular smc apoptosis . conversely , the apoptosis and antigen exposure as a result of the extracellular matrix degradation are also likely to contribute to the immune and/or inflammatory responses . the mechanism underlying the inflammatory responses in the outer media and adventitia of the vascular wall remains to be well defined . recent researches have shown that the increased angiogenesis in all layers of the aneurysmal wall is associated with inflammatory responses and related to aneurysmal rupture [ 2225 ] . that cigarette mainstream smoke enhanced the aaa formation in ang ii - treated apolipoprotein e - deficient mice as a result of the increased proteolytic activity of mmps . nicotine , a major alkaloid in tobacco leaves and a primary component in cigarette smoke , plays its pathophysiological roles partly through its receptor nicotinic acetylcholine receptors ( nachrs ) . in this paper , we will mainly discuss the pathogenesis of aaas involving inflammation , proteolysis , vascular smc apoptosis and angiogenesis , and the roles of nicotine and nachrs.we made the highlighted change according to the list of references . nicotine is a principal tobacco alkaloid occurring to the extent of about 1.5% by weight in commercial cigarette tobacco and comprising about 95% of the total alkaloid content . the nicotine in tobacco is largely the levorotary ( s)-nicotine , only 0.1 to 0.6% of total nicotine content is dextro - rotatory ( r)-nicotine . there are two major types of cholinergic receptors : the muscarinic and the nicotinic . the endogenous ligand , acetylcholine stimulates both receptor types , while the exogenous one , nicotine , preferentially stimulates nachrs . the nachrs were firstly identified in excitable cells , but later were identified in many other cell types including vascular and immune / inflammatory cells . there are 17 distinct isoforms ( 110 , 14 , , , and ) of the subunits , which form homomeric or heteromeric channels . among the subtypes , the muscle - type nachr1 , the five polypeptide subunits ( 1 , 1 , , and in a 2 : 1 : 1 : 1 ratio ) , and the homomeric cns - type , 7-nachrs , have been identified in a variety of non - neuromuscular cell types such as vascular ecs , vascular smcs , smooth muscle specific -actin positive myofibroblasts , t lymphocytes , and macrophages [ 2832 ] . muscle - type nachrs 1 and homomeric cns - type 7 nachrs had participated in the pathological processes of atherosclerosis and angiogenesis . inflammation plays a pivotal role in the formation and progression of aaas and aneurysm rupture [ 34 , 35 ] . the inflammatory cells including t , b lymphocytes , neutrophils , macrophages , and mcs mostly are recruited into the outer media and adventitia of the aneurysmal wall [ 13 , 22 , 24 , 36 , 37 ] . police et al . have demonstrated that the increased number of macrophages in periaortic adipose tissue surrounding the abdominal aortas of ang ii - infused obese mice was associated with the enhanced aaa formation . the inflammatory cells release not only photolytic enzymes to degrade elastin and other matrix proteins , but also inflammatory and chemotactic factors to recruit more inflammatory cells and stimulate the vascular smc synthetic phenotype by autocrine / paracrine . in previous studies , t lymphocytes are not indispensable in the aaas induced by ang ii in apolipoprotein e - deficient male mice , although which play a dominant role in atherosclerosis [ 39 , 40 ] . recently , the role of mcs in the aaa development has also been paid more attention by scientists . the specific granule contents from mcs are very important for the inflammatory cell recruitment , pro - mmp and renin - angiotensin system activation , angiogenesis , and vascular smc apoptosis [ 36 , 41 ] . it has been shown by few studies that nicotine played a proinflammatory role in vasculature in vivo and in vitro . two in vitro experiments have demonstrated that nicotine promoted the vcam-1 and icam-1 expression on human coronary artery endothelial cells and human umbilical vein endothelial cells [ 42 , 43 ] . in another study , chronic ( during 90 days ) nicotine exposure enhanced the production of pro - inflammatory cytokines such as tnf , interleukin 1 ( il-1 ) by macrophages and upregulates the mrna expression level of vcam-1 , cyclooxygenase-2 ( cox-2 ) , and platelet - derived growth factor ( pdgf- ) in the aortas from low - density lipoprotein receptor - deficient mice . it has been well known that vcam-1 and icam-1 were the key mediators of the inflammatory cell migration and infiltration into vascular wall . nevertheless , more evidences have demonstrated the anti - inflammatory role of nicotine via nachrs , that is , the so - called cholinergic anti - inflammatory pathway [ 4648 ] . if a hypothesis that nicotine can stimulate formation and progression of aaas through inflammation is true , is it the best explanation that the prolonged exposure to nicotine may induce desensitization and changes in the expression of nachrs and thus the beneficial effects of nicotine through its receptors may be halted ? it must be conceded that the aaas were usually detected in the older people with a longer smoking history . moreover , it has been indicated by some investigations that the inflammatory mediators including cox-2 and 5-lipoxygenase ( 5-lo ) were also associated with the development of aaas . cox-2 , a limiting enzyme converting arachidonic acidinto prostaglandin , plays an important role in the inflammatory diseases . in human aaas , the increased expression of cox-2 king et al . demonstrated the increased expression of cox-2 and the upregulated synthesis of pge2 selectively in the aortic aneurismal tissues by exposure to ang ii . the selective cox-2 inhibitor , celecoxib , decreased the incidence and severity of ang ii - induced aaas in apolipoprotein e - deficient mice and c57bl/6j mice . the above studies indicate that the increased cox-2 expression is one of the pathogenesis of aaa formation . it has been implied by limited studies that nicotine could stimulate the cox-2 expression likely through nachrs . in human umbilical vein endothelial cells , nicotine increases the cox-2 , icam-1 , and pge2 expression through nf - kappab activation which mediated by nachrs . in gastric cancer , nicotine stimulates the cox-2 expression to trigger tumor cell invasion and angiogenesis through the vegf activation , which subsequently modulates the mmp activity and plasminogen activators expression . activation of the 5-lo pathway contributes to the biosynthesis of proinflammatory leukotriene mediators in macrophages , mcs , and other inflammatory cells . 5-lo plays a role in promoting the aaa formation induced by an atherosclerotic diet in apolipoprotein e - deficient mice . 5-lo - positive macrophages localize in the adventitia of the diseased mouse and human arteries in the areas of neovascularization and constitute a major component of the aortic aneurysms . 5-lo deficiency attenuates the aortic aneurysms and reduces the aortic mmp-2 activity and diminished plasma macrophage inflammatory protein-1 ( mip-1 ) . it has been recently shown that the mrna levels for the three key enzymes / proteins in the biosynthesis of cysteinyl - leukotrienes , 5-lo , 5-lo - activating protein ( flap ) , and ltc4 synthase ( ltc4s ) , were significantly increased in the aneurysmal wall from the human abdominal aortas . 5-lo , flap , and ltc4s proteins express in the media and adventitia and localize in the areas rich in inflammatory cells including macrophages , neutrophils , and mcs . have similarly demonstrated that , in the aneurysmal wall of the human abdominal aortas , the leukotriene pathway mainly localized in the macrophage - rich adventitial areas . it has been recently indicated that nicotine could induce the 5-lo expression in colon neoplasm . a hypothesis : smoking promotes pathogenesis of aortic aneurysm through the 5-lipoxygenase pathway . had been proposed by takagi and umemoto in 2005 , but to date , it remains to be demonstrated . taken together , it has been demonstrated by compelling evidences that the inflammation in vascular wall is one of the pathogenesis of aaas . mediated by the inflammatory cells such as macrophages and mcs and inflammatory mediators including vcam-1 , icam-1 , cox-2 , and 5-lo , the inflammatory responses have a preference for the outer media and adventitia of the aneurysmal wall . currently , the notion that nicotine promotes the aaa formation by its receptor nachrs is still not supported by robust evidences . fortunately , in our recent animal experiment , the aaas have been successfully induced by both nicotine and ang ii in the older c57bl/6j mice , accompanied with the mc degranulation in the adventitia of the abdominal aortas . although the abundant connective tissue proteinases including mmps ( mmp-1 , -2 , -3 , -9 , -12 , and -13 ) , serine proteases ( tissue - type plasminogen activator ( t - pa ) ; urokinase - type plasminogen activator ( u - pa ) ; plasmin ; and neutrophil elastase ) , as well as cysteine proteases ( cathepsin d , k , l , and s ) have been described in the human aaa tissues , the most attentions have been kept on the members of the matrix metalloproteinase family [ 24 , 6265 ] . previous studies have focused on the 92-kd ( mmp-9 ; gelatinase b ) and 72-kd ( mmp-2 ; gelatinase a ) gelatinase / type iv collagenase , both most prominent elastolytic enzymes secreted by the aaa tissues in organ culture and in vivo , which are expressed by macrophages , vascular smcs , fibroblasts , or ecs , most often in the areas adjacent to the infiltrated inflammatory cells [ 62 , 6670 ] . therefore , it has been shown a close relationship between mmps and inflammatory responses in the aneurysmal tissues . the mmps are a group of zinc - mediated enzymes present in the extracellular matrix . it is a fundamental pathogenesis of aaas that the increased mmps in vascular wall degrade all kinds of extracellular matrix proteins , particularly elastin [ 14 , 71 ] . the mmps are inhibited by the specific endogenous timps , which comprise a family of four protease inhibitors : timp-1 , -2 , -3 and -4 . an imbalance in the proteolytic equilibrium between mmps and timps is a significant factor of the aaa formation . elastin and collagens type i / iii keep the integrality and elasticity of vascular wall , and resist stretch . under normal conditions , but in fact , the balance is principally maintained by the collagen metabolism , because elastin is synthesized and deposited in the early childhood and no further significant synthesis occurs in adult life . the content of collagens type i / iii increases compensatively in the early stage of the disease , while decreases dramatically in the advanced stage . degradation of elastin and loss of collagens during the advanced stage destroy the wall integrality and weaken the wall tensile strength , which promotes the development and rupture of aaas . it is supposed that the degradation of elastin is likely to exert a more significant role in the initiating process of aaas . it has been recently demonstrated that cigarette mainstream smoke could enhance the proteolytic activity of mmps including mmp-2 and -9 induced by ang ii and accelerate both formation and severity of aaas in the hypertensive apolipoprotein e - deficient mice , while cigarette smoke extract significantly downregulated timp-3 in aortic endothelial cells . similarly , nicotine increases the mmps ( especially mmp-2 and -9 ) expression and activities in the vascular wall components including ecs , vascular smcs and infiltrating inflammatory cells such as neutrophils and macrophages , [ 54 , 7580 ] and decreases the expression of timp-1 , -3 , and -4 in osteoblasts . moreover , the endogenic ligand of nicotine , 7-nachrs , is also involved in the mmp-2 and -9 upregulation in human retinal microvascular endothelial cells . taken together , nicotine and/or its ligantd 7-nachrs have been involved in the synthesis and release of proteolytic ingredients mmp-2 and -9 and decreased the timps expression in vivo and in vitro , thus very likely to be involved in the pathogenesis of aaas . histological examinations of both animal and human experimental aaas have revealed a paucity of medial vascular smcs in these specimens which are associated with the smc apoptosis [ 1521 ] . vascular smcs synthesize and release the extracellular matrix proteins including collagens , elastin , glycoproteins , and proteoglycans to provide the mechanical integrality to the vascular wall . in the aortic media , theoretically , a paucity of medial vascular smcs caused by apoptotic cell death , can reduce the synthesis of extracellular matrix proteins and collagens turnover , eventually attenuate the mechanical properties of the aortic wall and result in the formation and complications of aaas . henderson et al . have demonstrated that vascular smc apoptosis , macrophages , and t lymphocytes coexisted in the aortic mediaand companied by the upregulation of proapoptotic initiators , such as fas / fasl in the human aaa segments obtained from the patients undergoing open repair . recently , yamanouchi et al . firstly reported the direct links between medial vascular smc apoptosis and pathogenesis of aaas . in an ang ii - induced aneurysm model in apolipoprotein e - deficient mice , a novel caspase inhibitor q - vd - oph , inhibited apoptosis by blocking activation of caspases , drastically reduced the number of infiltrating macrophages and cd3 t cells and remarkably decreased the interleukin-6 level as well as the elastase activity . these findings suggested that inhibition of apoptosis may attenuate the aneurysm formation not only by preventing the vascular smc depletion but also by affecting the vascular inflammation and matrix degradation . in in vitro studies , cigarette smoke extract causes apoptosis of aortic smcs , human umbilical vein endothelial cells , pulmonary artery endothelial cells , and aortic endothelial cells from human and rodent animals [ 8386 ] . beyond our expectations , as a major ingredient of cigarette smoke , nicotine inhibits apoptosis of aortic smcs and ecs through nachrs in several investigations [ 87 , 88 ] . anyway , the viewpoint that nicotine is involved in the formation and progression of aaas through apoptotic mechanism seems to be not supported by existing evidences . angiogenesis is the new blood vessel formation from pre - existing blood vessels and is a prominent feature in both atherosclerosis and aaas [ 23 , 25 , 89 ] . in 1996 , thompson et al . had demonstrated that the density of the newly formed vessels was increased in all layers of the aneurysmal wall compared with the control samples . angiogenesis is associated with the inflammatory responses in the aneurysmal wall and accelerate the aneurysm rupture [ 2225 ] . usually , angiogenic stimuli ( e.g. , hypoxia or inflammatory cytokines ) may induce the expression and release of angiogenic growth factors such as vascular endothelial growth factor ( vegf ) and fibroblast growth factor ( fgf ) . these growth factors stimulate the proliferation of ecs in the existing vasculature and migration through the tissue to form new endothelialized channels [ 90 , 91 ] . almost all subunits of nachrs are expressed on ecs , whereas the most abundant receptor subunit is 7-nachrs . accumulated evidences have shown that , through excitable 7-nachrs on the plasma membrane of vascular smcs or ecs , nicotine could stimulate the proliferation and migration of ecs , increase the vegf and fgf release by vascular smcs and ecs and promote angiogenesis [ 29 , 54 , 79 , 9396 ] . moreover , the vegf receptor and 7-nachrs appear to mediate the distinct but interdependent pathways of angiogenesis . neovascularization is not the exclusive characteristic of aaas , which also exists in the atherosclerotic lesion , chronic ischemia , tumor and other illnesses . therefore , angiogenesis may not be a causative but a contributing to progression and rupture of aneurysm factor in the pathological process of aaas . it has been recently implied by a chronic nicotine exposure experiment in an hindlimb ischemic mice model that chronic nicotine exposure impaired the angiogenic response to ischemia mediated partly by downregulation of the vascular 7-nachrs , as well as by a reduction in plasma vegf level . however , because the researches did not assess the functional measures of limb perfusion , the conclusion remains to be further confirmed . it is irrefutable that cigarette smoking is the principal risk factor for aaas , but as a primary ingredient of cigarette smoke , nicotine , which has role in aaas is undefined . the increased mmps expression and degradation of all kinds of extracellular matrix proteins , particularly elastin , in the aneurysmal wall , has been demonstrated by compelling evidences , and nicotine may be involved in the process . the angiogenesis stimulated by nicotine may be the important factor for the progression or rupture of aaas rather than the causative factor . inflammation and apoptosis of vascular smcs , two important pathogenesis of aaas , are seemingly irrelevant to nicotine . whether nicotine is the key component in cigarette smoke promoting the formation and progression of aaas remains equivocal . in a chronic nicotine exposure experiment during 90 days it may be explained as the desensitization and changes in the expression of nachrs and thus the beneficial effects of nicotine through its receptors may be halted [ 49 , 50 ] . moreover , the adverse events of nicotine can be attributed to its dose - dependent effects , with the toxic cardiovascular effects at higher doses [ 49 , 93 , 99 ] . today nicotine replacement therapy is often used in smoking cessation , although there are little evidences on the safety and efficacy of long - term use . therefore , it is absolutely necessary to clarify the exact roles of nicotine in aaas , especially the adverse effects of chronic exposure .

inflammation , proteolysis , smooth muscle cell apoptosis , and angiogenesis have been implicated in the pathogenesis of abdominal aortic aneurysms ( aaas ) , although the well - defined initiating mechanism is not fully understood . matrix metalloproteinases ( mmps ) such as mmp-2 and -9 and other proteinases degrading elastin and extracellular matrix are the critical pathogenesis of aaas . among the risk factors of aaas , cigarette smoking is an irrefutable one . cigarette smoke is practically involved in various aspects of the aaa pathogenesis . nicotine , a major alkaloid in tobacco leaves and a primary component in cigarette smoke , can stimulate the mmps expression by vascular smcs , endothelial cells , and inflammatory cells in vascular wall and induce angiogenesis in the aneurysmal tissues . however , for the inflammatory and apoptotic processes in the pathogenesis of aaas , nicotine seems to be moving in just the opposite direction . additionally , the effects of nicotine are probably dose dependent or associated with the exposure duration and may be partly exerted by its receptors nicotinic acetylcholine receptors ( nachrs ) . in this paper , we will mainly discuss the pathogenesis of aaas involving inflammation , proteolysis , smooth muscle cell apoptosis and angiogenesis , and the roles of nicotine and nachrs .

os is a primary malignant bone tumor characterized by the direct formation of immature bone or osteoid tissue by the tumor cells . the classic os is a rare ( 45% of all malignant bone tumors ) highly malignant tumor [ 1 , 2 ] , with an estimated incidence of 3 cases / million population / year . os arises predominantly in the metaphysis of long bones , the most common sites of involvement are femur ( 42% , 75% of which are distal femur ) , tibia ( 19% , 80% of which are proximal tibia ) , and humerus ( 10% , 90% of which are proximal humerus ) . other significant locations are the skull and jaw ( 8% ) and pelvis ( 8% ) . the pretherapeutic diagnostic workup usually involves physical examination , radiological examination ( plain radiograph , computed tomography , magnetic resonance imaging , angiography ) , radionuclide imaging ( bone scintigraphy , tc - dmsa ( v ) , f - fdg - pet imaging ) , biopsy and lab tests . the aim of our study was to evaluate the role of pentavalent tc - dimercaptosuccinic acid [ tc - dmsa ( v ) ] and f - fluoro-2-deoxy - glucose f - fdg pet - ct in the management of patients with osteosarcoma and to compare results of tc - dmsa ( v ) with f - fdg pet - ct scan in these patients . a total of 22 patients ( 14 males and 8 females ) with biopsy - proven os were included in this study . age of the patients ranged from 866 yrs ( mean age = 22.2 yrs ) . pre- or postchemotherapy patients ( both the types of patients ) before surgery were included . patients excluded were those who had undergone surgery before the nuclear medicine examinations and patients with diabetes mellitus or pathologic glucose tolerance . we used two types of imaging modalities with two different radiopharmaceuticals , tc - dmsa ( v ) for whole - body scintigraphy using gamma camera and f - fdg for pet - ct imaging . tc is a gamma emitter and generator produced while f is a positron emitter and cyclotron produced . after fasting for at least 6 hrs , verifying the serum glucose level ( that should be below 140 mg / dl ) and with patients in a resting state , in a quiet room , a dose of 510 mci of f - fdg was injected intravenously depending on the age and weight of the patient . initial ct acquisition was done without oral or intravenous contrast injection ; followed by pet scan . on the pet / ct scanner , ct scan acquisition was performed on spiral dual slice ct with a slice thickness of 4 mm and a pitch of 1 . images were acquired using a matrix of 512 512 pixels and pixel size of about 1 mm . after completing the ct , the table is moved towards the field of view of pet . after transmissions scan , 3d pet acquisition was taken for 35 minutes per bed position for 57 bed positions depending on the patient length . pet data was acquired using a matrix of 128 128 pixels , and pet acquisition of the same axial range , as was with ct , was taken with the patient in the same position on the table . separate scans of the primary site were taken if it was not coming in the fov of the whole - body pet - ct acquisition . data obtained from the ct acquisition was used for low noise attenuation correction of pet emission data and for fusion of attenuation corrected pet images with the corresponding ct images . after completion of pet acquisition , images were reconstructed by iterative method ordered subset expectation maximization ( 2 iterations and 8 subsets ) with a filter of 5 mm . the reconstructed attenuation corrected pet images , ct images and fused images of matching pairs of pet and ct images was available for review in axial , coronal , and sagittal planes and in maximum intensity projections , three dimensional cine mode . after image reconstruction , a region of interest ( roi ) was carefully drawn around the site of the abnormal fdg uptake on lesion / s in the consequent 46 pet - ct scan slices . the slice with a maximal fdg uptake in the roi was chosen for quantitative measurement of metabolic activity of the tracer ( suv ) . from these rois , the suv was calculated according to the formula described below : ( 1)suv = mean roi activity ( mbq / g)injected dose ( mbq)/body weight ( g ) , whereas mbq is the mega - becquerel and g is the grams . patients were injected with 1520 mci of tc - dmsa ( v ) , depending upon the age and weight of the patients . dual head gamma camera was used and whole - body scans were taken 3 - 4 hours after the injection . whole - body planar images were acquired using the continuous mode with a table speed of 20 cms / sec from head to toe . spect images of the primary site and chest region were also takenspect images were taken with the following protocol.matrix size : 64 64,frame time : 1518 sec / frame , angular rotation : 3/frame , circular orbit : 360. spect images were taken with the following protocol.matrix size : 64 64,frame time : 1518 sec / frame , angular rotation : 3/frame , circular orbit : 360. matrix size : 64 64 , frame time : 1518 sec / frame , angular rotation : 3/frame , circular orbit : 360. two experienced nuclear medicine physicians evaluated the scan findings independently and they were blinded to the structural imaging findings and clinical findings . images were looked for area of increased radiotracer uptake and corresponding areas in the ct images , and fused pet - ct images were corroborated . multiple regression analysis was performed to find the significance of correlation ( r > 0.5 and p < 0.05 were considered significant ) . the intensity and the extent of uptake in f - fdg - pet - ct versus tc - dmsa ( v ) at the primary site accompanying lung lesions absent versus present and their uptake imperceptible versus perceptible were compared visually in both the modalities . all of them underwent thoracic ct within the two weeks before or after f - fdg - pet / ct and tc - dmsa ( v ) scan . those patients who were taken after chemotherapy had undergone therapy at least two weeks before the f - fdg - pet / ct and tc - dmsa ( v ) scan . characteristics of all patients are given in table 1 with graphical representation in figure 1 . agein 22 patients considered for this study , range of age was 866 years , with a mean age of 22.2 years . maximum number of patients falls within the range of 1020 years , with 10 patients ( 45.5% ) falling in this interval . out of these patients maximum number of patients as males 8/10 ( 80% ) and only 2/10 ( 20% ) were females . next higher number of patients , as in the range of 2030 years with 6/22 patients ( 27.3% ) , including 5/6 ( 83% ) males and 1/6 females ( 16.7% ) . interval of 110 years also included 3 patients ( 13.6% ) ; all were > 5 years in age with 100% females . interval 3040 years and 4050 years included 1 patient ( 4.5% ) each and both were females . another interval of 6070 years included 1 patient ( 4.5% ) , who was male . in 22 patients considered for this study , range of age was 866 years , with a mean age of 22.2 years . maximum number of patients falls within the range of 1020 years , with 10 patients ( 45.5% ) falling in this interval . out of these patients maximum number of patients as males 8/10 ( 80% ) and only 2/10 ( 20% ) were females . next higher number of patients , as in the range of 2030 years with 6/22 patients ( 27.3% ) , including 5/6 ( 83% ) males and 1/6 females ( 16.7% ) . interval of 110 years also included 3 patients ( 13.6% ) ; all were > 5 years in age with 100% females . interval 3040 years and 4050 years included 1 patient ( 4.5% ) each and both were females . another interval of 6070 years included 1 patient ( 4.5% ) , who was male . sexout of 22 patients , 14 patients ( 63.6% ) were males , 8 were females ( 36.4% ) . out of 14 males , maximum number of males was in the age group of 1020 years . in higher age groups of 3050 , 2 females ( 9% ) out of 22 patients were diagnosed with os . out of 22 patients , 14 patients ( 63.6% ) were males , 8 were females ( 36.4% ) . out of 14 males , maximum number of males was in the age group of 1020 years . in higher age groups of 3050 , 2 females ( 9% ) out of 22 patients were diagnosed with os . of 22 patients , 11 patients had femur as primary site ( 10 cases ( 90% ) of distal femur and 1/11 patient ( 10% ) of proximal femur ) , 3 patients had humerus as primary site ( with 100% proximal humerus involvement ) , and 3 patient had tibial involvement of os ( all the cases of proximal tibia ) . 2 patients ( 9.1% ) were having fibula inclusion ( 100% proximal fibula ) . out of these 3 ( 13.6% ) , 1 had chest involvement ( patient 6 ) , another patient had os in clavicle ( patient 11 ) and the last one was with jaw involvement ( patient 22 ) with each having a percentage contribution of 4.5% to all the patients ( table 2 ) . in all 22 patients , a baseline f - fdg - pet - ct examination was performed without any chemotherapy or after a minimum of two weeks of chemotherapy ; the primary or residual tumor site demonstrated a clear or moderate increased glucose uptake . abnormal fdg uptake was seen at the primary site in all the 22 patients showing the involvement of the primary site . f - fdg - pet - ct showed a mean of suvmax = 7.104 4.7 ( range of suvmax = 2.218.9 ) of the primary site . the details of individual f - fdg uptake are given in all patients in table 3 . since they were already histopathologically proven , so the sensitivity of f - fdg - pet - ct was 100% in detecting the primary site . no correlation was found between the size of the tumor and the suvmax ( r = 0.1209 ) ( figure 2(a ) ) . analyzing the results of whole - body f - fdg - pet - ct scans each patient underwent , foci with increased pulmonary / pleural tracer uptake was found in 11/22 patients . by correlating clinical and other imaging findings , 10/11 ( 90% ) of the examinations were classified as true positive , one as false positive . out of 11 patients , 8 had bilateral lung nodule with variable degree of fdg uptake ( mean suvmax = 1.1 ) . one patient ( patient 9 ) had bilateral lung nodule with no fdg uptake in one nodule . the other remaining two patients had solitary lung nodules ( patients 1 , 21 ) . 1 had mild [ f]fdg uptake while the other found to be inflammatory lesion on hrct ( patient 21 ) . so f - fdg - pet - ct was found to have a sensitivity of 100% in detecting the lung mets and specificity of 91% in comparison to the other clinical and imaging modalities findings . although pet alone was not able to detect many lesions but with the incorporation of ct , sensitivity of pet increased tremendously . with regard to other metastatic locations , abnormal uptake was also detected in one patient ( patient 10 ) at the site of left scapula ( coracoid process ) . all the 22 patients underwent tc - dmsa ( v ) scanning within one week , before or after , f - fdg - pet - ct scans . whole - body planar imaging and spect images of the primary site of os in all the patients revealed variable degree of uptake of tc - dmsa ( v ) at the primary tumor site . large variation was seen in the tumor - to - nontumor ratios ( t / nts ) . the mean of maximum t / nt or t / ntmax was found to be 4.446 2.27 ( ranging from 1.14810.689 ) . the mean value of t / ntavg came out to be 4.125 ( ranging from 1.88112.01 ) . individual patient values of t / ntmax and t / ntavg are given in table 3 . so tc - dmsa ( v ) showed 100% sensitivity in detecting the primary site like f - fdg - pet - ct findings . t / nts of tc - dmsa ( v ) showed no statistically significant correlation with spect to the size of the primary tumor . t / ntmax ( r = 0.0311 ) ( figure 2(b ) ) and t / ntavg ( r = 0.2490 ) ( figure 2(c ) ) were nowhere correlated to the size of the tumor . increased blood pool activity was evident in all the scans , which decreased with the delayed imaging . spect of chest in all the patients were taken along with the whole body scans to detect the deep seated metastasis . of all the 10/22 patients already proved to have lung metastasis 7 patients ( 70% ) showed abnormal uptake in the lungs . increased uptake of tc - dmsa ( v ) could be easily identified as focal , sharply defined regions with high lesion - to - background ratio ( l / n ) . the mean of maximum lesion - to - background ratio ( l / nmax ) was 1.8154 ( 1.520.23 ) with patient 4 showing the highest value of 20.23 . the mean of average lesion - to - background ratio ( l / navg ) was found to be 1.1987 ( 1.29812.166 ) in 6 out of 7 patients . intensity of tc - dmsa ( v ) uptake was either more ( figure 6 ) or comparable to the fdg uptake in f - fdg - pet - ct , while one patient ( patient 16 ) showed less uptake in comparison to f - fdg - pet - ct . patient 16 had large number of nodules with intense fdg uptake on f - fdg - pet scan while tc - dmsa ( v ) showed only one lesion with less intensity of uptake . lesions in rest of the 3 patients were not detected on the tc - dmsa ( v ) scan . 3/10 patients ( 33% ) ( patient 1 , 9 , 10 ) who did not show any tc - dmsa ( v ) uptake either had sub centimeter lung nodule ( patient 1 and 9 ) which because of the limited resolution of gamma camera ( 0.91.3 cm ) and the increased blood pool activity of tc - dmsa ( v ) in the major cardiovascular structures , were not visible in spect or was post chemotherapy ( patient 10 ) who showed significant response to the chemotherapy . also the overall number of lung lesions detected with tc - dmsa ( v ) was less in all the 7 patients . abnormal uptake was also detected in patient 10 in the left scapula region , as was seen in the f - fdg - pet - ct images tc - dmsa ( v ) could also show the amount of necrosis and soft tissue involvement at primary site . patient 9 also showed uptake in the right axillary lymph node . in case of children , there was an increased uptake at the growth plates because of increased growth at these points ( patients 3 , 5 , 7 , 11 , 14 , 16 , 17 , and 20 ) . no statistically significant correlation was found between suvmax of f - fdg - pet - ct and the t / nts ratios of tc - dmsa ( v ) of primary site with suvmax and t / ntmax ( r = 0.2711 , p = 0.2223 ) , and suvmax and t / ntavg ( r = 0.30 , p = 0.18 ) ( figures 3(a ) and 3(b ) ) . os is a rare form of bone cancers , which mainly affects the people in the age group of 1025 years . the 5-year relative survival for children with bone cancer has improved from 49% to 63% in the recent time with the development of improved diagnostic modalities , which helps in the early detection of disease . so it is essential to know about the metastatic involvement of the disease . in the present study we tried to assess the role of tc - dmsa ( v ) in detecting the primary as well as the metastatic involvement of the os by comparing it with the f - fdg - pet - ct . role of f - fdg - pet in characterizing the tumor as malignant and benign has already been studied extensively and it was found to be a promising tool to study the nature of tumor noninvasively [ 47 ] . tc - dmsa ( v ) , which is a tumor targeting radiopharmaceutical , has also found application in detection of medullary thyroid carcinoma , amyloidosis , head and neck cancers , and soft tissue tumors . it was found to have higher sensitivity in detecting the bone metastasis and soft tissue tumors than tc - mdp [ 8 , 9 ] . no study has been done till now to compare the role of tc - dmsa ( v ) and f - fdg - pet - ct in imaging the primary tumor of os . however , one study which compared the tc - dmsa ( v ) , tc - mdp , and ct found the scans of tc - dmsa ( v ) superior to the tc - mdp scans and the ct scans in identifying the metastases of os . in our study we found that both the f - fdg - pet - ct and tc - dmsa ( v ) showed intense uptake at the primary site in all the patients ( figures 4 , 5 , and 7 ) . tc - dmsa ( v ) showed equal sensitivity to f - fdg - pet in detecting primary tumor sites . the extent of localization and the intensity of uptake were comparable in both the cases . although no significant statistical correlation was found between the suvmax and t / nt ratios of f - fdg and tc - dmsa ( v ) , respectively . we found in our study that there is no significant statistical correlation between the suvmax or the t / nt ratios and the size of the primary tumors . this could be due to the association of primary tumors of os with extensive tumor necrosis and calcification which causes the nonuniform uptake of radiopharmaceutical . f - fdg - pet has also shown a higher sensitivity ( 98% ) in detecting lung mets , equivalent to ct . a positive f - fdg - pet result can be used to confirm any abnormalities seen on thoracic ct scans as metastatic . in our study f - fdg - pet - ct showed 100% sensitivity in detecting the lung mets . in case of tc - dmsa ( v ) the decreased sensitivity of tc - dmsa ( v ) could be attributed to the limited resolution of gamma camera ( 0.91.3 cm ) as most of the nodules , which were not seen , were subcentimetre in size . however , pet alone showed abnormal lung uptake in 8 patients only out of 10 proved patients with lung mets . this could be due to the partial - volume averaging effect ( which is a reduction in detected activity that occurs when the object size is smaller than the axial or transaxial spacing ) . the signal of small lesions could be diluted during reconstruction and malignant lesions can result in false negative findings on pet images . although with the introduction of pet - ct , its sensitivity has improved tremendously with the anatomic localization of various structures especially in case of evaluation of childhood sarcomas . similarly detection efficiency of tc - dmsa ( v ) can also be enhanced with the incorporation of spect - ct . in most of the cases , uptake in the normal bone was not detectably greater than the surrounding soft tissue . in the entire patients , both the excretion and retention in kidneys were apparent , with excreted radioactivity exclusively in the form of tc - dmsa ( v ) , but the degree of retention was variable . although no correlation was found between the extent of disease and the renal and hepatic uptake , no correlation could also be established between the extent of metastasis and renal and liver uptake . f - fdg - pet has a disadvantage of showing a high number of false positive results because of the increased uptake in many inflammatory and benign nodules ( as we had one patient which had false positive uptake in the inflammatory fibronodular lesion on f - fdg scan ) . tumor metabolism and , consequently tumor detection with f - fdg - pet , while tc - dmsa ( v ) is a tumor seeking agent and is not metabolism dependent the extent of tumor uptake can differentiate malignant and chondrogenic tumors . tc - dmsa ( v ) has an advantage of detecting bone as well as soft tissue tumor [ 9 , 14 ] . although several instances of false - positive uptake have occurred in both the soft - tissue as well as skeletal disease but the lesions latter showed relatively low uptake on bone scans . however , no false positive result was obtained in our study with tc - dmsa ( v ) . we could also detect abnormal uptake in bone mets at the scapula by both the imaging modalities . apart from application in imaging tumors , interest in the biodistribution of tc - dmsa ( v ) is increasing these days . since the -emitting analogues 186/188redmsa ( v ) offer the potential for targeted radiotherapy , the avidity with which the tracer is taken up in most bone metastasis suggests that this could be applied to palliative treatment . being a technetium labeled compound , tc - dmsa ( v ) is more economical and more accessible in comparison to the f - fdg - pet - ct . there is no report of any false positive uptake anywhere else in our study , which further increases its reliability . so the use of tc - dmsa ( v ) can be thought over for scanning the patients with os in detecting the primary as well as the metastatic involvement at least in those areas where f - fdg - pet - ct is not available . pentavalent tcdimercaptosuccinic acid appears to be a promising radiopharmaceutical for detecting primary and metastases in patients with osteosarcoma . f - fdg pet - ct also plays an important role in detecting primary and metastases in these patients . when tc - dmsa ( v ) is compared with 18f - fdg - pet - ct , it has lower sensitivity in detecting smaller metastatic lesions due to lower spatial resolution of gamma camera as compared to pet - ct . however , it requires costly setup including cyclotron , 18f - fdg synthesizing cell and a pet / ct camera , thereby the overall cost of an f - fdg - pet / ct scan is also more , which may be as much as 15 to 20 times the cost of a tc- dmsa ( v ) scan . although tc - dmsa ( v ) was not as sensitive as f - fdg - pet - ct was , in detection of subcentimetre nodules , but it seems to be an good alternative to f - fdg - pet / ct in those areas where pet / ct is not available . also , the sensitivity of tc - dmsa ( v ) scan may be improved for detection of sub centimeter nodules by using the spect - ct , which further improves the anatomical location , keeping the cost of scan still significantly less compared to an f - fdg - pet / ct scan . 99mtc - dmsa ( v ) also has another advantage over f - fdg - pet / ct . the average whole - body dose delivered to the patient in a tc - dmsa ( v ) is found to be approximately 3msv , while in a f - fdg - pet / ct scan , the average dose delivered has been found to be 11 - 12 msv . thus a tc - dmsa ( v ) scan delivers 4 times less radiation dose to the patient as compared to a f - fdg - pet / ct scan .

to evaluate the role of 99mtc - dmsa ( v ) and [ 18f]fdg pet - ct in management of patients with osteosarcoma , 22 patients were included in our study . all patients underwent both 99mtc - dmsa ( v ) and whole - body [ 18f]fdg pet - ct scans within an interval of 1 week . 555740 mbq of 99mtc - dmsa ( v ) was injected i.v . the whole - body planar , spect images of primary site and chest were performed after 3 - 4 hours . [ 18f]fdg pet - ct images were obtained 60 minutes after i.v . injection of 370 mbq of f-18 fdg . both fdg pet - ct ( mean suvmax = 7.1 ) and dmsa ( v ) scans showed abnormal uptake at primary site in all the 22 patients ( 100% sensitivity for both ) . whole - body pet - ct detected metastasis in 11 pts ( lung mets in 10 and lung + bone mets in 1 patient ) . whole - body planar dmsa ( v ) and spect detected bone metastasis in one patient , lung mets in 7 patients and ln in 1 patient . hrct of chest confirmed lung mets in 10 patients and inflammatory lesion in one patient . 7 patients positive for mets on dmsa ( v ) scan had higher uptake in lung lesions as compared to fdg uptake on pet - ct . three patients who did not show any dmsa uptake had subcentimeter lung nodule . resuts of both 99mtc - dmsa ( v ) ( whole - body planar and spect imaging ) and [ 18f]fdg pet - ct were comparable in evaluation of primary site lesions and metastatic lesions greater than 1 cm . though 99mtc - dmsa ( v ) had higher uptake in the lesions as compared to [ 18f]fdg pet - ct , the only advantage [ 18f]fdg pet - ct had was that it could also detect subcentimeter lesions .

intellectuals solve problems ; geniuses prevent them ! ! caries occur five times more frequently in occlusal fissures , than on smooth surfaces . sealant not only prevents tooth decay before it starts , but also halts the progress . this effect relies on the sealant 's ability to fill the fissure and not detach . retention is provided by resin tags that form an effective mechanical interlock between resin material and enamel surface . however it still remains to be evaluated as to how exactly do the factors , such as , resin tag length , microleakage and viscosity affect the clinical efficacy of these sealants . this study aims at evaluating the relationship between sealant viscosity , resin tag length and prevention of microleakage . test materials used in the study , helioseal , guardian seal and embrace wetbond guardian seal kerr orange ca 92867helioseal assortment , ivoclar vivadent ag , fl -9494 schaan/ liechtenstienembrace wetbond pulpdent corporation watertown na 02471 - 0780 usa guardian seal kerr orange ca 92867 helioseal assortment , ivoclar vivadent ag , fl -9494 schaan/ liechtenstien embrace wetbond pulpdent corporation watertown na 02471 - 0780 usa stereomicroscope ( leica wild m3z , germany)scanning electron microscope ( iisc bangalore)brooks field viscometer ( dv - e viscometer brookfield , analytical technologies , bangalore ) stereomicroscope ( leica wild m3z , germany ) scanning electron microscope ( iisc bangalore ) brooks field viscometer ( dv - e viscometer brookfield , analytical technologies , bangalore ) the collected teeth were cleaned off the calculus , plaque and debris with an ultrasonic scaler and were stored in thymol solution . the teeth were then randomly divided into three groups of 10 samples each : group e : embrace wetbond sealant.group h : helioseal sealant.group g : guardian seal sealant . occlusal surfaces of the teeth were etched with 37% phosphoric acid ( enamel preparator , ivoclar ) for 30 seconds and rinsed with water . the teeth were then dried with a mild oil - free air stream to achieve a characteristic frosty white , chalky appearance of enamel . two coats of bonding agent ( g bond , gc ) were applied ( using applicator on the etched tooth surface ) and light cured for 20 seconds using visible light curing unit ( 470 nm to 420 nm wavelength ) . occlusal surfaces of the teeth were etched with 37% phosphoric acid ( enamel preparatory , ivoclar ) for 30 seconds and rinsed with water . with embrace wetbond , rather , the surface should be lightly dried and very slightly moist with a glossy appearance . to accomplish this , the sealant embrace wet bond was applied as per manufacturer 's instructions [ figure 2 ] , followed by light curing for 40 seconds . application of sealant embrace wet bond as per the manufacturer 's instructions in the study the teeth were coated with double layer of nail varnish to prevent the leakage of the dye . the occlusal surface was excluded . a different colour nail varnish was used for each group for easy distinguishing . red - helioseal sealantblue - guardian sealantorange - embrace wetbond sealant red - helioseal sealant blue - guardian sealant orange - embrace wetbond sealant all the groups were then immersed inverted , ( such a way that unpainted surface was in contact with the dye ) in 1% methylene blue for 24 hours . [ figure 3 ] the samples were removed and gently brushed to remove excess dye . all the three groups were then subjected to thermocycling at a temperature range of 5 - 55c for 500 cycles , with a dwell time of 30 seconds . the procedure used to determine marginal leakage was similar to the one described by theodoridou each tooth was sectioned longitudinally in a mesio distal direction through the center of the sealant with a diamond wheel measuring 0.02 mm in thickness . one half of every tooth section was utilized for microleakage testing and the other half for evaluating the length of resin tags . teeth placed inverted into the dye for microleakage testing in the study the sections for microleakage evaluation were cleaned and examined under stereomicroscope for the dye penetration and were scored by 2 independent examiners as follows . [ figure 3 ] 0= no dye penetration.1= dye penetration down the mesial or distal wall.2= dye penetration down the mesial and distal walls.3= dye penetration underneath the sealant and down the mesial or distal wall.4= dye penetration all around the sealant . 0= no dye penetration . the other half of the tooth section was prepared for evaluation under scanning electron microscopy . the polished sections were then decalcified using 37% phosphoric acid for 15 seconds to etch away any enamel mineral component not protected by sealants and then rinsed and stored in distilled water . the tooth sections were dried thoroughly under the heat lamp , and then mounted on brass rings using a non conductor tape made of carbon . this was then applied to the sections , in the areas that did not need scanning . these mountings were then placed inside an ion spluttering device for 30 minutes using vacuum evaporation at 200 -300 a. the gold spluttered sections were then placed inside the scanning electron microscope of 20 kv capacity and photographs of the sections were obtained . scanning electron microscopy image showing the resin tag ( t ) and surface ( s ) , and arrow pointing to the resin tag the viscosity was checked by diluting the sealant with methyl methacrylate monomer liquid . the liquid was placed in the sample holder of the brooke 's field viscometer and was calibrated at 60 revolutions per minute , 100% torque . further , s-18 spindle was used and the reading was noted . 1 ml of the sealant was drawn out of its container and poured into a 10 ml test tube to which the methyl methacrylate liquid was added and mixed till the total liquid amounted to 10 ml ( since the minimum amount needed to evaluate the viscosity was 10 ml ) . each sample was individually placed in the brook 's field viscosity meter at room temperature and the viscosity was measured in cp ( centipoise ) . test materials used in the study , helioseal , guardian seal and embrace wetbond guardian seal kerr orange ca 92867helioseal assortment , ivoclar vivadent ag , fl -9494 schaan/ liechtenstienembrace wetbond pulpdent corporation watertown na 02471 - 0780 usa guardian seal kerr orange ca 92867 helioseal assortment , ivoclar vivadent ag , fl stereomicroscope ( leica wild m3z , germany)scanning electron microscope ( iisc bangalore)brooks field viscometer ( dv - e viscometer brookfield , analytical technologies , bangalore ) stereomicroscope ( leica wild m3z , germany ) scanning electron microscope ( iisc bangalore ) brooks field viscometer ( dv - e viscometer brookfield , analytical technologies , bangalore ) the collected teeth were cleaned off the calculus , plaque and debris with an ultrasonic scaler and were stored in thymol solution . the teeth were then randomly divided into three groups of 10 samples each : group e : embrace wetbond sealant.group h : helioseal sealant.group g : guardian seal sealant . occlusal surfaces of the teeth were etched with 37% phosphoric acid ( enamel preparator , ivoclar ) for 30 seconds and rinsed with water . the teeth were then dried with a mild oil - free air stream to achieve a characteristic frosty white , chalky appearance of enamel . two coats of bonding agent ( g bond , gc ) were applied ( using applicator on the etched tooth surface ) and light cured for 20 seconds using visible light curing unit ( 470 nm to 420 nm wavelength ) . occlusal surfaces of the teeth were etched with 37% phosphoric acid ( enamel preparatory , ivoclar ) for 30 seconds and rinsed with water . with embrace wetbond , rather , the surface should be lightly dried and very slightly moist with a glossy appearance . to accomplish this , the sealant embrace wet bond was applied as per manufacturer 's instructions [ figure 2 ] , followed by light curing for 40 seconds . occlusal surfaces of the teeth were etched with 37% phosphoric acid ( enamel preparator , ivoclar ) for 30 seconds and rinsed with water . the teeth were then dried with a mild oil - free air stream to achieve a characteristic frosty white , chalky appearance of enamel . two coats of bonding agent ( g bond , gc ) were applied ( using applicator on the etched tooth surface ) and light cured for 20 seconds using visible light curing unit ( 470 nm to 420 nm wavelength ) . occlusal surfaces of the teeth were etched with 37% phosphoric acid ( enamel preparatory , ivoclar ) for 30 seconds and rinsed with water . with embrace wetbond , rather , the surface should be lightly dried and very slightly moist with a glossy appearance . to accomplish this , the sealant embrace wet bond was applied as per manufacturer 's instructions [ figure 2 ] , followed by light curing for 40 seconds . the teeth were coated with double layer of nail varnish to prevent the leakage of the dye . red - helioseal sealantblue - guardian sealantorange - embrace wetbond sealant red - helioseal sealant blue - guardian sealant orange - embrace wetbond sealant all the groups were then immersed inverted , ( such a way that unpainted surface was in contact with the dye ) in 1% methylene blue for 24 hours . [ figure 3 ] the samples were removed and gently brushed to remove excess dye . all the three groups were then subjected to thermocycling at a temperature range of 5 - 55c for 500 cycles , with a dwell time of 30 seconds . the procedure used to determine marginal leakage was similar to the one described by theodoridou each tooth was sectioned longitudinally in a mesio distal direction through the center of the sealant with a diamond wheel measuring 0.02 mm in thickness . one half of every tooth section was utilized for microleakage testing and the other half for evaluating the length of resin tags . teeth placed inverted into the dye for microleakage testing in the study the sections for microleakage evaluation were cleaned and examined under stereomicroscope for the dye penetration and were scored by 2 independent examiners as follows . [ figure 3 ] 0= no dye penetration.1= dye penetration down the mesial or distal wall.2= dye penetration down the mesial and distal walls.3= dye penetration underneath the sealant and down the mesial or distal wall.4= dye penetration all around the sealant . 0= the other half of the tooth section was prepared for evaluation under scanning electron microscopy . the polished sections were then decalcified using 37% phosphoric acid for 15 seconds to etch away any enamel mineral component not protected by sealants and then rinsed and stored in distilled water . the tooth sections were dried thoroughly under the heat lamp , and then mounted on brass rings using a non conductor tape made of carbon . this was then applied to the sections , in the areas that did not need scanning . these mountings were then placed inside an ion spluttering device for 30 minutes using vacuum evaporation at 200 -300 a. the gold spluttered sections were then placed inside the scanning electron microscope of 20 kv capacity and photographs of the sections were obtained . scanning electron microscopy image showing the resin tag ( t ) and surface ( s ) , and arrow pointing to the resin tag the liquid was placed in the sample holder of the brooke 's field viscometer and was calibrated at 60 revolutions per minute , 100% torque . further , s-18 spindle was used and the reading was noted . 1 ml of the sealant was drawn out of its container and poured into a 10 ml test tube to which the methyl methacrylate liquid was added and mixed till the total liquid amounted to 10 ml ( since the minimum amount needed to evaluate the viscosity was 10 ml ) . each sample was individually placed in the brook 's field viscosity meter at room temperature and the viscosity was measured in cp ( centipoise ) . the recorded values were represented as mean standard deviation and range values , and were statistically analyzed using one way analysis of variance ( anova ) for multiple group comparison , post hoc tukey 's test for group wise comparison , the spearman 's rank correlation coefficient for correlation analysis and the mann whitney test for multiple group comparison of the microleakage scores . table 1 and graph 1 show the comparison of values obtained for microleakage between group e , group h and group g. the mean value of microleakage for group e is 0.4 , group h is 1.0 and group g is 1.6 . no statistically significant differences were found between the group e v / s group h and between group h v / s groups g. statistically significant difference ( p < 0.05 ) was seen between group e v / s group g. group e showed the least microleakage followed by group h and g. mean microleakage scores and inter group comparison of microleakage scores of group e , group h , group g showing percentage of microleakage scores and inter group comparison of microleakage scores of the three groups in the study table 2 and graph 2 show the comparison of values obtained for resin tag length between group e , group h and group g. the mean resin tag length for group e is 10.14 4.84 ; for group h is 9.65 4.28 and for group g is 5.86 1.85 . the mean difference of group e v / s group h is 0.49 , group g v / s group h is 3.79 and group e v / s group g is 4.28 . no statistically significant differences were found between group e v / s group h and group h v / s group g. statistically significant difference ( p < 0.05 ) was seen between group e v / s g. the mean resin tag length was highest for group e. mean difference of the resin tag lengths and inter group comparison of the resin tag length between group e , group h and group g showing the mean lengths of the resin tags and inter group comparison of the resin tag length of the three groups in the study table 3 shows the relationship between the microleakage and resin tag length in group e , group h and group g. statistically significant differences were seen in all the three groups . group e showed a p - value of p < -0.85 , group h showed a p - value of p < - 0.94 and group g showed a p - value of p < -0.96 . relationship between resin tag length and microleakage of group e , group h and group g ( correlation analysis ) table 4 and graph 3 show the compiled data showing the mean difference and range of resin tag length , microleakage scores and viscosity of group e , group h and group g. the viscosity of the group e was the lowest at 0.96 cp , group h was 1.59 cp and the group g was 1.92 cp . compiled data showing the mean difference and range of resin tag length , microleakage scores and viscosity of group e , group h and group g compiled data showing the mean difference and range of the resin tag length , microleakage scores and the viscosity of the three groups in the study the occlusal surface , especially the pits and fissures of posterior teeth have been recognized for their high caries susceptibility over many years . it is undoubtedly more vulnerable due to the unique morphology of the pits and fissures . occlusal pits and fissures vary in shape , but are generally narrow ( ~ 0.1 mm wide ) and tortuous , and are considered to be an ideal site for the retention of bacterial and food remnants . this is because the morphology renders the mechanical means of debridement inaccessible as an average tooth brush bristle ( 0.2 mm ) is too large to penetrate most of the fissures . other factors such as lack of salivary access to the fissures , the close proximity of fissure base to the dentino - enamel junction and remnants of debris and pellicle in the fissures increase caries susceptibility of fissures by many folds . therefore , to prevent initiation of caries in these fissures , the concept of pit and fissure sealants evolved . the cariostatic properties of sealants are attributed to the physical obstruction of the pit and fissures . this prevents colonization of the pits and fissures with new bacteria and also prevents the penetration of fermentable carbohydrates to any bacteria remaining in the pits and fissures . requisites of an efficient sealant include : a viscosity allowing penetration into deep and narrow fissures even in maxillary teeth , adequate working time , rapid cure , good and prolonged adhesion to enamel , low sorption and solubility , resistance to wear , minimum irritation to tissues , and cariostatic action . the third molars extracted for therapeutic purpose , which were free of caries , developmental defects , enamel micro - fractures and discoloration were included in this study , as previous studies have revealed that any preexisting alteration of surface morphology of the tooth directly influenced the caries progression . as compared to the formaldehyde , thymol has no effect on the protein structure and neither does it alter the enamel structure . hence in this study , 0.01% thymol solution was used as a storage solution and disinfectant . some studies show that pumice prophylaxis does not completely and consistently remove the pellicle and debris , especially in the depth of the fissure . studies by blackwood ja et al . showed that between enameloplasty , air abrasion and pumice prophylaxis , the least microleakage was seen with the conventional pumice prophylaxis . in the present study , despite of all the controversies , conventional pumice method was used for cleaning prior to etching . the teeth that have newly erupted are the ones that are most susceptible to caries and hence need protection from pit and fissures . but isolating them is the most difficult . until now their mechanism of adhesion is ionic bonding , not micromechanical retention to an acid etched enamel surface . paradi and co workers reported low sealant retention rates with glass ionomer cement ( gic ) . while traditional sealants are hydrophobic , embrace wetbond is hydrophilic . on light - curing , embrace wetbond incorporates di- , tri- and multifunctional acrylate monomers into an advanced acid - integrating chemistry that is activated by moisture . when placed in the presence of moisture , the sealant spreads over the enamel surface . because of its unique chemistry , embrace wetbond is miscible with water and flows into moisture - containing etched enamel and combines with it . additionally , a longitudinal study done in vivo with embrace wetbond has shown a retention rate of 90% . only 32 teeth out of the 334 teeth in the study required replacement of the sealant . early in the development of sealants , it was recognized that the addition of fluoride to a sealant could have the potential benefits of additional caries protection . the fluoride release achieved high levels initially , with salivary fluoride concentration being high within 30 minutes after sealant placement ; however , returned to baseline levels within 1 - 2 days after the sealant placement . evaluation of the combined use of fluoride and dental sealants has showed retention of 92% after 4 years . this suggests that pit and fissure sealants confer additional caries preventive benefits beyond those of fluoride therapy alone . a sealant is effective in preventing caries , only when it is retained in the fissures . the retention in resin based pit and fissure sealant is through micromechanical interlocking between the resin and the enamel . mechanical retention of sealant is the direct result of resin penetration into the porous enamel forming tags . monomer in the material polymerizes and the material becomes interlocked with the enamel surface . in the present study the mean length of tags obtained was in the range of 10 m to 5 m . other studies by gomez et al . and it was seen in our study that the pit and fissure sealant with the longer resin tags had lesser microleakage . thus , it can be postulated that , the longer resin tags formed , lesser will be microleakage . other than surface tension , the viscosity of the sealant also influences the penetration of sealants . this resistance of the fluid to flow is controlled by internal frictional forces within the liquid . the viscosity is measured in units of mpa ( mega pascal ) per second or cp ( centipoise ) . higher viscosity may cause poorer adaptation and incomplete penetration to the bottom of the pit and fissures resulting in decreased retention . with low viscosity sealants , there is a greater potential of the sealant to flow , spread more rapidly over the surface and penetrate . at times faster penetration rates are found with larger holes , less dense liquids and those with high surface tension . on the contrary , the study by dm barnes , p kihn , a elsabach and ja von fraunhofer has shown that the viscosity and flow properties of the fissure sealants do not affect their sealing ability . hence , we tried to analyze the nature of relationship between viscosity and the other factors of pit and fissure sealants . the viscosity of sealants can be assessed in centipoise units using ultrasonic vibratile viscometer or by conventional capillary tube method . the ultrasonic vibratile viscometer is a more accurate method as compared to the capillary tube method , as it gives a digital read out . the viscometer rotates the spindle in the liquid to overcome the viscous resistance to the induced movement , and thus a reading is obtained . in this study , 0.2% , thus allowing the test results to be duplicated anywhere in the world when the same model instrument is used . in the present study , the viscometer utilized required a minimum volume of 10 ml sample . to achieve this , 1ml of each sealant hence , the relative viscosities of the three sealants were obtained . in the present study , it has been shown that sealant containing fluoride tends to be thicker than those without fluoride . urethane monomer based sealant may confer more elasticity and adhesiveness to the resin than does bisphenol a - glycidyl methacrylate ( bis - gma ) . in this study , two of the sealants helioseal and guardian seal contained bis - gma , where as the embrace wet bond contained di , tri and multiacrylate monomers . that may be the reason why the viscosity of embrace wetbond was lower as compared to the other groups . microleakage is defined as the passage of bacteria and their toxins between restorative margins and the tooth preparation walls . clinically microleakage is important when one considers that pulpal inflammation is more likely caused by bacteria than by chemical toxicity of restorative material . microleakage may support the caries process beneath the sealant , hence the ability of the sealant to adequately seal the pits and fissures and prevent microleakage is important . microleakage assessment may be qualitative or quantitative with different systems , including both simple and computer based methods . dye penetration has been used in several studies , to assess the presence of marginal leakage around the sealant enamel surface . in the present study , the presence of a dye in the under penetration zone of the etched enamel can indicate a susceptible microleakage pathway . clinically , this may imply that the remaining etched area could be a factor for development of caries by microleakage , if the sealant was partially or completely lost . all the sealants used in this study showed some degree of microleakage and these factors agreed with previous studies . the least microleakage was seen with the embrace wetbond sealant , followed by helioseal and guardian seal . complete penetration of sealant into complex fissure systems is difficult due to the phenomenon of closed end capillaries or isolated capillaries . some lateral fissures arising from the main fissures also fail to be filled with sealant . hence , further research on the effect of fissure morphology on sealant microleakage and penetration are necessary . the sem was used to measure the length of the resin tags as the sem can produce very high - resolution images of a sample surface , and reveal details about less than 1 to 5 nm in size . due to the very narrow electron beam , sem micrographs have a large depth of field yielding a characteristic three - dimensional appearance useful for understanding the surface structure of a sample . it should be noted that the results of the present study are valid for in vitro conditions . depending on the environment , all pit and fissure sealants may act differently due to other variables like type of fissures , preparation of fissures , enamel etching and conditioning , application of bonding agent and contamination of prepared surfaces of fissures . appropriate method of application of sealants and viscosity of the sealant , are also a factors influencing the microleakage , and if a proper application method is followed , it can increase the length of resin tag and thus improve the efficiency of the sealant in preventing caries . this study clearly indicated that , there does exists a relationship between the resin tag length and microleakage , and the longer the resin tags formed , the lesser is the microleakage , and the better will the cariostatic action of the pit and fissure sealant be . the viscosity too plays a role in the formation of these all important resin tags . the lower viscosity sealants are better . also , with the newly developed hydrophilic sealant embrace wetbond , it is now possibly to go ahead and seal the newly erupted teeth that were previously left unprotected due to moisture control problems .

introduction : in this era of preventive dentistry , many techniques are available for prevention of caries , such as plaque control , use of systemic and local fluorides and pit and fissure sealants . the rationale of pit and fissure sealants is that , when they are applied into the caries prone fissures , they penetrate and seal them from the oral environment . this study aims to correlate the relationship between the viscosity of the sealant , resin tag length and microleakage.materials and methods:30 third molars were selected for study . the teeth were randomly divided into 3 groups . group e : embrace wetbond , h : helioseal , g : guardian seal . teeth were cleaned with pumice prophylaxis and pretreated with acid etching and bonding agent . the respective pit and fissure sealants were applied . teeth were placed in 1% methylene blue dye and sectioned mesio - distally into two halves . these were used to assess the microleakage using stereomicroscope and resin tag length using sem . viscosity was assessed using brooke 's field viscometer.results:viscosity was lowest for embrace wetbond and highest for guardian seal . microleakage scores were highest with guardian seal and lowest with embrace wetbond . resin tag lengths were longer with embrace wetbond as compared to other groups . there is a definite negative correlation between viscosity , resin tag length and microleakage . lower the viscosity , the longer were the resin tags and the microleakage decreased . embrace wetbond pit and fissure sealant had lowest viscosity , longest resin tag length and lowest microleakage scores.conclusion:embrace wetbond appears to be compatible with residual moisture and ideal for use in children , where isolation is a problem .

x - linked charcot - marie - tooth type 1 ( cmt1x ) disease is a hereditary chronic progressive disease , which is caused by mutations in the gap junction beta-1 protein ( gjb1 ) gene encoding the gap junction protein connexin 32 ( cx32 ) . clinically , the disease is characterized by the chronic progressive wasting and weakness of distal muscles in the lower limbs with sensory disturbance usually from the second decade . nerve conduction velocity test shows a moderate neuropathy , and sural biopsy usually shows axonal or mixed axonal - demyelinating neuropathy pathology . most of these were case reports , summarized electrophysiological findings , or pathological features in a few cases . here , we reported the clinical features and genetic mutations of gjb1 gene in a large chinese patient cohort . this study was conducted in accordance with the declaration of helsinki and was approved by the local ethics committee of peking university first hospital . informed written consent was obtained from all patients prior to their enrollment in this study . ninety - two patients with cmt from unrelated families were diagnosed by next - generation sequencing ( ngs ) at the department of neurology , peking university first hospital from january 2005 to january 2016 . among them , a total of 22 patients with gjb1 mutations were recruited in this study , which comprised 23.9% of the total cmt patients . as for the other cmt genes , the mutation frequency was 35.9% in peripheral myelin protein 22 gene , 17.4% in mitofusin 2 gene , 2.2% in myelin protein zero gene , 5.4% in inverted formin , fh2 and wh2 domain containing ( inf2 ) gene , 4.3% in ganglioside - induced differentiation associated protein 1 gene , 2.2% in neurofilament light gene , 2.2% in periaxin gene , 1.1% in sh3 domain and tetratricopeptide repeats 2 gene , 1.1% in early growth response 2 gene , 1.1% in glycyl - trna synthetase gene , 1.1% in leucine rich repeat and sterile alpha motif containing 1 gene , 1.1% in fig4 phosphoinositide 5-phosphatase ( fig4 ) gene , 1.1% in alanyl - trna synthetase gene , and 1.1% in dehydrogenase e1 and transketolase domain containing 1 gene . the clinical records and electrophysiological characteristics of these 22 cmt1x patients with gjb1 mutations were retrospectively collected and reviewed . ngs panel covered all of the exons and their flanking sequences of genes known to be associated with hereditary neuropathies ( gene list available on request ) . the exons and their flanking splice sites were captured and subsequently sequenced on an illumina hiseq 2500 sequencer ( illumina , san diego , ca , usa ) . the sequencing files were mapped to reference sequences with burrows - wheeler aligner and picard tools and then called with control samples with the gatk 3.0 haplotypecaller ( broad institute , usa ) . the segregation analyses of the mutations were confirmed in the parents and the affected family members . for the novel mutations , the biological relevance of the novel amino acid changes was studied using both polyphen-2 ( http://www.genetics.bwh.harvard.edu/pph2/ ) and mutation taster ( http://www.mutationtaster.org/ ) programs . this study was conducted in accordance with the declaration of helsinki and was approved by the local ethics committee of peking university first hospital . informed written consent was obtained from all patients prior to their enrollment in this study . ninety - two patients with cmt from unrelated families were diagnosed by next - generation sequencing ( ngs ) at the department of neurology , peking university first hospital from january 2005 to january 2016 . among them , a total of 22 patients with gjb1 mutations were recruited in this study , which comprised 23.9% of the total cmt patients . as for the other cmt genes , the mutation frequency was 35.9% in peripheral myelin protein 22 gene , 17.4% in mitofusin 2 gene , 2.2% in myelin protein zero gene , 5.4% in inverted formin , fh2 and wh2 domain containing ( inf2 ) gene , 4.3% in ganglioside - induced differentiation associated protein 1 gene , 2.2% in neurofilament light gene , 2.2% in periaxin gene , 1.1% in sh3 domain and tetratricopeptide repeats 2 gene , 1.1% in early growth response 2 gene , 1.1% in glycyl - trna synthetase gene , 1.1% in leucine rich repeat and sterile alpha motif containing 1 gene , 1.1% in fig4 phosphoinositide 5-phosphatase ( fig4 ) gene , 1.1% in alanyl - trna synthetase gene , and 1.1% in dehydrogenase e1 and transketolase domain containing 1 gene . the clinical records and electrophysiological characteristics of these 22 cmt1x patients with gjb1 mutations were retrospectively collected and reviewed . ngs panel covered all of the exons and their flanking sequences of genes known to be associated with hereditary neuropathies ( gene list available on request ) . the exons and their flanking splice sites were captured and subsequently sequenced on an illumina hiseq 2500 sequencer ( illumina , san diego , ca , usa ) . the sequencing files were mapped to reference sequences with burrows - wheeler aligner and picard tools and then called with control samples with the gatk 3.0 haplotypecaller ( broad institute , usa ) . the segregation analyses of the mutations were confirmed in the parents and the affected family members . for the novel mutations , the biological relevance of the novel amino acid changes was studied using both polyphen-2 ( http://www.genetics.bwh.harvard.edu/pph2/ ) and mutation taster ( http://www.mutationtaster.org/ ) programs . the mean age of onset was 15.6 8.7 years ( ranging from 1 year to 42 years ) . two patients ( 9.1% ) had an earlier onset before 10 years old , including one from infancy . the mean duration from onset to diagnostic time was 8.2 4.5 years ( ranging from 2 years to 18 years ) . the clinical and electrophysiological characteristics of 22 chinese cmt1x patients in this study cmt1x : x - linked charcot - marie - tooth type 1 ; ll : low limbs ; ul : upper limbs ; + : positive ; : negative ; cns : central nervous system ; mcv : motor conduction velocity ; cmap : compound motor action potential ; scv : sensory conduction velocity ; snap , sensory nerve action potential ; ne : not elicited ; na : not available . all patients showed distal muscle wasting and weakness of the lower limbs , with the involvement of the upper limbs in 13 cases . muscle strength ranged from iii to v in distal upper limbs and from 0 to v in distal lower limbs . cns involvement appeared in five patients ( 22.7% ) , including four cases with transient cns symptoms and one case with constant cns signs . four cases ( 18.2% ) with transient symptoms presented with sudden onset of cerebral symptoms including aphasia , dysphagia , quadriplegia , or paralysis induced by fever or infection . the symptoms usually lasted from hours to days , and the patients completely recovered without special treatment . one case with constant cns involvement additionally presented with nystagmus and ataxia on physical examination . in median nerves , the mean motor nerve conduction velocity ( mcv ) was 35.0 4.8 m / s ( range : 27.343.9 m / s ) , the mean amplitude of compound muscle action potential ( camp ) was 2.3 1.5 mv ( range : 0.25.2 mv ) , the mean sensory nerve conduction velocity ( scv ) was 42.0 9.6 m / s ( range : 32.166.0 m / s ) , and the mean sensory nerve action potential ( snap ) amplitude was 5.0 4.2 mv ( range : 1.412.1 mv ) . in ulnar nerves , the mean mcv was 33.6 5.7 m / s ( range : 27.746.7 m / s ) , the mean camp amplitude was 3.7 3.2 mv ( range : 0.310.3 mv ) , the mean scv was 40.4 12.8 m / s ( range : 30.371.0 m / s ) , and the snap mean amplitude was 3.3 3.3 mv ( ranging from 1.47 mv to 9.6 mv ) . in tibial nerves , the mean mcv was 32.5 5.5 m / s ( range : 23.039.2 m / s ) and the mean camp amplitude was 1.1 1.7 mv ( range : 0.04.8 mv ) . the cranial magnetic resonance imagings ( mris ) of patient 2 revealed abnormal signals in the splenium and genu of the corpus callosum in the first episode and in the bilateral posterior limbs of the internal capsule and splenium of the corpus callosum in the second episode . the cranial mris of patient 3 revealed abnormal signals in the centrum semiovale bilaterally in the first episode , the signals enlarged at the same location in the second episode , and widened at the third episode ; the abnormal signals were resolved at intervals . the cranial mris of patient 4 revealed abnormal signals in the posterior limb of the internal capsule and the periventricular area bilaterally . the mris of patient 13 revealed abnormal signals in the bilateral centrum semiovale and splenium of the corpus callosum [ figure 1 ] . brain magnetic resonance imagings of a male patient with x - linked charcot - marie - tooth type 1 ( patient 13 ) revealed abnormal signals in the bilateral centrum semiovale ( a d ) and splenium of the corpus callosum ( e h ) . ( a and e ) : t1 weighted ; ( b and f ) : t2 weighted ; ( c and d ) : fluid attenuated inversion recovery ; and ( d and h ) : diffusion - weighted magnetic resonance imaging . this study identified 19 different heterozygous mutations in these 22 cmt1x patients [ table 2 ] . the c.44g > t , c.59t > g , c.62g > a , c.194a > g , c.379a > t , c.403_404inst , c.424c > t , c.425g > a , c.490c > t , c.533a > g , c.548g > a , c.547c > t , c.556g > a , c.590c > t , and c.614a > g mutations were reported previously . patients with c.115g > t , c.380t > a , and c.263c > a mutations were sporadic , and the mutation was not found in their parents . all novel mutations were not found in 1000 healthy controls and also not found in ncbi snp database , indicating that they were not benign polymorphisms . the novel missense mutations ( c.115g > t , c.380t > a , and c.263c > a ) were highly conserved in the cx32 proteins across all mammalian species . the pathogenicities of the novel missense mutations were predicted to be possibly damaging ( c.380t > a ) and probably damaging ( c.115g > t and c.263c > a ) by polyphen-2 and disease causing by mutation taster software . patient 12 with pure peripheral neuropathy and patient 13 with cns impairment had the same c.425g > a mutations . gjb1 mutations in 22 cmt1x patients from unrelated families in this study cmt1x : x - linked charcot - marie - tooth type 1 ; ec : extracellular domain ; tm : transmembrane domain ; ic : intracellular loop ; gjb1 : gap junction beta-1 protein . among 22 patients , the frequency of the gjb1 mutations was 4.5% in transmembrane domain 1 ( tm1 ) , 4.5% in tm2 , 22.7% in tm3 , 9.1% in tm4 , 4.5% in extracellular 1 ( ec1 ) , 27.3% in ec2 , 9.1% in intracellular loop , 13.6% in the n - terminal domain , and 4.5% in the c - terminal domain [ figure 2 ] . distribution of the respective amino acid changes on the connexin 32 protein structure identified in 22 patients with x - linked charcot - marie - tooth type 1 . the mean age of onset was 15.6 8.7 years ( ranging from 1 year to 42 years ) . two patients ( 9.1% ) had an earlier onset before 10 years old , including one from infancy . the mean duration from onset to diagnostic time was 8.2 4.5 years ( ranging from 2 years to 18 years ) . the clinical and electrophysiological characteristics of 22 chinese cmt1x patients in this study cmt1x : x - linked charcot - marie - tooth type 1 ; ll : low limbs ; ul : upper limbs ; + : positive ; : negative ; cns : central nervous system ; mcv : motor conduction velocity ; cmap : compound motor action potential ; scv : sensory conduction velocity ; snap , sensory nerve action potential ; ne : not elicited ; na : not available . all patients showed distal muscle wasting and weakness of the lower limbs , with the involvement of the upper limbs in 13 cases . muscle strength ranged from iii to v in distal upper limbs and from 0 to v in distal lower limbs . cns involvement appeared in five patients ( 22.7% ) , including four cases with transient cns symptoms and one case with constant cns signs . four cases ( 18.2% ) with transient symptoms presented with sudden onset of cerebral symptoms including aphasia , dysphagia , quadriplegia , or paralysis induced by fever or infection . the symptoms usually lasted from hours to days , and the patients completely recovered without special treatment . one case with constant cns involvement additionally presented with nystagmus and ataxia on physical examination . in median nerves , the mean motor nerve conduction velocity ( mcv ) was 35.0 4.8 m / s ( range : 27.343.9 m / s ) , the mean amplitude of compound muscle action potential ( camp ) was 2.3 1.5 mv ( range : 0.25.2 mv ) , the mean sensory nerve conduction velocity ( scv ) was 42.0 9.6 m / s ( range : 32.166.0 m / s ) , and the mean sensory nerve action potential ( snap ) amplitude was 5.0 4.2 mv ( range : 1.412.1 mv ) . in ulnar nerves , the mean mcv was 33.6 5.7 m / s ( range : 27.746.7 m / s ) , the mean camp amplitude was 3.7 3.2 mv ( range : 0.310.3 mv ) , the mean scv was 40.4 12.8 m / s ( range : 30.371.0 m / s ) , and the snap mean amplitude was 3.3 3.3 mv ( ranging from 1.47 mv to 9.6 mv ) . in tibial nerves , the mean mcv was 32.5 5.5 m / s ( range : 23.039.2 m / s ) and the mean camp amplitude was 1.1 1.7 mv ( range : 0.04.8 mv ) . the cranial magnetic resonance imagings ( mris ) of patient 2 revealed abnormal signals in the splenium and genu of the corpus callosum in the first episode and in the bilateral posterior limbs of the internal capsule and splenium of the corpus callosum in the second episode . the cranial mris of patient 3 revealed abnormal signals in the centrum semiovale bilaterally in the first episode , the signals enlarged at the same location in the second episode , and widened at the third episode ; the abnormal signals were resolved at intervals . the cranial mris of patient 4 revealed abnormal signals in the posterior limb of the internal capsule and the periventricular area bilaterally . the mris of patient 13 revealed abnormal signals in the bilateral centrum semiovale and splenium of the corpus callosum [ figure 1 ] . brain magnetic resonance imagings of a male patient with x - linked charcot - marie - tooth type 1 ( patient 13 ) revealed abnormal signals in the bilateral centrum semiovale ( a d ) and splenium of the corpus callosum ( e h ) . ( a and e ) : t1 weighted ; ( b and f ) : t2 weighted ; ( c and d ) : fluid attenuated inversion recovery ; and ( d and h ) : diffusion - weighted magnetic resonance imaging . this study identified 19 different heterozygous mutations in these 22 cmt1x patients [ table 2 ] . the c.44g > t , c.59t > g , c.62g > a , c.194a > g , c.379a > t , c.403_404inst , c.424c > t , c.425g > a , c.490c > t , c.533a > g , c.548g > a , c.547c > t , c.556g > a , c.590c > t , and c.614a > g mutations were reported previously . four ( c.115g > t , c.263c > a , c.380t > a , and c.818_819insgggct ) were novel mutations . patients with c.115g > t , c.380t > a , and c.263c > a mutations were sporadic , and the mutation was not found in their parents . all novel mutations were not found in 1000 healthy controls and also not found in ncbi snp database , indicating that they were not benign polymorphisms . the novel missense mutations ( c.115g > t , c.380t > a , and c.263c > a ) were highly conserved in the cx32 proteins across all mammalian species . the pathogenicities of the novel missense mutations were predicted to be possibly damaging ( c.380t > a ) and probably damaging ( c.115g > t and c.263c > a ) by polyphen-2 and disease causing by mutation taster software . patient 12 with pure peripheral neuropathy and patient 13 with cns impairment had the same c.425g > a mutations . gjb1 mutations in 22 cmt1x patients from unrelated families in this study cmt1x : x - linked charcot - marie - tooth type 1 ; ec : extracellular domain ; tm : transmembrane domain ; ic : intracellular loop ; gjb1 : gap junction beta-1 protein . among 22 patients , the frequency of the gjb1 mutations was 4.5% in transmembrane domain 1 ( tm1 ) , 4.5% in tm2 , 22.7% in tm3 , 9.1% in tm4 , 4.5% in extracellular 1 ( ec1 ) , 27.3% in ec2 , 9.1% in intracellular loop , 13.6% in the n - terminal domain , and 4.5% in the c - terminal domain [ figure 2 ] . distribution of the respective amino acid changes on the connexin 32 protein structure identified in 22 patients with x - linked charcot - marie - tooth type 1 . this study confirmed that cmt1x was a common form of inherited neuropathy in chinese patients with cmt because 23.9% of all 92 cmt families were cmt1x . this study observed that the proportion of cmt1x in chinese population was greater than other reports . the proportion of cmt1x patients for all cmts was 10.7% in europe ( n = 997 ) , 15.2% in the usa ( n = 527 ) , 12.0% in australia ( n = 224 ) , and 10.9% in japan ( n = 128 ) . in a study of multi - ethnic malaysian patients with cmts ( n = 25 ) , cmt1x patients were all chinese and accounted for 24% of the total cmt patients . in this study , all cmt1x patients showed distal muscle weakness predominantly in the lower limbs with a mean onset age of 15.6 8.7 years , similar to other reports . patients with an earlier onset before 10 years old or in infancy were rare in the present study and other reports . several types of hereditary neuropathies were associated with other organ involvement , such as distal motor neuropathy with optic atrophy , hereditary transthyretin amyloidosis with cardiomyopathy , and dominant inherited intermediate cmt with focal segmental glomerulosclerosis . dysarthria and hemiparesis were the main symptoms of our patients , and other similar symptoms have been occasionally reported in other countries . cns symptoms developed after peripheral neuropathy symptoms in some patients in the present study , which has also been reported in previous studies . however , the involvement of the corpus callosum which was frequently reported did not appear in all cases in the present study . the isolated involvement of the centrum semiovale , internal capsule , and the periventricular area appeared in the present study as well as in other reports . we found that the ec2 domain of cx32 protein was more affected in our patients . the ec2 domain was a hotspot mutation domain and was affected in 44% of korean patients , which was more frequent compared to our patients . mutations in the ec1 and ec2 domains of cx32 were in 65% of the patients with spanish or portuguese descent . however , ec1 and ec2 were not hotspot mutation domains in japanese and malaysian patients . we found no relationship between the position of mutations and cns involvement in cmt1x . in the present series , mutations of five patients with cns involvement were located in the n - terminal , tm1 , and tm3 domains of gjb1 gene . the c.425g > a mutation was found in patients with pure peripheral neuropathy as well as in patients with additional cns involvement . four novel mutations in gjb1 gene were found in our cohort , which expands the spectrum of mutations in cmt1x . in summary , the cmt1x diagnosis should be considered in children with transient cns impairment with or without any signs of peripheral neuropathy . mutations in the ec2 domain of the gjb1 gene were hotspot in chinese cmt1x patients . this study was supported by a grant from the national science foundation of china ( no . this study was supported by a grant from the national science foundation of china ( no .

background : x - linked charcot - marie - tooth type 1 ( cmt1x ) disease is one of the most common forms of inherited neuropathy caused by mutations in the gap junction beta-1 protein ( gjb1 ) gene ( also known as connexin 32 ) . this study presented the clinical and genetic features of a series of chinese patients with gjb1 gene mutations.methods:a total of 22 patients from unrelated families , who were referred to department of neurology , peking university first hospital from january 2005 to january 2016 , were identified with gjb1 mutations . their clinical records and laboratory findings were retrospectively collected and reviewed . mutations in the gjb1 gene were analyzed by targeted next - generation sequencing ( ngs ) . nucleotide alternations were confirmed with sanger sequencing.results:the cmt1x patients predominantly showed distal muscle weakness of lower limbs with mild sensory disturbance . the mean age of onset was 15.6 8.7 years ( ranging from 1 year to 42 years ) . the sudden onset of cerebral symptoms appeared in four patients ( 18.2% ) ; two were initial symptoms . one case had constant central nervous system ( cns ) signs . there were 19 different heterozygous mutations , including 15 known mutations and four novel mutations ( c.115g > t , c.380t > a , c.263c > a , and c.818_819insgggct ) . among the 22 chinese patients with cmt1x , the frequency of the gjb1 mutation was 4.5% in transmembrane domain 1 ( tm1 ) , 4.5% in tm2 , 22.7% in tm3 , 9.1% in tm4 , 4.5% in extracellular 1 ( ec1 ) , 27.3% in ec2 , 9.1% in intracellular loop , 13.6% in the n - terminal domain , and 4.5% in the c - terminal domain . cmt1x with cns impairment appeared in five ( 22.7% ) of these patients.conclusions:this study indicated that cns impairment was not rare in chinese cmt1x patients . mutations in the ec2 domain of the gjb1 gene were hotspot in chinese cmt1x patients .

, we will review the evidence for the association between mitochondrial disease and gi disorders without specific reference to asd . this will provide a framework to further discuss the link between gi symptomatology and mitochondrial dysfunction in asd discussed later in this article . several well - defined mitochondrial diseases with genetic underpinnings are strongly associated with gi abnormalities . we review the link between specific mitochondrial diseases and gi disorders , the importance of mitochondrial function for enterocyte function , and the evidence for gi abnormalities in mitochondrial disease in general . mitochondrial neurogastrointestinal encephalopathy syndrome is characterized by progressive gi dysmotility leading to pseudo - obstruction and severe gi symptomatology . this disorder is caused by a mutation in the tymp gene , a gene for thymidine phosphorylase , which results in nucleotide pool imbalances and mitochondrial deoxyribonucleic acid ( mtdna ) depletion . interestingly , pseudo - obstruction is also caused by mutations in the nuclear polg and tmem70 genes . the polg gene codes for the mtdna polymerase gamma that is responsible for replication of human mtdna , and mutations in polg also cause mtdna depletion . the tmem70 gene codes for etc complex v ; complex v is responsible for making the energy carrier of the cell known as adenosine triphosphate ( atp ) . researchers examining the prevalence of mitochondrial dysfunction in adults with chronic intestinal pseudo - obstruction have found that 15 of the 80 adult patients studied ( 19% ) demonstrated biochemical and/or histological findings consistent with a mitochondrial disease ( 54 ) . five demonstrated tymp mutations , five demonstrated polg mutations , and two demonstrated mtdna mutations consistent with mitochondrial myopathy , encephalopathy , lactic acidosis , and stroke - like episodes syndrome ( melas ) . genetic defects were not found in the remaining three patients with biochemical and/or histological evidence of mitochondrial disease . another study examining eight children with chronic intestinal pseudo - obstruction could not find a genetic mutation that could account for a mitochondrial disease ( 55 ) so it was presumed unlikely in this cohort . in addition , the absence of a genetic mutation does not rule out mitochondrial disease . indeed , functional testing of mitochondrial enzymes , which was not performed , would be needed to evaluate the true function of the mitochondria . cyclic vomiting syndrome has been associated with melas ( 56 , 57 ) , kearns sayre syndrome ( 58 ) , large mtdna deletions ( 59 ) , 3010a and 16519 t mtdna polymorphisms ( 60 , 61 ) , the a3243 g mtdna mutation ( 62 ) , and greater homoplasmic sequence variants in the mtdna termination associated sequence ( 63 ) . it also has a pattern of maternal inheritance consistent with mitochondrial disease ( 64 ) . several studies have suggested that the genetic basis of this disorder is different in children as compared to adults ( 64 , 65 ) . one study that looked at the prevalence of mitochondrial disorders in patients with cyclic vomiting syndrome found that 38% of patients had abnormalities in blood and/or urine suggesting evidence of mitochondrial dysfunction ( 66 ) . finally , effective treatments for cyclic vomiting syndrome overlap treatments used for mitochondrial disease including co - enzyme q10 , riboflavin , niacin , l - carnitine , and lipoic acid ( 6769 ) . thus , cyclic vomiting syndrome appears to be strongly linked to mitochondrial dysfunction in a significant number of cases . in fact , about 1020% of childhood mitochondrial diseases involve liver dysfunction ( 71 , 72 ) . perhaps , the most well known is alpers huttenlocher syndrome , a syndrome caused by polg mutations that presents with refractory seizures , developmental regression , and liver dysfunction ( 73 ) . this disorder is well known because valproic acid can trigger acute liver failure leading to death ( 74 ) . like polg mutations , other genetic abnormalities that impair mtdna replication , including c10orf2 , dguok , mpv17 , peo1 , and suclg1 , are part of the hepatocerebral mitochondrial dna depletion syndromes , which are syndromes that include prominent liver failure ( 70 , 75 , 76 ) . genetic defects resulting in defects of mitochondrial protein synthesis also cause liver failure ( 70 , 77 ) . one of these mitochondrial protein synthesis abnormalities caused by trmu gene mutations is unique in that the infantile - onset acute liver failure can spontaneously remit in many cases ( 78 ) . the best known of these is , perhaps , pearson syndrome , which is a mitochondrial disorder caused by large - scale mtdna rearrangements , which are characterized by exocrine pancreatic insufficiency , macrocytic anemia , and lactic acidosis ( 79 ) . however , other mitochondrial disorders caused by mtdna mutations have also been reported to involve pancreatic dysfunction ( 80 ) . endocrine pancreatic dysfunction in the form of diabetes mellitus has also been linked to several mitochondrial diseases ( 81 , 82 ) and research has suggested that insulin resistance , the basic abnormality in type 2 diabetes , involves mitochondrial dysfunction ( 83 ) . interestingly , the organic acidurias , which are disorders involving metabolic enzymatic defects resulting in accumulation of organic acids , appear to directly or indirectly involve mitochondrial dysfunction ( 38 ) and commonly involve gi symptoms . for example , propionic acidemia and other branched - chain organic acidurias , such as methylmalonic aciduria , isovaleric aciduria , and maple syrup urine disease , commonly manifest vomiting during acute crises and can manifest pancreatitis in severe cases ( 8486 ) , whereas ethylmalonic encephalopathy is characterized by chronic diarrhea ( 87 ) . interestingly , propionic acid , which is significantly elevated in propionic acidemia , can have direct effects on gi physiology . in animal models , propionic acid reduces gastric motility ( 88 ) , directly stimulates longitudinal colonic smooth muscle contractions ( 89 ) , induces rapid large amplitude phasic contractions followed by tonic contractions in the distal colon via serotonin and prostaglandin release ( 90 ) , and dilates colonic arteries resulting in a trophic effect on intestinal mucosa ( 91 ) . this suggests that non - specific gi symptoms associated with propionic acidemia such as feeding refusal , vomiting , weight loss , and abdominal distension could be a direct effect of propionic acid on gi motility through a disruption in mitochondrial function . perturbed enterocyte mitochondrial function is believed to initiate inflammation and relapses in inflammatory bowel disease , potentially through disrupting the balance between the enterocyte and the endogenous enteric microbiome ( 92 ) or by disrupting the active transport of luminal substrates or ion flux across the cell membrane . disruption of mitochondrial function may be responsible for a wide range of disorders involved in enterocyte dysfunction , including ( a ) enterocyte dysfunction following traumatic brain injury ( 93 , 94 ) ; ( b ) small bowel injury related to non - steroidal anti - inflammatory drug exposure ( 9597 ) , surgical manipulation ( 98 , 99 ) , intestinal allergic reaction ( 100 ) , and carnitine deficiency ( 101 ) ; ( c ) cyclooxygenase inhibitor - induced enteropathy ( 102 ) ; ( d ) small bowel dysfunction in chronic alcoholics with liver disease ( 103 ) , and rodent models of cirrhosis ( 104 ) and obstructive jaundice ( 105 ) ; ( e ) clostridium perfringens enterotoxin - mediated enterocyte cell death ( 106 , 107 ) ; ( f ) clostridium difficile toxin a ( 108 ) ; ( g ) methotrexate - induced enteritis ( 109 ) ; and finally , ( h ) cases of villous atrophy ( 110 , 111 ) . several studies have taken a broader look at gi function in mitochondrial disorders in children . a case series described six children who initially presented with gi dysmotility within 2 weeks of life who experienced later onset of neurological symptoms . all were found to have significant etc abnormalities without identifiable mtdna mutations or neuropathic gi abnormalities ( 112 ) . in another case series of 36 children with diagnosed mitochondrial disorders , 20 ( 56% ) were found to have gi abnormalities ( 113 ) . lastly , a rather elegant study found that , of 26 children with mitochondrial disorders , gastric emptying was delayed in 69% and intestinal transit time was prolonged in 46% ( 114 ) . interestingly , variations in mitochondrial function that do not cause frank disease may also be related to gi abnormalities . for example , one study found that specific mtdna polymorphisms were associated with variations in satiation , rate of gastric emptying , gi pain symptoms , and specific types of irritable bowel syndrome ( 115 ) . thus , the connection between mitochondrial dysfunction and gi abnormalities is rather strong . given that it is likely that a large proportion of children with asd have abnormal mitochondrial function , it is possible that many of the comorbid gi abnormalities associated with asd could be related , at least in part , to mitochondrial dysfunction . both the mitochondria and gi tract are sensitive to environmental and iatrogenic exposures , and some of the exposures that can affect both the mitochondria and gi tract have been linked to asd . in this section , we will review the known iatrogenic and environmental exposures that are associated with asd and affect both gi and mitochondrial function . there are many examples of iatrogenic exposures that have been linked to gi abnormalities , mitochondrial dysfunction , and asd . valproic acid is also known to cause hepatotoxicity ( 116 ) and pancreatitis ( 117 ) as well as being linked to mitochondrial disease and dysfunction ( 118 ) , and exposure in utero increases the risk of developing asd ( 119 ) . in fact , the prenatal valproic acid exposure rodent model of asd has been shown to manifest mitochondrial dysfunction ( 41 ) . however , valproic acid is one of the few medications with good evidence for treating seizures and behavioral symptoms in children with asd with a good safety profile ( 4 ) , so its mechanism and effect on the mitochondria are likely to be complex and dependent on age or stage of development . both prenatal and perinatal acetaminophen exposure ( 120 ) as well as acetaminophen exposure during childhood ( 121 ) have been associated with the development of asd . acetaminophen has also been associated with gi problems , including acute upper gi emergencies ( 122 ) and liver toxicity ( 123 ) , and is believed to cause hepatotoxicity through its effect on the mitochondria ( 124 ) . exposures to antibiotics , either early in life ( 125 ) or during pregnancy ( 126 ) , have been linked to the later developmental of asd , and some have suggested that early antibiotic exposure could cause asd ( 127 , 128 ) . many believe that mitochondria are evolutionarily derived from bacteria , leading to the potential for some antibacterial agents such as antibiotics to negatively influence host mitochondria ( 129131 ) . indeed , antibiotics , particularly quinolones , aminoglycosides , and beta - lactams , some of which are not uncommonly used to treat childhood and maternal infections , can cause mitochondrial dysfunction ( 132 ) and are well known to cause adverse gi effects as well as altering the developing infant gut microbiome ( 132134 ) . lastly , proton - pump inhibitors , which are used to treat gastroesophageal reflex , a disorder not uncommonly associated with asd , impair the transportation of carnitine , potentially interrupting carnitine metabolism ( 135 ) . in addition , proton - pump inhibitors can alter the ph of the gi tract . since various bacteria grow optimally at a narrow ph range , proton - pump inhibitors can alter the enteric microbiome . several environmental exposures that have been linked to asd can cause mitochondrial dysfunction and gi abnormalities . pesticides and heavy metals that have been linked to asd ( 13 ) also cause mitochondrial dysfunction and gi abnormalities . for example , in rodents , chlorpyrifos induces mitochondrial ultrastructure changes ( 136 ) and reduces mitochondrial enzyme function ( 137 , 138 ) , while chronic chlorpyrifos exposure increases gut permeability and bacterial translocation in rodents ( 139 ) . mercury induces mitochondrial - dependent apoptosis in human ( 140 ) and murine ( 141 ) cells , induces mitochondrial dysfunction in rodent liver and brain tissue ( 142 ) , impairs the carnitine transporter ( 143 ) , and reduces mitochondrial enzyme function in cells from murine ( 141 , 144 , 145 ) and zebrafish ( 146 ) . mercury causes dysfunction of human intestinal epithelium cells ( 147 ) and has been linked to chronic atrophic gastritis ( 148 ) while it also appears to significantly change the bacterial community structures in the gut microbiome in animal models ( 149 ) . thus , from this brief review of some environmental exposures that have been linked to asd , it is clear that certain such exposures have also been associated with both mitochondrial dysfunction and gi abnormalities . although most studies have not looked at a causative link between gi abnormalities and mitochondrial function , given the fact that mitochondrial dysfunction can cause gi abnormalities , including gastric dysmotility , hepatic and pancreatic dysfunction , and abnormalities in enterocyte function , it is possible that mitochondrial dysfunction is the primary abnormality leading to the gi dysfunction associated with these exposures . the human digestive tract is host to a complex array of intestinal bacterial florae that outnumber host cells by a factor of at least 10 to 1 , and over 100 to 1 regarding the amount of genetic material . this ecosystem , termed the enteric microbiome , behaves as a functional organ and produces a diverse array of bioactive metabolic products capable of entering the systemic circulation . it is important to note that the enteric microbiome and its metabolic products are not static and can be altered throughout the life cycle of the individual , with the first 18 months of life being an especially important time for the development of a stable enteric microbial ecosystem ( 150 ) . the metabolic products from the enteric microbiome can have profound and dynamic effects on host metabolism , immune function , and gene expression in many organ systems , including the gut and brain ( 151153 ) . many authors have proposed a connection between the gut microbiome and asd ( 39 , 133 , 134 , 151 , 154156 ) . there are several studies that point to overrepresentation of enteric bacteria , particularly clostridia spp . , in children with asd ( 157161 ) , especially those with a regressive asd phenotype ( 162 , 163 ) and/or those children who present with gi symptoms at or before the onset of asd symptoms ( 164 ) . in addition , a small clinical trial demonstrated that treatment with vancomycin , an antibiotic aimed at decreasing clostridia spp . are producers of the short - chain fatty - acid propionic acid ( 39 , 40 ) following the fermentation of dietary carbohydrates and some proteins . propionic acid , as well as other short - chain fatty - acid bacterial fermentation products ( e.g. butyric and acetic acid ) , are compounds that are increasingly recognized as being important in the maintenance of health and have been implicated as possible contributing factors for certain disease processes ( 40 , 166 , 167 ) . in particular , propionic acid can modulate cell signaling ( e.g. specific free fatty acid g - protein - coupled receptors ) ( 168 , 169 ) , cell cell interactions ( e.g. gap junctions ) ( 170 ) , gene expression ( e.g. histone deacetylase inhibition ) ( 171 , 172 ) , immune function ( 173 ) , and neurotransmitter synthesis and release ( 174 ) as well as influence mitochondrial ( 175 ) and lipid ( 176 , 177 ) metabolism . interestingly , neurodevelopmental abnormalities that include asd features are seen in individuals with impaired propionic acid metabolism ( 175 , 178 , 179 ) . furthermore , elevated propionic acid levels are present in the stool from individuals with asd ( 180 ) . interestingly , propionic acid is also endogenously present or added as a food preservative to a wide variety of foods including refined wheat and dairy products ( 181187 ) . of note , propionic acid and its chemical derivatives have increasing use in agriculture and the food industry ( 166 ) and occur naturally in many foods ( e.g. swiss cheese ) . it is a major animal silage and food preservative in wheat and dairy products , either as a sodium or calcium salt ( 188 , 189 ) . propionic acid is also produced by adding high fructose corn syrup substrate to propionibacteria cultures , which are then inoculated into foods . inulin propionate has recently been suggested as a weight loss agent ( 190 , 191 ) , and aspartame is known to increase propionic acid levels in rodent gut flora . nitropropionic acid , a derivative of propionic acid produced by many plants and fungi , is a potential contaminant of processed rice and also produced in ruminant gut . it is a potent mitochondrial toxin , capable of causing neurotoxicity and administration in rodents is an acceptable model for huntington 's chorea ( 192 ) . in addition , propionate is an important naturally occurring intermediate of odd - chain length fatty - acid oxidation . recently , we have developed an animal model of asd ( 39 , 40 ) . in the initial model , brief intracerebroventricular pulsed infusions of propionic acid into adult animals produced reversible ( ~30 min ) bouts of asd - type behaviors including altered social interactions ( 193 ) , stereotyped behavior ( 194 ) , tics ( 194 ) , and hyperactivity ( 194 , 195 ) as well as other cognitive and sensorimotor deficits ( 196 ) [ see fig . 1 of ( 40 ) for behavioral videos of propionic acid rodent model of asd , link : http://www.ncbi.nlm.nih.gov/pmc/articles/pmc3747729/figure/f0001/ ] . the propionic acid rodent model of asd also demonstrates biological abnormalities associated with asd such as reactive astrocytosis and activated microglia ( 193 , 194 , 196 ) as well as abnormalities in redox , lipid , phosphatidylethanolamine , mitochondrial , acyl - carnitine , and carnitine metabolism ( 177 , 194 , 195 , 197 ) . electrographic abnormalities are also seen , specifically epileptiform - like spikes in the hippocampus , neocortex , and basal ganglia , with discharges in the basal ganglia associated with measurable extrapyramidal behavioral abnormalities ( 194 ) . further development of this model shows that brief intracerebroventricular infusions of propionic acid into adolescent rats results in similar abnormalities in behavior , including abnormal restricted and repetitive behavior , altered social behavior , object vs. social preference , and cognitive abnormalities as well as asd - like immunohistochemical evidence of innate neuroinflammation ( 198 ) . to further validate this developmental model of asd such exposure was found to alter conditioned taste and place avoidance ( 199 ) , acoustic startle response and pre - pulse inhibition ( 200 ) and induced an increase in anxiety , repetitive , and impaired social behavior ( 201 , 202 ) , in rats as adolescents in a sexually dimorphic manner . in addition , propionic acid , and to a lesser extent butyric acid , modulates the expression of genes associated with asd , including cell adhesion molecules ( neurexin 1 and neuroligin ) , neurotransmitter systems , mitochondrial , redox , immune , and fmr1 genes in pc12 cells ( 172 ) . one of the unique biochemical markers of this rodent model was also independently reported in children with asd ( 18 ) , suggesting that this model has predictive validity . a pattern of abnormalities in fatty - acid metabolism characterized by elevations in short - chain and long - chain , but not medium - chain , acyl - carnitines were found in brain tissue of adult rats intracerebroventricularly infused with propionic acid ( 177 ) . a similar pattern of elevation in acyl - carnitines was reported in children with asd in a study that reviewed a wide range of metabolic markers from 133 consecutive patients evaluated in a medically based autism clinic ( 18 ) . a standardized metabolic screening algorithm was used ( 203 , 204 ) and abnormalities were verified with repeat testing ( 203 ) . the workup included screening for fatty - acid oxidation defects by measuring a fasting acyl - carnitine panel . consistent acyl - carnitine abnormalities were present in 24% of the patient sample . when the individuals with acyl - carnitine abnormalities were pooled , a specific pattern of abnormalities in acyl - carnitines was found that paralleled the acyl - carnitine elevation in the adult rodent propionic acid model of asd : short - chain and long - chain acyl - carnitines but not medium - chain acyl - carnitines were elevated . interestingly , 67% of this subset of children with asd demonstrated neurodevelopmental regression , which is a particularly high rate compared to the general asd population [ about 25% ( 8) ] . to further verify this pattern of abnormalities , we reviewed 213 patients with asd seen in a medically based clinic who underwent a metabolic evaluation similar to the one noted above ( 19 ) . of the 213 patients screened , 17% were found to have consistent elevations in acyl - carnitines . when the data were combined across patients , c4oh , c14 , and c16:1 were found to be significantly elevated at 186% , 226% , and 131% of the upper limit of normal . this subset of asd patients with consistent elevations in short and long acyl - carnitines ( ceslac ) was evaluated further . further testing of individuals with asd and ceslac ruled - out secondary causes of fatty - acid oxidation deficiencies including multiple carboxylase deficiencies , zinc deficiency , elevated copper , generalized hyperlipidemia or hypercholesterolemia , and hypoglycemia . citrate and/or isocitrate was elevated in most ceslac patients , and lactate was elevated in about half of the ceslac patients . genetic testing demonstrated a 22q13.1-q13.33 duplication in one ( 31 ) and suspicious novel maternally inherited homoplasmic variants of unknown significance in the cytochrome b gene in two ( 205 ) . thus , overall , no clear genetic factors seem to be consistent within these ceslac patients that would explain their pattern of metabolic abnormalities . five ceslac patients who underwent muscle biopsy had abnormal muscle histology , and electron microscopy of the muscle demonstrated abnormal mitochondria . etc studies conducted on the muscle ( 206 ) demonstrated a partial defect in etc complex i and complex i + iii . the reason for the pattern of etc abnormalities , particularly a decrease in etc complex i function , is not exactly clear , but one of the hypothesized mechanisms in the context of elevated propionic acid has been outlined in our recent publication ( 19 ) . the significant inhibition of etc complex i may be closely related to a decrease in the production of the reduced form of nicotinamide adenine dinucleotide ( nadh ) since nadh is the driving force for etc complex i ( fig . 1 ) . we believe that this is due to decreased activity of two enzymes in the citric acid cycle that produce nadh , specifically , isocitrate dehydrogenase and -ketoglutarate dehydrogenase ( fig . the reason for this is outlined below . the tricarboxylic acid cycle during typical metabolism . carbohydrates and fatty acids enter the cycle as acetyl - coa and through a series of enzymatic steps produce energy utilizing two electron carriers , nicotinamide adenine dinucleotide ( nadh ) and flavin adenine dinucleotide ( fadh2 ) . nadh and fadh2 are metabolized by complex i and complex ii , respectively , of the electron transport chain ( etc ) . complex v of the etc produces adenosine triphosphate ( atp ) , the energy carrier of the cell . using acetyl - coa , propionic acid can produce propionyl - coa , a compound that can be further metabolized to methylmalonyl - coa ( fig . methylmalonyl - coa is important because it can enter the citric acid cycle after being metabolized into succinyl - coa ( fig . thus , high levels of propionic acid can be used to bypass the first four enzymes of the citric acid cycle , thereby functionally this results in a bypass of important steps of the citric acid cycle ( including two enzymes that make nadh ) and elevates succinyl - coa concentrations that can further negatively modulate these bypassed enzymes through several mechanisms . first , high levels of succinyl - coa will result in high levels of citric acid cycle intermediates that precede it in the cycle , such as -ketoglutarate , isocitrate , and citrate ( the latter two were found to be elevated in this asd patient cohort ) because their breakdown is stoichiometrically inhibited by high levels of their breakdown products . second , succinyl - coa directly inhibits citrate synthase , the first step in the citric acid cycle ( fig . 2 ) as well as -ketoglutarate dehydrogenase , the citric acid cycle enzyme that produces it and an enzyme that produces nadh ( fig . , elevated levels of -ketoglutarate will inhibit isocitrate dehydrogenase , the citric acid cycle enzyme that produces it and another enzyme that produces nadh ( fig . 2 ) . the tricarboxylic acid cycle during high levels of propionic acid . propionic acid , presumably derived from clostridia spp . , is metabolized to propionyl - coa using acetyl - coa . propionyl - coa is further metabolized into methylmalonyl - coa , which enters the tricarboxylic acid cycle as succinyl - coa . succinyl - coa inhibits the first and fourth enzyme in the tricarboxylic acid cycle . in this manner , short circuit the tricarboxylic acid cycle , thereby reducing the production of nicotinamide adenine dinucleotide ( nadh ) . this decrease in nadh is hypothesized to cause the decrease in complex i activity measured in the patients with consistent elevations in short and long acyl - carnitines ( ceslac ) . thus , an increased succinyl - coa concentration as a result of high levels of propionic acid can shut down the first half of the citric acid cycle and prevent the production of two of the three nadh molecules typically produced by the citric acid cycle . as etc complex i transfers electrons from nadh , whereas etc complex ii transfers electrons from flavin adenine dinucleotide in the hydroquinone form ( fadh2 ) , it is not surprising that etc complex i is found to be underactive ( especially relative to etc complex ii ) in the muscle of ceslac patients . the elevations in acyl - carnitines in ceslac patients suggest that at least some specific parts of fatty - acid oxidation are inhibited . this is important as each turn of -oxidation ( the key reaction of fatty acid oxidation ) produces three nadh for each two fadh2 , so inhibition of -oxidation will further decrease nadh relative to fadh2 . the products of fatty - acid oxidation and how they enter the citric acid cycle are dependent on the length of the fatty acid , specifically whether the fatty acid is odd or even in length . each turn of -oxidation shortens the fatty acid by a length of two carbons and produces one acetyl - coa . this is the same for both odd and even length fatty acids until the last step when the fatty acid is reduced to either a two- or three - carbon fatty acid . if the fatty acid is even in length , then the last step in the breakdown of the fatty acid produces an acetyl - coa , while the last step in the breakdown of an odd length fatty acid results in propionyl - coa . the significant elevations in the acyl - carnitines in this cohort were composed of even length fatty acids , suggesting that the breakdown of acetyl - coa is inhibited relative to propionyl - coa . this is consistent with the notion that the alternative pathway that short circuits the citric acid cycle is upregulated . the fact that acetyl - coa breakdown may be dependent on the availability of propionic acid when this alternative pathway is upregulated means that -oxidation may be stoichiometrically inhibited if propionic acid is not available . if the source of propionic acid is the metabolic fermentation products of certain enteric bacteria , then the level of propionic acid will depend on enteric factors that may vary from day - to - day and hour - to - hour , such as diet , transcolonal uptake , or gi transport time . the reason why medium - chain fatty acids are not particularly elevated in this cohort is not clear at this time . examination of the -oxidation pathway in fibroblasts did not demonstrate any consistent abnormalities across patients examined ( 207 ) , again pointing to an acquired functional inhibition of the fatty - acid oxidation pathway rather than a fixed deficit in this pathway . overall , the elevation in acyl - carnitines in ceslac patients appear to not be due to enzymatic defects but rather due to a functional abnormality related to altered metabolism . ceslac patients also demonstrated significant changes in glutathione metabolism suggesting increased oxidative stress and increased cellular vulnerability to reactive oxygen and nitrogen species . first , aconitase , the second enzyme in the citric acid cycle , is redox sensitive and its activity has been shown to correlate with the glutathione redox ratio in the brain of individuals with asd ( 22 ) . thus , the abnormal redox state of the cell could also be inhibiting the specific mitochondrial enzymes that appear to be dysfunctional in ceslac patients and in a similar manner could affect the mitochondria in the brain tissue in the propionic acid animal model of asd ( 19 , 40 , 195 ) . interestingly , etc function in fibroblasts from the ceslac patients was almost opposite from the results from the muscle , with complex i activity being elevated rather than depressed . however , fibroblasts are grown in culture for approximately 6 weeks , thereby representing mitochondrial function without the influences of any systemic metabolites or modulators , whereas muscle etc function is derived from freshly frozen muscle that essentially reflects the influences of the body in situ . thus , the fibroblast results suggest that the enzymes themselves are not dysfunctional , but rather that they are being actively inhibited by the in situ environment of the human body . thus , the detailed examination of ceslac patients reveals unique changes in etc and citric acid cycle function consistent with the excess metabolic flux of propionic acid ( 19 ) . theoretically , propionic acid can be overproduced by the overrepresented species of clostridia found in the gi tract of children with asd ( 39 , 157 , 180 ) and could result in mitochondrial dysfunction . systematic effects of propionic acidemia , an organic acidemia in which propionic acid builds up excessively , are being recognized to be due , in part , to mitochondrial dysfunction ( 38 ) . the propionic acid rodent model of asd demonstrates asd - like behavioral abnormalities as well as other biochemical and immune abnormalities associated with asd ( 40 ) . our recent study demonstrated that short - chain fatty acids , such as propionic acid , can modulate the expression of many asd implicated genes , including those involved in mitochondrial function ( 172 ) . propionic acid can have direct effects on gi motility ( 8890 ) and perfusion ( 91 ) reminiscent of gi abnormalities associated with asd ( 40 , 194 ) . interestingly , behavioral and gi symptoms in asd have been reported to improve when simple carbohydrates , which can be fermented by enteric bacteria to produce propionic acid , are eliminated from the diet ( 40 , 208 ) or when propionic acid producing bacteria are transiently eradicated by antibiotics ( 165 ) . this is clearly a novel area of asd research that requires further study in patients with asd and with additional animal studies . given these insights into the relationship between mitochondrial dysfunction and gi abnormalities in asd , there may be therapeutic avenues that can be explored for at least some children with asd and these underlying conditions . given that propionic acid is a short - chain fatty acid that can complex with l - carnitine , increased propionic acid production could also potentially explain the common relative carnitine deficiency documented in children with asd ( 19 , 39 , 40 ) . interestingly , clinical trials , including two double - blind placebo - controlled trials , have demonstrated that asd symptoms can be improved with l - carnitine treatment ( 33 , 209215 ) , with one trial demonstrating that the improvement in symptoms was related to the improvement in the l - carnitine levels in the blood . however , l - carnitine also increases the transport of fatty acids across the enterocytes into the body and has been suggested to be potentially detrimental if unhealthy fatty acids are ingested ( 216 ) . thus , it is important to consider that supplementation with l - carnitine could increase the flux of propionic acid from the gut into the body in the presence of bacteria that produce this metabolite . this could theoretically explain some of the adverse effects seen with l - carnitine clinical trials in some children with asd . acetyl l - carnitine , which transports the beneficial acetate moiety for driving tca function , may theoretically be superior , but this remains to be explored . still , it is possible that propionic acid could potentially be used as a fuel for the mitochondria , if mitochondrial metabolism was able to adapt accordingly , and this might be dependent on the form in which it is delivered . for example , propionyl - l - carnitine , but not l - carnitine or propionate , is therapeutic to intestinal pathology in ulcerative colitis ( 217 ) . lastly , the gi pathology in a rodent model of colitis induced by early exposure to propionic acid or trinitrobenzene sulfonic acid was reduced by pre - administration of l - carnitine ( 218 , 219 ) . interestingly , the ketogenic diet has been shown to improve asd - like symptoms in mouse models of asd , including the btbr ( 220 ) , el ( 221 ) , and prenatal valproic acid exposure ( 41 ) mouse models . the ketogenic diet has been rated as very effective for controlling seizures and improving behavior and cognition in children with asd ( 222224 ) and has been recommended for children with asd , especially those with epilepsy , in several systematic and expert literature reviews ( 4 , 225 ) . the ketogenic diet has been found to regulate key signaling pathways that both enhance mitochondrial function and promote neuroprotection ( 226 ) and is recommended for a wide variety of mitochondrial disorders ( 227 , 228 ) . with special reference to the novel mitochondrial disorder we have described in the ceslac patients , several lines of research suggest that the ketogenic diet may be particularly useful for complex i defects ( 229 , 230 ) . interestingly , the finding that the main high affinity blood brain barrier transporter of propionic acid can be competitively inhibited by hydroxybutyrate ( 231 ) gives a potential mechanism where ketogenic diets may work through competitive inhibition of propionic acid transport - mediated entry into the brain . effective treatments for cyclic vomiting syndrome , a disorder that most likely has heavy mitochondrial underpinnings , especially in children , overlap with treatments used for mitochondrial disease including co - enzyme q10 , riboflavin , niacin , l - carnitine , and lipoic acid ( 6769 ) . some of these same treatments have reportedly been found to improve asd symptoms , including l - carnitine ( 33 , 209215 ) , a multivitamin containing riboflavin , niacin and coenzyme q10 ( 232 , 233 ) , and ubiquinol ( 234 ) . there is growing interest in using typical enteric microbiome flora such as lactobacillus , which have a natural ability to bind to toxic heavy metals as a means for protecting against exposures ( 235 ) . in an open - label trial , lactobacillus rhamnosus gr was protective against increasing levels of blood mercury and arsenic in pregnant women , but not children ( 236 ) . given the wide use of probiotics in children with asd and the growing excitement as a result of recent animal models of asd ( 237 ) , further research in this area might be promising . melatonin is widely used for assisting with sleep initiation in children with asd and has been shown to be effective for improving sleep in several controlled clinical trials ( 238 , 239 ) . besides the hormonal effects of melatonin , it also appears to have antioxidant and anti - inflammatory properties as well as modulating mitochondrial function ( 240 ) . interestingly , melatonin is produced in and has receptors throughout the gi tract ( 240 ) and has been shown to modulate serotonin - induced contractions and gastric glandular mucosal blood flow ( 241 ) . melatonin is being investigated for its role in protection and healing of the gi tract ( 242 ) . thus , melatonin could be especially helpful for children with asd who have comorbid gi and mitochondrial abnormalities . small bowel injury related to surgical manipulation appears to involve mitochondrial dysfunction within enterocytes ( 98 , 99 ) . several animal studies have suggested that supplementation with metabolic intermediates can protect the bowel from injury in animal models through a mitochondrial mechanism . supplementation with -ketoglutarate improves intestinal morphology , antioxidant capacity , and parameters of mitochondrial energy production in the lipopolysaccharide - induced intestinal damage animal model ( 243 ) . interestingly , -ketoglutarate can be converted into glutamate , another metabolic intermediate and a neurotransmitter , by mitochondrial glutamate dehydrogenase in the gi tract . glutamine is an important nutrient and fuel for the gi tract during illness , stress , and injury and can be produced by glutamate and ammonia . pretreatment with glutamine prior to surgical gut manipulation in rodents prevents gut and lung injury due to oxidative and inflammatory processes and protects enterocyte mitochondria ( 244 ) . interestingly , the protective effect of enterocyte mitochondria during surgical stress may also be related to nitric oxide production as pretreatment with l - arginine protects enterocyte mitochondria from the effects of intestinal manipulation , but not when nitric oxide synthase is inhibited ( 245 ) . this manuscript outlines several of the connections between mitochondrial dysfunction , enteric microbiome abnormalities , and gi abnormalities in relation to asd . there are many potential interactions between basic biological mechanisms and disease etiologies , which can result in symptoms of asd and gi tract abnormalities as well as mitochondrial dysfunction . considering the effect of the microbiome and its metabolic products adds another layer of complexity onto an already complex story . understanding the interaction between asd and gi symptoms in the context of mitochondrial dysfunction can provide a greater understanding of the underlying pathophysiology and how biological abnormalities are related to the complex behavioral manifestations characteristic of asd . considering the potential etiologies that can cause mitochondrial dysfunction , asd and/or gi tract dysfunction can provide insight into the possible etiological factors contributing to asd . lastly , considering treatments that can address these underlying abnormalities may lead to the development of new treatments that reduce symptoms and improve cellular metabolism . this research was supported by the arkansas biomedical institute ( ref ) , autism canada ( dfm ) , autism research institute ( dfm , ref ) , and goodlife children 's charities ( dfm ) .

autism spectrum disorder ( asd ) affects a significant number of individuals worldwide with the prevalence continuing to grow . it is becoming clear that a large subgroup of individuals with asd demonstrate abnormalities in mitochondrial function as well as gastrointestinal ( gi ) symptoms . interestingly , gi disturbances are common in individuals with mitochondrial disorders and have been reported to be highly prevalent in individuals with co - occurring asd and mitochondrial disease . the majority of individuals with asd and mitochondrial disorders do not manifest a primary genetic mutation , raising the possibility that their mitochondrial disorder is acquired or , at least , results from a combination of genetic susceptibility interacting with a wide range of environmental triggers . mitochondria are very sensitive to both endogenous and exogenous environmental stressors such as toxicants , iatrogenic medications , immune activation , and metabolic disturbances . many of these same environmental stressors have been associated with asd , suggesting that the mitochondria could be the biological link between environmental stressors and neurometabolic abnormalities associated with asd . this paper reviews the possible links between gi abnormalities , mitochondria , and asd . first , we review the link between gi symptoms and abnormalities in mitochondrial function . second , we review the evidence supporting the notion that environmental stressors linked to asd can also adversely affect both mitochondria and gi function . third , we review the evidence that enteric bacteria that are overrepresented in children with asd , particularly clostridia spp . , produce short - chain fatty acid metabolites that are potentially toxic to the mitochondria . we provide an example of this gut brain connection by highlighting the propionic acid rodent model of asd and the clinical evidence that supports this animal model . lastly , we discuss the potential therapeutic approaches that could be helpful for gi symptoms in asd and mitochondrial disorders . to this end , this review aims to help better understand the underlying pathophysiology associated with asd that may be related to concurrent mitochondrial and gi dysfunction .

the health status of any community is influenced by the interplay of health consciousness of the people , socio - cultural , demographic , economic , educational and political factors . the common beliefs , traditional customs , myths , practices related to health and disease in turn influence the health seeking behavior of autochthonous people . a tribe is defined as a collection of families bearing a common name , speaking a common dialect , observing certain taboos regarding marriage , occupying or professing to a common territory and is not usually endogamous though originally it might have been so . nutrition is an essential and important component in human growth , development and in maintaining healthy life . tribal communities are highly disease prone and do not have required access to basic health facilities which leads to high degree of malnutrition . this is further aggressive and critical because of 8% ethnic share in 70% of rural population as reported by nnmb ( 1978 ) to take unbalanced diet because of poverty stress . it is reported that , these tribal societies are very much ignorant about their oral health problems . dental caries is an irreversible microbiologic disease of calcified tissues of teeth characterized by demineralization of inorganic portion and destruction of the organic substance of the tooth which often leads to cavitation . dental caries have historically been considered an important component of the global disease burden especially in developing countries , affecting 6090% of the children and the vast majority of adults . in india , the prevalence of dental caries is reported to be 5060% . the data of comprehensive national health survey conducted in 2004 in india showed 51.9% of 5-year - old children , 53.8% of 12-year - old children and 63.1% of 15-year - old teenagers are affected by dental caries . madhya pradesh is a home land of many tribal races namely bhil , bhilala , pateilia have been identified as special primitive tribal groups . the other main tribal groups include gond followed by baiga , korku , bhariya , halba , kaul , mariya , and sahariya , katkaris , koknas , kolis and warlis . among these ethnic races , the bhil , bhilala , pateilia are more sensitive to nutrition and health issues because of their diet , dependence on forest , forest produce , traditional unproductive agricultural practices only in monsoon , unbalanced diet deficient in micronutrient s and non availability of modern health services resulting into burden of various diseases targeting specifically to children . prevalence of hunger and malnutrition among the children are always there in their hamlets for years together due to their different linguistic , cultural and geographical environment , their forest habitats , extreme poverty , nutritional taboos and its peculiar complications ; the diverse tribal world of madhya pradesh has been largely cut - off from the mainstream of development the subject gains more importance in tribal children , due to certain adverse realities like insufficient food intake , frequent infections , lack of access to health services , illiteracy , unhygienic personal habits , adverse cultural practices etc . health of people is strongly influenced by the social and economical environment in which they live . burden of all diseases was more in disadvantaged and socially marginalized individuals oral diseases are the most common of chronic diseases and an important public health problem . till to date , there are no reports found in the scientific literature on oral health of these tribes of madhya pradesh . hence , this study was undertaken to determine the association of body mass index with dental caries among malnourished tribal children of indore division ( m.p . ) . a descriptive cross - sectional house to house survey was carried out among 275 study subjects , 615 years old tribal children in two major tribal districts of indore division , madhya pradesh , india . the sample size estimation was based on the pilot study conducted to assess the feasibility and standardization of data collection . considering the caries prevalence of 60 percent among 20 subjects of pilot study , 5% level of confidence and 0.8 power the estimated sample size was 256 . a 10 percent increase in sample size was done taking into account the remote places and non - cooperation of study participants . the ethical clearance was obtained by the institutional ethical committee of sri aurobindo college of dentistry , indore . permissions for conducting the study and clinical examinations on tribal children were also obtained from administrative authorities of both the districts . only children with confirmed malnourishment were considered for the study . only those participants whose parents or caregivers provided consent and were willing to participate were included in the study . any condition which contraindicated or did not allow oral examination in the study subject was considered for exclusion . anthropometric measurements such as height and weight for each tribal child were recorded for calculating body mass index ( bmi ) . the who international growth chart was used to evaluate the nutritional status . z scores for boys and girls aged 519 years were generated in the who growth chart . a cut off of 2 sd was used to identify children at significant risk for either inadequate growth or excessive growth . so a child who s bmi was-2 sd was considered malnourished and children below -3 sd were considered severely malnourished . also , who classification of malnutrition ( also called chronic energy deficiency ) using body mass index ( bmi ) was used to categorize malnutrition into mild , moderate and severe . a house - to - house survey with the help of local authorities was planned in the villages selected for the study . convenience sampling was employed to select the required sample size of 275 tribal children aged between 6 to 15 years . oral examination was performed under adequate illumination ( type iii ) , using a mouth mirror and who cpi probe . the data on periodontal status , dentition status and treatment needs were recorded using who 1997 proforma . the subjects were grouped into excellent , good and watch out zone based upon sugar sweet score . the data collected was entered into microsoft excel and analyzed using spss 20.0 software ( ibm , chicago ) . caries experience and gingival findings were compared among age and gender using analytical tests like anova and chi - square test . independent effects of age , gender , bmi , oral hygiene status and daily intake sugar on caries status were tested using logistic regression analysis . anthropometric measurements such as height and weight for each tribal child were recorded for calculating body mass index ( bmi ) . z scores for boys and girls aged 519 years were generated in the who growth chart . a cut off of 2 sd was used to identify children at significant risk for either inadequate growth or excessive growth . so a child who s bmi was-2 sd was considered malnourished and children below -3 sd were considered severely malnourished . also , who classification of malnutrition ( also called chronic energy deficiency ) using body mass index ( bmi ) was used to categorize malnutrition into mild , moderate and severe . a house - to - house survey with the help of local authorities was planned in the villages selected for the study . convenience sampling was employed to select the required sample size of 275 tribal children aged between 6 to 15 years . oral examination was performed under adequate illumination ( type iii ) , using a mouth mirror and who cpi probe . the data on periodontal status , dentition status and treatment needs were recorded using who 1997 proforma . the subjects were grouped into excellent , good and watch out zone based upon sugar sweet score . the data collected was entered into microsoft excel and analyzed using spss 20.0 software ( ibm , chicago ) . caries experience and gingival findings were compared among age and gender using analytical tests like anova and chi - square test . independent effects of age , gender , bmi , oral hygiene status and daily intake sugar on caries status were tested using logistic regression analysis . the results are based on the data collected from 275 tribal children of dhar and jhabua district of indore division ( m.p ) . the caries prevalence among permanent dentition of malnourished children was 61.4% with a mean of 1.61 ( 0.48 ) . severity of malnourishment can be seen more in bhil tribes ( 60.6% ) compared to bhilala ( 25.5% ) and patelia tribes ( 13.8% ) . the oral hygiene behavior in the majority of study subjects were cleaning tooth with toothpowder and finger ( 49% ) . only 20 % malnourished tribal children uses toothbrush and toothpaste as an oral hygiene measure . there is a statistically significant difference found among age , bmi , oral hygiene means and sugar intake in relation to their tribes ( p<0.05 ) as shown in table i. children belonging to bhilala tribes suffering from severe malnourishment exhibited higher mean caries experience ( 1.820.38 ) ( p<0.01 ) . increase in caries experience was seen with increase in severity of malnourishment ( p<0.005 ) as illustrated in table ii . there is a statistically significant difference found between age , dental caries and cpi score in relation to body mass index ( p<0.05 ) . children belonging to younger age group ( 68.4% ) had severe malnourishment compared to children belonging to older age group ( 44.9% ) . children suffering from severe malnourishment had more dental caries ( 43.8% ) ( p<0.05 ) . the cpi scores indicating the presence of bleeding conditions in 61% severely malnourished children followed by calculus ( 45% ) ( p<0.05 ) are shown in table iii . the regression model adjusting for all non - significant factors indicated age , gender and daily sugar intake to be significantly associated with dental caries ( p<0.05 ) shown in table iv . the treatment needs among the study subjects indicated that out of 275 malnourished tribal children 61.5% required some forms of treatment ; the major treatment required was two surface filling ( 23.8% ) followed by one surface filling ( 16.2% ) , pulp care / restoration ( 11.7% ) , extraction ( 7.3% ) and others ( 2.5% ) as illustrated in figure 1 . the present study was carried out to determine the association of body mass index with dental caries among the major tribal community of madhya pradesh who have been discriminated and marginalized due to several historical , cultural and socio - economic reasons . the malnourished tribal children population aged 6 to 15 years belonging to the dhar and jhabua tribal districts were studied and reported . these children were mainly belonging to three main tribal communities namely : bhil , bhilala and patelia tribes . among them , bhil tribe children were more common . it is an establish fact that both malnutrition and dental caries are complex conditions and many biologic , genetic , environmental and behavioral factors are known to be involved . our study reported that increase in caries prevalence was seen with increase in severity of malnourishment . have also reported that the children with less bmi score tend to have more caries affected teeth than children with normal bmi . the reason could be low literacy rates , no access to dental care , lack of balanced diet , lack of awareness about proper oral hygiene . malnutrition reduces the secretion rate of stimulated saliva and chronic malnutrition in growing children enhances the cariogenic potential stemming from fermentable carbohydrate . above all , a collaboration of etiological factors causing dental caries and already hypothesized immunological complex mainly lead to the occurrence of dental caries . on the contrary , pinto a et al ; our study also found female were more malnourished and had more occurrence of dental caries compared to males ( p<0.05 ) . the female children are underfed due to gender inequalities and it is a well known fact that female infanticide is prevalent in india . the results of the present study should be carefully generalized to other similar populations as this study may have some limitation of small study population and convenience sampling . the cross - sectional design of this study is also one of the limitations of this study as it does not allow establishment of the direction of the association . additionally , the children included in the study were all coming from deprived areas ; hence , the variation in the impact of socioeconomic position on the children s growth was not fully captured . therefore , research findings that link oral health to general health problems specific to low - income developing countries present an opportunity to put oral health on the agenda of health care policies . the association of untreated dental caries with undergrowth among deprived children in madhya pradesh observed in this study highlights the importance of integrating oral health policies with general health policies and with different social , political and environmental policies to address a cluster of health problems that share common determinants . this study found a high prevalence of dental caries among tribal children of madhya pradesh . caries experience was more prevalent in malnourished tribal children and was seen to increase with severity of malnourishment . underweight and dental caries are public health issues of high priority affecting children at an important phase of their development . therefore the oral health professionals should focus on meeting the need of this population and improve the knowledge and behavior regarding oral health maintenance .

background and aimnutrition is an essential component in human growth , development and maintenance of healthy life . tribal communities are highly disease prone and do not have the required access to basic health facilities , also having a high degree of malnutrition.the aim of this study was to determine the association of body mass index with dental caries among malnourished tribal children of indore division ( m.p.)methodsa cross - sectional house to house survey was carried out among 275 study subjects , 615 years old tribal children in two major tribal districts of indore division . permissions and consent was obtained from local administrative authorities , ethical committee and parents respectively . anthropometric measurements like height , weight , mid - arm and head circumference were recorded . children with confirmed malnourishment on basis of bmi index were considered for data collection . oral examination for caries was conducted according to who 1997 survey methods . descriptive tables and analytical tests such as anova and chi - square test were employed . independent effects of age , gender , bmi , oral hygiene status and daily intake sugar on caries status were tested using logistic regression analysis.resultsthe mean age was 9.75 ( 2.43 ) years . the caries prevalence among permanent dentition of malnourished children was 61.4% with a mean of 1.61 ( 0.48 ) . among tribes , malnourished children of bhilala tribe showed significantly higher caries prevalence ( 1.820.46 , p<0.01 ) . increase in caries prevalence was seen with increase in severity of malnourishment ( p<0.05 ) . logistic regression indicated gender ( males or=1.19 ) , age ( 510years or=1.11 ) and high sugar intake ( or=1.49 ) were significantly associated with caries occurrence ( p=0.001).conclusioncaries was more prevalent in malnourished tribal children and was seen to increase with severity of malnourishment .

presbycusis develops and is exacerbated by various factors , including heredity , medical disease and environmental factors , showing various and complex clinical manifestations . there are still debates on which part of the inner ear is most affected by the aging process and which area is the most important for hearing .

age - related hearing loss ( presbycusis ) refers to bilaterally symmetrical hearing loss resulting from aging process . presbycusis is a complex phenomenon characterized by audiometric threshold shift , deterioration in speech - understanding and speech - perception difficulties in noisy environments . factors contributing to presbycusis include mitochondria dna mutation , genetic disorders including ahl , hypertension , diabetes , metabolic disease and other systemic diseases in the intrinsic aspects . extrinsic factors include noise , ototoxic medication and diet . however , presbycusis may not be related to the intrinsic and extrinsic factors separately . presbycusis affects not only the physical , cognitive and emotional activities of patients , but also their social functioning . as a result , patients ' quality of life deteriorates , compounded by various symptoms including depression , social isolation and lower self - esteem . presbycusis is classified into six categories , as based on results of audiometric tests and temporal bone pathology , established by schuknecht ( 1993 ) : sensory , neural , metabolic or strial , cochlear conductive , mixed and indeterminate types . among these , metabolic presbycusis is the mainstay of presbycusis types . age - related changes also develop in the central hearing system . functional decline of the central auditory system , caused by aging , reduces speech - understanding in noisy background and increase temporal processing deficits in gap - detection measures . this study reviews the literature on the age - related hearing loss .

they represent an increasing challenge to the gynaecologists , and ovarian cancer being the most lethal of all gynaecological cancers , characterized by late presentation and poor response to treatment ( gaughan et al . , 2006 ) . adnexal masses can be present in patients being evaluated for a gynaecological complaint or in asymptomatic patients . most of patients with ovarian cancer stay asymptomatic for long time . when symptoms develop , they are usually nonspecific . in early - stage disease , moreover , in advanced - stage disease , patients may have symptoms related to the presence of ascites or intestinal metastases ( olson et al . , 2004 ) . preoperative suggestion of ovarian cancer can conduct the physician to refer women with doubtful adnexal masses to a skilled gynaecological oncologist for appropriate therapy and optimized debulking , while patients with benign adnexal masses can undergo more conservative surgical treatment ( yazbek et al . , no single diagnostic tool ( ultrasonography , magnetic resonance imaging , and computerized tomography ) is good enough in preoperative determination of malignancy , but there is an agreement that ultrasound assessment of adnexal mass morphology by an experienced sonographer can properly estimate the risk of malignancy ( yazbek et al . , 2008 ) . doppler ultrasound is usually combined with 2d ultrasound evaluation aiming to predict malignancy more appropriately through obtaining intratumoural blood flow velocity waveforms to calculate the resistance and pulsatility indices . also , evaluation of gray - scale ultrasound morphology with colour doppler findings of an adnexal mass is highly accurate in predicting its nature ( van calster et al . , 2007 ) . prediction models have been developed to assist clinicians to triage patients to appropriate treatment pathways ; however , none has gained universal acceptance in routine daily practice . the international ovarian tumour analysis ( iota ) group has developed and validated two ultrasound - based logistic regression models ( lr1 and lr2 ) that estimate the risk of malignancy in adnexal masses ( timmerman et al . , 2005 ) , but the performance of such models was tested by expert sonographers not less experienced ones besides the need of a computer for risk estimation using an excel file that can not be present in most hospitals and clinics in developing countries ( van calster et al . , 2012 ) . so , there was a need for a prediction model that has a high sensitivity and specificity combined with simplicity and low cost to be valid for application in developing countries for triaging patients with adnexal masses . the aim of our study was to determine the best two - dimensional and doppler sonographic features that allow distinction between benign and malignant adnexal masses and to develop a scoring model enables accurate triaging of patients with malignancy using those features . the study period was one year starting from the 1st of october 2012 till the 30th of september 2013 , 146 women diagnosed as having an adnexal mass and scheduled for surgical management at woman s health center , assiut university , egypt , were included in this prospective study . the study was approved by the ethical review board of assiut faculty of medicine and all women gave written informed consent . diagnostic work - up included a complete medical history , physical examination , 2d ultrasound and doppler examination . 2d ultrasound and doppler evaluation was done using a sono - ace x8 machine ( medison , korea ) with multifrequency transabdominal and transvaginal volumetric probes . first , the volume of the mass was calculated from the three diameters obtained in the two perpendicular planes . then , morphological evaluation was performed according to the international ovarian tumour analysis ( iota ) protocol ( timmerman et al . , 2000 ) attending the following features : type of mass ( all masses were classified into one of five categories : unilocular cyst , unilocular - solid cyst , multilocular cyst , multilocular - solid cyst , and solid mass ) internal wall ( smooth or with papillae < 3 mm or 3 mm length ) septae ( not present , incomplete , complete ; thin < 3 mm or thick 3 mm ) echogenicity ( anechoic , low - level echogenic , ground glass appearance , haemorrhagic or mixed echogenic ) presence of internal shadows presence of free fluid in douglas pouch after 2dus evaluation was performed a subjective semi quantitative assessment of the amount of blood flow within the examined lesion ( colour score ) was made according to the iota protocol ( timmerman et al . , 2000 ) . the amount of blood flow within the mass was scored as follows : if there is no blood flow detected in the lesion , a score of 1 was given ; if minimal blood flow could be detected , a score of 2 was given ; a score of 3 was given when moderate flow was present , and a score of 4 was given when the adnexal mass appeared highly vascularized with marked blood flow . the area distribution of visualized vessels in the adnexal mass was also recorded as in the centre of the mass , in the septum , in the papillae , at tumour wall or peri - tumour areas . malignancy was suspected if the penetrating vessels were visualized within papillary projections , solid areas , or central areas of a solid tumour . spectral pulsed wave doppler analysis was done after that , resistance index ( ri ) and pulsatility index ( pi ) were calculated for each mass . when no blood flow was detectable within the tumour , a signal was recorded from peripheral areas or the adnexal branch of the ovarian artery . the doppler variables used for diagnosing malignant adnexal mass was ri 0.42 and pi 1.0 obtained from papillary projections , thick septa , solid parts or cystic walls ( weiner et al . , 1992 ) . all surgically removed masses were examined histopathologically to assess their nature as the final diagnosis was based on histopathological reports . in our study , we chose the 2d sonographic and doppler features of adnexal masses that are best fitted to predict the pathological outcome of benignity or malignancy . then we combined those features together to develop a new scoring model , assiut scoring model ( asm ) . analysis of data was done using spss inc . , chicago , il , usa , version 21 . quantitative variables were presented in terms of mean , standard deviation and range , compared by mann - whitney test for non - parametric data and student s t - test for parametric data . sensitivity , specificity , positive and negative predictive values were calculated for 2dus and doppler evaluation , and finally for the new scoring model . the mean ( sd ) age of patients included in the study was 35.2 13.9 years ( range 12 - 70 years ) . one hundred twenty - nine patients ( 88.4% ) were in the reproductive age , 13 were postmenopausal ( 8.9% ) , and 4 of them ( 2.7% ) were in the premenarche period . overall , 104 patients ( 71.2% ) confirmed to have benign masses , and 42 patients ( 28.8% ) with malignant masses according to their final histopathological analysis . number of the evaluated masses was 161 masses ( due to bilaterality of masses in 15 patients ) ; 115 proved to be benign and 46 malignant . almost all of the evaluated 2d ultrasound features differed significantly between benign and malignant masses except presence of internal shadows ( table i ) . * ( n = 92 ) in benign masses , ( n = 33 ) in malignant masses ; sd , standard deviation ; dp , douglas pouch . doppler examination revealed that the mean pi and ri values of arteries in malignant masses were significantly lower than benign masses . blood flow signals commonly obtained from papillary projections , septae or central parts of the malignant masses ( table ii ) . sd , standard deviation ; pi , pulsatility index ; ri , resistance index ; psv , peak systolic velocity ; cm / sec , centimetre / second . assiut scoring model , a new model evaluated four gray - scale sonographic features used to predict the adnexal mass status : volume of the mass , type , presence and thickness of septae , and presence and length of papillary projections . also , there are two colour doppler features ; location of vessels and colour score . the different features were rated on a scale from 0 to 3 points depending upon the degree of expression observed for the individual characteristics ( table iii ) . the highest value of reliability in differentiation of malignant and benign adnexal masses was attaining score 6 or more ( fig . 1 , 2 ) . all parameters of the score showed significant p - values , thus demonstrating a statistically significant correlation between score point numbers and histological findings . although ovarian cancer is the second most common female genital cancer , preceded only by cancer body of the uterus , more women die from ovarian cancers . it is the most lethal of all the gynaecologic cancers , killing more women each year than both of cervical and endometrial cancers ( zoorob et al . , 2001 ) . good preoperative discrimination between benign and malignant ovarian tumours results in more women being correctly referred for gynaecologic oncology care and more women with benign masses undergoing conservative management ( yazbek et al . , 2008 ) . in the study conducted by jokubkiene et al . ( 2007 ) on 106 patients with adnexal masses , they concluded that almost all ultrasound features differed significantly between benign and malignant masses except presence of papillary projections , internal shadows , thick septae and bilaterality of masses . our results agreed with them regarding presence of internal shadows and bilaterality , but differed in presence of papillary projections and thick septae . the malignant cystic masses were often had papillary projections ( 42.4% vs. 4.3% , p < 0.001 ) , and thick septae ( 68% vs. 6.3% , p < multilocular - solid mass was the most common pattern of ovarian malignancy ( 30.4% ) followed by solid mass ( 28.3% ) . ( 2005 ) who found that multilocular - solid masses represent 43.6% of ovarian malignancy followed by solid masses ( 31.6% ) . ( 2007 ) who found that multilocaular - solid and solid masses represented 52% and 41% respectively of malignant ovarian masses . regarding the echogenicity of cyst fluid in malignant masses , our results showed that most of them were anechoic ( 72.2% ) followed by low level echogenicity ; 15.2% . ( 2005 ) , malignant cysts were anechoic in 40.2% of masses followed by low level echogenicity ; 22.6% . also in jokubkiene et al . ( 2007 ) series ; anechoic pattern was found in 67% of masses . as regards to doppler indices in the present study , ri and pi values were calculated for each mass . the mean value of ri was 0.79 for benign masses and 0.50 for malignant masses , while the mean value of pi was 1.66 for benign masses and 0.82 for malignant masses . these values , in correlation to various studies using 0.42 and 1 values for ri and pi respectively as a cut - off value for prediction of malignancy reflected that , ri had a sensitivity of 44% and a specificity of 99% , while pi had a sensitivity of 80% and a specificity of 84% . p - value was < 0.001 for both ri and pi among both groups , being of significant value in predicting malignancy of adnexal masses . in the previous studies , some authors suggested the existence of clear cut - off values for ri and pi of benign and malignant tumours ; timor - tritsch et al . ( 1993 ) reported the ri value of 0.4 had sensitivity 93.8% and specificity of 98.7% . ( 2007 ) also reported that the best ri cut - off value was < 0.48 giving a sensitivity , specificity , and accuracy of 90% , 89% , and 90% . ( 2009 ) in their systematic review showed that doppler can detect malignant masses when the ri was < 0.50 . ( 2003 ) reported sensitivity and specificity of 52.8% and 77.6% respectively using cut - off value of pi < 1 . in spite that we found that low pi < 1 was an important feature of malignancy ( 80.4 % ) but pi < 1 was also found in 15.7% of benign masses . out of 115 benign masses , colour doppler study could diagnose 107 masses as benign ( colour score 2 ) but labelled eight masses as malignant that were actually benign , while out of 46 malignant masses , only 29 masses were diagnosed as malignant ( colour score > ( 2005 ) showed that colour score sensitivity and specificity were 80% and 66.6% , respectively . colour doppler results showed predominantly peripheral localization of vessels in benign masses ( 69.6% ) and predominantly central or septal vessel localization ( 39.1% and 34.8% ) was observed in malignant masses . there were 20 benign masses showed absence of blood flow ( 17.4% ) , on the other hand nearly all the malignant masses showed vascularity ( 97.8% ) . ( 2007 ) who found that 57% of benign masses showed peripheral vascularization versus 70% of malignant masses showed central vascularization . there have been several different sonographic scoring systems developed for evaluation of particular characteristics of ovarian tumours in order to classify malignant diseases in the most objective way . ( 1991 ) stated a scale for morphological features , including inner wall structure , wall thickness , presence of septa and echogenicity of the mass , and were able to differentiate benign from malignant masses with specificity of 83% , sensitivity of 100% . 1993 ) used the weighted sum of tumour volume , wall structure , and septal structure . the weighted sum of these three features varied between 0 and 12 , with the cut - off point set at 5 . at this cut - off point , both of the previous two scoring systems had a low specificity besides of not using doppler features in their models in 1994 , lerner et al . tried to simplify the scoring system by excluding the parameter of wall thickness from sassone scoring system and added another one ; shadowing in order to discriminate dermoid cysts from malignant adnexal masses . lerner scoring system using a cut - off at score 3 was reported to have 96.8% sensitivity and 77% specificity . twickler et al . ( 1999 ) integrated the patient s age , ovarian volume , doppler velocimetry and vessel location , and echogenic predominance of the mass with the morphology scale of sassone to calculate the ovarian tumour index , a calculated probability of malignancy based on the weighting of each of the previous features . when we applied our new scoring model on the evaluated masses in the study we found that 43 out of 46 malignant and 106 out of 115 benign masses were accurately identified . the preliminary results of application of our scoring model are promising showing a sensitivity of 93.5% , specificity 92.2% , ppv 82.7% and npv 97.3% with overall accuracy 92.6% , but a larger prospective study is a must to validate our new scoring model . based on the results of our score , patients with masses score < 4 can be managed in gynaecological unit by a gynaecologist ; either conservatively or surgically according to their features . patients with masses score 8 must be referred to gynaecological oncologist and to be managed in specialized oncology centres . patients with masses score 4 - 8 are suspicious with high possibility of malignancy if score 6 so further investigations may be ordered as mri or ca-125 . in conclusion , assiut scoring model ( asm ) is simple , non time consuming , easy to applied and interpreted with reasonable accuracy and high sensitivity and specificity , so less experienced sonographers should be trained on its application and further studies should be conducted to test its validity to be incorporated in hospitals guidelines of work for accurate identification of ovarian malignancy and appropriate referral to gynaecologic oncologists .

objective : to determine the most discriminating two - dimensional gray - scale and colour doppler sonographic features that allow differentiation between malignant and benign adnexal masses , and to develop a scoring model that would enable more accurate diagnosis with those features.methods : a cross sectional prospective study was conducted on patients scheduled for surgery due to presence of adnexal masses at woman s health center , assiut university , egypt between october 2012 and october 2013 . all patients were evaluated by 2d ultrasound for morphological features of the masses combined with colour doppler examination of their vessels . the final diagnosis , based on histopathological analysis , was used as a gold standard.results : one hundred forty - six patients were recruited , 104 with benign masses , 42 with malignant masses . features that allowed statistically significant discrimination of benignity from malignancy were ; volume of mass , type of mass , presence and thickness of septae , presence and length of papillary projections , location of vessels at colour doppler and colour score . a scoring model was formulated combining these features together ; assiut scoring model ( asm ) . the cut - off level with the highest accuracy in detection of malignancy , was 6 , had a sensitivity of 93.5% and specificity of 92.2%.conclusion : our scoring model ; a multiparameter scoring using four gray - scale ultrasound and two colour doppler features , had shown a high sensitivity and specificity for prediction of malignancy in adnexal masses compared with previous scoring systems .

the french entomologist leon dufour 's statement in which he hypothesized that the remarkable diversity in genital morphology observed among dipterans is important for maintaining reproductive isolation ( ri ) between species is one of the notable ideas in speciation research that has generated both considerable controversy and much experimental work . indeed , among different mechanisms of ri , structural isolation involving genitalia appears to be one of the first mechanisms of speciation to be experimentally tested . however , despite the widespread differences in genital morphology observed among many animal species , and the intuitive nature of the so - called lock - and - key hypothesis that structural differences in the genitalia prevent species from hybridizing the vast majority of experimental tests have failed to find convincing evidence that the differences in genitalia between species have a substantial role in preventing hybridization . most of these tests , however , have focused on one specific mechanism of genital lock - and - key ri , and recent studies suggest that differences in genital morphology might in fact contribute to ri in more cryptic ways that reduce the reproductive success of heterospecific matings . lock - and - key reproductive isolation can operate via two different mechanisms [ 3 , 4 ] . the first is the classic mechanical or structural lock - and - key mechanism of dufour where differences in genital morphology between species prevent or reduce the success of copulation and/or insemination as a direct result of mechanical incompatibilities that occur during genital coupling . ri caused by structural lock - and - key also includes postmating fitness losses such as physical damage to either parent that might prevent any future matings . the second mechanism is sensory lock - and - key first posited by de wilde and later formalized by eberhard where differences in genital morphology are perceived by one or both sexes and evoke behavioral or physiological responses that result in premature termination of mating attempts or postcopulatory reproductive fitness problems . these two mechanisms are not mutually exclusive , and both can operate in concert to give rise to ri . because genital morphology and its role in ri have received much attention in literature , it will be helpful to make clear what i will discuss in this review . several hypotheses about the forces that drive the rapid evolution of genital morphology in animal taxa have been put forth , and there is good evidence that sexual selection often operates to drive the evolution of genital morphology within species . these hypotheses and the data supporting them have been reviewed thoroughly elsewhere [ 2 , 68 ] , and i refrain from doing so here . i also refrain from reviewing the evidence testing genital lock - and - key ri using comparative or phylogeographic data . my reason for doing this is that without detailed knowledge of the evolutionary histories of species , it is often difficult to draw firm conclusions about the role of genitalia in ri from these patterns alone . for example , although patterns like reproductive character displacement are consistent with reinforcement acting on genital morphology where species exist either sympatrically or parapatrically , an absence of reproductive character displacement does not preclude a history of differences in genital morphology contributing to ri between species , as other ri mechanisms ( e.g. , mate discrimination , ecological niche divergence , temporal isolation , etc . ) instead , my goal here is to examine evidence of hybridizations between species where differences in genital morphology appear to contribute to ri either by structural lock - and - key and/or sensory lock - and - key mechanisms . although many studies exist that present indirect evidence to suggest a possible role for genitalia in contributing to ri between species , i focus my discussion on only those studies that have either directly observed species crosses in nature , or performed laboratory crosses between species , and recorded postcopulatory reproductive fitness in heterospecific pairs . in each of these cases it is important to emphasize that other mechanisms of ri beside genital lock - and - key also exist between these species pairs , and i do not suggest that genital isolation is the primary cause of speciation in any of these examples . rather , i present these data to illustrate the possibility that ri between species can occur as a byproduct of differences in genital morphologies that have evolved in response to evolutionary forces such as sexual selection acting within species . in describing each of the heterospecific crosses below , i use the convention of always presenting the female parental species first ( e.g. , species a female species b male ) . observations of genitalia and structural isolation have enjoyed a long history in the study of speciation and systematics ( for a detailed history of the structural lock - and - key hypothesis and tests of the hypothesis , see ) . much of the appeal of the structural lock - and - key hypothesis is that it offers an intuitively obvious mechanism of ri and an appealing visual image . the idea that incompatibilities between genitalia of different species caused ri was perhaps considered so obvious that it appears few careful tests of structural isolation were performed in the years following dufour . among some of the early proponents of the idea was one of dufour 's students , prez , who made similar observations that genital morphology among several hymenoptera species was incredibly diverse , and supported the notion that these differences in male genital morphology were the likely cause of ri among species ( although prez seemed to accept that differences genital morphology are important for ri , he does urge some caution in his interpretation as he notes that in some groups the genital morphology among males varies very little . it is a bit unclear , however , if his caution is in regard to the potential for genitalia to cause ri , or for their usefulness in systematic classification . ) . another early champion of the idea , and one of the first to make careful observations of genital coupling between males and females in a laboratory setting , was jordan . jordan performed crosses between species of papilio and observed that for crosses within species , the male and female external genitalia are well - matched and fit together with tight coupling , but in crosses between species , the coupling between the male and female genitalia is not quite as good . specifically , jordan found that although the external genitalia of papilio memnon males and females fit to provide tight genital coupling , in crosses between p. helenus and p. memnon and between p. podalirius ( the modern classification of this species is iphiclides podalirius ) and p. machaon , the male and female genitalia did not fit as precisely to secure the copulating pairs as they did within species . jordan also observed that , similar to males , the females of different species also possess genital structures that appear different , but these differences are not as dramatic as those observed among male genital structures . despite these early observations that seemed to support structural lock - and - key ri , many subsequent observations called the ubiquity , and even the existence , of ri as a result of differences in genital morphology into question . boulang pointed out that in many hymenoptera species , the most divergent structures of the male genitalia are those that are not involved in intromission and insemination . instead , these structures make contact with female structures that possess essentially identical morphology across species , for example , the sides of the female abdomen . he also observed that in comparisons between species where females do possess divergent genital morphology , the differences are usually minor . richards and later robson and richards made several observations in bombus ( subgenus p sithyrus ) that also did not support the structural lock - and - key hypothesis . among the many criticisms they level against structural lock - and - key , they found that females usually do not differ greatly in genital morphology , so the required structural locks simply do not exist , the morphological differences in male genitalia of some species are so small as to likely have no effect on reducing the tight coupling between male and female genitalia , and there appears to be no correlation between the degree of diversity in genital morphology among species and the species richness of most groups . robson and richards also presented what is perhaps the most serious criticism against the importance of structural lock - and - key in ri : species that possess dramatic differences in genital morphology can often mate and produce offspring . from these observations , they state with regard to ri , we are forced to regard specific differences in the genitalia as of essentially the same nature as other apparently useless specific characters [ 14 , page 297 ] . they conclude that divergent genital morphologies are more likely the result of ri rather than its cause , and other isolating mechanisms ( in particular mate discrimination ) probably play more important roles in speciation . for many years following , tests for genital structural isolation were performed in a variety of animal taxa and almost all of these tests failed to find convincing evidence of structural lock - and - key ri . implies has also become a bit muddled throughout the literature since the introduction of the idea . a common notion of lock - and - key ri describes the phenomenon as natural selection acting on genital morphology to prevent species from hybridizing . when considering the case of reinforcement , this is certainly an applicable definition , but as a more general definition to describe genital lock - and - key ri , it is incorrect . just as some other ri mechanisms such as intrinsic postzygotic isolation evolve as a byproduct of evolutionary processes that occur within populations evolving in isolation , genital lock - and - key ri can also evolve as a byproduct of evolutionary processes that occur within isolated populations , such as sexual selection , that can act to drive genital morphological evolution . the divergence in morphology between two populations might consequently give rise to either structural or sensory isolation as a byproduct when they attempt to hybridize . a history of sympatry or parapatry is not required for lock - and - key ri to evolve under this scenario , and i use this broader definition of lock - and - key in evaluating the contribution of genitalia to ri . for structural isolation to operate two criteria need to be satisfied . first , the genitalia of both males and females are required to bear substantial , species - specific differences in morphology of structures important for successful copulation , intromission , and/or insemination . second , male and female genitalia within species are required to show strong correlated evolution in the morphology of these structures . tests of structural isolation are often difficult as they require both incomplete premating ri between species and careful measurements of parental fitness loss after heterospecific matings . despite these challenges , crosses between species in a handful of arthropod and arachnid taxa suggests that structural lock - and - key may , in fact , contribute to ri in at least some hybridizations . standfuss [ 15 , pages 60 - 61 ] observed crosses between 24 heterospecific pairs of the lackey moths bombyx franconica and b. neustria ( modern day classifications for these two species are malacosoma franconica and m. neustria , resp . ) . this large number of heterospecific matings suggests that little premating isolation exists between these two species , and indeed , courtship and copulation appear to proceed normally . after mating , the m. franconica females also appear to exhibit the normal postcopulatory behavior by walking around to identify locations to oviposit . within the next three to four hours of observation , however , most of these females die . among those that survive , some are unable to lay eggs . others do lay eggs , but egg lethality ranges from 50100% and larvae never reach adulthood . although no description is given of the morphological differences in male or female genitalia between these two species , standfuss speculates that the probable cause of lethality of m. franconica females was injuries suffered as a result of the m. neustria male genitalia . he is , however , cautious in his interpretation , and hypothesizes that other species differences might have been the cause of female lethality ( in a remarkably insightful passage for the time period , standfuss hypothesizes that some of the other possible causes of egg mortality and female lethality might include sperm - egg incompatibility and what he describes as molecular differences between the two species . ) . federley [ 16 , pages 371 - 372 ] performed crosses between the hawk moths metropsilus porcellus and chaerocampa elpenor ( modern day classifications for these two species are deilephia porcellus and d. elpenor , resp . ) . upon intromission , d. elpenor males become stuck and are unable to withdraw their aedeagus from the d. porcellus females . in the case of copulating species pairs that do manage to separate , females never lay eggs , although sperm is found in the bursa copulatrix , which suggests the possibility that damage to the female reproductive tract might prevent fertilization and/or oviposition . males of these two species bear substantial morphological differences in their external genitalia : d. elpenor males possess a long and slender aedeagus , whereas d. porcellus males possess a shorter and thicker aedeagus compared to those of d. elpenor males . these differences in male genitalia are consistent with structural isolation in the d. porcellus d. elpenor cross , and also agree with the results from the reciprocal cross males and females of several species of crab spider in the genus misumenops possess genital morphologies that are both highly divergent among species and show strong correlated evolution between males and females within species . during copulation , the male intromittant organ , the palpus , is guided into the reproductive tract by the female epigynum to successfully complete intromission and insemination . in the cross m. rothi m. gabrielensis , m. gabrielensis males initiate courtship and are received by the female , but are unable to orient their palpus and complete intromission because the m. gabrielensis palpus is much larger than the opening to the epigynum in m. rothi . ( it seems possible that the reciprocal cross might proceed through intromission and insemination , but was not attempted . ) in the cross m. lepidus m. californicus attempts at intromission were unsuccessful because the m. californicus male failed to correctly align the palpus with the epigynal structures of the m. lepidus female presumably because of the structural incompatibilities between the two structures . the reciprocal cross results in successful intromission and insemination , but does not produce offspring . individual females have been collected from nature that show genital morphology intermediate to those of m. lepidus and m. californicus , thus it does appear that the structural ri in the m. lepidus m. californicus cross is incomplete . one of the best - characterized examples of structural lock - and - key comes from hybridizations among beetles of the genus carabus ( subgenus ohomopterus ) . many species in the genus exist parapatrically and form hybrid zones . in the c. iwawakianus and c. maiyasanus species pair , males of both species do not discriminate against heterospecific females , and hybrid individuals that possess intermediate genital morphology between these two species have been found at low frequencies in the hybrid zone . males of each species possess striking differences in genital morphology : c. iwawakianus possesses a short and wide copulatory piece , whereas c. maiysanus possesses a long and thin copulatory piece ( figure 1 ) . the vaginal appendix of the females in each species also shows striking correlated morphology with the copulatory piece of their conspecific males ( figure 1 ) . during intromission , the copulatory piece of the male and vaginal appendix female lock together . sota and kubota performed reciprocal crosses between these two species and measured male and female fitness after intromission . in the cross c. iwawakianus c. maiyasanus 50% ( 18 of 36 ) of c. maiyasanus males suffer broken copulatory pieces that were likely to prevent future matings . female c. iwawakianus mortality is high ( 60% , 20 days postmating ) and postmortem dissections revealed ruptured vaginal appendices and torn bursae in the majority of cases . in the reciprocal cross c. maiyasanus c. iwawakianus , none of the c. iwawakianus males suffer injuries to the copulatory piece ( 0 of 27 ) , and female c. maiyasanus mortality is lower compared to the c. iwawakianus c. maiyasanus cross ( ~30% versus 60% ) . however , a substantial fraction of c. maiyasanus females still suffer damage to the vaginal appendix that appear severe enough to prevent future mating . in both directions of crosses between these species , mated females lay fewer eggs and the egg hatch rates are lower , although it is unclear whether this is a direct consequence of damage to the female reproductive tract by the male genitalia . structural isolation might prove to be a common mechanism of ri among carabus species as female mortality following heterospecific crosses has also been reported between c. albrechti and c. iwawakianus and between c. albrechti and c. yamato . another well - characterized example of a species group where structural lock - and - key appears to have an important role in ri occurs among some species of millipedes . the parafontaria tonominea species complex are also endemic to japan , and many species exist parapatrically ( some species exist sympatrically ) . tanabe and sota performed crosses between parafontaria tonominea sp . a and parafontaria tonominea sp . b. , two species that differ in their overall genital morphology , particularly in the size of their genital structures . they also possess notable differences in body size with parafontaria sp . a possessing a larger body size than parafontaria sp . courtship is initiated by the male with antennal contact , the male aligns head - to - head with female , and a preliminary intromission occurs without sperm transfer . if the female remains receptive following preliminary intromission , true intromission and insemination occur , which is then followed by postcopulatory behavior by the male . b , as males are equally likely to mate with a female of either species . in the cross parafontaria sp . b parafontaria sp . a , preliminary intromission fails in every cross ( 10 of 10 ) despite repeated contact between the male and female genitalia . the cause of the failed intromission is the difference in size between the much larger parafontaria sp . b female gonopore . in the reciprocal cross parafontaria sp . a parafontaria sp . b , preliminary intromission fails in ~65% of the attempts ( 9 of 14 ) because the smaller parafontaria sp . b male is unable to align his gonopod correctly even after repeated attempts due to his smaller overall body size . b crosses resulted in successful preliminary intromission , and true intromission occurred in three of the five . in each of these cases , however , the parafontaria sp . b male terminated postcopulatory behavior prematurely . a recent study has also identified structural isolation between two members of the drosophila melanogaster species subgroup . d. yakuba and d. santomea inhabit the island of sa tom off the western coast of africa . although these two species occur primarily at different elevations , their ranges overlap and form a hybrid zone at mid - elevation locations around the island [ 22 , 23 ] . d. yakuba males possess a pair of sclerotized spikes that project from the lateral portion of the aedeagus . d. santomea males also possess sclerotized projections in the same location , but the morphology of the projections is rounded and more nub - like . females of each of these species show correlated evolution of genital structures with those of their conspecific males : d. yakuba females possess a pair of heavily sclerotized cavities that receive the male aedeagus spikes during copulation , whereas d. santomea females lack these cavities . kamimura and mitsumoto studied the consequences of these morphological differences on structural isolation between d. santomea and d. yakuba . in the cross d. santomea d. yakuba , d. yakuba males successfully mount the d. santomea females , but usually fail to insert their aedeagus during mating , which results in insemination of only 20% ( 3 of 15 ) of the females in heterospecific matings . in 11 of the 12 remaining crosses that did not result in insemination , the male ejaculate was observed as a white , sperm - bearing mass on the external genitalia of either the d. yakuba male or d. santomea female in each pair . this mass appears to bind the genitalia of mating pairs together as heterospecific pairs are often observed struggling to separate from each other . moreover , severe copulatory injuries were observed in mated d. santomea females that match the pattern of aedeagus spikes from the d. yakuba male . the reciprocal cross d. yakuba d. santomea proceeds through copulation and insemination , although it is easy to dislodge mating pairs , which suggests that the spikes in d. yakuba may have evolved to secure genital coupling . similar modifications of male genitalia that function to secure genitalia during copulation within species have also been documented in d. bipectinata . it has been thought that the highly specific shape of structures and appendages of male and female copulatory apparatus constitutes a decisive structural factor in species isolation , acting as a system of key and lock . but it would rather seem that intraspecific matings are assured by precopulatory behavior , and probably by the mutual stimulation of specific sensory sites during the copulatory act . j. de wilde , 1964 j. de wilde , 1964 the possibility that genital incompatibilities might cause ri between species through behavioral or physiological responses has recently received renewed interest . in contrast to the requirements for structural lock - and - key ri where both male and female genital structures are required to possess both complementary morphologies within species and divergent morphologies between species , sensory lock - and - key ri requires that only one of the sexes possesses divergent morphology between species . the morphology of the opposite sex , whether species - specific or not , requires innervation with sensory neurons capable of relaying information about species identity . compared to structural lock - and - key mechanisms , sensory lock - and - key is perhaps more difficult to detect for the primary reason that the phenotype being studied is more complex rather than studying a simple structural incompatibility , it is necessary to study a structurally induced behavior , or structurally induced changes in physiology . although ri via sensory lock - and - key has not been as extensively studied as structural lock - and - key , there is growing evidence that it operates in some species crosses . in laboratory crosses among several genera of lepidoptera , forced matings often show no evidence of structural lock - and - key preventing successful insemination in heterospecific matings , despite that fact that male genital morphology often differs dramatically among closely related species ( e.g. , ) . although differences in male genital morphology do not appear to hinder the mechanics of copulation and intromission , they may relay species identity between the sexes . two species of brush - footed butterflies erebia nivalis and e. cassioides form hybrid zones along altitudinal gradients at some locations in the alps . although strong mating discrimination exists between them , heterospecific crosses can be obtained in the laboratory . in conspecific crosses , mating pairs will remain in copula ~1830 minutes after a male successfully courts a female . however , in the heterospecific cross e. nivalis e. cassioides , the male terminates copulation after only 0.57 minutes . during this time the female remains receptive to the heterospecific male , and presumably the male perceives the female is heterospecific and terminates copulation prematurely , although it is also possible that the female fails to cooperate with the male and as a result he terminates the mating attempt . the duration of copulation in the reciprocal cross e. cassioides e. nivalis appears normal . macrodactylus costulatus , m. sylphis , and m. sericinus females each possess relatively similar genital morphologies , but males of these species possess substantial differences in their genitalia . the differences in male morphology are most pronounced not in the hard cuticular structures of the external genitalia , but rather in the soft tissues involved in intromission . among these three species , eberhard observed 37 male mounting attempts between heterospecific pairs out of total 160 mounting attempts in the field . all attempts to mount by heterospecific males were short ( < 5 seconds ) and intromission never occurred . macrodactylus females are known to reject unwanted males in conspecific crosses by contracting their vaginal walls to prevent intromission , thus it seems likely that females perceive heterospecific males and reject their copulation attempts using a similar behavior . however , it also seems possible that because the male morphological differences occur in soft and possibly sensory tissues , the male might perceive species identity of females via these differences in genital morphology , and terminate mating attempts prematurely . damselflies present an interesting case where both structural and sensory lock - and - key mechanisms appear to contribute to ri . during courtship , the male damselfly grasps the female with his legs and brings his terminal appendages into contact with two mesostigmal grooves on the back of the female thorax . both the male appendages and the female mesostigmal grooves within species show structural complementary and species - specific morphologies [ 29 , 30 ] . ablation experiments show that the male terminal appendages are important for mate recognition and copulation ; females perceive the superior appendages , whereas the inferior appendages are used primarily by males for grasping the female . unreceptive females resist copulation by vigorously beating their wings and orienting their abdomen to prevent the males from achieving genital coupling . if the female is receptive , she bends her abdomen to make contact between her genitalia and those of the male . when damselflies occur in large groups , males will usually mate with conspecific females , but heterospecific matings do occur in nature . in several genera of damselflies , the fit between the male appendages and the female mesostigmal grooves appears to be used by the female to assess species identity of the male . in heterospecific crosses among different sympecma and lestes species , heterospecific females vigorously resist copulation shortly after the male grasps the female and makes contact with his terminal appendages . similar observations have also been made in crosses between ischnura elegans males mated to females of species belonging to different damselfly genera . paulson performed several laboratory crosses among enallagma , argia , ischnura , and telebasis species . he observed that in contrast to conspecific pairs , in heterospecific pairs , the male appendages are unable to grasp the female thorax correctly . roughly 66% of the crosses ( 8 of 12 ) show nearly complete inability of grasping by heterospecific males , which suggests structural incompatibilities between the morphologies of the male appendages and the female mesostigmal grooves also contribute along with sensory isolation to ri among these species . evidence of sensory lock - and - key is also found in some drosophila species . among members of the d. melanogaster species complex ( d. mauritiana , d. melanogaster , d. sechellia , d. simulans ) , males possess two bilaterally symmetrical sclerotized cuticular genital structures called the posterior lobes of the genital arch , which insert between the eight and ninth tergites of the female during copulation , and differ dramatically in both size and shape among species ( figure 2 ; [ 35 , 36 ] ) . females of these species show no apparent differences in external genital morphology , although the posterior lobe likely comes into contact with , or distends soft abdominal tissues during copulation . no evidence exists of a structural lock - and - key mechanism among the d. melanogaster complex species that involves the posterior lobe , but the posterior lobe does appear important for mounting and genital coupling [ 37 , 38 ] . ( it is worth pointing out , however , that the posterior lobe in d. sechellia and d. simulans is known to cause some damage to the soft abdominal tissues at the insertion site in conspecific crosses . ) heterospecific crosses among d. mauritiana , d. sechellia , and d. simulans show two cryptic defects that reduce the reproductive success of heterospecific pairs . the first defect is that copulation is generally shorter when females mate with heterospecific males compared to copulation duration when females mate with conspecific males . within species , copulation duration lasts an average of 1517 minutes in d. mauritiana , 30 minutes in d. sechellia , and 2530 minutes in d. simulans [ 37 , 40 , 41 ] . in the d. simulans d. mauritiana cross , copulation is much shorter than either pure species cross and lasts only 511 minutes [ 37 , 4042 ] . during copulation , the d. simulans females also actively resist mounting attempts of d. mauritiana males . in the d. mauritiana d. simulans cross , copulation duration is slightly shorter than that of d. simulans heterospecific crosses , but abnormally long compared to d. mauritiana conspecific crosses . in the d. simulans d. sechellia cross , copulation duration varies from as short as ~16 minutes to the normal 2530 minutes observed in d. sechellia conspecific crosses . in each heterospecific pair ( including heterospecific crosses involving the other species in the group , d. melanogaster ; ) , the duration of copulation is more similar to the copulation duration typical of the male species . this suggests the possibility that differences in posterior lobe morphology might allow males to maintain copulation duration for times that are characteristic of that species , which could be important for successful insemination . the second defect observed among these heterospecific crosses is abnormal sperm transfer and lower offspring production . in the d. simulans d. mauritiana cross , a smaller fraction of the d. mauritiana sperm are stored by the d. simulans female compared to either pure species cross , and the cross produces 40% fewer offspring compared to pure species d. mauritiana and 70% fewer compared to pure species d. simulans [ 37 , 40 ] . in the d. mauritiana d. simulans cross , d. simulans males transfer an abnormally large number of sperm during copulation , however , the female loses the heterospecific sperm rapidly from her storage organs . lastly , in the d. simulans d. sechellia cross , d. sechellia males transfer few or no sperm to d. simulans females even when copulation duration last the full 30 minutes . although the morphology of the posterior lobe might contribute to these postinsemination reproductive defects , these reproductive fitness problems might also result from differences in molecular incompatibilities between male seminal fluid proteins and proteins in the female reproductive tract , which are known to cause postcopulatory reproductive problems between other drosophila species [ 43 , 44 ] . because of their rapidly evolving morphology and their importance in reproductive fitness , animal genitalia have attracted the attention of evolutionary geneticists . aside from presenting a good model to study the genetics of rapidly evolving morphological traits , there is reason to suspect that genitalia might possess a particular genetic architecture if sexual selection drives the evolution of morphology within species . in particular , we might expect that several genes would be necessary to specify differences in genital morphology ( or in the case of sensory lock - and - key , differences in behavioral preferences ) in both males and females , reflective of the step - wise coevolution of phenotypes between the sexes . we might also predict to find the molecular signature of selection at loci important for specifying morphological ( behavioral ) differences . many closely related species possess divergent genital structures thus making it possible to construct interspecific hybrid genotypes to dissect the genetics of genital morphology . although experiments mapping differences in genital morphology have been performed in only two species groups where genitalia contribute to ri , the results of these mapping experiments suggest that the genetic architecture of species differences in genital morphology bears some similar characteristics across taxa . the results also support at least one of our predictions : species differences in genital morphology appear to be specified by many genes , and the phenotypic effects of species alleles act in the direction of the species phenotype , consistent with the idea that sexual selection drives the evolution of morphology within species . moreover , the genomic locations that have large effects on morphological differences are similar among species within some species groups , which suggests the possibility that change at some of the same loci might be involved in the evolution of species - specific genital morphology . recent work on c. maiyasanus and c. iwawakianus has identified several genomic regions between these two species that carry loci specifying the species differences in both the male and female genital morphology involved in structural lock - and - key ri [ 45 , 46 ] . sasabe and colleagues measured a panel of reciprocal f1 and backcross hybrids for genital morphology and performed quantitative trait loci ( qtl ) mapping experiments to identify the minimum number of genes that specify male and female morphology . they measured two genital phenotypes for each sex : in males , they measured copulatory piece length and copulatory piece width , and in females they measured vaginal appendix length and vaginal appendix width . the results of their mapping identified 15 qtl that reside across 8 of the 14 linkage groups in these species : three qtl specify differences in copulatory piece length , three qtl for copulatory piece width , four qtl for vaginal appendix length , and five qtl for vaginal appendix width . several genetic studies have also been performed that map the genomic regions that specify the differences in posterior lobe morphology among the d. melanogaster complex species . because crosses to d. melanogaster usually result in dead or sterile offspring [ 47 , 48 ] , most of these mapping studies focus on comparisons among d. mauritiana , d. sechellia , and d. simulans where it is easier to obtain f2 and backcross hybrid genotypes . qtl mapping experiments between d. mauritiana and d. simulans identified 20 qtl that map across each of the major chromosomes in these species underlying the posterior lobe morphological differences . in the d. sechellia - d . simulans species pair , qtl mapping revealed a minimum of 13 qtl that have effects on posterior lobe morphology . an introgression mapping approach was used to map loci specifying morphology between d. mauritiana and d. sechellia to small genomic regions across roughly 50% of the genome . interestingly , some of these genetic regions have morphological effects on posterior lobe size , but not posterior lobe shape , whereas others have morphological effects on posterior lobe shape , but not posterior lobe size . this result suggests that these two posterior lobe phenotypes are specified , in part , by different loci . transcriptome sequencing experiments in the larval tissue that gives rise to the male genitalia also reveal a possible role for gene expression differences in the insulin / insulin - like signaling pathway in specifying morphological differences between d. mauritiana and d. sechellia . although it has long been thought that differences in genital morphology had little or no importance for speciation , it appears that in some hybridizations lock - and - key mechanisms do in fact contribute to ri ( table 1 ) . however , despite the widespread diversity of genital morphologies among many animal taxa , it is clear that genitalia usually do not cause structural lock - and - key ri in the strict sense [ 4 , 9 ] . in most species crosses where structural lock - and - key has been tested , it seems reasonable to suspect that the criteria for structural isolation were not satisfied correlated differences of genital morphology between males and females within a species appear to occur much less frequently than the case of substantial diversity of male genital morphology among closely related species , but relatively little diversity of morphology among females of those species . this common sexual asymmetry in the degree of genital morphological divergence suggests the possibility that sensory lock - and - key ri mechanisms , however , could be quite common in limiting gene flow between species . thus , two major challenges face the study of genital evolution and its role in speciation for the near future . the first is to identify the frequency with which ri via genital sensory lock - and - key occurs among different taxa . this might be most easily tested in insect and arachnid species , although the potential for genital sensory lock - and - key ri in some vertebrate systems is currently being explored ( b. langerhans , personal communication , i. schlupp , personal communication ) . the second challenge will be to dissect the mechanistic basis both phenotypically and molecularly of morphologically induced behaviors or physiological responses that result from genital incompatibilities . this problem will require the availability of sophisticated measurement and molecular tools to manipulate genital morphology , but some experimental systems such as carabus and drosophila appear poised to begin work on determining the sensory consequences of genital morphology and its effect on ri .

