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conserved domains in proteins have crucial roles in protein interactions , dna binding , enzyme activity and other important cellular processes . with recently released predictions of the number of genes in the human genome being less than many previous predictions , interactions among protein domains protein domains are often conserved across many species and , as such , they offer an interesting dataset for analyzing how genomes maintain any given domain in relation to other conserved domains , as well as for analyzing the relationship of conserved domain occurrence to proteome size . many groups have attempted to find , document and annotate these conserved domains . whereas most groups use a form of hidden markov models for profiling , each group approaches the problem in a unique way , yielding a wide range of databases that can be used to verify each other . for this study i used the smart cd database to collect data on the number of genes containing each conserved domain in each genome . the study was restricted to the five eukaryote genomes sequenced so far : homo sapiens , drosophila melanogaster , arabidopsis thaliana , caenorhabditis elegans and saccharomyces cerevisiae . results were confirmed using a repository of databases called the proteome analysis database ( abbreviated here as pad ) . it has been possible to compare conserved domains across different genomes , and to validate the approach by using a repository of databases ( pad ) and one database from this group ( smart ) . a close link is revealed between numbers of genes with a given conserved domain and the total number of genes in each genome . data were gathered as follows : a perl script was written to submit requests to the smart database for the number of genes with each of 519 conserved domains in each genome . information in pad is already in genome - specific columns for the 200 most frequent conserved domains in humans and was downloaded directly . the information was parsed and stored for each genome . from the smart database , 211 conserved domains were selected on the basis of the fact that they occurred at least once in each of the five genomes ( see additional data files ) . from pad , 122 conserved domains were selected on the basis of the fact that they occurred at least once in each of the five genomes ( see additional data files ) . my initial observation was that for many conserved domains , the ratio of the sum of genes in genome 1 containing the conserved domain to the total number of predicted genes in genome 1 was proportional to the ratio of the sum of genes in genome 2 containing the conserved domain to the total number of predicted genes in genome 2 . given that : a = sum of proteins with given conserved domain ( cd ) in genome 1 ; b= sum of proteins with given cd in genome 2 ; e= sum of predicted genes in genome 1 ; f= sum of predicted genes in genome 2 , then on average : upon rearranging equation 1 , it was noted that for many conserved domains the ratio of the number of genes containing the given conserved domain in each genome accurately reflected the ratio of the total predicted number of genes ach genome . or , given the variables in equation 1 , then on average : to normalize the data i used a ratio of the sum genes with a given conserved domain in a genome to the sum genes with the given conserved domain in all five genomes . this was used to minimize the effect that the predicted number of genes may be significantly wrong for one of the genomes whereas the others may be more accurate . equation 1 was rewritten to reflect this normalization . given that a= sum proteins with given cd in genome 1 ; g= sum proteins with given cd in five genomes ; e = sum predicted genes in genome 1 ; h= sum predicted genes for all five genomes , then on average : the sums of conserved domains in each equation 3 ratio range were depicted graphically for each genome , and are displayed in figure 1 ( smart database ) and figure 2 ( pad ) . the average ratio for each genome was calculated and multiplied against the sum predicted genes of all five genomes , yielding a number close to the number of predicted genes in each respective genome ( table 1 ) . equation 2 could be used to predict total genes in a genome given that the other variables are reasonably well known , such as from expressed sequence tag ( est ) data . more important , this suggests the possibility that these conserved domains are maintained in this ratio as a result of functional constraints on interacting domains . the fact that this ratio is maintained fairly well in all five eukaryotic genomes attests to its potential importance . although there is much disagreement on the total number of genes for the different genomes , similar gene - finding methods were used for each of the five published eukaryotic genomes . it can therefore be assumed that ratios of predicted genes between the genomes will remain similar to present ratios , as the gene numbers for each genome are clarified . likewise , neither smart nor pad claim to have found all occurrences of each conserved domain in each genome . however , because of similar strategies used for finding conserved domains in different genomes within each database , the ratio of total genes found with a given