the divergent genital morphology observed among closely related animal species has long been posited as a mechanism of reproductive isolation . despite the intuitive appeal that rapidly evolving genitalia might cause speciation , evidence for its importance or even its potential in reproductive isolation is mixed . most tests of genital structural isolation between species often fail to find convincing evidence that differences in morphology prevent copulation or insemination between species . however , recent work suggests that differences in genital morphology might contribute to reproductive isolation in less obvious ways through interactions with sensory mechanisms that result in lowered reproductive fitness in heterospecific matings . in this paper , i present a brief history of the lock - and - key hypothesis , summarize the evidence for the involvement of genital morphology in different mechanisms of reproductive isolation , discuss progress in identifying the molecular and genetic bases of species differences in genital morphology , and discuss prospects for future work on the role of genitalia in speciation.l'armure copulatrice est un organe ou mieux un instrument ingnieusement compliqu , destin s'adapter aux parties sexuelles externes de la femelle pour l'accomplissement de l'acte copulatif ; elle est la garantie de la conservation des types , la sauvegarde de la lgitimit de l'espce . [ the copulation armor is an organ or better an instrument ingeniously complicated , destined to adapt to sexual parts external to the female for the completion of copulation ; it is the guarantee of the preservation of the standards , the safeguard of the legitimacy of the species . ] l. dufour , 1844

of these , approximately 300 000 immigrated to israel in two main periods : shortly after the establishment of the state of israel , and between 1989 and 1992 when large groups of jews immigrated from the former ussr . it is estimated that 200 000 survivors are now living in israel , most of whom are now elderly . in the 1950s , nearly 2000 holocaust survivors were repeatedly or chronically hospitalized in psychiatric hospitals in israel . the most common diagnosis then was that of schizophrenia . in 1998 , there were 700 such patients hospitalized in long - stay wards . ihe center 's psychogeriatric division consists of three wards encompassing 110 inpatient beds . from january to june 1998 , for the purpose of the present study , all aging holocaust survivors were interviewed . holocaust survivors were defined as subjects that were in eastern or western rurope under the nazi regime during the years 1933 to 1945 . inclusion criteria for the study were : ( i ) age 65 years ; ( ii ) being a holocaust survivor . rxclusion criteria were : ( i ) dsm - iv diagnosis of dementia ; ( ii ) inability ( cognitive impairment or language difficulties ) to endorse the impact of event scale ( ies ) ; and ( iii ) patient 's refusal to participate in the study . all patients had previously been diagnosed according to dsm - iv criteria as part of an ongoing study project ( the data relevant to this project are detailed in reference 16 ) . for purposes of the present study , the rptsd inventory is based on endorsement ( by the interviewing researcher ) of dsm criteria for the presence of ptsd . both these instruments were previously used and validated in studies of holocaust survivors and trauma victims . we used these simple statistical measures as the aim of the study was to present a descriptive audit . during the period january to june 1998 , 93 holocaust survivors were being treated at the abarbanel mental health center psychogeriatric wards . of these all 61 participating patients underwent a semistructured interview . after the interview all endorsed the ies . mean age for the group was 77.1 years ( 6.8 ; range 65 - 91 ) . ihe majority of subjects were in eastern rurope during the nazi regime ( 43 of 61 ; 70.5% ) . axis i ( dsm ) psychiatric diagnoses for the group were as follows : 32 of 61 ( 52.5% ) schizophrenia , 17 of 61 ( 27.9% ) affective disorders , and 12 of 61 ( 19.6% ) other psychotic disorders . the r - ptsd inventory facilitated diagnosis of comorbid ptsd in 91.8% of patients ( 56 of 61 ) . as previously shown , the inventory correlated well with the schizophrenia clinical interview for diagnosis ( scid ) . thus , comorbid ptsd can be said to be reliably diagnosed in the overwhelming majority of subjects in the present study . while both subscales were scored as significantly higher than the reported means for the normal population , intrusions were scored as notably more prominent than avoidance . mean intrusion score was 42.7 4.1 ( range 3651 ) and mean avoidance score was 29.73.4 ( range 2731 ) ; < 0.01 [ paired student /-test ] ) . the ies scores in the present study are in the range of a previous study of elderly subjects suffering from ptsd reported by our group . our sample represents a unique group of elderly holocaust survivors who show a high comorbidity of chronic ptsd ( 91.8% ) , with psychotic disorders more than 50 years after the experience of the massive psychic trauma of the holocaust . the occurrence of chronic ptsd of such magnitude for an extremely prolonged period is striking . it is significantly higher than the rate reported for war veterans , ranging from 12.4% to 45% . this difference may be related to the unique nature of the holocaust trauma , combining dehumanization , confrontation with death , and massive loss for a prolonged period . beal demonstrated that the co - occurrence of imprisonment in addition to the experience of combat led to a higher incidence of ptsd and other psychological symptoms , compared to combat experience alone . furthermore , kidson ct al show that the specific nature of the traumatic experience , such as taking of casualties , or the experience of combat stress , resulted in more pronounced severity , and was significantly associated with the occurrence of ptsd in wwii veterans . thus , the specific nature of the traumatic experience may influence the occurrence of ptsd and its persistence over time . beyond this aspect , the coexistence of a severe psychotic disorder in our scries of patients seems to bc decisive . as demonstrated by kidson et al , even minor pathologies , such as anxiety and depressive disorders , therefore , this seems to suggest that the severity of the coexistent psychiatric morbidity , such as schizophrenia , may explain the high incidence of chronic ptsd present for such a prolonged period . it is difficult to say whether the occurrence of ptsd in our group represents lifelong suffering , beginning close to the end of the traumatic experience and persisting for more than 50 years , or whether it represents a phase of symptomatic reactivation occurring in wwii veterans in their old age , as demonstrated by macleod . the exposure to trauma puts into effect mechanisms of coping ( directed towards the environment ) , as well as defense mechanisms ( directed towards traumatic memory and its psychic repercussions ) . it is generally acknowledged that most holocaust survivors have succeeded in mobilizing effective skills for coping , manifesting themselves by recreating families and adapting to social roles . however , the impact of the holocaust on ego functions led to the activation of defense mechanisms , mostly of splitting and ensuing encapsulation , numbing , and avoidance . as in psychosis , the coexistence of psychosis in our patients led to processes of fragmentation of the ego , thus impairing the exercise of these ego functions . again , this may have been a decisive factor in the occurrence of active ptsd symptomatology . furthermore , the primary process ( psychosis ) may have uncovered the core memories of the traumatic experience and prevented the possibility of masking . one of our patients only described a cannibalistic experience during extreme starvation in the theresienstadt concentration camp when in the manic - psychotic phase of his bipolar disorder . while euthymic or depressed , he was unable to recall or recollect this experience . this finding may also be related to the disorganizing effect of psychosis on the ego . our findings show that the comorbidity of psychosis and ptsd in holocaust survivors leads to a lifelong debilitating illness . nonpsychotic survivors usually succeeded in achieving a sense of integrity through historicizing their memories ( establishing a continuity between early , positive pre - holocaust memories , through traumatic memories during the holocaust and memories of reestablishing the fabric of life in the post - holocaust period ) . in contrast , the psychotic survivors were unable to reach this equilibrium and , for them , memory is a lifelong burden .

despite the fact that 50 years have passed since the nazi regime and the holocaust the psychic sequelae are far from being overcome . the majority of holocaust survivors and world war ii veterans still list their experiences as the most significant stressors of their lives . the literature provides ample evidence that posttraumatic stress disorder among survivors persists into old age . however , there is still a need to define the differences in frequency , clinical presentation , severity , and comorbid conditions among aging holocaust survivors . age at the time of trauma , cumulative lifetime stress , and physical illness are reported to have a positive association with more severe posttraumatic symptomatology . the presence of comorbid axis i psychiatric disorders ( diagnostic and statistical manual [ dsivi ] ) , has been the focus of research by our group , demonstrating that their interaction with earlier trauma leads to a course of chronic , debilitating disease . despite reactivation of traumatic symptoms during aging and continuous mental suffering , the majority of holocaust survivors show good instrumental coping and preserved functioning .

previous research has shown that both acute and chronic physical exercise can induce positive effects on brain function and this is associated with improvements in cognitive performance . however , there is currently little understanding regarding neurophysiological mechanisms underlying the effects of exercise on cognitive processing and further investigation is required . one approach to understand processes that underlie the beneficial effects of exercise on brain function and cognition has been to measure electrical brain activity using electroencephalography ( eeg ) . past research in this field focused on either a change in spectral power and/or event - related potentials ( erps ) . however , to the best of our knowledge , there are no studies examining the relationship between physical exercise and the individual alpha peak frequency ( iapf ) . the iapf corresponds to the discrete frequency showing the highest power within the alpha oscillation range ( ~713 hz ) . it is considered a putative marker of an individual 's state of arousal and attention , and it is positively related to the speed of information processing . a number of studies have shown a positive relationship between iapf and cognitive task performance . for example , individuals with higher iapf show shorter reaction times , better working memory scores [ 7 , 8 ] , and superior memory performance . in contrast , the iapf is significantly reduced in alzheimer 's disease ( ad ) , with the degree of the reduction being associated with the specific stage of ad . other diseases that are typically accompanied by cognitive decline are also characterized by a reduction of the iapf . these include major depression , attention deficit hyperactivity disorder , cerebral ischemia , and carotid artery occlusion . more recent studies identified that iapf correlates with higher cerebral blood flow ( cbf ) in brain areas involved in attentional modulation and preparedness for external input . in addition , subjects with higher iapf show increased fractional anisotropy values in fascicles connecting networks of brain areas associated with working memory functions and attentional modulation . previous research showed that acute exercise increases cbf and it was suggested that exercise activates arousal mechanisms within the brain , leading to improvements in implicit information processing . apart from these acute effects , aerobic exercise training has been shown to increase cerebral blood volume , perfusion , and the adult hippocampal volume . in addition , a cross - sectional study revealed that aerobic fitness is positively associated with functional connectivity in the default mode network , and this , in part , mediates better performance on tasks requiring set - shifting , task switching , and spatial working memory . consistent with this , one year of regular walking increased functional connectivity between parts of the frontal , posterior , and temporal cortices within the default mode network and a frontal executive network . moreover , greater aerobic fitness derived from a walking program was associated with increases in white matter integrity in the frontal and temporal lobes and greater improvement in memory performance . taken together , the above described literature suggests that physical exercise induces changes in the brain that should be associated with an increase in iapf . therefore , in a recent study , we analyzed the effect of an acute bout of exercise on the iapf in young children . therefore , the aim of the present study was to investigate whether exercise intensity has an influence on the postexercise iapf in healthy young adults . moreover , a recent study suggests that even shorter term aerobic exercise training with minimal improvements in fitness can facilitate neuroplasticity in sedentary adults . therefore , another aim of this study was to explore the effects of a short term exercise training program on both baseline iapf ( before exercise ) and the iapf response to acute exercise . it was hypothesized that exercise intensity will have an influence on the effect of acute exercise on iapf . furthermore , baseline iapf ( before exercise ) should be increased following exercise training and , thus , after training , the iapf response to an acute bout of physical exercise may be attenuated . 10 male volunteers ( age : 22.7 2.0 years , body mass : 79.6 6.9 kg , and height : 180 4.8 cm ) participated in the study . all of the subjects participated in regular exercise for the last two years ( > 30 min / day on > 3 days / week ) achieving current recommendations of the american college of sports medicine prior to the study and were able to undertake exhaustive exercise . however , highly trained endurance athletes from sports such as long distance running , road cycling , and triathlon were excluded from the study . a preliminary medical assessment confirmed that participants were of good health and had no overt cardiovascular , orthopaedic , or neurological disorders . they were informed about the intention and procedure of the study and their written consent was obtained . the study was reviewed and approved by the research ethics committee of the german sport university cologne in accordance with the declaration of helsinki . on the first laboratory visit , participants were asked to adjust the seat and handlebars of the ergometer and to take a comfortable seated position . individual ergometer settings obtained during this familiarization appointment were recorded and applied to all occasions in order to standardize the individuals ' cycling position . there were two blocks of assessments : one before ( t1 ) and one after four weeks of cycling training ( t2 ) . in each block , subjects were asked to complete an exhaustive exercise ( ee ) and a steady state exercise ( sse ) protocol with one leg . the ee was a ramp exercise protocol with initial intensity set at 30 w which was increased by 5 w every 30 sec until the subjects reached their point of exhaustion . this protocol was conducted 3648 h before and after the last training session in order to evaluate the effects of training on the subjects ' maximum exercise capacity . all subjects completed three training sessions per week over a four - week period , resulting in a total of 12 training sessions . each training session consisted of 30 min of one - legged cycling at 50% of the peak power output ( ppo ) . the ppo of each leg was derived from a graded exercise protocol ( starting at 40 w and increasing by 10 w every 3 min ) on a separate occasion that allowed for determining the subjects ' anaerobic lactate threshold . this was done to select an adequate training intensity slightly below the anaerobic lactate threshold to ensure it could be maintained for 30 min . cycling intensity was increased weekly by 5% to account for training effects . however , cycling intensity was identical in the first and the last training sessions to represent the sse protocol before and after training , respectively . at the end of each protocol , the subjects ' heart rate ( hr ) ( s810i , polar electro , kempele , finland ) and rate of perceived exertion were recorded . in addition , 20 l capillary blood was sampled from the earlobe to determine lactate concentration ( bla ) according to the enzymatic - amperometric principle of the biosen c - line ( ekf diagnostic , barleben / magdeburg , germany ) . continuous resting state eeg was recorded for 2 min before exercise ( pre ) , immediately after ( post ) , and after 10 min of rest ( post'10 ) ( see figure 1 ) . during eeg recording ( brain products gmbh , munich , germany ) participants were asked to take a relaxed sitting position on the cycling ergometer , to close their eyes , and to avoid any movement . eeg was recorded from 15 scalp locations equally distributed over the scalp ( fp1 , fp2 , f3 , fz , f4 , t7 , c3 , cz , c4 , t8 , p3 , pz , p4 , o1 , and o2 ) according to the international 1020 system . the electrodes were placed on a modular elastic cap with the electrical reference and ground electrode being located on positions fcz and afz , respectively . one electrooculographic ( eog ) electrode was placed in horizontal line of the eyelid to identify eye movement artefacts . eeg data were sampled at 1000 hz and were digitally band - pass filtered ( high - pass 2 hz , low - pass 120 hz ) . the signal was rereferenced to the average potential of the 13 recording electrodes and segmented into epochs of 4 s. all epochs were baseline corrected and those epochs contaminated by eye or muscular artefacts were excluded from further analysis . using fast fourier transformation ( fft ) , the time domain data were transferred into power values in the frequency domain with a frequency resolution of 0.244 hz . the iapf was defined as the frequency bin showing the highest power value within 713 hz at occipital sites ( means o1 and o2 ) . the occipital sites have been chosen according to numerous previous studies based on the rationale that alpha oscillations are the strongest over the parietooccipital cortical areas allowing reliable detection of the alpha peak [ 7 , 3032 ] . one subject was excluded from further analyses due to absence of a clear alpha peak . statistical comparisons of performance and physiological data , recorded after sse and ee at t1 and t2 , were performed using a repeated measurement analysis of variance ( anova ) with the factors block ( t1 versus t2 ) and protocol ( ee versus sse ) . the iapf was analyzed calculating a repeated measurement analysis of variance ( anova ) with the factors block ( t1 versus t2 ) , protocol ( ee versus sse ) , and time ( pre versus post versus post'10 ) . significant main effects and interactions were further analyzed using fischer 's lsd post hoc test . means and standard deviations ( sd ) of selected parameters recorded after the ee and sse are summarized in table 1 . an anova with the factors block ( t1 versus t2 ) and protocol ( ee versus sse ) was calculated for bla , hr , ppo , and rpe . as expected , a main effect for protocol was found for all parameters indicating higher values during ee when compared to sse thus , confirming that ee was more intense than sse ( all p < 0.000 ) . furthermore , a significant block main effect for ppo ( p < 0.000 ) , as well as significant protocol block interactions for bla , subsequent post hoc analyses on the protocol block interactions revealed significantly higher values for bla , hr , and ppo ( all p < 0.05 ) after ee when compared to sse at t1 and t2 , respectively . in addition , ppo in the ee protocol increased by 13.95 8.95% from t1 to t2 ( p = 0.000 ) . finally , hr was significantly reduced and bla showed a trend towards a reduction after sse at t2 when compared to t1 ( hr : p = 0.028 ; bla : p = 0.084 ) . an anova with the factors block ( t1 versus t2 ) , protocol ( ee versus sse ) , and time ( pre versus post versus post'10 ) yielded a significant main effect for time ( f2,16 = 7.232 , p = 0.006 ) , as well as a significant protocol time interaction ( f2,16 = 3.995 , p = 0.039 ) . post hoc analysis on the protocol time interaction revealed a significant increase in iapf from pre to post ( p = 0.012 ) , as well as from pre to post'10 ( p = 0.003 ) , for the ee protocol . although physical exercise has been shown to have positive effects on brain function and cognition across the lifespan , the underlying neurophysiological processes require further study . the present study examined the effects of an acute bout of physical exercise as well as four weeks of exercise training on the iapf . the acute effect was examined following exercising at two different intensities , sse and ee . the main finding is that the iapf significantly increased immediately after ee and remained elevated for a minimum of 10 minutes . furthermore , four weeks of exercise training at steady state intensity did not result in any changes of the iapf . the iapf is considered a putative marker of an individual 's state of arousal and attention , as well as speed of information processing . therefore , the results of the present study indicate an acute bout of strenuous physical exercise activating mechanisms in the brain which facilitate information processing . this is in line with previous research on the effect of acute exercise on cognitive task performance . a recent metaregression analysis indicated that , following exercise , cognitive task performance improved by a mean effect of 0.20 . in addition , some experiments measured young adults ' sensory sensitivity before and after exhaustive cycling using the critical flicker fusion ( cff ) frequency . cff threshold and , in contrast , steady state exercise induces an increase in cff discrimination mainly during exercise , but it quickly returns to baseline levels immediately after exercise has ended . in the same vein , reaction times ( rt ) are shorter during both maximal and submaximal exercises ; however , the effect of submaximal exercise on rt disappears very quickly after exercise cessation [ 17 , 39 ] . the results of the present study support previous research suggesting that acute exercise modulates arousal and activation , which remain temporarily elevated immediately after exercise . however , this effect depends on influencing variables such as intensity , duration , type of exercise , age , and the fitness level of the subjects as well as the time elapsed after exercise has ended . it has been proposed that the arousing effect of acute exercise is mediated by changes in neural activity in the ascending reticular - activating system . within the brainstem , neurons of the reticular formation stimulate cortical activation by exciting the widespread projecting neurons of the nonspecific thalamocortical projection system . these thalamocortical feedback loops of excitatory and inhibitory neurons are thought to be the primary generator of the alpha rhythm . therefore , the modulation of the iapf following acute exercise is probably linked to exercise - induced activation of the brain 's arousal mechanisms . in addition , acute exercise has been shown to increase cbf and higher cbf correlated with iapf in brain areas associated with working memory functions and modulation of attention [ 4 , 15 ] . in sum , these results indicate that the increase in iapf represents a potential neurophysiological mechanism underlying improvements in cognitive task performance following acute physical exercise . the temporal characteristics of brain adaptations in response to exercise training are not well understood because only few intervention studies have been conducted so far . previous aging studies focused largely on the effects of medium to long term ( > 6 months ) exercise training programs , whereas the shorter term effects have not been studied until recently . 2013 ) have shown that even shorter term ( 12 weeks ) aerobic exercise training can facilitate neuroplasticity and promote brain health in sedentary adults . moreover , in the literature , changes in brain structure and function are often attributed to changes in fitness . therefore , in the present study , we were interested whether a very short exercise training intervention ( just enough to induce an improvement in physical fitness ) is associated with a change in iapf . the iapf is functional measure , and therefore , adaptations may occur already after shorter training periods ; before any structural changes take place . however , although exercise training for four weeks at steady state intensity was effective to induce an improvement in physical fitness , it was not sufficient to change iapf . this is indicated by the absence of significant main effects and interactions for the factor block . it remains to be determined in future studies whether longer and/or more intensive periods of physical training may induce changes in iapf . although it is well established that iapf is correlated with cognitive performance , the proposed relationship between physical exercise , iapf , and cognition in this study remains speculative as behavioral measures were not collected . we abstained from behavioral tests because cognitive engagement has also been shown to induce a transient increase in iapf [ 43 , 44 ] . therefore , effects of physical exercise and cognitive engagement on iapf could interfere . to avoid such interference , in a first step , we focused on the relationship between physical exercise and iapf to provide a functional framework for future research such as addressing the significance of exercise - induced changes in iapf on cognitive abilities in different populations ( e.g. , males and females , healthy and diseased , and young and old ) . it is concluded that the iapf , a neurophysiological marker for the individual 's state of arousal and attention , increased following intense physical exercise . in contrast to intense physical exercise , the iapf remained unchanged after 30 min of steady state exercise , and it is not altered following four weeks of steady state exercise training despite improvements in physical fitness . the cumulative pattern of results indicates a dose - response relationship between physical exercise and iapf which needs to be further studied as it may have important implications for exercise recommendations to promote brain health and cognition .

previous research has shown that both acute and chronic physical exercises can induce positive effects on brain function and this is associated with improvements in cognitive performance . however , the neurophysiological mechanisms underlying the beneficial effects of exercise on cognitive processing are not well understood . this study examined the effects of an acute bout of physical exercise as well as four weeks of exercise training on the individual resting state electroencephalographic ( eeg ) alpha peak frequency ( iapf ) , a neurophysiological marker of the individual 's state of arousal and attention , in healthy young adults . the subjects completed a steady state exercise ( sse ) protocol or an exhaustive exercise ( ee ) protocol , respectively , on two separate days . eeg activity was recorded for 2 min before exercise , immediately after exercise , and after 10 min of rest . all assessments were repeated following four weeks of exercise training to investigate whether an improvement in physical fitness modulates the resting state iapf and/or the iapf response to an acute bout of sse and ee . the iapf was significantly increased following ee ( p = 0.012 ) but not following sse . it is concluded that the iapf is increased following intense exercise , indicating a higher level of arousal and preparedness for external input .

the ultimate goal of cleft lip and palate surgery is that the patients look well , feed well , and speak well . to achieve these goals , much attention has been paid in the literature to the technique of repair and clinical outcomes of surgery , but little attention has been paid to morbidity and mortality accompanying cleft surgery.1 in any case , some authorities believe that complication following cleft lip surgery is unusual.2 the factors influencing the overall outcome of cleft repair are multiple and complex . timing of cleft lip and palate repair remains controversial in the literature,1 and compromise must be reached between the age of the patient at surgery and the surgical outcome with respect to facial growth , scarring , language development , and psychological factors.3 morbidity and mortality rates for cleft lip and palate repairs are , however , difficult to compare because of the absence of a uniform classification of morbidity.4 improvement in pediatric anesthesia and surgical techniques has been reported to decrease the incidence of intraoperative and postoperative complications and has allowed more complex operations to be carried out at an earlier age.5 one hundred and fifteen patients consisting of children ( below 12 years ) and adult ( 12 and above ) patients who were operated for cleft lip alone , cleft palate alone , and cleft lip and palate combined within a 2-year period at the university of maiduguri teaching hospital ( a tertiary institution in the north eastern nigeria ) were followed up at 1 week , 2 weeks , 1 month , 3 months , 6 months , and 1 year intervals , respectively . the study protocol was approved by the research and ethics committee of the hospital , and informed consent forms were obtained from parents of participating children and the adult patients . a total of 120 cleft surgeries consisting of 90 cleft lip surgeries and closure of 30 cleft palates were done . unilateral cleft lips ( ucls ) were repaired using millard 's rotation advancement flap technique6 and bilateral cleft lips ( bcls ) were closed by millard 's myoplastic technique6 at the earliest age of 3 months . cleft palates were closed from 18 months using von langenbeck technique.6 all surgeries for children with cleft lip and palate were carried out under general anesthesia , while cleft lip surgeries in adults were operated under conscious sedation using local anesthesia . all palate surgeries in adult and cleft lip surgeries in apprehensive adults were also operated under general anesthesia . postoperatively , patients who had cleft lip repair were discharged after 57 days , while those who had palate repair were discharged after 2 weeks . postoperative complications were categorized as minor , major , and general1 ( complications not related to the operation ) . in the major category were excessive postoperative bleeding ( in cleft lip and palate surgeries ) and complete wound breakdown ( only in cleft lip surgery and airway obstruction following palate surgery ) . in the minor category were partial wound separation , vermilion notching , stitch marks formation , hypertrophic / keloid scar formation following cleft lip surgery , and also fistula formation following cleft palate surgery . general complications include diarrhea , malaria , upper respiratory tract infection , and lower respiratory tract infection . collected data included age , sex , type of cleft , and types of complication associated were analyzed using statistical package for social sciences ( spss ) for windows student version 13.0 ( spss inc . one hundred and twenty cleft surgeries were performed on 115 patients ( 85 children , and 30 adults ) and patients followed up over a period of 1-year . the ages of the patients ranged from 3 months to 65 years . of these surgeries , 18 were unilateral lip repair , 12 bilateral lip repair , while 13 were cleft palate surgeries ( only 3 of these were isolated soft palate surgeries ) [ table 1 ] . complications in 120 cleft surgeries in children and adult a total of 43 complications ( 31 in children , 12 were adults ) were recorded over the study period , 18 ( 41.9% ) of these complications were noticed in ucl repair , while 12 ( 27.9% ) and 13 ( 30.2% ) complications were observed in bcl and cleft palate surgeries respectively . twelve ( 27.9% ) minor complications occurred in 62 patients following ucl operation , and 9 ( 20.9% ) was observed in bilateral cleft repair [ table 2 ] . a high complication rate of 72% was recorded among children which consist substantially of minor and general complications ( complications due to cross - infection ) . however , more major complications were observed among adult patients . there was a significant statistical difference between children and adult with respect to all the complications ( major , minor , and general complication ) ( p < 0.05 ) [ table 3 ] . vermilion notching 38.1% was found to be the most common minor complication in cleft lip surgeries seen in both children and adult , 6 and 2 cases , respectively [ table 4 ] . number of surgeries by types of complication types of postoperative complications recorded after 120 cleft repair in children and adult minor complications following cleft lip repair in children and adult postoperative bleeding was the only major complication observed in both unilateral and bilateral lip closure in adults , whereas in children complete wound breakdown was seen in three cases [ table 5 ] . of the 30 palate surgeries performed , two patients had fistula in the anterior aspect of the hard palate and both were children [ table 6 ] . there were two postoperative bleeding from the soft palate in two adults , and a case of airway obstruction was seen in an adult [ table 6 ] . major complications following cleft lip repair in children and adult types of complications following cleft palate surgeries in children and adult table 7 revealed a high incidence ( 25.6% ) of general complications among children that underwent cleft surgery . upper respiratory tract infection 46% was found to be the most frequent general complication recorded . the presence of cleft lip and palate has both esthetic and functional implications for patients in their social interactions , particularly on their ability to communicate effectively and on their facial appearance . many surgical techniques have been advocated to improve the result of cleft surgery.7 it is obvious that every surgeon attempts to prevent complications following these surgical procedures . the incidence of surgical complications has been found out to be greater in patients who had lip repair in the 1 week of life . death from complications due to lip repair is not frequently reported in the literature which indicates an insignificance.1 some authorities even believe that complication following cleft lip surgery is unusual.2 our study revealed a high complication rate of 35.8% , most of which are minor complications 53.4% . this contrast the report of wilhelmsen and musgrave , which was as low as 13%.1 this high rate may be due to small sample size recorded in this study . wilhelmsen and musgrave , however , urged that surgery should be delayed until the patient is 10 weeks , weighs at least 10 pounds , had a hemoglobin of at least 10 g , and a white cell count of < 10,000 . he observed that when these rules were followed complications were 5 times less.1 in previous studies , the type of cleft lip makes no demonstrable difference in the number or the nature of subsequent complications.1 however , in this study , some notable difference was observed in bcl which has been brought about by proclined premaxilla , shortened prolabium , and the columella causing some tension at the suture line increasing the incidence of wound breakdown and scar formation . it has been the practice in our institution to commence elastic traction for patients with bcl , but the hindrance to this has been late presentation of patients and inadequate use of the appliance when the patients are at home . slight notching at the vermilion of the lip occurred in six children who had lip repair , and two cases were recorded in the adult [ figure 1 ] . mccarthy and court8 attributed vermillion notching after cleft lip repair to excessive sacrifice of vermillion during primary repair or inadequate approximation of orbicularis oris muscle fibers.9 hypertrophic scar was noticed at 2 weeks postoperative review in five children [ figure 2 ] and two adults , respectively . although the development of hypertrophic scar and keloids at incision sites following surgery have been reported to be relatively common among blacks when compared with caucasians , the etiology of this condition is still not clear.10 satisfactory improvement of some of the scars was noticed in the majority of children at 3 months of postoperative review . this probably maybe due to the greater healing potential commonly observed in the very young.11 the partial wound breakdown observed among children in this study was at the reconstructed nasal floor . contamination of surgical site from mucous secretion has been reported to be common in children.6 adekeye and lavery,6 however , attributed the wound dehiscence observed in his study to contamination of surgical site by the attending mothers . sowemimo12 opined that wound dehiscence in cleft surgery may sometimes be due to nosocomial infections from attending medical team . this informed the reason for early exposure of wound dressing after 72 h in our center . notching at the vermilion following cleft lip repair in an adult a child with hypertrophic scar following bilateral cleft lip repair the incidence of fistula formation following palate repair has been reported to be as high as 34% , and the severity of the original cleft has been shown to correlate with the risk of the fistulas.13 in this study , fistula was only noticed among children . this , however , is attributed to insufficient palatal tissue available for advancement to the midline . however , some authors have reported infection in the wound as the major concern because it may be responsible for dehiscence of the tissues and development of fistulas.14 postoperative bleeding was noticed more in the adult patients . this may be due to the large size of vessels supplying the surgical site . in any case , sustained digital pressure general complications were observed among children only at the rate of 25.6% , of which upper respiratory tract infection was most prominent . these are complications not directly related to the operation , majority of these general complications were as a result of long hospital stay on admission . this may be due to high susceptibility to infection of the children with low immunity common among children . our findings contrast with those of other authors.5 a large proportion of these complications was observed among patients who had cleft palate surgery and had to stay in the hospital for long . demay established in their study that reduction of hospital admissions and length of stay do not affect the outcome of cleft lip and palate surgery . they , however , hypothesized that the functionality of the patient family in postoperative care is an additional factor.5 a reduction in hospital stay may reduce the number of general complications following cleft lip and palate surgery considerably . wilhelmsen and musgrave reported 0.34% mortality.1 the mortality ever following cleft repair occurred in 1953 in a 3-month - old white male child who died on the 2 postoperative day due to viral pneumonia . we strongly advise a reduction in the length of hospital stay , particularly after surgery . we also , however , advice early contact and treatment for patients with cleft because of the good healing potentials among children . the authors certify that they have obtained all appropriate patient consent forms . in the form the patient(s ) has / have given his / her / their consent for his / her / their images and other clinical information to be reported in the journal . the patients understand that their names and initials will not be published and due efforts will be made to conceal their identity , but anonymity can not be guaranteed . the authors certify that they have obtained all appropriate patient consent forms . in the form the patient(s ) has / have given his / her / their consent for his / her / their images and other clinical information to be reported in the journal . the patients understand that their names and initials will not be published and due efforts will be made to conceal their identity , but anonymity can not be guaranteed .

background : this paper seeks to investigate the incidence of short - term postoperative complications in children and adult patients undergoing primary surgery of cleft lip and palate.patients and methods : one hundred and fifteen patients consisting of children ( below 12 years ) and adult ( 12 years and above ) who were operated for both cleft lip and palate within a 2-year period at the university of maiduguri teaching hospital were reviewed postoperatively at 1 week , 2 weeks , 1 month , 3 months , 6 months , and 1 year intervals , respectively . the complications encountered within the periods of the review were recorded.results:one hundred and twenty surgeries were performed on 115 patients ( 85 children 30 adults ) . a total of 43 complications ( 31 in children and 12 in adults ) were recorded over the study period . eighteen ( 41.9% ) of these complications were noticed in unilateral cleft lip repair , while 12 ( 27.9% ) and 13 ( 30.2% ) complications were observed in bilateral cleft lip and cleft palate surgeries , respectively . a higher complication rate ( 72.0% ) was recorded in children compared with adults . major complications ( 13.9% ) were , however , observed more in adults than children.conclusion:although every surgeon attempts to prevent complications during surgery , they may still occur . the high complication rate observed in our study may be due to a small sample size . general complications observed among children are due to cross infection during a hospital stay , contributing immensely to the higher rate of complications in children . moreover , this may be reduced by short hospital stay following surgery . we also advocate early contact with children with cleft , and early surgical intervention in other to prevent some of the major complications encountered in adult patients .

osteoporosis is a chronic progressive disease characterized by low bone mass , micro - architectural bone deterioration , and decreased bone strength that lead to increased bone fragility and a consequent increase in fracture risk . the world health organization ( who ) came up with definitions of osteoporosis and osteopenia in postmenopausal white women based on bmd to help physicians classify degrees of bone loss . in current clinical practice , the diagnosis of osteoporosis is based on either a health outcome like a fragility fracture , or an intermediate outcome like a low bmd . osteoporosis is generally considered a disorder of postmenopausal women , but low bone mass and accelerated bone loss can also occur early in life and contribute to pre - menopausal osteoporosis . certain groups of premenopausal women are at higher risk of osteoporosis than their peers , and these include women with disease states like primary hyperparathyroidism , cushing 's syndrome , and thyrotoxicosis , that promote accelerated bone loss . premenopausal osteoporosis is defined as low bone mineral density ( a z score below -2.0 ) in combination with risk factors such as chronic malnutrition , eating disorders , hypogonadism , glucocorticoid exposure , and previous fractures . longitudinal studies have shown that calcium utilization increases during early puberty and that the highest rates of calcium accrual may occur at a mean age of 12.5 years in girls and 14 years in boys . factors affecting the attainment of peak bone mass include genetic background , nutritional status , and activity level . family studies have shown that 50 - -80% of variance in bone mass is heritable . bone mineral density follows a normal distribution , and low bone density , defined as a t - score of less than 1.0 standard deviation below the young adult mean is present in about 15% of young , healthy women aged between 30 and 40 years . around 0.5% of these women . currently , there are insufficient data regarding the relationship between bmd and fracture risk in the premenopausal female population . therefore , it is not possible to make recommendations regarding the appropriate bmd criteria for a diagnosis of osteoporosis in premenopausal women in the absence of secondary causes . the who definition of osteoporosis based on a t - score cut - off point of -2.5 is applicable only to the postmenopausal female and can not be applied to the premenopausal female in the absence of secondary causes of bone loss . low peak bone mass without the presence of fragility fractures or height loss may be reflective of the normal variation in bmd . risk factors of premenopausal osteoporosis include the following : genetic influences , ethinicity , hormonal influences , nutritional factors , physical activity , disease factors , medications , and smoking . racial and ethnic differences in bmd values have been reported , and population norms have been established for use as dxa reference standards . bone loss can also occur due to prolonged amenorrhea and estrogen deficiency . in a study of 200 women , aged 16 to 40 with 6 months to 24 years of amenorrhea , it was found that lumbar spine bmd was 15% lower compared to 57 age matched controls . as estrogen has antiresorptive properties in bone , it is thought that oral contraceptive ( oc ) use can increase bone mineral density . however , prospective studies on oc use in premenopausal women failed to show consistent gains in bmd . progestational agents used for contraception in premenopausal women can cause bone loss due to the associated oestrogen deficiency . increased calcium intake is generally recommended in osteoporosis . in studies looking at the association of bmd and calcium intake in healthy premenopausal women , statistically significant correlations were found between calcium intake and bmd at three femoral sites . this is prevalent in female athletes as they have a higher incidence of eating disorders than their peers and are at a much higher risk of stress fractures . in a cohort study of 56 women with eating disorders , it was found that the bmd in the femur of these women were below the critical fracture threshold in 75% of patients . the factors involved include estrogen deficiency and resultant amenorrhea , leptin levels are usually low in patients with anorexia nervosa , and may have an effect on igf-1 , especially in low body weight states . physical activity has been shown to increase bmd . a longitudinal study was done to examine the bmd of the lumbar spine and femur in premenopausal caddies and desk workers . the change in bmd was significantly better in the caddies than desk workers , suggesting that regular intense activity resulted in gain in bmd . glucocorticoids are commonly used in the treatment of many conditions , including inflammatory and autoimmune diseases . glucocorticoid induced osteoporosis is the most common cause of drug - induced osteoporosis , and occurs as a result of decreased bone formation secondary to impaired osteoblastic differentiation and function . selective serotonin reuptake inhibitors ( ssri ) can cause bone loss by affecting functional serononin receptors and transporters that are present in osteoblasts and osteocytes . anticonvulsants accelerate the metabolism of vitamin d and may also have direct inhibitory effects on osteoblast differentiation . two meta - analyses have reported significant lowering of bmd at the hip in long term smokers compared to nonsmokers . although most young people with osteoporosis have an identifiable cause , some may have idiopathic osteoporosis in which no etiology can be found . this has been variably defined in the literature and most of the reported cases are in caucasians , with the usual clinical history being one of multiple atraumatic fractures , involving mainly cancellous bone . a retrospective study on young healthy women who presented with fragility fractures was carried out who had trans - iliac bone biopsies undertaken as part of their evaluation . all secondary causes of osteoporosis were excluded by means of history taking , physical examination and biochemical testing . it was demonstrated that women with idiopathic osteoporosis had evidence of decreased bone formation and altered bone resorption . thus , it was concluded that women with idiopathic osteoporosis have uncoupling of resorption and formation , as well as osteoblast dysfunction . weight bearing exercises , adequate nutrition and calcium intake , smoking cessation as well as maintenance of a normal body mass index are of value in maintaining bmd . antiresorptive therapy has only been evaluated in premenopausal women receiving glucocorticoid therapy or in secondary causes of bone loss such a primary hyperparathyroidism . there are no data regarding the use of bisphosphonates in the treatment of premenopausal osteoporosis in the absence of these secondary causes . in women who are oestrogen replete who have normal bone turnover , bisphosphonates may be harmful as they have long term skeletal and may have adverse effects on future pregnancies . they have been shown to pass through the placenta in animal studies and accumulate in the fetus , and their effects on the developing fetal skeleton are still unknown . hence , bisphosphonates should be limited in premenopausal women until further research is done to clarify its safety and efficacy . the national osteoporosis foundation states that the current available data are insufficient to formulate specific recommendations for premenopausal women . premenopausal women with disease states associated with accelerated bone loss should be managed on a case - by - case basis . the advantages of early detection have to be weighed against the risk of potential harm , including treatment associated morbidity . osteoporosis is a chronic progressive disease characterized by low bone mass , micro - architectural bone deterioration , and decreased bone strength that lead to increased bone fragility and a consequent increase in fracture risk . the world health organization ( who ) came up with definitions of osteoporosis and osteopenia in postmenopausal white women based on bmd to help physicians classify degrees of bone loss . in current clinical practice , the diagnosis of osteoporosis is based on either a health outcome like a fragility fracture , or an intermediate outcome like a low bmd . osteoporosis is generally considered a disorder of postmenopausal women , but low bone mass and accelerated bone loss can also occur early in life and contribute to pre - menopausal osteoporosis . certain groups of premenopausal women are at higher risk of osteoporosis than their peers , and these include women with disease states like primary hyperparathyroidism , cushing 's syndrome , and thyrotoxicosis , that promote accelerated bone loss . premenopausal osteoporosis is defined as low bone mineral density ( a z score below -2.0 ) in combination with risk factors such as chronic malnutrition , eating disorders , hypogonadism , glucocorticoid exposure , and previous fractures . longitudinal studies have shown that calcium utilization increases during early puberty and that the highest rates of calcium accrual may occur at a mean age of 12.5 years in girls and 14 years in boys . factors affecting the attainment of peak bone mass include genetic background , nutritional status , and activity level . family studies have shown that 50 - -80% of variance in bone mass is heritable . bone mineral density follows a normal distribution , and low bone density , defined as a t - score of less than 1.0 standard deviation below the young adult mean is present in about 15% of young , healthy women aged between 30 and 40 years . around 0.5% of these women . currently , there are insufficient data regarding the relationship between bmd and fracture risk in the premenopausal female population . therefore , it is not possible to make recommendations regarding the appropriate bmd criteria for a diagnosis of osteoporosis in premenopausal women in the absence of secondary causes . the who definition of osteoporosis based on a t - score cut - off point of -2.5 is applicable only to the postmenopausal female and can not be applied to the premenopausal female in the absence of secondary causes of bone loss . low peak bone mass without the presence of fragility fractures or height loss may be reflective of the normal variation in bmd . risk factors of premenopausal osteoporosis include the following : genetic influences , ethinicity , hormonal influences , nutritional factors , physical activity , disease factors , medications , and smoking . racial and ethnic differences in bmd values have been reported , and population norms have been established for use as dxa reference standards . bone loss can also occur due to prolonged amenorrhea and estrogen deficiency . in a study of 200 women , aged 16 to 40 with 6 months to 24 years of amenorrhea , it was found that lumbar spine bmd was 15% lower compared to 57 age matched controls . as estrogen has antiresorptive properties in bone , it is thought that oral contraceptive ( oc ) use can increase bone mineral density . however , prospective studies on oc use in premenopausal women failed to show consistent gains in bmd . progestational agents used for contraception in premenopausal women can cause bone loss due to the associated oestrogen deficiency . increased calcium intake is generally recommended in osteoporosis . in studies looking at the association of bmd and calcium intake in healthy premenopausal women , statistically significant correlations were found between calcium intake and bmd at three femoral sites . this is prevalent in female athletes as they have a higher incidence of eating disorders than their peers and are at a much higher risk of stress fractures . in a cohort study of 56 women with eating disorders , it was found that the bmd in the femur of these women were below the critical fracture threshold in 75% of patients . the factors involved include estrogen deficiency and resultant amenorrhea , leptin levels are usually low in patients with anorexia nervosa , and may have an effect on igf-1 , especially in low body weight states . physical activity has been shown to increase bmd . a longitudinal study was done to examine the bmd of the lumbar spine and femur in premenopausal caddies and desk workers . the change in bmd was significantly better in the caddies than desk workers , suggesting that regular intense activity resulted in gain in bmd . glucocorticoids are commonly used in the treatment of many conditions , including inflammatory and autoimmune diseases . glucocorticoid induced osteoporosis is the most common cause of drug - induced osteoporosis , and occurs as a result of decreased bone formation secondary to impaired osteoblastic differentiation and function . selective serotonin reuptake inhibitors ( ssri ) can cause bone loss by affecting functional serononin receptors and transporters that are present in osteoblasts and osteocytes . anticonvulsants accelerate the metabolism of vitamin d and may also have direct inhibitory effects on osteoblast differentiation . two meta - analyses have reported significant lowering of bmd at the hip in long term smokers compared to nonsmokers . although most young people with osteoporosis have an identifiable cause , some may have idiopathic osteoporosis in which no etiology can be found . this has been variably defined in the literature and most of the reported cases are in caucasians , with the usual clinical history being one of multiple atraumatic fractures , involving mainly cancellous bone . a retrospective study on young healthy women who presented with fragility fractures was carried out who had trans - iliac bone biopsies undertaken as part of their evaluation . all secondary causes of osteoporosis were excluded by means of history taking , physical examination and biochemical testing . it was demonstrated that women with idiopathic osteoporosis had evidence of decreased bone formation and altered bone resorption . thus , it was concluded that women with idiopathic osteoporosis have uncoupling of resorption and formation , as well as osteoblast dysfunction . weight bearing exercises , adequate nutrition and calcium intake , smoking cessation as well as maintenance of a normal body mass index are of value in maintaining bmd . antiresorptive therapy has only been evaluated in premenopausal women receiving glucocorticoid therapy or in secondary causes of bone loss such a primary hyperparathyroidism . there are no data regarding the use of bisphosphonates in the treatment of premenopausal osteoporosis in the absence of these secondary causes . in women who are oestrogen replete who have normal bone turnover , bisphosphonates may be harmful as they have long term skeletal and may have adverse effects on future pregnancies . they have been shown to pass through the placenta in animal studies and accumulate in the fetus , and their effects on the developing fetal skeleton are still unknown . hence , bisphosphonates should be limited in premenopausal women until further research is done to clarify its safety and efficacy . the national osteoporosis foundation states that the current available data are insufficient to formulate specific recommendations for premenopausal women . premenopausal women with disease states associated with accelerated bone loss should be managed on a case - by - case basis . the advantages of early detection have to be weighed against the risk of potential harm , including treatment associated morbidity . low bone mass density in premenopausal women warrants further evaluation and secondary causes should be sought for and treated . antiresorptive therapy has only been evaluated in premenopausal women receiving glucocorticoids or in secondary causes of bone loss such as primary hyperparathyroidism , and should be used with caution in premenopausal women because of the lack of safety data for future pregnancies . lifestyle modification is of value in preventing progressive bone loss in premenopausal women and should be actively encouraged .

osteoporosis has traditionally been considered a disorder of postmenopausal women , but low bone mass and accelerated bone loss can also occur early in life causing premenopausal osteoporosis . there are a few risk factors that increase a woman 's risk of premenopausal osteoporosis , including drugs , hormonal and nutritional factors , and physical in - activity , which need to be identified and managed accordingly . lifestyle modification is of importance in preventing progressive bone loss in premenopausal women and should be actively encouraged .

nanoparticles with the capacity to increase drug solubility , stability , and specificity and , therefore , achieve enhanced therapeutic efficacy and attenuated off - target side effects have been explored extensively for the treatment of various types of diseases . nanoparticulate drug carriers include micelles , liposomes , polymer drug conjugates , dendrimers , and inorganic particles . generally , a drug / small molecule can be loaded in a nanocarrier either by being encapsulated in a core with a similar hydrophobicity or by being conjugated through covalent bonds . for instance , hydrophobic drug doxorubicin can be conveniently encapsulated into the hydrophobic domain of a micelle fabricated from amphiphilic copolymers or conjugated to water - soluble polymers via various bonds . correspondingly , the loaded doxorubicin can be released from the carrier by diffusion , the erosion of the carrier , or both , or via the breakage of the linker . depending on the requirements of their applications , the extended drug release can decrease the dosing frequency , while the stimulus responsive drug release can potentially attenuate drug side effects . although tremendous efforts have been devoted to the development of nanomedicine for cancer chemotherapy , how to safely deliver active drugs into targeted cancer cells and kill them remains a challenge . there are at least two prerequisites for a safe nanocarrier : ( i ) a nanocarrier should be free of premature release before reaching its targeted cells , and ( ii ) the nanocarrier should enter only target cells . because conventional encapsulation approaches are commonly associated with burst release due to the detachment of a surface - adsorbed payload , polymer drug conjugates have attracted more and more attention recently because they can potentially eliminate the off - target effects by preventing initial burst release . drug conjugate , which allows the nanoparticle to be stable under extracellular physiological conditions while unloading all payloads in a very short period of time due to the cleavage of the linker by intracellular stimuli ; ( ii ) contain stealth poly(ethylene glycol ) ( peg ) blocks , which can extend the circulation time of nanocarriers by preventing opsonization and protecting them from being captured by the mononuclear phagocytic system ( mps ) ; and ( iii ) be functionalized with a targeting ligand that can specifically bind receptors overexpressed on the membrane of cancer cells . hereby , with the help of redox potential sensitive disulfide bonds and herceptin , a her2 antibody , we designed a camptothecin aldrithiol-2 , camptothecin ( cpt ) , and silica gel ( spherical , 100 m ) were purchased from tokyo chemical industry co. , ltd . 2-mercaptoethanol , dl - dithiothreitol ( dtt ) , doxorubicin ( dox ) , thiazolyl blue tetrazolium bromide ( mtt ) , 2,2-azobis(isobutyronitrile ) ( aibn ) , and cysteamine hydrochloride were purchased from sigma - aldrich ( st . louis , mo ) . peg - sh ( mpeg - sh ) ( mw = 5000 da ) was purchased from laysan bio , inc . 3-(tritylthio)propionic acid was obtained from santa cruz biotechnology ( santa cruz , ca ) . trypsin - edta , penicillin , streptomycin , dulbecco s modified eagle s medium ( dmem ) , and fetal bovine serum ( fbs ) were obtained from american type culture collection ( atcc , manassas , va ) . all the other solvents used in this research were purchased from sigma - aldrich and used without any further purification unless otherwise noted . poly[2-(pyridin-2-yldisulfanyl ) ] ( pds ) was synthesized by free radical polymerization of 2-(pyridin-2-yldisulfanyl)ethyl acrylate ( pdsa ) using aibn as an initiator ( figure 1 ) . briefly , 500 mg ( 2.7 mmol ) of pdsa was dissolved in 10 ml of anisole into a 100 ml round - bottom flask and degassed for 30 min at room temperature . after that , 0.09 mmol of the aibn anisole solution was added to the mixture . then the flask was immersed in an oil bath maintained at 65 c and stirred for 24 h. the resulting product was collected by precipitation ( three times ) into ice - cold diethyl ether and dried under vacuum for 48 h at room temperature . the structural composition of the polymer was analyzed by h nuclear magnetic resonance ( nmr ) ( mercury varian 400 nmr , varian inc . , palo alto , ca ) using cdcl3 as a solvent and tms as an internal standard . the molecular weight ( mw ) and polydispersity of pds were characterized by gpc ( viscotek gpcmax ve 2001 gpc solvent / sample module , viscotek ve 3580 ri detector , and 270 dual detector ) using tetrahydrofuran ( thf ) as the mobile phase . the pds polymer was modified to yield an amphiphilic polymer by grafting peg through the thiol briefly , the pds polymer ( 50 mg , 7.14 mol ) was dissolved in 5 ml of methylene chloride and mixed with 2 ml of a mpeg - sh ( 50 mg , 10 mol , mw of 5 kda ) solution , and the solution was stirred for 12 h at room temperature . the progress of the reaction was evidenced by the appearance of a yellow color . the final product was precipitated ( three times ) in ice - cold diethyl ether and vacuum - dried for 48 h to yield pdsg . syntheses of thiolated camptothecin ( cpt - sh ) ( a ) and the polymer drug conjugate ( cpdsg - nh2 ) ( b ) and fabrication of a camptothecin nanoparticle ( cn ) and a herceptin - functionalized camptothecin nanoparticle ( hcn ) ( c ) . the modification was confirmed by h nmr and hplc [ waters model 2695 attached to a waters 2996 photodiode array detector and column ( c18 , dimensions of 25 cm 4.6 mm ) ] using a 1:1 ( v / v ) acetonitrile / water mixture [ supplemented with 0.1% trifluoroacetic acid ( tfa ) ] as the mobile phase . the cpt - grafted pdsg polymer ( cpdsg ) was prepared by the thiol disulfide exchange reaction between cpt - sh and the pdsg polymer ( figure 1 ) . briefly , pdsg ( 50 mg ) was dissolved in dimethylformamide ( dmf ) ( 2 ml ) and mixed with cpt - sh ( 10 mg in 1 ml of dmf ) . the reaction was conducted at room temperature for 12 h. the final product was precipitated ( three times ) in ice - cold ether and dried under vacuum for 48 h. h nmr ( figure s1 of the supporting information ) , and hplc confirmed the formation of the cpt polymer conjugate . drug conjugate - based micelle nanoparticles of cpdsg were prepared in pbs by the cosolvent dialysis method . briefly , cpdsg ( 2 mg in 200 l of dmso ) was dropped into 2 ml of ddh2o while it was being stirred and equilibrated for 30 min . then the solution was dialyzed ( three times ) using a spectra / por dialysis tube [ molecular weight cutoff ( mwco ) of 1.0 kda ] against pbs for 8 h , and the nanoparticles were collected and analyzed . for surface functionalization , cpdsg ( 2 mg ) was reacted with cysteamine hydrochloride ( 200 g ) in 500 l of a dichloromethane / methanol mixture [ 1:1 ( v / v ) ] for 3 h at room temperature . after that , the solvent was removed under vacuum , and the residue was redissolved in dmso ( 100 l ) and dropped into 2 ml of ddh2o while it was being stirred . the nanoparticle solution was dialyzed ( three times ) through a spectra / por dialysis tube ( mwco of 1.0 kda ) against phosphate buffer ( ph 8.0 ) for 8 h to yield cn ( figure 1c ) . pds and pdsg nanoparticles were fabricated directly from pds and pdsg polymers in parallel as a control . to endow cn with the specific targeting effect for her2-positive cancer cells , cn was further functionalized with a her2 antibody , herceptin ( roche ) , at a ratio of 50 herceptin molecules / cn with the help of 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide ( edc ) and n - hydroxysuccinimide ( nhs ) to yield hcn . unreacted herceptin was removed from the nanoparticle solution by sephadex column filtration ( three times ) using pbs as a mobile phase . the morphology , size distribution , and surface charge ( potential ) of the cpdsg nanoparticles ( cn ) or cpdsg her nanoparticles ( hcn ) were studied via transmission electron microscopy ( tem ) using a hitachi h-800 instrument operated at an accelerating voltage of 200 kv and dynamic light scattering ( dls ) ( malvern zetasizer , nano - zs ) . for tem , the nanoparticle solution ( 1.0 mg / ml ) was dropped on a carbon - coated copper grid and dried using tissue paper , and the images were taken without any further processing . dls measurements were taken using a 1.0 mg / ml nanoparticle solution in pbs . in vitro release of the drug from cn was performed in pbs ( ph 7.4 ) with and without dtt at 37 c using a spectra / por dialysis bag ( mwco of 1.0 kda ) . three milliliters of a cn solution ( 200 g of cpt ) was transferred into the tubing and immersed in pbs buffer ( 40 ml ) . one hundred microliters of the solution from the bag was sampled at different time intervals , and the same amount of fresh buffer was replaced to keep the volume constant . after dialysis for 24 h , dtt ( final concentration of 10 mm ) was added to the release buffer , which was then sampled every 10 min for an additional 6 h. finally , the samples were lyophilized and redissolved in 1 ml of a dmso / ddh2o mixture [ 1:1 ( v / v ) ] , and the content of cpt - sh was quantified via hplc as described above . cancer cells , hct-116 and kb cells , obtained from atcc were maintained as a monolayer culture in dulbecco s modified eagle s medium ( dmem ) supplemented with 10% fetal bovine serum ( fbs ) and 100 units / ml penicillin - streptomycin at 37 c under a humidified atmosphere of 95% air and 5% co2 . cells were subcultured when the confluence reached 75% , and the medium was replaced every other day . the cells were lysed in 1 tritonx lysis buffer at 80% confluence with 1 sodium orthovanadate ( alfa aesar ) and 1 proteinase inhibitor ( roche ) . the protein lysate was then quantified by the bca method ( thermo scientific ) , and equal concentrations of proteins were mixed with laemmli buffer and loaded onto a gradient polyacrylamide gel ( 4 to 14% ) from bio - rad . the membrane was blocked using 5% blotting grade milk ( bio - rad ) for 45 min . after a brief wash with 1 phosphate - buffered saline tween 20 ( pbst ) , the primary antibody was added to the membrane at 1:5000 her2 ( cell signaling ) and 1:1000 gapdh ( santa cruz ) dilutions . the membranes were incubated with the antibody for 90 min at room temperature on a shaker . the membranes were then incubated in the hrp - conjugated secondary antibody at a 1:4000 dilution ( ge ) for 1 h at room temperature to detect target protein . signals were detected using an ecl kit ( amersham biosciences ) and recorded on x - ray film ( phenix ) . cellular uptake of the cpdsg nanoparticles was observed using confocal microscopy . to produce the fluorescence signal for the detection of nanoparticles , doxorubicin was encapsulated in cn or hcn during the fabrication of nanoparticles as described in our previous report . hct-116 and kb cells ( 2 10 cells / well ) was seeded in 35 mm glass - bottom dishes ( mattek ) and grown overnight . the medium were replaced with medium ( supplemented with 10% fbs ) containing the nanoparticle solution ( final dox concentration of 5 m ) and incubated for 3 h. then the cells were washed ( three times ) with prewarmed pbs , fixed for 25 min with formaldehyde ( 3.5% in pbs ) , and washed ( three times ) with pbs , and the nuclei were counterstained with hoechst 33342 ( final concentration of 1 g / ml ) for 5 min . finally , the cells were washed ( three times ) with pbs and analyzed under a confocal microscope ( lsm 510 , carl - zeiss inc . , then the unconjugated cy3 was removed by gel filtration chromatography using sephadex 25 ( three times ) . cy3-labeled hcn was prepared similarly by adding cy3-nhs to the cn suspension before the addition of activated herceptin as described in section 2.4 . cells were seeded at a density of 3.5 10 cells / well in 12-well plates . after being grown overnight , cells were incubated with different treatment groups ( cn and hcn ) for 4 h at a concentration of 1.8 g / ml for cy3 . trypsin was used to detach the cells from the plate , and the cells were collected by centrifugation and then redispersed with 500 l of pbs . facs analysis , cell debris and free particles were excluded by setting a gate on the plot of side - scattered light ( ssc ) versus forward - scattered light ( fsc ) . anticancer activities of cn and hcn nanoparticles were studied in different cancer cells ( hct-116 and kb cells ) in 96-well microplates . serial dilutions of the nanoparticles with a corresponding camptothecin concentration from 0.5 to 20 m were prepared in culture medium supplemented with 10% fbs , and 150 l of each concentration was directly added to the cells and incubated for 24 h in a 95:5 air / co2 mixture at 37 c . the medium was replaced with fresh medium , and the cells were grown for an additional 24 h. then the medium was replaced with 100 l of medium containing mtt reagent ( final concentration of 0.1 mg / ml ) and incubated for 3 h. mtt crystals were dissolved with 100 l of a mtt stop solution ( 20% sds in dmf and water at a 1:1 ratio and 2% acetic acid ) by incubation for 4 h at room temperature , and the optical density was measured ( = 595 nm ) using a microplate reader ( elx808 , bio - tech instrument , inc . ) . because of its easy postpolymerization functionalization , the pds polymer built from pyridyldisulfide - functionalized acrylate monomers was adopted and synthesized by free radical polymerization according to our published protocol ( mw = 7.1 kda ; pdi = 1.45 ) . thiolated poly(ethylene glycol ) ( peg5000-sh ) was conjugated to the pds polymer to yield the amphiphilic pds the emergence of the proton peak at 3.6 ppm in the h nmr spectrum after peg conjugation proved the formation of pdsg ( figure s1 of the supporting information ) . the right shifting of the gel permeation chromatography ( gpc ) curve ( retention volume decreased from 9.4 to 8.65 ml ) further confirmed the grafting of peg to pds ( figure s2 of the supporting information ) . the shallow shoulder in the gpc curve of pdsg indicated the presence of a small portion of unreacted pds . the molecular weight of pdsg was 11.9 kda ( pdi of 1.38 ) as determined by gpc , suggesting each pdsg polymer chain contained approximately one peg molecule . the hydrophobic anticancer drug camptothecin ( cpt ) was thiolated ( figure 1a ) and then grafted onto the pdsg polymer to form cpdsg through thiol disulfide exchange ( figure 1b ) . researchers have confirmed that thiolation of cpt does not significantly affect its anticancer efficacy . the successful yield of thiolated cpt ( cpt - sh ) was confirmed by h nmr , hplc , and mass spectroscopy . the ethylene proton resonance peaks corresponding to cpt at = 1.85 ppm slightly shifted to = 2.2 ppm . along with the characteristic proton peaks of cpt , a new ethyl proton peak ( = 2.84 ppm ) was clearly visible in the h nmr spectra ( figure s1 of the supporting information ) . the change in the chemistry of cpt after thiolation and conjugation onto the polymer was also observed in the hplc chromatogram . the retention time of cpt significantly shifted from 3.2 to 8.0 min after thiolation ( figure 2b ) . hplc was also employed to monitor the cpt conjugation process by quantifying the remaining free cpt - sh and showed that 3.5 h was enough to complete the reaction and achieve 100% conjugation efficiency ( figure 2a ) . hplc spectra evidenced the successful conjugation of cpt onto pdsg , an emerging peak with an elution time of 10.5 min ( figure 2b ) . the amount of cpt - sh selected for the preparation of cpdsg was optimized by incubating different amounts of cpt - sh with polymer , and 20% ( w / w ) cpt - sh was selected for the study based on the balancing between high drug loading content and appropriate particle size formed thereafter . the feeding ratio for grafting cpt - sh onto pdsg was 5:1 [ repeating unit of pds / cpt - sh ( molar ratio ) ] . because no residual cpt - sh signal was observed during hplc for the cpdsg reaction product ( see the spectrum of cpdsg in figure 2b ) , each polymer should have around six cpt - sh molecules grafted . to evaluate the ability of cpt - sh to be released from the cpdsg polymer in a reducing environment , 10 mm dithiothreitol ( dtt ) was added to the cpdsg polymer dmso solution . as expected , hplc showed that all conjugated cpt - sh could be liberated from cpdsg as evidenced by the complete disappearance of the absorbance peak at 10.5 min and the recovery of the peak at 8.0 min ( figure 2b ) . the critical micelle concentration of cpdsg was 12 g / ml as determined by fluorescence spectroscopy ( figure s3 of the supporting information ) . reaction kinetics of cpt - sh conjugation ( a ) and hplc spectra of cpt , cpt - sh , and cpt - conjugated polymer ( cpdsg ) and the release of cpt - sh from cpdsg in an environment containing 10 mm dtt ( b ) . hydrodynamic size ( a ) , potential ( b ) , and tem images ( c ) of nanoparticles and the kinetics of release of cpt - sh from cn in different environments ( d ) . scale bars are 500 nm in panel c. dynamic light scattering ( dls ) and phase analysis light scattering ( pals ) were employed to investigate the physicochemical characteristics of the nanoparticles prepared as described above . for the cpdsg polymer , because of the amphiphilicity of the polymer , cpdsg self - assembles into micelles with a core made of pds as well as cpt , and peg corona ( figure 1c ) . dls revealed that the conjugation of peg significantly reduced the size of pds from 168 to 107 nm ( figure 3a ) . because of the formation of a more condensed hydrophobic cpt core , cns with a low cpt grafting ratio were smaller than pdsg . our pilot study found that the size of cn increased proportionally with the increase in the cpt - sh grafting ratio . the goal for the cpt - sh loading optimization was to achieve a cn with the highest cpt loading content while keeping its size smaller than 100 nm . via the optimization , a cn with a 16.7% cpt - sh loading content and a size of 85 nm was selected . cn was functionalized with herceptin at a feeding ratio of 50 molecules per cn . the bradford protein assay was conducted to evaluate the herceptin conjugation efficiency . however , because of the relatively low concentration of herceptin used and the interference of the nanoparticle , the absolute number of herceptin molecules per nanoparticle could not be determined . cn doubled its size to 187 nm after herceptin functionalization . because the hydrodynamic size of herceptin was < 20 nm , we postulate that the increased size ( 102 nm ) was partially due to the cross - linking effect of herceptin in addition to surface functionalization . because of the contribution of pyridine groups , all nanoparticles were slightly negatively charged with ranging from 3.08 0.15 to 0.63 0.15 mv ( figure 3b ) , suggesting that hcn could be a good carrier for targeted drug delivery because of its ability to escape detection by the mps system . the addition of peg could further protect hcn from the capture of the mps system as well as decrease the size of pds and slightly attenuate the surface charge . tem images revealed that both cn and hcn nanoparticles were spherical and further confirmed the size increase for hcn after herceptin modification ( figure 3c ) . studies have revealed that the difference in redox potential level between the intracellular and extracellular environments is several orders of magnitude , < 0.01 mm glutathione ( gsh ) for the extracellular fluid and 111 mm gsh for the cytoplasm . to investigate the redox potential sensitivity of cn , dtt was employed instead of gsh because the addition of gsh would decrease the ph of the release buffer , which could potentially introduce false redox potential sensitivity . nanoparticles were dialyzed against pbs and pbs supplemented with 10 mm dtt to mimic extracellular and intracellular environments , respectively . figure 3d shows that before the addition of dtt only 38% of cpt - sh was released over 24 h. then 10 mm dtt was added to mimic the intracellular reducing environment . to our surprise , the remaining 62% of cpt - sh was released in only 20 min , indicating that cn was supersensitive to the endogenous stimulus , a high redox potential . cytotoxicity of cn and hcn for her2-positive hct-116 cells ( a ) and her2-negative kb cells ( c ) after treatment for 48 h and western blotting of her2 expression in hct-116 and kb cells ( b ) . ( student s t test ; * p < 0.05 ; p < 0.01 ) . her2-positive cancers are generally aggressive and closely associated with poor prognostic outcome . to endow cn with specificity in killing her2-positive cancer cells , herceptin , a her2 antibody , was conjugated to the surface of cn to yield hcn . to investigate the selective cell killing effect of hcn , her2-posivive hct-116 colon cancer cells and her2-negative kb cells were treated with cn and hcn for 48 h and evaluated by the mtt assay . western immunoblotting analysis confirmed the expression of her2 in hct-116 but not in kb cells ( figure 4b ) . panels a and c of figure 4 show that thiolated cpt ( cpt - sh ) retained its anticancer activity and exhibited dose responsive inhibition of proliferation for both cell lines , which is consistent with the reports of others . the mtt assay also revealed that cn was almost nontoxic for both hct-116 and kb cells , killing less than 12 and 7% of the hct-116 and kb cells , respectively , at the equivalent cpt - sh concentration of 20 m , at which free cpt - sh killed more than 80 and 50% of the cells , respectively . this suggested that the premature release of cpt from cn ranged from minimal to none . unlike cn , hcn exhibited a potency similar to that of free cpt - sh in killing her2-positive hct-116 cells ; 68% of the cells were killed after the hcn treatment at a concentration of 20 m . to our surprise , hcn barely killed her2-negative kb cells ( only 20% of the cells were killed at a concentration of 20 m ) , suggesting that hcn did highly selectively inhibit the growth of her2-positive cancer cells . to examine whether this her2 specific cell killing was solely due to the inhibitory effect of herceptin , a her2 antibody used clinically for the treatment of her2-positive breast cancer , a mtt assay was conducted to measure the cell viability after treatment with herceptin at concentrations of 0.18.7 g / ml . figure 5a shows that herceptin was nontoxic in the tested concentration range . because the corresponding herceptin dose in hcn at a cpt - sh concentration of 20 m was 2.4 g / ml , which was less than 8.7 g / ml and far less than its clinical concentration , we think that the her2 specific cell killing effect was due to herceptin enhancing hcn uptake rather than the cell growth inhibitory effect of herceptin itself . the mtt assay also proved that the pdsg polymer was not toxic for hct-116 cells ( figure 5b ) . cytotoxicity of herceptin ( a ) and pdsg ( b ) for her2-positive hct-116 cells after treatment for 48 h. data represent means the standard deviation ( n = 3 ) . to investigate how hcn achieved a selective killing effect for her2-positive cells , doxorubicin ( dox ) , an anticancer drug that is also a convenient fluorescent probe , was encapsulated into cn and hcn . the addition of dox into cn and hcn slightly decreased their hydrodynamic size ( 5 nm ) . there was no difference in surface charge between the cns or hcns with and without doxorubicin encapsulation . after treatment for 3 h , there were only very dim red fluorescent signals shown in both cell lines treated with cn ( figure 6 ) , indicating that cn had hardly been taken up by either cell line . as expected , a much stronger red fluorescent signal appeared in hcn - treated hct-116 cells than in cn - treated ones . however , there was no observable increase of the red fluorescent signal in kb cells cocultured with hcn , suggesting that hcn can effectively enter only her2-positive cells , not her2-negative ones . flow cytometry data ( figure 6c , d ) further revealed that the addition of herceptin to cn did significantly enhance its entry into hct-116 cells ( her - positive cells ) but not kb cells ( her - negative cells ) . confocal images and flow cytometry spectra of hct-116 cells ( a and c ) and kb cells ( b and d ) treated with cn and hcn nanoparticles . doxorubicin was encapsulated into cn and hcn nanoparticles as a fluorescence reporter ( red , dox ) ; nuclei of the cells were stained with hoechst 33342 ( blue , dapi ) ( a and b ) . cells were cocultured with cy3-labeled cn and hcn ( c and d ) for 4 h. to achieve a specific cell killing effect , a drug carrier system should meet at least the following three conditions . first , the loaded drug should be released only after entering the targeted cell . disulfide bonds have been incorporated into various drug carriers with the aim of achieving a desired stability during circulation and quick release in a reducing intracellular environment because of the high gsh concentration . in our design , because peg is hydrophilic while both cpt and pds segments are hydrophobic , cpdsg self - assembled into the nanoparticle cn in an aqueous solution ( figure 3c ) . the pds polymer is biodegradable and can be degraded through the hydrolysis of ester bonds at acidic ph ( inside the lysosomes ) and the cleavage of disulfide bonds ( in reducing cytosol ) . the molecular weight of pds will decrease to < 3000 da after the cleavage of all side chains , which can be easily excreted from the body by renal clearance . figure 3d proves that in a buffer with no reducing agent cpt - sh could be only slowly released ( 38% released in 24 h ) . as a consequence , the off - target effect induced by premature release would be significantly reduced . cell killing results shown in panels a and c of figure 4 also revealed that . hplc demonstrated that cpt - sh could be released from cn without any modification ( figure 2b ) . furthermore , the mtt assay showed that cpt - sh was still active in killing cancer cells ( figure 4 ) , which is consistent with the literature report . figure 6 validates that without the help of targeting ligand cn could enter neither her2-positive nor her2-negative cells . confocal microscopy also verified that herceptin modification facilitated the entry of hcn into only her2-positive cells , not her2-negative ones . flow cytometry data further proved that the modification of herceptin to cn facilitated the entry of hcn into hct-116 cells , not kb cells ( figure 6c , d ) . moreover , figure 3d also confirmed that cpt - sh could be liberated from the nanoparticle in a superfast manner under conditions similar to those of the intracellular environment ( 62% in 20 min ) , suggesting cn was far more sensitive to the reducing environment than other developed redox potential sensitive systems , which generally need several hours to release all payloads . the difference in cell viability between two cell lines was only observed in hcn and not cn treatment , indicating the observed cell type specific killing effect was not caused by the difference in free thiols produced in the culture media between two cell lines . the integration of the characteristics of hcn mentioned above endows it with the ability to specifically kill her2-positive cancer cells , as shown in figure 4 . her2 is overexpressed in approximately 2030% of invasive breast cancer and is associated with poor disease - free survival and poor response to chemotherapy . one of the significant advances in the application of monoclonal antibodies in oncology was the introduction and approval of herceptin , a humanized anti - her2 antibody , for the treatment of her2-positive breast cancer and her2-positive metastatic gastric or gastresophageal junction adenocarcinoma . despite initial successes and encouraging results , the development of monoclonal antibody - based therapies faces several challenges . up to 74% of her2-positive breast cancer patients are resistant to anti - her2 antibody herceptin , and a majority of patients with her2-positive breast cancer develop acquired resistance within one year . a majority of these herceptin - resistant cells still overexpress her2 , suggesting that resistance to herceptin is not due to the loss of her2 overexpression . the herceptin - conjugated nanoparticle developed in this study may provide a unique system that can be used to specifically deliver therapeutic drugs to her2-positive cancer cells regardless of their response to herceptin treatment . in addition , in response to chemotherapy , cancer cells can develop multidrug resistance ( mdr ) ; mechanisms for this include compartmentalization , enhanced metabolism , and efflux pumps . the p - glycoprotein ( p - gp ) pump ( efflux pump ) can remove drug molecules from the cytoplasm and decrease the intracellular drug concentration to a level lower than its effective dose , which is the primary reason for the failure of chemotherapy . to overcome the mdr effect , an effective drug carrier should have the ability to quickly release its payload intracellularlly to saturate the processing capacity of p - gp pumps . because hcn could discharge all payloads in 20 min , a her2-targeted nanoparticle , hcn , has been developed using the polymer drug conjugates of pdsg and camptothecin . the release kinetic study found that hcn was supersensitive to the trigger of elevated redox potential , a characteristic of the intracellular environment , releasing all payloads in less than 30 min . with the help of her2 antibody herceptin because of its supersensitivity to the intracellular environment , hcn could open a new paradigm in overcoming mdr of cancer .

ideal smart nanoparticles for drug delivery should enhance therapeutic efficacy without introducing side effects . to achieve that , we developed a drug delivery system ( hcn ) based on a polymer drug conjugate of poly[2-(pyridin-2-yldisulfanyl)]-graft - poly(ethylene glycol ) and camptothecin with an intracellularly cleavable linker and human epidermal growth factor receptor 2 ( her2 ) targeting ligands . an in vitro drug release study found that hcn was stable in the physiological environment and supersensitive to the stimulus of elevated intracellular redox potential , releasing all payloads in less than 30 min . furthermore , confocal microscopy revealed that hcn could specifically enter her2-positive cancer cells . as a consequence , hcn could effectively kill her2-positive cancer cells while not affecting her2-negative cells .

bacterial strains : a total of 123 strains of s. aureus isolated in 2007 and 2008 from subclinical bovine mastitis ( scm ) milk samples were included in this study . the bacterial strains were identified as s. aureus by colonial and microscopic morphology examination and tube tests for coagulase and catalase activity . the strains were additionally tested by using pcr analysis to amplify the part of the nuc gene , encoding thermostable nuclease , specific for s. aureus . the primer sequences and pcr conditions are presented in table 1table 1.sequences of starters used for pcr analysisno.detected geneprimer sequencessize of thepcr product ( bp)references1nucnucf : 5 gcgatagatggtgatacggttnucr : 5 agccaagccttgacgaactaaagc2702mecamec1 : 5aaaatcgatggtaaaggttggmec2 : 5agttctgcagtaccggatttgc5333blazblaz1 : 5aagagatttgcctatgcttcblaz2 : 5 gcttgaccacttttatcagc517 . this part of the analyses was performed during our previous research aimed at analysis of the biofilm production of these strains . antimicrobial susceptibility testing : bacteria were grown at 37c for 18 to 24 hr on a nonselective luria agar medium ( a&a biotechnology , gdynia , poland ) , and their antimicrobial susceptibility was evaluated using the disk diffusion method on mueller - hinton agar ( sigma - aldrich corp . , st . louis , mo , u.s.a . ) according to the guidelines of the clinical and laboratory standards institute ( clsi ) . lenexa , ks , u.s.a . ) were tested : penicillin ( p ; 10 i.u ) , amoxicillin ( aml ; 25 g ) , amoxicillin with clavulanic acid ( amc ; 30 g ) , ampicillin ( amp ; 10 g ) , cephalothin ( kf ; 30 g ) , clindamycin ( da ; 2 g ) , cloxacillin ( ob ; 5 g ) , cefoperazone ( cfp ; 75 g ) , erythromycin ( e ; 15 g ) , gentamicin ( cn ; 10 g ) , enrofloxacin ( enr ; 5 g ) , lincomycin ( my ; 15 g ) , linezolid ( lzd ; 30 g ) , neomycin ( n ; 30 g ) , vancomycin ( va , 30 g ) , oxacillin ( ox ; 1 g ) , rifampicin ( rd ; 5 g ) , streptomycin ( s ; 10 g ) , tetracycline ( te ; 30 g ) and trimethoprim / sulfamethoxazole ( sxt ; g ) . the inoculated agar plates with discs were left at room temperature for 30 min and then incubated at 35c for 24 hr . following the incubation , the diameters of the inhibition zones were measured in millimeters and compared with the ranges suggested by the manufacturer of the disks ( oxoid , thermo fisher scientific inc . ) . the strains were classified as resistant , intermediate or susceptible on the basis of the size of the inhibition zone . mic assay : the strains that exhibited resistance to at least one tested antibiotic ( n=37 ) and two strains susceptible to all chemotherapeutics were additionally analyzed for resistance to lysostaphin , nisin , polymyxin b and lysozyme ( all from sigma - aldrich corp . ) . the minimal inhibitory concentration ( mic ) values of these agents were determined using the broth microdilution method according to the standard protocol of the clsi with a slight modification proposed by kusuma and kokai - kun . briefly , twofold dilutions of tested agents were performed in cation - adjusted mueller - hinton broth ( sigma - aldrich corp . ) supplemented with 2% of nacl and 0.1% of bovine serum albumin ( bsa ; sigma - aldrich corp . ) . wells of a 96-well polystyrene plate were inoculated with 5 10 cfu / ml of s. aureus strains per well diluted from the overnight culture of the bacteria grown on luria agar . a control comprised of growth of strains with no tested agents was included in each assay . the determination of the mic values of the tested agents was performed by measuring the absorbance at 531 nm using a vector microplate reader ( perkinelmer , inc . , the lowest concentration of antibiotic yielding inhibition of growth equal or higher than 90% of growth control was taken as the mic value . the presence of 0.1% of bsa in each well was necessary to inhibit nonspecific lysostaphin ( and probably other agents ) adherence to the polystyrene plate , which was previously observed by climo et al . . -lactamase production : the production of -lactamases , the enzymes responsible for resistance to -lactam antibiotics , was analyzed using nitrocefin discs according to the manufacturer s instructions ( remel , thermo fisher scientific inc . , the strains were additionally tested for presence of the blaz gene coding for these enzymes . isolation of dna : each isolate was subcultured overnight in 1 ml of luria broth ( a&a biotechnology ) . after centrifugation ( 12,000 g , 1 min ) , dna was purified from bacterial cells using a bacterial & yeast genomic dna purification kit ( eurx , gdask , poland ) according to the manufacturer s instruction with minor modifications . namely , 10 l of lysostaphin ( 1 u ; sigma - aldrich corp . ) solution was added to the cell suspension for enzymatic lysis of s. aureus cell wall murein . the mixture was incubated at 37c for 30 min . obtained dna solutions were stored at 20c until further analysis . pcr conditions : the amplification reactions were performed using an automated thermocycler ( eppendorf mastercycler gradient ; eppendorf poland , warszawa , poland ) . three different targets were amplified : nuc , meca and blaz . in all three cases , the same composition of reaction mixtures was used : 2 l of dntps ( 2.5 mm each ) , 2.5 l of 10 pcr reaction buffer ( 100 mm tris - hcl , ph 8.8 , 1 mm dtt , 0.1 mm edta , 100 mm kcl , 0.5% nonidet p40 and 0.5% tween 20 ) , 2 l of mgcl2 solution ( 50 mm ) , 1 l of each of the 2 required primer solutions ( 10 m ) , 1 l of dna solution ( prepared as described above ) , 0.2 l ( 1 u ) of polymerase delta from pyrococcus woesei(dna - gdask ii s.c . , poland ) and deionized sterile water to adjust the mixture volume to 50 l . the primer sequences applied are presented in table 1 . in the case of all three genes , the following pcr conditions were used to generate the amplicons : 94c for 240 sec ; 35 cycles of 94c for 30 sec , 55c for 30 sec and 72c for 30 sec ; and then 72c for 300 sec for final extension . the gels were stained with ethidium bromide ( 1 g / ml ) , and bands were visualized in uv light . the level of antibiotic resistance of the investigated group of strains was low overall ( table 2table 2.the resistance rates of s. aureus subclinical mastitis isolates for particular antibioticsno.antibioticnumber of resistant / intermediatelyresistant strains% of resistant/ intermediatelyresistant strainscode number of resistant / intermediatelyresistant isolate1p29/023.6/01 , 5 , 9 , 11 , 29 , 34 , 38 , 39 , 42 , 54 , 70 , 71 , 72 , 74 , 75 , 78 , 83 , 84 , 86 , 93 , 94 , 95 , 101 , 102 , 103 , 104 , 105 , 112 , 113 / -2amp28/022.8/01 , 5 , 9 , 11 , 29 , 34 , 38 , 39 , 42 , 54 , 70 , 71 , 72 , 74 , 75 , 78 , 83 , 84 , 86 , 93 , 94 , 95 , 102 , 103 , 104 , 105 , 112 , 113 / -3aml22/117.9/0.81 , 5 , 9 , 11 , 29 , 54 , 70 , 71 , 72 , 74 , 75 , 83 , 84 , 86 , 93 , 94 , 95 , 102 , 103 , 104 , 105 , 113 / 784s13/010.6/06 , 11 , 27 , 66 , 76 , 84 , 88 , 93 , 94 , 95 , 101 , 116 , 119 / -5e3/02.4/027 , 101 , 112 / -6my2/01.6/027 , 101 / -7te2/01.6/053 , 70 / -8n2/01.6/027 , 101 / -9amc2/01.6/01 , 11 / -10da2/01.6/027 , 101 / -11sxt1/00.8/012 / -12cfp0/50/4.1- / 1 , 83 , 84 , 86 , 103 , 10513ox0/10/0.8- / 101p : penicillin , amp : ampicillin , aml : amoxicillin , s : streptomycin , e : erythromycin , my : lincomycin , te : tetracycline , n : neomycin ; amc : amoxicillin with clavulanic acid , da : clindamycin , sxt / trimethoprim / sulfamethoxazole , cfp : cefoperazone , ox : oxacillin . ) . eighty - six out of 123 examined isolates were susceptible to all 20 tested antibiotics ( 70% ) . cephalothin , cloxacillin , enrofloxacin , gentamicin , linezolid , rifampicin and vancomycin were active against all strains tested . the highest percentage of resistance was observed in the case of streptomycin ( n=13 ; 10.6% ) and -lactam antibiotics : amoxicillin ( n=22 , 17.9% ) , ampicillin ( n=28 , 22.8% ) and penicillin ( n=29 , 23.6% ) . in fact , all strains resistant to amoxicillin and ampicillin were also resistant to penicillin ( n=22 , 17.9% ) . twenty - five of the penicillin - resistant strains were also found to carry the blaz gene coding for -lactamases , which was generally in concordance with the nitrocefin test . although the level of antibiotic resistance was low overall , the worrisome problem is isolation of several strains that could be classified as multidrug resistant ( mdr ) strains ( table 3table 3.the antibiotic resistance profile of s. aureus mastitis isolatesno.number of antibioticsfor resistance / intermediate resistancephenotype of resistancenumber of strains11/0sxt1te1p1s522/0amp+p432/1amp+p / aml143/0amp+p+aml11amp+p+e153/1amp+p+aml / cfp364/0amp+p+aml+s4amp+p+aml+te174/1amp+p+aml+amc / cfp1amp+p+aml+s / cfp185/0amp+p+aml+amc+s1da+e+my+s+n196/1p+da+e+my+s+n / ox1p : penicillin , amp : ampicillin , aml : amoxicillin : s : streptomycin : e : erythromycin , my : lincomycin : te : tetracycline , n : neomycin : amc : amoxicillin with clavulanic acid , da : clindamycin , sxt : trimethoprim / sulfamethoxazole , cfp : cefoperazone , ox : oxacillin . ) carrying resistance to 6 antibiotics and intermediate resistance to 1 agent ( n=1 , 0.8% ) , five antibiotics ( n=2 , 1.6% ) , four antibiotics and intermediate resistance to 1 agent ( n=2 , 1.6% ) and four antibiotics ( n=5 , 4.0% ) . the strains resistant to 6 and 5 antibiotics were found to be meca positive , so they could also be classified as mrsa ( methicillin - resistant s. aureus ) ; however , only the first one was phenotypically intermediately resistant to oxacillin in the disc diffusion assay . in the case of nonantibiotic agents tested by the microdilution method , the best results were obtained for lysostaphin ( table 4table 4.bactericidal activity of the alternative agents against s. aureus isolates resistant to at least one of the tested antibiotics . sensitive strains , with numbers 2 and 3 , were used as a controlcode numberof the strainlysostaphin mic(g / ml)nisin mic(g / ml)polymyxin b mic(g / ml)lysozyme mic(g / ml)10.12551.264>204850.12551.264>204890.03151.264>2048110.01612.832>2048120.01612.864>2048270.01651.264>2048290.12551.264>2048340.551.232>2048380.12551.264>2048390.01625.664>2048420.03112.864>2048530.00825.664>2048540.01625.664>2048660.00812.864>2048700.06325.664>2048710.06312.864>2048720.01651.264>2048740.03112.864>2048750.00825.664>2048760.01625.632>2048780.01625.664>2048830.01651.264>2048840.06351.232>2048860.03125.664>2048880.06325.664>2048930.00825.664>2048940.01625.632>2048950.01651.2128>20481010.00851.264>20481020.00851.2128>20481030.00851.264>20481040.06351.264>20481050.06351.264>20481120.5>51.264>20481130.00851.264>20481160.03112.864>20481190.01625.632>204820.06325.664>204830.00812.864>2048 ) . there is no defined or officially accepted mic value for this protein that could be used as a susceptibility breakpoint . however , some authors , for example , kusuma and kokai - kun , classify staphylococci as resistant to lysostaphin , if the mic value is higher than 32 g / ml . the lysostaphin mic values for all strains tested were definitely lower and were in the range from 0.008 to 0.5 g / ml , so all of them can be classified as susceptible . polymyxin b was able to inhibit the growth of most of the strains tested ( n=31 , 79.5% ) at a concentration of 64 g / ml ; for 6 isolates , the mic value for this agent was 32 g / ml , and 2 strains required a higher concentration , 128 g / ml , for growth inhibition . p : penicillin , amp : ampicillin , aml : amoxicillin , s : streptomycin , e : erythromycin , my : lincomycin , te : tetracycline , n : neomycin ; amc : amoxicillin with clavulanic acid , da : clindamycin , sxt / trimethoprim / sulfamethoxazole , cfp : cefoperazone , ox : oxacillin . p : penicillin , amp : ampicillin , aml : amoxicillin : s : streptomycin : e : erythromycin , my : lincomycin : te : tetracycline , n : neomycin : amc : amoxicillin with clavulanic acid , da : clindamycin , sxt : trimethoprim / sulfamethoxazole , cfp : cefoperazone , ox : oxacillin . taking into account this value , 18 out of 39 isolates from our collection were resistant to this agent s activity . however , in the case of 17 of them , the mic value was 51.2 g / ml and was not much higher than the susceptibility breakpoint value , and only in the case of 1 strain ( assigned as 112 ) , the nisin mic value was above 51.2 g / ml . the same strain was less susceptible to lysostaphin activity , which suggests a modified composition of the cell wall . however , the detailed mechanism of higher resistance to both agents was not determined and in our opinion requires further investigation . as expected , all strains tested were resistant to lysozyme activity ( mic above 2048 g / ml ) . the presented results indicated a considerable prevalence of antibiotic resistant strains among s. aureus isolated from bovine mastitis in the eastern part of poland . similar to studies of other authors from different regions of the world , the highest rate of resistance was detected for -lactam antibiotics . the resistance to other tested agents was less common , which is also in agreement with the general trend observed worldwide . however , earlier published reports aimed at analysis of the antibiotic resistance of s. aureus strains isolated from mastitis in poland indicated markedly higher levels of antibiotic resistance . in comparison , among the isolates examined by malinowski et al . , the rates of resistance to penicillin , tetracycline and lincomycin were 62.3% , 41.7% and 39.4% , respectively ( in each case , over 800 strains were tested ) . additionally , the authors found that over 20% of the investigated strains were resistant to bacitracin and cephalexin . higher occurrence of resistance among s. aureus isolates was also observed by sachanowicz et al . , who analyzed strains from the same region , although earlier , in 2005 and 2006 . high percentages of resistance against -lactam antibiotics were also observed in several different geographical regions . among 103 strains isolated in turkey , the rates of resistance to penicillin , ampicillin and amoxicillin were 62.1% , 56.3% and 45.6% , respectively . in the same study , about 50% resistance was found in the case of gentamicin ( 56.3% ) and trimethoprim / sulfamethoxazole ( 45.6% ) . additionally , eighteen of these strains were found to be phenotypically resistant to methicillin . however , aslanta et al . , who analyzed a group of 104 strains isolated from subclinical bovine mastitis cases during 2006 to 2008 in hatay province , turkey , found that 25% of them were resistant to macrolide and lincosamide ( ml ) antibiotics . resistance to -lactam antibiotics , ampicillin ( 59.5% ) and penicillin ( 61.4% ) , was very common in a group of over 170 strains isolated in estonia . in the same study , markedly high resistance other tested antibiotics , i.e. , tetracycline , erythromycin and gentamicin , were much more active with resistance rates below 10% . according to results presented by klimiene et al . , among 176 strains isolated in lithuania , the percentages of resistance to penicillin , ampicillin and amoxicillin were 76.7% , 78.4% and 81.3% , respectively , and 38.1% of isolates were resistant to the combination of amoxicillin and clavulanic acid . the same strains were highly sensitive to cephalosporin s activity , and the rates of sensitivity rate to cephalothin and cephalexin were 95.5% and 93.2% , respectively . antibiotic susceptibility testing of 236 strains isolated from china showed an overall high level of resistance to tested antimicrobial agents . among twenty tested antibiotics , the most active ones were two aminoglycosides , kanamycin and gentamicin with sensitivity rates of only 74.6% and 69.9% , respectively . the highest rate of resistance was observed in the case of penicillin ( 87.3% ) . recently , gao et al . analyzed antibiotic resistance in a group of 52 strains isolated from one chinese herd . interestingly , nearly all of the isolates were resistant to penicillin ( 96.3% ) and tetracycline ( 98.1% ) , and all of them were susceptible to oxacillin , cefazolin and ciprofloxacin . a high level of resistance to penicillin ( 82.4% ) was also observed in the case of s. aureus isolated from mastitis in ethiopia ; however , a very limited group , only 17 strains , was analyzed . the isolates were even more resistant to clindamycin ( 88.2% ) and were highly resistant to erythromycin ( 58.8% ) , whilst the rates of sensitivity to chloramphenicol and nalidixic acid were 58.8% and 82.4% , respectively . a high prevalence of mrsa ( 13.1% ) was observed in a group of 107 strains isolated in india . the bacteria were also highly resistant to most of the other tested antibiotics , i.e. , 36.4% were resistant to streptomycin , 33.6% were resistant to oxytetracycline , 29.9% each were resistant to gentamicin and ampicillin , 28.9% were resistant to penicillin and 26.2% each were resistant to chloramphenicol , pristinamycin and ciprofloxacin . the authors also revealed a high prevalence of genes coding for different pathogenicity factors , mainly adhesins and toxins , among the tested isolates . a very similar pattern of resistance was observed among 193 strains from switzerland and 150 isolates from france . the resistance to most of the 16 antibiotics tested was low overall in both countries ; however , in the case of penicillin , the susceptibility rates were 77.7% and 70% , respectively . six and 8 isolates ( from switzerland and france , 3.1% and 5.3% , respectively ) were resistant to tetracycline . even lower rates of antibiotic - resistant isolates were identified in sweden . among a group of 109 strains , persson et al . tested the activities of 9 antibiotics and found only 4 ( 3.7% ) strains resistant each to kanamycin and penicillin and only 3 strains characterized by resistance to tetracycline . the observed worldwide predominance of resistance for -lactam antibiotics is probably the consequence of the fact that they are still one of the most widely used classes of agents for treatment of bovine mastitis . based on a questionnaire completed by 109 practicing veterinarians representing all regions of poland , krasucka et al . proved that penicillins are the antimicrobials most often used in treatment of cattle infections ( about 35% of cases ) . additionally , in a study carried out by the same authors , a comprehensive analysis of available statistic data revealed that -lactams are the most popular agents used in veterinary service in several other european countries including the czech republic , denmark and norway . frequent contact of bacteria with a specific antibiotic can cause an increase in resistance and decrease the effect of treatment . in fact , s. aureus pathogens have developed a broad spectrum of mechanisms of antibiotic resistance , which make them difficult targets even for treatment using agents from different chemical groups or combine therapy with more than 1 antibiotic from different chemical groups . the most common mechanism of -lactam resistance is based on production of -lactamases encoded by blaz , which was confirmed in this report . the ability to produce low - affinity penicillin binding protein 2a ( pbp2a ) determined by the presence of the chromosomal gene meca is still found incidentally among staphylococci isolated from mastitis , which is in concordance with the presented results . the current situation in the dairy industry and veterinary service require limitation of spreading of s. aureus antibiotic resistance and urgent development of new antimicrobial agents that would not covered by existing mechanisms of resistance . the first goal can be achieved only if the treatment is preceded by an antimicrobial susceptibility test and selection of the most accurate agent . such practice should be in fact commonly used in human medicine and veterinary service . given the current state of knowledge , the most promising alternative strategies in the case of s. aureus diseases seem to be therapies with antimicrobial proteins and peptides , bacteriophages and plant- ( e.g. , stilbenoids and flavonoids ) and animal - derived compounds ( e.g. , chitosan and propolis ) as well as usage of vaccines and photodynamic therapy . the results of our examination clearly indicate that polymyxin b , nisin and especially lysostaphin should be considered potential agents to treat infections caused by staphylococci including bovine mastitis . polymyxin b is very effective in treatment of infections caused by g - negative bacteria . it s breakpoint mic value for pseudomonas aeruginosa is 4 g / ml . the mechanism of bactericidal activity of this agent is based on destabilization of outer and inner cell membranes surrounding the cells of g - negative bacteria . g - positive bacteria are protected by a thick cell wall composed of peptidoglycan ; therefore , they are more resistant to polymyxin b. among the analyzed group of isolates , the mic values for polymyxin b are in the range from 32 to 64 g / ml ( except for 2 strains with an mic value of 128 g / ml ) . quite similar results were obtained by boyen et al . , who tested the activity of polymyxin b against 24 canine mrsa isolates and observed mic values in the range of 864 g / ml . the observed mic values for all strains tested in both investigations are definitely higher than the susceptibility breakpoint for this agent , established for p. aeruginosa , which in our opinion disqualifies using this peptide as a potential chemotherapeutic in infections caused by staphylococci , including bovine mastitis . quite satisfactory bactericidal anti - staphylococcal activity was also found in the case of nisin ( 54% strains classified as susceptible ) . in our opinion , identification of 17 resistant strains does not disqualify this peptide as a potential agent for treatment of bovine mastitis caused by staphylococci . in fact , only in the case of one strain was the mic value higher than 51.2 g / ml , and the mic value for other resistant strains was 51.2 g / ml and close to the susceptibility breakpoint ( 32 g / ml ) . definitely , the most promising results were obtained in the case of lysostaphin . all strains tested were susceptible to its activity , and the determined mic values were very low ( from 0.008 to 0.5 g / ml ) and much lower the susceptibility breakpoint of 32 g / ml established by kusuma and kokai kun . the high anti - staphylococcal bactericidal activity of this protein has been also confirmed in many animal models of different types of infections , such as keratitis , endocarditis and many others , that have been widely discussed in review articles by kumar and szweda et al . . the high potential of this protein as an alternative s. aureus mastitis agent has been also confirmed by other authors . zhang et al . revealed high in vitro bactericidal activity of lysostaphin against s. aureus isolated from mastitis in china . but , the most promising results have been published by wall et al . , who produced transgenic cows secreting lysostaphin at concentrations ranging from 0.9 to 14 g / ml of their milk . protection against s. aureus mastitis appears to be achievable with as little as 3 g / ml of lysostaphin in milk . the positive therapeutic effects of using nisin in treatment of bovine mastitis have been earlier confirmed previously by several authors . analyzing a group of 90 lactating holstein cows with subclinical mastitis , which were randomly divided into nisin - treated ( n=46 ) and control ( n=44 ) groups , wu et al . indicated that nisin therapy had bacteriological cure rates of 90.1% for streptococcus agalactiae ( 7 of 14 ) , 41.2% for coagulase - negative staphylococci ( 7 of 17 ) and 65.2% for all cases ( 30 of 46 ) . meanwhile , only 15.9% ( 7 of 44 ) of untreated cows spontaneously recovered . similar results were also presented by cao et al . , who revealed that nisin therapy for mastitis offered a clinical cure rate similar to gentamicin . however , it has been shown that polymyxin b is well distributed throughout the bovine mammary gland and is absorbed to a significant degree into the systemic circulation from the acutely inflamed udder . less data are available about trials that have considered use of polymyxin b as a mastitis therapeutic agent . our investigation revealed low potential for antistaphylococcal activity of this peptide . the results of the present research indicate that lysostaphin and nisin are promising alternative agents to combat staphylococcal infections . without doubt

abstractthe aim of this study was to analyze the resistance of staphylococcus aureus isolates from bovine mastitis in the eastern part of poland to a set of 20 antibiotics and three alternative agents : lysostaphin , nisin and polymyxin b. eighty - six out of 123 examined isolates were susceptible to all 20 tested antibiotics ( 70% ) . the highest percentage of resistance was observed in the case of -lactam antibiotics : amoxicillin ( n=22 , 17.9% ) , ampicillin ( n=28 , 22.8% ) , penicillin ( n=29 , 23.6% ) and streptomycin ( n=13 ; 10.6% ) . twenty - five of the penicillin - resistant strains were found to carry the blaz gene coding for -lactamases . two strains were found to be meca positive and a few strains were classified as multidrug resistant ( mdr ) , one of them was simultaneously resistant to six antibiotics . all strains , resistant to at least one antibiotic ( n=37 ) and two control strains , were susceptible to lysostaphin with mic values of 0.0080.5 g / ml ( susceptibility breakpoint 32 g / ml ) . twenty - one ( 54% ) isolates were susceptible to nisin . the mic value of this agent for 17 ( 44% ) strains was 51.2 g / ml and was not much higher than the susceptibility breakpoint value ( 32 g / ml ) . polymyxin b was able to inhibit the growth of the strains only at a high concentration ( 32128 g / ml ) . the presented results confirmed the observed worldwide problem of spreading antibiotic resistance among staphylococci isolated from bovine mastitis ; on the other hand , we have indicated a high level of bactericidal activity of nisin and especially lysostaphin .

each year 10 million children < 5 years of age die , the majority from infectious diseases [ 1 , 2 ] . many deaths could be prevented if appropriate treatment , such as a broad spectrum antibiotic , could be administered early on in the course of the illness . children in the developing world are at most risk because of poor healthcare systems and distance from facilities able to provide medical care . many infants , and in particular neonates , die at home before medical care can be given . community - based health care workers have been shown to be effective at teaching mothers to recognize unwell infants and those in need of treatment . however , availability and technical expertise to initiate treatment such as parental antibiotics is often impossible to introduce and maintain at a village level . a large multicentre study looking at pre - referral malaria treatment showed that a rectal suppository of artesunate given at the time of referral significantly reduced death and disability in patients who lived hours away from the nearest health facility . if a rectal formulation of a broad spectrum antibiotic could be developed , it could be deployed in the community and would have the potential to reduce early deaths in infants from sepsis . in order to develop such a preparation that would rely critically on rectal absorption , the milieu of the rectum and in particular the ph must be known . there is very little information on the rectal ph of adults and children in the literature . a german study described the rectal ph in 100 well children undergoing elective surgery : children up to the age of 14 years were examined but no neonates were included . the paper reported a mean ph of 9.6 with a surprisingly wide range of results ( 7.212.1 ) . previously , bitterman et al . studied the rectal ph in humans and dogs : they determined that the mean ph was 7.9 ( sd 0.07 ) in 121 healthy adults . the aims of the current study were to determine the rectal ph of well and unwell infants from birth up to 1 year of age . maela camp for displaced persons is located in north west thailand in the hills adjoining the myanmar border , the shoklo malaria research unit ( smru ) clinic provides care for unwell infants as well as follow up for infants after discharge from hospital . vital sign measurements are routinely taken at these visits and include a rectal temperature for all children < 1 year . over a 6-month period infants presenting to the clinic had a rectal ph measurement taken at the same time as the rectal temperature was taken . these infants were categorized as being an unwell neonate ( 28 days ) , well neonate , unwell infant ( 29365 days ) or a well infant . concurrently , infants ( 0365 days ) who were unwell and required admission had sequential rectal ph measurements taken ( at the same time as a rectal temperature was taken ) over the course of their illness . the ph was taken using a beckman coulter phi 410 handheld ph / mv meter with a calomel - phree sealed glass probe . for measurement , the non - lubricated probe was inserted 2 cm into the rectum and kept in situ until a stable ph reading was obtained . the probe was cleaned with 70% isopropanol between measurements and was calibrated on a daily basis . student 's t - test was used to compare mean ph values between the groups . the wilcoxon signed - rank test was used to compare longitudinal changes in ph within an individual . the median age of the infants was 83 days ( range 0366 days ) and 59% were male . the ph was not significantly different between well and unwell infants : the mean ph in the 100 well infants was 6.69 ( 95% ci 6.556.83 ) and in the 45 unwell infants 6.88 ( 95% ci 6.647.12 ) ( p = 0.15 ) . however , rectal ph was significantly lower in infants 28 days compared with those > 28 days ( table 1 ) . table 1comparison of rectal ph in well infants aged 28 days and infants aged > 28 dayswell neonatal ( 28 days ) n = 50well infant ( > 28 days ) n = 50mean ( 95% ci)6.47 ( 6.296.65)6.90 ( 6.687.12)p = 0.003 comparison of rectal ph in well infants aged 28 days and infants aged > 28 days fourteen infants who were admitted to the in - patient department had sequential rectal ph measurements taken during their hospital stay ( fig . 1 ) . there was no significant change in rectal ph from hour 0 ( admission ) to hour 12 , 24 and 36 within the individuals ( p = 0.11 , 0.92 and 0.81 , respectively ) . we showed that the mean intrarectal ph in infants was 6.75 and that this did not change significantly during an illness episode . we found a similar mean rectal ph to that described previously in healthy adults despite obvious differences in diet . there has been only one previous study looking at the rectal ph of children and infants . in this study there were methodological differences between the studies which may account for the difference in results . in our study infants were not under anaesthesia and a digital exam was not performed prior to the ph measurement , both of which may alter the normal ph of the rectum , and may explain the much lower variance in recorded values in the present study . an example of the relevance of our findings is that artesunate has been shown to be unstable in acidic conditions . reassuringly none of the study infants were found to have a low intrarectal ph , supporting the use of rectal artesunate formulations in infancy . these results will assist in the development of rectally formulated drugs for pre - referral use in resource - limited settings .

prompt antibiotic treatment for infants with sepsis has the potential to save lives . a rectal formulation of an antibiotic could be used at a village level before referral to hospital . the development of such a preparation needs to take into account the rectal ph of infants that will affect drug partitioning and absorption . rectal ph measurements were taken in 100 well and 45 unwell infants . we also measured rectal ph in 14 infants sequentially over the course of their illness . the mean rectal ph was 6.75 with no significant difference in well or unwell infants . the mean ( 95% ci ) rectal ph of well neonates was significantly lower than that of older infants ( > 28 days ) : 6.47 ( 6.296.65 ) vs. 6.90 ( 6.68 to 7.12 ) p = 0.003 .

needle lengths for subcutaneous injections started out as long as 16 mm in 1985 , and 12.7-mm needles were introduced in the early 1990s . over time , with growing evidence of longer needles increasing risks for intramuscular injections and improved technology , shorter needles of 4 , 5 , 6 , and 8 mm have been developed . when evaluating skin thickness in 388 patients with bmis varying from 19.4 to 64.5 kg / m , gibney et al . ( 8) found that a 10-kg / m difference accounted for a 4-mm difference in subcutaneous tissue thickness . the average subcutaneous thickness ranged from 10.35 mm to 15.45 mm across all injection sites . this study concluded that 1 ) the thickness does not vary much between under- , normal- , or overweight individuals and that 2 ) bmi differences do not cause much variation in the level of skin thickness . the bmi differences were statistically significant but did not make a clinical impact ( p < 0.001 ) . the skin thickness averages were 1.92.4 mm across several different injection sites ( i.e. , arm , thigh , abdomen , and buttocks ) , as well as across several bmi levels . with the upper end of skin thickness falling at 2.4 mm , it is logical to assume that a 4-mm needle would be effective in all patients . therefore , the study further concluded that 4-mm needles would be able to successfully deliver insulin in most adult patients . a later multisite crossover study by hirsch et al . patients in this study had a1c levels ranging from 5.5 to 9.5% and were randomized into two groups : 1 ) 32 g , 4-mm/31 g , 5-mm pen needle group ( further separated by low [ 20 units ] or regular [ 2140 units ] doses of insulin ) or 2 ) 32 g , 4-mm/31 g , 8-mm pen needle ( also further separated into low- or regular - dose insulin ) . the patients within each group used the two assigned needles for a 3-week period each . the study did not find a statistically significant difference with change in fructosamine ( 1113 mmol / l difference ) , nor did it find that bmi affected fructosamine changes . ( fructosamine measures glucose concentrations over the previous 23 weeks . ) other studies have also found that patients were able to maintain glycemic control similarly with longer pen needles ( 12.7 mm ) or shorter ( 58 mm ) pen needles ( 9,10 ) . ( 9 ) specifically evaluated the use of a 31 g , 5-mm compared to a 31 g , 8-mm pen needle , as well as needle preference for 130 patients with type 1 or type 2 diabetes and a bmi range of 30.162.5 kg / m ( 9 ) . the patients were divided into two groups , and each group used a specific needle length for 3 months before switching to the other needle length for an additional 3 months . although a favorable , statistically significant difference was found in a1c of 0.12% ( p = 0.02 ) between the 5- and 8-mm pen needles , there was no statistical difference between the two groups for fructosamine levels , hypoglycemic episodes , site bruising , or pain perception . unique to this study is the evaluation of 1,5-anhydroglucitol for possible differences in postprandial elevations , which found no changes . the patients in this study did not express preference for the 5- or 8-mm needle lengths ( 46 and 41% , respectively ) . ( 10 ) also demonstrated that glycemic control could be maintained with either a 31 g , 6-mm or a 29 g , 12.7-mm needle in their multicenter , crossover study of 62 patients with type 1 or type 2 diabetes and a bmi range of 3064 kg / m ( 10 ) . ( 9 ) , the patients used one needle length for a 3-month period before switching to the other needle length for an additional 3 months . there was no statistical difference in the final a1c between the two needle lengths ( 7.6% with the 5-mm needle and 7.9% with the 12.7-mm needle ) . patients in the studies described rated the shorter pen needles as much less painful and , not surprisingly , mostly preferred over longer pen needles ( 5,10 ) . in the schwartz study ( 10 ) , patients also completed several surveys , including the world health organization diabetes treatment satisfaction questionnaire ; insulin treatment satisfaction questionnaire ; and , specifically asking questions related to needles , the needle handling questionnaire and the needle preference questionnaire . their results show that patients repor - ted greater satisfaction ( p < 0.001 ) with shorter needles and greater preference for shorter needles ( 89% , p < 0.001 ) and rated shorter needles more favorably with regard to ease of use , perceived pain , and glycemic control ( p < 0.001 ) . despite concern that shorter needles are more apt to cause insulin leakage , especially with larger doses of insulin or with obese patients , the evidence seems to demonstrate the contrary ( 2,4,5,9,10 ) . in addition to evaluating glycemic control with a 4-mm pen needle , hirsch et al . ( 5 ) also looked at insulin leakage and concluded that there was no difference between 4- , 5- , and 8-mm needles in the amount of leakage . this study found that 58% of the leaks reported were with the 5- and 8-mm pen needles , and a smaller proportion reported leaks with the 4-mm pen needle . ( 9 ) found no correlation between insulin leakage , bmi , and insulin dose with the 5- versus the 8-mm needles . no comparable difference in the volume of leakage was found between the 6- and 12.7-mm needles ( 2.7 vs. 3.2 mm ) by schwartz et al . a difference in leakage , however , was found , although minimal and not clinically significant , when using a vertical injection technique as opposed to an angled technique ( 65 vs. 59% , p < 0.001 ) with 5-mm needles ( 2 ) . additionally , wittmann et al . ( 4 ) found increasing leakage , in absolute terms , with larger doses and shorter needles when various amounts of medium were injected into pork rind . but , once again , comparing the percentage of leakage against the larger volume , the difference was not found to be significant . it can be concluded that shorter pen needles can be used without a significant increase in insulin leakage . the patients participating in the described studies did not have any physical or cognitive limitations noted . special populations such as patients with physical limitations that are related to hand movement or dexterity ( e.g. , osteoarthritis , rheumatoid arthritis , tremors , carpal tunnel syndrome , or tendonitis ) , the elderly , vision impaired , or pregnant patients may be limited in their capacity to perform accurate self - injections and may require innovative approaches for insulin injection ( 11,12 ) . patients with physical limitations or those who use an injection technique in which a shorter needle is not inserted completely , despite education , may not be ideal candidates for the shorter needles ( 11 ) . these special circumstances underline the crucial role of diabetes educators in providing diabetes self - management education and support . needle lengths for subcutaneous injections started out as long as 16 mm in 1985 , and 12.7-mm needles were introduced in the early 1990s . over time , with growing evidence of longer needles increasing risks for intramuscular injections and improved technology , shorter needles of 4 , 5 , 6 , and 8 mm have been developed . when evaluating skin thickness in 388 patients with bmis varying from 19.4 to 64.5 kg / m , gibney et al . ( 8) found that a 10-kg / m difference accounted for a 4-mm difference in subcutaneous tissue thickness . the average subcutaneous thickness ranged from 10.35 mm to 15.45 mm across all injection sites . this study concluded that 1 ) the thickness does not vary much between under- , normal- , or overweight individuals and that 2 ) bmi differences do not cause much variation in the level of skin thickness . the bmi differences were statistically significant but did not make a clinical impact ( p < 0.001 ) . the skin thickness averages were 1.92.4 mm across several different injection sites ( i.e. , arm , thigh , abdomen , and buttocks ) , as well as across several bmi levels . with the upper end of skin thickness falling at 2.4 mm , it is logical to assume that a 4-mm needle would be effective in all patients . therefore , the study further concluded that 4-mm needles would be able to successfully deliver insulin in most adult patients . a later multisite crossover study by hirsch et al . patients in this study had a1c levels ranging from 5.5 to 9.5% and were randomized into two groups : 1 ) 32 g , 4-mm/31 g , 5-mm pen needle group ( further separated by low [ 20 units ] or regular [ 2140 units ] doses of insulin ) or 2 ) 32 g , 4-mm/31 g , 8-mm pen needle ( also further separated into low- or regular - dose insulin ) . the patients within each group used the two assigned needles for a 3-week period each . the study did not find a statistically significant difference with change in fructosamine ( 1113 mmol / l difference ) , nor did it find that bmi affected fructosamine changes . ( fructosamine measures glucose concentrations over the previous 23 weeks . ) other studies have also found that patients were able to maintain glycemic control similarly with longer pen needles ( 12.7 mm ) or shorter ( 58 mm ) pen needles ( 9,10 ) . ( 9 ) specifically evaluated the use of a 31 g , 5-mm compared to a 31 g , 8-mm pen needle , as well as needle preference for 130 patients with type 1 or type 2 diabetes and a bmi range of 30.162.5 kg / m ( 9 ) . the patients were divided into two groups , and each group used a specific needle length for 3 months before switching to the other needle length for an additional 3 months . although a favorable , statistically significant difference was found in a1c of 0.12% ( p = 0.02 ) between the 5- and 8-mm pen needles , there was no statistical difference between the two groups for fructosamine levels , hypoglycemic episodes , site bruising , or pain perception . unique to this study is the evaluation of 1,5-anhydroglucitol for possible differences in postprandial elevations , which found no changes . the patients in this study did not express preference for the 5- or 8-mm needle lengths ( 46 and 41% , respectively ) . ( 10 ) also demonstrated that glycemic control could be maintained with either a 31 g , 6-mm or a 29 g , 12.7-mm needle in their multicenter , crossover study of 62 patients with type 1 or type 2 diabetes and a bmi range of 3064 kg / m ( 10 ) . ( 9 ) , the patients used one needle length for a 3-month period before switching to the other needle length for an additional 3 months . there was no statistical difference in the final a1c between the two needle lengths ( 7.6% with the 5-mm needle and 7.9% with the 12.7-mm needle ) . patients in the studies described rated the shorter pen needles as much less painful and , not surprisingly , mostly preferred over longer pen needles ( 5,10 ) . in the schwartz study ( 10 ) , patients also completed several surveys , including the world health organization diabetes treatment satisfaction questionnaire ; insulin treatment satisfaction questionnaire ; and , specifically asking questions related to needles , the needle handling questionnaire and the needle preference questionnaire . their results show that patients repor - ted greater satisfaction ( p < 0.001 ) with shorter needles and greater preference for shorter needles ( 89% , p < 0.001 ) and rated shorter needles more favorably with regard to ease of use , perceived pain , and glycemic control ( p < 0.001 ) . despite concern that shorter needles are more apt to cause insulin leakage , especially with larger doses of insulin or with obese patients , the evidence seems to demonstrate the contrary ( 2,4,5,9,10 ) . in addition to evaluating glycemic control with a 4-mm pen needle , hirsch et al . ( 5 ) also looked at insulin leakage and concluded that there was no difference between 4- , 5- , and 8-mm needles in the amount of leakage . this study found that 58% of the leaks reported were with the 5- and 8-mm pen needles , and a smaller proportion reported leaks with the 4-mm pen needle . ( 9 ) found no correlation between insulin leakage , bmi , and insulin dose with the 5- versus the 8-mm needles . no comparable difference in the volume of leakage was found between the 6- and 12.7-mm needles ( 2.7 vs. 3.2 mm ) by schwartz et al . a difference in leakage , however , was found , although minimal and not clinically significant , when using a vertical injection technique as opposed to an angled technique ( 65 vs. 59% , p < 0.001 ) with 5-mm needles ( 2 ) . additionally , wittmann et al . ( 4 ) found increasing leakage , in absolute terms , with larger doses and shorter needles when various amounts of medium were injected into pork rind . but , once again , comparing the percentage of leakage against the larger volume , the difference was not found to be significant . it can be concluded that shorter pen needles can be used without a significant increase in insulin leakage . the patients participating in the described studies did not have any physical or cognitive limitations noted . special populations such as patients with physical limitations that are related to hand movement or dexterity ( e.g. , osteoarthritis , rheumatoid arthritis , tremors , carpal tunnel syndrome , or tendonitis ) , the elderly , vision impaired , or pregnant patients may be limited in their capacity to perform accurate self - injections and may require innovative approaches for insulin injection ( 11,12 ) . patients with physical limitations or those who use an injection technique in which a shorter needle is not inserted completely , despite education , may not be ideal candidates for the shorter needles ( 11 ) . these special circumstances underline the crucial role of diabetes educators in providing diabetes self - management education and support . a 63-year - old , morbidly obese ( bmi 46.58 kg / m ) woman with type 2 diabetes and arthritis presented to the clinic diabetes service with an initial complaint of sporadic yet large - volume insulin leakage from her injection site while using 5-mm pen needles . her a1c was 7.6% , and her blood glucose log revealed wide glucose excursions ( 92371 mg / dl ) . during the education visit , the patient was able to demonstrate proper injection technique ( e.g. , angle of injection , complete depression of insulin dose , and appropriate duration of needle insertion ) . to help reduce insulin leakage , she was counseled to split the dose into two separate injections of 42 units each and provided additional education on injecting the insulin slowly and rotating the injection site . at her follow - up visit her glucose values continued to show wide excursions ranging from 83 to 354 mg / dl , and her a1c remained at 7.6% . the patient also complained at this visit of her injection site bubbling up immediately after the injections . taking her bmi into consideration , it was decided that a longer pen needle length may help prevent insulin leakage despite evidence that skin thickness is not increased in obesity ( 4 ) . therefore , the patient was switched to an 8-mm pen needle length . at a 2-week follow - up visit , she also stated that the needles were often bent when she pulled them out , which she attributed to a bad injection site and scar tissue . upon reassessment of her injection technique , the patient demonstrated and admitted to always using both hands to deliver the insulin due to her arthritis and decreased mobility . she also stated that she would reinsert the needle in different spots on her abdomen to find a good spot . in addition , unbeknownst to the patient , it was observed that she was unintentionally pulling the needle part of the way out while maneuvering the insulin pen and injecting the insulin . after re - education on injection technique , the patient could not successfully demonstrate a clean her arthritic condition made it challenging for her to use one hand and to keep the needle in while maneuvering and depressing the pen with the other hand awkwardly stretched across her body . subsequently , she was switched to 12.7-mm pen needles , which , upon repeat demonstration and most likely due to the longer length and 90-degree insertion , stayed in the subcutaneous space despite her unorthodox technique . seven months later , the patient confirmed that the leakage problem had resolved and denied any bubbling up of the skin , which could possibly be attributed to intradermal delivery of the insulin while the needle was being pulled out . her a1c increased slightly to 7.8% , but her blood glucose excursions improved ( 140260 mg / dl ) , implying more consistent insulin delivery . after receiving a consistent insulin dose and allowing for proper titration , her a1c improved to 6.7% . insulin injection technique directly affects glycemic control , medication adherence , and , ultimately , quality of life . alarmingly , an international survey assessing patients on multiple factors related to insulin injection found that many patients do not recall learning various concepts such as site rotation , mixing insulin , and duration of injection ( 13 ) . diabetes educators are in a key position to ensure that patients receive thorough education and periodic assessments of their injection technique . a teach - back method is an approach that should be employed to confirm proper understanding and technique ( 1518 ) . a plethora of literature describes the negative impact of low health literacy on clinical outcomes of chronic disease states such as diabetes and how communication methods such as the teach - back help improve patient outcomes ( 16 ) . in addition to assessing patients self - injection for appropriate technique and approach ( e.g. , injection location and injecting through clothing ) , educators have the responsibility of working together with patients using shared decision - making to select an appropriate and manageable needle length to ensure accurate delivery of insulin . additionally , injections that are less painful and allow for accurate delivery would most likely help to improve patient motivation for better diabetes self - management and medication adherence . the patient case described here illustrates just one example of a unique circumstance inhibiting successful use of a shorter pen needle . studies have demonstrated that bmi does not affect efficacy of particular needle lengths , and consensus panels have published guideline recommendations supporting shorter needles . nonetheless , this article describes a situation in which insulin delivery and , ultimately , glycemic control was improved with a longer pen needle length , demonstrating that there may be unique circumstances in which a longer pen needle should be used ( 1,5,811 ) . longer pen needles may be a better option for some patients with higher bmis and coordination challenges resulting from comorbid health conditions such as arthritis . although shorter needle lengths should be the standard , diabetes educators and providers should assess their appropriateness and effectiveness on a case - by - case basis and still consider longer needles when needed .

ensuring the correct delivery of insulin is essential in the treatment of diabetes . both proper injection technique and needle length are important considerations for adequate insulin delivery . there have been several studies demonstrating that bmi does not affect efficacy or insulin leakage with shorter pen needles ( e.g. , 4 or 5 mm vs. 12.7 mm ) . additionally , the international scientific advisory board for the third injection technique workshop released recommendations in 2010 on best practices for injection technique for patients with diabetes , which , with regard to needle length , concluded that 4-mm pen needles were efficacious in all patients regardless of bmi . however , regardless of patients bmi , insulin injection technique should always be assessed and physically disabling comorbid conditions taken into consideration when choosing a needle length that will be manageable for patients . the purpose of this article is to raise awareness of unique patient circumstances that may warrant the use of the longer 12.7-mm needle .

we obtained clinical samples and background information from 15 glanderous equids in punjab , pakistan from 1999 through 2007 ( table ; appendix table ) . research on equine subjects was approved by the synopsis scrutiny committee and animal ethics committee , faculty of veterinary science , university of agriculture , faisalabad , pakistan . samples were plated on brain - heart infusion ( bhi ) agar with 4% glycerol and incubated for 2430 hours at 37c . individual colonies were inoculated into bhi broth containing 4% glycerol and were incubated with shaking for 36 h at 37c . an aliquot of broth ( 1.5 ml ) was centrifuged at 13,000 rpm for 15 min . genomic dna was extracted from the resulting pellets using standard digestion buffer and phenol - chloroform extraction protocols ( 6 ) . samples from equines with similar histories were assigned to the same epidemiologic group ( e.g. , group 1 , group 2 ) . subgroups were defined based on vntr data ; samples with identical vntr genotypes were assigned to the same subgroup ( e.g. , prl11 and prl13 in group 1b ) . data are comparing subgroups 3a and 3b to each other ( prl1 and prl41 ) and excluding subgroup 3c ( prl7 ) . data are comparing subgroups 3a and 3b combined ( prl1 with prl41 ) to subgroup 3c ( prl7 ) . for genotyping , we screened 23 loci ( appendix table ) from a previously established 32-marker multiple locus variable number of tandem repeats ( vntr ) analysis system designed for b. pseudomallei and b. mallei ( 7 ) . in silico genotyping of the same loci was also performed for 10 whole genome sequences ( wgs ) of b. mallei ( 8 ; appendix table ) . vntr markers have higher mutation rates than other genetic markers which make them inappropriate for determining deep levels of evolutionary relatedness , however vntrs are appropriate for 1 ) discrimination between closely related isolates , 2 ) determination of the degree of relatedness among isolates , and 3 ) discernment of population structure on a spatial scale ( 7,9,10 ) . this utility is especially important for b. mallei because it is a recently emerged clone of b. pseudomallei and has been shown to be genetically monomorphic with typing methods such as multilocus sequence typing ( 11 ) . to compare the genetic diversity of our punjab isolates to that of sequenced strains , we performed a phylogenetic analysis on the 23 loci using the neighbor - joining algorithm in paup * 4.0b ( 9 ) . to determine the genetic relationships among the punjab population itself , we performed the same analysis using only the punjab isolates and polymorphic loci ( n = 15 loci ) . combined analysis of the punjab isolates and wgs showed that the punjab isolates are phylogenetically distinct from wgs ( figure 1 ) . this finding was also demonstrated in the values for average pairwise distance ( apd ) , where the apd among punjab isolates is 2 lower than the apd calculated for either the entire phylogeny or the wgs alone ( figure 1 ) . the punjab isolates represent only a small amount of the genetic diversity demonstrated in this pathogen . phylogenetic analysis of the punjab isolates alone placed 14 of the 15 samples into 3 distinct clades with 1 sample standing alone ( figure 2 ) . most samples ( 9/15 ) belong to clade a , whose isolates are both temporally and geographically diverse , suggesting that this lineage is ecologically established in punjab . unrooted neighbor - joining tree based on 23 variable number tandem repeat loci demonstrating that the punjab isolates ( black text and prl-20 ) are genetically distinct from and less diverse than available whole genome sequences ( wgs , red text ) ( 8) . statistical supports for branches based on 1,000 bootstrap iterations are shown . sample prl-20 is shown in red text because it is also available as a whole genome sequence ; therefore , it was used in all 3 situations where an average pairwise distance ( apd ) was calculated . among 10 wgs , the average pairwise distance was 0.607 ; between 10 wgs and punjab isolates , average pairwise distance was 0.627 ; and among 15 punjab isolates , average pairwise distance was 0.312 . these results indicate that the punjab isolates are more closely related to each other than to the sequenced strains because the apd among punjab isolates is 2 lower than the apd calculated in the other 2 situations . unrooted neighbor - joining tree showing phylogenetic relationships among 15 samples of burkholderia mallei from the punjab province , pakistan . sample names are color - coded to match their district of origin in reference to the inset map of the punjab province . approximate linear distances between districts are faisalabad to lahore 120 km , faisalabad to sargodha 84 km , lahore to sargodha 168 km . because of the limited sample size , many of the patterns observed from these data may result from sampling bias . however , even a limited amount of genotypic data can be useful in formulating hypotheses regarding the dispersal of b. mallei . for example , the presence of samples from faisalabad in each clade suggests that this district may be a center of diversity in the province ( figure 2 ) but this does not indicate a lesser degree of diversity in other districts where fewer samples were collected . the diversity seen in the district of faisalabad may result from either 1 ) the industrial nature of faisalabad or 2 ) from high endemism of b. mallei in the region . currently , the district has 10,000 horses and mules and > 44,000 donkeys , plus other transient equines ( 12 ) . many equines move through and work in the city , potentially introducing strains from surrounding regions . because horses and mules can be positive but asymptomatic for glanders ( 13 ) , many hosts are available to maintain strains throughout the region . communal stables and water troughs are common throughout the district and b. mallei has been isolated from these water troughs ( a. naureen , unpub . furthermore , b. mallei can remain viable in contaminated stables for up to 6 weeks ( 14 ) and in sterile tap water for up to 4 weeks ( 15 ) , which provides an environment for establishment and retention of b. mallei populations in faisalabad . combining phylogenetic with epidemiologic data for example , epidemiologic data suggests that 2 horses from a farm in the district of sargodha ( prl3 and prl4 ) contracted glanders while at a polo club in the lahore district . this is supported by vntr data , as these 2 isolates clustered phylogenetically with one of the samples obtained from an outbreak that occurred at the same polo club 3 months prior ( groups 5 and 6 , table ) . furthermore , at the time of the prl3 and prl4 infections , a co - resident mule with no previous travel history ( prl44 ) was negative for glanders , making it unlikely that these horses acquired glanders from their farm . this mule was positive for glanders 1.5 years later , and the isolate obtained from its infection clustered phylogenetically with the samples from the polo club and sargodha horses . therefore , we hypothesize that the infected horses either directly transferred the disease to the mule or they contaminated a source on the farm which subsequently led to the mules infection . environmental sampling would be required to identify the original infection source for the horses and subsequent transmission route to the mule . nevertheless , this case shows a strain that was transferred a distance of 168 km , demonstrating that human - mediated movement of equines can influence the distribution of b. mallei genotypes . other cases in the province demonstrate that infections either stem from similar strains or are caused by multiple strains . for example , samples that were placed in the same epidemiologic group cluster together phylogenetically ( groups 1 , 2 , and 4 ; table ) , indicating communal infections similar to the cases described above . in contrast , epidemiologic group 5 ( prl19 and prl20 ) was separated into 2 distinct clades ( figure 2 ) , indicating that this outbreak was caused by multiple strains . therefore , it should not be assumed that an outbreak of glanders is always caused by a single strain . our study suggests that numerous lineages of burkholderia mallei are present in punjab , pakistan , and that these lineages persist across geographic space and time . despite this the economics and use of equines likely contribute to the persistence of glanders in this region because modern methods for control of this disease ( monitoring and euthanasia ) are not viable options . we suggest that a focus on finding methods to improve the sanitary conditions of communal water troughs and stables may lead to a practical solution for disease reduction and containment . finally , our study demonstrates the utility of vntrs paired with extensive epidemiologic data for analyzing the distribution of b. mallei genotypes throughout endemic regions . epidemiologic and 23-locus vntr data for 15 isolates of burkholderia mallei from the punjab province , pakistan , and 9 burkholderia mallei whole genome sequences *

we collected epidemiologic and molecular data from burkholderia mallei isolates from equines in punjab , pakistan from 1999 through 2007 . we show that recent outbreaks are genetically distinct from available whole genome sequences and that these genotypes are persistent and ubiquitous in punjab , probably due to human - mediated movement of equines .

o tratamento endodntico tem seus procedimentos clnicos fundamentados em conhecimentos bsicos , principalmente biolgicos , cujo xito do tratamento depende das condies dos tecidos da regio apical e periapical , dos mecanismos de defesa orgnicos e da potencialidade de reparao das clulas . diferentes materiais obturadores , com os mais diferentes componentes qumicos , tm sido testados em busca de uma substncia que tenha propriedades fsico - qumicas e principalmente biolgicas ideais para perfeita vedao do canal radicular . um fato importante referente aos insucessos nos tratamentos endodnticos diz respeito aos componentes txicos dos cimentos obturadores de canal , includos com o propsito de neutralizar as falhas de desinfeco durante o preparo do canal . avaliar histologicamente o comportamento tecidual subcutneo de ratos aps implantes de tubos de polietileno preenchidos com cimentos obturadores de canal base de hidrxido de clcio ( sealapex , apexit e sealer 26 ) . foram utilizados para este estudo 60 ratos , os quais foram divididos em oito grupos : quatro grupos experimentais , cada um com 10 ratos , e quatro grupos controle , com cinco ratos . resultados os resultados da anlise histolgica mostraram que todos os cimentos foram irritantes , porm em intensidade que variaram entre si e em funo do tempo . nos perodos iniciais ( 48 horas e sete dias ) todos os materiais exibiram resultados prximos , com quadro inflamatrio entre severo e moderado , respectivamente . nos perodos finais ( 21 e 60 dias ) , as reaes teciduais aos implantes contendo o cimento sealapex apresentaram quadro ativo , mas em fase involutiva , em relao aos cimentos apexit e sealer 26 . os grupos controle apresentaram nmero de clulas inflamatrias inferior aos grupos experimentais , mantendo quadro reacional em fase involutiva . o mtodo estatstico utilizado foi a anlise de varincia para que fosse feita uma comparao entre as mdias . conclui - se que o cimento sealapex foi o mais biocompatvel seguido pelo apexit e sealer 26 . the field of endodontics , especially as regards the materials and techniques , has been presenting considerable advances in the last years , providing the professionals with more safety for accomplishment of root canal filling . the basis of clinical procedures of endodontic techniques is the basic knowledge , and a successful treatment depends on the conditions of the apical and periapical tissues , immune mechanisms and potential of cell regeneration . different filling materials with diverse chemical components have been tested in the search for some substance that presents ideal physicochemical and mainly biological properties for a perfect root canal sealing . an important aspect related to unsuccessful endodontic therapies is related to the toxic components of root canal sealers , which are included in an attempt to neutralize any deficiencies in the disinfection accomplished during root canal preparation . ingle and taintor5 ( 1985 ) stated that the initial reaction of the periapical tissues to all sealers is inflammatory , yet cell regeneration occurs as the sealers harden , unless they continue to disintegrate with release of any toxic components . root canal sealers are needed to fill unavoidable gaps between the solid filling materials and the root canal walls , and should be both biologically acceptable and able to bond to dentin15 . the zinc oxide - eugenol - based root canal sealers , long employed for root canal filling , display quite satisfactory clinical outcomes . however , the biocompatibility of these materials is impaired by the presence of eugenol , which has cytotoxic properties . the modern endodontics has been struggling to eliminate certain empirically based myths with the search for a material that meets the ideal requirements as regards the biological and physicochemical properties . within this context , the calcium hydroxide - based sealers were introduced and have been increasingly employed ; however , the presence of calcium hydroxide in a product does not assure its therapeutic properties , since there are also other components in root canal sealers . thus , the aim of this investigation was to histologically evaluate the behavior of the subcutaneous tissue of wistar rats after contact with calcium hydroxide - based root canal sealers with different chemical compositions ( sealapex , sealer 26 , apexit ) , in order to provide useful information for clinical studies addressing the selection of a more biocompatible material . the present investigation utilized 60 male wistar rats ( rattus norvegicus , albinus ) , all young adult animals ( about 60 days ) weighing 180 to 220 grams , obtained from the laboratory animal of the research center aggeu magalhaes ( fiocruz ) , recife , pe , brazil . the use of animals for this research was approved by the local ethics committee for animal research , and nih guidelines stated in the " principles of laboratory animal care " were followed ; the study design was approved by the ethics committee under approval report n. 183/03 . the animals were divided into eight groups , being four experimental and four control groups . the experimental groups contained 10 rats each , and the control groups had five rats each . the groups were analyzed in 48 hours ( group 1 ) ; 7 days ( group 2 ) ; 21 days ( group 3 ) and 60 days ( group 4 ) . the animals were anesthetized with intraperitoneal sodium thiopental ( cristalia produtos qumicos e farmaceuticos ltda , itapira , sao paulo , brazil ) , 50mg / kg of weight , one at a time in order to reduce suffering , followed by trichotomy on the dorsal area . longitudinal incisions measuring approximately 5 mm were performed on three predetermined areas outside the midline ( two at the scapular and one at the right pelvic area ) . thereafter , tissue dissection was laterally accomplished with blunt - pointed scissors with the achievement of three surgical areas with subcutaneous access , in order to allow subcutaneous placement of the implants and thus avoid dilaceration ( at 2 cm from the incision line ) . the sealers were then prepared according to the manufacturers ' instructions and placed inside polyethylene tubes measuring 10 mm in length and 1.5 mm in internal diameter , with one end closed . following , the specimens with the study materials were inserted with pliers in the surgical areas according to the type of material to be analyzed , as follows : sealapex ( sybron / kerr , industria e comercio ltda . sao paulo , brazil ) at the right scapular area ; sealer 26 ( dentsply , petrpolis , rio de janeiro , brazil ) at the left scapular area ; apexit ( vivadent , schaan , liechenstein ) at the right dorsal pelvic area . thus , three dorsal implants were placed in each animal of the eight groups , being two in the scapulae and the other in the pelvis . the tubes in the experimental group contained the root canal sealers under investigation , and the control group received implantation of empty tubes in the same predetermined areas . afterwards , the rats were returned to their cages and kept under observation until sacrifice . after the experimental periods of 48 hours ( experimental group 1 , control group 1 ) , 7 ( experimental group 2 , control group 2 ) , 21 ( experimental group 3 , control group 3 ) and 60 days ( experimental group 4 , control group 4 ) , the rats were anesthetized and sacrificed and the tissue around the implant was surgically removed , including removal of enough surrounding tissue . after fixation of the specimens in bouin 's liquid ( histology laboratory of upe / pe ) , the properly identified tissue blocks containing the polyethylene tubes were trimmed and embedded in paraffin after removal of the tubes . from the 180 identified blocks achieved , the 120 blocks from the experimental group were separated according to the period and material analyzed , and the control blocks were separated according to the period . the blocks were submitted to microtomy on a leitz wetzlar microtome ( germany ) from the department of morphological sciences ( histology laboratory of upe / pe ) . five non - adjacent sections from different areas were randomly selected , which corresponded to five slabs . the 900 slabs obtained and prepared were stained with hematoxylin and eosin , gomori 's trichrome and picrosirius as described by maia9 , ( 1979 ) bancroft and stevens1 ( 1982 ) and mota10 ( 1985 ) . microscopic analysis was conducted on a binocular light microscope ( olympus - japan ) with immersion objective ( 1,000x ) . the reactions were investigated at the interface between tissue and material at the open end of the tube and analyzed by an examiner ( histologist ) blinded to the study . reading of the number of inflammatory cells and fibroblasts was performed by means of division of each slab into five fields . it should be noticed that , depending on the extension of reaction , one or two fields might not present inflammation and thus the number zero would be recorded for each inflammatory cells . means were recorded for each type of cell for each animal after reading of five microscopic fields on each slab related to each material and group . evaluation of the degree of cell infiltrate was conducted by sum of the means of the different types of inflammatory cells for all five fields for each group and material , following the classification suggested by wolfson and seltzer18 ( 1975 ) and adopted by leal , et al.7 ( 1988 ) with slight modifications : up to 49 cells : mild ; from 50 to 99 cells : medium or discreet ; from 100 to 249 cells : moderate ; equal to or above 250 cells : severe . microscopic evaluation at the study periods did not reveal any condition of inflammatory and/or infectious nature on the surgical sites prepared to receive the implants . analysis of the skin surrounding the tubes revealed absence of exudate , as well as adherence of tissues in groups 3 and 4 . the specimens containing the implants with sealers sealapex , apexit and those of the control group presented normal color around them ; however , the implants filled with sealer 26 were often surrounded by an hyperemic and edematous area , besides the presence of a black material inside the tube . the mean and standard deviation between groups were calculated from the data achieved by using the variance analysis method . at 48 hours ( group 1 ) , the mean number of neutrophils was remarkably higher for sealer 26 ( image 3a ) and less high for apexit ( image 2a ) . this group did not exhibit giant cells , being plasma cells observed only for the material apexit , whose mean value was 1.00 . the variability was high between fields in this group for the plasma cells for the material apexit . at 7 days ( group 2 ) , the mean number of neutrophils was lower for the sealapex ( image 1b ) and higher for sealer 26 ( image 3b ) , with respective values of 1.00 and 22.40 . the presence of plasma cells and giant cells was only observed for sealapex ( image 1b ) . the variability between fields was very high in this group for neutrophils , plasma cells and giant cells for the sealapex . at 21 days ( group 3 ) , the mean number of neutrophils was quite reduced , with a maximum value of1.60 for sealer 26 ( image 3c ) . the highest mean numbers were 14.40 for lymphocytes for sealer 26 , and 13.00 for macrophages for the apexit ( image 2c ) , with no observation of presence of neutrophils for the sealapex ( image 1c ) . there was a large variability in this group for the plasma cells for the sealapex , and neutrophils and plasma cells for the apexit . at 60 days ( group 4 ) , the mean number of cells was relatively low , except for the macrophages for sealer 26 ( image 3d ) , with a mean of 30.33 . the variability was large in this group for the plasma cells for sealapex and giant cells for the apexit . comparison between the experimental groups for the mean of fields reveals the following : the mean number of neutrophils was significantly higher for the animals in group 1 than the other groups , for any material ; the mean number of lymphocytes and macrophages was slightly higher for the group of animals killed at 7 days ( group 2 ) compared to the animals in the other three experimental groups , except for the macrophages in group 3 for apexit and lymphocytes for sealer 26 , which also had reasonably high values . the maximum mean number of lymphocytes was found for the sealapex in group 2 , with a value of 16.20 ; for the macrophages , this maximum value was observed for apexit in group 2 , with a value of 23.67 ; plasma cells were not observed for the sealapex and sealer 26 in group 1 nor for apexit and sealer 26 in groups 2 and 4 ; giant cells were only observed for the sealapex in group 2 , all three materials in group 3 and apexit and sealer 26 in group 4 . with regard to the means of fields for the control groups , the following was observed : the mean occurrence of neutrophils was higher for group 1 , followed by group 2 with respective values of 25.4 and 16.33 . for group 3 and 4 , the occurrence of this type of cell was reduced ; the mean presence of lymphocytes with values of 12.00 was slightly higher for group 3 than for the other groups ; there were no plasma cells and giant cells in any control group ; the variability between fields was remarkably high for the neutrophils in group 1 . table 1 presents the total number of inflammatory cells according to group and material , and the classification of cell infiltrate . thus , it was possible to observe that , for the experimental groups , the results of cell infiltrate were as follows : for group 1 , all three sealers were classified as severe inflammatory reaction ; for group 2 as moderate ; for group 3 as medium or discreet for sealapex and moderate for the other two sealers ; and for group 4 , as mild inflammatory reaction for sealapex and moderate for the other two sealers . the sealer 26 presented the highest amounts of inflammatory cells in all groups , thus presenting the worst performance . for groups 1 and 2 , the apexit had the lowest amounts of inflammatory cells , whereas for groups 3 and 4 the lowest amounts of cells were found for the sealapex . all control groups were classified as moderate at most . in these groups , the results according to the criteria established were moderate inflammatory reaction for group 1 , medium for group 2 and mild for groups 3 and 4 . the figures present the histological sections for the sealers sealapex ( figure 1 ) , apexit ( figure 2 ) and sealer 26 ( figure 3 ) , at the four study periods . the development of scientific knowledge in medical and related areas has long been employing animal investigations . the frequent utilization of rats as a research tool is based on aspects such as easy handling , relatively short vital cycles , and well - known anatomy , physiology and genetic variations.12 according to leal , et al.7 , ( 1988 ) yesilsoy , et al.19 ( 1988 ) and soares , et al.14 , ( 1990 ) who analyzed the performance of the sealapex sealer and observed the occurrence of diffuse calcifications in the presence of this sealer , an axiom for every material for biological application is not to be cytotoxic , and thus the ideal response of the periapical tissues to the endodontic therapy would be the formation of biological sealing of the apical foramen by deposition of newly formed cementum . leal et al.7 ( 1988 ) investigated the biocompatibility of sealapex , as well as crcs , fill canal and n - rickert in the subcutaneous tissue of rats and demonstrated that , in general , all sealers were fairly irritant to the connective tissue ; however , the sealapex at seven days yielded a moderate inflammatory reaction , with a mild inflammatory reaction at 21 and 60 days , indicating a tendency toward repair . in agreement with these findings , silva , et al.13 ( 1997 ) studied the biological behavior of sealapex , crcs , apexit and sealer 26 sealers in the connective tissue and peritoneal cavity of isogenic mice and revealed a large increase in the number of polymorphonuclear leukocytes at the initial 2-to 4-day period in all sealers analyzed when compared to the control group . comparison of the sealers to each other revealed fewer neutrophils at the initial stage for the sealapex sealer in relation to the other sealers analyzed . despite somewhat different from the present outcomes at the initial 48-hour period , on which the apexit sealer displayed fewer neutrophils when compared to the sealapex , these findings are in agreement with the present outcomes at the subsequent periods , which displayed a progressive reduction in the number of inflammatory cells , characterizing an evolution toward repair . on the other hand , the findings of nassri , et al.11 , ( 2003 ) who investigated the biocompatibility of sealapex and apexit sealers in the subcutaneous connective tissue of rats , fully corroborate the present results , on which the apexit displayed a much more aggressive tissue reaction than the sealapex . differently , zmener , et al.19 ( 1988 ) conducted a quantitative study on which the tissue response to the sealapex sealer was compared to the crcs sealer , a calcium hydroxide - based sealer containing eugenol , and observed that the tissue reaction to the sealapex sealer displayed a progressive increase after 30 and 90 days , with the persistence of a granulomatous reaction with presence of black particles in the cytoplasm of macrophages , which the authors believed to be particles of titanium dioxide and which would be associated to these outcomes because of the high phagocytic activity . however , holland4 concluded that the sealapex was less toxic when compared to zinc oxide - eugenol - based sealers , yet also highlighted the presence of titanium dioxide in the cytoplasm of macrophages . even though the results of zmener , et al.21 ( 1990 ) have confirmed that the black particles observed inside macrophages and giant cells in investigations on sealapex are in fact composed of titanium and barium dioxides , the same investigations concluded that removal of these components from the sealer composition surprisingly increased the inflammatory reaction . histological analysis of this study clearly demonstrates the more aggressive reaction of the apexit sealer compared to sealapex , corroborating the findings of nassri , et al.11 ( 2003 ) . however , the in vitro analysis conducted by beltes , et al.2 ( 1995 ) stated that the apexit sealer is less cytotoxic when tested in cell suspension , demonstrating a better performance compared to the sealapex sealer . in agreement with the present study , silva , et al.13 ( 1997 ) observed the presence of tissue necrosis in contact with material remnants and a reduced number of polymorphonuclear cells at the intermediate and final periods , which was more pronounced for the sealapex sealer , followed by apexit and sealer 26 . on the other hand , sealer 26 is derived from ah26 , to which calcium hydroxide was added . the ah26 was a widely investigated root canal sealer , especially as regards its biological properties . the most irritant among all components of ah26 was found to be the a bisphenol ether.8 the unsatisfactory results obtained in the present investigation for sealer 26 , which displayed a severe inflammatory reaction at the initial 48-hour period and moderate reaction at the further 7- , 21- and 60-day periods were in accordance with the findings of silva , et al.13 ( 1997 ) on which the histological outcomes demonstrated that the reactions to sealer 26 yielded an intense neutrophilia at the initial periods , inducing severe inflammation and degeneration . however , these findings are in disagreement with those of jacobovitiz6 ( 1996 ) on the subcutaneous tissue of rats , which found that sealer 26 was less irritant than the crcs and apexit . this corroborates the outcomes observed by valera16 , ( 1995 ) who investigated the biocompatibility of sealapex , sealer 26 , apexit and ketac endo sealers , and found that sealer 26 presented the mildest inflammatory reaction at 90 days . this study highlighted the need to increase the experimental periods up to 90 days , since all experimental groups at the 60-day period exhibited material remnants contacting the tissues , thus indicating that they were still being repaired . an interesting aspect is the fact that the biological properties reflect the ability of the material to be inert , yet if this is not true , the material must contain substances that may be more easily phagocyted and digested and less dispersed , thus leading to a better response as to tissue repair . all sealers were irritant , yet the intensity varied between each sealer and as a function of time ; the sealapex sealer was the most biocompatible , followed by the apexit and sealer 26 ; the sealer 26 induced a larger number of inflammatory cells at all study periods .

objective : to histologically evaluate the behavior of the subcutaneous tissue of rats after placement of polyethylene tubes filled with calcium hydroxide - based root canal sealers ( sealapex , apexit e sealer 26).study design : the study employed 60 rats , which were divided into eight groups , being four experimental groups with 10 rats each , and four control groups with five rats . analysis was conducted at 48 hours , 7 , 21 and 60 days . results : histological analysis revealed that all sealers were irritant ; however , the intensity varied between each sealer and as a function of time . at the initial periods ( 48 hours and seven days ) , all materials demonstrated similar outcomes , with inflammatory reaction from severe to moderate , respectively . at the final periods ( 21 and 60 days ) , the tissue reactions to the implants containing sealapex displayed an active status yet with regression , compared to the apexit and sealer 26. the control groups exhibited less inflammatory cells than the experimental groups , keeping a reaction status with regression.conclusions:the results achieved in this study revealed that the sealapex sealer was the most compatible , followed by apexit and sealer 26

a 47-year - old man was admitted with the chief complaint of persistent high fever and transient loss of consciousness . although his general condition improved after intravenous acyclovir administration , the patient presented with visual loss in both eyes 4 days after admission . visual acuity in his right eye was 20/200 and his left eye had light perception alone . progression of retinal detachment was prevented in both eyes ; however , visual acuity of the left eye was totally lost because of neovascular glaucoma . although cases of bilateral acute retinal necrosis have been reported after herpetic encephalitis , this condition is rare after herpetic meningitis . prophylactic acyclovir therapy and early panretinal photocoagulation may prevent retinal detachment and improve the prognosis . neurologists and ophthalmologists should be aware that not only herpetic encephalitis but also herpetic meningitis can lead to acute retinal necrosis within a very short interval . acute retinal necrosis ( arn ) was first described as a rapidly progressive unilateral necrotizing retinitis in 1971 by urayama et al.1 arn usually presents unilaterally and has a poor prognosis . bilateral cases have also been reported.2 to prevent a poor outcome , arn requires aggressive management.3 although several case reports have described the occurrence of arn after herpetic encephalitis,48 arn after herpetic meningitis is rare.9 here , we report the case of a healthy immunocompetent man with bilateral arn that occurred in combination with herpetic meningitis during the administration of intravenous acyclovir . a 47-year - old man was admitted with the chief complaint of persistent high fever and transient loss of consciousness . the patient was somnolent and complained of posterior cervical pain but nuchal rigidity was unclear . a painless erythematous macule with blistering was observed on both upper eyelids and several painless erythematous macules with blisters were observed on the left side of the chest . glucose was maintained at a normal level and cerebrospinal fluid was negative for herpes simplex virus ( hsv ) dna and varicella zoster virus ( vzv ) dna . brain t2-weighted magnetic resonance imaging revealed no findings of herpetic encephalitis , such as hyperintensity corresponding to edematous changes in the temporal lobes ( figure 1 ) . therefore , viral meningitis was suspected and administration of intravenous acyclovir was initiated at 750 mg every 8 hours ( 10 mg / kg every 8 hours ; the patient s body weight was 75 kg ) . three days after admission , the patient s consciousness status slightly improved but binocular diplopia and blurred vision suddenly occurred . in addition , several new sharply circumscribed erythematous macules appeared on the right and left upper and lower extremities . four days after admission , the visual acuity was 20/200 oculus dexter ( od ) and light perception oculus sinister ( os ) . the right and left corneas were clear , but flare and many cells were observed in both anterior chambers and vitreous bodies . optic disc swelling , macular edema , exudation along blood vessels , narrowing of arterioles , dilatation of venules , and superficial retinal hemorrhages were observed in both retinas ( figure 2a ) . oral administration of prednisolone and ocular instillation of betamethasone sodium phosphate and tropicamide were initiated and subsequently tapered over several weeks . five days after admission , panretinal photocoagulation was performed for both eyes . although the patient s body temperature had returned to normal 5 days after admission , medical treatment was continued . inflammation , optic disc swelling , macular edema , and exudation along blood vessels were alleviated in the right eye and the visual acuity of this eye had improved to 20/20 4 months later . however , massive retinal hemorrhage derived from central vein occlusion appeared in the left eye 19 days after panretinal photocoagulation and the hemorrhage extended to vitreous 26 days after photocoagulation . all sight was lost due to neovascular glaucoma approximately 1 month after the occurrence of the hemorrhage ; however , rhegmatogenous retinal detachment was prevented by photocoagulation in both eyes ( figure 2b ) . by 25 days after admission , the patient s general condition and ophthalmoscopic findings had improved , and he was treated as an outpatient . the onset of arn occurred a few days after the administration of intravenous acyclovir , although the patient was not immunocompromised . watanabe et al11 reported a case in which bilateral arn occurred after hsv type 1 meningitis . in their case , the retinal findings deteriorated and retinal detachment occurred in both eyes . in our case , we prevented rhegmatogenous retinal detachment in both eyes . this may be partly due to the administration of intravenous acyclovir before the onset of retinal symptoms as well as early panretinal photocoagulation . serological test results were negative for vzv antibodies and the results of cerebrospinal examination were negative for hsv dna and vzv dna . administration of acyclovir improved the patient s general condition , suggesting that herpes viruses were the causal agent . ganatra et al12 reported six out of seven patients diagnosed with arn in whom hsv type 1 dna was detected had a history of hsv encephalitis and three out of six patients with hsv type 2 dna had a likely history of hsv meningitis . therefore , they concluded that herpes simplex virus is likely to be the viral cause of central nervous system infection in a patient with acute retinal necrosis syndrome . it is possible that isolated meningitis can be accidentally coincident with arn ; however , in our case , we believe this was a part of the disseminated herpetic virus infection . vandercam et al13 reported an overall interval of 5 weeks ( 14 days to 2 months ) between vzv encephalitis and arn . however , as far as the authors of the present report are aware , a bilateral arn occurring in an immunocompetent patient following vzv encephalitis has not been previously described . in this patient , arn occurred in both eyes simultaneously and the occurrence of arn after encephalitis was very short . our patient showed a very rapid and rare clinical course of arn occurring immediately after herpetic meningitis . neurologists and ophthalmologists should be aware that not only herpetic encephalitis but also herpetic meningitis can lead to arn within a very short interval .

purposethe report of a case of bilateral acute retinal necrosis after herpetic meningitis.case reporta 47-year - old man was admitted with the chief complaint of persistent high fever and transient loss of consciousness . although his general condition improved after intravenous acyclovir administration , the patient presented with visual loss in both eyes 4 days after admission . visual acuity in his right eye was 20/200 and his left eye had light perception alone . both eyes showed panretinal arteritis diagnosed as acute retinal necrosis . panretinal photocoagulation was performed for both eyes . progression of retinal detachment was prevented in both eyes ; however , visual acuity of the left eye was totally lost because of neovascular glaucoma . visual acuity of the right eye recovered to 20/20.conclusionalthough cases of bilateral acute retinal necrosis have been reported after herpetic encephalitis , this condition is rare after herpetic meningitis . prophylactic acyclovir therapy and early panretinal photocoagulation may prevent retinal detachment and improve the prognosis . neurologists and ophthalmologists should be aware that not only herpetic encephalitis but also herpetic meningitis can lead to acute retinal necrosis within a very short interval .

the incidence of infection after primary total knee arthroplasty ( tka ) has been reported to be 1% with an increase to 5% in patients with revision tka12 ) . for many years , the gold standard for treatment of chronic periprosthetic joint infection ( pji ) has been two - stage revision . static cement antibiotic spacers are well known to be an effective treatment for infection in tka34 ) . static cement spacers provide local antibiotic therapy while maintaining soft - tissue tension until the second stage procedure . with the use of static spacers , this may lead to stiffness of the knee and problems such as bone loss , pain , scarring , and instability56 ) . to overcome these problems , articulating spacers have been developed . articulating spacers have the advantage that they allow weight bearing and maintain joint movement , thereby improving patient 's satisfaction , reducing bone loss , facilitating the second - stage procedure , and resulting in a greater range of motion ( rom ) in the end7 ) . several studies have shown articulating spacers have the same reinfection rates as static spacers while , to some extent , preserving the function of the knee in between stages89 ) . two types of cement - on - cement articulating spacer have been presented : either those that can be made during operation using antibiotic - loaded cement and silicon mold components or prefabricated and commercially available cement spacers910 ) . articulating spacers can also be made reusing the original autoclaved femoral component with a new tibial polyethylene insert or using a new femoral component and a new tibial polyethylene insert ( nfc spacer ; pfc sigma , depuy synthes , warsaw , in , usa ) . the aim of this paper is to report our experience with an nfc spacer in terms of both eradication of infection and functional improvement . from december 2010 to march 2013 ( 28 months ) , a consecutive series of 32 two - stage revisions using nfc spacers were performed in 31 patients suspected of having a chronic infected tka . we only included patients with a primary tka who had not received any prior two - stage revision for pji . in all cases , patient 's medical records and radiographs were reviewed retrospectively after approval was obtained by the danish data protection agency . the diagnostic criteria for pji are difficult and numerous definitions have been proposed . in our study , patients were diagnosed with pji based on clinical suspicion ( pain , swelling , redness , or warmth ) , radiography , white blood cell count ( wbc ) , and creactive protein ( crp ) level or microbiologic examination of preoperative joint fluid , although knee aspiration was not performed routinely . the diagnosis was verified from microbiologic examination of joint tissue at the time of first - stage surgery . in general , a positive culture result of 2 or more than 5 intraoperative cultures was considered an infection12 ) . a pji may be present although none or only one out of 5 intraoperative cultures is positive . several other factors such as presence of purulence in the affected joint , elevated synovial leukocyte count , and elevated crp concentration also need to be considered in order to determine the presence of pji . retrospectively , all our cases were confirmed to have pji according to the universally adopted new definition for pji13 ) used to ensure consistency in definition in this study . thorough debridement of the infected and devitalized tissue and bone was performed , which was followed by pulsating saline lavage . five intraoperative cultures were taken from inflamed synovial tissue or bone - cement membrane before the administration of intravenous antibiotics12 ) . nfc spacers were cemented with refobacin revision bone cement ( biomet , valance , france ) . a sachet of 40 g refobacin revison bone cement contains 1 g gentamicin and 1 g clindamycin ( fig . 1 ) . the cement was in a late doughy stage so it would not adhere well to the bone , thereby allowing removal without any major bone loss at the time of second stage procedure . according to a standard protocol , patients who were considered positive for pji received a minimum of six - week antibiotic treatment including two postoperative weeks of intravenous administration and oral administration thereafter . patients were encouraged to mobilize with full weight bearing and perform rom exercises as tolerated immediately after the first stage procedure with the help of a physiotherapist . the second stage procedure in the revision tka using the pfc sigma tc3 knee system ( depuy synthes ) was scheduled minimum 2 weeks after the first stage procedure without antibiotic administration . there was no rise in the inflammatory blood markers combined with the clinical appearance of the soft tissue ( fig . one patient was excluded due to prior two - stage surgery with a static cement spacer immediately before the two - stage revision using an nfc spacer and two patients died from unrelated medical reasons and were lost to follow - up . one patient with a bilateral nfc spacer had a dislocation tendency in the left knee and a chronic resistant infection in the left charcot foot . an above knee amputation of the left knee with an nfc spacer was required and thus this patient was excluded from the analysis . another 6 patients who had two - stage revisions using nfc spacers were excluded for the following reasons . first , they had negative tissue cultures in all 5 samples collected during the first stage of the surgery despite clinical signs of infection prior revision surgery such as pain , swelling and increased crp . second , they did not meet the other criteria for pji according to new definition for pji13 ) . therefore , they only received antibiotic treatment ranging from 413 days postoperatively until negative tissue cultures were obtained . they all had a successful second stage procedure within 412 weeks after the first stage surgery when soft tissue allowed the procedure . all patients were followed up with an outpatient visit at 6 weeks and 1 year after surgery . from almost all patients , american knee society knee score ( akss ) , function score , and rom were obtained prior to the first stage surgery and one year after the second stage surgery , which were registered in the danish knee arthroplasty registry . all patients were reviewed at " e - journal " , which is an internet - based medical journal for physicians where all contacts with the danish health care system is stored , to ensure that no patient had any additional knee surgery at other institutions after the second stage procedure at our institution . the result after the 2-stage revision using an nfc spacer was considered successful when eradication of infection was achieved using only one nfc spacer . the 22 cases meeting the diagnostic criteria for pji and included in the follow - up consisted of 12 females 10 males with an average age of 65.7 years ( range , 49 to 82 years ) . all had a two - stage revision using an nfc spacer with a mean follow - up of 37.6 months ( range , 25 to 52 months ) . cases 115 ( table 1 ) had 2 or more positive tissue cultures out 5 samples except one patient ( case 5 ) that had negative test results in all 5 tissue cultures but presented obvious purulent material in the knee joint at the first stage surgery along with other signs of pji . all 15 cases , therefore , received our standard antibiotic regimen with a minimum 6-week antibiotic treatment before the second stage procedure . eradication of infection was achieved using only one nfc spacer in all 15 cases and no reinfection was observed . cases 1618 were primarily not considered positive for pji because none or only one out of 5 intraoperative cultures was positive . therefore , they only received antibiotic treatment for a few days postoperatively until negative tissue cultures were obtained . retrospectively , pji was confirmed by the new definition for pji13 ) in the patients , and , therefore , they should have received a standard antibiotic regimen for a minimum of six weeks before the second stage procedure . however , they all had a successful second stage procedure and no reinfection was observed in these 3 cases . cases 1922 were positive for pji with 2 or more positive tissue cultures and had a recurrence of infection after their first second stage procedure . cases 19 and 20 were treated with multiple spacers ( more than 2 ) at our institution and still remained infected , and consequently they were referred to other institutions for further treatment and , therefore , they were lost to follow - up . case 21 was infected with coagulase - negative staphylococcus and had a successful two - stage procedure and no reinfection was observed for several years . but due to urine retention , the patient had numerous urological procedures and therefore became reinfected with escherichia coli as a result of hematogenous dissemination . the patient has undergone two - stage revision for a second time and just recently received a new revision tka . case 22 became reinfected with the same species ( coagulase - negative staphylococcus ) and was treated with two - stage revision for a second time but has shown no sign of reinfection after receiving a second revision tka more than a year ago . the average time from primary tka to the first stage procedure was 29.1 months ( range , 3 to 115 months ) . the average value of crp was 81.7 mg / l ( normal reference < 8 mg / l ) before the first stage surgery and the average value of wbc was 8.810/l ( normal reference 3 , 58 , 810/l ) before the first stage surgery . the akss was 29.3 ( range , 3 to 59 ) , function score was 29.5 ( range , 0 to 70 ) , and the average rom was 82 ( range , 40 to 120 ) before the first stage surgery . the average time from the first stage procedure to the final second stage procedure was 12.7 weeks ( range , 4 to 45 weeks ) . at the last scheduled follow - up in our outpatient clinic 1 year postoperatively from the second stage surgery , the akss had improved to 66 ( range , 40 to 94 ) and the function score was 64 ( range , 45 to 80 ) . successful eradication of infection was achieved in 18 ( 82% ) out of the 22 cases that were positive for pji using only one nfc spacer . periprosthetic infection remains a devastating and challenging complication of tka for both the patient and the surgeon . two - stage revision with an articulating spacer has shown good results in terms of infection control79 ) , but very few studies have been reported on the use of an nfc spacer as an articulating spacer in two - stage revision of chronic pji in tka111415 ) . the purpose of the present study was to present our short - term results using an nfc spacer as an articulating spacer . successful eradication of infection was achieved in 18 ( 82% ) of 22 cases using only one nfc spacer and this is comparable to other studies describing the use of an nfc spacer11 ) ; however , none of our patients needed long - term antibiotic suppression after the second stage procedure . eradication rate of 82% is comparable to that in the study by park et al.3 ) and fehring et al.6 ) where the use of static cement spacers in two - stage revision resulted in the eradication rate of 85% and 88% , respectively . although our results in terms of the eradication of infection seems to be no better than that in the knees with static spacers , early ambulation immediately after the first stage surgery , which is possible due to the use of an nfc spacer , helps patients to perform activities of daily living , thereby improving patient 's satisfaction and compliance . studies comparing articulating spacers with static spacers report greater postoperative function scores and rom in the knees with articulating spacers916 ) . brunnekreef et al.16 ) reported significantly greater postoperative rom and faster recovery in the group with articulating spacers . limitations of our study include the relatively small study population and the lack of comparison with a control group . however , our study demonstrated good functional results of two - stage revision with an nfc spacer : rom increased from 82 to 104 and function score increased from 29.5 to 64 postoperatively . previous studies have described good results with the use of an autoclaved femoral component as an articulating spacer 1718 ) . compared to an nfc spacer , the autoclaved original femoral component is cost - effective11 ) ; however , we are concerned about reusing an implant designed for single use only19 ) . at our institution , therefore , the total price for an nfc spacer is $ 723 , which is less than the price of our prefabricated articulating cement spacer ( spacer k with gentamicin , tecres , verona , italy ) , which costs $ 1,055 albeit we are aware that regional differences in pricing can occur . another benefit of the nfc spacer is that it gives the surgeon the opportunity to balance the knee by adjusting the thickness of the polyethylene insert compared to the prefabricated articulating cement spacers that often only come in a few sizes . our study demonstrated a significantly improvement in both akss and rom in the knees with an nfc spacer . thus , the present spacer model seems to be a safe device for the treatment of infection of the knee offering good functional results . short - term results from our study provide evidence that the use of a nfc spacer as an articulating spacer is effective in two - stage revision of chronic infected tka with regard to eradication of infection and functional improvement .

purposeto report our experience with two - stage revision using a new femoral component ( nfc ) spacer ( depuy synthes ) as an articulating spacer.materials and methodsin this retrospective study , we reviewed 22 two - stage revisions that were performed using an nfc spacer in 22 patients suspected of having an infected total knee arthroplasty ( tka ) from december 2010 to march 2013 . the result was considered successful when eradication of infection was achieved using only one nfc spacer.resultsthe average time from primary tka to the first stage procedure was 29.1 months and the average time from the first stage procedure until the final second stage procedure was 12.7 weeks . the average range of motion increased from 82 preoperatively to 104 postoperatively . the american knee society knee score increased from 29.3 points to 66 points . the function score increased from 29.5 points to 64 points . four cases were reinfected after two - stage revision . the mean follow - up was 37.6 months.conclusionsthe new articulating spacer showed promising short - term results both with regard to eradication of infection and functional improvement .

from january to june 2008 , 1,422 pig blood samples were collected on 39 pig farms in northern italy . ten farms were farrow - to - finish , 17 farrow - to - weaning , and 12 were fattening operations . on average , 10% of animals per farm were sampled and the sera were analysed for anti - hev iggs using an indirect enzyme - linked immunosorbent assay ( elisa ) . the elisa test was a human commercial kit ( hev - ab , diagnostic bioprobes , milan , italy ) , modified with a specific tracer ; that is , goat anti - swine instead of goat anti - human igg . this test is based on the use of plates coated with a recombinant antigen containing immunodominat epitopes from the orf2 and orf3 regions of mexican ( genotype 2 ) and burmese ( genotype 1 ) viral human strains . ( 50 l / well ) was examined at a fixed dilution ( 1:100 in pbs containing 1% yeast ) . the peroxidase - conjugated goat anti - swine igg ( goat anti - pig igg , serotec , oxford , uk ) were used at 1:3000 dilution . the absorbance value was measured at 492 nm wavelength and the results expressed as optical density ( od ) . the pre- and post - infection serum from pigs experimentally infected with hev were included as positive and negative controls , respectively . the cut - off value used was 0.274 and was calculated as the mean od value plus three standard deviation ( sd ) , of 80 antibody - negative pig sera . the chi - squared test was performed on contingency tables to find p - values . the od values of pig sera for anti - hev igg values ranged from 0.045 to 3.369 with an average od of 0.52 ( sd 0.62 ) and a median value of 0.352 . using the cut - off value of 0.274 , 38 out of 39 farms had at least one seropositive sample ( 97.43% , 95% ci : 92.5100% ) and 714/1422 serum samples ( 50.21% , 95% ci : 47.752.8% ) were positive for anti - hev igg . the sows presented the highest seroprevalence ( 70.6% , 95% ci : 6774.1% ) and the risk of developing seroconversion was about four times higher than all the other groups put together ( or=4.7 ; ic=3.75.9 ; table 1 ) . number of positive serum samples displayed by productive age considering the different type of farms , the mean seroprevalence value was 70.5% ( ranged 21.6100% ) in farrow - to - weaning farms , 61.2% ( 094.1% ) in farrow - to - finish farms , and 30.3% ( 3.681.3% ) in fattening farms ( table 2 ) . a direct correlation between farm size and seroprevalence was also evident ( table 3 ) . seroprevalence in different type of herd , displayed by productive age seroprevalence in farms according to the herd size the data indicate a high seroprevalence for anti - hev antibodies in an italian pig population , even if the percentage of seropositive animals varied widely among herds , being around 0% on some farms and almost 100% on others . in particular , the seroprevalence was higher in larger herds and it varied greatly among different age classes . in fact , we found the highest and the lowest seroprevalence in sows and in weaners , respectively ; in piglets ( 816 weeks of age ) we got a lower seroprevalence than in fattenings ( up to 24 weeks ) . unexpectedly , seroprevalence decreased in finishers ( 30.8% ) , perhaps due to the mixing of pigs from different farms and to the absence of sows in these herds . such differences are probably due to the dynamics of the infection in swine , which is influenced by maternal immunity . passive immunity protects piglets up to 2 months old and , after the infection , seroconversion occurs with igg increase mainly at 15 weeks old . this infection dynamic is supported by studies based on detected viral rna in faeces , with the highest values in pigs around 6 months old and also at slaughter time , suggesting that hev can infect at any age ( 8) . seminati and collaborators ( 9 ) had similar results in spain : the total anti - hev igg seroprevalence was 41.9% in sera collected from 1998 to 2000 and 60.8% in sera of gilts and sows collected from 1998 to 1999 . other studies conducted on small - sized pig sera samplings showed seroprevalences in developed countries : united kingdom ( 85% ) , sweden ( 58% ) , germany ( 23% ) , and the united states ( 34.5% ) ( 6 ) . in canada , 594 out of 998 ( 59.5% ) human genotype 1 and 2 antigens have previously been used to test human samples with good specificity and sensitivity ( 11 ) . due to the existence of common immunodominant epitopes in human and swine hev ( 3 ) , these antigens are efficient in capturing pig antibodies produced against the hev swine strain circulating in northern italy ( 7 ) . in conclusion , although there are a few cases recognising human infection , hev is widespread on pig farms in northern italy . the high seroprevalence in pigs should raise concern as it has been described previously that seropositive animals , despite developing an immunological response , could still contain hev at slaughter age ( 12 ) , representing a risk for food security and for persons in contact with pig or pork products . the authors declare no conflict of interest and they have not received any funding or benefits that could have affected this study .

the prevalence of the hepatitis e virus ( hev ) infection in pigs in northern italy was serologically examined . the survey was carried out on 39 farms : 17 farrow - to - feeder , 10 farrow - to - finish , and 12 fattening enterprises . there were 1,422 sera that were tested using commercial indirect elisa . this method originally developed for testing human sera was adapted for the analysis of pig sera . all farms except one ( 97.43% ) and 714 sera samples ( 50.21% ) resulted positive for anti - hev igg antibodies . this study confirms that hev is widespread in pigs in italy and might be endemic on most farms .

cervical radiculopathy could be caused by disc herniation , spondylosis , instability , and trauma or on rare occasions by tumors . majority of cervical radiculopathies are caused by cervical spondylosis , while 25% are the result of disc herniation . cervical radiculopathy point prevalence and annual incidence have been reported at a population of 3.5/1,000 and 83/100,000 , respectively . approximately 83% of patients with cervical radiculopathy respond to conservative treatment methods , while an approximate one - third of patients will suffer from persistent symptoms . surgery is indicated for cases that have signs / symptoms of severe / progressive neurological deficits and persistence of radicular pain despite 12 weeks of conservative treatment . surgery is mostly performed via an anterior approach with or without fusion , although traditionally posterior approach is another method . outcome of surgical management for cervical disc herniation has shown a success rate of 66 to 98% . from april 2003 to june 2014 , 1280 cases of spine surgeries were performed by the senior author at a single centre . surgical indications for cervical disc herniation ( cdh ) were ( 1 ) progressive myelopathy ; ( 2 ) persistence or worsening of radiculopathy despite 12 weeks of medical treatment ; and ( 3 ) motor deficit or intractable pain . our inclusion criteria were ( 1 ) single- or multilevel cdh and ( 2 ) more than 12 months of postoperative follow - up . cases were excluded due to ( 1 ) coexistent spine pathologies ; ( 2 ) history of previous spine surgery ; and ( 3 ) less than 12 months of postoperative follow - up . sixty - eight cases of cdh that were managed by acdf were included in this series . the outcome instruments were ( 1 ) study - designed questionnaire that addressed residual and/or new complaints and subjective satisfaction with the operation ; ( 2 ) recent ( one week prior to the interview ) postoperative vas for neck and upper extremity radicular pain ; ( 3 ) japanese orthopaedic association myelopathy evaluation questionnaire ( joacmeq ) ( standard persian version ) ; and ( 4 ) follow - up cervical magnetic resonance imaging ( mri ) and lateral x - ray . preoperative medical information which consisted of preoperative symptoms , duration of pain ( from onset up to surgery ) , physical examination , and pain severity using visual analogue scale ( vas ) was recorded at the time of operation by the senior author . our study population was contacted by phone and informed about the research project and invited for a follow - up visit . a physician working in the field of spine research carried out the follow - up visits . this is the first study that addresses the long - term clinical and radiographic outcome and influential factors among iranian patients who underwent acdf for herniated cervical disc using polyetheretherketone ( peek ) cage stand - alone technique . for descriptive statistics , central and dispersion tendencies were performed . for comparison between qualitative variables , nonparametric test ( chi - square ) our mean follow - up time was 52.93 ( months ) 31.89 sd ( range : 13131 months ) . 42 cases had one - level disc herniation and 21 and 5 patients were operated on for two- and three - level involvements , respectively . mean preoperative vas ( one week prior to operation ) for neck pain and radicular pain were 9.32 2.40 sd and 9.29 2.41 sd , respectively . mean postoperative vas ( at the time of follow - up ) for neck and radicular pain were 1.28 2.50 sd and 1.03 2.22 sd . most ( 22 , 32.4% ) of our cases had disc herniation at the level of c5-c6 . two - level disc herniation was mostly ( 10 , 47.6% ) encountered at c5-c6/c6-c7 . fusion was confirmed with imaging studies in all 42 cases that came for follow - up imaging study ( 100% fusion rate ) . furthermore , radiologic findings were not shown to affect the severity of residual complaints statistically . success rates for clinical outcome with regard to vas for neck and radicular pain were 88.2% and 89.7% , respectively ( mean vas for neck pain : 8.04 3.27 sd ; mean vas for radicular pain : 8.26 3.10 sd ) . early postoperative complications were hoarseness ( 3 cases , 4.4% ) , c5 root palsy ( one case , 1.5% ) , and dysphagia ( one case , 1.5% ) . late postoperative complications were 8 ( 19% ) cases of subsidence , 6 ( 14.3% ) cases of adjacent segment degeneration , 2 ( 2.94% ) cases of adjacent segment disease , 1 ( 1.5% ) case of right upper broken screw , 1 ( 1.5% ) case of screw loosening , and one ( 1.5% ) case of graft extrusion . among cases who were diagnosed with subsidence , 6 ( 75% ) had undergone single - level acdf ( c5-c6 , c6-c7 ) and 2 had undergone 2-level acdf . during the follow - up time , none of the patients complained of symptoms recurrence . 47.1% of patients complained of residual complaints at the time of the follow - up . the most common ( 7 , 10.3% ) residual complaint was sensory deficits , followed by cases ( 6 , 8.8% ) who still suffered from both upper extremity radicular pain and sensory deficits at the final follow - up . these were neck pain ( 1 case ; cervical mri showed 10% cage subsidence ; vas : 5 ) , contralateral to preoperative side of radicular pain ( 1 case ; cervical mri demonstrated different level disc herniation ) , limitation of shoulder abduction ( 1 case ; diagnosed with c5 root palsy ) , and sensory deficits ( 1 case , vas : 8) . the outcome of surgery did not differ by type of job , smoking , preoperative neck pain or sensory complaints , preoperative hypoesthesia , duration of pain , or level of disc herniation . the surgical outcome was significantly ( p value : 0.039 ) better for men ( mean rank : 38.09 ) in comparison to women ( mean rank : 30.91 ) . older patients had lower scores in uef ( p value : 0.043 ) , lef ( p value : 0.001 ) , bf ( p value : 0.001 ) , and qol ( p value : 0.01 ) . presence of preoperative radicular pain affected uef ( p value : 0.046 ) ( mean uef score for those with preoperative radicular pain was 96.69 10.43 sd and for individuals without preoperative radicular pain was 88.30 17.99 sd ) . preoperative hoffmann 's sign affected lef ( p value : 0.008 ) , bf ( p value : 0.007 ) , and qol ( p value : 0.029 ) . preoperative babinski sign had an effect on lef and bf ( p value : 0.01 ) . in this study , we achieved improvement with regard to neck and arm pain of 88.2% and 89.7% of our study population . with an average follow - up period of 18 months , kwon et al . demonstrated rates of 96.1% and 82.1% for neck and arm pain , respectively , based on vas . at mean follow - up of 25.6 months , liu et al . demonstrated significant clinical improvement with regard to vas for arm and neck pain . furthermore , dagli et al . reported a significant decrease in vas at two - year follow - up . a statistically significant decrease for vas , both for neck and arm pain , was achieved for study population in park et al . 's series at mean follow - up period of 12 months . except qol functional score , the other four success rates calculated with joameq ranged between 70.6 and 83.8% . with regard to residual complaints , we observed that 47.1% of our cases complained of minor residual symptoms at the final follow - up . peolsson reported that 70% of their study population suffered from persistent pain and disability at 6-year follow - up . studies of bohlman et al . , lied et al . , and gaetani et al . in concordance with other studies [ 1921 ] , we showed that men with younger age achieved better outcome . in agreement with study of bohlman et al . , we also could not find a relation between smoking status and outcome . in our series , duration of preoperative symptoms . demonstrated no correlation between duration of symptoms and surgical outcome in their evaluation on cases with cervical spondylotic radiculopathy who were treated with acdf . lied et al . also achieved no significant correlation between preoperative duration of pain and pain relief acdf has been advocated as a safe procedure , but complications could still arise . among its complications are nonunion , postoperative dysphagia , recurrent laryngeal nerve palsy , esophageal tear , carotid artery injury , vertebral artery injury , neurologic deficit , postoperative respiratory embarrassment , and disc space infection . injury to rln was found by flynn to be the most frequently encountered neurologic complication . two studies [ 23 , 26 ] reported dysphagia as the most common acdf - related complication . we had one case ( 1.5% ) with dysphagia , which is lower than the incidence of dysphagia reported in other studies that ranged between 2.5 and 21.3% [ 2730 ] . in a study conducted by chen et al . , incidence of 0.16% was reported for hoarseness , while this rate was reported higher in baron et al . 's series at a rate of 4.9% , which is close to the incidence of 4.4% , which we observed among our study population . an average rate of 4.3% ( range : 1.6%12.1% ) has been documented in literature for the incidence of c5 root palsy after anterior decompression and fusion . in our series , we had one case ( 1.5% ) with c5 root palsy which is lower than the incidence of kim et al . 's series which reported a rate of 4.3% . incidence of graft extrusion has ranged between 0 and 0.88% [ 12 , 26 , 35 ] . prevalence of this complication ( 1.5% ) in our series was higher than other previously reported studies . with an average follow - up duration of 18 months , kulkarni et al . reported that none of their study population had cage extrusion or migration . . demonstrated no cage extrusion on average follow - up period of 28.4 months . in a study conducted by nanda et al . , cases with graft extrusion had persistent neurological symptoms after the operation , but graft extrusion in our patient was associated with a new - onset neck pain . incidence of adjacent segment degeneration ( asdeg ) after acdf has been reported to range from 16 to 51 [ 38 , 39 ] . study with 4.5-year follow - up showed that 41% of their series developed asdeg . in our series , we showed a rate of 14.3% after a follow - up period of 4.41 yrs 2.67 sd . asdx progression to a degree that requires additional surgery was the case for one of our patients ( 1.5% ) . with an average follow - up period of 6 years , bohlman et al . described that 9% of their patients required additional surgery for asdx . in another series , 17% of the study population required additional surgery for asdx at an average of 4.5 years of follow - up . the prevalence of subsidence in our study was 19% , which is higher than cabraja et al . 's series that reported peek cage subsidence in 14.3% of their cases at mean follow - up of 28.4 months . at 2-year follow - up period follow - up periods , rate of peek cage associated subsidence of 8.1% has been observed at mean follow - up time of 18.9 months by ha et al . . and with an average follow - up of 12 months , park et al . reported that 22.6% of their cases had subsidence . in a study conducted by song et al . . found that subsidence was significantly associated with gender and number of treatment levels and treatment at c57 . in kast we achieved no correlation between age , gender , number of treatment levels , and subsidence ; although with no statistically significant difference , the majority ( 75% ) of cases diagnosed with subsidence had undergone single - level acdf ( c5-c6 , c6-c7 ) . reported a rate of 93.33% for peek cage fusion at 6 months . at a mean follow - up of 10 months 's cases . with an average of 18 months of follow - up , kulkarni et al . 's study population fusion was maintained at their last follow - up . at mean follow - up of 28.4 months , cabraja et al . achieved a fusion rate of 88.1% for peek cage . at mean follow - up of 25.6 months , liu et al . , fusion rate at 12-month follow - up was 100% for peek cage group . with mean follow - up period of 18.9 months we achieved 100% of fusion rate at mean follow - up time close to 53 months . acdf is a successful surgical technique for the management of cervical disc herniation among iranian population .

background and aim . first - line treatment strategy for managing cervical disc herniation is conservative measures . in some cases , surgery is indicated either due to signs / symptoms of severe / progressive neurological deficits , or because of persistence of radicular pain despite 12 weeks of conservative treatment . success for treatment of cervical disc herniation using acdf has been successfully reported in the literature . we aim to determine the outcome of acdf in treatment of cervical disc herniation among iranians . methods and materials / patients . in a retrospective cohort study , we evaluated 68 patients who had undergone acdf for cervical disc herniation from march 2006 to march 2011 . outcome tools were as follows : ( 1 ) study - designed questionnaire that addressed residual and/or new complaints and subjective satisfaction with the operation ; ( 2 ) recent ( one week prior to the interview ) postoperative vas for neck and upper extremity radicular pain ; ( 3 ) japanese orthopaedic association myelopathy evaluation questionnaire ( joacmeq ) ( standard persian version ) ; and ( 4 ) follow - up cervical magnetic resonance imaging ( mri ) and lateral x - ray . results . with mean follow - up time of 52.93 ( months ) 31.89 sd ( range : 13131 months ) , we had success rates with regard to vas for neck and radicular pain of 88.2% and 89.7% , respectively . except qol functional score of joameq , 100% success rate for the other 4 functional scores of joameq was achieved . conclusion . acdf is a successful surgical technique for the management of cervical disc herniation among iranian population .

together , the world health organization 's policy framework for active aging and its guide for the development of age - friendly cities have set a policy and research agenda in which the built environment is conceived to play a crucial role as a facilitator or inhibitor of older people 's capacity to age actively . this theoretical standpoint assumes that built environments which are conducive to the outdoor mobility of older people will help provide them with ongoing opportunities for both participation within society and maintenance of their health through the gaining of physical exercise as they move around their neighborhoods . it remains unclear , however , whether or not older people 's self - assessments of being active are linked to either their levels of participation within society or their engagement in outdoor physical activity , and , to date , there is no convincing evidence of a direct relationship between the built environment and older people 's walking behavior . active aging is defined by the who [ 1 , page 12 ] as the process of optimizing opportunities for health , participation , and security in order to enhance quality of life as people age . this concept presents both opportunities and challenges to policy makers and researchers alike . in terms of policy , active aging has been acknowledged as providing a sound basis for responding to the challenges posed by population aging in industrialized nations by linking a number of key policy domains including employment , pensions , retirement , health , and citizenship [ 3 , page 121 ] . yet within europe , for example , the focus on active aging has so far been largely limited to a crude reduction in terms of working longer [ 4 , page s117 ] . walker [ 3 , page 124 ] noted more than a decade ago that active aging does not amount to a coherent strategy and is sometimes just a slogan used to cover anything that seems to fit under it . more recently , he has stressed that this concept is more of an aspiration for both individuals and collectivities than it is a process that exists in practice [ 4 , page s126 ] . the usefulness of the active aging concept for policy makers aiming to comprehensively address issues associated with the growing aging population will necessarily rest on their capacity to devise a coherent set of strategies that effectively deal with all of the three key determinants of active aging identified by the who participation , health , and security . one key opportunity presented to researchers by the development of the active aging concept is its potential to act as a driver for greater attention on the older person - macroenvironment relationship . indeed , there has been unprecedented research interest in this area since the release of the who 's policy framework ; prior to the early 2000s , it was the microenvironments of older people which predominated as the main focus of investigations into the older person - environment relationship ( see ) . nevertheless , the complex , multifaceted nature of the active aging concept is problematic , especially since it effectively encompasses dimensions covered by a number of earlier concepts . these include successful ageing which walker traces back to the early 1960s ( a concept emphasizing the importance of maintaining activity patterns and values of middle age into old age ) and productive aging which emerged in the 1980s ( mostly focused on removing barriers to older persons engaging in work paid or unpaid and thereby extending their productive capacity beyond retirement age ) . walker [ 3 , page 123 - 124 ] notes that active aging emerged in the 1990s , giving recognition to both the connection between activity and health and the importance of healthy aging . his account of the development of the active aging concept clearly demonstrates that active aging is much broader in scope than all of these earlier concepts . nevertheless , a review of empirical literature recently undertaken by hung and colleagues grouped active aging with successful , productive , positive , and robust aging as cognates of healthy agingdespite the fact that the active aging concept encompasses more dimensions than any of these other concepts ( including continued participation in society ; the maximization of social , mental , and physical health ; the maintenance of dignity , self - efficacy , and human rights ; and the creation of age - friendly physical environments to facilitate autonomy and independence ) ( see [ 3 , 7 ] ) . coherency of the accumulating evidence on active aging may well be undermined by both the overlap between this concept and its predecessors and the treatment of them as interchangeable terms . it is also noteworthy that successful aging has now been a focus of research for around half a century and there is still no widely agreed definition for this term or consistency between studies in the measures used to investigate this subject area , and the first rigorous analysis of measures was only undertaken recently ( see ) . active aging is operationally defined by researchers given the broad scope of this concept . if active aging objectives are to be met , it would seem crucial that laypersons ascribe meanings to this term that are in line with the who 's policy framework and fully grasp the nature and importance of all of the constituents of active aging as laid out in this framework . to date research undertaken in the united kingdom by bowling found this term was most commonly conceived by a sample of older people aged 60 years and over , to involve having / maintaining physical health and functioning ( 43% ) , leisure and social activities ( 34% ) , mental functioning and activity ( 18% ) , and social relationships and contacts ( 15% ) . comparison with academic conceptualizations of active aging showed that there was overlap between lay perceptions of the constituents of active ageing and those used in theoretical models within the literature , although the latter entailed additional components ( productivity , empowerment , human rights , and dignity ) to those identified by laypersons . multiple regression analyses revealed that health status , longstanding illness , and quality of life ( all based on self - report ) explained 41% of the variance in self - ratings of active aging , while sociodemographic and economic variables were not significantly associated with this outcome [ 7 , page 298 ] . comparison of data gathered on the perceived constituents of active aging in this particular study with data obtained from an earlier survey of older people 's perceptions of successful aging , also administered in the united kingdom but with adults aged 50 years and over ( for further details see ) , showed that there was considerable overlap in the meanings ascribed to a later study by bowling compared the perceptions of minority ethnic groups with those of the general population in the united kingdom , revealing that ethnically diverse respondents were less likely to define active aging in terms of good physical health and fitness ( and exercise to promote these two aims ) and also less likely to rate themselves as aging actively than respondents within the general population . from bowling 's research , it would seem that individual factors such as health status and longstanding illness impact on older people 's self - assessments of aging actively , but we currently have little understanding of whether or not there is any relationship between these self - assessments and the actual behaviors of older persons with respect to their actual levels of participation and physical activity outside of the home domain . older people 's participation in the wider community necessarily relies on their capacity for remaining mobile in their out - of - home environments . the accumulated body of evidence suggests that individual factors as well as multiple aspects of the built environment affect older people 's potential out - of - home mobility and thus their capacity to participate in society . driving status [ 11 , 12 ] , quality and availability of public transport [ 13 , 14 ] , and numerous other features of the built environment ( see [ 15 , 16 ] ) are all implicated in this regard . walkable environments are particularly important , given that neighborhood walking increases not only older people 's opportunities for physical exercise but also social interaction . engagement in outdoor physical activity by older people is crucial to them remaining ambulatory and also provides numerous other health benefits . in the united states , lack of physical activity among older people has been described as a major public health issue ; the release of surgeon - general 's report in 1996 indicated that less than one - third of adults aged 65 years and over met health recommendations of moderate intensity exercise ( e.g. , brisk walking ) for 30 minutes , five or more days per week [ 19 , s267 ] . more recent research conducted in new jersey shows a similar pattern , with just 28.6 percent of 50- to 74-year - olds being found to meet exercise recommendations . the proportion of older people who meet exercise guidelines has been shown to be highly variable , however , both within and between different countries . a systematic review of studies investigating physical activity among older adults ( limited to those studies which reported their findings in terms of older people meeting exercise guidelines ) revealed that this proportion ranges between 2.4 and 83.0 percent , with the majority of the 53 studies reviewed being found to report proportions of between 20 and 60 percent . only 6 of these studies ( undertaken in the united states , australia , the united kingdom , brazil , china , canada , new zealand , columbia , south africa , greece , cyprus , sweden , and switzerland ) used an objective measure of physical activity ( i.e. , accelerometer ) , with the remainder being based on self - reported activity . research efforts over the past decade have provided insight into many features of the built environment that encourage or constrain older people 's walking decisions . yet , the extent to which older people 's walking behavior actually differs as a function of the particular built environment in which they live remains unclear . much of the research literature related to the built environment and walking activity is based on random samples , with findings being largely representative of those living in urbanized areas ( i.e. , similar built environments ) . in addition , the vast majority of available studies of walking behavior among older people are based on self - report , making the findings subject to bias ( e.g. , participant recall and selection of socially acceptable responses ) . a recent review of empirical literature that examined mobility or disability among older people and also utilized objective measures of the built environment identified 17 studies published between 1990 and 2010 that met these criteria . of these , 14 studies investigated walking behavior , with all but one being based on self - reports of walking . rosso et al . concluded that although evidence of a direct relationship between the built environment and elderly walking behavior was lacking , of all of the features of the built environment investigated , high density of intersections , street and traffic conditions , proximity to destinations , and green space were the factors most likely to have a direct effect on older people 's out - of - home mobility . nevertheless , a study of the consumption patterns and mode of travel used by older people ( via gps tracking ) for accessing goods and services within two different countries ( suburbs of canada and france ) showed that older people in both locations were highly reliant on travel by motor vehicle , despite substantial differences in the built environment and availability of public transport in the two study sites . this raises questions about the assumption that levels of walking outdoors among the older segment of the population will naturally follow modifications to the built environment that make them more age - friendly , without a shift away from sedentary lifestyles and a change in the propensity of the elderly to rely predominantly on motor vehicles for movement outside of the home domain . older people have identified particular urban design features such as green and open spaces [ 25 , 26 ] as being conducive to walking . other features like poorly maintained pedestrian infrastructure and traffic , however , heighten their fears about personal safety . evidence suggests that these fears about safety are well grounded , given that most falls occur outside of the home domain with the majority taking place on curbs , sidewalks , and streets and that older people are the most likely group to present at hospital for injuries sustained from pedestrian - cyclist collisions . unfortunately , however , fear of moving outdoors also increases older people 's risk of diminished capacity to walk outdoors in the longer term . longitudinal research has shown that older people who fear walking outdoors are 4.6 times more likely than those without this fear to develop difficulties in walking a distance of 0.5 kms . older people 's personal expectations about aging also influence their walking behavior . a pilot test of a behavioral intervention aimed at altering belief in the inevitability of becoming sedentary as a consequence of the aging process demonstrated that older people not only increased their walking after intervention but also experienced multiple other benefits including ease in performing daily life tasks and improvements in mental health - related quality of life , pain , energy , and quality of sleep . meeting the objective of maximising people 's opportunities for ongoing participation within society as they proceed into older age is likely to be a major challenge for policy makers in industrialized nations . cross - country analyses ( of canada , finland , germany , italy , japan , the netherlands , sweden , the united kingdom , and the united states ) indicate that there is a remarkably similar pattern in the age profile of work and passive and active leisure activities across countries . despite considerable variation in hours spent doing paid work between countries for adults in the 4554 age group , intercountry differences seem to vanish by the age of 75 years and over , with time that was previously spent doing paid work being predominantly reallocated to passive rather than active leisure activities . much of older people 's time also appears to be spent at home . research undertaken in berlin with community - based and institutionalized adults aged 70 years and over , for example , indicates that older people spent the majority of their day alone at home ( 80% of awake time ) and less than a fifth of their day outdoors ( 18.7% ) . similarly , more recent australian research indicates that only a small proportion of time is spent away from home ( 90.2% spent at home ) by community - based adults aged 65 years and over , with the mean number of episodes spent away from home in the previous week being 6.3 ( sd = 4.5 ; range 019 ) . in addition , the average daily time spent away from home by older people who have had a stroke and those who have not appears to very similar ( 0.9 versus 1.1 hours , resp . ) . age- and/or health - related issues are implicated , however , in a substantial difference observed in older australians ' engagement in volunteering . twice as many current drivers ( 66% ) as retired drivers ( 30% ) reported involvement in this particular form activity . the current study used a mixed - methods approach to explore areas of interest within the area of active aging that are currently underresearched , including ( 1 ) the connection between older people 's self - perceptions of being active and indicators of their health and participation ( domains which are proposed by the who to be two of the three key determinants of active aging ) ; ( 2 ) older people 's perceptions of characteristics of their respective communities , which are generally conceived to facilitate aging in place ; and ( 3 ) the extent to which older people 's out - of - home physical activity differs across different built environments , based on high quality objective measures of active travel ( walking and biking ) . a convenience sampling method was used to recruit a total sample of 48 adults aged 55 years and over , comprising 4 subsamples of equal numbers ( n = 12 ) of individuals living in ( 1 ) inner city suburbs and ( 2 ) suburbs outside the inner city area of a capital city , ( 3 ) a regional city , and ( 4 ) a rural town in queensland , australia . the first involved residents of inner city suburbs of brisbane ( within 5 kms of the central business district ) who had participated in a previous project ( living in the city ) and also indicated their willingness to be contacted for future research being invited to participate as representatives of high density living environments . the second recruitment method involved key community organisations and groups being approached to assist in identifying potential participants living in brisbane suburbs situated outside the inner city , toowoomba ( a regional city ) , and roma ( a rural town ) , as well as one individual to complete the brisbane inner city subsample . each individual was subsequently contacted via phone and/or email and invited to participate in the study . the total sample comprised 24 males and 24 females with each of the four subsamples being comprised of equal numbers of males and females ( a thirteenth individual from roma who volunteered to participate in the study jointly with her spouse was excluded from the study to ensure both an equal gender ratio and subsample size ) . all participants were ambulatory and the age range for the whole sample was 56 to 93 years ( average age of 72.02 years ; sd = 8.46 ) . table 1 provides a demographic profile of the sample across the four study locations with respect to age , income , marital status , and current housing ( derived from survey data ) . the population density for each of the four study locations the mean age for three of the subsamples was almost identical , but the mean age of the regional city ( rc ) subsample was between 2.5 and 2.7 years higher than the inner city ( ic ) , city suburban ( cs ) , and rural town ( rt ) groups . nearly all of those from the ic group had high incomes , while the majority living in the cs , rc , and rt locations had incomes that fell in the low range . the most common source of income across the whole sample was the old age pension ( 33.3% ) , but superannuation also provided whole or part of the annual incomes of one - third of the sample . only a small proportion of the whole sample was engaged in full- or part - time work ( 15.2% ) . the majority of participants in each subsample were married and also owned the homes in which they resided . all members of the ic group were living in a flat or unit ( interview data revealed that most lived in a unit ) , while the majority of the other three subsamples were living in a house . most of the older people in each of the four study locations owned their own dwelling . quantitative data were gathered from a gps device ( which captured one logged position every one minute and was used to track all out - of - home travel over seven consecutive days ) as well as responses to a brief survey contained in the front section of a travel diary . the gps device , gps charger , and diary were posted to each participant , which they returned to the project site by post or courier at the end of the tracking period . qualitative data were obtained from daily travel diary entries made by each participant ( documenting all out - of - home travel , activities undertaken outside the environment , and the mode of transport used for each trip ) . in a few cases , participants forgot to either recharge their gps device or take the device when they left home on one or two days and so were asked to continue completing their travel diaries and using the gps to ensure that their out - of - home travel was monitored for the required seven days . following the return of the completed diaries , gps device , and charger , the gps data were converted into individual time / space maps for each participant using google earth software . diary entries were used to color - code each trip line shown on the maps to indicate the means of travel used for each trip made ( by car , bus , ferry , train , taxi , and bicycle and on foot ) . these maps were subsequently used to direct discussion and verify correspondence between the gps data and diary entries during in - depth , semistructured interviews held with each participant approximately two weeks after the tracking period . survey items tapped demographic characteristics ( see table 1 ) as well as attributes of the built environment in which participants lived . the latter were assessed from endorsement of statements that represented reasons for them living in their current community and included proximity to destinations ( it is close to shops , etc . , and close to my family / friends ) and safety ( it is a safe area ) . participants ' perceptions of the age - friendliness and disability - friendliness of their communities were captured by responses to two separate items with a shared lead - in question ( do you think your community is : ( 1 ) age - friendly ; and ( 2 ) disability - friendly . available options were the same for each item ( yes , no , do not know , and never thought about it ) . self - perceptions of being healthy and being active were tapped from responses to two single - item , five - level likert scales ( options and coding for these variables are reported in table 3 ) . survey forms also included a question asking about all modes of transport normally used for moving around one 's neighbourhood ( for details of survey item and available options , see footnotes of table 4 ) . travel diary entries were used in conjunction with gps data for determining the number of days when participants ventured out of home , their use of public transport , time spent travelling on foot and by bicycle outside of the home environment , and the total time spent traveling outside home over the seven - day tracking period . in combination , travel diary entries and gps tracking produce high quality data on travel behavior . a new variable was created which measured the total time spent travelling on foot and by bicycle to ensure that all of participants ' travel by active means over the tracking period was captured . the continuous variables for time spent walking and travelling by active means were converted into a six - level measure of 30-minute intervals ( ranging from zero to a maximum of over 2 hours ) for the purpose of correlational analysis because of the wide variation in minutes travelled on foot and walking / biking by those who walked or used active means for a period of more than two hours . travel time captured by gps in off - road open spaces ( such as parks and golf courses ) was included , but any time spent inside buildings ( such as shopping centers ) was excluded from travel calculations . results are reported in tables in the form of cell counts due to the small size of each subsample , with percentages being shown in the totals column of each table . missing values are reported in the tables ( as no response ) and are included in the row totals . pearson correlation analyses were performed to identify relationships between select variables for the entire sample for exploratory purposes to determine relationships between self - perceptions of being active and being healthy and objective measures of participation ( number of days when participants travelled out of their homes ) and travel by active means outside the home environment . comparative analyses of the four study locations were undertaken using a nonparametric approach , the kruskal - wallis test of significance , due to its appropriateness for three or more independent , small , and nonrandom samples . the statistical significance of differences between groups determined by this test is based on ordinal information only , with observations being ranked and the mean rankings of the various groups being compared . the original continuous variable minutes spent walking and minutes spent walking / biking were used for these analyses to ensure true ranking of data ( actual minutes ) and minimization of tied ranks . table 2 shows that the majority ( 64.6% ) of the whole sample believed that their communities were age - friendly but less than half ( 41.4% ) agreed that they were interestingly , a similar proportion ( 41.7% in total ) neither agreed nor disagreed about the disability - friendliness of their communities and either did not know ( 18.8% ) , had never thought about it ( 10.4% ) , or did not respond to the question ( 12.5% ) . more individuals within the rc and rt subsamples rated their communities as being age- and disability - friendly than the two capital city subsamples ( ic and cs ) . participants ' reasons for living in their current community are shown in the lower part of table 2 . one - third of the whole sample identified their respective communities as being safe areas ( 33.3% ) and similar proportions reported proximity as a factor that contributed to their reasons for living in their current communities ( close to family / friends more older people living in the rc location identified being close to family / friends than those in the ic , cs , and rt groups ( 7 versus 3 ) as a reason for living in their current community , while similar numbers selected being close to shops , and so forth . only two older people living in the cs area agreed that safeness of the area was a reason for them living in their communities ( compared to 4 or 5 in the other three groups ) . the patterns of self - ratings of being active and of being healthy were each found to follow a normal distribution with a left skew ( prone to being healthy and active ) , results that are to be expected for a community - based older sample . ( 47.9% ) , and just under one - third of the sample considered themselves to be somewhat active ( 45.8% ) . less than one - fifth rated themselves as unhealthy ( 14.6% ) and none of the participants reported being very unhealthy ( see table 3 ) . no relationship was found to exist between self - ratings of being active and self - rated health status ( r = 0.24 ; p = 0.098 ) . self - perceptions of being active were also found to be unrelated to age ( r = 0.15 ; p = 0.317 ) , but a statistically significant , negative correlation was found between self - rated health status and age ( r = 0.32 ; p = 0.027 ) . only five of the total sample of 48 travelled outside of their home in four days or less over the monitored week . the number of days on which participants ventured out of home was positively associated with their self - ratings of being active ( r = 0.36 ; p = 0.011 ) but was unrelated to both self - ratings of health ( r = 0.11 ; p = 0.468 ) and age ( r = 0.26 ; p = 0.072 ) , and the subsample means and medians for days travelled outside home were remarkably similar ( see table 3 ) . the first part of table 4 summarizes self - report travel - related information derived from survey to provide context to the gps tracking results for time actually spent walking and walking / biking combined . most drove cars but one person drove a motorcycle and another two drove both a car and a motorcycle . another six ( 12.5% ) travelled by motor vehicle as a passenger , and only three reported that they did not travel by motor vehicle ( as either drivers or passengers ) . less than half ( 41.7% ) of the whole sample reported ever using public transport , and all but one of this group were from the ic and cs locations . none of the rc sample reported using public transport even though bus services are available in their location . both the rc and rt locations only have access to train travel for the purpose of traveling outside of their local areas ( rail link extends eastbound to brisbane and westbound to charleville ) , and only one participant from the rt group reported ever using the train . gps data showed that only a quarter ( 25.0% ) of the sample actually used public transport during the tracking period . the actual time spent walking and time spent walking and biking combined by individuals living within the four study locations ( based on gps data ) are shown in the lower section of table 4 . with respect to walking , one - third of the whole sample did not travel on foot out of their home environments at all over the tracking period . considering this group and the next two categories ( 130 mins and 3160 mins ) , more than half ( 56.2% ) of the whole sample travelled out of home on foot for an hour or less during the monitored week . just under one - third ( 31.3% ) travelled on foot for more than two hours over the seven days . no correlation was found between time spent walking and self - perceptions of being active ( r = 0.12 ; p = 0.401 ) and being healthy ( r = 0.12 ; p = 0.414 ) or age ( r = 0.06 ; p = 0.688 ) . in table 4 , it can also be seen that the number of nonwalkers in the lower density suburbs of brisbane was twice that of their counterparts living in the inner city suburbs of brisbane , with the numbers of nonwalkers in the ic group being similar to both the rc and rt groups . considering the actual time spent walking ( in minutes ) by each individual across each of the four groups , however , the kruskal - wallis test indicated there was no significant difference in the mean rankings by study location ( h(3 ) = 0.892 ; p = 0.827 ) . once time spent traveling by bicycle was taken into account , only three individuals in the cs group were found to have spent no time using active means of travel ( on foot or biking ) , but the remainder of the findings were similar to those observed for walking . time spent travelling by active means was found to be unrelated to both self - ratings of being active ( r = 0.16 ; p = 0.279 ) and healthy ( r = 0.18 ; p = 0.216 ) and to age ( r = 0.16 ; p = 0.284 ) . no significant difference between the four study locations was identified in the mean rankings of active travel ( based on actual minutes ) either ( h(3 ) = 0.520 , p = 0.915 ) . a visual representation of the lack of correlation between self - perceptions of being active and use of active means of travel is shown in figure 1 . note , for example , that self - ratings of being active among those who spent no time walking or biking are distributed across four categories , from very inactive to very active . a visual representation of the proportion of all time travelled over the tracking period spent using active forms of transport is shown in figure 2 . the similarity in the overall pattern that emerged for each of the four study locations is clearly evident and the kruskal - wallis test confirmed that there was no significant difference in the mean rankings of the proportion of overall time spent travelling by active means across the four study locations ( h(3 ) = 0.457 , p = 0.928 ) . overall , the findings contribute to a relatively small but growing empirical literature focused on active aging , by way of whole sample analysis and equal - sized subsample comparison . they add value to this body of evidence in five main ways . firstly , the whole sample analyses enabled determinations to be made about the distribution of self - ratings of being active among community - based adults living in australia . like bowling 's study undertaken in the united kingdom ( based on a larger sample than the current study ) , the most common self - rating for being active was the fourth level of a five - level single - item likert scale ( active in this study and fairly actively in bowling 's study ) . however , while the second most common rating in bowling 's study was the fifth - level option very actively , our research found the third - level option somewhat active to be the second most common , followed by very active . this difference may be attributable in part to the wording and response options used by each study but may reflect either true difference in activity levels between older people in the united kingdom and australia or difference in the benchmark used by older people in these two nations when making this form of self - assessment . replication studies of bowling 's [ 7 , 10 ] research are needed in australia and elsewhere in order to further understand older people 's perceptions about the key constituents of active aging from their own perspective if we are to gauge similarities and differences across different cultures and countries . secondly , the current study was able to investigate correlates of self - ratings of being active beyond those examined by previous studies . our finding that age was unrelated to self - ratings of being active is consistent with bowling 's study . our finding that self - rated health status was unrelated to self - perceptions of being active differs however . this inconsistency may rest on the nature of the sample used in the current study , whereby three - quarters were recruited through community organisations ( and thus were actively participating in community activities ) and around two - thirds reported being healthy or very healthy or differences in the measure of health status used in each study ( bowling 's study used presence of a limiting longstanding condition or disability while this study used self - rated health status ) . the negative correlation we found between age and self - rated health is consistent with previous research showing an association between increasing age and more chronic health problems and declining functional ability . thirdly , this study had the capacity to explore whether self - ratings of being active were connected with actual behavior . the fact that these self - ratings were positively correlated with the number of days when time was spent out of home but unrelated to levels of out - of - home physical activity raises the possibility that older people in this study have embraced the idea that active aging is primarily about one 's current participation within society . future research efforts need to be directed at exploring the degree that self - assessments of being active as people age correspond with objective measures of both participation and outdoor physical activity . active aging objectives are unlikely to be met if a gap exists between the meanings ascribed to active aging by older people and their actual behavior with respect to the latter . more than half of the sample ( all ambulatory and mostly healthy ) spent a daily average of between zero and 8.6 minutes walking outside of home over seven days . if this level of outdoor physical activity is representative of these participants ' normal pattern of walking outdoors , their long - term prospects of remaining ambulatory and capable of performing daily life activities could potentially be compromised , with this in turn eventually undermining their capacity for living independently , aging in place , and participating in society . our finding that less than one - third of older people in this study had walked for more than 2 hours over seven days is consistent with findings from studies undertaken in the united states [ 19 , 20 ] , where a similar car culture to australia 's prevails . given the accumulated evidence about barriers to walking ( see [ 16 , 27 ] ) and the difficulties associated with using public transport experienced by older people , as well as this group 's reliance on cars [ 12 , 24 ] , it may be that the developments of community - based programs that encourage walking behavior among older people with a specific focus on recreational walking in spaces where pedestrian infrastructure is in good condition and free of traffic ( e.g. , parks and cyclist - free pedestrian walkways ) are needed . these programs could target individuals as well as groups ( such as senior citizens organizations ) and may require the use of motorized transport ( e.g. , private motor vehicles and public or private bus ) for gaining access to well - maintained , safe walking tracks . this approach could potentially create opportunities for older people to overcome their fears about moving outdoors , increase their number of social interactions , maintain or improve their overall health and well - being through the gaining of regular exercise [ 18 , 31 ] , and also change expectations that an increasingly sedentary life is a natural part of the aging process . fourthly , this study was able to explore whether different built environments ( with varying public transport services and population densities ) were associated with higher or lower levels of active travel . while the lack of difference in time spent walking ( or walking / biking combined ) between locations observed in this study could potentially be due to the size of the groups in the subsample , there is no reason to suspect that this particular finding is simply spurious in nature . we were able to gather a range of information about each location , including older people 's perceptions of the age- and disability - friendliness of their respective communities , their safeness and closeness to amenities and family / friends , and differences in public transport services . interestingly , similar numbers of participants in each location identified being close to shops , and so forth as a reason for living in their current communities . thus this particular feature of the environment was not one that differentiated the inner city subsample from the other three lower density areas , even though higher density areas are often touted to be places that provide greater and easier access to amenities , goods , and services than lower density areas . previous research indicates that it is proximity to particular destinations ( especially shopping malls , retail outlets , and places of employment ) and not population density that is associated with the walking behavior of older people ( see ) . despite differences in public transport services in each study location and in older residents ' perceptions of the age- and disability - friendliness of their respective communities , the emergent pattern of the proportion of time traveled by active means over seven days by older people was remarkably similar this along with the finding from the whole sample analysis that only one - third engaged in active travel for more than two hours , a proportion that is consistent with findings from large population studies of older people 's walking behaviour [ 19 , 20 ] , raises the possibility that variability in the walking behavior of older people has more to do with normal variation within populations ( as a consequence of numerous factors ) than it does to features of the surrounding environment . together with the absence of any convincing evidence that there is a direct relationship between the built environment and older people 's walking behavior and the many barriers identified in the extant empirical literature which deter older people from venturing out of home on foot , our study 's findings give grounds for questioning any taken - for - granted assumption that older people will walk outdoors more frequently if the surrounding environment is made more a universal approach may need to be taken with respect to the provision of community - based walking programs for older people ( discussed earlier ) to ensure they are widely available , irrespective of the nature of the surrounding built environment of their homes , if a substantial increase in the outdoor physical activity of this segment of the population is to be achieved . fifthly , the mixed - methods approach used in this study provided the opportunity to explore connections between self - ratings of being active and objective measures of physical activity outside of the home domain via gps tracking , as well as comparison of physical activity across four different settings that differ by population density and built environment ( including public transport services ) . the capturing of time spent biking is an additional strength of this study , since research that relies on walking behavior alone ignores physical activity undertaken through alternative means . by instructing participants to document all modes of transport used for their out - of - home travel in combination with gps tracking , this research avoided the circumstance where the research process itself leads participants to modify their walking behavior . this is a limitation of studies based on accelerometer - based information , the results of which do not necessarily reflect people 's usual pattern of walking , because participation procedures ( i.e. , wearing an accelerometer ) potentially prompt individuals to walk more frequently than normal for the monitored period . finally , further research based on objective measures of walking ( as well as larger samples recruited from urban and rural settings ) is needed if evidence - based determinations are to be made about the extent to which particular built environments foster greater levels of walking among older people ( as proposed by giles - corti et al . ) . the constraints imposed on the size of this study 's sample by the nature of its methodological approach may be averted by future studies , given that technological advancements in recent years are making this approach increasingly feasible for larger samples . the developments of digital diaries , which are able to be used by participants at the same time and on the same device as gps tracking takes place , hold great promise in their capacity to streamline data collection of movement and activity information , as well as data management and analysis , and may also reduce participant burden ( see draijer et al . ) . whether or not these new devices are useful for older samples ( who may resist making diary entries in a technological device in place of paper and pencil diaries ) remains to be seen however . the high quality of data produced by gps / travel diary methods suggests significant benefits could accrue from the trialing of these new technologies with older samples . if successful , substantial headway could be made in expanding the body of evidence on the relationship between the built environment and older people 's walking behavior gathered from high quality measures of walking . active aging is being promoted as a key policy agenda for dealing with the growing aging populations in industrialized nations , this particular subject area warrants much greater attention than it has attracted to date . it is imperative that older people understand the nature of all the constituents of the active aging concept as outlined by the who if the objectives of its policy framework are to be met . currently , evidence related to the meanings older people ascribe to this concept as well as the particular constituents of active aging that inform this group 's self - ratings of being active is limited . more research is needed to determine if older populations are prone to connect active aging with getting out and about and doing things in the present more than they do behaviors such as walking outdoors as a means to maintain their health into the future . active aging is extremely broad in scope compared to its conceptual predecessors ( successful , it may therefore be necessary for messages communicated to the public under the banner of active aging to clearly articulate and emphasize the need for physical activity as a means to maintain health and prolong older people 's participation within society . furthermore , the flurry of research interest in the relationship between the built environment and older people 's walking behavior that appears to have been sparked by the who 's global age - friendly cities publication has not been matched by similar interest in other areas pertaining to active aging . researchers and policy makers alike need to remain mindful that the built environment is just one of many factors affecting older people 's opportunities to age actively and that modifications to the built environment may be insufficient to the task of overturning older people 's unwillingness to move outdoors on foot through fear of falls and injuries from traffic and poorly maintained pedestrian infrastructure , their expectations about aging , or the perceived comfort and convenience of the motor vehicle .

we examined whether self - ratings of being active among older people living in four different settings ( major city high and lower density suburbs , a regional city , and a rural area ) were associated with out - of - home participation and outdoor physical activity . a mixed - methods approach ( survey , travel diary , and gps tracking over a one - week period ) was used to gather data from 48 individuals aged over 55 years . self - ratings of being active were found to be positively correlated with the number of days older people spent time away from home but unrelated to time traveled by active means ( walking and biking ) . no significant differences in active travel were found between the four study locations , despite differences in their respective built environments . the findings suggest that additional strategies to the creation of age - friendly environments are needed if older people are to increase their levels of outdoor physical activity . active aging promotion campaigns may need to explicitly identify the benefits of walking outdoors to ambulatory older people as a means of maintaining their overall health , functional ability , and participation within society in the long - term and also encourage the development of community - based programs in order to facilitate regular walking for this group .

pulmonary arterial hypertension ( pah ) is a common complication of connective tissue diseases ( ctd ) such as systemic sclerosis ( ssc ) , systemic lupus erythematosus ( sle ) , mixed connective tissue disease ( mctd ) , rheumatoid arthritis ( ra ) , and primary sjogren 's syndrome ( pss ) . in updated clinical classification of pulmonary hypertension , this type of pulmonary hypertension ( ph ) was defined as follows : pah associated with connective tissue diseases belongs to group one . most of ctd - associated pah ( ctd - apah ) presented itself as the type of ssc - associated pah ( ssc - apah ) . the prevalence of pah in patients with ssc , on the condition that the diagnosis is based on right heart catheterization for assessment of filling pressures , is about 814% [ 2 , 3 ] . not only the prevalence of ctd - apah is higher than idiopathic pah ( ipah ) , but also its prognosis is worse than ipah [ 4 , 5 ] . th17 cells expressing retinoic acid - related orphan receptor t ( ror t ) are a recently - identified subset of cd4 t cells . meanwhile , this cell subset , having the capacity to produce potent proinflammatory cytokines including il-17a ( il-17 ) , il-17f , il-21 , and il-22 , is likely to play an important role in autoimmunity [ 6 , 7 ] , while cd4cd25foxp3regulatory t cells ( treg ) cells expressing forkhead / winged helix transcription factor ( foxp3 ) are capable of modulating the function of effector t cells , maintaining immunological homeostasis , and preventing autoimmunity [ 8 , 9 ] . however , the detection of tregs by specific surface marker staining ( cd4cd25 cd127 ) is also widely - used in recent researches [ 10 , 11 ] . the dynamic balance between th17 and treg cells involved in the pathogenesis of ctd is damaged [ 1215 ] . it is acknowledged that a possible role of the th17/treg axis in ctd - apah has never been elucidated . besides , the relevant researches about the prognosis influence of th17/treg axis disorder as pathogenic factor on patients with ctd - apah remain to be discovered . we hypothesized that imbalance of circulating th17/treg cells may also present themselves in patients with ctd - apah , which is related with pathogenesis of ctd - apah and important to the prognosis . thus , the purpose of this prospective cohort study is to verify the hypothesis that the balance of th17 and treg cells frequencies and absolute counts in the peripheral circulation is disturbed in patients with varying degrees of ctd - apah and to prove the influence of th17/treg imbalance on prognosis . the plasma levels of th17- and treg - related cytokines and mrna expression of relevant transcription factors ( ror t and foxp3 ) in peripheral blood mononuclear cells ( pbmcs ) were evaluated and the potential correlation of th17/treg cells imbalance with the severity and prognosis of ctd - apah was also explored . n - terminal probrain natriuretic peptide ( nt - probnp ) , as a sensitive marker of cardiovascular diseases , is independently associated with mortality in ipah and ssc - apah [ 16 , 17 ] . therefore , the potential correlation of th17/treg imbalance with serum nt - probnp level of ctd - apah was explored . ctd was classified into subtypes such as ssc , sle , mctd , ra , and pss according to the american college of rheumatology criteria . all patients were evaluated in the aspects of renal , cardiac , hepatic , and pulmonary functions . chest high - resolution computed tomography ( hrct ) was used to detect the existence of pulmonary embolisms or pulmonary interstitial disease . being younger than 18 years or older than 75 years ; with ctd being at active stage ; with a history of chronic infection or recent ( < 1 month ) infection with clinically significant or inflammatory conditions including trauma , vaccination , any invasive medical tests or surgery ( <3 months ) ; with heart function belonging to world health organization functional class iv ; with other comorbidities , such as severe hepatic or kidney failure , pulmonary fibrosis , obstructive sleep apnoea syndrome , cancer , uncontrolled hypertension , diabetes , obesity ( body mass index ( bmi ) > 30 kg / m ) , asthma , chronic obstructive pulmonary disease , inflammatory bowel disease , and pregnancy ; being treated with corticosteroid or other immunosuppressant in last three months . the rest of the potential subjects who haven't met the exclusion criteria were checked with hemodynamics . systolic pulmonary artery pressure ( spap ) > 40 mmhg , estimated by echocardiograph , was used as a screening threshold for catheterization . patients identified by noninvasive tests were operated with catheterization of right side of the heart . confirmed pah was defined as mean pulmonary artery pressure ( mpap ) 25 mmhg , with a pulmonary capillary wedge pressure of 15 mmhg at a resting state by using a catheter method . fifty - three patients with pah , whose leading causes were excluded such as inborn diseases , medicines , and infection , examined by catheter check , were admitted into ctd - apah group . subjects with ctd - apah were classified into two subgroups according to their systolic pulmonary artery pressure as mild to moderate ctd - apah ( 40 < spap 70 mmhg , n = 22 ) and severe ctd - apah ( spap > 70 mmhg , n = 31 ) . the remaining 117 patients with ctd , whose pulmonary artery pressures were at normal state , were admitted into ctd group . 6-minute walk test and world health organization functional class statistics from two groups were recorded . forty - eight healthy volunteers with the feature of matched sex and age ( 39 females , 9 males , 61.9 10.6 years old ) were recruited into control group . peripheral blood mononuclear cells ( pbmcs ) were isolated from heparinized peripheral venous blood by ficoll - hypaque lymphoprep ( nyegaard , norway ) density centrifugation for analysis of flow cytometry and real - time quantitative polymerase chain reaction ( rt - qpcr ) . plasma was obtained after centrifugation and stored at 80c for assay of the cytokines and nt - probnp . for analysis of treg cells , pbmcs were surface - labeled with cd4-pe / cy5 , cd25-pe followed by fixation and permeabilization and intracellularly stained with foxp3-alexa fluro488 or were surface - labeled with cd4-pe / cy5 , cd25-pe , and cd127-fitc ( ebioscience , usa ) . for analysis of th17 cells , pbmcs was suspended in complete culture medium ( rpmi1640 was supplemented with 10% heat - inactivated fetal calf serum , ( gibco brl , usa ) ) . cultures were stimulated for 1 hour using 50 ng / ml phorbol myristate acetate ( pma ) and 1 g / ml ionomycin ( sigma - aldrich , usa ) and then stimulated using 500 ng / ml monensin ( alexis biochemicals , usa ) for 3 hours at environment of 37c and 5% co2 . cells were then washed in pbs and surface - labeled with cd4-pe / cy5 ( ebioscience , usa ) . following surface staining , cells were fixed and permeabilized using fixation / permeabilization buffer ( ebioscience , usa ) and then stained with il-17a - pe ( ebioscience , usa ) . labeled cells were washed and analyzed with facs calibur flow cytometer ( becton - dickinson , usa ) using flowjo ( tree star , usa ) v7.6.1 software . in each case , staining was compared with that of the appropriately labeled isotype control antibody . total rna was extracted from individual pbmcs preparation by using trizol reagent ( invitrogen , usa ) . the following primer pairs were used : foxp3 : f : 5-cacgcatgtttgccttcttcaga-3 ; r : 5-gtagggttggaacacct gctggg-3 ; and ror t : f : 5-gcaatggaagtggtgctggtt-3 ; r : 5-aggatgctttggcgatgagtc-3. real - time quantitative pcr for foxp3 , ror t were performed in abi prism 7900 sequence detection system ( applied biosystems , usa ) with the sybr green master mix kit ( takara , china ) . both foxp3 and ror t expression data were , then , normalized relative to glyceraldehyde-3-phosphate dehydrogenase ( gapdh ) . the plasma levels of il-17a , il-6 , ifn- , il-2 , tgf- , and tnf- were measured by radioimmunoassay method utilizing radioimmunoassay kit ( nibt , china ) , abiding by the protocol of department of nuclear medicine of huashan hospital , fudan university . all samples were measured in duplicates . as previously described , the nt - probnp concentration was determined with the method of an elecsys nt - probnp sandwich immunoassay by an elecsys 2010 instrument ( roche diagnostics , switzerland ) . the analytical range was extended from 20 pg / ml to 35000 pg / ml . comparisons among groups were made using students ' t - test or pearson chi - square test . nonparameter analysis was performed by using kolmogorov - smirnov ( k - s ) test to analyze the distribution . we compared the discrimination of tregs by detection of cd4cd25foxp3 t cells and cd4cd25cd127 t cells but could not detect significant differences between both methods which were utilized in 18 patients with ctd , 23 patients with ctd - apah , and 20 healthy controls ( p > 0.05 ) ( as shown in figures 1(a ) and 1(b ) ) . we utilized cd4 , cd25 and foxp3 as the markers to detect treg cells in this study . the prevalence of treg cells was expressed as a ratio of cd4cd25foxp3/cd4 t cells and absolute counts . as shown in figure 1(c ) , the frequencies and absolute counts of treg cells were significantly decreased in the peripheral blood of patients with ctd ( 2.12 0.20% ; 39.97 22.98 cells/l ) and ctd - apah ( 1.55 0.38% ; 28.30 21.01 cells/l ) compared with those of healthy controls ( 2.96 0.29%,p < 0.01 , p < 0.01 ; 53.40 25.35 cells/l , p < 0.01 , p < 0.01 ) . circulating treg cells percentages and absolute counts were markedly higher in patients with ctd than those patients with ctd - apah ( 2.12 0.20% versus 1.55 0.38% , p < 0.01 ; 39.97 22.98 cells/l versus 28.30 21.01 cells/l , p = 0.011 ) . moreover , significant differences of percentages were found between patients with severe ctd - apah and patients with mild to moderate ctd - apah ( 1.30 0.24% versus 1.90 0.25% , p < 0.01 ) ( as shown in figure 1(f ) ) . as shown in figures 1(a ) and 1(d ) , the prevalence of th17 cells was expressed as a ratio of cd4 il-17 t cells / cd4 t cells and absolute counts . the frequencies and absolute counts of th17 cells were evidently increased in the peripheral blood of patients with ctd ( 1.65 0.28% ; 10.29 5.52 cells/l ) and ctd - apah ( 2.19 0.40% ; 13.06 7.19 cells/l ) than those in normal control group ( 0.87 0.14% , p < 0.01 , p < 0.01 ; 7.40 3.60 cells/l , p = 0.012 , p < 0.01 ) . significant differences of percentages and absolute counts were also found between ctd - apah and ctd group ( 2.19 0.40% versus 1.65 0.28% , p < 0.01 ; 13.06 7.19 cells/l versus 10.29 5.52 cells/l , p = 0.013 ) . furthermore , the percentages of th17 cells were markedly higher in patients with severe ctd - apah than those in subgroup with mild to moderate ctd - apah ( 2.42 0.36% versus 1.87 0.18% , p < 0.01 ) ( as shown in figure 1(f ) ) . the significance of increased th17 cells and decreased treg cells was further explored by calculation of th17/treg cells percentage ratio of patients in ctd - apah , ctd , and healthy controls groups ( as shown in figure 1(e ) ) . we demonstrated that the ratio of th17 to treg cells was highest in patients with ctd - apah ( 1.53 0.54 ) , lower in those patients with ctd ( 0.77 0.06 ) and lowest in subjects of control group ( 0.29 0.02 ) . as a result , the th17/treg cells ratio was significantly increased in patients with ctd - apah compared with patients in ctd and control group ( 1.53 0.54 versus 0.77 0.06 , p < 0.01 ; 1.53 0.54 versus 0.29 0.02 , p < 0.01 ) . in ctd - apah group , the similar significant difference also presented in two subgroups between severe ctd - apah and mild to moderate ctd - apah ( 1.91 0.38 versus 1.00 0.12 , p < 0.01 ) ( as shown in figure 1(f ) ) . the specific transcription factors of both t subsets in ctd - apah , ctd , and control group were evaluated to confirm cytology observations . ror t is an important transcription factor for the differentiation of th17 cells , while foxp3 is the master transcription factor in treg cells . the levels of these transcription factors for th17 and treg cells were analyzed in pbmcs from included subjects by rt - qpcr . as shown in figure 2(a ) , the levels of ror t expression were upregulated in ctd group ( 3.79 0.52 ) and ctd - apah ( 4.91 0.93 ) than those in the healthy control group ( 3.45 0.45 ; p < 0.01 , p < 0.01 ) , while there was also significant difference between previous two groups ( p < 0.01 ) . in contrast , the expression of foxp3 was markedly lower in patients with ctd ( 4.63 0.61 ) and ctd - apah ( 3.74 0.67 ) as compared with patients in the control group ( 5.13 0.69 ; p < 0.01 , p < 0.01 ) . furthermore , ctd - apah patients had a significantly lower expression than patients with ctd ( p < 0.01 ) ( as shown in figure 2(b ) ) . moreover , ror t and foxp3 levels in mild to moderate ctd - apah subgroup ( 4.14 0.55 ; 4.29 0.58 ) were significantly different from those of the subgroup with severe ctd - apah ( 5.45 0.74 , p < 0.01 ; 3.34 0.40 , p < 0.01 ) ( as shown in figure 2(c ) ) . t - helper and regulatory t cells - related cytokines and nt - probnp in serum of patients in ctd and ctd - apah and healthy controls were measured by radioimmunoassay or elecsys nt - probnp sandwich immunoassay . as shown in table 2 , patients with ctd exhibited higher levels of serum il-17a and nt - probnp than those patients in healthy controls ( p < 0.01 ) but lower than those patients with ctd - apah ( p < 0.01 ) . meanwhile , the expression of tgf- was highest in subjects of control group , lower in patients with ctd , and lowest in patients with ctd - apah . patients in both ctd and ctd - apah group had higher levels of serum il-6 , ifn- , il-2 , and tnf- when compared with patients in healthy controls ( p < 0.01 ) , while no significant difference was observed between ctd and ctd - apah group ( p > 0.05 ) . in subgroups , there were obvious differences between mild to moderate ctd - apah group and severe ctd - apah group on serum il-17a , tgf- , and nt - probnp ( p < 0.01 ) . there were significant correlations between th17 and treg cells in the peripheral circulation of ctd - apah group ( r = 0.353 , p = 0.01 ) ( as shown in figure 3(a ) ) . th17/treg ratios were positively correlated with serum concentrations of il-17a ( r = 0.387 , p < 0.01 ) and expression of ror t mrna ( r = 0.602 , p < 0.01 ) ( as shown in figures 3(b ) and 3(c ) ) in pbmcs and negatively related with serum concentrations of tgf- ( r = 0.338 , p = 0.013 ) and expression of foxp3 mrna ( r = 0.521 , p < 0.01 ) ( as shown in figures 3(d ) and 3(e ) ) in pbmcs . the relationship between th17/treg ratios and serum levels of il-6 , ifn- , il-2 , and tnf- in patients with ctd - apah was also studied , but no correlations were identified ( p > 0.05 ) . spap and serum levels of nt - probnp were used as markers of disease severity of patients with ctd - apah . in ctd - apah group , spap and serum levels of nt - probnp were both positively correlated with ratio of th17/treg ( r = 0.342 , p = 0.012 ; r = 0.344 , p = 0.012 ) ( as shown in figures 3(f ) and 3(g ) ) . to evaluate th17 and treg axis association with clinical outcomes , k - s test showed that th17/treg ratio in patients with ctd - apah exhibited skewed distribution ( p = 0.032 ) . therefore , the median value ( 1.474 ) , which was calculated as ratio of th17/treg of 53 patients in this research , was chosen as division standard for subgroup . twenty - six patients with ctd - apah whose th17/treg ratios were less than 1.474 were admitted into low th17/treg ratio group . in contrast , 27 patients with ctd - apah whose th17/treg ratios were equal to or more than 1.474 were admitted into high th17/treg ratio group . the evaluation period was defined from the date that patients with ctd - apah were admitted into abovementioned two subgroups till october 2012 . the overall survival time for all 53 patients with ctd - apah was 22.63 1.64 months . for patients in low th17/treg ratios group , the overall survival time was 25.79 2.46 months . for patients in high th17/treg ratios group , the overall survival time was 19.52 2.00 months . for all patients , the overall survival rates were 71.70% , 41.51% , and 16.98% at 1 , 2 , and 3 years , respectively . for patients in low th17/treg ratios group , the overall survival rates were 76.92% , 53.85% , and 23.08% at 1 , 2 , and 3 years , respectively . for patients in high th17/treg ratios group , the overall survival rates were 66.67% , 29.63% , and 11.11% at 1 , 2 , and 3 years , respectively ( as shown in figure 4 ) . there was a significant difference between the two subgroups ( x-2=4.10 , p = 0.043 ) . in the steady state , th17 and treg cells may remain in a dynamic balance . this balance is destroyed in autoimmune diseases such as ctd . despite the similarities in hemodynamic perturbations , outcomes in patients with ctd - associated pah previous studies have confirmed that il-17 concentrations and th17 cells were elevated in patients with ctd [ 15 , 18 , 19 ] . most reports have shown that the prevalence of treg weakened in pbmcs compartment in ctd , while some studies have reported normal or elevated treg levels [ 2023 ] . nevertheless , it is generally thought that immune suppression capacity of treg diminished in patients with ctd [ 2429 ] . these discrepancies are likely to be related to the variability of the series of patients included in the different studies in relation to type and stage of disease as well as administered treatment . although the frequency of pah in the patients with ctd was high , the researches about th17/treg axis in ctd - apah were very rare . tamosiuniene et al . demonstrated that vascular endothelial growth factor receptor-2 ( vegfr-2 ) blockade induced significant pulmonary endothelial apoptosis in t cell - deficient rats but not in immune reconstituted ( ir ) rats . ir with cd4 cd25 t cell subset prior to vascular injury attenuated the development of pah . in addition , after exposing to endothelial cells , treg cells upregulate the surface expression of programmed death-1 ( pd-1 ) receptor as well as secretion of il-10 and tgf- , whose reactions promote vasodilatation , whereas more direct evidences showed that ph conditions are also associated with aberrant treg cells numbers , activity or simply reduced cd4 t cell numbers [ 30 , 32 ] . in summary , these studies suggest that treg cells function is normally to limit vascular injury and may play a protective role against the development of pah . there is a lack of related researches in this field as for th17 cells , which are closely related with inflammation or autoimmune disorder , as well as counter - interacting to treg cells subset . in our research , th17/treg axis disorder in different stages of ctd - apah and efficiency of th17/treg imbalance as biomarker for predicting prognosis of patients with ctd - apah the present statistics provide direct evidence that the frequencies and absolute counts of treg cells and the expression of foxp3 mrna were significantly lower in patients with ctd - apah when compared to patients with ctd or healthy controls . meanwhile , we found out that patients with ctd - apah exhibited a significant increase in peripheral th17 numbers , th17-related cytokines , and ror t mrna levels when compared to patients with ctd or healthy controls , even though in patients with ctd - apah , the frequencies of th17/treg cells , th17-related cytokines , and ror t / foxp3 mrna levels showed significant difference between mild to moderate ctd - apah subgroup and severe ctd - apah subgroup . in addition , the ratio of th17/treg cells had a linear positive correlation with ctd - apah severity indicated by spap and nt - probnp . therefore , the th17/treg ratio could be an important tool in detecting and describing various immunological conditions in patients with ctd - apah . the cytokine milieu plays a pivotal role in the differentiation from naive cd4 t cells to treg or th17 cells [ 3335 ] . in the present study , results showed that the concentrations of il-6 and il-17 , both of which elevated inflammation , were all significantly higher in patients with ctd and ctd - apah , and il-17 concentrations were positively correlated with th17/treg ratios . several large - scaled prospective studies have shown that spap and nt - probnp levels are independent predictors of severity and prognosis of ipah and ctd - apah . a highly - connected link between the ratios of th17/treg and spap or serum nt - pro bnp levels had been proved in this study . these evidences indirectly suggested that the ratio of th17/treg has been effective in evaluating the severity of ctd - apah and in predicting the prognosis of patients with ctd - apah . the distribution frequency of th17/treg ratio through k - s test in patients with ctd - apah showed skewed distribution . we choose median values according to levels of th17/treg ratios in patients with ctd - apah as basis of group division in the survival analysis . survival analysis showed that patients with ctd - apah , who have lower th17/treg ratios , have better survival tendency than the patients with higher th17/treg ratios . these findings directly suggested that the dynamic balance between th17 and treg cells was likely to influence prognosis of patients with ctd - apah and to evaluate prognosis of patients with ctd - apah as the predicting factor . our study demonstrated that the imbalance of th17/treg existed in the peripheral blood of patients with ctd - apah . we also confirmed that the ratio of th17/treg not only correlated with inflammatory markers but also associated with severity and prognosis of ctd - apah . all these suggested a potential role for th17/treg imbalance in the pathogenesis and progression of ctd - apah . the abovementioned results needed proving in larger cohorts . in addition , the further studies could be designed to observe the change of th17/treg axis in patients with ctd - apah at an active stage and to investigate the impact of corticosteroid or immune suppressant treatment on the th17/treg balance in patients with ctd - apah .

this prospective cohort study is to verify the hypothesis that the balance of th17 and treg cells frequencies in the peripheral circulation is disturbed in patients with varying degrees of connective tissue diseases - associated pulmonary arterial hypertension ( ctd - apah ) and to prove the influence of th17/treg imbalance on prognosis . we detected the frequencies and absolute counts of th17 and treg cells and related serum cytokines secretion and expressions of key transcription factors in 117 patients with connective tissue diseases ( ctd ) , 53 patients with ctd - apah , and 48 healthy volunteers . moreover , the median value according to levels of th17/treg ratios in patients with ctd - apah was chosen as basis of group division for survival analysis . ctd - apah patients revealed significant increase in peripheral th17 cells , th17-related cytokines , and ror t mrna levels . they also presented a significant decrease in treg cells , treg - related cytokines , and foxp3 mrna levels as compared with ctd patients and healthy controls . more importantly , the th17/treg ratio was significantly related to the severity and prognosis of ctd - apah . this study indicated that the th17/treg axis disorder plays a critical role in ctd - apah . furthermore , the dynamic balance between th17 and treg cells was likely to influence prognosis of patients with ctd - apah .

two drastically different approaches to understanding the driving forces behind cancer onset and proliferation have crystallized through years of research . these are the somatic mutation theory ( smt ) and the tissue organization field theory ( toft ) . the essence of smt is that cancer is derived from a single somatic cell which has successively accumulated multiple dna mutations , and that those mutations occur on genes which control cell proliferation and cell cycle . thus , according to smt the neoplastic lesions that destroy normal tissue architecture are the results of dna - level events . conversely , according to toft , carcinogenesis is primarily a problem of tissue organization : carcinogenic agents ( eg , environmental chemicals , inflammation , viruses ) destroy the normal tissue architecture thus disrupting cell - to - cell signaling and compromising genomic integrity . hence , in toft the dna mutations are the effect , and not the cause , of the tissue - level events.1 the smt and toft have a long history of development , perhaps under the disguise of constantly evolving terminologies . the best source of relevant information is the book by sonneschein and soto.2 a concise , yet comprehensive , overview of this history can also be found in the review by baker.3 arguments in favor of both toft and smt are numerous and strong , as seen , for example , from two papers4,5 recently published next to each other . at the same time , a large grey zone of biological facts and clinical cases exists which poses the questions that are difficult to resolve from either of these viewpoints.6 essentially , for a long time the theories conceptually close to smt and toft were considered as two different facets ( along with many others ) of the complex phenomenon of carcinogenesis . it is a comparatively new development that smt and toft began to be regarded as irreconcilable alternatives . thus , according to sonnenschein and soto,7 toft and smt belong to distinct levels of biological complexity and therefore , are incompatible , as are their philosophical stances ( reductionism versus organicism ) . indeed , the cornerstone of smt is the notion that carcinogenesis is triggered by a single aberrant cell which happened to acquire multiple dna mutations , and that these mutations predominantly damage the genes responsible for the cell cycle and apoptosis.8 frequently observed monoclonality of the initial cancer cell population is an important evidence supporting smt . in particular , as observed by nowell , cytogenetic studies have demonstrated that in many primary tumors all cells show the same abnormal karyotype ; the immunoglobulin produced by plasma cell tumors has in almost every case the homogeneity characteristic of a single clone.9 as an ultimate manifestation of this paradigm , direct evidence of a single catastrophic event triggering carcinogenesis has been presented by stephens et al.10 the authors report : the model to explain the distinctive genomic structures described here is that the overwhelming majority of rearrangements occur in a single catastrophic event . in this scenario , the chromosome or chromosomal region shatters into tens to hundreds of pieces , some ( but not all ) of which are then stitched together by the dna repair machinery in a mosaic patchwork of genomic fragments ... a cell suffering tens to hundreds of dna breaks in a single cataclysmic event would be expected to undergo apoptosis . that a cell can survive such an insult and progress to become cancerous suggests that the extensive remodeling of the genome may confer significant selective advantage to that clone . in direct opposition to these views , toft depicts carcinogenesis as general deterioration of the tissue microenvironment due to extracellular causes . this deterioration hinders normal cell - to - cell signaling thus making normal functioning of the intracellular machinery impossible and eventually leading it to the breaking point . in such a scenario , the deleterious mutations should be scattered all over the genome randomly and incoherently with little chance for clonal homogeneity . according to smt , cancer progression is a unidirectional and mostly irreversible process ; the disease can not be cured unless the entire tumor mass is surgically removed , radiationally killed , or deprived of its aggressive nature by carefully targeted chemotherapeutic agent . paramount importance of toft for cancer biology , for the practice of clinical oncology and for cancer prevention , dwells in the fact that according to this view carcinogenesis is not a unidirectional process ; rather , it is curable and reversible.11 in this paper , we do not take sides in this clash of seemingly irreconcilable views . our goal here is to outline a plausible scenario in which a single event , insignificant on its own , may trigger a system - wide catastrophic restructuring . such a scenario may be envisioned and conceptualized within the framework of the so called self - organized criticality ( soc ) . self - organized critical phenomena are wide - spread in nature and society . for example , a large - scale devastating forest fire may be sparked by a single cigarette butt , but abundant availability of flammable dry wood is the prerequisite . a minor shock to overstressed tectonic plates may destroy their precarious equilibrium and cause a large - scale earthquake followed by a tsunami . a single micron - size dust particle dropped into a vessel with overcooled liquid may cause rapid crystallization of the entire mass of liquid . in a society overburdened by internal strife and misery , a charismatic self - motivated leader may grab attention of a disoriented crowd and become a seed for rapid transition to new modalities of existence.12,13 all these dissimilar phenomena have one fundamental feature in common : the system resides in a metastable state and its collapse may be triggered by a small - scale event insignificant on its own . in what follows , we attempt to amass systemic arguments and known biological facts in favor of the proposed scenario . soc is an avalanche - like system - wide transformation which rapidly moves a system into a new state.1416 a popular metaphor for soc is the sandpile paradigm . if additional sand grains are randomly added to a sand pile then inevitably an instance will occur when local steepness of the slope surpasses a certain critical threshold thus causing local failure of structural stability . the excess of material will cascade into adjacent areas of the pile causing their failure as well . thus an avalanche will occur , shifting the entire sandpile into a new stable state . what is fundamentally important in this process is that a random local event quickly propagates through the entire system thus establishing long - range correlations within the system . suppose that there exists a two - dimensional lattice of cells in which the state of each cell is characterized by a time - dependent load , ij ( t ) . suppose also that there exists a limiting capacity of each cell , ij , and every time when ij ( t ) > ij , the excess of load , that is , ij ( t) ij , is randomly redistributed among the neighboring sites . after one step of random redistribution , one or more neighboring sites may become overloaded , thus causing the domino effect of subsequent instabilities resulting in an avalanche . the actual meaning of the quantity symbolized by the word load may vary . in particular , it may represent a certain amount of information ; in this case , occurrence of the system - wide coherence may be interpreted as rapid information transfer ( recall rapid rumor propagation in a community anticipating some breaking news ) . obviously , the rules of the game would remain essentially the same if , instead of a lattice of the cells , one considers a network of interacting units with an arbitrary topology . it is of crucial importance to realize that the network - wide information transfer and coherent restructuring is not a result of long - range exchange of signals , neither is it a result of collective thinking or following the orders of some sort of command center . the role of soc in rapid system - wide restructuring has been studied in many works ( see 1719 and references therein ) . slight variation in the rules of updating may drastically change the overall dynamics of the system . for example , in the model15 proposed by this author , the loads , per reaching critical capacity , are randomly redistributed not only among the neighbors ( as in the sandpile paradigm ) but also among all members of the community . this simple modification of rules leads to a fundamentally different behavior of the system ; that is , to the excitation of self - organized self - sustained oscillations . in physics , avalanches are known under the name phase transition , with the subcritical states preceding the transition being called metastable . the transition itself may be triggered by a minor event with little significance of its own ; due to this insignificance the phase transition may appear to be spontaneous . however , spontaneity does not mean that there is no reason for phase transition ; the fundamental reason is that the system resides in a subcritical state and is ripe for collapse . a brief list of examples of soc from other disciplines include earthquakes , wild fires , landslides , revolutions , epidemics , crowd stampedes , stock market crashes , rumor propagation , bird flock self - organization , snow avalanches , and , of course , sandpile avalanches and dune formation . a common feature among all these diverse examples is that prior to a catastrophic event , the system resides in subcritical state and is ripe for restructuring . after the catastrophe , the system moves itself into a new stable state , but this state is again subcritical . the long - term dynamics of such systems represents various patterns of intermittency . as to the period of transition between the sequential critical states , an avalanche itself is an essentially random process ; in this sense the temporary stability is often said to exist on the edge of chaos . a fundamental property of soc is that a minor event may trigger a large - scale system - wide response , thus serving as a natural amplifier of weak and insignificant signals . genetic regulatory network ( grn ) in an individual cell is an excellent example of a system permanently existing on the edge of chaos . this aspect of intracellular regulatory dynamics has been extensively covered in the literature,2022 including publications by this author.15,2326 a brief synopsis of relevant ideas is as follows . each transcription event , ie , synthesis of a single mrna molecule , requires participation of a large number of transcription factors ( tfs ) which essentially are the proteins expressed by other genes . in turn , these supporting proteins can not come into existence unless their parent genes have complete teams of their own tfs coming from yet another set of genes . this tight interdependence of genes ( often metaphorically referred to as gene - to - gene interactions ) creates the situation when each gene may be expressed only with the support of many other genes , essentially of the entire grn . the situation may be likened to a large number of assembly lines working in parallel and each requiring a large number of parts produced by other assembly lines . ideally , the system can only work in a perfectly synchronized manner , with each of thousands of parts being produced and delivered where needed in a timely manner . if , however , at least one part fails to arrive in time to its destination , the corresponding assembly line then comes to a stop , thus triggering the domino effect of secondary failures and ultimately driving the entire system to a complete halt . this domino effect is similar to what earlier in this paper has been called an avalanche . the work may only be resumed by starting everything anew , again in a perfectly synchronized manner . in mathematical terms , the question which is discussed here is the question of dynamic stability . if a multidimensional dynamical system consists of many tightly interacting units then the probability that such a system may reside in a steady state equilibrium is negligibly small.27 the only mode of motion conceivable in such a system is sporadic jumping from one instance of perfect synchronization to another . a well - documented and frequently occurring phenomenon of burstiness is a vivid manifestation of this sporadicity.2831 the concept of stochastic cooperativity introduced by this author32 helps to conceptualize this important phenomenon . a deep insight into critical dynamics of grn has been given by balleza , et al.22 using a reasonably well - established model of boolean network for representation of the grn , the authors demonstrate existence of a dynamical phase transition from ordered to chaotic dynamics . furthermore , using microarray data gleaned from the organisms belonging to four distinct kingdoms ( saccharomyces cerevisiae , escherichia coli , bacillus subtilis , drosophila melanogaster , arabidopsis thaliana ) , they also demonstrate that critical dynamics of their grns is largely similar to that envisioned by the boolean model . based on empirical data and simulation experiments , balleza et al.22 also conclude that critical behavior observed in the dynamics of the genetic networks of the organisms under study is mainly produced by the network architecture rather than by the specific nature of the regulatory functions . it essentially states that studying the network topology , that is , the arrangements of the links between its nodes , is of superior importance as compared to studying the specific biochemical details of the interactions symbolized by these links . grn is just one example of a biological system which permanently resides in subcritical regime on the edge of chaos . there is a well - rooted paradigm in theoretical biology that the phenomenon of life as a whole , from the level of cells through the level of populations and up to the level of society , is a perpetual existence on the verge of collapse.12,3336 comparatively simple and universal forces driving the living systems towards critical conditions are always present behind the scenes in all soc phenomena . in very general terms , a hallmark of living entities is their ability to replicate and proliferate themselves . in a community of such entities , unstoppable proliferation will inevitably drive the community towards exhaustion of common resources , whatever these resources are , thus bringing the populations to the verge of extinction . nice illustrations of the basic mechanisms leading to soc in living systems has been given by adami.37 soc plays an important role in dna damage and transition of cellular machinery into chaotic state . a meticulously well - studied example of this kind has been given in the work by yarosh38 which focused on the dna damage induced by the uv radiation . according to this work , the sequence of molecular events in such a process unfolds as follows : the first cellular factor to sustain the damage is the rna polymerase ii during the transcription of an active gene ; this damage leads to a stalled transcription fork . the stalled fork triggers dna repair mechanisms by attracting a large number of proteins which , in turn , allosterically modify binding affinities of many other proteins . it may happen that the damage occurs in the so called hub proteins ( such as p53 protein)38 which are capable of modifying a large number of vital cellular functions simultaneously . up to a certain level of mutagenic load , the repair mechanisms are capable of containing damage , thus maintaining a generally healthy cell population . however , the last straw effect may also occur when the cell , after an insult , remains unrepaired yet undestroyed , thus giving rise to a genetically aberrant sub - population . this last straw event is analogous to the last grain of sand in the sandpile avalanche because it fires up multiple , very complex , and mostly irreversible pathways . such a massive complex response to a seemingly minor event is a hallmark of soc . it would be an obvious misjudgment to regard any particular minor event as a cause of the system s collapse . rather , one may expect that a mutationally overloaded system would collapse anyway , whatever a minor event actually happens to be the trigger . another example in which soc plays an important role is the autoimmune disease known as systemic lupus erythematosus ( sle)39 ; much of what is said with respect to sle is also applicable to autoimmune diseases in general.40 the authors write : we therefore conclude that systemic autoimmunity necessarily takes place when host s immune system is overstimulated by external disturbance , ie , repeated exposure to antigen , to the levels that surpass system s self - organized criticality , and propose here self - organized criticality theory explaining the cause of autoimmunity . recent developments in cancer research reveal deep connections between autoimmunity and carcinogenesis both disturbing and promising . thus , the authors of a detailed review41 indicate : complex relationship between autoimmunity and cancer has been reported in numerous studies over the past years , based on the assumption that autoimmune disease and malignancies share several common features . clinical observations suggest that autoimmunity and malignancy are linked in a bidirectional way as clinical features resembling autoimmune disease are frequently encountered in paraneoplastic syndromes . in the context of soc , these conclusions may have quite an ominous connotation : overstressing the immune system beyond a certain breaking point may cause a disproportionally massive response in the form of a cluster of autoimmune diseases , including sle and cancer . soc is a fundamental , all - pervading principle manifesting itself in a large variety of forms throughout all the levels and types of biological organization . soc is a core mechanism governing spontaneous transition of an organism or biomolecular system towards higher levels of complexity . an excellent discussion of this aspect of soc may be found in the paper by suki,42 in which the author argues that sudden and unexpected improvement in the functionality of an organism is enabled by a phase transition in the network structure associated with that function . major transitions of life include , but are not limited to , emergence of living matter , eukaryotic cells , photosynthesis , sex , multicellularity , vision , consciousness , language , culture , and society . this view of avalanche - like qualitative leaps is somewhat alternative to the vision of evolution as a slow accumulation of beneficial traits through the generations of an organism . rather , they represent the patterns of punctuated evolution , as proposed in the seminal paper by gould and eldredge.43 according to the views expressed by suki,42 when the link density in a network increases , whatever the network is or schematically represents , a critical state will inevitably occur beyond which a new property of the network would spontaneously emerge . this property would belong to a higher level of complexity and brings about new functionality . transplanted into the context of carcinogenesis , one may hypothesize that up to a certain critical level of population , the aberrant cells behave quasi - independently of each other and as a whole do not constitute a community of tightly interdependent entities . at this stage , however , when the link density crosses the critical threshold , the network of aberrant cells collapses into a coherent self - organized mode , and subsequently behaves as a community sharing the common purpose of survival . as pointed out by sonnenschein and soto,7 the smt versus toft dichotomy may be boiled down to the following core question : what is the default state of the cell , proliferation , or quiescence ? according to smt , cancer cells develop the ability to defy the mechanisms of apoptosis , to outtrick the immune system responsible for their elimination , and as a result , to obtain exclusive capabilities to survive , to proliferate , and to transfer deleterious mutations to progeny . thus , in smt , proliferation is claimed to be a distinctive feature and the default state of cancer cells ; in contrast , the default state of normal cells is assumed to be quiescence . this is because cancer is the tissue level disease , and proliferation is the default state of any cell . the authors7 indicate : based on an evolutionary perspective and on our experience using a variety of cell culture models and their animal counterparts , we favor the concept that the default state of cells in metazoa , like those of unicellular organisms and metaphyta , is proliferation . in a recent revisiting of the subject , we became aware that at the end of the 19th century , the famed pathologist h. ribbert postulated that cancer cells , freed from the restraint of tissue structure , would express their constitutive property to proliferate . this conceptual view was further elaborated as follows : implicitly , the smt also adopts the premise that , unlike in unicellular organisms , quiescence is the default state of cells in metazoa . in effect , such claims ignore the fundamental fact that cancer can only arise in metazoa in the context of complex and highly differentiated tissue structures.4 and further : switching premises regarding the default state of cells from quiescence , as adopted by the proponents of the smt , to proliferation , as stated by the toft , qualifies as a paradigmatic change in both a narrow ( limited to the field of carcinogenesis ) and a broad sense because it proposes incorporating a novel evolutionary perspective into the field of carcinogenesis and in its relationship with that of biology at large . ( italicized by sr)4 due to the extraordinary importance emphasized above of the concept of default state for cancer biology and biology at large , it seems worthwhile to explore this concept in more detail . to the best of the author s knowledge , there has been no attempt in the literature to provide a more or less crisp definition of the concept of default state . therefore , when applying this concept one should mostly rely on one s own intuition , as well as on the analogies and metaphors borrowed from other domains of science and social experience . on the other hand , since the concept of default state has been placed in the center of argument of paradigmatic proportion , it is vitally important to reach some sort of consensus regarding the meaning of this concept . at first glance , it may be thought that the words default state are somewhat equivalent to the natural or naturally - predisposed state . although it sounds approximately right , the drawback is that there is not much of an illuminating potential in such a definition ; this is because everything in the nature is , of course , natural . a little more specificity may be found in huang,44 where the default state is equated to the state that needs not to be actively maintained . this definition is fairly ambiguous ; it provokes more questions than it is able to resolve . indeed , one conceivable way the aforementioned active maintenance may be realized is through the interactions with other cells and extracellular matrix ; hence , it could be said that the community of cells is the entity taking responsibility for the individual cell s maintenance . such definition , however , lacks specificity ; under this definition , there is no difference between the default state and any other arbitrary non - default state . an alternative logical possibility is that active maintenance is governed by some external layer of control or by supervisory authority . obviously , this possibility should be ruled out in the in vivo biology ; however , active maintenance by external forces is indeed conceivable and always occurs in the in vitro biology . adherence to such definition would automatically mean that the very definitions of the default states in the in vivo and in vitro biology are not consistent with each other . therefore , it would not be logically justifiable to observe the default states in the laboratory and then to extrapolate the observations towards in vivo conditions . the originators of toft propose the following definition of the default state : by default state we mean the state in which the cells are found when they are freed from any active control.11 the reservations expressed in the previous paragraph are mostly applicable to this definition as well . with this definition , the crux of the issue is what should be regarded as an active control ? it is not just a terminological hair - splitting ; a fundamental question is at stake : how is the cell expected to behave when it is allowed to act on its own in accordance with its natural propensity ? implicitly , existence of such a natural propensity assumes that the cell is a self - sustained and self - motivated organism capable of living on its own and independently of the society it belongs to . in vivo , the active control mentioned above can be only thought of as the control coming from other cells in the tissue . freedom from any active control ( stipulated by the above cited definition)11 is a hypothetical situation in which the cell lives outside its native environment but nevertheless retains the same intrinsic properties that it would have when being tightly embedded in it . essentially , such a notion equates a cell with a unicellular organism whose constitutive properties , according to the above cited definition by h. ribbert , are best seen if the cell is freed from any active control of other cells . we would like to re - emphasize that the discussion here is about determining what the definition of the concept of default state is , rather than about what the cells actually do while residing in their hypothesized default states . as seen from the above considerations , a non - trivial ( ie , bearing some reasonable level of specificity ) and yet logically self - consistent definition of the concept of the default state is elusive . on the other hand , ample experimental evidence is available which casts a shadow of doubt on the very existence of the default states . as demonstrated in many works , the phenotypic traits of individual cells are shaped by interactions within their respective communities.4550 therefore , the default states of the cells freed from the restraints of tissue structure may not be identical , or even similar , to those that are densely packed and immobilized in tissue . numerous examples of phenotypic dependencies between the cellular states and specific circumstances in which the cells are functioning come from microbiology . thus , it has been shown in a landmark work47 that bacterial communities in biofilms ( closely mimicking somatic cells in the tissue ) are capable of maintaining self - identity , purposeful alterations of the colony structure , and recognition and identification of other colonies . this is an amazing example demonstrating that unicellular organisms are capable of modifying their phenotypic traits ( default states ) to the extent that make them capable of distinguishing between allies and enemies , and mounting collective defense against the genotypically identical rivals . examples of this kind are numerous ; they indicate that the default states of individual cells are largely shaped by their roles and positions within the community . essentially , all this means that the concept of default state is an abstract idealization and simplification with limited applicability to real states of the cells , either in tissue or in culture . in a larger context , it is not self - evident why such an entity as the cell deserves the honor to have some sovereign default state , but a community of the cells , as a whole , would not . astounding coherence observed between all the elementary processes on various levels of biological organization allows one to see a community of cells as a superorganism or even as a separate organ,51,52 and to talk about its defensive tactics.53 the most fundamental property of a superorganism is the shared purpose of its existence.54 therefore , if we continue to ascribe specific default states to various biological entities , it seems legitimate to ask : what is the default state of a superorganism ? patterns of highly organized behavior are observed in intra - cellular processes.55 after having postulated existence of some natural default state of the cell , should we now continue inward by ascribing some default states to individual genes , to individual mitochondrions , to individual rna polymerases , or to individual proteins ? if one starts making such a priori statements at some hierarchical level and then logically extends this process to adjacent hierarchical levels , then ultimately the entire system of knowledge will be transformed into the collection of a priori postulated descriptors of the default states . hence , no scientific inquiry would be necessary if all the conceivable default states and behaviors are appropriately postulated and cataloged a priori . all this is to say that assumptions regarding the very existence of the default states may be a shaky basis for erecting a massive edifice of the theory of carcinogenesis . it is our view that the roots of logical difficulties in giving a crisp definition to the concept of default state stem from the fact that the cells possess a rich gamut of functions widely varying in the degree of their autonomy . some of these functions are fully automatic and work in the same way under any circumstances , whether within or outside the network of cells . others depend on the inputs from other cells and extracellular sources ; these functions retain a high degree of autonomy but are modifiable by external forces . still other functions are completely dependent on interactions with other cells ; these functions are unthinkable if the cell is freed from any active control ( as stipulated by the definition cited above ) . all these numerous properties and behaviors , separately or in combination , form an intricate mosaic of possible default states . we conclude this section with the conjecture that the concept of default state may receive a specific meaning only within a specific operational context . therefore , the question what is the default state of the cell ? does not make much sense out of the context of a dynamic environment to which it belongs . the tendency to proliferate , or to be quiescent , or to follow a more elaborate patterns of phenotypic behavior would strongly depend on the specific stimulus - response rules imposed by the community of cells . another persistent motif in the toft discourse is that carcinogenic agents , whatever they are , destroy the normal tissue architecture thus disrupting the intercellular communications and depriving the intracellular machinery of necessary resources for seamless functioning . for example , sonneschein and soto claim : the second premise [ of toft ] is that carcinogenesis are defects of tissue architecture . specifically , the targets of carcinogens would be all the morphogenetic fields comprising interacting tissues . in this context , physical , chemical , and biological carcinogens qualify as disruptors of tissue architecture.1 this general characterization is echoed and further detailed in many other works . for example , as indicated by potter : metaplasias and cancer are characterized , at the earliest stages , by disruption of the standing - wave templates , which leads to a shift in phenotypes and a change in tissue microarchitecture.56 obviously , the word architecture used in this context is a metaphor borrowed from a completely different domain of experience . for reference purposes , here is what the merriam - webster dictionary tells us regarding the meanings of the word architecture : i ) the art or science of building ; specifically : the art or practice of designing and building structures and especially habitable ones ; ii ) formation or construction resulting from or as if from a conscious act ; iii ) a unifying or coherent form or structure ; iv ) architectural product or work ; and v ) a method or style of building . apparently , definition iii ) provides the meaning which best fits into the biological context . basically , structural integrity of the tissue is the key element brought about by invoking the metaphor of architecture . there is another , distinctly different aspect of the toft : cell - to - cell communication . we find in soto and sonnenschein : altered communication among cells is at the core of the toft.11 in the above - mentioned review by baker,3 importance of cell - to - cell communications for the entire toft paradigm is summarized as follows : the tissue organization field theory says that cancers arise from a disruption of cell communication needed to maintain normal tissue architecture . although the general intuitive meaning of the above statements seems to be quite clear , it is nevertheless fairly obvious that the concept of communication transplanted into the bimolecular context is a metaphor that should not be taken too literally and pushed too far along the ways of analogy . taken literally , the disruption of communications can not be an immediate cause of ruining the architecture . using an analogy , when the internet connection in one s home is down , the home itself remains standing and does not immediately collapse . it is useful again to check with the merriam - webster dictionary regarding the authentic meaning of the word communication : communication is a process by which information is exchanged between individuals through a common system of symbols , signs , or behavior . information , in turn , is a coded message transmitted from an entity called transmitter ( t ) to an entity called receiver ( r ) using a language common to both t and r. fundamentally , neither the language nor the algorithms of coding and decoding of its symbols are the creations of t or r ; they should be in existence prior to the process of information transfer . this is a big difference with molecular communications where the t , the r , and the language they use belong to the same realm on organic molecules and biochemical reactions . it should also be noted that information transfer by itself is not sufficient to cause any action ; some sort of mechanism is still required for reading information and transforming it into mechanical or chemical changes . the point we are making here is that a fair amount of imagination is required in order to envision the parallels between the information transfer and cascading biochemical reactions constituting the cells life . such parallels are helpful but not reliable in creating a logically self - consistent theory . the metaphor of cell - to - cell communication implicitly elevates the cells to the status of self - motivated individuals each possessing some knowledge and communicating with each other in the business of maintaining the tissue architecture . all these metaphorical constructions are nothing else than the crutches for the human mind to cope with biomolecular complexity and to somehow succinctly conceptualize a big picture of myriads of biochemical processes inside the cells . in summary , it does not seem sufficient just to say that the disruption of cell - to - cell communication and damage to tissue architecture are the primary causes of carcinogenesis . there are great many of biochemical contexts and structural characterizations in which the evolution of tissue , from a healthy state to a precancerous state and further to tumorigenesis , may be described and analyzed . in particular , chronic systemic inflammation has been widely recognized to be among the leading factors in progression of healthy tissue towards precancerous and cancerous lesions . the specific mechanisms of such progression include sustained cell proliferation in an environment rich in inflammatory cells and molecular agents causing dna damage.57 as observed by lee et al . : excessive and pathologic inflammation causes dna damage , genomic instability , epigenetic dysregulation , and alteration of intracellular signaling , all of which are involved in neoplastic transformation.58 it is important to realize that inflammation - triggered carcinogenesis can not be reduced to just cell proliferation and conquering new tissue territories ( as simple ecological analogies would suggest ) . a number of complex molecular mediators facilitate proliferation of genomic damage , among which an important role belongs to inflammasomes , ie , the multi - protein complexes that mediate immune response.59 as seen from the above description , there is a striking similarity between the pictures of inflammatory damage proliferation through tissue and flame propagation through dry forest ( as the very word inflammation would suggest . ) , gene methylation is mostly localized in the coding region whereas the promoter region remains mostly unmethylated . a different pattern is observed in neoplasia : the genome - wide hypomethylation is accompanied by localized hypermethylation . evidence suggests that methylation is an important factor in carcinogenesis since genome - wide hypomethylation can trigger the chromosome instability and increase the mutation rates.60 laird et al observed that dna methylation changes in cancer cells are not mere by - products of malignant transformation , but can play an instrumental role in the cancer process . it seems clear that dna methylation plays a variety of roles in different cancer types and probably at different stages of oncogenesis.61 generally , abnormal patterns of methylation signify elevated cancer risk due to heightened susceptibility to cancer cell proliferation . according to vendramini - costa and carvalho,62 tumor initiation involves irreversible changes in dna through activation of oncogenes or inactivation of tumor suppressor genes . further development leads mutated cells to expansion through increased proliferation and suppression of cell death . in the process of invasion of adjacent tissues cancer cells again , the process is quite similar to the forest fire propagation , which accumulates additional strength while invading new territories . as briefly touched upon above , disruption of cell - to - cell communication is an important aspect characterizing precancerous tissue.51 in toft , this disruption is seen as a central component of a bigger process of tissue disorganization . the viewpoint being advanced in the previous paper by this author63 is that a community of cells is not simply a collection of units dwelling within certain architectural structures . swarm intelligence is the organized behavior of large communities without global organizer and without mapping the global behavior onto the cognitive / behavioral abilities of the individual members of the community . ) with the destruction of signaling pathways , not only the normal regulation of individual cellular processes is damaged , but also a blow is dealt , so to speak , to the mental capabilities of the community as a whole . its collective memory is wiped out or distorted , customary division of labor between subpopulations is shifted towards aberrant modalities , and community - wide self - defense mechanisms are weakened or broken . these processes in turn cause a shift in expression profiles and metabolic dynamics , eventually penetrating to the level of dna and causing multiple mutations . an important aspect of swarm intelligence is the faculty of quorum sensing ( qs ) . there is a growing consensus in the cancer research community regarding the fundamental importance of disruption of qs in cancer onset and proliferation . agur et al.65 provide a brief review of relevant biological facts and propose a mathematical model of qs boiled down to its simplest mechanistic elements . they conclude that cancer initiation is driven by disruption of the qs mechanism , either by genetic mutations , complying with the current notion of cancer evolution , or purely by the environment , genetic mutations being only a side - effect of excessive proliferation . disruption of qs aggravates weaknesses of the tissue defenses thus moving the system closer to the verge of collapse . as seen from the above discussion , the mechanisms of tumor initiation play a prominent role in carcinogenesis , and a single catastrophic event indeed can make a fundamental impact on all subsequent events . however , it goes without saying that not every event that may be seen as catastrophic on the level of individual cell would necessarily lead to carcinogenesis . vast majority of those events would fade and disappear without traces . when tissue homeostasis is perturbed , sentinel macrophages and mast cells release cytokines , chemokines , reactive oxygen species ( ros ) , and other bioactive mediators that induce mobilization of additional leukocytes.66 this means that the mutant cell capable of starting the domino - effect of subsequent failures should be able to overcome the tissue s natural defenses ; this may happen only if the tissue is already preconditioned for failure and resides on the verge of systemic collapse . in the section above , we attempted to provide just a glimpse of extremely complex and tangled transition of healthy tissue towards precancerous state . obviously , even a complete knowledge of each and every process contributing to this transition does not automatically lead to understanding the process as a whole . resorting again to the sandpile analogy , it would be as difficult as understanding the phenomenon of avalanche from observations of each and every sand grain trajectory . this is why systemic approaches are not simply helpful , they are absolutely necessary and unavoidable for synthesizing existing biomolecular knowledge into a coherent picture of carcinogenesis . , it follows that neither a single catastrophic event nor a persistent damage to the tissue architecture and to the cell - to - cell communications , taken separately , are sufficient for triggering carcinogenesis . the carcinogenesis scenario outlined above is characteristic for the manifestations of soc : from the systemic point of view , it is quite analogous to wild forest fires , to economic collapses , to electric grid blackouts , to rumor propagations , and to many other phenomena uniformly conceptualized by the theory of soc . within the soc scenario , toft and smt do not contradict each other but come into confluence and complement each other in a single unified theory of carcinogenesis .

two drastically different approaches to understanding the forces driving carcinogenesis have crystallized through years of research . these are the somatic mutation theory ( smt ) and the tissue organization field theory ( toft ) . the essence of smt is that cancer is derived from a single somatic cell that has successively accumulated multiple dna mutations , and that those mutations occur on genes which control cell proliferation and cell cycle . thus , according to smt , neoplastic lesions are the results of dna - level events . conversely , according to toft , carcinogenesis is primarily a problem of tissue organization : carcinogenic agents destroy the normal tissue architecture thus disrupting cell - to - cell signaling and compromising genomic integrity . hence , in toft the dna mutations are the effect , and not the cause , of the tissue - level events . cardinal importance of successful resolution of the toft versus smt controversy dwells in the fact that , according to smt , cancer is a unidirectional and mostly irreversible disease ; whereas , according to toft , it is curable and reversible . in this paper , our goal is to outline a plausible scenario in which toft and smt can be reconciled using the framework and concepts of the self - organized criticality ( soc ) , the principle proven to be extremely fruitful in a wide range of disciplines pertaining to natural phenomena , to biological communities , to large - scale social developments , to technological networks , and to many other subjects of research .

the furanose ring links phosphate groups into an extended linear backbone and anchors the heterocyclic bases to form the unique chemical structure of rna , dna , tna and related nucleic acids . it has long been recognized from x - ray crystallographic studies that the furanose moiety is not planar but can take many distinct puckered conformations . proton nmr studies of nucleosides and mononucleotides suggested a rapid interconversion of these pucker states . in conventional 35-linked rna , the optimal pucker state can shift from c3-endo in a standard a - form duplex to c2-endo in other structural motifs . this inherent flexibility of the ribose backbone allows rna to fold into specific tertiary structures with exquisitely specific molecular recognition and catalytic activities . the catalytic abilities of folded rna structures lend strong credibility to the hypothesis of an rna world , in which rna served as both the genetic material and the principal catalysts of primitive life . experimental efforts to synthesize a self - replicating protocell have been hindered by a series of problems in achieving nonenzymatic rna replication . one of these apparent problems is the relatively poor regiospecificity of nonenzymatic rna copying . however , recent studies in this laboratory revealed that folded rna structures can tolerate a significant amount of backbone heterogeneity . certain aptamers and ribozymes can retain their functions even when 25% of the original 35 linkages are substituted by 25 linkages . this shifts the paradigm from the view that 25 linkages are deleterious for rna replication to one in which a certain level of backbone heterogeneity is advantageous because it may facilitate thermal denaturation and therefore rna replication , while remaining compatible with the biological functions of rna . subsequent x - ray crystallographic studies and molecular dynamics ( md ) simulations showed that in rna duplexes , the structural perturbations caused by 25 linkages are largely confined to the two nearest base pairs and have minimal effects upon the rest of the duplex . furthermore , 25-linked nucleotides can adopt either the c3-endo or c2-endo conformation , with a preference for the latter . this is in sharp contrast with conventional 35-linked rnas and prompted us to systematically calculate the free energy landscape of sugar puckering and to investigate how this landscape is modulated by distinct backbone linkages . calculating a free energy landscape requires a set of judiciously chosen collective variables ( cv ) that reduces the high - dimensional phase space to a low - dimensional cv space while still distinguishing all significant conformers . the free energy for each point in the cv space is then calculated by advanced simulation methods such as umbrella sampling and metadynamics . these free energy calculations have provided critical insights into many fundamental chemical or biophysical problems by allowing one to determine not only the population of any given conformer but also the free energy along a transition path that connects two stable conformers . the ideal cvs for studying the conformational changes of sugar pucker are the two pseudorotation parameters , the phase angle p and the amplitude m , which were first proposed by altona and sundaralingam and subsequently generalized by cremer and pople . these two parameters analytically describe the complete spectrum of all possible puckered states . following the cremer pople definition , early theoretical studies calculated the potential energy surface of the phase angle p for several nucleoside analogues in vacuo and revealed that the conversion between c2-endo and c3-endo occurs via the o4-endo pathway , with an energy barrier of 23 kcal / mol . the alternative o4-exo pathway has a much higher barrier and is thus less favorable . the high computational cost , however , precluded the possibility of applying such ab initio methods to nucleic acids in explicit solvent . for these larger systems typically with no less than tens of thousands of atoms , md simulation based on empirical force fields is an attractive approach that balances computational cost and accuracy . indeed , starting with a minimized conformation , benchmark simulations of various ribo- and deoxyribo - nucleosides as well as dna , rna , and dna / rna hybrid duplexes reproduced the stable sugar pucker modes observed in experiments . the sampling from these unrestrained simulations , however , is often insufficient to identify high free energy barriers or other minima that are separated by such barriers . as such , a complete free energy landscape of the entire pseudorotation cycle has not yet been described even for simple nucleosides . as a step toward elucidating the structural effects of 25 linkages upon functional rnas , and to provide a unified description of the various puckered conformations of nucleic acids , we developed a computational method that directly used p and m as the cvs to calculate pseudorotation free energy . compared to previous work that used root - mean - square - displacement as an indirect cv , this new approach overcame the insufficiency of sampling and allowed us to sample the entire two - dimensional pseudorotation cycle . in two nucleoside model systems , the calculation not only accurately predicted the free energy difference between the two main pucker states , c2-endo and c3-endo , but also determined the free energy of key transition intermediates , the o4-endo and o4-exo states . application to a native rna duplex revealed the c3-endo state as the single dominant free energy minimum . for a 25-linked nucleotide in an otherwise identical rna duplex , our free energy calculation revealed a flattened free energy landscape , in which the c2-endo state is only 3 kcal / mol more stable than the c3-endo conformation . the interconversion between these two states is coupled to a switch of hydrogen bonds between o3h and the pro - sp - oxygen or pro - rp - oxygen . the free energy barrier corresponds to the c4-exo state , with no intramolecular hydrogen bond between 3-oh and either nonbridging oxygen of the downstream phosphate . this conclusion is supported by 420 unrestrained md simulation trajectories in which the nucleotide spontaneously migrates to the more stable c2-endo state from the initial c3-endo conformation . from these simulations , we estimated that the transition from c3-endo to c2-endo occurs in nanoseconds , whereas the reverse transition requires approximately microseconds . such a rapid interconversion will allow 25-linked rna to swiftly sample various pseudorotation states across the flattened energy landscape , and such an expanded conformational flexibility could allow a single rna sequence to sample multiple functional conformations . guanosine ( rg ) and 2-deoxyguanosine ( dg ) nucleosides were solvated in 29 29 34 tip3p water boxes with a total of 2.6 10 atoms for each system . the native rna duplex ( 5-ccggcgccgg-3 ) simulation was based on the reported 1.32 resolution x - ray crystal structure ( pdb code : 4ms9 ) , and the c525-linked rna duplex ( 5-ccggc*gccgg-3 , where the asterisk represents a 25 phosphodiester bond ) was based on the reported 1.55 resolution structure ( pdb code : 4msb ) . the final systems contained 1.2 10 atoms including rna , water , and ions . a total of 1.4 s of unrestrained md simulations ( table s1 , supporting information ) were performed using the program namd 2.9 with the charmm36 parameter set . langevin dynamics was used to keep the temperature at 298 k with a damping constant of 5 ps , and a langevin piston was applied to maintain the pressure at 1 atm . the bonded , nonbonded , and electrostatic interactions were calculated at time steps of 1 , 2 , and 4 fs , respectively . the switching ( cutoff ) distance for nonbonded interactions was set at 10 ( 12 ) . to compute long - range electrostatic interactions , the particle mesh ewald method with a grid density of at least 1 was used . to provide a benchmark for the pseudorotation of nucleosides , 300 ns unrestrained simulations were performed for both rg and dg ( table s1 , supporting information ) . both systems were first subjected to 10 000 steps of minimization using the conjugate gradient method . to enhance the sampling as well as estimate the sampling variability , five replica runs were set up using the same minimized initial structure but different initial velocities . the coordinates of the simulation trajectories were saved at 0.5 ps intervals and used to calculate p(p,m ) , the probability of observing a given pseudorotation conformation . the pseudorotation free energy is then calculated by f(p,m ) = kbt ln p(p,m ) . to study the kinetics of the c3-endo to c2-endo transition of c5 in the c525-linked rna duplex , 40 distinct configurations sampled at 100 ps intervals were extracted from a 4 ns simulation in which the pucker state of c5 was restrained at the c3-endo conformation . each configuration was used as the initial coordinate for 20 1-ns unrestrained simulations with different initial velocities . the aggregate simulation time used to study this conformational change was 800 ns ( table s1 , supporting information ) . we implemented p and m as the cv for umbrella sampling and metadynamics in a modified version of namd 2.9 using the cremer pople pseudorotation definition , from which the gradient of p and m can be computed analytically . the derivation of these equations , together with a brief introduction to the cremer pople definition , is provided in the supporting information . in the results and discussion section , the p and m values calculated according to the cremer sundaralingam definition that is more widely used in the literature using the following equations:12 figure 1 illustrates the relationship between p and various sugar pucker conformations . sundaralingam definition , are given in multiples of 36. the corresponding structures of the furanose ring are shown on the periphery of the circle . the umbrella sampling calculation of nucleoside pseudorotation comprises five replicas of simulations at 298 k with a biasing harmonic potential centered on p ( varying successively from 180 to 180 every 10 with a force constant of 0.007 kcal mol deg ) and m ( varying successively from 0.2 to 0.55 every 0.05 with a force constant of 280 kcal mol ) . each simulation was 400 ps , and the distribution of ( p , m ) from the last 280 ps trajectory was used as the input to reconstruct the unbiased free energy surface . the weighted histogram analysis method ( wham ) with bayesian bootstrapping was applied to generate 200 bootstrapped free energy surfaces from 1440 histograms of the five replica runs ( table s1 , supporting information ) . a reliable free energy reconstruction requires significant overlap between the histograms from adjacent simulation windows . indeed , extensive overlap was observed for both rg ( figure s3 , supporting information ) and dg ( figure s4 , supporting information ) . the average and standard deviation of the free energy was calculated on the basis of these 200 bootstrapped free energy surfaces . the source code for the analyses was developed on the basis of the one - dimensional wham code written by david minh , and has been deposited to simtk.org . for c5 in the native rna duplex , the umbrella sampling calculation focused on the east half of the pseudorotation space , with biasing potential centered on p ( varying successively from 90 to 90 every 10 with a force constant of 0.008 kcal mol deg ) and m ( varying successively from 0.15 to 0.50 every 0.05 with a force constant of 320 kcal mol ) . for c5 in c525-linked rna duplex , a similar setup was used except p was sampled at every 7.5 and m was sampled from 0.10 to 0.50 with 0.05 intervals . each simulation was performed for 800 ps , and the last 500 ps was used for free energy calculation . as above , the distributions from adjacent simulation windows overlap extensively ( figure s5 , supporting information , for native rna duplex ; figure s6 , supporting information , for c525-linked rna duplex ) . for each duplex , five replicas were performed , and the free energy surface was reconstructed using the same protocol as for free nucleosides . we first calculated the free energy surface defined by p and m for guanosine ( rg ) in solution by directly using p and m as the collective variables in umbrella sampling ( see materials and methods for computational details ) . the resulting free energy surface represents the intrinsic properties of guanosine pseudorotation in the absence of other structural constraints imposed by secondary or tertiary structures of rna , and therefore , it serves as an important reference for understanding nucleic acid pseudorotation . previous nmr and crystallographic studies provide critical experimental data for testing the accuracy and validity of our computational method . the lowest free energy state corresponds to the c2-endo conformation with p from 160 to 180 and m between 35 and 42 ( figure 2d ) . this agrees well with the one conformer observed in the crystal structure of guanosine dihydrate ( p = 161.4 , m = 36.2 ) . the other conformer adopts a c1-exo conformation ( p = 139.2 , m = 44.3 ) that is different from many other purine nucleosides or their derivatives . our free energy calculation also located a second minimum around p = 20 that corresponds to the c3-endo conformation ( figure 2b ) . the free energy difference between these two states is 1 kcal / mol , in excellent agreement with 0.8 kcal / mol derived from previous nmr measurements based on jh1h2. for 5-guanosine monophosphate , our previous nmr measurements yield a free energy difference of 0.5 kcal / mol , suggesting that the equilibrium between these two states is largely unaffected by the c5 exocyclic substituents . ( a ) the pseudorotation free energy of guanosine is plotted in polar coordinates with p , the phase angle , increasing clockwise from a vertical value of p = 0 , and m , the puckering amplitude , increasing radially from the central dot ( a completely planar ribose ring ) . the contour lines are drawn every 0.5 kcal / mol , and the standard deviation ( figure s7 , supporting information ) is in general less than 0.2 kcal / mol . ( b e ) representative structures of key states along the pseudorotation cycle . the calculated free energy landscape also predicts the free energy barriers of the transitions between the two minima and the corresponding pseudorotation states . the main transition pathway , via an o4-endo intermediate , has a barrier of 2 kcal / mol , which can be attributed to the eclipsed o2c2c3o3 torsion angle ( figure 2c ) . the height of this barrier is low enough to be sampled by sufficiently long unrestrained md simulations and serves as an additional benchmark for our free energy calculations . five independent unrestrained simulations ( 60 ns each ) were performed . during the 250 ns aggregate production time , 848 transitions between c2-endo and c3-endo were observed , providing extensive sampling over the east half of the pseudorotation cycle . the calculated free energy surface from these unrestrained simulations ( figure s8 , supporting information ) agrees well with the one from umbrella sampling , with an identical 2 kcal / mol free energy barrier . detailed analyses of these unrestrained simulations regarding the nucleobase orientation and the intramolecular 5-ohn3 hydrogen bond during the pseudorotation cycle are provided in the supporting information . the agreement between umbrella sampling results and independent computational as well as experimental data suggests that our implementation of pseudorotation collective variables allows the accurate calculation of the phase angle , amplitude and the energetics of pseudorotation . umbrella sampling can accurately determine the free energies of high free energy conformations , which are otherwise poorly represented or even absent , in unrestrained simulations . previous ab initio calculations of the conformations of a nucleoside analogue predicted that the o4-exo conformer is such an unstable intermediate . this state , together with its adjacent c1-endo and c4-endo states , were not observed in our 300-ns unrestrained simulations ( figure s8 , supporting information ) nor in a previous 50-ns benchmark simulation with a slightly different force field . by employing umbrella sampling with pseudorotation parameters as the collective variables , we could sample these rare high energy states and the o4-exo state has the highest free energy of 9.5 kcal / mol , and the origin of this high barrier is illustrated in figure 2e : in addition to the eclipsed o2c2c3o3 torsion angle , the nucleobase and c5 exocyclic substituents are also in steric conflict in the o4-exo conformation . the calculated two - dimensional free energy surface also revealed that the o4-exo state prefers a smaller m ( figure 2a ) because a more puckered conformation will reduce the c1c4 distance and aggravate the steric conflict . to elucidate how the absence of a 2-hydroxyl group affects nucleoside pseudorotation , we computed the pseudorotation free energy surface of 2-deoxyguanosine ( dg ) in solution . the free energy landscape of dg is generally similar to that of rg but with noticeable alterations ( figure 3a ) . the absence of the 2-hydroxyl group releases the constraint imposed by the eclipsed o2c2c3o3 torsion angle , stabilizing both the o4-exo ( figure 3e ) and o4-endo states by approximately 2.5 kcal / mol . consequently , the free energy of the former drops to 7 kcal / mol , whereas the latter merges into a broad basin that includes the c2-endo and adjacent c1-exo states . the peak of the main transition path shifts to the c4-exo state ( figure 3c ) , with a free energy barrier of 1.5 kcal / mol . this broad basin is 1.0 kcal / mol more stable than the c3-endo state ( figure 3b ) , and the free energy difference again agrees well with previous nmr results as well as with our unrestrained md simulations ( figure s10 , supporting information ) . ( a ) the pseudorotation free energy of 2-deoxyguanosine is plotted in polar coordinates as defined in figure 2 . the contour lines are drawn every 0.5 kcal / mol , and the standard deviation is in general less than 0.2 kcal / mol ( figure s9 , supporting information ) . ( b e ) representative structures of key states along the pseudorotation cycle . to delineate how distinct backbone connectivities can influence the pseudorotation free energy landscape of rna , it is critical to compare two regioisomers of a nucleotide at the same position within a duplex to avoid potential interference by positional and sequence effects . our previous crystallographic studies provided one such opportunity through the high - resolution structures of a native 5-ccggcgccgg-3 rna duplex and a regioisomeric 5-ccggc*gccgg-3 rna duplex ( c525-linked rna ) , where the asterisk represents a 25-phosphodiester bond . here , we first calculated the pseudorotation free energy landscape of c5 within the native rna duplex using umbrella sampling to cover the east half of the pseudorotation cycle , including both c2-endo and c3-endo states as well as the more preferable o4-endo transition pathway . the resulting free energy landscape ( figure 4a ) has a deep minimum corresponding to the c3-endo conformation , with compact stacking of planar base pairs ( figure 4b ) . the other minimum matches the c2-endo conformation and is significantly less stable by 6 kcal / mol ( figure 4a ) . adoption of the c2-endo pucker mode destabilizes the a - form duplex because it disrupts the planar base pair structures ( figure 4d ) , therefore weakening both the stacking and hydrogen - bonding interactions . in some trajectories , the c5 base transiently flipped out of the helix ( figure 4d ) . the large free energy penalty explains why in all known a - form rna duplex structures , only the c3-endo conformation has been observed . ( a ) the pseudorotation free energy landscape of c5 in an a - form rna duplex , plotted in polar coordinates as defined in figure 2 . the contour lines are drawn every 1 kcal / mol , and the standard deviation is in general less than 0.4 kcal / mol ( figure s11 , supporting information ) . the most stable conformation corresponds to the c3-endo state , whose calculated structure is depicted in ( b ) . a second minimum matches the c2-endo state , which can adopt two different conformations depending on the orientation of the base ( d ) . the conversion between these two minima occurs via the c1-exo state , and ( c ) shows one representative snapshot of this barrier . strand a of the rna duplex is shown in green , and nucleotide c5 is highlighted . the free energy calculation further revealed that the c1-exo state is the least stable puckering state between the c2-endo and c3-endo conformations ; the free energy barrier of this transition is approximately 12 kcal / mol ( figure 4a ) . compared to the ribonucleosides , the free energy barrier is significantly higher because of the geometrical constraints imposed by the rna duplex . the c1-exo state is even less stable than the c2-endo state because in addition to the nonplanar base pair , it is further destabilized by the heavily eclipsed o2c2c3o3 torsion angle ( figure 4c ) . experimental measurement of the sugar puckering barrier in an rna molecule requires site - specifically labeling ribose with c ( ref ( 40 ) ) and so far has not been performed on an a - form duplex . therefore , there is no direct experimental data to compare with our calculated barrier . in the gcaa tetraloop , the measured sugar puckering barrier varies from 10 to 18 kcal / mol depending on the local structural context . our calculated value falls within the same range and provides a testable prediction that may stimulate further experimental investigations . to elucidate the structural effect of 25 linkages on the pseudorotation of nucleotides in an rna duplex , we performed a pseudorotation free energy calculation for 25-linked c5 in an rna that is regioisomeric to the native rna duplex described above . as shown in figure 5a , the most stable conformation of the resulting free energy surface corresponded to the c2-endo state ( figure 5d ) , with p between 150 to 180 and m around 35 , in excellent agreement with the crystallographic result ( p = 160 6 , m = 36 2 ) . previous nmr studies also showed that the c2-endo state is preferred in homogeneous 25-linked rna duplexes and branched trinucleotides containing 25-linkages , suggesting that such a preference may be an intrinsic property of 25-linked rnas . the free energy calculation also reveals that the other minimum corresponds to the c3-endo conformation ( p around 10 , m around 35 , figure 5b ) , with a slightly higher free energy of only 3 kcal / mol , which could be overcome with favorable molecular interactions . indeed , the calculated pseudorotation parameters agree well with a c3-endo conformation ( p = 16.7 , m = 40.8 ) that we serendipitously captured in g3 of an rna duplex with identical sequence but three 25-linkages ( 5-ccg*gc*gc*cgg-3 , where the asterisks represent the 25 phosphodiester bonds , hereafter referred to as triple 25-linked rna ) . compared to the native duplex , which has a deep free energy minimum at the c3-endo state , a 25 linkage profoundly flattens the pseudorotation free energy landscape and shifts the minimum to the c2-endo state . ( a ) the pseudorotation free energy landscape of 25-linked c5 in an rna duplex , plotted in polar coordinates as defined in figure 2 . the contour lines are drawn every 0.5 kcal / mol , and the standard deviation is in general less than 0.2 kcal / mol ( figure s12 , supporting information ) . the most stable conformation corresponds to the c2-endo state , whose calculated structure is depicted in ( d ) . the hydrogen bond between the 3-hydroxyl group and the pro - sp - oxygen is indicated by black dashed lines . the c3-endo conformation forms a second minimum that is 3 kcal / mol less stable ( b ) . in the c3-endo state , the hydrogen bond shifts to o3hpro - rp - oxygen ( indicated by the black dashed lines ) . the conversion between these two minima occurs via the c4-exo state , and ( c ) shows a representative snapshot of this barrier , in which there is no hydrogen bond between the 3-hydroxyl group and the downstream phosphate . strand a of the rna duplex is shown in green , and nucleotide c5 is highlighted . hydrogen atoms , except on the 3-hydroxyl group , are omitted for clarity . to understand what destabilized the c3-endo state , we performed a 50-ns md simulation in which the ribose of c5 was restrained in the c3-endo state ( p = 15 5 , table s2 , supporting information ) . a conformational ensemble of the c525-linked rna duplex with c5 in the c3-endo pucker state was generated using 20 000 frames of the last 40-ns trajectory sampled at 2 ps intervals , and was subsequently used to calculate structural parameters including pseudorotation , base pair , and base - pair step parameters . most of the parameters are indistinguishable from those of the c2-endo conformation , although the c5 g6:g6 c5 base - pair step in the c3-endo state has a smaller overlap area ( tables s2 and s3 , supporting information ) . this may weaken the base - stacking and partly explain the instability of the c3-endo state . the md simulation also confirmed that the conformational shift from c2-endo to c3-endo concomitantly altered the hydrogen bond between the 3-oh and the downstream phosphate from o3hpro - sp - oxygen to o3hpro - rp - oxygen ( figure 5b , d ) , a result that was initially inferred on the basis of the oxygen oxygen distance in the crystal structures . a close examination of the conformational ensemble from our restrained simulations further revealed that this hydrogen bond in the c3-endo state is weaker than its counterpart in the c2-endo state ( figure s13 , supporting information ) , with a longer average o o distance ( 2.94 in c3-endo versus 2.86 in c2-endo ) , and a larger deviation from the desired in - line conformation ( o h o angle is 136 20 in c3-endo versus 152 12 in c2-endo ) . therefore , the relative instability of the c3-endo state can be attributed to both a weaker hydrogen bond and weaker stacking , although we can not rule out other possibilities . according to our free energy calculation , the peak of the c2-endo to c3-endo transition path is at the c4-exo state ( p is from 40 to 60 , and m is around 30 ) , and the corresponding free energy barrier is approximately 6 kcal / mol ( figure 5c ) . in this c4-exo state , the intramolecular hydrogen bond between 3-oh and either nonbridging oxygen of the downstream phosphate is broken ( figure 5c ) . a similar conformation is observed in c7 of the triple 25-linked rna crystal structure , albeit with a slightly larger amplitude ( p = 42.8 , m = 42.8 ) . in this case the 3-oh group forms a hydrogen bond with a nearby water molecule instead of phosphate ( figure s14 , supporting information ) . this supports the notion that in the c4-exo state there is no intramolecular hydrogen bond between the 3-oh and the downstream phosphate . to study the kinetics of the interconversion between the c2-endo and c3-endo conformations of a 25-linked nucleotide in an rna duplex , 800 1-ns unrestrained simulations were carried out from 40 distinct initial coordinate sets with c5 in the c3-endo conformation . among these , 420 trajectories ( 52.5% ) successfully reached the more stable c2-endo conformation with 144 < p < 180. to further extract kinetic details of these spontaneous conformational changes from c3-endo to c2-endo , we focused on 5 ps trajectories immediately prior to forming a stable c2-endo state . analysis of these trajectories confirmed that during the spontaneous transition from the c3-endo to c2-endo state , the breaking of the o3hpro - rp - oxygen hydrogen bond precedes the formation of the o3hpro - sp - oxygen hydrogen bond , and both hydrogen bonds are absent in the c4-exo state ( figure 6 ) . we also noticed that in the initial stage of these spontaneous transitions , the heavy atom distance of the o3hpro - rp - oxygen hydrogen bond is already significantly elongated to approximately 3.4 , even though c5 remains in the c3-endo state ( figure 6 ) . this suggests that the transition would first occur among a subpopulation of c3-endo states with weakened o3hpro - rp - oxygen hydrogen bonds . these unrestrained simulations also suggest that t1/2 of the transition is approximately 1 ns . given that the c2-endo state is about 3 kcal / mol more stable than the c3-endo state , t1/2 of the reverse transition ( c2-endo to c3-endo ) should be approximately 150 ns . it should be noted that estimating transition rates from unrestrained md simulations is still a great challenge in computational chemistry . indeed , the error of the calculated binding and unbinding rates of benzamidine to trypsin was about an order of magnitude , even though the extensive sampling accurately predicted the ligand binding mode and the binding free energy . therefore , the rates from these calculations should be interpreted cautiously : they only suggest that the transition rates between these two conformations can occur on roughly the ns and s time scale , respectively . nevertheless , these calculations suggest a rapid interconversion between the c2-endo and c3-endo states of a 25-linked nucleotide in an rna duplex . dynamics of hydrogen - bond switching during the pseudorotation conformational change of a 25-linked nucleotide . the spontaneous transition from the c3-endo to the c2-endo state was observed in 420 out of 800 1-ns unrestrained trajectories . a 5-ps trajectory immediately prior to forming a stable c2-endo state was extracted for each of the 420 trajectories and used to calculate two - dimensional histograms of p versus o3pro - rp - oxygen distance ( a ) and o3pro - sp - oxygen distance ( b ) . the reported free energy calculations and unrestrained md simulations provide important insights into the thermodynamic and kinetic properties of native rna duplexes and those containing 25 linkages . our study highlights a flattened free energy landscape for pseudorotation in 25-linked nucleotides , which can switch rapidly between the c2-endo and c3-endo conformations . this is in contrast with a single dominant c3-endo minimum found in the native rna duplex . therefore , in addition to lowering the melting temperature , the presence of 25 linkages may expand the conformational space accessible for an rna duplex . mechanistically , our calculations demonstrate that hydrogen bonding between the 3-hydroxyl group and the downstream phosphate serves as a molecular switch for this backbone conformational change . our study establishes the feasibility of an atomic - level description of the pseudorotation free energy for free nucleosides , as well as nucleotides in rna duplexes in solution . even though some intermediates are too unstable to be sampled efficiently with conventional md simulation techniques , we were able to gain insights into these structures by implementing a set of collective variables for use in conjunction with advanced free energy calculation methods . the methodology we developed in this study may be further applied to investigate other processes in which pseudorotation plays a significant role , such as nonenzymatic primer extension reactions . furthermore , applications to other synthetic nucleic acid systems ( e.g. , threose nucleic acids ) may lead to a fuller understanding of the chemical etiology of nucleic acid structure .

the five - membered furanose ring is a central component of the chemical structure of biological nucleic acids . the conformations of the furanose ring can be analytically described using the concept of pseudorotation , and for rna and dna they are dominated by the c2-endo and c3-endo conformers . while the free energy difference between these two conformers can be inferred from nmr measurements , a free energy landscape of the complete pseudorotation cycle of nucleic acids in solution has remained elusive . here , we describe a new free energy calculation method for molecular dynamics ( md ) simulations using the two pseudorotation parameters directly as the collective variables . to validate our approach , we calculated the free energy surface of ribose pseudorotation in guanosine and 2-deoxyguanosine . the calculated free energy landscape reveals not only the relative stability of the different pseudorotation conformers , but also the main transition path between the stable conformations . applying this method to a standard a - form rna duplex uncovered the expected minimum at the c3-endo state . however , at a 25 linkage , the minimum shifts to the c2-endo conformation . the free energy of the c3-endo conformation is 3 kcal / mol higher due to a weaker hydrogen bond and a reduced base stacking interaction . unrestrained md simulations suggest that the conversion from c3-endo to c2-endo and vice versa is on the nanosecond and microsecond time scale , respectively . these calculations suggest that 25 linkages may enable folded rnas to sample a wider spectrum of their pseudorotation conformations .

allergic conjunctival diseases ( acd ) are conjunctival inflammatory disorders , characterized by an immediate hypersensitivity reaction associated with antigen - specific ige antibodies . patients with acd are usually categorized into the following types based on clinical criteria : seasonal allergic conjunctivitis ( sac ) , perennial allergic conjunctivitis ( pac ) , vernal keratoconjunctivitis ( vkc ) , atopic keratoconjunctivitis ( akc ) , and giant papillary conjunctivitis ( gpc ) . sac and pac are characterized by conjunctival hyperemia , conjunctival edema , and papillary hyperplasia of tarsal conjunctiva and are classified as mild acd . in contrast , vkc is characterized by the development of proliferative lesions of the conjunctiva , including giant papillary proliferation of the tarsal conjunctiva and gelatinous cell infiltration of the limbal conjunctiva . vkc also complicates corneal disorders , such as shield ulcer and punctate corneal keratitis , and is classified as a severe acd , because the visual prognosis may be poor . akc usually develops in older patients with atopic dermatitis and also complicates severe ocular surface diseases , including giant papillary conjunctivitis , shield ulcer , and dry eye . in shield ulcers of patients with vkc and akc , depositions of major basic protein and eosinophil cationic protein ( ecp ) , which comprise specific granules of eosinophils , have reportedly been observed histologically in the corneal ulcerative lesions [ 3 , 4 ] . furthermore , ecp levels in the tears of vkc and akc patients are reportedly increased , in comparison with those in controls [ 57 ] . therefore , the pathophysiological characteristics of these severe acd , including vkc and akc , include eosinophilic inflammation in the conjunctiva . eotaxin is a member of the cc chemokine family and is divided into three subfamilies , namely , ccl11/eotaxin-1 , ccl24/eotaxin-2 , and ccl26/eotaxin-3 . eotaxin-1 , eotaxin-2 , and eotaxin-3 interact with the cc chemokine receptor 3 ( ccr3 ) . representative inflammatory cells expressing ccr3 on their cell surfaces are eosinophils , type-2 helper t cells ( th2 ) , and basophils . il-13 , which is a th2-derived cytokine , induces eotaxin in vitro through activation of the il-4r receptor / stat-6 pathway [ 8 , 9 ] . therefore , eotaxin-1 , eotaxin-2 , and eotaxin-3 are thought to be allergic inflammation - related chemokines . in the eotaxin subfamily , it has been reported that increased eotaxin-2 levels in tears and expression of ccl24 ( eotaxin-2 ) mrna on the ocular surface are more common in vkc patients than those of eotaxin-1 and eotaxin-3 [ 10 , 11 ] . however , the relationship between the expression levels of ccl24 ( eotaxin-2 ) mrna on the ocular surface and the severity of severe acd , including vkc and akc , has not been fully investigated . in this study , we evaluated the clinical efficacy of using ccl24 ( eotaxin-2 ) mrna expression levels on the ocular surface in patients with severe acd , including vkc and akc , as a biomarker for severe acd . this study was approved by the institutional review board of the nihon university school of medicine and adhered to the tenets of the declaration of helsinki . this study included 18 consecutive patients with vkc or akc treated at the department of ophthalmology , nihon university itabashi hospital , tokyo , japan , from august 2012 to january 2015 ( akc / vkc group ) , and 12 healthy volunteers who did not have any personal or family history of atopic disease , were not affected by ocular surface diseases , or have a history of wearing contact lenses as controls ( control group ) . objective diagnoses were made in vkc and akc patients by means of slit - lamp clinical examination and serum examination for antigen - specific ige antibodies , according to the japanese guidelines for acd . patients with ocular surface disease other than acd , including lagophthalmos , blepharospasm , conjunctival chalasis , dry eye , infectious conjunctivitis , infectious keratitis , stevens - johnson syndrome , and ocular pemphigoid , were excluded . nontreated patients or patients treated with antiallergic ophthalmic solutions alone , such as mast cell stabilizers , histamine h1 receptor antagonists , corticosteroids , and immunosuppressive agents , were included in the study . patients who used oral medicines or injections for treating allergic diseases and those who received immunotherapy were excluded from the study . clinical scores of objective findings in the akc / vkc group were determined using the 5 - 5 - 5 exacerbation grading scale for acd . the akc / vkc group was divided into two subgroups depending on the clinical score : the active stage subgroup with clinical scores of 100 points or more ( n = 6 ) and the stable stage subgroup with 100 points or less ( n = 12 ) . modified impression cytology was performed after instillation of topical oxybuprocaine 0.4% ( benoxil , santen , osaka , japan ) . schirmer 's test strips ( schirmer tear production measuring strips , showa yakuhin kako , tokyo , japan ) were applied to the upper tarsal conjunctiva , pressed gently using a glass rod , and then removed . the membrane was preserved in rnalater rna stabilization reagent ( qiagen , hilden , germany ) until analysis . total rna was harvested from each schirmer tested paper using an rneasy mini kit ( qiagen , hilden , germany ) following the manufacturer 's instructions . cdna was then synthesized using a high - capacity cdna reverse transcription kit ( life technologies japan , tokyo , japan ) , according to the manufacturer 's instructions . to detect expression of ccl24 ( eotaxin-2 ) mrna , real - time reverse transcription polymerase chain reaction ( real - time rt - pcr ) was performed using a commercial pcr master mix ( taqman universal pcr master mix ; life technologies , tokyo , japan ) and predesigned primers ( life technologies ) for ccl24 ( eotaxin-2 ; hs00171082_m1 ) . samples were analyzed using the step one plus real - time pcr system ( life technologies ) and comparative threshold ( ct ) values were obtained . target ct values were normalized to those of gapdh ( hs99999905_m1 ) in the same sample . differences between akc / vkc and control groups were identified using welch 's t - test or the chi - square test . the results for ccl24 ( eotaxin-2 ) mrna expression on the ocular surface were evaluated using the nonparametric steel - dwass test . spearman 's rank correlation coefficient was used to evaluate whether ccl24 ( eotaxin-2 ) mrna expression correlated with the clinical score for ccl24 ( eotaxin-2 ) mrna expression on the ocular surface , 6 of 6 patients in the active stage subgroup of akc / vkc group were ccl24- ( eotaxin-2- ) positive , with median ( range ) levels of 133 ( 27.6232 ) . eleven of 12 patients in the stable stage subgroup of akc / vkc group were ccl24- ( eotaxin-2- ) positive , with median ( range ) levels of 5.99 ( 0.14033.2 ) ; the remaining patient had levels below the lower limit of detection . eight of 12 patients in the control group were ccl24- ( eotaxin-2- ) positive , with median ( range ) levels of 0.98 ( 0.0820.2 ) , while 4 patients had levels below the lower limit of detection . the expression levels of ccl24 ( eotaxin-2 ) mrna on the ocular surface were significantly higher in the active stage than in the stable stage akc / vkc subgroup and the control group ( both p < 0.01 ; steel - dwass test ; figure 1 ) . the median value ( range ) of the clinical scores in the active and stable stage subgroups of akc / vkc group were 13 ( 233 ) and 128 ( 343112 ) , respectively . in patients with akc / vkc , clinical scores were significantly correlated with the levels of ( ccl24 ) eotaxin-2 mrna expression on the ocular surface ( = 0.795 , p < 0.01 , spearman 's rank correlation coefficient ; figure 2 ) . a 10-year - old boy was diagnosed as having vkc and had been under treatment for vkc by a local doctor for 6 years . his clinical observation of vkc repeated exacerbation and remission , and his severity of vkc was different in right and left active giant papillae and exfoliative epithelial keratopathy was present in his right eye ; his clinical score was 212 , and his ccl24 ( eotaxin-2 ) mrna expression levels on the ocular surface were 203.2 . in his left eye , papillary lesions and hyperemia at the upper palpebral conjunctiva were observed , and the clinical score was 2 , while the ccl24 ( eotaxin-2 ) mrna expression level on the ocular surface was 17.2 ( figure 3 ) . a 10-year - old girl was diagnosed as having vkc and had been under treatment for vkc by a doctor for 1 year . she experienced pain in her right eye and also had recurrence of corneal plaque and was referred to our hospital . at her first examination , she demonstrated shield ulcer in her right eye , giant papillae , and palpebral conjunctiva with a velvety appearance . topical 0.1% tacrolimus ophthalmic suspension ( talymus ophthalmic suspension 0.1% , senju pharmaceutical , co. , ltd . , osaka , japan ) and 2% sodium cromoglicate ophthalmic solution ( intal ophthalmic solution 2% , sanofi , tokyo , japan ) were administered . at commencement of treatment with tacrolimus ophthalmic suspension , her clinical score was 213 and the level of ccl24 ( eotaxin-2 ) mrna expression on the ocular surface was 349.6 . during the first 10 weeks of treatment , the clinical score was 3 points and the ccl24 ( eotaxin-2 ) mrna expression level on the ocular surface was 9.6 ( figure 4 ) . in this study , we assessed the usefulness of ccl24 ( eotaxin-2 ) mrna expression levels on the ocular surface as a biomarker of the severity of acd . we found that these levels correlated well with the clinical score reflecting objective findings in patients with akc and vkc . as a method for sampling the ocular surface to test the expression levels of ccl24 ( eotaxin-2 ) mrna this method entailed a membrane biopsy technique , as used for impression cytology , but used filter paper instead of nitrocellulose membrane for the biopsy . in the conventional impression cytology method , the specimen obtained using a nitrocellulose membrane is examined histologically . however , the major concern of this method is the ocular sensation of a foreign body and the ocular pain experienced after the examination ; repeated examinations for follow - up of the biomarker may cause discomfort for the patients . changing to the filter paper for impression cytology sampling and using a quantitative method ( real - time rt - pcr ) allowed assessment of the biomarker on the ocular surface . the specimens obtained by modified impression cytology most likely included conjunctival epithelial and invading inflammatory cells , in addition to tears and mucin . it was therefore considered useful for investigating inflammation - associated factors expressed by conjunctival epithelial cells and inflammatory cells as potential biomarkers of allergic inflammation at the ocular surface . in this study , we elucidated a significant correlation between clinical observations and ccl24 expression on the ocular surface of akc / vkc patients with or without treatment with ophthalmic solutions . these results suggest that ccl24 expression on the ocular surface is suitable as a biomarker of severe allergic conjunctival diseases . in previous reports , it has been demonstrated that eotaxin-1 plays a critical role in eosinophilic infiltration in the conjunctiva and cornea of patients with acd [ 10 , 11 , 13 , 14 ] . however , the concentration of eotaxin-2 in tears has been reported to be higher than those of eotaxin-1 in acd patients . therefore , in this study , we investigated the usefulness of ccl24 ( eotaxin-2 ) mrna expressed on the ocular surface as a biomarker for patients with akc / vkc . we found that expression levels of ccl24 ( eotaxin-2 ) mrna on the ocular surface were significantly increased and correlated with the clinical score in the active stage of vkc . eotaxin-1 is reportedly produced by corneal keratinocytes [ 13 , 14 ] , conjunctival fibroblasts , cd68-positive cells in the conjunctiva , and eosinophils in the conjunctiva . previously , we have reported that the tear levels of eotaxin-2 correlated significantly with those of eosinophil cationic protein and that epithelial cells in conjunctival smears of patients with vkc expressed eotaxin-2 , based on immunohistochemistry . leonardi and colleagues reported that tear levels of eotaxin-1 and eotaxin-2 significantly correlated with the percentage of eosinophils in tears . therefore , conjunctival epithelial cells and eosinophils are thought to be candidate eotaxin-2-producing cells on the ocular surface . furthermore , expression levels of ccl24 ( eotaxin-2 ) mrna in modified impression cytology may be a good biomarker for evaluating allergic inflammation in the conjunctivas of patients with akc / vkc . in these case reports , we showed differences between the right and left eye in the severity of vkc based on the quantitative analysis of ccl24 ( eotaxin-2 ) mrna levels . furthermore , in the 10-year - old girl with vkc , we were able to show the therapeutic effect of treatment by tacrolimus instillation by the reduction of both clinical scores and expression levels of ccl24 ( eotaxin-2 ) mrna . therefore , in akc / vkc patients undergoing treatment , monitoring of the expression levels of ccl24 ( eotaxin-2 ) mrna may provide a useful index of exacerbation and therapeutic response . the limitation of this study included the small sample size , and a lack of patients with mild acd , such as sac and pac . further investigation is necessary for verifying the usefulness of ccl24 ( eotaxin-2 ) mrna levels as a biomarker of acd in a large sample that includes patients with sac and pac . another limitation of this study was that akc / vkc patients not receiving treatment and those only receiving treatment with ophthalmic solutions were enrolled . the ccl24 mrna expression on the ocular surface may be affected by treatment with ophthalmic solutions . however , the efficacy of the therapeutic agent is one of the items measured by a biomarker . therefore , further investigation on the therapeutic effect of antiallergic treatment in a large cohort , including untreated patients with allergic conjunctival diseases , using eotaxin-2 expression as a biomarker , will be necessary in the future . expression levels of ccl24 ( eotaxin-2 ) mrna on the ocular surface are a useful biomarker of the clinical severity of akc / vkc .

purpose . this study aimed to evaluate the clinical efficacy of using expression levels of ccl24 ( eotaxin-2 ) mrna on the ocular surface as a biomarker in patients with vernal keratoconjunctivitis ( vkc ) and atopic keratoconjunctivitis ( akc ) . methods . eighteen patients with vkc or akc ( vkc / akc group ) and 12 control subjects ( control group ) were enrolled in this study . the vkc / akc clinical score was determined by objective findings in patients by using the 5 - 5 - 5 exacerbation grading scale . all subjects underwent modified impression cytology and specimens were obtained from the upper tarsal conjunctiva . expression levels of ccl24 ( eotaxin-2 ) mrna on the ocular surface were determined using real - time reverse transcription polymerase chain reaction . results . the vkc group was divided into two subgroups , depending on the clinical score : the active stage subgroup with 100 points or more of clinical scores and the stable stage subgroup with 100 points or less . ccl24 ( eotaxin-2 ) mrna expression levels in the active vkc / akc stage subgroup were significantly higher than those in the stable vkc / akc subgroup and the control group . clinical scores correlated significantly with ccl24 ( eotaxin-2 ) mrna expression levels in the vkc group . conclusions . ccl24 ( eotaxin-2 ) mrna expression levels on the ocular surface are a useful biomarker for clinical severity of vkc / akc .

a combination of photoredox and palladium catalysis has been employed to facilitate the room temperature decarboxylative allylation of recalcitrant -amino and phenylacetic allyl esters . this operationally simple process produces co2 as the only byproduct and provides direct access to allylated alkanes . after photochemical oxidation , the carboxylate undergoes radical decarboxylation to site - specifically generate radical intermediates which undergo allylation . a radical dual catalysis mechanism is proposed . free phenylacetic acids were also allylated utilizing similar reactions conditions .

the murine ( m ) glutag l - cell line was cultured in dulbecco 's modified eagle 's medium ( dmem ) containing 25 mmol / l glucose and supplemented with 10% fbs ; the medium was changed every 23 days and cells were passaged by trypsinization and reseeding at a 1:3 dilution ( 17,34,35 ) . the human ( h ) nci - h716 l - cell line was obtained from the american type culture collection ( manassas , va ) . fetal rat intestinal cultures ( frics ) were prepared by enzymatic dispersal of term fetal intestines from 19 to 20 days ' pregnant wistar rats , and cells were maintained overnight in dmem containing 25 mmol / l glucose , 10% fbs , and penicillin - streptomycin , as previously reported ( 15,17,35,37 ) . all three models of the intestinal l - cell have been validated with respect to the regulation of glp-1 secretion , such that glp-1 is secreted in response to known secretagogues , including muscarinic / cholinergic agonists , leptin and long - chain fatty acids in all models , as well as glucose - dependent insulinotropic peptide ( gip ) in the rodent cells ( 17,3438 ) . coated 24-well culture plates and grown to 80% confluence . for experiments with hnci - h716 cells , adhesion of the cells was initiated by plating on matrigel matrix ( becton dickinson , bedford , ma ) in dmem medium , supplemented with 10% fbs , 2 days before the experiment , as described ( 35,38 ) . adenosine 3,5-cyclic monophosphate ( camp ) responses to oea were determined by washing mglutag cells with hanks ' balanced salt solution followed by treatment for 30 min with fbs - free medium containing 1% dmso alone ( negative control ) , 10 mol / l forskolin ( positive control ) , or 1015 mol / l oea , as previously described ( 39 ) . to prevent camp degradation , all media contained 10 mol / l 3-isobutyl-1-methylxanthine ( sigma - aldrich , oakville , on , canada ) . mglutag , hnci - h716 , and fric cells were washed and then incubated for 2 h with fbs - free dmem containing 1% dmso alone ( negative control ) , 10 mol / l forskolin ( sigma - aldrich ) , or 1 mol / l gip ( positive controls ) ( bachem , torrance , ca ) or with different concentrations of oea , palmitoylethanolamide ( pea ) , or lpc ( all from sigma - aldrich ) . some cells were preincubated for 30 min with 10 mol / l or 30 mol / l h89 ( sigma - aldrich ) to inhibit protein kinase a ( pka ) or with 1 mol / l urb597 ( calbiochem , mississauga , on , canada ) , an faah inhibitor ( 29 ) . dmso was used as a solvent to prepare stock solutions of fatty acid derivates and inhibitors . for secretion experiments , medium and cells were collected separately and peptides were extracted by reversed - phase adsorption using c18 silica cartridges ( sep - pak ; waters scientific , mississauga , on , canada ) , as previously described ( 17,3436 ) , for ria of glp-1 content . glp-1 secretion was calculated as total glp-1 content of medium normalized for the total amount of glp-1 in the medium plus cells . average basal secretion of mgluta , hnci - h716 , and fric cells was 3.4 0.3% ( n = 31 ) , 2.7 0.4% ( n = 28 ) , and 2.86 0.6% ( n = 4 ) , respectively , of total glp-1 . no changes in total glp-1 content were found under any of the experimental conditions ( data not shown ) . mglutag cells were plated in a 24-well plate , as described above . scrambled small interfering rna ( sirna ) ( control ) and two sirnas targeting murine gpr119 coding sequences were purchased from ambion ( austin , tx ) . transfection was performed by 5-h incubation in opti - mem medium using 20 pmol / l sirna and 1 l lipofectamine 2000 ( invitrogen , burlington , on , canada ) as instructed by the manufacturer . total rna was extracted from mglutag cells using an rneasy kit as instructed by the manufacturer ( qiagen , mississauga , on , canada ) and subjected to reverse transcription using superscript ii and random hexamers ( invitrogen ) , followed by real - time pcr using taqman gene expression assays ( applied biosystems , foster city , ca ) for gpr119 ( mm00731497_s1 ) and 18s ( hs99999901_s1 ; endogenous control ) . relative quantification of gpr119 mrna expression was calculated using the cycle threshold method ( 40 ) . viability of the mglutag cells after treatment was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide ( mtt ) ( sigma - aldrich ) assay , as reported ( 17 ) . cells were plated in poly - d - lysine coated 96-well plates and treated for 2 h with medium containing 1% dmso alone ( control ) , 5 mmol / l h2o2 ( positive control ) , or fatty acid derivates or inhibitors at concentrations used for the secretion experiment , after which the mtt reaction was carried out . the resulting absorbance was measured at 570 nm ; higher absorbance correlates with cell viability and lower absorbance with cell death . camp was measured by ria of ethanol extracts as described ( 39 ) ( biomedical technologies , stoughton , ma ) . ria for cooh - terminal glp-1 immunoreactivity was conducted using an established lab assay ( 17,3436 ) . as there is currently no good gpr119 antiserum commercially available , gpr119 expression was determined by rt - pcr . human jejunum and colon total rna was purchased from ambion , and human placental rna was a kind gift from dr . challis ( university of toronto , toronto , on , canada ) . total rna from mglutag , hnci - h716 , and fric cells , the primer pairs used for amplification of human gpr119 were described by soga et al . ( 17 ) ; all other primer pairs were designed using primerquest ( idt , coralville , ia ) ( sequences are shown in table 1 ) . pcr were carried out at 57c for 35 cycles , and pcr without rna template was used as the negative control . products were analyzed by agarose gel electrophoresis and visualized with ethidium bromide . in vivo animal protocols male wistar rats ( 200300 g ) were obtained from charles river laboratories ( st . constant , qc , canada ) and maintained on a standard laboratory diet with free access to water under a 12-h light - dark schedule . following an overnight fast , rats were anesthetized with isoflurane ( baxter , mississauga , on , canada ) . one hour before blood sampling , some rats were intraperitoneally injected with 3 mg / kg urb597 to inhibit faah ( 29,41 ) . urb597 alone did not affect the levels of glucose , insulin , or bioactive glp-1 in these studies ; therefore , data from rats with and without urb597 injection were combined . the carotid artery was cannulated for blood sampling and the jugular vein for injections . to prevent bioactive glp-1 degradation by dipeptidylpeptidase ( dpp ) iv , rats received 5 mg / kg sitagliptin ( januvia ; msd sharp & dohme , haar , germany ) , a selective inhibitor of dpp - iv ( 10,42 ) , intravenously 30 min before blood collection . some rats also received a femoral vein cannula for continuous infusion of 37.5% glucose to maintain glycemia at 13 mmol / l for at least 30 min . the glucose infusion rate was adjusted based on frequent ( every 510 min ) blood glucose measurements , as described in goh et al . rats undergoing a hyperglycemic clamp were not pretreated with sitagliptin in order to reduce the number of variables that might affect glycemia . the abdominal cavity of all rats was then opened and a 10-cm section of the distal ileum was cleansed by perfusion with saline and tied off to create a luminally distinct compartment that retained vascular perfusion . subsequent to collection of the basal blood sample at t = 0 min , either the luminal compartment was filled with 2 ml of 10 mol / l oea or vehicle ( 0.9% saline/10% tween 80 ; sigma - aldrich ) , or 5 mg / kg oea or vehicle ( 0.9% saline/10% tween 80 ) was administered intravenously , and additional blood samples were collected at 5 , 15 , 30 , and 60 min . all samples ( 1 ml each ) were collected into 10% ( vol / vol ) trasylol ( 5,000 kalikrein inactivating units / ml ; bayer , toronto , on , canada ) , edta ( 12 mg / ml ) , and diprotin a ( a dpp - iv inhibitor , 68 mg / ml ; sigma - aldrich ) , and plasma was stored at 80c until analysis . plasma glucose levels were analyzed on a beckman analyzer ii ( beckman , fullerton , ca ) , and plasma insulin levels were measured by enzyme - linked immunosorbent assay ( crystal chem , downers grove , il ) in normoglycemic animals and by ria ( millipore , billerica , ma ) in hyperglycemic animals due to the wider insulin detection range . plasma levels of bioactive glp-1 were determined by electrochemiluminescence - based detection assay ( meso scale discovery , gaithersburg , md ) . area under the curve ( auc ) was determined using the trapezoidal rule , and the data are expressed per min . statistical analysis was performed using sas software ( sas institute , cary , nc ) . one- and two - way anova was followed by student 's t test or n-1 custom hypotheses post hoc tests , as appropriate . to reduce interassay variations , some data were normalized to basal levels . the murine ( m ) glutag l - cell line was cultured in dulbecco 's modified eagle 's medium ( dmem ) containing 25 mmol / l glucose and supplemented with 10% fbs ; the medium was changed every 23 days and cells were passaged by trypsinization and reseeding at a 1:3 dilution ( 17,34,35 ) . the human ( h ) nci - h716 l - cell line was obtained from the american type culture collection ( manassas , va ) . fetal rat intestinal cultures ( frics ) were prepared by enzymatic dispersal of term fetal intestines from 19 to 20 days ' pregnant wistar rats , and cells were maintained overnight in dmem containing 25 mmol / l glucose , 10% fbs , and penicillin - streptomycin , as previously reported ( 15,17,35,37 ) . all three models of the intestinal l - cell have been validated with respect to the regulation of glp-1 secretion , such that glp-1 is secreted in response to known secretagogues , including muscarinic / cholinergic agonists , leptin and long - chain fatty acids in all models , as well as glucose - dependent insulinotropic peptide ( gip ) in the rodent cells ( 17,3438 ) . coated 24-well culture plates and grown to 80% confluence . for experiments with hnci - h716 cells , adhesion of the cells was initiated by plating on matrigel matrix ( becton dickinson , bedford , ma ) in dmem medium , supplemented with 10% fbs , 2 days before the experiment , as described ( 35,38 ) . adenosine 3,5-cyclic monophosphate ( camp ) responses to oea were determined by washing mglutag cells with hanks ' balanced salt solution followed by treatment for 30 min with fbs - free medium containing 1% dmso alone ( negative control ) , 10 mol / l forskolin ( positive control ) , or 1015 mol / l oea , as previously described ( 39 ) . to prevent camp degradation , all media contained 10 mol / l 3-isobutyl-1-methylxanthine ( sigma - aldrich , oakville , on , canada ) . mglutag , hnci - h716 , and fric cells were washed and then incubated for 2 h with fbs - free dmem containing 1% dmso alone ( negative control ) , 10 mol / l forskolin ( sigma - aldrich ) , or 1 mol / l gip ( positive controls ) ( bachem , torrance , ca ) or with different concentrations of oea , palmitoylethanolamide ( pea ) , or lpc ( all from sigma - aldrich ) . some cells were preincubated for 30 min with 10 mol / l or 30 mol / l h89 ( sigma - aldrich ) to inhibit protein kinase a ( pka ) or with 1 mol / l urb597 ( calbiochem , mississauga , on , canada ) , an faah inhibitor ( 29 ) . dmso was used as a solvent to prepare stock solutions of fatty acid derivates and inhibitors . for secretion experiments , medium and cells were collected separately and peptides were extracted by reversed - phase adsorption using c18 silica cartridges ( sep - pak ; waters scientific , mississauga , on , canada ) , as previously described ( 17,3436 ) , for ria of glp-1 content . glp-1 secretion was calculated as total glp-1 content of medium normalized for the total amount of glp-1 in the medium plus cells . average basal secretion of mgluta , hnci - h716 , and fric cells was 3.4 0.3% ( n = 31 ) , 2.7 0.4% ( n = 28 ) , and 2.86 0.6% ( n = 4 ) , respectively , of total glp-1 . no changes in total glp-1 content were found under any of the experimental conditions ( data not shown ) . mglutag cells were plated in a 24-well plate , as described above . scrambled small interfering rna ( sirna ) ( control ) and two sirnas targeting murine gpr119 coding sequences were purchased from ambion ( austin , tx ) . transfection was performed by 5-h incubation in opti - mem medium using 20 pmol / l sirna and 1 l lipofectamine 2000 ( invitrogen , burlington , on , canada ) as instructed by the manufacturer . total rna was extracted from mglutag cells using an rneasy kit as instructed by the manufacturer ( qiagen , mississauga , on , canada ) and subjected to reverse transcription using superscript ii and random hexamers ( invitrogen ) , followed by real - time pcr using taqman gene expression assays ( applied biosystems , foster city , ca ) for gpr119 ( mm00731497_s1 ) and 18s ( hs99999901_s1 ; endogenous control ) . relative quantification of gpr119 mrna expression was calculated using the cycle threshold method ( 40 ) . viability of the mglutag cells after treatment was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide ( mtt ) ( sigma - aldrich ) assay , as reported ( 17 ) . cells were plated in poly - d - lysine coated 96-well plates and treated for 2 h with medium containing 1% dmso alone ( control ) , 5 mmol / l h2o2 ( positive control ) , or fatty acid derivates or inhibitors at concentrations used for the secretion experiment , after which the mtt reaction was carried out . the resulting absorbance was measured at 570 nm ; higher absorbance correlates with cell viability and lower absorbance with cell death . camp was measured by ria of ethanol extracts as described ( 39 ) ( biomedical technologies , stoughton , ma ) . ria for cooh - terminal glp-1 immunoreactivity was conducted using an established lab assay ( 17,3436 ) . as there is currently no good gpr119 antiserum commercially available , gpr119 expression was determined by rt - pcr . human jejunum and colon total rna was purchased from ambion , and human placental rna was a kind gift from dr . total rna from mglutag , hnci - h716 , and fric cells , as well as from rodent tissues , was extracted as described above . the primer pairs used for amplification of human gpr119 were described by soga et al . ( 17 ) ; all other primer pairs were designed using primerquest ( idt , coralville , ia ) ( sequences are shown in table 1 ) . pcr were carried out at 57c for 35 cycles , and pcr without rna template was used as the negative control . male wistar rats ( 200300 g ) were obtained from charles river laboratories ( st . constant , qc , canada ) and maintained on a standard laboratory diet with free access to water under a 12-h light - dark schedule . following an overnight fast , rats were anesthetized with isoflurane ( baxter , mississauga , on , canada ) . one hour before blood sampling , some rats were intraperitoneally injected with 3 mg / kg urb597 to inhibit faah ( 29,41 ) . urb597 alone did not affect the levels of glucose , insulin , or bioactive glp-1 in these studies ; therefore , data from rats with and without urb597 injection were combined . the carotid artery was cannulated for blood sampling and the jugular vein for injections . to prevent bioactive glp-1 degradation by dipeptidylpeptidase ( dpp ) iv , rats received 5 mg / kg sitagliptin ( januvia ; msd sharp & dohme , haar , germany ) , a selective inhibitor of dpp - iv ( 10,42 ) , intravenously 30 min before blood collection . some rats also received a femoral vein cannula for continuous infusion of 37.5% glucose to maintain glycemia at 13 mmol / l for at least 30 min . the glucose infusion rate was adjusted based on frequent ( every 510 min ) blood glucose measurements , as described in goh et al . rats undergoing a hyperglycemic clamp were not pretreated with sitagliptin in order to reduce the number of variables that might affect glycemia . the abdominal cavity of all rats was then opened and a 10-cm section of the distal ileum was cleansed by perfusion with saline and tied off to create a luminally distinct compartment that retained vascular perfusion . subsequent to collection of the basal blood sample at t = 0 min , either the luminal compartment was filled with 2 ml of 10 mol / l oea or vehicle ( 0.9% saline/10% tween 80 ; sigma - aldrich ) , or 5 mg / kg oea or vehicle ( 0.9% saline/10% tween 80 ) was administered intravenously , and additional blood samples were collected at 5 , 15 , 30 , and 60 min . all samples ( 1 ml each ) were collected into 10% ( vol / vol ) trasylol ( 5,000 kalikrein inactivating units / ml ; bayer , toronto , on , canada ) , edta ( 12 mg / ml ) , and diprotin a ( a dpp - iv inhibitor , 68 mg / ml ; sigma - aldrich ) , and plasma was stored at 80c until analysis . plasma glucose levels were analyzed on a beckman analyzer ii ( beckman , fullerton , ca ) , and plasma insulin levels were measured by enzyme - linked immunosorbent assay ( crystal chem , downers grove , il ) in normoglycemic animals and by ria ( millipore , billerica , ma ) in hyperglycemic animals due to the wider insulin detection range . plasma levels of bioactive glp-1 were determined by electrochemiluminescence - based detection assay ( meso scale discovery , gaithersburg , md ) . area under the curve ( auc ) was determined using the trapezoidal rule , and the data are expressed per min . statistical analysis was performed using sas software ( sas institute , cary , nc ) . one- and two - way anova was followed by student 's t test or n-1 custom hypotheses post hoc tests , as appropriate . to reduce interassay variations , some data were normalized to basal levels . significance was assumed at p < 0.05 . rt - pcr for rodent gpr119 mrna transcripts was performed on total rna extracted from mglutag and fric cells , as well as from mouse ileum and rat colon tissue . as shown in fig . 1a , gpr119 mrna was detected in both rodent l - cell models , as well as in the intestinal samples . additionally , gpr119 mrna was detected in the hnci - h716 cells and in human colon and placental tissue ( fig . in contrast to previous reports ( 22 ) , we did not detect gpr119 mrna in human jejunum , possibly due to a low number of l - cells in this tissue ( 11 ) . consistent with our previous findings ( 17 ) , mrna transcripts for both gpr40 and gpr120 were detected in the mglutag cells , and both receptor mrnas were also found to be expressed in the hnci - h716 cells ( fig . expression of gpr119 mrna in l - cell models . a : total rna from mglutag cells , frics , and b : total rna from hnci - h716 cells , human intestinal tissues , and human placenta was analyzed for expression of gpr119 mrna by rt - pcr . c : total rna from mglutag cells and hnci - h716 cells was analyzed for expression of gpr40 and gpr120 mrna by rt - pcr . all products were separated on agarose gels and visualized with ethidium bromide , with the molecular - size ladder on the left . possible effects of gpr119 receptor activation on glp-1 secretion were first investigated in mglutag cells . release of glp-1 was increased to 3.1 0.4fold of basal values by treatment with forskolin , a direct activator of adenylyl cyclase and strong l - cell secretagogue ( 34 ) . mol / l ) , a known ligand of gpr119 ( 24 ) , significantly increased glp-1 secretion to a maximum of 2.1 0.2fold of basal levels at 10 mol / l ( p < 0.001 ) ( fig . the same concentrations of pea , a saturated fatty acid ethanolamide ( 16:0 ) that is a very weak agonist of gpr119 ( 23,24 ) , did not increase glp-1 secretion from mglutag cells . importantly , oea treatment did not affect the viability of the glutag cells ( fig . in contrast , although lpc treatment of mglutag cells at concentrations reported to activate gpr119 ( 1015 mol / l ) also enhanced glp-1 release ( to a maximum of 10.4 0.5fold of control values ; data not shown ) , lpc was found to markedly reduce cell viability by up to 60.0 6.8% compared with control cells ( p < 0.05 and p < 0.001 ) ( fig . the effects of oea on glp-1 secretion were also confirmed in hnci - h716 and fric cells ( fig . 2c and d ) , wherein oea treatment significantly increased glp-1 secretion to 2.6 0.2 and 5.8 2.5fold of basal levels at 10 mol / l ( p < 0.001 and p < 0.01 ) , respectively . interestingly , higher concentrations of oea were associated with diminished glp-1 release in all cell models , suggestive of desensitization . mglutag cells ( n = 912 ) ( a ) , hnci - h716 cells ( n = 8) ( c ) , and fric cells ( n = 4 ) ( d ) were incubated with medium alone ( 1% dmso , negative control ) , forskolin ( 10 mol / l , positive control ) , oea ( 220 mol / l ) , or pea ( 1015 mol / l , negative control ) for 2 h. glp-1 content of media and cells was determined by ria . b : to determine potential effects on cell viability , mglutag cells were incubated with medium alone ( 1% dmso , negative control ) , h2o2 ( 5 mol / l ) for 2 h , followed by mtt assay ( n = 816 ) . * p < 0.05 ; * * p < 0.01 ; * * * p < 0.001 vs. control . to verify the activity of gpr119 in the l - cell , mglutag and psn632408 increased glp-1 secretion by both cell lines to 2.1 0.2 and 2.9 0.5fold of basal values ( p < 0.01 for mglutag and p < 0.05 for hnci - h716 cells ) ( fig . 3 ) , demonstrating that gpr119 is functional and can initiate glp-1 secretion in these cells . to determine possible function of the other fatty acid receptors expressed in the l - cell lines gpr40 and gpr120 ( fig . 1c ) , both mglutag and hnci - h716 cells were also treated with the combined gpr40/gpr120 agonist gw9508 ( 10 mol / l ) ( 24 ) . consistent with previous findings from our group ( 17 ) , gpr40 and gpr120 were not found to be involved in glp-1 secretion from mglutag cells , as treatment with gw9508 did not increase glp-1 secretion ( fig . , gw9508 increased glp-1 secretion by hnci - h716 cells to 4.5 0.6fold of basal values ( p < 0.01 ) , providing indication for a role of gpr40 and/or gpr120 in human l - cells . mglutag ( n = 4 ) ( a ) and hnci - h716 ( n = 4 ) ( b ) cells were incubated with medium alone ( 1% dmso , negative control ) , oea ( 10 mol / l ) , the gpr119 agonist psn632408 ( 10 mol / l ) , or the combined gpr40/gpr120 agonist gw9508 ( 10 mol / l ) for 2 h. glp-1 content of media and cells was determined by ria . prevention of oea degradation with urb597 significantly increased oea - induced glp-1 secretion to 3.2 0.4 and 3.3 0.3fold of control values at 10 and 15 mol / l oea in mglutag cells ( p < 0.001 compared with urb597 alone and p < 0.010.001 compared with oea treatment without urb597 ) ( fig . 4a ) , as well as in hnci - h716 cells ( to 3.5 0.2 and 5.5 0.7fold of control values at 10 and 15 mol / l oea ( p < 0.001 compared with urb597 alone and p < 0.050.001 compared with oea treatment without urb597 ) ( fig . these findings indicate that degradation by faah limits the effects of oea on glp-1 secretion in both of the intestinal l - cell lines utilized . mglutag ( n = 618 ) ( a ) and hnci - h716 ( n = 12 ) ( b ) cells were pretreated for 30 min with urb597 ( 1 mol / l ) to inhibit faah and prevent oea degradation before incubation with medium alone ( 1%dmso , negative control ) or oea ( 1015 mol / l ) for 2 h. glp-1 content of media and cells was determined by ria . * * * p < 0.001 vs. control ; # p < 0.05 ; # # p < 0.01 ; # # # p < 0.001 vs. oea treatment alone . as ligand binding to gpr119 leads to activation of adenylyl cyclase , increased production of camp , and enhanced pka activity ( 24 ) , mglutag cells were first examined for camp responses to oea ( 1015 mol / l ) and gip , which is known to signal through camp- and pka - dependent pathways ( positive control ) ( 44 ) . oea treatment alone caused a small but significant increase in intracellular camp to 1.11 0.03 and 1.12 0.04fold of control values at 10 and 15 this effect was enhanced when oea degradation was prevented with urb597 , such that camp levels increased by an additional 1.3 0.04 and 1.3 0.03fold , respectively ( p < 0.001 compared with urbb597 alone and p < 0.050.01 compared with oea treatment without urb597 ) . furthermore , pretreatment of the mglutag cells with the pka inhibitor h89 ( 10 mol / l ) completely abolished oea ( 1015 mol / l)-induced glp-1 secretion ( fig . similar results were found in hnci - h716 cells , although an increased concentration of h89 ( 30 mol / l ) was required to abrogate oea - induced glp-1 release ( fig . 5c , inset ) , demonstrating the specificity of this inhibitor for pka - mediated signaling . mglutag ( n = 69 ) ( a and b ) and hnci - h716 ( n = 46 ) ( c ) cells were pretreated for 30 min with medium alone ( 1% dmso , negative control ) , h89 ( 10 mol / l for mglutag and 30 mol / l for hnci - h716 ) , or urb597 ( 1 mol / l ) to inhibit pka or faah , respectively , followed by incubation with medium alone ( 1% dmso , negative control ) , gip ( 1 mol / l , positive control ) , or oea ( 1015 mol / l ) for 2 h. c , inset : hnci - h716 cells were pretreated for 30 min with media alone ( 1% dmso ) or with h89 ( 30 mol / l ) , followed by incubation with the gpr40/120 agonist gw9508 ( 10 mol / l ) . camp content of cells and glp-1 content of media and cells were determined by ria . * p < 0.05 ; * * * p < 0.001 vs. appropriate control ; # p < 0.05 ; # # p < 0.01 ; # # # p < 0.001 vs. paired treatment alone . to determine whether the effects of oea on camp production and glp-1 secretion are dependent upon gpr119 , mglutag cells were transfected with specific gpr119 sirna or scrambled sirna ( control ) , resulting in 23% knockdown of gpr119 mrna , as determined by real - time rt - pcr ( fig . oea ( 10 mol / l ) failed to enhance camp levels in cells treated with gpr119 sirna , whereas the camp response to oea treatment was preserved in the control cells ( p < 0.05 ) ( fig . furthermore , gpr119 knockdown led to a 45% reduction in the glp-1 secretory response to oea ( p < 0.05 ) ( fig . these data therefore provide support for a role of gpr119 and the pka signaling pathway in oea - induced glp-1 secretion . pmol / l , control ) or gpr119 sirna ( 20 pmol / l ) 2 days before the experiment . cells were then incubated with medium alone ( 1% dmso , negative control ) or oea ( 1015 mol / l ) for 2 h. camp content of cells ( n = 6 ) ( a ) and glp-1 content of media and cells ( n = 9 ) ( b ) were determined by ria . a , inset : gpr119 mrna transcript levels were determined by quantitative rt - pcr relative to 18s transcript levels . * p < 0.05 ; * * * p < 0.001 vs. control or vs. the change in control cells , as indicated by the lines . # p < 0.05 vs. oea treatment with scrambled sirna . to establish the effects of oea on the l - cell in vivo , intraluminal application of oea ( 10 mol / l ; e.g. , 20 nmol / rat ) to euglycemic rats significantly increased plasma bioactive glp-1 concentrations to 1.5 0.2fold of basal values ( p < 0.05 ) within 5 min of administration , and this stimulation was maintained throughout the entire 60-min time course of the experiment ( fig . thus , the auc for the bioactive glp-1 response was significantly increased to 1.6 0.1fold of vehicle - infused rats ( p < 0.001 ) ( fig . , intravenous administration of oea at a 200-fold higher dose ( e.g. , 4 mol/250 g rat ) than that used intraluminally demonstrated no effect on bioactive glp-1 concentrations compared with rats treated with vehicle alone . throughout the 60-min experiment , the plasma levels of glucose ( fig . 7d ) remained stable at basal levels and did not differ between treatment and control groups . in vivo effect of oea on glp-1 secretion . anesthetized rats received intraluminal or intravenous injections of vehicle ( saline/10% tween 80 ; combined controls ) , intraluminal oea ( 2 ml of 10 mol / l ) , or intravenous oea ( 5 mg / kg ) , and blood samples were collected over a 1-h period . plasma concentrations of bioactive glp-1 ( a ) , glucose ( c ) , and insulin ( d ) were determined by elisa and glucose analyzer , as appropriate ( n = 511 ) . b : auc for the absolute plasma bioactive glp-1 concentrations was determined using the trapezoidal rule and is expressed per min . d , inset : rats ( n = 45 ) were maintained at 13 mmol / l plasma glucose ( hyperglycemic clamp ) for a minimum of 30 min , and this was maintained throughout the oea treatment procedure . a : to reduce interassay variations due to use of separate kits , bioactive glp-1 concentrations were calculated as fold increase over basal glp-1 levels ( control : 29.4 8.6 pg / ml ; intraluminal oea : 18.9 4.9 pg / ml ; and intravenous oea : 32.7 3.4 pg / ml ; p = ns between the basal values ) . * p < 0.05 vs. control ; * * p < 0.01 vs. control ; * * * p < 0.001 vs. control ; # p < 0.05 and # # p < 0.01 vs. basal values . as glp-1 is known to lose its insulinotropic effects under normoglycemic conditions , changes in insulin levels upon oea treatment were also measured under hyperglycemic conditions mmol / l , the upper physiological level in rats , for at least 30 min before the start of oea application and throughout the remainder of the procedure . the basal concentration of insulin before oea application was 1.4 0.4 pg / ml and did not differ between the groups . intraluminal application of oea caused a 3.9 0.7fold increase in insulin plasma levels within 5 min of application ( 11.2 2.1 vs. 2.8 2.0 pg / ml in the control group ; p < 0.01 ) ( fig . 7d , inset ) . in contrast , intravenous infusion of oea did not affect insulin levels during the entire treatment period in the hyperglycemic rats . rt - pcr for rodent gpr119 mrna transcripts was performed on total rna extracted from mglutag and fric cells , as well as from mouse ileum and rat colon tissue . as shown in fig . 1a , gpr119 mrna was detected in both rodent l - cell models , as well as in the intestinal samples . additionally , gpr119 mrna was detected in the hnci - h716 cells and in human colon and placental tissue ( fig . in contrast to previous reports ( 22 ) , we did not detect gpr119 mrna in human jejunum , possibly due to a low number of l - cells in this tissue ( 11 ) . consistent with our previous findings ( 17 ) , mrna transcripts for both gpr40 and gpr120 were detected in the mglutag cells , and both receptor mrnas were also found to be expressed in the hnci - h716 cells ( fig . expression of gpr119 mrna in l - cell models . a : total rna from mglutag cells , frics , and b : total rna from hnci - h716 cells , human intestinal tissues , and human placenta was analyzed for expression of gpr119 mrna by rt - pcr . c : total rna from mglutag cells and hnci - h716 cells was analyzed for expression of gpr40 and gpr120 mrna by rt - pcr . all products were separated on agarose gels and visualized with ethidium bromide , with the molecular - size ladder on the left . possible effects of gpr119 receptor activation on glp-1 secretion were first investigated in mglutag cells . release of glp-1 was increased to 3.1 0.4fold of basal values by treatment with forskolin , a direct activator of adenylyl cyclase and strong l - cell secretagogue ( 34 ) . mol / l ) , a known ligand of gpr119 ( 24 ) , significantly increased glp-1 secretion to a maximum of 2.1 0.2fold of basal levels at 10 mol / l ( p < 0.001 ) ( fig . the same concentrations of pea , a saturated fatty acid ethanolamide ( 16:0 ) that is a very weak agonist of gpr119 ( 23,24 ) , did not increase glp-1 secretion from mglutag cells . importantly , oea treatment did not affect the viability of the glutag cells ( fig . in contrast , although lpc treatment of mglutag cells at concentrations reported to activate gpr119 ( 1015 mol / l ) also enhanced glp-1 release ( to a maximum of 10.4 0.5fold of control values ; data not shown ) , lpc was found to markedly reduce cell viability by up to 60.0 6.8% compared with control cells ( p < 0.05 and p < 0.001 ) ( fig . the effects of oea on glp-1 secretion were also confirmed in hnci - h716 and fric cells ( fig . 2c and d ) , wherein oea treatment significantly increased glp-1 secretion to 2.6 0.2 and 5.8 2.5fold of basal levels at 10 mol / l ( p < 0.001 and p < 0.01 ) , respectively . interestingly , higher concentrations of oea were associated with diminished glp-1 release in all cell models , suggestive of desensitization . mglutag cells ( n = 912 ) ( a ) , hnci - h716 cells ( n = 8) ( c ) , and fric cells ( n = 4 ) ( d ) were incubated with medium alone ( 1% dmso , negative control ) , forskolin ( 10 mol / l , positive control ) , oea ( 220 mol / l ) , or pea ( 1015 mol / l , negative control ) for 2 h. glp-1 content of media and cells was determined by ria . b : to determine potential effects on cell viability , mglutag cells were incubated with medium alone ( 1% dmso , negative control ) , h2o2 ( 5 mol / l ) for 2 h , followed by mtt assay ( n = 816 ) . * p < 0.05 ; * * p < 0.01 ; * * * p < 0.001 vs. control . to verify the activity of gpr119 in the l - cell , mglutag and hnci - h716 cells were treated with a specific gpr119 agonist , psn632408 ( 10 mol / l ) ( 24 ) . psn632408 increased glp-1 secretion by both cell lines to 2.1 0.2 and 2.9 0.5fold of basal values ( p < 0.01 for mglutag and p < 0.05 for hnci - h716 cells ) ( fig . 3 ) , demonstrating that gpr119 is functional and can initiate glp-1 secretion in these cells . to determine possible function of the other fatty acid receptors expressed in the l - cell lines gpr40 and gpr120 ( fig . 1c ) , both mglutag and hnci - h716 cells were also treated with the combined gpr40/gpr120 agonist gw9508 ( 10 mol / l ) ( 24 ) . consistent with previous findings from our group ( 17 ) , gpr40 and gpr120 were not found to be involved in glp-1 secretion from mglutag cells , as treatment with gw9508 did not increase glp-1 secretion ( fig . 3 ) . in contrast , gw9508 increased glp-1 secretion by hnci - h716 cells to 4.5 0.6fold of basal values ( p < 0.01 ) , providing indication for a role of gpr40 and/or gpr120 in human l - cells . mglutag ( n = 4 ) ( a ) and hnci - h716 ( n = 4 ) ( b ) cells were incubated with medium alone ( 1% dmso , negative control ) , oea ( 10 mol / l ) , the gpr119 agonist psn632408 ( 10 mol / l ) , or the combined gpr40/gpr120 agonist gw9508 ( 10 mol / l ) for 2 h. glp-1 content of media and cells was determined by ria . * p < 0.05 ; * * p < 0.01 vs. control . prevention of oea degradation with urb597 significantly increased oea - induced glp-1 secretion to 3.2 0.4 and 3.3 0.3fold of control values at 10 and 15 mol / l oea in mglutag cells ( p < 0.001 compared with urb597 alone and p < 0.010.001 compared with oea treatment without urb597 ) ( fig . 4a ) , as well as in hnci - h716 cells ( to 3.5 0.2 and 5.5 0.7fold of control values at 10 and 15 mol / l oea ( p < 0.001 compared with urb597 alone and p < 0.050.001 compared with oea treatment without urb597 ) ( fig . these findings indicate that degradation by faah limits the effects of oea on glp-1 secretion in both of the intestinal l - cell lines utilized . mglutag ( n = 618 ) ( a ) and hnci - h716 ( n = 12 ) ( b ) cells were pretreated for 30 min with urb597 ( 1 mol / l ) to inhibit faah and prevent oea degradation before incubation with medium alone ( 1%dmso , negative control ) or oea ( 1015 mol / l ) for 2 h. glp-1 content of media and cells was determined by ria . * * * p < 0.001 vs. control ; # p < 0.05 ; # # p < 0.01 ; # # # p < 0.001 vs. oea treatment alone . as ligand binding to gpr119 leads to activation of adenylyl cyclase , increased production of camp , and enhanced pka activity ( 24 ) , mglutag cells were first examined for camp responses to oea ( 1015 mol / l ) and gip , which is known to signal through camp- and pka - dependent pathways ( positive control ) ( 44 ) . oea treatment alone caused a small but significant increase in intracellular camp to 1.11 0.03 and 1.12 0.04fold of control values at 10 and 15 this effect was enhanced when oea degradation was prevented with urb597 , such that camp levels increased by an additional 1.3 0.04 and 1.3 0.03fold , respectively ( p < 0.001 compared with urbb597 alone and p < 0.050.01 compared with oea treatment without urb597 ) . furthermore , pretreatment of the mglutag cells with the pka inhibitor h89 ( 10 mol / l ) completely abolished oea ( 1015 mol / l)-induced glp-1 secretion ( fig . similar results were found in hnci - h716 cells , although an increased concentration of h89 ( 30 mol / l ) was required to abrogate oea - induced glp-1 release ( fig . 5c , inset ) , demonstrating the specificity of this inhibitor for pka - mediated signaling . mglutag ( n = 69 ) ( a and b ) and hnci - h716 ( n = 46 ) ( c ) cells were pretreated for 30 min with medium alone ( 1% dmso , negative control ) , h89 ( 10 mol / l for mglutag and 30 mol / l for hnci - h716 ) , or urb597 ( 1 mol / l ) to inhibit pka or faah , respectively , followed by incubation with medium alone ( 1% dmso , negative control ) , gip ( 1 mol / l , positive control ) , or oea ( 1015 mol / l ) for 2 h. c , inset : hnci - h716 cells were pretreated for 30 min with media alone ( 1% dmso ) or with h89 ( 30 mol / l ) , followed by incubation with the gpr40/120 agonist gw9508 ( 10 mol / l ) . camp content of cells and glp-1 content of media and cells were determined by ria . * p < 0.05 ; * * * p < 0.001 vs. appropriate control ; # p < 0.05 ; # # p < 0.01 ; # # # p < 0.001 vs. paired treatment alone . to determine whether the effects of oea on camp production and glp-1 secretion are dependent upon gpr119 , mglutag cells were transfected with specific gpr119 sirna or scrambled sirna ( control ) , resulting in 23% knockdown of gpr119 mrna , as determined by real - time rt - pcr ( fig . oea ( 10 mol / l ) failed to enhance camp levels in cells treated with gpr119 sirna , whereas the camp response to oea treatment was preserved in the control cells ( p < 0.05 ) ( fig . furthermore , gpr119 knockdown led to a 45% reduction in the glp-1 secretory response to oea ( p < 0.05 ) ( fig . these data therefore provide support for a role of gpr119 and the pka signaling pathway in oea - induced glp-1 secretion . mglutag cells were transfected with scrambled sirna ( 20 pmol / l , control ) or gpr119 sirna ( 20 pmol / l ) 2 days before the experiment . cells were then incubated with medium alone ( 1% dmso , negative control ) or oea ( 1015 mol / l ) for 2 h. camp content of cells ( n = 6 ) ( a ) and glp-1 content of media and cells ( n = 9 ) ( b ) were determined by ria . a , inset : gpr119 mrna transcript levels were determined by quantitative rt - pcr relative to 18s transcript levels . * p < 0.05 ; * * * p < 0.001 vs. control or vs. the change in control cells , as indicated by the lines . to establish the effects of oea on the l - cell in vivo , rats were treated with oea either intraluminally or intravenously . intraluminal application of oea ( 10 mol / l ; e.g. , 20 nmol / rat ) to euglycemic rats significantly increased plasma bioactive glp-1 concentrations to 1.5 0.2fold of basal values ( p < 0.05 ) within 5 min of administration , and this stimulation was maintained throughout the entire 60-min time course of the experiment ( fig . thus , the auc for the bioactive glp-1 response was significantly increased to 1.6 0.1fold of vehicle - infused rats ( p < 0.001 ) ( fig . , intravenous administration of oea at a 200-fold higher dose ( e.g. , 4 mol/250 g rat ) than that used intraluminally demonstrated no effect on bioactive glp-1 concentrations compared with rats treated with vehicle alone . throughout the 60-min experiment , the plasma levels of glucose ( fig . 7d ) remained stable at basal levels and did not differ between treatment and control groups . in vivo effect of oea on glp-1 secretion . anesthetized rats received intraluminal or intravenous injections of vehicle ( saline/10% tween 80 ; combined controls ) , intraluminal oea ( 2 ml of 10 mol / l ) , or intravenous oea ( 5 mg / kg ) , and blood samples were collected over a 1-h period . plasma concentrations of bioactive glp-1 ( a ) , glucose ( c ) , and insulin ( d ) were determined by elisa and glucose analyzer , as appropriate ( n = 511 ) . b : auc for the absolute plasma bioactive glp-1 concentrations was determined using the trapezoidal rule and is expressed per min . d , inset : rats ( n = 45 ) were maintained at 13 mmol / l plasma glucose ( hyperglycemic clamp ) for a minimum of 30 min , and this was maintained throughout the oea treatment procedure . a : to reduce interassay variations due to use of separate kits , bioactive glp-1 concentrations were calculated as fold increase over basal glp-1 levels ( control : 29.4 8.6 pg / ml ; intraluminal oea : 18.9 4.9 pg / ml ; and intravenous oea : 32.7 3.4 pg / ml ; p = ns between the basal values ) . * p < 0.05 vs. control ; * * p < 0.01 vs. control ; * * * p < 0.001 vs. control ; # p < 0.05 and # # p < 0.01 vs. basal values . as glp-1 is known to lose its insulinotropic effects under normoglycemic conditions , changes in insulin levels upon oea treatment were also measured under hyperglycemic conditions . mmol / l , the upper physiological level in rats , for at least 30 min before the start of oea application and throughout the remainder of the procedure . the basal concentration of insulin before oea application was 1.4 0.4 pg / ml and did not differ between the groups . intraluminal application of oea caused a 3.9 0.7fold increase in insulin plasma levels within 5 min of application ( 11.2 2.1 vs. 2.8 2.0 pg / ml in the control group ; p < 0.01 ) ( fig . 7d , inset ) . in contrast , intravenous infusion of oea did not affect insulin levels during the entire treatment period in the hyperglycemic rats . previous studies have indicated that the fatty acid derivate receptor gpr119 is present on pancreatic -cells and intestinal l - cells , and its stimulation by a gpr119 agonist increases insulin and glp-1 secretion , respectively ( 21,22,24 ) . however , the role of physiologically occurring ligands of gpr119 , such as oea , and the intracellular mechanisms underlying gpr119-dependent glp-1 secretion from the intestinal l - cell have remained undefined . the results of the present study demonstrate , for the first time , that oea stimulates glp-1 secretion from both mouse and human intestinal l - cell lines , as well as from primary rat l - cells in vitro . additionally , application of oea directly into the intestinal lumen in rats induced a significant and persistent increase in bioactive glp-1 levels over 1 h , supporting the in vitro findings and demonstrating a role for oea as a glp-1 secretagogue in vivo . to further establish the role of oea in glp-1 secretion , the intracellular signaling mechanisms underlying its effects on the l - cell were investigated . oea induced a small , but significant , increase in intracellular camp concentration in both the mglutag and hnci - h716 cells , comparable with findings in oea - treated rinm5 and min6 pancreatic -cell lines ( 45 ) . furthermore , glp-1 secretion in oea - treated cells was strictly dependent on pka in both the human and mouse l - cells , as indicated by complete abrogation of the response in h89-treated cells . these findings are consistent with studies by our lab and others showing that increased camp levels , in response to camp analogs as well as to secretagogues such as gip , stimulate glp-1 release by the intestinal l - cell ( 16,34,46 ) . furthermore , while initial studies on oea identified the intranuclear receptor ppar and transient receptor potential vanilloid type 1 ( trpv1 ) as targets for oea ( 27,33,47 ) , neither of these receptors has been reported to stimulate the camp / pka signaling pathway . in contrast , the deorphanization of gpr119 as a camp - linked oea receptor ( 24 ) implicated gpr119 as more likely target for oea in the intestinal l - cell . consistent with this hypothesis , treatment of human and mouse intestinal l - cells with a gpr119-specific agonist psn632408 significantly increased glp-1 secretion in both cell lines . furthermore , transfection of mglutag cells with gpr119 sirna significantly diminished both the camp and glp-1 responses to oea . when taken together , these findings provide support for both the presence of functional gpr119 in the intestinal l - cell and the requirement for this novel g - protein coupled receptor in oea - induced glp-1 secretion . we have previously reported that oleic acid is a strong l - cell secretagogue , increasing glp-1 secretion both in vivo and in vitro ( 17,18,48 ) . it is therefore interesting that oea is rapidly degraded to oleic acid and ethanolamide via faah - dependent hydrolysis ( 29 ) , thereby providing a possible gpr119-independent mechanism underlying oea - induced glp-1 secretion . therefore , to exclude the possible effects of oleic acid on the actions of oea , l - cells were pretreated with the faah inhibitor urb597 to reduce oea hydrolysis and resultant oleic acid accumulation . the increase in oea - induced glp-1 secretion found with urb597 treatment provides support for a direct role of oea , and not of oleic acid , in oea - induced glp-1 secretion . while intraluminal application of oea in vivo significantly increased glp-1 secretion in normal rats , intravenous injection of oea at a 200-fold higher dose failed to increase circulating levels of bioactive glp-1 . additionally , while intraluminal oea application clearly increased insulin levels in hyperglycemic rats , intravenous injection of oea did not affect insulin levels in these animals under either euglycemic or hyperglycemic conditions . taken together , this data suggests that circulating oea does not cause significant increases in hormone release from either of its known target tissues ( the intestinal l - cell and the pancreatic -cell ) . first , the volume of distribution for oea as a lipophilic substance should be high , therefore increasing the concentration of oea required for administration into the jugular vein to reach distant target tissues at stimulatory concentrations . however , higher levels of oea can not be achieved in the circulation of the rat due to the limited solubility of oea in solvents . furthermore , whether oea is cleared through the liver and/or lungs is unknown and may be significant . the actions of both oea and a grp119 agonist on insulin secretion have previously been reported to be glucose dependent ( 21,45 ) , as further confirmed in the present study . previous studies have also demonstrated that 45% of the glucose - lowering effect of an oral gpr119 agonist given concurrently with oral glucose is mediated through enhancement of glp-1 release ( 22 ) , suggesting that the insulinotropic effect of gpr119 activation is mediated both directly , through gpr119 expressed on the -cell , and indirectly , through enhanced release of glp-1 . nonetheless , although both the l - cell and the -cell are responsive to oea , the glucose sensitivity of the -cell response , compared with the glucose insensitivity of the l - cell response that we have observed , suggests that oral administration of this fatty acid derivate alone to enhance glp-1 secretion , without influencing insulin release , will permit the insulin - independent biological actions of glp-1 . in contrast , administration of oea in the setting of a glucose - containing meal would facilitate release of both glp-1 and insulin , thereby also modulating glycemic responses ( 22,49 ) . the observation of apparent desensitization in all of the dose - response curves was unexpected , as this has not previously been reported for gpr119 . however , gs - coupled receptors are well established to undergo homologous desensitization ( 50 ) ; indeed , preliminary studies in which hnci-716 cells were pretreated with the gpr119 agonist psn632408 ( 10 mol / l for 6 h ) demonstrated a 70.9 4.6% and 50.7 11.7% decrease in subsequent oea- and psn632408-induced glp-1 secretion , respectively ( data not shown ) . collectively , these findings indicate that gpr119 may undergo homologous desensitization , a phenomenon that clearly warrants further investigation . in summary , the results of this study establish , for the first time , the role of gpr119 in oea - induced glp-1 secretion and add gpr119 to the growing list of fatty acid responsive pathways that function to modulate release of glp-1 , including gpr40 , gpr120 , and pkc. when combined with the reported insulinotropic effects of gpr119 agonists , these findings implicate gpr119 as a potential pharmacological target , as well as oea as a nutriceutical approach to enhance glp-1 in patients with type 2 diabetes .