conserved domain in each genome is likely to remain near constant as gene prediction improves . an interesting finding from this research was that while the ratios for h. sapiens , a. thaliana , and s. cerevisiae related closely to the total predicted genes for each organism , both databases gave a peak ratio that exchanged total predicted gene numbers between d. melanogaster and c. elegans ( figures 1,2 ) . from figure 2 it can be seen that it is outlying conserved domain ratios that cause the average in table 1 to be shifted closer to actual predicted total gene numbers for c. elegans . while this exchange can not be explained at present , it may offer insights into the distinctions between the genomes , and genes that remain unidentified . it is important to note that by mainly analyzing conserved domains occurring most frequently , conserved domains that occur only once in each genome are , for the most part , excluded from the analysis . it has been shown that conserved domains in proteins are maintained in proteome - specific ratio for the five eukaryotic genomes sequenced so far . the reasons for this ratio are unclear , but it would not be unreasonable to suspect that the functional interactions of these protein domains require that they be kept in a specific ratio . further research may reveal that conserved domains outside of this ratio are critical to the organism 's unique functions , and will be necessary to understand the reasons for , and universality of this ratio in eukaryotic genomes . the smart database was searched for conserved domains occurring at least once in each of the five genomes . for pad the search was restricted to those conserved domains listed in the top 200 domains occurring in humans for which there was at least one occurrence in each of the four other genomes . this strategy of limiting the study to more global conserved domains was used to increase the chance that the conserved domains were constructed correctly and to increase the statistical reliability of the results . the total number of predicted genes for each genome was as follows : h. sapiens , 35,000 ; d. melanogaster , 14,100 ; a. thaliana , 26,000 ; c. elegans , 19,100 ; s. cerevisiae , 6,300 . this yielded a total of 100,500 genes for all five genomes , and a total of 39,500 for d. melanogaster , c. elegans , and s. cerevisiae alone . the number of genes in each genome is approximate because it is an estimate that is continually being updated . smart_cds.txt is a text , tab - delimited file containing all 211 conserved domain names from the smart database used in this study . for each conserved domain name , pad_cds.txt is a text , tab - delimited file containing all 122 interpro entry numbers for the domains in pad used in this study . for each interpro entry number , the corresponding number of genes containing the conserved domain in each genome is listed . smart_cds - a text , tab - delimited file containing all 211 conserved domain names from the smart database used in this study . click here for additional data file pad_cds - a text , tab - delimited file containing all 122 interpro entry numbers for the domains in pad used in this study . i thank those at tigr who reviewed the ideas presented here and b. parvizi and j. vamathevan for help with the writing and analysis . thank you to s. malek for critical review of the manuscript . sum of conserved domains ( cds ) in each ratio range of cds in a genome ( see equation 3 ) compared to their occurrence in all five genomes ( 211 cds considered ) . equation 3 was used for all cds for each genome . the number of cds in each ratio range for each genome was summed and graphed . it is apparent that the number of cds peaks at a particular ratio for each genome , with an average near the respective proteome size ( multiply average ratio for each genome by 100,500 as in table 1 ) . sum of cds in each ratio range of cds in a genome ( see equation 3 ) compared to their occurrence in all five genomes ( 122 cds considered ) . the number of cds in each ratio range for each genome was summed and graphed . it is apparent that the number of cds peaks at a particular ratio for each genome , with an average near the respective proteome size ( multiply average ratio for each genome by 100,500 as in table 1 ) .

backgroundconserved domains in proteins have crucial roles in protein interactions , dna binding , enzyme activity and other important cellular processes . it will be of interest to determine the proportions of genes containing such domains in the proteomes of different eukaryotes.resultsthe average proportion of conserved domains in each of five eukaryote genomes was calculated . in pairwise genome comparisons , the ratio of genes containing a given conserved domain in the two genomes on average reflected the ratio of the predicted total gene numbers of the two genomes . these ratios have been verified using a repository of databases and one of its subdivisions of conserved domains.conclusionsmany conserved domains occur as a constant proportion of proteome size across the five sequenced eukaryotic genomes . this raises the possibility that this proportion is maintained because of functional constraints on interacting domains . the universality of the ratio in the five eukaryotic genomes attests to its potential importance .

it is freely available to any one who allows adequate exposure of skin to the ultraviolet radiation present in the sunrays . however , vitamin d deficiency is not uncommon even in the sunny land of india . the clinical syndrome vitamin d deficiency consists of osteomalacia in adults and rickets in children . other manifestations of vitamin d deficiency include nonspecific backache , joint pains , and generalized body ache . with the availability of a method to accurately measure 25-hydroxyvitamin d [ 25(oh ) d ] , a state of hypovitaminosis d without overt signs and symptoms of vitamin d deficiency has become apparent [ 1 , 2 ] . the amount of vitamin d provided by sun exposure and diet is difficult to determine as the duration and intensity of sunlight exposure depends on age , clothing , and sunscreens . the insufficient intake of vitamin d has been implicated as one of the factors in the development of bone disease [ 46 ] . various elements contribute to this insufficient vitamin d intake such as skin pigmentation , which presumably interferes with ultraviolet ray transmission through epidermis , genetic factors [ 8 , 9 ] , social customs such as avoidance of sun exposure , consumption of chapattis ( flattened rounded wheat bread ) that is high in phytates which bind calcium in the gut and interfere with calcium absorption , lack of sun exposure and malnutrition to name a few important contributing factors . this is exemplified by the high prevalence of vitamin d deficiency in house bound and elderly patients [ 1113 ] . while osteomalacia and rickets have been studied extensively , there is little information on vitamin d deficiency without obvious bone disease . the aims and objectives of this study were ( a ) to study the clinical symptoms and signs of vitamin d deficiency and their correlation with 25-hydroxyvitamin d [ 25(oh ) d ] levels , ( b ) to study the bone profile of patients of hypovitaminosis d , and ( c ) to observe the response to treatment with vitamin d on these individuals . the patients were seen between 1996 and 2001 at the rheumatology services of the hinduja hospital and medical research centre , mumbai , india . a proforma was prepared to record relevant details ( appendix a - proforma : profile of vitamin d deficiency ) . this included details regarding symptom and signs , dietary history , duration and severity of illness , associated illnesses , sun exposure , renal disease , gastrointestinal tract disease and intake of anticonvulsants such as diphenylhydantoin sodium . the treatments given to these patients were recorded and their responses to treatment were noted . for the details not available in the medical chart , an attempt was made to contact the patients on telephone or in person to get complete information . of these 38% ( n = 27 ) were in the age group of 3140 years . vitamin d deficiency in this population , while symptomatic , was without any of florid signs ( 63% showed no tenderness as shown in table 3 ) . the bone profile of patients with vitamin d deficiency is shown in table 4 . the radiological data showing various vitamin d associated changes in the study 's patients are shown in table 6 . taking the normal range of vitamin d , 25(oh ) d in our laboratory as 9.941.5 ng / ml , we classified our patients into 3 grades of vitamin d deficiency . the grades were mild 25-hydroxyvitamin d [ 25(oh ) d ] levels6 to 9.9 ng / ml , moderate 25-hydroxyvitamin d [ 25(oh ) d ] levels2 to 5.9 ng / ml , and severe 25-hydroxyvitamin d [ 25(oh ) d ] levels < 2 ng / ml . the distribution of severity of vitamin d amongst these patients is shown in table 7 using these parameters . vitamin d deficiency is variably defined as a 25(oh ) d level less than 1015 g / l [ 16 , 17 ] . the commonness of vitamin d deficiency in an otherwise healthy population was an eye - opener as well as a teaser . at the hinduja hospital and medical research center , mumbai , india , the awareness of vitamin d deficiency started with appreciation of vitamin d deficiency as a complicating factor in rheumatic diseases . a case is briefly described to make the point . a 59year - old male patient with ankylosing spondylitis of more than 30 years duration was admitted with a recurrence of left - sided hemiparesis and a recent increase in joint pains . he had diabetes for the past 10 years which was controlled on oral hypoglycemic agents . a year before his admission , he had developed left - sided hemiparesis from which he had recovered completely . the increase in joint pains started six months before he was admitted but he reported no swelling or stiffness at that time . there was no evidence of synovitis of peripheral joints , but he had severe diffuse bony tenderness , especially of his spine and ribs . investigations