objectiveintestinal l - cells secrete the incretin glucagon - like peptide-1 ( glp-1 ) in response to ingestion of nutrients , especially long - chain fatty acids . the gs - coupled receptor gpr119 binds the long - chain fatty acid derivate oleoylethanolamide ( oea ) , and gpr119 agonists enhance glp-1 secretion . we therefore hypothesized that oea stimulates glp-1 release through a gpr119-dependent mechanism.research design and methodsmurine ( m ) glutag , human ( h ) nci - h716 , and primary fetal rat intestinal l - cell models were used for rt - pcr and for camp and glp-1 radioimmunoassay . anesthetized rats received intravenous or intraileal oea , and plasma bioactive glp-1 , insulin , and glucose levels were determined by enzyme - linked immunosorbent assay or glucose analyzer.resultsgpr119 messenger rna was detected in all l - cell models . oea treatment ( 10 mol / l ) of mglutag cells increased camp levels ( p < 0.05 ) and glp-1 secretion ( p < 0.001 ) in all models , with desensitization of the secretory response at higher concentrations . glp-1 secretion was further enhanced by prevention of oea degradation using the fatty acid amide hydrolase inhibitor , urb597 ( p < 0.050.001 vs. oea alone ) , and was abolished by h89-induced inhibition of protein kinase a. oea - induced camp levels and glp-1 secretion were significantly reduced in mglutag cells transfected with gpr119-specific small interfering rna ( p < 0.05 ) . application of oea ( 10 mol / l ) directly into the rat ileum , but not intravenously , increased plasma bioactive glp-1 levels in euglycemic animals by 1.5-fold ( p < 0.05 ) and insulin levels by 3.9-fold ( p < 0.01 ) but only in the presence of hyperglycemia.conclusionsthe results of these studies demonstrate , for the first time , that oea increases glp-1 secretion from intestinal l - cells through activation of the novel gpr119 fatty acid derivate receptor in vitro and in vivo .

after selecting the type of a process and determining the specifications of all equipment , the next step is to design a plant layout how to determine the location of each process units in an area with significant engineering creativities , heuristics , prior knowledge , and so on1,2,3,4 . thus , the total construction cost is the combination of the purchasing equipment cost , the piping cost and the site cost . moreover , a plant layout should secure enough maintenance and safety spaces for efficient accessibilities and safety requirements to repair process units and prevent domino impacts . in addition , in case of off - shore plants , multi - floor processes have to be installed in the limited site . these issues make the plant layout problems very difficult and complex . to solve this problem , suzuki et al.1 developed a heuristic rules for the two - dimensional layout problems . mixed integer linear programming ( milp ) models have been employed as considering various sizes and geometries of equipment based on the assumption that all of equipments and connections are rectangular shapes and rectilinear2 , 3 . some researchers have transformed this problem into milp or mixed integer non - linear programming ( minlp ) models to find the optimal plant layout via conventional optimization solvers such as general algebraic modeling system ( gams ) with the consideration of safety issues7 . castell et al.5 proposed the genetic algorithm - based method with the mond index . however , since the most previous researches have been focused on only single - floor problems , it is urgently needed to develop an efficient method handling multi - floor plant layouts such as floating production , storage and offloading ( lng - fpso ) . han et al.9 proposed the minlp model with the safety considerations including the impact of possible accidents and the individual risk factors . but , it is really hard to evaluate the objective individual frequencies of accidents and their consequences . to tackle these difficulties in this study , the safety distance between equipments is fixed as a constant to maintain the sufficient empty spaces for safety , maintenance and repairs . however , it would be possible that there are other constraints according to the type of process and it is not always possible to represent these with mathematical formula . thus , it is impossible to make this problem suitable mathematical formula for the conventional minlp solvers which use the derivatives of equations in case the equations are very complex and severe nonlinear . in this study , the pso ( particle swarm optimization ) technique , which is one of the representative sampling approaches , pso is a population based algorithm introduced by kennedy et al.10 which mimics flocks of bird . many researchers demonstrated that pso is more efficient than other heuristic optimization methods and is cheaper to implement11 . in third and fourth section describe the concept of the proposed objective function and an optimization solver . to build a mathematical equations for finding an optimal multi - floor plant layout , the following information have to be fixed previously : a set of n equipment items and their sizethe number of floors , their sizes and heightsthe cost data of pipe lines and pumpsprocess flow diagramthe fixed minimum safety distance between equipment items a set of n equipment items and their size the number of floors , their sizes and heights the cost data of pipe lines and pumps the fixed minimum safety distance between equipment items firstly , it is assumed that the shape of all equipments is rectangular and they are allowed to rotate by 90 degrees . ai and bi are the length of both sides for an equipment , i. the equation for equipment orientations is as the following : where oi is the binary parameter to determine the rotation for equipment item , i. if the value of oi is 1 , it means that there is no rotation . otherwise , i.e. the value of oi is 0 , i should be rotated by 90 . based on this equation , next , two binary parameters , which indicate the locations of equipments at a specified floor , are employed . the binary parameter , vik , is assigned as 1 if the equipment item i is installed on k - th floor ; otherwise vik would be 0 . if fij is 1 , two equipments , i and j , are on the same floor , otherwise , fij is 0 and it means that two equipments are installed on the different floor . in case fij is 1 , the overlapping problem between i and j has to be considered . otherwise , there is no overlapping between i and j. nf is the total number of floors . since most equipments of onshore or offshore plants have to be repaired onsite , enough maintenance spaces for all equipments should be required . in this study , esi is defined with the maintenance space for equipment , i , and it is assumed that the maintenance space for each equipment is set to 30% of each equipment size . moreover , in order to prevent the propagation of accidents , the minimum safety distances between equipments should be also defined . in this study , the minimum safety distance , m , is defined and the distances between equipments have to be larger than m. throughout the consideration of maintenance spaces and safety distances , the impact of problems due to maintenances , repairs and accidents can be reduced . according to the property of problems , the equations for considering the size can be changed . if the size is fixed previously , the equipments should be located into the area . otherwise , the objective function should include a term to minimize the land size . these equations can be derived as the following ; the next equations are to calculate the piping distances between equipments i and j as avoiding the overlapping and satisfying the constraints . in this study , it is assumed that all connections between the equipment can be evaluated based on the geometry center of equipment . the total rectilinear distance between equipments i and j is evaluated based on the center of equipments by the considering relative distances in x , y , and z coordinates . the vertical lengths between equipments should be evaluated as considering the differences of floors and their original heights from a ground . xij and yij are the linear distances between equipments , i and j in x and y axis . hi is the height of a floor where i is installed and ui is the total height of i including the height of floor and the center of i in z coordinate . uij and tdij are a vertical distance and the total rectilinear distance between i and j. moreover , there are many constraints associated with the working spaces for the maintenances and the passages for the operators . it is very hard to generalize the model and represent as the mathematical formula , since these conditions vary according to the types and circumstances of plants . however , these constraints should be included . to construct a general model , a penalty function if the location of equipments item violates these constraints , a large amount of the penalty is assigned to the total costs . this helps the optimal solution to minimize a penalty and to satisfy all assumed constraints . the objective function is the sum of piping , pumping , land and risk and the optimal solution have to satisfy various constraints . in equation ( 4 ) , ccij is the piping cost per unit distance . also , the pumping cost should be considered , since the vertical pumping cost is much expensive than the horizontal one . chij and cvij are the horizontal and vertical pumping cost per unit distance . in case the size of land is not fixed , the land cost has to be also included in the objective function . a is the cost per unit square meter . as explained in a previous section , the penalty factor , p ( x , y ) , if there is the violation of constraints , a large amount of penalty would be assigned . design variables of the objective function are as the following : oi : the rotation binary parameter.vik : the floor binary parameter for evaluating the location of equipment.xi , yi : the position of equipment according to x and y axis . xi , yi : the position of equipment according to x and y axis . if the number of equipments is n , the total number of designed variables should be 4n , respectively . the locations of each equipment items should be determined to minimize the total costs . as explained in the previous section , there are many constraints for the working spaces and passages , which have many limitations for the mathematical formulations . in addition , the type of constrains vary according to the floor and the type of a process . thus , it is not always possible to use conventional tools such as gams for solving this problem , since the derivatives of the constraints are not available . as an alternative , pso ( pso is a population based sampling optimization technique motivated by the social behaviour of collection of animals10 . it starts with randomly generated swarms , called particles , remember the best solution found . the particles move around the solution space with adjusted velocities and have a tendency to fly towards the global optimal solution over the optimal procedure . the attractive features of pso are that it does not need to evaluate derivatives of objective function and constraints . moreover , there are relatively a small number of parameters to adjust12 . many researchers have modified pso to solve the minlp problem , since the original pso can not handle the integer variable . in this study , decision variable , oi and vik are integer and the value of these the proposed algorithm is tested with ethylene oxide ( eo ) plant in fig . 1fig . 1.the pfd of ethylene oxide plant .. the eo plant is well - known due to its recent accident histories . this plant consists of eo reactor , eo absorber , the co2 absorber , and so on . it is assumed that two potential floors are available and the floor heights are two types ( 5 and 7 m ) . the floor size is fixed as 3,600 m ( 60 m 60 m ) . the minimum safety distance , m , is fixed as 4 m. table 1table 1.basic information of the eo plant taken from han et al.9eq.no.dim . bcostconnectionccij(m)chij(m)cvij(m)15.225.22335,00022004004,000211.4211.4211,00032004004,00037.687.68107,00042003003,00048.488.484,00052003003,00057.687.6881,30012001001,00062002002,00062.602.605,00072002001,50072.402.4015,00052001501,500 shows basic data including the all of equipments connection , pumping costs and each equipment s sizes . tables 2table 2.the optimal solution in case the height of floor is 5 mnumberequipmentfloorx ( m)y ( m)1reactor2nd24.4029.702heat exchanger2nd11.0943.023eo absorber1st11.1042.994heat exchanger1st24.9129.745co2 absorber2nd34.8519.256flash drum1st41.2819.917pump1st34.7713.41 and 3table 3.the optimal solution in case the height of floor is 7 mnumberequipmentfloorx ( m)y ( m)1reactor2nd38.3214.702heat exchanger1st29.9014.683eo absorber1st45.1829.964heat exchanger2nd27.3529.965co2 absorber2nd14.4242.916flash drum1st9.08342.927pump1st15.5949.43 show the summarizations of the results calculated by pso . in case the floor height are 5 m and 7 m , the best costs of objective function are 1.42208 10 , and 1.80894 10 . 2.the results of optimal solution calculated by pso in case the height of floor is 5 m. and 3fig . 3.the results of optimal solution calculated by pso in case the height of floor is 7 m. show the best plant layout results satisfying all assumed constraints . according to various tests with many conditions , it is concluded that the vertical pumping cost is the most important factor to determine the plant layout . 4.the cost ratios according to piping cost , horizontal and vertical pumping costs . shows the portion of cost according to piping cost , horizontal and vertical pumping costs . the results of optimal solution calculated by pso in case the height of floor is 5 m. the results of optimal solution calculated by pso in case the height of floor is 7 m. the cost ratios according to piping cost , horizontal and vertical pumping costs . in the next case , only one floor is available and the floor size is not fixed previously . therefore , the goal of this case is to find the best plant layout with the smallest area , since , the cost of land accounts for the largest portion for building a plant in general . in this study , it is assumed that the land cost per unit square is 26.69 . 6.the optimal solution in case the only one floor is available and the land size is not fixed . and table 4table 4.the optimal solution in case the only one floor is available and the land size is not fixednumberequipmentx ( m)y ( m)1reactor15.5656.812heat exchanger28.9370.163eo absorber44.2888.094heat exchanger84.1345.705co2 absorber69.1132.776flash drum59.9517.037pump53.4123.71 show the best results satisfying all assumed constraints . in this case , it is verified that the best cost of objective function is 3.8835 10 and the land cost accounts for the largest portion in fig . 7.the ratios according to piping cost , horizontal pumping cost , vertical pumping cost and land cost .. the horizontal and vertical length of a land is 90 m and 94 m. minimum cost during pso iterations . the optimal solution in case the only one floor is available and the land size is not fixed . the ratios according to piping cost , horizontal pumping cost , vertical pumping cost and land cost . to handle a multi - floor optimal layout problem , a minlp model with the consideration of safety distances , maintenance spaces , multi - floor conditions , etc . are transformed into mathematical equations and the objective function is represented in terms of cost . finally , the minimum value of objective function is to reduce the construction cost as much as possible . to tackle the limitations of conventional optimization solvers , pso , which is available without the derivatives of equations , this means that the optimal solution can be investigated even if the mathematical model is very complex and highly nonlinear . to verify the efficacy of the proposed algorithm , two cases of eo plant are tested and the results show that the proposed algorithm provide a reasonable benefits . it is expected that the proposed algorithm would contribute to reduce the costs and find the optimal layout of various compact multi - floor processes such as floating production and storage offloading . i , j : process equipments , i and j oi : binary parameter for orientation of i ai bi : length of each side of i li : horizontal length of i di : vertical length of i vik : binary parameter . if i is installed on the k - th floor , 1 . otherwise , 0 . fij : binary parameter for comparing the floors of equipments i and j cij : a parameter for comparing for safety and maintenance distances sij : binary parameter . 1 if cij is positive . esi : maintenance space for equipment i xi : x coordinate of center of i yi : y coordinate of center of i zi : z coordinate of center of i m : minimum safety distance n : total number of equipments nf : total number of floors xij : distance in x - axis between the center of i and j yij : distance in y - axis between the center of i and j hi : the height of a floor where i is installed ui : the total height of i including hi and zi uij : distance in z - axis between the center of i and j tdij : total rectilinear distance between i and j ccij : piping cost per unit distance cvij : vertical pumping cost per unit distance chij : horizontal pumping cost per unit distance xmax : length in x direction of process site ymax : length in y direction of process site

in the fields of researches associated with plant layout optimization , the main goal is to minimize the costs of pipelines and pumping between connecting equipment under various constraints . however , what is the lacking of considerations in previous researches is to transform various heuristics or safety regulations into mathematical equations . for example , proper safety distances between equipments have to be complied for preventing dangerous accidents on a complex plant . moreover , most researches have handled single - floor plant . however , many multi - floor plants have been constructed for the last decade . therefore , the proper algorithm handling various regulations and multi - floor plant should be developed . in this study , the mixed integer non - linear programming ( minlp ) problem including safety distances , maintenance spaces , etc . is suggested based on mathematical equations . the objective function is a summation of pipeline and pumping costs . also , various safety and maintenance issues are transformed into inequality or equality constraints . however , it is really hard to solve this problem due to complex nonlinear constraints . thus , it is impossible to use conventional minlp solvers using derivatives of equations . in this study , the particle swarm optimization ( pso ) technique is employed . the ethylene oxide plant is illustrated to verify the efficacy of this study .

when an individual is deprived of a sensory modality , the other senses can compensate for the loss with behavioural enhancements . this effect has been demonstrated in both deaf and blind humans , as well as in animal models of sensory deprivation ( for a review , see [ 1 , 2 ] ) . generally , the sensory enhancements that occur after deprivation are attributed to the extra processing power that is afforded by the recruitment of the deprived sensory cortex , which is thought to reorganize to support the enhancement . support for this relationship between enhanced sensory behaviour and cross - modal processing comes from human research on blindness , where enhanced performance on various tasks correlates with task - related activity [ 37 ] , and cortical thickness of visual regions in the occipital cortex . this relationship has also been demonstrated in congenitally deaf cats , where enhancements to visual motion detection and peripheral localization are abolished when the cat 's auditory cortices are deactivated [ 9 , 10 ] . while the evidence for this relationship is convincing , no research to date has established a direct connection between cross - modal plasticity and enhanced sensory behaviour in deaf people . in deaf people , research on enhanced sensory behaviour and cross - modal plasticity has progressed mostly independently . in terms of sensory compensation , much research has focused on the role of vision . while some behavioural enhancements have been attributed to changes in visual attention ( for a review , see ) , others appear to be due to changes to basic sensory processing . these include enhancements to motion detection , discrimination of the angle of motion direction , a larger field of view [ 14 , 15 ] , and faster reaction times to visual stimuli [ 1618 ] , with a possible bias for peripheral visual fields [ 19 , 20 ] ( but see [ 17 , 18 ] ) . some of these behavioural enhancements may be supported by changes to both the peripheral and early cortical components of the visual system . for example , visual field area in deaf people correlates with neural rim area on the retina , denotative of increased retinal ganglion cells , and changes to the retinal neural fiber layer distribution . additionally , reaction times for target detection correlate with early event - related potentials in the visual cortex . however , none of these behavioural enhancements have been directly associated with plasticity in the auditory cortex . in terms of cross - modal plasticity for visual processing , multiple functional neuroimaging studies have identified visually evoked activity in the auditory cortices of deaf people , especially in response to stimuli that evoke visual motion , such as moving dots [ 2124 ] , gratings , and hands and/or lips [ 23 , 26 , 27 ] . in early - deaf people , this activity consistently occurs in the right hemisphere planum temporale and adjoining superior temporal gyrus [ 2127 ] . the left planum temporale [ 25 , 26 ] and primary auditory cortex [ 21 , 22 ] also show activity in response to motion versus static stimuli , although these regions are not activated in every study [ 23 , 26 , 27 ] . while these studies clearly demonstrate the responsivity of the deprived sensory cortex to the nondeprived stimuli , they do not assess its association with enhanced sensory performance , as has been done in the human blind population with correlation and regression analyses ( e.g. , [ 3 , 8 ] ) and in deaf cats by manipulation of cortical function [ 9 , 10 ] . testing the relationship between the auditory cortex and vision is necessary to demonstrate that cross - modal reorganization in deaf people supports enhanced visual abilities . in the current study , we hypothesized that compensatory visual enhancements in deaf people are supported by plasticity in auditory cortex . based on parallel research questions in the blind , we reasoned that if a cortical region supports sensory enhancement in the deaf , then its cortical thickness will vary in relation to behavioural performance . although much previous research has examined anatomical changes in the deaf brain , results have concentrated on changes that are associated with sensory deprivation [ 2937 ] rather than compensatory plasticity . in auditory regions , these changes include decreased white matter volume [ 30 , 33 , 34 , 38 ] and white matter integrity , as measured by diffusion - weighted mri [ 2931 , 35 ] . in contrast , grey matter volume in auditory regions appears to be preserved after deafness [ 3338 ] . few studies have examined cortical thickness , and no changes have been documented between deaf and hearing adults in auditory regions with this measure . given the lack of evidence for atrophy of grey matter after deafness , we expected that cortical thickness might capture compensatory plasticity , rather than disuse - related atrophy . to test our hypothesis , we used visual motion detection thresholds as a gauge for enhanced visual abilities , based on evidence for improved performance in the deaf as compared to hearing on this task . with t1-weighted magnetic resonance imaging , we measured cortical thickness and tested for a correlation with behaviour in eight regions of interest ( rois ) : the planum temporale ( pt ) , heschl 's gyrus and sulcus ( hgs ) , the middle temporal area ( mt+ ) , and the calcarine sulcus , bilaterally . our primary prediction was that the cortical thickness of the right pt would correlate with enhanced visual abilities , given its consistent involvement in cross - modal processing of visual motion . based on mixed results from previous research , we also explored the involvement of the left pt and bilateral primary auditory cortex , located within hgs . finally , in addition to auditory rois , we considered the possibility that enhanced visual motion detection in deaf people is supported by changes to the visual system rather than , or in addition to , cross - modal processing in auditory regions . this consideration was inspired by previous research that demonstrates a correlation between visual ability and activity in the early visual system after deafness and increased activity in mt+ for peripheral visual stimulation [ 39 , 40 ] . as such , we included the calcarine sulcus , which encompasses primary visual cortex , and the motion processing area mt+ . the experiment was approved by the research ethics board at the montreal neurological institute and all participants gave written informed consent . a sign language interpreter was present throughout all testing sessions to translate ( either langue des signes qubcoise or american sign language ) between the experimenter and participant . eleven bilaterally , profoundly , and early - deaf people ( 5 men and 6 women ; mean age = 28.2 years old ; age range = 2137 years old ) participated in the study . all participants took part in an earlier study in our laboratory , which identified enhanced visual motion detection thresholds in deaf people . ten participants reported congenital deafness and one became deaf at six months of age due to meningitis . two participants confirmed that their deafness was hereditary , and the remaining eight had unknown or unconfirmed etiologies . we used standard pure tone audiometry to measure monaural hearing thresholds in both ears of each deaf participant , confirming a hearing loss of greater than 90 db at 500 , 1000 , 2000 , and 8000 hz in all participants but four , who were able to sense 500 hz at 80 or 85 db . six participants were native sign language users who had typical language acquisition through early - life interaction with deaf family members , and five participants learned sign language in school around the age of five years and used a combination of signed french , home signs , and gestures to communicate prior to this . all participants used sign language as their primary language of communication once learned and used hearing aids during their childhood but stopped during their adolescence or earlier . threshold measures for visual motion detection were taken from our earlier study , and the details of the psychophysical procedure have already been published . we used a two - alternative forced - choice procedure , in which participants maintained central fixation while viewing two simultaneously presented sinusoidal gratings ( grating size : 6 6 , spatial frequency : 0.33 cycle/ , and michelson contrast : 50% ) . the gratings were presented for 500 ms in the left and right visual fields , centered at 10 and + 10. in each trial , one of the two gratings was randomly selected to move while the other remained stationary , and participants were instructed to indicate , by button press , which of the two gratings was moving and to guess if uncertain . the speed of the motion varied according to a one - up one - down adaptive staircase procedure , with a 1 : 3 weighting in step size . eye movements were monitored with an eyelink 1000 eye tracker ( sr research , mississauga , on , canada ) , and trials were discarded from the staircase if fixation was broken . the staircase terminated after 15 reversals , which were averaged to give the threshold measure for that run . a run was discarded if the participant broke fixation in more than 18% of the trials ( representing 2 standard deviations above the mean number of times that fixation was broken across all participants and runs ) . participants completed 8 runs , and the median threshold across these runs was used as the final threshold measure . we used a 3-t siemens trio scanner with a 32-channel head coil to acquire t1-weighted mprage scans ( 1.0 1.0 1.0 mm resolution , 176 slices , 256 256 matrix , and repetition time / echo time = 2300/2.98 ) . we used the freesurfer image analysis suite ( http://surfer.nmr.mgh.harvard.edu/ ) to parcellate the regions of interest and automatically calculate cortical thickness across the brain . the details of this procedure are described in previous publications . in brief , the steps include removal of nonbrain tissue , intensity normalization , tessellation of grey and white matter borders , automated topology correction [ 44 , 45 ] , surface deformation [ 46 , 47 ] , surface inflation , and registration to a spherical atlas . each brain surface was automatically parcellated into 56 regions in each hemisphere , according to the destrieux atlas [ 5052 ] . from this atlas , we extracted the mean cortical thickness in the pt , hgs , and the calcarine sulcus , bilaterally . cortical thickness of mt+ was extracted via freesurfer 's built - in probabilistic map . in previous research , cross - modal activations of the pt in deaf people typically include portions of the laterally adjoining posterior superior temporal gyrus [ 2123 , 2527 ] . the expansiveness of these activations is not surprising , considering that functional activations of the pt in general are not constrained by the gross anatomical borders of this region , which are in any case often difficult to identify . the spatially extensive activity of the pt is consistent with the fact that the cytoarchitectonic fields of this area also extend into adjacent areas , including parietal operculum , superior temporal sulcus , and supramarginal gyrus . with this in mind , we chose to expand the borders of our planum temporale roi by five vertices , increasing the surface area from 532.7 to 950.3 mm ( in the freesurfer standard space ) . in order to distinguish this roi from the standard planum temporale output of freesurfer , we will herein refer to it as the planum temporale region ( ptr ) . for each of our eight rois , we tested for a pearson partial correlation between visual motion detection thresholds and mean cortical thickness with age as a covariate . this covariate was included based on evidence that both motion detection ability and cortical thickness decline with age during adulthood . specifically , a linear decrease of 10.5% has been documented in the cortical thickness of the superior temporal cortex from eight to thirty years of age , and a linear increase in coherent motion detection thresholds from nineteen to ninety - two years of age . additionally , in an earlier study from our lab that used the same task as used here to measure visual motion detection thresholds in 36 hearing and deaf adults , thresholds increased from twenty - one to fifty - six years of age ( r = 0.48 ; p = 0.003 ; unpublished statistic with data from ) . based on this evidence , we reasoned that age may explain some of the variance in our hypothesized relationship between cortical thickness and visual motion detection thresholds , and thus its inclusion has the potential to strengthen the predicted effect . for our primary hypothesis concerning the right ptr , we considered correlations where p < 0.05 ( two - tailed ) to be significant . we made no prediction about the direction of the relationship , given that both increased and decreased cortical thickness have been associated with cross - modal plasticity in previous research in the blind . the remaining rois were exploratory , with mixed support for their involvement in cross - modal activity ( see introduction ) , and thus we applied a bonferroni correction for multiple comparisons , where we considered correlations of p < 0.007 ( two - tailed ) to be significant . this threshold is equal to the p < 0.05 threshold used for our primary hypothesis , divided by 7 , which is the number of exploratory comparisons that we pursued . we also carried out a vertex - wise analysis within our rois in order to explore whether or not specific subregions of these areas were related to our behavioural measure . this was particularly relevant to the case of the ptr , which is thought to consist of several functional subregions , and the calcarine sulcus , where effects might be specific to the areas that represent peripheral visual space . for this analysis , we smoothed the data with a 15 mm fwhm gaussian kernel and performed a vertex - wise regression of visual motion detection thresholds to generate a z - statistic map and considered all vertices within our rois that had a probability of p < 0.01 , uncorrected for multiple comparisons . it should be noted that this second analysis differs from the first because it strived to localize changes within the rois , rather than identify which rois correlated with cortical thickness . with this vertex - wise analysis we uncovered a subregion within the right ptr that varied in cortical thickness according to visual motion detection thresholds ( see results ) . to further characterize the location of this subregion , we expanded it to include all adjacent vertices that passed a threshold of p < 0.01 uncorrected , unconstrained by the boundaries of our roi . this was necessary to ensure that our result was primarily within the ptr , rather than an overlap from a cluster centered on an adjacent region . in an earlier fmri study from our lab , we identified an area centered in the posterior superior temporal gyrus where deaf individuals showed activity in response to visual motion . five participants from the current study took part in this earlier experiment , which tested early - deaf people with varying degrees of residual hearing . we wanted to assess if this previous fmri result could be the functional equivalent of the current study 's anatomical result . to do so , we transformed the results from the previous study into the average surface space and calculated the percentage overlap of the two regions . finally , in order to fully describe our effect , we compared its mean cortical thickness to that of 11 hearing controls from our earlier dataset that were selected to match the age and gender distribution of the current study . the cortical thickness of the hearing control participants was measured with identical imaging and analysis parameters to those of the current study , described above . our primary hypothesis was that the cortical thickness of the right ptr would correlate with visual motion detection thresholds . we found a negative partial correlation ( figure 1 , r = 0.66 , p = 0.026 , two - tailed , n = 11 , degrees of freedom = 8) after controlling for participant age . there was no correlation between visual motion detection thresholds and cortical thickness in any other region ( left ptr : r = 0.01 , p = 0.987 ; left hgs : r = 0.43 , p = 0.218 ; right hgs : r = 0.51 , p = 0.131 ; left mt+ : r = 0.32 , p = 0.21 ; right mt+ : r = 0.27 , p = 0.22 ; left calcarine sulcus : r = 0.42 , p = 0.230 ; right calcarine sulcus : r = 0.14 , p = 0.693 ) . one - tailed paired - sample student 's t - test on the fisher - transformed correlation coefficients from the right and left ptr indicated that the correlation in the right ptr was stronger than that in the left ( figure 1 , t = 3.1 , p = 0.01 ) . mean cortical thickness values for each participant in each roi are listed in table 1 . in the vertex - wise regression within the rois , we uncovered a subregion of 238.5 mm within the right ptr , where cortical thickness predicted behavioural performance ( p < 0.01 , uncorrected for multiple comparisons , maximum z - statistic = 2.72 at mni152 coordinates 63 , 37 , 17 ) . when unbounded by the ptr roi , this subregion expanded to 273.6 mm ( p < 0.01 , uncorrected for multiple comparisons ) and remained centered in the ptr roi ( figure 2 ) . nearly half of this cluster ( 47% ) overlapped with a region that demonstrated cross - modal activity in deaf people in a previous fmri experiment from our lab ( figure 3 ) . this expanded region had an average cortical thickness of 2.73 mm ( 0.19 mm standard deviation ) , which did not differ from hearing controls ( figure 4 , mean = 2.68 mm , standard deviation = 0.14 mm ; t = 0.623 , p = 0.54 ) . consistent with our prediction , we found that cortical thickness in the right ptr correlates with enhanced performance on a visual motion detection task in early - deaf people : greater cortical thickness was associated with better thresholds , when age was controlled for ( figure 1 ) . our finding supports the idea that compensatory visual enhancements are supported by cross - modal structural plasticity after deafness , establishing the first direct evidence for this relationship in deaf humans . the finding is consistent with prior data because it was detected in a region where cross - modal activations in deaf people have been reported in previous studies [ 2124 , 26 , 27 ] , including one from our lab ( figure 2 ) . a direct comparison of the regions of effect in our current and previous studies shows a partial overlap , with the current study 's effect localized more medially , centered on the superior bank of the superior temporal gyrus rather than on its lateral surface ( figure 3 ) . the relative closeness of these regions of effect supports the idea that they represent corresponding functional and anatomical cross - modal plasticity , particularly in the subregion where they overlap . however , we can not draw definitive conclusions from their comparison , as the studies used different participant groups and image processing strategies . regardless of their correspondence with one another , both results implicate posterior regions of the superior temporal lobe , confirming a role of this area in cross - modal reorganization after deafness . the structure - behaviour relationship uncovered here is consistent with findings from congenitally deaf cats , which show that cross - modal activity supports enhanced visual abilities . in deaf cats , motion detection thresholds were associated with activity in a region that extends dorsally from primary auditory cortex , known as the auditory dorsal zone ( dz ) . given their covariation ( in activity and thickness , resp . ) with visual motion detection thresholds across species , we propose that the cat 's dz and the current study 's region of effect may be similarly reorganized after early deafness . one prediction from this idea is that the cortical thickness of the dz and motion detection thresholds of deaf cats will correlate . a comparison of cortical volume of the dz in deaf and hearing cats found no global differences ; however , neither cortical thickness nor the potential correlation between levels of activity and visual ability was examined . our finding mirrors what has been identified in the blind population , where increased cortical thickness in the occipital lobe was associated with enhanced performance on pitch and melody discrimination tasks . however , anatomical mri research in blind people differs from the deaf , in that the blind show widespread differences from the sighted in the cortical thickness of visual areas [ 6163 ] . in contrast , anatomical comparisons between deaf and hearing adults report null differences in grey matter measures of auditory regions in the temporal lobe for either cortical thickness or volume [ 33 , 34 , 36 ] and likewise for cortical volume in deaf and hearing cats . similar to this previous work , we found no global difference between deaf and hearing in the cortical thickness of our region of effect ( figure 4 ) . despite this lack of difference between deaf and hearing groups , we have confirmed that cortical thickness of auditory regions in humans is indeed altered after deafness and that these alterations are identified only when examined in the context of enhanced visual behaviour and age . our finding suggests that many different factors influence the cortical thickness of the planum temporale region , such that no global difference arises between deaf and hearing , but when relevant variables can be identified , such as visual motion detection abilities , then some of the variance can be accounted for . this complexity may reflect the fact that cortical thickness captures the interaction of numerous different cellular - level mechanisms , which can reflect both adaptive and nonadaptive plasticity ( for a review , see ) . in the case of cross - modal plasticity after deafness , recent research on deaf cats demonstrates one possible adaptive mechanism : in a cross - modally active subregion of auditory cortex , early - deaf cats show increased dendritic spine density as compared to hearing cats . speculatively , this cellular - level change may occur in tandem with increased axonal branching , which could in turn increase cortical thickness . the relationship between cortical thickness and visual motion detection was not found in the left pt , nor in either hemisphere 's periprimary auditory areas on hgs , the motion processing area mt+ , or primary visual cortex in the calcarine sulcus . our vertex - wise regression analysis within the rois helps mitigate the risk that only subregions of these areas had an effect . however , we can not rule out the possibility that an effect may have been found with a more individualized definition of these rois , such as by mapping the retinotopy of v1 and using only regions that represent peripheral visual space or by defining mt+ within each participant via a functional localizer . as they are , our results provide no evidence that plasticity in these regions is related to enhanced visual motion detection . interestingly , in our study the correlation between visual ability and cortical thickness occurred exclusively in the right hemisphere . this implies that cross - modal plasticity for visual motion in the deaf may be lateralized and is consistent with suggestions from previous research where the cross - modal activity for moving stimuli was exclusive to the right hemisphere [ 2124 , 27 ] or appeared stronger in the right than left [ 25 , 66 ] . this lateralization is in contrast to deaf cats , where only bilateral deactivation was effective at inhibiting enhanced behaviour , and highlights the relevance of cross - species comparisons . the right hemisphere is widely believed to be specialized for spatial processing , an idea inspired by this hemisphere 's role in spatial neglect disorders ( for a review , see ) . key to these disorders is the right temporoparietal junction ( tpj ) , an area just posterior to our region of effect . the tpj is implicated in reorienting attention to behaviourally relevant sensory targets . as part of this attention module , activity that is related to auditory stimulus - driven attention is localized to the anterior portion of the tpj , extending into the posterior superior temporal lobe . given our region of effect 's proximity to these functions , we suggest that our effect may reflect reorganization of an area involved in auditory sensory reorienting . following this idea , the enhancement of visual motion detection in deaf people may be due to a global enhancement to detect changes in the environment for the purpose of sensory reorienting . there is also a region in the posterior pt that shows sensitivity to auditory motion stimuli ( e.g. , ) , which has led to the suggestion that auditory motion sensitivity could be coopted to support visual motion sensitivity after deafness [ 21 , 22 ] . this explanation may be complementary to our suggestion of reorganized sensory reorienting , as motion sensitivity in this area may interact with sensory reorienting . both the sensory reorienting and motion processing interpretations are consistent with evidence that cross - modal reorganization exploits the homology of functions across different sensory modalities [ 9 , 10 ] . our investigation is limited within the deaf population to early - deaf sign language users with minimal hearing aid use . previous research indicates that the age of onset of deafness , duration of deafness , early language experience [ 27 , 7375 ] , and duration of hearing aid use can each affect neural organization after deafness . thus , future research needs to investigate how our results generalize to different deaf populations , such as those with residual hearing or adult - onset deafness . importantly , since all of our participants used sign language as their primary mode of communication , we can not separate the effects of deafness and language use . although there is some evidence that sign language experience ( versus oral language experience ) alters neuroanatomy ( e.g. , ) , we think it is unlikely that our effect is due to sign language alone , considering its parallelism with research in deaf cats that have no language experience . future research should also investigate whether motion detection thresholds are related to structural variation in hearing people . relationships between visual ability and grey matter structure of the visual cortex have been demonstrated in the typical hearing population in previous research ( e.g. , ) . since we only examined one measure of enhanced vision in deaf people , future research may also investigate whether or not other behavioural enhancements are related to cross - modal plasticity . if , as we have proposed , the involvement of our region of effect has to do with changes to the cortical system for sensory reorienting , then performance on tasks that involve target detection at unattended locations , such as those where deaf people show an advantage in reaction times [ 1620 ] , should show a similar relationship with cortical thickness in the right ptr . since these sensory enhancements may also reflect changes to early visual processing [ 15 , 17 ] , an important step will be to understand how plasticity affects the interactions between auditory and visual cortices . this research provides evidence that the right posterior superior temporal cortex reorganizes to support enhanced visual motion detection abilities in early and profoundly deaf people and that this plasticity is marked by increased cortical thickness .

after sensory loss , the deprived cortex can reorganize to process information from the remaining modalities , a phenomenon known as cross - modal reorganization . in blind people this cross - modal processing supports compensatory behavioural enhancements in the nondeprived modalities . deaf people also show some compensatory visual enhancements , but a direct relationship between these abilities and cross - modally reorganized auditory cortex has only been established in an animal model , the congenitally deaf cat , and not in humans . using t1-weighted magnetic resonance imaging , we measured cortical thickness in the planum temporale , heschl 's gyrus and sulcus , the middle temporal area mt+ , and the calcarine sulcus , in early - deaf persons . we tested for a correlation between this measure and visual motion detection thresholds , a visual function where deaf people show enhancements as compared to hearing . we found that the cortical thickness of a region in the right hemisphere planum temporale , typically an auditory region , was greater in deaf individuals with better visual motion detection thresholds . this same region has previously been implicated in functional imaging studies as important for functional reorganization . the structure - behaviour correlation observed here demonstrates this area 's involvement in compensatory vision and indicates an anatomical correlate , increased cortical thickness , of cross - modal plasticity .

a 55-year - old man , presented to our hospital , complained of a large pelvic mass , which was found incidentally during a routine ultrasonography . the patient had no history of abdominal or pelvic discomfort , nor any abdominal or pelvic surgery . in addition , the patient had no relevant medical or family history except for diabetes mellitus . upon a digital rectal examination , a large mass with a stone - like hardness was palpated at 5 - 6 cm above the anal verge . a single - phase helical ct ( lightspeed ultra ; ge healthcare , milwaukee , wi ) , spanning the whole abdominopelvic cavity , was performed . the ct scanning parameters included a beam collimation of 8 1.25 mm , a reconstruction interval of 1.25 mm , a pitch of 1.35 , a rotation time of 0.8 seconds , a table speed of 13.5 mm / rotation , 120 kv and 160 mas . transverse ct images were obtained 70 seconds after the intravenous injection of non - ionic contrast material ( 120 ml ) at a rate of 3 ml / sec ( ultravist 300 ; bayer schering pharma , berlin , germany ) . the ct images revealed a large , well - defined mass in the presacral space of the pelvic cavity , which seemed to have a long stalk arising from the left gluteus medius muscle ( fig . the stalk traversed along the surface of the left iliac bone , and proceeded through the left sciatic foramen , and ended up connecting with the mass . the ct attenuation generated an easily recognizable mass , which was mainly composed of fatty tissue and calcification , with most of the stalk becoming calcified . some focal areas of intermediate attenuation were identified as a mixture between fatty tissue and calcification , and occupied only a limited portion of the mass . mr imaging ( horizon 1.5 t ; ge healthcare , milwaukee , wi ) was also performed with the use of a torso coil . on both the t1-weighted ( 366/8 , echo train length of zero , 5-mm slice thickness , 2-mm gap , 256192 matrix , 24-cm field of view , 2 signal acquired , and sequence duration of 3 - 4 minutes ) and t2-weighted ( 4300/84 , echo train length of ten , 5-mm slice thickness , 2-mm gap , 512256 matrix , 24-cm field of view , 2 signal acquired , and sequence duration of 3 - 4 minutes ) mr images , the mass was revealed to mainly consist of the hyperintense and signal void areas that represent fatty tissue and calcification , respectively ( fig . 1c - e ) . also seen are the focal areas of intermediate signal intensity , as shown on the ct images . the gadolinium - enhanced t1-weighted mr images obtained 30 seconds , 60 seconds , and 3 minutes after the intravenous injection of contrast material ( gadovist ; bayer schering pharma , berlin , germany ) , and the focal areas of the intermediate signal intensity around the calcifications observed at 30 and 60 seconds post - injection , became isointense as the surrounding fat tissue at 3 minutes post - injection . the main mass was resected using a transabdominal approach , whereas the stalk was removed through the sciatic foramen . the surgical specimen consisted of a 13 cm , well - circumscribed , hard mass , with a severely calcified external surface . the cut surface of the resected specimen revealed a mass mixed with fatty tissue and calcification ( fig . a histopathological examination revealed that the mass was composed of lipoma with chondroid metaplasia ( fig . the term mesenchymoma was originally defined by stout ( 7 ) in 1948 to describe tumors containing at least two mesenchymal tissues not normally found together . the tumors can be classified as benign or malignant , and most of the benign varieties have been called angiomyolipomas , angiolipomas , chondrolipomas , osteolipomas , and other names depending on the predominant tissue form in the tumor . however , fibrous tissue is found in all mesenchymal tumors , and is not counted as one of the elements ( 6 ) . conversely , if a mesenchymal tumor is well - defined or encapsulated and composed predominantly of one type of mesenchymal tissue , along with one or more minor element , the diagnosis should reflect the predominant mesenchymal tissue ( 6 ) . in our case , the diagnosis of a chondrolipoma was appropriate , since the mass was composed of a sufficiently high proportion of both fatty tissue and extensively calcified chondroid tissue , though it was well - defined and encapsulated . two possible explanations for the pathogenesis of cartilage and bone formation in benign mesenchymomas have been proposed ( 5 , 6 , 8 , 9 ) . the first is that cartilage arises from chondro - osseous metaplasias of adipose tissue , presumably as a result of mechanical stress or trophic disturbance . the close proximity or contact with bone and a large joint , myxoid background , or a lipodystrophy - like change , which are often associated with these tumors , suggest a metaplastic process caused by a trophic disturbance or mechanical stress ( 8 - 10 ) . a previous immunohistochemical study supported the explanation that the pattern of expression for growth factor- , latent transforming growth factor- binding protein-1 transforming , and bone morphogenetic protein might play an important role in the transformation of multipotential cells into the chondrolipoma ( 11 ) . considering the proximity of the identified chondrolipoma to the iliac bone of our case study , we agree with the first explanation of chondroid - osseous metaplasia , which states that the mass may be a result of a micro - trauma . our mass occupied both the inside and outside of the pelvic cavity ; however , we could not clarify whether its primary site was the pelvic cavity or the left buttock . according to previously reported cases the only reported case of an abdominal chondrolipoma in a human occurred in the small bowel ( 8) , whereas a chondrolipoma was once documented in the pelvic cavity of a dog ( 12 ) . considering these reports , we thought that in our case , a chondrolipoma in the buttock might be extended to the pelvic cavity through the sciatic foramen , though the proportion of the mass in the pelvis was greater than in the buttock . there are few reports pertaining to the imaging features of chondrolipomas of various organs , with most cases describing their mr features ( 5 , 9 , 13 , 14 ) . for these reports , chondroid and fatty tissues were described as intermediate and high signal intensity areas on t1-weighted images , respectively . both chondroid and fatty tissues were identified as having a higher signal intensity area than the surrounding muscle , whereas chondroid tissue was depicted as having a higher signal intensity area than fatty tissue on t2-weighted images . signal void areas corresponding to calcifications were only found in a small portion of these reported cases . for the ct images , fatty tissue was depicted as a low attenuation area ( less than -30 hounsfield units ) , whereas chondroid tissue was depicted as an intermediate attenuation area . in our case , the mass was depicted to be primarily composed of fatty tissue and calcification , which matched the ct attenuation and mr signal intensity . also , we found that additional focal areas of intermediate attenuation or signal intensity corresponded to chondroid tissue . these findings were different from those described in previously reported cases whereby , in our case , the calcification was much more extensive , and the signal intensity of chondroid tissue on the t2-weighted images was intermediate . moreover , we found extensive calcification located peripherally in a linear and rosary pattern . a previous report also cited the observation of chondroid tissue in a rosary pattern ( 9 ) . despite the very limited number of reported cases , we propose that the rosary pattern of either the chondroid tissue or calcification could be a characteristic finding of chondrolipomas , and in our case , the calcification in a rosary pattern was likely a consequence of the dystrophic change involving the chondroid tissue . moreover , the signal intensity of chondroid tissue was intermediate on the t2-weighted images , and could be explained by the presence of fibrous tissue within the chondroid tissue ( fig . 1i ) , which reduced the signal intensity of chondroid tissue below the fatty tissue . it is interesting that the focal areas of intermediate signal intensity around the calcifications showed delayed enhancement on the gadolinium - enhanced mr images . ( 9 ) described the presence of enhancing foci between the fatty tissue and chondroid tissue in a chondrolipoma , without clarifying the histopathological findings . also , there have been no reports of chondrolipomas with delayed enhancement . in our case , however , keeping in mind that the location of these focal areas corresponded to chondroid tissue , and that chondroid tissue is usually not enhanced , even on the delayed phase , the delayed enhancement of these focal areas may be attributed due to sufficient fibrous tissue within the chondroid tissue . despite the characteristic imaging findings of chondrolipomas , fatty tumors such as lipomas or liposarcomas , which contain chondro - osseous differentiation , the treatment of choice for chondrolipomas is surgical excision ( 5 , 8 , 14 ) . conclusively , we experienced the only reported human case of a chondrolipoma in the pelvic cavity , and found that it had characteristic imaging features consistent with fatty tissue and calcification , as well as the areas corresponding to cartilage .

a chondrolipoma is an extremely rare form of a benign mesenchymal tumor containing mature cartilage and fatty tissue . chondrolipomas may be found in almost any part of the body , particularly in the connective tissue of the breast , head and neck area , as well as in the skeletal muscle . however , to the best of our knowledge , chondrolipomas located in the pelvic cavity have not been reported . in this case report , we describe a case of a chondrolipoma in the pelvis , and show that it has its own characteristic imaging findings , which included the composition of fatty tissue and calcification in most parts , as well as some focal areas of chondroid tissue based on the ct and mr findings .

motoharu seiki ( university of tokyo ) and maintained in c57bl/6 background ( 11 ) . mmp14 haploinsufficient ( mmp14 ) and wild - type ( mmp14 ) male mice , aged 12 weeks , were used for all experiments . each mouse was housed individually and placed on a high - fat ( 45% fat ) or control ( 10% fat ) diet ( research diets ) for the indicated periods of time . in selected studies , mice were individually housed in oxymax / clams metabolic chambers ( columbus instrument ) for metabolic studies . after 2 days ' acclimation , vo2 , food consumption , and x - y ambulation were measured consecutively for 4 days . mmp - dependent collagen degradation products were detected with c1 - 2c antibody ( ibex ) ( 12 ) . fibrillar collagens were stained with sirius red and quantified with imagej ( nih ) ( 7 ) . student 's t test ( unpaired and two tailed ) was used for statistical analysis . primary preadipocytes were isolated from the inguinal fat pads of 3- to 4-week - old male wild - type and haploinsufficient ( mmp14 ) mice ( 7 ) . type i collagen was extracted from rat tails and conjugated with oregon green 488 ( molecular probe ) ( 13 ) . preadipocytes were cultured atop fluoresceinated type i collagen polymers with or without an adipogenic mix ( 0.2 mol / l insulin , 0.5 mmol / l 3-isobutyl-1-methylxanthine , and 0.25 mol / l dexamethsone ) ( 7 ) . nuclei were stained with dapi and zones of collagenolysis identified with a fluorescent microscope ( model no . ix71 ; olympus ) using an objective lens 40/na 0.65 ( olympus ) at 25c . gene expression data were obtained by hybridizing labeled crna to affymetrix mouse genome 430 2.0 array . for analysis , data were standardized using the robust multiarray average expression measure ( 14 ) . the paired t test was two tailed , and p < 0.01 was considered statistically significant . scanning electron microscopy was used to examine the architecture of fat pad associated collagen . inguinal fat pads were fixed with 2% glutaraldehyde/1.5% paraformaldehyde in 0.1 mol / l na cacodylate buffer and postfixed in 1% osmium tetroxide . samples were immersed in liquid n2 , fractured to expose the inner mass of adipose tissues , and imaged with an amray 1910 field emission scanning electron microscope . japanese healthy individuals with no obvious medical conditions ( n = 3,653 ) were recruited through random sampling ( 15 ) . all subjects gave written informed consent prior to the study , and the study design ( millennium genome project ) was approved by the institutional review board and the ethics committee of the national cardiovascular center , osaka , japan . mmp14 gene variations were detected by taqman pcr ( abi prism 7900ht ) and verified in a subset of samples by direct sequencing ( abi 3700 ) . the association of mmp14 gene haplotypes with age- and sex - adjusted quantitative traits was tested with the qtlhaplo program ( 17 ) using logistic regression analysis ( 18 ) . the genotype - phenotype association with rs2236302 was performed with one - way ancova adjusted for age , history of smoking , drinking , diabetes , hypertension , and hyperlipidemia . motoharu seiki ( university of tokyo ) and maintained in c57bl/6 background ( 11 ) . mmp14 haploinsufficient ( mmp14 ) and wild - type ( mmp14 ) male mice , aged 12 weeks , were used for all experiments . each mouse was housed individually and placed on a high - fat ( 45% fat ) or control ( 10% fat ) diet ( research diets ) for the indicated periods of time . in selected studies , mice were individually housed in oxymax / clams metabolic chambers ( columbus instrument ) for metabolic studies . after 2 days ' acclimation , vo2 , food consumption , and x - y ambulation were measured consecutively for 4 days . mmp - dependent collagen degradation products were detected with c1 - 2c antibody ( ibex ) ( 12 ) . fibrillar collagens were stained with sirius red and quantified with imagej ( nih ) ( 7 ) . student 's t test ( unpaired and two tailed ) was used for statistical analysis . primary preadipocytes were isolated from the inguinal fat pads of 3- to 4-week - old male wild - type and haploinsufficient ( mmp14 ) mice ( 7 ) . type i collagen was extracted from rat tails and conjugated with oregon green 488 ( molecular probe ) ( 13 ) . preadipocytes were cultured atop fluoresceinated type i collagen polymers with or without an adipogenic mix ( 0.2 mol / l insulin , 0.5 mmol / l 3-isobutyl-1-methylxanthine , and 0.25 mol / l dexamethsone ) ( 7 ) . nuclei were stained with dapi and zones of collagenolysis identified with a fluorescent microscope ( model no . ix71 ; olympus ) using an objective lens 40/na 0.65 ( olympus ) at 25c . gene expression data were obtained by hybridizing labeled crna to affymetrix mouse genome 430 2.0 array . for analysis , data were standardized using the robust multiarray average expression measure ( 14 ) . the paired t test was two tailed , and p < 0.01 was considered statistically significant . scanning electron microscopy was used to examine the architecture of fat pad associated collagen . inguinal fat pads were fixed with 2% glutaraldehyde/1.5% paraformaldehyde in 0.1 mol / l na cacodylate buffer and postfixed in 1% osmium tetroxide . samples were immersed in liquid n2 , fractured to expose the inner mass of adipose tissues , and imaged with an amray 1910 field emission scanning electron microscope . japanese healthy individuals with no obvious medical conditions ( n = 3,653 ) were recruited through random sampling ( 15 ) . all subjects gave written informed consent prior to the study , and the study design ( millennium genome project ) was approved by the institutional review board and the ethics committee of the national cardiovascular center , osaka , japan . mmp14 gene variations were detected by taqman pcr ( abi prism 7900ht ) and verified in a subset of samples by direct sequencing ( abi 3700 ) . the association of mmp14 gene haplotypes with age- and sex - adjusted quantitative traits was tested with the qtlhaplo program ( 17 ) using logistic regression analysis ( 18 ) . the genotype - phenotype association with rs2236302 was performed with one - way ancova adjusted for age , history of smoking , drinking , diabetes , hypertension , and hyperlipidemia . to characterize the impact of nutritionally induced obesity on the remodeling of the extracellular matrix ( ecm ) in adipose tissues , 3-month - old c57bl/6 mice were placed on either a low - fat ( control ) or high - fat diet for 1 week . subsequently , inguinal fat pads were isolated and type i collagen architecture was assessed by sirius red staining . as expected , adipose tissues recovered from mice placed on a control diet displayed a web - like network of interlocking collagen fibrils ( fig . in marked contrast , a high - fat diet induced significant decreases in sirius red staining consistent with an unexpectedly rapid activation of collagenolytic activity ( fig . 1a and b ) . given the dominant role assigned to matrix metalloproteinases in type i collagen turnover in vivo ( 7,9 ) , adipose tissues prepared from control or high - fat diet challenged mice were probed for the generation of collagen cleavage products with a polyclonal antibody that recognizes type i collagen neoepitopes exposed following mmp - specific hydrolysis ( 12 ) . while control fat pads contained only small amounts of immunoreactive material , collagen cleavage products increased more than threefold in the high - fat diet challenged group ( fig . consistent with these results , scanning electron microscopy confirmed that the high - fat diet challenge induces a marked loss in the adipocyte - associated meshwork of fibrillar collagen ( fig . a : type i collagen cleavage induced by a 1-week regimen of high - fat diet feeding . fibrillar collagen is detected with sirius red staining in inguinal fat pads of 3-month - old c57bl/6 mice under a control diet ( cd ) ( top row ) or high - fat diet ( hfd ) ( bottom row ) feeding . type i collagen cleavage products are detected with c1 , 2c antibody ( cleaved collagen , red ) . the fibrillar status of type i collagen fibrils encircling adipocyte clusters is disrupted when mice are placed on an hfd ( scanning electron microscopy [ sem ] ) . disrupted collagen fibers ( arrowhead ) are found in association with variably sized adipocytes ( asterisks ) . b and c : quantification of sirius red positive staining and cleaved collagen in the inguinal fat pads of mice after 1 week of control diet or high - fat feedings . for each group , images were collected from more than six randomly selected fields . * p < 0.01 ; * * p < 0.01 . d : genome - wide transcriptome change induced by a 1-week high - fat diet challenge . while more than 99% of transcripts did not display significant changes ( x - axis , control diet ; y - axis , high - fat diet ) , a specific subset of genes display a significant increase in expression . . slopes with y - intercepts 0 , 1 , and 1 ( log 2 scale ) are shown . dependent transcriptome changes after a 1-week high - fat diet challenge are categorized following validation in two or more independent experiments . bar = 100 m . * lipid / cholesterol biosynthesis pathways ; * * ecm remodeling pathways . ( a high - quality digital representation of this figure is available in the online issue . ) to assess the impact of high - fat diet initiated ecm remodeling on the adipose tissue transcriptome , mrna was isolated from inguinal fat pads of control or high - fat diet challenged mice and analyzed with cdna microarrays . following 1 week 's feeding of a high - fat diet , a subset of 113 transcripts was upregulated by twofold or greater with 34 transcripts suppressed ( fig . uniquely up- and downregulated genes in the inguinal fat pads of wild - type mice can be found in supplemental table 1 ( available in an online appendix [ http://diabetes.diabetesjournals.org/cgi/content/full/db10-0073/dc1 ] ) . gene ontology analysis of upregulated transcripts revealed the enrichment for biologic processes related to collagen catabolism ( p = 0.004 ) , collagen fibril organization ( p = 0.003 ) , and integrin - mediated signaling ( p = 0.002 ) , a major transduction pathway for adipocyte - type i collagen interactions ( 19,20 ) . interestingly , gene programs consistent with acute changes in lipid biosynthesis ( p = 0.001 ) , steroid metabolism ( p = 0.003 ) , and biosynthesis ( p = 0.003 ) were also upregulated in tandem with the recruitment of ecm - remodeling transcripts ( fig . , these data support a model wherein high - fat diet induced changes in ecm remodeling are closely linked to the early transcriptional programs responsible for regulating lipid and cholesterol biosynthesis a conclusion corroborated by recent studies linking a collagen subfamily member to the regulation of adipose tissue mass ( 21 ) . while a number of secreted mmps express type i collagenolytic activity ( 22 ) , mmp-13 , mmp-8 , and mmp-2 expression were not altered following challenge with a high - fat diet . by contrast , expression levels of the membrane - anchored collagenase , mmp14 , were increased twofold during the 1-week - long high - fat diet challenge as assessed by quantitative pcr ( relative mrna levels : high - fat diet 7.2 0.95 vs. control 3.4 0.10 ; n = 4 ) . mmp14 is a membrane - tethered matrix metalloproteinase that has been identified as the dominant pericellular collagenase used by mesenchymal cells ( 7,13,23 ) . as the collagenolytic activity of isolated mouse preadipocytes is enhanced in the presence of an adipogenic cocktail ( 7 ) , the impact of mmp14 dosage on collagenolytic activity was first assessed in vitro . under resting conditions , mmp14 or mmp14 preadipocytes cultured atop a three - dimensional bed of type i collagen fibrils degraded the underlying substrate comparably ( fig . when stimulated with an adipogenic mix , however , mmp14 cells displayed a two - fold increase in collagenolysis while mmp14 cells were unable to upregulate their collagen degrading activity ( fig . loss of adipogenic collagenolysis in the haploinsufficient state is consistent with a quantitative requirement for the full complement of mmp14 protein on the cell surface ( 24 ) . preadipocytes isolated from the inguinal fat pads of wild - type ( mmp14 ) and haploinsufficient ( mmp14 ) mice were cultured atop a bed of fluorescent type i collagen ( green ) , and subjacent degradation was monitored by the disappearance of fluorescent signal after a 3-day culture period in the absence or presence of an adipogenic cocktail . cells were isolated from a cohort of three mice for each group ( n = 3 ) . induced collagenolysis is almost completely blocked by mmp14 haploinsufficiency ( fibrillar collagen detected with sirius red staining and cleaved collagen by immunofluorescence ) ( red ) . scanning electron microscopy revealed intact collagen fibers enwrapping adipocytes in mmp14 , but not mmp14 , inguinal fat pads . d : cleaved collagen and fibrillar collagen contents in inguinal fat pads of mmp14 and mmp14 mice following a 1-week control diet or high - fat diet challenge . results are expressed as means 1 sd ( n = 6 ) . * p < 0.01 . e : following a 2-week high - fat diet , the inguinal and perigonadal fat pads were isolated from mmp14 or mmp14 mice and tissue weights determined . ( a high - quality digital representation of this figure is available in the online issue . ) given these in vitro findings , the role of mmp14 in regulating collagen turnover in adipose tissues in situ was assessed in haploinsufficient mice because mmp14-null mice fail to thrive from birth and exhibit a marked decrease in life span ( 9 ) . mmp14 haploinsufficient mice were indistinguishable from wild - type littermates , and no significant differences in adipose tissue size or morphology could be detected between mmp14 and mmp14 mice ( supplemental fig . 1 ) . furthermore , gene expression patterns of key adipogenic factors including peroxisome proliferator activated receptor , insulin receptor , glut4 , and lipoprotein lipase ( 25 ) were similar between the two groups ( supplemental fig . when , however , mmp14 mice were placed on a high - fat diet , only small decreases in fibrillar collagen content were detected relative to littermate controls ( fig . furthermore , significant increases in collagen cleavage products following high - fat challenge were not observed in mmp14 mice , while the fibrillar collagen architecture ( as determined by scanning electron microscopy ) remained unchanged ( fig . in concert with the diminished ability of mmp14 haploinsufficient mice to remodel fat pad collagen architecture during high - fat feeding , the mmp14 mice were also unable to expand their adipose mass comparably with control ( fig . whereas the weight of inguinal or perigonadal adipose tissue of mmp14 mice increased two- to threefold during the 1-week high - fat diet challenge , the haploinsufficient mice failed to mount a similar increase in adipose tissue mass ( fig . 2e ) . to probe the functional impact of mmp14 in regulating the acute phase gene response to a high - fat diet , we examined the transcriptome profile of inguinal fat pads of mmp14 haploinsufficient mice placed on a high - fat diet for 1 week . in contrast with mmp14-sufficient mice , the haploinsufficient animals failed to induce the gene sets enriched for ecm remodeling or lipid biosynthesis ( fig . 3a and b ) . further , as opposed to wild - type mice , 42 genes were uniquely upregulated and 91 genes downregulated in the heterozygote animals ( fig . in these mice , the high - fat diet challenge led to a paradoxical deregulation in the gene expression profile of transcripts involved in a wide range of biological processes , including glycerol-3-phosphate metabolism , acetyl - coa biosynthesis , and both humoral immune and acute phase responses ( fig . these results indicate that a reduction in mmp14 gene dosage leads to a disruption in the transcriptional link existing between ecm remodeling and lipid biosynthesis that coordinates the expansion of adipose tissue . a and b : disrupted high - fat diet induced transcriptome changes in mmp14 haploinsufficient mice . a scatter plot is shown of mrna expression values ( log2 scale ) in inguinal fat pads isolated from mmp14 mice fed a control diet ( x - axis ) vs. high - fat diet ( y - axis ) . in comparison with gene expression profiles obtained in wild - type mice ( fig . 1b ) , genes involved in a diverse set of metabolic pathways critical to adipose tissue function ( asterisks ) are misregulated in mmp14 mice . c and d : mmp14 haploinsufficiency protects mice from diet - induced increase of fat mass after 3 or 6 months of high - fat diet feeding ( means sd ; n = 8) . representative images of inguinal and perigonadal fat pads are shown following isolation from mmp14 and mmp14 mice that had been placed on high - fat feeding for 3 months . e and f : feeding bouts ( g / day ) and metabolic rates adjusted for lean mass ( vo2/kg lean mass ) were determined in metabolic cages during high - fat diet feeding . ( a high - quality digital representation of this figure is available in the online issue . ) to next assess the long - term consequences of mmp14 haploinsufficiency on high - fat diet induced obesity , wild - type and haploinsufficient mice were placed on a high - fat diet for 36 months . the average percentage of fat mass of mmp14 haploinsufficient and wild - type mice placed under a control diet was comparable ( mean sd 5.29 0.58 vs. 5.07 0.7% ; n = 6 ) . on the high - fat diet , however , whereas wild - type mice displayed an approximate 20 and 60% weight gain after 3 and 6 months , respectively , mmp14 mice increased in total weight by less than one - half of that observed in the control ( fig . as expected , the dramatic changes in weight gain that occured in the long - term , high - fat feeding of mmp14 mice was also reflected in the attenuated expansion of tissue mass in isolated inguinal and perigonadal fat pads ( fig . changes in mmp14 expression did not , however , affect whole - body energy balance during high - fat feeding because the respiration rate , daily food intake , and locomotion of wild - type and heterozygous mice were not significantly different ( fig . these results stress the role of mmp14 as a proteolytic modifier that acts locally within adipose tissues without overtly affecting the hypothalamic regulation of metabolism . obesity is driven by a complex process that is coordinated by a host of genetic , epigenetic , and environmental factors ( 2628 ) . to extend the findings of the genotype - phenotype link found in mice , the association of mmp14 snp genotypes with human obesity and diabetes traits the human mmp14 gene is located at chromosome 14q1112 , comprising 10 exons and spanning a 10-kb region that contains 157 known snps ( ncbi dbsnp ) . using a preliminary group ( n = 48 ) randomly sampled from a japanese population ( 15 ) , we assessed the minor allele frequencies of candidate mmp14 snps ( fig . 16 snps spanning human mmp14 gene ( from rs17211964 at chr14:23,304,272 through rs2236307 at chr14:23,312,554 ) were screened to determine their pairwise linkage disequilibrium coefficients ( supplemental tables 1 and 2 ) . three snps located in exon 5 ( rs2236302 ; allele 2 , c > allele 1 , g ) , intron 5 ( rs2236304 ; allele 2 , c > allele 1 , g ) , and exon 6 ( rs2236307 ; allele 2 , t > allele 1 , c ) were chosen based on their frequency ( > 10% ) , proximity to the catalytic domain of mmp14 , and pairwise linkage disequilibrium that allow diverse haplotypes in combination . the minor allele ( allele 1 ) frequency for the three snps among the study population was 11.0 , 44.8 , or 38.5% , respectively ( fig . a : human mmp14 snps located in the proximity of the region encoding mmp14 catalytic domain . b : mmp14 haplotype determinations were based on the combination of rs2236302 , rs2236304 , and rs2236307 . c : association of human mmp14 haplotypes with bmi . haplotype associations with bmi of the total population ( n = 3,531 ) , women ( n = 1,944 ) , and men ( n = 1,587 ) were assessed in both dominant and recessive inheritance models . d : association of rs2236302 genotype with bmi and waist - to - hip ( w - h ) ratio . means sem of bmi ( left panel ) and waist - to - hip ratio ( right panel ) are shown for the total population ( n = 3,647 ) , women ( n = 1,944 ) , and men ( n = 1,703 ) , respectively . using a study population that included 3,653 individuals consisting of 1,708 men and 1,945 women , we assessed mmp14 haplotypes . the analysis of the pairwise linkage disequilibrium among the three snps suggested that they constitute a block of haplotypes ( d > 0.977 ) . however , the estimated square of the correlation coefficient ( r ) among the studied snps were sufficiently low ( table 1 ) to allow for the assembly of at least four major haplotypes ( fig . the association of mmp14 haplotypes with obesity and diabetes traits , i.e. , bmi , waist - to - hip ratio , body fat , a1c , fasting blood glucose and insulin levels , homeostasis model assessment ( homa ) of insulin resistance and -cell function , was examined in the dominant or recessive genetic model with multiple logistic regression analysis ( 17,18,28 ) . among the four major haplotypes of the mmp14 gene ( 212 , 221 , 122 , and 222 ) , the haplotype 122 ( gct ) was found to positively associate with bmi ( p = 0.0017 ) ( fig . 4c ) and waist - to - hip ratio ( p = 0.0079 ) ( table 2 ) . because of the dominant role played by rs2236302 in defining the link between obesity traits and mmp14 haplotypes , rs2236302 genotype was used to further delineate the link between mmp14 genotype and quantitative obesity traits . the distribution of c / c , c / g , and g / g genotypes were 79.7% ( n = 2,908 ) , 19.1% ( n = 695 ) , and 1.2% ( n = 44 ) among the study population . due to the low frequency of the g / g genotype , the quantitative association study was performed by comparing between homozygote c / c and heterozygote c / g genotype groups . the association with obesity traits was then examined in a genotype or dominant model with ancova . in the multivariate analyses of the total population , bmi and waist - to - hip ratio were associated with age ( p < 0.0001 ) , sex ( p < 0.0001 ) , history of smoking ( p = 0.0129 ) , hypertension ( p < 0.0001 ) , diabetes ( p = 0.0003 ) , and hyperlipidemia ( p < 0.0001 ) . when analyzed with adjustment for these variables , 4d ) ( mean sem bmi , c / c 22.74 0.06 vs. c / g 23.16 0.11 kg / m ; waist - to - hip ratio , c / c 0.901 0.001 vs. the increase of bmi caused by rs2236302 minor allele was 0.42 kg / m ( cohen d = 0.13 ) . homa- , homa of -cell function ; homa - ir , homa of insulin resistance . of note , the positive effect of rs2236302 genotype on bmi and waist - to - hip ratio was preferentially observed in women ( fig . 4d ) , suggesting the existence of sexual dimorphism in the link between mmp14 and obesity phenotype ( women p = 0.0004 , d = 0.199 , vs. men p = 0.5419 , d = 0.059 ) . finally , human mmp14 snps were found to be weakly associated with a1c in men ( p = 0.0685 ) ( table 3 ) , suggesting a paradoxical relationship of mmp14 snps with diabetes predisposition in males . consistently , mmp14 haplotype ( 121 ) associated with increased fasting blood glucose ( p = 0.0069 ) and homa of insulin resistance ( p = 0.0386 ) ( table 3 ) , supporting a potential link between mmp14 and diabetes in men . sexual dimorphism in the link between mmp14 and obesity / diabetes traits * any genotype . homa- , homa of -cell function ; homa - ir , homa of insulin resistance ; ns , p > 0.1 . to characterize the impact of nutritionally induced obesity on the remodeling of the extracellular matrix ( ecm ) in adipose tissues , 3-month - old c57bl/6 mice were placed on either a low - fat ( control ) or high - fat diet for 1 week . subsequently , inguinal fat pads were isolated and type i collagen architecture was assessed by sirius red staining . as expected , adipose tissues recovered from mice placed on a control diet displayed a web - like network of interlocking collagen fibrils ( fig . in marked contrast , a high - fat diet induced significant decreases in sirius red staining consistent with an unexpectedly rapid activation of collagenolytic activity ( fig . 1a and b ) . given the dominant role assigned to matrix metalloproteinases in type i collagen turnover in vivo ( 7,9 ) , adipose tissues prepared from control or high - fat diet challenged mice were probed for the generation of collagen cleavage products with a polyclonal antibody that recognizes type i collagen neoepitopes exposed following mmp - specific hydrolysis ( 12 ) . while control fat pads contained only small amounts of immunoreactive material , collagen cleavage products increased more than threefold in the high - fat diet challenged group ( fig . consistent with these results , scanning electron microscopy confirmed that the high - fat diet challenge induces a marked loss in the adipocyte - associated meshwork of fibrillar collagen ( fig . a : type i collagen cleavage induced by a 1-week regimen of high - fat diet feeding . fibrillar collagen is detected with sirius red staining in inguinal fat pads of 3-month - old c57bl/6 mice under a control diet ( cd ) ( top row ) or high - fat diet ( hfd ) ( bottom row ) feeding . type i collagen cleavage products are detected with c1 , 2c antibody ( cleaved collagen , red ) . the fibrillar status of type i collagen fibrils encircling adipocyte clusters is disrupted when mice are placed on an hfd ( scanning electron microscopy [ sem ] ) . disrupted collagen fibers ( arrowhead ) are found in association with variably sized adipocytes ( asterisks ) . b and c : quantification of sirius red positive staining and cleaved collagen in the inguinal fat pads of mice after 1 week of control diet or high - fat feedings . for each group , images were collected from more than six randomly selected fields . * p < 0.01 ; * * p < 0.01 . d : genome - wide transcriptome change induced by a 1-week high - fat diet challenge . while more than 99% of transcripts did not display significant changes ( x - axis , control diet ; y - axis , high - fat diet ) , a specific subset of genes display a significant increase in expression . . slopes with y - intercepts 0 , 1 , and 1 ( log 2 scale ) are shown . dependent transcriptome changes after a 1-week high - fat diet challenge are categorized following validation in two or more independent experiments . bar = 100 m . * lipid / cholesterol biosynthesis pathways ; * * ecm remodeling pathways . ( a high - quality digital representation of this figure is available in the online issue . ) to assess the impact of high - fat diet initiated ecm remodeling on the adipose tissue transcriptome , mrna was isolated from inguinal fat pads of control or high - fat diet challenged mice and analyzed with cdna microarrays . following 1 week 's feeding of a high - fat diet , a subset of 113 transcripts was upregulated by twofold or greater with 34 transcripts suppressed ( fig . uniquely up- and downregulated genes in the inguinal fat pads of wild - type mice can be found in supplemental table 1 ( available in an online appendix [ http://diabetes.diabetesjournals.org/cgi/content/full/db10-0073/dc1 ] ) . gene ontology analysis of upregulated transcripts revealed the enrichment for biologic processes related to collagen catabolism ( p = 0.004 ) , collagen fibril organization ( p = 0.003 ) , and integrin - mediated signaling ( p = 0.002 ) , a major transduction pathway for adipocyte - type i collagen interactions ( 19,20 ) . interestingly , gene programs consistent with acute changes in lipid biosynthesis ( p = 0.001 ) , steroid metabolism ( p = 0.003 ) , and biosynthesis ( p = 0.003 ) were also upregulated in tandem with the recruitment of ecm - remodeling transcripts ( fig . , these data support a model wherein high - fat diet induced changes in ecm remodeling are closely linked to the early transcriptional programs responsible for regulating lipid and cholesterol biosynthesis a conclusion corroborated by recent studies linking a collagen subfamily member to the regulation of adipose tissue mass ( 21 ) . while a number of secreted mmps express type i collagenolytic activity ( 22 ) , mmp-13 , mmp-8 , and mmp-2 expression were not altered following challenge with a high - fat diet . by contrast , expression levels of the membrane - anchored collagenase , mmp14 , were increased twofold during the 1-week - long high - fat diet challenge as assessed by quantitative pcr ( relative mrna levels : high - fat diet 7.2 0.95 vs. control 3.4 0.10 ; n = 4 ) . mmp14 is a membrane - tethered matrix metalloproteinase that has been identified as the dominant pericellular collagenase used by mesenchymal cells ( 7,13,23 ) . as the collagenolytic activity of isolated mouse preadipocytes is enhanced in the presence of an adipogenic cocktail ( 7 ) , the impact of mmp14 dosage on collagenolytic activity was first assessed in vitro . under resting conditions , mmp14 or mmp14 preadipocytes cultured atop a three - dimensional bed of type i collagen fibrils degraded the underlying substrate comparably ( fig . when stimulated with an adipogenic mix , however , mmp14 cells displayed a two - fold increase in collagenolysis while mmp14 cells were unable to upregulate their collagen degrading activity ( fig . loss of adipogenic collagenolysis in the haploinsufficient state is consistent with a quantitative requirement for the full complement of mmp14 protein on the cell surface ( 24 ) . preadipocytes isolated from the inguinal fat pads of wild - type ( mmp14 ) and haploinsufficient ( mmp14 ) mice were cultured atop a bed of fluorescent type i collagen ( green ) , and subjacent degradation was monitored by the disappearance of fluorescent signal after a 3-day culture period in the absence or presence of an adipogenic cocktail . cells were isolated from a cohort of three mice for each group ( n = 3 ) . induced collagenolysis is almost completely blocked by mmp14 haploinsufficiency ( fibrillar collagen detected with sirius red staining and cleaved collagen by immunofluorescence ) ( red ) . scanning electron microscopy revealed intact collagen fibers enwrapping adipocytes in mmp14 , but not mmp14 , inguinal fat pads . d : cleaved collagen and fibrillar collagen contents in inguinal fat pads of mmp14 and mmp14 mice following a 1-week control diet or high - fat diet challenge . e : following a 2-week high - fat diet , the inguinal and perigonadal fat pads were isolated from mmp14 or mmp14 mice and tissue weights determined . ( a high - quality digital representation of this figure is available in the online issue . ) given these in vitro findings , the role of mmp14 in regulating collagen turnover in adipose tissues in situ was assessed in haploinsufficient mice because mmp14-null mice fail to thrive from birth and exhibit a marked decrease in life span ( 9 ) . mmp14 haploinsufficient mice were indistinguishable from wild - type littermates , and no significant differences in adipose tissue size or morphology could be detected between mmp14 and mmp14 mice ( supplemental fig . 1 ) . furthermore , gene expression patterns of key adipogenic factors including peroxisome proliferator activated receptor , insulin receptor , glut4 , and lipoprotein lipase ( 25 ) were similar between the two groups ( supplemental fig . when , however , mmp14 mice were placed on a high - fat diet , only small decreases in fibrillar collagen content were detected relative to littermate controls ( fig . 2c and d ) . furthermore , significant increases in collagen cleavage products following high - fat challenge were not observed in mmp14 mice , while the fibrillar collagen architecture ( as determined by scanning electron microscopy ) remained unchanged ( fig . 2c ) . in concert with the diminished ability of mmp14 haploinsufficient mice to remodel fat pad collagen architecture during high - fat feeding , the mmp14 mice were also unable to expand their adipose mass comparably with control ( fig . whereas the weight of inguinal or perigonadal adipose tissue of mmp14 mice increased two- to threefold during the 1-week high - fat diet challenge , the haploinsufficient mice failed to mount a similar increase in adipose tissue mass ( fig . to probe the functional impact of mmp14 in regulating the acute phase gene response to a high - fat diet , we examined the transcriptome profile of inguinal fat pads of mmp14 haploinsufficient mice placed on a high - fat diet for 1 week . in contrast with mmp14-sufficient mice , the haploinsufficient animals failed to induce the gene sets enriched for ecm remodeling or lipid biosynthesis ( fig . 3a and b ) . further , as opposed to wild - type mice , 42 genes were uniquely upregulated and 91 genes downregulated in the heterozygote animals ( fig . in these mice , the high - fat diet challenge led to a paradoxical deregulation in the gene expression profile of transcripts involved in a wide range of biological processes , including glycerol-3-phosphate metabolism , acetyl - coa biosynthesis , and both humoral immune and acute phase responses ( fig . these results indicate that a reduction in mmp14 gene dosage leads to a disruption in the transcriptional link existing between ecm remodeling and lipid biosynthesis that coordinates the expansion of adipose tissue . a and b : disrupted high - fat diet induced transcriptome changes in mmp14 haploinsufficient mice . a scatter plot is shown of mrna expression values ( log2 scale ) in inguinal fat pads isolated from mmp14 mice fed a control diet ( x - axis ) vs. high - fat diet ( y - axis ) . in comparison with gene expression profiles obtained in wild - type mice ( fig . 1b ) , genes involved in a diverse set of metabolic pathways critical to adipose tissue function ( asterisks ) are misregulated in mmp14 mice . c and d : mmp14 haploinsufficiency protects mice from diet - induced increase of fat mass after 3 or 6 months of high - fat diet feeding ( means sd ; n = 8) . representative images of inguinal and perigonadal fat pads are shown following isolation from mmp14 and mmp14 mice that had been placed on high - fat feeding for 3 months . e and f : feeding bouts ( g / day ) and metabolic rates adjusted for lean mass ( vo2/kg lean mass ) were determined in metabolic cages during high - fat diet feeding . ( a high - quality digital representation of this figure is available in the online issue . ) to next assess the long - term consequences of mmp14 haploinsufficiency on high - fat diet induced obesity , wild - type and haploinsufficient mice were placed on a high - fat diet for 36 months . the average percentage of fat mass of mmp14 haploinsufficient and wild - type mice placed under a control diet was comparable ( mean sd 5.29 0.58 vs. 5.07 0.7% ; n = 6 ) . on the high - fat diet , however , whereas wild - type mice displayed an approximate 20 and 60% weight gain after 3 and 6 months , respectively , mmp14 mice increased in total weight by less than one - half of that observed in the control ( fig . as expected , the dramatic changes in weight gain that occured in the long - term , high - fat feeding of mmp14 mice was also reflected in the attenuated expansion of tissue mass in isolated inguinal and perigonadal fat pads ( fig . changes in mmp14 expression did not , however , affect whole - body energy balance during high - fat feeding because the respiration rate , daily food intake , and locomotion of wild - type and heterozygous mice were not significantly different ( fig . these results stress the role of mmp14 as a proteolytic modifier that acts locally within adipose tissues without overtly affecting the hypothalamic regulation of metabolism . obesity is driven by a complex process that is coordinated by a host of genetic , epigenetic , and environmental factors ( 2628 ) . to extend the findings of the genotype - phenotype link found in mice , the association of mmp14 snp genotypes with human obesity and diabetes traits the human mmp14 gene is located at chromosome 14q1112 , comprising 10 exons and spanning a 10-kb region that contains 157 known snps ( ncbi dbsnp ) . using a preliminary group ( n = 48 ) randomly sampled from a japanese population ( 15 ) , we assessed the minor allele frequencies of candidate mmp14 snps ( fig . 16 snps spanning human mmp14 gene ( from rs17211964 at chr14:23,304,272 through rs2236307 at chr14:23,312,554 ) were screened to determine their pairwise linkage disequilibrium coefficients ( supplemental tables 1 and 2 ) . three snps located in exon 5 ( rs2236302 ; allele 2 , c > allele 1 , g ) , intron 5 ( rs2236304 ; allele 2 , c > allele 1 , g ) , and exon 6 ( rs2236307 ; allele 2 , t > allele 1 , c ) were chosen based on their frequency ( > 10% ) , proximity to the catalytic domain of mmp14 , and pairwise linkage disequilibrium that allow diverse haplotypes in combination . the minor allele ( allele 1 ) frequency for the three snps among the study population was 11.0 , 44.8 , or 38.5% , respectively ( fig . a : human mmp14 snps located in the proximity of the region encoding mmp14 catalytic domain . b : mmp14 haplotype determinations were based on the combination of rs2236302 , rs2236304 , and rs2236307 . haplotype associations with bmi of the total population ( n = 3,531 ) , women ( n = 1,944 ) , and men ( n = 1,587 ) were assessed in both dominant and recessive inheritance models . d : association of rs2236302 genotype with bmi and waist - to - hip ( w - h ) ratio . means sem of bmi ( left panel ) and waist - to - hip ratio ( right panel ) are shown for the total population ( n = 3,647 ) , women ( n = 1,944 ) , and men ( n = 1,703 ) , respectively . using a study population that included 3,653 individuals consisting of 1,708 men and 1,945 women , we assessed mmp14 haplotypes . the analysis of the pairwise linkage disequilibrium among the three snps suggested that they constitute a block of haplotypes ( d > 0.977 ) . however , the estimated square of the correlation coefficient ( r ) among the studied snps were sufficiently low ( table 1 ) to allow for the assembly of at least four major haplotypes ( fig . the association of mmp14 haplotypes with obesity and diabetes traits , i.e. , bmi , waist - to - hip ratio , body fat , a1c , fasting blood glucose and insulin levels , homeostasis model assessment ( homa ) of insulin resistance and -cell function , was examined in the dominant or recessive genetic model with multiple logistic regression analysis ( 17,18,28 ) . among the four major haplotypes of the mmp14 gene ( 212 , 221 , 122 , and 222 ) , the haplotype 122 ( gct ) was found to positively associate with bmi ( p = 0.0017 ) ( fig . 4c ) and waist - to - hip ratio ( p = 0.0079 ) ( table 2 ) . because of the dominant role played by rs2236302 in defining the link between obesity traits and mmp14 haplotypes , rs2236302 genotype was used to further delineate the link between mmp14 genotype and quantitative obesity traits . the distribution of c / c , c / g , and g / g genotypes were 79.7% ( n = 2,908 ) , 19.1% ( n = 695 ) , and 1.2% ( n = 44 ) among the study population . due to the low frequency of the g / g genotype , the quantitative association study was performed by comparing between homozygote c / c and heterozygote c / g genotype groups . the association with obesity traits was then examined in a genotype or dominant model with ancova . in the multivariate analyses of the total population , bmi and waist - to - hip ratio were associated with age ( p < 0.0001 ) , sex ( p < 0.0001 ) , history of smoking ( p = 0.0129 ) , hypertension ( p < 0.0001 ) , diabetes ( p = 0.0003 ) , and hyperlipidemia ( p < 0.0001 ) . when analyzed with adjustment for these variables , a highly significant correlation was observed between rs2236302 genotype and obesity traits ( fig . 4d ) ( mean sem bmi , c / c 22.74 0.06 vs. c / g 23.16 0.11 kg / m ; waist - to - hip ratio , c / c 0.901 0.001 vs. the increase of bmi caused by rs2236302 minor allele was 0.42 kg / m ( cohen d = 0.13 ) . homa- , homa of -cell function ; homa - ir , homa of insulin resistance . of note , the positive effect of rs2236302 genotype on bmi and waist - to - hip ratio was preferentially observed in women ( fig . 4d ) , suggesting the existence of sexual dimorphism in the link between mmp14 and obesity phenotype ( women p = 0.0004 , d = 0.199 , vs. men p = 0.5419 , d = 0.059 ) . finally , human mmp14 snps were found to be weakly associated with a1c in men ( p = 0.0685 ) ( table 3 ) , suggesting a paradoxical relationship of mmp14 snps with diabetes predisposition in males . consistently , mmp14 haplotype ( 121 ) associated with increased fasting blood glucose ( p = 0.0069 ) and homa of insulin resistance ( p = 0.0386 ) ( table 3 ) , supporting a potential link between mmp14 and diabetes in men . sexual dimorphism in the link between mmp14 and obesity / diabetes traits * any genotype . homa- , homa of -cell function ; homa - ir , homa of insulin resistance ; ns , p > 0.1 . in this study , we have demonstrated that a high - fat diet acutely initiates the mmp14-dependent degradation of the type i collagen network found in adipose tissues and induces a selective set of transcripts that link ecm - related remodeling to lipid / cholesterol biosynthesis . while one allele of mmp14 is sufficient for postnatal adipose tissue development , our results highlight the quantitative requirement of this protease for diet - induced expansion of adipose tissues in vivo . mmp14 gene expression , however , is not confined to preadipocyte / adipocyte cell population but can be found in vascular endothelial cells , pericytes , or fibroblasts ( 13,29,30 ) . consequently , mmp14-dependent remodeling of fat pad architecture may well involve the cooperative interplay of multiple cell populations . nevertheless , the direct physical association of the collagenous web with preadipocytes and adipocytes , coupled with the deposition of collagen degradation products in the periadipocyte space , supports a dominant role for these cells in diet - induced collagen remodeling . further , our in vitro results also support the importance of adipogenic regulation on preadipocyte - mediated collagenolysis . of note , human mesenchymal stem cells have recently been shown to mobilize mmp14 for trafficking and differentiation in three - dimensional collagen environments ( 31 ) . given that adipocyte progenitors can reside in perivascular stromal tissues ( 32,33 ) , it is intriguing to speculate that mmp14 may likewise support the migration and differentiation of adipocyte progenitors within adipose tissues . in addition to the ability of mmp14 to remodel collagen in a diet - dependent manner , the enzyme 's role in regulating the high - fat diet high - fat diet challenge rapidly within a week induces a selective set of genes enriched for ecm remodeling and lipid / cholesterol biosynthesis , including the previously described genes , mest and npr3 ( 34 ) . by contrast , the enrichment of genes associated with ecm remodeling and lipid / cholesterol biosynthesis is ablated in mmp14 haploinsufficient mice , though induction of mest and npr3 gene expression remains intact ( supplemental tables 1 and 2 ) . the precise molecular mechanism by which mmp14 gene selectively regulates the fat pad transcriptome is unknown ; however , mmp14-dependent collagenolysis may regulate transcriptional programs by modulating cell shape and tension in collagen - rich microenvironments ( 7,20 ) . despite the marked changes in fat pad size and function observed in mmp14 haploinsufficient mice , vo2 consumption , food intake , and physical activity in these mice appear to be comparable with controls . while white adipose tissues of high - fat diet challenged mmp14 haploinsufficient mice are small in size , the efficiency with which they oxidize fat versus carbohydrate may have undergone adaptive alterations in the in vivo setting . indeed , respiratory ratio ( vco2/vo2 ) , was relatively lower in mmp14 haploinsufficient mice , which is consistent with a preferred utilization of fat over carbohydrate . while severe lipodystrophy increases the risk of fatty liver ( 35 ) , we were unable to detect increased triglyceride accumulation in the livers of mmp14 haploinsufficient mice ( total glycerol 14.4 2.7 vs. 11.8 1.7% in wild - type and mmp14 mice , respectively ; n = 6 ) . in parallel with our findings in a mouse model of diet - induced obesity , human mmp14 snps were found to be associated with quantitative traits of obesity and diabetes in a japanese population . the link between mmp14 snps and obesity or diabetes traits found in this study may well be due to altered mmp14 gene expression , catalytic activity , exocytosis ( 36 ) , or unknown effects on neighboring genes that are in linkage disequilibrium . interestingly , however , a rare nonsynonymous polymorphism identified in exon 5 of mmp14 appears to alter collagenolytic activity and adipogenic potential in vitro ( t .- h.c . , unpublished observations ) , supporting its potential link with mmp14 catalytic activity . a sib - pair linkage analysis of black and caucasian nuclear family volunteers ( 364 sib pairs ) has pointed to chromosome 14q11 , where mmp14 resides , as one of three candidate loci linked with bmi and fat mass ( 37 ) . while genetic risks for obesity have recently been highlighted by the identification of fto ( 38 ) and mc4r ( 39 ) gene variants by genome - wide association studies ( gwas ) , candidate - gene approaches are still needed to bridge the gaps that remain unfilled by gwas analysis alone ( 4042 ) . our study of a japanese cohort is of moderate size ( n = 3,653 ) but demonstrates a significant increase of bmi with rs2236302 heterozygosity . the effect size of this risk allele is modest ( bmi 0.42 kg / m ) , which may not allow for its detection by gwas . though multiple genes affect obesity traits in mice without relevant findings in humans , mmp14 gene dose or polymorphism may define a genetic susceptibility for obesity traits that spans the gulf between mice and humans . in humans , obesity and diabetes phenotypes of mmp14 gene variants the stronger association of mmp14 genotype / haplotypes with obesity traits in women may relate their higher subcutaneous fat volume ( 43 ) . because subcutaneous fat depots contain higher concentrations of collagen fibers relative to other tissues , mmp14-dependent collagenolysis may contribute more to the size regulation of subcutaneous fat pads in women . female mice in the c57bl/6 background , whether mmp14 wild - type or haploinsufficient , did not significantly change their fat mass in response to diet . however , unlike male mice , mmp14 haploinsufficient female mice displayed a 30% reduction of fat mass even under the conditions of a control diet ( mean sd 13.3 1.9 vs. 9.5 0.9% for mmp14 and mmp14 mice , respectively ; n = 6 ) . of note , basal fat mass of female mice is more than two times higher than that of male mice . as such , mmp14 might be fully engaged in maintaining adipose tissue mass in female mice and perhaps in women as well . under these conditions , additionally , the association of rs2236302 with bmi found in women was reproduced in postmenopausal women ( n = 1,503 , p = 0.0008 ) , suggesting that constitutional but not hormonal differences contribute to the sexual dimorphism . conversely , in men , mmp14 gene variants are associated with diabetes but not obesity traits ( table 3 ) . the genetic or epigenetic predisposition that determines obesity or diabetes phenotypes in men , therefore , given the diverse biological functions of mmp family members interacting with an array of substrates ( 44 ) , it is often difficult to pin down the causal relationship between a specific mmp and a selective substrate . for example , mmp3 gene expression and variations are associated with body fat in pima indian population ( 45 ) . while mmp3 gene expression decreases in obesity , mmp3 has been shown to be necessary for adipocyte differentiation in a manner independent of ecm context ( 46 ) . indeed , mmp3 is not required for type i collagen hydrolysis ( 47 ) , and the substrate targets for mmp3 that regulates adiposity are unknown . by contrast , mmp14-dependent regulation of adipocyte differentiation is restricted to collagenous microenvironments ( 7 ) , suggesting that the mmp14type i collagen axis is the dominant pathway operative in adipocytes in vitro as well as in mouse and human adipose tissues . however , additional interactions with other mmp14 substrates , or the involvement of additional mmp family members in this process , can not be ruled out . indeed , mmp13 and mmp2 , whose latent forms can be activated by mmp14 ( 10,48 ) , may play cooperative roles in regulating adipocyte function . additional studies will be required to identify other pathogenic links that may exist between mmp family members and their respective substrates during obesity progression . along these lines , the metabolic impact of undigested collagen in mmp14 wild - type and haploinsufficient male mice also bears consideration . for example , the transgenic expression of hypoxia - inducible factor-1 leads to increased fibrosis , inflammatory response , and insulin resistance ( 49 ) . hence , while targeting mmp14 in adipose tissue may prevent diet - induced fat expansion , the overall impact of reduced collagen remodeling on inflammation and metabolism remains to be determined . nonetheless , our data lend further support to a model wherein mmp14 functions as a metabolic rheostat that controls the rate of collagen turnover in adipose tissues . our in vitro and mouse studies , combined with snp association analyses , point to a critical role for the mmp14/type i collagen axis in regulating adipose tissue mass in states of nutritional challenge .

objectivein white adipose tissue , adipocytes and adipocyte precursor cells are enmeshed in a dense network of type i collagen fibrils . the fate of this pericellular collagenous web in diet - induced obesity , however , is unknown . this study seeks to identify the genetic underpinnings of proteolytic collagen turnover and their association with obesity progression in mice and humans.research design and methodsthe hydrolysis and degradation of type i collagen at early stages of high - fat diet feeding was assessed in wild - type or mmp14 ( mt1-mmp)-haploinsufficient mice using immunofluorescent staining and scanning electron microscopy . the impact of mmp14-dependent collagenolysis on adipose tissue function was interrogated by transcriptome profiling with cdna microarrays . genetic associations between mmp14 gene common variants and obesity or diabetes traits were examined in a japanese cohort ( n = 3,653).resultsin adult mice , type i collagen fibers were cleaved rapidly in situ during a high - fat diet challenge . by contrast , in mmp14 haploinsufficient mice , animals placed on a high - fat diet were unable to remodel fat pad collagen architecture and display blunted weight gain . moreover , transcriptional programs linking type i collagen turnover with adipogenesis or lipogenesis were disrupted by the associated decrease in collagen turnover . consistent with a key role played by mmp14 in regulating high - fat diet induced metabolic programs , human mmp14 gene polymorphisms located in proximity to the enzyme 's catalytic domain were closely associated with human obesity and diabetes traits.conclusionstogether , these findings demonstrate that the mmp14 gene , encoding the dominant pericellular collagenase operative in vivo , directs obesogenic collagen turnover and is linked to human obesity traits .

eruption disorders are frequently encountered in dental practice ; these include ectopic eruption , impaction , over - eruption , infra - occlusion , transmigration , and failure of eruption . there are two types of failure of eruption ; ( 1 ) mechanical failure of eruption due to ankylosis or obstruction of the eruption pathway by hard or soft tissue , or ( 2 ) primary failure of eruption ( pfe ) which is failure of the eruptive mechanism of the tooth . proffit and vig described primary or idiopathic failure of eruption as a condition in which failure of full or partial eruption of nonankylosed teeth is attributed to the disturbance in the eruption process with a lack of mechanical interferences or other recognizable disorders . as an outcome to the absence of teeth in the posterior part of the dental arch , a posterior open - bite results , which does not respond to orthodontic treatment , complicating the overall management . transmigration of teeth is a rare disorder defined as the pre - eruptive intra - osseous migration of a tooth across the midline of the jaw . javid suggested that a canine should be classified as transmigrated when more than half of the length of the tooth had passed the midline . these transmigrated canines can either erupt ectopically at the midline or on the opposite side of the arch , or they just can remain asymptomatically impacted which is the more common outcome . the first case of canine transmigration reported in the literature was in 1951 , since then the number of reported transmigrated canines has increased and well exceeded the 127 cases reported by mupparapu . it was previously believed that the incidence of transmigration occurred distinctively in mandibular canines , this is now refuted as several cases of maxillary canine transmigration have been reported . in view of the fact that unilateral transmigration of mandibular canines is considered a rare phenomenon , the occurrence of bilateral transmigration of mandibular canines aktan et al . observed that only 9% of transmigrated mandibular canines were bilateral while the rest were unilateral . to date , the number of cases reported in the literature with bilateral mandibular canine transmigration has reached only 17 cases . both anomalies , pfe and bilateral canine transmigration are challenging to treat separately , and even more challenging when combined together in a single patient . we report the first case that combines pfe , bilateral mandibular canine transmigration , transposition , torus palatinus , and class iii incisor relationship . a 33-year - old female patient presented to the prosthodontic department requesting a denture to be made due to missing posterior teeth . upon revealing that she had never extracted any of her posterior teeth , a panoramic radiograph was required on which the presence of all of her missing teeth was revealed , she was then referred to the orthodontic department for further evaluation . her past medical history was completely normal with no history of radiation or unusual drug therapy . her family history was also normal with none of the family members having features of unerupted teeth . investigations were carried out to exclude any associated syndromes ; thyroid function tests , parathormone levels , hormone assays , serum calcium , and phosphorous levels . intra - oral examination [ figure 1 ] revealed 2 retained deciduous teeth 54 and 55 . the only permanent teeth present were 11 , 12 , 21 , and all 4 lower incisors . a torus palatinus existed and bulging related to the unerupted teeth with no signs of pain and crackling . a class iii incisor relationship with deep bite and bilateral posterior open - bite were present . examination of mandibular movements revealed no displacements excluding the presence of pseudo class iii malocclusion . ( a ) intraoral frontal photograph showing class iii incisor relationship with erupted upper permanent central incisors , upper right lateral incisor and lower incisors . ( b ) intraoral occlusal photograph of maxillary dental arch showing erupted 11 , 12 , 21 , retained upper right d and e and torus palatinus . ( c ) intraoral occlusal photograph of mandibular dental arch showing erupted 31 , 32 , 41 and 42 . ( d ) intraoral photograph showing posterior open - bite on the right side and bulging in the lower ridge . ( e ) intraoral photograph showing posterior open - bite on the left side and bulging in the lower ridge panoramic radiograph [ figure 2 ] revealed two retained deciduous teeth , 13 unerupted permanent teeth in the maxilla and 12 unerupted permanent teeth in the mandible . a total of 25 unerupted teeth : 13 , 14 , 15 , 16 , 17 , 18 , 22 , 23 , 24 , 25 , 26 , 27 , 28 , 33 , 34 , 35 , 36 , 37 , 38 , 43 , 44 , 45 , 46 , 47 , 48 . the upper left lateral incisor was located above the root apex of the upper left central incisor . however , the lower canines were horizontally impacted below the apices of the lower incisors and have transmigrated across the midline , with the right canine lying above the left canine . the 34 , 35 , and 45 were labially inclined . moreover , the lower molars on both sides were near to the inferior border of the mandible . panoramic radiograph showing 25 unerupted permanent teeth , transposition of upper left canine and the first premolar with mandibular canines horizontally impacted beneath the root apices of the lower central incisors and transmigrated across the midline the presence of class iii incisor relationship entailed investigation of the skeletal and dental factors that contributed to this malocclusion , therefore , a cephalometric radiograph was requested , which revealed normal skeletal features , increased proclination of lower incisors [ figure 3 and table 1 ] . cephalometric radiograph revealing increased lower incisor proclination ( l1-mp ) , protrusion of lower lip ( ll - e ) , reduced labiomental angle and posterior open - bite pretreatment cephalometric analysis of skeletal , dental and soft tissue parameters on reviewing the literature , this is the first case that combines both failure of eruption and bilateral canine transmigration . most of the reported cases either report failure of eruption or transmigration . moreover , the patient also presented with torus palatinus and anterior crossbites , which were not previously reported in the literature , ultimately adding bizarre features to this case . recent studies have shown that pfe is largely an inherited anomaly and that mutations in pth1r genes explain several familial cases of pfe . a number of known human syndromes give a clear evidence of the genetic origin of the eruption defect which manifests as part of the clinical features of the syndrome . these syndromes include cleidocranial dysplasia , osteoglophonic dysplasia , osteopetrosis , rutherford syndrome , and gapo syndrome [ growth retardation , alopecia , pseudoanodontia ( failure of tooth eruption ) , and progressive optic atrophy ] . nevertheless , proffit and vig observed that their patients did not have similarly affected close relatives , and none had any apparent medical problems or associated syndrome . proffit and vig concluded that the characteristics of pfe involve ( 1 ) more commonly affected posterior teeth than anterior ; ( 2 ) partial or complete failure of involved teeth ; ( 3 ) both deciduous and permanent molars may be affected ; ( 4 ) involvement may be unilateral or bilateral ; ( 5 ) affected permanent teeth are likely to become ankylosed ; ( 6 ) application of orthodontic force in an effort to extrude the teeth leads to their ankylosis ( 7 ) isolated cases and the absence of affected family members with this condition . most of the features presented in this patient were in line with the characteristics suggested by proffit and vig . disturbances of canine eruption are suggested to be under strong genetic control having shown to affect some family members . some believe that ectopic canines show signs of a multifactorial inheritance pattern with high phenotypic variance and low penetrance . studies have shown the involvement of the msx 1 gene in the developmental position of tooth buds , consequently , mutations in this gene can lead to disturbances in tooth positions . moreover , an association between pax 9 transcription factor and repositioning of tooth buds on the mesenchymal level has been established . proposed a classification for bilateral mandibular canine transmigration [ table 2 ] . in this patient the radiographic appearance of the transmigrated canines were in accordance with type iii subtype a , in which both mandibular canines had transmigrated across the midline , having a long - axis inclination of about 90 , and were positioned within the symphysis region , one above the other . mupparapu 's proposed classification of bilateral mandibular canine transmigration pfe presents an extremely challenging condition for orthodontic treatment planning . teeth with pfe do not respond to orthodontic extrusive mechanics and become ankylosed when subjected to orthodontic force . in view of the fact that an ankylosed tooth is considered as a method for absolute orthodontic anchorage , an effort to pull down a tooth which ankyloses soon after force this worsens the situation by converting an isolated posterior open - bite into a more general lateral open - bite . surgical procedures such as distraction osteogenesis or segmental osteotomy are useful treatment options to move unerupted teeth into occlusion ; however , their use in routine practice is limited due to the associated complications of high risks of damaging vital structures . moreover , the position of the nonerupting teeth may preclude surgery to bring the teeth into occlusion . regarding the transmigrated mandibular canines , the proposed treatment options are surgical removal , auto - transplantation , surgical exposure with orthodontic alignment and observation . surgical removal is the preferred treatment approach for migrated canines . as for transplantation , it can be considered if the mandibular incisors are in a normal position and there is sufficient space for the transmigrated canines . surgical exposure and orthodontic alignment can be carried out if it is feasible to bring the teeth into normal position . however , it will be mechanically impossible to bring them into place if the crown of such a tooth migrates past the opposite incisor area or if the apex is seen to have migrated past the apex of the adjacent lateral incisor . at last , observation of symptomless transmigrated canines might be the best and simplest solution with periodical successive radiographs taken for these patients . this report presents the first case of pfe combined with bilateral transmigration of mandibular canines , transposition of upper canine and the first premolar , torus palatinus , and class iii incisor relationship . both dental anomalies ; pfe and bilateral canine transmigration are extremely rare and even more rare when occurring together , making treatment planning and management very challenging for the dental practitioner . there is little understanding of both conditions because they are not well presented in textbooks , due to little number of documented cases . further documentation of these anomalies will lead to improvement and better understanding of these rare conditions .

eruption disorders are numerous varying from delayed to complete failure of eruption . primary failure of eruption ( pfe ) is a rare condition that involves arrested eruption of teeth with the absence of local or general contributory factors . another rare and clinically challenging phenomenon is canine transmigration which is the intra - osseous movement of impacted canines across the midline . this report presents the first case of combined failure of eruption of multiple teeth with bilateral mandibular canine transmigration , transposition of upper canine and the first premolar , torus palatinus , and class iii incisor relationship in a 33-year - old asymptomatic and nonsyndromic female patient .

human taeniasis results from an intestinal infection with the parasitic tapeworm of the genus taenia . the disease is caused by 3 species including taenia asiatica , taenia saginata , and taenia solium . taeniasis is zoonotic because they involve pigs ( t. solium and t. asiatica ) or cattle ( t. saginata ) as the intermediate host and humans as the definitive host . in case of t. saginata , when cattle digest the eggs , oncospheres hatch and then invade the intestinal mucosa and migrate via circulation to muscles where they develop into metacestodes ( = cysticerci ) . humans can become infected by ingesting raw or undercooked infected beef . usually in humans , a single t. saginata worm is infected , grows to become an adult in about 3 months . the adult tapeworm produces gravid proglottids , which crawl out of the anus of the infected humans or are diffused in the feces . abdominal discomfort , mild diarrhea , and loss of body weight can also occur due to the presence of the tapeworm in the intestine . before 1993 , when t. asiatica was first reported as a new species in the republic of korea ( = korea ) , all t. asiatica had been considered as t. saginata because of the almost indistinguishable morphology of the 2 species . therefore , human taenia tapeworms in korea were diagnosed as either t. solium or t. saginata , or undetermined until 1993 . in 1993 , it was reported that t. asiatica could be distinguished from t. saginata by the existence of the rostellum on its scolex , presence of small posterior protuberances in gravid proglottids , higher numbers of uterine twigs , and wart - like formations on the larval bladder surface . however , these distinctions are difficult to observe in each individual strobila , and both morphological and genetic analyses were required to clearly distinguish the 2 species . until the 1980s , in korea , human taeniases had been one of the not uncommon parasitic infections . an old report in 1924 documented that the prevalence of taenia spp . after 1927 , the prevalence declined gradually until it became 0% in a national survey of 20,541 randomly selected korean people in 2004 . however , in a subsequent national survey performed in 2012 which targeted 23,956 people , 0.04% ( 10 egg - positive cases ) prevalence of taenia spp.was detected . it was suggested that human taeniasis might be resurging recently . in this article , we report 4 cases of t. saginata infection all of which were found during 6 months in 2013 ( march to september ) , based on adult tapeworm recovery from the patients ( neither t. asiatica nor t. solium cases during the same period ) . all the patients had the history of travel abroad to asian countries , although no direct relationship was recognizable between the overseas travel and taenia tapeworm infections . the specific identification of the worms was based on molecular analyses , including pcr - rflp and partial sequencing of the cytochrome c oxidase subunit 1 ( cox1 ) gene of the worms . the 4 patients ( cases a - d ) found yellowish white tapeworm proglottids ( 4 - 5 in number in each patient ) moving in their underwear or feces and consulted to the department of parasitology and tropical medicine , seoul national university , seoul , korea . one of them complained of lower abdominal discomfort and anal itching ; however , the other 3 had no special clinical complaints . all of them had experiences of visiting other countries including china , cambodia , japan , dubai , or turkey . two of them tried flubendazole and albendazole based on wrong prescription , before prescribed with praziquantel in our department . all the 4 patients had the history of eating raw beef or rarely cooked beef steak recently ( table 1 ) . in case b ( 48-year - old male ) , an almost complete strobila ( about 1.1 m long ) without scolex was obtained after treatment with praziquantel ( 15 mg / kg in a single dose ) and purging with 40 g magnesium sulfate ( mgso4 ) . the gravid proglottids from the 4 patients revealed 16 - 20 lateral uterine branches and grossly looked like either t. saginata or t. asiatica . it was needed to do molecular analysis to obtain a specific diagnosis [ 9 - 11 ] . we performed pcr - rflp and gene sequencing of the mitochondrial cox1 for specific identification of the 4 taenia tapeworms . in order to use as positive controls , we obtained 3 known samples from the parasite resource bank in chungbuk national university ( cheongju , chungbuk province , korea ) ; t. asiatica ( cat . the genomic dna was extracted from a single segment by using the dneasy blood & tissue kit ( qiagen , hilden , germany ) as recommended by the manufacturer . the pcr primers used were t1f ( 5'-ata ttt act tta gat cat aag cgg-3 ' ) and t1r ( 5'-acg aga aaa tat att agt cat aaa-3 ' ) . pcr was carried out in a 30 l reaction mixture containing 15 l of smart 2x pcr pre - mix ( solgent co. , ltd , daejeon , korea ) , 2 l of template dna , 10 m of each primers , and 11 l of distilled water . pcrs were progressed under 1 cycle of initial denaturation at 94 for 3 min followed by 40 cycles of denaturation ( 94 for 1 min ) , annealing ( 52 for 1 min ) , and extension ( 72 for 1 min ) , with a final extension at 72 for 10 min . for pcr - rflp , the pcr products ( 10 l ) were digested with the restriction endonuclease ( 1 l of nco1 ) in a final volume of 13 l containing 2 l of ez - one 10x buffer for 1 hr at 37 , and then analyzed by electrophoresis on 2% agarose gels . the sequences of cox1 from taenia tapeworm specimens were aligned using the genieous program ( version 6.1.4 ) ( biomatters ltd . , the taenia tapeworms were identified based on the similarity of nucleotide sequences and phylogenetic relationships with those of t. asiatica from korea ( genbank no . the neighbor - joining tree was constructed under the kimura 2 parameter model by mega program version 5.2 to figure phylogenetic relationships . when the pcr products were digested with nco1 restriction enzyme , all the cases ( a , b , c , and d ) revealed identical pcr - rflp patterns which were consistent with the known t. saginata ( fig . 1 ) . only 1 band ( 491 bp ) was observed for t. saginata , whereas 2 different bands ( 152 bp and 339 bp ) were obtained for t. solium and t. asiatica . sequencing of the 491 bp cox1 gene showed 99.8 - 100% identity with t. saginata ; however , only 94.7 - 94.9% and 87.8 - 88.0% identity was seen with t. asiatica and t. solium , respectively ( table 2 ) . furthermore , the neighbor - joining tree revealed that our 4 specimens ( cases a - d ) were phylogenetically compatible to t. saginata but far from t. asiatica or t. solium reported from various asian countries and deposited in genbank ( fig . we used nco1 restriction enzyme , which recognizes and digests c^catgg sites , to analyze the mitochondrial cox1 gene of our taenia specimens . the nco1 was previously used for digestion of the cox1 gene of t. solium which was divided into 2 fragments . genes . in the present study , we observed for the first time that t. asiatica ( from the parasite resource bank , chungbuk national university ) was also divided into 2 fragments just like t. solium ( from the parasite resource bank ) both having the same recognition sequences by nco1 . however , in case of t. saginata ( our specimens ) , there was no site for enzyme recognition by nco1 , and thus the pcr products were preserved retaining only 1 band . therefore , the nco1 enzyme appears to be useful to clarify the distinction between t. saginata and t. asiatica which are morphologically difficult to distinguish . korea had been one of the endemic areas for taenia spp . until the 1980s . however , several documents announced that the taenia egg positive rate became as low as 0.02% nation - widely in 1997 and finally dropped to 0% in 2004 [ 15 - 17 ] . thereafter , only 5 sporadic cases were diagnosed during 2006 - 2011 , and 10 additional cases were detected in 2012 in a national survey of 23,956 randomly selected people . with regard to the occurrence of t. saginata , jeon et al . reported 14 specimens of t. saginata out of 68 taenia spp . tapeworm specimens collected during 1935 - 2005 in korea based on morphological and genetic analyses . after 2008 , 2 cases of t. saginata have been distinguished in chungbuk national university . however , case reports with species identification of t. saginata , including molecular analysis , have not been published since 2008 . in this study , we reported 4 patients of t. saginata infection who were referrred to our department during march - september 2013 . all the 4 patients had the history of travel abroad but no evidence for contracting taeniasis abroad could be identified . no matter what the source of infection is , our findings may suggest resurgence of t. saginata infection among the people in korea . however , in korea , the infection status of cattle with t. saginata metacestodes has seldom been documented except in a few old articles which described 17.8 - 21.3% prevalence in 1924 among cattle in seoul and gongju , up to 37.6% in 1926 among cattle from different localities , and 30.9% ( 1936 ) and 5.1% ( 1943 ) among cattle in jeju - do . particularly , after 1945 , not a single official document has been published on the prevalence of t. saginata metacestodes among the korean cattle . only one episode concerning a measled cattle ( = infected with t. saginata metacestodes ) was available from a rural village of gangwon - do in 1975 based on a personal communication . this study is significant to collect 4 cases of t. saginata infection within a short time period of 6 months in a university parasitology department in 2013 . neither t. asiatica nor t. solium cases were detected during the same period in the department . considering that only a limited number of t. saginata cases occurred previously in korea compared to t. asiatica which occurred 4 times more frequently than t. saginata , there might be a resurgence of t. saginata infection recently in korea . reactivation of the domestic life cycle of t. saginata , if it was remained , and increased overseas travel of people , as well as import of beef from foreign countries , may be the possible responsible factors . public attention should be paid to prevent human taeniasis avoiding intake of raw beef in korea as well as in asian countries endemic for taeniasis .

human taeniases had been not uncommon in the republic of korea ( = korea ) until the 1980s . the prevalence decreased and a national survey in 2004 revealed no taenia egg positive cases . however , a subsequent national survey in 2012 showed 0.04% ( 10 cases ) prevalence of taenia spp . eggs suggesting its resurgence in korea . we recently encountered 4 cases of taenia saginata infection who had symptoms of taeniasis that included discharge of proglottids . we obtained several proglottids from each case . because the morphological features of t. saginata are almost indistinguishable from those of taenia asiatica , molecular analyses using the pcr - rflp and dna sequencing of the cytochrome c oxidase subunit 1 ( cox1 ) were performed to identify the species . the pcr - rflp patterns of all of the 4 specimens were consistent with t. saginata , and the cox1 gene sequence showed 99.8 - 100% identity with that of t. saginata reported previously from korea , japan , china , and cambodia . all of the 4 patients had the history of travel abroad but its relation with contracting taeniasis was unclear . our findings may suggest resurgence of t. saginata infection among people in korea .

cancer is a disease that is difficult to treat , and novel drugs are still highly demanded . synthesis of metal complexes with biologically active ligands is a promising approach in developing anticancer drugs , as metal ions can significantly alter the physical and biological properties of these ligands . indolo[3,2-d]benzazepines , also referred to as paullones , are one class of potential cyclin - dependent kinase ( cdk ) inhibitors , identified in a comparative database search at the national cancer institute ( nci ; nci60 screen ) . thereby , the lead compound kenpaullone exhibited an activity profile similar to that of flavopiridol , the first clinically studied cdk inhibitor . within a series of paullones , however , the antiproliferative activity did not parallel the cdk inhibitory potencies . as a result , other intracellular targets for this class of compounds , e.g. , glycogen synthase kinase 3 ( gsk3 ) or mitochondrial malate dehydrogenase ( mmdh ) , have been suggested . despite marked efforts to develop these compounds as anticancer drugs , paullones remain at an early preclinical stage mainly because of their low aqueous solubility and bioavailability . however , the original paullones did not contain suitable binding sites for metal ions , and these had to be introduced by chemical modification . a library of paullone - based ligands with a broad structural diversity and the respective complexes with copper(ii ) , gallium(iii ) , ruthenium(ii ) , and osmium(ii ) have been reported . in an effort to elucidate novel structure activity relationships ( sars ) , the folded seven - membered azepine ring of paullones has been replaced by a pyridine ring , leading to another class of biologically active compounds , namely , indolo[3,2-c]quinolines , with an essentially planar structure . indolo[3,2-c]quinolines and the structurally related indolo[3,2-b]quinolines are known phytochemicals found in the roots of the west african climbing shrub cryptolepis sanguinolenta that is used in traditional african medicine . both exhibit a broad spectrum of biological properties , including antibacterial , antitumor , as well as anti - inflammatory activity . in contrast to indolo[3,2-b]quinolines , few studies have addressed indolo[3,2-c]quinolines . like paullones , they can , however , be introduced with synthetic tools essentially different from those applied for paullones . the first ruthenium(ii ) , osmium(ii ) , and copper(ii ) complexes with modified indolo[3,2-c]quinoline ligands were derived from structurally related paullone complexes using distinct chemical transformations . in particular , it has been found that complexes of indolo[3,2-c]quinolines exhibit higher cytotoxicity than their paullone counterparts , thus clearly establishing the effect of replacing the azepine ring in paullones by a pyridine ring in indoloquinolines . in addition , it was shown that sars of complexes with modified paullones do not necessarily apply to indolo[3,2-c]quinoline - based compounds . current efforts by us are focused on investigation of the underlying mechanisms of their antiproliferative activity by exploiting the intrinsic fluorescence of indolo[3,2-c]quinolines . recently , we reported on the syntheses of highly antiproliferative copper(ii ) complexes with modified indolo[3,2-c]quinolines . herein , we report on the synthesis of a more elaborate bioconjugate hl with two distinct binding sites and the dinuclear copper(ii ) and zinc(ii ) complexes 1 and 2 , respectively . the new ligand is sufficiently soluble in biological media and intrinsically fluorescent when light irradiated at ex = 395 nm . these properties permitted us to track the intracellular distribution of hl and 2 . moreover , the ligand design led to assembly of homometallic dinuclear complexes with distinct compartments ( scheme 1 ) , a feature not explored by us so far in the development of anticancer metal complexes . hcl ; ( ii ) diethyl-2,2-iminodiacetate , triethylamine , dry thf , room temperature , 3 h ( 95% ) ; ( iii ) methanol , room temperature , 2 h ( 95% ) ; ( iv ) copper(ii ) acetate monohydrate or zinc(ii ) acetate dihydrate , methanol , room temperature , 30 min [ 1 ( 37% ) , 2 ( 33% ) ] . hydrochloric acid , 2-hydroxy-5-methylbenzaldehyde ( a ) , diethyl-2,2-iminodiacetate , 4-((2-hydroxyethyl)-piperazin-1-yl)ethanesulfonic acid ( hepes ) , and guanosine 5-triphosphate were received from sigma - alrdich . l - histidine , formaldehyde solution ( 35% ) , copper(ii ) acetate monohydrate , and zinc(ii ) acetate dihydrate were received from merck , while tetrahydrofuran ( thf ) and methanol ( both analytical reagent grade ) were received from fisher scientific . dimethyl sulfoxide ( dmso ) was received from acros , ammonium bicarbonate , formic acid , and l - glutamic acid were received from fluka , and l - aspartic acid was received from serva . milli - q water ( 18.2 m , millipore advantage a10 , 185 uv ultrapure water system , molsheim , france ) and methanol ( fisher , hplc grade ) were used for esi - ms experiments . 6-hydrazinyl-11h - indolo[3,2-c]quinolone ( d , scheme 1 ) was synthesized according to the published protocol . details of the synthesis and h nmr characterization of d are given in the supporting information . 3-(chloromethyl)-2-hydroxy-5-methylbenzaldehyde ( b ) was obtained from 5-methyl-2-hydroxybenzaldehyde ( a ) via a previously described chloromethylation reaction . to a stirred solution of 3-(chloromethyl)-2-hydroxy-5-methylbenzaldehyde ( 0.10 g , 0.54 mmol ) in dry thf ( 30 ml ) was added diethyl-2,2-iminodiacetate ( 85 l , 0.48 mmol ) under argon atmosphere . upon addition of triethylamine ( 281 l , 2.0 mmol ) the colorless solution turned bright yellow and a white precipitate formed . after stirring for 3 h at room temperature , the yellow filtrate was concentrated under reduced pressure to give an orange oil , which was dried in vacuo overnight . h nmr 500.13 mhz ( dmso - d6 , h , ppm ) : 10.60 ( s , 1h , oh ) , 10.23 ( s , 1h , c1 ) , 7.43 ( d , 1h , j(hc5 ) = 2.0 hz , c7 ) , 7.32 ( d , 1h , j(hc7 ) = 2.1 hz , c5 ) , 4.12 ( q , 4h , j(hc16 , c18 ) = 7.1 hz , c15 , c17 ) , 3.90 ( s , 2h , c9 ) , 3.54 ( s , 4h , c11 , c13 ) , 2.26 ( s , 3h , c8 ) , 1.20 ( t , 6h , j(hc15 , c17 ) = 7.1 hz , c16 , c18 ) . c{h } nmr 125.76 mhz ( dmso - d6 , c , ppm ) : 192.9 ( c1 ) , 171.2 ( c12 , c14 ) , 158.3 ( c3 ) , 137.9 ( c5 ) , 129.1 ( c7 ) , 128.6 ( c6 ) , 125.2 ( c4 ) , 122.3 ( c2 ) , 60.8 ( c15 , c17 ) , 54.4 ( c11 , c13 ) , 53.6 ( c9 ) , 20.3 ( c8 ) , 14.5 ( c16 , c18 ) . to a solution of c ( 0.78 g , 2.3 mmol ) in methanol ( 40 ml ) the reaction mixture turned into a bright yellow suspension , which was stirred for 2 h under argon atmosphere and allowed to stand at 4 c overnight . the yellow product was filtered off , washed with cold methanol ( 2 4 ml ) , and dried in vacuo overnight . calcd for c32h33n5o50.75h2o ( mr = 581.15 ) : c , 66.14 ; h , 5.98 ; n , 12.05 . h nmr 500.13 mhz ( dmso - d6 , h , ppm ) : 12.46 ( s , 1h , n11 ) , 10.72 ( s , 1h , n5 ) , 10.63 ( s , 1h , oh ) , 8.77 ( s , 1h , c14 ) , 8.43 ( d , 1h , j(hc8 ) = 7.8 hz , c7 ) , 8.11 ( d , 1h , j(hc2 ) = 7.8 hz , c1 ) , 7.80 ( d , 1h , j(hc3 ) = 8.3 hz , c4 ) , 7.61 ( d , 1h , j(hc9 ) = 8.3 hz , c10 ) , 7.59 ( s , 1h , c20 ) , 7.507.44 ( m , 1h , c3 ) , 7.407.35 ( m , 1h , c9 ) , 7.307.22 ( m , 2h , c8 , c2 ) , 7.10 ( s , 1h , c18 ) , 4.13 ( q , 4h , j(hc29 , c31 ) = 7.1 hz , c28 , c30 ) , 3.94 ( s , 2h , c22 ) , 3.57 ( s , 4h , c24 , c26 ) , 2.30 ( s , 3h , c21 ) , 1.21 ( t , 6h , j(hc28 , c30 ) = 7.1 hz , c29 , c31 ) . c{h } nmr 125.76 mhz ( dmso - d6 , c , ppm ) : 171.2 ( c25 , c27 ) , 154.0 ( c16 ) , 152.1 ( c14 ) , 150.1 ( c6 ) , 138.6 ( c10a ) , 138.5 ( c11a ) , 138.3 ( c4a ) , 132.1 ( c18 ) , 129.5 ( c3 ) , 129.2 ( c20 ) , 127.8 ( c19 ) , 124.3 ( c9 ) , 124.2 ( c17 ) , 124.1 ( c6b ) , 122.9 ( c7 ) , 122.2 ( c1 ) , 121.9 ( c2 ) , 121.2 ( c8 ) , 120.8 ( c15 ) , 117.1 ( c4 ) , 113.6 ( c11b ) , 112.0 ( c10 ) , 105.2 ( c6a ) , 60.6 ( c28 , c30 ) , 54.5 ( c24 , c26 ) , 53.1 ( c22 ) , 20.7 ( c21 ) , 14.6 ( c29 , c31 ) . esi - ms ( methanol ) , positive m / z 379 [ hl n(ch2cooet)2 ] , 568 [ hl + h ] , 590 [ hl + na ] ; negative m / z 566 [ hl h ] , 603 [ hl + cl ] . vis ( methanol ) , max ( , m cm ) : 226 ( 43 300 ) , 260 ( 32 350 ) , 273 sh ( 25 300 ) , 306 ( 22 400 ) , 348 ( 15 250 ) , 365 sh ( 15 900 ) , 382 ( 17 300 ) . atr - ir , selected bands , cm : 3640 , 3375 , 2976 , 1730 , 1608 , 1461 , 1192 , 1002 . to a suspension of hl ( 0.20 g , 0.35 mmol ) in methanol ( 15 ml ) was added copper(ii ) acetate monohydrate ( 0.16 g , 0.78 mmol ) . after stirring for 30 min the dark - green solution was allowed to stand at 25 c to evaporate slowly . after 3 days , green crystals formed were filtered off , dried in vacuo overnight , and stored under argon atmosphere . calcd for c33h31cu2n5o91.5h2o ( mr = 795.72 ) : c , 49.81 ; h , 4.31 ; n , 8.80 . found : c , 49.57 ; h , 4.30 ; n , 8.64 . esi - ms ( methanol ) : positive m / z 604 unidentified , 648 [ 1 ( hoac ) ( oac ) ] , 680 [ 1 ( oac)2 + ( ch3o ) ] . uv vis ( methanol ) , max ( , m cm ) : 235 ( 60 800 ) , 272 ( 41 000 ) , 296 ( 24 540 ) , 354 ( 18 900 ) , 420 sh ( 20 800 ) , 441 ( 22 700 ) . atr - ir , selected bands , cm : 1737 , 1583 , 1540 , 1385 , 1217 , 1028 . x - ray diffraction - quality single crystals were picked from the reaction vessel prior to filtration . to a suspension of hl ( 0.14 g , 0.25 mmol ) in methanol ( 15 ml ) was added zinc(ii ) acetate dihydrate ( 0.12 g , 0.57 mmol ) . after stirring for 30 min after 4 days cold pentane was added and the mixture allowed to stand at 4 c for 3 h. the yellow precipitate formed was filtered off , dried in vacuo overnight , and stored under argon atmosphere . calcd for c33h31n5o9zn2ch3ohh2o ( mr = 822.46 ) : c , 49.65 ; h , 4.53 ; n , 8.52 . h nmr 500.13 mhz ( dmso - d6 , h , ppm ) : 12.3311.60 ( bs , 2h , n11 , n12 ) , 8.626.71 ( bm , 11h , c14 , 710 , 14 , 18 , 20 ) , 4.003.53 ( bm , 9h , c22 , 24 , 26 , 28 ) , 2.22 ( s , 3h , c21 ) , 1.88 ( bs , 6h , ch3coo ) . esi - ms ( methanol ) , positive : m / z 668 [ 2 ( oac)2 ( ch3 ) + ( ch3oh ) ] , 682 [ 2 ( oac)2 + ( ch3o ) ] , 710 [ 2 ( oac ) ] , 724 unidentified , 784 unidentified . uv vis ( methanol ) , max ( , m cm ) : 230 ( 44 400 ) , 258 ( 45 700 ) , 290 ( 27 900 ) , 309 ( 31 000 ) , 330 ( 17 900 ) , 346 ( 18 200 ) , 394 ( 18 900 ) . atr - ir , selected bands , cm : 1744 , 1706 , 1583 , 1407 , 1216 , 1012 . x - ray diffraction - quality single crystals were picked from the reaction vessel prior to addition of pentane . c hsqc , and h c hmbc nmr spectra were recorded on a bruker avance iii spectrometer ( ultrashield magnet ) in dmso - d6 at 25 c using standard pulse programs at 500.13 ( h ) and 125.76 ( c ) mhz . h and c nmr chemical shifts are quoted relative to the residual solvent signals . elemental analyses were carried out at the microanalytical service of the faculty of chemistry , university of vienna . electrospray ionization mass spectrometry was performed on a bruker esquire 3000 instrument ( bruker daltonic , bremen , germany ) on samples dissolved in methanol . vis spectra were recorded with an agilent 8453 spectrophotometer in the 1901000 nm window using samples dissolved in methanol at 10 m concentrations . ir spectra were measured with a bruker vertex 70 fourier transform ir spectrometer by means of the attenuated total reflection ( atr ) technique . fluorescence excitation and emission spectra were recorded with a horiba floromax-4 spectrofluorimeter and processed using the fluoressence v3.5 software package . samples of hl and 2 were prepared from a 1 mm solution of each in dmso and dilution with hepes buffer ( 20 mm , ph = 7.4 ) to give samples at 10 m concentrations with a maximum content of 1% dmso ( v / v ) . x - ray diffraction measurements were performed on a bruker x8 apexii ccd diffractometer . single crystals were positioned at 40 mm from the detector , and 1312 and 722 frames were measured , each for 60 and 90 s over 1 scan width for 13ch3oh and 22ch3oh , correspondingly . crystal data , data collection parameters , and structure refinement details are given in table 1 . structures were solved by direct methods and refined by full - matrix least - squares techniques . non - hydrogen atoms were refined with anisotropic displacement parameters , while h atoms were inserted in calculated positions and refined with a riding model . the following software programs were used : structure solution , shelxs-97 ; refinement , shelxl-97 ; molecular diagrams , ortep ; computer , intel coreduo . wr2 = { [w(fo fc)]/[w(fo)]}. gof = { [w(fo fc)]/(n p ) } , where n is the number of reflections and p is the total number of parameters refined . magnetic measurements were carried out on a microcrystalline sample of 1 with a quantum design squid magnetometer ( mpms - xl ) . variable - temperature ( 2300 k ) direct current ( dc ) magnetic susceptibility was measured under an applied magnetic field of 0.1 t. all data were corrected for the contribution of the sample holder and diamagnetism of the samples estimated from pascal s constants . analysis of the magnetic data was carried out by fitting the mt(t ) and m(t ) thermal variations including temperature - independent paramagnetism ( tip ) , impurity contribution ( ) , and intermolecular interaction ( zj ) according to the expression ( eq 1)1 complex formation was studied by uv vis titration of 10 and 250 m solutions of hl in methanol with 10 l aliquots of 0.5 and 6.25 mm stock solutions of copper(ii ) acetate monohydrate , respectively . one aliquot was added at 2 min intervals followed by homogenization of the solutions as within this period the equilibrium could be reached . an agilent 8453 spectrophotometer was used to record uv vis spectra in the 1901000 nm window . stability constants and molar absorbance spectra of the individual copper(ii ) complexes were calculated by the computer program psequad . electrospray ionization mass spectra were recorded on an amazon sl ion trap mass spectrometer ( bruker daltonics gmbh , bremen , germany ) . experimental data and provided simulations were acquired using compass 1.3 software and processed using data analysis 4.0 ( bruker daltonics gmbh , bremen , germany ) . the experimentally obtained mass signals include a maximum standard deviation of m / z 0.06 for each species . general instrument parameters were set as follows : positive - ion mode ( hv 4.5 kv , rf level 89% , trap drive 74.4 , dry temperature 250 c , nebulizer 8 psi , dry gas 6 l / min and average accumulation time 144 s ) , negative - ion mode ( hv 4.5 kv , rf level 89% , trap drive 63.8 , dry temperature 250 c , nebulizer 8 psi , dry gas 6 l / min and average accumulation time 2 ms ) . samples were diluted with water : methanol ( 50:50 ) or water : methanol : formic acid ( 50:50:0.2 ) to a final metal concentration of 510 m and measured by direct infusion into the mass spectrometer at a flow rate of 4 l / min . stock solutions of 1 and 2 in dmso ( 10 mm ) were prepared and stored at 20 c in the dark . each compound was diluted in ammonium carbonate buffer ( 20 mm , ph = 7.95 ) to give a solution of 100 m of each compound ( with 1% dmso content ) . furthermore , a solution containing l - histidine ( his ) , l - aspartic acid ( asp ) , l - glutamic acid ( glu ) , and guanosine 5-triphosphate ( gtp ) in equimolar amounts ( 100 m each ) and a solution containing his , asp , glu , and gtp ( each 100 m ) and ascorbic acid ( asc , 400 m ) were prepared in the same buffer . metal - containing solutions were diluted with buffer or mixed with the solutions containing the amino acids and asc at equimolar ratios to give a final metal concentration in each incubation mixture of 50 m . reaction mixtures were incubated at 37 c , and aliquots were measured directly after mixing and after 1 , 3 , 5 , and 24 h after 10-fold dilution of each with water : methanol ( 1:1 ) . the slightly acidic tetramethylammonium acetate buffer ( 20 mm , ph = 6 ) was avoided because partial release of the metal was observed . finally , dilution with water only resulted in a low ionization in the positive- and negative - ion modes . for cytotoxicity determination , three different human cancer cell lines were used : a549 ( nonsmall cell lung cancer ) and sw480 ( colon carcinoma ) from the american type culture collection ( atcc , manassas , va ) , both kindly provided by brigitte marian , institute of cancer research , department of medicine i , medical university vienna , austria , as well as ch1 ( ovarian carcinoma ) , established and kindly provided by the laboratory of lloyd r. kelland , crc centre for cancer therapeutics , institute of cancer research , sutton , u.k . cells were grown as adherent monolayer cultures in 75 cm culture flasks ( starlab , cytoone ) in minimal essential medium supplemented with 10% heat - inactivated fetal bovine serum ( invitrogen ) , 1 mm sodium pyruvate , 1% ( v / v ) nonessential amino acids ( from 100 ready - to - use stock ) , and 4 mm l - glutamine but without antibiotics at 37 c under a moist atmosphere containing 5% co2 and 95% air . all cell culture media and reagents were purchased from sigma - aldrich austria unless indicated otherwise . cytotoxicity was determined by the colorimetric mtt assay ( mtt = 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2h - tetrazolium bromide ) as described previously . briefly , cells were harvested by trypsinization and seeded in medium ( vide supra ) into 96-well plates in volumes of 100 l / well . depending on the cell line , different cell densities were used to ensure exponential growth of the untreated controls during the experiment : 1.0 10 ( ch1 ) , 2.0 10 ( sw480 ) , and 3.0 10 ( a549 ) cells per well . in the first 24 h , the cells were allowed to settle and resume exponential growth . then the test compounds were dissolved in dmso , serially diluted in medium , and added to the plates in volumes of 100 l / well so that the dmso content did not exceed 1% . due to limited solubility of hl and 1 , the highest concentration was applied in volumes of 200 l / well after replacing the original medium . after continuous exposure for 96 h ( in the incubator at 37 c and under 5% co2 ) , the medium was replaced with 100 l / well rpmi 1640 medium ( supplemented with 10% heat - inactivated fetal bovine serum and 4 mm l - glutamine ) and mtt solution ( mtt reagent in phosphate - buffered saline , 5 mg / ml ) in a ratio of 6:1 and plates were incubated for further 4 h. then the medium / mtt mixture was removed and the formed formazan was dissolved in dmso ( 150 l / well ) . optical densities at 550 nm were measured ( reference wavelength 690 nm ) with a microplate reader ( elx880 , biotek ) . the quantity of viable cells was expressed as a percentage of untreated controls , and 50% inhibitory concentrations ( ic50 ) were calculated from the concentration effect curves by interpolation . every test was repeated in at least three independent experiments , each consisting of three replicates per concentration level . sw480 cells were seeded in medium on coverslips in 6-well plates and allowed to settle and resume exponential growth for 24 h. then cells were incubated for 12 h with 5 m of 2 or 10 m of hl in medium . co - staining with er - tracker red and lyso - tracker red ( invitrogen ) was performed according to the manufacturer s instructions . after staining , each slide was washed three times in pbs . a fluorescence microscope bx40 ( olympus ) with f - view ccd camera ( olympus ) , cellf fluorescence imaging software ( olympus ) , and 60 magnification oil immersions objective lens were used . syntheses of the ligand hl and the copper(ii ) and zinc(ii ) complexes 1 and 2 , respectively , were carried out as shown in scheme 1 . we prepared a potentially hexadentate nonsymmetric ligand consisting of two chelating arms , one flexible able to provide facial coordination to an octahedral metal ion , while the second is rigid and provides a meridional binding . ester functionalities are frequently introduced into the structure of organic molecules to improve their aqueous solubility and bioavailability . recently , our group reported on the conjugation of l- and d - proline to 3-(chloromethyl)-2-hydroxy-5-methylbenzaldehyde ( b ) after chloromethylation of 2-hydroxy-5-methylbenzaldehyde ( a ) ( scheme 1 ) . similarly , we reacted 3-(chloromethyl)-2-hydroxy-5-methylbenzaldehyde ( b ) with diethyl-2,2-iminodiacetate and triethylamine in dry thf at room temperature , obtaining diethyl-2,2-((3-formyl-2-hydroxy-5-methylbenzyl)azanediyl)-diacetate ( c ) as an orange oil in excellent yield ( 95% ) . the ligand hl was obtained by reacting c with 6-hydrazinyl-11h - indolo[3,2-c]quinoline ( d ) in methanol at room temperature , again in excellent yield ( 95% ) . complexes 1 and 2 were synthesized in 37% and 33% yields starting from the ligand hl and copper(ii ) acetate monohydrate and zinc(ii ) acetate dihydrate , respectively , in methanol at room temperature . the complexation reaction is in both cases accompanied by hydrolysis of one ethyl ester group and transesterification of another ethyl ester function with formation of a new ligand hl . both generated donor arms are involved in coordination to copper(ii ) and zinc(ii ) in 1 and 2 , respectively , via the deprotonated carboxylate group and the carbonyl oxygen of the methyl ester group ( see scheme 1 , figures 1 and 2 ) . the ligand hl and its zinc(ii ) complex 2 have been characterized by one- and two - dimensional nmr spectroscopy , esi mass spectrometry , elemental analysis , uv vis , and atr - ir spectroscopy , while copper(ii ) complex 1 was studied by magnetic susceptibility measurements , esi mass spectrometry , and optical spectroscopy . h and c nmr spectral data of intermediate c , ligand hl , and zinc(ii ) complex 2 along with their assignments are given in the experimental section . the presence of a proton at n in the h nmr spectra and the chemical shift of neighboring c in the c nmr spectra indicate that the ligand adopts a configuration with an exocyclic c = n double bond . moreover , esi mass spectra of 1 and 2 in methanol showed peaks that confirmed formation of dimetal complexes . the most abundant peaks at m / z 680 and 682 for 1 and 2 , correspondingly , were assigned to [ 1/2 ( oac)2 + ( ch3o ) ] . uv vis spectra of hl , 1 , and 2 in methanol are depicted in figure s1 ( supporting information ) . metal coordination led to pronounced changes in the visible range of the ligand spectrum , namely , to evolution of an absorption band at ca . 400 nm for 2 and formation of a broad charge - transfer band at 440 nm for 1 . results of x - ray diffraction studies of 13ch3oh and 22ch3oh shown in figures 1 and 2 , respectively , confirm formation of dinuclear complexes with the two copper(ii ) ions and zinc(ii ) ions bridged by the phenolate oxygen and two exogenous 2-: acetato ligands . both copper(ii ) ions in 1 are distorted square pyramidal with = 0.27 for cu1 with the bridging phenolate oxygen o1 in an apical position and tertiary amine n23 , atoms o6 and o8 of the two bridging acetates , and one aminoacetate o2 in the basal plane . we do not describe the coordination environment around cu1 as octahedral , since the interaction between cu1 and o5 of the dangling methyl ester group is extremely weak ( cu1o5 2.946(2 ) ) . for cu2 a distorted square - pyramidal coordination geometry ( = 0.22 ) was realized with a bridging phenolate oxygen o1 , quinoline nitrogen n5 , hydrazinic nitrogen n13 , one oxygen atom o9 of bridging acetate in a basal plane , and another bridging acetate oxygen atom o7 in apical position . unlike 1 , the coordination environments of zinc(ii ) ions in 2 differ from each other . zn1 has an octahedral environment comprised of the bridging phenolate oxygen o1 , tertiary amine donor n23 , methyl ester oxygen o5 , and atom o6 of the bridging acetate in equatorial positions and two oxygen atoms , one aminoacetate o2 , and a second o8 of a bridging acetate in apical positions . zn2 in contrast to cu2 shows a more pronounced tendency toward a trigonal - bipyramidal coordination geometry ( = 0.47 ) of the same donor atoms . cu2 lies in the mean plane through cu2n5c6n12n13 in 1 , while cu1 comes out from this plane by 1.307 . in 2 the deviation of zn1 from the mean plane through zn2n5c6n12n13 is markedly smaller ( 0.820 ) , while distortion from planarity of the indoloquinoline moiety is more evident than in 1 . the bridging of copper(ii ) ions via phenolate oxygen results in distinct cu1o1 and cu2o1 bond distances . the difference between them ( 0.39 ) is larger than in other nonsymmetrically -phenoxido bridged dicopper(ii ) complexes , in which the two copper(ii ) ions in addition are bridged by at least one exogenous 2-: acetato group . the cu1o6 bond distance is markedly shorter than cu2o7 , and cu1o8 is also shorter than cu2o9 ( see caption to figure 1 ) . the cu1cu2 distance in the complex is 3.2897(6 ) , which is comparable with cucu distances of 3.297(3 ) and 3.263(2 ) in dicopper(ii ) complexes with symmetric dinucleating ligands , containing a di--acetato--phenolatodicopper(ii ) core . ortep view of [ cu2(l)(ch3coo)2 ] with thermal ellipsoids drawn at the 50% probability level . selected bond distances ( angstroms ) and bond angles ( degrees ) : cu1o1 2.323(2 ) , cu1o2 1.937(2 ) , cu1o6 1.946(2 ) , cu1o8 1.936(2 ) , cu1n23 2.093(3 ) , cu1o5 2.946(2 ) , cu2o1 1.913(2 ) , cu2o7 2.194(2 ) , cu2o9 2.002(2 ) , cu2n5 2.038(3 ) , cu2n13 1.956(3 ) , cu1o1cu2 101.47(10 ) . the zn1o1 bond distance is only slightly longer than zn2o1 , as also observed in other complexes with nonsymmetrical dinucleating ligands with a di--acetato--phenolatodizinc(ii ) core . the zn1o6 bond distance is only slightly longer than zn2o7 , while the difference between zn1o8 and the shorter bond zn2o9 is more pronounced ( see caption to figure 2 ) . the interaction between zn1 and o5 of the dangling methyl ester group is markedly stronger than comparable interaction in 1 . the zn1zn2 distance in the complex is at 3.2154(7 ) , which is similar to the znzn distance of 3.29(1 ) in a dizinc(ii ) complex with a nonsymmetrical hybrid ligand . ortep view of [ zn2(l)(ch3coo)2 ] with thermal ellipsoids drawn at the 50% probability level . selected bond distances ( angstroms ) and bond angles ( degrees ) : zn1o1 2.057(3 ) , zn1o2 2.105(3 ) , zn1o6 2.004(3 ) , zn1o8 2.079(3 ) , zn1n23 2.149(4 ) , zn1o5 2.322(3 ) , zn2o1 2.027(3 ) , zn2o7 1.991(3 ) , zn2o9 1.993(3 ) , zn2n5 2.087(4 ) , zn2n13 2.097(4 ) , zn1o1zn2 103.86(14 ) . the magnetic behavior of a polycrystalline sample of 13ch3oh in the temperature range 2300 k in a field of 0.1 t is shown in figure 3 . the value of mt is 0.952 cm k mol at 300 k. this value is slightly higher than the expected mt value ( 0.750 cm k mol ) for two noninteracting copper(ii ) ions ( d , g = 2.0 , s = 1/2 ) . the value of mt continuously increases with decreasing temperature and reaches a value of 1.174 cm k mol at 3 k. this behavior suggests the presence of ferromagnetic interactions in 13ch3oh . according to x - ray diffraction data , complex 13ch3oh has a dinuclear structure , in which the two copper(ii ) ions are connected by a phenolate oxygen atom and two bidentate bridging acetato ligands ( figure 1 ) . therefore , the magnetic behavior can be analyzed by using the classical spin hamiltonian ( eq 2):2where j is the exchange coupling constant and s1 = s2 = 1/2 . plots of mt vs t and magnetization vs h ( inset ) at 2 and 3 k for 13ch3oh . solid lines correspond to the best fit with parameters quoted in the text . in this case , the van vleck equation leads to the following analytical expression ( eq 3)3the fitting procedure results in an excellent agreement between the experimental data and the calculated curve ( r = 1.4 10 ; figure 3 ) . the parameters extracted from the fit are j = 3.49(3 ) cm , g = 2.24(1 ) , and zj = 0.08(1 ) cm and correspond to ferromagnetic interaction between copper(ii ) ions . the temperature - independent paramagnetism ( tip ) and impurity contribution ( ) have values close to zero , and both were fixed at zero in the final fit . the presence of ferromagnetic interaction was confirmed by magnetization measurements at low temperature ( see inset picture in figure 3 ) . the fitting of magnetization vs field using the brillouin function indicates the presence of spin ground state s = 1 ( g = 2.203(2 ) ) in 13ch3oh , which is consistent with the results obtained from analysis of the temperature dependence of magnetic susceptibility . the nature of magnetic interaction in dinuclear copper(ii ) complexes has been extensively studied from both theoretical and experimental points of view . the magnetic interaction in 13ch3oh occurs via three bridges : two 2-: acetato ligands and one bridging phenolate . . according to the literature , the acetate bridges mediate the antiferromagnetic interactions , while the phenolate bridge in dinuclear copper(ii ) complexes can promote both antiferromagnetic as well as ferromagnetic interactions . the character of magnetic interaction depends on geometrical features , especially on the cu o cu angle , out - of - plane deviation angle ( see figure 4 ) , and torsion angle cu o cu o . for angles < 99 and angles > 30 , a strong ferromagnetic interaction can be expected . in the case of 13ch3oh with = 101.47 and = 30.04 , the presence of a weak ferromagnetic interaction ( j = 3.49 cm ) is justified . we can conclude that due to the out - of - plane deviation of the phenol group relative to cu o cu plane the resulting magnetic interaction between the triple - bridged cu(ii ) ions is weakly ferromagnetic . comparable weakly ferromagnetic interactions were reported for other dinuclear copper(ii ) complexes with two or three different bridges . to elucidate whether the two binding sites in hl show different affinities to copper(ii ) , vis titrations of the ligand hl at two different concentrations with copper(ii ) acetate monohydrate in methanol at room temperature ( figures 5 and s2 , supporting information ) . uv vis absorbance spectrum of the ligand hl ( dashed trace ) and its changes by addition of copper(ii ) acetate monohydrate ( solid traces ) in methanol ( cl = 10 m ; ccu = 022.5 m ; t = 298 k ; l = 1 cm ) . 440 nm was observed upon addition of up to 1.5 mol equiv of copper(ii ) . a wide band with max at 664 nm overlapped partly with the charge - transfer band was seen upon addition of copper(ii ) . this absorption band is slightly red shifted upon addition of more than 1 equiv of copper(ii ) . on the basis of the spectral changes in the wavelength range 230520 nm ( figure 5 ) , overall stability constants have been calculated for the mono- [ cul ] ( log = 7.17 0.08 ) and dinuclear [ cu2l ] species ( log = 13.13 0.24 ; log k = 5.96 ) . the molar absorbance spectra of the ligand , [ cul ] , and [ cu2l ] complexes were also calculated ( figure 5 ) . the goodness - of - fit between measured and calculated absorbance values is shown in figure 6 . d transition bands were in good agreement with those obtained by monitoring the charge - transfer band within 0.2 log unit . stepwise formation constant of the [ cu2l ] species is merely 1 log unit lower than that of the [ cul ] showing the overlapping binding of the metal ions . therefore , it can be concluded that both binding sites in ligand hl coordinate with a similar affinity and no preference for either of them can be perceived . measured and calculated ( dashed lines ) absorbance values at 382 ( ) and 440 nm ( ) at various hl -to - copper(ii ) ratios ( cl = 10 m ; ccu = 022.5 m ; t = 298 k ; l = 1 cm , methanol ) . the stability of complexes 1 and 2 in aqueous solution and their reactivity toward small biomolecules was studied by esi mass spectrometry ( esi - ms ) since it proved to be effective for characterizing also complex metallodrug interactions with biomolecules . both complexes display a very similar aqueous solution behavior , which is characterized by ester hydrolysis of the ligand and partial metal release over time . products of ester hydrolysis are detected directly after dissolving the compounds in buffer , and the ester is quantitatively hydrolyzed within 24 h. the major thermodynamic products after this period correspond to ions [ m2(l me)(oh ) h ] and [ m(l me ) h ] , where m = cu or zn and l = l , detected in the negative - ion mode ( figure 7 ) . the latter mass signal suggests that release of specifically one metal can occur from both 1 and 2 . interestingly , these signals are detected at 95% and 38% intensities relative to [ m2(l me)(oh ) h ] for 1 and 2 , respectively , i.e. , the cu complex 1 releases the metal to a greater extent . therefore , complex 2 appears to be slightly more stable in aqueous solution , which also seems to be of relevance for the cytotoxicity . additionally , 2 does not ionize in the positive - ion mode , suggesting stable bonds between zn ions and the acetato ligands ( figure s3 , supporting information ) . note that acetato complexes were not detected in the mass spectra of 1 or 2 . furthermore , the isotopic distributions of the major mass signals of 1 and 2 are in good agreement with simulated patterns ( figure s4 , supporting information ) . both complexes were exposed to mixtures containing equimolar amounts of l - histidine ( his ) , l - aspartic acid ( asp ) , l - glutamic acid ( glu ) , and guanosine 5-triphosphate ( gtp ) . the complexes did not react with any of the biological nucleophiles , and similar mass spectra were observed compared to solutions containing only the respective metal . addition of 4 equiv of ascorbic acid ( asc ) to the amino acids resulted in transient formation of glu and asc adducts with 1 in a small amount ; however , they were only detected immediately after mixing ( figure 7c ) and absent for 2 . free ascorbate was consumed within 1 h but had no impact on the overall reactivity of the complexes . esi mass spectra in negative - ion mode are shown for 1 ( a ) and 2 ( b ) in methanol over a period of 24 h. ( c ) glu- and asc - adducts of 1 , which were only detected directly after mixing . an interesting feature of both compounds is their ability to release a metal ion also in a ph - dependent manner ( figure s5 , supporting information ) . the samples incubated at ph = 7.95 for 24 h displayed only partial metal release . lowering the ph of this incubation solution by dilution with 0.1% formic acid resulted in immediate and quantitative release of one metal from both dimetallic complexes . it is suggested that the carboxylates are prone to protonation under these conditions , leading to release of the coordinated metal . fluorescence spectra of hl and 2 were recorded in hepes - buffered solutions ( 20 mm ; ph = 7.4 ) with a 1% ( v / v ) content of dmso ( figure s6 , supporting information ) . fluorescence excitation spectra ( em = 470 nm ) were measured in the range between 260 and 460 nm and emission spectra ( ex = 395 nm ) in the range from 410 to 710 nm . coordination to zinc(ii ) led to a blue shift of the emission band by 54 nm , with the maximum at 466 nm in the spectra of 2 . hl was found fluorogenic , as excitation and emission spectra strongly increased in intensity upon binding to zinc(ii ) . the cytotoxicity of hl , 1 , and 2 was determined by the mtt assay in three human cancer cell lines , namely , a549 ( nonsmall cell lung carcinoma ) , ch1 ( ovarian carcinoma ) , and sw480 ( colon adenocarcinoma ) , all yielding ic50 values in the micromolar concentration range ( table 2 ) . values for a simple copper(ii ) salt , cucl2 , are given for comparison . fifty percent inhibitory concentrations ( means standard deviations from at least three independent experiments ) , as obtained by the mtt assay using exposure times of 96 h. ch1 is the most sensitive cell line to all tested compounds , whereas a549 , a more chemoresistant cell line equipped with multidrug - resistance - mediating proteins , is the least sensitive one , with ic50 values up to 13 times higher than in ch1 cells . whereas complexation with copper(ii ) has either little effect on cytotoxicity ( a549 , sw480 cells ) or yields 3-fold decreased potency ( ch1 cells ) , complexation with zinc(ii ) results in about 2-fold enhancement of cytotoxicity , compared to the metal - free ligand hl in all three cell lines . in comparison to the dicopper(ii ) complex 1 , the dizinc(ii ) complex 2 is up to three times more active in sw480 and four times more active in ch1 cells ( see also figure s7 , supporting information ) . this might be directly related to the lower tendency of 2 to release a metal in aqueous media compared to 1 as observed in the esi - ms experiments . on the basis of these observations , it can be concluded that complexation to zinc(ii ) results in higher cytotoxicity but also better solubility in biocompatible media compared to the metal - free ligand as well as copper(ii ) complex 1 . cytotoxic potency of a simple copper(ii ) salt , cucl2 , is lower , with ic50 values being at least five times higher than those of complex 1 . on the basis of the fluorescence properties of hl and dizinc(ii ) complex 2 , their subcellular localization was studied by fluorescence microscopy in human cancer cells including their colocalization with organelle - specific dyes . for visualization of the compounds in live sw480 cells , the u - mwu2 filter ( olympus japan , excitation filter bp330 - 385 , emission filter ba420 ) was used , while the costaining dyes were recorded using the u - mwg2 filter ( olympus japan , excitation filter bp510 - 550 , emission filter ba590 ) . the compounds do not show interference in the u - mwg2 channel , and autofluorescence of the cells was not observed with the used filters . microscopic images of cells treated with hl and 2 , as shown in figure 8 , revealed localization of fluorescence in diffuse voluminous as well as distinct small cytoplasmic structures but no discernible uptake into the nucleus . the highest accumulation matches with both the er - tracker red and the lyso - tracker red staining , suggesting that the endoplasmic reticulum as well as lysosomes are potential target compartments of hl or that lysosomes are involved in sequestration and/or detoxification of the compound . the same may apply to 2 , provided that the complex is sufficiently stable throughout its passage through the cell , as it can not be ruled out that the fluorescence distribution originates from dissociated ligand molecules . cells were costained with 10 m of hl ( a ) or 5 m of 2 ( b ) and er - tracker red ( 500 nm ) and lyso - tracker red ( 1 m ) , respectively . condensation of 6-hydrazinyl-11h - indolo[3,2-c]quinoline with diethyl-2,2-((3-formyl-2-hydroxy-5-methylbenzyl)azanediyl)diacetate afforded a new nonsymmetric dinucleating ligand hl with increased aqueous solubility and fluorescence properties . complexes 1 and 2 were obtained upon treatment of the ligand with 2 equiv of cu(ch3coo)2h2o and zn(ch3coo)22h2o in methanol , respectively . complexation reaction in both cases is accompanied by hydrolysis of one ethyl ester group and transesterification of another ethyl ester function with formation of hl . dinuclear structure in 13ch3oh and 23ch3oh is supported by three bridges : two acetato ligands and one phenolato bridge from nonsymmetric hl ligand . the temperature dependence and field dependence magnetic measurements for 13ch3oh indicate a weak ferromagnetic interaction ( j = 3.49 cm ) between copper(ii ) ions . all three compounds show respectable antiproliferative activity in human cancer cell lines ( a549 , ch1 , sw480 ) with ic50 values in the low micromolar concentration range . it seems that the increased resistance of 2 toward metal release in aqueous solution compared to 1 may be responsible for the higher cytotoxicity . localization of hl and 2 in cytoplasmic structures has been found by fluorescence microscopy , suggesting that the endoplasmic reticulum as well as the lysosomes can be potential target compartments of these compounds .

dicopper(ii ) and dizinc(ii ) complexes [ cu2(meooclcoo)(ch3coo)2 ] ( 1 ) and [ zn2(meooclcoo)(ch3coo)2 ] ( 2 ) were synthesized by reaction of cu(ch3coo)2h2o and zn(ch3coo)22h2o with a new nonsymmetric dinucleating ligand etoochlcooet prepared by condensation of 6-hydrazinyl-11h - indolo[3,2-c]quinoline with diethyl-2,2-((3-formyl-2-hydroxy-5-methylbenzyl)azanediyl)diacetate . the design and synthesis of this elaborate ligand was performed with the aim of increasing the aqueous solubility of indolo[3,2-c]quinolines , known as biologically active compounds , and investigating the antiproliferative activity in human cancer cell lines and the cellular distribution by exploring the intrinsic fluorescence of the indoloquinoline scaffold . the compounds have been comprehensively characterized by elemental analysis , spectroscopic methods ( ir , uv vis , 1h and 13c nmr spectroscopy ) , esi mass spectrometry , magnetic susceptibility measurements , and uv vis complex formation studies ( for 1 ) as well as by x - ray crystallography ( 1 and 2 ) . the antiproliferative activity of etoochlcooet , 1 , and 2 was determined by the mtt assay in three human cancer cell lines , namely , a549 ( nonsmall cell lung carcinoma ) , ch1 ( ovarian carcinoma ) , and sw480 ( colon adenocarcinoma ) , yielding ic50 values in the micromolar concentration range and showing dependence on the cell line . the effect of metal coordination on cytotoxicity of etoochlcooet is also discussed . the subcellular distribution of etoochlcooet and 2 was investigated by fluorescence microscopy , revealing similar localization for both compounds in cytoplasmic structures .

a choroidal hemangioma is an uncommon benign vascular tumor of the choroid that can be either circumscribed or diffuse . in our experience , diffuse choroidal hemangiomas in asian patients often require multiple photodynamic therapy ( pdt ) treatment sessions . five sessions of pdt treatment were required over a period of 1 year and a final optical coherence tomogram 3 months later revealed resolution of subretinal fluid and the choroidal hemangioma . this case report illustrates that a single application of pdt using standard published parameters was insufficient to achieve the destruction of the enlarged vessels . the decision for repeat treatment was based on subretinal fluid recurrence , rather than complete tumor regression . our case report supports previous suggestions that larger dilated vessels in the vascular network of a choroidal hemangioma might affect the efficacy and selectivity of pdt in treating the eyes of asian patients which may explain the need for multiple treatments . choroidal hemangioma is an uncommon benign vascular tumor of the choroid that can be either circumscribed or diffuse.1 diffuse choroidal hemangiomas , usually associated with sturge weber syndrome ( sws ) , are more likely to develop secondary retinal detachment.2 diffuse choroidal hemangiomas have been treated using many modalities including radiotherapy , antivascular endothelial growth factor , and photodynamic therapy ( pdt).2 theoretically , pdt has ideal properties for such treatment as it offers site - specific tumor destruction while sparing the overlying retina and retinal vasculature.3 to date , there have been only six case reports of successful single pdt treatment for diffuse choroidal hemangiomas.2 we present a case report to illustrate a case of diffuse choroidal hemangioma that was refractory to a single treatment . a 7-year - old boy with sws presented with blurring of vision in the left eye ( os ) with increasing hypermetropia ( 8.0 d to 11.0 d ) over a period of 6 months . at presentation , his best - corrected visual acuity ( bcva ) was 20/20 right eye ( od ) and 20/150 os . fundus examination of the left eye revealed a diffuse , orange choroidal tumor with overlying exudative retinal detachment ( rd ) . b - scan ultrasound confirmed the presence of a diffuse choroidal hemangioma with thickening of the choroid and associated rd ( figure 1 ) . verteporfin was infused at a concentration of 6 mg / m , and an 83-second treatment was conducted with a 689 nm visulas 690s photodynamic therapy laser system ( carl zeiss meditec , inc . , ag , jena , germany ) that was delivered at 50 j / cm with an intensity of 600 mw / cm . five partially overlapping , maximally large spots ( 5200 m ) were applied to the diffuse choroidal hemangioma . optical coherence tomography was used to monitor treatment response ( figure 1a and figure 2 ) . however , because of persistent choroidal thickening and subretinal fluid , additional sessions of pdt , as described , were applied 2 , 3 , and 4 months post - presentation ( a total of four sessions ) , with persistent subretinal fluid at ocular coherence tomography ( oct ) thicknesses of 557 m , 507 m , and 407 m , respectively . these further treatments finally resulted in elimination of the subretinal fluid and the overlying exudative rd ( oct retinal thickness at fovea 337 m ) , and bcva improved to 20/40 os . a year later , on routine follow - up , the patient presented with recurrence of subretinal fluid diagnosed on oct and a reduction in bcva to 20/100 os . a fifth session of pdt treatment applied and a final oct 3 months later revealed resolution of subretinal fluid and the choroidal hemangioma ( oct retinal thickness at fovea 300 m ) with a stable final bcva of 20/100 os . this case report exemplifies our experience with asian patients with diffuse choroidal hemangiomas , who often require multiple treatments with pdt ( unpublished ) . a single application of pdt using currently prescribed parameters is insufficient to destroy the enlarged vessels . this experience is similar to previous reports from china describing circumscribed choroidal hemangiomas.4 the decision for repeat treatment was based on subretinal fluid recurrence , rather than on complete tumor regression . however , our patient was refractory to treatment , requiring a total of five pdt sessions . factors such as timing of laser application , absorption wavelength , and laser dose characteristics influence the efficacy of pdt.5 however , it has been suggested that patients of asian descent may respond differently to pdt treatment,6 possibly due to inherent differences in the retina , such as increased pigmentation in the retinal pigment epithelium . in another study involving chinese patients , it was suggested that the larger dilated vessels in the vascular network of a choroidal hemangioma might affect the efficacy and selectivity of pdt.4 diffuse choroidal hemanogiomas associated with sws are uncommon but require aggressive treatment to prevent visual loss from exudative rd , and possibly , subsequent sequelae such as amblyopia

backgrounda choroidal hemangioma is an uncommon benign vascular tumor of the choroid that can be either circumscribed or diffuse . in our experience , diffuse choroidal hemangiomas in asian patients often require multiple photodynamic therapy ( pdt ) treatment sessions.methodswe here provide a case report of a 7-year - old boy with sturge weber syndrome who presented with diffuse choroidal hemangioma in the left eye . five sessions of pdt treatment were required over a period of 1 year and a final optical coherence tomogram 3 months later revealed resolution of subretinal fluid and the choroidal hemangioma.resultsfinal visual acuity was 20/100 in the left eye with resolution of subretinal fluid . this case report illustrates that a single application of pdt using standard published parameters was insufficient to achieve the destruction of the enlarged vessels . this experience is similar to previous chinese reports on circumscribed choroidal hemangiomas . the decision for repeat treatment was based on subretinal fluid recurrence , rather than complete tumor regression.conclusionour case report supports previous suggestions that larger dilated vessels in the vascular network of a choroidal hemangioma might affect the efficacy and selectivity of pdt in treating the eyes of asian patients which may explain the need for multiple treatments .

the previous issue of critical care includes a report of a multicenter study in which cardiac index and gastric mucosal ph targets were compared during resuscitation of patients with septic shock . similar to all other hemodynamic variables with the exception of central venous oxygen saturation , the value of a cardiac index target in the resuscitation of patients in septic shock has not been established . in contrast , achievement of other goals in the treatment of septic patients ( for instance , early and adequate antibiotic treatment ) has been associated with improved outcomes . because ' normal ' systemic hemodynamics do not necessarily guarantee adequate tissue oxygen supply , monitoring organ perfusion or function it has been shown that persistent microcirculatory alterations are associated with organ failure and mortality in patients with septic shock . gastric mucosal ph ( phi ) is an interesting parameter that has been shown to predict outcome in many patient groups . phi is a composite variable that reflects not just adequacy of local perfusion but also systemic metabolic acid - base homeostasis and ventilation . the interaction of the components of phi and their response to treatment may confound the interpretation and prognostic relevance of gastric mucosal acidosis . in the study conducted by palizas and coworkers , resuscitation to a phi goal conceptually , there are four interpretations of these findings : the tested variables ( cardiac index and phi ) are not relevant to survival ; the defined targets were not adequate ; the targets were relevant but only in combination with the achievement of other goals ; and finally , the efforts to achieve the goals were not adequate , and the goal therefore was not achieved . cardiac output determines tissue oxygen delivery and is certainly an important variable with respect to the resolution of septic shock . however , other variables contribute to tissue oxygen transport , and the demands may also vary . in that sense , as the authors state themselves , a goal representing a presumed adequate relationship between oxygen delivery and consumption ( for example , mixed venous oxygen saturation ) would have been preferable . its strong association with mortality makes phi an ideal target variable for improving outcomes . in the study reported by palizas and coworkers , the target cardiac index was achieved at baseline in nearly 90% of patients . because the mortality was 30% , it is clear that the target should have been higher , if increasing cardiac index is believed to be beneficial . it is a relatively common practice to increase minute ventilation in order to normalize arterial ph when bicarbonate is low . whether a ( normal ) phi of 7.32 can be achieved at all during the early phase of septic shock is questionable . the use of vasoconstrictors can decrease mesenteric perfusion , and metabolic effects of adrenaline ( epinephrine ) may increase pco2 further . there is no information on how other aspects of septic shock treatment were addressed ( antibiotics , source control , ventilator settings , and so on ) the authors indicate that crystalloids and colloids were given , but in their protocol only saline is listed . although colloids can be harmful in sepsis , normal saline ( or colloids in saline ) can cause hyperchloremic metabolic acidosis and thereby decrease mucosal phi . adrenaline ( epinephrine ) , noradrena - line ( norepinephrine ) , dopamine , and dobutamine were used to improve blood pressure and flow . although adrenaline can impair splanchnic perfusion in septic shock , dobutamine appears to be able to reverse these changes , at least in part . phi was measured every 6 hours , and no data are given on the frequency of cardiac index assessments . four daily assessments of the treatment 's influence on the target variable may be too few , especially in view of the fact that splanchnic blood flow is highly variable in septic patients . it would have been interesting to see how the individual therapeutic interventions influenced the target variables . without this information , nevertheless , the work by palizas and coworkers reminds us that mucosal acidosis persists in the majority of patients who ultimately will die after resuscitation from septic shock . monitoring and treating cardiac output determines tissue oxygen delivery and is certainly an important variable with respect to the resolution of septic shock . however , other variables contribute to tissue oxygen transport , and the demands may also vary . in that sense , as the authors state themselves , a goal representing a presumed adequate relationship between oxygen delivery and consumption ( for example , mixed venous oxygen saturation ) would have been preferable . in the study reported by palizas and coworkers , the target cardiac index was achieved at baseline in nearly 90% of patients . because the mortality was 30% , it is clear that the target should have been higher , if increasing cardiac index is believed to be beneficial . in contrast , the incidence of low phi was high . however , the variable is influenced by arterial carbon dioxide tension ( pco2 ) . it is a relatively common practice to increase minute ventilation in order to normalize arterial ph when bicarbonate is low . thus , the mucosalarterial pco2 gradient would have been a better choice . whether a ( normal ) phi of 7.32 can be achieved at all during the use of vasoconstrictors can decrease mesenteric perfusion , and metabolic effects of adrenaline ( epinephrine ) may increase pco2 further . there is no information on how other aspects of septic shock treatment were addressed ( antibiotics , source control , ventilator settings , and so on ) the authors indicate that crystalloids and colloids were given , but in their protocol only saline is listed . although colloids can be harmful in sepsis , normal saline ( or colloids in saline ) can cause hyperchloremic metabolic acidosis and thereby decrease mucosal phi . adrenaline ( epinephrine ) , noradrena - line ( norepinephrine ) , dopamine , and dobutamine were used to improve blood pressure and flow . although adrenaline can impair splanchnic perfusion in septic shock , dobutamine appears to be able to reverse these changes , at least in part . phi was measured every 6 hours , and no data are given on the frequency of cardiac index assessments . four daily assessments of the treatment 's influence on the target variable may be too few , especially in view of the fact that splanchnic blood flow is highly variable in septic patients . it would have been interesting to see how the individual therapeutic interventions influenced the target variables . without this information , nevertheless , the work by palizas and coworkers reminds us that mucosal acidosis persists in the majority of patients who ultimately will die after resuscitation from septic shock . monitoring and treating the department of intensive care medicine at inselspital has or has had research contracts and/or collaboration contracts with edwards lifescience , ge healthcare , pulsion , orion pharma , berna biotech and b. braun medical .

the need to achieve adequate tissue oxygen delivery early in patients with septic shock is well established . however , it is less well recognized that tissue hypoperfusion can exist despite normalization of systemic hemodynamics . efforts to resuscitate septic patients until adequate tissue perfusion has been achieved can potentially improve outcome . in a multicenter study , 130 patients with septic shock were resuscitated within 12 hours of diagnosis using a protocol including goals for mean arterial and pulmonary artery occluded pressures , urinary output , arterial ph , and hemoglobin goals . they were then randomly assigned to further resuscitation with either a cardiac index ( 3 l / minute per m2 ) or a gastric mucosal ph ( 7.32 ) target . the intensive care unit length of stay and 28-day mortality did not differ between groups , but more patients in the cardiac index group were in the target range , both at baseline and after resuscitation , as compared with the gastric mucosal ph group . in contrast to cardiac index , gastric mucosal ph at baseline and at 24 and 48 hours predicted mortality . whether other targets for the chosen variables , or different and in particular earlier resuscitation efforts would have favored one group can not be concluded from the data provided .

immunocompromised patients are at a high risk of invasive fungal infection associated with high morbidity.1 among the fungi , candida is the most common organism causing infection in neutropenics or after organ transplantation.2 musculoskeletal involvement , especially vertebrae and intervertabral disc space , is uncommon . still rarer is aspergillus vertebral osteomyelitis3 and only few case reports of this disease have been described in literature occurring in immunocompetent men.34 a 25-year - old man , hailing from bihar and working in a multinational company , presented with pain in lower back since last 6 months , which was dull aching , nonradiating , and relieved by rest . physical examination was unremarkable except for tenderness present over the spine in lower lumbar region ( l2-l5 ) . he was diagnosed as having pott 's spine and was taking antitubercular treatment , prescribed by referral hospital ; however , no improvement was noted in the patient . general and systemic evaluation revealed no manifestation of acquired immunodeficiency syndrome ( aids ) or any underlying immunosuppressive disorder . chest radiographs revealed no pulmonary lesion and sputum culture was negative for acid - fast bacilli ( afb ) . x - ray of the spine revealed lytic lesion in the l4-l5 vertebral region [ figure 1 ] . magnetic resonance imaging ( mri ) of the spine revealed destruction of l4-l5 vertebrae , with abscess formation and cauda equina compression [ figure 2 ] . he underwent l4-l5 instrumentation , with posterior decompression and interbody fusion with tricortical bone graft from the iliac crest [ figure 3 ] . direct fluorescence for afb was negative and the culture and sensitivity for afb and pyogenic organism were also negative . the biopsy tissue was received in multiple fragments together measuring 4 3 1 cm . microscopy revealed fibrocollagenous tissue , normal , necrotic and new bone , degenerated cartilage , and dense inflammatory infiltrate composed of lymphocytes , plasma cells , histiocytes , few eosinophils , polymorphs , and several granulomas containing giant cells [ figure 4a and b ] . numerous fungal profiles were seen in the granulation tissue , which were septate and showed acute angle branching . after the diagnosis of fungal osteomyelitis , the patient was started on itraconazole 200 mg twice a day for 3 months . at 1 year followup , he was doing well with no complaints of back pain . x - ray of lumbar spine ( lateral view ) depicting lytic lesion in the l4-l5 region ( a ) t1wi sagittal mr image showing l4 and l5 vertebral body with intervertebral disc involvement . ( b ) t2wi showing l4 and l5 vertebral body hyperintensity with intervertebral disc involvement posterior instrumented interbody fusion ( l4-l5 ) with autogenous bone grafting photomicrograph showing ( a ) ill - formed granulomas with many giant cells ( h and e , 200 ) . ( b ) inflammatory infiltrate comprising numerous eosinophils and ill - formed granuloma ( h and e , 400 ) . ( c ) gomori methenamine stain showing septate and acute angle branching fungal hyphae and few spores in the giant cells ( gms , 400 ) a 19-year - old college student was apparently healthy 9 months ago , when he developed pain in the midback , which radiated to both the flanks and had difficulty in walking . pain gradually increased with bending , walking , or any movement and was relieved by rest and medication . three days after the pain he noticed a swelling in the midback region , which was increasing continuously . physical examination was unremarkable , except for mild tenderness and gibbus formation at the d10-d11 vertebral level . general and systemic examination in this patient also did not reveal any manifestation of aids or any underlying immunosuppressive disorder . x - ray / mri of the spine showed d10-d11 vertebral collapse with kyphosis and cord compression . the patient underwent transthoracic d10-d11 corpectomy and fusion with d9-d12 expandable cage with staple and rod fixation . biopsy tissue revealed fragments of normal and necrotic bone with extensive and diffuse necrosis and numerous surrounding eosinophils [ figure 5 ] . grocott 's methenamine silver ( gms ) stain revealed numerous septate and branching fungal profiles having morphological features of aspergillus species [ figure 6 ] . the patient was postoperatively started on anti - fungal treatment ( itraconazole 200 mg bd ) for 2 months ; however , no followup was available in this case . photomicrograph showing necrotic bony trabaeculae with giant cell reaction in marrow spaces ( h and e , 4 ) gms stain showing narrow and acute angle branching fungal hyphae consistent with aspergillus ( gms , 1000 ) a 25-year - old man , hailing from bihar and working in a multinational company , presented with pain in lower back since last 6 months , which was dull aching , nonradiating , and relieved by rest . physical examination was unremarkable except for tenderness present over the spine in lower lumbar region ( l2-l5 ) . he was diagnosed as having pott 's spine and was taking antitubercular treatment , prescribed by referral hospital ; however , no improvement was noted in the patient . general and systemic evaluation revealed no manifestation of acquired immunodeficiency syndrome ( aids ) or any underlying immunosuppressive disorder . chest radiographs revealed no pulmonary lesion and sputum culture was negative for acid - fast bacilli ( afb ) . x - ray of the spine revealed lytic lesion in the l4-l5 vertebral region [ figure 1 ] . magnetic resonance imaging ( mri ) of the spine revealed destruction of l4-l5 vertebrae , with abscess formation and cauda equina compression [ figure 2 ] . he underwent l4-l5 instrumentation , with posterior decompression and interbody fusion with tricortical bone graft from the iliac crest [ figure 3 ] . direct fluorescence for afb was negative and the culture and sensitivity for afb and pyogenic organism were also negative . the biopsy tissue was received in multiple fragments together measuring 4 3 1 cm . microscopy revealed fibrocollagenous tissue , normal , necrotic and new bone , degenerated cartilage , and dense inflammatory infiltrate composed of lymphocytes , plasma cells , histiocytes , few eosinophils , polymorphs , and several granulomas containing giant cells [ figure 4a and b ] . numerous fungal profiles were seen in the granulation tissue , which were septate and showed acute angle branching . after the diagnosis of fungal osteomyelitis , the patient was started on itraconazole 200 mg twice a day for 3 months . at 1 year followup , he was doing well with no complaints of back pain . x - ray of lumbar spine ( lateral view ) depicting lytic lesion in the l4-l5 region ( a ) t1wi sagittal mr image showing l4 and l5 vertebral body with intervertebral disc involvement . ( b ) t2wi showing l4 and l5 vertebral body hyperintensity with intervertebral disc involvement posterior instrumented interbody fusion ( l4-l5 ) with autogenous bone grafting photomicrograph showing ( a ) ill - formed granulomas with many giant cells ( h and e , 200 ) . ( b ) inflammatory infiltrate comprising numerous eosinophils and ill - formed granuloma ( h and e , 400 ) . ( c ) gomori methenamine stain showing septate and acute angle branching fungal hyphae and few spores in the giant cells ( gms , 400 ) a 19-year - old college student was apparently healthy 9 months ago , when he developed pain in the midback , which radiated to both the flanks and had difficulty in walking . pain gradually increased with bending , walking , or any movement and was relieved by rest and medication . three days after the pain he noticed a swelling in the midback region , which was increasing continuously . physical examination was unremarkable , except for mild tenderness and gibbus formation at the d10-d11 vertebral level . general and systemic examination in this patient also did not reveal any manifestation of aids or any underlying immunosuppressive disorder . x - ray / mri of the spine showed d10-d11 vertebral collapse with kyphosis and cord compression . the patient underwent transthoracic d10-d11 corpectomy and fusion with d9-d12 expandable cage with staple and rod fixation . biopsy tissue revealed fragments of normal and necrotic bone with extensive and diffuse necrosis and numerous surrounding eosinophils [ figure 5 ] . grocott 's methenamine silver ( gms ) stain revealed numerous septate and branching fungal profiles having morphological features of aspergillus species [ figure 6 ] . the patient was postoperatively started on anti - fungal treatment ( itraconazole 200 mg bd ) for 2 months ; however , no followup was available in this case . photomicrograph showing necrotic bony trabaeculae with giant cell reaction in marrow spaces ( h and e , 4 ) gms stain showing narrow and acute angle branching fungal hyphae consistent with aspergillus ( gms , 1000 ) staphylococcus aureus , accounts for 60% of pyogenic vertebral osteomyelitis.5 other causes of spinal infection include mycobacterium tuberculosis , and rarely fungi . the rate of occurrence of fungal infections has risen over the last decade , as the population of immunocompromised patients has increased . the diagnosis and treatment of fungal infection of the spine is often delayed , as clinical suspicion is often low , particularly in immunocompetent individuals.6 the most common organism causing mycotic infections are candida and , rarely , aspergillus . these infections mainly present as discitis or osteomyelitis in the spine.7 most reviews of fungal spinal infections have included a limited number of patients and short followup . in a study by eismont et al.,2 of 65 cases of spondylitis were caused by fungal organisms.8 fungal organisms are slow growing and difficult to identify by culture . therefore , histopathological examination is important to obtain correct diagnosis.6 the deficiency in the immune system secondary to infection with the human immunodeficiency virus , the widespread and injudicious use of broad - spectrum antibiotics , the use of corticosteroids and other immunosuppresive drugs , and the use of parenteral hyperalimentation are listed as risk factors for fungal infection.2578 in the series by frazier , 9 of 11 patients with spinal osteomyelitis were immunocompromised and 7 had infection due to candida.2 spinal infection by aspergillus is uncommon.910 aspergillus species are ubiquitous saprophytic organisms . more than 300 species are known , but only a few of them are of importance regarding opportunistic infection . invasive aspergillosis ( ia ) usually involves the sinopulmonary tract , with the lung being the most common site of infection , while osteomyelitis due to aspergillus species is rare.11 it affects bone tissues by contiguity , hematogenous spread , and direct implantation , eg , traumatic or iatrogenic . the incidence of aspergillus affecting the bone among all cases of invasive aspergillosis ( ia ) is estimated to be 3%.11 in a series by horn , the incidence of ia was 2.6% , similar to those reported by denning and steven in 1990.712 nandeesh et al . described less than 50 cases with spinal aspergillus osteomyelitis , and most of these patients were immunocompromised.13 a comprehensive literature search yielded approximately 15 cases of aspergillus osteomyelitis affecting the spine in immunocompetent young adults without any underlying disorders , including the present 2 cases [ table 1 ] . aspergillus spinal osteomyelitis cases have a bimodal age distribution pattern ; children with chronic granulomatous disease and the older adults with other underlying disorder . back pain is an almost universal initial symptom of vertebral osteomyelitis caused by aspergillus species . unfortunately , some patients have also presented with paraparesis.20 reported cases of aspergillus vertebral osteomyelitis in immunocompetent patients spinal osteomyelitis forming abscess and causing compressive myelopathy is not common , and current literature is largely limited to case reports only . typical complications of spinal osteomyelitis include osteolytic destruction of body endplates , paravertebral soft tissue swelling , disc space narrowing , and spinal cord compression.11 the overall 12-week mortality among the patient with aspergillus osteomyelitis was 25%.7 choice of treatment in fungal infection , especially aspergillus osteomyelitis , is debridement of necrotic bone in addition to the use of antifungal like voriconazole , itraconazole , amphotericin b , and , possibly , 5-fluorocytosine or rifampin.710 in advanced cases , antimycotic drug therapy may not be effective and emergency surgical debridement may be indicated when the destruction is progressive and spinal cord compression is imminent or manifest . in such cases , spinal instrumentation can help maintain and restore spinal stability and spinal alignment.10 aspergillus vertebral osteomyelitis and abscess is a rare cause of compressive myelopathy .

fungal infections are one of the important cause of morbidity and mortality in immunocompromised patients . aspergillus vertebral osteomyelitis is extremely rare . we report two cases of aspergillus vertebral osteomyelitis in immunocompetent men in the absence of an underlying disorder . the clinical and radiological findings were suggestive of pott 's spine . the absolute cd4 , cd8 counts and their ratio were normal . the hiv status was negative in both patients . both patients underwent surgical decompression . the histopathology of tissue obtained were suggestive of aspergillus osteomyelitis . one patient had antifungal treatment for 3 months and was doing well at 1 year followup , whereas other patient did not turnup after 2 months .

the prevalence of diabetes is high and increasing over the world . especially in developing countries , the total number of adults with diabetes will increase 69% from 2010 to 2030 . diabetes mellitus ( dm ) prevention is recognized as a major public health priority in developing nations . therefore , there is a great interest in identifying individuals at high risk of developing diabetes . currently , it is well known that risk scoring models and anthropometric measures could predict future incidence of dm [ 28 ] . for example , the finnish diabetes risk score and the framingham offspring study simple clinical model , based on more than 10 years follow - up , have been used as a tool to predict the risk for diabetes . however , it seems that the predictive value of risk scoring models is time - varying . stern et al . published a clinical model to predict diabetes risk using fasting glucose and other routine clinical data , including age , sex , and ethnicity . this clinical model predicted 7.5-year incidence of diabetes better than the 2 h glucose load during oral glucose tolerance test in mexican - american and non - hispanic white participants from san antonio heart study . had found that the validity of this clinical model to predict incident diabetes was inconsistent between 5 - 6 years and 10 years in japanese americans aged less than 55 . on the other hand , we do not know whether this phenomenon also exits in anthropometric measures , especially in chinese han nationality . to our knowledge , the duration of studies using anthropometric measures for predicting diabetes in chinese han nationality was mostly less than 15 years , and a little literature had been reported for more than 15 years . therefore , we compared the predictive ability of anthropometric measures for predicting dm incidence based on a 15-year prospective study in chinese adults . the study sample population was obtained from a chinese multi - provincial cohort study ( cmcs ) approved by the beijing institute of heart , lung and blood vessel diseases for the entire duration , a nationwide , multicenter prospective cohort study on cvd risk factors in the chinese . a total of 30121 chinese subjects aged 3564 and free of cvd at commencement was included by a multistage sampling method in which detailed information regarding the methodology had been previously reported [ 1315 ] . of these , 27003 subjects were recruited from 16 centers in 11 chinese provinces between 1992 and 1993 . at one of the centers ( sichuan , china ) , 711 individuals in an urban community located in chengdu , sichuan province , china , were included . the cohort also accepted a health examination in 2007 for a study supported by megaprojects of science research for the 11th five - year plan ( trends in the incidence of metabolic syndrome and integrated control in china ) with the same methods . twenty - four individuals were excluded from this analysis for being diagnosed with diabetes in 1992 . therefore , 687 individuals with complete data were available ( figure 1 ) . in 1992 , medicine doctors and nurses did a survey of cardiovascular disease ( cvd ) risk factors according to the multinational monitoring of trends and determinants in cardiovascular disease ( monica ) protocol . standardized questionnaire , physical examination , and laboratory tests were included in this survey . a standardized questionnaire was used for collecting information on subjects ' demographic characteristics , cvd risk factors , such as smoking status , alcohol consumption levels , physical activity , and family history of cvd . anthropometric measurements included height , weight , wc , and hip circumference measured while the patients were lightly clothed and without shoes after approximately 12 h of overnight fasting . measurements were conducted using a calibrated weighing scale , wall - mounted stadiometer , and tape measure . the results were recorded to the nearest 0.2 kg , 1 cm , and 0.5 cm . waist circumference ( wc ) was taken with the subject in a standing position , at the level midway between the lateral rib margin and the iliac crest . bmi was calculated using the following formula : weight / height ( kg / m ) . whpr was calculated as wc divided by hip circumference , and whtr as wc divided by height . blood pressure ( bp ) was measured in the right arm with subjects in a sitting position using a regular mercury sphygmomanometer after resting for 15 minutes . the three consecutive blood pressure readings were used for mean value of the bp . fasting plasma glucose ( fpg ) , fasting serum total cholesterol ( tc ) , low - density lipoprotein cholesterol ( ldl - c ) , high - density lipoprotein cholesterol ( hdl - c ) , and triglyceride ( tg ) were included . biochemistry parameters were measured at the laboratory of west china hospital ( chengdu , china ) . tc , tg , and fpg were all determined using the enzymatic method , while hdl - c was measured using the phosphotungstic acid / magnesium chloride precipitation method . the level of non - hdl cholesterol was calculated by subtracting hdl cholesterol from total cholesterol determined . the concentration of ldl - c was estimated using the friedewald formula , and very low - density lipoprotein cholesterol was calculated by subtracting serum ldl - c and hdl - c from tc [ 13 , 14 , 17 ] . this survey was approved by the ministry of health of china as well as the ethics committee of west china hospital of sichuan university . the study protocol conforms to the ethical guidelines of the 1975 declaration of helsinki as reflected in a priori approval by the institution 's human research committee . hypertension was defined as having systolic blood pressure ( sbp ) of at least 140 mm hg and/or diastolic blood pressure ( dbp ) of at least 90 mm hg and/or currently taking antihypertensive medications . diabetes mellitus was defined as one of the following at follow - up assessment : ( 1 ) fasting plasma glucose 7.0 mmol / l , ( 2 ) a positive response to the question , has a doctor ever told you that you have diabetes ? , or ( 3 ) current use of insulin or oral hypoglycemic agents . alcohol intake was defined as average intake of alcohol of at least 50 g / d . physical activity was defined as exercise one or more times per week , at least 20 minutes for each time [ 15 , 17 , 18 ] . continuous variables were expressed as the mean sd or median ( interquartile range ) as appropriate . differences of baseline characteristics between participants with and without diabetes were tested by independent t - test for normally distributed variables and by the nonparametric mann - whitney or wilcoxon test for skewed variables . this study population was stratified into quartiles of bmi , wc , whpr , and whtr . to assess the effects of baseline anthropometric measures on the new onset of diabetes , cox 's proportional hazards regression models were used to estimate the hazard ratios ( hrs ) of them , and the discriminatory power of anthropometric measures for diabetes was assessed by the area under the receiver operating curve ( aroc ) . the hazard ratios ( hrs ) were computed for quartiles 2 , 3 , and 4 as compared with the lowest quartile in different cox 's proportional hazards regression models . covariates including age , systolic blood pressure ( sbp ) , tg , hdl - c , and fpg were fitted as continuous variables in the multivariate analyses and alcohol intake , smoking , regular physical exercise , and family history of diabetes were fitted as categorical variables . the point represented the largest sum of sensitivity and specificity on the receiver operating curve ( roc ) was chosen to obtain these four measurements in predicting diabetes . 687 eligible subjects studied at baseline completed the 7 - 8 years and the 15-year follow - up . the incidence of diabetes was 2.8% ( n = 19 ) at 8 years and 10.8% ( n = 74 ) at 15 year . the percentage of self - reported history was 67.6% ( 50/74 ) during the 15-year follow - up . among the self - reported participants , 37 individuals were on antidiabetic drugs . for the other 24 diagnosed dm individuals , the average fpg was 9.5 mmol / l . mmol / l and the maximum and minimum were 16.2 mmol / l and 7.1 mmol / l , respectively . characteristics of participants at baseline were shown in table 1.compared with subsequent nondiabetic subjects , the demographic data in 1992 showed that subsequent diabetic subjects had a higher bmi , wc , hip circumference , whpr , whtr , fasting plasma glucose , and triglycerides at 7 - 8 years and 15-year follow - up ( all p < 0.001 , table 1 ) . the univariate cox 's proportional hazards regression analysis presented that these anthropometric measures ( bmi , wc , whpr , and whtr ) could statistically increase the risk for the new onset of diabetes with a 15-year follow - up ( table 2 ) . in the multivariate cox 's proportional hazards regression models , all measurements were significantly associated with risk of diabetes after adjustment for potential risk factors including age , sex , alcohol intake , smoking , regular physical exercise , family history of diabetes , and fpg . after further adjustment for sbp , tg , and hdl , the associations were reduced , but remained to be significant ( table 2 ) . figure 2 showed the cumulative incidence of dm in different anthropometric variables groups in multivariate cox - regression in model 2 . the areas under the roc curves were 0.668 ( 95% ci : 0.6310.703 ) for bmi , 0.781 ( 95% ci : 0.7480.811 ) for wc , 0.769 ( 0.7360.800 ) for whpr , and 0.768 ( 0.7340.799 ) for whtr at 7 - 8-year follow - up , respectively ( all p < 0.05 ) , while 0.668 ( 95% ci : 0.6010.734 ) for bmi , 0.701 ( 95% ci : 0.6410.760 ) for wc , 0.691 ( 95% ci : 0.6370.748 ) for whpr , and 0.715 ( 95% ci : 0.6570.774 ) for whtr ( all p < 0.05 , table 3 ) at 15-year follow - up . table 3 indicated that wc , whpr , and whtr were better than bmi for predicting incident diabetes at 7 - 8 years in chinese han nationality population from chengdu community . however , the four anthropometric measurements had no significant difference for predicting 15-year incidence of diabetes . the goal of the study was to assess whether bmi , wc , whpr , and whtr could predict future diabetes on the basis of data collected from a general chinese group during 15-year follow - up and to assess whether the validity of anthropometric measures to predict incident diabetes were stable as time goes by , especially in chinese han nationality . after adjusting for potential confounders , our findings showed that both general obesity measures ( bmi ) and central obesity measures ( wc , whpr , whtr ) could predict future diabetes in a chinese population after a long follow up . on the other hand , wc , whpr , and whtr were better than bmi for predicting incident diabetes at 7 - 8 years in chinese han nationality population from chengdu community . the four anthropometric measurements had not significant difference for predicting 15-year incidence of diabetes . in recent years , some studies showed a more close association between diabetes and central obesity measures than bmi [ 1923 ] , whereas other studies showed no significant differences between the obesity measures [ 2426 ] . meanwhile , meta - analysis studies also showed inconsistent results [ 27 , 28 ] . however , we reviewed literatures of previous prospective studies comparing bmi , wc , whpr , and whtr for predicting incidence of diabetes and found that many studies reporting a difference between the obesity measures did not use appropriate study statistical methods or that it did not investigate all indicators with data on only 1 or more of the 4 indicators ( i.e. , bmi , wc , and whpr ) with regard to the association with diabetes risk . more importantly , length of the follow - up periods might influence study results [ 2946 ] ( see supplementary table 1 available online at http://dx.doi.org/10.1155/2013/239376 ) . firstly , we compared the predictive ability of the four anthropometric measures for predicting dm incidence based on a 15-year prospective study in chinese adults . similar to our findings , in 610 years and 1115 years longitudinal follow - up groups in supplementary table 1 , the majority of trials had shown no significant difference between obesity measures . thought that different methods of collecting numbers of onset diabetes and obesity measures might result in this result . diabetes incidence was self - reported and anthropometric indicators were self - measured by the participants in the two studies . but , investigators from the san antonio heart study and epic - potsdam study , with similar methods , had demonstrated higher risk with wc or whpr than bmi in predicting diabetes incidence . our findings revealed that wc , whpr , and whtr were better than bmi for predicting incident diabetes at 7 - 8 years in the same population . however , the four indictors did not have a significant difference in our study at 15 years . a study reported that over 10 years , total fat mass and fat mass except subcutaneous fat increased , whereas body weight did not change . it was possible that many subjects tend to become abdominally obese rather than become totally obese during follow - up , and the impact of baseline body fat distribution on diabetes risk was weakened with lengthening of the follow - up period . on the other hand , racial and regional differences in the association between obesity and the risk for diabetes might also have influenced the results . although the prevalence of obesity is much lower in asia than in western countries , the prevalence of diabetes is similar between the two regions , and diabetes occurs in asians who are less obese . therefore , we reviewed studies that compared anthropometric measures in predicting risk of diabetes in chinese mainland [ 5053 ] ( supplementary table 2 ) . there might be a greater bias risk in data collection ( i.e. , potential detection bias ) , and the cross - sectional data showed only the association with present risk factor conditions but did not directly predict the future risk of dm . a long follow - up time was to avoid not only insufficiency of new onset diabetes cases in short - term follow - up that underestimated the predictive power of the measurement indexes but also potential detection bias in cross - sectional studies that exaggerate the statistical power of the indexes . taking into account the sample size , sex , and region , we can not extend our findings to general population . but we need to know that the length of follow - up period is a factor that can not be ignored . another advantage was that a standardized protocol was used and all anthropometric variables were collected using direct measurement by trained doctors and nurses rather than self - report . this limited the study , and we could not analyze the association between anthropometric measures with diabetes risk classified by gender . the results of our study may have limited statistical power ; but we still can get some clues . the second was the way to diagnose diabetes mellitus . during the investigation , taken into account the financing and feasibility , we had diagnosed diabetes by measuring fasting plasma glucose and patient self - reports rather than oral glucose tolerance test means . some individuals would have developed dm that was not detectable by changes in fasting glucose alone or by clinical history , leading to the underestimation of the experimental results . , our findings showed that anthropometric measures could predict diabetes with a long - time follow - up . however , the validity of anthropometric measures to predict incident diabetes may change with time . future researches may be warranted to assess whether this phenomenon still exists in mainland china with different duration of follow - up time in a large population .

objective . to examine whether anthropometric measures could predict diabetes incidence in a chinese population during a 15-year follow - up . design and methods . the data were collected in 1992 and then again in 2007 from the same group of 687 individuals . waist circumference , body mass index , waist to hip ratio , and waist to height ratio were collected based on a standard protocol . to assess the effects of baseline anthropometric measures on the new onset of diabetes , cox 's proportional hazards regression models were used to estimate the hazard ratios of them , and the discriminatory power of anthropometric measures for diabetes was assessed by the area under the receiver operating curve ( aroc ) . results . seventy - four individuals were diagnosed with diabetes during a 15-year follow - up period ( incidence : 10.8% ) . these anthropometric measures also predicted future diabetes during a long follow - up ( p < 0.001 ) . at 7 - 8 years , the aroc of central obesity measures ( wc , whpr , whtr ) were higher than that of general obesity measures ( bmi ) ( p < 0.05 ) . but , there were no significant differences among the four anthropometric measurements at 15 years . conclusions . these anthropometric measures could still predict diabetes with a long time follow - up . however , the validity of anthropometric measures to predict incident diabetes may change with time .

majority of the cases are reported in the dorsal spine due to more blood circulation than the other parts of the spinal cord . we are presenting a 6-year - old girl with solitary icc at the level c4c6 presented with neck stiffness without neurological deficit , diagnosed by visualization of scolex on magnetic resonance imaging ( mri ) and was treated successfully by albendazole for 1 month . a 6-year - old female child of low socioeconomic status , was referred to department of orthopedics with the history of insidious onset stiffness of neck with minimal pain for 1 week . patient 's parent gave a history of tuberculosis contact , but had no history of trauma , evening increase of temperature , decrease appetite , and significant weight loss . noncontrast mri of the neck showed a well - defined intramedullary cystic lesion at c4c6 vertebral levels , which was hypointense on t1 weighted and hyperintense on t2 weighted images with peripheral , spinal cord edema having an intracystic hypointense on t2 weighted target like lesion suggestive of scolex . screening of whole spine and brain did not show any other lesion [ figure 1a c ] . sonography of abdomen and computed tomography ( ct ) scan of the chest was also unremarkable . hence , on the basis of this absolute diagnostic criterion , final diagnosis of solitary icc was made . as the patient had no neurological deficit , conservative treatment was opted for this patient . before the start of treatment , serum ag - elisa the patient was given albendazole 15 mg / kg body weight per day orally for 1 month . oral prednisolone 1 mg / kg body weight / day , was also started 2 days before the start of albendazole , for 2 weeks . ag - elisa was repeated after 2 months of completion of treatment , and observed negative suggestive of no residual lesion . ( a ) t2-weighted magnetic resonance imaging image of cervical spine showing a hyperintense cystic lesion with intracystic hypointense ystic lesion with intracystic hypointense target like scolex . cysticercosis of central nervous system by taenia solium characteristically involves the brain parenchyma , intracranial subarachnoid space , or ventricular system . . the possible mechanisms of spinal involvement of cysticercosis are migration of the cysticercus through the ventriculo - ependymal pathway and hematogenous dissemination . the blood flow to the brain is approximately 100-times more than the spine making the brain more vulnerable for cysticercosis than the spine . in the spine , the thoracic cord has higher blood flow and has reported higher incidence . since limited space in the spinal canal , mass effect of this lesion all reported cases of icc had a neurological deficit , to best of our knowledge , this is the first case of icc without neurological involvement . the symptomatology of spinal cysticercosis depends on location , spinal level , lesion size , and the presence or absence of inflammation . these patients usually present with radiculopathy , paresthesia , paresis , bowel and bladder incontinence , and sensory loss . the absolute criteria to make diagnosis of neurocysticercosis are ( 1 ) histological demonstration of the parasite from biopsy of the brain or spinal cord , ( 2 ) cystic lesion showing scolex on ct / mri , and ( 3 ) direct visualization of subretinal parasites on fundoscopic examination . any one of the above criteria is sufficient for the diagnosis . in our case , mri is a better diagnostic tool than the ct scan , and far more sensitive in picking up the parenchymal lesion . often what is thought to be a solitary lesion turns out to be one of many such lesions . another advantage of mri is the detection of scolex which is often missed on ct scan . it is differentiated from tuberculoma as latter loses signal intensity on t2-weighted . when a patient presenting with a history of cysticercosis or cames from an endemic region and along with mri features suggestive of cystic lesion , the diagnosis of cysticercosis should always be kept in mind , which should be further strengthen by visualization of scolex and positive serology preferably antibody elisa either in cerebrospinal fluid or in serum . serum level of antigen tends to decrease drastically after the treatment , hence it is a valuable tool to assess the effectiveness of treatment and recurrence . the rarity of spinal cysticercosis case has precluded the evolution of treatment algorithm as compared to cerebral neurocysticercosis . it is agreed that medical management of intramedullary spinal cysticercosis should be considered in patients with no neurological deficit and in cases diagnosed by cerebrospinal fluid assay . however , in patients presenting with acute or progressive neurological deterioration , and in those cases where the diagnosis is in dilemma , surgical removal if possible should be the treatment of choice . early surgery not only provides sample for early diagnosis , but it also provides recovery before irreversible cord damage accomplished . albendazole is often used with corticosteroids because its blood level could be synergistically increased by the latter and also decrease the cord edema that is anticipated after the death of parasite due to the release of inflammatory mediators . it has been demonstrated that monoclonal antibody - based ag - elisa is a promising technique to monitor neurocysticercosis patients after treatment and it is much cheaper than ct - scan , particularly for developing countries . in this case report , ag - elisa was used to monitor the effectiveness of the treatment and was negative after 2 months of treatment and at the latest follow - up . the diagnosis can be clinched on mri , and it can be treated successfully by albendazole if there is no neurological deficit .

neurocysticercosis is commonly seen intracranially and its incidence in the spinal cord is very low . among spine dorsal region is more common due to more blood flow in this region and it is usually associated with lesion at other sites . the intramedullary location is rarer than extramedullary . hence , solitary intramedullary cervical spine cysticercosis ( icc ) is extremely rare entity . only a handful numbers of cases are reported in the literature . all reported cases are presented with the neurological deficit as spinal canal diameter is very low and any space occupying lesion is not tolerated . we are presenting a 6-year - old girl having solitary icc with intact neurology , diagnosed by an appreciation of scolex on magnetic resonance imaging and were treated successfully with albendazole . follow - up was performed by the estimation of ag - elisa which was negative after 2 months of completion of treatment and patient was asymptomatic at 2 years of follow - up .

these disorders are more prevalent in early puberty among girls , especially during the first 2 years of menstruation in which many periods are without ovulation . one of the most common disorders at this time is menstruation signs , which include irritability , tender breasts , low back pain , skin outbreaks , fatigue , palpitation , social isolation , nausea and vomiting , abdominal pain ( cramps ) , and general weakness occurring before , during , or after menstruation . retrospective and prospective studies have reported the prevalence of these signs over 60% and 23 - 67% , respectively . these signs not only affect the quality of life but also are the main reasons for adolescents and teenagers school absenteeism . the intensity and frequency of menstruation - related signs differ based on menstruation cycle 's stages and the cultures , so the lowest prevalence is for western countries like yugoslavia . based on world health organization ( who ) report , menstruation signs have higher prevalence in asian countries compared to western countries . based on biopsychosocial model , menstruation - related signs are under the influence of not only biological factors such as hormonal disorders and lifestyle ( sports and nutrition ) but also environmental and social factors like interaction with friends , family , and colleagues and the attitude toward menstruation and psychological factors including anxiety , depression , and stress . psychological disorders may activate corticotrophin releasing hormone ( crh ) from the nervous system followed by increased cortisol and prolactin which lead to the incidence of menstruation signs . in addition to their effect on quality of life , these psychological disorders may lead to suicide , addiction , early sexual experience , adulthood depression , crimes , educational decline , low self - confidence and its consequences , and eventually , occupational , familial , and social dysfunctions . prevalence of these psychological disorders is rare in the pre - adolescence period and shows no gender - based difference , while in the adolescence period , these disorders increase with a steep slope and are more prevalent among girls who are more vulnerable to various psychosocial factors , so the ratio of girls involvement in these disorders compared to boys is 1.3:1 . with regard to previous studies conducted on psychological disorders and menstruation signs , lonelli ( 2010 ) showed a significant association between pre - menstruation signs and stress , anxiety , and depression . meanwhile , balaha ( 2005 ) reported that about 22% of the students had acute pre - menstruation signs , but there was no significant association between pre - menstruation signs and stress , anxiety , and depression . there are controversial studies in this regard as adolescence period ( 10 - 19 years ) is an important period of human 's life . there are also a higher number of adolescents in today 's world ( 21.8% of total population in iran based on national census of 2006 ) , and major changes occur in appearance , social behavior , and psychological status at puberty . with regard to the aforementioned facts and as menstruation signs and psychological disorders including anxiety , depression , and stress vary in different societies and have various prevalence rates in different cultures on the one hand , and since iran is a country with a young population and such a study had not been already conducted in mashhad on the other hand , the researcher decided to conduct a study aimed to define the association between menstruation signs and anxiety , depression , and stress among high school girls . this is a cross - sectional study conducted on 407 governmental school girls in the school year 2011 . the researcher got research approval from ethical considerations committee of the university and obtained a letter of introduction from mashhad nursing and midwifery school . she delivered the letter to the authorities of schools , and after explaining about the research goals and obtaining students written informed consent with respect to ethical codes and making the necessary co - ordination concerning the time of questionnaire distribution , she conducted sampling and performed the study . after a pilot study on 10 students , the sample size was calculated as 384 based on correlation index formula : the total number of subjects was estimated to be 420 with respect to 10% increase as a result of random sampling ( z1 = 1.96 with regard to confidence coefficient of 95% , z2 = 84% with regard to test power of 80% , and r based on the correlation coefficient between menstruation signs and depression , anxiety , and stress estimated as 0.15 in the pilot study ) . the method of sampling was such that a high school or vocational school was randomly selected from each of the six educational districts of khorasan razavi province using a random number table . then , with regard to the population of each district , a certain number of students in various courses and grades were selected from the related high school or vocational school . data collection tool included a questionnaire of demographic and menstruation characteristics , menstrual distress questionnaire ( mdq ) , and depression , anxiety , and stress scale ( dass ) . this questionnaire is scored a four - point likert scale ( no sign = 0 , very severe = 4 ) and contains 16 questions in four dimensions [ pain , control , autonomic reactions , and ( water ) weight gain ] , and records the menstrual signs a week before menstruation , during bleeding , and a week after menstruation in the recent year . a score of 16 is given for minor menstruation signs , 17 < score < 32 for moderate menstruation signs , and 33 < score < 48 is for acute menstruation signs . dass-21 : this questionnaire contains 21 questions measuring depression ( 7 questions ) , anxiety ( 7 questions ) , and stress ( 7 questions ) . the questions are scored by a four - point likert scale : never ( zero ) to very much ( three ) . the depression - related questions were questions 17 , 16 , 13 , 10 , 5 , and 3 , anxiety - related questions were 20 , 19 , 15 , 9 , 7 , 4 , and 2 , and stress - related questions were 18 , 14 , 12 , 11 , 8 , 6 , and 1 . a score of 5 - 6 showed minor depression , 7 - 10 moderate , 11 - 13 acute , and over 14 showed very acute depression . scores 4 - 5 were given for minor anxiety , 6 - 7 moderate , 8 - 9 acute , and over 10 were given for very acute anxiety . scores 8 - 9 were given for minor stress , 10 - 12 moderate , 13 - 16 acute , and over 17 for very acute stress . questionnaires of menstruation distress and scale of depression , anxiety and stress are valid and reliable tools that have been used in the studies of kordi ( 2011 ) and mollahadi ( 2010 ) . inclusion criteria were giving an informed consent to attend the study , iranian nationality , residing in mashhad , being student of grades 1 - 4 of high school or vocational school , body mass index ( bmi ) < 30 , and at least 2 years should have passed after the first menstruation . exclusion criteria were being a professional athlete , experiencing an awful or stressful major event within the recent 6 months of study ( death of immediate relatives , acute familial conflicts , financial problems , and a major change in life ) , taking antipsychotic medication , being on a special diet , being married , the history of psychiatric diseases during the recent year ( a psychotic disease diagnosed by a psychiatrist or taking antipsychotic medications ) , and presence of any medical disease ( diabetes , thyroid disorders , cushing 's disease , hypophysis tumors , reproductive system diseases like myoma , endometriosis , ovarian cysts , pelvic inflammatory diseases ) . after the researcher referred to research environment and distributed informed consent forms among the students , the goal of study was explained to them . if the subjects agreed to attend and were qualified based on the inclusion and exclusion criteria , the demographic and menstruation questionnaire was given to them to fill up , and then they were asked to complete the mdq and dass . after the data of 407 subjects were collected , they were analyzed by the statistical tests of student 's t - test , mann whitney 's one - way analysis of variance ( anova ) , chi - square , pearson correlation coefficient , and linear regression tests through spss version 14 . based on the findings , the mean ( sd ) value for age was 16.24 ( 1.3 ) years , for bmi was 19.6 ( 1.6 ) , and for the first menstruation was 12.78 ( 1.23 ) . a total of 357 ( 87.7% ) subjects were at moderate economic status , 339 ( 82.2% ) were exposed to cigarette smoke , 386 ( 94.8% ) had mothers without university education , and 38 ( 9.4% ) had working mothers . in addition , 21.4% of subjects had bleeding for more than 7 days , 87% had a period of 21 - 42 days , and 51% had polymenorrhea . with regard to menstruation signs , 301 ( 74% ) of subjects reported having the signs a week before menstruation , 383 ( 94.1% ) had signs during bleeding , and 166 ( 4.8% ) had them a week after menstruation . in addition , the mean ( sd ) scores of pre - menstruation signs , during bleeding , and post - menstruation signs were 20.12 ( 5.70 ) , 24.86 ( 7.58 ) , and 17.82 ( 4.38 ) , respectively . the intensities of menstruation signs in various stages of menstruation and in various dimensions have been presented in tables 1 and 2 , respectively . intensity of menstruation signs during three stages of menstruation frequency distribution of subjects based on various dimensions of menstruation signs the most common signs were low back pain , early tiredness , and sore muscles pre - menstruation ; low back pain , early tiredness , and headaches during bleeding ; and low back pain , early tiredness , and dizziness post - menstruation . the total mean ( sd ) scores of depression , anxiety , and stress were 7.02 ( 0.02 ) , 4.4 ( 0.01 ) , and 5.5 ( 0.04 ) , respectively . a total of 186 subjects ( 45.7% ) had no anxiety , 181 ( 44.5% ) were not depressed , and 215 ( 52.8% ) were not stressed [ table 3 ] . pearson correlation coefficient showed a positive correlation between the signs a week before , during bleeding , and a week after menstruation and depression , anxiety , and stress [ table 4 ] . frequency distribution of subjects based on the intensity of anxiety , depression , and stress pearson correction coefficient results based on menstruation signs and depression , anxiety , and stress it was such that higher intensity of menstruation signs increased the subjects depression , anxiety , and stress levels . meanwhile , pearson correlation test showed no significant association between menstruation bleeding pattern ( duration , length of period , and amount of bleeding ) and menstruation signs ( anxiety , depression , and stress ) . pearson correlation test results showed a positive correlation between the signs and depression , anxiety , and stress scores a week before , during bleeding , and a week after menstruation [ table 3 ] , therefore a higher intensity of menstruation signs and increased depression , anxiety , and stress . in order to control the effective variables , it was such that effective variables like menstruation signs and depression , anxiety , and stress including age , bmi , educational level , the age of the first menstruation , exposure to cigarette smoke , mothers job and education , and economic status were entered as independent variables , and then in the next step , the main variables of the study including menstruation signs , stress , anxiety , and depression were separately entered as dependent variables in several steps . finally , variables of menstruation signs score , depression , stress , and anxiety had a significance level < 0.05 and were considered as predictive variables . family and social health depends on the fulfillment of their health and socio - cultural needs . one of the most critical periods of women 's life is adolescence in which menstruation starts . nowadays , the research on and consideration of menstruation signs are widely spread and organized and are counted as an important research issue . meanwhile , although menstruation signs are of great importance , they are not yet discussed enough . the present study showed that a high percentage of the students suffer from psychological disorders despite the menstruation signs in pre - menstruation , during bleeding , and post - menstruation periods . the obtained results also showed a significant association between menstruation signs and anxiety , depression , and stress . with regard to the prevalence of menstruation signs in various parts of the world with different cultures and races , derman 's ( 2004 ) study reported the prevalence of pre - menstruation signs as 61.4% among female students in turkey . in addition , vichin ( 2006 ) reported the prevalence of pre - menstruation signs as 59% among 13 - 18-year - old students in the usa . the findings of the present study showed that the highest prevalence of signs was for during bleeding period ( 94% ) and the lowest was for post - menstruation period ( 40.8% ) . prevalence of menstruation signs a week before menstruation , during bleeding , and a week after menstruation was observed to be higher in the present study compared to other studies conducted . differences in the cultures , races , religions , and societies can be a reason for the observed difference in prevalence and intensity of menstruation signs . the obtained results also showed that low back pain , early tiredness , and sore muscles were the most prevalent pre - menstruation signs , while low back pain , early tiredness , and headaches were common during bleeding . also , low back pain , early tiredness , and dizziness were prevalent in the post - menstruation period . meanwhile , the most reported menstruation signs among students were abdominal cramps ( 46.5% ) , low back pain ( 28.4% ) , acne ( 21.4% ) , and tender breasts ( 17.5% ) in lee 's ( 2011 ) study . chang et al . ( 2009 ) reported the most common signs , obtained by retrospective 12-question mdq , as dysmenorrhea , acne , and tiredness . in the study of chen et al . ( 2005 ) , abdominal cramps , tiredness , low back pain , abdominal bloated feeling , and tender breast were reported as the symptoms . bakhshani ( 2009 ) in her study on 18 - 28-year - old students in zahedan using a questionnaire with 20 questions reported low back pain , abdominal bloated feeling , and tender breasts as the most prevalent pre - menstruation signs . in order for an absolute diagnosis of menstruation signs to be made , especially the pre - menstruation signs . in the present study , menstruation distress signs in pre - menstruation , during bleeding , and post - menstruation periods of the year before were evaluated through retrospective recorded data . meanwhile , on the contrary , vichni ( 2006 ) studied the signs of the latest menstruation , and takeda ( 2010 ) studied those of the past three cycles . in addition to aforementioned issues , the obtained findings of the present study showed that 21.7% of the subjects suffered from depression , 24.3% from anxiety , and 21.8% had acute or very acute stress , which shows a higher prevalence of these disorders among the studied group compared to other individuals . meanwhile , the results of health and disease project investigating the mental health among iranian individuals over 15 years of age reported the prevalence of mental disorders to be 21% in the total population of iran . parvaresh ( 2011 ) reported a prevalence of mental disorders over 34% among 15-year - old women in kerman . the difference can be as a result of various adopted methods and tools , socio - economic , cultural , and geographic conditions , as well as different age groups . the results of the present study showed a positive correlation between menstruation signs in pre - menstruation , during bleeding , and post menstruation periods and signs of depression , anxiety , and stress ; therefore , an increase in menstruation signs increased the intensity of depression , anxiety , and stress . ionelli ( 2010 ) showed a positive correlation between pre - menstruation signs and depression , anxiety , and stress disorders . although these results are in line with our obtained results , lower sample size in that study ( 150 subjects ) and investigation of just pre - menstruation signs compared to the higher sample size of the present study and investigation of the signs in three time intervals , namely , pre - menstruation , during bleeding , and post - menstruation reveal the positive points of the present study . in balaha 's cross - sectional study ( 2005 ) on 250 medical students in saudi arabia , linear regression test showed no significant association between pre - menstruation signs and anxiety , depression , and stress . differences in race , culture , and age of the studied subjects ( age 18 years ) , the sample size [ 250 subjects compared to our study ( 407 subjects ) ] , as well as the differences in the adopted questionnaire to record menstruation signs can be among the reasons for the inconsistency observed between these two studies . despite the researcher assuring the subjects about the confidentiality of their information , in some cases , some information might have not been revealed by them . personal , personality , and genetic differences affecting the intensity of menstruation have been relatively controlled by the researcher . prospective recording of the data is more precise compared to retrospective recording due to the confounding elements of memory , lack of students cooperation , and interference of the study with their school assignments , which were among the limitations of the present study ; therefore , future studies are suggested to be conducted prospectively . with regard to high prevalence of low back pain , early tiredness , sore muscles , and dizziness before , during bleeding , and after menstruation , as well as high incidence of depression , anxiety , and stress signs among the students , investigation , prevention , and treatment of these disorders by the authorities in education and training and the ministry of health are essential , and educational programs and counseling services should be conducted to tackle these problems .

background : menstruation signs are among the most common disorders in adolescents and are influenced by various environmental and psychosocial factors . this study aimed to define the association between menstruation signs and anxiety , depression , and stress in school girls in mashhad in 2011-2012.materials and methods : this was a cross - sectional study on 407 high school girls in mashhad who were selected through two - step random sampling . the students completed a questionnaire concerning demographic characteristics , menstruation , depression , anxiety , and stress scale of 21 questions ( dass-21 ) , and menstruation signs in three phases of their menstruation . data were analyzed by the statistical tests of pearson correlation coefficient , student 's t - test , one - way analysis of variance ( anova ) , and regression through spss version 14.results:based on the findings , 74% of the subjects reported pre - menstruation signs , 94% reported signs during bleeding , and 40.8% reported post - menstruation signs . about 44.3% of the subjects had anxiety , 45.5% had depression , and 47.2% had stress . in addition , pearson correlation coefficient test showed a significant positive correlation between menstruation signs and depression , anxiety , and stress ( p < 0.05).conclusion : with regard to the association between menstruation signs and psycho - cognitive variables , prevention and treatment of these disorders by the authorities of education and training and the ministry of health are essential .

nontuberculous mycobacteria are usually recovered from the environment and can cause disease through respiratory , cutaneous , parenteral and gastrointestinal exposure . we describe a case of a young , immunocompetent patient that developed symptomatic mycobacterium gordonae pulmonary infection . a computed tomography of the chest revealed hilar lymphadenopathy and nodular densities on the left side . treatment consisted of a short course of rifampin and ethambutol , which resulted in a clinical and radiographic improvement . mycobacterium gordonae is capable of causing clinically significant disease in both immunocompetent and immunosuppressed individuals . of all the mycobacteria species , mycobacterium gordonae ( m. gordonae ) is the least pathogenic and its isolation is typically regarded as a contaminant . nosocomial transmission has also been reported due to tap water used for rinsing of medical instruments and dye added to topical anesthetic used in bronchoscopy . despite its non - virulent nature , there have been reports of clinically significant disease caused by m. gordonae . m. gordonae pulmonary disease occurring in a patient without a previous pulmonary abnormality is extremely uncommon . a 26 year old female with no previous medical conditions presented to the emergency room for acute worsening of a non - productive cough , dyspnea and chest tightness . the patient denied any fevers , night sweats , weight loss , hemoptysis , and exposure to sick contacts or a history of cigarette smoking . the patient had been empirically treated as an outpatient by her primary care physician with albuterol and singulair with no improvement . on admission laboratory data including comprehensive metabolic panel and complete blood count with differential was unrevealing . contrasted computed - tomography ( ct ) of the chest revealed left sided hilar adenopathy and several nodular densities throughout the left lower lobe . tuberculin skin test was non - reactive and a blood test for hiv was negative . the patient was started on anti - tuberculous medication consisting of rifampin , isoniazid , pyrazinamide and ethambutol . rifampin was discontinued after only one week because the patient developed fever and a diffuse rash . two months after starting tb therapy , the cultures from the tissue biopsy grew m. gordonae . at this time a repeat ct scan of the chest was done 6 months after initial diagnosis and showed complete resolution of the adenopathy and only one nodule was present in the left lower lobe . m. gordonae , formerly called mycobacterium aquae , is slow growing mycobacteria , usually requiring up to 3 weeks to reach mature growth . the organism grows best at a temperature range of 35 - 37 c and can be recovered from pipelines , fresh water and laboratory faucets . m. gordonae is typically thought to be a contaminant ; however there are numerous reports of the organism causing disease particularly in immunosuppressed individuals . corticosteroid therapy , hiv infection , malignancy , organ transplant recipients , and those in extreme of age groups are all underlying conditions and patient populations in which the bacteria have caused significant disease . there have only been rare reports of immunocompetent individuals such as our patient that have developed symptomatic disease . infection involving the peritoneum , soft tissue , cornea , genitourinary system and disseminated disease has been described ; but pulmonary infection is the most likely site of symptomatic disease . in a review of 24 cases of infection with m. gordonae , all of the patients had at least one antecedent pulmonary abnormality including a history of infection with tuberculosis , pulmonary malignancy , chronic bronchitis and smoking . symptomatic pulmonary infection in individuals with no pulmonary abnormalities as seen in our patient is unusual . the presence of clinical symptoms and radiographic abnormalities are required for the diagnosis of m. gordonae pulmonary infection . a variety of radiographic finding are possible including pulmonary nodules , cavities , infiltrates , bronchiectasis and consolidation . in fact , one study concluded that regardless of the ntm species , the most common pulmonary findings on a ct scan were small bilateral nodules or branching centrilobular lesions . in addition to clinical symptoms and radiographic abnormalities , positive cultures from sputum , bronchial wash or transbronchial or lung biopsy are necessary . positive cultures of sputum and bronchial wash should be interpreted with caution since these cultures are more likely to represent contamination in the absence of clinical and radiographic evidence . the treatment regimen for m. gordonae infection is not well defined but antimicrobial agents that have the most consistent in vitro activity include ethambutol , rifamycins , clarithromycin , linezolid and the flouroquinolones . duration of therapy is also ill defined and in one review ranged from 9 to 22 months with a median of 15 months . cure is seldom achieved even with effective treatment but the disease can be stabilized and result in symptomatic improvement . in our patient rifampin and ethambutol provided effective coverage against the organism , but treatment with rifampin was discontinued after only one week due to allergy development and treatment with ethambutol was continued for 2 months . despite the short treatment course and the use of only ethambutol as effective coverage , our patient achieved symptomatic improvement and almost complete resolution of the pulmonary lesions 6 months after initial diagnosis . although commonly regarded as a contaminant , m.gordonae is capable of causing significant infections in both immunocompromised and immunocompetent hosts and its isolation should not completely be dismissed without further evaluation .

context : nontuberculous mycobacteria are non - communicable organisms and currently there are over 125 species . nontuberculous mycobacteria are usually recovered from the environment and can cause disease through respiratory , cutaneous , parenteral and gastrointestinal exposure.case report : we describe a case of a young , immunocompetent patient that developed symptomatic mycobacterium gordonae pulmonary infection . a computed tomography of the chest revealed hilar lymphadenopathy and nodular densities on the left side . definitive diagnosis was made by a culture of a transbronchial biopsy . treatment consisted of a short course of rifampin and ethambutol , which resulted in a clinical and radiographic improvement.conclusions:mycobacterium gordonae is capable of causing clinically significant disease in both immunocompetent and immunosuppressed individuals .

maternal mortality , which is widely accepted as a key indicator of population health and of socioeconomic development,1 is high in many african communities . africa , particularly sub - saharan africa ( ssa ) , has made insignificant progress toward reducing maternal mortality rate in the continent where almost half of the maternal deaths still occur . according to babalola and fatusi,2 africa has the highest burden of maternal mortality in the world and ssa is largely responsible for the dismal maternal death figure for the region , contributing ~66% of the maternal deaths for the region.3 despite the progress made toward reduction in maternal mortality since the late 1980s , such as the international safe motherhood initiative ( smi ) in 1987,4 international conference on population and development ( icpd ) in 1994,5 fourth world conference on women , beijing 1995,6 united nations millennium development goals ( mdgs ) 20007 and sustainable development goals ( sdgs ) 2015,8 in addition to other country - specific programs , many women and babies still die during pregnancy and childbirth in developing countries , including nigeria . the worrisome aspect of this situation is that in ssa , there has been no discernible or perceptible change over the past 2 or more decades.9 akokuwebe and okafor10 concluded that for real sustainable transformation of nigeria , maternal health should be accorded priority through reducing the maternal mortality rate by the government and other stakeholders . the overwhelming majority of maternal deaths each year occur in developing countries in stark contrast to the situation in developed regions . according to the world health organization,3 developing regions accounted for ~99% ( 302,000 ) of the global maternal deaths in 2015 , with ssa alone accounting for roughly 66% ( 201,000 ) , followed by southern asia ( 66,000 ) . the problem of maternal mortality may be due to the interplay of many antecedent factors that could be sociocultural , economic or related to health care services and logistics . as observed by some authors,11,12 sociocultural factors and economic barriers may limit women s health seeking behavior , making pregnancy and childbirth precarious . poor or broken down health systems hinder the progress of maternal mortality reduction and lead to high maternal deaths among mothers due to complications of pregnancy and childbirth.13,14 there is no single intervention measure that will considerably decrease maternal mortality , which indicates the need to examine factors other than medical causes , which are least studied , in order to understand comprehensively the factors affecting maternal health and mortality . shah and say9 noted that the trend in developing countries is much worse now than when the smi was launched in kenya in 1987 . the increasing burden of poor institutional health care services delivery presents mothers with multiple challenges during pregnancy and delivery , leading to high maternal mortality in developing countries . maternal mortality statistics remain very high in nigeria ; in fact , it is one of the highest in the world after india.1517 nigeria needs immediate intervention to reduce her unacceptably high levels of maternal deaths . the situation is even grave in rural areas in nigeria where the health care system is poor and overstretched , in addition to the poverty level being high . according to the nigeria demographic and health survey ( ndhs ) , 63% of deliveries occur at home and only a small proportion ( 38% ) of women in nigeria is attended to at delivery by a skilled attendant.18 the health of mothers is very crucial because it has a bearing on the health of every member of the household , particularly that of children and aged persons . undoubtedly , regional variation in factors affecting the utilization of maternal health services across the 6 geopolitical zones of nigeria has been observed;19,20 the situation perhaps may be worse in rural and semiurban areas where there is a low proportion of functional primary health care facilities and patronage . several studies have documented the scarcity of reliable and accurate data on maternal mortality,16,17 particularly in developing countries , where the maternal mortality is high . according to ujah et al,21 knowledge of maternal mortality statistics on nonmedical factors , such as information on social , economic , cultural and behavioral factors , contributing to maternal mortality are rarely available , especially at the community level . one important fact is that the dynamics of maternal health and mortality are yet to be fully comprehended in nigeria . understanding the real situation of a mother s disease condition and death is vital in dealing with the challenge of uncompromising high maternal mortality rates in nigeria . although community - based studies are few in nigeria,17 most of the available studies provide mainly fragmentary hospital - based information , which is not representative in nature , as the majority of deliveries are conducted at home by traditional birth attendants ( tbas ) or relatives as indicated earlier . hence , an innovative community - based approach to unearth the nonmedical factors influencing deaths and morbidity among mothers in communities in ado - odo / ota local government area ( lga ) in ogun state , nigeria , becomes imperative . the main objective of this study was to determine the nonmedical factors that influence maternal mortality in the study area and to identify the nonmedical determinants that will provide baseline data for future investigation into maternal mortality and its contributing factors in the region . it will also aid in adopting interventions on maternal health services and policies for improving the management and reduction of maternal mortality in ado - odo / ota lga and , by extension , the entire country . this study engaged a multistage design and an informant - based survey because of its sensitive nature . ado - odo / ota lga was selected purposively out of 20 lgas in ogun state , nigeria . in the second stage , all the political wards were listed , and 11 were randomly selected out of 16 wards . interviews were conducted in these 11 randomly selected wards ; identified households in these wards were visited to collect information from eligible respondents for the quantitative study . eligible respondents consisted of husbands whose wives had died during pregnancy , in the course of delivery or 42 days thereafter in the 3 years preceding the survey . the study used a structured questionnaire designed to elicit the required information from eligible respondents that met the above criteria . informants were employed to aid in locating respondents through the assistance of health workers in hospital / primary health centers and tbas facilities . where the husband was not available , a close sister or head of household who was fully aware of the case was approached for eliciting information on maternal death and issues related to it as contained in the research instrument . the data collection instrument was pretested for reliability and validity , and necessary modifications and revisions were carried out to perfect its reliability . interrater reliability was used , and ratings from 2 independent raters were correlated ; the index yielded r=0.64 , whereas the cronbach s alpha index yielded a value of 0.68 . the construct validity of the instrument was guaranteed by a demographer and health care personnel . the data were analyzed with the aid of ibm spss statistics 19 ( ibm corporation , armonk , ny , usa ) . in addition , for qualitative data collection , focus group discussions ( fgds ) were conducted among stakeholders the fgd participants ( fgdps ) were drawn from 4 groups , namely , 1 ) tbas and faith - based birth attendants ( fbbas ) , 2 ) community health workers ( chws ) comprising nurses and midwives , 3 ) pregnant women attending the antenatal care ( anc ) clinic and 4 ) community development associations ( cdas ) . these stakeholders were considered to be associated with pregnancy ( pre- and postpregnancy issues ) and child delivery in the study area . the essence was to harvest the experiences of the participants on maternal mortality and morbidity in the study location . common discussion guidelines that were designed to gather information on the factors influencing maternal mortality based on maternal health care practices and problems , decision on choice of place of institutional delivery , payment of treatment costs , awareness of pregnancy complications and knowledge of place of anc treatment were engaged in the exercise , and all the rules of fgds were followed . the discussions were led by a good moderator who had some experience in the conduct of fgds . a notetaker and a video recording device were used to capture all information , and the moderator followed a discussion guide . transcripts were coded according to participants responses to each question , and the most salient themes emerged across the set of discussions . the data in local language were translated by a skilled translator into english language and analyzed using content analysis . this study did not require ethics committee approval . however , informed consent was obtained from all respondents before the interview . respondents were informed of the purpose of the study , assured of confidentiality of information supplied . in addition , a request was made not to answer any question they were not willing to and could stop the interview at will . the age at first marriage of respondents was highest between ages 25 and 44 years with a proportion of 64.2% ( table 1 ) . the occupational status of the husbands interviewed ranged from unskilled labor ( 15% ) , skilled artisans ( 32.2% ) , traders ( 23.3% ) and farmers ( 11.4% ) to civil servants ( 10.6% ) . overall , 89.4% belonged to the working class , while only 7.5% were not working as at the time of the survey . the highest level of education attained by the majority of the respondents interviewed was primary education ( 49.7% ) , followed by secondary education ( 29.2% ) . however , respondents with no school and with postsecondary education accounted for 16.1% and 5% , respectively . incidentally , the age at first marriage of deceased spouses was highest between ages 15 and 29 years with the same proportion of 64.2% . regarding the occupational status of the deceased spouse , those working comprised the highest proportion ( 74.5% ) , followed by full - time housewives ( 25.5% ) . when reference was made to the education of their deceased spouses , a similar scenario was observed : 50% of them had had only primary education , followed by those who attained secondary school ( 27.5% ) and those who never went to school ( 18.9% ) . respondents who had health facilities close to their homes ( < 23 km ) accounted for the least ( 10.3% ) , those who had the health facility at a distance of 45 km accounted for 14.5% and about three - fourths ( 75.2% ) of the respondents had to travel 6 km and beyond to access health care services . on examination of the social class of the respondents , it was observed that respondents in the study area belonged to the poor social class , as majority of them belonged to the low class category ( 66.6% ) , followed by a reasonable proportion in the middle class category ( 32.8% ) and negligible proportion in the upper class ( 0.6% ) cadre . an overwhelming proportion ( 80.3% ) agreed that malaria and fever were the most common ailments in the study locations ( table 2 ) . the remaining proportion was accounted for by typhoid ( 13.9% ) , headache ( 1.9% ) , cold / cough ( 3.3% ) , diarrhea ( 0.3% ) and diabetes ( 0.3% ) . in the study area , 49.7% of the respondents stated that it was the husband s responsibility to pay for the treatment costs . this was followed by relatives ( 25% ) , others ( 11.7% ) , both respondent and spouse ( 8.9% ) and finally spouse ( wife ) ( 4.7% ) . places of consultation visited by the respondents showed that slightly more than half of them approached medical personnel ( 51.4% ) , and nonmodern consultation accounted for 48.6% . the noninstitutional consulting homes included homes of traditional healers ( 12.2% ) , pastors ( 2.8% ) , family members ( 5.8% ) and self ( 27.8% ) . an overwhelming proportion of the respondents ( 90.8% ) admitted knowledge of place of anc treatment . the assistance during pregnancy and childbirth is also encouraging as 71.9% of respondents acknowledged the services of medical personnel at delivery as against 28.1% who still availed themselves of the services of nonmedical personnel during delivery . while a preponderance of the respondents wives had institutional delivery ( 71.7% ) , most of these deliveries were concentrated in the private hospitals / clinics ( 42.2% ) . however , just more than half the respondents ( 50.6% ) had no awareness of pregnancy complications . the multivariate analysis results further buttress these findings from the study ( table 3 ) . the results showed that place of consultation ( p=0.000 ) , who pays the treatment costs ( p=0.000 ) , awareness of pregnancy complications ( p=0.002 ) and knowledge of place of anc treatment ( p=0.000 ) are significant to the outcome of the place of delivery , which is a proxy for maternal mortality . from the analysis of variance ( anova ) , there was also a significant relationship between these variables and the dependent variable , the place of delivery of last birth . the f - statistic ( 15.100 ) confirmed the hypothesis that nonmedical factors were significantly related to maternal mortality among the women in the study area . the correlation of predictor variables ( table 4 ) was significant at the 0.01 level and at the 0.05 level ( 2-tailed ) . the report from the qualitative study showed the factors influencing maternal mortality as reflected by awareness of pregnancy complications , knowledge of place of anc treatment , who pays the treatment costs and place of delivery practices among others . the fgdps were drawn from 4 groups , namely 1 ) tbas and fbbas , 2 ) chws comprising nurses and midwives , 3 ) pregnant women attending the anc clinic and 4 ) cdas . it is an advocacy association that popularizes health programs among communities in the study area . all these categories were considered to be associated with pregnancy ( pre- and postpregnancy issues ) and child delivery . certainly , the majority of participants ( 50.6% ) had no knowledge of pregnancy complications ; almost half ( 49.4% ) of them , however , had awareness regarding the same ( table 2 ) . the evolved theme was as follows : higher knowledge of pregnancy complications reduced pregnancy complications and enhanced the use of institutional delivery . the problem of hypertension and malaria that make me lost 2 pregnancies . sometimes pregnant woman get stress , high blood pressure , baby in the stomach , bleeding , restlessness , lost of pregnancy , swelling for face and legs , and lack of blood are common problems of pregnancy . when i get pregnant i have miscarriage because of stress and lack of using the drugs given to me in the clinic . some people when they are pregnant will start vomiting and will be advised not to have sexual intercourse until about 4 or 5 months to avoid losing the baby . [ sic ] [ fgdp , 37 year old from ota ] the problems of pregnancy are plenty . sometimes pregnant woman get stress , high blood pressure , baby in the stomach , bleeding , restlessness , lost of pregnancy , swelling for face and legs , and lack of blood are common problems of pregnancy . when i get pregnant i have miscarriage because of stress and lack of using the drugs given to me in the clinic . some people when they are pregnant will start vomiting and will be advised not to have sexual intercourse until about 4 or 5 months to avoid losing the baby . [ sic ] [ fgdp , 37 year old from ota ] another participant revealed : i do nt know whether pregnancy complication exist in this area but know that not all women that get pregnant survive the journey . [ sic ] [ fgdp , 35 year old from ota ] i do nt know whether pregnancy complication exist in this area but know that not all women that get pregnant survive the journey . [ sic ] [ fgdp , 35 year old from ota ] another participant , corroborating , said that pregnancy complications in this area are common and hypertension and bleeding and baby outside womb sometime prolonged labor occurs . in some cases they leads to death , and [ sic ] [ fgdp , 36 year old from ota ] pregnancy complications in this area are common and hypertension and bleeding and baby outside womb sometime prolonged labor occurs . in some cases they leads to death , and tba provider wo nt do anything . [ sic ] [ fgdp , 36 year old from ota ] the knowledge of place of anc treatment is interesting as an overwhelming proportion of the respondents admitted knowledge of it ( 90.8% ) , but a small proportion posited that they were unaware ( table 2 ) . however , most of them visited the anc treatment place to get a card in case of emergency and not because they use the facility . perhaps this might be the reason why noninstitutional delivery and providers exist , as seen in table 2 . we go there sometimes to book and get card because nurse will demand your card for emergency . if you do nt come with card the nurses will talk rudely to you and you can not see the doctor . [ sic ] [ fgdp , 30 year old from ota ] we know about health clinic for anc treatment . we go there sometimes to book and get card because nurse will demand your card for emergency . if you do nt come with card the nurses will talk rudely to you and you can not see the doctor . [ sic ] [ fgdp , 30 year old from ota ] the majority of the respondents preferred institutional delivery ( 71.7% ) and anc providers ( 71.9% ) ( table 2 ) . assistance by nonmedical providers leads to low quality of services , which could have resulted in pregnancy complications and maternal deaths among the deceased wives of respondents in the study area . several reasons , such as cost , quality of service , satisfaction , as well as proximity and transportation issues , were given for the use of nonmedical facilities . fgdps mentioned husbands and relatives , and sometimes friends , as sources of their money . the theme derived is that the chance of maternal mortality is higher among nonusers of institutional facilities due to poor knowledge of pregnancy complications , among others . we like local delivery because they treat us fine and when pregnancy come we walk to baba doctor s place ( tba s home ) no body walk to clinic when delivery come . [ sic ] [ fgdp , 33 year old from ota ] our health center is far and no transport . we like local delivery because they treat us fine and when pregnancy come we walk to baba doctor s place ( tba s home ) no body walk to clinic when delivery come . [ sic ] [ fgdp , 33 year old from ota ] another participant was quick to add : baba doctor give big medicine and collects money small by small . his home is close by walk and often he comes to conduct delivery in our houses . [ sic ] [ fgdp , 39 year old from ota ] baba doctor give big medicine and collects money small by small . his home is close by walk and often he comes to conduct delivery in our houses . [ sic ] [ fgdp , 39 year old from ota ] a cda participant revealed that noninstitutional deaths are not uncommon in this community due to complications of pregnancy and delivery . my friend s wife died when giving birth because bleeding was high and blood was not available . [ sic ] [ fgdp , 38 year old from ota]the hospital people will ask us pay big money and we will ask our husbands because they provide money us and women do nt have money . sometimes our relatives and friends give us money for clinic . if we do not get the money from our husbands we do nt go to hospital because they will send us back and not treat us even in serious condition . [ sic ] [ fgdp , 35 year old from ota ] noninstitutional deaths are not uncommon in this community due to complications of pregnancy and delivery . my friend s wife died when giving birth because bleeding was high and blood was not available . [ sic ] [ fgdp , 38 year old from ota ] the hospital people will ask us pay big money and we will ask our husbands because they provide money us and women do nt have money . sometimes our relatives and friends give us money for clinic . if we do not get the money from our husbands we do nt go to hospital because they will send us back and not treat us even in serious condition . [ sic ] [ fgdp , 35 year old from ota ] another participant revealed that even when formal health fees are low , other informal costs such as buying complete delivery items , drugs and food pose barriers to the utilization of available health services . [ sic ] [ fgdp , 41 year old from ota ] even when formal health fees are low , other informal costs such as buying complete delivery items , drugs and food pose barriers to the utilization of available health services . [ sic ] [ fgdp , 41 year old from ota ] corroborating this claim , a nursing sister from one of the health facilities observed that some of the women who delivered in noninstitutional homes can not afford the minimum charges of government health centers but the risk of complications and maternal deaths may be very high for those attending noninstitutional homes . [ sic ] [ fgdp , 40 year old from ota ] some of the women who delivered in noninstitutional homes can not afford the minimum charges of government health centers but the risk of complications and maternal deaths may be very high for those attending noninstitutional homes . the age at first marriage of respondents was highest between ages 25 and 44 years with a proportion of 64.2% ( table 1 ) . the occupational status of the husbands interviewed ranged from unskilled labor ( 15% ) , skilled artisans ( 32.2% ) , traders ( 23.3% ) and farmers ( 11.4% ) to civil servants ( 10.6% ) . overall , 89.4% belonged to the working class , while only 7.5% were not working as at the time of the survey . the highest level of education attained by the majority of the respondents interviewed was primary education ( 49.7% ) , followed by secondary education ( 29.2% ) . however , respondents with no school and with postsecondary education accounted for 16.1% and 5% , respectively . incidentally , the age at first marriage of deceased spouses was highest between ages 15 and 29 years with the same proportion of 64.2% . regarding the occupational status of the deceased spouse , those working comprised the highest proportion ( 74.5% ) , followed by full - time housewives ( 25.5% ) . when reference was made to the education of their deceased spouses , a similar scenario was observed : 50% of them had had only primary education , followed by those who attained secondary school ( 27.5% ) and those who never went to school ( 18.9% ) . respondents who had health facilities close to their homes ( < 23 km ) accounted for the least ( 10.3% ) , those who had the health facility at a distance of 45 km accounted for 14.5% and about three - fourths ( 75.2% ) of the respondents had to travel 6 km and beyond to access health care services . on examination of the social class of the respondents , it was observed that respondents in the study area belonged to the poor social class , as majority of them belonged to the low class category ( 66.6% ) , followed by a reasonable proportion in the middle class category ( 32.8% ) and negligible proportion in the upper class ( 0.6% ) cadre . an overwhelming proportion ( 80.3% ) agreed that malaria and fever were the most common ailments in the study locations ( table 2 ) . the remaining proportion was accounted for by typhoid ( 13.9% ) , headache ( 1.9% ) , cold / cough ( 3.3% ) , diarrhea ( 0.3% ) and diabetes ( 0.3% ) . in the study area , 49.7% of the respondents stated that it was the husband s responsibility to pay for the treatment costs . this was followed by relatives ( 25% ) , others ( 11.7% ) , both respondent and spouse ( 8.9% ) and finally spouse ( wife ) ( 4.7% ) . places of consultation visited by the respondents showed that slightly more than half of them approached medical personnel ( 51.4% ) , and nonmodern consultation accounted for 48.6% . the noninstitutional consulting homes included homes of traditional healers ( 12.2% ) , pastors ( 2.8% ) , family members ( 5.8% ) and self ( 27.8% ) . an overwhelming proportion of the respondents ( 90.8% ) admitted knowledge of place of anc treatment . the assistance during pregnancy and childbirth is also encouraging as 71.9% of respondents acknowledged the services of medical personnel at delivery as against 28.1% who still availed themselves of the services of nonmedical personnel during delivery . while a preponderance of the respondents wives had institutional delivery ( 71.7% ) , most of these deliveries were concentrated in the private hospitals / clinics ( 42.2% ) . however , just more than half the respondents ( 50.6% ) had no awareness of pregnancy complications . the multivariate analysis results further buttress these findings from the study ( table 3 ) . the results showed that place of consultation ( p=0.000 ) , who pays the treatment costs ( p=0.000 ) , awareness of pregnancy complications ( p=0.002 ) and knowledge of place of anc treatment ( p=0.000 ) are significant to the outcome of the place of delivery , which is a proxy for maternal mortality . from the analysis of variance ( anova ) , there was also a significant relationship between these variables and the dependent variable , the place of delivery of last birth . the f - statistic ( 15.100 ) confirmed the hypothesis that nonmedical factors were significantly related to maternal mortality among the women in the study area . the correlation of predictor variables ( table 4 ) was significant at the 0.01 level and at the 0.05 level ( 2-tailed ) . the report from the qualitative study showed the factors influencing maternal mortality as reflected by awareness of pregnancy complications , knowledge of place of anc treatment , who pays the treatment costs and place of delivery practices among others . the fgdps were drawn from 4 groups , namely 1 ) tbas and fbbas , 2 ) chws comprising nurses and midwives , 3 ) pregnant women attending the anc clinic and 4 ) cdas . it is an advocacy association that popularizes health programs among communities in the study area . all these categories were considered to be associated with pregnancy ( pre- and postpregnancy issues ) and child delivery . certainly , the majority of participants ( 50.6% ) had no knowledge of pregnancy complications ; almost half ( 49.4% ) of them , however , had awareness regarding the same ( table 2 ) . the evolved theme was as follows : higher knowledge of pregnancy complications reduced pregnancy complications and enhanced the use of institutional delivery . sometimes pregnant woman get stress , high blood pressure , baby in the stomach , bleeding , restlessness , lost of pregnancy , swelling for face and legs , and lack of blood are common problems of pregnancy . when i get pregnant i have miscarriage because of stress and lack of using the drugs given to me in the clinic . some people when they are pregnant will start vomiting and will be advised not to have sexual intercourse until about 4 or 5 months to avoid losing the baby . [ sic ] [ fgdp , 37 year old from ota ] the problems of pregnancy are plenty . sometimes pregnant woman get stress , high blood pressure , baby in the stomach , bleeding , restlessness , lost of pregnancy , swelling for face and legs , and lack of blood are common problems of pregnancy . when i get pregnant i have miscarriage because of stress and lack of using the drugs given to me in the clinic . some people when they are pregnant will start vomiting and will be advised not to have sexual intercourse until about 4 or 5 months to avoid losing the baby . [ sic ] [ fgdp , 37 year old from ota ] another participant revealed : i do nt know whether pregnancy complication exist in this area but know that not all women that get pregnant survive the journey . [ sic ] [ fgdp , 35 year old from ota ] i do nt know whether pregnancy complication exist in this area but know that not all women that get pregnant survive the journey . [ sic ] [ fgdp , 35 year old from ota ] another participant , corroborating , said that pregnancy complications in this area are common and hypertension and bleeding and baby outside womb sometime prolonged labor occurs . in some cases they leads to death , and [ sic ] [ fgdp , 36 year old from ota ] pregnancy complications in this area are common and hypertension and bleeding and baby outside womb sometime prolonged labor occurs . in some cases they leads to death , and tba provider wo nt do anything . the knowledge of place of anc treatment is interesting as an overwhelming proportion of the respondents admitted knowledge of it ( 90.8% ) , but a small proportion posited that they were unaware ( table 2 ) . however , most of them visited the anc treatment place to get a card in case of emergency and not because they use the facility . perhaps this might be the reason why noninstitutional delivery and providers exist , as seen in table 2 . we go there sometimes to book and get card because nurse will demand your card for emergency . if you do nt come with card the nurses will talk rudely to you and you can not see the doctor . [ sic ] [ fgdp , 30 year old from ota ] we know about health clinic for anc treatment . we go there sometimes to book and get card because nurse will demand your card for emergency . if you do nt come with card the nurses will talk rudely to you and you can not see the doctor . the majority of the respondents preferred institutional delivery ( 71.7% ) and anc providers ( 71.9% ) ( table 2 ) . assistance by nonmedical providers leads to low quality of services , which could have resulted in pregnancy complications and maternal deaths among the deceased wives of respondents in the study area . several reasons , such as cost , quality of service , satisfaction , as well as proximity and transportation issues , were given for the use of nonmedical facilities . fgdps mentioned husbands and relatives , and sometimes friends , as sources of their money . the theme derived is that the chance of maternal mortality is higher among nonusers of institutional facilities due to poor knowledge of pregnancy complications , among others . we like local delivery because they treat us fine and when pregnancy come we walk to baba doctor s place ( tba s home ) no body walk to clinic when delivery come . [ sic ] [ fgdp , 33 year old from ota ] our health center is far and no transport . we like local delivery because they treat us fine and when pregnancy come we walk to baba doctor s place ( tba s home ) no body walk to clinic when delivery come . [ sic ] [ fgdp , 33 year old from ota ] another participant was quick to add : baba doctor give big medicine and collects money small by small . his home is close by walk and often he comes to conduct delivery in our houses . [ sic ] [ fgdp , 39 year old from ota ] baba doctor give big medicine and collects money small by small . his home is close by walk and often he comes to conduct delivery in our houses . [ sic ] [ fgdp , 39 year old from ota ] a cda participant revealed that noninstitutional deaths are not uncommon in this community due to complications of pregnancy and delivery . my friend s wife died when giving birth because bleeding was high and blood was not available . [ sic ] [ fgdp , 38 year old from ota]the hospital people will ask us pay big money and we will ask our husbands because they provide money us and women do nt have money . sometimes our relatives and friends give us money for clinic . if we do not get the money from our husbands we do nt go to hospital because they will send us back and not treat us even in serious condition . [ sic ] [ fgdp , 35 year old from ota ] noninstitutional deaths are not uncommon in this community due to complications of pregnancy and delivery . my friend s wife died when giving birth because bleeding was high and blood was not available . [ sic ] [ fgdp , 38 year old from ota ] the hospital people will ask us pay big money and we will ask our husbands because they provide money us and women do nt have money . sometimes our relatives and friends give us money for clinic . if we do not get the money from our husbands we do nt go to hospital because they will send us back and not treat us even in serious condition . [ sic ] [ fgdp , 35 year old from ota ] another participant revealed that even when formal health fees are low , other informal costs such as buying complete delivery items , drugs and food pose barriers to the utilization of available health services . [ sic ] [ fgdp , 41 year old from ota ] even when formal health fees are low , other informal costs such as buying complete delivery items , drugs and food pose barriers to the utilization of available health services . [ sic ] [ fgdp , 41 year old from ota ] corroborating this claim , a nursing sister from one of the health facilities observed that some of the women who delivered in noninstitutional homes can not afford the minimum charges of government health centers but the risk of complications and maternal deaths may be very high for those attending noninstitutional homes . [ sic ] [ fgdp , 40 year old from ota ] some of the women who delivered in noninstitutional homes can not afford the minimum charges of government health centers but the risk of complications and maternal deaths may be very high for those attending noninstitutional homes . marriage exposes a woman to the risk of pregnancy , and early marriage puts a woman into longer exposure to pregnancy and child delivery complications.18 the age at first marriage of respondents was highest between ages 25 and 44 years with a proportion of 64.2% ( table 1 ) . incidentally , the age at first marriage of deceased spouses was highest between ages 15 and 29 years with the same proportion of 64.2% . this means that the age at first marriage of deceased spouses was relatively low compared to that of their husbands . the low age at first marriage has also been noted in earlier studies.2226 comparatively , education and occupation , though low , showed that respondents were better off than their deceased spouses , a strong indication of the low status of women in the study area . the poor socioeconomic class of the respondents also buttressed the poor social class of respondents ( 66.6% ) . the association of maternal education with empowerment and knowledge of safe motherhood practices , leading to reduction in maternal mortality , has been observed by other scholars.27,28 the social status of respondents in the study population needs urgent attention as this might adversely affect access to health care services , leading to high maternal morbidity and mortality . the study revealed that distance to the health facility was also a major retarding factor in accessing health care services among the wards in the study area,13,2931 and the situation is disturbing as an overwhelming proportion ( 75.2% ) of respondents indicated that they had to travel 6 km and to access health care services . a pregnant woman would prefer to visit the next - door tba rather than walk for kilometers to the health center where she had no confidence in the service,31 as equally revealed during the fgds . similar observations were reported by other researchers previously.30,32 factors influencing maternal morbidity and mortality ( table 2 ) revealed interesting findings . an overwhelming majority ( 80.3% ) agreed that malaria and fever were the most common ailments in the study locations . payment of hospital bills was exclusively within the domain of the respondents ( husbands ) . culturally , in a patriarchal society such as nigeria , male dominance and women subjugation are normal ways of life . in the study area , 49.7% of the respondents stated that it was their responsibility to pay for the treatment costs . this is also a restraining force to accessing health care facilities by pregnant women as it increases the dependency syndrome of wives . this relates to the statement from fgdps that if their husbands did not give them money for the clinic , they would not avail the services of the anc center , the consequence of which is grave . the payment of cost of anc services has great bearing on utilizing the health facility . cost may reduce women s use of maternal health services and refrain them from having hospital - based deliveries or seeking care even when complications arise.31 information gathered through fgds revealed that even when formal fees are low , other informal costs such as buying complete delivery items , drugs , food , and so on pose barriers to the utilization of available health services . this calls for government subsidizing the health care cost and bringing health facility closer to the masses . places of consultation chosen by the respondents showed that slightly more than half of them used medical personnel ( 51.4% ) , and nonmodern consultation , including self , accounted for 48.6% . the noninstitutional consulting homes included homes of traditional healers , pastors and relatives , which attracted a reasonable proportion ( 20.8% ) . it is noteworthy that 27.8% of the respondents diagnosed their illness by themselves without recourse to institutional consultation . this is worrisome , particularly when we consider the dangerous consequences of such practice in nigeria . the awareness regarding place of anc treatment is fascinating and encouraging as an overwhelming proportion of the respondents ( 90.8% ) admitted knowledge of place of anc treatment . this level of knowledge should be made to translate into practice the terms of reducing pregnancy complications and home delivery in the study area . the assistance during pregnancy and childbirth is also encouraging as 71.9% of respondents acknowledged the services of medical personnel at delivery , as against 28.1% who still availed themselves of the services of nonmedical personnel during delivery . undoubtedly , this proportion of mothers patronizing home delivery services is high , just as for the whole of nigeria,18 considering the low quality of services rendered by these categories of health providers in a country beset with dismal health services,31 which could have resulted in pregnancy complications and maternal deaths among the deceased wives of respondents in the study area . this high preference for higher family size exposed women to higher risk of maternal deaths and apparently portrays the low status of women . this is in line with the observation on the prevailing situation in ssa nations.3 despite high institutional delivery ( 71.7% ) among respondents wives , both at government and private hospitals , the study revealed that a reasonable proportion of wives still patronized noninstitutional deliveries as in the case of the larger society,18 due to several factors ranging from difficulty in payment of hospital bills , distance to the health facility and unfriendly attitude of health care workers to poor quality of services , among others . these findings from both quantitative and qualitative dimensions corroborate the results of other previous studies within and outside nigeria.2,3336 hence , considering the findings herein and the level of poverty in nigeria , the government should subsidize rural health care services and establish midway service delivery points ( msdps ) to reduce the problem of distance and further bring grass roots health care services closer to the rural population . the study revealed some basic awareness of pregnancy complications among respondents ( 49.4% ) , as well as during fgds ; slightly more than half of the respondents ( 50.6% ) had no awareness of pregnancy complications . this situation might lead to serious issues during pregnancy and calls for health education in order to reduce the hazards that accompany ignorance of pregnancy complications . the findings from the multivariate analysis survey also revealed that place of consultation ( p=0.000 ) , who pays the treatment costs ( p=0.000 ) , awareness of pregnancy complications ( p=0.002 ) and knowledge of place of anc treatment ( p=0.000 ) are significant to the place of delivery , which is a proxy to maternal mortality . the place of consultation reflects the tendency to use the place for delivery , thereby influencing the provision of assistance during pregnancy and after childbirth . the absence of these maternal ingredients was tantamount to poor survival chances among pregnant women in the study community . in addition , modern health care services cost money , and the lower social status of the household as well as that of women discourage the wife from seeking modern medical services . results of the anova showed that there is also a significant relationship between these variables and the dependent variable , the place of delivery of last birth . the f - statistics ( 15.100 ) confirmed the hypothesis that nonmedical factors were significantly related to maternal mortality among the women in the study area . the correlation of predictor variables ( table 4 ) was significant at the 0.01 level and at the 0.05 level ( 2-tailed ) . maternal mortality in nigeria continues to be a serious public health problem and contributes to the low life expectancy among women in nigeria . this study has identified several factors that have an important influence on maternal mortality in the study area . among these are variables such as place of consultation / diagnosis , the person who pays the treatment costs , awareness of pregnancy complications and knowledge of the place of anc treatment , among others . the fgd technique was beneficial and provided qualitative insight on factors influencing maternal mortality and prospects of pregnancy care intervention programs . many women preferred noninstitutional delivery . long distance was mentioned as one of the reasons for respondents preference for home delivery or noninstitutional delivery , in addition to the cost of medical treatment , poor treatment at health care clinics and the harsh attitudes of health care providers . the findings from this study may help government , civil society organizations / nongovernmental organizations and private organizations to design comprehensive interventions toward reducing the high maternal mortality in the study area and , by extension , the entire country . the government should subsidize health care services and establish msdps to reduce the problem of distance and further bring grass roots health care services closer to the rural population . health education and mass enlightenment should be strengthened to create greater awareness on pregnancy complications and zero noninstitutional delivery tolerance . empowering and improving the status of women may reduce maternal mortality , prompt better utilization and increase the survival chances of women in the study area . in addition , government should train tbas to reduce their limitations and improve on their performance . the outcome of this study will be made known to the local government and equally disseminated to the communities in the study area .

backgroundmaternal mortality and morbidity reflect the status of population health and quality of life across nations . poor understanding of the interplay of many antecedent factors , including sociocultural , economic and logistic factors , combined with an overwhelming poor health services delivery , is a basic challenge in several countries , particularly in rural settings where functional health care services are relatively scarce . there are still uncertainties as to the extent of this burden , owing to current challenges with information and data collation . this study aimed at identifying nonmedical factors associated with maternal mortality in rural and semiurban communities of southwestern nigeria.methodologythe study was carried out in ado - odo / ota local government area of ogun state . a multistage sampling technique and an informant survey approach were used in the study . a total sample of 360 eligible respondents were selected randomly from 11 out of 16 political wards in the study area and interviewed through the administration of questionnaires . the data were processed using descriptive statistics and regression analyses.resultsplace of consultation ( p=0.000 ) , who pays the treatment costs ( p=0.000 ) , awareness of pregnancy complications ( p=0.002 ) and knowledge of the place of antenatal care treatment ( p=0.000 ) significantly influenced maternal mortality ( proxy by place of delivery of last birth ) . the f - statistic ( 15.100 ) confirmed the hypothesis that nonmedical factors influence maternal mortality . the correlation of predictor variables was significant at both the 0.01 level and the 0.05 level ( 2-tailed).conclusionour findings suggest that in a rural community setting with a depleted health care system , health education tailored toward community culture , subsidized maternal health care services by the government and operators of private clinics , as well as empowering and improving the status of women may reduce maternal mortality and prompt better utilization and survival chances of women in the study area as well as in all of nigeria .

the pectoralis major muscle in humans is a large fan shaped muscle situated at the anterior upper part of the chest . it is complex in that it has two separate muscular bellies and a dual nerve supply . the clavicular portion of the pectoralis major muscle arises from the medial one half to two thirds of the clavicle , and passes downward and laterally to its humeral insertion . the sternocostal portion arises from the anterior surface of the sternum 's second to the sixth costal cartilages , and from the aponeurosis of the external oblique muscle . from these two origins , the fibers converge toward their insertions : those arising from the clavicle pass obliquely downward and laterally , and are usually separated from the rest by a slight interval ; those from the lower part of the sternum , and the cartilages of the lower true ribs , run upward and laterally , while the middle fibers pass horizontally . they all end in a flat tendon , which is inserted into the lateral lip of the intertubercular sulcus of the humerus ( 1 ) . the pectoralis major muscle is innervated by lateral ( c5 and c6 ) and medial pectoral ( c7 , c8 , and t1 ) nerves . the pectoralis major muscle adducts and medially rotates the humerus , and draws the scapula anteriorly and inferiorly . the clavicular head acts to flex the humerus , and the muscle is supplied by the pectoral branch of the thoracoacromial trunk artery , and by branches of the lateral thoracic artery . it is also a dynamic stabilizer of the shoulder joint ; therefore , it is important in upper limb mobility , and the presence of supernumerary large heads may provide additional strength to the biceps brachii as described by swieter and carmichael ( 2 ) . here we report a unique case of unilateral four - head pectoralis major muscle in an 83 year - old cadaver ( figure 1 ) , that was otherwise anatomically and functionally unremarkable . we observed the accessory tendon of the pectoralis major muscle on the left side of this male cadaver . the accessory tendon has approximately the same dimensions and position as the tendon of the long head of the biceps brachii muscle . in fact , the accessory tendon travels alongside the tendon of the long head of the biceps brachii muscle ( figure 2 . ) . this accessory tendon is located directly lateral to the tendon of the long head of the biceps brachii muscle . the position of this accessory tendon obscures the view of the biceps tendon when the pectoralis major is not retracted . proximally , the accessory tendon blends into the fibers of the capsule of the shoulder joint . distally , the accessory tendon attaches to and blends in with the superior fibers of the pectoralis major tendon , which inserts into the lateral lip of the intertubercular sulcus of the humerus . no abnormality was observed in the nerve and blood supply either to the pectoralis major muscle or to the accessory tendon . gray described some variations associated with the pectoralis muscle such as greater or lesser extent of attachment to the ribs and sternum , varying size of the abdominal part or its absence , greater or lesser extent of separation of sternocostal and clavicular parts , fusion of clavicular part with deltoid , and decussation in front of the sternum . the sternocostal origin is the most commonly missing part , lack of which causes weakness in the adduction and medial rotation of the shoulder joint ( 4 ) . in addition to the variations within the pectoralis muscle , gray also described the rare presence of additional muscles such as costocoracoideus , chondro - epitrochlearis , and sternalis , all of which are closely associated with the pectoralis major ( 1 ) . although most of the variations mentioned above are associated with the surface anatomy of the bellies of the pectoralis muscle , the presence of an accessory tendon in the pectoralis muscle has never been described . the presence of an accessory tendon to the pectoralis major muscle might stabilize the muscle origins ( 2 ) . however , premortem clinical history was unremarkable regarding impairment of shoulder movements . this indicates that the presence of such an anomaly may not impede normal movements of the shoulder . most of the reported pectoralis major abnormalities focused on its complete or partial absence due to congenital reasons ( 510 ) , or as a result of diseases such as leukemia ( 11 ) and poland 's syndrome , where absence of the costosternal portion of the pectoralis major has been reported ( 12 , 13 ) . poland 's syndrome is a group of unilateral congenital abnormalities of the chest wall with or without involvement of the arm on the same side . the right side is affected twice as often as the left side and there is a male preponderance . pectoralis major abnormalities included bilateral ( 14 ) or unilateral absence of the pectoralis major ( 15 ) . however , regarding the presence of accessory tendons of the pectoralis muscle , studies reported accessory fibrous bands arising from the lateral border of the pectoralis that have intimate contact with the median nerve ( 16 ) . we believe that the variation reported in this region is important both functionally and clinically . this finding is of clinical importance in transplant surgery , giving additional options for insertion of tendons in the shoulder joint area . additionally , the fact that we found this unique case in a male cadaver raises the question whether it is sex - specific , and encourages a more thorough search to find out if a similar pattern of variation exists in females . should this pattern be present in females , it would be of clinical importance in cases of breast or transplant surgeries .

a unilateral four - headed pectoralis major muscle was observed on the left side of an 83 year - old caucasian male cadaver . the accessory tendon arises from the lateral aspect of the pectoralis major muscle , travels along with the tendon of the long head of the biceps brachii and blends into the capsule of the shoulder joint . this anomaly is rare . a complete or partial absence of pectoralis major muscle is normally reported . additionally , supernumerary heads of muscles other than pectoralis major have been documented . however , the existence of an accessory tendon to the pectoralis major muscle is unique . the implications of such a finding are discussed .

the importance of selenium for the life of animals has been known since the 1950s , while the essentiality of this element for humans was revealed in 1970s with the discovery of its ability to prevent the keshan disease , first described in 1935 in china in a province of the same name . subsequently , it was discovered its important role as part of the body 's antioxidant mechanisms and also its role in protecting the body against heavy metals as well as its importance in the immune system [ 1 , 2 ] . cow 's milk and dairy products are usually a poor source of selenium in human nutrition . however , human milk is a poor source of selenium , so recently manufacturers of dairy - based infant formulas have added selenium to these products . selenium has a very narrow range between dietary deficiency ( < 40 g / day for adults ) and toxic levels ( > 400 g / day ) . for this reason , it is very important to have control of the selenium intake by human and animals . several analytical techniques are suitable for determining total selenium in milk , such as neutron activation analysis ( instrumental neutron activation analysis ( inaa ) ) , fluorimetry , gas chromatography ( gc ) , and atomic absorption spectrometry ( aas ) . the neutron activation technique is not available in most laboratories ; it is generally used for determinations of selenium in certified reference materials as an alternative method and in proficiency testing . it has the advantage of being a nondestructive technique , but the detection limits are usually higher than for other techniques [ 3 , 4 ] . other methods usually are based on the generation of a piazselenol complex , which is subsequently submitted to different detection techniques . for instance , a fluorimetric method is recommended in the bulletin of the international dairy federation ( idf ) and in the aoac 1996 compendium as an official method , thus it has been widely studied . the method is based on the measurement of fluorescence of a piazselenol formed from selenite and dan ( 2,3-diaminonapthalene ) which is a carcinogenic reagent . detection limits as low as 0.2 g l can be obtained ; this is suitable for this kind of determinations but it involves a stage of prereduction and extraction of the complex in an organic solvent [ 3 , 4 ] . another method for selenium determination is based on the reaction with ( 3,3-diaminobenzidine ) in acidic medium to form the corresponding piazselenol , which is measured spectrophotometrically . in this method , the necessary time for color development is 50 minutes . as an example of determination of selenium by gas chromatography , we can mention the method based on selective complexation of selenium to form the piazselenol . this is extracted into an organic phase before being injected in a chromatograph fitted with an electron capture detector . . the determination of total selenium in milk at trace and ultratrace levels is often performed by electrothermal atomic absorption spectrometry ( et - aas ) [ 8 , 9 ] . other popular technique is atomic absorption spectrometry with hydride generation ( hg - aas ) [ 1012 ] . this technique also presents some important advantages , including the separation of the analyte from the matrix which reduces the number of interferences that may occur . the generation of the hydride can be carried out in batch or can be automated using different flow systems . continuous flow systems have been used extensively for hydride generation for over 20 years , and there are several commercial models with accessories for hydride generation [ 1315 ] . automation by flow injection analysis ( fia ) technique [ 16 , 17 ] has proved to be useful and efficient for this purpose ; several articles published report the coupling of fia to the generation of hydrides with excellent results ( fia - hg - aas ) [ 12 , 18 ] . both continuous flow and fia systems have also been coupled to atomic florescence detection ( fia - afs ) [ 1921 ] . there are also some reports of flow systems coupled with atomic emission spectrometry ( hg - icp - oes ) [ 22 , 23 ] . reported an application for total inorganic selenium determination by hydride generation - atomic fluorescence spectrometry with a multisyringe flow injection system ( msfia ) . on the other hand , multicommutated flow analysis ( mcfa ) is a technique based on flow networks built around electrically operated solenoid valves which are turned on or off under computer control [ 2528 ] . it has been already used by rdenas - torralba et al . for the determination of tellurium in milk by hydride generation but coupled to atomic fluorescence detection . in a previous work we developed and validated a multicommutated flow system for the determination of total selenium in milk and infant formulas . when comparing the techniques of fia and mcfa , it is evident that fia systems tend to be simpler and can be implemented with a manually operated valve , although such a system is not useful if some degree of automation is desired . for this purpose , however , an electrically operated valve can be used connected to a computer or even to an appropriate timer with an electric switch . mcfa on the other hand requires some electronic and computer skills but the user ends up with a much more flexible system which is intrinsically amenable to automation . given that the solenoid valves are controlled by the software , it is easy to modify the time when each valve is energized and the duration of that condition . this enables to change sample and reagent volumes , reaction times , and also , by resorting to binary sampling , the form in which samples and reagents are mixed . the aim of this work is the comparison and evaluation of the performance of two flow systems based on different techniques : a fia system developed , optimized , and validated , presented in this work and a mcfa system developed and validated in a previous work . this comparison is proposed in order to investigate the benefits that either technique could provide for the determination of total selenium in infant formulas . the two flow systems were operated using detection by atomic absorption with hydride generation , allowing the determination of selenium as se(iv ) which is the species that can be detected as seh2 . the se(iv ) was obtained by quantitative reduction of total inorganic selenium present in the matrix after mineralization . interferences due to the presence of transition metals were not expected due to the low concentrations of these potential interferents in this matrix ; a fact that has been confirmed in the literature [ 3032 ] . all glassware was soaked overnight in 10% ( v / v ) nitric acid and then rinsed exhaustively with deionized water . connections and doubly - helical mixing coils were made from 0.8 mm internal diameter teflon pfa tubing . a u - shaped lab - built glass gravitational gas - liquid phase separator was used . the carrier gas was nitrogen ( dried and purified by a combined drierite / molecular - sieve trap ) . a 0.5% ( w / v ) solution was prepared daily by dissolving the solid in 0.05% ( w / v ) naoh . purified water ( astm type i ) was obtained from a millipore ( so paulo , brazil ) simplicity 185 purifier fed with glass - distilled water . a 1000 mg l selenium standard solution was prepared from selenium metal ( aldrich , 99.99% ) , dissolved in nitric acid , and made up to volume with 10% ( v / v ) hydrochloric acid . an intermediate standard solution ( 0.8 mg l ) was prepared daily by stepwise dilution with 1.5% ( v / v ) hydrochloric acid . measurements were carried out with a perkin elmer ( norwalk , ct , usa ) model 5000 atomic absorption spectrometer fitted with a 10 cm burner ( air - acetylene flame ) and operated at the 196.0 nm analytical line . atomization was carried out in a t - shaped quartz atomization cell ( precision glassblowing , centennial , co , usa ) . the light source was a photron ( narre warren , australia ) superlamp intensified emission hollow - cathode lamp operated as recommended by the manufacturer . calibration solutions were prepared by accurately diluting aliquots of the 0.8 mg l intermediate standard solution , to which 20 ml of water and 10 ml of concentrated hydrochloric acid were added . the mixture was heated on a hot plate for 1 hour at gentle boiling to carry out the prereduction of se(vi ) to se(iv ) then cooled down to room temperature and diluted to 30.0 ml with purified water . the sample used for this comparative study was a standard reference material ( srm ) of infant formula ( nist 1846 infant formula ) . these were prepared as follows : 0.50 g of the sample was accurately weighed in a 30 ml screw - capped teflon pfa vessel ( savillex , minnetonka , mn , usa ) . then , 6 ml of concentrated nitric acid was added , the vessel was loosely capped , placed in a modified polypropylene fast cooker , and heated in a household microwave oven ( ariston model mo991b ) . the cooker was modified in order to vent all acid vapors and other gases via a piece of tubing to a flask containing sodium hydroxide solution which acted as a trap for acidic vapors . the oven was programmed to heat for 5 minutes at 30% and then for 3 minutes at 40% of the maximum power . it was then cooled down to room temperature , 1 ml of 30% hydrogen peroxide was added , and the vial ( loosely capped ) was heated again for 2 minutes at 40% power . afterwards , the contents of the vial were transferred quantitatively to a 50 ml erlenmeyer flask containing 10 ml of 10% ( w / v ) sulfamic acid solution and 10 ml of concentrated hcl ; the prereduction step was carried out by heating at gentle boiling on a hot plate for 1 hour and then cooled down to room temperature and diluted with water to 20.0 ml . the fia system ( figure 1 ) was based upon a gilson ( villiers - le - bel , france ) minipuls 2 multichannel peristaltic pump fitted with either tygon or viton tubing . injection of the sample was made by means of a 6-port valco cheminert valve with microelectric actuator controlled from a personal computer via the serial rs232 serial port using a program compiled in quickbasic 4.0 . the mcfa system ( figure 2 ) was based on a peristaltic pump and two 3-way solenoid valves ; it has been already described elsewhere . for both systems , the analytical signal ( absorbance ) was obtained from the analog output connector of the spectrometer ( 1-v full scale ) and digitized via a 12-bit analog to digital interface ( measurement computing , model usb 1208ls ) connected to a usb port and operated at a sampling rate of 1 s. a program was compiled in visual basic 6.0 for this purpose . the sample is introduced into the flow stream of a carrier using a two - position injection valve . the loading time of the loop and subsequent injection of the sample by changing the position of the valve automatically were programmed in the software . once the analytical signal returns to the baseline and the recording finishes , a new cycle of injection starts . for optimization , two multivariate experiments ( based on a central composite design ) and several univariate experiments were carried out . in table 1 , the influence of the flow rate of the carrier ( hcl ) , the reducing agent ( nabh4 ) , and the mixing coil length in the peak height ( signal ) are presented . for these experiments a solution of se(iv ) of 50 g l was prepared . the fixed variables were the concentrations of the carrier and the reducing agent ( hcl : 10% ( v / v ) ; nabh4 : 0.2% ( m / v ) ) , the sample volume ( 500 l ) , and the flow rate of the carrier gas ( n2 : 0.20 l min ) . according to the results in table 1 , experiment 3 determined that the best conditions were mixing coil length 50 cm , carrier flow rate 3.5 ml min , and flow rate of the reducing agent 4.5 ml min . to complete the final optimization , the fixed conditions were mixing coil length ( 50 cm ) , carrier gas flow rate ( n2 : 0.20 l min ) , and sample volume ( 500 l ) . experiment number 11 proved to give the best results ; this meant a significant decrease in the concentration of hcl and nabh4 with respect to the initial conditions . once the optimal operative conditions were reached , the sampling frequency was 70 hours . a rigorous description of the optimization of this system is presented in a previous work using multivariate experiments . figure 2 shows the scheme of the mcfa system . under the conditions shown in figure 2 , the sampling frequency was 160 samples per hour ( hour ) . linearity was studied by means of an 8-point calibration curve in the range of 1.050.0 g l ( for both systems : n = 5 ) . the linearity range was evaluated by visual inspection of the graphical representation and by means of the regression coefficient . the routine calibration curve covered a smaller range because selenium concentrations expected following the preparation of these samples did not exceed 5 g l. for this reason a routine calibration curve in the range of 110 g l was adequate . precision ( s r ( % ) ) was estimated by analytical repetition of the complete analysis of the reference material ( n = 5 ) . detection ( ld , 3 ) and quantification ( lq , 10 ) limits were estimated by measuring ( n = 10 ) the dispersion of the blank and signal referring the measurements to the calibration curve . the figures of merit are presented in table 4 . to establish the trueness of the proposed methods , a certified reference material of infant formula ( nist 1846 infant formula ) was analyzed . trueness was evaluated by comparison of the value of total selenium obtained for each system ( n = 5 ) with the reference value of the srm by means of a student 's t - test ( table 5 ) . this can be justified because of the low concentrations of potential interferents ( transition metals ) , in this kind of matrix . table 4 shows the figures of merit obtained from the validation of the fia and mcfa systems for the determination of total selenium in reference material of infant formula . the results in table 5 show that values obtained using both systems do not present bias , thus the trueness was demonstrated . the most impressive figures of merit were the limits of detection and quantification in solutions for mcfa - hg - aas , which were almost one order of magnitude lower than the values for fia - hg - aas , and the sampling frequency that was more than twice using the mcfa system . the sampling rate was even higher than the reported by semenova et al . for a msfia system . this added to the lower reagent and sample consumption ( and hence waste generation ) of the mcfa system which is in accordance with the principles of green chemistry . in terms of precision and accuracy , the fia method exhibited a greater linear range , but the difference is not significant for this application . commercial continuous flow systems are widely used , but they consume a large amount of sample and reagents since they are continuously circulating through the system while the determination is carried out . the rigorous assessment of the figures of merit for validation after the optimization of a flow system for a particular application is important to develop new fast , reliable , and environmentally friendly analytical methods . the two flow systems developed were successful for the determination of total selenium in infant formulas by hydride generation atomic absorption spectrometry . the multicommutated flow system ( mcfa ) showed advantages over the flow injection system ( fia ) , presenting a much better detection limit and a higher analytical throughput . the mcfa system also demonstrated to be more flexible because of the possibility to easily change operating parameters such as sample volume by means of the software user interface , without the need of physical modifications of the flow system .

two flow methods , based , respectively , on flow - injection analysis ( fia ) and on multicommutated flow analysis ( mcfa ) , were compared with regard to their use for the determination of total selenium in infant formulas by hydride - generation atomic absorption spectrometry . the method based on multicommutation provided lower detection and quantification limits ( 0.08 and 0.27 g l1 compared to 0.59 and 1.95 l1 , resp . ) , higher sampling frequency ( 160 versus . 70 samples per hour ) , and reduced reagent consumption . linearity , precision , and accuracy were similar for the two methods compared . it was concluded that , while both methods proved to be appropriate for the purpose , the mcfa - based method exhibited a better performance .

endovascular interventional procedures of intracranial circulation have increased significantly over the past 20 years , because they can treat serious diseases with a significant decrease in morbidity and mortality . these procedures are often associated with long and recurrent periods of fluoroscopy that may cause serious skin lesions associated with radiation . in this study , we report a case of a patient who developed a square shaped alopecia after an endovascular embolization procedure for arteriovenous malformation ( avm ) . the case we present here is about a 38-year - old man who came to the emergency room in february 2013 , due to nausea and diplopia of sudden onset , 1 day prior to admission . physical examination revealed a limitation of ocular supraversion movements and bilateral absence of pupillary light reflex , consistent with dorsal midbrain syndrome . a ct scan showed a left dorsal midbrain hematoma with ventricular hemorrhage and incipient signs of hydrocephalus . subsequently an intracranial arteriography confirmed the presence of a left posterior parasagittal avm with contributions from posterior choroidal artery branches [ figure 1 ] . endovascular treatment was performed with selective embolization with glubran20% without any complications . in may 2013 , an angiographic control identified remains of the malformation , with bilateral posteromedial choroidal afferents . a new selective embolization was performed in july 2013 , which achieved a complete devascularization of the avm with excellent angiographic results . angiogram ( anteroposterior view ) demonstrate a left posterior parasagittal arteriovenous malformation with contributions from posterior choroidal artery branches ( black arrow ) a month later , he presented to the dermatology department for hair loss localized in the occipital region of the head . physical examination showed an alopecic square plaque of 5 cm 5 cm with almost total hair loss , but without signs of inflammation [ figure 2 ] . sharply defined square alopecia in the occipital region with almost total hair loss and no signs of inflammation . the lesion occurred 4 weeks after the last endovascular procedure in total , the patient was exposed to four angiographic procedures , two of them with endovascular therapy . the total radiation exposure time was approximately 150 - 200 min and the absorbed radiation dose after the last session was estimated at more than 4 gy . radiation - induced transient alopecia diagnosis was established and there was no need to start treatment . after an interval of more than half a century , reports of fluoroscopically induced skin lesions reappeared in the literature in the 1990s . these reports coincide with the introduction of fluoroscopically guided interventional procedures in the clinical arsenal . although the effects of radiation therapy are well - documented , the skin effects of radiation by fluoroscopically endovascular procedures are scarce . in 1994 , huda et al . published the first case of alopecia secondary to an endovascular procedure guided by fluoroscopy , since then there have been few reports in dermatology journals . the scalp is very resistant to radiation exposure , but paradoxically alopecia presents with lower doses of radiation than the rest of the body . this radiodermatitis has been attributed to the high sensitivity of hair follicles to radiation in the anagen phase that particularly affects rapidly dividing cells of the follicular matrix . at low doses ( 2 - 5 gy ) , mild dermatitis may occur and later evolve into a temporary hair loss that manifests as geographical pattern alopecia . in medium doses ( 5 - 10 gy ) a more prolonged erythema and permanent partial alopecia appear . at high doses ( 10 - 15 gy ) permanent scarring alopecia and late radiation skin changes such as skin induration other factors that may favor the development of alopecia are : the total time of the procedure , residual effects of previous radiation procedures and some patient biological factors ( age , smoking , malnutrition , tissue oxygenation , capillary density , hormonal status , genetic factors , lesion location , and ethnicity ) . it starts at 2 - 5 weeks after the endovascular procedures and has a good prognosis of hair reappearing from 2 to 6 months later . the main differential diagnoses are pressure - induced ( postoperative ) alopecia secondary to hypoxia , which occurs in areas of prolonged pressure on the scalp during operative procedures , and alopecia areata , especially with an ophiasis pattern . transient radiation alopecia is a difficult diagnosis and often is misdiagnosed by dermatologists . it does not require specific treatment , and most patients recover hair in few months ; therefore , the most important thing is to give adequate information to the patient about this self - limiting process .

endovascular interventional procedures are the first choice of treatment for many vascular intracranial lesions , especially those with complex anatomy . these procedures may cause numerous skin lesions depending on the dose of radiation to which patients have been exposed . in this report , we presented a case of a 38-year - old man who developed a square plaque of alopecia in the occipital area after two selective embolization of a left posterior parasagittal arteriovenous malformation . the alopecia was transient and the hair grew up 2 months later after the last procedure . this case illustrates one of the effects of radiation on the scalp and expands our knowledge about the clinical manifestation of this transient disease .

on december 7 , 2005 , a previously healthy 17-year - old boy with no history of recent travel became ill ; symptoms were headache , rhinorrhea , low back pain , and cough without fever . he had received inactivated influenza vaccine administered intramuscularly on november 11 , 2005 . during an outpatient clinic visit on december 8 , 2005 , a nasal wash specimen was obtained and tested positive for influenza a by rapid influenza diagnostic test ( binaxnow a&b , binax , inc . , the specimen was sent to the wisconsin state laboratory of hygiene ( wslh ) , and an influenza a virus was isolated by shell vial tissue cell culture ( mdck cells , wslh , madison , wi , usa ) . real - time reverse transcription pcr ( rrt - pcr ) was positive for influenza a virus but negative for human subtypes h1 , h3 , and asian avian h5 . at the centers for disease control and prevention ( cdc ) , rrt - pcr testing of the shell vial viral culture material was positive for influenza a virus , but negative for human subtypes h1 and h3 , as well as avian subtypes h5 , h7 , and h9 . complete genomic sequencing of the virus at cdc identified it as a swine influenza a ( h1n1 ) triple reassortant virus , a / wisconsin/87/2005 h1n1 . investigation by the wisconsin division of public health and the sheboygan county division of public health showed that the patient had assisted his brother - in - law in butchering pigs at a custom slaughterhouse 3 days before illness onset . thirty - one swine were delivered to the facility that morning by a distributor who had acquired the animals from multiple sources . the patient helped hold and abduct the forelimbs of 1 freshly killed pig while his brother - in - law eviscerated it . a few chickens were housed at the slaughterhouse premises , but no poultry were slaughtered on site . the patient denied any other contact with swine , poultry , or other animals during the 7 days before becoming ill . eight days before illness onset , the patient s father obtained a live chicken that was kept in the home for 1 day before it was sacrificed during a ritual ceremony . the patient was never within 10 feet of the chicken and did not attend the ceremony . none of the patient s household members or any slaughterhouse employees reported illness during the 2 weeks before or after the patient became ill . paired serum specimens were obtained from the patient and 4 family members on december 13 , 2005 , and january 9 , 2006 . a single serum specimen was obtained from the patient s brother - in - law on december 19 , 2005 . serologic testing was conducted at cdc by a standard hemagglutinin inhibition ( hi ) antibody assay and reference antisera against influenza a ( h1 ) , a ( h3 ) , b , and swine a ( h1n1 ) viruses a / swine / wisconsin/238/97 , a / swine / wisconsin / nj56371/99 , a / swine / minnesota/593/99 , and a / wisconsin/87/2005 ( isolated from the patient ) . hi antibody testing was negative for influenza a ( h1 ) , a ( h3 ) , and swine subtype h1n1 on all serum specimens , but 1 family member had evidence of a 4-fold rise in hi antibody titer to influenza b / hong kong/330/2001 ( b / victoria/2/87 lineage ) virus , which suggested an acute influenza b virus infection . all serum samples were also tested by microneutralization assay at cdc using the patient s swine influenza a ( h1n1 ) virus isolate and a human influenza a virus ( a / new caledonia/20/99 h1n1 ) . the patient s serum specimens demonstrated a 2-fold increase in neutralizing antibody titer against swine influenza a / wisconsin/87/2005 subtype h1n1 virus , but the level of neutralizing antibodies to a / new caledonia/20/99 ( h1n1 ) virus was unchanged in acute- and convalescent - phase serum specimens , which is consistent with his history of influenza vaccination in mid - november 2005 . we report a human case of swine influenza a ( h1n1 ) triple reassortant virus infection in the united states . swine influenza a / wisconsin/87/2005 ( h1n1 ) virus was isolated from an upper respiratory specimen obtained from the patient , and serologic testing suggested , but was not diagnostic of , an immune response to acute infection . epidemiologic investigation showed the patient had direct and close exposure to freshly killed pigs and their organs while assisting his brother - in - law in butchering them . although the pigs did not appear ill , the most plausible source of the patient s swine influenza a virus infection was respiratory secretions of freshly killed pigs . surveillance data suggest that triple reassortant subtype h1n1 viruses are the predominant genotype of subtype h1n1 viruses in north american pigs ( 14 ) . persons having direct contact with swine are at greatest risk of infection with swine influenza viruses ( 47,13 ) , but such contact is not documented in all cases ( 7 ) . human - to - human transmission of swine influenza virus is rare , but evidence suggests that it has occurred ( 13,7,8,13 ) . human illness caused by infection with swine influenza viruses is often indistinguishable clinically from infections caused by other influenza viruses ( 1,7,12,13 ) ; complications , including pneumonia and death , have been documented ( 3,4,7 ) . asymptomatic infections in humans caused by swine influenza viruses may occur ( 5,8,13 ) and therefore , the true frequency of swine - to - human influenza virus transmission is unknown ( 3,57 ) . we were limited in assessing other possible swine influenza a ( h1n1 ) virus infections and in confirming swine influenza in the pigs . pigs delivered to the slaughterhouse the day of the patient s exposure originated from multiple farms , but specimens were unavailable for testing due to delays during animal traceback . our findings suggest that microneutralization assay may be more sensitive than a standard hi assay in detecting human antibodies to swine influenza a viruses . primers and probes for detection of human influenza a viral rna by rrt - pcr identified a nonhuman influenza a virus , triggering further analyses that specifically identified the virus . we could not confirm whether the patient s influenza vaccination and high levels of vaccine - derived subtype h1n1 neutralizing antibody influenced his relatively mild clinical course of illness . the patient did not have a 4-fold increase in neutralizing antibody titer to the swine influenza a ( h1n1 ) virus isolated from his respiratory specimens , which would be more suggestive of acute infection . further studies are needed to understand the human immune response to infection caused by swine influenza a viruses and to interpret serologic test results . human infections with novel influenza a subtype viruses are now nationally notifiable in the united states . clinicians should inquire about exposure to animals ( including pigs ) and visits to petting zoos and county fairs when evaluating patients with unexplained influenza - like illnesses ( 15 ) . ideally , joint animal health and public health investigations should be conducted promptly to identify and control the source of swine influenza . investigations should attempt to specifically identify the virus in animals and persons ; define the scope and clinical spectrum of human illnesses , including appropriately timed collection of serum specimens from ill persons and exposed individuals ; determine risk factors for human infection ; and assess the potential for human - to - human transmission of swine influenza a viruses .

zoonotic infections with swine influenza a viruses are reported sporadically . triple reassortant swine influenza viruses have been isolated from pigs in the united states since 1998 . we report a human case of upper respiratory illness associated with swine influenza a ( h1n1 ) triple reassortant virus infection that occurred during 2005 following exposure to freshly killed pigs .

acute respiratory distress syndrome ( ards ) is a common and devastating complication which is mainly caused by direct lung injuries , such as inhalation of toxic substances , and indirect systemic diseases including sepsis and severe trauma . ards leads to high morbidity , high mortality , and exorbitant health care costs . despite adoption of lung - protective ventilation and overall intensive care unit ( icu ) , sepsis is believed to be one of the most important causes of occurrence and development of ards . numerous researches have declared that inflammation may directly and indirectly affect lung endothelia and the pulmonary hemodynamics by activating inflammatory cells , such as neutrophils , monocytes , and lymphocytes , and proinflammatory mediators , including cytokines , proteases , and cyclooxygenases [ 3 , 4 ] . although there is a variety of anti - inflammatory interventions and treatments against the inflammatory process in lungs , the mortality of ards remains higher than acceptable . endotoxin , also known as lipopolysaccharide ( lps ) , is one of the main ingredients formatting cell wall of gram - negative bacteria which has also been recognized as the most important pathogen for incurable inflammation . evidences have revealed that lps could activate neutrophils , airway epithelial cells , and alveolar macrophages followed by excessive generation of chemokines , such as no , cox-2 , tnf- , il-1 , and il-6 . several studies have addressed that activation of nuclear factor- ( nf- ) b and mitogen - activated protein kinase ( mapk ) pathways was responsible for the excessive generation of proinflammatory cytokines [ 6 , 7 ] . what was more , plenty of evidence indicated that tnf- and il-1 may degrade ikbs ( ikb in particular ) and translocate nf-b from the cytoplasm into the nucleus in minutes , which would further amplify the inflammatory process . besides , overgeneration of inflammatory mediators played a key role in damaging the alveolar - capillary barrier and permeability . therefore , it may be of great significance to find new ways to interfere with the inflammatory process in treatment of ards . resveratrol ( rsv ) has been known for hundreds of years as powdered root of polygonum cuspidatum and nowadays widely known to exist in grapes , nuts , and red wine . also , resveratrol is one of the most intensively researched natural compounds since it exhibited protecting effects on multiple diseases , such as cardiovascular disorders [ 10 , 11 ] , cancers [ 12 , 13 ] , and inflammation [ 14 , 15 ] . there are also plenty of evidences indicating that resveratrol exhibited its pharmacological properties by interfering with the expression and activity of silent information regulator type-1 ( sirt1 ) , which has been demonstrated to play a key role in transcriptional and posttranscriptional regulation of gene expression through the deacetylation of histone and nonhistone proteins . although resveratrol preserved multiple bioactivities , it has never been adopted as a clinical drug for its poor pharmacokinetic and bioavailability properties , while 3,5,4-tri - o - acetylresveratrol ( ac - rsv ) , a prodrug of resveratrol firstly reported in 2002 , has overcome some of the shortages and results in the accumulation of rsv in lung . more importantly , researches from our laboratory and other teams have revealed that ac - rsv attenuated seawater inhalation induced lung injury in rat and reduced -irradiation related death in mice [ 20 , 21 ] . in the present research , we have shown that ac - rsv exerted protective roles in lps exposure induced ards in mice by modulating sirt1 expression . furthermore , our results have demonstrated that the protective effects of ac - rsv on lung tissue might be through attenuating the pulmonary edema and lung inflammation by inhibiting the p38 mitogen - activated protein kinase ( mapk ) pathway . adult male kunming mice ( 1822 g ) were obtained from the animal center of the fourth military medical university ( fmmu , xi'an , china ) . the mice were captured in air - filtered , temperature - controlled units with equaled light - dark cycles and had free access to food and water . all experimental processes and treatments to animals have been approved by the institutional animal care and use committee of the fmmu according to the declaration of the national institutes of health guide for care and use of laboratory animals ( publication number 85 - 23 , revised 1985 ) . lps ( escherichia coli lipopolysaccharide , 055:b5 ) and ex527 were obtained from the sigma chemical company ( st . resveratrol ( 3,5,4-trihydroxystilbene ) was purchased from xi'an grass plant technology corporation ( xi'an , china ) with purity above 98% . 3,5,4-tri - o - acetylresveratrol ( ac - rsv , structure showed in figure 1 ) was synthesized by the pharmacy department of medicinal chemistry of fmmu with hplc purity > 99% . enzyme - linked immunosorbent assay ( elisa ) kits for tnf- , il-6 , and il-1 have been purchased from the r&d corporation ( r&d systems inc . ) . myeloperoxidase ( mpo ) activity analyzing kit has been purchased from jiancheng bioengineering institute ( nanjing , china ) . antibodies , including anti - p - nf-b , anti - nf-b , anti - p - p38 mapk , anti - p38 mapk , anti - sirt1 , anti - p - erk , anti - erk , and anti--actin , have been purchased from the santa cruz biotechnology inc . ( santa cruz , ca , usa ) . in order to explore the protecting effects of ac - rsv on the mortality , mice received an intraperitoneal injection of 20 mg / kg lps ( dissolved in 0.9% saline and filtered through a 0.22 mm membrane ) with or without pretreatment of different doses of ac - rsv ( 25 , 50 , or 100 mg / kg body weight ) for 7 days . the mortality of mice from different groups ( n = 20 ) was recorded every 12 h for 84 h after lps exposure . in the research on the protecting effects of ac - rsv on lps - induced ards , mice were randomly divided into 4 groups : control ; lps ( 5 mg / kg ) only ; lps ( 5 mg / kg ) + ac - rsv ( 50 mg / kg ) ; ac - rsv ( 50 mg / kg ) . mice in the lps + ac - rsv group were pretreated with 50 mg / kg ac - rsv for 7 days and lps was injected 90 min after the last administration of ac - rsv since our previous results have indicated that concentration of rsv in blood reached the peak 90 min after oral administration of ac - rsv . in addition , the concentration of lps ( 20 mg / kg and 5 mg / kg ) was selected according to previous researches . lung tissues of the same lobe from different groups were fixed with 4% paraformaldehyde for 24 h and then embedded in paraffin . lung samples were cut into 5 m thick sections after being deparaffinized and dehydrated . finally , all slices were examined and captured under microscope ( leica , germany ) . lung samples were obtained 12 h after injection of lps and weighed as soon as possible ; after that , all samples were dried to constant weight in an oven at 70c for 72 h and weighed again . at last , the wet - to - dry weight ratios were calculated by dividing the wet weight of each sample by the dry one . at the end of experiments , lungs were removed intactly from mice and lavaged 5 times with 1 ml ice - cold pbs ( phosphate buffered saline ) . then , the balf was centrifuged at 1000 rpm for 5 min at 4c . the supernatant was collected and protein concentration was determined by bca method , the results were calculated according to a standard curve , and data were expressed as g protein per ml ( g / ml ) . on the other hand , the cell mass was resuspended in red blood cell - lysis buffer and total cell amount was determined by using a hemocytometer . mpo activity in lung tissues was evaluated to represent the accumulation of neutrophils in lungs challenged by lps or not . briefly , lung samples were homogenized in cold pbs ( lung tissue to pbs 1 : 10 ) , and homogenate supernatant was collected by centrifuging at 1000 rpm for 5 min at 4c . mpo activity was evaluated according to the manufacturer 's instructions and values were measured with a spectrophotometer at 460 nm . the alveolar macrophage cell line , nr8383 , was purchased from the atcc ( usa ) and maintained in ham 's f12 medium containing 10% fetal calf serum at 37c in a humidified atmosphere containing 5% co2 and 95% air . in order to evaluate the protecting effects of rsv on lps stimulated nr8383 cells , cells were divided into four groups : control ; lps ( 1 g / ml ) only ; lps ( 1 g / ml ) + resveratrol ( 40 g / ml ) ; resveratrol ( 40 g / ml ) . cells from different groups were collected for further investigation after being treated for 12 h. the concentration of lps in this experiment was selected according to previous researches . in the research of exploring the relationship between sirt1 expression and the protecting effects of rsv on lps stimulated nr8383 cells , cells were divided into four groups and treated with normal ham 's f12 medium ( control ) , lps ( 1 g / ml ) , lps ( 1 g / ml ) + ex527 ( 1 m ) + resveratrol ( 40 g / ml ) , and lps ( 1 g / ml ) + resveratrol ( 40 g / ml ) for 12 h. cells were then collected for further examination . by the way , ex527 was first dissolved in dimethyl sulfoxide ( dmso ) to 1 mm and then diluted to the working concentration ( 1 m ) with ham 's f12 medium . although it is usually concerning for the distortion of results , elisa was chosen to analyze the content of tnf- , il-6 , and il-1 in lungs and cells in order to provide supplementary data for the current research . briefly , lung tissues from each group were homogenized in cold pbs ( lung tissue to pbs 1 : 5 ) by using a tissue - tearor and cells treated as described above were homogenized by repeated frozen and dissolved method . homogenates from tissue and cells were centrifuged at 12000 rpm for 5 min at 4c . contents of tnf- , il-6 , and il-1 in supernatants from tissue and cell samples were measured according to the manufacturer 's instructions . at the end of the experiment , cell and tissue samples were collected and total proteins were extracted according to the manufacturer 's instructions ( beyotime institute of biotechnology , jiangsu , china ) . protein concentrations were determined by bca method with an assay kit ( beyotime ) . after boiling , equal amounts of proteins from each group were separated on sds - page gel and transferred to polyvinylidene fluoride membranes by wet transfer method . the membrane was blocked with 5% nonfat dry milk in tris - buffered saline with tween 20 followed by incubation with monoclonal antibodies overnight at 4c against p - nf-b ( 1 : 200 ) , p - p38 mapk ( 1 : 300 ) , sirt1 ( 1 : 200 ) , p - erk ( 1 : 200 ) , and -actin ( 1 : 5000 ) . following the incubation of antibodies were repeated washing and incubation of secondary antibody for 2 h at room temperature . finally , immune complexes were visualized via the enhanced chemiluminescence ( ecl ) system ( amersham pharmacia biotech , arlington heights , il , usa ) . statistical analysis was performed with spss 17.0 for windows . differences between groups were performed by one - way analysis of variance ( anova ) followed by dunnett 's test after the distribution of data was confirmed to be a normal distribution . the kaplan - meier method and the log - rank test were used to analyze survival data . in order to assess the protecting effects of ac - rsv on endotoxemia , we firstly evaluated the effects of ac - rsv on lps - induced mortality in mice . as shown in figure 2 , the accumulative mortalities of mice in middle - dose ( 50 mg / kg ) and high - dose ( 100 mg / kg ) groups were 55% and 65% , respectively , which were significantly lower than that of lps group ( 80% mice dead ) ( p < 0.05 ) . those data indicated that ac - rsv protected mice from lps - induced death and 50 mg / kg of ac - rsv exhibited the best protecting effects which was adopted to carry out further researches . histopathological results showed that lung samples from control ( figure 3(a ) ) and ac - rsv ( figure 3(d ) ) groups exhibited a normal structure with clear pulmonary alveoli , while lps exposure led to infiltration of inflammatory cells , damage of alveoli , thickened alveolar wall , and formation of hyaline membranes ( figure 3(b ) ) . however , lps - induced changes in lung structure have been dramatically attenuated by ac - rsv pretreatment ( figure 3(c ) ) . the wet - to - dry weight ratios of lung samples from different groups have been calculated in order to assess the pulmonary edema in mice challenged by lps with or without pretreatment of ac - rsv . as shown in figure 3(e ) , the wet - to - dry ratio was significantly increased in lungs from lps group compared with that of control ( n = 6 , p < 0.05 ) . however , administration of ac - rsv dramatically reduced water content in lungs 12 h after lps injection . the result also indicated that ac - rsv treatment alone did not affect water content in lungs . cell and protein content in balf from all four groups were evaluated in order to assess the vascular leakage in lungs stimulated with lps , as well as determine the protecting effects of ac - rsv in this respect . as shown in figures 3(f ) and 3(g ) , lps injection increased the cell and protein content in balf compared with those of control ( p < 0.05 ) , while ac - rsv restricted cell and protein leakage from pulmonary vessels into the alveoli when stimulated with lps ( p < 0.05 ) . also , there was no significant difference between control and ac - rsv group . in order to evaluate the effects of ac - rsv on lps - induced inflammation in lungs , mpo activity ( figure 4 ) and contents of tnf- , il-6 , and il-1 ( figures 5(a)5(c ) ) the results revealed that lps injection significantly upregulated the activity of mpo and increased the contents of tnf- , il-6 , and il-1 in lungs compared with those of control ( p < 0.05 ) . however , pretreatment of ac - rsv dramatically decreased mpo activity and inhibited the concentration of tnf- , il-6 , and il-1 in lungs ( p < 0.05 ) . also , the results indicated that ac - rsv treatment alone did not affect the mpo activity and content of cytokines in lungs . in order to further illustrate the mechanisms of how ac - rsv exhibited the protecting effects against the endotoxemia induced by lps the results ( figure 6 ) showed that administration of lps significantly decreased the sirt1 expression and increased the expression of p - p38 mapk , p - erk , and p - nf-b ( p < 0.05 ) . however , pretreatment of ac - rsv dramatically decreased p - p38 mapk , p - erk , and p - nf-b expression and enhanced the expression of sirt1 in lungs stimulated with lps ( p < 0.05 ) . based on the findings that ac - rsv attenuated lps - induced lung injury , we further evaluated the effects of its intermediate metabolite , resveratrol , on nr8383 cells the results ( figures 5(d)5(f ) ) showed that lps stimulation increased the generation of tnf- , il-6 , and il-1 in nr8383 cells compared with that of control ( p < 0.05 ) , while resveratrol significantly reversed this trend and decreased the formation of tnf- , il-6 , and il-1 ( p < 0.05 ) in nr8383 cells exposed to lps . what was more , there were no significant differences in the content of chemokine between control and resveratrol groups . we further evaluated whether resveratrol could modulate the expression of mapk / sirt1 pathway in nr8383 cells like ac - rsv did in lps challenged lungs . the expressions of sirt1 , p - p38 mapk , p - erk , and p - nf-b have been evaluated in cells stimulated by lps together with treatment of rsv or not , and the results ( figure 7 ) showed that lps stimulation inhibited the expression of sirt1 and upregulated p - p38 mapk , p - erk , and p - nf-b expression in nr8383 cells ( p < 0.05 ) , while resveratrol treatment reversed this trend just like ac - rsv did in lps stimulated lungs . nr8383 cells were exposed to normal culture , lps , lps + ex527 + rsv , and lps + rsv for 12 h , and then the expression of sirt1 and generation of cytokines were evaluated by western blot and elisa method , respectively . the results ( figure 8(a ) ) showed that sirt1 expression was dramatically suppressed by lps compared with that of control , while rsv treatment effectively reversed the decrease of sirt1 expression . cotreatment of ex527 and rsv counterbalanced the elevating effects of rsv on the expression of sirt1 . on the other hand , the results ( figures 8(b)8(d ) ) from the evaluation on the generation of cytokines revealed that lps stimulation increased the generation of tnf- , il-1 , and il-6 , and rsv treatment reversed this trend . similar to the effects on sirt1 expression , coincubation of ex527 and rsv did not inhibit the generation of those cytokines . ards is a serious manifestation of systemic inflammation induced by varied factors which may further result in multiple organs dysfunction syndromes ( mods ) with a very high mortality . previous researches have revealed that overactivated inflammatory cells and secretion of inflammatory mediators were the primary pathogenesis for ards [ 23 , 24 ] . there were also researches on the mechanism and treatment of ards in vitro and in vivo and substantial progression has been made in understanding of this disease . however , available treatments were still limited in clinic , and it was thus meaningful to explore new pharmacological treatment for ards . as a natural polyphenol , resveratrol ( rsv ) has been confirmed to possess a number of pharmacological functions [ 1015 ] . rsv has never been adopted as a clinical drug for its short half - life , low bioavailability , and poor targeting . however , those shortages have been overcome to some extent by ac - rsv and administration of ac - rsv increased the concentration of rsv in lungs 20-fold compared with that of administration of rsv . therefore , we investigated the protecting effects of ac - rsv on lps - induced ards and the results revealed that ac - rsv could reduce the mortality rate of mice challenged by lps and attenuate lung injury by restricting leakage of fluid from blood vessels into pulmonary alveoli , inhibiting the concentration of cytokines and alleviating the abnormal expression of mapk / sirt1 expression induced by lps . as a hallmark of ards , pulmonary edema was caused by dysfunction of alveolar - capillary barriers and increased filtration of protein - rich fluid into the alveolar spaces . in the present research , cell content and protein concentration in balf and lung edema have been measured in order to quantify the magnitude of pulmonary edema and small vascular permeability . the results revealed that administration of lps increased water content in lung tissues and enhanced infiltration of cells and leakage of protein - rich fluid from blood vessels into alveoli , while pretreatment of ac - rsv significantly reduced the lung w / d ratio , decreased the protein concentration in balf , and inhibited the exudation of cells from blood vessels into alveoli induced by lps . it has been confirmed that inflammation was another character for ards ; in addition , neutrophils and macrophages were the main executive cells in lps - induced pulmonary inflammation . myeloperoxidase ( mpo ) was a unique constituent of neutrophil cytoplasmic granules which was indispensable for the killing of phagocytosed pathogens . therefore , mpo activity was markedly relevant to neutrophils accumulation and served as the marker of inflammation . in the current research , it has been found that mpo activity was dramatically increased in lungs 12 h after lps exposure , while pretreatment of ac - rsv significantly decreased the mpo activity . in addition , histopathological study has also revealed that ac - rsv pretreatment markedly reduced the neutrophil infiltration from blood vessels into pulmonary alveoli . lps stimulation as well as activated neutrophils could further induce the generation and secretion of numerous inflammatory mediators and chemotactic cytokines , among which tnf- , il-1 , and il-6 were characterized mediators participating in the occurrence and development of ards . those chemokines were also related to the influx , accumulation , and activation of highly destructive cells involved in inflammation ; more importantly , the positive feedback between tnf- and il-1 and nf-b would facilitate more nf-b expression , phosphorylation , and translocation into nucleus which could further elicit the inflammatory cascade leading to severe damage to lung tissues [ 30 , 31 ] . in the present research , it has been found that tnf- , il-1 , and il-6 increased remarkably in lungs and nr8383 cells after lps exposure , while treatment of ac - rsv and rsv significantly decreased the generation and secretion of tnf- , il-1 , and il-6 in lung tissues and nr8383 cells , respectively . in addition to regulating the expression of inflammatory cytokines , ac - rev should have affected the activation of enzymes and mediators involved in pathogenesis of inflammation . mitogen - activated protein kinase ( mapk ) was a conserved cellular energy status sensor which could be activated by lps [ 32 , 33 ] . what was more , activation of mapks may further lead to secretion of cytokines such as tnf- and il-1 and expression of inflammatory mediators such as inducible nitric oxide synthase ( inos ) and cyclooxygenase-2 ( cox-2 ) by enhancing transcription of nf-b . together with mapks , sirt1 has also been regarded as a key sensor and metabolic modulator for inflammation , especially ards . besides , the corporation between sirt1 and mapk in mediating the molecule response to stimuli without or within pretreatment was closely related to nf-b [ 35 , 36 ] . based on those clues , we explored the expression of sirt1 and mapk pathway in lps - induced ards ; the results showed that lps exposure inhibited sirt1 expression and enhanced the phosphorylation of p38 mapk and erk followed by increased p - nf-b expression in vitro and in vivo . however , pretreatment of ac - rsv relieved lps - induced inhibition on sirt1 expression and restrained the effects of lps on p38 mapk , erk , and nf-b in lungs and nr8383 cells . to further confirm the protective role of ac - rsv on lps - induced ards via interfering with the expression of sirt1 , in vitro experiment has been carried out by cotreatment of ex527 and rsv for lps stimulated nr8383 cells . ex527 is a new type of highly selective inhibitor for sirt1 which could effectively inhibit the acetylation enzyme activity of sirt1 ( ic50 38 as expected , lps stimulation decreased sirt1 expression and increased the generation of tnf- , il-1 , and il-6 , while rsv treatment could reverse this trend to some extent . however , cotreatment of ex527 and rsv counterbalanced the attenuating effects of rsv on decreased expression of sirt1 and accelerated generation of tnf- , il-1 , and il-6 resulting from lps stimulation . taken together , those results indicated that the activation of sirt1 by rsv attenuated the inflammation in nr8383 cells stimulated by lps , while the inhibition of sirt1 by ex527 could neutralize the protecting effects of rsv on lps - induced inflammation and lung injury . in summary , results from the present research indicated that ac - rsv pretreatment therapeutically attenuated lps - induced ards by suppressing lung edema , inhibiting leakage of protein - rich fluid from blood vessels into pulmonary alveoli , and depressing inflammatory process in mice . the mechanisms underlying the therapeutic effects of ac - rsv were probably via the mapk / sirt1 pathways . although further researches were needed , the present research indicated that ac - rsv may be a potential therapeutic agent for ards .

the aim of the present research was to investigate the protecting effects of 3,5,4-tri - o - acetylresveratrol ( ac - rsv ) on lps - induced acute respiratory distress syndrome ( ards ) . lung injuries have been evaluated by histological examination , wet - to - dry weight ratios , and cell count and protein content in bronchoalveolar lavage fluid . inflammation was assessed by mpo activities and cytokine secretion in lungs and cells . the results showed that ac - rsv significantly reduced the mortality of mice stimulated with lps . pretreatment of ac - rsv attenuated lps - induced histological changes , alleviated pulmonary edema , reduced blood vascular leakage , and inhibited the mpo activities in lungs . what was more , ac - rsv and rsv treatment reduced the secretion of tnf- , il-6 , and il-1 in lungs and nr8383 cells , respectively . further exploration revealed that ac - rsv and rsv treatment relieved lps - induced inhibition on sirt1 expression and restrained the activation effects of lps on mapks and nf-b activation both in vitro and in vivo . more importantly , in vivo results have also demonstrated that the protecting effects of rsv on lps - induced inflammation would be neutralized when sirt1 was in - hibited by ex527 . taken together , these results indicated that ac - rsv protected lung tissue against lps - induced ards by attenuating inflammation via p38 mapk / sirt1 pathway .

the national cervical cancer coalition ( nccc ) estimates that worldwide , 473,000 new cases of cervical cancer are diagnosed and 253,500 patients die of this disease annually . this makes cervical cancer the twelfth most common cancer and the fifth cause of cancer - related deaths in the world . human papillomavirus ( hpv ) infection is the most important risk factor for the development of cervical cancer . treatment of cervical cancer consists of surgery at the early stages and chemotherapy and radiation therapy for advanced disease . if diagnosed early , the 5-year survival rate of the patient is more than 90% . therefore , early detection is currently the most effective means to improve survival of patients . the papanicolaou ( pap smear screening ) test followed by colposcopy is the most frequently used means to detect precancerous and cancerous changes in the cervix . however , the current most practical pap smear screening plus cytology test has a low sensitivity , especially in discriminating high - grade squamous intraepithelial lesions ( hsils ) from other abnormalities and normal tissues . in routine clinical practice , thus , development of a more sensitive and specific method to detect cervical cancer early and accurately is urgently needed . for several past decades , we have witnessed the rapid development and use of optical methods to investigate changes in cell behaviors ( such as migration and intracellular protein movement ) and for disease diagnosis ; optical techniques enable the detection of changes in biochemical and morphological features that are concurrent with precancerous conditions . the advantage of optical techniques is that in living cells visible light does no harm or is relatively benign . optical techniques such as confocal reflectance microscopy , optical coherence tomography , light - scattering spectroscopy , and elastic scattering spectroscopy are able to distinguish and identify intrinsic optical properties of tissues . during the transition from normal to cancerous cells , biochemical , molecular , and morphologic alterations may lead to changes in the cells ' optical properties . indeed , recent studies have shown that elastic light - scattering spectroscopy may permit the earliest detection of carcinogenesis . light - scattering spectroscopy ( lss ) is an optical technique in which quantitative information on cell organelle morphology is extracted via measurement of the spectra and angular distribution of backscattered light . therefore , lss could detect precancerous and early cancerous changes in tissues . in this study , we investigated the novel technique confocal light absorption and scattering spectroscopic ( class ) microscopy to detect the light scattering differences in liquid - based cervical cell samples . class combines lss with confocal microscopy for early cancer detection [ 7 , 8 ] . class provides not only size information but also the biochemical and physical properties of the cells . it images tissue at greater depth than conventional confocal microscopy and provides enhanced image contrast . in addition , by utilizing backscattered light to detect subcellular structures , class can detect changes in living cells without introducing exogenous labels . in the present study , we demonstrate that class microscopy could distinguish between populations of normal , low - grade squamous intraepithelial lesion ( lsil ) , and hsil cells ex vivo , and therefore could be developed for clinical use in the accurate and precise diagnosis of pap smear samples . forty women aged twenty and older who had a history of abnormal cervical cytology were enrolled in this study at the department of pathology , cancer hospital of fudan university , between 2009 and 2010 . of these 40 patients , 20 had cytologically confirmed hsil and 20 were non - hsil . all cytological samples were free of blood clot and stored at 4c for experimental procedures within 24 h. to perform class analysis , the cytological specimens were first put into thinprep preservation solution containing deionized water , 0.1% polysorbate 20 ( tween-20 ) detergent , and 54% ethanol . the pathologist then mapped a region of interest , designating the region that contained the worst cell lesions . cells were suspended at 10 cells / ml to avoid overlapping nuclei in the analysis . cells in thinprep or in saline ( as the control ) were kept in an open volume container or dipped onto glass slides . measurements were made with a fiber optic probe placed on the surface of the cell suspension . to perform the class analysis , we used a prototype class microscope as described in a previous report . after the optical signal was collected by the class receiver , the data was analyzed using spectral data acquisition bwspec software ( b&w , newark , de , usa ) . before every experiment , the data was first normalized to the saline control samples and then comparisons were made between the hsil and non - hsil samples . sensitivity and specificity were calculated by percentage of cases correctly identified by class as compared to those confirmed by cytology , and then presented as mean standard deviation ( sd ) . from patients with abnormal pap smear cytology results , we collected 20 cervical cytology samples of hsil , and 20 of non - hsil . in the hsil samples , the pathology report showed 6 cases with lesions of cervical intraepithelial neoplasia ( cin ) grade ii , and 14 cases with lesions of cin grade iii . in the non - hsil samples , 8 cases showed normal cytology , and 12 cases were positive for lsil ( table 1 ) . one representative image each of normal , lsil , and hsil cells is shown in figure 1 ( left to right , resp . ) . we chose cell samples from 4 , 7 , and 9 confirmed diagnosed normal , lsil , and hsil cytology specimens , respectively , for class analysis . using class microscopy , these cells show a unclear edge , but bright nuclei . in the class method of cell imaging , enlarged nuclei become brighter ( figure 2 ) . thus , nuclei that have changed in size and condensation are detected by lss and are early indications of malignant transformation . data collected from class analyses of hsil and non - hsil cytology samples were then analyzed by bwspec spectral data acquisition software and plotted ( figure 3 ) . spectrum wavelengths ( nm ) are represented on the abscissa , and the relative intensity is plotted on the ordinate scale . the data summarizes the spectra of the scattering light reflections from cell nuclei , which were normalized to the control and liquid - based normal cells . as illustrated in figure 3 , the red curve shows the trend of back scattering lights of non - hsil cells , and the blue curve represents the trend of back scattering lights of hsil cells . between 600 and 800 nm wavelengths , there were two peaks in the spectra curves , but the intensity differed in each . the hsil cells ( blue ) trended toward a higher relative intensity , and the non - hsil ( red ) trended relatively lower ( table 2 ) . however , although hsil cells trended higher in relative intensity , 4 of the 20 hsil cases did not have higher intensity compared to the non - hsil group . conversely , 2 of the 20 cases of non - hsil were higher in intensity compared to the hsil group . comparing the trends of the hsil and non - hsil curves , the relative intensity of the hsil cells was obviously higher than that of the non - hsil at most wavelengths . there was a significant difference between intensities of the hsil and non - hsil at 600 and 800 nm . however , there was no significant difference between them at wavelengths > 800 nm . sensitivity and specificity for hsil and non - hsil were 80% and 90% , respectively , compared to confirmed diagnosis by cytology . cervical cancer , unlike most other cancers , is relatively easy to prevent and diagnose early , which contributes to reduced incidence and a better long - term survival rate . however , advanced stages of cervical cancers continue to be a serious health problem worldwide , especially in developing countries . indeed , the incidence and mortality rates of cervical cancer are increasing in young women in china , although mortality for this cancer has significantly decreased in the united states and western europe . this may be due to the widespread use of the pap smear screening test and safer sex practices . in an effort to improve the sensitivity and specificity of the pap cytology test our data demonstrates that class microscopy was able to distinguish among normal , lsil , and hsil cells at specific wavelengths . class microscopy is thus a useful and reliable technique for the detection of early cervical epithelial abnormality and could be used to help pathologists diagnose early cervical cancer . clinically it is crucial to distinguish precancerous lesions from normal and lsil , because precancerous lesions could progress to invasive carcinoma if left untreated . many methods have been developed and used preclinically and clinically for the detection of early cervical cancer [ 10 , 11 ] . the goals of investigations of new techniques are to reduce the cost of screening , enable the practical surveillance of populations , or to improve the sensitivity to hsils , which are considered cervical cancer precursors . elastic light scattering spectroscopy can provide a valuable , noninvasive means to quantitatively probe tissue morphology and even detect biochemical changes in cells . however , despite growing evidence of the clinical utility of a variety of light scattering - based optical techniques , the biological bases of differences in scattering signals between normal and neoplastic tissues are not well understood . the rationale for using optical techniques is to detect biochemical and morphological features that are concurrent with precancerous conditions . therefore , this kind of noninvasive method has many advantages , including the ability to measure changes in nuclear size , which is one of the recognized morphological changes occurring during the transformation of normal cells to cancerous . some studies have demonstrated that the intensity of light scattering increases with progression of atypia . the most dramatic differences in scattering occur between non - hsil and hsil cells because changes in nuclear size and dna content are most pronounced between these two categories of cells . studies have suggested that changes in scattering properties at high cin grades are dominated by the effects of increased nuclear size and increased dna content . in fact , a complex spatial pattern is formed that is dependent on cell size , shape , refraction index , density , and morphology . in this study , we used the optical method class to distinguish hsil of precancerous cervix cells from normal and lsil samples in liquid - based specimens ex vivo . we found that class was able to differentiate these in confirmed cytology samples , by the non - hsil and hsil - relative intensity curves generated by the bwspec software . we showed that there was a significant difference in relative intensity between non - hsil and hsil cells in the 600800 nm wavelength ranges . we could discriminate hsil from non - hsil cervical epithelial cells by differences in the curves , which represented the median light - scattering intensities of confirmed samples . sixteen of the 20 hsil cases demonstrated higher trends in relative intensity , and 18 of the 20 non - hsil conformed to a lower trend in relative intensity . therefore , in this study we established a novel model for diagnosis of liquid - based cervical cytological samples through the use of class microscopy . as reported previously , this method is noninvasive with high sensitivity . in addition , liquid - based specimens are easy to collect and preserve for analysis . in addition , four cases of hsil did not differ from the lsil optimized curve . in the future , we will collect a larger number of samples to confirm our current data before translating this technique to the clinic .

confocal light absorption and scattering spectroscopic ( class ) microscopy can detect changes in biochemicals and the morphology of cells . it is therefore used to detect high - grade cervical squamous intraepithelial lesion ( hsil ) cells in the diagnosis of premalignant cervical lesions . forty cervical samples from women with abnormal pap smear test results were collected , and twenty cases were diagnosed as hsil ; the rest were normal or low - grade cervical squamous intraepithelial lesion ( lsil ) . the enlarged and condensed nuclei of hsil cells as viewed under class microscopy were much brighter and bigger than those of non - hsil cells . cytological elastic scattered light data was then collected at wavelengths between 400 and 1000 nm . between 600 nm to 800 nm , the relative elastic scattered light intensity of hsil cells was higher than that of the non - hsil . relative intensity peaks occurred at 700 nm and 800 nm . class sensitivity and specificity results for hsil and non - hsil compared to cytology diagnoses were 80% and 90% , respectively . this study demonstrated that class microscopy could effectively detect cervical precancerous lesions . further study will verify this conclusion before the method is used in clinic for early detection of cervical cancer .

a human retinal pigment epithelial ( arpe-19 ) cell line was obtained from atcc ( manassas , va , usa ) . cells were maintained in dulbecco 's modified eagle 's medium / ham 's f-12 nutrient medium ( invitrogen - gibco , carlsbad , ca , usa ) supplemented with 10% fetal bovine serum , penicillin , streptomycin , and amphotericin b. the arpe-19 cells were used for four to six passages and plated in six - well plates at 1.5 10 cells per well . they were incubated for 24 hours in a humidified 5% co2 atmosphere at 37 after reaching approximately 70% confluence . the cells were serum - starved for four hours before h2o2 exposure and treated with h2o2 ( 100 to 400 m ) for 16 hours to induce oxidative stress before they were harvested for cell death analysis . the supernatants were collected at baseline ( 0 hours ) and at 16 hours , centrifuged to remove cell debris , and stored at -70 until enzyme - linked immunosorbent assay was performed according to the manufacturer 's instructions . in this analysis , a monoclonal antibody specific for vegf - a was pre - coated onto a microplate . standards and samples were pipetted into the wells , and any vegf - a present was bound by the immobilized antibody . after washing any unbound substances , an enzyme - linked polyclonal antibody specific for vegf - a was added to the wells . following a wash to remove any unbound antibody- enzyme reagent , a substrate solution was added to the wells , and color developed in proportion to the amount of vegf - a bound in the initial step . the color development was then stopped , and the intensity of the color was measured . to determine the optical density of each well the concentrations of vegf - a standard numbers 1 to 8 were 0 , 15.6 , 31.2 , 62.5 , 125 , 250 , 500 , and 1,000 pg / ml of recombinant vegf - a in a buffered protein base with preservatives , respectively . the level of vegf - a protein was measured in cell - free supernatant using a human vegf - a quantikin elisa kit ( catalog no . dve00 ; r&d systems , minneapolis , mn , usa ) . the cells were washed with pbs and incubated in serum- free dulbecco 's modified eagle 's medium in the presence of h2o2 ( 100 , 200 , 300 , and 400 m ) for 16 hours . bevacizumab ( 0.33 , 0.67 , 1.33 , and 2.67 mg / ml , respectively ) was added 2 hours before h2o2 treatment . an annexin v - fluorescein isothiocyanate ( fitc ) apoptosis kit ( bd biosciences , franklin lakes , nj , usa ) was used to detect phosphatidylserine externalization as an index of apoptosis . the cells were washed and incubated for 15 minutes at room temperature in the presence of annexin v labeled with fitc and propidium iodide ( pi ) . in total , 10,000 cells were excited at 488 nm , and emission was measured at 530 and 584 nm to assess fitc and pi fluorescence , respectively . the number of gated cells was plotted on a dot plot with reference to both annexin v and pi staining . the cells were diluted to 1 10 cell / ml and were incubated for 24 hours in six - well plates ( falcon , bd biosciences ) . after washing the culture media twice with pbs , h2o2 and bevacizumab were challenged at different concentrations ( 0 , 100 , 200 , 300 , 400 m and 0.33 , 0.67 , 1.33 , 2.67 mg / ml ) and incubated for 16 hours . isolated rna was quantified , and 1 g of total rna and 100 pmol of oligo dt were added to the reverse transcription ( rt ) premix ( bioneer , seoul , korea ) to prepare 20 l of cdna . preformed cdna , bcl-2 primer , and glycerol-3-phosphate dehydrogenase ( gapdh ) primer were mixed with polymerase chain reaction ( pcr ) premix ( bioneer ) , and pcr was performed . quantification of bcl-2 mrna content was performed using computer - assisted video densitometry ( eagle eye ii - system ; stratagene , la jolla , ca , usa ) . data are expressed as percentage of control or mean standard deviation of results in three or more independent experiments . chicago , il , usa ) . the kruskal - wallis test was used to compare the difference between control and experimental groups . a human retinal pigment epithelial ( arpe-19 ) cell line was obtained from atcc ( manassas , va , usa ) . cells were maintained in dulbecco 's modified eagle 's medium / ham 's f-12 nutrient medium ( invitrogen - gibco , carlsbad , ca , usa ) supplemented with 10% fetal bovine serum , penicillin , streptomycin , and amphotericin b. the arpe-19 cells were used for four to six passages and plated in six - well plates at 1.5 10 cells per well . they were incubated for 24 hours in a humidified 5% co2 atmosphere at 37 after reaching approximately 70% confluence . the cells were serum - starved for four hours before h2o2 exposure and treated with h2o2 ( 100 to 400 m ) for 16 hours to induce oxidative stress before they were harvested for cell death analysis . the supernatants were collected at baseline ( 0 hours ) and at 16 hours , centrifuged to remove cell debris , and stored at -70 until enzyme - linked immunosorbent assay was performed according to the manufacturer 's instructions . in this analysis , a monoclonal antibody specific for vegf - a was pre - coated onto a microplate . standards and samples were pipetted into the wells , and any vegf - a present was bound by the immobilized antibody . after washing any unbound substances , an enzyme - linked polyclonal antibody specific for vegf - a was added to the wells . following a wash to remove any unbound antibody- enzyme reagent , a substrate solution was added to the wells , and color developed in proportion to the amount of vegf - a bound in the initial step . the color development was then stopped , and the intensity of the color was measured . to determine the optical density of each well the concentrations of vegf - a standard numbers 1 to 8 were 0 , 15.6 , 31.2 , 62.5 , 125 , 250 , 500 , and 1,000 pg / ml of recombinant vegf - a in a buffered protein base with preservatives , respectively . the level of vegf - a protein was measured in cell - free supernatant using a human vegf - a quantikin elisa kit ( catalog no . dve00 ; r&d systems , minneapolis , mn , usa ) . the cells were washed with pbs and incubated in serum- free dulbecco 's modified eagle 's medium in the presence of h2o2 ( 100 , 200 , 300 , and 400 m ) for 16 hours . bevacizumab ( 0.33 , 0.67 , 1.33 , and 2.67 mg / ml , respectively ) was added 2 hours before h2o2 treatment . an annexin v - fluorescein isothiocyanate ( fitc ) apoptosis kit ( bd biosciences , franklin lakes , nj , usa ) was used to detect phosphatidylserine externalization as an index of apoptosis . the cells were washed and incubated for 15 minutes at room temperature in the presence of annexin v labeled with fitc and propidium iodide ( pi ) . in total , 10,000 cells were excited at 488 nm , and emission was measured at 530 and 584 nm to assess fitc and pi fluorescence , respectively . the number of gated cells was plotted on a dot plot with reference to both annexin v and pi staining . the cells were diluted to 1 10 cell / ml and were incubated for 24 hours in six - well plates ( falcon , bd biosciences ) . after washing the culture media twice with pbs h2o2 and bevacizumab were challenged at different concentrations ( 0 , 100 , 200 , 300 , 400 m and 0.33 , 0.67 , 1.33 , 2.67 mg / ml ) and incubated for 16 hours . isolated rna was quantified , and 1 g of total rna and 100 pmol of oligo dt were added to the reverse transcription ( rt ) premix ( bioneer , seoul , korea ) to prepare 20 l of cdna . preformed cdna , bcl-2 primer , and glycerol-3-phosphate dehydrogenase ( gapdh ) primer were mixed with polymerase chain reaction ( pcr ) premix ( bioneer ) , and pcr was performed . quantification of bcl-2 mrna content was performed using computer - assisted video densitometry ( eagle eye ii - system ; stratagene , la jolla , ca , usa ) . data are expressed as percentage of control or mean standard deviation of results in three or more independent experiments . chicago , il , usa ) . the kruskal - wallis test was used to compare the difference between control and experimental groups . the cells were treated with bevacizumab for 16 hours ( 0 , 0.33 , 0.67 , 1.33 , and 2.67 mg / ml ) . cell apoptosis was not significantly different at control , clinical ( 0.33 and 0.67 mg / ml ) , or high ( 1.33 and 2.67 mg / ml ) doses of bevacizumab without h2o2 ( fig . the cells were cultured with h2o2 for 16 hours ( 0 , 100 , 200 , 300 , and 400 m ) . cell apoptosis was 3.26% for rpe at 100 m h2o2 compared to 3.82% for the controls , but the difference was not significant . cell apoptosis decreased at 200 m h2o2 ( 2.65% ) but increased at 300 and 400 m h2o2 ( 10.42% , 17.99% ) . the means at these concentrations were significantly different than the control ( p < 0.05 ) ( fig . vegf - a expression was increased after addition of 50 , 100 , and 200 m h2o2 to rpe cells . however , after being treated with 300 and 400 m h2o2 , vegf - a expression decreased . means at these concentrations were significantly different from the control ( p < 0.05 ) ( fig . the cells were cultured with h2o2 for 16 hours ( 0 , 100 , 200 , and 300 m ) . expression of bcl-2 mrna decreased as oxidative stress increased ( p < 0.05 ) ( fig . 2c ) . arpe-19 cells were treated with bevacizumab ( 0 , 0.33 , 0.67 , 1.33 , and 2.67 mg / ml ) under low oxidative stress conditions ( 100 m of h2o2 ) . cell apoptosis was not significantly different at any concentration of bevacizumab at 100 m h2o2 . expression of bcl-2 mrna decreased under low oxidative stress conditions , and the decrease was proportional to the increase in bevacizumab . there was a statistically significant difference at all concentrations compared to the result of the control . each bar shows the mean standard deviation of results in three or more independent experiments ( p < 0.05 ) ( fig . arpe-19 cells were treated with bevacizumab ( 0 , 0.33 , 0.67 , 1.33 , and 2.67 mg / ml ) under moderate oxidative stress conditions ( 200 m of h2o2 ) . as mentioned previously , cell apoptosis decreased at 200 m h2o2 ( 2.65% ) compared with the control ( 3.82% ) . cell apoptosis increased to 3.59% , 3.50% , and 4.13% at bevacizumab concentrations of 0.33 , 0.67 , and 1.33 mg however , cell apoptosis increased significantly to 10.16% at a high dose of bevacizumab ( 2.67 mg / ml ) under the same oxidative stress conditions . there was a statistically significant difference at all concentrations ( p < 0.05 ) ( fig . arpe-19 cells were treated with bevacizumab ( 0 , 0.33 , 0.67 , 1.33 , and 2.67 mg / ml ) under high oxidative stress conditions ( 300 m h2o2 ) . as mentioned previously , cell apoptosis increased at 300 m h2o2 ( 10.42% ) compared with the control ( 3.82% ) . cell apoptosis increased to 12.65% and 10.97% at clinical doses of bevacizumab ( 0.33 , 0.67 mg / ml ) under high oxidative stress conditions , but there was no statistically significant difference compared to the control . cell apoptosis did significantly increase to 19.83% and 24.85% at high doses of bevacizumab ( 1.33 , 2.67 mg / ml ) under the same oxidative stress . expression of bcl-2 mrna decreased under high oxidative stress conditions ( 300 m h2o2 ) , but there was no trend of increase or decrease as the dose of bevacizumab increased ( fig . we suggest that the reverse transcription - pcr results might be a result of severe cellular apoptosis under high oxidative stress conditions . the cells were treated with bevacizumab for 16 hours ( 0 , 0.33 , 0.67 , 1.33 , and 2.67 mg / ml ) . cell apoptosis was not significantly different at control , clinical ( 0.33 and 0.67 mg / ml ) , or high ( 1.33 and 2.67 mg / ml ) doses of bevacizumab without h2o2 ( fig . the cells were cultured with h2o2 for 16 hours ( 0 , 100 , 200 , 300 , and 400 m ) . cell apoptosis was 3.26% for rpe at 100 m h2o2 compared to 3.82% for the controls , but the difference was not significant . cell apoptosis decreased at 200 m h2o2 ( 2.65% ) but increased at 300 and 400 m h2o2 ( 10.42% , 17.99% ) . the means at these concentrations were significantly different than the control ( p < 0.05 ) ( fig . vegf - a expression was increased after addition of 50 , 100 , and 200 m h2o2 to rpe cells . however , after being treated with 300 and 400 m h2o2 , vegf - a expression decreased . means at these concentrations were significantly different from the control ( p < 0.05 ) ( fig . the cells were cultured with h2o2 for 16 hours ( 0 , 100 , 200 , and 300 m ) . expression of bcl-2 mrna decreased as oxidative stress increased ( p < 0.05 ) ( fig . 2c ) . arpe-19 cells were treated with bevacizumab ( 0 , 0.33 , 0.67 , 1.33 , and 2.67 mg / ml ) under low oxidative stress conditions ( 100 m of h2o2 ) . cell apoptosis was not significantly different at any concentration of bevacizumab at 100 m h2o2 . expression of bcl-2 mrna decreased under low oxidative stress conditions , and the decrease was proportional to the increase in bevacizumab . there was a statistically significant difference at all concentrations compared to the result of the control . each bar shows the mean standard deviation of results in three or more independent experiments ( p < 0.05 ) ( fig . arpe-19 cells were treated with bevacizumab ( 0 , 0.33 , 0.67 , 1.33 , and 2.67 mg / ml ) under moderate oxidative stress conditions ( 200 m of h2o2 ) . as mentioned previously , cell apoptosis decreased at 200 m h2o2 ( 2.65% ) compared with the control ( 3.82% ) . cell apoptosis increased to 3.59% , 3.50% , and 4.13% at bevacizumab concentrations of 0.33 , 0.67 , and 1.33 mg / ml , respectively , but the differences were not statistically significant . however , cell apoptosis increased significantly to 10.16% at a high dose of bevacizumab ( 2.67 mg / ml ) under the same oxidative stress conditions . there was a statistically significant difference at all concentrations ( p < 0.05 ) ( fig . arpe-19 cells were treated with bevacizumab ( 0 , 0.33 , 0.67 , 1.33 , and 2.67 mg / ml ) under high oxidative stress conditions ( 300 m h2o2 ) . as mentioned previously , cell apoptosis increased at 300 m h2o2 ( 10.42% ) compared with the control ( 3.82% ) . cell apoptosis increased to 12.65% and 10.97% at clinical doses of bevacizumab ( 0.33 , 0.67 mg / ml ) under high oxidative stress conditions , but there was no statistically significant difference compared to the control . cell apoptosis did significantly increase to 19.83% and 24.85% at high doses of bevacizumab ( 1.33 , 2.67 mg / ml ) under the same oxidative stress . expression of bcl-2 mrna decreased under high oxidative stress conditions ( 300 m h2o2 ) , but there was no trend of increase or decrease as the dose of bevacizumab increased ( fig . we suggest that the reverse transcription - pcr results might be a result of severe cellular apoptosis under high oxidative stress conditions . in the present study , we tested the possible toxicity of bevacizumab on rpe cells under various oxidative stress conditions , and we examined the expression of the anti - apoptotic protein bcl-2 and the apoptosis of rpe cells . we used h2o2 to induce oxidative stress in order to replicate in vivo conditions that are implicated in the pathogenesis of amd and the annexin v - fitc apoptosis kit to detect phosphatidylserine externalization as an index of apoptosis . intravitreal injections of anti - vegf agents such as bevacizumab are widely used for the treatment of various retinal disorders associated with new vessels such as amd or proliferative diabetic retinopathy in order to reduce angiogenesis . bevacizumab is a full - length , recombinant humanized immunoglobulin g antibody that binds to and inhibits all biologically active forms of vegf - a isoforms . despite ongoing clinical trials with intravitreal bevacizumab in many neovascular ocular disorders , hypothetically , repeated injections with bevacizumab could interfere with the neuroprotective and survival actions of vegf under oxidative stress . in amd patients , several studies have reported cases of rpe tears after intravitreal injections of bevacizumab for neovascular amd . atrophy of the choriocapillaries or loss of endothelial cell fenestrations impairs nutritional support , which can lead to functional and morphologic damage to the rpe and photoreceptors , with particular adverse effects if the macular lesion is affected . the development of geographic atrophy contributed to visual acuity loss after repeated anti - vegf injections . in addition , in a recent study in which a retinal neovascularization model was developed , it was suggested that caution is warranted in the treatment of patients with acute or severe retinal neovascularization using anti - vegf drugs such as bevacizumab in order to prevent capillary nonperfusion and macular ischemia . according to many recent reports , bevacizumab at concentrations at or above the dose normally used in clinical practice is not toxic to rpe cells . several preclinical experimental toxicity studies have reported the histopathological effects of bevacizumab on retinal cells , retinal neovascular membranes , and capillaries in paraffin - embedded sections or organotypic culture , as well as in ultrastructural evaluations . based on in vitro assays , bevacizumab has little toxic effect on ganglion cells , neuroretinal cells , rpe cells , choroidal endothelial cells , or corneal epithelial cells . however , there are limited previous preclinical studies associated with rpe under oxidative stress conditions . in our study , rpe cell apoptosis did not show a significant difference at 100 m h2o2 but decreased at 200 m h2o2 and increased with higher oxidative stress conditions ( 300 and 400 m h2o2 ) ( fig . it seems that rpe cells can tolerate up to 200 m h2o2 due to cell - protective mechanisms , but higher oxidative stress conditions caused in vitro mitochondrial dna damage and promoted apoptosis . reported that exposure of rpe cells to concentrations of h2o2 that cause in vitro mitochondrial dna damage also promotes apoptosis , and this correlates well with our results under high oxidative stress conditions . in different cell types , including vascular smooth muscle cells , vegf expression is triggered by oxidative stress . other studies of vegf expression are consistent with our results when h2o2 in concentrations up to 200 m was added to rpe cells . however , vegf expression decreased at 300 and 400 m h2o2 , suggesting that vegf production decreases according to the decrease in live rpe cells . oxidative stress triggers multiple signaling pathways , including some that are cytoprotective and others that contribute to cell damage and eventually cell death . among these are the pathways of the bcl-2 family proteins , which have subcellular locations on the mitochondrial outer membrane , nuclear envelope , and endoplasmic reticulum . expression of bcl-2 mrna decreased as oxidative stress increased , which was consistent with another study . the retina is a highly oxygen - consumptive tissue that always functions under high oxygen tension ; rpe cells are therefore exposed to oxidative stress . chronic oxidative stress can lead to impairment and death of rpe cells , implicating it as a risk factor of amd . in our study , low to high oxidative stress conditions of rpe cells were implicated in the phase of amd . have reported that addition of a high concentration ( 2.5 mg / ml ) of bevacizumab to the culture medium did not affect the survival of either control rpe cells or cells under a low level of oxidative stress ( 150 m h2o2 ) . however , under higher oxidative stress levels ( 200 or 300 m h2o2 ) , pretreatment with bevacizumab ( 2.5 mg / ml ) induced a significantly higher level of rpe cell death . addition of clinically applicable ( 0.33 and 0.67 mg / ml ) and high concentrations ( 1.33 and 2.67 mg / ml ) of bevacizumab did not affect the survival of rpe cells under low oxidative stress ( 100 m h2o2 ) ; nevertheless , bcl-2 expression decreased in a dose - dependent manner ( fig . although bcl-2 is an important survival factor , it is sensitive to oxidative stress , and a decrease in bcl-2 precedes cell apoptosis . under moderate oxidative stress conditions ( 200 m h2o2 ) , bcl-2 expression decreased with increasing concentration of bevacizumab ; however , cell apoptosis did not show significant change until 1.33 mg / ml of bevacizumab . it then increased to 10.16% at high doses of bevacizumab ( 2.67 mg / ml ) under the same oxidative stress ( fig . 4 ) . under high oxidative stress conditions ( 300 m h2o2 ) , rpe cell apoptosis increased at high bevacizumab concentrations ( 1.33 and 2.67 mg / ml ) , but this might be due to multiple survival factors in rpe cells or experimental error due to low bcl-2 expression . in our experiment , rpe apoptosis did not increase with increased concentration of bevacizumab in the absence of h2o2 ; however , as the oxidative stress level increased , apoptosis of rpe cells increased . the higher was the oxidative stress , the lower was the concentration of bevacizumab that induced cellular apoptosis . although cellular apoptosis at clinically applicable levels of bevacizumab ( 0.33 and 0.67 mg / ml ) did not show significant increase , care should be taken since bcl-2 expression showed a significant decrease even at lower levels of oxidative stress . to our knowledge , we have shown for the first time that bevacizumab influences bcl-2 expression and apoptosis of rpe cells under oxidative stress . first , our conclusions are drawn from in vitro studies , which are not the best approximation of in vivo conditions . moreover , some previous studies have shown that non - confluent arpe-19 cells behave quite differently in terms of oxidative stress compared to confluent monolayers . further studies are needed to examine these findings in porcine retina - rpe - choroid cultures or on mouse retinae . second , it is difficult to determine the degree of h2o2 simulating oxidative stress conditions of human eyes . further studies are needed to examine the intracellular pathway or cumulative effects at repeated doses . fourth , we could not determine the additional mechanism of action of bevacizumab in our study . bevacizumab might bind rpe cell surface receptors in addition to known vegf receptors . in our experiment , we demonstrated the possibility that addition of the vegf inhibitor bevacizumab might block the protective effect of vegf in our in vitro model under high oxidative stress conditions mimicking those in amd eyes . under increased oxidative stress conditions , high doses of bevacizumab furthermore , bevacizumab might affect the expression of anti - apoptotic genes such as bcl-2 under all oxidative conditions . since the oxidative stress levels of each patient are unknown , repeated injections of intravitreal bevacizumab , as in eyes with amd , might influence rpe cell survival . therefore close monitoring is needed in patients who receive repeated injections of intravitreal bevacizumab .