revealed the following statistics : hemoglobin : 12.2 gm / dl , white blood cell count : 16,700/mm , platelets : 2.3 lac / mm , esr : 28 mm / h , serum calcium : 8.3 mg / dl ( normal range : 8 to 10.4 mg / dl ) , phosphorus 2.9 mg / dl ( normal range : 2.54.6 mg / dl ) , alkaline phosphatase : 590 iu / l ( normal range : 40120 iu / l ) , 25(oh ) vitamin d : 4.3 ng / ml ( normal range : 9.941.5 ng / ml ) and parathormone 333 pg / ml ( normal range : 1272 pg / ml ) . a routine urine test showed trace proteins , 24 hours urinary proteins : 792 mg ( 0165 mg ) and creatinine 1 mg / dl ( normal range : 0.6 to 1.01 mg / dl ) . he was diagnosed with vitamin d deficiency - related osteomalacia with secondary hyperparathyroidism being made and he was initiated on a vitamin d treatment regimen ( alpha d3 and vitamin d 60 000 units ) , along with one gram supplemental calcium . within one week his pains started to decrease and were significantly more manageable in three weeks time and he was discharged . at this man 's medical case was followed by the publication of a small series of letters to the editor in the journal of association of physicians of india . soon patients with a variety of nonspecific musculoskeletal complaints were tested for vitamin d status . to our surprise thus of the samples tested for vitamin d levels in our hospital laboratory , 80% had low serum 25-hydroxyvitamin d [ 25(oh ) d ] levels , that is , < 9.9 ng / ml . it therefore became imperative that we study the significance of low vitamin d levels in adults for clinical , therapeutic , and other aspects . thus the case selection was based upon the presence of low vitamin d levels in individuals attending the clinic , for various nonspecific musculoskeletal symptoms . the present study has its inherent limitations but was thought necessary to understand the phenomenon of low vitamin d levels . all the study patients had musculoskeletal complaints and were suspected to suffer from rheumatoid arthritis , ankylosing spondylitis , or vertebral disc disease . although it is not possible to judge the exact proportion of such patients , vitamin d deficiency is an important differential diagnosis of patients with such complaints . in rheumatology parlance , fibromyalgia and hypothyroidism closely resemble this symptomatology , and hence myalgia and nonspecific pain may be due to vitamin d deficiency . muscle weakness led to inability to get up from squatting position and waddling gait in some patients . vitamin d deficiency is an important consideration in a patient with proximal muscle weakness , ( and may additionally complicate a case of polymyositis - dermatomyositis ) . vitamin d deficiency in this population was without florid signs ( 63% showed no tenderness ) . this is important , as without bony tenderness ( which would suggest osteomalacia ) and without proper awareness on the part of physicians , vitamin d deficiency can easily be overlooked . when we tried to correlate the vitamin d levels with the symptoms ( as shown in table 7 ) , we came to the conclusion that although patients with low vitamin d levels present many nonspecific symptoms like backache , body ache , leg pain , and thigh pain , not all are osteomalacic . how does one explain the absence of clinical osteomalacia in individuals with very low vitamin d levels ? is it likely that even at low vitamin d levels , there is enough 1 , 25 ( oh)2 d3 to maintain homeostasis ? although this current study does not possess the funding to answer this question , it remains an interesting line of inquiry . there may be ( over ) active conversion of 25 ( oh ) d to 1 , 25 ( oh ) 2 d3 to maintain bone mass . a parallel can be drawn with iron deficiency anemia ; until body iron stores are exhausted iron deficiency does not manifest as anemia . an attempt was made to see if the severity of vitamin d deficiency correlated with the number of symptoms recorded ( as shown in table 7 ) . ( unfortunately , the severity of individual symptoms can not be analyzed as this was not recorded using a visual analogue scale . ) still there does not seem to be any such correlation given that only one patient had more than four out of the seven symptoms analyzed . we compared the records of 12 patients who were diagnosed as osteomalacic and found that 50% of the patients had vitamin d levels that measured at levels below 5 ng / ml . it is apparent that individuals with vitamin d deficiency may not suffer from clinical osteomalacia but can have nonspecific musculoskeletal symptoms accompanied with morbidity . it is likely that some may go on to develop frank nutritional osteomalacia unless treated . this is because , compared with the commonness of vitamin d deficiency , clinical osteomalacia is not a common disorder . it is difficult to guess the prevalence of vitamin d deficiency in the community , but it does seem logical to assume that osteomalacia ( as well as rickets ) are only the tip of the iceberg . the common etiological factors of vitamin d deficiency are lack of exposure to sun rays ( solar ultraviolet - b exposure to as much of body surface as possible produces vitamin d ) and decreased intake of foods rich in vitamin d. a specific history was not recorded in all patients studied , but a majority of these patients were vegetarians , with poor intake of milk or milk products and little outdoor activity . none of the patients belonged to a low socioeconomic group and hence likely had access to plenty of food . thus paradoxically , vitamin d deficiency may be a disease of the rich ! the case involves a 50-year - old gentleman , a businessman and an epileptic on diphenylhydantoin sodium living in a house with drawn curtains , and traveling in a car with tainted glass . when seen by the physician , he was nearly crippled by back pain with frank evidence of osteomalacia . the pain was treated as spondylosis , and he was even suspected to be suffering from tuberculosis!. vitamin d , calcium , and regular sun exposure brought him back to normalcy . when we checked the response to vitamin d and calcium therapy , we found that out of 20 patients placed on calcium and/or vitamin d , 13 patients showed significant clinical improvement . also followup information was not provided for 51 of these patients , and so it is impossible to speculate about their response to treatment . it should be noted that the cause for nonresponse to treatment in some patients may be due to factors other than vitamin d deficiency . additionally , the symptoms these patients report , symptoms that indicate a lack of vitamin d , may also be caused by other incidental factors . for example a recent controlled study found that diffuse musculoskeletal pain may not be associated with low vitamin d and that this pain does not respond to treatment with vitamin d . as already stated vitamin d deficiency can complicate a rheumatic disease and make diagnosis difficult [ 18 , 2023 ] . g , a 46-year - old female patient suffering from rheumatoid arthritis for more than 15 years had tried a few disease modifying agents at the initial stages of the disease without beneficial effects . eight years prior to being seen , she had undergone a left hip replacement . for five of those years , she was bed ridden with deformities and complained of severe , debilitating , unbearable aches and pains . she could not even turn in bed , move her legs , or lift her arms due to the pain . investigations revealed undetectable levels of vitamin d , low serum calcium , phosphorus , and increased alkaline phosphatase . a diagnosis of concomitant osteomalacia was made , and she was placed on three weekly bolus of 60 000 units of oral vitamin d followed by a daily supplement of 1.5 grams of calcium . over a period of 1 month , her bone pains reduced significantly . she was able to lift her arms , eat with her own hands , and sit without support . the case was an eye - opener . in every case with pains out of proportion to clinical findings , it would be wise to check for vitamin d deficiency symptoms . since this was a retrospective study where the study participants had to recall their dietary history , type of indoor and outdoor activities performed , and history of sun exposure , a recall bias is evident . furthermore the data on assessment of muscle weakness were incomplete and were not evaluated by another independent rater . this small retrospective study shows that vitamin d deficiency is not uncommon but frank osteomalacia is uncommon . a subclinical vitamin d state exists which is characterized by nonspecific musculoskeletal symptoms . this state may complicate a rheumatic disorder , and it is important to consider this diagnosis as the results of treatment are most gratifying . three conclusions can be drawn from this study : ( a ) vitamin d deficiency runs across the board ; importantly it affects individuals in the prime of their life and affects the quality of life and the productivity of those suffering from it ; ( b ) the preponderance of female patients compared to male patients suffering from vitamin d deficiencies possibly reflects social factors like the avoidance of milk and milk products and staying indoors ; ( c ) a large chunk of vitamin d deficiency is subclinical . if estimation of vitamin d is not possible , a trail of oral vitamin d supplements and calcium is indicated to be beneficial . there is a new and emerging body of literature showing relationship between vitamin d deficiencies and respiratory infections , along with obstetrical conditions such as pre - eclampsia .