purposeto evaluate the effects of bevacizumab on expression of b - cell leukemia / lymphoma ( bcl)-2 and apoptosis in retinal pigment epithelial ( rpe ) cells under oxidative stress conditions.methodsrpe cells were treated with h2o2 ( 0 , 100 , 200 , 300 , and 400 m ) and bevacizumab at or above the doses normally used in clinical practice ( 0 , 0.33 , 0.67 , 1.33 , and 2.67 mg / ml ) . cell apoptosis was measured using flow cytometry with annexin v - fluorescein isothiocyanate . the expression of bcl-2 mrna was determined using reverse transcription polymerase chain reaction.resultsunder low oxidative stress conditions ( h2o2 100 m ) , cell apoptosis was not significantly different at any concentration of bevacizumab , but bcl-2 mrna expression decreased with increasing concentration of bevacizumab ( 0.33 , 0.67 , 1.33 , and 2.67 mg / ml ) . under moderate oxidative stress conditions ( h2o2 200 m ) , bcl-2 mrna expression decreased with increasing concentration of bevacizumab ( 0.33 , 0.67 , 1.33 , and 2.67 mg / ml ) , but cell apoptosis increased only at 2.67 mg / ml of bevacizumab . under high oxidative stress ( 300 m ) conditions , cell apoptosis increased at high concentrations of bevacizumab ( 1.33 and 2.67 mg / ml ) , but it did not correlate with bcl-2 expression.conclusionswithdrawal of vascular endothelial growth factor can lead to rpe cell apoptosis and influences the expression of anti - apoptotic genes such as bcl-2 under oxidative stress conditions . since oxidative stress levels of each patient are unknown , repeated injections of intravitreal bevacizumab , as in eyes with age - related macular degeneration , might influence rpe cell survival .

during the 20th century , laparoscopic procedures replaced most traditional abdominal operations and achieved high - quality standards . it seemed that the optimal surgical method had been achieved ; however , a new concept , which might possibly become even safer and simpler is now being developed , the concept of natural orifice surgery ( nos ) . the existing natural openings of the body started to be used for introduction of surgical instruments for diagnostic purposes and surgical procedures , avoiding penetrating the abdominal wall . parallel to the american natural orifice surgery consortium for assessment and research ( noscar ) group , is the new european surgical academy ( nesa ) established in berlin on june 23 , 2006 . it is the first european - based nos working group with participation of scientists and surgeons from different disciplines and countries . after the published experimental achievements had been presented and discussed , the working group decided to concentrate mainly on the transvaginal / transdouglas access in women . a new surgical instrument , the transdouglas endoscopic device ( ted ) has been designed . this is a flexible multichannel instrument enabling single - entry surgical , urological , and gynecological operations . ted respects the anatomy of the pelvis . to get to the upper abdomen , an s - shaped device was designed , bending first to the front , and then backwards . for the lower abdomen , the wide diameter of the device ( 35 mm ) and its multichannel design enables simultaneous use of different instruments , therefore avoiding hybrid procedures . various surgical and gynecological procedures have been successfully simulated , and the manufacturing of the device is in progress . transvaginal / transdouglas surgery is expected to be a valid alternative to traditional endoscopic procedures in women . it seems that nos will create a spectrum of innovative and high - quality procedures performed by an interdisciplinary team and will improve patient safety . laparoscopic operations are expected to have the same outcome as laparotomies , but with fewer intraor postoperative complications . in comparison with patients undergoing laparotomy , patients having endoscopic surgery need less postoperative analgesics and have decreased morbidity and shorter hospital stay . is it , despite the high standards achieved , still possible to make abdominal surgery simpler and safer ? a new surgical concept , natural orifice surgery , seems to be the next step in the evolution of minimally invasive surgery . , gastroenterologists have used natural openings for various procedures , such as gastroscopy , duodenoscopy , rectoscopy , and colonoscopy . furthermore , for the last few years , surgeons have performed the transanal endoscopic microsurgery procedure routinely , and since the end of the 19th century , gynecologists have used the vaginal route for hysterectomies in benign cases and in malignancies . appendicectomies following vaginal hysterectomies have already been performed , and the pouch of douglas has also been used to remove a gallbladder at the end of an endoscopic cholecystectomy and as an entry point for some gynecological procedures , such as the removal of fibroids . recently , peritoneoscopy , appendicectomy , liver biopsy , splenectomy , and partial hysterectomy have been done experimentally transgastrically with endoscopes . claimed advantages of nos include less invasiveness , elimination of abdominal incision , reduction in postoperative abdominal wall pain , wound infection , hernia formation , and adhesions . parallel to the american natural orifice surgery consortium for assessment and research ( noscar ) group is the new european surgical academy ( nesa ) established in berlin on june 23 , 2006 , the first european - based nos working group . while the natural orifice transluminal endoscopic surgery ( notes ) working group includes transgastric peritoneal access , nesa is exploring the transdouglas route . the difference between the terms nos and notes is not accidental . nos includes notes because it refers to all surgical procedures performed through all body openings . members of the nos working group are scientists , surgeons , gynecologists , anesthesiologists , urologists , physiologists , and pharmacologists from germany , the netherlands , england , denmark , austria , italy , france , switzerland , israel , the united states , and canada as well as representatives from the industry ( table 1 ) . during meetings , the concept of natural orifice surgery and published experimental achievements have been presented and discussed , and the pharmacological and physiological challenges concerning the transgastric approach have been considered . three main problems concerning transgastric access have been identified : the risk of bacteriological contamination when instruments are introduced into the peritoneal cavity after passing through contaminated areas.the optimal way of the gastric wall repair has to be defined.the existing multichannel endoscopes have to be improved . the risk of bacteriological contamination when instruments are introduced into the peritoneal cavity after passing through contaminated areas . members of the natural orifice surgery ( nos ) working group the nos working group decided to focus on the use of the transdouglas approach in women because ( table 2 ) : in transdouglas operations , there is little risk for bacterial contamination compared with the transgastric pathway . unlike the mouth cavity , oesophagus , and stomach , the vagina can be cleaned and disinfected more easily , thus minimizing the risk of intraperitoneal infection.the opening and closure of the vaginal wall is safe and is done under vision.the vaginal wall repairs itself without leaving any visible scars and without causing long - term dysfunction.the introduction of the instruments will be done under vision and parallel to the major blood vessels , thus minimizing the risk of them being injured.because the traditional 15 mm hg co2 pressure is not needed for the introduction of the device , we expect that the working pressure will not exceed 8 mm hg or 9 mm hg . this will enable procedures to be performed with epidural anesthesia.the transdouglas approach improves operation ergonomics . the surgeon can sit comfortably during the procedure.there is no risk for postoperative herniation or eventration.the pouch of douglas enables the introduction of wide multichannel devices , so that no extra entry and no additional trocars are necessary . in transdouglas operations , there is little risk for bacterial contamination compared with the transgastric pathway . unlike the mouth cavity , oesophagus , and stomach , the vagina can be cleaned and disinfected more easily , thus minimizing the risk of intraperitoneal infection . the opening and closure of the vaginal wall is safe and is done under vision . the vaginal wall repairs itself without leaving any visible scars and without causing long - term dysfunction . the introduction of the instruments will be done under vision and parallel to the major blood vessels , thus minimizing the risk of them being injured . because the traditional 15 mm hg co2 pressure is not needed for the introduction of the device , we expect that the working pressure will not exceed 8 mm hg or 9 mm hg . the pouch of douglas enables the introduction of wide multichannel devices , so that no extra entry and no additional trocars are necessary . comparison of different minimally invasive accesses an unavoidable step in the development of the transdouglas approach is the establishment of a multidisciplinary team . surgeons , gynecologists , and urologists have to learn from one another and work as a team . the main aim of our group is the development of a device that will enable the performance of the complete surgical procedure by using a single entry , the transvaginal one , thus avoiding hybrid procedures . that means no need for any additional abdominal wall incision or puncture ( one entry , one device ! ) . such an instrument , the transdouglas endoscopic device ( ted ) is actually being developed . its diameter is 35 mm , and it consists of working channels in sizes from 3 mm to 5 mm in diameter , integrated with insufflation and a control system . to insert the ted , just a pincer and scissors are necessary to perform the posterior colpotomy . for this purpose , the instruments pincer and scissors together with the arms on which they are controllably and movably mounted are hidden in the head of the device . there are no sharp edges or any other obstacles allowed at the outer shell of the head that could lead to any injury ( figure 1 ) . after the ted has been inserted into the body , the arms with the pincer and scissors will be deployed ( figure 2 ) . besides the 2 instruments , there will be housed in the head a camera , a light , and the instrument for flushing and sucking . light and camera both will remain operative when the instruments are hidden in the head . thus , light and camera are already usable during the insertion of the ted into the body . this central channel corresponds to the working channel of endoscopy . the transdouglas endoscopic device ( ted ) in closed state . the transdouglas endoscopic device ( ted ) with 2 instruments driven out what makes the ted special , in comparison with other known endoscopy or video - endoscopy devices , are the fold - out arms in the small narrow head of this new surgery device . these fold - out arms allow movements in nearly every direction and at the same time a hand - like approach from opposing sides to a surgery exist , until now only an idea or concept . , gastroenterologists have used natural openings for various procedures , such as gastroscopy , duodenoscopy , rectoscopy , and colonoscopy . furthermore , for the last few years , surgeons have performed the transanal endoscopic microsurgery procedure routinely , and since the end of the 19th century , gynecologists have used the vaginal route for hysterectomies in benign cases and in malignancies . appendicectomies following vaginal hysterectomies have already been performed , and the pouch of douglas has also been used to remove a gallbladder at the end of an endoscopic cholecystectomy and as an entry point for some gynecological procedures , such as the removal of fibroids . recently , peritoneoscopy , appendicectomy , liver biopsy , splenectomy , and partial hysterectomy have been done experimentally transgastrically with endoscopes . claimed advantages of nos include less invasiveness , elimination of abdominal incision , reduction in postoperative abdominal wall pain , wound infection , hernia formation , and adhesions . parallel to the american natural orifice surgery consortium for assessment and research ( noscar ) group is the new european surgical academy ( nesa ) established in berlin on june 23 , 2006 , the first european - based nos working group . while the natural orifice transluminal endoscopic surgery ( notes ) working group includes transgastric peritoneal access , nesa is exploring the transdouglas route . the difference between the terms nos and notes is not accidental . nos includes notes because it refers to all surgical procedures performed through all body openings . members of the nos working group are scientists , surgeons , gynecologists , anesthesiologists , urologists , physiologists , and pharmacologists from germany , the netherlands , england , denmark , austria , italy , france , switzerland , israel , the united states , and canada as well as representatives from the industry ( table 1 ) . during meetings , the concept of natural orifice surgery and published experimental achievements have been presented and discussed , and the pharmacological and physiological challenges concerning the transgastric approach have been considered . three main problems concerning transgastric access have been identified : the risk of bacteriological contamination when instruments are introduced into the peritoneal cavity after passing through contaminated areas.the optimal way of the gastric wall repair has to be defined.the existing multichannel endoscopes have to be improved . the risk of bacteriological contamination when instruments are introduced into the peritoneal cavity after passing through contaminated areas . . members of the natural orifice surgery ( nos ) working group the nos working group decided to focus on the use of the transdouglas approach in women because ( table 2 ) : in transdouglas operations , there is little risk for bacterial contamination compared with the transgastric pathway . unlike the mouth cavity , oesophagus , and stomach , the vagina can be cleaned and disinfected more easily , thus minimizing the risk of intraperitoneal infection.the opening and closure of the vaginal wall is safe and is done under vision.the vaginal wall repairs itself without leaving any visible scars and without causing long - term dysfunction.the introduction of the instruments will be done under vision and parallel to the major blood vessels , thus minimizing the risk of them being injured.because the traditional 15 mm hg co2 pressure is not needed for the introduction of the device , we expect that the working pressure will not exceed 8 mm hg or 9 mm hg . this will enable procedures to be performed with epidural anesthesia.the transdouglas approach improves operation ergonomics . the surgeon can sit comfortably during the procedure.there is no risk for postoperative herniation or eventration.the pouch of douglas enables the introduction of wide multichannel devices , so that no extra entry and no additional trocars are necessary . in transdouglas operations , there is little risk for bacterial contamination compared with the transgastric pathway . unlike the mouth cavity , oesophagus , and stomach , the vagina can be cleaned and disinfected more easily , thus minimizing the risk of intraperitoneal infection . the opening and closure of the vaginal wall is safe and is done under vision . the vaginal wall repairs itself without leaving any visible scars and without causing long - term dysfunction . the introduction of the instruments will be done under vision and parallel to the major blood vessels , thus minimizing the risk of them being injured . because the traditional 15 mm hg co2 pressure is not needed for the introduction of the device , we expect that the working pressure will not exceed 8 mm hg or 9 mm hg . the pouch of douglas enables the introduction of wide multichannel devices , so that no extra entry and no additional trocars are necessary . an unavoidable step in the development of the transdouglas approach is the establishment of a multidisciplinary team . surgeons , gynecologists , and urologists have to learn from one another and work as a team . the main aim of our group is the development of a device that will enable the performance of the complete surgical procedure by using a single entry , the transvaginal one , thus avoiding hybrid procedures . that means no need for any additional abdominal wall incision or puncture ( one entry , one device ! ) . such an instrument , the transdouglas endoscopic device ( ted ) is actually being developed . its diameter is 35 mm , and it consists of working channels in sizes from 3 mm to 5 mm in diameter , integrated with insufflation and a control system . to insert the ted , just a pincer and scissors are necessary to perform the posterior colpotomy . for this purpose , the instruments pincer and scissors together with the arms on which they are controllably and movably mounted are hidden in the head of the device . there are no sharp edges or any other obstacles allowed at the outer shell of the head that could lead to any injury ( figure 1 ) . after the ted has been inserted into the body , the arms with the pincer and scissors will be deployed ( figure 2 ) . for a surgeon to perform surgical procedures , these instruments need to be moveable . besides pivoting , they will be partly rotatable and move forwards and backwards . besides the 2 instruments , there will be housed in the head a camera , a light , and the instrument for flushing and sucking . light and camera both will remain operative when the instruments are hidden in the head . thus , light and camera are already usable during the insertion of the ted into the body . the transdouglas endoscopic device ( ted ) in closed state . the transdouglas endoscopic device ( ted ) with 2 instruments driven out . what makes the ted special , in comparison with other known endoscopy or video - endoscopy devices , are the fold - out arms in the small narrow head of this new surgery device . these fold - out arms allow movements in nearly every direction and at the same time a hand - like approach from opposing sides to a surgery exist , until now only an idea or concept . transvaginal / transdouglas surgery seems to be a valid alternative to traditional endoscopic procedures in women . we strongly believe that ted will create a spectrum of innovative and high - quality operations performed by an interdisciplinary team and will improve patient safety .

background : during the 20th century , laparoscopic procedures replaced most traditional abdominal operations and achieved high - quality standards . it seemed that the optimal surgical method had been achieved ; however , a new concept , which might possibly become even safer and simpler is now being developed , the concept of natural orifice surgery ( nos ) . the existing natural openings of the body started to be used for introduction of surgical instruments for diagnostic purposes and surgical procedures , avoiding penetrating the abdominal wall . parallel to the american natural orifice surgery consortium for assessment and research ( noscar ) group , is the new european surgical academy ( nesa ) established in berlin on june 23 , 2006 . it is the first european - based nos working group with participation of scientists and surgeons from different disciplines and countries . after the published experimental achievements had been presented and discussed , the working group decided to concentrate mainly on the transvaginal / transdouglas access in women.database:a new surgical instrument , the transdouglas endoscopic device ( ted ) has been designed . this is a flexible multichannel instrument enabling single - entry surgical , urological , and gynecological operations . ted respects the anatomy of the pelvis . to get to the upper abdomen , an s - shaped device was designed , bending first to the front , and then backwards . for the lower abdomen , the u - shaped mode of the instrument was designed . the wide diameter of the device ( 35 mm ) and its multichannel design enables simultaneous use of different instruments , therefore avoiding hybrid procedures . various surgical and gynecological procedures have been successfully simulated , and the manufacturing of the device is in progress . preclinical studies will start soon.conclusions:transvaginal/transdouglas surgery is expected to be a valid alternative to traditional endoscopic procedures in women . it seems that nos will create a spectrum of innovative and high - quality procedures performed by an interdisciplinary team and will improve patient safety .

lymph node and bone are the two most common metastatic sites of pa while lung is the other common site of metastasis . however , cutaneous metastasis of pa is rare , occurring in < 1% cases . diagnosis of cutaneous metastasis of pa by fine - needle aspiration cytology ( fnac ) has been rarely reported . we herein report the rare cytodiagnosis of cutaneous metastases of pa in an elderly man . a 55-year - old gentleman presented with papulonodular lesions and urinary symptoms since last 8 months . the papulonodular lesions were present in the suprapubic region , inner aspects of both the thighs and the scrotum . the lesions were subcutaneous in location , ranging from 0.3 to 1.5 cm in diameter , erythematous , firm , smooth surfaced and nontender [ figure 1 ] . fnac was done from multiple nodules and smears were air - dried and fixed in 90% ethanol for giemsa and papanicolaou stains , respectively . microscopic examination showed cellular smears comprised of loose clusters and scattered malignant cells having hyperchromatic nuclei , mild nuclear pleomorphism , occasional conspicuous nucleoli and scanty cytoplasm . formation of glands by few overlapping clusters of malignant cells was also seen [ figure 2a ] . a cytological diagnosis suggestive of adenocarcinoma was given , and a biopsy for confirmation was advised . since the patient had urinary symptoms , skin metastasis of prostate cancer was suspected , and further investigations were done accordingly . digital rectal examination of the patient revealed an enlarged prostate which was hard , nodular , and fixed to the rectal wall . his serum prostate specific antigen ( psa ) level was 45.4 ng / ml ( normal value < 4.0 ng / ml ) . subsequently , skin and prostatic needle core biopsies were taken and submitted for histopathological examination . hematoxylin and eosin - stained sections of skin biopsy showed infiltration by malignant cells and few atypical glands in the mid dermis . fused ill - formed glands with ragged margins were found infiltrating in the deeper dermis and subcutis [ figure 2b - c ] . prostatic needle core biopsy showed adenocarcinoma of gleason 's score 7 ( 3 + 4 ) . later on , radical prostatectomy was done , which was also diagnosed as adenocarcinoma of the prostate , gleason 's score 7 ( 3 + 4 ) . immunohistochemistry of skin and prostatic biopsies showed strong cytoplasmic expression of pancytokeratin [ figure 2d ] and negative expression of high molecular weight cytokeratin 34e12 . a diagnosis of pa gleason score 7 ( 3 + 4 ) with metastasis to the skin was made . clinical photograph of the patient showing multiple papulonodular lesions ( a ) fine - needle aspiration cytology of skin lesions showing malignant cells with formation of glands ( giemsa , 400 ) . ( b ) skin biopsy showing subepidermal grenz zone and malignant epithelial cells infiltrating mid - dermis and deeper dermis ( h and e , 40 ) . ( c ) sheets of malignant epithelial cells infiltrating dermal collagen ( h and e , 200 ) . ( d ) immunostaining with pancytokeratin highlighting epidermis and scattered malignant cells of mid - dermis ( ihc , 400 ) prostatic adenocarcinoma is a common visceral malignancy of elderly men . in more than 81,000 reported cases of primary visceral malignancies , the incidence of cutaneous metastasis is 2.9% whereas cutaneous metastasis of pa is very rare with an incidence of 0.36% . therefore , because of its rarity , most of the practicing cytopathologists are not aware of this entity . this case report highlights utility of fnac as an initial investigation and role of cytopathologist in the diagnosis of cutaneous metastases of pa . in this case , fnac provided earliest clue for diagnosis , which was later confirmed by serum psa level along with biopsy and immunohistochemistry . presence of skin metastases may be the initial sign of underlying primary genitourinary malignancy as noticed , in this case . on clinical examination , metastatic lesions of primary genitourinary malignancies typically appear on the skin as discrete , round to oval plaques or nodules of variable sizes . metastatic lesions of the skin are seen with primary pa as well as with carcinomas of the urinary bladder and kidney . in these cases , metastatic dissemination to the skin occurs by one of the following four mechanisms : direct invasion from underlying cancer;implantation from operative scar;hematogenous spread and / or;spread through the lymphatics . direct invasion from underlying cancer ; implantation from operative scar ; hematogenous spread and/or ; spread through the lymphatics . cutaneous metastasis to abdominal skin occurs due to spread through dermal lymphatic or blood vessels . although rare , cytology of cutaneous metastasis of pa has been reported.[23 cytological examination of pa shows scattered malignant cells with formation of glands at places and cells showing hyperchromatic nuclei and prominent nucleoli . histopathogical examination of cutaneous metastases often involves dermis with sparing of the superficial dermis from the epidermis forming grenz zone . histopathological examination should distinguish cutaneous metastasis of primary visceral malignancies from primary skin adnexal tumors . in comparison to primary skin adnexal tumors , metastatic carcinomas show infiltrative growth pattern of malignant epithelial cells in collagen bundles and are arranged singly , in narrow cords or in large clusters . demonstration of lymphovascular invasion favors a diagnosis of cutaneous metastasis over primary skin adnexal tumor . since cutaneous metastasis of pa is uncommon , a high index of suspicion is required for diagnostic work - up . fnac may prove to be easy , cost - effective and reliable investigation for an early diagnosis . cutaneous metastasis , of underlying primary malignancy , represents an advanced stage of the disease and associated with poor prognosis . thus , cutaneous metastasis of pa must be considered in an elderly male patient with raised serum psa levels and multiple papuonodular lesions over the lower abdomen , pelvic and pubic areas .

prostatic adenocarcinoma ( pa ) is a common visceral malignancy of elderly men . cutaneous metastasis of pa is rare . the incidence is < 1% . a 55-year - old man presented with urinary symptoms and multiple cutaneous nodules around suprapubic region , inner aspect of both thighs and scrotum . fine - needle aspiration cytology ( fnac ) of cutaneous nodules was suggestive of metastatic adenocarcinoma . skin and prostatic biopsies confirmed the cytological diagnosis . serum level of prostate specific antigen was raised . total prostatectomy revealed adenocarcinoma of gleason 's score 7 ( 3 + 4 ) . though rare , cutaneous metastases of pa must be known to cytopathologists . meticulously performed fnac in such cases may help in early diagnosis .

interstitial pulmonary fibrosis induced by intratracheal instillation of bleomycin ( bl ) involves an excess production of reactive oxygen species , unavailability of adequate levels of nad and atp to repair the injured pulmonary epithelium , and an overexuberant lung collagen reactivity followed by deposition of highly cross - linked mature collagen fibrils resistant to enzymatic degradation . in the present study , we have demonstrated that dietary supplementation with taurine and niacin offered almost complete protection against the lung fibrosis in a multidose bl hamster model . the mechanisms for the protective effect of taurine and niacin are multifaceted . these include the ability of taurine to scavenge hocl and stabilize the biomembrane ; niacin 's ability to replenish the bl - induced depletion of nad and atp ; and the combined effect of taurine and niacin to suppress all aspects of bl - induced increases in the lung collagen reactivity , a hallmark of interstitial pulmonary fibrosis . it was concluded from the data presented at this conference that the combined treatment with taurine and niacin , which offers a multipronged approach , will have great therapeutic potential in the intervention of the development of chemically induced interstitial lung fibrosis in animals and humans.imagesfigure 4 . afigure 4 . b

pilon fractures are challenging to manage because of the complexity of the injury pattern and the risk of significant complications . the soft tissue injury and handling of the soft tissue envelope are crucial in pilon fracture outcomes . the purpose of this study was to evaluate the early rate of complications using the strategy of soft tissue control for operative treatment of complex pilon fractures . patients followed the standard staged protocol , anterolateral approach to the distal tibia , the no - touch technique and incisional negative pressure wound therapy for pilon fractures . patients were examined clinically at 2 - 3 weeks and then 8 weeks for complications associated with the surgical technique . all fractures were ao / ota ( orthopaedic trauma association ) type c fractures ( 61% c3 , 22% c2 and 16% c1 ) . the strategy of soft tissue control for treatment of pilon fractures resulted in relatively low incidence of early wound complications in patients with complex pilon fractures . pilon fractures are among the most serious fractures that involve the ankle joint and continue to present a challenge to the orthopedic surgeon . they are often the result of violent trauma and are associated with significant soft tissue damage , osseous comminution and articular surface disruption . management principles were originally outlined by redi and allgwer1 and included reconstruction of the fibula as well as the articular surface of the tibia . the subsequent literature initially revealed high soft tissue complication rates when using these techniques and principles in high - energy injuries.23456 current opinion is that the high rates of infection and wound healing problems may be secondary to operating through swollen , compromised soft tissues . this has led to a staged approach to management for high - energy pilon fractures with initial restoration of fibular length and tibial external fixation followed by delayed open reduction internal fixation of the tibia when soft tissue swelling subsides.7891011 in addition to using a staged protocol for high - energy injuries , meticulous handling of soft tissues is mandatory to minimize soft tissue complications.611 careful attention must be directed to soft tissue retraction and wound management . we recommend the staged protocol , anterolateral approach to the distal tibia , the no - touch technique where there is no retractor placed in the soft tissues yet adequate visualization is present to permit reduction of the fracture and applying incisional negative pressure wound therapy ( npwt ) , which we called soft tissue control , for treatment the complex pilon fractures . the purpose of this study was to evaluate the early rate of complications with using the strategy of soft tissue control for operative treatment of complex pilon fractures . 36 close pilon fractures were treated with the two stage protocol at our institution between february 2008 and february 2011 . the interval between injury and presentation ranged from 1 h to 24 h ( average , 6.7 h ) . patients eligible for inclusion were between ages 18 and 65 years with closed pilon fractures ota ( orthopaedic trauma association ) 43c . patients were excluded if they had a chronic disorder of their soft tissues , since this study evaluated soft tissue outcomes . the mechanisms of injury were high - energy falls ( n=21 ) , motor vehicle accidents ( n=11 ) , crushing injury ( n=4 ) . the fractures were classified according to the ao / asif ( association for the study of internal fixation ) system : 6 were classified as type c1 , 8 type c2 and 22 type c3 . after the initial evaluation in the emergency department , the limb was pulled out to length and placed in a well padded splint . as soon as the patient was medically cleared , he or she was brought to the operating room . immediate open reduction and internal fixation of fibular fracture with wound closure was performed . the fibula posterolateral approach at least 6 cm away from the planned anterolateral pilon incision , if radiographs revealed a pilon fracture with an associated fracture of the posterior malleolus that was displaced , a posterolateral incision was made midway between the posterior border of the fibula and lateral aspect of the achilles tendon . the posterolateral approach gave excellent visualization of the posterior aspect of the tibia and posterior malleolar fragment as well as the fibula without creating extensive surgical flaps.12 through the same incision , open reduction and internal fixation ( orif ) of the posterolateral fragment of the tibia and fibula fractures were achieved . once completed , the tourniquet was deflated and a medially placed external fixator that spans the ankle joint was applied . the external fixation pins were placed well away from the subsequent surgical incision both proximally and distally . after reduction , the purpose of so doing was to maintain tibial length and make the distal tibial fractures relative stable ; thus , allowing soft tissue healing . once edema had resolved , generally within 7 - 14 days postinjury , surgery could be performed . the limb was cleaned and draped in the sterile field from the level of the tourniquet to the toes . the frame was initially removed and flashed for sterility so that could be reused intraoperatively as a distractor . the anterolateral approach was useful for many complex and complete articular and partial articular fracture patterns . the incision was centered at the ankle parallel to the fourth metatarsal distally and between the tibia and fibula proximally as described by herscovici et al.13 full - thickness skin flaps were maintained while dissecting through the subcutaneous tissues . the superficial peroneal nerve would likely cross proximal to the ankle in the surgical field and was identified , mobilized and protected throughout the exposure . the fascia over the anterior compartment of the distal tibia and the extensor compartment was incised sharply and the anterior compartment tendons were all retracted medially . during the procedure , the full - thickness skin flap was gently lifted with a small skin retractor and k - wire was placed distally on the anterior medial portion of the ankle joint proximal to the articular fracture fragments . when placed , the k - wires were bent at a right angle to protect any assistants during this procedure . the k - wires used for retraction permitted excellent visualization of the metaphyseal - diaphyseal region and exposure to the medial aspect of the distal tibia . reduction should proceed to reconstruct the joint surface . during this time , with the retracting k - wires in place , there was no excess traction on the skin and soft tissues . the retraction k - wires were then removed once the anterolateral plate was applied [ figure 1c ] . if needed , a second , medially based incision was used for reduction of separate medial malleolar fractures and for percutaneous medial plate or screw application [ figure 2 ] . wound closure then beginning with deep vicryl sutures ( ethicon , somerville , new jersey ) . the subcutaneous tissue was closed with interrupted 2.0 vicryl sutures and the skin was closed with interrupted 2.0 nylon vertical mattress sutures . the vacuum assisted closure system ( kinetic concepts inc . , san antonio , tx ) was used to achieve npwt over the surgical incision including anterolateral and medial incision if existed . npwt were applied in the operating room and then changed on postoperative day 2 and every 1 - 2 days thereafter until wound drainage was minimal . the limb was placed in a splint for the first 1 - 2 weeks to maximize soft tissue recovery and then ankle mobilization was started . anteroposterior and lateral x - rays of anke joint showing ( a ) c2 pilon fracture ( b ) internal fixation of the fibula and application of a medial external fixator ( c ) anterolateral plate in situ through the anterolateral incision of pilon fracture ( a ) anteroposterior and lateral x - rays of ankle joint showing of c3 pilon fracture ( b ) anteroposterior and lateral x - rays of ankle joint showing internal fixation of the fibula and application of a medial external fixator ( c ) preoperative clinical photograph showing the anterolateral incision and a second , medially incision used for percutaneous medial plate or screw application in the second stage ( d ) fluoroscopic view showing reduction of pilon fracture through the anterolateral approach with applying external fixator and percutaneous medial plate application after reduction ( e ) anteroposterior and lateral x - ray of the pilon fracture showing internal fixation through the anterolateral and a second , medial incision 36 patients with pilon fractures were operated on by a single surgeon ( hx ) using this technique . the average time from external fixator placement to definitive orif was 10 days ( range 7 - 22 days ) . all patients who underwent an anterolateral approach were treated using the no touch technique during definitive orif . patients in the study had npwt applied to their wound for a mean of 60 h ( range 20 - 219 h ) . all patients received preoperative intravenous antibiotics and postoperative intravenous antibiotics for 24 - 48 h. only one patient developed superficial infection and resolved with antibiotics and local wound care . historically , surgical treatment of pilon fractures has been fraught with wound complications and many authors have reported poor results particularly when primary orif was used . they noted skin slough in 27% of their cases and a 37% infection rate in their high - energy fractures . similarly , in a study by mcferran and smith4 evaluating complications encountered in the treatment of plafond fractures , the local complication rate was 54% . of these patients , 13 had wound breakdown , 3 had inadequate soft tissue coverage , 9 had a deep soft tissue infection / osteomyelitis and 6 had superficial wound infections . eighteen soft tissue coverage procedures were required in addition to multiple debridements and closure attempts . this growing body of literature led to the concept that the soft tissue injury can be more severe than the fracture classification implies , leading to an underestimation of the wound complication risk . subsequently , a staged approach to management for high - energy plafond fractures was developed . during the first stage and by means of immediate external fixation , the fracture was provisionally stabilized , allowing soft tissue edema to resolve before formal orif . definitive fixation was delayed for 7 - 22 days until swelling had diminished , fracture blisters had resolved and the skin displayed wrinkling . this delay was identical to the findings of both sirkin et al . and patterson and cole.78 furthermore , the frame was initially removed and flashed for sterility so that it can be reused intraoperatively as a distractor during the definitive surgery . the reason for this was that the fixator could help maintain length and the definitive plating could then take place with less manipulation of the soft tissues . incision placement affects the surgeon 's ability to see , reduce and stabilize bony and articular fragments and can further traumatize overlying soft tissues . the use of the anterolateral approach to the distal tibia and ankle has become accepted for orif of pilon fractures . this is because of improved understanding of pilon fracture patterns , availability of precontoured anterolateral plates and most importantly because it provides an extensile exposure of the distal tibia and tibiotalar joint . several reports1415 have presented evidence that pilon fracture patterns are predictable and that most comminution occurs in the anterolateral aspect of the distal tibia . herscovici et al.13 described the use of an anterolateral surgical approach to the ankle and stated that this approach has the advantage of providing not only good exposure of the distal tibia , but also easy access to distal anterolateral ( chaput ) fracture fragments , thereby allowing direct reduction of the articular surface . another potential advantage of this approach is that it provides greater soft tissue coverage over the implants when compared with the traditional anteromedial approach . we found that the anterolateral approach provided excellent exposure to the distal tibia for intraarticular pilon fracture reduction and fixation . as a result of the location of the anterolateral incision , a second , medially based incision can be used for reduction of separate medial malleolar fractures and for percutaneous medial plate or screw application . however , to adequately visualize the fracture during fixation , oftentimes the soft tissues are retracted for a significant amount of time . the retraction of compromised soft tissues and swelling that is often present at the time of fixation may lead to complications . the swelling may be decreased from the initial injury with staged protocol , but is still present at fixation and can contribute to a poor outcome . with the no - touch technique , there is no retraction of the soft tissues , with the exception of the initial placement of the k - wires . thus , the use of the no - touch technique resulted in a low complication rate . although , the concept of the application of npwt to open and dehisced wounds is well - established in the literature , there is little literature regarding the potential application over incisions to prevent complications in high risk wounds . a recently published study by timmers et al.16 applied npwt to patients forearms with an intact soft tissue envelope . these data suggest a statistically significant increase in microvascular blood flow to the skin.16 a number of clinical experience and animal data also suggest increased blood flow as a result of application of npwt.17181920212223 the mechanism of action of npwt to augment wound healing is not completely understood . there are at least three proposed mechanisms : increased blood flow through the capillary vessels ; edema reduction ; and mechanical stretching of cells leading to cell growth and expansion.1724 based on the results of the above study , application of npwt to surgical incisions immediately after surgical fixation and closure should be considered after high - energy pilon fractures to decrease wound complications ; although , good closure was achieved.25 the limitations of the study are : this is a retrospective study . it does not provide a statistical comparison with a control group for operative treatment of complex pilon fractures . the focus of the study was on wound healing and soft tissue complications ; therefore , this duration of followup was deemed to be sufficient as opposed to complications arising at a later time caused by nonunion , loss of fixation , malunion or late osteomyelitis . further study is required to examine these late complications and whether they may be related to the use of the strategy of soft tissue control . finally , our sample size is relatively small and it is therefore difficult to predict trends that may occur with a larger cohort of patients with pilon fractures treated using the strategy of soft tissue control . to conclude , this study represents a case series of ao / ota ( orthopaedic trauma association ) type c pilon fractures treated with the strategy of all fractures reviewed were ao / ota ( orthopaedic trauma association ) type c fractures with 61% being type c3 . we feel that this played a large role in our ability to manage the soft tissue postoperatively and limit a relatively low incidence of soft tissue complications despite being at a high risk for complication . as successful management of pilon fractures relies in part on avoidance of complications , this series provides evidence that the strategy of soft tissue control can reliably be used for fixation of these complex injuries .

background : pilon fractures are challenging to manage because of the complexity of the injury pattern and the risk of significant complications . the soft tissue injury and handling of the soft tissue envelope are crucial in pilon fracture outcomes . the purpose of this study was to evaluate the early rate of complications using the strategy of soft tissue control for operative treatment of complex pilon fractures.materials and methods:36 complex pilon fractures were treated with the soft tissue control strategy . patients followed the standard staged protocol , anterolateral approach to the distal tibia , the no - touch technique and incisional negative pressure wound therapy for pilon fractures . patients were examined clinically at 2 - 3 weeks and then 8 weeks for complications associated with the surgical technique.results:all fractures were ao / ota ( orthopaedic trauma association ) type c fractures ( 61% c3 , 22% c2 and 16% c1 ) . only one patient developed superficial infection and resolved with antibiotics and local wound care . none developed deep infection.conclusions:the strategy of soft tissue control for treatment of pilon fractures resulted in relatively low incidence of early wound complications in patients with complex pilon fractures .

forty - two adult male japanese white rabbits ( hokudo , sapporo , japan ) weighing approximately 3.0 kg were used in this study . the animal protocol was approved by the animal committee of health sciences university of hokkaido . the rabbit nasal ostium has previously been used as an experimental model ( asai et al . the sinus , extending about 2.0 cm anteroposteriorly , was approximately 1.0 cm wide and 1.5 cm high . the sinus mucosa consisted of pseudostratified ciliated epithelium , and numerous serous glands were found in the submucosal tissue . a sagittal view of a dissected rabbit skull showing the location can be seen in fig.1 . experimental site ( dorsal meatus , red arrow ) from the sagittal view ab , alveolar bone ; ms , maxillary sinus ; vn , ventral nasal meatus ; nm , nasopharyngeal meatus ; mm , middle meatus . sinus augmentation surgery was performed bilaterally , using the right side as the experimental site and the left side as the control site . the rabbits were anesthetized intravenously with pentobarbital sodium ( 0.5 mg / kg ) , and the head was shaved and swabbed with 10% benzethonium chloride solution . at the midline of the nasal dorsum , 0.5 ml of 1% lidocaine with epinephrine ( 1 : 100,000 ) was injected subcutaneously . a 50 mm midline incision was made with a # 25 scalpel , and the skin and periosteum were elevated sufficiently to expose the nasal bone and the nasoincisal suture line . using a round bur , two rectangular nasal bone windows ( 10 5 mm ) were outlined bilaterally on the nasal bone . the window was located approximately 10 mm anterior to the nasofrontal suture line and 5 mm lateral to the midline . the windows were completed by osteotomy with continuous cooling with a sterile saline solution ( fig.2 ) . care was taken to avoid damage of the antral membrane , which moved back and forth with the respiratory rhythm . the membrane was then elevated from the bony walls of the antrum to provide a large compartment approximately 5 10 10 mm , which was filled with 250 mg -tcp ( osferion olympus terumo biomaterial corp . , tokyo , japan ) . the surgical field was covered with a copolymer of cellulose acetate and nitrocellulose membrane ( millipore co. , billerica , ma , usa ) using medical cyanoacrylate adhesive . the periosteum was then closed using medicinal cyanoacrylate adhesive , and the skin was closed using a 4 - 0 suture . lipus therapy began on the day after sinus augmentation surgery and was applied to the right ( experimental ) side . the surgical gingival area was painted with a healing gel , and ultrasound was applied at a frequency of 1 or 3 mhz at an intensity of 240 mw using the br - sonic - pro device ( ito co. , tokyo , japan ) device ( fig.3 ) . the lipus therapy was performed for 15 min every day for 2 , 4 and 8 weeks . the rabbits were anesthetized intravenously with pentobarbital sodium and euthanized at 2 , 4 and 8 weeks after surgery . the animals were exsanguinated , and 10% neutral buffered formalin was perfused through the jugular veins . after decapitation and dissection , the entire nose complex , including the nasal ostium , samples were embedded in polyester resin ( rigolac , ohken , tokyo , japan ) and sliced into sections of various thickness , perpendicular to the direction of nasal dorsum as much as possible , using an exakt cutting grinding system ( bs 3000 , mg 4000 ; exakt co. , hamburg , germany ) . each section was further polished to a thickness of 150 m . specimens were double stained with basic fuchsin and methylene blue . the staining solution consisted of 2% fuchsin and 0.1% methylene blue in sodium hydroxide - alcohol , and the sections were stained as described previously ( murai et al . the specimens were observed at low magnification under a light microscope ( s2h10 bh-2 ; olympus , tokyo , japan ) . using this method , original bone stained pink and new bone stained reddish - violet . the resulting low magnification images were scanned into a computer and analysed using digital image software ( gimp , gnu general public licence : gnome foundation inc , boston , ma , usa ) . each histological area of the experimental and control sites was divided into upper and lower parts from the parietal region to a depth of 5 mm ( fig.4 ) . the area of new bone was measured in the experimental and control sites using image j ver 1.44 software ( national institutes of health , bethesda , md , usa ) . spss ver.19.0 ( spss inc , chicago , il , usa ) was used for data analysis . forty - two adult male japanese white rabbits ( hokudo , sapporo , japan ) weighing approximately 3.0 kg were used in this study . the animal protocol was approved by the animal committee of health sciences university of hokkaido . the rabbit nasal ostium has previously been used as an experimental model ( asai et al . the sinus , extending about 2.0 cm anteroposteriorly , was approximately 1.0 cm wide and 1.5 cm high . the sinus mucosa consisted of pseudostratified ciliated epithelium , and numerous serous glands were found in the submucosal tissue . a sagittal view of a dissected rabbit skull showing the location can be seen in fig.1 . experimental site ( dorsal meatus , red arrow ) from the sagittal view ab , alveolar bone ; ms , maxillary sinus ; vn , ventral nasal meatus ; nm , nasopharyngeal meatus ; mm , middle meatus . sinus augmentation surgery was performed bilaterally , using the right side as the experimental site and the left side as the control site . the rabbits were anesthetized intravenously with pentobarbital sodium ( 0.5 mg / kg ) , and the head was shaved and swabbed with 10% benzethonium chloride solution . at the midline of the nasal dorsum , 0.5 ml of 1% lidocaine with epinephrine ( 1 : 100,000 ) was injected subcutaneously . a 50 mm midline incision was made with a # 25 scalpel , and the skin and periosteum were elevated sufficiently to expose the nasal bone and the nasoincisal suture line . using a round bur , two rectangular nasal bone windows ( 10 5 mm ) were outlined bilaterally on the nasal bone . the window was located approximately 10 mm anterior to the nasofrontal suture line and 5 mm lateral to the midline . the windows were completed by osteotomy with continuous cooling with a sterile saline solution ( fig.2 ) . care was taken to avoid damage of the antral membrane , which moved back and forth with the respiratory rhythm . the membrane was then elevated from the bony walls of the antrum to provide a large compartment approximately 5 10 10 mm , which was filled with 250 mg -tcp ( osferion olympus terumo biomaterial corp . , tokyo , japan ) . the surgical field was covered with a copolymer of cellulose acetate and nitrocellulose membrane ( millipore co. , billerica , ma , usa ) using medical cyanoacrylate adhesive . the periosteum was then closed using medicinal cyanoacrylate adhesive , and the skin was closed using a 4 - 0 suture . lipus therapy began on the day after sinus augmentation surgery and was applied to the right ( experimental ) side . the surgical gingival area was painted with a healing gel , and ultrasound was applied at a frequency of 1 or 3 mhz at an intensity of 240 mw using the br - sonic - pro device ( ito co. , tokyo , japan ) device ( fig.3 ) . the lipus therapy was performed for 15 min every day for 2 , 4 and 8 weeks . low - intensity pulsed ultrasound stimulation irradiation equipment . the rabbits were anesthetized intravenously with pentobarbital sodium and euthanized at 2 , 4 and 8 weeks after surgery . the animals were exsanguinated , and 10% neutral buffered formalin was perfused through the jugular veins . after decapitation and dissection , the entire nose complex , including the nasal ostium , samples were embedded in polyester resin ( rigolac , ohken , tokyo , japan ) and sliced into sections of various thickness , perpendicular to the direction of nasal dorsum as much as possible , using an exakt cutting grinding system ( bs 3000 , mg 4000 ; exakt co. , hamburg , germany ) . the staining solution consisted of 2% fuchsin and 0.1% methylene blue in sodium hydroxide - alcohol , and the sections were stained as described previously ( murai et al . 2005 ) . the specimens were observed at low magnification under a light microscope ( s2h10 bh-2 ; olympus , tokyo , japan ) . using this method , original bone stained pink and new bone stained reddish - violet . the resulting low magnification images were scanned into a computer and analysed using digital image software ( gimp , gnu general public licence : gnome foundation inc , boston , ma , usa ) . each histological area of the experimental and control sites was divided into upper and lower parts from the parietal region to a depth of 5 mm ( fig.4 ) . the area of new bone was measured in the experimental and control sites using image j ver 1.44 software ( national institutes of health , bethesda , md , usa ) . the rabbits were anesthetized intravenously with pentobarbital sodium and euthanized at 2 , 4 and 8 weeks after surgery . the animals were exsanguinated , and 10% neutral buffered formalin was perfused through the jugular veins . after decapitation and dissection , the entire nose complex , including the nasal ostium , samples were embedded in polyester resin ( rigolac , ohken , tokyo , japan ) and sliced into sections of various thickness , perpendicular to the direction of nasal dorsum as much as possible , using an exakt cutting grinding system ( bs 3000 , mg 4000 ; exakt co. , hamburg , germany ) . the staining solution consisted of 2% fuchsin and 0.1% methylene blue in sodium hydroxide - alcohol , and the sections were stained as described previously ( murai et al . 2005 ) . the specimens were observed at low magnification under a light microscope ( s2h10 bh-2 ; olympus , tokyo , japan ) . using this method , original bone stained pink and new bone stained reddish - violet . the resulting low magnification images were scanned into a computer and analysed using digital image software ( gimp , gnu general public licence : gnome foundation inc , boston , ma , usa ) . each histological area of the experimental and control sites was divided into upper and lower parts from the parietal region to a depth of 5 mm ( fig.4 ) . the area of new bone was measured in the experimental and control sites using image j ver 1.44 software ( national institutes of health , bethesda , md , usa ) . sagittal section of the rabbit nose . spss ver.19.0 ( spss inc , chicago , il , usa ) was used for data analysis . at 2 weeks after implantation , no evidence of inflammation was found in any of the groups . newly formed bone was observed along the bone walls and under the lifted antral membrane . early signs of centripetal new bone formation were observed projecting from the bone walls . in both groups , the bone substitute was almost completely intact . in the 1 mhz group , newly formed bone bridged the particles of -tcp . in the 3 mhz group of rabbits , new bone formation was only visible at the periphery of the created space on both sides ( fig.5 ) . sagittal section of the upper part at 2 weeks newly formed bone was observed along the antral membrane at all sites ( arrows ) . nb , newly formed bone ; t , -tcp ; b , bone wall . in both the 1 and 3 mhz experimental sites , newly formed bone was observed along the bony walls in the upper part , but not the lower part . there was no difference in the histological findings between the upper and lower parts of the 3 mhz experimental sites ( figs5 and 6 ) . the differences in the area of newly formed bone were not significant between the experimental sites and the control sites of the 3 mhz group , but significantly more new bone formed at the experimental sites than at the control sites of the 1 mhz group . when the upper and lower parts of each area were measured in combination , there was a statistical difference between the experimental and control sites in the 1 mhz group at 2 weeks ; however , there was no statistical difference between the experimental and control sites in the 3 mhz group ( fig.7 ) . sagittal section of the lower part at 2 weeks there were no histological differences between any of the sites ( arrows ) . total area of upper and lower parts of newly formed bone area of newly formed bone in the upper and lower parts at 2 , 4 and 8 weeks . a , b values with different letters within a group are significantly different ( nonparametric test ) . at 4 weeks after implantation , newly formed bone was observed surrounded by -tcp particles in the 1 and 3 mhz groups . although in the 3 mhz group newly formed bone was found in both the experimental sites and the control sites , there were no significant differences between them ( fig.8 ) . newly formed bone was present underneath both the upper and lower parts in the 1 and 3 mhz groups . there was no difference in histological findings between the upper and lower parts in the 1 and 3 mhz groups . there was more newly formed bone in the experimental sites than in the control sites in the 1 and 3 mhz groups ( figs8 and 9 ) . the experimental sites exhibited significantly more new bone growth than the control sites in both the upper and lower parts of the 1 mhz group , but there was no significant difference between the experimental sites and the control sites in both the upper and lower parts of the 3 mhz group . the experimental sites of the 1 mhz group exhibited significantly more new bone growth than the experimental sites of the 3 mhz group in the lower part . ( fig.10 ) . when the upper and lower parts of each area were measured in combination , there was a statistical difference between the experimental and control sites in the 1 mhz group at 4 weeks ; however , there were no statistical differences between the experimental and control sites in the 3 mhz group ( fig.7 ) . sagittal section of the upper part at 4 weeks the 1 mhz group manifested advanced bone formation and calcification . although in the 3 mhz group newly formed bone was found in both the experimental and control sites , there was no significant difference in the newly formed bone ( arrows ) . sagittal section of the lower part at 4 weeks there was no difference in the newly formed bone between any of sites ( arrows ) . area of newly formed bone area of newly formed bone in the upper and lower parts at 2 , 4 and 8 weeks . a , b values with different letters within a group are significantly different ( nonparametric test ) . at 8 weeks after implantation , both the experimental and the control sites in the 1 and 3 mhz groups contained fewer -tcp particles than at 4 weeks ( fig.11 ) . in the experimental sites of the 1 mhz group , we did not find any differences between the upper and lower parts in the 1 and 3 mhz groups ( figs11 and 12 ) . the experimental sites in the 1 mhz group exhibited significantly more new bone growth than the control sites in the upper parts . there were no significant differences between the experimental sites and the control sites in both the upper and the lower parts of the 3 mhz group , and also no significant differences between the experimental sites of the 1 and 3 mhz groups ( fig.10 ) . when the upper and lower parts of each area were measured in combination , there was no statistical difference between the experimental and control sites in the 1 and 3 mhz group at 8 weeks . sagittal section of the upper part at 8 weeks in the 1 mhz group , the area of newly formed bone at the experimental site was greater than at the control site ( arrows ) . sagittal section of the lower part at 8 weeks in the 1 mhz group the area of newly formed bone at the experimental site was greater than at the control site as well as at both sites in the 3 mhz group ( arrows ) . sinus augmentation has emerged as a good option for implant site development to ensure long - term biomechanical stability in cases with anatomical limitations ( aparicio et al . 2001 ) . however , a disadvantage of this technique is that it takes 45 months for completion of the repair cycle ( tatum 1986 ) , during which time the patient is unable to masticate . lipus has been shown to accelerate new bone formation in animals not only in orthopedics but also in oral implantology ( walsh et al . 2008 ; kim & hong 2010 ; nakanishi et al . the healing processes around dental implants are similar to those that occur during repair of a fractured bone . because ultrasound stimulation can accelerate the rate of bony healing , lipus may offer a new technique to shorten the duration of osseointegration of dental implants and to improve outcomes ( liu et al . ultrasound , a form of mechanical energy that is transmitted through and into biological tissues as an acoustic pressure wave at frequencies above the limit of human hearing , is used widely in medicine as a therapeutic , operative and diagnostic tool ( rubin et al . it has been hypothesized that the micromechanical strains produced in biological tissues may result in biochemical events that could stimulate fracture healing ( pilla et al . 1990 ; gleizal et al . the intensity of lipus used for the treatment of fractures is 3040 mw / cm , which is considerably lower than the intensity of 10003000 mw / cm used in physiotherapy . moreover , the increase in temperature of the deep tissue is assumed not to be caused by pulse waves ( saito et al . although therapeutic ultrasound has been used for over 40 years , its current use in the clinical environment has changed significantly over this period . whereas in the past , its use was primarily for its thermal effect , it is now more widely employed for its non - thermal effects , especially in relation to bone fracture healing ( sapareto & dewey 1984 ; uchiyama et al . 2007 ; watson & young 2008 ; fu et al . only one clinical study to date has suggested that lipus has a significant effect on new bone formation in sinus augmentation ( kim & hong 2010 ) . we therefore aimed to evaluate the efficacy of lipus and to determine the optimal frequency for enhancing bone regeneration in sinus augmentation using a rabbit model . our results showed that lipus has the potential to accelerate bone regeneration in sinus augmentation within certain parameters . studies investigating the optimal lipus setting for enhancing fracture healing have examined several different low intensities . bone injury has been shown to occur if the intensity level is more than 1.5 w / cm . the device used in our study was applied at approximately 27 mw / cm because this study designed to have a fixed parameter . ( pilla et al . 1990 ; heckman et al . 1994 ) . the effect of lipus on the repair process at different frequencies has not been clarified ; however , many studies have demonstrated that lipus enhances fracture healing ( speed 2001 ) . ultrasound at a frequency of 1 mhz penetrates 35 cm into the tissue and is therefore recommended for deeper injuries and in patients with more subcutaneous fat . a frequency of 3 mhz is recommended for more superficial injuries at depths of 12 cm ( speed 2001 ) . there are no reports comparing lipus at frequencies of 1 and 3 mhz in sinus augmentation in rabbit models . consequently , in this study , each histological area of the experimental and control sites was divided into upper and lower parts from the parietal region to a depth of 5 mm to detect specific differences between the frequencies of 1 and 3 mhz . our results showed that lipus at a frequency of 3 mhz did not even penetrate all of the upper parts at 2 , 4 and 8 weeks , because the energy was absorbed in the superficial tissue and did not reach the deep tissue . it is possible that the penetration was affected by the position and angle of the probe at the experimental site , which varied because it was held in the researcher s hand and not fixed in place , by movement of the experimental animals , which were also not fixed in place . however , lipus at a frequency of 1 mhz penetrated all upper and lower sites , despite the existence of factors that may have affected lipus penetration . the characteristics of certain tissues have been shown to affect the depth of penetration ( duarte 1983 ) . for example , tissues such as blood and fat , which have a high water content and a low protein content , absorb little of the ultrasound energy , but those with a lower water content and a higher protein content absorb far more ultrasound energy . it is difficult to predict the thickness of each of these layers in individual patients . there was no significant difference in maximum torque or torsional stiffness in rat femora with lipus treatment ( 30 mw / cm ) using a frequency of 0.5 mhz compared with 1.5 mhz . after 21 days of healing , both frequencies led to increased maximum torque and torsional stiffness compared with untreated controls ( wang et al . another researcher observed significantly greater mineral apposition rates at 2 and 3 weeks post - fracture in rabbit fibulae that had been treated with 1.5 mhz compared with 3 mhz frequencies , both using a lipus intensity of 500 mw / cm ( tsai et al . , we showed that there were significant differences between the experimental sites and control sites of the 1 mhz group in the upper and lower part at 2 or 4 weeks . these findings are in contrast with another study which demonstrated that new bone growth around an implant was significantly greater at 3 mhz than at 1 mhz in dogs jaws ( fujii et al . the reason for this discrepancy may be that lipus at frequency of 3 mhz was effective over the short distance between the probe and dental implants because lipus was applied to the side of the implants rather than to the long axis . it was hypothesized that the high - frequency ultrasound waves were reflected by the oral implants , thus reducing the acceleration of bone regeneration . further research is needed to determine the optimal parameters for applying lipus in sinus augmentation , relating not only to frequency but also to intensity , pulse width , pulse cycle and exposure time . this study suggests that lipus stimulates bone regeneration in sinus augmentation although it is thought that the areas of new bone formation at the control sites in the 1 and 3 mhz groups were potentially subject to bias by administering lipus at the experimental site although all the control sites in the 1 and 3 mhz group were involved negligible lipus exposure as well . challenges for the future of this study need to be modified so that the control sites are not subject to bias . in conclusion , our study shows that lipus stimulates bone regeneration in sinus augmentation and has a significant effect on the development of newly formed bone at a frequency of 1 mhz .

objectivesthe objective of the present study was to evaluate the efficacy of low intensity pulsed ultrasound stimulation ( lipus ) and to determine the optimal frequency for enhancing bone regeneration in sinus augmentation using a rabbit model.material and methodsthirty male rabbits underwent sinus augmentation . two rectangular nasal bone windows were outlined bilaterally . lipus was applied at two different frequencies ( 1 mhz and 3 mhz ) on experimental sites daily for 2 , 4 and 8 weeks . each histological area of the experimental and control sites was divided into upper and lower parts from the parietal region to a depth of 5 mm . each area of new bone was measured.resultsat 2 , 4 and 8 weeks , the experimental sites in the 1 mhz group exhibited significantly more new bone growth than the control sites in both the upper and lower parts . when the upper and lower parts of each area were measured in combination there was a statistical difference between the test and control sites in the 1 mhz group at 2 , 4 and 8 weeks ; however , there were no statistical differences between the test and control sites in the 3 mhz group.conclusionsthe results suggest that clinical application of lipus for sinus augmentation may promote new bone formation , and that the effect of lipus for sinus augmentation at a frequency of 1 mhz was greater than at 3 mhz until 8 weeks after sinus augmentation .

the kaiser permanente southern california ( kpsc ) medical care program is a large prepaid group - practice managed health care organization with over 3.2 million members . members receive their health care in kpsc - owned facilities throughout the seven - county region . the study population consisted of women who had one or more singleton births at 20 weeks gestation in kpsc hospitals , who were identified as having gdm using the 100-g oral glucose tolerance test ( ogtt ) from 1 january 1999 through 31 december 2006 , and who remained kpsc members for at least 6 months postpartum . one randomly selected pregnancy was included in the analyses for women who had had more than one gdm - affected pregnancy during the study period . of the women in the final sample , 3% had more than one gdm - affected pregnancy during the study period . an algorithm using outpatient encounter and inpatient hospitalization codes , laboratory test results , and pharmacy prescription records was applied to identify women with evidence of diabetes before pregnancy ( 2,18 ) . after excluding these women , we used the results from the 100-g 3-h ogtt to identify women who had had gdm based on at least two abnormal plasma glucose measurements greater than or equal to the carpenter and coustan threshold values recommended by the ada ( fasting 95 mg / dl , 1-h 180 mg / dl , 2-h 155 mg / dl , 3-h 140 mg / dl ) ( 19 ) . women who had a 2-h 75-g ogtt or fasting plasma glucose ( fpg ) alone in the period from 7 days to 6 months postpartum were considered tested , while women without testing or those with only a random ( nonfasting ) plasma glucose during this period were considered not tested for the purposes of this study . time to first ogtt or fpg after delivery was calculated by determining the number of days from the infant birth date to the first testing date . we further categorized tested women based on timing of their first test as 7 days postpartum up to 6 weeks postpartum ( early testing window ) , 612 weeks postpartum ( ada - recommended testing window ) ( 9,10 ) , and after 12 weeks through 6 months postpartum ( late testing window ) . using the ada criteria , we defined women with an fpg ( whether alone or as part of a 75-g ogtt ) < 100 mg / dl as normal , 100125 mg / dl as ifg , and 126 mg / dl as having a provisional diagnosis of diabetes ( 1 ) . categories based on the glucose concentration 2 h after a 75-g post - glucose load were as follows : < 140 mg / dl normal , 140199 mg / dl igt , and 200 mg / dl provisionally diabetic . women with ifg and/or igt ( ifg / igt ) were combined into one category . for women with more than one test during the follow - up period during separate visits , the outcome is reported based on the most abnormal test result . information derived from the infants ' birth certificates that were included in these analyses included maternal age , race / ethnicity ( categorized as hispanic [ regardless of race ] , white , black , asian , or pacific islander [ asian / pi ] and all others ) , country of birth ( u.s . or foreign born ) , infant birth weight , and gestational age at delivery . median household income was based on the estimated median household income in the census block group in which the woman resided . pharmacotherapy for gdm was defined as having one or more prescriptions filled for oral hyperglycemia agents ( metformin or glyburide ) and/or insulin at a kpsc pharmacy during pregnancy . gdm was considered noted in the medical record if the international classification of diseases ( icd ) code 648.8 was included among the discharge diagnoses after delivery . the postpartum visit was defined as a clinical visit with a physician , certified nurse midwife , or nurse practitioner in the obstetrics and gynecology department from 612 weeks postpartum . we estimated the prevalence of postpartum glucose testing by dividing the number of women with postpartum glucose testing giving birth that year by the total number of women with gdm pregnancies giving birth during that year . we examined the associations between demographic , clinical , and health care related characteristics and postpartum glucose testing ( yes / no ) and time to first postpartum glucose test after delivery ( 7 days to < 6 weeks , 612 weeks , > 12 weeks to 6 months ) . multiple logistic regression models were used to calculate the adjusted odds ratio ( or ) and corresponding 95% cis to assess the associations between demographic , clinical , and health care related characteristics and postpartum glucose testing . among women with a postpartum glucose test , we examined the associations between these characteristics and postpartum test results ( normal , ifg / igt , or diabetes ) . we also explored the mean fasting , 1-h , 2-h , and 3-h values from the 100-g 3-h ogtt during pregnancy and postpartum glucose testing ( yes / no ) , timing of postpartum testing , and outcomes of postpartum testing . differences were evaluated using the student 's t tests and one - way anova in conjunction with tukey 's honestly significant difference to account for multiple comparisons . the associations between the postpartum test result ( normal , ifg / igt , diabetes ) and the timing of postpartum testing and pharmacological gdm treatment , respectively , were examined using multinomial logistic regression to adjust for maternal demographic characteristics as well as year of delivery . analyses were performed with sas version 9.1 ( sas institute , cary , nc ) . the kaiser permanente southern california ( kpsc ) medical care program is a large prepaid group - practice managed health care organization with over 3.2 million members . members receive their health care in kpsc - owned facilities throughout the seven - county region . the study population consisted of women who had one or more singleton births at 20 weeks gestation in kpsc hospitals , who were identified as having gdm using the 100-g oral glucose tolerance test ( ogtt ) from 1 january 1999 through 31 december 2006 , and who remained kpsc members for at least 6 months postpartum . one randomly selected pregnancy was included in the analyses for women who had had more than one gdm - affected pregnancy during the study period . of the women in the final sample , 3% had more than one gdm - affected pregnancy during the study period . an algorithm using outpatient encounter and inpatient hospitalization codes , laboratory test results , and pharmacy prescription records was applied to identify women with evidence of diabetes before pregnancy ( 2,18 ) . after excluding these women , we used the results from the 100-g 3-h ogtt to identify women who had had gdm based on at least two abnormal plasma glucose measurements greater than or equal to the carpenter and coustan threshold values recommended by the ada ( fasting 95 mg / dl , 1-h 180 mg / dl , 2-h 155 mg / dl , 3-h 140 mg / dl ) ( 19 ) . women who had a 2-h 75-g ogtt or fasting plasma glucose ( fpg ) alone in the period from 7 days to 6 months postpartum were considered tested , while women without testing or those with only a random ( nonfasting ) plasma glucose during this period were considered not tested for the purposes of this study . time to first ogtt or fpg after delivery was calculated by determining the number of days from the infant birth date to the first testing date . we further categorized tested women based on timing of their first test as 7 days postpartum up to 6 weeks postpartum ( early testing window ) , 612 weeks postpartum ( ada - recommended testing window ) ( 9,10 ) , and after 12 weeks through 6 months postpartum ( late testing window ) . using the ada criteria , we defined women with an fpg ( whether alone or as part of a 75-g ogtt ) < 100 mg / dl as normal , 100125 mg / dl as ifg , and 126 mg / dl as having a provisional diagnosis of diabetes ( 1 ) . categories based on the glucose concentration 2 h after a 75-g post - glucose load were as follows : < 140 mg / dl normal , 140199 mg / dl igt , and 200 mg / dl provisionally diabetic . women with ifg and/or igt ( ifg / igt ) were combined into one category . for women with more than one test during the follow - up period during separate visits , information derived from the infants ' birth certificates that were included in these analyses included maternal age , race / ethnicity ( categorized as hispanic [ regardless of race ] , white , black , asian , or pacific islander [ asian / pi ] and all others ) , country of birth ( u.s . or foreign born ) , infant birth weight , and gestational age at delivery . median household income was based on the estimated median household income in the census block group in which the woman resided . pharmacotherapy for gdm was defined as having one or more prescriptions filled for oral hyperglycemia agents ( metformin or glyburide ) and/or insulin at a kpsc pharmacy during pregnancy . gdm was considered noted in the medical record if the international classification of diseases ( icd ) code 648.8 was included among the discharge diagnoses after delivery . the postpartum visit was defined as a clinical visit with a physician , certified nurse midwife , or nurse practitioner in the obstetrics and gynecology department from 612 weeks postpartum . we estimated the prevalence of postpartum glucose testing by dividing the number of women with postpartum glucose testing giving birth that year by the total number of women with gdm pregnancies giving birth during that year . we examined the associations between demographic , clinical , and health care related characteristics and postpartum glucose testing ( yes / no ) and time to first postpartum glucose test after delivery ( 7 days to < 6 weeks , 612 weeks , > 12 weeks to 6 months ) . multiple logistic regression models were used to calculate the adjusted odds ratio ( or ) and corresponding 95% cis to assess the associations between demographic , clinical , and health care related characteristics and postpartum glucose testing . among women with a postpartum glucose test , we examined the associations between these characteristics and postpartum test results ( normal , ifg / igt , or diabetes ) . we also explored the mean fasting , 1-h , 2-h , and 3-h values from the 100-g 3-h ogtt during pregnancy and postpartum glucose testing ( yes / no ) , timing of postpartum testing , and outcomes of postpartum testing . differences were evaluated using the student 's t tests and one - way anova in conjunction with tukey 's honestly significant difference to account for multiple comparisons . the associations between the postpartum test result ( normal , ifg / igt , diabetes ) and the timing of postpartum testing and pharmacological gdm treatment , respectively , were examined using multinomial logistic regression to adjust for maternal demographic characteristics as well as year of delivery . analyses were performed with sas version 9.1 ( sas institute , cary , nc ) . of the 11,825 women with a gdm - affected pregnancy during the 8-year study period , just over half ( 50.2% ; n = 5,939 ) had at least one fpg or 75-g ogtt during the 6-month postpartum period ( table 1 ) . of the tested women , 2,458 ( 41.4% ) were first tested during the early window , 2,749 ( 46.3% ) during the ada recommended 6- to 12-week window , and 732 ( 12.3% ) during the late window ( table 1 ) ; 79.1% were tested with fpg only , 18.2% were assessed with a 75-g ogtt , and 2.7% were given both tests . approximately 7% ( n = 408 ) of these women were tested more than once in the 6 months postpartum , although repeated testing was not found to be contingent upon the results of the first test ( data not shown ) . characteristics of 11,825 women with gdm , proportion of women having postpartum glucose testing , timing of first test , and adjusted or and 95% ci for testing during the 6 months postpartum in kpsc clinics , 19992006 * p value based on test for categorical variables ; fisher 's exact test used for variables with any cell count < 10 . the distribution of each variable within the population is shown as a column percent . the proportions tested and not tested are shown as row percentages within each strata of the variable . in the unadjusted analyses , demographic , clinical , health care characteristics , and year of delivery timing of the first postpartum test was significantly associated with all demographic variables except age and income tertile , all clinical variables except parity , and year of delivery . in the multiple logistic regression model ( table 1 ) , odds of testing increased with increasing maternal age , education , and income and were higher for foreign - born women compared with u.s .- born women . the odds of testing decreased with increasing parity and were lower for women who were black or white compared with hispanic women , for women with cesarean deliveries , and for women with babies weighing 4,000 g. women who were treated with insulin ( oral agents ) had the same odds of being tested as those who did not receive pharmacotherapy , whereas women treated with oral agents alone were less likely to be tested . finally , women who did not have gdm coded at discharge had 39% lower odds of being tested than those who were so coded . in contrast , the odds of postpartum testing were over three times greater for women who had a postpartum visit than for those who had not . women who had blood glucose testing postpartum had slightly lower mean fasting ( 91.4 vs. 92.8 mg / dl , p < 0.0001 ) and 3-h values ( 124.9 vs. 126.6 mg / dl , p < 0.001 ) on their 100-g ogtt performed during pregnancy than women who were not tested postpartum ( supplementary fig . the mean fasting glucose on the 100-g ogtt during pregnancy was similar for women tested during the early postpartum period and those tested during the 6- to 12-week period ( 90.9 and 90.8 mg / dl , respectively ) , whereas women tested in the late window had a higher mean fasting value during pregnancy ( 95.1 mg / dl , p < 0.0001 ) ( supplementary fig . results were available ( n = 5,857 ; 99% ) , 16.3% ( n = 956 ) were classified as ifg / igt and 1.1% ( n = 66 ) as having diabetes . of the 1,154 women who were given a 2-h 75-g ogtt postpartum , 810 ( 70.2% ) had normal fasting and postchallenge values , 172 ( 14.9% ) ifg only , 75 ( 6.5% ) igt only , 71 ( 6.2% ) both ifg and igt ( for a total of 318 women [ 27.6% ] with ifg and/or igt ) , and 26 ( 2.3% ) were found to have diabetes based on the fasting and/or 2-h values . of the 1,065 women who were given a 2-h 75-g ogtt postpartum without a prior fpg test , 8.1% of 835 women with a normal fpg had an abnormal 2-h value ( igt = 7.9% , diabetes = 0.2% ) , and 16.7% of 921 women with normal 2-h values had an abnormal fpg ( ifg = 16.5% , diabetes = 0.2% ) . of the women who had an fpg only , 12.8% had ifg and 0.8% were found to have diabetes . in the unadjusted analyses , demographic characteristics significantly associated with test result categories included all except country of birth ( table 2 ) . ogtt values observed during pregnancy all differed significantly ( p < 0.001 ) between the three postpartum glucose categories ( supplementary fig . 3 ) . both pharmacotherapy and time to postpartum test were highly associated with abnormal outcomes in the multinomial logistic regression analysis ( table 3 ) . women treated with insulin during pregnancy had 57% higher odds of ifg / igt and were over three times as likely to have diabetes as women who did not take medication for gdm . women who took oral agents only had 40% higher odds of having ifg / igt and were over two times as likely to have diabetes . in addition , women tested before 6 weeks postpartum were 26% less likely to have ifg / igt and 58% less likely to have diabetes than women tested between 6 and 12 weeks postpartum . in contrast , women who were tested from 12 weeks to 6 months postpartum were 55% more likely to have ifg / igt and over three times as likely to have diabetes as were women tested from 6 to 12 weeks postpartum . the associations between postpartum testing period and gdm treatment and the test result categories remained highly significant , even after adjustment for the prenatal ogtt fasting values ( data not shown)the only prenatal value that differed by testing period ( supplementary fig . 2 ) . demographic , clinical , and health care related characteristics associated with postpartum glucose test results among 5,857 women * who had gdm and who had postpartum glucose testing , 19992006 * excludes 82 women who were tested but for whom test results were not available . p value is for the test for each variable separately for the three categories ; monte carlo simulations for exact p values were used when any cell size was < 10 . all percentages are shown as row percentages for outcome distribution within each covariate strata . adjusted or and 95% ci for characteristics associated with postpartum glucose test result categories among 5,508 women with a history of gdm who had postpartum glucose testing , 19992006 * data are adjusted or ( 95% ci ) for the outcome of ifg and/or igt and presumptive diabetes versus normal test results . final model includes 5,508 observations with complete data for each covariate ( 94% of the sample with test results ) . of the 11,825 women with a gdm - affected pregnancy during the 8-year study period , just over half ( 50.2% ; n = 5,939 ) had at least one fpg or 75-g ogtt during the 6-month postpartum period ( table 1 ) . of the tested women , 2,458 ( 41.4% ) were first tested during the early window , 2,749 ( 46.3% ) during the ada recommended 6- to 12-week window , and 732 ( 12.3% ) during the late window ( table 1 ) ; 79.1% were tested with fpg only , 18.2% were assessed with a 75-g ogtt , and 2.7% were given both tests . approximately 7% ( n = 408 ) of these women were tested more than once in the 6 months postpartum , although repeated testing was not found to be contingent upon the results of the first test ( data not shown ) . characteristics of 11,825 women with gdm , proportion of women having postpartum glucose testing , timing of first test , and adjusted or and 95% ci for testing during the 6 months postpartum in kpsc clinics , 19992006 * p value based on test for categorical variables ; fisher 's exact test used for variables with any cell count < 10 . the distribution of each variable within the population is shown as a column percent . the proportions tested and not tested are shown as row percentages within each strata of the variable . in the unadjusted analyses , demographic , clinical , health care characteristics , and year of delivery timing of the first postpartum test was significantly associated with all demographic variables except age and income tertile , all clinical variables except parity , and year of delivery . in the multiple logistic regression model ( table 1 ) , odds of testing increased with increasing maternal age , education , and income and were higher for foreign - born women compared with u.s .- born women . the odds of testing decreased with increasing parity and were lower for women who were black or white compared with hispanic women , for women with cesarean deliveries , and for women with babies weighing 4,000 g. women who were treated with insulin ( oral agents ) had the same odds of being tested as those who did not receive pharmacotherapy , whereas women treated with oral agents alone were less likely to be tested . finally , women who did not have gdm coded at discharge had 39% lower odds of being tested than those who were so coded . in contrast , the odds of postpartum testing were over three times greater for women who had a postpartum visit than for those who had not . women who had blood glucose testing postpartum had slightly lower mean fasting ( 91.4 vs. 92.8 mg / dl , p < 0.0001 ) and 3-h values ( 124.9 vs. 126.6 mg / dl , p < 0.001 ) on their 100-g ogtt performed during pregnancy than women who were not tested postpartum ( supplementary fig . the mean fasting glucose on the 100-g ogtt during pregnancy was similar for women tested during the early postpartum period and those tested during the 6- to 12-week period ( 90.9 and 90.8 mg / dl , respectively ) , whereas women tested in the late window had a higher mean fasting value during pregnancy ( 95.1 mg / dl , p < 0.0001 ) ( supplementary fig . of the women tested for whom results were available ( n = 5,857 ; 99% ) , 16.3% ( n = 956 ) were classified as ifg / igt and 1.1% ( n = 66 ) as having diabetes . of the 1,154 women who were given a 2-h 75-g ogtt postpartum , 810 ( 70.2% ) had normal fasting and postchallenge values , 172 ( 14.9% ) ifg only , 75 ( 6.5% ) igt only , 71 ( 6.2% ) both ifg and igt ( for a total of 318 women [ 27.6% ] with ifg and/or igt ) , and 26 ( 2.3% ) were found to have diabetes based on the fasting and/or 2-h values . of the 1,065 women who were given a 2-h 75-g ogtt postpartum without a prior fpg test , 8.1% of 835 women with a normal fpg had an abnormal 2-h value ( igt = 7.9% , diabetes = 0.2% ) , and 16.7% of 921 women with normal 2-h values had an abnormal fpg ( ifg = 16.5% , diabetes = 0.2% ) . of the women who had an fpg only , 12.8% had ifg and 0.8% were found to have diabetes . in the unadjusted analyses , demographic characteristics significantly associated with test result categories included all except country of birth ( table 2 ) . ogtt values observed during pregnancy all differed significantly ( p < 0.001 ) between the three postpartum glucose categories ( supplementary fig . 3 ) . both pharmacotherapy and time to postpartum test were highly associated with abnormal outcomes in the multinomial logistic regression analysis ( table 3 ) . women treated with insulin during pregnancy had 57% higher odds of ifg / igt and were over three times as likely to have diabetes as women who did not take medication for gdm . women who took oral agents only had 40% higher odds of having ifg / igt and were over two times as likely to have diabetes . in addition , women tested before 6 weeks postpartum were 26% less likely to have ifg / igt and 58% less likely to have diabetes than women tested between 6 and 12 weeks postpartum . in contrast , women who were tested from 12 weeks to 6 months postpartum were 55% more likely to have ifg / igt and over three times as likely to have diabetes as were women tested from 6 to 12 weeks postpartum . the associations between postpartum testing period and gdm treatment and the test result categories remained highly significant , even after adjustment for the prenatal ogtt fasting values ( data not shown)the only prenatal value that differed by testing period ( supplementary fig . 2 ) . demographic , clinical , and health care related characteristics associated with postpartum glucose test results among 5,857 women * who had gdm and who had postpartum glucose testing , 19992006 * excludes 82 women who were tested but for whom test results were not available . p value is for the test for each variable separately for the three categories ; monte carlo simulations for exact p values were used when any cell size was < 10 . all percentages are shown as row percentages for outcome distribution within each covariate strata . adjusted or and 95% ci for characteristics associated with postpartum glucose test result categories among 5,508 women with a history of gdm who had postpartum glucose testing , 19992006 * data are adjusted or ( 95% ci ) for the outcome of ifg and/or igt and presumptive diabetes versus normal test results . final model includes 5,508 observations with complete data for each covariate ( 94% of the sample with test results ) . among the membership of a large managed health care plan with a high prevalence of gdm , approximately one - half of women identified as having gdm had postpartum glucose testing in the 6 months after delivery . the majority of women ( 79% ) tested in the 6 months postpartum had an fpg only . only one - half of women who had postpartum glucose testing had their tests during the 6- to 12-week postpartum period recommended by the ada . postpartum testing and timing of testing were associated with demographic , clinical , and health care related factors . women whose treatment included insulin alone or insulin plus oral hypoglycemic agents had the same odds of being tested postpartum as those whose treatment did not include any pharmacologic agents . however , individuals treated with oral agents alone had lower odds for being tested than those who did not receive pharmacotherapy during pregnancy . of the women tested , a relatively small proportion ( 1% ) met the criteria for diabetes , while a greater proportion ( 16% ) had ifg and/or igt . however , detection of ifg / igt or diabetes was independently and positively associated with having been treated with insulin or oral agents during pregnancy and timing of postpartum glucose testing . while women tested between 12 weeks and 6 months postpartum had higher mean fpg during pregnancy compared with those tested in the early and ada - recommended windows , the association between being tested in the later period and having abnormal results postpartum persisted after controlling for the prenatal fpg value . the observation that women not tested postpartum had higher fasting and 3-h values during pregnancy suggests that if a larger number of women had been tested postpartum , a greater proportion of the entire sample of gdm women would have been found to be glucose intolerant postpartum . if a 75-g ogtt postpartum test had been consistently administered , the prevalence of diagnosed diabetes would most likely have increased ( 20 ) . of the women in our sample who had an ogtt postpartum with normal fpg results , 8% had igt or diabetes based on their 2-h value . there has been significant heterogeneity across studies that have examined rates and correlates of postpartum glucose testing after gdm with respect to calendar years studied , time between delivery , and postpartum testing population ( hospital - based samples [ 1214,17 ] versus managed care ) and the approach used to identify women with gdm . the present study included all women who met the ada criteria for gdm based on the results from the 100-g ogtt test performed during pregnancy , since icd-9 codes alone was found to be an unreliable means of identifying women with gdm in our health plan database ( 2 ) . indeed , while 16% of the women identified as having gdm based on their prenatal 100-g ogtt results did not have a discharge diagnosis of gdm , over one - third ( 38.5% ) of these women had postpartum glucose testing . in spite of the differences and consistent with the present study , approximately half of the women with gdm in the earlier studies underwent postpartum testing ( 12,13,1517 ) . of the studies that examined gdm pharmacological treatment in relation to whether or not postpartum testing was performed , two found a positive association ( 14,16 ) , one found a negative association ( 13 ) , and one found no association ( 15 ) . in the present study , we found that while 50.3% of the women with no pharmacotherapy and 51.1% of the women on insulin were tested , only 42.6% of women on oral agents had postpartum glucose testing . given that the women who took oral agents only or insulin during their gdm pregnancy and received postpartum testing were 40 or 57% as likely to have ifg / igt and two to three times as likely to have diabetes , respectively , as women not treated with these medications , women who require pharmacotherapy to control their blood glucose during pregnancy should be tested in the postpartum period . in the three other studies reporting the results of postpartum glucose testing ( 12,13,16 ) , the prevalence of diabetes ranged from 2 to 8% compared with 1% reported in the present study . additionally , the prevalence of ifg and/or igt ranged from 11 to 33% , compared with 16% in the present study . in the two studies ( 12,13 ) that used the same ada criteria to identify women as having gdm in their clinical center as were used in the present study , 72% of those tested for glucose intolerance in one of the centers had an ogtt postpartum ( 13 ) compared with only 21% in the present study . this may have resulted in a higher proportion of women identified as having impaired glucose regulation in their population . one study used the higher nddg blood glucose threshold ( 21 ) to identify women with gdm and included tests conducted from 6 weeks through 1 year postpartum ( 16 ) . either or both of these factors may have accounted for the higher prevalence of impaired glucose regulation in their population compared with our findings . we were unable to assess the occurrence of postpartum glucose testing among the 15% of women with gdm who disenrolled from the health plan within 6 months after their deliveries . we could not determine whether women were advised by a health care professional to have postpartum testing or whether the test was ordered but not completed , since this information is not retained in the laboratory database during the study period . we could also not determine if the patient received a reminder to report for testing by mail or telephone . thus , missed tests could be a result of lack of physician orders or women not returning for postpartum testing . additionally , we were not able to explore the associations between obesity , postpartum testing , and ongoing glucose abnormalities in the postpartum period because maternal height and weight were not available in clinical databases or the california birth certificates for the period of this study . the strengths of this study include the use of multiple clinical and administrative databases to identify and characterize women with gdm , including pharmacotherapy for gdm during pregnancy from a diverse insured cohort and to determine whether members of that cohort had been tested for glucose abnormalities postpartum . because 95% ( n = 11,187 ) of the women in the sample had kpsc prescription drug coverage during pregnancy , we were able to examine the associations requiring anti - hyperglycemic agents , postpartum testing , and test results with a high level of accuracy . our sample included all women from 1999 through 2006 identified as having gdm using a standard laboratory test ( 100-g 3-h ogtt ) using ada criteria to assess the presence of gdm . additionally , we were able to identify those women found to have igt / ifg and those having a provisional diagnosis of diabetes using the results of their 75-g ogtt and fpg tests done during the postpartum period and to describe the demographic , clinical , and health service utilization characteristics of those women . testing for ongoing glucose dysregulation after a gdm - affected pregnancy may allow the opportunity for clinical interventions to reduce the risk of developing diabetes or treatment to reduce the risk of diabetes - related complications among women found to have diabetes ( 22 ) . the risk of pregnancy - related complications among women with diabetes , a population that is growing ( 2,23 ) , may be reduced by optimal use of preconception care ( 24 ) . the findings from this and other studies suggest that the rates of postpartum glucose testing after gdm are suboptimal . rates of postpartum glucose testing after gdm pregnancies may be increased by incorporating alerts into electronic medical records so that postpartum glucose testing orders are automatically generated or physician - generated . automated live or recorded telephone or e - mail messages could also be used to remind women to obtain testing after the tests are ordered . postpartum glucose testing is an important first step in attempting to prevent both recurrence of gdm and the development of nongestational diabetes subsequent to a pregnancy complicated by gdm . there has been significant heterogeneity across studies that have examined rates and correlates of postpartum glucose testing after gdm with respect to calendar years studied , time between delivery , and postpartum testing population ( hospital - based samples [ 1214,17 ] versus managed care ) and the approach used to identify women with gdm . the present study included all women who met the ada criteria for gdm based on the results from the 100-g ogtt test performed during pregnancy , since icd-9 codes alone was found to be an unreliable means of identifying women with gdm in our health plan database ( 2 ) . indeed , while 16% of the women identified as having gdm based on their prenatal 100-g ogtt results did not have a discharge diagnosis of gdm , over one - third ( 38.5% ) of these women had postpartum glucose testing . in spite of the differences and consistent with the present study , approximately half of the women with gdm in the earlier studies underwent postpartum testing ( 12,13,1517 ) . of the studies that examined gdm pharmacological treatment in relation to whether or not postpartum testing was performed , two found a positive association ( 14,16 ) , one found a negative association ( 13 ) , and one found no association ( 15 ) . in the present study , we found that while 50.3% of the women with no pharmacotherapy and 51.1% of the women on insulin were tested , only 42.6% of women on oral agents had postpartum glucose testing . given that the women who took oral agents only or insulin during their gdm pregnancy and received postpartum testing were 40 or 57% as likely to have ifg / igt and two to three times as likely to have diabetes , respectively , as women not treated with these medications , women who require pharmacotherapy to control their blood glucose during pregnancy should be tested in the postpartum period . in the three other studies reporting the results of postpartum glucose testing ( 12,13,16 ) , the prevalence of diabetes ranged from 2 to 8% compared with 1% reported in the present study . additionally , the prevalence of ifg and/or igt ranged from 11 to 33% , compared with 16% in the present study . in the two studies ( 12,13 ) that used the same ada criteria to identify women as having gdm in their clinical center as were used in the present study , 72% of those tested for glucose intolerance in one of the centers had an ogtt postpartum ( 13 ) compared with only 21% in the present study . this may have resulted in a higher proportion of women identified as having impaired glucose regulation in their population . one study used the higher nddg blood glucose threshold ( 21 ) to identify women with gdm and included tests conducted from 6 weeks through 1 year postpartum ( 16 ) . either or both of these factors may have accounted for the higher prevalence of impaired glucose regulation in their population compared with our findings . we were unable to assess the occurrence of postpartum glucose testing among the 15% of women with gdm who disenrolled from the health plan within 6 months after their deliveries . we could not determine whether women were advised by a health care professional to have postpartum testing or whether the test was ordered but not completed , since this information is not retained in the laboratory database during the study period . we could also not determine if the patient received a reminder to report for testing by mail or telephone . thus , missed tests could be a result of lack of physician orders or women not returning for postpartum testing . additionally , we were not able to explore the associations between obesity , postpartum testing , and ongoing glucose abnormalities in the postpartum period because maternal height and weight were not available in clinical databases or the california birth certificates for the period of this study . the strengths of this study include the use of multiple clinical and administrative databases to identify and characterize women with gdm , including pharmacotherapy for gdm during pregnancy from a diverse insured cohort and to determine whether members of that cohort had been tested for glucose abnormalities postpartum . because 95% ( n = 11,187 ) of the women in the sample had kpsc prescription drug coverage during pregnancy , we were able to examine the associations requiring anti - hyperglycemic agents , postpartum testing , and test results with a high level of accuracy . our sample included all women from 1999 through 2006 identified as having gdm using a standard laboratory test ( 100-g 3-h ogtt ) using ada criteria to assess the presence of gdm . additionally , we were able to identify those women found to have igt / ifg and those having a provisional diagnosis of diabetes using the results of their 75-g ogtt and fpg tests done during the postpartum period and to describe the demographic , clinical , and health service utilization characteristics of those women . testing for ongoing glucose dysregulation after a gdm - affected pregnancy may allow the opportunity for clinical interventions to reduce the risk of developing diabetes or treatment to reduce the risk of diabetes - related complications among women found to have diabetes ( 22 ) . the risk of pregnancy - related complications among women with diabetes , a population that is growing ( 2,23 ) , may be reduced by optimal use of preconception care ( 24 ) . the findings from this and other studies suggest that the rates of postpartum glucose testing after gdm are suboptimal . rates of postpartum glucose testing after gdm pregnancies may be increased by incorporating alerts into electronic medical records so that postpartum glucose testing orders are automatically generated or physician - generated . automated live or recorded telephone or e - mail messages could also be used to remind women to obtain testing after the tests are ordered . postpartum glucose testing is an important first step in attempting to prevent both recurrence of gdm and the development of nongestational diabetes subsequent to a pregnancy complicated by gdm .

objectiveto estimate the prevalence of postpartum glucose testing within 6 months of pregnancies complicated by gestational diabetes mellitus ( gdm ) , assess factors associated with testing and timing of testing after delivery , and report the test results among tested women.research design and methodsthis was a retrospective study of 11,825 women who were identified as having gdm using the 100-g oral glucose tolerance test ( ogtt ) from 1999 to 2006 . postpartum testing ( 75-g 2-h ogtt or fasting plasma glucose [ fpg ] ) within 6 months of delivery and test results from laboratory databases are reported . postpartum test results are categorized as normal , impaired fasting glucose ( ifg ) and/or impaired glucose tolerance ( igt ) , and provisionally diabetic.resultsabout half ( n = 5,939 ) the women were tested with either a fpg or 75-g ogtt from 7 days to 6 months postpartum . of these women , 46% were tested during the 6- to 12-week postpartum period . odds of testing were independently associated with age , race / ethnicity , household income , education , foreign - born status , parity , mode of delivery , having a postpartum visit , having gdm coded at discharge , and pharmacotherapy for gdm . of the 5,857 women with test results , 16.3% ( n = 956 ) had ifg / igt and 1.1% ( n = 66 ) had provisional diabetes . after adjustment for demographic and clinical factors , abnormal postpartum test results was associated with having required insulin , glyburide , or metformin during pregnancy and with longer period from delivery to postpartum testing.conclusionsafter a pregnancy complicated by gdm , automated orders for postpartum testing with notification to physicians and electronically generated telephone and e - mail reminder messages to patients may improve the rates of postpartum testing for persistence of glucose intolerance .

malaria is a global health problem with more than 1 billion people estimated to be at risk . this infectious disease is caused by plasmodium parasites transmitted to humans through bites of infected anopheles mosquitos . geographically , plasmodium falciparum ( p. falciparum ) parasites predominate in sub - saharan africa while plasmodium vivax ( p. vivax ) is the major infectious agent in south america and southeast asia . according to the latest world malaria report , disease mortality and risk have been steadily decreasing in the last decade to the point that many countries are already targeting malaria elimination and eradication [ 25 ] . this decreasing trend in malaria transmission intensity , although highly beneficial to the affected populations , brings additional challenges to disease surveillance and elimination ( reviewed in ) . one of these challenges is related to the use of the current metrics of malaria risk in populations where disease transmission intensity is low and potentially affected by seasonal effects . the popular parasite rate is determined by the proportion of infected individuals at time of the survey . however , in low transmission settings , this measure is critically affected by the different performance of current diagnostic tools to detect the presence of infection while screening asymptomatic individuals . another difficulty of using such measure is the high chance of finding only a few infected individuals in the sample , thus , having limited power to discriminate disease hotspots from other less - affected sites , as demonstrated in studies from brazil or somalia . it is defined by the frequency at which people are bitten by infectious mosquitoes , thus , being informative on the direct interaction between the human and mosquito populations . the gold standard to estimate this measure is to use human - landing catches where mosquitoes are caught as they attempt to land on the exposed limbs of field workers [ 9 , 10 ] . although alternative methods exist in the literature , the estimation of the entomological inoculation rate is in general a laborious and time - consuming task in low transmission settings owing to a low number of infected mosquitoes . it is also affected by seasonal effects and mosquito population dynamics and the degree of mosquito attractiveness to the human hosts or the chemicals used in the study . to tackle the limitations of the above malaria risk measures , alternative indicators based on antibodies against different malaria antigens have been proposed and tested in different epidemiological contexts [ 7 , 8 , 1316 ] . the rationale of using antibody data is that the antibody concentrations in the serum are a direct correlate of parasite exposure , thus , providing information on current and recent infections . the temporal stability in antibody concentrations is an important advantage to reduce any seasonal effect on malaria transmission . in seroepidemiological studies , the most popular antibodies are those against the blood - stage apical membrane antigen-1 ( ama1 ) and merozoite surface protein-1 ( msp1 ) [ 7 , 8 , 1316 ] owing to their broad immunogenicity and putative role in malaria vaccine development [ 17 , 18 ] . recent research identified other parasite targets [ 19 , 20 ] but these remain to be tested in different epidemiological settings . experimentally , antibody quantification is usually done by means of traditional enzyme linked immunosorbent assays . optical densities or titres in arbitrary units are then used for the subsequent data analysis . the most popular approach is to first define the serological status , seropositive or seronegative , of each individual . one then calculates the so - called seroprevalence that is defined by the proportion of seropositive individuals in the sample . several studies showed an increased resolution of seroprevalence in discriminating sites with different endemicity levels in relation to parasite rate [ 7 , 8 ] . since seroprevalence tends to increase with age as a result of augmenting immunity against malaria parasites , different stochastic models can be constructed for the data using age as a proxy of time . the common assumption to all these models is that individuals transit between seronegativity and seropositivity states upon malaria exposure or absence of it . in this scenario , one typically estimates the rate by which seronegative individuals become seropositive , the so - called seroconversion rate ( scr ) . scr was found to correlate well with the parasite rate or the entomological inoculation rate , thus , capturing the underlying malaria transmission intensity . moreover , scr also strongly correlates to the annual parasite index ( the number of confirmed cases during 1 year / population under surveillance ) 1000usually calculated by official health authorities . this paper aims to review the mathematical and statistical aspects underlying the analysis of antibody data for inferring malaria transmission intensity . special attention will be given to current methods aiming to define seropositivity and the subsequent mathematical models for estimating scr under different epidemiological settings : stable malaria transmission intensity , abrupt reduction in scr due to a malaria control intervention , change in scr due to a putative age - dependent behavior , detection of migration effects , and detection of individual level heterogeneity through a set of covariates . three different data sets from bioko island in equatorial guinea , jacarecanga from the brazilian amazonia region , and western highlands from kenya are used to illustrate the application of these models to real - world problems . finally , future analytical challenges will be discussed in the context of malaria elimination and eradication . in practice , there are two popular approaches to determine the serological status of an individual . the first approach uses an additional sample of nonexposed individuals in order to determine the distribution of the antibody levels referring to the underlying seronegative population . statistically , the antibody levels of this sample are usually log transformed in order to approximately obtain a gaussian distribution for the data . the serological classification of each individual in the sample is done by the 3 rule for gaussian distributions described in any introductory textbook of statistics . in more detail , this rule defines the range of antibody levels containing a 0.999 probability under the assumption of a gaussian random variable for the data . one then classifies the individuals as seropositive if the respective antibody levels exceed the mean plus 3 times the standard deviation of the seronegative population , otherwise the individuals are considered as seronegative . this simple approach , despite ensuring a high probability of correctly classifying exposed individuals , has the disadvantage of underestimating seroprevalence . the basic assumption is that the sample is composed of a mixture of latent seronegative or seropositive populations . the respective data is then analyzed by the so - called two - component mixture gaussian model invoking a gaussian distribution with average value 0 and standard deviation 0 for the seronegative population and another one with average value 1 and standard deviation 1 for the seropositive population . for independent and identically distributed random sample of n individuals , the corresponding sampling distribution is described by the following equation:(1)fxi 0,1,0,1,=i=1n1fn0,0xi+fn1,1xi , where xi is the antibody level of the ith individual in the sample , fn(0,0)(xi ) and fn(1,1)(xi ) are probability density functions of the gaussian distributions associated with seronegative and seropositive populations , respectively , and is the probability of sampling a seropositive individual from the population . maximum likelihood estimation is facilitated by using the expectation - maximization ( em ) algorithm that can be found in the mixtools package for the r software . the next stage of the analysis is to assign each individual to each corresponding serological population . again , one can use the 3 rule as described above . an alternative way to perform such classification is to jointly use the probabilities of classifying an individual with antibody level x as either seropositive or seronegative and then specify appropriate cut - off values to determine the serological status of each individual . the probabilities of classifying an individual with antibody level x as seropositive and seronegative are , respectively , given by(2)p+ x=fn1,1x1fn0,0x+fn1,1x , p x=1p+ x.the classification rule of the ith individual in the sample is then described as follows:(3)ci = seronegative , if xicindeterminate , if c<xi < c+seropositive , if xic+,where c and c are the cut - off values in the antibody distribution that ensure a given classification probability , for instance , 90% . note that individuals with antibody levels between c and c are deemed indeterminate due to the uncertainty in the corresponding serological classification . besides checking whether model assumptions hold true on the data under analysis , an additional assessment of the quality of the classification rule is to report the size of this indeterminate region and the proportion of indeterminate individuals in the sample . the health authority of equatorial guinea launched integrated treatment and mosquito control programs in the bioko island . after 4 years of their initiation , a large cross - sectional survey was conducted at 18 sentinel sites in the island in order to assess the impact of these programs on malaria transmission . the antibody levels as measured by arbitrary titres range from 116.3 to 2618.9 , suggesting a wide breadth of immune responses to this malaria antigen ( figure 1(a ) ) . as expected from data of a malaria endemic region , the corresponding quantile - quantile plot showed a strong departure of the data in relation to the gaussian distribution due to presence of recently or currently exposed individuals with high antibody levels ( figure 1(b ) ) . by fitting the above two - component gaussian mixture model to the data , the serological status of each individual was determined by ( 3 ) with c = 97.0 and c = 200.8 ( figure 1(c ) ) . these cut - off values suggested that 31.2% and 56.1% of the sample consisted of seronegative and seropositive individuals , respectively . the remaining 12.7% of the sample had unclear serological classification ( table 1 ) . the above gaussian mixture model can be extended to the setting where there are more than two components . immunologically , such extension is in line with the notion that antibody levels can be boosted by frequent malaria exposure . in this scenario , each component can be interpreted as corresponding to a specific degree of malaria exposure : not exposed , once exposed , twice exposed , three times exposed , and so forth . under the assumption of a known number of components for the data ( say k + 1 ) , the corresponding sampling distribution is given by(4)fxi k,k,k=i=1nk=0kkfnk,kxi , where 0 < 1 < 2 < <k are the averages of the population not exposed , once exposed , twice exposed , , and k times exposed , respectively , 0 , 1 , 2 , , k are the corresponding standard deviations , and 0 , 1 , 2 , , k are the corresponding sampling probabilities ( with 0 = 1 k=1 ) . the conditional classification probabilities of seropositive and seronegative individuals given antibody level x can be generalized as follows:(5)p+ x=k=1kkfnk,kxk=0kkfnk,kxi , p x=1p+ x.the corresponding classification rule is also given by ( 3 ) but now the cut - off values must be recalculated according to these new classification probabilities . as for the two - component gaussian mixture model , maximum likelihood estimation via em algorithm can also be performed to estimate the unknown parameters { k , k , k , k = 0 , , k}. starting this estimation algorithm with different initial conditions is recommended to obtain the correct convergence to the global maxima of the log - likelihood function . an important question in practice is to know how many components one must consider to describe the data well . in terms of maximum likelihood estimation , this question can be answered by using the profile likelihood method . this method proceeds as follows : ( i ) start the analysis with k = 1 , ( ii ) obtain the corresponding maximum likelihood estimates and then calculate the respective value of the likelihood function , ( iii ) add another component into the analysis and repeat step ( ii ) , and ( iv ) keep increasing the number of components until reaching a realistic maximum value for that parameter . the optimal number of components is the one providing the maximum value of all maximum likelihood values calculated for each number of components considered in the analysis . the profile likelihood method , despite estimating the total number of components , brings potential problems of model overfitting and uncertainty in the classification rule . this problem can be surpassed by using different information measures with the aim of weighting the quality of the data fitting with the intrinsic complexity of a model . the most popular information measure is the akaike 's information criterion ( aic ) defined by twice the absolute value of the log - likelihood function evaluated at the maximum likelihood estimates ( measuring the quality of the respective data fitting ) plus twice the total number of estimated parameters ( estimating the intrinsic model complexity ) . since models are penalised in this criterion as function of the total number of parameters , one should choose the model that shows the lowest aic value among all models tested . uncertainty in the classification rule can arise from data where the different serological populations are tight together in the antibody distribution . a simple solution is to choose the model with the highest likelihood but implying a sufficiently clear serological classification of the individuals in the sample . an additional difficulty in using a gaussian mixture model with more than two components is the ambiguity in linking each component to the corresponding serological status . let us consider the three - component mixture model for the moment . in this setting , the components with the lowest and highest average titres are easily interpreted as related to seronegative and seropositive populations , respectively . on the one hand , the component with intermediate average titres can be interpreted as a seronegative population if one assumes two populations with different genetic backgrounds . this interpretation agrees with studies from burkina faso where the fulani typically have higher antibody concentrations at baseline in comparison to other ethnic groups living in the same area [ 25 , 26 ] . on the other hand , this intermediate component can also be interpreted as a seropositive population under the assumption of immunity boosting upon recurrent malaria exposure as described above . this and the component with the highest average concentrations are then related to exposed and boosted populations , respectively . similar reasoning can easily be applied to the scenario of a higher number of components . for that one just needs to consider the putative existence of more than one seronegative and seropositive population . in absence of additional information about the populations under study , a possible solution is to first understand how the performance of the classification is affected by changing the interpretation of the components and then make a judgement call upon the reasonability of the corresponding results . models with three and four components seemed to describe the data better than the one with two components only , according to aic ( table 1 ) . despite providing a good balance between data fitting and model complexity , the four - component models implied high percentages of individuals with unclear classification ( > 22% ) . the best model would appear to be the one with three components where the second and the third components were interpreted as referring to seropositive populations . this model improves the quality of the data fitting and implied a percentage of individuals with unclear classification ( 14% ) similar to the one obtained from the two - component model . comparing to previous results for the two - component model , the inclusion of a third additional component suggested that the seropositive population could in fact be split into exposed and boosted individuals with average antibody titres of 214.0 and 848.3 , respectively . the new cut - off values for the classification rule led to the classification of 19.3% and 66.8% of the sampled individuals as seronegative and seropositive , respectively . recent research has highlighted the great potential of using bayesian approaches in gaussian mixture models . the major advantage of these methods is to provide a coherent and elegant analytical framework for estimating the total number of components from the data . since this number is unknown quantity , it is considered as random variable with a given probability distribution before conducting data analysis the so - called prior distribution . bayes theorem then allows linking the prior distributions for all unknown parameters with the sampling distribution of the data . as a result , prior distributions of the parameters are updated by the data , giving rise to the so - called posterior distributions . the current success of bayesian approaches is intimately related to the use of powerful simulation methods in order to determine the posterior distributions given the data . in gaussian mixture models , the markov chain monte carlo with reversible jumps is a popular choice for posterior estimation . these models are particularly suitable for analyzing data of more than one malaria antigen simultaneously ( e.g. , for analyzing ama1 and msp1 data together ) . a bayesian two - component mixture model using arbitrary probability distributions for the latent populations was proposed for classifying fever and nonfever malaria cases according to the underlying parasitaemia [ 29 , 30 ] . up to now a single seroepidemiological study in malaria is known to have analyzed data via bayesian methods and , thus , little can be said about their performance in practice . after classifying individuals into their serological status , the corresponding data analysis proceeds by estimating stochastic models that aim to inform about the underlying malaria transmission intensity . the most popular models belong to the class of the reversible catalytic models ( rcms ) [ 3133 ] . when applied to serological data from infectious diseases that do not induce long - lasting immunity , such as the case of malaria , these models assume that age is deemed an appropriate proxy of the historical time so that data from each individual can be seen as a random realization of a seroconversion - seroreversion stochastic law . more precisely , individuals are born as seronegative but can be converted into seropositive upon malaria exposure . in the absence of frequent malaria exposure mathematically , this idea can be described as a markov chain model where one must specify the average rates by which the individuals become seropositive and return to the seronegative , the seroconversion , and seroreversion rates ( scrs and srrs ) , respectively . epidemiologically , scr is related to the underlying disease transmission intensity as it correlates well with typical malariometrics , such as parasite rate or entomological inoculation rate . in contrast , srr reflects host factors ( e.g. , genetics or age ) affecting antibody decay in absence of malaria infection . the simplest model for the data is to assume stable and constant malaria transmission intensity over time . a fixed srr is also assumed because seropositivity data has limited power to describe variations in that parameter . for mathematical simplicity , the seroconversion - seroreversion dynamics of each individual is easily described by a markov chain with two states , seronegative ( s ) and seropositive ( s ) . the resulting rcm is described by the following probability of an individual aged t being seropositive:(6)ps+t=+1e+t , where and are the scr and srr , respectively . it is worth noting that the above probability is an increasing function of age reaching a plateau at /( + ) when age goes to infinite . the above model can be extended to the so - called superinfection model ( sim ) , where immunity boosting can occur owing to recurrent malaria infections . in line with the gaussian mixture models with more than 2 components for antibody titre data , the notion of boosting can be translated into distinct seropositive states , for instance , s , s , and s , depending on the cumulative level of malaria exposure ( figure 2(b ) ) . in particular , this same individual while still being first - order seropositive can evolve to a second - order seropositive upon an additional malaria exposure and so forth . a practical implication of this idea is a longer sojourn time in the seropositive state(s ) in relation to the one predicted by rcm . moreover , since there are multiple latent seropositive states , the estimates of the seroconversion rate tend to be higher in this model than in its reversible catalytic counterpart for the same data . the probability of an individual aged t being at any seropositive state is now given by(7)pst=1e/1et , where s represents the set of all possible seropositive states an individual can belong to . , the application of this model to real - world problems shows limitations in terms of estimation . on the one hand , sim and rcm are approximately equivalent to each other in low transmission settings due to the rarity of boosting events . on the other hand , thus , for the matter of simplicity , a model considering seroconversion only is more reasonable for that situation . interestingly , ( 6 ) and ( 7 ) when 0 can be rewritten as the classical complementary log - log model : ( 8)loglog1ps+t = log+logt.despite having limited application in malaria research , the complementary log - log model has been used in nonmalaria immunological settings where a single immunization is thought to exert a permanent seropositive phenotype [ 37 , 38 ] . with respect to model estimation , seropositive data adjusted for age is organized as a two - way frequency table with a rows and two columns , where a is the total number of different age values in the sample and the two columns refer to the serological status of the individuals ( i.e. , seronegative and seropositive ) . in this data format , the sampling distribution is assumed to be a binomial - product sampling distribution , an independent binomial distribution per age value and probability of success given by the model under fitting ; that is,(9)fmt nt,,=t=1antmttmt1tntmt , where mt and nt are the frequency of seropositive and all individuals aged t years , respectively , and (t ) is the expected seroprevalence at age t described by ( 6 ) , ( 7 ) , or ( 8) if estimating rcm , sim , or the complementary log - log model , respectively . stata and r scripts for data fitting are currently available from the authors upon request . example i ( bioko island continued ) . as mentioned earlier , the cross - sectional survey from bioko island consisted of 18 sentinel sites spread over the island . to increase statistical power , the corresponding data was analyzed by considering 5 major geographical regions : northeast , northwest , southeast , southwest , and malabo . a comprehensive analysis of this data set can be found in the original study report . for illustrative purposes , the statistical analysis was carried out on data from northeast and northwest regions specifically . according to the seropositivity determination step , there are 1332 and 877 individuals with an assigned serological status from northwest and northeast regions , respectively . the corresponding overall seroprevalence was estimated at 86.7% ( 95% ci : 84.8%88.5% ) and 69.9% ( 95% ci : 66.7%72.9% ) . these estimates are higher than the ones reported in the original study ( 69.2% and 46.6% , resp . ) because this study used a two - component gaussian mixture model for titre data , thus , predicting a higher cut - off value for seropositivity ( table 1 ) . as expected from a malaria endemic area , the seroprevalence increased with age in both regions ( figures 2(c ) and 2(d ) ) . with respect to northwest region , both models described the seroprevalence curve well ( figure 2(c ) ) . however , sim provided a slightly better fit to the data than rcm ( log - likelihood = 69.71 and 71.31 , resp . ) , a result in line with the use of a three - component mixture model for seropositivity determination . also in agreement with theoretical expectations was the higher scr obtained from sim in relation to the one predicted by rcm ( 0.359 versus 0.286 ; table 2 ) . for northeast region , the overall seroprevalence is decreased , thus , implying lower scr estimates for rcm and sim ( 0.124 and 0.139 for rcm and sim , resp . ) . although rcm showed a better fit to the data than its sim counterpart ( log - likelihood = 96.87 and 102.95 , resp . ) , both models overestimated the seroprevalence of young aged individuals ( up to 10 years ago ) ( figure 2(d ) ) and , thus , they could not be considered as good candidate models for the data . such overestimation suggested that young aged and older individuals have different serological dynamics that can not easily be captured by a stable malaria transmission assumption . an easy explanation is the putative reduction in malaria transmission intensity after the initiation of malaria control programs in 2004 in the island . a unique advantage of using serology data is the possibility of detecting heterogeneity in disease transmission across different epidemiological situations . this advantage has been demonstrated in several studies where seroprevalence taken as a function of age qualitatively changes at a given age value . such change might be attributed to an abrupt reduction in malaria transmission after the initiation of a malaria control or elimination program [ 15 , 16 ] . another possible explanation for that change is related to distinct malaria risk between young and older individuals owing to behavioral factors . a third and last explanation is the occurrence of migration waves over time , as observed in chagas disease . the commitment of many national health authorities in reducing or targeting elimination in future years brings future challenges in assessing the real impact of the designed interventions on the target populations . for that one assumes there was an abrupt reduction of malaria transmission intensity at some time point before data collection . it is expected that an abrupt reduction in malaria transmission intensity would translate in a similar effect on the scr . sampled individuals are then split according to their date of birth in relation to the calendar time when the reduction in malaria transmission intensity actually occurred . more precisely , the serological history of individuals born before that reduction contemplates a first time period where the past scr operates followed by a second period where the current scr sets the rules . in contrast , individuals born after the reduction would lie down on that second time period and , thus , their serological dynamics are simply described by previous rcm and sim for stable malaria transmission . to calculate the seroprevalence of an individual with age t that experienced a reduction in malaria transmission intensity at time before sample collection , one must consider the sum of two probabilities associated with the following mutually exclusive events : ( i ) an individual became seropositive between birth and t and remained so after that and ( ii ) an individual remained seronegative between birth and t and became seropositive after that . since rcm can be formulated as a two - state markov chain , the seroprevalence for individuals with age t is calculated by multiplying the vector of probabilities associated with an individual being seropositive and seronegative at time t ( see ( 6 ) ) by the probability transition matrix of the second markov chain associated with the current scr and evaluated at time . the resulting expected seroprevalence is then given by(10)ps+t=21e2+11e1te2,if t>21e2t , if t,where i = i/(i + ) , i = i + , i = 1 , 2 , 1 and 2 are the past and current scr under the restriction of 2 < . similar argument can be applied to the superinfection model , leading to the following seroprevalence:(11)ps+t=1e1/eet2/1e,if t>1e2/1et , if t.with respect to parameter estimation , the sampling distribution is again assumed to be a binomial - product distribution ( ( 9 ) , where (t ) is described by ( 10 ) or ( 11 ) ) . to estimate all parameters ( 1 , 2 , , and ) via maximum likelihood , a profile likelihood approach is usually applied to the data under analysis : ( i ) set = 1 , ( ii ) determine the respective maximum likelihood estimates for the remaining parameters , ( iii ) calculate the corresponding log - likelihood function , ( iv ) increase one unit to and repeat steps ( ii - iii ) , and ( v ) keep increasing until reaching the maximum expected value for that parameter . the overall maximum likelihood estimates are those associated with the value of that provides the maximum value of all log - likelihood values . although statistically sound , this method tends to overestimate the true change point ( i.e. , estimates located further in past than they should ) , even if using a large sample size ( our own results ) . this suggests that seropositivity data might not have enough information to estimate that parameter with high precision . therefore , the interpretation of a specific estimate for the reduction time point should be done with caution . in practice , models assuming a stable or an abrupt reduction in malaria transmission intensity must compare to each other for the same data . a log - likelihood ratio test can then be applied to the corresponding results using the following test statistic under the null hypothesis:(12)l=2stablereduction22,where stable and reduction are the log - likelihood functions evaluated at the maximum likelihood estimates for the models assuming a stable or an abrupt reduction in malaria transmission intensity , respectively , and (2 ) is a chi - square distribution with the two degrees of freedom resulting from the difference in the total number of parameters of the models under testing ( and versus 1 , 2 , , and ) . for a 5% significance level , p values < 0.05 show evidence for a significant change in disease transmission . with the increasing complexity of the models under analysis , statistical inference via maximum likelihood methods becomes more cumbersome due to possible lack of convergence of the numerical algorithms leading to maximum likelihood estimates and the inaccuracy of large sample approximations for the confidence intervals and test statistics . , each parameter in a model has an associated prior distribution that , in turn , is updated with the observed data by means of bayes theorem . posterior mean and median of this distribution are two possible bayesian estimates for the same parameter . credible intervals are the bayesian equivalent to the confidence intervals of classical statistics and calculated by the appropriate quantiles of the posterior distribution that ensure a given probability mass ( i.e. , 95% ) . model comparison can be performed via aic or other bayesian information measures , such as the deviance information criterion ( dic ) . theoretically , dic is defined by the posterior mean of the deviance function ( twice the absolute value of log - likelihood function ) plus the effective number of parameters of a given model . in turn , the effective number of parameters is calculated by the difference between the posterior mean of the deviance function and the same function evaluated at the posterior means of the parameters . likewise with aic , one should choose the model that shows the lowest dic value among all models tested . the first one relates to how to choose the prior distributions for the unknown parameters . one solution is to use noninformative prior distributions in situations where prior information about the parameters of interest is limited or scarce . popular choices for noninformative prior distributions are the uniform distribution for parameters defining probabilities or gaussian distributions with mean 0 and sufficiently large standard deviation for parameters defined in real space . in contrast , if one has strong prior beliefs about the parameters of interest , informative prior distributions can be elicited . prior elicitation is generally based on a convenient probability distribution ( e.g. , a gaussian distribution ) upon which one determines the corresponding prior parameters the so - called hyperparameters by conjugating the expected prior mean with a set of prior quantiles set for that distribution . although informative prior distributions are in line with the permanent dialogue between inductive and deductive reasoning intrinsic to the scientific method , most researchers adopt a conservative strategy to data analysis by using noninformative prior distributions for the unknown parameters . the second difficulty concerns the calculation of the posterior distributions . however , this is greatly reduced by the powerful markov chain monte carlo ( mcmc ) that , virtually , can deal with any kind of model complexity . in practice , r / jags is an easy - to - use package for mcmc computing . illustrative scripts for the above rcm and sim are available from the authors upon request . as highlighted earlier , the fits of rcm and sim assuming stable malaria transmission intensity suggested a variation in malaria risk between younger and older individuals living in the northeast region of the island ( figure 2(d ) ) . such variation might be attributed to a reduction in malaria transmission intensity owing to a known malaria control initiative launched in 2004 . to test this hypothesis , rcm and sim with an abrupt reduction in malaria transmission intensity the most likely reduction point for both models was 6 years before data collection ( figure 3(a ) ) ; the corresponding 95% confidence intervals were 4.28.4 and 4.87.7 for rcm and sim , respectively . both models were in close agreement with the data visually ( figure 3(b ) ) and better than the previous ones assuming stable transmission , according to likelihood ratio test ( p values < 0.001 ) . sim led to a higher log - likelihood value than its rcm counterpart ( table 2 ) and , thus , it might be deemed the best model for the data . again , this result is consistent with the choice of three - component gaussian mixture model for the corresponding titre data . previous and current scrs were estimated at 0.900 and 0.098 for sim and at 0.274 and 0.077 for rcm . these implied a reduction in malaria transmission intensity of around 89% and 72% for sim and rcm , respectively . note the putative overestimation of the time point for the reduction event ( 6 years before sampling versus the time when the bioko malaria control initiative started ) . this result is in line with ongoing research where the profile likelihood method overestimated the true change point from simulated data typically found in african population ( our own results ) . a very similar age - adjusted seroprevalence curve to previous case can be found for populations where older individuals have a higher malaria transmission intensity compared to the one for younger individuals due to an age - dependent behavior factor . a typical example is the commute of adults to working sites that are malaria transmission hotspots in contrast to children and adolescents who do not travel to those sites . this situation was reported for some populations living in the forests of cambodia and indonesia [ 41 , 45 ] . more precisely , both younger and older individuals share the same scr until a certain age . then scr abruptly increases to a new level in a similar way as previous case . therefore , ( 10 ) for an abrupt reduction in malaria transmission intensity can be adapted as follows:(13)ps+t=11e1+21e2te1,if t>11e1t , if t,where i = i/(i + ) , i = i + , i = 1 , 2 , 1 and 2 are the scr for younger and older individuals , respectively , under the restriction of 1 < 2 . for the superinfection assumption , the resulting model can be expressed as follows:(14)ps+t=1e2/eet1/1e,if t>1e1/1et , if t.parameter estimation and model comparison can be performed via maximum likelihood and bayesian methods as described above for the models with an abrupt change in malaria transmission intensity . a recent study was conducted in the brazilian amazonia region where p. vivax is currently the major malaria threat opposed to what occurred in the past where p. falciparum infections predominated . a total of around 1300 individuals were sampled from 7 different municipalities in par state . previous analysis suggested stable malaria transmission for p. vivax infections but detected a putative abrupt reduction of p. falciparum transmission intensity estimated to have occurred around 2530 years before sampling . although this change is in line with the intensification of malaria control initiatives by brazilian health authorities in the area , alternative explanations were also discussed but not formally tested . more precisely , gold mining is one of the key economic activities in the area but also an important risk factor for malaria transmission . mining was also a determinant factor of the known migration wave from nonendemic states to the region since 1970s . in this line of thought , the detection of a change occurred 2530 years before sampling might be confounded by the increased malaria risk of the older population that are typically miners . this hypothesis is now tested against the one assuming an abrupt reduction of malaria transmission intensity . the analysis is focused on the p. falciparum seropositivity data from the municipality of jacareacanga where the past reduction in scr seemed more pronounced ( i.e. , from 0.514 to 0.017 ) . data under analysis comprised a total of 172 individuals of which 2.3% were infected with malaria parasites at the day of the survey . the seroprevalence for any p. vivax and p. falciparum antigens was estimated at 69.2% and 59.3% , respectively , using a two - component gaussian mixture model for the corresponding titre data . these estimates suggested a high malaria endemicity for that municipality as issued by the brazilian authority for malaria control but using the recorded annual parasite index . in contrast to previous example , bayesian methods were alternatively applied to the data using the following noninformative prior distributions for the parameters : ( i ) gaussian distributions with mean 0 and standard deviation 103 for all scrs and srrs in log scale and ( ii ) a discrete uniform distribution between 1 and 40 for the age cut - off . since seropositivity data was previously derived from a two - component gaussian mixture model , this analysis is based on the rcm only . mcmc simulation via r / jags package was used to obtain the posterior estimates for the parameters ; a long chain of 1,050,000 iterations was generated where the first 50,000 were discarded as the burn - in period and a lag of 100 was used to reduce correlation between simulated values . as previously reported , the model assuming an abrupt reduction in scr captures data well ( figure 4(a ) ) . however , there was some degree of uncertainty on the time in which that reduction had occurred ( figure 4(b ) ) . the posterior mean and median were consistent with a sudden drop in p. falciparum transmission intensity 28 years before data collection ( e.g. , around 1980 ) . the posterior mean for past and current scr was 0.436 and 0.019 , respectively , while the corresponding posterior medians were 0.386 and 0.019 ( table 2 ) . in agreement with a bayesian analysis using noninformative prior distribution , these posterior estimates implied a reduction in scr in the same order of magnitude to that obtained in the original study . the model assuming a behavioral factor also fitted the data well ( figure 4(a ) ) with a slightly higher age cut - off for the occurrence of such behavior ( around 29 years old ) . again , there was some uncertainty associated with the age value where that behavior becomes epidemiologically relevant . in absence of that putative behavioral factor , the baseline scr was estimated at 0.051 or 0.046 if one chooses the posterior mean or median , respectively ( table 2 ) . this scr increased to the posterior mean and median of 0.654 and 0.693 at older ages . the respective dic estimates are 89.72 and 96.67 for the rcms assuming an abrupt reduction and a change in scr due to an age - dependent behavioral factor . since the best model is the one that shows the lowest value for dic , the change in scr seemed to be better explained by an abrupt reduction in p. falciparum transmission intensity rather than the existence of a putative risk factor dependent on age , such as those related to gold mining activities in the heart of the amazonia forest . up to now all models were analyzed under the assumption of stable populations with no migration effects . this assumption is reasonable in most studies because individuals living shortly in a given study area are typically excluded from the survey . however , in the current era of facilitated movement between populations , it might be difficult to recruit locally born individuals only , thus , affecting the estimation of the scr . in one extreme , the easiest migration setting is the importation of malaria cases to nonendemic regions where there are no sufficient conditions for efficient malaria transmission . in this case , there is no strong rationale to use any of the above models since scr would reflect the disease transmission intensity of the places where the sampled individuals come from . on the other extreme , migration from nonendemic region to endemic ones more precisely , at the time of migration , individuals are immunologically naive to malaria parasites in comparison to those with the same age but locally born in the region . this is the case of the history of malaria in brazil where a gold rush in 1970s led to the migration of thousands of people from nonendemic states to the heart of the amazonia forest . another known example from the literature is the founder effect in a peruvian community affected by chagas disease where locally born individuals and founders have distinct seroprevalence histories . until now age was considered a proxy of the total exposure time of each individual to malaria antigens . in the situation where individuals migrated from a nonendemic region to an endemic one , age used in all above models is simply replaced with the total residence time of each individual in the endemic area if available . in practice , such information is not routinely collected , thus , requiring additional estimation . without lack of generality , the same model including migration effects is described as follows:(15)ps+t=+1e+t,where t and t are the age and the total residence time of an individual living in an endemic area , respectively . in absence of information of the total residence time , the estimation of t can be done by considering t = t pt , where pt ( 0,1 ] is the proportion of residence time of individuals with age t. in practice , estimation of each pt might be cumbersome by maximum likelihood methods . firstly , the above rcm and sim are intrinsically nonlinear and these additional unknown parameters might lead to convergence problems of the respective maximization algorithms . secondly , sample information might be insufficient to provide accurate estimation of the residence time of each individual . as mentioned earlier , bayesian inference is nowadays facilitated by the availability of powerful mcmc simulators that can estimate any kind of statistical model . moreover , bayesian inference can also coherently integrate external information on the residence time by describing the prior distribution accordingly . in so doing , one can consider the following family of prior distributions for pt:(16)ppt = x = p0,if x=1,1p0xt11xt1bet,t , if 0<x<1,where p0 is the prior probability of an individual with age t being locally born and pt for a migrant is modeled a priori by a beta distribution with hyperparameters t and t . if little information is known about the migrant population , one can specify t = 1 and t = 1 in order to obtain uniform distribution . this is particularly useful to capture migration effects of specific age groups , such as adults who tend to migrate for work reasons . as mentioned above , the history of malaria in brazil is intimately related to a gold rush in 1970s from nonendemic regions to endemic ones in the heart of the amazonia forest . since mining is the main economic activity of jacarecanga , it is possible that the above past and current scr estimates can be improved in order to take into account any past migration effects . unfortunately data concerning time of residence were not consistently recorded across individuals and study sites in the original study and , thus , the parameters pts were directly estimated from seropositivity data . the above data analysis was then extended to the situation of rcm assuming an abrupt reduction in the disease transmission intensity together with putative migration effects described by ( 10 ) . little information was known about the migrant population and , thus , a prior uniform distribution for the parameters pt 's was specified for the bayesian analysis . with respect to the prior probability p0 , the brazilian office for geography and statistics states that 14.4% of the population living in jacareacanga in the 2010 census were not born in the north states comprising the amazonia region . therefore , it seemed unlikely that the percentage of the migrant population from jacareacanga was lower than 15% . to understand the impact of p0 on the subsequent inferences , different values for that hyperparameter were tested , specifically , 0.25 , 0.50 , 0.75 , and 0.9 . the best one appeared to be 0.75 because it implied the highest posterior median and mean of the log - likelihood function ( results not shown ) . the introduction of migration effects in the rcm with an abrupt reduction resulted in a seroprevalence curve with a more complex pattern ( figure 4(a ) ) . however , this higher complexity in the seroprevalence curves augmented the uncertainty associated with the posterior distributions of the time in which that reduction in scr actually occurred and of the ratio between current and past scr ( figures 4(b ) and 4(c ) ) . adjusting for putative migration effects , the posterior median and mean for the changing point are around 24.5 and 26 years before sampling , two estimates close to the previous ones assuming no migration ( around 28 years ; table 3 ) . for the reduction in scr itself , the respective posterior mean and medians are 75.8% and 75.9% , two estimates slightly more conservative than those obtained for the rcm with no migration effects ( 93.5% and 95.3% , resp . ) . finally , notwithstanding the limited sample size , it was possible to borrow information from the sample in order to update the prior distributions of the residence time of each individual ( figure 4(d ) ) . many individuals could be assumed as locally born in jacareacanga because the respective posterior median for the fraction of time living in area was close to 100% ( figure 4(d ) ) . in the remaining cases , there was evidence for the presence of migrants in the sample . in conclusion , although model complexity increased uncertainty of the subsequent parameter estimation , the results provided a more realistic snapshot of the p. falciparum malaria history of jacareacanga . all above models for seropositivity data provide a broad description of the scr at the population level . their utility is then limited if one aims to understand more granular , individual level heterogeneities inherent to malaria transmission . for example , a recent study from cambodia has highlighted the role of age , ethnicity , village of residence , or forest work on the seroconversion of each individual during rainy season . other examples are the effect of elevation in scr in northeast tanzania [ 12 , 13 ] or the impact of malaria control interventions in western kenyan highlands . although age is an intrinsic variable of the above rcm and sim , the effect of other types of covariates affecting seropositivity suggests adopting a regression - type approach to tackle putative individual level heterogeneity in scr . this can be easily done by considering the following log - linear model for the scr of the ith individual with a set of p covariates x1,i , x2,i , , xp , i:(17)logi=0+1x1,i+2x2,i++pxp , i , where 0 is the overall effect in absence of covariates and 1 , , p are the regression coefficients associated with each covariate . this regression model is then coupled with rcm or sim ( see ( 6 ) and ( 7 ) ) with stable transmission intensity but describing with the above model . in the unrealistic situation that malaria infections induce lifelong immunity ( see ( 8) ) , the inclusion of covariates is facilitated because the resulting model is integrated in the well - known generalized linear model framework via a complementary log - log model for binary variables . since the analysis must take into account the data from each individual , previous binomial - product for the sampling distribution ( see ( 9 ) ) is now reconverted into a bernoulli - product , one bernoulli distribution per individual ; that is,(18)fyi i=i=1niyi1i1yi , where yi is the serological status of the ith individual , i is the probability of the ith individual being seropositive , and n is the sample size . in theory , maximum likelihood and bayesian methods can be applied to estimate all unknown parameters of the above model . in practice , computationally efficient maximum likelihood methods still need to be developed and , therefore , bayesian methods via mcmc are the most pragmatic approach to data analysis . in absence of prior information about the regression parameters , the usual choice for the respective a priori distribution is to use gaussian distribution with mean 0 and a sufficiently large standard deviation ( e.g. , 100 ) . if any prior information exists for the regression parameters , one can alternatively use any eliciting method for bayesian regression analysis as described elsewhere [ 48 , 49 ] . the western kenyan highlands are currently characterized by low - level endemic and highly heterogeneous p. falciparum malaria transmission . to ensure high resolution to detect heterogeneity in malaria exposure , approximately one - third of the total population , around 17,500 individuals , were sampled from a 100 km area in rachuonyo south in the western kenyan highlands . the analysis was focused on p. falciparum seropositivity data from about 13,000 individuals with complete data . combined seropositivity for ama1 and msp1 antigens was calculated using the two - component gaussian mixture model approach for determining seropositivity to each antigen . rcm with stable malaria transmission was then fitted to the data using maximum likelihood methods . when the observed seropositivity of each individual was aggregated to the household level and plotted on a map , there is a substantial variation within the study area ( figure 5(a ) ) . however , the large amount of variation renders it difficult to delineate hotspots of seroprevalence . with this in mind , the previous analysis was then refined in order to take into account available information on gender , elevation , residing or not in a house that received indoor residual spraying in the previous 12 months , and sleeping or not under a bednet the previous night . similar to the jacareacanga example , bayesian methods were applied to the data using noninformative prior distributions for the regression coefficients and srr . since a two - component gaussian mixture model was used for determining seropositivity to each antigen , this extended analysis focused on the rcm model given by ( 6 ) where scr was described by a log - linear regression model including the above - mentioned covariates . posterior estimates highlighted a significant role of elevation on scr while the remaining covariates , despite explaining some individual variation in seropositivity , were not statistically significant in the regression model ( results not shown ) but were maintained due to their known impact on malaria . a new map based on the posterior means of scr for each individual aggregated to the household level was then generated ( figure 5(b ) ) . this map suggested that significant variation in scr exists within this 100 km study area and identifies households with high scrs . the identification of these putative hotspots of exposure may be instrumental to design future interventions in the study area . in all models described above , the information on antibody titres is reduced to the proportions of seropositive and seronegative . alternatively one can analyze data of antibody titres themselves using the antibody acquisition models [ 50 , 51 ] . in these models , one assumes that the rate at which antibody levels are acquired can be used as a marker for transmission intensity . if an individual 's antibody level a is boosted at rate (t ) and decays at rate r then antibody levels can be described by the following ordinary differential equation : ( 19)dadt=tra.when malaria transmission is constant over time , the same is assumed for the rate of generation of antibodies in response to infection ; that is , (t ) = . under the initial condition of a(0 ) = 0 , the above equation can be solved analytically to give(20)at=r1ert , where t is again regarded as the age of an individual at data collection . likewise for seropositivity - based models , historical changes in malaria transmission intensity can also be accounted for . for example , if there was an abrupt reduction in transmission years before data collection such that the rate of acquisition of antibodies changed from 1 to 2 , then the expected antibody titre of an individual with age t is(21)at=2r1ert , t,1r1ertert+2r1er,t>.the above equation is explained as follows . for individuals born after the change in transmission ( t ) , the expected antibody dynamics follow exactly as in the constant transmission scenario but with boosting rate 2 ( see ( 20 ) ) . for the individuals born before the change in transmission , one can partition the antibody levels into two terms , the first one referring to the expected antibody levels produced until the change point with boosting rate 1 discounted by an exponential decay with rate r until present time and the second one referring simply to the antibody counts expected to be produced since the change point . equations ( 20 ) and ( 21 ) provide expressions for an individual 's antibody titre as a function of age . however , in a population of individuals there is likely to be substantial variation in antibody titres . as mentioned earlier for seropositivity determination , antibody titre data are often approximately gaussian distributed on a log scale . therefore , when constructing the sampling distribution for the comparison of the antibody acquisition model with data , one can assume that at age t antibody data is log - normally distributed with parameters = log(a(t ) ) and . note that in this interpretation a(t ) is the geometric mean titre ( gmt ) at age t ( corresponding to the mean on a log scale ) . for a random sample of n individuals , the respective sampling distribution is given by(22)fxi ,n=i=1n1xi2elogxilogati2/22,where xi and ti are the antibody titres and age of the ith individual , respectively , and is the parameter vector associated with antibody acquisition model under analysis . this parameter vector is given by = ( , r , ) or = ( 1 , 2 , , r , ) if fitting the model with constant malaria transmission intensity or fitting the model with an abrupt reduction in transmission , respectively . since the above models are nonlinear , parameter estimation can be performed by nonlinear least squares available for the r software or by bayesian methods via mcmc . for cases where the data are not well described by a log - normal distribution , alternative sampling distributions will need to be constructed . for example , if a proportion of the population has never been exposed to malaria ( i.e. , their antibody titres are just background responses ) , then a zero - inflated log - normal distribution could be used as an alternative sampling model . statistical methods to fit that distribution to data can be found elsewhere [ 5254 ] . example i ( bioko island continued ) . to extend previous analysis based on seropositivity data , antibody titre data from northeast and northwest regions of bioko island the respective data is shown in figures 6(a ) and 6(b ) . to compare with previous results , the above antibody acquisition models assuming a constant malaria transmission intensity ( figures 6(c ) and 6(d ) ) and an abrupt reduction in malaria transmission ( figures 6(e ) and 6(f ) ) were fitted to each data set separately . again , there was evidence for a constant malaria transmission intensity in the northwest region of the island ( figure 6(c ) ) with an average increase of antibody titres of around 60.4 units per year of exposure ( table 4 ) . in contrast , the antibody acquisition model with constant transmission intensity showed some fitting deficiencies at younger ages for the northeast region ( figure 6(d ) ) , which were eliminated by assuming a drop in malaria transmission intensity ( figure 6(f ) ) . that drop in malaria transmission intensity seemed to have occurred 7 years before sampling , an estimate consistent with the one obtained from seroprevalence data ( 6 years before sampling ; table 2 ) . according to posterior estimates in table 4 , these estimates suggested a reduction of 84% in malaria transmission intensity , which is in close agreement with a reduction in scr of 89% estimated from the superinfection model ( table 2 ) . malaria eradication and elimination are currently in the agenda of various countries worldwide , such as sri lanka or haiti . with this mind , an important question naturally arises : how can one declare if elimination or eradication was actually achieved ? again , serology can help answering this question due to its capacity to detect recent malaria exposure in an apparently asymptomatic population . as discussed above , the first step of serology data analysis is typically to determine seropositivity from titre data . however , in a malaria elimination setting , seroprevalence is supposedly low in the population , thus , making it difficult to discriminate whether the data comes from a single gaussian distribution or from a gaussian mixture model . in this context , the presence of a single gaussian distribution in the data can be interpreted as indicative of a seronegative population only , thus , suggesting malaria elimination ( or eradication ) . on the other hand , the detection of a mixture distribution indicates the presence of seropositive individuals that might be on their way to seronegativity but also might have been exposed to malaria after a putative elimination event . hence , sample size determination before data collection ensures to some extent the accuracy of the findings . however , sample size determination is theoretically challenging in the context of mixture distributions because standard asymptotic theory for hypothesis testing does not hold . this implies using less - known statistical methods , such as the bootstrap approach proposed by mclachlan or the adjusted likelihood ratio test derived by lo et al . . limited sample size guidelines exist for these alternative methods and , therefore , future research is needed to better help designing malaria surveillance based on serology data . under the assumption of detecting a mixture distribution in the antibody titre data , it was shown above how to infer different sources of heterogeneity in malaria transmission intensity as long as the correct antibodies are analyzed . in this regard , recent research produced a list of putative antigens that can be used in malaria elimination studies for being highly informative on the time to most recent infection . although malaria elimination and eradication are more likely to be achieved by a slow decrease of malaria transmission intensity towards 0 , a reasonable analytical starting point is to aim at using the above rcm and sim under the assumption of an abrupt reduction in scr at a given time point before sampling collection . in a malaria elimination setting , current scr of these models is set at 0 while past scr should be estimated from the data . the corresponding age - adjusted seroprevalence curve shows a distinctive pattern where children born after the time of malaria elimination are all seronegative while the remainder might or not be seropositive depending on the past malaria exposure before malaria elimination ( figure 7(a ) ) . these latter individuals would slowly revert to a seronegative state over time . for certification purposes , these models must be compared to the ones assuming a ( very low ) stable malaria transmission intensity for the same data . therefore , one needs to understand whether data has enough statistical power for detecting disease elimination . to overcome eventual problems later in a study this problem has recently been tackled for estimating the scr in stable malaria transmission intensity settings . general guidelines for sample size determination are difficult to put forward because estimation precision and statistical power are intimately related to the age distribution associated with a given study design . in this regard , african studies are more facilitated than those conducted elsewhere because the age distribution tends to be consistent across populations with a decreasing trend from newborns to elders ( figure 7(b ) ) . if such distribution is combined with the expected age - adjusted seroprevalence , one can have an idea of the evolution of overall seroprevalence over time ( figure 7(c ) ) . since age distribution can vary from one study to another , the minimum sample size for detecting malaria elimination is better calculated by means of data simulation using the expected age distribution for the sample together with the most likely age - adjusted seroprevalence curve for the malaria elimination . as an example , figure 7(d ) shows the power to detect malaria elimination as a function of sample size under the assumption of simple random sampling from a typical african population where the past scr was conceptually equivalent to 0.1 infectious bites per person per year . as expected , the required sample size decreases with time of the malaria elimination event . for a 90% power , detecting malaria elimination occurring 3 , 5 , and 10 years before data collection requires sample sizes of 1,000 , 500 , and < 250 individuals , respectively . it is worth noting that the choice of a particular sample strategy involves weighting ethical issues , availability of human and economic resources , and so forth , in order to be officially approved and feasible in real time . moreover , targeting or oversampling specific age groups might be alternative sampling strategies to reduce sample size . in conclusion , serology data in conjunction with mathematical modelling provides a powerful approach to inform epidemiologists on malaria transmission intensity and its putative changes over time . however , serology - based analysis needs to be complemented with any additional data available that would provide an external validation to the serological findings . imagining the best model for a given data set assumes a constant malaria transmission intensity over time . checking the official statistics of the malaria cases if available might shed some light on whether such assumption holds true in reality . similar rationale can be applied to situations where a drop in malaria transmission intensity was detected on the data . this approach of using official data to consolidate serological finding was indeed followed by the study in the brazilian amazonia region here analyzed . in this study , the seroconversion rates for p. vivax antigens from several sites with different malaria endemicity levels were highly correlated with the corresponding annual parasite indexes compiled by the brazilian health authorities , suggesting that the serological analysis was capturing the epidemiology of the study sites . in the same line of thought , another study found that seroconversion rates were highly correlated with the parasite rates in northeast tanzania . notwithstanding this good agreement between serological findings and other data , it is worth mentioning that the mathematical models for serology data are a simplified abstraction of the real world ; a discussion about how robust these models are in practice can be found elsewhere . however , in the words of the statistician george box , all models are wrong but some are useful and that seems to be the case for the mathematical models here presented . two main limitations can be pinpointed to the use of a serological approach to malaria epidemiology . the most obvious one is related to which antigens are epidemiologically informative . with this respect , the two antigens most used in practice are msp1 and ama1 due to their immunogenicity together with the existence of optimised experimental protocols . research efforts are currently carried out in order to identify the panel of antigens that would provide the best characterization of the epidemiological status quo of a population [ 20 , 58 ] . however , these new identified antigens remained to be fully tested in subsequent field studies . one less obvious limitation is the putative lack of statistical power to estimate the seroreversion rate and its putative changes throughout life . this is very clear in low transmission settings where only a few seronegative individuals might result from seroreversion events . however , in practice , seroreversion rate is estimated indirectly via cross - sectional data , possibly leading to poor estimation precision as discussed in depth elsewhere . possible solutions for this problem include using prior information in the analysis or analyzing data from different populations together in order to borrow information from samples where the estimation of seroreversion rate is facilitated . several future challenges were identified in particular in the context of malaria elimination and eradication . it is worth noting that a serology approach should not be seen as strict to malaria epidemiology with the potential of being applicable to other infectious diseases as long as these are capable of triggering an antibody - mediated immune response in the host . in particular , antibody data is particularly useful to track down the transmission intensity of some neglected tropical diseases , such as trachoma , chagas [ 40 , 43 ] , or dengue [ 57 , 59 ] , due to their low endemicity and the lack of clinical symptoms of most infections . however , this requires a deeper knowledge of the antibodies with the highest potential of informing the underlying disease transmission intensity . an interesting idea with public health potential is to use a panel of multidisease antibodies that can be instrumental to know what the infectious agents are in circulation in a given population and their putative dynamics . this idea has not been tested in practice but definitely will require extending the above mathematical models to fully account for the immunological interaction between different diseases .

the last decade has witnessed a steady reduction of the malaria burden worldwide . with various countries targeting disease elimination in the near future , the popular parasite infection or entomological inoculation rates are becoming less and less informative of the underlying malaria burden due to a reduced number of infected individuals or mosquitoes at the time of sampling . to overcome such problem , alternative measures based on antibodies against specific malaria antigens have gained recent interest in malaria epidemiology due to the possibility of estimating past disease exposure in absence of infected individuals . this paper aims then to review current mathematical models and corresponding statistical approaches used in antibody data analysis . the application of these models is illustrated with three data sets from equatorial guinea , brazilian amazonia region , and western kenyan highlands . a brief discussion is also carried out on the future challenges of using these models in the context of malaria elimination .

tillet and francis ( 1930 ) discovered the presence of crp in the serum of patients with pneumonia , but it was not actually isolated until 1941 . the name is derived due to the ability of the crp to react with c - polysaccharide isolated from pneumococcal cell walls . crp is synthesized by hepatocytes and is classified as an acute phase protein on the basis of its increase in plasma concentration during infection and inflammation and is a golden marker of inflammation . pepys and baltz ( 1983 ) suggested that crp is synthesized by the liver in response to diverse inflammatory stimuli , including heat , trauma , infection , and hypoxia . a major function of crp , a component of innate immune system , is its ability to bind phosphocholine and recognize some foreign pathogens as well as phospholipids of damaged cells . it can activate complement system when bound to one of its ligands and can also bind to phagocytic cells , an observation suggesting that it can initiate the elimination of targeted cells by its interaction with both humoral and cellular effector systems of inflammation . other proinflammatory effects of crp include induction of inflammatory cytokines and tissue factor in monocytes . it is thought to assist in complement binding to foreign and damaged cells and affect the humoral response to disease . crp has the ability to prevent the adhesion of neutrophils to endothelial cells by decreasing the surface expression of l - selectin , inhibit the generation of superoxide by neutrophils , and stimulate the synthesis of interleukin-1 ( il-1 ) receptor antagonist by mononuclear cells . crp has also been reported to stimulate tissue factor production by human peripheral blood monocytes and has a procoagulant effect . it has also been reported that crp recruits monocytes by receptor - mediated chemotaxis into the arterial wall . the plasma levels of crp in most healthy subjects is usually 1 mg / l , with the normal being defined as < 10 mg / l . plasma levels increase within 4 - 6 h after initial tissue injury and continue to increase several hundredfold within 24 - 48 h. crp remains elevated during the acute phase response and returns to normal with restoration of tissue structure and function . the rise in crp is exponential , doubling every 8 - 9 h. the half - life is less than 24 h. the amount of crp produced by the body varies from person to person , and this is affected by an individuals genetic makeup ( accounting for almost half of the variation in crp levels between different people ) and lifestyle . after stimulation of the hepatocytes by cytokines , levels of crp in the blood start to increase within 6 h. these concentrations can increase up to 1000-fold or even more . smoking and obesity are positively correlated with crp levels , whereas weight loss and cessation of smoking decrease crp values . the clearance rate of crp is constant , therefore the level of crp in the blood is regulated solely by synthesis . a single high crp value must be followed by re - sampling when it is above 1.75 mg / l for men , above 1.00 mg / l for no oral contraceptives ( oc)-using women , and above 2.00 mg / l for oc - using women . elevated crp levels reflect certain low - grade infections associated with chd , periodontitis , and infections caused by cytomegalovirus and chlamydia pneumoniae . early laboratory methods were only qualitative in nature until the late 1970s when significant advances in isolating crp and measuring to the picogram range were made . most clinical laboratories now use laser nephelometric assay because of its ease of use , speed , and reproducibility . various analytical methods are available of crp determination , such as enzyme - linked immunosorbent assay ( elisa ) , immunoturbidometry , rapid immunodiffusion , and visual agglutination . people with normal body mass index showed twofold difference in average crp between those who had periodontal disease and those who did not , but the difference decreased with increasing body mass index and was negligible among severely obese people . interleukin-6 ( il-6 ) and crp represent the most sensitive markers used to evaluate the inflammatory status of an individual . established risk factors for high - normal values of crp within the general population include older age , cigarette smoking , chronic bacterial infections , and chronic bronchial inflammation . higher crp levels tend to be found in individuals who smoke , have high blood pressure , are overweight , and do not exercise , whereas lean , athletic individuals tend to have lower crp levels . large amounts of crp are produced by hepatocytes in response to circulating cytokines , such as tumor necrosis factor ( tnf)- and il-1 , produced at the site of tissue destruction . this crp production by hepatocytes occurs at the expense of albumin and other constitutive proteins , a process labeled reprioritization of hepatic protein synthesis . however , competing demands for protein synthesis in cases of acute , overwhelming inflammation can lead to anomalous short - term changes in acute - phase reactant . napoli et al . showed elevated levels of crp in conditions such as acute stroke , surgery , and cancer . localized infections resulting in increased inflammation and tissue loss in the periodontium elicit systemic host changes manifested by an increase in acute phase reactants . several inflammatory mediators are elevated in peripheral blood in subjects with periodontal disease , suggesting that periodontal inflammation either contributes directly to the elevation of the concentration of these substances in peripheral blood or signals distant organs ( e.g. the liver ) to produce them . these proteins may have deleterious effects on other target organs ( e.g. heart , brain ) by modulating disease processes such as atherosclerosis . this is significant because crp is a widely accepted measure of the level of systemic inflammation , and increases in crp levels are associated with an increased risk of atherosclerosis . in patients with both atherosclerosis and periodontal disease , periodontal disease also may lead to transient increase in circulating levels of il - i , tnf- , and prostaglandin e2(pge2 ) . this may be the first step in the contribution of periodontal diseases to systemic inflammation . elevated levels of crp , il-6 , and neutrophils in patients with peridontitis may occur when bacteria and bacterial products , and cytokines enter the ciruculatuion . the total volume of inflamed periodontal tissue may also play a role and there is tendency for higher crp levels in generalized periodontitis compared to localized periodontitis . it has been established that the extent of bacteremia is directly related to the severity of the periodontal inflammation . subsequently , the systemically dispersed bacteria and lipopolysaccharide ( lps ) , as well as cytokines from the periodontal lesion , may stimulate hepatocytes and circulating leukocytes to produce crp and il-6 , respectively . dentate people with extensive periodontal disease had an increase of approximately one - third in mean crp and a doubling in the prevalence of elevated crp , compared with periodontally healthy people . one pathway of the model proposes that periodontal disease occurs as a joint response to local pathogens and to an underlying hyperinflammatory trait , which also causes elevation of systemic inflammatory mediators . however , an additional synergistic mechanism is proposed in which local periodontal infection creates an elevated systemic inflammatory response that potentiates the increase in crp caused by systemic risk factors . the presence of periodontal pathogens porphyromonas gingivalis , prevotella intermedia , camphylobacter rectus , and bacteroides forsythus in subgingival samples was positively associated with elevated crp levels . the lps and inflammatory cytokines that are present in periodontal disease may increase the expression of leukocyte adhesion molecules such as intercellular adhesion molecules or vascular cell adhesion molecules , or by endothelial cells , which in turn is associated with atheroma formation . crp localizes with complement in human heart during myocardial infection , suggesting that it binds to diseased muscle tissue , fixes complement , and hence triggers complement - mediated inflammation that contributes to atheroma formation . periodontal infections may be associated with an increased risk of atherosclerotic processes and strokes , in part via the association of periodontal infections with elevated levels of crp . intraoral source of infection can create a systemic inflammatory response , therefore placing apparently healthy patients at increased risk of cardiovascular disease ; such an association could also represent a mechanism underlying recent epidemiological findings that oral diseases appear to be risk factors for cardiovascular disease . moderate increase in crp ( i.e. > 2.11 mg / l ) in healthy population was a risk factor for myocardial infarction and stroke . crp , il-6 , and neutrophils may contribute in part to the mechanism for the observed associations between chronic infections and cardiovascular diseases . il-6 has pro - inflammatory properties and procoagulant effects ; these properties may contribute to the pathogenesis of coronary syndromes . the elevated levels of neutrophils affect blood rheology ; they may adhere to endothelial membranes and release harmful oxygen radicals and proteolytic enzymes , and in this way also contribute to increase inflammatory activity in atherosclerotic lesions . the clinical relevance of much smaller increase in crp has been highlighted recently in epidemiological studies demonstrating that individuals with so , moderate elevation of crp in apparently healthy individual is a risk factor for cardiovascular disease . lps and other bacterial components can activate an impressive cascade of inflammatory cytokines that play a role in atherosclerotic heart disease , either through a direct action on the vessel wall or by inducing the liver to produce acute phase proteins . combination of chronic infection and elevated crp level is associated with a significantly higher chd risk than either of these factors alone . it also induces complement activation , leading to an increased inflammatory response that could increase the likelihood of lethal arrhythmias or the volume of ischemic tissue . crp production by the liver is also stimulated by interleukin and promotes leukocyte adhesion that results in enhanced recruitment of monocytes to atherosclerotic plaques , thus supporting thrombus formation . this is significant because crp is a widely accepted measure of the level of systemic inflammation and increases in crp levels are associated with an increased risk of atherosclerosis ( acs ) . in patients with both acs and periodontal disease , crp levels were elevated above the level seen with only one disease or the other . periodontal disease also may lead to transient increase in circulating levels of il - i , tnf- , and prostaglandine2(pge2 ) . this may be the first step in the contribution of periodontal diseases to systemic inflammation . crp , the classical marker of acute phase response , is an indicator of a variety of pathological processes including infection , tissue damage , and chronic inflammatory disease . as the goal of modern health care continues to shift from an attitude of treatment to one of prevention , investigations will increasingly be directed toward elucidating predisposing factors that lead to atherosclerosis and developing appropriate early intervention . periodontitis itself is endemic in many countries , despite the fact that treatment modalities have proven successful over the long term and preventive measures are well understood . periodontal diseases , like atherosclerosis , are an inflammatory disease . herein may lie the most plausible link between the two diseases , in that periodontal disease may be contributing to a heightened systemic inflammatory state that , in turn , contributes to the progression or exacerbation of atherosclerosis . it will be incumbent on all dental professionals to take appropriate measures to both counsel patients in the prevention of periodontal diseases and , where necessary , arrange treatment for affected individuals to receive appropriate care in a specialist setting .

periodontitis is a local inflammatory process mediating the destruction of periodontal tissues , triggered by bacterial insult . recent evidence suggests the presence of chronic inflammatory periodontal disease may significantly affect systemic health conditions such as coronary heart disease , stroke , or adverse pregnancy outcome . c - reactive protein ( crp ) is an acute phase protein which reflects a measure of the acute phase response . crp is used as one of the markers of choice in monitoring the acute phase response because it increases to a relatively high concentration compared to basal concentration . crp has been shown to predict cardiovascular ( cv ) mortality in recent studies , and elevated crp levels have been observed in middle - aged patients with periodontitis . combination of chronic infections like periodontitis with elevated crp is associated with higher chronic heart diseases . the recognition of the relationship between periodontal diseases and atherosclerotic events is relatively recent and mostly based on the inflammatory hypothesis of atherosclerosis . periodontal disease is one of the risk factors for cardiovascular disease and possibly one of its causes . hence , even associations of modest magnitude have a large impact . the cost to the society directly attributable to atherosclerotic sequelae is very large . periodontitis is treatable ; moreover , it is preventable . experimental conformation of this shows that another widely prevalent and preventable contributor to the burden of cardiovascular disease would be added to the options available of the clinicians and public health practitioners for the control of the epidemic of cardiovascular disease .

the mathematics covered by earnshaw s theorem are based on a system where magnetic materials are attracted to each other by a force that is inversely proportional to the distance squared between them . for such a situation , earnshaw s theorem states that the laplacian of a magnetic particle s potential energy in the applied magnetic field is at best equal to zero ( u = dx2u + dy2u + dz2u = 0 ) . thus , the curvature of the potential energy for any particle at any location can not be positive ( u can not be greater than 0 ) and so earnshaw s theorem states that it is not possible to form a stable equilibrium ( an energy well ) between magnets . in earnshaw s words , with parenthetical text added for clarity : it may be observed also that the instability can not be removed by arrangement [ of the particles or external magnets ] for though the values of dx2u , dy2u , dz2u depend upon the arrangement of the particles , the fact that one at least must be positive and one negative depends only upon the equation dx2u + dy2u + dz2u = 0 which is true for every arrangement . earnshaw s mathematical formulation has been applied to a magnetizable particle under the influence of a static magnetic field . the potential energy of a magnetic particle is u = 0mh , where the dot product of the particle magnetization m and the applied magnetic field h is multiplied by the permeability of free space 0 . without magnetic saturation , the particle magnetization is m = vh , where the magnetic field is multiplied by the material magnetic volume susceptibility and the particle volume . in other words , the potential energy of the particle typically , small particles align with the magnetic field first before moving along the magnetic field gradient . by using maxwell s magnetostatic equations , it can be shown that the energy of a small particle that has undergone such an alignment is u = (|hx| + |hy| + |hz| ) , where = 0v . since the elements within the parentheses are always positive , and since is always positive for ferri- , ferro- , or paramagnetic particles , the resulting system is unstable ( the curvature of the magnetic potential energy can never be positive : u 0 ) . in the case of diamagnetic materials ( e.g. , water , pyrolytic graphite ) , however , the magnetic constants of diamagnetic materials are orders of magnitude smaller than for ferromagnetic materials , implying that extremely strong magnetic fields and magnetic field gradients are required in order to push or concentrate diamagnetic materials . the instability stated in earnshaw s theorem implies that a distribution of particles can never be focused to a central target by using external static magnets . this implication has been cited by investigators in the field of magnetic particle therapeutics as a major challenge . figure 1 illustrates how various potential energy shapes impact a distribution of ferri- , ferro- , or paramagnetic particles . under the u 0 curvature constraint stated by earnshaw , magnetic field configurations can be made to generate magnetic forces that spread particles out by creating a magnetic energy peak ( figure 1a ) . alternatively , a magnetic energy saddle point can be made that generates magnetic forces pushing particles together in one direction , but as described by earnshaw s theorem , this saddle point will also create forces spreading the particles out in another direction ( figure 1b ) . to date , there has been no demonstration of how to create a magnetic energy well that generates forces capable of focusing all particles to a central location ( figure 1c ) . if such a magnetic energy well was generated , it is possible that it could be used to focus particles to a central target deep within the body . a magnet configuration creates a magnetic potential energy surface ( top row ) that generates the magnetic forces . particles will move from locations of high - energy states ( white ) to low - energy locations ( black ) . equivalently , particles will move due to either divergent forces ( blue arrows ) or convergent forces ( red arrows ) . by applying earnshaw s theorem to static magnetic fields , only unstable static magnetic potential energy configurations were theorized to be possible , e.g. , ( a ) a peak energy configuration and ( b ) a saddle . ( c ) through the use of pulsed magnetic fields , we have achieved the creation of a magnetic potential energy well on - average that is capable of concentrating particles to a central target . if we no longer consider the case of static magnetic fields and instead broaden our consideration to include the possibility of transient magnetic fields , then it becomes possible to choose a magnetic field configuration that can focus magnetic materials to a central target . if instead of using spherical particles we use rods , which align with the magnetic field only after they have already begun moving along the magnetic field gradient , we can effectively reverse the sign in earnshaw s curvature constraint and achieve an energy well ( a stable equilibrium ) . in this work , we experimentally show in vitro that , by quickly pulsing magnetic fields , ferromagnetic rods can be forced to temporarily invert their magnetic potential energy shape , thereby concentrating an arbitrary number of ferromagnetic rods to a central target . rods have been shown by previous reports to have in vitro and in vivo efficacy of nanorod - based gene delivery . we are currently working to demonstrate our approach in vivo , and also to show the safety of our method , and those results will be reported in future publications . if successful , combined with clinically available magnetic fields and gradients , this method opens the possibility of effectively delivering therapies to target areas deep within the body by using external magnets . the main idea behind our technique can be conceptualized by observing the behaviors of toy bar magnets . at short time when these small rods are near a large magnet , the rods poles closest to the magnet will determine the rods behavior ( see figure 2 ) . in one configuration , if the poles of the rods nearest the magnet are opposite , then the rods will be attracted toward the magnet ( figure 2a ) . in another configuration , if the poles closest to each other are the same , then the magnet will repel or push the rods away ( figure 2b ) . regardless of whether the rods are being attracted or repelled , the magnet is simultaneously rotating the rods so that the rods closest poles will be opposite to the magnet s closest pole . therefore , given enough time , ( a ) when the rods are aligned with the magnet ( e.g. , the north pole of the magnet near to the south poles of the rods ) , then the rods are attracted to the magnet . ( b ) when the rods are antialigned with the magnet ( e.g. , north poles of the magnet near to the north pole of the rods ) , then the rods are pushed away from the magnet . our technique capitalizes on the ability of magnets to repel ferromagnetic rods that are antialigned with the magnet ( figure 2b ) . specifically , our technique has first been applied to ferromagnetic rods due to their stabilized magnetic domains , which allows their behavior to more closely resemble the behavior of tiny bar magnets . we first apply a uniform magnetic field that orients the rods in one direction . we then apply a magnetic field gradient that is antialigned with the rods ( e.g. , the closest pole of the magnet matches the closest pole of the rod ) so as to ensure the initial movement of the rods is away from the magnet . mathematically , the potential energy ( u = 0mh ) of such antialigned rods will be inverted since the rod magnetization is now opposite that of the magnetic field ( e.g. , m/m = h/h ) . this type of inversion ultimately allows for the creation of a magnetic energy well , as the laplacian of the magnetic potential energy will now initially be greater than zero , u > 0 . through this technique of dynamically inverting the magnetic forces , we attain well - like curvatures of magnetic potential energy surfaces . the key technical component of our technique is to invert the magnetic potential energy by the exploitation of the rotational dynamics of the ferromagnetic rods . this is accomplished by actuating two electromagnets around the rods to transiently generate a uniform polarizing magnetic field that rotates the rods so that the rods alignment matches the magnetic field alignment ( figure 3a ) . this places the north poles of the rods to match to the north pole of the external field and the south poles of the rods to the south pole of the field . since this uniform field has no spatial field gradient , it does not translate the rods . the uniform field is removed once the rods are aligned , at which time a transient gradient field ( from only a single electromagnet ) is applied that is antialigned with the polarizing field . since the new gradient field is antialigned with the polarizing field , it is also antialigned with the rods prior orientations . as a result , the rods will be repelled by this applied gradient field ( figure 3b ) . ( a ) using two electromagnets , a uniform magnetic field is applied that rotates the rods to match the field . ( b ) a gradient that is opposite in alignment from the rods is then applied causing the rods to move away from the magnet . the sequential application of two magnetic fields , one to polarize and the other to push , can momentarily repel rods away from a magnet . however , the rods can only be repelled while they remain anti - aligned with the gradient field . during the application of the transient gradient field , the rods will be repelled and will also begin to rotate to match the alignment of the gradient field . once rods match the gradient field alignment , they will no longer be repelled and will begin to be attracted toward the magnet . therefore , there exists a maximum time duration of the gradient field before rods eventually rotate their magnetization and are attracted to the electromagnet . the transient repulsion described above ( instead of the usual attraction of particles to magnets ) switches the sign in earnshaw s formulation . it converts u 0 to u 0 and allows the inversion of an energy peak ( figure 1a ) into an energy well ( figure 1c ) . to achieve such an energy well on average , we use pulsed electromagnets arranged in two dimensions around a collection of ferromagnetic rods ( see figure 4 ) . the sequence of activations shown in figure 4 is designed to concentrate rods in two - dimensions by subsequently aligning and repelling rods in four directions . as each act of repulsion has a magnetic potential energy curvature greater than zero , the magnetic potential energy curvature on average will also be greater than zero , and an energy well will be formed . it follows that , by applying a repulsive force in both the positive and negative z direction , the rods can be focused in three dimensions . these 8 steps can be repeated multiple times to concentrate the ferromagnetic rods at a central location . the particles are ( 1 ) rotated to match a horizontal field , ( 2 ) repelled to the right , ( 3 ) rotated to match a vertical field , ( 4 ) repelled upward , ( 5 ) rotated to match a horizontal field , ( 6 ) repelled to the left , ( 7 ) rotated to match a vertical field , and then ( 8) repelled downward . to better understand the maximum allowable duty cycle of the repulsive gradient field , it is important to discuss the two types of effects behind the rotation of the magnetization of the rods : brownian and nel relaxation . brownian relaxation describes the alignment of a particles magnetization with the field due to the physical rotation of the particles within the fluid . nel relaxation describes the shifting of the particles magnetic domains so that they match the external field . usually , the hydrodynamic resistance for reorienting the particles in space is less than the magnetostatic resistance for reorienting the particles poles ( e.g. , for ferromagnetic particles ) . typically , small superparamagnetic particles have short nel and brownian relaxation times ( < 100 ns ) , while larger ferromagnetic and paramagnetic rods have much longer relaxation times > 100 s . to achieve effective continuous repulsion of the ferromagnetic rods , the pulse duration of the magnetic gradient field should be less than the time it takes for the rods to realign with the gradient field . this is easier to achieve with rods compared to spheres , where the elongated shape of a rod grants the particles a higher moment of inertia . under brownian relaxation , cobalt rods ( with a length of 200 m , diameter of 200 nm , and a magnetic susceptibility of 0.65 ) would take approximately 500 s to rotate and match a perpendicular magnetic field of 0.5 t. in the experiments described below , we applied the gradient field for 50 s , which is a much shorter time than it takes the rods to rotate . since the rod magnetization will be opposite to the gradient field during the time the transient gradient field is applied , the rods will be repulsed by each electromagnet . consequently , the magnetic potential energy can be inverted and , therefore , allows for the rods to be focused . to demonstrate the dynamic magnetic inversion technique , we first built a system to push rods in one dimension . this system was used to confirm our hypothesis that the forces produced from the pulsed magnetic fields acting upon the ferromagnetic rods would indeed invert the magnetic potential energy shape . both the one - dimensional and two - dimensional experiments are detailed in the supporting information . next , we built a system to experimentally demonstrate that dynamic magnetic inversion could focus cobalt rods in two dimensions ( 2-d ) . the 2-d magnetic focusing experimental system consisted of two helmholtz coils ( square diameter of 5 cm with 44 turns per side powered by a maximum of 11 a ) for polarizing the rods , four gradient coils ( 2 cm diameter coils , 2 cm in length , with 120 turns powered by a maximum of 66 a ) for transiently repulsing the rods , a usb camera ( celestron model 44302-a ) for optical visualization of the rods , and a custom - built system using high voltage relays ( gigavac gh1 ) that controlled which coils received current . all coils surrounded a 25 mm 25 mm sample region ( see figure 5 ) . experimental system for dynamic magnetic inversion that repulses and concentrates rods in two - dimensions . ( a ) schematic showing a computer controlling both the square pulse signal generator and the relay controller . pulsed power supplies provided current to the high voltage relays , which passed the current into a specified pair of coils . any axis can therefore be polarized in any direction as dictated by the computer graphical user interface . ( b ) pulse sequence element for inverting the energy surface of ferromagnetic rods and concentrating them at the center of the sample area . this pulse sequence element is repeated many times for the four directions to push and concentrate the rods to the center . the applied sequence of magnetic fields and gradients used to concentrate rods to the center is shown schematically in figure 4 . cobalt rods ( 5 mg of 200 m long 200 nm , from plasmachem gmbh , cat . pl - cow200 ) were suspended within a 12 mm vial containing a 1:5 solution of hexane : isopropanol ( solution used for reduced viscosity , however water can alternatively be used ) . the rods were dispersed by gently shaking the vial , making them initially undetectable to the unaided eye . the vial was placed within the sample region of the 2-d pulsed magnet system ( figure 5a ) . last , we applied a train of magnetic pulse sequence elements shown in figure 5b . each pulse sequence element comprises the polarizing pulse followed by a short 5 s delay time and finished with the gradient pulse . this pulse sequence element was repeated continuously and oscillated in all four directions ( + x , x , + y , and y ) as shown above in figure 4 . the applied sequence of magnetic fields and gradients concentrated the ferromagnetic rods tightly to the central target , as shown in the final panel of figure 6 . focusing has also been demonstrated for custom grown 250 nm diameter nickel rods that are 28 m in length and either without a coating or with a coating of 1 kda peg . focusing has been accomplished for all particle types in three solutions : a 1:5 hexane to isopropanol solution ; tap water ; and 1 phosphate buffered saline ( sigma aldrich ) . focusing of ferromagnetic rods to a central target . four snapshots of concentrating cobalt rods to the center of the sample area using dynamic magnetic inversion . after 09:06 min , the rods were concentrated at the center of the sample area . the experiment results shown in figure 6 illustrate that pulsed magnetic focusing worked in vitro . development of the method for safe and effective operation in vivo is the subject of ongoing work and will be reported in future publications . magnetic forces have been used to manipulate therapies in human patients , but the scope and utility of magnetic targeting using external magnets has been limited to superficial targets . we have shown in vitro that pulsed magnetic fields can exploit the rotational dynamics of ferromagnetic rods and thus attain well - like curvature of the magnetic potential energy . by applying transient magnetic gradients before the rods have had an opportunity to realign with the magnetic field , we can reverse the direction of forces and can create a stable energy well ( i.e. , inward pointing magnetic forces ) . using this approach , we focused a disperse concentration of ferromagnetic rods to a central target between eight external electromagnets . in future work , we hope to demonstrate pulsed focusing to central targets between magnets in vivo , to validate safety , and to scale - up the method to the distances anticipated in human patients . success would grant clinicians a tool capable of delivering therapy where it is needed , a key goal in magnetic drug targeting .

the ability to use magnets external to the body to focus therapy to deep tissue targets has remained an elusive goal in magnetic drug targeting . researchers have hitherto been able to manipulate magnetic nanotherapeutics in vivo with nearby magnets but have remained unable to focus these therapies to targets deep within the body using magnets external to the body . one of the factors that has made focusing of therapy to central targets between magnets challenging is samuel earnshaw s theorem as applied to maxwell s equations . these mathematical formulations imply that external static magnets can not create a stable potential energy well between them . we posited that fast magnetic pulses could act on ferromagnetic rods before they could realign with the magnetic field . mathematically , this is equivalent to reversing the sign of the potential energy term in earnshaw s theorem , thus enabling a quasi - static stable trap between magnets . with in vitro experiments , we demonstrated that quick , shaped magnetic pulses can be successfully used to create inward pointing magnetic forces that , on average , enable external magnets to concentrate ferromagnetic rods to a central location .

systolic heart failure ( hf ) is a major cause of death in adults in the united states , and although it is less commonly encountered in children , it is highly lethal , leading to death or transplantation in 46% of children within 5 years of diagnosis . children listed for heart transplantation face prolonged waiting times for organ availability and experience mortality of up to 17% while on a 30day waiting list among patients requiring mechanical ventilation . over the past decade , ventricular assist devices ( vads ) have emerged as a major tool for the treatment of endstage hf in adults , but until recently , no comparable devices were approved for use in children . this situation changed in 2011 , when the berlin heart excor pediatric vad ( excor ) was approved by the us food and drug administration for use in the united states as a bridge to heart transplantation for children with severe ventricular dysfunction ( clinicaltrials.gov identifier nct00583661 ) . the decision by the us food and drug administration was informed to a large degree by a clinical investigational device exemption ( ide ) trial using historical controls with extracorporeal membrane oxygenation . the ide trial demonstrated a successful bridge to transplantation or recovery in 88% to 92% of children supported with excor , but a high incidence of neurological dysfunction was noted in this trial ( 29% ) . this finding was substantiated in a subsequent analysis of a cohort of 204 children composed of the ide cohort and a larger compassionateuse cohort enrolled contemporaneously . in both of these reports , a recent publication of a cohort of 102 children treated at 2 sites in the united kingdom describes a similarly high incidence of 25.4% . neurological injury was also the most common cause of death in the excor ide trial . the purpose of this report is to provide a detailed analysis of the neurological adverse events for all 204 participants in the excor ide trial . the major objectives of this analysis are to describe the neurological injury , including frequency , severity , and clinical impact , and to attempt to develop a riskstratification model for neurological adverse events during support . all children who received excor between may 9 , 2007 , and december 31 , 2010 , in the united states and canada were identified using data collected by the study sponsor for its us regulatory trial . the trial design , inclusion criteria , and end points have been reported previously , as have survival rates on vad compared with extracorporeal membrane oxygenation and incidence and risk factors for mortality . the major inclusion criteria for the primary ide study cohort were age of 16 years , weight of 3 to 60 kg , 2ventricle circulation , severe hf despite optimized medical treatment , and inclusion on a waiting list for cardiac transplantation . compassionateuse access to excor was available to children at the ide sites who did not meet the inclusion criteria for the trial . compassionateuse access was also available to children who were implanted at any of 27 nonide trial sites in north america . data were gathered by study coordinators at study sites using standard data definitions and a data platform adapted from the interagency registry for mechanically assisted circulatory support ( intermacs ) database . intermacs is a federally funded us postmarket vad registry with data elements and definitions developed jointly by the academic medical community , industry , the national heart lung and blood institute , the us food and drug administration , and the centers for medicare and medicaid services . all children were followed from the time of excor implant until transplant , death , or recovery . at the 17 ide study sites , a study monitor visited each site regularly ; an independent clinical events committee adjudicated the adverse events and causes of death for each patient . for nonide sites , adverse events were reported but were not centrally adjudicated . an independent data and safety monitoring board monitored the study and reviewed outcome data for the primary study cohorts . the study protocol was approved by each center 's institutional review board , and written informed consent was obtained for all patients . nonide sites followed the ide protocol when possible and as allowed by their individual compassionateuse site approvals ; these patients were consented under compassionateuse regulations . the trial protocol specified that prior to undergoing implantation of excor , patients would receive a baseline head computed tomography ( ct ) scan and a standardized neurological examination , the pediatric stroke outcome measure ( psom ) . during the period of support , standardized medical evaluations were performed at 1 week , 2 weeks , 4 weeks , 6 weeks , and 3 months after implant and every 3 months while the participant remained on support . psom examinations were performed at the 1week , 4week , and 3month evaluations and every 3 months while the participant remained on support . if a neurological event was judged to have occurred by study site personnel , then head ct and psom were obtained . after explant , psom was obtained at 30 days or at the time of hospital discharge , whichever was longer . neurological dysfunction was defined as any new , temporary or permanent , focal or global neurological deficit ascertained by a standard neurological examination ( administered by a neurologist or other qualified physician and documented with appropriate diagnostic tests and consultation note ) . all neurological events were adjudicated by a committee that included 2 pediatric stroke neurologists and were subcategorized as follows : transient ischemic attack : an acute event that resolved completely within 24 hours , with no evidence of infarctionischemic stroke : an event that persisted > 24 or < 24 hours and that was associated with infarction on an imaging studyhemorrhagic stroke : an event that persisted > 24 or < 24 hours and that was associated with parenchymal hemorrhage or hemorrhagic infarction on an imaging studynew abnormality of head ultrasound ( if patient was aged < 6 months)electroencephalogram positive for seizure activity with or without clinical seizure ( if patient was aged < 6 months)other : adverse events that did not meet 1 of the other protocol neurological dysfunction categories ; this category included subdural and epidural hematomas and asymptomatic or clinically covert strokes transient ischemic attack : an acute event that resolved completely within 24 hours , with no evidence of infarction ischemic stroke : an event that persisted > 24 or < 24 hours and that was associated with infarction on an imaging study hemorrhagic stroke : an event that persisted > 24 or < 24 hours and that was associated with parenchymal hemorrhage or hemorrhagic infarction on an imaging study new abnormality of head ultrasound ( if patient was aged < 6 months ) electroencephalogram positive for seizure activity with or without clinical seizure ( if patient was aged < 6 months ) other : adverse events that did not meet 1 of the other protocol neurological dysfunction categories ; this category included subdural and epidural hematomas and asymptomatic or clinically covert strokes time on the device was defined as the duration of time from excor implant until transplant , recovery , or death . all clinical and demographic baseline variables were collected within a 48hour window prior to the time of excor implantation . the psom is a validated outcome scale for infants and children with stroke and is based on a conventional clinical neurological examination by a child neurologist , the findings of which are recorded and scored in a standardized manner so as to provide information about the localization , nature , and functional severity of the abnormalities in an ageappropriate manner . psom subscores are assigned in 5 areas : right sensorimotor , left sensorimotor , expressive language , receptive language , and cognition / behavior . psom subscores are graded 0 ( no deficit ) , 0.5 ( mild deficit that does not interfere with function ) , 1 ( moderate deficit that interferes with function ) , and 2 ( severe deficit with loss of function ) . , a total psom of 0.5 to 1.0 was classified as a mild deficit , 1.5 to 2.0 was classified as a moderate deficit , and 2.5 was classified as a severe deficit . an unacceptable neurological outcome was defined as any 1 or a combination of the following : comatose state , quadriparesis ( psom of 3 to 4 on motor scales ) , severe global aphasia ( psom of 3 to 4 on language scales ) , or a severe cognitive deficit ( psom of 2 on the cognition / behavior subscale ) . in this analysis , for those children with neurological events , deidentified head ct scans that were gathered during the study from the ide sites were reviewed by an independent pediatric neuroradiologist ( s.p . ) who was blinded to clinical information to determine stroke location , size , and presence of midline shift , tonsillar herniation , and extraaxial hemorrhage . summary statistics are presented as median ( interquartile range ) or number ( percentage ) . patient characteristics were compared across study cohorts using the chisquare test or fisher exact test when any expected frequency was < 5 for categorical variables and the kruskal multivariable analysis was performed using backward stepwise logistic regression with a p value of 0.05 to retain variables in the final model . all children who received excor between may 9 , 2007 , and december 31 , 2010 , in the united states and canada were identified using data collected by the study sponsor for its us regulatory trial . the trial design , inclusion criteria , and end points have been reported previously , as have survival rates on vad compared with extracorporeal membrane oxygenation and incidence and risk factors for mortality . the major inclusion criteria for the primary ide study cohort were age of 16 years , weight of 3 to 60 kg , 2ventricle circulation , severe hf despite optimized medical treatment , and inclusion on a waiting list for cardiac transplantation . compassionateuse access to excor was available to children at the ide sites who did not meet the inclusion criteria for the trial . compassionateuse access was also available to children who were implanted at any of 27 nonide trial sites in north america . data were gathered by study coordinators at study sites using standard data definitions and a data platform adapted from the interagency registry for mechanically assisted circulatory support ( intermacs ) database . intermacs is a federally funded us postmarket vad registry with data elements and definitions developed jointly by the academic medical community , industry , the national heart lung and blood institute , the us food and drug administration , and the centers for medicare and medicaid services . all children were followed from the time of excor implant until transplant , death , or recovery . at the 17 ide study sites , a study monitor visited each site regularly ; an independent clinical events committee adjudicated the adverse events and causes of death for each patient . for nonide sites , adverse events were reported but were not centrally adjudicated . an independent data and safety monitoring board monitored the study and reviewed outcome data for the primary study cohorts . the study protocol was approved by each center 's institutional review board , and written informed consent was obtained for all patients . nonide sites followed the ide protocol when possible and as allowed by their individual compassionateuse site approvals ; these patients were consented under compassionateuse regulations . the trial protocol specified that prior to undergoing implantation of excor , patients would receive a baseline head computed tomography ( ct ) scan and a standardized neurological examination , the pediatric stroke outcome measure ( psom ) . during the period of support , standardized medical evaluations were performed at 1 week , 2 weeks , 4 weeks , 6 weeks , and 3 months after implant and every 3 months while the participant remained on support . psom examinations were performed at the 1week , 4week , and 3month evaluations and every 3 months while the participant remained on support . if a neurological event was judged to have occurred by study site personnel , then head ct and psom were obtained . after explant , psom was obtained at 30 days or at the time of hospital discharge , whichever was longer . neurological dysfunction was defined as any new , temporary or permanent , focal or global neurological deficit ascertained by a standard neurological examination ( administered by a neurologist or other qualified physician and documented with appropriate diagnostic tests and consultation note ) . all neurological events were adjudicated by a committee that included 2 pediatric stroke neurologists and were subcategorized as follows : transient ischemic attack : an acute event that resolved completely within 24 hours , with no evidence of infarctionischemic stroke : an event that persisted > 24 or < 24 hours and that was associated with infarction on an imaging studyhemorrhagic stroke : an event that persisted > 24 or < 24 hours and that was associated with parenchymal hemorrhage or hemorrhagic infarction on an imaging studynew abnormality of head ultrasound ( if patient was aged < 6 months)electroencephalogram positive for seizure activity with or without clinical seizure ( if patient was aged < 6 months)other : adverse events that did not meet 1 of the other protocol neurological dysfunction categories ; this category included subdural and epidural hematomas and asymptomatic or clinically covert strokes transient ischemic attack : an acute event that resolved completely within 24 hours , with no evidence of infarction ischemic stroke : an event that persisted > 24 or < 24 hours and that was associated with infarction on an imaging study hemorrhagic stroke : an event that persisted > 24 or < 24 hours and that was associated with parenchymal hemorrhage or hemorrhagic infarction on an imaging study new abnormality of head ultrasound ( if patient was aged < 6 months ) electroencephalogram positive for seizure activity with or without clinical seizure ( if patient was aged < 6 months ) other : adverse events that did not meet 1 of the other protocol neurological dysfunction categories ; this category included subdural and epidural hematomas and asymptomatic or clinically covert strokes time on the device was defined as the duration of time from excor implant until transplant , recovery , or death . all clinical and demographic baseline variables were collected within a 48hour window prior to the time of excor implantation . the psom is a validated outcome scale for infants and children with stroke and is based on a conventional clinical neurological examination by a child neurologist , the findings of which are recorded and scored in a standardized manner so as to provide information about the localization , nature , and functional severity of the abnormalities in an ageappropriate manner . psom subscores are assigned in 5 areas : right sensorimotor , left sensorimotor , expressive language , receptive language , and cognition / behavior . psom subscores are graded 0 ( no deficit ) , 0.5 ( mild deficit that does not interfere with function ) , 1 ( moderate deficit that interferes with function ) , and 2 ( severe deficit with loss of function ) . for the purposes of this study , a total psom of 0.5 to 1.0 was classified as a mild deficit , 1.5 to 2.0 was classified as a moderate deficit , and 2.5 was classified as a severe deficit . an unacceptable neurological outcome was defined as any 1 or a combination of the following : comatose state , quadriparesis ( psom of 3 to 4 on motor scales ) , severe global aphasia ( psom of 3 to 4 on language scales ) , or a severe cognitive deficit ( psom of 2 on the cognition / behavior subscale ) . in this analysis , for those children with neurological events , deidentified head ct scans that were gathered during the study from the ide sites were reviewed by an independent pediatric neuroradiologist ( s.p . ) who was blinded to clinical information to determine stroke location , size , and presence of midline shift , tonsillar herniation , and extraaxial hemorrhage . summary statistics are presented as median ( interquartile range ) or number ( percentage ) . patient characteristics were compared across study cohorts using the chisquare test or fisher exact test when any expected frequency was < 5 for categorical variables and the kruskal multivariable analysis was performed using backward stepwise logistic regression with a p value of 0.05 to retain variables in the final model . during the 4year study period , 204 children were enrolled in the excor ide trial . this included 48 participants ( 24% ) who were enrolled in the original us food and drug administration ide trial cohort and 20 participants ( 10% ) who met all trial criteria but were implanted after trial enrollment was complete under a continued access protocol . an additional 136 patients ( 67% ) were implanted under compassionateuse criteria , including 95 who received excor at 1 of 27 centers not participating in the trial and 41 who were implanted at an ide center but who met at least 1 clinical trial exclusion criterion . the recorded diagnosis was cardiomyopathy in 71% ( 145 participants ) , specified as dilated cardiomyopathy in 110 , myocarditis in 28 , restrictive cardiomyopathy in 4 , and other cardiomyopathies in 3 . at the time of implantation , intermacs status was level 1 in 52% ( 107 participants ) and level 2 in 44% ( 90 participants ) , indicating very high severity of illness in the study population at the time of excor implantation . characteristics of study participants ( n=204 ) bsa indicates body surface area ; ecmo , extracorporeal membrane oxygenation ; iqr , interquartile range ; vad , ventricular assist device . in total , 73 neurological events were recorded in 59 participants ; therefore , 29% of the cohort experienced 1 neurological events . of these neurological events , fourteen participants were known to have experienced a stroke prior to implantation of excor , of whom only 1 had an additional event during the time on excor support . preimplantation strokes were evaluated as a potential risk factor in predicting strokes following implantation of excor but were not included in the postimplantation neurological event counts reported in this paper . although all sites underwent training and were provided with an anticoagulation protocol ( published as supplementary material to the primary ide trial ) , event adjudication was performed only for events reported at ide sites . there was no difference in the neurological event rate based on whether the implanting center was an ide site ( 29 affected participants of 109 total , 27% ) or a nonide site ( 30 affected participants of 95 total , 32% , p value not significant ) . the daily neurological event rate for the entire cohort was 0.51 events per 100 patientdays ; however , many of the neurological events occurred early in the course of support , with 30 events recorded during the first 14 days of support and an additional 15 events during the next 14 days . the distribution of events partly reflects the greater number of patients at risk in the first 30 days because the median duration of support for the entire cohort was 38.5 days . a hazard function analysis confirms that the risk per patientday is generally higher in the early period of support , decreasing to a comparatively low baseline by day 50 . the late spikes in hazard function seen in figure 2 are due to isolated neurological events occurring in a small residual sample size . temporal distribution of neurological events after implantation with the berlin heart excor ventricular assist device . hazard rate of first neurological event compared with the number of days of excor support . the overall freedom from neurological events was 73% at 30 days , falling further to 64% at 90 days , as shown in the kaplan meier curve shows the survival function for the probability of children implanted with excor to remain free from neurological events . of the 73 documented neurological events , 12 were defined as transient ischemic attack , 7 were described as a new abnormality of head ultrasound , 1 was an electroencephalogram finding of seizure activity , and 52 events were strokes . considering only those events described as strokes , the vast majority ( n=47 , 89% ) were ischemic , and there were comparatively few hemorrhagic strokes ( n=5 , 9% ) . head cts were available for review for all 29 children with neurological events at ide sites . of these children , 59% ( 17 of 29 ) had baseline head ct prior to implantation , and 12% ( 2 of 17 ) of the baseline head cts documented prior ischemic stroke . these 29 children had 35 neurological events : 28 were ischemic strokes , including 1 covert stroke ( ie , a symptom prompted evaluation with head ct , which demonstrated an ischemic stroke , but the neurological examination was nonfocal ) ; 4 were hemorrhagic strokes ; 2 were transient ischemic attacks ; and 1 was progressive global cerebral volume loss ( atrophy ) . in 52% of children with ischemic stroke , multiple new infarcts were present on the head ct at the time of the first recorded neurological event . the majority of ischemic infarcts ( 57% ) were in the anterior circulation , 25% were in the anterior and posterior circulation , 3% were in the posterior circulation only , and 14% were border zone or watershedtype infarcts . in 10% of the strokes , the infarcts were large , occupying more than one third of the middle cerebral artery territory . hemorrhagic transformation of ischemic strokes occurred in 3% and occurred only in the setting of a large infarct ( ie , stroke in more than one third of the middle cerebral artery territory ) . cerebral edema causing midline shift was present in 4 children , and tonsillar herniation was seen in 2 children . indicators of the severity of the neurological events can be found in the psom scores and by considering the impact of the stroke on subsequent likelihood of successful transplantation . unfortunately , for the majority of participants , a preimplantation baseline psom assessment was not performed because the patient was too ill and/or was sedated and intubated . baseline psom scores were obtained in 23 of the 68 patients ( 34% ) at ide sites who met the primary enrollment criteria and in 6 of the 41 patients ( 15% ) at ide sites who did not meet enrollment criteria . at the nonide sites , care was provided according to existing standard of care , and preimplantation psom examinations were rare ( 4% ) . of the 59 participants who had neurological events , 29 were implanted at ide sites , and of those , 25 had at least 1 psom completed , whereas 15 had psom data collected within 7 days following the event . for each of these participants , the psom following the event and the last available psom are shown in table 3 . as can be seen , in this subset of 15 patients , psom data capture and followup were essentially complete , with followup values lacking only for the 2 participants who died . for this group , the median value of the psom within 7 days after the event was 3.0 ( interquartile range 1 to 5 ) , whereas the last psom obtained for each of these participants had a median value of 1.00 ( interquartile range 0 to 3 ) . the time between the event and the last psom was a median of 136 days ( interquartile range 79 to 171 ) . the distribution of psom scores according to clinical severity is shown in figure 4 for both the initial and final psom scores . this figure includes all psom data available , even if no psom was administered immediately following the event . the proportion of patients with a psom score indicating severe neurological deficit was 53% based on the psom within 7 days of the event and 39% based on the last psom following the event ( p value not significant for change over time ) . psom scores at investigational device exemption sites following first neurological event n / a indicates not applicable due to death ; psom , pediatric stroke outcome measure . the bar graph shows neurological outcome severity as measured by the psom , when available , for children within 7 days of a neurological event and long term , at a median of 136 days ( interquartile range 79 to 171 ) after a neurological event . severity of neurological injury was further assessed by evaluating the clinical course subsequent to the injury . table 4 provides the subsequent clinical outcome for the 59 patients who experienced 1 neurological event . overall , 56% of patients who experienced a neurological event continued to successfully undergo heart transplantation . an additional patient had successful weaning from excor because of recovery of ventricular function with good neurological outcome . the remaining patients either died ( n=23 ) or were weaned from the device with subsequent mortality ( n=2 ) . the mortality rate for patients who had at least 1 neurological event was 42% ( 25 of 59 ) , significantly greater than the rate of 18% ( 26 of 145 ) for those who did not have a neurological event ( p=0.0006 ) . as can also be seen in table 4 , the mortality ( including weaning failure ) associated with neurological events did not appear to be evenly distributed between the study sites , with mortality of 17% at ide sites compared with 67% at nonide sites ( p=0.0002 ) . to determine whether this result was more likely a site effect or a difference in patients , mortality was compared between patients who did and did not meet the primary study enrollment criteria , regardless of site of care . this analysis demonstrated 58% mortality in patients who did not meet primary enrollment criteria compared with 31% for those who did meet these criteria . the findings fell slightly short of achieving the specified statistical significance ( p=0.06 ) . clinical outcome following neurological injury ide indicates investigational device exemption . among the 59 children with neurological events , 42 children had 47 ischemic strokes , and 14 of those had support withdrawn five children had hemorrhagic strokes , and 1 had support withdrawn because of the severity of the neurological injury . surgical intervention for hematoma evacuation was performed in 2 of 29 children ( 1 parenchymal hematoma evacuation and 1 subdural hematoma evacuation ) . in view of the clinical importance of the neurological events , we attempted to construct a riskstratification model using demographic and clinical variables available at the time of implantation . the variables selected and the univariate test results are shown in table 5 . these included sex , weight , cardiac diagnosis , baseline renal and hepatic function , use of extracorporeal membrane oxygenation prior to excor , use of left vad versus bivad , and pump size . in univariate analysis , sex , diagnosis of congenital heart defect , use of extracorporeal membrane oxygenation prior to excor , device type ( left vad or bivad ) , prior stroke , and any pump replacement because of thrombus all had p<0.25 and were included in the multivariate model . in the multivariate analysis , only sex and pump replacement were significant risk factors for a neurological event , with female participants and those with a pump change because of thrombus more likely to experience a neurological event . because of the manner in which the data were collected , it was not possible to reconstruct the temporal relationship between the pump exchange and the neurological event . potential risk factors for occurrence of neurological events in univariable and multivariable analyses bsa indicates body surface area ; bvad , biventricular assist device ; chd , congenital heart defect ; cns , central nervous system ; ecmo , extracorporeal membrane oxygenation ; ide , investigational device exemption ; intermacs , interagency registry for mechanically assisted circulatory support ; iqr , interquartile range ; lvad , left ventricular assist device ; or , odds ratio . during the 4year study period , 204 children were enrolled in the excor ide trial . this included 48 participants ( 24% ) who were enrolled in the original us food and drug administration ide trial cohort and 20 participants ( 10% ) who met all trial criteria but were implanted after trial enrollment was complete under a continued access protocol . an additional 136 patients ( 67% ) were implanted under compassionateuse criteria , including 95 who received excor at 1 of 27 centers not participating in the trial and 41 who were implanted at an ide center but who met at least 1 clinical trial exclusion criterion . the recorded diagnosis was cardiomyopathy in 71% ( 145 participants ) , specified as dilated cardiomyopathy in 110 , myocarditis in 28 , restrictive cardiomyopathy in 4 , and other cardiomyopathies in 3 . at the time of implantation , intermacs status was level 1 in 52% ( 107 participants ) and level 2 in 44% ( 90 participants ) , indicating very high severity of illness in the study population at the time of excor implantation . characteristics of study participants ( n=204 ) bsa indicates body surface area ; ecmo , extracorporeal membrane oxygenation ; iqr , interquartile range ; vad , ventricular assist device . in total , 73 neurological events were recorded in 59 participants ; therefore , 29% of the cohort experienced 1 neurological events . of these neurological events , fourteen participants were known to have experienced a stroke prior to implantation of excor , of whom only 1 had an additional event during the time on excor support . preimplantation strokes were evaluated as a potential risk factor in predicting strokes following implantation of excor but were not included in the postimplantation neurological event counts reported in this paper . although all sites underwent training and were provided with an anticoagulation protocol ( published as supplementary material to the primary ide trial ) , event adjudication was performed only for events reported at ide sites . there was no difference in the neurological event rate based on whether the implanting center was an ide site ( 29 affected participants of 109 total , 27% ) or a nonide site ( 30 affected participants of 95 total , 32% , p value not significant ) . the daily neurological event rate for the entire cohort was 0.51 events per 100 patientdays ; however , many of the neurological events occurred early in the course of support , with 30 events recorded during the first 14 days of support and an additional 15 events during the next 14 days . the distribution of events partly reflects the greater number of patients at risk in the first 30 days because the median duration of support for the entire cohort was 38.5 days . a hazard function analysis confirms that the risk per patientday is generally higher in the early period of support , decreasing to a comparatively low baseline by day 50 . the late spikes in hazard function seen in figure 2 are due to isolated neurological events occurring in a small residual sample size . temporal distribution of neurological events after implantation with the berlin heart excor ventricular assist device . hazard rate of first neurological event compared with the number of days of excor support . the overall freedom from neurological events was 73% at 30 days , falling further to 64% at 90 days , as shown in the kaplan meier curve shows the survival function for the probability of children implanted with excor to remain free from neurological events . of the 73 documented neurological events , 12 were defined as transient ischemic attack , 7 were described as a new abnormality of head ultrasound , 1 was an electroencephalogram finding of seizure activity , and 52 events were strokes . considering only those events described as strokes , the vast majority ( n=47 , 89% ) were ischemic , and head cts were available for review for all 29 children with neurological events at ide sites . of these children , 59% ( 17 of 29 ) had baseline head ct prior to implantation , and 12% ( 2 of 17 ) of the baseline head cts documented prior ischemic stroke . these 29 children had 35 neurological events : 28 were ischemic strokes , including 1 covert stroke ( ie , a symptom prompted evaluation with head ct , which demonstrated an ischemic stroke , but the neurological examination was nonfocal ) ; 4 were hemorrhagic strokes ; 2 were transient ischemic attacks ; and 1 was progressive global cerebral volume loss ( atrophy ) . in 52% of children with ischemic stroke , multiple new infarcts were present on the head ct at the time of the first recorded neurological event . the majority of ischemic infarcts ( 57% ) were in the anterior circulation , 25% were in the anterior and posterior circulation , 3% were in the posterior circulation only , and 14% were border zone or watershedtype infarcts . in 10% of the strokes , the infarcts were large , occupying more than one third of the middle cerebral artery territory . hemorrhagic transformation of ischemic strokes occurred in 3% and occurred only in the setting of a large infarct ( ie , stroke in more than one third of the middle cerebral artery territory ) . cerebral edema causing midline shift was present in 4 children , and tonsillar herniation was seen in 2 children . indicators of the severity of the neurological events can be found in the psom scores and by considering the impact of the stroke on subsequent likelihood of successful transplantation . unfortunately , for the majority of participants , a preimplantation baseline psom assessment was not performed because the patient was too ill and/or was sedated and intubated . baseline psom scores were obtained in 23 of the 68 patients ( 34% ) at ide sites who met the primary enrollment criteria and in 6 of the 41 patients ( 15% ) at ide sites who did not meet enrollment criteria . at the nonide sites , care was provided according to existing standard of care , and preimplantation psom examinations were rare ( 4% ) . of the 59 participants who had neurological events , 29 were implanted at ide sites , and of those , 25 had at least 1 psom completed , whereas 15 had psom data collected within 7 days following the event . for each of these participants , the psom following the event and the last available psom are shown in table 3 . as can be seen , in this subset of 15 patients , psom data capture and followup were essentially complete , with followup values lacking only for the 2 participants who died . for this group , the median value of the psom within 7 days after the event was 3.0 ( interquartile range 1 to 5 ) , whereas the last psom obtained for each of these participants had a median value of 1.00 ( interquartile range 0 to 3 ) . the time between the event and the last psom was a median of 136 days ( interquartile range 79 to 171 ) . the distribution of psom scores according to clinical severity is shown in figure 4 for both the initial and final psom scores . this figure includes all psom data available , even if no psom was administered immediately following the event . the proportion of patients with a psom score indicating severe neurological deficit was 53% based on the psom within 7 days of the event and 39% based on the last psom following the event ( p value not significant for change over time ) . psom scores at investigational device exemption sites following first neurological event n / a indicates not applicable due to death ; psom , pediatric stroke outcome measure . the bar graph shows neurological outcome severity as measured by the psom , when available , for children within 7 days of a neurological event and long term , at a median of 136 days ( interquartile range 79 to 171 ) after a neurological event . severity of neurological injury was further assessed by evaluating the clinical course subsequent to the injury . table 4 provides the subsequent clinical outcome for the 59 patients who experienced 1 neurological event . overall , 56% of patients who experienced a neurological event continued to successfully undergo heart transplantation . an additional patient had successful weaning from excor because of recovery of ventricular function with good neurological outcome . the remaining patients either died ( n=23 ) or were weaned from the device with subsequent mortality ( n=2 ) . the mortality rate for patients who had at least 1 neurological event was 42% ( 25 of 59 ) , significantly greater than the rate of 18% ( 26 of 145 ) for those who did not have a neurological event ( p=0.0006 ) . as can also be seen in table 4 , the mortality ( including weaning failure ) associated with neurological events did not appear to be evenly distributed between the study sites , with mortality of 17% at ide sites compared with 67% at nonide sites ( p=0.0002 ) . to determine whether this result was more likely a site effect or a difference in patients , mortality was compared between patients who did and did not meet the primary study enrollment criteria , regardless of site of care . this analysis demonstrated 58% mortality in patients who did not meet primary enrollment criteria compared with 31% for those who did meet these criteria . clinical outcome following neurological injury ide indicates investigational device exemption . among the 59 children with neurological events , 42 children had 47 ischemic strokes , and 14 of those five children had hemorrhagic strokes , and 1 had support withdrawn because of the severity of the neurological injury . surgical intervention for hematoma evacuation was performed in 2 of 29 children ( 1 parenchymal hematoma evacuation and 1 subdural hematoma evacuation ) . in view of the clinical importance of the neurological events , we attempted to construct a riskstratification model using demographic and clinical variables available at the time of implantation . the variables selected and the univariate test results are shown in table 5 . these included sex , weight , cardiac diagnosis , baseline renal and hepatic function , use of extracorporeal membrane oxygenation prior to excor , use of left vad versus bivad , and pump size . in univariate analysis , sex , diagnosis of congenital heart defect , use of extracorporeal membrane oxygenation prior to excor , device type ( left vad or bivad ) , prior stroke , and any pump replacement because of thrombus all had p<0.25 and were included in the multivariate model . in the multivariate analysis , only sex and pump replacement were significant risk factors for a neurological event , with female participants and those with a pump change because of thrombus more likely to experience a neurological event . because of the manner in which the data were collected , it was not possible to reconstruct the temporal relationship between the pump exchange and the neurological event . potential risk factors for occurrence of neurological events in univariable and multivariable analyses bsa indicates body surface area ; bvad , biventricular assist device ; chd , congenital heart defect ; cns , central nervous system ; ecmo , extracorporeal membrane oxygenation ; ide , investigational device exemption ; intermacs , interagency registry for mechanically assisted circulatory support ; iqr , interquartile range ; lvad , left ventricular assist device ; or , odds ratio . this analysis of the excor ide database represents the most detailed evaluation to date of neurological adverse events associated with use of excor . the present report includes > 200 children treated at multiple centers using a common protocol . the overall incidence of neurological events was high , with 29% of patients experiencing at least 1 neurological event . half of all patients who would ultimately experience a neurological event did so within the first 14 days of support , with an additional 25% occurring in the next 14 days . consequently , the initial 28 days of support defined a period of particularly high risk for the development of a neurological event . the freedom from neurological events was 64% after 90 days of support . this report does not answer the question of why the neurological event rate is initially high , but several possibilities may be considered . the perioperative period involves inhibited platelet function and surgical bleeding , both of which complicate the initiation of effective anticoagulation . in addition , given the severity of hf prior to implantation , renal and hepatic function may both be in flux in this period , further complicating the ability to balance thrombotic and hemorrhagic risk . a further contribution may be cardiovascular instability following initiation of vad support . in recent pivotal trials of adult continuousflow vads , the observed rate of 29% with the pulsatileflow excor is quite high , using a comparable set of data definitions . there are significant differences between continuousflow and pulsatile pumps , so a more pertinent comparison might be the neurological injury rate reported for adults supported on pulsatile devices . the thoratec xve left vad is associated with a very low neurological event rate ( 0% in 1 study ) , whereas the more recently developed syncardia total artificial heart has been reported to have a rate of 8% . the greater stroke risk with excor likely reflects a combination of device risks such as smaller pump size and inherent difficulties associated with anticoagulation in children , including the lack of adequate normative data and the complex interplay of developmental factors . although a small number of events were transient ischemic attacks , and thus were not associated with permanent neurological injury , 71% of neurological events were strokes . with a single exception , these strokes produced overt neurological symptoms . the preponderance of ischemic strokes was striking ( 47 ischemic compared with 5 hemorrhagic ) . this raises the question of whether a more aggressive anticoagulation protocol might have reduced the frequency of neurological events . given that the incidence of major bleeding was 44% , increased intensity of anticoagulation would be likely to carry significant risk of increased hemorrhage . because the definition of major bleeding included the requirement for blood product transfusion without surgical exploration ( but excluded hemorrhagic stroke from the definition ) , it may be appropriate to accept a higher bleeding risk in exchange for a reduction in ischemic stroke risk ; however , if the consequence of more intensive anticoagulation is hemorrhagic stroke , that would be a less desirable exchange . the clinical severity of the neurological events was difficult to establish because of data limitations . psom data were intended to characterize stroke severity , but in many instances , these data were not collected because of the patient 's medical condition . the majority of patients were sedated prior to implantation and sometimes up to 2 weeks after implantation , and this precluded a detailed neurological assessment . the available psom data demonstrate that the clinical burden from stroke is initially consistent with severe impairment ( median psom within 7 days following event 3.0 ) but is lower at last available followup ( median psom at last assessment 1.0 ) , which is indicative of mild to moderate impairment . in addition , as judged by the crude measure of associated mortality rates , the strokes that occurred at ide trial sites were comparatively well tolerated , with 17% mortality . in contrast , children treated at nonide sites had mortality of 67% after a first neurological event despite a very similar overall rate of neurological events . potentially , the differences might relate to differences in patient selection for study enrollment , in severity of stroke , or in site decision making following a neurological event . our analysis suggests that patient mix is likely a significant contributor to this finding , with patients who did not meet primary study enrollment showing a trend toward higher mortality rates following stroke compared with those patients who met enrollment criteria , regardless of site of treatment . the importance of severity of illness at the time of stroke onset is supported by studies of children with stroke from all causes : the presence of critical illness prior to stroke onset is associated with increased mortality , and the need for intensive care unit care after stroke is associated with higher mortality . neither severity of stroke nor decision making following stroke can be compared between ide and nonide sites , given the available data , although there is no reason to suspect a difference between ide and nonide sites in this regard . nonide sites were provided with the anticoagulation protocol , but adherence was not required or tracked , so it is possible that this played a role in modulating outcome after a neurological injury . the significant neurological injury rate and severity observed in this study led to the question of what clinical care might be indicated following treatment with excor . a recent american heart association scientific statement urged periodic developmental surveillance , screening , evaluation , and reevaluation of children with congenital heart defect throughout childhood . in children with hf requiring mechanical circulatory support children who have a stroke should be considered candidates for standardized developmental assessments at least annually until aged 5 years and also should undergo cognitive assessments ( usually aged > 5 years ) . neuropsychological testing should be considered at intervals thereafter to understand the effects of stroke and to allow targeted educational interventions for these children . in view of the important role of neurological injury in shaping both survival and longterm outcomes after device support contrary to what many investigators believe , this analysis does not demonstrate a relationship between use of the 10ml excor pump and the risk of neurological injury . in fact , the only variables that emerged as predictive of increased risk of neurological injury are female sex and vad pump change because of thrombus . female sex has also emerged as a stroke risk factor in the adult experience reported by intermacs , but the reason for this remains unclear . pump exchange as a risk factor is difficult to interpret because the study database does not allow us to determine the temporal relationship between the pump replacement and the neurological injury . furthermore , although the neurological events had a defined onset time , the recognition of an event may be delayed because of factors associated with critical care , such as systemic sedation for intubation or pain relief . it is possible that visible thrombus precedes neurological injury and represents a therapeutic target , either via pump exchange or alteration of anticoagulation . it is also possible that clinical care is altered by an antecedent neurological injury , leading to more frequent exchange of the vad . there is also complex interplay between center experience and threshold for vad pump exchange ; there were no established criteria for replacement of the vad , either at the time of the trial or even today . this trial provided other important lessons that can be applied to future clinical trials of vads in children . the adult definitions of neurological events from intermacs were difficult to apply to critically ill children in this trial . the accepted definition of an arterial ischemic stroke , for example , specifies a focal deficit that conforms to an arterial territory with a corresponding infarct on brain imaging ; however , in a child aged < 6 months , brain injury may be associated only with seizures or altered mental status rather than a focal deficit , so the imaging determines the event classification . in addition , seizures as a defined neurological injury were problematic because they are best regarded as a symptom rather than as a specific type of neurological injury . future trials of mechanical circulatory support might be better supported by a revised set of neurological definitions for pediatric stroke that are consistent with the national institutes of health common data elements . this study also illustrates the difficulty of obtaining unambiguous neurological baseline information , given the acute and severe presentation of hf in a large number of the children in this study . imaging data are likely to continue to be required to provide adequate baseline data in future trials . ongoing assessment of neurological injury also proved problematic , as shown by the erratic collection of psom data . this may reflect limited access to skilled pediatric neurologists , even at sites that were able to enroll children in a vad trial . solutions to this problem will be difficult to implement . to the degree that is possible , use of instruments that can be completed at the bedside by the primary treating physician will ameliorate that problem , but such instruments have not been validated for assessing neurological injury in children . first , there is a need for preimplantation imaging for future device trials in which clarity is needed around timing and attribution of neurological events relative to device use . second , neurological morbidity is part of the underlying disease process in children with hf and requires attention and research at the preimplant stage of disease , separate from the postimplant stage of disease . third , the longterm functional outcome reflects the totality of neurological injury that these children experience , including the strokes that occur before device use and in those children who come close to needing a device but who are eventually managed without device support . if possible , more rigorous assessment of baseline neurological function in children with severe hf is warranted prior to the point of requiring mechanical circulatory support . of children treated with excor as a bridge to transplant , 29% experienced a neurological injury . the majority of neurological injuries were ischemic strokes and occurred in the first 28 days of support . neurological injury does not preclude successful transplantation in the majority of cases but nonetheless ranks as the leading cause of death after excor implantation . of children treated with excor as a bridge to transplant , 29% experienced a neurological injury . the majority of neurological injuries were ischemic strokes and occurred in the first 28 days of support . neurological injury does not preclude successful transplantation in the majority of cases but nonetheless ranks as the leading cause of death after excor implantation .

backgroundthe berlin heart excor ventricular assist device has been approved for use in the united states as a bridge to heart transplantation in children . we sought to characterize neurological events in children supported with the berlin heart excor device.methods and resultsthe multicenter prospective cohort consisted of all 204 children implanted with the berlin heart excor device at 47 centers in north america between may 2007 and december 2010 . there were 73 neurological events in 59 patients , with 29% of the cohort experiencing 1 neurological event . events included 52 strokes in 43 patients ( 21% of the cohort ) . the neurological event rate was 0.51 events per 100 patientdays . many of the neurological events occurred early in the course of support , with 30 events recorded during the first 14 days of support . the mortality rate in participants with at least 1 neurological event was 42% ( 25 of 59 ) , significantly higher than the 18% mortality rate ( 26 of 145 ) for those who did not have a neurological event ( p=0.0006 ) . riskfactor analysis did not identify significant preimplantation predictors of neurological injury.conclusionsof children treated with the berlin heart excor device as a bridge to transplant , 29% experienced at least 1 neurological event . the majority of neurological events were ischemic strokes , and many of those occurred early in the course of support . neurological injury was the leading cause of death after implantation of the berlin heart excor device . risk stratification for stroke or neurological injury is not possible based on baseline preimplantation characteristics.clinical trial registrationurl : www.clinicaltrials.gov . unique identifier : nct00583661 .

in recent years , there has been greater interest in the role of arterial stiffness in the development of cardiovascular disease ( cvd ) . several patient- and population - based cohort studies have proved a strong relationship between augmented arterial stiffness and increased risk of cvd , such as stroke , myocardial infarction , and heart failure.15 by means of pulse - wave velocity ( pwv ) measurement , arterial stiffness can be assessed noninvasively . with a large amount of epidemiological evidence , the velocity of carotid femoral pwv ( cf - pwv ) seems to be very important in prognosis , requires little technical expertise , and is considered to be the gold standard for assessing central arterial stiffness.57 cvd is one of the leading causes of morbidity and mortality in patients with chronic kidney disease ( ckd ) . patients with ckd are more likely to die from cvd than progress to end - stage renal disease ( esrd ) , let alone patients who have already progressed to esrd or dialysis.8 in addition to traditional cv risk factors , including advanced age , diabetes , hypertension , dyslipidemia , and smoking , large - vessel arterial stiffness , which is a common issue in ckd , is considered another newfound mechanism accounting for the increased risk of cvd in ckd.9 several studies have demonstrated that increased arterial stiffness is a strong and independent predictor of mainly cv and all - cause mortality in patients with esrd.1013 these population - specific studies focused much of their attention on advanced stages of ckd . however , after adjusting for other traditional cv risk factors , even mild impairment of renal function ( estimated glomerular filtration rate [ egfr ] < 90 ml / min/1.73 m ) becomes an independent cv risk factor.14 therefore , a new problem arises : is arterial stiffness predictive in individuals with mild renal impairment ? we hypothesize that arterial stiffness is an independent predictive factor of cvd , which is replaced by major adverse cv events ( maces ) as major end points in individuals with mildly impaired renal function . therefore , the present study investigated the predictive value of arterial stiffness ( cf - pwv ) on cvd ( maces ) by studying the association between levels of cf - pwv at baseline and the incidence of maces during the follow - up period in a large , community - based , longitudinal sample grouped by renal function ( egfr ) from beijing , people s republic of china . this community - based cohort study was carried out in the pingguoyuan area of shijingshan district , beijing , people s republic of china . subjects with severe systemic diseases , including severe liver , renal , endocrine , metabolic ( except for diabetes mellitus , dm ) , and neoplastic diseases , collagenosis , inflammation , heart failure , and a history of ischemic heart disease were excluded . in total , 1,680 participants ( age 18 years ) were initially eligible for the study after a routine health checkup between september 2007 and january 2009 . this community - based population was followed prospectively , and the second visit for these participants was conducted from february 1 to september 30 , 2013 . during the second visit , all participants came to the community medical center and received a questionnaire survey . 181 participants were excluded from the study on account of failure to follow - up . finally , integrated data were obtained from 1,499 participants , and the rate of follow - up was 89.2% . the study protocol was approved by the ethics committee of the people s liberation army ( pla ) general hospital ( beijing , people s republic of china ) , and each participant provided informed written consent . information that included lifestyle factors , prevalent diseases , family history , and medication use was collected with standardized self - reporting questionnaires , which were carried out by a face - to - face counseling method . trained physicians from the department of geriatric cardiology at the pla general hospital height and weight were measured by a wall - mounted measuring tape and a digital scale , respectively . systolic blood pressure ( sbp ) and diastolic bp ( dbp ) were measured on the right arm twice in a sitting position after 5 minutes of rest . blood samples of participants were collected from 8 am to 10 am after 12 hours of fasting . these samples were preserved at 4c for 2 hours at most , before being centrifuged at 1,200 g for 15 minutes . before the assays were performed , plasma aliquots were frozen at 80c . concentrations of fasting blood glucose , triglyceride , total cholesterol , high - density lipoprotein cholesterol , and low - density lipoprotein cholesterol were detected by using roche enzymatic assays ( roche diagnostics gmbh , mannheim , germany ) on a roche autoanalyzer . concentrations of plasma creatinine were detected by using an enzymatic assay ( roche ) on a hitachi 7600 autoanalyzer ( hitachi , tokyo , japan ) . all blood samples were analyzed in the same laboratory , following the criteria of the world health organization lipid reference laboratories . measurement of arterial stiffness was conducted in a quiet environment in the morning at a stable temperature . all participants were asked to avoid alcohol , smoking , and caffeine for at least 12 hours before measurement . after resting in the supine position for 5 minutes , arterial stiffness was measured by automatic cf - pwv measurement using a complior sp device ( cratech sas , besanon , france ) , which achieves online recording of pw and automatic calculating of pwv . two ty-306 fukuda pressure - sensitive transducers ( fukuda denshi co ltd , tokyo , japan ) were used : one transducer over the common carotid artery at the bottom of the neck , and the other transducer over the femoral artery . we collected pws of both the carotid artery and the femoral artery simultaneously at two different sites , and repeated at least ten independent and complete cardiac cycles . pwv was calculated from the measurement of the pulse - transit time and the distance traveled by the pulse between the two recording sites ( measured on the surface of the body in meters ) , and on the basis of the formula : cf - pwv(m / s)=distance(m)transit time(s)(1 ) hypertension was defined as 1 ) sbp 140 mmhg , 2 ) dbp 90 mmhg , and/or 3 ) the use of antihypertensive medication . smoking status was defined as smoking one or more cigarettes per day for at least 1 year . body mass index was defined as weight in kilograms divided by height in meters squared ( kg / m ) . egfr was calculated using the following chronic kidney disease epidemiology collaboration equation : gfr=141min(scr/,1)max(scr/,1)1.2090.993age1.018(if female)1.159(if black)(2)where scr is plasma creatinine ( mg / dl ) , is 0.7 for females and 0.9 for males , is 0.329 for females and 0.411 for males , min indicates the minimum of scr/ or 1 , and max indicates the maximum of scr/ or 1 . , maces included cardiocerebrovascular death ( including both cv death and cerebrovascular death ) , nonfatal myocardial infarction , and stroke ( including both ischemic and hemorrhagic stroke ) . death was ascertained from legal death records.15 results are expressed as numbers and percentages for dichotomous variables and mean standard deviation or median ( interquartile range ) for continuous variables . the continuous variables were tested for normality before being tested , and were normalized by natural logarithm transformation as necessary . we chose 90 ml / min/1.73 m as a cutoff point for egfr , and 30 ml / min/1.73 m as a lower cutoff point for renal function , in order to exclude severe renal dysfunctions . in our study population , subjects with severe liver and renal diseases had already been excluded at the very beginning , and thus the renal function of all participants was above the lower cutoff point . participants were classified into two groups : a normal renal function group and an impaired renal function group . then , we chose 12 m / s as a cutoff point for cf - pwv , in accordance with the upper third of cf - pwv reported by blacher et al.11 cf - pwv 12 m / s is considered elevated and < 12 m / s is considered normal . finally , according to egfr and cf - pwv levels , participants were classified into four groups : group 1 ( egfr 90 ml / min/1.73 m , cf - pwv < 12 m / s ; n=744 ) , group 2 ( egfr 90 ml / min/1.73 m , cf - pwv 12 m / s ; n=230 ) , group 3 ( egfr < 90 ml / min/1.73 m , cf - pwv < 12 m / s ; n=256 ) , and group 4 ( egfr < 90 ml / min/1.73 m , cf - pwv 12 m / s ; n=242 ) . statistical comparison of these groups was analyzed by student s t - test ( continuous variables ) or test ( dichotomous variables ) . a multivariate cox proportional - hazard regression model was used to calculate hazard ratios ( hrs ) and 95% confidence intervals ( cis ) to estimate the relative risks of the incidence of maces . regression models were adjusted for age and sex as independent variables ( model 1 ) , and additionally adjusted for body mass index , sbp , dbp , fasting blood glucose , triglyceride , total cholesterol , high - density lipoprotein cholesterol , and low - density lipoprotein cholesterol as independent variables ( model 2 ) . all analyses were conducted using spss software for windows , version 13.0 ( spss , chicago , il , usa ) . this community - based cohort study was carried out in the pingguoyuan area of shijingshan district , beijing , people s republic of china . subjects with severe systemic diseases , including severe liver , renal , endocrine , metabolic ( except for diabetes mellitus , dm ) , and neoplastic diseases , collagenosis , inflammation , heart failure , and a history of ischemic heart disease were excluded . in total , 1,680 participants ( age 18 years ) were initially eligible for the study after a routine health checkup between september 2007 and january 2009 . this community - based population was followed prospectively , and the second visit for these participants was conducted from february 1 to september 30 , 2013 . during the second visit , all participants came to the community medical center and received a questionnaire survey . 181 participants were excluded from the study on account of failure to follow - up . finally , integrated data were obtained from 1,499 participants , and the rate of follow - up was 89.2% . the study protocol was approved by the ethics committee of the people s liberation army ( pla ) general hospital ( beijing , people s republic of china ) , and each participant provided informed written consent . information that included lifestyle factors , prevalent diseases , family history , and medication use was collected with standardized self - reporting questionnaires , which were carried out by a face - to - face counseling method . trained physicians from the department of geriatric cardiology at the pla general hospital completed this investigation . height and weight were measured by a wall - mounted measuring tape and a digital scale , respectively . systolic blood pressure ( sbp ) and diastolic bp ( dbp ) were measured on the right arm twice in a sitting position after 5 minutes of rest . blood samples of participants were collected from 8 am to 10 am after 12 hours of fasting . these samples were preserved at 4c for 2 hours at most , before being centrifuged at 1,200 g for 15 minutes . before the assays were performed , plasma aliquots were frozen at 80c . concentrations of fasting blood glucose , triglyceride , total cholesterol , high - density lipoprotein cholesterol , and low - density lipoprotein cholesterol were detected by using roche enzymatic assays ( roche diagnostics gmbh , mannheim , germany ) on a roche autoanalyzer . concentrations of plasma creatinine were detected by using an enzymatic assay ( roche ) on a hitachi 7600 autoanalyzer ( hitachi , tokyo , japan ) . all blood samples were analyzed in the same laboratory , following the criteria of the world health organization lipid reference laboratories . measurement of arterial stiffness was conducted in a quiet environment in the morning at a stable temperature . all participants were asked to avoid alcohol , smoking , and caffeine for at least 12 hours before measurement . after resting in the supine position for 5 minutes , arterial stiffness was measured by automatic cf - pwv measurement using a complior sp device ( cratech sas , besanon , france ) , which achieves online recording of pw and automatic calculating of pwv . two ty-306 fukuda pressure - sensitive transducers ( fukuda denshi co ltd , tokyo , japan ) were used : one transducer over the common carotid artery at the bottom of the neck , and the other transducer over the femoral artery . we collected pws of both the carotid artery and the femoral artery simultaneously at two different sites , and repeated at least ten independent and complete cardiac cycles . pwv was calculated from the measurement of the pulse - transit time and the distance traveled by the pulse between the two recording sites ( measured on the surface of the body in meters ) , and on the basis of the formula : cf - pwv(m / s)=distance(m)transit time(s)(1 ) hypertension was defined as 1 ) sbp 140 mmhg , 2 ) dbp 90 mmhg , and/or 3 ) the use of antihypertensive medication . smoking status was defined as smoking one or more cigarettes per day for at least 1 year . body mass index was defined as weight in kilograms divided by height in meters squared ( kg / m ) . egfr was calculated using the following chronic kidney disease epidemiology collaboration equation : gfr=141min(scr/,1)max(scr/,1)1.2090.993age1.018(if female)1.159(if black)(2)where scr is plasma creatinine ( mg / dl ) , is 0.7 for females and 0.9 for males , is 0.329 for females and 0.411 for males , min indicates the minimum of scr/ or 1 , and max indicates the maximum of scr/ or 1 . the major end points assessed were maces . in our study , maces included cardiocerebrovascular death ( including both cv death and cerebrovascular death ) , nonfatal myocardial infarction , and stroke ( including both ischemic and hemorrhagic stroke ) . results are expressed as numbers and percentages for dichotomous variables and mean standard deviation or median ( interquartile range ) for continuous variables . the continuous variables were tested for normality before being tested , and were normalized by natural logarithm transformation as necessary . we chose 90 ml / min/1.73 m as a cutoff point for egfr , and 30 ml / min/1.73 m as a lower cutoff point for renal function , in order to exclude severe renal dysfunctions . in our study population , subjects with severe liver and renal diseases had already been excluded at the very beginning , and thus the renal function of all participants was above the lower cutoff point . participants were classified into two groups : a normal renal function group and an impaired renal function group . then , we chose 12 m / s as a cutoff point for cf - pwv , in accordance with the upper third of cf - pwv reported by blacher et al.11 cf - pwv 12 m / s is considered elevated and < 12 m / s is considered normal . finally , according to egfr and cf - pwv levels , participants were classified into four groups : group 1 ( egfr 90 ml / min/1.73 m , cf - pwv < 12 m / s ; n=744 ) , group 2 ( egfr 90 ml / min/1.73 m , cf - pwv 12 m / s ; n=230 ) , group 3 ( egfr < 90 ml / min/1.73 m , cf - pwv < 12 m / s ; n=256 ) , and group 4 ( egfr < 90 ml / min/1.73 m , cf - pwv 12 m / s ; n=242 ) . statistical comparison of these groups was analyzed by student s t - test ( continuous variables ) or test ( dichotomous variables ) . a multivariate cox proportional - hazard regression model was used to calculate hazard ratios ( hrs ) and 95% confidence intervals ( cis ) to estimate the relative risks of the incidence of maces . regression models were adjusted for age and sex as independent variables ( model 1 ) , and additionally adjusted for body mass index , sbp , dbp , fasting blood glucose , triglyceride , total cholesterol , high - density lipoprotein cholesterol , and low - density lipoprotein cholesterol as independent variables ( model 2 ) . all analyses were conducted using spss software for windows , version 13.0 ( spss , chicago , il , usa ) . demographic characteristics , cv risk factors , and related laboratory test results in each group are also shown in table 1 . in normal renal function groups , the incidence of maces significantly increased from 2.69% in group 1 to 9.57% in group 2 . baseline cf - pwv was associated with the occurrence of maces ( hr 1.862 , 95% ci 1.0543.287 ; p=0.032 ) in the unadjusted model , but the association disappeared when the models were additionally adjusted for age , sex , bp , plasma lipids , and other traditional risk factors ( hr 1.398 , 95% ci 0.7482.613 ; p=0.293 ) ( model 2 in table 2 ) . furthermore , the association remained insignificant after adjustment for stroke ( hr 0.453 , 95% ci 0.01711.867 ; p=0.635 ) ( model 3 in table 2 ) . in the impaired renal function groups , the incidence of maces significantly increased from 8.2% in group 3 to 14.88% in group 4 . a higher baseline cf - pwv level was associated with a higher risk of maces after adjustment for age , sex , bp , plasma lipids , and other traditional risk factors . although the risk ratio was reduced after adjustment , the participants in group 4 still had a significantly higher risk of maces ( hr 2.334 , 95% ci 1.0825.036 ; p=0.031 ) compared to group 3 ( model 2 in table 3 ) . furthermore , after adjustment for stroke , the risk ratio was still higher in group 4 ( hr 2.4 , 95% ci 1.1065.208 ; p=0.027 ) ( model 3 in table 3 ) . demographic characteristics , cv risk factors , and related laboratory test results in each group are also shown in table 1 . in normal renal function groups , the incidence of maces significantly increased from 2.69% in group 1 to 9.57% in group 2 . baseline cf - pwv was associated with the occurrence of maces ( hr 1.862 , 95% ci 1.0543.287 ; p=0.032 ) in the unadjusted model , but the association disappeared when the models were additionally adjusted for age , sex , bp , plasma lipids , and other traditional risk factors ( hr 1.398 , 95% ci 0.7482.613 ; p=0.293 ) ( model 2 in table 2 ) . furthermore , the association remained insignificant after adjustment for stroke ( hr 0.453 , 95% ci 0.01711.867 ; p=0.635 ) ( model 3 in table 2 ) . in the impaired renal function groups , the incidence of maces significantly increased from 8.2% in group 3 to 14.88% in group 4 . a higher baseline cf - pwv level was associated with a higher risk of maces after adjustment for age , sex , bp , plasma lipids , and other traditional risk factors . although the risk ratio was reduced after adjustment , the participants in group 4 still had a significantly higher risk of maces ( hr 2.334 , 95% ci 1.0825.036 ; p=0.031 ) compared to group 3 ( model 2 in table 3 ) . furthermore , after adjustment for stroke , the risk ratio was still higher in group 4 ( hr 2.4 , 95% ci 1.1065.208 ; p=0.027 ) ( model 3 in table 3 ) . in the current longitudinal study , we observed that after adjusting for age and sex , as well as other traditional cv risk factors , cf - pwv 12 m / s had a higher predictive value on maces in individuals with egfr < 90 ml / min/1.73 m , compared with cf - pwv < 12 m / s . furthermore , we also found that the risk of maces did not increase with incremental cf - pwv , according to adjusted models in individuals with egfr 90 ml / min/1.73 m. pwv measurement offers a simple and noninvasive assessment of regional arterial stiffness . intermittent ventricular ejection can lead to oscillatory changes in bp , and a very important function of the aorta and large arteries is to buffer these changes . the high distensibility of the arterial system helps most tissues avoid peak systolic pressures and receive near - steady flow . impairment of distensibility makes the aorta and large arteries become rigid and unable to buffer the oscillatory changes caused by intermittent ventricular ejection , which results in elevated systolic pressure . therefore , important organs , including the heart , brain , and kidneys , suffer greatly from higher systolic pressures and greater - pressure oscillatory changes as arterial stiffness increases , leading to myocardial hypertrophy and fibrosis , renal impairment , microvascular damage , and an increased risk of maces.1619 taken together , pwv measurements are fit for large - scale studies , and several studies2022 have already demonstrated the predictive ability of arterial stiffness on the risk of maces . however , many new questions appear as more results become available . one of these concerns the fact that although a common conception based on the results of individual studies proved that arterial stiffness has an important predictive role , these studies involved different populations . we would like to know more clearly whether arterial stiffness exerts a different predictive ability in populations with different characteristics and estimated baseline cv risks.23 a meta - analysis of 17 longitudinal studies performed by vlachopoulos et al found that arterial stiffness offered higher predictive ability in individuals with a higher baseline cv risk.23 among all the cv risk factors , as previous studies have shown , ckd leads to an elevated risk of cv , with an inverse graded relationship with egfr < 60 ml / min/1.73 m , independently of other traditional cv risk factors.2426 however , most attention has been focused on the predictive ability of arterial stiffness in patients with esrd or even requiring dialysis.2729 moreover , current guidelines accept egfr < 60 ml / min/1.73 m as a cutoff point for an increased risk of ckd complications . individuals with egfr between 60 and 89 ml / min/1.73 m are not considered to have ckd unless there is also a marker of renal impairment , eg , proteinuria.2426 therefore , the key issue we investigated in the current work was whether arterial stiffness is predictive in individuals with mildly impaired renal function . in our present community - based cohort study , we chose 90 ml / min/1.73 m as the cutoff point for egfr , and found that cf - pwv 12 m / s was a moderate predictor of maces in individuals with egfr < 90 ml / min/1.73 m ( hr 2.334 , 95% ci 1.0825.036 ; p=0.031 ) . this result suggests that arterial stiffness is predictive even in individuals with mildly impaired renal function , and not just in individuals with esrd or on dialysis . this predictive ability remains after adjustment for other established traditional cv risk factors . understanding the association between increased arterial stiffness , mild renal impairment , and the risk of maces is vital to guide preventive strategies for screening and treatment.14 although cv risk in esrd is extreme , the public health burden of cv disease caused by early stage ckd is much greater.3032 improvement in arterial stiffness might be helpful for a better prognosis in individuals with slightly reduced renal function , and our results highlight the role of arterial stiffness as a potential therapeutic target in individuals with mildly impaired renal function . another interesting finding of our study was that in individuals with egfr 90 ml / min/1.73 m , the risk of maces did not increase with incremental cf - pwv according to adjusted models ( hr 1.398 , 95% ci 0.7482.613 ; p=0.293 ) . that is to say , the predictive value of arterial stiffness was limited in individuals with normal renal function subclinical impairment might not have been observed or evaluated with the insufficient follow - up period , and thus the predictive value of increased arterial stiffness might have been affected to some extent . to the best of our knowledge , the present investigation is the first study evaluating the association between arterial stiffness and maces in individuals with mild impaired renal function ; therefore , it differs from those studies that focused on the predictive value of arterial stiffness on cvd only in advanced ckd . as such , active monitoring and treatment of arterial stiffness would be required to reduce the risk of maces in certain individuals . first , the present study was carried out only among the chinese from a community in beijing . therefore , these results may not represent the chinese population from other areas of the people s republic of china and might not be applicable to other ethnic groups . second , although the results were adjusted for multiple covariates , other residual confounding factors may remain . to the best of our knowledge , the present investigation is the first study evaluating the association between arterial stiffness and maces in individuals with mild impaired renal function ; therefore , it differs from those studies that focused on the predictive value of arterial stiffness on cvd only in advanced ckd . as such , active monitoring and treatment of arterial stiffness first , the present study was carried out only among the chinese from a community in beijing . therefore , these results may not represent the chinese population from other areas of the people s republic of china and might not be applicable to other ethnic groups . second , although the results were adjusted for multiple covariates , other residual confounding factors may remain . the present community - based prospective study clearly demonstrated that arterial stiffness is a moderate and independent predictive factor for maces in individuals with mildly impaired renal function ( egfr < 90 ml / min/1.73 m ) . furthermore , in individuals with normal renal function ( egfr 90 ml / min/1.73 m ) , the risk of maces does not increase with arterial stiffening .

objectivesdespite growing evidence that arterial stiffness has important predictive value for cardiovascular disease in patients with advanced stages of chronic kidney disease , the predictive significance of arterial stiffness in individuals with mildly impaired renal function has not been established . the aim of this study was to evaluate the predictive value of arterial stiffness on cardiovascular disease in this specific population.materials and methodswe analyzed measurements of arterial stiffness ( carotid femoral pulse - wave velocity [ cf - pwv ] ) and the incidence of major adverse cardiovascular events ( maces ) in 1,499 subjects from a 4.8-year longitudinal study.resultsa multivariate cox proportional - hazard regression analysis showed that in individuals with normal renal function ( estimated glomerular filtration rate [ egfr ] 90 ml / min/1.73 m2 ) , the baseline cf - pwv was not associated with occurrence of maces ( hazard ratio 1.398 , 95% confidence interval 0.7482.613 ; p=0.293 ) . in individuals with mildly impaired renal function ( egfr < 90 ml / min/1.73 m2 ) , a higher baseline cf - pwv level was associated with a higher risk of maces ( hazard ratio 2.334 , 95% confidence interval 1.0825.036 ; p=0.031).conclusionarterial stiffness is a moderate and independent predictive factor for maces in individuals with mildly impaired renal function ( egfr < 90 ml / min/1.73 m2 ) .

igg4-related disease ( igg4-rd ) is an autoimmune condition characterized by increased serum igg4 levels and infiltration of igg4 plasma cells into the tissues . igg4 is a subtype of igg and comprises approximately 3 - 6% of total igg . increase in serum igg4 levels has been associated with various conditions like autoimmune pancreatitis , sclerosing cholangitis , mikulicz disease , and retroperitoneal fibrosis , which have now been encompassed together as igg4-rd . the organs predominantly involved in this condition are pancreas , salivary glands , and biliary tract . in the head and neck region , , we report a case series of two patients of pediatric age group ( adolescents 12 - 16 years ) diagnosed with igg4-rd involving the sinonasal region . in addition , sinonasal igg4 involvement in pediatric age group has not been reported previously in the literature to the best of our knowledge . a 15-year - old girl presented with recurrent epistaxis and bilateral progressive nasal obstruction of 9 months duration . on rhinoscopy , the nasal cavity was crowded with thickening of the nasal septum and inferior turbinates on both sides . magnetic resonance imaging ( mri ) showed t1-isointense , t2-hypointense [ figure 1a and b ] soft tissue thickening involving the nasal septum , anterior part of the right inferior turbinate , right lateral nasal wall , and uncinate process . erosion of the uncinate process , inferior turbinate , and septum was seen with extension of soft tissue thickening along the medial wall of the right maxillary sinus [ figure 1b ] . in addition , the mass was seen to extend along the right naso - lacrimal duct . on post - contrast study , moderate heterogeneous enhancement of the soft tissue serum igg4 levels were raised ( 206 mg / dl ; 95 percentile range = 6 - 112 mg / dl ) . histopathology showed dense fibrosis with lymphoplasmocytic infiltrate and immunohistochemistry revealed igg4 plasma cells constituting 50% of igg plasma cells , confirming the diagnosis of igg4-rd . mri t2w axial and coronal ( a , b ) images revealed t2-hypointense soft tissue thickening ( arrows in a , b ) involving the nasal septum and right lateral nasal wall , with extension into the right maxillary sinus ( arrow in b ) . post - contrast t1w axial image ( c ) revealed heterogeneous enhancement of lesion ( arrow ) with central hypoenhancing regions . biopsy with immunohistochemistry of lesion and raised serum igg4 levels confirmed the diagnosis of igg4-related disease a 15-year - old girl presented with recurrent right - sided blood stained nasal discharge of 7 years duration and right - sided facial swelling with difficulty in opening her mouth for 3 months . mri revealed a t1-isointense , t2-hypointense [ figure 2a d ] sheet - like soft tissue thickening extending along the right lateral nasal wall , nasal roof , nasal septum , right maxillary , ethmoid and sphenoid sinus . erosion of the lamina papyracea , floor of orbit , and posterolateral wall of the maxilla was seen with extension of the lesion into the pterygopalatine fossa , medial orbit , orbital apex , and right cavernous sinus [ figure 2d ] . the medial rectus muscle was involved and there was enhancement of the optic nerve sheath . serum igg4 levels were elevated ( 579 mg / dl ; 95 percentile range = 6 - 112 mg / dl ) . she underwent biopsy of the sinonasal mass which revealed prominent sclerosis and dense lymphoplasma cell infiltrate . immunohistochemistry revealed igg4 plasma cells constituting 50% of igg plasma cells and igg4-positive cells > 30/hpf , which was consistent with igg4-related sclerosing disease . computed tomography ( ct ) abdomen showed ill - defined sheet - like retroperitoneal soft tissue along the right iliac vessels encasing the right ureter , which was consistent with a diagnosis of retroperitoneal fibrosis . patient was started on prednisolone ( 1 mg / kg / day ) . on follow - up after 4 months , there was significant reduction in her serum igg4 levels ( 138 mg / dl ) . repeat mri , however , revealed persistent sino - orbital disease with additional increase in soft tissue thickening in the right cavernous sinus with resultant narrowing of the right internal carotid artery ( ica ) [ figure 2e ] . patient was started on rituximab ( two intravenous infusions of 600 mg- 15 days apart ) due to inadequate response to prednisolone and is currently on follow - up . mri t2w and t1 high - resolution isotropic volume examination ( thrive ) coronal images ( a , b ) showed t2-hypointense ( arrow in a ) , t1-isointense ( arrow in b ) sheet - like soft tissue thickening extending along the right lateral nasal wall , nasal septum , and right maxillary sinus . post - contrast t1w coronal image ( c ) showed homogenous contrast enhancement ( arrow ) . t2w coronal image ( d ) showed soft tissue extension into the right cavernous sinus ( arrow ) . on follow - up mri imaging after 4 months , t2w coronal image ( e ) showed increase in soft tissue thickening in right cavernous sinus with narrowing of right ica ( arrow ) a 15-year - old girl presented with recurrent epistaxis and bilateral progressive nasal obstruction of 9 months duration . on rhinoscopy , the nasal cavity was crowded with thickening of the nasal septum and inferior turbinates on both sides . magnetic resonance imaging ( mri ) showed t1-isointense , t2-hypointense [ figure 1a and b ] soft tissue thickening involving the nasal septum , anterior part of the right inferior turbinate , right lateral nasal wall , and uncinate process . erosion of the uncinate process , inferior turbinate , and septum was seen with extension of soft tissue thickening along the medial wall of the right maxillary sinus [ figure 1b ] . in addition , the mass was seen to extend along the right naso - lacrimal duct . on post - contrast study , moderate heterogeneous enhancement of the soft tissue serum igg4 levels were raised ( 206 mg / dl ; 95 percentile range = 6 - 112 mg / dl ) . histopathology showed dense fibrosis with lymphoplasmocytic infiltrate and immunohistochemistry revealed igg4 plasma cells constituting 50% of igg plasma cells , confirming the diagnosis of igg4-rd . mri t2w axial and coronal ( a , b ) images revealed t2-hypointense soft tissue thickening ( arrows in a , b ) involving the nasal septum and right lateral nasal wall , with extension into the right maxillary sinus ( arrow in b ) . post - contrast t1w axial image ( c ) revealed heterogeneous enhancement of lesion ( arrow ) with central hypoenhancing regions . biopsy with immunohistochemistry of lesion and raised serum igg4 levels confirmed the diagnosis of igg4-related disease a 15-year - old girl presented with recurrent right - sided blood stained nasal discharge of 7 years duration and right - sided facial swelling with difficulty in opening her mouth for 3 months . mri revealed a t1-isointense , t2-hypointense [ figure 2a d ] sheet - like soft tissue thickening extending along the right lateral nasal wall , nasal roof , nasal septum , right maxillary , ethmoid and sphenoid sinus . erosion of the lamina papyracea , floor of orbit , and posterolateral wall of the maxilla was seen with extension of the lesion into the pterygopalatine fossa , medial orbit , orbital apex , and right cavernous sinus [ figure 2d ] . the medial rectus muscle was involved and there was enhancement of the optic nerve sheath . serum igg4 levels were elevated ( 579 mg / dl ; 95 percentile range = 6 - 112 mg / dl ) . she underwent biopsy of the sinonasal mass which revealed prominent sclerosis and dense lymphoplasma cell infiltrate . immunohistochemistry revealed igg4 plasma cells constituting 50% of igg plasma cells and igg4-positive cells > 30/hpf , which was consistent with igg4-related sclerosing disease . ultrasound ( usg ) abdomen detected mild right hydrouretronephrosis . computed tomography ( ct ) abdomen showed ill - defined sheet - like retroperitoneal soft tissue along the right iliac vessels encasing the right ureter , which was consistent with a diagnosis of retroperitoneal fibrosis . patient was started on prednisolone ( 1 mg / kg / day ) . on follow - up after 4 months , there was significant reduction in her serum igg4 levels ( 138 mg / dl ) . repeat mri , however , revealed persistent sino - orbital disease with additional increase in soft tissue thickening in the right cavernous sinus with resultant narrowing of the right internal carotid artery ( ica ) [ figure 2e ] . patient was started on rituximab ( two intravenous infusions of 600 mg- 15 days apart ) due to inadequate response to prednisolone and is currently on follow - up . mri t2w and t1 high - resolution isotropic volume examination ( thrive ) coronal images ( a , b ) showed t2-hypointense ( arrow in a ) , t1-isointense ( arrow in b ) sheet - like soft tissue thickening extending along the right lateral nasal wall , nasal septum , and right maxillary sinus . post - contrast t1w coronal image ( c ) showed homogenous contrast enhancement ( arrow ) . t2w coronal image ( d ) showed soft tissue extension into the right cavernous sinus ( arrow ) . on follow - up mri imaging after 4 months , t2w coronal image ( e ) showed increase in soft tissue thickening in right cavernous sinus with narrowing of right ica ( arrow ) igg4 disease is a systemic disorder characterized by infiltration of igg4 plasma cells into the tissues resulting in fibrosis and resultant organ dysfunction . initially reported with autoimmune pancreatitis , it was subsequently associated with sclerosing cholangitis , retroperitoneal fibrosis , interstitial pneumonia , and mediastinal fibrosis . this entity has various synonyms like igg4-related sclerosing disease , igg4-related autoimmune disease , systemic igg4 plasmacytic syndrome , hyper igg4 disease , and igg4-related multiorgan lymphoproliferative syndrome , but igg4-rd is the preferred terminology now . it was interesting to note that both patients in our series were young patients in the pediatric age group and this further expands our understanding of the spectrum of this unique condition . igg4-rd involves multiple organs synchronously or metachronously , and sometimes may show isolated organ involvement [ table 1 ] . clinical symptoms vary depending on the organs involved , and organ dysfunction is a result of cellular infiltration and fibrosis . commonly involved regions in this condition are pancreas , biliary tract , kidney , retroperitoneum , lungs , head and neck , and blood vessels . in the head and neck region , involvement of salivary glands , lacrimal gland , common imaging manifestations of igg4-rd in the head and neck include enlargement of the salivary and lacrimal glands , thyroid lesions , and inflammatory pseudotumors in the orbit . involvement of sinonasal cavity is considered rare , with very few reports describing the imaging findings of this entity . systemic manifestations of igg4-related disease presenting symptoms in cases of sinonasal involvement are chronic rhinitis , blood - stained nasal discharge , and nasal obstruction , as was noted in our series . on ct , igg4 involvement is seen as homogenous soft tissue lesions with or without bone destruction . on mri , involvement is seen as t2-hypointense soft tissue in the nasal and paranasal sinuses , which shows homogenous enhancement on post - contrast scans . in our patients , igg4 involvement was seen as t2-hypointense sheet - like soft tissue thickening involving the nasal and paranasal sinuses , extending along the walls of contiguous structures , using existing structures as scaffolds with surrounding bone destruction . in one case , although homogenous contrast enhancement is described commonly in this condition , lesions may show slightly heterogeneous enhancement due to dense fibrosis . have described a case of igg4-related sclerosing disease in the sinonasal region which showed perineural and bone marrow infiltration . similarly in our study , infiltration of the pterygopalatine fossa and superior orbital fissure with cavernous sinus extension was seen in one case . cavernous sinus involvement resulted in narrowing of the ipsilateral ica , which was probably due to fibrotic nature of the lesion . toyoda et al . reported that when t2-hypointense lesions are noted in the head and neck region , investigation of igg4 levels and imaging of chest and abdomen should be done to look for systemic manifestations of igg4 disease . systemic manifestation noted in one of our cases was retroperitoneal fibrosis . differentials which need to be considered for t2-hypointense sinonasal lesions include lymphoma , invasive aspergillosis , wegner 's granulomatosis , and solitary fibrous tumor . these can be differentiated based on clinical presentation and by estimation of serum igg4 levels . have proposed comprehensive clinical diagnostic criteria ( ccd ) for the diagnosis of igg4-rd [ table 2 ] . studied patients with igg4-related chronic rhinosinusitis and compared them with a control group of non - igg4related chronic rhinosinusitis . they noted that nasal mucosa positive staining of igg4 is not specific for igg4 disease and there should be elevated serum igg4 levels for diagnosis of igg4 disease . in our study , all cases showed elevated serum igg4 levels . however , in some cases of igg4-rd , treatment might be ineffective or there may be relapse during steroid tapering . , there was progression of the disease on corticosteroid therapy , with increase in soft tissue in the cavernous sinus despite reduction in serum igg4 levels . igg4 is an autoimmune condition associated with infiltration of igg4 plasma cells into the tissues , resulting in fibrosis . sinonasal involvement is seen as t2-hypointense sheet - like soft tissue thickening extending along the walls of contiguous structures with variable enhancement and surrounding bone destruction . a possibility of igg4-related sinonasal disease should be considered in a patient with history of nasal obstruction and blood - stained nasal discharge having a locally invasive moderately enhancing t2-hypointense lesion on imaging with elevated serum igg4 levels . radiologists should be aware of the imaging findings of this entity to ensure early diagnosis and treatment .

we present the imaging findings in two cases of igg4-related disease involving the sinonasal region in the pediatric age group . imaging findings in igg4-related disease affecting the nasal cavity and paranasal sinuses have been rarely reported in literature . the diagnosis is made by a combination of clinical , imaging , and histopathologic findings . radiologists should be aware of the imaging findings of this condition to ensure early diagnosis and treatment .

recently , the number of newly diagnosed patients with hiv infection is increasing in japan and eastern asia ( even though it is decreasing in most western countries ) while amebiasis is also increasing in japan as a sexually transmitted disease , especially among males who admit to having sex with men ( msm ) . there is also a high frequency of asymptomatic entamoeba histolytica infection , especially among msm . amebiasis is characterized by diarrhea and it sometimes induces the development of liver abscesses as a complication with amebic colitis.1 pleural empyema is rare as a complication in cases of amebiasis . trimethoprim - sulfamethoxazole is often administered to patients with an hiv infection to prevent pneumocystis pneumonia . unfortunately , trimethoprim - sulfamethoxazole may also induce agranulocytosis in patients with an hiv infection , thus leading to the development of severe infectious diseases . a 41-year - old male was admitted to the hospital with symptoms of diarrhea , fever , dyspnea and right pleural effusion . the illness began in the latter part of august 2008 , initially presenting with diarrhea and fever . he had no history of traveling abroad , alcoholism , any medication or intravenous drug use . his vital signs on admission were ; conscious , blood pressure 95/60 mmhg , pulse late 146 beats / min , respiration , 45 breaths / min , saturation o2 ( room air ) , 88% and body temperature 39c . the notable findings of a physical examination included emaciation ( bmi 16.1 ) , oral candidasis and decreased breath sounds on the right side of his chest . a chest radiograph and computed tomography ( ct ) of the chest showed a large amount of right pleural effusion ( figure 1 ) . the laboratory data included a leukocyte count of 10,320/l with 89% neutrophils , 8% lymphocytes , 3% monocytes , hemoglobin 8.8 g / dl , c - reactive protein 23.6 mg / dl , aspirate aminotransferase 95 u / l , alanine aminotransferase 74 u / l , alkaline phosphatase 478 u / l , -glutamyl transpeptidase 134 u / l , albumin 2.1 g / dl , total cholesterol 57 mg / dl , blood urea nitrogen 18.4 mg / dl , creatine 0.57 mg / dl and hyponatremia ( 123 meq / l ) . the findings of human immunodeficiency virus type 1 antibody tests were positive for enzyme immunoassays ( eia ) and also based on the western blot method . thoracentesis revealed milk chocolate or caf au lait colored pleural fluid ( figure 3 ) . in an examination of the pleural fluid , cytology , bacterial culture , smear and polymerase chain reaction to detect mycobacterium tuberculosis dna were negative , the adenosine deaminase activity was 240 the pleural fluid showed a cell count of 40125/ml ( 74.3% neutrocytes , 25.7% monocytes ) . other examinations of the laboratory findings detected cysts of entamoeba histolytica in the patient s stool . the cd4 lymphocyte count in the peripheral blood was 179/l ( repeated counts for cd4 lymphocytes ranged from 286 to 359/l ) and the amount of hiv - rna was 3700 copies/l ( repeated counts for hiv - rna ranged from 43,000 to 90,000 copies / ml ) . although e. histolytica was not identified from the pleural fluid , antibodies ( fluorescence antibody technique ) against e. histolytica were demonstrated in the serum ( 200 ) . the patient was thus diagnosed to have amoebic colitis , amoebic liver abscess and amoebic empyema complicated with an hiv infection . the right side pleural effusion was drained using a chest tube and he was administered metronidazole ( 2250 mg / day ) orally for 28 days in total . the patient was therefore administered trimethoprim - sulfamethoxazole to prevent pneumocystis pneumonia and itraconazole to prevent fungal infections . agranulocytosis in this case was therefore considered to have been caused by the administration of trimethoprim - sulfamethoxazole . after the discontinuation of trimethoprim - sulfamethoxazole , the recombinant human granulocyte colony - stimulating factor ( g - csf ) drug filgrastim was administered daily at a dose of 200 g / m intravenously . following 4 days of treatment with g - csf , the patient s absolute neutrophil count was above 9/l , while after 7 days of treatment it was 2990/l ( white blood cell count 4600/l , neutrophils 65% ) . amebiasis may become invasive depending on various host immune factors , especially in patients with an hiv infection.2 three to 9% of patients with amoebic colitis develop amoebic liver abscess3 and furthermore 7% to 20% of amoebic liver abscess develop amoebic empyema.4,5 however , about a third of all cases of pleuropulmonary amebic disease demonstrate the presence of exudative or serous , reactive pleural effusion.6,7 this means that some of intrathoracic complications of amebiasis are thus included reactive pleural effusion other than amoebic empyema . some mechanisms of amoebic empyema have been previously described . for example , a rupture of an amoebic liver abscess into the pleural space through the diaphragm , hepatobronchial fistula , lymphatic spread , hematogenous spread from the primary intestinal lesion have all been reported.68 the most common pathogenetic mechanism of pleural amebic empyema is that the liver abscess advances to and across the diaphragm or bursts into the right pleural space . in this case , there was no hepato - bronchial fistula , no sign of lymphatic spread and no perihepatic ascites detected by ct , thus indicating that there was no rupture of a liver abscess . amoebic empyema was suggested to occur by extension or inflammatory invasion of an amoebic liver abscess directly into the pleural cavity . the detection of e. histolytica from pleural fluid is rare in the diagnosis of amoebic empyema . amebas are detected in less than 10% of cases.5 the diagnosis of amoebic empyema is made by the identification of antibodies against e. histolytica in the serum , and a negative bacterial culture of the pleural fluid . the color of the pus aspirated from amoebic liver abscesses also tends to be described as chocolate brown or anchovy - sauce color in the literature.6 however , the features of amoebic empyema remain to be fully elucidated . the color of the pleural fluid in the current case was noteworthy . furthermore , the pleural fluid in this case did not have a foul odor , although most pleural fluid specimens with bacterial empyema tend to have a foul odor because the majority of empyema are caused by anaerobes . the color and smell of the pleural fluid may therefore be a unique diagnostic feature of amoebic empyema and may thus be helpful in making a differential diagnosis to distinguish common bacterial empyema from amoebic empyema . the administration of trimethoprim - sulfamethoxazole to prevent pneumocystis pneumonia is common in the management of patients with hiv infection . therefore , trimethoprim - sulfamethoxazole was administered after diagnosing the patient to have an hiv infection . although agranulocytosis is a potentially life - threatening condition , especially under conditions of severe amebiasis , the patient was able to successfully recover after the administration of g - csf and oral metronidazole . trimethoprim - sulfamethoxazole is known to sometimes induce agranulocytosis as a side effect . as a result , an increasing number of such cases are expected in the future because many patients with hiv infection require the administration of trimethoprim - sulfamethoxazole in order to prevent pneumocystis pneumonia , and the occurrence of amebiasis is also increasing . the management of amoebic empyema in most cases involves the administration of parenteral metronidazole,9 followed by drainage . metronidazole was administered orally in the current patient , followed by drainage , and this yielded a good outcome . the incidence of amebiasis is not as high in aids patients as in hiv - seronegative patients.10 however , whether risk of invasive amebiasis is higher among hiv - infected persons than uninfected persons remains . hung and coworkers described persons infected with hiv are at an increased risk for invasive amebiasis.2,11 furthermore the epidemiology demonstrated that invasive amebiasis occurs much more frequently in taiwan than reported in western countries . interestingly , amebiasis is a relatively common parasitic disease at a time when cd4 counts are relatively high in taiwan in comparison to western countries.2,11 extraintestinal amebiasis is also treated by metronidazole , even in aids patients who have low cd4 cell counts and receiving anti - hiv drugs.8,11,12 the estimated mean survival is not significantly different between the hiv - infected patients with and without invasive amebiasis.11 even though the population of newly diagnosed patients with hiv infection is decreasing in most western countries , the number is , in contrast , increasing in japan , korea , hong kong and taiwan . in addition , the occurrence of invasive amebiasis is therefore also expected to increase in these countries .

a 41-year - old male was admitted to the hospital with symptoms of diarrhea , fever and rapidly progressive respiratory distress . a chest radiograph and computed tomography ( ct ) of the chest and the abdomen showed a large amount of right pleural effusion and a large liver abscess . the patient was thus diagnosed to have amoebic colitis , amoebic liver abscess and amoebic empyema complicated with an hiv infection . the patient demonstrated agranulocytosis caused by the administration of trimethoprim - sulfamethoxazole . however , the administration of granulocyte colony - stimulating factor made it possible for the patient to successfully recover from agranulocytosis , and he thereafter demonstrated a good clinical course .

there has been a growing need for total knee replacement arthroplasty ( tka ) as a life - changing surgery . in elderly patients with osteoarthritis of the knee , tka is the choice of treatment in advanced stages of the disease . metabolic syndrome , clustering of obesity , hypertension , and diabetes ( dm ) are not only considered as risk factors for osteoarthritis , but are also related with postoperative morbidity and mortality . although tka is known to be a safe surgery , a considerable number of patients undergoing tka are susceptible to postoperative morbidity and mortality due to metabolic syndrome . the incidence of acute kidney injury ( aki ) varies from 0.8% to 10% in patients with noncardiac surgery , depending on which aki definition is used . recently , the international kidney disease improving global outcomes ( kdigo ) criteria combined risk , injury , failure , loss , and end - stage kidney disease ( rifle ) and acute kidney injury network ( akin ) criteria . postoperative aki is related with elongated hospital stay and increased morbidity and mortality after noncardiac surgery . it has been shown that there are multiple risk factors for aki , including perioperative hypotension , advanced age , high body mass index ( bmi ) , emergency surgery , use of vasopressor infusion , and administration of diuretics in various settings . the physiologic functions of albumin include free - radical scavenging , control of plasma volume by maintenance of colloid oncotic pressure , and effects on capillary membrane permeability . although the positive effect of albumin on renal function is not clearly understood , its renoprotective effect has been suggested in many previous studies . recently , it has been shown that early postoperative low albumin level was associated with aki after liver transplantation . however , little is known about the prevalence and risk factors for aki following tka . moreover , therefore , the aim of this study was to assess the incidence and risk factors for aki following tka based on the kdigo criteria . in addition , we assessed the influence of early postoperative serum albumin level on the development of aki following tka . the medical records of patients who underwent primary unilateral tka between january 2008 and december 2014 in single center , a tertiary teaching hospital in seoul , south korea , were retrospectively reviewed . a total of 2579 consecutive patients who underwent primary unilateral tka surgery ( emergency and revision operations were excluded ) were recognized through our electronic medical records system . of these , 1270 were excluded , including those who underwent bilateral tka , 1 or 2 weeks apart from each other ( n = 1,056 ) , those who underwent a combined operation ( n = 3 ) , those whose preoperative serum creatinine ( scr ) level was > 1.5 mg / dl or those who had chronic kidney disease ( n = 30 ) , and those with incomplete data ( n = 181 ) . there were no patients who had severe liver dysfunction . in the final analysis , 1309 the included patients were divided into 2 groups by the lowest serum albumin level within 2 postoperative days ( pod2_alb level ) ( 3.0 g / dl [ group 1 , n = 839 ] and < 3.0 g / dl [ group 2 , n = 470 ] ) ( fig . this study was accepted by the institutional review board of the asan medical center ( 2015 - 0472 ) and informed consent was waived as we only reviewed the electronic medical records in this current retrospective study . study flow diagram . iptw = inverse probability of treatment weighting , pod2_alb level = the lowest serum albumin level within 2 postoperative days , scr = serum creatinine , tka = total knee arthroplasty . we obtained the demographic and laboratory and perioperative data of patients from electronic medical records at our institution . the demographic data included age , sex , bmi , underlying disease ( dm , hypertension , cerebrovascular accident , pulmonary disease , and ischemic heart disease ) , american society of anesthesiologists physical status ( asa ps ) , smoking history , and preoperative medications . laboratory data included hemoglobin , platelet count , albumin , scr , and uric acid ( ua ) . perioperative data included anesthetic technique ( general vs regional anesthesia ) , intraoperative crystalloid and colloid amount , use of diuretics and vasopressors , the lowest mean blood pressure ( mbp ) , transfusion ( packed red blood cells [ prbc ] , fresh - frozen plasma [ ffp ] , and platelets ) , urine output , anesthetic time , and tourniquet time . the main result of the present study was to define the incidence of aki analyzed by kdigo criteria . the incidence of aki was described by a change in the scr level on postoperative days 1 to 7 compared with the baseline scr level , described as the latest concentration measured before surgery . in relation to kdigo criteria , aki was diagnosed by alteration of scr level 0.3 mg / dl within 48 hours , or rise in the scr level 50% within the prior 7 days . aki was staged for severity according to the kdigo criteria ; stage 1 , 1.5 to 1.9-fold increase or 0.3 mg / dl increase in the scr level from baseline ; stage 2 , 2.0 to 2.9-fold increase in the scr level from baseline ; stage 3 , 3-fold increase in the scr level from baseline or increase to 4.0 mg / dl in the scr level or initiation of renal replacement therapy . the information about in - hospital mortality was identified by the review of electronic medical records . all data are presented as the mean standard deviation , median ( interquartile range [ iqr ] ) , or number ( percentages ) . baseline characteristics and perioperative data were compared between the 2 groups by the chi - square or fisher 's exact test for categorical variables . multivariate logistic regression analysis by backward elimination included all the variables with a p value of < 0.1 in univariate analysis . the hosmer lemeshow test ( 2 = 10.557 , df = 8 , p = 0.228 ) was identified for the calibration of the multivariate logistic regression models in table 2 . multivariate cox proportional hazard regression analysis was utilized to calculate the adjusted hazard ratios ( hr ) of the associations between the albumin concentration and outcomes to evaluate risk factors for mortality . cumulative survival rates were analyzed using the kaplan meier analysis , and alterations between curves were estimated by the log - rank test . the influence of possible confounding factors was reduced by performing inverse probability of treatment weighting ( iptw ) based on the propensity score ( ps ) analysis . for this technique , the weights for patients having 3.0 g / dl for postoperative albumin value were the inverse of 1 minus the propensity score , and weights for patients being < 3.0 g / dl were the inverse of the propensity score . the propensity scores were estimated using multiple logistic regression with postoperative albumin value as dependent variable . model discrimination was assessed with c statistics ( 0.712 ) , and model calibration was assessed with hosmer lemeshow statistics ( = 6.389 ; df = 8 , p = 0.604 ) . after the adjustment by iptw , the outcome variables including incidence of aki and survival probability were compared using weighted logistic regression and weighted cox proportional hazards model with robust standard errors respectively , and the length of hospital stay was analyzed by a weighted t - test after log transformation . cary , nc ) or r software version 2.10.1 . was used in handling and analyzing data . the medical records of patients who underwent primary unilateral tka between january 2008 and december 2014 in single center , a tertiary teaching hospital in seoul , south korea , were retrospectively reviewed . a total of 2579 consecutive patients who underwent primary unilateral tka surgery ( emergency and revision operations were excluded ) were recognized through our electronic medical records system . of these , 1270 were excluded , including those who underwent bilateral tka , 1 or 2 weeks apart from each other ( n = 1,056 ) , those who underwent a combined operation ( n = 3 ) , those whose preoperative serum creatinine ( scr ) level was > 1.5 mg / dl or those who had chronic kidney disease ( n = 30 ) , and those with incomplete data ( n = 181 ) . there were no patients who had severe liver dysfunction . in the final analysis , 1309 the included patients were divided into 2 groups by the lowest serum albumin level within 2 postoperative days ( pod2_alb level ) ( 3.0 g / dl [ group 1 , n = 839 ] and < 3.0 g / dl [ group 2 , n = 470 ] ) ( fig . this study was accepted by the institutional review board of the asan medical center ( 2015 - 0472 ) and informed consent was waived as we only reviewed the electronic medical records in this current retrospective study . study flow diagram . iptw = inverse probability of treatment weighting , pod2_alb level = the lowest serum albumin level within 2 postoperative days , scr = serum creatinine , tka = total knee arthroplasty . we obtained the demographic and laboratory and perioperative data of patients from electronic medical records at our institution . the demographic data included age , sex , bmi , underlying disease ( dm , hypertension , cerebrovascular accident , pulmonary disease , and ischemic heart disease ) , american society of anesthesiologists physical status ( asa ps ) , smoking history , and preoperative medications . laboratory data included hemoglobin , platelet count , albumin , scr , and uric acid ( ua ) . perioperative data included anesthetic technique ( general vs regional anesthesia ) , intraoperative crystalloid and colloid amount , use of diuretics and vasopressors , the lowest mean blood pressure ( mbp ) , transfusion ( packed red blood cells [ prbc ] , fresh - frozen plasma [ ffp ] , and platelets ) , urine output , anesthetic time , and tourniquet time . the main result of the present study was to define the incidence of aki analyzed by kdigo criteria . the incidence of aki was described by a change in the scr level on postoperative days 1 to 7 compared with the baseline scr level , described as the latest concentration measured before surgery . in relation to kdigo criteria , aki was diagnosed by alteration of scr level 0.3 mg / dl within 48 hours , or rise in the scr level 50% within the prior 7 days . aki was staged for severity according to the kdigo criteria ; stage 1 , 1.5 to 1.9-fold increase or 0.3 mg / dl increase in the scr level from baseline ; stage 2 , 2.0 to 2.9-fold increase in the scr level from baseline ; stage 3 , 3-fold increase in the scr level from baseline or increase to 4.0 mg / dl in the scr level or initiation of renal replacement therapy . the information about in - hospital mortality was identified by the review of electronic medical records . all data are presented as the mean standard deviation , median ( interquartile range [ iqr ] ) , or number ( percentages ) . baseline characteristics and perioperative data were compared between the 2 groups by the chi - square or fisher 's exact test for categorical variables . multivariate logistic regression analysis by backward elimination included all the variables with a p value of < 0.1 in univariate analysis . the hosmer lemeshow test ( 2 = 10.557 , df = 8 , p = 0.228 ) was identified for the calibration of the multivariate logistic regression models in table 2 . multivariate cox proportional hazard regression analysis was utilized to calculate the adjusted hazard ratios ( hr ) of the associations between the albumin concentration and outcomes to evaluate risk factors for mortality . meier analysis , and alterations between curves were estimated by the log - rank test . the influence of possible confounding factors was reduced by performing inverse probability of treatment weighting ( iptw ) based on the propensity score ( ps ) analysis . for this technique , the weights for patients having 3.0 g / dl for postoperative albumin value were the inverse of 1 minus the propensity score , and weights for patients being < 3.0 g / dl were the inverse of the propensity score . the propensity scores were estimated using multiple logistic regression with postoperative albumin value as dependent variable . model discrimination was assessed with c statistics ( 0.712 ) , and model calibration was assessed with hosmer lemeshow statistics ( = 6.389 ; df = 8 , p = 0.604 ) . after the adjustment by iptw , the outcome variables including incidence of aki and survival probability were compared using weighted logistic regression and weighted cox proportional hazards model with robust standard errors respectively , and the length of hospital stay was analyzed by a weighted t - test after log transformation . cary , nc ) or r software version 2.10.1 . was used in handling and analyzing data . the median follow - up of these patients was 4.2 years ( iqr 2.45.7 ) . the incidences of stage 1 and 2 aki were 53 ( 4.0% ) and 4 ( 0.3% ) , respectively . there was no stage 3 aki in our study population . the overall survival rates for 1 year , 3 years , and 5 years were 99.3 ( 95% ci : 98.799.6 ) , 97.6 ( 95% ci : 96.598.4 ) , and 94.9 ( 95% ci : 93.296.2 ) , respectively . table 1 shows the baseline characteristics including demographic and perioperative data of the patients categorized on the basis of pod2_alb level . group 1 patients had higher incidence of dm , to be on preoperative aspirin and statin medication , and to be younger than group 2 patients . moreover , patients in group 1 had higher bmi , hemoglobin , and albumin levels . as the patients who underwent elective tka were relatively healthy ( the majority of patients were asa ps 1 or 2 [ 95.8% , table 1 ] ) , 91.1% of the study population had their preoperative albumin levels within normal range ( normal range : 3.55.2 g / dl in our center ) . anesthetic time and tourniquet time were prolonged in group 1 . after reducing the effect of confounding factors by the iptw method , multivariate logistic regression analysis revealed that age ( or , 1.05 ; 95% ci , 1.011.09 ; p = 0.030 ) , dm ( or , 3.12 ; 95% ci , 1.655.89 ; p < 0.001 ) , preoperative beta blocker use ( or , 2.65 ; 95% ci , 1.484.73 ; p = 0.001 ) , ua level ( or , 1.51 ; 95% ci , 1.261.82 ; p < 0.001 ) , intraoperative diuretic use ( or , 16.42 ; 95% ci 3.0887.68 ; p = 0.001 ) , and pod2_alb level < 3.0 g / dl ( or , 1.92 ; 95% ci 1.093.37 ; p = 0.023 ) were risk factors for aki by the kdigo criteria ( table 2 ) . in multivariate cox proportional hazards analysis , aki ( hr , 3.41 ; 95% ci 1.696.86 ; p = 0.001 ) , general anesthesia ( hr , 2.27 ; 95% ci 1.094.72 ; p = 0.028 ) , and age ( hr , 1.09 ; 95% ci 1.051.13 ; p < the intergroup differences of postoperative outcomes stratified by the pod2_alb level are shown in table 4 . crude ( univariate ) analysis indicated that the pod2_alb level < 3.0 g / dl was related with the occurrence of aki ( or , 1.77 ; 95% ci , 1.043.01 ; p = 0.036 ) . the hospital stay was longer in patients with pod2_alb level < 3.0 g / dl than that of the pod2_alb level 3.0 g / dl group ( 16.6 5.6 days vs 16.0 4.2 days , respectively , p = 0.024 ) . after iptw adjustment , the pod2_alb level < 3.0 g / dl was also associated with aki ( or , 1.82 ; 95% ci , 1.033.24 ; p = 0.041 ) and longer hospital stay than pod2_alb level 3.0 g / dl ( 16.7 5.5 days vs 15.9 4.2 days , respectively , p = 0.001 ) . meier survival curve demonstrated that the incidence of overall mortality was higher in patients with aki than in those without aki ( log - rank test , p < 0.001 , fig . however , no correlation was found between pod2_alb level < 3.0 g / dl and overall mortality after iptw adjustment in patients who underwent tka ( hr , 0.99 ; 95% ci 0.581.68 ; p = 0.973 ) . comparison of the aki incidence and overall mortality by the lowest serum albumin level within 2 postoperative days . the survival rate of patients with acute kidney injury was significantly lower than in those without acute kidney injury ( log - rank test , p < 0.001 ) . in this study , we demonstrated that the prevalence of aki was 4.4% in patients who underwent unilateral tka . multivariate analysis revealed that age , dm , preoperative beta blocker use , high ua level , intraoperative diuretic use , and pod2_alb level < 3.0 g / dl were related with aki by kdigo criteria . even after iptw adjustment , the pod2_alb level was an independent risk parameter for aki based on the kdigo criteria and longer hospital stay . the prevalence of aki in this study was within the range of previous reports . in spite of the previous studies , kateros et al . and grams et al . demonstrated that the occurrences of aki after orthopedic surgeries were 8.9% and 11.2% , respectively . however , due to disparity of the surgery types , the incidence of aki in current study was 50% less than the above 2 studies . we only included tka and excluded emergent and revision surgery , which are known to be significant predictors of aki . although the mechanism has not been fully elucidated , studies have shown that albumin has a renoprotective effect . albumin can act as an antioxidant by ligand - binding and free radical - trapping properties . a previous study reported that albumin plays an important role in survival of renal tubular cells and macrophages by scavenging of reactive oxidative species . albumin has been shown to possess a reservoir function for signaling molecules and donors of nitric oxide ( no ) . no generated from l - arginine raises renal blood flow and the glomerular filtration rate by dilation of vessels , and improves renal function . additionally , albumin tends to improve microcirculatory performance , thus supporting maintenance of major organ function . notably , the endothelial glycocalyx layer , an albumin - rich layer made of glycosaminoglycans , is known to be crucial to the endothelial barrier , and the disruption of the glycocalyx layer is associated with protein extravasation , tissue edema , and accelerated inflammation . a recent study demonstrated that the endothelial glycocalyx layer might be damaged in patients with aki in liver transplantation . in relation to postoperative hypoalbuminemia , injury of the endothelial glycocalyx layer may contribute to change in oncotic pressure gradients , and albumin and fluid leakage into the interstitium , all of which could possibly result in aki occurrence following tka . wiedermann et al provided evidence that hypoalbuminemia is a significant predictor of both aki and mortality following aki development in various settings . lee et al also reported that preoperative hypoalbuminemia is a major risk factor for aki after off - pump coronary artery bypass surgery . in addition , sang et al showed that the pod2_alb level was a potent predictor of aki defined according to both the akin and rifle criteria , and that aki was related to overall mortality in patients with living donor liver transplantation . however , there is little information about the association with early postoperative albumin level and aki occurrence in tka . to our knowledge , the present study is the first to demonstrate the association between postoperative low albumin level and aki in patients undergoing tka surgery . we selected early postoperative hypoalbuminemia as a main factor for the development of aki because early detection of risk factors for aki would be beneficial in perioperative management and could improve the clinical outcome . in this current study , the pod2_alb level turned out to be an independent risk parameter for aki based on the kdigo criteria in patients who underwent primary unilateral tka . although administration of albumin had beneficial effects on renal function in a recent meta - analysis , conflicting results have been demonstrated . response relationship between albumin administration and the risk for aki in cardiac surgery patients . nonetheless , caironi et al showed that maintenance of albumin level > 3 mg / l did not have an advantage in reducing renal dysfunction or in increasing overall survival in patients with severe sepsis . further multicenter randomized controlled trials will be required to investigate whether administration of albumin could prevent renal injury . in this study , general anesthesia , age , and aki were risk factors for overall mortality following tka . among these , aki was the most potent predictor of overall mortality . moreover , in the present study , the pod2_alb level was an independent risk factor for aki . as a result , the pod2_alb level may have indirectly influenced overall mortality by preceding aki in patients undergoing tka . though we tried to take into account confounding factors and reduce bias through the iptw method , it was impossible to exclude the effect of hidden factors . additionally , because our study was an observational analysis , we could not determine a causal relationship between the pod2_alb level and the risk of postoperative aki and overall mortality . further prospective study will be necessary to approve the causal relationship between albumin and aki in patients with tka .

abstracthypoalbuminemia has been reported to be an independent risk factor for acute kidney injury ( aki ) . however , little is known about the relationship between the albumin level and the incidence of aki in patients undergoing total knee arthroplasty ( tka ) . the aim of our study was to assess incidence and risk factors for aki and to evaluate the relationship between albumin level and aki following tka.the study included a retrospective review of medical records of 1309 consecutive patients who underwent tka between january 2008 and december 2014 . the patients were divided into 2 groups according to the lowest serum albumin level within 2 postoperative days ( pod2_alb level < 3.0 g / dl vs 3.0 g / dl ) . multivariate logistic regression analysis was used to assess risk factors for aki . a comparison of incidence of aki , hospital stay , and overall mortality in the 2 groups was performed using propensity score analysis.of 1309 patients , 57 ( 4.4% ) developed aki based on kidney disease improving global outcomes criteria . factors associated with aki included age ( odds ratio [ or ] 1.05 ; 95% confidence interval [ ci ] 1.011.09 ; p = 0.030 ) , diabetes ( or 3.12 ; 95% ci 1.655.89 ; p < 0.001 ) , uric acid ( or 1.51 ; 95% ci 1.261.82 ; p < 0.001 ) , beta blocker use ( or 2.65 ; 95% ci 1.484.73 ; p = 0.001 ) , diuretics ( or 16.42 ; 95% ci 3.0887.68 ; p = 0.001 ) , and pod2_alb level < 3.0 g / dl ( or 1.92 ; 95% ci 1.093.37 ; p = 0.023 ) . after propensity score analysis , pod2_alb level<3.0 g / dl was associated with aki occurrence ( or 1.82 ; 95% ci 1.033.24 , p = 0.041 ) and longer hospital stay ( p = 0.001).in this study , we demonstrated that pod2_alb level<3.0 g / dl was an independent risk factor for aki and lengthened hospital stay in patients undergoing tka .

nanowell - confined chemistry is developed and optimized using carbon nanotube transistors with a 4 m channel length between source and drain electrodes . nanowells , ranging from 5 nm to the full 4 m channel length , are patterned in a thin polymer layer using high - resolution electron - beam lithography . we design the masks to cover the electrodes and to expose only a portion of the nanotube channel as illustrated in figure 2a . nanotube segments outside the electrodes are kept fully covered on one side and fully exposed on the other side to act as negative and positive controls , respectively . figure 2b shows atomic force microscopy ( afm ) images of a 4-m - long nanotube device covered by a mask with a 20-nm - wide nanowell ( middle ) and the same nanotube after removal of the mask ( top ) . the corresponding height profile in figure 2b ( bottom ) reveals a polymer thickness of approximately 70 nm and confirms the position and width of the nanowell ( see additional afm characterization in supplementary figure 1 ) . to confirm full - depth opening of the nanowells down to substrate level , we evaporate an 8-nm - thick layer of titanium over test masks with nanowells of various widths . scanning electron microscopy of the resulting metal lines ( shown in supplementary figure 2 ) indicates that the smallest wells that can be reliably fabricated are 20 nm wide . after incubation for 24 h in saline buffer , we observe no significant deterioration in the polymer defining the nanowells ( see supplementary figure 3 ) . ( a ) mask design showing a nanowell of controlled position and width over the device channel . positive and negative controls are designed outside the device channel using full exposure and full protection of the nanotube , respectively . ( b ) atomic force microscopy image showing a carbon nanotube between metallic electrodes ( top ) and the same device covered with a protecting mask opened with a 20 nm nanowell ( middle ) , along with the corresponding height profile ( bottom ) . ( c ) distribution of conductance change g / g0 after functionalization within nanowells of different widths , compiled on n individual devices from the same nanotube ( ntotal = 196 ) . arrows indicate the mean conductance change obtained from a log - normal fit of the distribution . ( d ) conductance change and error bars ( s.d . ) obtained using 20 nm nanowell masks ( cyan ) compared to control devices ( black ) . ( e ) modeled probability of getting a number n of functional groups using small nanowells . circles represent measured points based on data in panel c ; others are extrapolated from a poisson distribution . in order to install functional groups on the nanotube sidewall , we perform covalent chemistry inside the nanowells formed in the polymer mask . aryl diazonium chemistry is chosen because it is a reliable , well - characterized reaction that forms stable adducts . this reaction also provides good electronic coupling with the nanotube , and attached single - molecule probe groups can be terminated with reactive functional groups . in this study , we functionalize carbon nanotubes by incubating mask - covered devices for 24 h in an aqueous solution of 4-carboxybenzenediazonium tetrafluoroborate salt ( cbdt ) ( see details on the functionalization protocol in the supporting information ) . afm profiles taken before and after functionalization confirm the absence of swelling or other alterations in the mask ( see figure 2b and supplementary figure 3 ) . after the reaction , we remove the mask by soaking devices in room - temperature acetone , thus making the functional groups available for chemical reactions in organic solvents and with larger , more complex molecules . we study the statistics of nanowell - confined chemistry by exposing hundreds of devices created from the same , ultralong nanotube to the cbdt reaction ( see supporting information and supplementary figure 4 ) . the width of the nanowells is varied from 20 nm to 4 m , and some devices are kept fully covered with resist to serve as negative controls . for each device and report the conductance change defined as g / g0 = gfunctionalized / ginitial at vds = 50 mv and vg = 10 v ( see supplementary figure 5 for examples of full current voltage characteristics ) . figure 2c shows histograms of the data for each nanowell width on a typical nanotube , as well as the mean conductance change obtained from a log - normal fit of the distributions . as expected , the fully exposed devices exhibit a more than an order of magnitude drop in conductance as well as a defect - related d band in their raman spectra ( see supplementary figure 6 ) . as the nanowell width is decreased , the conductance change and d - band intensities also decrease ( see supplementary figure 7 ) . these observations confirm that functionalization does occur on the nanotube and only inside the nanowell . in the case of the smallest 20-nm - wide nanowell , we observe a small but consistent conductance change when compared to the unexposed controls ( figure 2d ) , as tested on four different nanotubes . in contrast to the exposure through large nanowells , where current drops are highly variable for different nanotubes ( see supplementary figure 7 ) , reaction in 20-nm - wide nanowells generates a reproducible 20% average current drop on all tested nanotubes . this difference between large - scale and localized functionalization is likely due to the fact that , in the case of large nanowells , cbdt molecules have a high probability of encountering intrinsic , reactive defects along the nanotube , resulting in inhomogeneous reactions , whereas , in the case of 20 nm - wide nanowells , cbdt molecules have a very low probability of encountering such defects . consistency in the current drop for small nanowells across different devices and nanotubes suggests that it stems from the creation of a consistent number of functional groups . the actual number of functional groups is extracted from the population of unreacted devices for each nanowell width . we find that the overlap between the g / g0 distributions of 20 nm nanowell devices and unexposed control devices is about 1 in 4 , which is assigned to the population of unreacted devices . using a poisson distribution p(n ) = e / n ! to model the probability p(n ) to get a number n of individual functional groups , we first estimate p(0 ) from the fraction of devices with no or positive change in conductance , then extrapolate to find the average number of functional groups . for 20 nm nanowells , we find an average of = 1.39 functional groups per device and a population of devices having single - molecule probe attachment of p(n = 1 ) = 35% , which approaches the = 1 theoretical optimum for single - molecule devices illustrated in figure 2e . in contrast , larger nanowells allow for a wider distribution in the number of functional groups per device , as illustrated by the poisson distribution calculated for 40 nm nanowells in figure 2e and by the corresponding broadening of histograms in figure 2c . finally , we note that the 20% average conductance drop associated with this single - molecule reaction is consistent with recent experimental work on cbdt - induced defects and that this value is significantly less in amplitude than that obtained by oxidative defect - mediated single - point approaches , in agreement with hybrid density functional theory ( dft)/nonequilibrium green s function calculations that predict the oxidative method to generate a greater alteration than a single - point chemical bond . to confirm the presence of a single - molecule functional group and to test the ability of the platform to monitor sequential chemical reactions on this probe , we use an array of ten smfets functionalized using nanowell - confined chemistry and integrated with a microfluidic platform ( see supporting information and figure 3a ) . of 10 devices , seven are exposed through 20-nm - wide nanowells using cbdt chemistry ( exposed devices ) , and three are designed to be controls by fully covering them with the polymer mask ( unexposed raman spectroscopy is used on positive and negative controls to confirm , respectively , the reactivity of the nanotube to the diazonium chemistry and the effectiveness of the protective mask ( see figure 2a ) . after completing the reaction , five of the exposed devices experience an average drop in conductance of 20% , a signature consistent with that obtained above in the statistical analysis for the binding of single - molecule probes . the two other exposed devices show no conductance drop , consistent with remaining not functionalized , as discussed above . functionalized and unfunctionalized , respectively ( see supplementary table 1 ) . similarly , all unexposed devices preserve their conductance due to the protection provided by the mask . real - time sensitivity to secondary reactions on the primary single - molecule probe . ( a ) design of smfet devices , including carbon nanotube channel , drain , and source electrodes , as well as pseudoreference electrodes for electrolytic gating in the microfluidic cell . ( b ) reaction kinetics between an edc molecule and a single - molecule carboxyphenyl probe covalently attached on the nanotube . two levels of kinetics can be observed : slow transition between the unbound and bound states , corresponding to the reversible activation and hydrolysis reactions on the cooh probe , as well as fast kinetics due to fluctuations within a bound cooh / edc complex . ( c ) real - time response of a device in the presence of 50 m edc , after baseline correction , showing an active phase with two - state activity characteristic of rapid fluctuations in a single carboxy - edc adduct ( b1/b2 fluctuations ) . ( d ) zoomed region of the trajectory superimposed with an idealized trace obtained from a hidden markov model , revealing events resolved in the submillisecond range . ( e ) survival probability plot for the high- and low - conductance states , fitted with a single - exponential function to obtain average lifetimes high and low. ( f ) distribution of high/low ratios obtained from multiple , successive , 1-s - long segments of a long trajectory , showing kinetics centered on the symmetric case in which high = low , with a level of heterogeneity in the distribution that is typical of single - molecule experiments . all devices are immersed in aqueous 2-(n - morpholino)ethanesulfonic acid ( mes ) buffer solution ( 100 mm , ph 4.5 ) in the flow cell , and their drain - source current is measured in real - time using a constant dc applied bias of vsd = 100 mv and an electrolytic gating potential of vl = 300 mv , chosen to place the electrostatic potential of the nanotubes between their electrical on and off states ( see supporting information and supplementary figure 8) . under these conditions , all devices exhibit a baseline current of several hundreds of na with 1/f background noise and no distinguishable activity ( see supplementary figure 9 ) . the buffer solution is then switched to a 50 m solution of 1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide ( edc ) in aqueous mes buffer ( 100 mm , ph 4.5 ) . edc is an activating and dehydrating agent that forms an o - acylisourea derivative of a carboxylic acid . these intermediate derivatives hydrolyze quickly in the presence of water with a typical turnover period of 10100 s in such conditions , as illustrated by the slow kinetics between the unbound ( a ) and bound ( b ) states in figure 3b . in presence of edc , two of the functionalized devices are found to exhibit successive phases of random telegraph noise ( rtn ) in their conductance , separated by inactive phases as shown in supplementary figure 10 . following goldsmith et al . , we attribute the inactive phases to the unbound , hydrolyzed state ( a ) and the active phases with rtn to rapid fluctuations ( b1/b2 ) within a single cooh / edc complex . the period of active or inactive phases is observed to be longer than 5 s ( see supplementary figure 10 ) , which is consistent with the turnover rate of the activation / hydrolysis reaction . an example trajectory measured in an active phase is shown in figure 3c and d , after baseline correction for clarity ( see raw trajectories and baseline fit in supplementary figure 10 ) . the trajectory shows rapid two - state rtn activity , in which dwell times with submillisecond duration can be resolved ( see figure 3d ) . using a hidden markov model , we extract the distribution of dwell times in the high- and low - conductance states and build survival probability plots , presented in figure 3e . these plots can be fitted with a single - exponential function , indicating simple kinetics with average lifetimes of high = 4.5 ms and low = 4.7 ms for the high- and low - conductance states , respectively . the similarity between the lifetimes of the two states in that trajectory indicates that both states are almost equally probable , i.e. , that the equilibrium constant between b1 and b2 is close to 1 . when repeating this analysis on multiple , successive 1-s - long segments , we obtain a distribution of high/low ratios centered around the case in which high = low , as presented in figure 3f . the width of the distribution illustrates a level of heterogeneity in the kinetics which is commonly observed in single - molecule measurements . we also perform several controls to ensure that the active phases with rapid two - state rtn are strictly related to the cooh / edc adduct ( see supplementary figure 9 ) . first , the observed two - state rtn disappears after flushing the device with fresh buffer , consistent with the fact that the system remains in the unbound state ( a ) after the edc molecule is washed away . in addition , none of the three unexposed devices nor the two unfunctionalized devices exhibit any activity when exposed to edc ( see supplementary table 1 ) . examples of trajectories for these different types of controls are presented in supplementary figure 9 . the presence of multiseconds - long inactive and active phases , millisecond - scale fast rtn in the active phase , and heterogeneous kinetics centered on high = low are all consistent with results reported for the interaction of the edc molecule with a single carboxylic acid group . moreover , the ratio of exposed devices exhibiting this specific signature ( 2 out of 7 ) is compatible with the proportion of devices functionalized with a single - molecule probe as derived from the earlier , large - array study . this remarkable agreement obtained from two independent experiments ( i.e. , the conductance change experiment and the real - time reactivity experiment ) corroborates the ability of our nanowell - confined chemical approach to produce single - molecule probes with high and reproducible yields . more practically , the presence of this specific cooh / edc signature can be used as an indicator to identify , from a large array of devices , the set of devices holding a single - molecule probe , which can be further used as a template to support a variety of other individual molecules , as demonstrated in the following section . finally , we demonstrate the ability of the platform to support a second level of functionalization , by covalently tethering an individual biomolecule to the primary single - molecule probe and measuring its conformational dynamics in real - time . for these experiments , we use a 24-nucleotide , 5-amino - modified , single - stranded dna oligomer that models human telomeric dna in that it is composed of four repeats of the ttaggg guanine - rich sequence typically found in the terminal , single - stranded , telomeric region of human chromosomes ( see supporting information for full sequence ) . this sequence can fold into a structure called a g - quadruplex that is comprised of a series of stacked guanine tetrads as illustrated in figure 4a . monovalent cations , in particular k and , to a lesser extent , na , stabilize the folded form of the g - quadruplex sequence by binding between ( k ) or within ( na ) the plane of the guanine tetrads . on the same microfluidic - integrated array of ten smfets described above , we covalently attach the 5-amino - modified dna oligomer to the primary single - molecule probe on the carbon nanotube via an amide bond formed using two consecutive chemical reactions , as described in the supporting information . ( a ) representation of a folded g - quadruplex dna sequence capturing two k cations , bound to an initial single - molecule probe on a nanotube . ( b c ) real - time trajectories with baseline correction , showing two - state fluctuations in conductance in the presence of 10 mm kcl or nacl , respectively . ( d ) survival probability plots for each conductance state ( high - g / low - g ) and each ion type ( k / na ) are fitted with a double - exponential kinetic model . ( e ) average lifetime fast and error bars extracted from the survival plots in panel d for the high - g and low - g states as well as both k and na ions . ( f ) high - g and low - g conductance states are attributed to the unfolded and folded dna configurations , respectively . the folded configuration is found to have a longer lifetime in the presence of k cations than na . after functionalization with the dna oligomer , we measure the conductance of all ten devices in real time and at room temperature , with vds = 100 mv and vl = 300 mv . the conductance of the devices is recorded in real - time for 15 min in tris - hcl buffer ( 10 mm , ph 7.5 ) supplemented with 10 mm kcl . under these conditions , one device exhibits fluctuations between a low - conductance state ( low - g ) centered at 1.49 s and a high - conductance state ( high - g ) centered at 1.61 s , as shown in the baseline - corrected trajectory in figure 4b ( raw traces are provided in supplementary figure 11 ) . after once again flushing the flow cell thoroughly , we collect real - time data for 15 min and at room temperature in tris - hcl buffer , supplemented this time with 10 mm nacl . as observed in the presence of k , the trajectory recorded in the presence of na shows fluctuations between a low - g state and a high - g state , respectively centered at conductance values of 1.64 s and 1.73 s ( figure 4c ) . a control experiment recorded using the same device , but in the presence of only the tris - hcl buffer ( i.e. , in the absence of k or na ions ) , does not exhibit such fluctuations , which is consistent with other studies reporting only weak interaction between pristine carbon nanotubes and g - quadruplex dna . similarly , measurements using an unexposed control device do not exhibit such fluctuations , either in the presence of kcl or nacl . each of these controls is presented in supplementary figure 12 . in order to rigorously assign the low - g and high - g states to configurations of the g - quadruplex oligomer , we perform a statistical analysis of the transitions between these states . to do so , we use a hidden markov model as previously described to calculate the most probable sequence of transitions between the low - g- and high - g states forming the trajectories ( i.e. , the idealized paths ) ( see supporting information and supplementary figure 11 ) . survival probability plots of the dwell times spent in low - g state prior to transitioning to the high - g state and , conversely , of the dwell times spent in high - g state prior to transitioning to the low - g state are shown in figure 4d . consistent with previous single - molecule biophysical studies of g - quadruplex sequences , each survival probability plot is best described by a double exponential decay function ( see supplementary figure 13 ) , yielding two characteristic lifetimes ( fast and slow ) for both the low- and high - g states ( see supplementary table 2 ) . while the small number of long dwells that predominantly contribute to slow render this lifetime particularly susceptible to the effects of possible missed transitions , the much larger number of short dwells that predominantly contribute to fast render this lifetime significantly resistant to the effects of possible missed transitions ( see supplementary figure 14 ) . consequently , fast is a much more robust lifetime measurement and is therefore presented in figure 4e for each state and each ion type . for the low - g state , we find that fast obtained in the presence of k is 10 times longer than that obtained in the presence of na . in contrast , for the high - g state , we find that fast obtained in the presence of k is within error of that obtained in the presence of na . previous studies of g - quadruplex sequences similar to that investigated here have demonstrated that , at any particular monovalent ion concentration , the folded form of the g - quadruplex sequence should be more stable in the presence of k versus na , whereas the stability of the unfolded form should be independent of the identity of the ion . given that all of our experiments are conducted at the same monovalent ion concentration and given our finding that k stabilizes the low - g state by a factor of 10 over na , whereas the stability of the high - g state is independent of the identity of the ion , we unambiguously assign the low - g and high - g states of the trajectories to the folded and unfolded forms of the g - quadruplex sequence , respectively ( see figure 4f ) . we note that the values of fast for both the folded and unfolded forms of the g - quadruplex sequence reported here are in close agreement with the corresponding lifetimes of the folded and unfolded forms of a similar g - quadruplex sequence obtained from single - molecule fluorescence resonance energy transfer ( smfret ) studies performed under similar experimental conditions to those used here . in addition , the existence of two classes of dwells ( i.e. , fast and slow ) in both the folded- and unfolded forms of the g - quadruplex sequence is fully consistent with previous smfret studies of similar g - quadruplex sequences . the close correspondence between our results and the results of smfret studies of analogous g - quadruplex sequences suggests that tethering of the g - quadruplex to the surface of the carbon nanotube device does not impair the folding / unfolding dynamics of the g - quadruplex . this observation validates the use of the smfet devices described here for single - molecule studies of biomolecular folding and structural dynamics . notably , the label - free- , increased time - resolution- , and expanded observation time aspects of the smfet approach described here should enable investigations of biomolecular folding reactions and dynamic processes that are currently difficult or impossible to investigate using more conventional single - molecule biophysical approaches such as smfret , single - molecule force spectroscopy , or single - molecule tethered - particle motion approaches . nanowell - confined chemistry on carbon nanotube devices provides a versatile platform to support and monitor individual chemical reactions . we obtain a high yield for the attachment of the primary single - molecule probe , and the covalent chemistry ensures strong nanotube - molecule coupling and long - term stability of the adduct . the method is independent of a specific chemical reaction and generalizable to any aqueous nanotube chemistry . it is also easily scalable to the wafer scale , enabling the production of a large number of devices and robust statistical analysis . once the mask defining the nanowells is removed , the platform supports an even larger variety of reactions and reagents , for instance reactions in organic solvents or bioconjugation with large macromolecules . successive secondary single - molecule reactions on the same single - molecule probe can be recorded in real time , with submillisecond resolution and for many hours . in particular , this platform can measure the conformational dynamics of individual dna g - quadruplexes , which are important components of the biological mechanisms underlying cell aging and the proliferation of cancer cells , in that their formation is known to inhibit the replication of the terminal ends of chromosomes . more generally , this platform opens routes to investigate a variety of other fundamental chemical mechanisms such as lifetimes of intermediates in catalytic reactions and to improve chemical sensor and lab - on - chip technology with localized functionality .

a new approach to synthetic chemistry is performed in ultraminiaturized , nanofabricated reaction chambers . using lithographically defined nanowells , we achieve single - point covalent chemistry on hundreds of individual carbon nanotube transistors , providing robust statistics and unprecedented spatial resolution in adduct position . each device acts as a sensor to detect , in real - time and through quantized changes in conductance , single - point functionalization of the nanotube as well as consecutive chemical reactions , molecular interactions , and molecular conformational changes occurring on the resulting single - molecule probe . in particular , we use a set of sequential bioconjugation reactions to tether a single - strand of dna to the device and record its repeated , reversible folding into a g - quadruplex structure . the stable covalent tether allows us to measure the same molecule in different solutions , revealing the characteristic increased stability of the g - quadruplex structure in the presence of potassium ions ( k+ ) versus sodium ions ( na+ ) . nanowell - confined reaction chemistry on carbon nanotube devices offers a versatile method to isolate and monitor individual molecules during successive chemical reactions over an extended period of time .

antimicrobial substances comprise , in addition to pharmacological products ( antibiotics ) , also a series of antimicrobial chemical compounds marketed as biocides , disinfectants , antiseptics , or preservatives . given that both antibiotics and biocides are characterized primarily by their antimicrobial action it is obvious that the selective pressure exerted by these compounds on bacteria may have analogies , and thus might converge in similar mechanisms of resistance or co - existence of resistance . in such a way , selective pressure exerted by any of these types of antimicrobial agents on bacterial populations translates into selection of those cells which are resistant , with the literature being full of examples for both co- and cross - resistance [ 1 , 2 ] . taking into consideration these studies , national and international agencies and consumer associations have recently raised awareness on a risk of widespread biocide use in health care settings , in the environment , and in cosmetic and food industry towards selection of antibiotic - resistant bacteria . this has been witnessed by the 2009 report of the scientific committee on emerging and newly identified health risks on biocides in general , the report of the scientific committee on consumer safety in 2010 , and the most recent report ( december 2013 ) of the food and drug administration ( fda ) . all these reports pose significant questions highlighting one of the main issues that , despite the information obtained in vitro on laboratory strains , there exists a lack of coherent and relevant epidemiological data linking utilization of compounds to toxicity , resistance and aspects of co- and cross - resistance to antibiotics . we have recently reported epidemiological cut - off values of biocide susceptibility profiles for over 3300 strains of different species . here , we report the correlation of the susceptibility profiles between antibiotics and biocides in the largest group of these strains in order to provide a clear dataset onto which to base future risk evaluations . this is particularly relevant because biocides are usually utilized at bactericidal concentrations , and hence changes on mbc can reflect on the actual susceptibility to these antimicrobial agents . to investigate the relationship between susceptibility profiles of biocides and antibiotics , we determined the susceptibility profiles of the most commonly used antibiotics in 1632 clinical s. aureus isolates with known susceptibility profiles of the biocides chlorhexidine , benzalkonium chloride , sodium hypochlorite and triclosan [ 8 - 10 ] . the evaluation of the relationship between biocide and antibiotic susceptibility profiles was performed using non - linear correlations . spearman 's correlation coefficient was computed for each bivariate combination of these variables in order to find non - linear associations between the biological variables . for each computed spearman 's correlation coefficient , a hypothesis test was performed in order to test the statistical association between each pair of variables and a p - value calculated followed by a bonferroni correction to control the family - wise error rate due to multiple testing . using the non - linear correlation approach , no strong relationship between any biocide and antibiotic phenotypes was evidenced . indeed , the data analysed showed weak to moderate bivariate correlations . the result of this study matches with that of a previous study of a smaller group of antibiotics where only the profiles of both benzalkonium chloride and chlorhexidine were associated with multi - drug resistance [ 10 , 11 ] . in this dataset , we found , after performing the correction for family - wise error rate , a series of pairs of variables with a statistically significant spearman s correlation coefficient . the highest correlation coefficient characterised the compounds of the same class to other compounds of the same class ( i.e. resistance to one beta - lactam is correlated to resistance to another beta - lactam ) . as expected high correlation coefficients for macrolides ( > 0.86 ) , quinolones ( > 0.77 ) and beta - lactams ( > 0.67 ) were yielded . the shading in table 1 also clearly indicates the pair - wise relationship between macrolides , beta - lactams , quinolones , and also quinupristin / dalfopristin . such association is reflected by the well - known occurrence of multi - drug resistance in methicillin - resistant staphylococcal clones . with respect to the biocides , a series of observations have to be made which include ( i ) that whether the mics to chlorhexidine and benzalkonium chloride have a statistically significant coefficient of 0.5 in accordance with the fact that both compounds are effluxed by the nora and qacabcghj efflux pumps ; on the contrary , absence of any correlation between mics and mbcs for both chlorhexidine and benzalkonium chloride is in accordance with the absence of correlation of any known death - preventing and mbc - increasing resistance mechanisms , and ( iii ) a correlation coefficient of 0.6 between the mics and mbcs for triclosan which are in accordance with the molecular characterisation of phenotypes conferred by fabi - related resistance mechanisms , influencing both inhibition and cell death . the main outcome relative to the scope of this work was the characterisation of bivariate correlations between biocide and antibiotic susceptibility profiles of a collection of staphylococcal bacteria , where we compared both the mics and mbcs of biocides to the mics of antibiotics ( table i ) . a previous study was conducted in this direction but with a smaller group of antibiotics . the analysis of the same set of strains using machine learning techniques , has shown that a simultaneous reduced susceptibility to chlorhexidine and to benzalkonium chloride is associated with resistance to at least one antibiotic . however , using the same approach we were unable to find specific biocide / antibiotic associations . in this study , we expand our analysis by studying the independent correlations on the susceptibilities of a large group of antibiotics and a set of the most frequently used biocides : chlorhexidine , triclosan hypochloride and benzakonium . the data obtained in this study did not show any strong or moderate statistically significant correlation when comparing the susceptibilities of either triclosan or sodium hypochlorite for any of the tested antibiotics . on the other hand , correlation coefficients for mics of benzalkonium chloride were found to be above 0.4 associated with the susceptibility of quinolones , beta - lactams and also macrolides . this correlation might be due to the well described association of qac determinants and the beta - lactamase transposon tn552 which are located on psk41-like plasmids , widespread among clinical multi drug resistant s. aureus clones since early 50s and also among animal isolates of different staphylococcal species . the somewhat weaker data for chlorhexidine are due to the less evident phenotype conferred by the qaca efflux pump . a large survey on over 3300 isolates of s. aureus , salmonella spp . enterococcus faecium , and enterococcus faecalis has recently shown that biocide resistant subpopulations are uncommon in natural populations of clinically relevant microorganisms . in this study , s. aureus exhibited discrete subpopulations with defined genetic mechanisms for reduced susceptibility to triclosan , benzalkonium chloride and chlorhexidine [ 7 - 9 ] . we here performed a correlation analysis of the biocide susceptibility profiles to antimicrobial drug resistance in general by testing over 1600 clinical s. aureus isolates . data clearly show an absence of correlation of susceptibility profiles for the biocides triclosan or sodium hypochlorite and any clinically relevant antibiotic . in contrast , our data show a significant relationship with a moderate correlation between susceptibility profiles to chlorhexidine and benzalkonium chloride . although a relationship between mdr efflux pumps and quinolone resistance is known , it should be noted that while in vitro mutants in the promoter of the nora efflux pump gene confer resistance to norfloxacin and fluoroquinolones , which is not observed for all clinical isolates with nora promoter mutations . more importantly , resistance to benzalkonium chloride has been attributed to the activity of qac efflux pumps in the vast majority of clinical isolates and none of the qacabcg pumps was shown to confer fluoroquinolone resistance , still not ruling out that gyrase or topoisomerase mutation may be facilitated in strains carrying qac efflux pumps . these observations would thus favour the hypothesis that cross - resistance between benzalkonium chloride and chlorhexidine on one side and fluoroquinolones could be involved to some extent in co - selection of strains . in the case of beta - lactams , the fact that the blaz gene is carried by most if not all qaca plasmids would indicate that co - resistance could be the driver of co - selection of resistant strains . in this context , it should be noted that the recently identified is256 associate sh - fabi gene which confers reduced susceptibility to triclosan was found in few occasions on qaca carrying plasmids and plasmids which also harbour cadmium resistance pumps . these data indicate that there are numerous distinct mechanisms potentially responsible for the association of the qaca carrying psk41-like plasmids to mdr staphylococcal clones and that this association can not be pinpointed to a single mechanism . the data here show that in s. aureus there is no correlation of susceptibility profiles to triclosan or sodium hypochlorite and any clinically relevant antibiotic . the data further show that there is in contrast a significant relationship with a moderate correlation between susceptibility profiles to the bis - biguanide chlorhexidine and the quaternary ammonium compound benzalkonium chloride and some classes of antibiotics . in the light of the recently published observations that most clinically relevant bacterial species do not show the presence of subpopulations with decreased biocide susceptibility , our data suggest that the global use of biocide to date appears not to have resulted in a clinically relevant impact on antibiotic resistance . while our data do not allow for inference as to the direction of selective pressure in the case of the association between susceptibility profiles to some biocides and antimicrobial resistance , they clearly rule out the possibility that such evidence exists at present for other compounds . while not addressing toxicity of the biocides , this report should answer some of the other questions relating to risk for human health raised by the recent fda report on the safety and effectiveness of consumer antiseptics .

there is a growing concern by regulatory authorities for the selection of antibiotic resistance caused by the use of biocidal products . we aimed to complete the detailed information on large surveys by investigating the relationship between biocide and antibiotic susceptibility profiles of a large number of staphylococcus aureus isolates using four biocides and antibiotics commonly used in clinical practice . the minimal inhibitory concentration ( mic ) for most clinically - relevant antibiotics was determined according to the standardized methodology for over 1600 clinical s. aureus isolates and compared to susceptibility profiles of benzalkonium chloride , chlorhexidine , triclosan , and sodium hypochlorite . the relationship between antibiotic and biocide susceptibility profiles was evaluated using non - linear correlations.the main outcome evidenced was an absence of any strong or moderate statistically significant correlation when susceptibilities of either triclosan or sodium hypochlorite were compared for any of the tested antibiotics . on the other hand , correlation coefficients for mics of benzalkonium chloride and chlorhexidine were calculated above 0.4 for susceptibility to quinolones , beta - lactams , and also macrolides . our data do not support any selective pressure for association between biocides and antibiotics resistance and furthermore do not allow for a defined risk evaluation for some of the compounds . importantly , our data clearly indicate that there does not involve any risk of selection for antibiotic resistance for the compounds triclosan and sodium hypochlorite . these data hence infer that biocide selection for antibiotic resistance has had so far a less significant impact than feared .

additional supporting information may be found in the online version of this article : video s1 .

key clinical messagelv pseudoaneurysm can be a late complication of mitral valve replacement . in our case , it was an early postoperative complication . this pseudoaneurysm was causing compression of lcx artery during systole , leading to presentation of nstemi two weeks after the surgery .

short bowel syndrome ( sbs ) is the most common cause of protracted intestinal failure . even though the causes of sbs are diverse , the management has identical pillars : parenteral nutrition ( pn ) and early enteral nutrition ( en ) . prognosis is associated with age - adjusted small bowel length , the presence of the ileocaecal valve ( icv ) and colon , the advent of pn complications , and the capacity of the remaining intestine to adapt . several hormonal and surgical methods to improve bowel adaptation have been reported [ 38 ] , but en remains an important and controllable factor . direct trophic effect of nutrients and stimulation of gastrointestinal secretions and hormonal factors enhances intestinal adaptation [ 3 , 6 , 8 ] . we present 4 cases of ultrashort bowel syndrome ( < 40 cm residual small bowel ) ( table 1 ) rapidly weaned of pn after introduction of an elemental formula ( neocate , nutricia , strombeek - bever , belgium ) . table 2 illustrates the composition of neocate compared to other elemental formulas available in other countries and to the semi - elementary formula used in 2/4 patients . all patients received a ppi ( omeprazole ) as additional medication as part of the sbs treatment . the diagnosis of congenital short bowel in patient 1 was made by explorative laparotomy at the age of 3 months . he had no treitz angle , a small bowel of 50 cm , and a colon of 30 cm length . en with semi - elemental formula was started following the en scheme used in our department as illustrated in table 3 . a third of his calories was tolerated by continuous en through a gastrostomy ( 43 kcal / kg / d ) , and home pn was organised for the remaining caloric requirements ( 86 kcal / kg / d ) . the advancement of en was limited by recurrent profuse diarrhoea , and at 3 years of age , neocate was introduced . he was started at 1/3 of his daily requirements ( total cals : 130 kcal / kg / d ) on continuous enteral infusion and upgraded following table 2 . after initial pn weaning , the patient needed another 5 days of tpn during an episode of d - lactate acidosis with severe dehydration . he was started on probiotics ( vsl3 : 450 bilion lactic acid bacteria per package in defined ratios of bifidobacterium breve , bifidobactium longum , bifidobacterium infantis , lactobacillus acidophilus , lactobacillus plantarum , lactobacillus paracasei , lactobacillus bulgarius , streptococcus thermophilus by vsl pharmaceuticals , inc . ) and remains well since . although rast testing remained negative , serum albumin dropped , and the patient stopped growing for 12 months ( 25th percentile to 10th percentile ) . he remains on step 4 of the weaning scheme ( table 2 ) receiving 46% of his calories by nocturnal feeds ( 1500 ml neocate 20% + 20 g protifar = 1527 kcal/58.5 g ew/69.2 g v/168 g kh ) , and reintroduction of cow 's milk and soy protein is planned . his motor development is normal , and his growth parameters ( height and weight ) follow the 10th percentile . patient 2 was born at 25 weeks gestation , with a birth weight of 700 g and suffered severe enterocolitis at day 27 . a second look operation revealed 32 cm of small bowel remaining to re - establish continuity . several attempts to start and increase en with semi - elemental formula ( alfare , nestle , brussels , belgium ) failed . continuous enteral feeding with neocate was started successfully in combination with pn and oral stimulation ( table 2 , step 1 ) . at the age of 5 months the patient reached step 2 ( table 2 ) when she left the hospital at 8 months of age ( total calories 128.4 cal / kg / day ) . she obtained complete oral gastrointestinal autonomy at the age of 1 year and has successfully been switched to semi - elemental formula ( nutramigen , mead johnson , braine - l'alleud , belgium and frisomel ; friesland coberco dairy foods , meppel , the netherlands ) . her growth is normal ( weight 10th percentile , height 10th25th percentile ) , and her vitamin status and electrolytes are checked regularly and are within the normal range without supplements . patient 3 , born at 33 weeks , 1700 g , had dark gastric aspirates five days after birth . a large bowel resection left 12 cm of jejunum and 8 cm of ileum with an intact colon and a preserved icv . en with neocate was started 14 days after surgery following table 2 . at the age of 2 months , the patient tolerated full en on neocate 20% ( step 2 , table 2 ) . a gastrostomy was placed , and pn was discontinued . at discharge , he was on daytime boluses and night - time continuous feeding ( step 3a table 2 ) which were adapted weekly following tolerance ( total calories 142 cal / kg / day , 10% po , 90% ps ) . he achieved oral autonomy ( step 4 table 2 ) at 6 months , and solids were introduced . his oral caloric intake from neocate remained 93 cal / kg / day . by that time , the patient caught up in length and weight , reaching normal growth percentiles ( 3rd percentile ) for his age . in patient 4 , born at term , the amniotic fluid was meconium stained , the abdomen was dilated , and vital signs were compromised . extensive resection was needed , leaving 5 cm of duodenum , 4 cm of terminal ileum , the icv , and the colon . postoperatively tpn was initiated . on day 12 , continuous enteral feeding with neocate following table 2 was started . when she was discharged home at the age of 3 months , the gastrostomy had been removed due to peritonitis , and she was fed by small oral boluses of neocate 20% ( 6 20 ml ) and nightly pn ( 130 kcal / kg / d , 36% po and 64% pn ) . although she made good progress with oral neocate , growth deviation from the 50th to the 25th percentile indicated the need for extra support and nocturnal feeding through a gastrostomy at 11 months . the patient maintained her growth with oral intake and supplemental enteral feeding ( total 121.5 kcal / kg / d , 73.5% po and 26.5% ps ) ( step 4 table 2 ) . frequent liquid stools without reducing substances were debilitating and persisted despite trials of cotrimoxazole , cholestyramin , and probiotics . she eats a varied diet during the day and has nightly enteral supplements with neocate ( total 121.5 kcal / kg / d , 48% po and 52% ps , 4.9 g prot cow 's milk and soy are excluded from her diet because of clinical intolerance and elevated rast tests . she was toilet trained at 30 months and has normal motor development and growth ( weight 50th percentile , height 25th percentile ) . sbs is defined as malabsorption and failure to thrive following extensive resection of the small bowel . surgical classification of sbs is based on the residual length of the small intestine measured peroperatively along the antimesenteric border . although residual length is an important element , it is not the only prognostic factor . the adaptive capacity of the remaining gut is a major component for obtaining enteral autonomy . enteral autonomy and pn weaning while maintaining adequate growth are the principal endpoints in this group of patients . continuous enteral feeding is the starting method of choice as it enhances intestinal absorption and improves tolerance . concentration of the continuous feed is progressively increased until total caloric requirements are tolerated . only then , pn is discontinued , and bolus feeding initiated . enteral tolerance is monitored by intake , growth , stool frequency , stool consistency , reducing substances , and biochemistry . three of the 4 infants reported have an ultrashort gut ( < 40 cm ) , and one has a congenital short bowel of 50 cm , in which function is known to be altered . all patients needed parenteral nutrition , but enteral autonomy was obtained and maintained relatively rapidly ( < 13 months ) after introduction of neocate ( table 1 ) . georgeson and breaux retrospectively described a group of 52 sbs patients receiving an elemental diet . pn weaning was obtained in 12/21 , and the mean weaning period was 24.1 months . not all patients could be weaned off pn , but the majority of this group had no icv ( 17/21 ) . more recently , goulet et al . reported 78 sbs patients , receiving initially a hydrolyzed diet , followed up for 15 years . ultrashort bowel , < 40 cm , was seen in 21/78 patients . in 9 patients ( 42.8% ) pn weaning failed , and in the 12 patients remaining ( 57.2% ) , enteral or parenteral support was necessary after initial pn weaning . the weaning period in georgeson 's study was considerably shorter with an elemental diet compared with goulets ' group , receiving a semi - elemental diet ( 24.1 versus 47.4 months ) . semi - elemental or elemental formulas are most frequently started [ 1 , 6 ] . where some studies suggest that a more complex diet with long chain fat ( lcf ) and complex carbohydrates enhances adaptation [ 1315 ] , others support the important influence of an elemental diet on the weaning of pn and enteral tolerance . the first 2 patients described in our series failed to thrive on a hydrolyzed formula as did 4 patients reported by bines et al . . although the ratio is different , both hydrolyzed and elemental formulas contain lcf and complex carbohydrates . therefore , another contributing factor needs to be considered : the elimination of a certain allergen or the presence of a yet unidentified stimulating substance . many children acquire sbs at a very young age ; it makes them naturally prone for cow 's milk intolerance ( cmi ) . in infants generally , cmi is considered to be 2%-3% below the age of 2 years , but there is no exact data about the incidence of cmi and food allergy in sbs patients , except for small case series [ 16 , 17 ] . an alteration in gut permeability , often present in sbs patients , can be a predisposing factor . first of all , the administration of tpn without enteral support increases intestinal permeability , whereas the administration of even small amounts of en can reverse this process . bacterial overgrowth on the other hand , a common complication of sbs , influences gut permeability through local gut inflammation . we think that the combination of these factors might make sbs patients more prone for cmi . in 2 of our patients ( patient 1 and 4 ) , even the late introduction of cow 's milk protein failed . this is an additional argument for the contribution of cmi to their initial feeding intolerance and explains the success of neocate in these patients . only 1 study compared intestinal permeability in sbs patients in a crossover study with hydrolized and whole protein formula . ten patients were included when 30% of enteral nutrition was tolerated , and no difference in intestinal permeability was found . although measurement of intestinal permeability in this patient group while investigating tolerance to different formulas is of interest , no comment was made about pn weaning , 30% of enteral nutrition had to be tolerated before inclusion , the study period was rather short ( 60 days ) , and no washout period in between formulas was considered . because of a parental choice , none of the patients received breast milk . cmi develops less frequently in breastfed babies , and breast milk has the additional trophic effect of probiotics and growth factors on the intestinal mucosa , beneficial to sbs patients [ 8 , 18 ] . unfortunately we realise that , even after reporting these 4 cases , the published data about the relationship between residual bowel length , bowel adaptation , formula , cmi , and pn weaning remain scarce . in all our patients icv and colon were preserved , which increases the chance of definite pn weaning [ 1 , 2 , 6 ] . evidence from randomised controlled trials ( rcts ) on this subject is lacking . because of limited patient numbers , large rcts are difficult to perform in a single centre and a multicentre approach is necessary . measurement of intestinal permeability , growth , allergic parameters , and pn duration should be included . if breast milk is not an option , we would recommend an amino - acid based formula as the initial enteral feeding for sbs infants . it may lead to reduction in pn duration , cost , and complications and benefit the patients ' quality of life . despite all , it remains important as a doctor to focus on the outcome for the patient and to continue investigating if the initial treatment fails .

clinical management of short bowel syndrome remains a multistage process . although pn is crucial , early introduction of enteral feeding is mandatory . we describe retrospectively 4 patients with an ultrashort bowel who could be weaned off pn on very short terms after introduction of an amino - acid - based formula ( neocate ) . patient 1 had congenital short bowel with 50 cm small bowel and 30 cm colon . he had persistent diarrhoea on a semielementary formula . when neocate was introduced he could be weaned from pn within 6 months . patient 2 needed multiple surgical interventions because of nec at d 27 . he maintained 40 cm small bowel and an intact colon and remained pn dependent on semielemental formula . after introducing neocate , pn could be weaned within 3 months . in the next 2 patients , neocate was introduced as initial enteral feeding after bowel resection following antenatal midgut volvulus . patient 3 had 20 cm small bowel and an intact colon . pn was weaned after 2 months . patient 4 had 9 cm small bowel and an intact colon . pn was weaned after 13 months . in all patients ileocaecal valve ( icv ) was preserved . no consensus is reached on the type of formula to use for short bowel syndrome . compared to recent data in the literature , the weaning period in these 4 patients was significantly shortened on an aminoacid based formula . the reason for this may lie in the antiallergic properties of this formula . we recommend the use of an amino - acid - based formula to induce earlier weaning of pn .

a hypersensitivity reaction ( hsr ) is a host - dependent idiosyncratic adverse drug reaction ( adr ) that can not be predicted by the dose , frequency , or length of the treatment . hsr is defined by the triad of fever , rash , and internal organ involvement [ 16 ] . hsrs occur with an incidence of 1 in 1000 to 1 in 10000 drug exposures , with a mortality rate approaching 10% [ 58 ] . symptoms of hsr can range from general manifestations , such as morbiliform rash , urticaria , angioedema , fever , malaise , anaphylaxis , bronchospasm , and erythema multiforme , to severe cutaneous adverse reactions ( scar ) such as drug - induced hypersensitivity syndrome ( dihs)/drug reaction with eosinophilia and systemic symptoms ( dress ) , and stevens - johnson syndrome ( sjs)/toxic epidermal necrolysis ( ten ) [ 2 , 47 , 911 ] . cutaneous eruptions are reported in the vast majority of dihs / dress cases , often presenting as maculopapular rash or generalized erythematous rash [ 7 , 8 ] . the internal organ most often affected is the liver , with drug - induced liver injury ( dili ) presenting as anomalies in liver function tests or the presence of hepatomegaly . aspartate aminotransferase ( ast ) and alanine aminotransferase ( alt ) were found to be increased an average of 9 - 10-fold above the upper normal limits in a literature review of 172 dress cases . the presence of serum bilirubin raised > 3 times over the upper limit of normal along with aminotransferase elevations is associated with a more severe set of symptoms than isolated aminotransferase abnormalities alone , an observation known as hy 's law [ 12 , 13 ] . other internal organs , such as kidneys , are less often affected [ 4 , 7 ] . skin rash , liver involvement , high - grade fever , hypereosinophilia , lymphadenopathy , and the increased presence of atypical lymphocytes are symptoms associated with dihs / dress cases classified as maculopapular rash , erythema multiforme , exfoliative dermatitis , acute generalized exanthematous pustular dermatosis - like eruptions , and erythroderma are types of cutaneous manifestations associated with dihs , while mucosal involvement is rare . on the other hand , skin and mucosal involvement characterizes sjs / ten . a further distinction between dihs / dress and sjs / ten is the time to onset of symptoms , which is often delayed ( 4 - 5 weeks ) in the former , while it occurs in the early stages of drug exposure ( approximately 3 weeks ) in the latter . carbamazepine was the drug most often associated with dress among 44 drugs linked to this reaction in 172 cases reported over a period of 12 years . anticonvulsants such as carbamazepine , phenytoin , phenobarbital , lamotrigine , zonisamide , and sodium valproate are the class of drugs most often related to dihs / dress . other pharmaceutical agents , including allopurinol , nonsteroidal anti - inflammatory drugs , chlormezanone , aminopenicillins , cephalosporins , quinolones cycline antibiotics , and antiretrovirals , as well as anti - infective sulfonamides , have also been implicated [ 68 , 11 , 1416 ] . scar development results in patient hospitalization , and the culprit drug is interrupted on the first day of hospitalization . the mean time to recovery was 6.4 weeks in 172 dress cases , while death was recorded in 9 ( 5.2% ) patients . death was connected with a more severe course of reaction , particularly observed in older patients , and often due to liver failure . while the precise mechanism of hsr is unknown , various theories involve the interplay between metabolic and immunologic factors [ 2 , 4 , 6 ] , genetic predisposition , and , more recently , infection with human herpes viruses ( hhv ) , particularly hhv-6 . drug metabolites bind to cellular macromolecules such as proteins , creating covalent adducts that can serve as antigenic stimuli for the immune system [ 4 , 6 ] . several genetic factors are believed to predispose certain individuals to this type of adverse reaction . the subset of the population that is susceptible to hsrs carries defects in drug detoxifying pathways ( e.g. , epoxide hydrolase ) , such that a greater amount of a reactive drug metabolite is produced than that which can be detoxified [ 2 , 4 , 6 ] . additional genetic predispositions that render certain individuals susceptible to hsrs include human leukocyte antigen ( hla ) alleles , which are part of the major histocompatibility complex ( discussed by neuman et al . ) . reactivation of latent viral infections has been linked to the development of more severe hsr symptoms , particularly in the context of dihs / dress . for example , hhv-6 reactivation was associated with more severe organ involvement and a prolonged course of disease in 62 of 100 dihs patients , compared with the remaining 38 patients who did not experience hhv-6 reactivation . in the same study , all five deaths and ten cases of renal failure were observed among the 62 patients with hhv-6 dna detected in the serum . hhv-6 is a lymphotropic dna virus belonging to the betaherpesviridae subfamily [ 19 , 20 ] . while little is known about hhv-6a , hhv-6b infection often occurs during the first 2 years of life and is associated with acute febrile illness in young children and exanthem subitum in infants [ 1926 ] . hhv-6 has been increasingly recognized as a cause of hepatitis and liver failure , as well as increased graft rejection and consequent decreased patient survival [ 2026 ] . following the initial infection , hhv-6b remains present in a latent phase , and infection can be reactivated during episodes of immunosuppression [ 20 , 22 , 24 , 26 ] . inherited forms of both hhv-6a and hhv-6b this condition , known as chromosomally integrated hhv-6 ( cihhv-6 ) , occurs in 0.8% of control populations but is found in approximately 2% of patient populations in europe , united states , and the united kingdom , with a lower rate in japan . cihhv-6 is characterized by the complete integration of the hhv-6 genome into the host germ line genome , such that cihhv-6 dna is found in every nucleated cell in the body [ 24 , 27 ] . persistently high viral copy numbers in whole blood or serum are observed in individuals with this condition [ 28 , 29 ] . the viral genome of cihhv-6 is transmitted vertically in the germline in a mendelian manner . approximately 1% of newborns are diagnosed with congenital hhv-6 , all of which are infected with cihhv-6 , directly or indirectly . in a sample of 43 infants with congenital infection , the majority ( 86.0% ) were born with inherited cihhv-6 , while the remaining ( 14.0% ) were not born with cihhv-6 but became infected with hhv-6 transplacentally from their cihhv-6-positive mothers . the cellular dna damage machinery responds to virus infection and the foreign genomes that accumulate in the nuclei of infected cells . many dna viruses have been shown to manipulate the cellular dna damage response pathways in order to create environments conducive to their own replication . arbuckle et al . showed that hhv-6 dna integrates specifically and efficiently into the telomere sequences of chromosomes . the integrated virus can be chemically activated with trichostatin a , a stimulatory compound known to reactivate latent herpesviruses . active hhv-6 replication is determined by the presence of hhv-6 dna in serum , plasma , or cerebrospinal fluid , or by reverse - transcriptase polymerase chain reaction ( rt - pcr ) in peripheral blood mononuclear cells ( pbmc ) . rt - pcr is the sole technique that allows active hhv-6 replication to be differentiated from inactive cihhv-6 . the present paper reviews recently published case reports of scar ( dihs / dress and sjs / ten ) and dili developed secondary to drug exposure , with a particular focus on the role of hhv-6 reactivation and cihhv-6 presence in the clinical outcome of the reaction . our principal aim is to address the role of hhv-6 reactivation in the course of hsrs , particularly in dili and dihs / dress . in addition , we will focus on the complex cellular responses triggered by hhv-6 reactivation . studies discussed in this paper were selected based on a pubmed search of english language papers published in the last 15 years ( 19972011 ) using keywords such as hhv-6 and hypersensitivity , hhv-6 reactivation and drug , hhv-6 and drug rash , and chromosomally integrated hhv-6 . the selected case reports are part of a larger database of hsr cases with hhv-6 reactivation . in addition , our laboratory has studied hsrs and their mechanisms for the last 25 years , and we discuss the cases based on our experience related to the laboratory and clinical manifestations . following initial infection in early childhood , hhv-6 continues to exist in a latent phase in the body . consequently , hhv-6 dna can be measured in different body compartments in hhv-6-positive individuals without active infection . trace amounts of hhv-6 viral dna were also detected by real - time quantitative pcr in 91 ( 26.8% ) of 339 pediatric patients diagnosed and treated for acute lymphoblastic leukemia . low rates of hhv-6b dna were detected in whole blood ( 8.0% ) , pbmcs ( 16.5% ) , and polymorphonuclear leukocytes ( 10.5% ) belonging to 200 volunteers . the mean observed hhv-6b viral loads were 81 copies/10 cells in whole blood , 62 copies/10 cells in pbmcs , and 34.5 copies/10 cells in polymorphonuclear leukocytes . hhv-6-dna was positive in only 27 ( 39.1% ) blood samples from 69 children undergoing elective tonsillectomy for moderate tonsillar hyperplasia or recurrent streptococcal infection without evidence of acute hhv-6 infection , while evidence of hhv-6 dna was found in 100% of tonsil samples in the same population . the common features of dihs identified in a sample of 7 patients were high fever ( 39c ) in all patients , facial edema in all patients , diffuse lymphadenopathy in 5 ( 71.4% ) patients , hypereosinophilia ( > 0.5 10/l ) in 4 ( 57.1% ) patients , atypical circulating lymphocytes in 4 ( 57.1% ) patients , alt elevated > 3 times the reference level in 5 ( 71.4% ) patients , and hypogammaglobulinemia in 4 ( 57.1% ) patients . the onset of dress took place after a mean period of 25 days ( 5 days6 weeks ) of therapy with the incriminated drug . similar findings are reported in other cohorts of dihs patients [ 15 , 56 ] . several studies suggest that hhv-6 reactivation can only occur in susceptible individuals under conditions of transient immunosuppression , such as those transiently associated with the onset of dihs [ 56 , 57 ] . lower serum igg levels were observed in 10 adult anticonvulsant - induced hsr patients ( mean 745 mg / dl ) compared to 15 controls ( p < 0.001 ) . similarly , circulating levels of b cells were also decreased in dihs patients , compared to controls [ 15 , 57 ] . in susceptible individuals , continuous therapy with anticonvulsants , which have been associated with dihs , moreover , hhv-6 reactivation may occur at the same time , but it can not always be detected in blood , since it takes 24 weeks for antibodies to appear after infection or reactivation . because decreases in circulating igg and b cell levels are not observed with other hypersensitivity conditions such as sjs / ten , depressed levels of igg and b cells can be used as biomarkers for the onset of dihs . hhv-6 reactivation may be partially explained by the immunosuppressive effects of dihs - related drugs . the magnitude of inflammation is often proportional to the clinical manifestations , while hypogammaglobulinemia and hhv-6 reactivation worsen the clinical course of the disease . a more detailed description of disease progression is presented in case reports , some of which are summarized in table 1 [ 3552 ] . edema , predominantly on the face , was observed with a relatively high frequency [ 35 , 37 , 3944 , 4749 , 52 ] , as were lymphadenopathy [ 35 , 36 , 39 , 4143 , 45 , 46 , 4951 ] , hypereosinophilia [ 3540 , 4252 ] , and atypical lymphocytes [ 35 , 36 , 3840 , 42 , 44 , 46 , 47 , 52 ] . internal organ involvement manifested itself largely as liver dysfunction , with elevated levels of liver enzymes [ 3546 , 4850 , 52 ] and cholestatic hepatitis with hepatocellular insufficiency . multiple organ failure was observed in a couple of studies [ 47 , 51 ] , one of which resulted in death . hypogammaglobulinemia , another symptom of dihs , was observed in a number of studies as well [ 35 , 38 , 39 , 45 , 49 ] . the offensive drug is immediately interrupted in dihs patients , and treatment with prednisolone is often used to reverse the condition . symptoms begin to improve gradually , but both fever and skin manifestations often relapse approximately 2 - 3 weeks after the onset of dihs . there is a delay until antibodies to the virus are generated . symptoms relapse coincides with detection of hhv-6 reactivation , measured by circulating anti - hhv-6 antibodies and hhv-6 dna [ 15 , 56 ] . anti - hhv-6 igg levels are elevated after 3 weeks following the onset of the reaction ( time of drug withdrawal ) , but not after 2 weeks [ 58 , 59 ] . increases in hhv-6 dna in pbmcs were found to precede the increases in anti - hhv-6 igg levels in anticonvulsant hsr patients and were absent in controls . the presence of hhv-6 dna in pbmcs was found to correlate with the magnitude of anti - hhv-6 antibody titers ( p = 0.0002 ) . intermittent detection of hhv-6 dna in pbmcs was not indicative of illness , such that hhv-6 dna can be found in the pbmcs at low levels in immunocompetent children , with and without symptoms . significant increases in anti - hhv-6 igg titers were noted in all 6 patients included in a study . hhv-6 reactivation was observed between 17 and 27 days after the onset of the reaction . the presence of hhv-6 dna in plasma or serum is evidence of active hhv-6 infection . in addition , the presence of serum anti - hhv-6 igm and increases in the levels of anti - hhv-6 igg levels are used to differentiate primary hhv-6 infection ( i.e. , seroconversion ) from hhv-6 reactivation . serum anti - hhv-6 igg was detected in 7 dress patients analyzed , with serum anti - hhv-6 igm detected in only 4 ( 57.1% ) patients . seroconversion was diagnosed in 2 patients , in which serum anti - hhv-6 igm titers were initially undetectable and became detectable in subsequent serum samples , coinciding with increases in anti - hhv-6 igg titers . seroconversion is described in detail in a 56-year - old male patient with aromatic anticonvulsant - induced hsr . aside from the presentation of fever and cutaneous symptoms , other features of hhv-6 reactivation can include lymphadenopathy , hepatosplenomegaly , encephalitis caused by the virus , and severe lymphocytopenia . hhv-6 replication in hepatocytes led to severe hepatitis , associated with elevated enzyme levels , in an 18-year - old female . higher odds of detecting hhv-6 dna in pbmcs and liver biopsies were found in cases of fulminant hepatic failure or acute decompensation of chronic liver disease , compared to chronic liver disease ( p = 0.02 ) , in a sample of 23 children ( median age 24 months ) with acute liver failure . similarly , higher hhv-6 dna levels were found in cases of fulminant hepatic failure or acute decompensation of chronic liver disease , compared to chronic liver disease cases ( p = 0.0043 ) . tumor necrosis factor- ( tnf ) and interleukin- ( il ) 6 levels were initially elevated prior to hhv-6 reactivation in 4 ( 66.7% ) of 7 patients in a study . il-6 levels were once again elevated after hhv-6 infection in 5 ( 83.3% ) of 6 patients . il-6 and tnf- are produced primarily by monocytes and macrophages , the likely reservoir cells of latent hhv-6 . these cytokines likely play an important role in viral reactivation , yet the low number of patients analyzed in this study makes a definite answer impossible . serum levels of il-5 , interferon ( ifn)- , and eosinophil cationic protein were increased on day 29 and decreased on day 39 during an episode of carbamazepine dihs . an interesting case of drug sensitization was observed in a patient who developed symptoms of dihs within a few days of being exposed to the antibiotic cefaclor . this patient was previously exposed to cefaclor during an episode of carbamazepine - induced dihs . slight increases in il-5 , il-6 , il-10 , ifn- , and eosinophil cationic protein were elevated in sera on day 6 of cefaclor dihs . a drug - induced lymphocyte stimulation test using pbmcs showed that the patient was sensitized to carbamazepine during the first episode , but not to cefaclor . results of the test showed sensitivity to cefaclor during the second episode . during the first episode , carbamazepine - induced hsr was diagnosed 17 days after the start of carbamazepine , consistent with dihs . also , symptoms persisted after the drug was interrupted , and the patient responded to pulsed intravenous ( iv ) methylprednisolone . in contrast , the patient developed the symptoms much quicker during the second episode , during which time the symptoms were milder and disappeared faster after interruption of cefaclor . the involvement of hhv-6 during the first episode was established with anti - hhv-6 igg titers and hhv-6 dna . the magnitude of inflammatory cytokine increase was higher , and eosinophilia was more pronounced during the more severe carbamazepine - induced dihs , prompting aihara et al . to argue that hsr is mediated by both elevated inflammatory cytokine levels and activated eosinophils . it is also possible that corticosteroids administered as initial treatment for drug toxicity are involved in the reactivation of hhv-6 , epstein - barr virus ( ebv ) , and cytomegalovirus ( cmv ) , which became active in the later stage of the disease . anti - hhv-6 igg titers were elevated in 7 dihs patients 2138 days after the onset of the reaction . an elevation in anti - cmv igg was observed in all patients 3554 days after the onset of the reaction . only 2 patients experienced an elevation in anti - ebv igg , 37 and 48 days after the onset of the reaction . antibodies to herpes simplex virus , varicella - zoster virus , human parvovirus b19 , rubella , and measles were not changed . viral dna was detected in whole blood in all patients for whom anti - viral igg was observed , and in serum for 6 of 7 hhv-6 patients , 5 of 6 hhv-7 patients , and all cmv and ebv patients . there appears to be a correlation between relapse of symptoms and detection of increased viral loads for the virus that gets reactivated first ( i.e. , hhv-6 or hhv-7 ) . further immune system perturbations brought about by the initial hhv reactivation are believed to be triggers for subsequent reactivation of other hhvs . cihhv-6 was described and quantified in healthy blood donors and various hospital populations ( e.g. , organ transplant recipients and immunosuppressed patients ) [ 28 , 6167 ] . the mean viral loads in whole blood ranged from 10 copies / ml to 10 copies / ml [ 28 , 62 , 64 ] . vertical transmission of inherited chromosomal integration in addition , there are reports of cihhv-6 acquisition through bone marrow transplant , followed by the presence of hhv-6 in every cell derived from hematopoietic stem cells [ 24 , 6971 ] . it is not known if cihhv-6 can activate from its integrated state in vivo , but several lines of evidence suggest that this is a significant possibility . integrated hhv-6 has been shown to be activated in vitro using histone deacetylase inhibitors and other compounds known to reactivate herpesviruses from latency . as previously discussed , transmission of cihhv-6 from infected mothers to their non - cihhv-6 infants , via the placenta , is another indicator that cihhv-6 may be activated from its integrated state in vivo . children suspected of encephalitis in the united kingdom had a four times greater rate of cihhv-6 than the general population , and liver transplant patients with cihhv-6 have higher rates of graft rejections and opportunistic infections . marek 's disease virus , a herpesvirus associated with tumors in chickens , can reactivate from its integrated state in vitro . if specific drugs can cause integrated virus to activate , then individuals with cihhv-6 may be at an increased risk for drug - induced hsr . report the case of a 47-year - old japanese male with fever ( 38.8c ) and generalized erythematous rash 78 days after carbamazepine exposure . mild hepatotoxicity ( alt 70 iu / l , ast 73 iu / l , -glutamyl transpeptidase 129 iu / l , and lactate dehydrogenase 686 u / l ) and hypogammaglobulinemia ( igg 609 mg / dl , iga 34 mg / dl and igm 27 mg / dl ) were measured . an abdominal skin biopsy revealed hydropic and vacuolar degeneration of epidermal basal cells , lymphocytic infiltration in the epidermis , and a dense upper dermal infiltrate consisting mainly of mononuclear cells . anti - hhv-6 igg titers were increased 128-fold 6 weeks after the reaction onset , while anti - hhv-6 igm titers remained unchanged throughout the course of the reaction , pointing towards hhv-6 reactivation . dna was persistently high in serum ( > 10000 copies / ml ) and whole blood . hhv-6 dna levels in primary infection and acute reactivation are typically below 5.5 10 copies . of interest , the only cases reported of viral loads above this level ( or in the same range as individuals with cihhv-6 ) appear to be those with graft - versus - host disease or dihs , two conditions that have very similar symptoms and courses [ 18 , 27 , 74 ] . in the present paper , we review cases of hsr associated with hhv-6 reactivation . in the vast majority of patients , hhv-6 infection occurs during infancy or early childhood . following resolution of the primary infection , the virus remains latent in salivary glands , pbmcs , and the central nervous system . reactivates in immunosuppressed individuals and is associated with fever , rash , encephalitis , bone marrow suppression , and transplanted organ rejection . primary hhv-6 infection can be identified by the presence of hhv-6 dna in the serum , plasma , or cerebrospinal fluid , or reverse transcription pcr in the absence of elevated antibody titers . reactivation is marked by detection of both viral dna and elevated anti - hhv-6 antibody titers . as anti - hhv-6 igg levels increase during the course of the reaction , anti - hhv-6 igm levels remain constant around < 1 : 20 in patients with hhv-6 reactivation [ 42 , 75 ] . hsrs seem to occur with almost a thousandfold higher incidence in aids patients compared to immunocompetent individuals exposed to the same medication , as there is a much higher exposure to drugs in these patients , as well as a much higher incidence of viral infections [ 76 , 77 ] . aside from a higher incidence of hsr , hiv positivity can also lead to more severe symptoms , compared to hiv - negative patients . in addition , an in vitro study showed increased dna viral replication in pbmcs belonging to dihs patients that were depleted for natural killer cells ( nk ) . these findings point toward an extensive immune barrier aimed against dihs development , while immune defects in components of these pathways can explain why only a relatively small amount of the population exposed to a drug will develop the reaction . suggest that hhv-6 reactivation is not a consequence , but rather a prerequisite for anticonvulsant hsr . the expansion of cd4 t cells and cd8 t cells in response to hhv-6 reactivation seems to be of extreme importance in the development of multiple organ failure during the course of anticonvulsant hsr . from their immunological findings , believe that continuous anticonvulsant therapy leads to transient immune dysfunction , marked by decreased igg production . while t cells protect against reactivation of viruses from a latent stage , antibodies prevent the dissemination of reactivating lytic virus . therefore , dihs may occur when transient drops in b cell counts and immunoglobulin production allow hhv-6 to be reactivated from latency , in conjunction with the presence of drug - specific t cells . the likely explanation for the lag between start of anticonvulsant therapy and onset of disease is that time is required for immunoglobulin levels to drop below a certain threshold . an in vitro study using the human t cell line mt4 found that therapeutic doses of both sodium valproate and carbamazepine led to enhanced hhv-6b replication in a dose - independent manner , whereas phenytoin and sulfasalazine had no effects on hhv-6b replication or cell proliferation . in vivo , there have been reports of phenytoin and sulfonamide antibiotics leading to hhv-6 reactivation in dihs patients [ 42 , 43 , 47 , 52 ] . as such , while widespread , hhv-6 infection is not encountered in all dihs cases . for example , hhv-6 dna was detected by pcr in blood or serum in a 25-year - old female treated with phenobarbital , while it was absent in a 21-year - old female treated with phenytoin . matsuda et al . describe 2 cases of carbamazepine - induced dihs in which no viral reactivation was observed . symptoms , including fever , skin eruptions , and liver involvement , occurred between 22 days and 4 weeks after carbamazepine initiation in the two patients , which is consistent with dihs . furthermore , eosinophilia and a drop in white blood cell count were observed in both patients . hhv-6 reactivation typically occurs 2 - 3 weeks after the development of rash in dihs patients [ 8 , 76 ] . hhv-6 reactivation is often accompanied by relapse of fever and hepatitis . a cascade of viral reactivation can then be started , with hhv-6 , ebv , and/or hhv-7 at the top , followed , with some delay , by other herpes viruses , particularly cmv . reactivation of these viruses is followed by development or exacerbation of clinical symptoms of dihs , including various organ failures [ 8 , 76 , 80 ] . of particular interest is cmv reactivation , which can be followed by transient fever , skin rash , myocarditis , pneumonia , or gastrointestinal bleeding . clinical symptoms ( fever , hepatitis , and/or skin rash ) correspond to the appearance of detectable hhv-6 dna levels . fever and/or hepatitis are the most common clinical features of hhv-6 reactivation in dihs patients . based on these findings , hhv-6 reactivation leads to a more severe course of disease in patients with dihs . hhv-6 reactivation is associated with more severe organ involvement and a prolonged course of the disease in patients with drug rash and systemic symptoms , compared with the remaining patients who did not experience hhv-6 reactivation . significant detection of hhv-6 dna corresponds to the presence of anti - hhv-6 igg titers , and there is a positive correlation between hhv-6 dna detection and the development of fever and hepatitis . while hhv-6 reactivation is a common feature of dihs / dress , hhv-6 dna was not detected in 15 patients with generalized morbilliform / maculopapular drug reactions . hhv-7 , cmv , or ebv dna was not detected in any of the patients included in this study . in situ hybridization revealed hhv-6 mrna in a small subset of infiltrating mononuclear cells around blood vessels and appendages in dihs patients only . similarly , only 1 ( 25.0% ) of 4 sjs / ten patients for whom data was available showed an increase in anti - hhv-6 igg titers from 1 : 10 to 1 : 1280 . aside from a lower likelihood of hhv-6 reactivation , sjs / ten is marked by a quicker onset of reaction than dihs / dress ( 2 - 3 weeks versus a mean 34 days ) . however , fever ( > 38.5c ) , leukocytosis , eosinophilia , and atypical lymphocytosis were common features of both sjs / ten and dihs / dress , whereas liver dysfunction was observed predominantly in sjs / ten patients and lymph node enlargement was observed predominantly in dihs / dress patients . treatments for sjs / ten included systemic corticosteroids in 7 ( 87.5% ) patients and iv immunoglobulin in 1 ( 12.5% ) patient . furthermore , teraki et al . warn that some of the clinical features of anticonvulsant - induced sjs / ten may differ from sjs / ten induced by other drugs , such as a more delayed onset of symptoms in the former category . additionally , the rate of hepatic dysfunction and hematological anomalies was also higher than that described with sjs / ten induced by other drugs . hhv-6 reactivation and atypical lymphocytosis were observed in dihs patients predominantly , compared to sjs / ten patients . the precise morphological nature of the skin reaction is another criterion used to differentiate dihs from sjs / ten . carbamazepine was associated with 12 ( 52.2% ) of 23 anticonvulsant hsr cases reported in a cohort study . maculopapular exanthematous eruptions were observed in 15 patients , erythroderma in 11 patients , exfoliative dermatitis in 3 patients , bullous eruption in 1 patients , urticaria in 1 patient , and purpura ( vasculitis ) in 2 patients . there was no relationship between the drug used and the length or severity of the reaction . in addition to the skin , 20 ( 87.0% ) patients had at least one other organ affected by the reaction , while 8 ( 34.8% ) patients had at least two other organs affected by the reaction . dili was observed in 12 ( 52.2% ) patients , and kidney involvement was observed in 8 ( 34.8% ) patients . other affected organs include the spleen ( splenomegaly ) , the lung , the thyroid ( hypothyroidism ) , the heart , and the pancreas . drug - drug interactions are particularly problematic when several pharmaceutical agents capable of inducting hsr are administered simultaneously . for example , conilleau et al . report the case of a child exposed intermittently to two anticonvulsants prior to developing dihs . despite ethosuximide being interrupted prior to hospitalization and sodium valproate being associated more often with dihs , it appears that the reaction was caused by the combination of these two structurally unrelated anticovulsants , as a patch test was positive for both ethosuximide and sodium valproate . in a separate case , an infant that was exposed to carbamazepine , phenytoin , and phenobarbital presented with initial hhv-6 infection . despite the patient being diagnosed with phenobarbital hypersensitivity , all three of these aromatic antiepileptic drugs have the potential to cause hsr . minocycline - induced dihs with ebv reactivation was reported in a 24-year - old african american female . sequential activation of hhv-6 , hhv-7 , herpes simplex virus , and cmv was observed in a 46-year - old male who developed dihs after being exposed to cyanamide , a drug used to control alcoholism . hhv-6 reactivation was observed in 2 elderly patients with allopurinol dihs [ 26 , 79 ] , and ebv reactivation was observed in a 40-year - old black male with allopurinol dihs and pancreatitis . the cause of the reaction was unknown in an 18-year - old female with sjs / ten exposed to valacyclovir , promethazine , synthroid , belladonna , and orthotricycline . in addition to anticonvulsants , patients can be exposed to additional medication for the treatment of comorbid diseases , opportunistic infections , or even for the management of hsr symptoms ( e.g. , antibiotics or nonsteroidal inflammatory drugs administered for the treatment of fever ) . furthermore , epilepsy patients are often exposed to more than one anticonvulsant , most of which are capable of causing an hsr . while medication is interrupted at the time of hsr diagnosis and the patient is encouraged to avoid rechallenge with the drug that was deemed to have initiated the reaction , it is still important that all medication is considered and tested individually for its capacity to initiate an hsr . investigators seldom check to see if there is an additive effect between the incriminated anticonvulsant and other medication , or if the anticonvulsant caused the reaction on its own . also , it is unlikely that hhv-6 reactivation is measured upon admission . as a result , it is difficult to establish whether the drug or the virus initiates the reaction . the same investigators report the case of a 46-year - old woman who developed dihs 4.5 months after initiating therapy with zonisamide and corticosteroids . since the reaction occurred well outside the time frame characteristic of dihs and since reactivation of herpes viruses was not measured , it is possible that this phenomenon could have created the conditions necessary for dihs to occur in this patient with long - term exposure to anticonvulsant therapy . the presence of proinflammatory cytokines during dili and scar is supported by findings that a significantly higher number of eosinophils was found both in the blood and tissue of patients with a drug - induced maculopapular exanthems , compared to control subjects with normal skin and skin from patients with psoriasis . the presence of hla - a*3101 allele and homozygosity for epoxide hydrolase 1 ( ephx1 ) single nucleotide polymorphism rs1051740 ( t - c ) in exon 3 , associated with modifications in epoxide hydrolase activity , were found in a patient with anticonvulsant hsr , possibly triggered by hhv reactivation , suggesting a genetic predisposition to hsr upon hhv reactivation . hhv-6 reactivation can be further observed in the absence of an hsr and is primarily associated with hepatotoxicity . phillips et al . were able to show with the help of transmission electron microscopy that full and empty virus capsids accumulate in the nucleus of hepatocytes , while viral particles bud out into the nuclear envelope and the cytoplasm . over the years , several drugs were used to treat hhv reactivations per se , as well as opportunistic infections in hsr patients with hhv reactivation , including idoxuridine , cytarabine , and vidarabine . vancomycin was used to treat an opportunistic infection with staphylococcus aureus in a dihs patient with hhv-6 reactivation . however , treatment against hhv-6 reactivation is often withheld out of fear of worsening the condition due to an increasing number of drugs that the patient is exposed to . drug - induced adverse reactions represent a concern for patients , clinicians , the pharmaceutical industry , and health providers . interestingly , very little mention of hhv-6 reactivation is made in a comprehensive article discussing dili , including acute liver failure in major dili registries from europe and the united states . in conclusion , this paper provides a multifaceted assessment of drugs implicated in hsr that have been reported to induce hhv-6 reactivation . this information may facilitate the possible link between the diagnosis of scar and dili , and hhv-6 reactivation .

background . there are challenges in the clinical diagnosis of drug - induced injury and in obtaining information on the reactivation of human herpes viruses ( hhv ) during idiosyncratic adverse drug reactions . objectives . ( i ) to develop a unified list of drugs incriminated in drug - induced hepatotoxicity and severe cutaneous reactions , in which drug hypersensitivity leads to hhv-6 reactivation and further complication of therapy and recovery and ( ii ) to supplement the already available data on reporting frequencies of liver- or skin - induced cases with knowledge of individual case reports , including hhv-6 reactivation and briefly introducing chromosomally integrated hhv-6 . data sources and extraction . drugs identified as causes of ( i ) idiosyncratic reactions , ( ii ) drug - induced hypersensitivity , drug - induced hepatotoxicity , acute liver failure , and stevens - johnson syndrome , and ( iii ) human herpes virus reactivation in pubmed since 1997 have been collected and discussed . results . data presented in this paper show that hhv-6 reactivation is associated with more severe organ involvement and a prolonged course of disease . conclusion . this analysis of hhv-6 reactivation associated with drug - induced severe cutaneous reactions and hepatotoxicity will aid in causality assessment and clinical diagnosis of possible life - threatening events and will provide a basis for further patient characterization and therapy .

the lsj2 strain and the n2 laboratory strain are descended from one ancestral hermaphrodite isolated by w. nicholas . lsj2 was grown continuously in liquid axenic media starting in 1957 at the kaiser foundation research institute , uc berkeley and san jose state university until a sample was frozen in 2009 . values report the average fraction of dauer animals 72 hours after eggs were laid on assay plates . with the exception of the mapping experiments , rils were generated from reciprocal crosses between lsj2 and an n2-derived strain and inbred for ten generations . two laboratory - derived polymorphisms that modify the npr-1 and glb-5 genes in n2 affect many c. elegans behaviors ; to eliminate their effects , the cross was initiated with a strain containing 99% n2 dna but the ancestral alleles of npr-1 and glb-5 from the cb4856 strain ( figure s1 ) . the fraction of animals forming dauers in response to 1 m c3 ascaroside was used as a phenotype for nonparametric qtl mapping . srg-36 , srg-37 , and cbg24690 were ectopically expressed in ash using the sra-6 promoter . vehicle control , 1 m of c3 , or 1 m of c6 was dissolved in m13 buffer and presented to animals using the drop test . each animal was scored three times for the ability to reverse in response to the stimulus . the genetically - encoded calcium indicator gcamp3.0 was expressed in ash using the sra-6 promoter .

evolution can follow predictable genetic trajectories1 , indicating that discrete environmental shifts can select for reproducible genetic changes2 - 4 . conspecific individuals are an important feature of an animal 's environment , and a potential source of selective pressures . we show here that adaptation of two caenorhabditis species to growth at high density , a feature common to domestic environments , occurs by reproducible genetic changes to pheromone receptor genes . chemical communication through pheromones that accumulate during high - density growth causes young nematode larvae to enter the long - lived but non - reproductive dauer stage . two strains of caenorhabditis elegans grown at high density have independently acquired multigenic resistance to pheromone - induced dauer formation . in each strain , resistance to the pheromone ascaroside c3 results from a deletion that disrupts the adjacent chemoreceptor genes serpentine receptor class g ( srg)-36 and -37 . through misexpression experiments , we show that these genes encode redundant g protein - coupled receptors for ascaroside c3 . multigenic resistance to dauer formation has also arisen in high - density cultures of a different nematode species , caenorhabditis briggsae , resulting in part from deletion of an srg gene paralogous to srg-36 and srg-37 . these results demonstrate rapid remodeling of the chemoreceptor repertoire as an adaptation to specific environments , and indicate that parallel changes to a common genetic substrate can affect life history traits across species .

patients with different chronic conditions are an economically significant patient group everywhere in the world . therefore , it is important that patient education is designed together with patients in order to decrease harmful side effects that are related to the knowledge deficiencies of the patients . patient education is undergoing a paradigm shift in which the perspectives of patients are increasingly being incorporated into learning programs . access to the users experience is now considered a prerequisite for the development of quality health services , but how this user experience is incorporated is somewhat unclear.14 it is widely believed that user involvement requires participation in shared decision making , with dialogue being a prerequisite.2,3,5,6 in an interview study , rise et al7 found that both users and health professionals describe user involvement as a dialogue where the goal is shared decisions . however , significant barriers to user involvement and shared decision making have been identified , including inequality , lack of power , and few genuine opportunities for contributing to decisions within the health service.812 furthermore , the inclusion of experiential knowledge and user involvement can challenge professional authority , roles , and working methods,13 which have not traditionally involved users in decision making . although dialogue and collaboration between professionals and users are essential to effective user involvement , little is understood about the characteristics of their collaboration . one model of user involvement in decision making is practiced at the learning and mastery centers ( lmcs ) in norway . based at hospitals , lmcs are competence centers that play a key role in the education and training of patients with different chronic conditions . the philosophy of the lmcs is grounded in equality between users and health professionals and the integration of user experience with professional knowledge . lmc patient education courses address the coping challenges for both patients and their relatives to enable them to live with the changes in their daily life . in lmc courses , the experiences of the users are regarded as equally important as the contributions of the professionals . thus , the user representatives are given a completely new role as equal contributors in the health service . a collaboration group is set up for each learning program being developed at the lmcs . the group normally consists of two user representatives from the relevant user organization , relevant professionals ( examples : doctor , nurse , physiotherapist ) , and the director of the center . the professionals are at work and return to their job site after the group meeting . the planning , implementation , and evaluation of each learning program is this group s responsibility . although professionals and users routinely collaborate to meet the lmcs patient education and user involvement goals , the characteristics of these collaborations have not been previously studied . thus , the purpose of this study was to describe the collaboration between professionals and users in the process of developing lmc learning programs for the chronically ill.14 the study included patients and next of kin ( both considered users ) , as well as various health professionals as they collaborated on the planning , implementation , and evaluation of patient education courses . this article describes two contrasting types of collaboration that emerged from the analyses : knowledge sharing and information exchange . a field study was conducted in lmcs at three different locations in norway ( oslo university hospital , oslo ; notodden hospital , notodden ; and nordland hospital , bod ) . these lmcs offered patient education based on the same ideology and had similar patient groups attending their courses . we observed the collaborations of 17 different groups of users and health professionals with altogether 100 participants . when the groups started to plan the course , some were meeting for the first time , while others had prior experience working together to develop courses . the field notes were based on open observations in 46 collaboration meetings , with most groups being observed several times . further , ten users and 14 health professionals were individually interviewed in accordance with a thematic interview guide focusing on their experience of participating and offering personal or professional experience and competence . the interviews were conducted soon after the field observations by the same researcher who also recorded and transcribed these verbatim . we used several analytical techniques , alternating between open reading and theme - focused reading.1518 the field notes and transcriptions were analyzed both separately and in light of each other . main themes were identified in the field notes and supported by a considerable number of sub - themes from the interview transcriptions . to further explore aspects of collaboration through a more integrated analysis , we developed a new method for systematic data integration ( figure 1).19 researchers are encouraged to view field notes and interview texts as a complete body of material for analysis.16,20 this new method involved interweaving observation data and interview data from group interactions and was inspired from a similar method used on analysis of events.21 the meetings in all of the collaboration groups focused on the planning , implementation , and evaluation of the course . this series of meetings is named as sequences of interaction . for each sequence of collaboration , raw data from field notes were first placed in the left side column in a document and subjected to thematic analysis . then the identified themes were placed in the middle column , and matching interview sequences in the right side column ( table 1 ) . the result of this first interweaving process was a reconstructed and integrated text from each sequence of collaboration . each reconstructed text was then read vertically and horizontally to more fully explore these sequences of collaboration . in the second interweaving process , all the reconstructed sequences of collaboration were put together in a large comprehensive text . what characterizes these collaboration processes?19 interweaving the texts in this manner was essential for creating an overall perspective and acquiring integrated material for further analyses . the data protection official for research in norway approved the study ( ref no 11247 ) . written informed consent a field study was conducted in lmcs at three different locations in norway ( oslo university hospital , oslo ; notodden hospital , notodden ; and nordland hospital , bod ) . these lmcs offered patient education based on the same ideology and had similar patient groups attending their courses . we observed the collaborations of 17 different groups of users and health professionals with altogether 100 participants . when the groups started to plan the course , some were meeting for the first time , while others had prior experience working together to develop courses . the field notes were based on open observations in 46 collaboration meetings , with most groups being observed several times . further , ten users and 14 health professionals were individually interviewed in accordance with a thematic interview guide focusing on their experience of participating and offering personal or professional experience and competence . the interviews were conducted soon after the field observations by the same researcher who also recorded and transcribed these verbatim . we used several analytical techniques , alternating between open reading and theme - focused reading.1518 the field notes and transcriptions were analyzed both separately and in light of each other . main themes were identified in the field notes and supported by a considerable number of sub - themes from the interview transcriptions . to further explore aspects of collaboration through a more integrated analysis , we developed a new method for systematic data integration ( figure 1).19 researchers are encouraged to view field notes and interview texts as a complete body of material for analysis.16,20 this new method involved interweaving observation data and interview data from group interactions and was inspired from a similar method used on analysis of events.21 the meetings in all of the collaboration groups focused on the planning , implementation , and evaluation of the course . this series of meetings is named as sequences of interaction . for each sequence of collaboration , raw data from field notes were first placed in the left side column in a document and subjected to thematic analysis . then the identified themes were placed in the middle column , and matching interview sequences in the right side column ( table 1 ) . the result of this first interweaving process was a reconstructed and integrated text from each sequence of collaboration . each reconstructed text was then read vertically and horizontally to more fully explore these sequences of collaboration . in the second interweaving process , all the reconstructed sequences of collaboration were put together in a large comprehensive text . what characterizes these collaboration processes?19 interweaving the texts in this manner was essential for creating an overall perspective and acquiring integrated material for further analyses . the data protection official for research in norway approved the study ( ref no 11247 ) . written informed consent the main finding from the systematic data integration revealed the collaboration process to be a complex phenomenon that varied from dynamic to passive , depending on different combinations of participant types . the first integrated analysis , the thematic analysis of interwoven field notes and interview texts , revealed several types of users and professionals ( tables 2 and 3 ) . three types of users were identified based on clear differences in how they shared their experiences and participated in the group . three types of professionals were identified based on differences in how they acted and what they shared in the group . in the second integrated analyses , we were looking for whether and how the core values of the lmc , equality in both partnership and forms of knowledge ( eg , experiential versus professional ) , were realized in the collaboration process . in the following sections , two contrasting collaboration processes , referred to as information exchange and knowledge sharing , are presented ( table 4 ) . the most prominent feature of this passive type of collaboration process was the absence of dialogue ( ie , egalitarian communication about a common cause ) . further , there were few signs of recognition of users knowledge , and a passive form of interaction characterized the group discussions . this may be due to the composition of the groups , which were primarily composed of novice users and the visitor type of health professional . the following extract from the field notes is a representative example of a first time group meeting : six people in white coats sit beside each other parallel to the row of windows . the users , the leader , and me as an observer sit on the other side . bleepers disturb us constantly , and the health professionals frequently have to leave the room . the leader welcomes the group , and asks everyone to introduce themselves and say something about their views on the learning program to be planned . the health professionals are more concise than the users , both when they introduce themselves and in their comments on the learning program . i wonder why there is so little commitment to what is to be created and decided on . suddenly something happens : one of the professionals has a strong opinion about what must be included and says , if there s any point in it at all , we ca nt leave this out [ ] . after some back and forth between this physiotherapist and the leader , the leader takes over : we have to make our minds up . the leader s eagerness to make a decision and the health professionals insistence on important elements that are to be included seems to form the outcome . the health professionals reluctance in relating to the users experiences as relevant knowledge , can be referred to as a waiting attitude . this attitude may portray a lack of commitment to collaborate . after a round of such presentations the users seemed withdrawn . the health professionals resembled visitors rather than co - workers ( table 3 ) . in the group meetings , they seemed to have no spare time to participate and appeared uninterested in the users comprehensive narratives . keeping their distance this passive collaboration process can best be described as an exchange of information : each participant gave their opinions , with little connection to others opinions . both user experience and professional knowledge were displayed in the group but as parallel dimensions that appeared to have little connection . most often , professional knowledge appeared to have prime importance , and consequently , the course program reflected a traditional focus on illness and treatment . the group leader engaged in facilitating the process , but several attempts to encourage the group members to exchange opinions were unsuccessful . when the time came to make decisions , the leader often became proactive with regard to what was to be included and who was to contribute to the course . when one of the leaders was asked if she thought that everyone had had a say in the decision , her position regarding reaching a result became clear : no . one goal could have been to get everybody to say something , but i think the goal is really more to reach a result . they seemed to lack the basic abilities and/or attitudes necessary for collaboration and behaved as some of these planning meetings gave an impression of a passive situation where little happened . even though both parties had their say , there was no logic that this created an intersection between the experiences of chronic illness and professional knowledge . most often , the statements of the health professionals and the users remained separate and disconnected . it was dynamic , occurred in both established and new groups , and was characterized by the participants engagement . a mixture of several types of users and health professionals as co - workers constituted these groups . the main impression of a typical dynamic collaboration was that the dialogue reflected the participants acknowledgment of each other as persons and each other s knowledge contributions . commitment and exploratory interaction seemed intertwined and particularly dependent on the health professionals being engaged in the users and their experiences . the health professionals attitude was typically both proactive with respect to exploring users knowledge and somewhat reserved regarding themselves and their own knowledge . the following field note from an introductory meeting exemplifies the exploration of each other as persons : the leader asks the participants to introduce themselves and to say whether they know anyone else in the group from before . the users take a little more time to tell about the date of their stroke , their reactions , the outcome , the practical consequences , and why they agreed to be part of this working group . it has cost so much to figure things out and to find solutions , one of the women says . the male user refers to the ergonomist and says , the last time i saw her she was with me in the shower . everyone laughs , and she replies , circumstances were quite different then . the following field note further exemplifies the exploration of significant knowledge that the parties bring into the planning of the course : the group leader asks the users if they have any special wishes for the meeting . the physiotherapist says that the brochure she has compiled on the topic disappeared when it was circulated for comments . the doctor asks the group leader : have you covered this topic before ? and what were people most interested in then ? a next of kin shares the view that there should be an interaction between users and health professionals . he thinks it would be a good idea if somebody summarized some of the literature and , as he puts it , added something of his own experience . the leader asks if this means that he will do it , and he agrees to do so . the doctor says , little knowledge exists of this area , mostly human experience . a younger user thinks that it must be presented in a way that gives people pegs to hang things on : we must dare to talk about the challenges . then the next of kin says it would be a good idea to include some facts for example hygiene . the younger user says , include depression [ ] the doctor wants to say something about bodily matters . the leader makes the program more specific : the next of kin is given 15 minutes to review the important points and his own experience . these descriptions of reciprocal engagement and exploration of knowledge exemplify the conceptualization of knowledge sharing . in this dynamic collaboration , the following example illustrates how an abruption of the discussion led to reflective views of health professionals own contribution : the leader asked , what type of reciprocal knowledge do we now have at our disposal for the job we are to do ? two health professionals responded : i ve only partly understood what the users are most interested in hearing about ; i must do this [ my lecture ] differently from what i had planned . when planning courses , decisions had to be made . during this process some users were listening to the dialogue , while some dared to disagree and raised questions , sometimes critical ones , about the suggested choices and solutions . the planning proceeded toward a conclusion by a clarification of the content , the methods , and who was to contribute . the health professionals generally took the lead in the concluding phase , but some users were definitively involved : after some discussion , the leader concluded that the group ought to distribute an information brochure . he suggests sending out a brochure from a medical company , and says that the language is much milder , which is important for helping people not to get depressed when they have been diagnosed . an example from a course illustrates how a physician reshaped professional expertise into knowledge for a lay audience in order to increase common comprehension : he starts with the feedback system . you have to understand this to be able to understand what happens to you , he says . you must know enough perhaps more than your doctor to be able to cope . . you must be able to say : i want to have a blood value of 1 to feel reasonably well . it s no use being within the reference area for blood values if you do nt feel comfortable and ok there , he tells them . suddenly there is a flood of testimonies about normal blood tests and feeling wretched . in addition to illustrating the importance of re - shaping language , this is an example of a health professional regarding user experience as trustworthy knowledge and perhaps questioning the meaning of scientific knowledge . this dynamic collaboration process was also characterized by a commitment by all to each other and to what was to be planned , an exploration of each other s viewpoints and not least the reciprocity in the decisions made . both the content and the methods of the course resulted from contributions by users , health professionals , and the group leaders . in summary , two contrasting collaboration processes were identified in this analysis . distribution of knowledge was central to both processes : the first one was characterized by indifferent display of knowledge , while in the second one , there was mutual sharing of both parties knowledge . hence , a key finding of our study was that knowledge sharing is a critical element of effective collaboration between professionals and users , with users involvement in knowledge sharing seemingly influenced by how health professionals relate to the users experiences and knowledge . the most prominent feature of this passive type of collaboration process was the absence of dialogue ( ie , egalitarian communication about a common cause ) . further , there were few signs of recognition of users knowledge , and a passive form of interaction characterized the group discussions . this may be due to the composition of the groups , which were primarily composed of novice users and the visitor type of health professional . the following extract from the field notes is a representative example of a first time group meeting : six people in white coats sit beside each other parallel to the row of windows . the users , the leader , and me as an observer sit on the other side . bleepers disturb us constantly , and the health professionals frequently have to leave the room . the leader welcomes the group , and asks everyone to introduce themselves and say something about their views on the learning program to be planned . the health professionals are more concise than the users , both when they introduce themselves and in their comments on the learning program . i wonder why there is so little commitment to what is to be created and decided on . suddenly something happens : one of the professionals has a strong opinion about what must be included and says , if there s any point in it at all , we ca nt leave this out [ ] . after some back and forth between this physiotherapist and the leader , the leader takes over : we have to make our minds up . the leader s eagerness to make a decision and the health professionals insistence on important elements that are to be included seems to form the outcome . the health professionals reluctance in relating to the users experiences as relevant knowledge , can be referred to as a waiting attitude . this attitude may portray a lack of commitment to collaborate . after a round of such presentations the users seemed withdrawn . the health professionals resembled visitors rather than co - workers ( table 3 ) . in the group meetings , they seemed to have no spare time to participate and appeared uninterested in the users comprehensive narratives . keeping their distance this passive collaboration process can best be described as an exchange of information : each participant gave their opinions , with little connection to others opinions . both user experience and professional knowledge were displayed in the group but as parallel dimensions that appeared to have little connection . most often , professional knowledge appeared to have prime importance , and consequently , the course program reflected a traditional focus on illness and treatment . the group leader engaged in facilitating the process , but several attempts to encourage the group members to exchange opinions were unsuccessful . when the time came to make decisions , the leader often became proactive with regard to what was to be included and who was to contribute to the course . when one of the leaders was asked if she thought that everyone had had a say in the decision , her position regarding reaching a result became clear : no . one goal could have been to get everybody to say something , but i think the goal is really more to reach a result . they seemed to lack the basic abilities and/or attitudes necessary for collaboration and behaved as the ones who know . openness and curiosity toward the users experience were uncommon . some of these planning meetings gave an impression of a passive situation where little happened . even though both parties had their say , there was no logic that this created an intersection between the experiences of chronic illness and professional knowledge . most often , the statements of the health professionals and the users remained separate and disconnected . it was dynamic , occurred in both established and new groups , and was characterized by the participants engagement . a mixture of several types of users and health professionals as co - workers constituted these groups . the main impression of a typical dynamic collaboration was that the dialogue reflected the participants acknowledgment of each other as persons and each other s knowledge contributions . commitment and exploratory interaction seemed intertwined and particularly dependent on the health professionals being engaged in the users and their experiences . the health professionals attitude was typically both proactive with respect to exploring users knowledge and somewhat reserved regarding themselves and their own knowledge . the following field note from an introductory meeting exemplifies the exploration of each other as persons : the leader asks the participants to introduce themselves and to say whether they know anyone else in the group from before . the users take a little more time to tell about the date of their stroke , their reactions , the outcome , the practical consequences , and why they agreed to be part of this working group . it has cost so much to figure things out and to find solutions , one of the women says . the male user refers to the ergonomist and says , the last time i saw her she was with me in the shower . everyone laughs , and she replies , circumstances were quite different then . the following field note further exemplifies the exploration of significant knowledge that the parties bring into the planning of the course : the group leader asks the users if they have any special wishes for the meeting . the physiotherapist says that the brochure she has compiled on the topic disappeared when it was circulated for comments . the doctor asks the group leader : have you covered this topic before ? and what were people most interested in then ? a next of kin shares the view that there should be an interaction between users and health professionals . he thinks it would be a good idea if somebody summarized some of the literature and , as he puts it , added something of his own experience . the leader asks if this means that he will do it , and he agrees to do so . the doctor says , little knowledge exists of this area , mostly human experience . a younger user thinks that it must be presented in a way that gives people pegs to hang things on : we must dare to talk about the challenges . then the next of kin says it would be a good idea to include some facts for example hygiene . the younger user says , include depression [ ] the doctor wants to say something about bodily matters . the leader makes the program more specific : the next of kin is given 15 minutes to review the important points and his own experience . these descriptions of reciprocal engagement and exploration of knowledge exemplify the conceptualization of knowledge sharing . in this dynamic collaboration , the following example illustrates how an abruption of the discussion led to reflective views of health professionals own contribution : the leader asked , what type of reciprocal knowledge do we now have at our disposal for the job we are to do ? i ve only partly understood what the users are most interested in hearing about ; i must do this [ my lecture ] differently from what i had planned . when planning courses , decisions had to be made . during this process some users were listening to the dialogue , while some dared to disagree and raised questions , sometimes critical ones , about the suggested choices and solutions . the planning proceeded toward a conclusion by a clarification of the content , the methods , and who was to contribute . the health professionals generally took the lead in the concluding phase , but some users were definitively involved : after some discussion , the leader concluded that the group ought to distribute an information brochure . he suggests sending out a brochure from a medical company , and says that the language is much milder , which is important for helping people not to get depressed when they have been diagnosed . an example from a course illustrates how a physician reshaped professional expertise into knowledge for a lay audience in order to increase common comprehension : he starts with the feedback system . you have to understand this to be able to understand what happens to you , he says . you must know enough perhaps more than your doctor to be able to cope . the textbook s level of reference does nt seem to be completely correct in practice . you must be able to say : i want to have a blood value of 1 to feel reasonably well . it s no use being within the reference area for blood values if you do nt feel comfortable and ok there , he tells them . suddenly there is a flood of testimonies about normal blood tests and feeling wretched . in addition to illustrating the importance of re - shaping language , this is an example of a health professional regarding user experience as trustworthy knowledge and perhaps questioning the meaning of scientific knowledge . this dynamic collaboration process was also characterized by a commitment by all to each other and to what was to be planned , an exploration of each other s viewpoints and not least the reciprocity in the decisions made . both the content and the methods of the course resulted from contributions by users , health professionals , and the group leaders . in summary , two contrasting collaboration processes were identified in this analysis . distribution of knowledge was central to both processes : the first one was characterized by indifferent display of knowledge , while in the second one , there was mutual sharing of both parties knowledge . hence , a key finding of our study was that knowledge sharing is a critical element of effective collaboration between professionals and users , with users involvement in knowledge sharing seemingly influenced by how health professionals relate to the users experiences and knowledge . we found that the health professionals acquired new perspectives regarding how illness , treatment , learning , and coping affect the users everyday life , while the users were able to continue reflecting on their experience and thus acquired new knowledge of their own . how the groups are led and how the health professionals participate is vital for both the reciprocal exchange of knowledge and consensus decisions about the course program . similar patterns between professional practice , patient participation , and meaningful learning processes have also been described in two studies of hospitalized persons with mental illness.22,23 the aim of these studies was to describe the subjective experiences of learning processes in a therapeutic setting , and they found that atmosphere , attitudes , interpersonal relationships , equality , and recognition were factors that promoted individual learning processes.22,23 a knowledge - sharing type of collaboration process created space that enabled everyone to learn and promoted new joint understandings and new alternatives for action . the health professionals participation in this type of collaboration is closely connected to what is called the teacher role in liberatory pedagogy.24 research into user involvement in the health service emphasizes the sharing of knowledge as the most realistic power dimension in the collaboration between users and health professionals.10,25 it is claimed that when users share their experience , they raise problems rather than solving them , and that this represents the users potential for power . in a review of the literature , aujoulat et al26 explored how the concept of empowerment ( which implicitly includes user involvement ) was applied in patient education for the chronically ill . one finding was that processes of involvement and empowerment required self - involvement from both users and health professionals . swift and dieppe27 concluded that recognition of the patients expertise can transform expert patients narratives into powerful educational resources that can support other patients in similar situations . users told their experiences in full measure but had little impact on the planning meetings due to health professionals who were neither committed nor allowed themselves to be influenced by the users stories . if the health professionals had been more engaged , the novice users may have participated more actively . some health professionals were determined to inform the group of their own viewpoints by emphasizing their professional position . such collaboration groups had access to different viewpoints , but little knowledge was shared in spite of the leaders efforts . this exchange of information is quite different from knowledge sharing which has a reciprocal aspect . tritter28 claims that user involvement in the health service primarily takes place in an indirect way by the users serving as information suppliers for professional decision makers . a systematic review argued the need for clarification of what collaboration and user involvement entails in the development of both health services and professional health education.25 this review and some case studies12 question whether user involvement is to be simply an exchange of information and consultation or an equal partnership with health professionals . our study can perhaps provide additional clarification in this area . in light of our findings , genuine shared decision making seems to require a mutual recognition of both parties as having relevant knowledge to contribute toward a good decision . we have previously debated the crucial importance of experience as insider knowledge in patient education for the chronically ill.29 insider knowledge is regarded as particularly reliable and is characterized by participation and first - hand experience . first - hand experience involves senses and feelings , thus providing concrete , visible , colorful , and subjectively convincing data.30 as a result , this involvement of the senses and emotions and the lively nature of the data have a strong impact on perceptions and reflections . insider knowledge can thus contribute to both an appropriate and relevant knowledge basis in patient education.29 achieving equivalence in asymmetric relationships and real knowledge sharing is not a straightforward procedure.31 knowledge sharing is different from persuasion , professional explanation , and consent . we found that the process appears to entail an attitude of recognizing the other party and their knowledge as meaningful to the goal of the process , as well as a mutual sharing of knowledge where the professionals toned down some of their knowledge dominance . health professionals have and must take responsibility for instigating knowledge exchange , promoting mutual reflection , and initiating decisions both during and when ending the process . the fact that knowledge sharing is dominant in our study , and similarly that shared decision making is an assisted consequence of knowledge sharing , may be due to the ideology of the lmc . decision making only based on professionals proposals is different from shared decision making based on a joint perception that both parties possess knowledge relevant to the decision - making process . we view both parties knowledge and perspectives as crucial for making good decisions in patient education including in one - on - one situations . thus , it is necessary to create settings that encourage the mutual exchange of different forms of knowledge . the findings of this study should be considered in light of its strengths and limitations . the collaboration groups differed in size , in combination of participants , and chronic condition . this might represent a limitation of the study . a single researcher conducted all observations and interviews . however , the researcher has considerable experience conducting such observations and interviews,32,33 and the data analysis processes were validated through detailed discussions with supervisors and other fieldwork researchers . in addition , the researcher s position as an outside observer may have facilitated the recognition and appreciation of themes that may be more blurred from an insider s position . given that the results of this study are supported by research in other areas , the findings are likely transferable beyond the current context . the findings of this study should be considered in light of its strengths and limitations . the collaboration groups differed in size , in combination of participants , and chronic condition . this might represent a limitation of the study . a single researcher conducted all observations and interviews . however , the researcher has considerable experience conducting such observations and interviews,32,33 and the data analysis processes were validated through detailed discussions with supervisors and other fieldwork researchers . in addition , the researcher s position as an outside observer may have facilitated the recognition and appreciation of themes that may be more blurred from an insider s position . given that the results of this study are supported by research in other areas , the findings are likely transferable beyond the current context . we explored collaboration processes in lmcs based on specific values of equality and the integration of different knowledge forms . we found knowledge sharing to be more than the exchange of information and consultation and also to be a prerequisite for shared decision making . the prerequisites for genuine sharing processes include several elements : mutual recognition of the other s knowledge and curiosity about participation in learning processes . also , for the professionals it is important to be somewhat reticent with information they think is important for users . this form of user involvement in patient education requires health professionals who have specific values that are reflected in their interaction with the users . health professionals have the power to ensure that knowledge sharing with users can take place and thus contribute to an extended perspective in patient education .

backgroundpatient education is undergoing a paradigm shift in which the perspectives of patients are increasingly being incorporated into learning programs . access to the users experience is now considered a prerequisite for the development of quality health services , but how this user experience is incorporated is somewhat unclear . the inclusion of experiential knowledge and user involvement can challenge professional authority , roles , and working methods because knowledge sharing is different from persuasion , professional explanation , and consent . dialogue and collaboration between professionals and users are essential to effective user involvement ; however , little is understood about the characteristics of their collaboration.objectiveto describe characteristics of the collaboration between users and health professionals in developing , implementing , and evaluating patient education courses in hospitals.design , setting , and methodsa field study was conducted in three different hospitals . data collection comprised open observations in meetings of 17 different collaboration groups with a total of 100 participants , and 24 interviews with users and professionals . the data analyses included both thematic and the systematic data integration approach.resultstwo contrasting types of collaboration emerged from the analyses ; knowledge sharing and information exchange . the first was characterized by mutual knowledge sharing , involvement , and reciprocal decision making . characteristics of the second were the absence of dialogue , meagre exploration of the users knowledge , and decisions usually made by the professionals.conclusioncollaboration between users and health personnel takes place in an asymmetric relationship . mutual knowledge sharing was found to be more than the exchange of information and consultation and also to be a prerequisite for shared decision making . in developing patient education when users are involved the health professionals have the power and responsibility to ensure that knowledge sharing with users takes place .

the database of neurointervention in asan medical center ( amc ) was reviewed retrospectively to identify all wingspan stent procedures which were used for the treatment of intracranial atherosclerotic disease . records were reviewed to identify cases of failure of stent deployment in daily practice . since 2007 , 80 wingspan stents were placed in our department for the treatment of intracranial atherosclerotic arterial stenosis . procedural details are the same as those described previously [ 3 , 7 - 9 , 19 ] . among 80 patients , we identified seven instances ( 8.8% ) of failure of stent deployment ( table 1 ) . mean age of the patients was 62 ( range 47~78 ) and male to female ratio was 6:1 . we excluded a patient because wingspan was used for an aneurysm coil embolization . in this patient , wingspan was inappropriately deployed proximal to the target lesion while treating a wide - necked paraclinoid internal carotid artery ( ica ) aneurysm with atherosclerotic change in parent artery . a neuroform ez ( stryker , kalamazoo , mi , usa ) was used for stent - assisted coil embolization after the wingspan stent was removed by a snare loop device successfully . we defined procedural failure as failure of any process in wingspan stent insertion , delivery failure as failed delivery of wingspan system into proper position for stenotic lesion , deployment failure as failed deployment of stent despite proper positioning of wingspan system , and retrieval failure as failed retrieval of dual tapered ( olive ) tip after deployment of the stent because of the friction between stent strut and bulky olive tip . wingspan delivery system consists of inner and outer bodies , and nitinol ( wingspan ) stent . when the delivery system is introducing , a olive tip protrudes distal to the outer body which has outer diameter of 3.5f . dual tapered tip has 9 mm length , entry profile of 0.027 and mid - profile of 0.046 inch . when the stent is being deployed , outer body is pulling back while maintaining the inner body in its own position . after deployment , dual tapered tip is retrieved by pulling the delivery system through the deployed stent . wingspan delivery system consists of inner and outer bodies , and nitinol ( wingspan ) stent . when the delivery system is introducing , a olive tip protrudes distal to the outer body which has outer diameter of 3.5f . dual tapered tip has 9 mm length , entry profile of 0.027 and mid - profile of 0.046 inch . when the stent is being deployed , outer body is pulling back while maintaining the inner body in its own position . after deployment , dual tapered tip is retrieved by pulling the delivery system through the deployed stent . we categorized procedural failures occurred in seven patients into three categories ( table 2 ) : delivery failure of the system ( n = 3 ) , deployment failure of stent ( n = 3 ) , retrieval failure of olive tip of the inner body through the deployed the stent ( n = 1 ) . there were three patients in whom delivery of the delivery system to the lesion has failed . in patient 1 , while treating a serious stenosis at right m1-m2 junction , successful balloon angioplasty was performed but the wingspan stent delivery system could not be introduced to the lesion because of excessive vascular tortuousness . we failed to deploy the stent despite multiple attempts . in patient 2 , we could not advance the stent delivery system to the target lesion beyond the tight carotid siphon even after using stiff guide wire while treating a focal severe stenosis at middle portion of left middle cerebral artery trunk . patient 3 revealed a severe stenosis of the right m1 segment just distal to the previously stented area by bare metal stent in which there was no restenosis . the olive tip of the delivery system could not introduce beyond the previous stent after balloon angioplasty . then an enterprise was used to relieve the stenosis . as in three patients of our experience the stent was not deployed even though the stent delivery system was in proper position to cover the lesion . we removed the whole system because there was too much resistance felt in the outer body , which was not pulling back smoothly . after removal of the stent , we found that there was partially deployed stent at the tip ( fig . this configuration revealed that the acute angulation between m1 - 2 segment prevented continuous and smooth deployment of the stent . finally , we introduce an enterprise and could open the occluded segment . in patient 5 , stent delivery system was finally advanced to the basilar artery through excessively tortuous right subclavian artery and vertebral artery , but the stent could not be deployed because the stabilizer was not as stiff as to provide enough supporting force . as to patient 6 , stent could not be deployed at the proper position because of marked resistance between inner and outer bodies caused by excessive proximal vascular tortuousness , even the olive tip of the delivery system had navigated over the lesion . we tried to advance the stent by pushing the inner body but finally failed to push the stent segment because olive tip at the end of the inner body was stretched out from the end of the outer body ( fig . when the delivery system was withdrawn , we found that the stretched portion of the inner body was exposed far beyond the stent loaded segment in the delivery microcatheter ( outer body ) . as in patient 7 , after successful deployment of wingspan stent , we met a little resistance when we tried to retrieve the olive tip of the stent delivery system . the outer body was readvanced through the deployed stent and then the olive tip was retrieved and repositioned to the distal end of the microcatheter ( outer body ) . reducing the size gap between olive tip and inner body shaft can make better smooth retrieval of the delivery system . there were three patients in whom delivery of the delivery system to the lesion has failed . in patient 1 , while treating a serious stenosis at right m1-m2 junction , successful balloon angioplasty was performed but the wingspan stent delivery system could not be introduced to the lesion because of excessive vascular tortuousness . , we could not advance the stent delivery system to the target lesion beyond the tight carotid siphon even after using stiff guide wire while treating a focal severe stenosis at middle portion of left middle cerebral artery trunk . patient 3 revealed a severe stenosis of the right m1 segment just distal to the previously stented area by bare metal stent in which there was no restenosis . the olive tip of the delivery system could not introduce beyond the previous stent after balloon angioplasty . as in three patients of our experience , there seemed to be different mechanism of deployment failure . in patient 4 , the angiogram showed complete occlusion of the distal left m1 . the stent was not deployed even though the stent delivery system was in proper position to cover the lesion . we removed the whole system because there was too much resistance felt in the outer body , which was not pulling back smoothly . after removal of the stent , we found that there was partially deployed stent at the tip ( fig . 1 ) . this configuration revealed that the acute angulation between m1 - 2 segment prevented continuous and smooth deployment of the stent . finally , we introduce an enterprise and could open the occluded segment . in patient 5 , stent delivery system was finally advanced to the basilar artery through excessively tortuous right subclavian artery and vertebral artery , but the stent could not be deployed because the stabilizer was not as stiff as to provide enough supporting force . as to patient 6 , stent could not be deployed at the proper position because of marked resistance between inner and outer bodies caused by excessive proximal vascular tortuousness , even the olive tip of the delivery system had navigated over the lesion . we tried to advance the stent by pushing the inner body but finally failed to push the stent segment because olive tip at the end of the inner body was stretched out from the end of the outer body ( fig . when the delivery system was withdrawn , we found that the stretched portion of the inner body was exposed far beyond the stent loaded segment in the delivery microcatheter ( outer body ) . as in patient 7 , after successful deployment of wingspan stent , we met a little resistance when we tried to retrieve the olive tip of the stent delivery system . the outer body was readvanced through the deployed stent and then the olive tip was retrieved and repositioned to the distal end of the microcatheter ( outer body ) . reducing the size gap between olive tip and inner body shaft can make better smooth retrieval of the delivery system . the published data on the use of the wingspan stent for intracranial atherosclerosis revealed that technical success rate was high between 96.7% and 98.8% [ 15 , 17 , 18 , 20 ] . when we review the literature , there were only few publications regarding wingspan deployment failure [ 13 - 17 ] being totally less than one dozen of cases . we reported seven cases of wingspan stent deployment failures in our experience and revealed 7.5% failure rate except the last patient in whom stent deployment was finally successful . the overall rate of technical failure in our study seems to be higher than other studies . this difference may reveal that the failure rate can be underestimated because information regarding stent deployment failure is not exactly reported . the other possible reason is that some studies did not include stent misplacement as technical failure . the main cause of wingspan stent delivery failures in the published studies was excessive cerebral vascular tortuousness . there were several reported cases of wingspan delivery failure because of excessively tortuous cerebrovasculature [ 13 , 15 - 17 ] . among them , a patient presented later with recurrent stenosis and successfully underwent stenting with wingspan . although vascular tortuousness was the delivery failure reason as in two out of three delivery failure patients , we experienced in a patient whom the olive tip of the stent could not pass the lesion beyond the previously stented m1 . this passage failure of the olive tip is due to 0.046 inch ( 1.17 mm ) mid - profile of the olive tip could not pass the rigid tube of previous bare metal stent as in patient 3 . the salvage technique for the delivery failure is to resort to stent with more flexible delivery system and thinner profile , such as enterprise and neuroform . such self - expanding stents designed for coiling of wide - neck aneurysm sometimes neither have proper size of stent length and diameter nor enough radial force to resist delayed restenosis in atherosclerotic lesions . lesley et al . introduced a catheter exchange technique for the wingspan stent deployment by cutting off the olive tip of the delivery system if the delivery system could not be advanced to the lesion . this concept is similar to use neuroform except that outer diameter of the delivery catheter is 2.8f in high - flow renegade for neuroform compared to 3.5f in the outer body for wingspan . although delivery failure was the main mechanism as in 3 out of our seven patients , we also met deployment or retrieval failure which has not been described in detail previously . two of them occurred due to proximal vessel tortuousness and the other occurred due to angled vessel configuration just distal to the stenotic lesion . acute distal vessel angle as in m1 - 2 junction probably made the olive tip buckled and precluded smooth stent deployment . two other cases revealed that the stent might not be delivered because of insufficient support of the inner body system in one and a part of the inner body had been pushed out of the delivery microcatheter ( outer body ) without introducing the stent segment in the other . retrieval failure of the olive tip did not lead to procedure failure because we could finally remove the system after resheathing the inner body by advancing the outer body over the inner body up to the olive tip . even though this failure can be managed by resheathing technique , incomplete understanding of the stent design might result in injury of the stent vessel by forceful pulling of the system despite the resistance of the olive tip against deployed wingspan stent . reported three cases of stents misplacement in the early phase of their experience . in conclusion , our study revealed that there were three mechanisms of technical failure in using wingspan stent : delivery , deployment and retrieval failures . these technical failures are related to tortuousness of the proximal as well as distal cerebral vessels to the stenotic lesion , rather large and bulky profile of the olive tip , tight and rigid lumen of previously stented vessel . to avoid device - related complication , complete understanding of the stent design is mandatory before using the stent .

purposewingspan is the only fda approved self - expanding stent for intracranial artery and known to have better delivery compared to balloon expandable stent . however , some delivery failure has been reported but incidence and mechanism of the failure have not been completely elucidated . we present the cause and mechanism of wingspan deployment failure experienced in our institute.materials and methodswe experienced deployment failure in seven patients ( 8.8% ) out of 80 patients who underwent wingspan stenting since 2007 . mean age of the patients was 62 ( range 47~78 ) and male to female ratio was 6:1 . we evaluated the cause and mechanism why the deployment was not successful and how we could manage it subsequently.resultswe categorized failures occurred in seven patients into three categories : delivery failure ( n = 3 ) , deployment failure of stent ( n = 3 ) , retrieval failure of dual tapered ( olive ) tip of the inner body through the deployed the stent ( n = 1 ) . the technical failure in using wingspan stent ( delivery , deployment and retrieval failures ) are related to tortuousness of the proximal ( n = 4 ) as well as distal ( n = 1 ) cerebral vessels to the stenotic lesion and bulky profile of the olive tip ( n = 2).conclusionthe technical failure in using wingspan stent ( delivery , deployment and retrieval failures ) are related to tortuousness of the proximal as well as distal cerebral vessels to the stenotic lesion and bulky profile of the olive tip . to avoid device - related complication , complete understanding of the stent design is mandatory before using the stent .

supplementary material is available for this article at 10.1007/s13659 - 013 - 0024 - 9 and is accessible for authorized users .

dendrowardol c ( 1)a novel sesquiterpenoid , with an unprecedented 4/5/6/6 tetracyclic carbon backbone , together with two known cyclopacamphane - type sesquiterpenoids ; dendronobilin i ( 2 ) and dendrobane a ( 3 ) were isolated from the stems of dendrobium wardianum warner . the structure of 1 was established on the basis of spectroscopic data and the absolute configuration was determined by single - crystal x - ray diffraction crystallography . the hypothetical biosynthetic pathway of 1 was postulated . compound 1 showed no cytotoxic activity against human tumor cell lines hl-60 , smmc-7721 , a-549 , mcf-7 , and sw480 . electronic supplementary materialsupplementary material is available for this article at 10.1007/s13659 - 013 - 0024 - 9 and is accessible for authorized users .

focal segmental glomerulosclerosis ( fsgs ) causes scarring or sclerosis of glomeruli that act as tiny filters in the kidneys , damage to which results in diminished ability to properly filter blood , resulting in the urinary loss of plasma proteins and subsequent proteinuria . a 60-year - old , white female with a history of intermittent proteinuria was referred by her primary care physician for renal dysfunction . she also had rheumatoid arthritis ( ra ) but no active synovitis and was maintained on prednisone 5 mg / d . she also complained of worsening vision in her right eye and was diagnosed with optic neuritis ( on ) . she remained stable for about 8 months when examination indicated fsgs relapse , and she reported painful ra flares . she was treated with acthar gel ( 40 mg biweekly ) for 6 months , after which proteinuria and urine protein - to - creatinine ratio decreased to about half . her on improved , and she reported that she had fewer ra flares and pain improved by 50% . this case of confirmed fsgs showed an improved response to treatment with acthar gel for fsgs with concomitant ra and on . this referral case is relevant to primary care practitioners who treat disorders that may be responsive to corticosteroid therapy . the antiproteinuric effects and ancillary improvement in ra and on symptoms during treatment with acthar gel are not entirely explained by its steroidogenic actions . acth is a bioactive peptide that , together with -melanocyte - stimulating hormone , exhibits biologic efficacy by modulating proinflammatory cytokines and subsequent leukocyte extravasation and may have autocrine / paracrine effects in joints . while acthar gel was primarily administered in this case to treat proteinuria , it also showed ancillary benefits in patients with concomitant inflammatory disease states . focal segmental glomerulosclerosis ( fsgs ) is named for the characteristic scarring or sclerosis of glomeruli that act as tiny filters in the kidneys . focal relates to the fact that only some of the filters are damaged , whereas damage to the glomeruli results in a diminished ability to properly filter blood , resulting in the urinary loss of plasma proteins ( ie , proteinuria ) , which is a hallmark of nephrotic syndrome ( ns ) . the etiology of fsgs is often unknown , but known risk factors include infection , drug toxicity , and systemic diseases such as diabetes , hypertension , and obesity.1 fsgs is a relatively common form of kidney disease in the united states and is now one of the most common patterns of glomerular injury encountered in human kidney biopsies , as well as the most common cause of proteinuria in the african american and us hispanic populations.1 in 2012 , a clinical practice guideline from the organization kidney disease : improving global outcomes ( kdigo ) was published based on a systematic literature review conducted in january 2011 , and supplemented by additional evidence through november 2011.1 the recommended initial treatment of idiopathic fsgs associated with clinical features of the ns is immunosuppressive therapy that usually begins with prednisone given at high dose for a minimum of 4 weeks to a maximum of 16 weeks as tolerated , to achieve remission , with subsequent tapering over 6 months.1 kdigo recommends consideration of calcineurin inhibitor as first - line therapy for patients with a relative contraindication to high - dose corticosteroids ( cs).1 rheumatoid arthritis ( ra ) is classified according to the 2010 american college of rheumatology and european league against rheumatism classification criteria . the current classification focuses on features at earlier stages that are associated with persistent / erosive disease.2 historically , treatment for most patients started with cs or nonsteroidal anti - inflammatory drugs . however , emerging evidence indicates that early and aggressive targeted - treatment strategies that provide consistent suppression of ra disease activity is linked to better long - term outcomes.3,4 optic neuritis ( on ) is an inflammatory , demyelinating condition that is highly associated with multiple sclerosis ( ms ) , and it may be the presenting feature in 15% to 20% of patients with ms.5 on typically affects young adults , and women are affected more often than men . on is second only to glaucoma as the most common acquired optic nerve disorder in persons younger than age 50.6 on usually improves on its own and although intravenous cs may speed recovery , it does not appear to provide long - term benefit and undergoing no treatment for acute on is an option.6,7 a meta - analysis of randomized controlled clinical trials using cs or adrenocorticotropic hormone ( acth ) showed that either modality produced significant improvement of visual disability and is therefore effective in accelerating short - term recovery.8 the author is not aware of a previous case that highlights an improved response to treatment with acthar gel ( repository corticotropin injection ) in a patient having these multiple disorders that are usually treated with cs therapy . a 60-year - old , white , obese woman was referred by her primary care physician for renal dysfunction . the patient , who has a history of intermittent proteinuria , had normal renal biopsy findings 14 years ago , and would likely benefit from another biopsy . she also has a history of hypertension , hyperlipidemia , hypothyroidism , peptic ulcer , chronic obstructive pulmonary disease , ra , and type 2 diabetes mellitus . proteinuria was 2.5 g at presentation , serum creatinine was at a normal level , and c - reactive protein level was elevated . glomerular immunofluorescence was uniformly negative for immunoglobulins igg , iga , complement c3 , c1q , and light chains . the quality of glomerular tissue preservation was adequate , with no evident cellular or proliferative changes or entrapment of immune complexes at any capillary or mesangial foci . foot processes of the visceral epithelial cells and capillary basement membranes were intact and free of defects . the patient was treated with an angiotensin - converting enzyme ( ace ) inhibitor for fsgs and was maintained on her current dosage of prednisone for ra . she also complained of worsening vision in her right eye and of seeing water bubbles . an exam with an ophthalmologist showed that the patient had orbital inflammation and on hyperemia and she was diagnosed with on . the patient s fsgs remained stable for approximately 8 months after presentation when examination showed worsening edema and highly probable fsgs relapse . serum creatinine concentration was 1.54 mg / dl , and she had 4 + proteinuria ( 319 mg / dl ) and a urine protein - to - creatinine ratio ( upcr ) of 9.3 g / g . the patient also experienced painful ra flares during ongoing treatment with 5 mg of prednisone once daily . she began treatment with a 6-month regimen of acthar gel ( 40 u twice weekly ) in april 2013 for fsgs . at her initial follow - up visit in september 2013 , proteinuria and upcr had decreased to about one - half ( 159 mg / dl and 4.3 g / g , respectively ) and serum albumin was 3.4 g / dl . at a follow - up visit later in september , spot urine upcr was 1.78 g / g and serum albumin was 3.7 g / dl . in addition , ophthalmic follow - up showed a decrease in orbital inflammation and reduction in swelling and thickness of the optic nerve in both eyes . although approved by the food and drug administration ( fda ) for treatment of on , there are limited data demonstrating the efficacy of acthar gel ( a highly purified preparation of acth ) for this condition . the patient reported that after starting treatment with acthar gel , she experienced fewer ra flares and pain had decreased by about 50% . she was also instructed to call in the case of worsening symptoms or adverse effects to medications . the patient did experience an elevation of her blood pressure and vitreous seeding was observed . this patient had a confirmed diagnosis of fsgs of unknown etiology but had a history of risk factors associated with fsgs . although less responsive than minimal - change ns , fsgs appears to respond to cs and treatment with cs is specifically recommended in kdigo guidelines.1 although not specifically studied in primary or secondary fsgs , ace inhibitors or angiotensin - receptor blockers ( arbs ) also reduce proteinuria and slow progression in proteinuric kidney disease , and patients with fsgs should receive renin - angiotensin system blockade and instructions to restrict dietary sodium.9 this patient was initially treated with the ace inhibitor lisinopril 20 mg and later switched to the arb telmisartan 80 mg . the empirical results shown in the patient in this case appear to support further consideration of acthar gel for treatment of fsgs . the role of acthar gel has not been adequately studied in ra , and it is fda - approved in ra only for short - term use . however , acth stimulates the adrenal cortex to produce cortisol in response to stress and also is an important physiologic agonist of the melanocortin ( mc ) system.10 the rationale for its use in ra is related to a study showing increased levels of -melanocyte - stimulating hormone ( -msh ) in the synovial fluid of patients with ra or juvenile ra , and it also has been shown to reduce joint pathology in rats with adjuvant - induced arthritis.11 acthar gel is a highly purified preparation of acth ( acth139 ) . acth139 is a bioactive peptide derived from a large precursor protein named pro - opiomelanocortin . acth is biochemically cleaved in vivo into another bioactive peptide , -msh , that corresponds to the first 13 amino acids of acth139 12 and exhibits biologic efficacy by modulating proinflammatory cytokines and subsequent leukocyte extravasation.13 investigations into the biologic distribution and function of mc receptors show that acth139 has anti - inflammatory or immunomodulatory functions related to the activation of mc1r , mc3r , and mc5r and is the only known mc peptide that will activate mc2r in the adrenal cortex.12 mechanisms of action now being considered for acthar gel hypothesize effects beyond steroidogenesis . the precise role of mcs in ra is uncertain , but evidence suggesting that mcs exert anti - inflammatory properties in ra is drawn from clinical studies that showed acth139 decreased joint pain , swelling , and rheumatic symptoms.13 mc peptides modulate inflammation through mc receptors and the activation of mcs initially reduce inflammatory cytokines and chemokines , later followed by the induction of anti - inflammatory mediators that facilitate the resolution of inflammatory response.13 in ra , the inflamed joint is characterized by infiltrating leukocytes as well as activated bone / cartilage cells , and studies suggest that mc peptides could act locally in an autocrine / paracrine manner.13 evidence for this is supported by an experimental murine model that mimics the flare of ra , in which the administration of an mc-3 agonist attenuated disease in incidence and severity in wild - type mice but not in mc-3 deficient mice , with sustained nf-b signaling in the osteoclasts of mc-3 deficient mice.14 this experimental model suggests that endogenous mc-3 controls the arthritic response in a model of passive serum - transfer arthritis.14 the mechanism of action of acthar gel comprises multiple components , including five mc receptors , each having varying biological activities.10 receptor - binding studies have revealed that all five mc receptors show a strong affinity for acth , which is one of four mc peptides , thereby establishing the potential for this hormone to activate these receptors.10 acth has shown efficacy as primary and secondary therapy for ns . two recent studies have prospectively examined the use of acthar gel in patients with fsgs.15,16 one was a prospective open - label trial of 15 subjects with resistant glomerular disease treated with acthar gel ( 80 u , subcutaneously , twice weekly).15 five subjects had resistant fsgs or minimal - change disease ( mcd ) and preserved renal function at baseline ( serum creatinine 0.61.2 mg / dl ) and had failed a traditional course of cs.15 all subjects with fsgs / mcd failed to achieve sustained remission with cs therapy and at least one other immunosuppressive therapy.15 among the five subjects , two subjects ( one with fsgs , one with mcd ) showed sustained improvement in upcr from baseline to 24 weeks ( 3.160.78 , mcd ; 1.940.43 , fsgs).15 of the remaining three subjects , two showed an increase in upcr from baseline to week 24 , and one showed no appreciable change.15 these results should be interpreted in the context of a population of treatment - resistant patients in whom the failure of first - line treatment increases the likelihood for the failure of second - line therapy , and so forth.15 given the small sample size , the study provides evidence to warrant further investigation in controlled trials . another study was a retrospective case series that evaluated the use of acthar gel in 21 adult patients with idiopathic ns , including one patient with fsgs.17 three patients received acthar gel as primary therapy , 18 patients had failed prior immunosuppressive therapy , nine of whom had failed at least three therapies . acthar gel was administered subcutaneously in varying regimens ranging between 80 iu and 160 iu per week.17 the patient with fsgs was a 63-year - old hispanic female who had been previously treated with cs , mycophenolate mofetil , and a calcineurin inhibitor and had proteinuria of 10,275 mg / d.17 the patient was treated with acthar gel , 80 u biweekly , for 6 months and achieved partial remission , with a decrease in proteinuria from 10,275 mg / d before treatment to 2,970 mg / d.17 in the largest retrospective case series to date , 44 patients with ns were treated with acthar gel , including 15 patients with fsgs.18 eligible cases underwent an assessment of 24-hour proteinuria level or spot upcr prior to and following 6 months of acthar gel therapy . complete remission was defined as proteinuria 500 mg / g and partial remission as proteinuria > 500 mg / g to 3,500 mg / g and with 50% reduction in proteinuria from baseline . clinical response was defined as 25% reduction in proteinuria level from baseline and the reduction in final proteinuria did not meet the definitions for complete or partial remission . twelve of the 15 patients with fsgs showed partial remission ( n=8 ) or a clinical response ( n=4 ) of approximately 30% reduction in proteinuria . fifteen of 44 patients had treatment - related side effects , mainly steroid - like such as hypertension , weight gain , and hyperglycemia . seven patients had early termination of treatment due to increased edema , fatigue , weight gain , hypertension , and seizures . therefore , all the adverse effects known to occur with elevated cortisol may occur with acthar gel administration as well.19 according to the existing experience of clinical use of acth as well as ongoing clinical trials of other mc analogs , the side effects of mc - based therapy seem mild , tolerable , and reversible.20 acth therapy through the cortisol effect might also induce mild hypertension . as an endogenous glucocorticoid , cortisol may directly elevate blood pressure by enhancing vascular tone or , because it has a fair amount of activity on the mineralocorticoid receptor , may cause sodium and water retention and volume expansion.20 for nonsteroidogenic mcs or analogs , mildly elevated blood pressure has been reported during phase ii or iii clinical trials , possibly attributable to central stimulation of sympathetic outflow mediated by the interaction of -msh with mc4r.2022 another recent study examined 24 subjects and the data for analysis were available for 23 patients.16 six subjects had steroid - dependent fsgs , 15 had steroid - resistant fsgs , and two additional subjects were treated with acthar gel as first - line therapy between january 2009 and april 2012.16 the trial included 16 subjects in a prospective investigator - initiated pilot study ( nct01155141 ) ; four had been described previously with shorter - term results and the remaining four were evaluated retrospectively in chart review.16 the classification of steroid - dependent or steroid - resistant fsgs was based on kdigo guidelines.1 treatment regimens were not entirely uniform : 12 subjects received 40 u of acthar gel subcutaneously , weekly for 2 weeks , 80 u , subcutaneously , weekly for 2 weeks , followed by 80 u , subcutaneously , twice weekly for 16 weeks of treatment . seven subjects received 40 u of acthar gel , subcutaneously , twice weekly for 2 weeks , followed by 80 u , subcutaneously , twice weekly for 6 months of treatment . the remaining five subjects received acthar gel with heterogeneous dosing as determined by the treating physician . the duration of therapy ranged from 12 to 56 weeks , with a mean follow - up time of 48 weeks.16 at the end of therapy , seven of 24 ( 29% ) patients had experienced remission ( two complete and five partial).16 in subjects achieving remission of proteinuria , median time to reduced proteinuria was 5 weeks and median time to remission of proteinuria was 16 weeks.16 although only 29% of the subjects experienced remission in this series , improved outcomes may encourage physicians to consider acthar gel in idiopathic fsgs cases that are refractory to previous cs therapy . , we recommend that acth be discontinued for patients with fsgs who have not demonstrated any significant decline in proteinuria by 1216 weeks . the pathological basis of fsgs and mcd appears to essentially be podocyte - related , as evidenced by podocyte foot processes effacement , microvillus transformation , and podocytopenia.23 knockout and transgenic models have provided proof of concept that mutations in specific podocyte proteins mediate genetic forms of fsgs.24 a study was conducted in rats with subtotal nephrectomy to examine the capability of acthar gel for providing podocyte protection.25 compared with the control group , acthar gel preserved kidney function as measured by increased renal plasma flow and glomerular filtration rate and lower serum creatinine levels . these data suggest that acthar gel reduces proteinuria and renal injury in a preclinical model of progressive glomerulosclerosis . lindskog investigated the expression of mc in human kidneys to test the hypothesis that synthetic acth directly exerts its antiproteinuric effects via mc receptors.26 gene expression of the mc receptor mc1r ( but no other mc receptor ) was identified in podocytes , glomerular endothelial cells , mesangial cells , and tubular epithelial cells , and analysis showed that podocytes expressed most of the mc1r protein.26 a specific mc1r agonist ( ms05 ) demonstrated that mc1r agonism reduced proteinuria , improved podocyte morphology , and reduced oxidative stress in rats with passive heymann nephritis ( phn).26 the nonspecific mc receptor agonist -msh ( n=8 ) also significantly reduced proteinuria by 52% compared with untreated phn ( n=8 ; p<0.05 ) . within the -msh group , this corresponds to a 55% decrease in proteinuria after 4 weeks of treatment compared with proteinuria at the start of treatment . acthar gel ( acth ) has antiproteinuric and immunomodulatory effects that are not entirely explained by its steroidogenic activity . acth is the parent molecule of -msh , and exhibits biologic efficacy by modulating proinflammatory cytokines and subsequent leukocyte extravasation , and evidence suggests that mcs also may have autocrine / paracrine effects in joints . acthar gel was primarily administered in this case to induce the remission of proteinuria in a patient with a history of intermittent proteinuria and a confirmed diagnosis of fsgs treated with ace inhibitor therapy , but who experienced worsening edema and serum creatinine with 4 + proteinuria . this case is interesting not only because of its effect in improving proteinuria , but also because it showed clinically beneficial ancillary effects on the patient s inflammatory disease states . the role of acth in the treatment of ra or on has not been adequately studied , but improvement in this patient s flares and pain in ra as well as orbital inflammation and reduction in the swelling of the optic nerve and thickness is noteworthy and may be related to its agonist effects on multiple mc receptors .

backgroundfocal segmental glomerulosclerosis ( fsgs ) causes scarring or sclerosis of glomeruli that act as tiny filters in the kidneys , damage to which results in diminished ability to properly filter blood , resulting in the urinary loss of plasma proteins and subsequent proteinuria.case presentationa 60-year - old , white female with a history of intermittent proteinuria was referred by her primary care physician for renal dysfunction . biopsy confirmed fsgs and she was treated with an angiotensin - converting enzyme inhibitor . she also had rheumatoid arthritis ( ra ) but no active synovitis and was maintained on prednisone 5 mg / d . she also complained of worsening vision in her right eye and was diagnosed with optic neuritis ( on ) . she remained stable for about 8 months when examination indicated fsgs relapse , and she reported painful ra flares . she was treated with acthar gel ( 40 mg biweekly ) for 6 months , after which proteinuria and urine protein - to - creatinine ratio decreased to about half . her on improved , and she reported that she had fewer ra flares and pain improved by 50% . this case of confirmed fsgs showed an improved response to treatment with acthar gel for fsgs with concomitant ra and on.conclusionthis referral case is relevant to primary care practitioners who treat disorders that may be responsive to corticosteroid therapy . the antiproteinuric effects and ancillary improvement in ra and on symptoms during treatment with acthar gel are not entirely explained by its steroidogenic actions . acth is a bioactive peptide that , together with -melanocyte - stimulating hormone , exhibits biologic efficacy by modulating proinflammatory cytokines and subsequent leukocyte extravasation and may have autocrine / paracrine effects in joints . while acthar gel was primarily administered in this case to treat proteinuria , it also showed ancillary benefits in patients with concomitant inflammatory disease states .

vitiligo is a non - contagious acquired pigmentation disorder characterized by defined white patches of variable shape and dimensions , increasing in size and number with time . the prevalence range in india found to be 0.46 - 8.8% with gujarat and rajasthan states have highest prevalence of approximately 8.8% . approximately 30% acquire the disease before the age of 20 years and 14% before the age of 10 years , with decreasing incidence in later life , and fewer than 10% develop vitiligo by the age of 42 years . infants 4 - 6 months of age may develop typical vitiligo mainly in the genital or perianal area . etiology of vitiligo is not known but the loss of melanocytes in epidermis is explained by certain pathogenetic hypothesis such as genetic hypothesis , autoimmune hypothesis , neural hypothesis , autocytotoxic hypothesis , novel microenvironment - related hypothesis and convergence theory . as a depigmenting disease vitiligo also has a greater impact on patients psychosocial aspects . persons affected with vitiligo especially children and females have greater difficulty in their social relationships due to psychological depression . our study group consists of 100 vitiligo patients . the demographic features including age and genders were included in this study . patients with vitiligo skin lesions were only included in this study . vitiligo associated with systemic conditions such as thyroid disorders , juvenile diabetes mellitus , pernicious anemia , addison 's disease was not included . the frequency of the occurrence of oral mucosal vitiligo and most commonly involved mucosal site were observed in this study . the minimum age of 4 years and the maximum age of 76 years were observed in this study . among the 100 vitiligo patients 18% were up to 25 years , 14% within 25 to 35 years , 30% within 35 to 45 years , 19% within 45 to 55 years and 19% more than 55 years ( graph 1 ) . in the present study of 100 vitiligo patients , oral mucosal depigmentation were presented in lip ( 42% ) , palate ( 8% ) , buccal mucosa ( 5% ) , labial mucosa ( 5% ) and gingiva ( 2% ) [ table 1 and graph 2 ] . the association of oral vitiligo with respect to age was 10% within 25 years , 6% among age group of 26 - 35 years , 14% among age group of 36 - 45 years , 6% within 46 - 55 years and 9% of patients above 55 years [ table 2 and graph 3 ] . regarding the gender , the association of oral presentation among our study group showed to be 18% in males and 27% in females [ table 3 and graph 4 ] . pie chart showing the age distribution among the 100 vitiligo patients presence of depigmentation in various sites of oral cavity percentage of presence and absence of oral depigmentation in lips , buccal mucosa , labial mucosa , palate and gingiva among the study group presence of oral findings with respect to age presence of oral findings with respect to age presence of oral findings with respect to gender presence of oral findings with respect to gender vitiligo is a common non - infectious dermatological disorder characterized by acquired , idiopathic , progressive , circumscribed hypomelanosis of the skin and hair , with the total absence of melanocytes microscopically . out of 100 patients in this study , 44 patients were males and 56 patients were females showing higher incidence . shajil et al . , pandya et al . showed 33.8 years , 25.59 years and 21 to 30 years , respectively . our study of vitiligo patients showed a minimum age of 4 years and a maximum age of 76 years . another study by gonul et al . showed minimum and maximum age as 8 years to 72 years respectively which is closely correlating with our study . the oral presentation of vitiligo in the present study showed depigmentation of lip in 42 patients , buccal mucosa in 5 patients , labial mucosa in 5 patients , palate in 8 patients , gingiva in 2 patients . in our study out of 100 vitiligo patients , 21 patients showed intra oral mucosal depigmentation . among the intraoral mucosal sites palate was found to be the common followed by labial and buccal mucosa , gingiva and alveolar mucosa . the number of vitiligo patients in our study showing oral depigmentation was about 9 up to 25 years , 8 in the age range of 25 to 35 years , 19 among the age of 35 to 45 years , 15 in the age range of 45 to 55 years and 11 above 55 years . thus , higher incidence of oral mucosal vitiligo was noted in patients of age 35 to 45 years of age in the present study . also , females showed higher oral presentation of about 33 patients and 29 patients were males . in a report of three vitiligo cases by dummett et al . , one patient had complete clinical depigmentation of all the oral tissues and another patient with history of heavy gingival pigmentation showed pink edentulous ridges at the time of examination . mucosal involvement occurring frequently in lips , genitals , gingival , areolar and nipples have been reported in the literature . solitary presentation of palatal depigmentation in a vitiligo patient was reported by lawoyin et al . in our study 42 age range up to 25 years with lip involvement was noted in 7 patients , 25 - 35 years of age range showed 6 patients , 10 patients with lip involvement were within 35 - 45 years , 45 - 55 years of age showing lip depigmentation in 11 patients and above 55 years 8 patients presented lip depigmentation [ figure 1 ] . depigmentation in lip among 100 patients in our study depigmentation of buccal mucosa was present in five patients in which three were males and two females . with age of range up to 25 years none , within 25 - 35 years of age two patients , among patients of age 35 - 45 years two patients , within age range of 45 - 55 years one patient and above 55 years of age none of the patients showed depigmentation in buccal mucosa [ figure 2 ] . depigmentation in palate in the present study labial mucosa showed depigmentation in a male patient and four female patients . the following age ranges of up to 25 years , 25 - 35 years , 35 - 45 years , 45 - 55 years , above 55 years presented depigmentation of labial mucosa in one patient , none , three patients , one patient and none , respectively [ figure 3 ] . depigmentation in buccal mucosa in our study palatal depigmentation was found in eight patients out of them five patients were males and three were females . up to age range of 25 years one patient had palatal depigmentation , 25 - 35 years of range showed none , within age of 35 - 45 years three patients , 45 - 55 years of age range two patients and above 55 years of age two patients showed depigmentation in palate [ figure 4 ] . with respect to age of up to 25 years , 25 - 35 years , 45 - 55 years age none showed gingival depigmentation . patients within the age range of 35 - 45 years and above 55 years showed one patient each with depigmentation of gingiva [ figure 5 ] . depigmentation in gingiva among the 100 patients in our study 2 patients showed oral depigmentation as the only manifestation of vitiligo both manifestating in the lip . in relation to age up to 25 years one patient and 35 - 45 years one patient showed oral depigmentation . depigmentation of oral mucosa can be noted as an early feature in oral submucous fibrosis . in reverse these depigmentations are excluded based on the clinical history . in our study no relation of previous dental procedures to the depigmentation was noted . as far as our knowledge no literature states about the koebner phenomenon as a cause for oral vitiligo . out of 100 patients in this study , 44 patients were males and 56 patients were females showing higher incidence . , nigam et al . , pandya et al . , chanda et al . the study conducted by shameer et al . showed male predilection . shajil et al . , pandya et al . showed 33.8 years , 25.59 years and 21 to 30 years , respectively . our study of vitiligo patients showed a minimum age of 4 years and a maximum age of 76 years . another study by gonul et al . showed minimum and maximum age as 8 years to 72 years respectively which is closely correlating with our study . the oral presentation of vitiligo in the present study showed depigmentation of lip in 42 patients , buccal mucosa in 5 patients , labial mucosa in 5 patients , palate in 8 patients , gingiva in 2 patients . in our study out of 100 vitiligo patients , 21 patients showed intra oral mucosal depigmentation . among the intraoral mucosal sites palate was found to be the common followed by labial and buccal mucosa , gingiva and alveolar mucosa . the number of vitiligo patients in our study showing oral depigmentation was about 9 up to 25 years , 8 in the age range of 25 to 35 years , 19 among the age of 35 to 45 years , 15 in the age range of 45 to 55 years and 11 above 55 years . thus , higher incidence of oral mucosal vitiligo was noted in patients of age 35 to 45 years of age in the present study . also , females showed higher oral presentation of about 33 patients and 29 patients were males . in a report of three vitiligo cases by dummett et al . , one patient had complete clinical depigmentation of all the oral tissues and another patient with history of heavy gingival pigmentation showed pink edentulous ridges at the time of examination . mucosal involvement occurring frequently in lips , genitals , gingival , areolar and nipples have been reported in the literature . solitary presentation of palatal depigmentation in a vitiligo patient was reported by lawoyin et al . in our study 42 patients presented lip depigmentation out of which 20 were males and 22 females . age range up to 25 years with lip involvement was noted in 7 patients , 25 - 35 years of age range showed 6 patients , 10 patients with lip involvement were within 35 - 45 years , 45 - 55 years of age showing lip depigmentation in 11 patients and above 55 years 8 patients presented lip depigmentation [ figure 1 ] . depigmentation in lip among 100 patients in our study depigmentation of buccal mucosa was present in five patients in which three were males and two females . with age of range up to 25 years none , within 25 - 35 years of age two patients , among patients of age 35 - 45 years two patients , within age range of 45 - 55 years one patient and above 55 years of age none of the patients showed depigmentation in buccal mucosa [ figure 2 ] . depigmentation in palate in the present study labial mucosa showed depigmentation in a male patient and four female patients . the following age ranges of up to 25 years , 25 - 35 years , 35 - 45 years , 45 - 55 years , above 55 years presented depigmentation of labial mucosa in one patient , none , three patients , one patient and none , respectively [ figure 3 ] . depigmentation in buccal mucosa in our study palatal depigmentation was found in eight patients out of them five patients were males and three were females . up to age range of 25 years one patient had palatal depigmentation , 25 - 35 years of range showed none , within age of 35 - 45 years three patients , 45 - 55 years of age range two patients and above 55 years of age two patients showed depigmentation in palate [ figure 4 ] . with respect to age of up to 25 years , 25 - 35 years , 45 - 55 years age none showed gingival depigmentation . patients within the age range of 35 - 45 years and above 55 years showed one patient each with depigmentation of gingiva [ figure 5 ] . among the 100 patients in our study 2 patients showed oral depigmentation as the only manifestation of vitiligo both manifestating in the lip . in relation to age up to 25 years one patient and 35 - 45 years one patient showed oral depigmentation . depigmentation of oral mucosa can be noted as an early feature in oral submucous fibrosis . in reverse these depigmentations are excluded based on the clinical history . in our study no relation of previous dental procedures to the depigmentation was noted . as far as our knowledge no literature states about the koebner phenomenon as a cause for oral vitiligo . vitiligo lesions presenting as skin depigmentation may also show oral mucosal depigmentation with lip and palate being the most common sites . sometimes oral depigmentation may be the first indicator of vitiligo which may manifest in unnoticeable areas . so educating patients regarding oral depigmentation and possibility of occurrence in other parts of body will help in the earlier commencement of treatment , thereby battling against a socially distressing disorder could be possible . very few articles have been published regarding oral manifestations of vitiligo . these articles report depigmentation of lip , palate and gingiva . in our study we have identified the presence of depigmentation of labial mucosa and buccal mucosa as well .

background : vitiligo is one of the disorder that has social impact . both skin and mucous membrane show depigmentation in vitiligo . depigmentation in oral cavity can be more easily observed and the patient can be given awareness regarding the condition if they are unaware of vitiligo elsewhere in their body and can be guided for treatment.aim and objectives : the aim of this study is to determine the frequency of occurrence of oral mucosal vitiligo in vitiligo patients and to determine the most commonly involved oral mucosal site.materials and methods : the study sample included 100 vitiligo patients . the patients of all age groups and both genders were included . vitiligo patients associated with systemic conditions such as thyroid disorders , juvenile diabetes mellitus , pernicious anemia , addison 's disease were excluded in this study.results:out of 100 vitiligo patients 44 % male and 56% were female . the oral presentation of vitiligo in this study showed depigmentation of buccal mucosa in 5% of patients , labial mucosa in 5% of patients , palate in 8% of patients , gingiva in 2% of patients and alveolar mucosa 1% . depigmentation of lip was seen in 42% of patients . lip involvement refers to depigmentation of both the lips or either lip . also vermilion border involvement was noted in majority of cases . in some cases , the depigmentation of lip extended to the facial skin also.conclusion:in this study 55 patients out of 100 patients showed depigmentation in the oral cavity . lip involvement was most common in this study showing about 42% of patients . intraoral mucosal involvement was found in 21% of patients . among intraoral mucosal site palate was common followed by buccal and labial mucosa , gingiva . two patients had lip pigmentation as the only manifestation without any depigmentation in the skin .

errors and mistakes in ultrasound diagnostics of the abdominal cavity in children result from many factors . the major one is lack of experience in examining children and lack of the knowledge on physiological processes in the developing body which translate into the ultrasound image of some organs . the diversity and non - specific nature of clinical symptoms in children , which may imitate congenital diseases , inflammations as well as mask the neoplastic process , constitute the next reason for difficulties in pediatric diagnostics . it is imperative in the diagnostic process to obtain the correlation of the ultrasound image with clinical data of the patient , the results of other imaging and the scope of the performed surgery . knowledge on the most common pathologies in particular periods of a child s life and their relation with clinical examinations and the results of laboratory tests constitute the basis for a reliable ultrasound examination . the following are of crucial importance : knowledge concerning the clinical state of the patient , results of laboratory tests , the past and current diseases and the information on what the examination is supposed to explain . errors and mistakes in interpreting the ultrasound image may result in lack of cooperation with the child during the examination . bad preparation for the examination , severe child s condition as well as a patient following a trauma or a surgery are the next factors affecting the quality of the assessment of the abdominal cavity organs . it is also of high importance to have a good class ultrasound equipment and the knowledge concerning its operation . the article presents errors and mistakes in pediatric diagnostics in accordance with the following division : errors resulting from the lack of knowledge / failure to adjust the ultrasound apparatus for examining children ( pediatric applications , proper heads ) ; errors resulting from inappropriate examination method ( lack of cooperation with the patient , patient s built , improper patient preparation to the examination ) ; errors resulting from the lack of knowledge on physiological varieties of organs construction in a child ; errors resulting from the lack of knowledge concerning the most common pathologies of the age of childhood and the lack of clinical data about the patient . insufficient quality of the ultrasound apparatus as well as lack of appropriate heads and software pose the risk of erroneous assessment of organs , misinterpreting the existing pathology or interpreting artifacts as pathologies . children ( especially younger ones ) have smaller organs , which are located closer to the abdominal integuments and a thinner subcutaneous adipose tissue , therefore depending on the child s age electronic heads of wide spectrum of frequency should be utilized ( preferably with changing frequency ) from 3.5 to 10 mhz and with various sizes ( shape and length of the head ) : convex head 2d with the frequency of 3.55 mhz and 47 mhz ; phased array head 2d with the frequency of 47 mhz and 58 mhz ; linear head 2d with the frequency of 510 mhz ; linear head 2d with the frequency of 712 mhz . ultrasonography of the abdominal cavity in newborns is performed with the use of heads having the frequency of 7.5 mhz , in infants and younger children 5 mhz , in older children and teenagers 3.5 mhz . in the case of newborns and infants , the ultrasound apparatus should be factory - equipped with the so - called pediatric software ( abdomen , kidney , pelvic , bowel ) and technical parameters ( e.g. : gain , tgc , focus ) proper for examining a child at every age , which will make it possible to precisely assess the parenchymatous organs of the abdominal cavity and intestinal loops and the retroperitoneal space . large physical activity and breathing a lot in the case of small patients connected with the need to perform measurements of the abdominal cavity organs require one to possess the registration option and going back to the examined cross - sections , the so - called sine loop . errors in the assessment of the examination may also result from improperly prepared ultrasonography room , interferences caused by the proximity of other electronic devices or fatigue of the examiner . lack of cooperation with the child as regards performing orders ( breathing in , keeping bladder full ) , crying and anxiety make the examination harder and prolong it . unfortunately , this creates the hazard of not observing the existing pathology or overinterpreting the artifacts , or e.g. stomach full of food or the residual stool in the intestine as being abnormalities . precise assessment of the urinary system requires examination with full bladder , which in the case of youngest children is very hard . the bladder not being filled sufficiently , or being filled poorly , the presence of echoless , smooth - walled ovarian cyst may be interpreted as correct bladder ( fig . 1 ) . echoless ovarian cyst assessed to be a correct bladder errors and mistakes in the assessment of the abdominal cavity organs also result from a large amount of gases filling the intestines , which hide internal organs . as in the case of intestines distended with a liquid mass residue or filled with stool , which displace the neighboring organs hindering their assessment . the ultrasound image of the abdominal cavity organs in children basically does not vary from the one of adults , apart from increasing the size of organs when the child grows ( the size of organs is assessed in correlation with the length / growth of the patient ) . however , there are organs which change their morphological image in ultrasound examination as a person matures : kidneys , adrenal glands , pancreas and ovaries . the kidneys of a newborn and infant have a different built than in children aged above 12 months of age . lack of the knowledge on this fact is unfortunately sometimes the reason for erroneous diagnosis of the presence of pathological lesions . correct kidney of a newborn or infant is characterized by the following features : echogenicity of the renal cortex is equal to or higher than the one of the liver and spleen ; in premature babies it is even strongly visible ( fig . 2 ) ; visible dominance of echoless renal pyramids , smaller volume of the cortex may be misinterpreted for the presence of cysts ( fig . 3 ) ; owing to a small amount of the adipose tissue , poor echogenicity of the renal sinus ; in some patients , there is a clearly visible hypertrophy of the column of bertin , which may be taken for a tumor ( fig . 4 ) ; renal pelvis ( with no widening of the calyxes ) in the ap cross - section should not exceed 10 mm . it should be borne in mind that especially in the case of children the physiological prominence of the left kidney is well visible , it is also sometimes taken to be a lesion with the nature of a tumor ( fig . . kidney of a newborn correct high echo of the parenchyma , echoless pyramids right kidney of a newborn correct higher echo of the parenchyma , hypoechogenic pyramids kidney , hypertrophy of the column of bertin : a. variant of standard ; b. color doppler imaging option , correct vascularization of the kidney physiological prominence of the left kidney : a. in a newborn with the presence of correct echoless pyramids ; b. in an older child adrenals , until the age of approximately 56 months , are large and usually are y- , v or l - shaped and have the typical built for that age with preserved cortex - medulla differentiation ( fig . the adrenal gland is prolonged and larger , which may be mistakenly diagnosed as an improperly developed kidney . in girls in the period of being a newborn and infants , it is frequent to make visible large ovaries with the presence of multiple follicles , the diameter of which may even amount to a dozen or so mm . this is a typical image related to hormone stimulation of the mother and should not be diagnosed as the formation of cysts in the ovaries ( fig . approximately at the age of 2 , the size of ovaries starts to decrease to develop again in puberty . uterus undergoes similar changes in newborns , it is large , with dominating cervix and well visible hyperechogenic cavity . right adrenal gland of a newborn , correct image correct ovary of a newborn with follicles . urinary bladder with residual amount of urine the next organ , whose ultrasound image changes at the time the child grows , is the pancreas . in younger children , a correct pancreas has the echogenicity equal to or lower than the echogenicity of the liver , and in the period of being a newborn it may be higher ( fig . 8) . in the period of being an infant , high echo of the pancreas proves a pathology . ultrasound diagnostics of the abdominal cavity in a child is difficult , especially when it concerns the youngest children , including newborns and infants . it requires knowledge in the field of the incidence of congenital defects ( of bile ducts , urinary and reproductive systems , intestinal loops ) , inflammations and the proliferative process . interpreting the ultrasound image always needs to be based on clinical data of the child , the results of laboratory tests and other imaging as well as the knowledge of the most common abnormalities in specific age periods of a child . in newborns , bleeding may occur as a result of antepartum hypoxia or perinatal trauma , most frequently to one adrenal gland ( fig . ultrasound image may be mistaken for the presence of a neoplastic lesion of a neuroblastoma type ( fig . clinical data and the assessment of the level of catecholamines and their products in urine may prove helpful in the assessment as well as the evolution and changes in the ultrasound image of the hematoma in time . tumor dislocating the kidney downwards the presence of liquid lesions with the nature of a cyst within the abdominal cavity and the pelvis of the child may also result in diagnostic difficulties . it mainly applies to small children owing to small sizes of the area being subject to examination , which in the majority of patients is filled by a large cyst . the assessment of the starting point of the cyst may be doubtful , it may be varied and most often covers : the ovary , alimentary canal , mesentery or the common bile duct cyst . ovarian cyst in the case of girls may be very large and reach as high as the liver . precise assessment of the parenchyma of the liver and the bile ducts and presenting the connection of the cyst and the bile duct make it possible to assess its nature ( fig . a cyst being a duplication of the alimentary canal is characterized by the presence of a wall typical of the intestinal wall structure ( fig . 12 ) , while the cyst of the mesentery with no pouch undergoes compression and small dislocation as a result of the head s pressure ( fig . a. common bile duct cyst , echoless cyst in the region of the hilum of the liver ; b. extended common bile duct cyst duplication of the alimentary canal ; echoless cyst with a wall typical of the intestinal structure cyst of the mesentery . echoless space undergoing compression under the pressure of the head , invisible pouch in the course of ultrasonography of the abdominal cavity in children , it is an error not to perform the assessment of the stomach , pylorus , alimentary canal , including vermiform appendix , and the retroperitoneal space , also with the use of linear heads . it is especially recommended for patients with abdominal pain and concomitant vomiting , diarrhea or constipation , subfebrile body temperature , or the presence of blood in the stool . diagnosing pylorostenosis , intussusception and intestinal inflammation , or enlarged lymph nodes in ultrasonography provide essential information and make it possible to narrow down the diagnostic process ( fig . a. thickened wall of the final part of the ileum with increased vascular flow ; b. enlarged lymph node in the right iliac fossa . enteritis caused by the yersinia bacteria errors in the assessment of the abdominal cavity may also result from the lack of accurate information concerning the treatment and the scope of the surgery performed , especially in the case of cancer patients . the application of chemotherapy and radiotherapy not only leads to changes in the echostructure and echogenicity of pathological lesions , but also affects the image of appropriate organs . in the case of the surgery performed , an essential piece of information is to what extent the lesion was removed and whether the visible abnormal image is a residual mass or post - operative hematoma ( fig . condition following hepatic tumor removal : a. area with mixed echo site following the surgery ; b. follow - up : liquid lesion evolution of the hematoma ultrasound examination of the abdominal cavity in children is a difficult one , requiring a lot of knowledge in the field of physiology of the developing body and the ability to connect the present symptoms and searching in ultrasonography for lesions typical of a given pathology . it entails a precise assessment of the whole abdominal cavity , true pelvis and the retroperitoneal cavity and the alimentary canal in order to minimize the possibility of an error . the article presents the principles to follow when performing and assessing the ultrasound examination of the abdominal cavity in children , paying particular attention to the youngest patients . when performing an ultrasound examination in older children , it is necessary to follow the same standards as in the case of examining adults , ones described in articles on the subject matter in the journal .

the diagnostics of the abdominal cavity in children , especially in the neonatal - infantile period , requires knowledge in the field of anatomical and physiopathological differences as well as clinical symptomatology and pathology at every stage of the child s development . errors and mistakes in ultrasound diagnostics of the abdominal cavity in children result from many factors , including lack of experience in examining children and the knowledge concerning most frequent ailments and pathologies as well as the incidence or no changes in the ultrasound image of the abdominal cavity organs . the assessment of the ultrasound image should be always based on clinical data of the patient , information on the past diseases , surgeries and the results of additional examinations and laboratory tests . particular attention should be paid to the occurrence of congenital diseases and inflammations , which may have varied clinical manifestation especially in the case of pediatric diagnostics . the variety and non - specific nature of clinical symptoms may also mask the developing neoplastic process . mistakes in ultrasound diagnostics , especially among the youngest children , may also be caused by technical difficulties related to carrying out the examination . the above situation results from lack of cooperation with the child , who is uneasy , wailing , fails to perform orders , which may lead to overlooking the existing lesion or overinterpreting , e.g. a full stomach or residual stool in the intestines to be a pathology . it is also of high importance to have a good class of the ultrasound equipment and technical knowledge concerning its operation . when performing an ultrasound examination in children , it is necessary to apply a wide range of phased - array , convex and linear heads and appropriate applications , the so - called pediatric software ( stomach , kidneys , true pelvis , organs at the surface ) .

chronic periodontitis is a multifactorial disease primarily caused by dental plaque microorganisms with modifying effects from other local and systemic factors . it results from the immunoinflammatory response to chronic infection , which leads to the destruction of periodontal tissues . recent studies have demonstrated that chronic periodontitis is a potential risk factor for systemic diseases such as cardiovascular disease , diabetes mellitus ( dm ) etc . diabetes influences the periodontium mainly due to the classic microvascular and macrovascular diabetic complications , changes in subgingival microbiota , and alterations in the host immunoinflammatory response to potential periodontal pathogens . diabetes may result in impairment of neutrophil adherence , chemotaxis , and phagocytosis , which may facilitate bacterial persistence in the periodontal pocket and progressive periodontal destruction . chronic periodontal inflammation and proinflammatory cytokines such as interleukin-1 ( il-1 ) , tumor necrosis factor alpha , and il-6 leads to insulin resistance in type 2 dm . the control of periodontal infection has an impact on improvement of glycemic control in diabetic patients . vitamin d plays an important role in many chronic inflammatory diseases by influencing the expression of inflammation related cytokines . recent evidences showed that persons with diabetes have lower serum concentrations of vitamin d and its supplementation has improved glycemic control and insulin sensitivity in patients with type 2 dm . vitamin d has potential anti - inflammatory effect and its active metabolite , 1 , 25 dihydroxyvitamin d inhibit cytokine production . recent data suggested that vitamin d deficiency may play a role both in periodontal disease and dm . the inflammatory status as well as vitamin d insufficiency may create an environment conducive for the progression of chronic diseases such as diabetes and periodontitis . since a bidirectional relationship exist between periodontitis and diabetes , we hypothesized that the serum vitamin d level in patients with chronic periodontitis with type 2 dm could be at a lower level than that of patients with chronic periodontitis and healthy subjects . surprisingly , this association has not been elucidated in the literature so far to the best of our knowledge . hence , the aim of our study was to find out the level of serum vitamin d in chronic periodontitis patients with ( chpdm ) and without type 2 dm . this cross - sectional hospital - based clinical study was conducted in the department of periodontics , government dental college , calicut , kerala , india in collaboration with the department of biochemistry and the department of internal medicine , government medical college calicut from january 2012 to june 2012 . one hundred and forty - one subjects were selected and written informed consent was obtained . the case groups included 93 subjects , of which 43 subjects ( male-16 , female-27 ) had chpdm and 50 subjects ( male-18 , female-32 ) had chronic periodontitis without type 2 diabetes mellitus ( chronic periodontitis patients [ chp ] ) . the mean age for chpdm and chp group was 43.44 and 40.76 years , respectively . the control group comprised of 48 subjects ( male-28 , female-20 ) and their mean age was 40.77 years . a decline in serum vitamin d is reported with aging and it may be attributed to an age - related reduction in the capacity of the skin to produce vitamin d3 . the diagnostic criteria for periodontitis was based on american academy of periodontology criteria 1999 , and clinical case definitions proposed by the center for disease control and prevention working group . chronic periodontitis subjects were selected from the outpatient wing of department of periodontics , government dental college , calicut . dm was defined based on standard medical care 2010 criteria ( serum glucose 126 mg / dl after fasting for a minimum of 8 h ) . patients with type 2 dm were recruited from the outpatient wing of diabetic clinic , department of internal medicine , medical college calicut and they were screened for chronic periodontitis . control group consists of 48 periodontally healthy subjects or those with mild periodontitis who were selected from patient 's bystanders and staff members of government dental college , calicut . history of periodontal therapy within 6 months , antibiotic intake within 3 weeks , postmenopausal woman , subjects with cardiovascular or renal diseases and disease associated with vitamin d deficiency including bone diseases , malignancies , and multiple sclerosis were excluded from the study . age , gender , educational status , family history of diabetes , smoking habits , duration of sun exposure , frequency of outdoor activity , history of diabetes , and history of oral hypoglycemic drug intake or insulin administration were assessed by an interview and questionnaire [ table 1 ] . oral hygiene and gingival status were assessed by silness and loe plaque index ( pi ) , calculus component of simplified greene and vermillion index ( ci ) and lobene et al . pocket depth ( pd ) , gingival recession , and the clinical attachment loss ( cal ) were measured for all teeth on six sites ( mesio - buccal , mid - buccal , disto - buccal , mesio - lingual , mid - lingual , and disto - lingual ) using a william 's graduated periodontal probe . all the study blood samples ( cases and controls ) were collected and assessed in the month of march 2012 . fasting blood sugar ( fbs ) , serum calcium , and serum 25-hydroxyvitamin d ( 25[oh ] d ) were assessed . serum 25(oh ) d concentrations is considered to be a suitable biomarker of vitamin d status that reflects both exposure to sunlight and vitamin d intake from food supplements . this form of vitamin d is biologically inactive with levels approximately 1000 fold greater than the circulating 1 , 25(oh)2d . it is the major storage form of vitamin d in the human body and the half - life of circulating 25(oh ) d is 23 weeks . ideally , the desirable concentration of 25(oh ) d for general health is 30 ng / ml and 2030 ng / ml is considered as vitamin d insuffiency . assessment of vitamin d and other laboratory parameters were done by a qualified and experienced laboratory person who was blinded about periodontal and diabetic status . both internal and external quality control had been done prior to the estimation of vitamin d level . measuring range was 10250 ng / ml ( defined by the lower detection limit and the maximum of the master curve ) . one - way anova test with post - hoc adjustment ( bonferroni test ) was used to compare the mean for periodontal parameters like gi , pi , ci , pd , cal and ( 25[oh ] d ) among the groups . the relation between clinical parameters of the whole study population ( control + chpdm + chp ) and serum vitamin d concentration was analyzed using spearman 's rank correlation coefficient test . both internal and external quality control had been done prior to the estimation of vitamin d level . measuring range was 10250 ng / ml ( defined by the lower detection limit and the maximum of the master curve ) . one - way anova test with post - hoc adjustment ( bonferroni test ) was used to compare the mean for periodontal parameters like gi , pi , ci , pd , cal and ( 25[oh ] d ) among the groups . the relation between clinical parameters of the whole study population ( control + chpdm + chp ) and serum vitamin d concentration was analyzed using spearman 's rank correlation coefficient test . the distributions of percentages of person for education status , smoking habits and outdoor activity , and sun duration were similar in these groups [ table 1 ] . body mass index ( bmi ) ( world health organization classification ) was statistically insignificant among the groups ( p > 0.05 ) . the mean fbs was 94.9 15.4 mg / dl , 145.8 52.4 mg / dl and 108 38.7 mg / dl for control , chpdm , and chp group respectively and there was significant difference for chpdm group as compared to control ( p = 0.001 ) and chp group ( p = 0.01 ) . the mean serum calcium level did not achieve statistical significance between chpdm and chp groups [ table 2 ] . oral hygiene habits did not differ among the groups when evaluated for their oral hygiene methods ( finger / brush ) , frequency ( once / twice / after every meal ) , and materials ( tooth paste / tooth powder / others ) , whereas poor oral hygiene predominated in the chp group . all periodontal clinical parameters were significantly higher in case groups as compared to healthy controls . post - hoc and bonferroni correction for mean pi and cal scores , showed no statistically significant difference between chpdm and chp groups , ( p > 0.05 ) , whereas the comparison of means of mgi , ci , and pd between the pairs of groups showed high significance [ table 3 ] . comparison of mean periodontal parameters mgi , pi , ci , pd , cal and mean serum 25(oh ) d level between control , chpdm and chp group the mean serum 25(oh ) d level was found to be 22.3 5.8 ng / ml , 14.1 4.6 ng / ml and 16.9 5.6 ng / ml for control , chpdm , and chp group respectively . the difference was statistically significant between chp and chpdm groups ( p = 0.001 ) , chpdm and control groups ( p = 0.001 ) and chp and control groups ( p = 0.001 ) [ table 3 ] . this showed that a strong association exists between chronic periodontitis with type 2 dm and serum 25(oh ) d levels . in chpdm groups a statistical significant difference was observed in vitamin d level between moderate periodontitis and severe periodontitis subjects , whereas in chp groups a significant association was not attained [ table 4 ] . the distribution of 25(oh ) d levels in different groups ( chpdm , chp and control ) was showed in scatter box plot [ figure 1 ] . the values pi , mgi , ci , pd , and cal for the whole study population ( control + chpdm + chp ) were plotted in scatter plot on x axis and serum 25(oh ) d values were plotted on y axis [ figure 2 ] . it was found that correlation of serum 25(oh ) d with mgi , pi , ci , pd , and cal ( control + chpdm + chp ) was highly significant [ figure 2 ] . linear regression analysis indicated that the serum vitamin d level was influenced by dm and periodontitis even after adjusting for age , smoking habits and outdoor activity [ table 5 ] . serum vitamin d level in cases with moderate and severe chronic periodontitis distribution of mean serum 25-hydroxyvitamin d level in groups ( a ) relation between mean serum 25-hydroxyvitamin d ( 25[oh]d ) level and clinical parameters ( control + chronic periodontitis patients + chronic periodontitis patients with type 2 diabetes mellitus ) serum 25(oh)d level versus modified gingival index , ( b ) serum 25(oh)d level versus plaque index , ( c ) serum 25(oh)d level versus calculus index , ( d ) serum 25(oh)d level versus probing depth , ( e ) serum 25(oh)d level versus clinical attachment loss linear regression analysis of serum 25(oh ) d level and demographic parameters chronic systemic inflammation from periodontal disease stimulates inflammatory cytokines leading to insulin resistance and impaired fasting glucose level . our findings suggested that periodontal disease could have an effect on increased risk for prediabetes . vitamin d stored in fat tissue causes decreased bioavailability and exposes obese patients to greater risk of developing vitamin d insufficiency . however , in the present study , bmi of the patients in case groups and control group did not differ significantly . in this study , even though the probing pocket depth and calculus index was significantly higher in chp group as compared to chpdm group , the cal was not statistically significant between them . this showed that the rate of tissue destruction was more in chpdm group and diabetes could have an effect on cal . factors other than calculus and pathogenic microorganism may influence the actual presentation of chronic periodontitis in diabetes subjects . this is in accordance with the previous studies showing that diabetes is a risk factor for periodontitis . one of the important observations found in this study : the mean serum vitamin d level was less than the normal range in all groups including control subjects . the insufficient vitamin d level ( 22.3 5.8 . ng / ml ) in control subject may be due to inadequate intake of vitamin d containing food substances coupled with inadequate sunlight exposure . with modernization the number of hours spent on outdoor activity is decreased . harinarayan et al . in 2008 reported low vitamin d status in urban and rural population of andhra pradesh state in south india . yu et al . in 2012 reported that in his study population , the majority of patients with type 2 diabetes were vitamin d deficient . recently , a meta - analysis for observational studies showed a relatively consistent association between low vitamin d status , and type 2 dm . from these studies , it is evident that a relationship exists between insufficient serum vitamin d level and type 2 diabetes . in contrast , hidayat et al . in 2010 suggested that there was no correlation between vitamin d deficiency and type-2 dm . in our study , the prevalence of vitamin d deficiency was high in chp group as compared to control group . dietrich et al . in 2005 evaluated the association between serum 25(oh ) d and gingival inflammation and found a strong negative association . they concluded that this inverse association may be due to the anti - inflammatory effect of vitamin d. boggess et al . in 2011 examined the relationship between maternal vitamin d status and periodontal disease and reported that pregnant women with moderate to severe periodontal disease had lower median 25(oh ) d levels than controls . however , literature also revealed a contrasting report regarding elevated plasma 25(oh ) d level in aggressive periodontitis patients . in our study , a negative correlation with vitamin d level and cal was observed . our result is consistent with the study conducted by dietrich et al . in 2004 who reported an inverse association between serum 25(oh ) d and periodontal attachment loss . its anti - inflammatory effect is mediated through inhibition of cytokine production , stimulation of monocytes and macrophages , and secretion of peptides with potent antibiotic activity . recently , it has been suggested that its effect on periodontal disease , tooth loss , and gingival inflammation is through its anti - inflammatory effect . the low vitamin d level observed in chp group in our study could have an effect on periodontal disease process and progression . the serum vitamin d level in chpdm group was at a lower level than that of patients with chronic periodontitis in this study . the inflammatory status as well as vitamin d insufficiency may create an environment conducive for the progression of diabetes and periodontitis in this group . studies have suggested that patients with chronic inflammatory diseases are deficient in 25(oh ) d. the vitamin d nuclear receptors ( vdr ) located in the nucleus of a variety of cells including immune cells . the body controls the activity of vdr through recognition of vitamin d metabolite 25(oh ) d , which antagonize or inactivate the receptor , while 1 , 25 dihydroxyvitamin d agonize or activates the receptor . when exposed to infection , the body begins to convert the inactive form of 25-d into the active form , 1 , 25-d . as cellular concentrations of 1 , 25-d increase , 1 , 25-d activates the vdr . however , bacteria create ligands , which like 25-d , inactivates the vdr and in turn , the innate immune response . an intraphagocytic bacterial microbiota is proposed to be the primary cause of vdr dysfunction which allows the microbes to proliferate . in response , the body increases the production of 1 , 25-d from 25-d , leading to low 25-d and a high 1 , 25-d , one of the hallmarks of chronic inflammatory disease . a low level of serum 25-d is seen as the result of down regulation , rather than a causal factor leading to illness . the present study reveals , it is possible that the low serum vitamin d level in the case groups ( chp and chpdm ) may be due to the diseases process rather than low vitamin d acting as a cause . this study observed a low level of serum vitamin d level in patients with chronic periodontitis and chronic periodontitis with type 2 diabetes . low serum vitamin d level in chp and chpdm group in our study may be due to the disease process rather than low vitamin d acting as a cause . so the underlying microbial disease process and diabetes could have a synergistic effect in lowering serum 25(oh ) d levels in chpdm patients . in this article , we are trying to put forward a new hypothesis , regarding the possibility of association between periodontal inflammation and low serum vitamin d level as future directions . multicenter study with large sample size is needed to study the serum vitamin d status in chronic periodontitis and diabetes patients .

aim : vitamin d is associated with inflammatory diseases such as periodontal disease and diabetes mellitus ( dm ) . the aim of our study was to find out the level of serum vitamin d in chronic periodontitis patients ( chp ) with and without type 2 dm.materials and methods : this study consists of 141 subjects , including 48 controls . case groups consisted of 43 chronic periodontitis patients with type 2 dm ( chpdm ) and 50 chp . pocket depth ( pd ) , clinical attachment loss ( cal ) , modified gingival index ( mgi ) , plaque index ( pi ) , and calculus index ( ci ) were taken . serum 25-hydroxyvitamin d ( 25[oh ] d ) level in g / ml was estimated by electrochemiluminescence immunoassay with elecsys and cobase e immunoassay analysers(cobase e 411 ) . other laboratory investigations including fasting blood sugar ( fbs ) and serum calcium were measured in all subjects.results:the mean serum 25(oh ) d level was 22.32 5.76 g / ml , 14.06 4.57 g / ml and 16.94 5.58 g / ml for control , chpdm and chp groups respectively . the difference was statistically significant ( p < 0.05 ) . the mean value of fbs was significantly high in chpdm group as compared to chp group . periodontal parameters like mgi , pi , pd , and ci showed significant difference between groups ( p < 0.05 ) and higher score was found in chp group , while cal and pi showed no statistically significant difference between chp and chpdm group ( p > 0.05).conclusions : this study observed a low level of serum vitamin d level in patients with chp and chpdm . low vitamin d level was observed in case groups may be due to the diseases process rather than low vitamin d acting as a cause for the disease .

the online version of this article ( doi:10.1007/s00267 - 011 - 9751-z ) contains supplementary material , which is available to authorized users . habitat fragmentation by transport networks and consequential secondary development has become one of the most serious global threats to biological diversity ( eea 2002 ; iuell and others 2003 ; laurance and others 2009 ; bentez - lpez and others 2010 ) . with more than 100 million km of roads worldwide ( cia 2008 ) , the road network plays a main role in shaping the environment . road effects have been widely studied and include biodiversity decline , environmental degradation , alteration of ecological processes and ecosystem services , and increases in both extinction and outbreak probabilities ( forman and alexander 1998 ; trombulak and frissell 2000 ; forman and others 2003 ; fahrig and rytwinski 2009 ) . by acting as a barrier , roads isolate populations , reduce the overall landscape connectivity and restrict ( or even block off ) gene flow for a wide variety of taxa ( keller and others 2004 ; epps and others 2005 ; riley and others 2006 ; balkenhol and waits 2009 ) . in a long term , this loss of genetic diversity and connectivity increases the extinction risk of populations and , reduces their ability to adapt to future global changes . in general , these effects are synergistic , extremely complex , cumulative and time - lagged ( forman and alexander 1998 ; findlay and bourdages 2000 ; mcgarigal and others 2001 ; forman and others 2003 ) . the spatial influence of these effects ( road - effect zone ) ranges from a few meters to kilometres ( e.g. , forman and deblinger 2000 ) . when taking into account this road area - of - influence , about one fifth of the land in usa and the netherlands is ecologically affected by the road system ( reijnen and others 1995 ; forman 2000 ) . estimates indicate that only 18% of the usa lands are more than 1 km away from the closest road ( riitters and wickham 2003 ) . not only major roads , but also minor and unpaved roads may have a considerable impact on the environment ( e.g. , van langevelde and others 2009 ) natural areas are being increasingly fragmented by a rapidly expanding transport network , together with urban sprawl ( eea 2006 , 2010 ) . in the case of usa forests , estimations yield about 11% of all forest located within 85 m of a road ( riitters and wickham 2003 ) . the secondary impacts of roads account for an even more serious threat to biodiversity and ecosystem resilience . land use and transport networks are interdependent in complex ways ( wilkie and others 2000 ; eea 2006 ; mller and others 2010 ) . roads facilitate human access into formerly remote areas , accelerating land use change , habitat degradation and biodiversity loss , due to an increase in hunting , poaching , fishing , tourism , logging , mining , fires and urbanization ( trombulak and frissell 2000 ; wilkie and others 2000 ; hawbaker and others 2006 ) . urban development and sprawl are strongly linked to transport networks , also in rural and natural environments ( wilkie and others 2000 ; eea 2006 ; mller and others 2010 ) . thus , the density of transport infrastructures is a good indicator of the intensity of human activities and their impacts on biological diversity , and can be taken as a proxy for general disturbance levels ( e.g. , wilkie and others 2000 ; sanderson and others 2002 ; hawbaker and others 2006 ; theobald 2008 ; laurance and others 2009 ) . anthropogenic disturbances , in general , cause stress and reduce resilience and adaptive capacity of populations and species . this is of special concern in the context of climate change , which is increasing local extinction rates and forcing latitudinal and elevational shifts in species ranges ( walther and others 2002 ; parmesan 2006 ) . europe , as the cradle of industrialization and vehicular transport , is probably the continent most highly fragmented by transport infrastructures . especially the eu-12 countries ( austria , belgium , denmark , finland , france , germany , ireland , italy , luxembourg , netherlands , sweden , united kingdom ) , historically highly developed and crowded , constitute the epicenter of habitat conversion and fragmentation ( pullin and others 2009 ) . it would be informative , for example , to differentiate between urban and rural roads , as well as among road types ( motorways , highways , minor roads ) . however , such specific data are available only regionally or even locally . taking into account these constraints ( tent - t , a subset of the continent s overall road network targeted to support the economical integration of the eu ) in 2005 was 98,500 km , of which 78% corresponded to roads in eu-15 countries ( eu-12 plus greece , portugal , spain , data from tina 2008 ) . in a 10-year period ( 19952005 ) the length of motorways has increased by more than 13,000 km in the eu countries ( steer davies gleave 2009 ) . while eu plans imply slight increases of its total length , planned upgradings would increase the proportion of motorways in the network from 49% ( 2005 ) to 63% ( 2020 ) . the land dedicated to transport networks continues to increase in the whole eu . in the period 19901998 approximately 10 ha of land were taken for new motorway construction every day in the eu-15 ( eea 2002 ) . for example , in the netherlands , on average 1 km of land is crossed by 3 km of asphalt road ( eea 2002 ) ; and the average size of polygons enclosed by the network of all roads is 1.14 km ( van langevelde and others 2009 ) . in belgium , transport infrastructure already occupies more than 4% of the country the environmental consequences of this process may be different in the already highly fragmented eu-15 and in the new member states ( accessions from 2004 ; bulgaria , cyprus , czech republic , estonia , hungary , latvia , lithuania , malta , poland , romania , slovakia , slovenia , eu-27 ) with a lower density of roads and generally harbouring relatively well - preserved biodiversity and ecosystems , but where transport infrastructures are developing quickly after their accession . fragmentation by transport infrastructures in hungary and in the czech and slovak republics are already more severe than the eu-15 average . in the 1990s , total motorway length doubled in the new members ( 2300 km built ) , while in the eu-15 it increased by almost one third ( 12000 km built , eea 2002 ) . major transport infrastructures in the new members ( 174 km , eea 2002 , data from 1998 ) is still above the average of the eu-15 ( 121 km ) . however , differences between individual eu member states are more pronounced ; finland , and scandinavian countries in general , being the least fragmented in the continent , followed by countries in the carpathian region . mountainous countries , like italy or austria , still maintain relatively large unfragmented patches ( eea 2002 ) . thus , it seems reasonable to hypothesize that the few areas of low or no road fragmentation still existing in europe might be an important focus of future conservation efforts . in this paper , the term roadless areas refers to relatively large areas without any roads , whereas areas containing only roads with low - traffic intensity , below an established limit , are termed low - traffic areas ( see definition below ) . our main goal is to bring attention to the conservation value of roadless and low - traffic areas in europe , specifically in the eu . we aim at ( 1 ) describing the benefits of roadless and low - traffic areas for biodiversity conservation ; ( 2 ) exploring how these areas are valued and considered in european legislation ; ( 3 ) assessing how low - traffic areas are protected as natura 2000 sites in germany , as a case study , comparing the western and eastern parts of the country ; and , ( 4 ) discussing the potential role of roadless and low - traffic areas to strengthen the effectiveness of the natura 2000 network , especially in the context of climate change and the unaccomplished goal of halting biodiversity loss by 2010 ( eu 2001 ; ec 2010 ) , as well as their integration into legal instruments and transport policies . roadless areas and , to a lesser extent , low - traffic areas , represent relatively undisturbed natural habitats and functioning ecosystems . as at low traffic intensity , road impacts , such as the barrier effect for fauna , wildlife disturbance or pollution , are dampened ( iuell and others 2003 ; jaeger and others 2006 ; theobald 2008 ; charry and jones 2009 ) , it is sensible to assume that low - traffic areas may also represent sites of high conservation value , especially in europe , a human - dominated landscape . under this scenario , the ecological benefits described for roadless areas , which have been the focus of most research , may well apply to low - traffic areas as well . scientific evidence shows that roadless areas are critical in maintaining biodiversity , ecosystem processes , connectivity and overall ecosystem integrity . large , well - connected patches increase landscape connectivity and complement the network of protected areas ( e.g. , develice and martin 2001 ; loucks and others 2003 ; crist and others 2005 ) . thus , roadless areas sustain important elements of ecosystem integrity , such as the ability of species to move and natural processes to function . they largely contribute to the preservation of native biodiversity and contain more species and individuals , species with large spatial requirements ( e.g. , top carnivores ) , and species sensitive to human disturbance ( haskell 2000 ; watkins and others 2003 ; angelstam and others 2004 ; blake and others 2008 ; chen and roberts 2008 ) . they have the potential to ensure sufficient habitat for viable populations of species of conservation concern , as well as to increase the representation of rare ecological communities ( strittholt and dellasala 2001 ; loucks and others 2003 ; crist and others 2005 ) . they serve as a barrier against pests , diseases ( of wildlife , livestock and humans , e.g. , the lyme disease ) and invasive species ( strittholt and dellasala 2001 ; allan and others 2003 ; gelbard and harrison 2003 ; holdsworth and others 2007 ; von der lippe and kowarik 2007 ) . it is in these large areas of unfragmented land that ecosystem services , vital for human societies , are rendered ( millennium ecosystem assessment 2005 ) . roadless and low - traffic areas are of special importance in the context of climate change because they are more resilient than areas more fragmented by roads , and because they have a vast buffering capacity ( mcgarigal and others 2001 ) . their ecosystem dynamics are still internally driven , as opposite to the dynamics of fragmented patches , which are predominantly driven by external forces ( saunders and others 1991 ) . ecosystems already fragmented and stressed by human activities will be more vulnerable to climatic threats , while large intact areas better resist and recover from climate change impacts ( markham 1996 ; laurance and williamson 2001 ; noss 2001 ; opdam and wascher 2004 ; ferguson and others 2008 ) . these relatively undisturbed habitat patches can facilitate movements of organisms in the case of climate - forced range shifting . in general , they may represent havens for many species that are displaced from former habitats ( noss 2001 ; lovejoy 2006 ) . by slowing the rates of changes , moderating local climate , and being more diverse and resilient , roadless and low - traffic areas may contribute to mitigate the effects of species phenology changes and trophic mismatches caused by climate change ( parmesan 2006 ) , and to facilitate species adaptation . especially when comprising forest ecosystems or peatlands , roadless and low - traffic areas can play an important role in carbon fixation . undisturbed habitats may be better players in carbon sequestration than their fragmented and degraded counterparts ( harmon and others 1990 ; laurance and williamson 2001 ; ferguson and others 2008 ; luyssaert and others 2008 ) . it should be a relevant hypothesis to test that relatively carbon - rich ecosystem types in roadless and low - traffic areas store more carbon for maintaining a less stressed and thus more functional status . in the case of forests , in areas with poor access ( e.g. , in remote mountain areas ) roadless and low - traffic areas also provide protection against the impacts of storm events , like flooding or landslides , and wildfires ( lower fire risk and higher resilience ; usda forest service 2000 , 2001 ; dellasala and frost 2001 ; laurance and williamson 2001 ; ferguson and others 2008 ) . these relatively intact areas contribute to floodplain protection and drought abatement , as well as to maintain local climates stables and buffering weather extremes . the social and economic benefits of roadless and low - traffic areas , such as recreation , have also been well documented ( noss 1991 ; loomis and richardson 2000 ; krieger 2001 ) . roadless and low - traffic areas , as lands with a relatively low human footprint and good conservation status , have been considered a priority in regional conservation planning in several countries outside europe . for example , in bolivia , various conservation planning exercises on regional and national scales followed a functional approach and integrated roadless areas as surrogates for functional conservation targets , such as ecosystem processes and emergent features of biodiversity , especially required in the face of environmental change ( viability , resilience and adaptive capacity ; ibisch and others 2005 ) . however , the most ground - breaking initiative and important precedent has been the u.s . roadless area conservation rule of 2001 . it stated that 237,000 km within the u.s . national forest system ( 2% of us continental land ) the two inventories of roadless areas , rare i and rare ii , included unfragmented patches larger than 2024 ha ( an important part of which was designated as wilderness ) and 405 ha , respectively ( see usda forest service 2001 ; strittholt and dellasala 2001 ; turner 2006 , 2009 for details ) . in europe , initiatives specifically restricting road development in natural areas and giving special attention to the protection of roadless and low - traffic areas have hardly been launched . the centerpiece of the eu nature conservation policy is the natura 2000 network , which consists of special protection areas and special areas of conservation delineated according to the provisions of the birds and habitats directives , respectively ( 79/409/eec and 92/43/eec , see pullin and others 2009 ) . although both european directives oblige the member states to take the appropriate measures to maintain the integrity of natura 2000 sites and to guarantee the long - term persistence of species and ecosystems , in practice they face enormous difficulties in avoiding habitat fragmentation . very illustrative are recent conflicts like the planned construction of the via baltica express - way through the biebrza marshes , the unique rospuda mire and two large natural forests in northeastern poland ( eea 2006 ) . moreover , a high proportion of natura 2000 sites is already in close proximity to major transport infrastructures and/or will be potentially affected by the future development of the european transport network ( fisher and waliczky 2001 ; eea 2002 ) . we conducted an exploration of european legal instruments , ranging from selected national laws to eu nature conservation directives and european conventions ( table 1 , see supplementary material ( appendix ) for detailed description of the laws ) . only one of the explored laws considered roadless or low - traffic areas as a conservation target , although , paradoxically , the majority of them aimed at protecting other targets which are inherently and intimately related to fragmentation like connectivity , ecosystem processes or integrity . one important finding is an apparent conceptual shift from mere species and habitat protection ( e.g. , bern convention 1979 ) to more holistic approaches of ecosystem conservation , including processes , functions and aspects of integrity . we especially consider the carpathian convention ( 2003 ) that explicitly addresses regulations of traffic impacts and development , and encourages the parties to develop sustainable transport policies . traffic and energy infrastructure and similar projects shall be integrated so that fragmentation and consumption of the landscape as well as ecological impairment is avoided or reduced to a minimum ( federal nature conservation act from 29 july 2009 ; table 1 ) . in spite of the apparent trend that european nature legislation is starting to consider minimising fragmentation by transport infrastructures , it is unfortunate that neither the eu nor the large majority of national laws recognise the significance of areas with low levels of fragmentation by roads in their conservation policies.table 1conservation targets and entities explicitly considered in selected european legal instruments , including conventions , european directives and national lawscountry or regionroadless and low - traffic areasecosystem servicesnatural resourcesecosystem integrityecosystem functioningecological processesconnectivitybiodiversitylandscapenatural ecosystemshabitatsnative species ( flora&fauna)natural heritagebern convention regionxx alpine convention region xxxxxxxxxxx carpathian convention region xxxxxxxxxxxxeuropean unionxxx xxxxxxxgermanyxxx 1 and supplementary material ( appendix ) for details of the countries included in each region and the respective laws . symbol x indicates inclusion of targets and symbol indicates exclusion ; ( ) except scotland , whose law includes those conservation targets conservation targets and entities explicitly considered in selected european legal instruments , including conventions , european directives and national laws see fig . 1 and supplementary material ( appendix ) for details of the countries included in each region and the respective laws . x indicates inclusion of targets and symbol indicates exclusion ; ( ) except scotland , whose law includes those conservation targets germany is one of the largest european countries , located in the center of the continent , including a wide variety of biogeographical regions . after world war ii , germany was divided into the federal republic of germany ( western germany ) and the german democratic republic ( eastern germany ) ; in 1990 they were unified again . differences in natural conditions , history , political regimes , social models and economic development between eastern and western germany illustrate well the differences between the so - called western and eastern europe , once separated by the iron curtain . germany lies in the transition zone between these two parts of europe ( fig . it reflects somehow different situations between the highly developed and industrialized countries in eu-15 and the new member states , more rural and less fragmented by transport infrastructures . these two german regions also show strong differences in population density ; the old federal states ( former federal republic of germany ) have almost twice the population density of the new federal states ( former german democratic republic).fig . 1map of europe indicating the members of the european union ( with a distinction of the new member states that have acceded since 2004 ) and the contracting parties under the alpine and carpathian convention map of europe indicating the members of the european union ( with a distinction of the new member states that have acceded since 2004 ) and the contracting parties under the alpine and carpathian convention germany is the first european country where data on the distribution and size of low - traffic areas have become available . the german federal agency for nature conservation conducted a first inventory of large areas not cut by major transport infrastructures ( bfn 2008 ) , whose data were used for the present study ( federal agency for nature conservation , technical data on natura 2000 from 2008 , unpublished ) . these low - traffic areas were defined as larger than 100 km and not dissected by roads with more than 1000 vehicles per day , by railway lines ( twin - track and single - track electrified lines ) or by human settlements , airports or channels ( with the status of a category iv federal waterway or above ) . these criteria are generally applied in europe ( e.g. , andel and others 2005 ; jaeger and others 2007 ; bfn 2008 ) . although further research to identify threshold values for traffic volumes are needed , the value of 1000 vehicles per day seems to be an acceptable synthesis of the current ecological evidence and models and may serve as a preliminary basis for further discussions . below this threshold many , though certainly not all , populations of conservation - relevant species dissected by a road are thought to remain viable by several authors ( e.g. , hels and buchwald 2001 ; iuell and others 2003 ; seiler 2003 , 2005 ; jaeger and others 2006 ; charry and jones 2009 ) . we superimposed the gis shape files of these low - traffic areas with those of natura 2000 sites in germany ( merging special areas of conservation and special protection areas , federal agency for nature conservation , technical data on moderately fragmented areas with low traffic intensity from 2006 , unpublished ) to analyze their spatial relationship . particularly , our aim was to determine to what extent low - traffic areas are protected within the natura 2000 network and to what extent natura 2000 sites lie outside these areas . as the ecological coherence of the natura 2000 network is one of the main goals of the habitats directive , we were also interested in determining the degree of fragmentation of natura 2000 sites . for this purpose , we counted the number of spatially isolated subareas each natura 2000 site is composed of and analyzed the class size distribution of subareas for special areas of conservation and special protection areas separately . although the natura 2000 network covers an important proportion of the country ( 16% ; table 2 ; fig . 2 ) , most low - traffic areas ( 75% ) lie outside the network and thus remain without protection . this proportion is higher for the old federal states ( western germany ) , where only one fifth of low - traffic patches are protected . the new federal states are much less fragmented , and about 45% of its surface consists of low - traffic areas , as against 18% in the old federal states . more than half of the natura 2000 sites in germany lie outside low - traffic areas , especially in the old federal states ( 72% of the sites ; table 2 ) . natura 2000 sites are highly fragmented , and often they consist of several subareas , many of which have a very small size . almost 64% of these subareas classified as special areas of conservation are less than 50 ha ( fig . 3 ) . in the case of the special protection areas these figures are better ( 27% ) . in general , special areas of conservation comprise smaller subareas ; only 4% of them cover more than 1000 ha , against 26% in the case of special protection areas ( fig . 3).table 2representation of low - traffic areas and natura 2000 sites in germany , with a distinction between the old and the new federal statesold federal statesnew federal stateswhole germanysurface248,884109,025357,909low - traffic areas45,161 ( 18%)48,843 ( 45%)94,004 ( 26%)natura 2000 sites33,662 ( 14%)23,753 ( 22%)57,415 ( 16%)low - traffic areas not covered by natura 2000 sites35,677 ( 79%)34,398 ( 70%)70,075 ( 75%)natura 2000 sites lying outside low - traffic areas24,178 ( 72%)9,308 ( 39%)33,486 ( 58%)the surface ( km ) and percentage of low - traffic areas , natura 2000 sites and their overlap are also indicated . low - traffic areas are defined as larger than 100 km and crossed only by small roads with less than 1000 vehicles per day ( data from 2007 , bfn 2008)fig . 3size class distribution of the subareas forming natura 2000 sites in germany : special protection areas ( birds directive ) and special areas of conservation ( habitats directive ) representation of low - traffic areas and natura 2000 sites in germany , with a distinction between the old and the new federal states the surface ( km ) and percentage of low - traffic areas , natura 2000 sites and their overlap are also indicated . low - traffic areas are defined as larger than 100 km and crossed only by small roads with less than 1000 vehicles per day ( data from 2007 , bfn 2008 ) natura 2000 network and low - traffic areas coverage in germany size class distribution of the subareas forming natura 2000 sites in germany : special protection areas ( birds directive ) and special areas of conservation ( habitats directive ) biodiversity continues to decline in europe in spite of considerable conservation efforts carried out by administrations as well as non - governmental organisations ( ec 2010 ) . the unaccomplished target of halting biodiversity loss by 2010 ( eu 2001 ; ec 2010 ) calls for additional conservation measures to be put into practice as soon . fruit of this concern are recent initiatives of the european commission on defining targets beyond 2010 , developing green infrastructures or scaling up efforts to protect wilderness ( ep 2008 , 2009 ; ec 2011a , b ) . the need to strengthen the natura 2000 network and adapt it to the rising challenges of climate change has also been highlighted . in this context , ecosystem resilience and landscape connectivity are key goals that should be reinforced ( ep 2010a , b ) . the macis report ( berry and others 2008 ) states that to prevent and minimise future impacts of climate change on biological diversity in the eu , the minimization of fragmentation and the creation of connectors between protected areas is of extreme importance . therefore , maintaining unfragmented large patches of natural habitats , i.e. , roadless and low - traffic areas , seems a prudent strategy under any climate change scenario . the capacity of an ecosystem to preserve its integrity and biodiversity increases in large habitats that are well connected and which are far from the influence of external disturbances ( e.g. , fahrig and merriam 1985 ) . we feel that there is enough evidence of the benefits of roadless and low - traffic areas for nature conservation , especially in the context of global change . they represent sites with low human footprint , a high level of ecological integrity , and thus , intact ecosystem functioning and ongoing ecological processes , which translates into higher resilience and adaptive capacity . even in densely populated and intensively used landscapes , like germany , the remaining roadless and low - traffic areas may be of enormous value in supporting resilience and adaptive capacity of the biodiversity . in many areas of europe , it is probably too late to conserve roadless areas sensu stricto to a substantial extent . in this context , the last remnants are of special value , much more if they are primeval or close to a natural state . although the importance of keeping unfragmented large patches of natural habitats is increasingly recognised ( e.g. , jaeger and others 2006 ; charry and jones 2009 ; bentez - lpez and others 2010 ) , more research on the ecological benefits of both roadless and low - traffic areas in europe is deemed necessary . such research efforts should be accompanied by improving the quality and availability of road data ( e.g. , across road types unpaved roads included or differentiating between urban and rural roads ) and securing data coverage for the entire continent . conservation policies should focus not only on tangible elements , like species or habitats , but also include surrogates ( such as roadless areas ) of more abstract conservation targets , like ecosystem functioning , ecological processes or ecosystem services . the identification and inventory of the roadless and low - traffic areas in europe a detailed inventory of these areas , based on sound and concrete criteria , and more detailed estimations of the level of fragmentation by roads in europe shall form the basis for a proper assessment of the magnitude of the problem . given the considerable impacts of minor and unpaved roads in natural ecosystems ( e.g. , reed and others 1996 ; riitters and wickham 2003 ; van langevelde and others 2009 ) , fragmentation and impact assessments should also consider them . as an example , in sweden , classically considered as one of the least fragmented countries in europe ( 400 km is the average size of non - fragmented land according to eea 2002 ) , the figures may look quite different when forest roads are taken into account . during the last decade , the rate of road construction in swedish forests has been approximately 1700 km per year , as a long - term goal of the forestry sector is to have no more than 500 m or 1000 m to the nearest road in southern and northern sweden , respectively ( swedish forest agency 2008 ) . data on the habitat types covered by roadless and low - traffic areas and their overlap with the natura 2000 network should be compulsory in europe . stronger scientific evidence on traffic and area thresholds following an ecosystem approach is needed ; in general , road research needs to effectively address questions of direct management relevance and design studies that have high inferential strength , e.g. evaluating impacts before and after road construction ( gontier and others 2006 ; roedenbeck and others 2007 ) . as a second step , inventoried roadless and low - traffic areas should be considered in european legal instruments in a more explicit way . fortunately , european legislation has just started to take them into account . in this sense , the recent carpathian convention and the german federal conservation act are among the most progressive legal instruments in europe ( see table 1 ) . the urgent need to protect large , undisturbed habitat patches in europe , the european parliament adopted the report on wilderness in europe ( ep 2008 , 2009 ) that calls on the commission for better protection of wild areas , as a means for climate protection and maintenance of ecosystem services and biodiversity . in this context , a another possible step would be to include roadless and low - traffic areas in the habitats directive , either within a new annex of functional targets complementing and facilitating the adaptation of the natura 2000 network to climate change , or even as a new category of site . whether a special protection category or designation of these areas is needed or not , large natural areas without roads are protected de facto . mirroring the us legislation , the main goal would be to protect roadless and low - traffic areas from further road development , namely building new roads , increase of traffic volume on existing small roads and use of off - road vehicles . inclusion of roadless and low - traffic areas in the national and eu biodiversity strategies would also be desirable . only after being inventoried and receiving a legal status , roadless and low - traffic areas could be properly integrated into transport and spatial planning . the environmental impact assessment ( eia , 85/337/eec ) and the strategic environmental assessment directives ( sea , 2001/42/eec ) are essential instruments to assess the impacts from transport infrastructures and for the integration of ecological issues into spatial planning ; however the quality of ecological assessments is still limited ( e.g. , inappropriate criteria and methods , local approach , lack of integration between ecological and landscape assessments , gontier and others 2006 ; joumard and nicolas 2010 ) . the consideration of biodiversity in environmental assessments requires a holistic approach , i.e. , to scale - up to the ecosystem level and to link local and global aspects ( gontier and others 2006 ; joumard and nicolas 2010 ) . a concrete step towards the accomplishment of this vision will be to consider roadless and low - traffic areas in environmental assessments . bearing in mind the strong meaning of the term ( e.g. , joumard and nicolas 2010 ) , a sustainable and conservation - sound planning of the european transport network should take into account the still existing roadless and low - traffic areas . especially in the new member states , containing highly valuable areas but where transport networks are developing at a brisk pace , more sustainable transport policies are necessary . research shows that , whenever possible , the design of new routes should avoid dissecting remote and roadless areas ( jaeger and others 2006 ; forman 2007 ; charry and jones 2009 ; bentez - lpez and others 2010 ) . studies on road animal mortality , population persistence and road configuration ( jaeger and others 2006 ; charry and jones 2009 ) support the bundling traffic concept , concluding that ( 1 ) the road network should leave areas as large as possible free from disturbances due to traffic , ( 2 ) traffic should be concentrated on highly travelled roads , and ( 3 ) when traffic can not be combined on one road , it is better to bundle roads close together than to distribute them evenly across the landscape . thus , in natural areas with low level of fragmentation and human footprint ( e.g. , crossed by small roads ) , the general recommendation is to prevent increases in traffic volume ( charry and jones 2009 ) . when avoidance of habitat fragmentation is not possible , mitigation measures and strategies to maintain landscape connectivity , like ecological corridors , should be designed ( iuell and others 2003 ) . so far , scientific evidence of their effectiveness from a genetic point of view is still needed ( corlatti and others 2009 ) . in minor roads , traffic calming is another type of intervention used to mitigate negative impacts by reducing traffic volumes and speeds , and which has been shown to increase the persistence on animal populations in areas with a dense road network ( van langevelde and jaarsma 2009 ) . road closure , especially in remote areas , has also been suggested as a measure to decrease fragmentation ( berry and others 2008 ; charry and jones 2009 ) . integrated solutions are obliged to include urban planning . given the magnitude of urban sprawl in europe , also affecting natural habitats ( eea 2006 ) , an equally important goal should be to impede secondary effects along existing roads in roadless and low - traffic areas . this is of high concern in countries lacking spatial plans in most of their surface ( e.g. , poland ) and where roads are inevitably followed by urban development . the implementation of sustainable development schemes at large spatial scales , linking long and short terms , as well as local and global issues , would prevent the degradation of the integrity of roadless and low - traffic areas and thus , of their contribution to ecosystem and landscape resilience . clearly , in the preservation of roadless areas and sustainable development of low - traffic areas , synergies of nature conservation and other societal goals are manifold . just to name a few examples , traffic calming also benefits health and well - being ( less accidents , noise and pollution ) , and water provision to society generally improves in volume and quality with the functionality of roadless and low - traffic areas ( e.g. , van langevelde and jaarsma 2009 ) . such synergies make roadless areas and low - traffic areas worth considering by a variety of stakeholders beyond the conservation community . with broader societal support , roads have brought benefits to human societies for centuries . in the current situation of road encroachment , biodiversity crisis and global and climate change , roadless and low - traffic areas may far exceed roaded areas in the benefits provided . we call for a pan - european conservation strategy defining relatively unfragmented and low- footprint areas ( roadless and low - traffic areas ) regardless of their biotic characteristics as conservation targets . further scientific evidence on their ecological benefits and further research to answer key questions in road ecology regarding low - traffic and roadless areas under the different conditions in european countries is urgently needed . conservation scientists and administrations should join forces to halt the loss of biodiversity in europe , to keep healthy and resilient ecosystems and to preserve the services they provide . preserving the below is the link to the electronic supplementary material . supplementary material 1 ( docx 19 kb )

with increasing road encroachment , habitat fragmentation by transport infrastructures has been a serious threat for european biodiversity . areas with no roads or little traffic ( roadless and low - traffic areas ) represent relatively undisturbed natural habitats and functioning ecosystems . they provide many benefits for biodiversity and human societies ( e.g. , landscape connectivity , barrier against pests and invasions , ecosystem services ) . roadless and low - traffic areas , with a lower level of anthropogenic disturbances , are of special relevance in europe because of their rarity and , in the context of climate change , because of their contribution to higher resilience and buffering capacity within landscape ecosystems . an analysis of european legal instruments illustrates that , although most laws aimed at protecting targets which are inherent to fragmentation , like connectivity , ecosystem processes or integrity , roadless areas are widely neglected as a legal target . a case study in germany underlines this finding . although the natura 2000 network covers a significant proportion of the country ( 16% ) , natura 2000 sites are highly fragmented and most low - traffic areas ( 75% ) lie unprotected outside this network . this proportion is even higher for the old federal states ( western germany ) , where only 20% of the low - traffic areas are protected . we propose that the few remaining roadless and low - traffic areas in europe should be an important focus of conservation efforts ; they should be urgently inventoried , included more explicitly in the law and accounted for in transport and urban planning . considering them as complementary conservation targets would represent a concrete step towards the strengthening and adaptation of the natura 2000 network to climate change.electronic supplementary materialthe online version of this article ( doi:10.1007/s00267 - 011 - 9751-z ) contains supplementary material , which is available to authorized users .

from january to may 2004 , a total of 600 salmonella isolates from 585 patients were obtained from 5 medical centers and 14 district hospitals throughout taiwan ; these isolates were serotyped with commercial antisera ( difco , detroit , mi , usa ) . the 4 most common serotypes of salmonella enterica ( enteritidis , typhimurium , stanley , and choleraesuis ) accounted for 66.8% of all isolates . two isolates were untypeable , and the remainder were typed into 42 serotypes ( data not shown ) , which were each represented by 1 to 23 isolates . resistance to ciprofloxacin ( mic 4 g / ml ) was seen in 50 ( 8.3% ) isolates ( table 1 ) ; 20 ( 3.3% ) were resistant ( mics ranging from 8 to > 64 g / ml ) to ceftazidime , ceftriaxone , cefotaxime , or aztreonam ( table 2 ) ; 6 isolates showed decreased susceptibilities to 1 or 2 of the 4 escs ( mics 0.52 g / ml ) ; 10 ( 1.7% ) isolates were resistant to both ciprofloxacin and escs . s. choleraesuis had high rates of resistance to ciprofloxacin ( 84.4% ) , escs ( 17.8% ) , and both ( 17.8% ) . none of the 26 salmonella isolates with resistance or decreased susceptibility to escs produced esbl , according to the double - disk synergy method ( 10 ) . among the 20 esc - resistant isolates , 10 isolates were ciprofloxacin - resistant , 4 isolates showed decreased susceptibility to ciprofloxacin ( mic 0.251 g / ml ) and resistance to nalidixic acid , and 6 isolates were susceptible to ciprofloxacin and nalidixic acid ( table 2 ) . all 20 esc - resistant isolates were susceptible to cefepime ( mic < 0.03 g / ml ) and imipenem ( mic < 1 g / ml ) , and 17 isolates were resistant to > 1 non-lactam agent . * am , ampicillin ; esc , extended - spectrum cephalosporins ; fx , cefoxitin ; cm , chloramphenicol , cp , ciprofloxacin ; na , nalidixic acid ; gm , gentamicin ; km , kanamycin ; sxt , trimethoprim - sulfamethoxazole ; tc , tetracycline . the s. albany isolate and the 3 s. cairo isolates showed decreased susceptibilities to ciprofloxacin ( mic 0.251 g / ml ) . for each isolate , the first letter indicates region ( c , central region ; n , northern region ; s , southern region ) , and the second letter represents hospital . isolates nc04.001 , nc04.002 , and nc04.003 were from the same patients ; all other isolates were from different patients . all 20 esc - resistant isolates expressed a -lactamase of pi 9.0 by isoelectric focusing ( 3,11 ) ; 11 of these isolates expressed an additional pi 5.4 -lactamase ( table 2 ) . blatem-1 was detected in the 11 isolates with the pi 5.4 -lactamase by polymerase chain reaction ( pcr ) and sequence analyses with the primers for the entire blatem - related and blacmy-2-related structural genes ( 2,3 ) . the qrdr sequences of gyra , gyrb , parc , and pare of the 20 esc - resistant salmonella isolates were determined by pcr and sequence analyses ( 5 ) . all 10 ciprofloxacin - resistant isolates showed 2 mutations at the ser-83 and asp-87 codons in gyra and a single mutation at the ser-80 codon in parc ( table 2 ) . four isolates with decreased susceptibility to ciprofloxacin had a single mutation at either the ser-83 or the asp-87 codon in gyra . all 20 esc - resistant isolates showed no mutations in the qrdrs of gyrb and pare . esc resistance was transferred from 18 of the 20 esc - resistant salmonella isolates to escherichia coli c600 in the liquid mating - out assay ( 3,12 ) . all transconjugants showed decreased susceptibilities to the 4 escs tested ( mics 1664 g / ml ) and cefoxitin ( mic 64128 g / ml ) and were susceptible to all non-lactam agents tested . a pi 9.0 vz -lactamase and blacmy-2 were detected by isoelectric focusing and pcr assays , respectively , in all transconjugants . restricted by the endonuclease ecori , the 18 transferred plasmids produced 9 major restriction patterns ( figure 1 and table 2 ) . the result of the hybridization assay with the blacmy-2 probe labeled with digoxigenin ( roche molecular biochemicals , mannheim , germany ) is shown below the gel , and arrowheads indicate the locations of the restriction fragments that were hybridized . lanes 221 , plasmids from transconjugants of salmonella isolates nb04.022 , sb04.003 , nl04.050 , sa04.028 , cg04.039 , sg04.039 , sg04.042 , se04.006 , sg04.047 , se04.005 , ng04.011 , ng04.016 , ng04.018 , nc04.001 , nc04.002 , nc04.003 , nc04.004 , and ce04.015 ; lanes 1 and 22 , molecular marker ii ( roche molecular biochemicals ) ; lanes 12 and 13 , a 1-kb molecular marker ( promega co. , madison , wi , usa ) . the 38 ciprofloxacin - resistant s. choleraesuis isolates were genotyped by pulsed - field gel electrophoresis on a chef mapper apparatus ( bio - rad laboratories , hercules , ca , usa ) according to the pulsenet protocol ( 13 ) . banding patterns generated by xbai restriction were compared with bionumerics software ( applied maths , kortrijk , belgium ) . the 38 isolates showed a close relationship ( dice correlation coefficient of 90% ) and had only 1 pulsotype , based on tenover criteria ( figure 2 ) ( 14 ) . five esc - resistant isolates displayed the same pulsosubtypes ( ia or ic ) as esc - susceptible isolates ( table 1 and figure 2 ) . dendrogram ( left ) obtained from cluster analysis of xbai - generated macrorestriction patterns of 38 ciprofloxacin - resistant salmonella enterica serotype choleraesuis ( right ) . asterisks indicate extended - spectrum cephalosporin - resistant isolates . a percent scale of similarity is shown above the dendrogram . pulsed - field gel electrophoresis ( pfge ) types are shown between the gel and the dendrogram . h9812 , s. enterica serotype braenderup strain h9812 , which was used as reference size marker . we describe the prevalence of resistance to ciprofloxacin and escs among salmonellae isolated from january to may 2004 in taiwan . we found widespread resistance of salmonella isolates to both escs and ciprofloxacin ; high prevalence of resistance to ciprofloxacin , escs , and both in s. choleraesuis ; and widespread prevalence of cmy-2producing salmonella isolates of various serotypes in taiwan . the prevalence of salmonella isolates resistant to both ceftriaxone and ciprofloxacin may pose a therapeutic problem . cmy-2 is one of the ampc enzymes , which are usually less active against cefepime and cefpirome than esbls ( 15 ) . accordingly , we have used cefepime to successfully treat several patients infected with cmy-2producing and ciprofloxacin - resistant s. choleraesuis ( 8) . therefore , ampc - producing strains should be differentiated from esbl - producing strains by phenotypic or genotypic methods when esc - resistant salmonella strains are isolated in the clinical microbiology laboratory ( 15 ) . the ciprofloxacin - resistant rate in s. choleraesuis in taiwan has been > 60% since 2001 ; the high prevalence was mainly due to clonal spread of resistant strains ( 46 ) . the ciprofloxacin - resistant rate in s. choleraesuis in this report ( 84.4% ) was higher than those reported previously ( 70% ) ( 46 ) . blacmy-2 in salmonella in taiwan was first reported in 2 s. typhimurium strains isolated in 2000 ( 3 ) . the first reported s. choleraesuis strain with blacmy-2 was a ciprofloxacin - resistant strain isolated in 2002 ( 7 ) . all our 38 ciprofloxacin - resistant s. choleraesuis isolates , including 8 esc - resistant isolates , were genetically related . moreover , we found possibly unrelated blacmy-2-positive plasmids ( lanes 3 , 4 , 7 , 8 , 10 , and 11 in figure 1 ) among closely related isolates ( figure 2 ) . these data together suggest that the development and rapidly increasing prevalence of esc and ciprofloxacin resistance in s. choleraesuis in taiwan might result from the extremely high prevalence of ciprofloxacin resistance followed by the horizontal transfer of blacmy-2 into ciprofloxacin - resistant epidemic strains rather than from the spread of a clone that had been resistant to ciprofloxacin and escs . all our ciprofloxacin - resistant salmonella isolates tested had mutations in the qrdrs of gyra and par , a finding consistent with previously reported results ( 1,4,5 ) . the rates of ciprofloxacin resistance in the 3 most common serotypes , enteritidis , typhimurium , and stanley , remained very low ( 0%0.6% ) . six of 11 ciprofloxacin - resistant isolates in the group of uncommon serotypes belonged to serotype schwarzengrund and accounted for 42.9% of all serotype schwarzengrund isolates . thus , the high rate ( 5.5% ) of ciprofloxacin resistance in this group was in part due to the high prevalence of ciprofloxacin resistance in serotype schwarzengrund .

we report the prevalence and characteristics of salmonella strains resistant to ciprofloxacin and extended - spectrum cephalosporins in taiwan from january to may 2004 . all isolates resistant to extended - spectrum cephalosporins carried blacmy-2 , and all ciprofloxacin - resistant salmonella enterica serotype choleraesuis isolates were genetically related .

lung cancer remains the most frequently diagnosed malignancy and the leading cause of cancer - related death worldwide.13 approximately 80%85% of all lung cancers are non - small cell lung cancer ( nsclc ) with ~55% of these presenting with locally advanced or metastatic disease causing the 5-year survival rate to be as low as < 5%.1,3 in recent years , the discovery of the epidermal growth factor receptor tyrosine kinase inhibitor ( egfr - tki ) brings a new hope for patients in the treatment of advanced nsclc . egfr is a known transmembrane receptor , which upon ligand binding , induces receptor dimerization and tyrosine autophosphorylation and leads to cell proliferation.46 mutation in this gene is significantly associated with lung cancer.7 in previous studies , the egfr is detected by immunohistochemistry on 40%80% of nsclc.8,9 accordingly , promising molecular inhibitors targeting the activating mutants of the egfr have been intensely pursued in the treatment of egfr - mutant positive nsclc . in the last decade , two small molecules , selective , orally bioavailable and reversible egfr - tkis gefitinib and erlotinib have been extensively developed in nsclc.3 gefitinib ( figure 1a ) is the first egfr - tki , which has been approved as early as 2002 for the treatment of advanced nsclc patients . in 2003 , gefitinib received accelerated approval by the us food and drug administration ( fda ) as third - line treatment for advanced egfr - mutated nsclc patients . then , in 2015 it was approved as the first - line treatment of patients with metastatic nsclc harboring activating egfr mutations by fda.1012 gefitinib is metabolized by the hepatic cytochrome p450 ( cyp450 ) enzyme cyp2d6 , cyp3a4 , and cyp3a5 . and cyp2d6 is the enzyme primarily responsible for the formation of o - desmethyl gefitinib ( figure 1b).13 previous studies indicate that among gefitinib metabolites , only o - desmethyl gefitinib can be identified in human plasma , whereas the plasma concentrations of other gefitinib metabolites , such as m537194 and m387783 , which are metabolized by cyp3a4 are very low or even undetectable.2,1316 there are several studies showing that patients with poor metabolizers ( pms ) of cyp2d6 and inhibition of cyp3a4 may lead to prominent overdose of gefitinib , which may finally cause gefitinib - induced hepatotoxicity.13,16 therefore , we believe that it is necessary to evaluate the effects of 24 cyp2d6 variants on the metabolism of gefitinib in vitro . as a member of cyp450 enzyme superfamily , cyp450 2d6 plays a vital role in the metabolism of ~20%30% of therapeutically important drugs , mainly including antipsychotic drugs , antiarrhythmic agents , tricyclic antidepressants , -blockers , and opioids.17,18 like other cyp450 enzymes , cyp2d6 exhibits significant genetic polymorphisms between individuals . to date , over 100 cyp2d6 alleles have been identified and named by the human cyp allele nomenclature committee ( http://www.cypalleles.ki.se/cyp2d6.htm ) . thus , individuals can be classified into four phenotypes : pms , intermediate metabolizers , extensive metabolizers ( ems ) , or ultrarapid metabolizers . genetic polymorphisms in cyp2d6 have been shown to have clinical consequences , resulting in toxicity and/or altered efficacy of many drugs . thus , the identification of the polymorphic allele , especially defective alleles , in different racial or ethnic populations , is of great importance to understand the differences in the clinical response to drugs.19 previously , 22 new nonsynonymous mutation sites among the 2,129 people of the chinese han population were discovered , 12 of them were defined as the novel alleles ( cyp2d6 * 87*93 , * 94a , * 94b , and * 95*98 ) by the human cyp allele nomenclature committee.20 in our study , we systematically assessed the enzymatic characteristics of these 22 cyp2d6 allelic variants and 2 typical variants ( cyp2d6.2 and cyp2d6.10 ) as well as the wild type cyp2d6.1 toward gefitinib . and we hope that our data can provide valuable information about cyp2d6 genetic polymorphisms and offer a reference to the personalized medication for cyp2d6-metabolized drugs . gefitinib and its metabolite o - desmethyl gefitinib were purchased from shanghai canspec scientific instruments co. , ltd ( perfemiker , shanghai , china ) , the reduced nicotinamide adenine dinucleotide phosphate ( nadph ) and carbamazepine ( internal standard , is ) were obtained from sigma - aldrich company ( st louis , mo , usa ) . p450 cytochrome b5 microsomes and recombinant human cyp2d6 microsomes were kind gifts from beijing hospital ( beijing , china).21 high performance liquid chromatography ( hplc ) grade organic solvents and lc - ms grade acetonitrile were purchased from merck ( darmstadt , germany ) , formic acid of hplc grade ( fa , purity 99.9% ) was purchased from j&k scientific ltd . ( shanghai , china ) , and all other reagents used were of analytical grade . samples were analyzed by ultra - performance liquid chromatography - tandem mass spectrometry ( uplc - ms / ms ) using a waters acquity i - class and a waters xevo tqd triple - quadrupole mass spectrometry ( waters corp . , instrument control and data acquisition were performed by masslynx 4.1 software ( waters corp . ) . recombinant microsomes expressing cyp2d6 variants generated in spodoptera frugioerda 21 ( sf21 ) insect cells were obtained according to the previously reported methods.21 western blotting was performed to determine protein expression levels and the content of microsomes was determined using a bca protein assay kit with bsa as a standard.21 the volume of incubation mixture is 200 l , which consisted of microsomes ( 5 pmol cyp2d6.1 or 5 pmol other cyp2d6 mutants , respectively ) , purified cytochrome b5 , gefitinib ( 1100 m ) and 0.1 m tris - hcl buffer ( ph 7.4 ) . the reaction was allowed to preincubate for 5 min in a fisher shaking water bath . then 400 l acetonitrile and 20 l carbamazepine ( internal standard , 1 g / ml ) were added to the incubation mixture , which was then vortexed for 2 min and centrifuged at 13,000 rpm at 4c for 10 min . the supernatant was 1:1 diluted with water , and 2 l of the mixture was injected into the uplc - ms / ms system for analysis . the chromatographic separation was carried out using an acquity uplc - ms / ms and performed on a waters acquity uplc beh c18 column ( 2.150 mm , 1.7 m , waters corp . ) at 40c . the initial mobile phase consisted of solvent a ( water containing 0.1% formic acid ) and solvent b ( acetonitrile ) with gradient elution at a flow rate of 0.4 ml / min , injection volume 2 l . a gradient elution program was as follows : 00.6 min ( 60%10% a ) , 0.61.2 min ( 10% a ) , 1.21.8 min ( 10%60% a ) . the total run time was 2.5 min . under above conditions , o - desmethyl gefitinib and carbamazepine were well separated and their retention times were 0.35 and 0.77 min , respectively . a waters xevo tqd ms was set to positive electrospray ionization in multiple reaction monitoring mode , with ionization conditions as follows : desolvation temperature 500c , ionization source temperature 150c , and capillary voltage 2,000 v. nitrogen was served as desolvation gas with flow rate of 1,000 l / h . the multiple reaction monitoring transitions were m / z 447.2128.1 , m / z 433.1128.1 , and m / z 237.1194.2 for gefitinib , o - desmethyl gefitinib , and carbamazepine , respectively . the collision energy for gefitinib , o - desmethyl gefitinib , and carbamazepine was set at 25 , 25 , and 20 v , respectively ; and the cone voltage for each was set at 40 v. prior to the experiments , the uplc - ms / ms method was validated . a seven - point standard curve in the range of 150 g / ml was used to quantify o - desmethyl gefitinib , with a correlation coefficient r>0.99 . the limit of quantification , defined as a signal / noise ratio of 3 , was 0.2 ng / ml for o - desmethyl gefitinib . the precision and accuracy of the method were assessed by determination of quality control samples at three concentrations . the relative standard deviations of intra - day and inter - day precisions were both below 10% ; the accuracy was from 87.77% to 93.73% . menten curves ( substrate vs velocity ) and enzyme kinetic parameters were obtained by using graphpad prism 5 ( graphpad software inc . , sandiego , ca , usa ) . one - way analysis of variance ( anova ) was used for intergroup comparison , and various variants were considered as factor whereas maximum initial velocity ( vmax ) , michaelis constant ( km ) or intrinsic clearance ( clint ) values were deemed as dependent list . statistical analysis was carried out with the statistical package for the social sciences ( version 17.0 ; spss inc . , chicago , il , usa ) by one - way anova together with the post hoc dunnet method , with * p<0.05 considered to be statistically significant . gefitinib and its metabolite o - desmethyl gefitinib were purchased from shanghai canspec scientific instruments co. , ltd ( perfemiker , shanghai , china ) , the reduced nicotinamide adenine dinucleotide phosphate ( nadph ) and carbamazepine ( internal standard , is ) were obtained from sigma - aldrich company ( st louis , mo , usa ) . p450 cytochrome b5 microsomes and recombinant human cyp2d6 microsomes were kind gifts from beijing hospital ( beijing , china).21 high performance liquid chromatography ( hplc ) grade organic solvents and lc - ms grade acetonitrile were purchased from merck ( darmstadt , germany ) , formic acid of hplc grade ( fa , purity 99.9% ) was purchased from j&k scientific ltd . ( shanghai , china ) , and all other reagents used were of analytical grade . samples were analyzed by ultra - performance liquid chromatography - tandem mass spectrometry ( uplc - ms / ms ) using a waters acquity i - class and a waters xevo tqd triple - quadrupole mass spectrometry ( waters corp . , instrument control and data acquisition were performed by masslynx 4.1 software ( waters corp . ) . recombinant microsomes expressing cyp2d6 variants generated in spodoptera frugioerda 21 ( sf21 ) insect cells were obtained according to the previously reported methods.21 western blotting was performed to determine protein expression levels and the content of microsomes was determined using a bca protein assay kit with bsa as a standard.21 the volume of incubation mixture is 200 l , which consisted of microsomes ( 5 pmol cyp2d6.1 or 5 pmol other cyp2d6 mutants , respectively ) , purified cytochrome b5 , gefitinib ( 1100 m ) and 0.1 m tris - hcl buffer ( ph 7.4 ) . the reaction was allowed to preincubate for 5 min in a fisher shaking water bath . l carbamazepine ( internal standard , 1 g / ml ) were added to the incubation mixture , which was then vortexed for 2 min and centrifuged at 13,000 rpm at 4c for 10 min . the supernatant was 1:1 diluted with water , and 2 l of the mixture was injected into the uplc - ms / ms system for analysis . the chromatographic separation was carried out using an acquity uplc - ms / ms and performed on a waters acquity uplc beh c18 column ( 2.150 mm , 1.7 m , waters corp . ) at 40c . the initial mobile phase consisted of solvent a ( water containing 0.1% formic acid ) and solvent b ( acetonitrile ) with gradient elution at a flow rate of 0.4 ml / min , injection volume 2 l . a gradient elution program was as follows : 00.6 min ( 60%10% a ) , 0.61.2 min ( 10% a ) , 1.21.8 min ( 10%60% a ) . the total run time was 2.5 min . under above conditions , o - desmethyl gefitinib and carbamazepine were well separated and their retention times were 0.35 and 0.77 min , respectively . a waters xevo tqd ms was set to positive electrospray ionization in multiple reaction monitoring mode , with ionization conditions as follows : desolvation temperature 500c , ionization source temperature 150c , and capillary voltage 2,000 v. nitrogen was served as desolvation gas with flow rate of 1,000 l / h . the multiple reaction monitoring transitions were m / z 447.2128.1 , m / z 433.1128.1 , and m / z 237.1194.2 for gefitinib , o - desmethyl gefitinib , and carbamazepine , respectively . the collision energy for gefitinib , o - desmethyl gefitinib , and carbamazepine was set at 25 , 25 , and 20 v , respectively ; and the cone voltage for each was set at 40 v. prior to the experiments , the uplc - ms / ms method was validated . a seven - point standard curve in the range of 150 g / ml was used to quantify o - desmethyl gefitinib , with a correlation coefficient r>0.99 . the limit of quantification , defined as a signal / noise ratio of 3 , was 0.2 ng / ml for o - desmethyl gefitinib . the precision and accuracy of the method were assessed by determination of quality control samples at three concentrations . the relative standard deviations of intra - day and inter - day precisions were both below 10% ; the accuracy was from 87.77% to 93.73% . michaelis menten curves ( substrate vs velocity ) and enzyme kinetic parameters were obtained by using graphpad prism 5 ( graphpad software inc . , sandiego , ca , usa ) . one - way analysis of variance ( anova ) was used for intergroup comparison , and various variants were considered as factor whereas maximum initial velocity ( vmax ) , michaelis constant ( km ) or intrinsic clearance ( clint ) values were deemed as dependent list . statistical analysis was carried out with the statistical package for the social sciences ( version 17.0 ; spss inc . , chicago , il , usa ) by one - way anova together with the post hoc dunnet method , with * p<0.05 considered to be statistically significant . in our study , the catalytic activities of the wild - type cyp2d6.1 and 24 allelic variants were detected using gefitinib as substrate and intrinsic clearance ( vmax / km ) was used as evaluation criteria for each variant of cyp2d6 in vitro metabolic activity of gefitinib . the michaelis menten curves and substrate inhibition plots for each of the cyp2d6 variants are shown in figure 2 , and the corresponding kinetic parameters ( vmax , km , ksi , intrinsic clearance , and relative clearance ) are summarized in table 1 . as shown in table 1 , among all these variants , cyp2d6.92 and cyp2d6.96 showed no enzymatic activity as no concentration of o - desmethyl gefitinib was detected . except for cyp2d6.92 and cyp2d6.96 , the remaining 22 variants displayed considerable differences in km , vmax , ksi , or intrinsic clearance values compared with wild - type cyp2d6.1 . three allelic variants ( cyp2d6.10 , cyp2d6.93 , and e215k ) exhibited lower vmax values and eight variants ( cyp2d6.2 , cyp2d6.10 , cyp2d6.98 , r25q , e215k , d336n , r344q , and r440c ) had higher km values than that of cyp2d6.1 ( * p<0.05 ) . according to the intrinsic clearance values when compared with the wild - type , only one variant ( cyp2d6.94 ) had increased catalytic activity to 184.78% , whereas the remaining 13 variants ( cyp2d6.2 , cyp2d6.10 , cyp2d6.87 , cyp2d6.90 , cyp2d6.93 , cyp2d6.95 , cyp2d6.98 , r25q , e215k , v327 m , d336n , r440c , and r497c ) had a significant decrease in enzyme activities compared with cyp2d6.1 ( * p<0.05 ) , for which the percentage varied from 3.11% to 69.42% . especially , the intrinsic clearance values for variants cyp2d6.10 , cyp2d6.93 , and e215k were decreased to 3.11% , 3.68% , and 9.79% compared with cyp2d6.1 , respectively . however , through the analysis of the kinetic parameters , there are eight variants ( cyp2d6.88 , cyp2d6.89 , cyp2d6.91 , cyp2d6.97 , v342 m , r344q , f219s , and f164l ) which showed no marked difference in intrinsic clearance values compared with wild type . in addition , different from the previous studies,19,2124 the metabolic ability toward gefitinib in vitro exhibited substrate inhibition trend , the inhibition constant of 25 tested cyp2d6 variants varied from nearly 5.9 to 158.1 m . cyp2d6 plays a major role in the oxidation of ~20%30% prescription drugs , and it is involved in the metabolism of gefitinib to its active metabolite , o - desmethyl gefitinib . according to the research , the formation of o - desmethyl gefitinib seems to be dependent on cyp2d6 activity . therefore , polymorphisms in the cyp2d6 gene can greatly influence the rate of elimination of gefitinib . individuals who are pms of cyp2d6 have twofold higher plasma concentrations of gefitinib than ems.14 up to now , the enzymatic characteristics of cyp2d6 alleles toward gefitinib have not been well studied both in vivo and in vitro . cyp2d6.2 and cyp2d6.10 tend to undergo the most study of the variants . in order to improve the credibility of our experiment , cyp2d6.2 and cyp2d6.10 , two typical variants , served as the positive controls for the functional analyses . cyp2d6 * 10 is the most common allele in asians , occurring in 42.86% of the chinese han population.20,25 several functional analyses have been shown that cyp2d6.10 has higher km , lower vmax and behave with the least vmax / km values for dextromethorphan , bufuralol , nortriptyline , atomoxetine , and debrisoquine in vitro.21,25 cyp2d6 * 10 is generally considered to be an allele associated with the intermediate metabolizer phenotype . for typical defective allele cyp2d6 * 2 , cai et al found it exhibited lower intrinsic clearance value for dextromethorphan and bufuralol , ~43.85% and 40.41% of wild type , respectively.21 as a consequence , in our study the relative clearance of cyp2d6.2 ( 23.43% of cyp2d6.1 ) and cyp2d6.10 ( 3.11% of cyp2d6.1 ) were both presented significantly decreased . these results were consistent with previous work and indicate that it is appropriate to use our cyp expression and in vitro metabolism system for analyzing the catalytic activities of the other cyp2d6 variants.21,22,26,27 to better understand the enzymatic characteristics of cyp2d6 allelic isoforms and their effects on the metabolism of gefitinib in vitro , we functionally analyzed the enzymatic activity of 22 novel cyp2d6 alleles toward gefitinib using recombinant insect microsomes . we found that some of the variants showed similar trend about the enzyme activity as studies reported,21,27 but some caused increased or no change in enzyme activity , which is not in accordance with the results of previous research . it indicates that cyp2d6 not only exhibits significant genetic polymorphisms between individuals and among different races and ethnicities , but also showed marked differences on the metabolism of various clinical drugs . therefore , it is of great necessity to undertake further research on the function of cyp2d6 alleles on the metabolism of gefitinib . in our study , both cyp2d6.92 and cyp2d6.96 had undetectable enzyme activity in gefitinib metabolism , similar to previous reports.19,21,23,24 the reason for this may be because both of these variants were expressed as truncated proteins due to the premature termination in protein translation process , which led to the function loss of enzymatic activity . particularly , only one variant cyp2d6.94 , with a decrease km and an increase vmax , showed significant increased relative clearance compared with cyp2d6.1 , suggesting higher catalytic activity than cyp2d6.1 . on the contrary , as reported in other studies , cyp2d6.94 exhibited markedly different decreased catalytic activity toward propranolol ( 31.37% ) , nebivolol ( 58.23% ) , dextromethorphan ( 67.03% ) , venlafaxine ( 71.9% ) , and bufuralol ( 74.96% ) in vitro.19,21,23,24 these data suggest that cyp2d6.94 may be reclassified as an ultrarapid metabolizer phenotype for gefitinib . moreover , cyp2d6.88 , cyp2d6.89 , cyp2d6.91 , cyp2d6.97 , f164l , f219s , and v342 m were found in chinese people and exhibited similar metabolic activity of cyp2d6.1 for gefitinib . cai et al showed that cyp2d6.91 , cyp2d6.97 , f164l , f219s , and v342 m exhibited markedly decreased catalytic activity toward bufuralol and dextromethorphan . in addition , cyp2d6.88 and cyp2d6.89 exhibited decreased intrinsic clearance for bufuralol as compared with wild type but no significant difference toward dextromethorphan.21 these results suggest that cyp2d6 had substrate - specific alterations in metabolic activity and that results obtained from one substrate can not necessarily be extrapolated to other substrates . we speculate that the different affinity of enzyme and substrate might be responsible for this inconsistency . it is important to point out that two allelic variants cyp2d6.93 and e215k exhibited a drastic decrease in enzymatic activity . cyp2d6.93 exhibited ~94.4% lower vmax , 1.53-fold higher km , and 96.32% lower intrinsic clearance values compared with wild type . as for this result , dai et al speculated that the normal entrance of the cyp2d6 substrate might be blocked , resulting from the replacement of the hydrophilic thr with the hydrophilic pro at position 249 , which would influence the spatial structure of the adjacent egress channel . e215k contains an amino acid switch from glu to lys at position 215 , which is located in f - helix in the active site cavity of the cyp2d6 protein.27 that may be related with e215k s extremely decreased intrinsic clearance values ( 9.79% ) of cyp2d6.1 owing to high km ( 5.67-fold ) . our data suggests that cyp2d6.93 and e215k may have to be associated with a pm phenotype in vivo , with gefitinib as a substrate . several studies have reported that patients with nsclc who possessed pm phenotype have severe hepatotoxicity after treatment with gefitinib.16,28 our work may serve as a reference for rational administration clinically , thereby can avoid adverse drug reactions such as gefitinib - induced hepatotoxicity . furthermore , the most meaningful difference between our study and previous reports19,2124 is that gefitinib exhibited a substrate inhibition trend of metabolic ability by cyp2d6 in vitro . twenty - three tested cyp2d6 allelic variants showed this substrate inhibition trend toward gefitinib and their ksi values varied from ~6 to 158 m . as reported in a study by swaisland et al , suggested that gefitinib produced a little inhibition of cyp2d6 isoenzyme activity which resulted in a small increase in the metoprolol area under the curve and the maximum serum concentration.29 hereby , we know that gefitinib was both an inhibitor and a substrate of cyp2d6 in addition , we believe that it is still valuable to pay more consideration to the dose of cyp2d6 substrates which have a narrow therapeutic window when in the presence of gefitinib . in summary , we systematically assessed the enzymatic activity of 24 cyp2d6 allelic isoforms on the metabolism of gefitinib . in addition , it has been reported that inhibition of cyp3a4 leads to a shunt to a cyp2d6-dependent metabolic pathway . therefore , we speculate that patients with these dysfunctional cyp2d6 alleles should reduce the dosage of gefitinib in order to avoid gefitinib - induced hepatotoxicity when functions of cyp3a4 are also inhibited . moreover , further studies in vivo or in vitro on the effects of cyp2d6 and cyp3a4 toward gefitinib are required to make sense of the relation between cyps and gefitinib clearance . as the first study of these alleles for gefitinib metabolism

backgroundcytochrome p450 2d6 ( cyp2d6 ) , a member of the cyp450 enzyme super family , is a polymorphic enzyme that metabolizes ~25% of therapeutic drugs . cyp2d6 exhibits significant genetic polymorphisms which might cause adverse effects and therapeutic failures of some drugs.objectivethe purpose of this study was to evaluate the catalytic activities of 22 novel cyp2d6 alleles ( cyp2d6 * 87 , * 88 , * 89 , * 90 , * 91 , * 92 , * 93 , * 94 , * 95 , * 96 , * 97 , * 98 , r25q , f164l , e215k , f219s , v327 m , d336n , v342 m , r344q , r440c , r497c ) on the metabolism of gefitinib in vitro.methods and resultscyp2d6 variants were incubated with 1100 m gefitinib for 60 min at 37c and the reaction was terminated by cooling to 80c immediately . gefitinib and its metabolite o - desmethyl gefitinib were analyzed by an ultra - performance liquid chromatography - tandem mass spectrometry system . compared to cyp2d6.1 , most cyp2d6 variants exhibited significantly decreased relative clearance values ( from 3.11% to 79.35% ) , whereas cyp2d6.92 and cyp2d6.96 displayed no detectable enzyme activity . only cyp2d6.94 exhibited a markedly increased intrinsic clearance value , and eight variants ( cyp2d6.88 , cyp2d6.89 , cyp2d6.91 , cyp2d6.97 , v342 m , r344q , f219s , and f164l ) showed no significant difference . in addition , 23 cyp2d6 allelic isoforms exhibited substrate inhibition trend toward gefitinib.conclusionas the first study of all the aforementioned alleles for gefitinib metabolism , these comprehensive data may help in the clinical assessment of the metabolism of gefitinib , and may also offer a reference for personalized treatment with gefitinib in clinical settings .