vitamin d deficiency is not uncommon even in the sunny land of india . lack of sun exposure and inadequate oral intake are both responsible for vitamin d deficiency . this article provides a retrospective , examining the effects of vitamin d deficiency in 71 patients . the study 's inclusion criterion was low vitamin d level combined with musculoskeletal symptoms but without the presence of osteomalacia . all patients in this study were suspected to have vitamin d deficiency . the data were retrieved from the case - charts of patients seen between 1996 and 2001 at the rheumatology services of hinduja hospital , mumbai , india . this study found no correlation between vitamin d levels and symptoms , or between the severity of vitamin d deficiency and the number of symptoms displayed . subclinical vitamin d deficiency or preosteomalacic state was the term coined for individuals with vitamin d deficiency producing nonspecific musculoskeletal symptoms in the absence of clinical osteomalacia .

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

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

in the last three decades , magnetic resonance imaging ( mri ) has been used for noninvasive assessment of the prostate and surrounding structures . initially , prostate mri was only based on morphologic assessment , using t1-weighted imaging ( t1wi ) and t2-weighted imaging ( t2wi ) sequences . its role was primarily for loco - regional staging of the prostate cancer as it provided limited capability to distinguish between benign pathological tissue and clinically insignificant tumors from significant prostate cancer . to increase the diagnostic accuracy , anatomic t2wi and functional mri sequences - such as dynamic contrast enhanced mri ( dce - mri ) , diffusion - weighted imaging ( dwi ) with its derivate apparent - diffusion coefficient ( adc ) map and hydrogen 1 mr spectroscopic imaging ( mrsi ) - were combined in an integrated multiparametric mri ( mp - mri ) examination . the technological advances , combined with a growing interpreter experience , have substantially improved the detection of clinically significant prostate cancer , which is critical for reducing mortality , and increased confidence in ruling out benign diseases and dormant malignancies , in order to reduce unnecessary biopsies and treatment . in 2012 the european society of urogenital radiology ( esur ) working group developed the guidelines for mp - mri of the prostate . the acquisition protocols were then proposed , in order to provide the prostate imaging reporting and data system ( pi - rads ) , which relays the probability of cancer risk and its aggression . recently , the pi - rads version 2 was developed , including the following changes : ( a ) the concept of a dominant sequence depending on the location of the lesion ; ( b ) the statement that dce - mri should be scored positive if early focal enhancement is present and negative , if not or if diffuse enhancement is noticed , instead of using the curve - type analysis described in pi - rads version 1 ; ( c ) for positive dce - mri results , the pi - rads score should be increased by one point , if it makes a clinically relevant difference ; ( d ) finally , an overall pirads score , on a scale of 15 is assigned , according to the revised rules from pi - rads version 2 . in the second version of pi - rads , clinically significant cancer is defined on pathology as gleason score 7 ( including 3 + 4 with prominent gleason 4 component ) , and/or volume 0.5 cc , and/or extraprostatic extension . this definition is intended to standardize reporting of mp - mri and correlation with pathology for clinical and research applications . the aim of this study is to evaluate the diagnostic performance of mp - mri at 1.5-tesla ( 1.5-t ) , for the detection of clinically significant prostate cancer . a total of 90 patients with clinically suspected prostate cancer were examined by mp - mri , between october 2013 and february 2016 , in a prospective single - center study . we included in this study patients with clinically significant prostate cancer proved on biopsy or prostatectomy . the patients with a history of positive prostate biopsy and the patients who were treated for prostate cancer were excluded . finally , 39 patients - mean age 68.02 years ( ranging from 51 to 78 years ) were included in this study . written informed consent was obtained from all the patients included in the study , in order to use their laboratory , imaging and histopathologic data . the study was carried out in agreement with the code of ethics of the world medical association ( helsinki declaration ) for experiments involving human subjects . all patients were examined by using a 1.5-tesla equipment ( magnetom avanto , siemens healthcare , erlangen , germany ) . the same mp - mri protocol was used for all patients : axial t2wi , sagittal t2wi , coronal t2wi , axial dwi with adc map , axial t2 fat - sat , coronal t1wi and axial dce - mri ( table i ) . for dce - mri a bolus injection of 0.1 mmol / kg body weight of gadolinium - based contrast agent followed by a saline flush of 20 ml was given . the examinations were read by a radiologist with 3 years of experience in prostate - mri . the images were analyzed using the syngo vb17 software with the commercially available applications ( siemens medical solutions , erlangen , germany ) and osirix viewer . suspected lesions were noticed in mri reports and were categorized according to the pi - rads 2 lexicon ; a pirads score was assigned for all mr abnormalities . for 17 patients examined between october 2013 and december 2014 , the version 1 pi - rads score was initially used . once the pi - rads version 2 scoring system was available , a revised pi - rads score was reported for these patients without modifying the initial mri report . the transition from pi - rads version 1 to pi - rads version 2 was made in order to use the same scoring system for all of the patients examined in the study . the mr findings were reported as positive if pirads 3 , pirads 4 or pirads 5 lesions were present and negative if only pirads 1 or pirads 2 findings were identified . a standardized multiparametric - mri reporting scheme - modified after rothke et al . - all 39 patients underwent a standard 12 core transrectal ultrasonography ( trus)guided biopsy ; additional targeted cores were picked up in 7 patients , from mr suspected lesions . during the study period , 4 of 39 patients with prostate cancer underwent radical prostatectomy . antibodies used were p63 ( clone 7jul , novocastra ) , high molecular weight cytokeratin ( clone 34betae12 , novocastra ) and also alpha - methylacyl - coa racemase ( amacr , p504s , clone epmu1 , novocastra ) . antigen retrieval was performed with a pressure cooker and hier method . a pathologist with 8 years of experience in prostate pathology examined the slides , using an olympus bx43 microscope . the clinically significant cancer was defined , according to pi - rads version 2 system , as gleason score 7 , and/or volume 0.5 cc , and/or extraprostatic extension . when prostatectomy was not performed and only biopsy result was available , we defined a clinically significant prostate cancer core as gleason score 7 , and/or having a cancer length greater than 5 mm . the standard of reference was settled by the results of systematic trus - guided biopsy : a patient was considered true positive if biopsy specimens showed pathologically positive results and true negative if biopsy result was negative . numerical variables were descriptive presented . for testing normality distribution of the numerical variables we used the kolmogorov - smirnov test . for comparison of numerical variables , the student test , mann - whitney and kruskal - wallis ( depending on its distribution ) were used . a total of 90 patients with clinically suspected prostate cancer were examined by mp - mri , between october 2013 and february 2016 , in a prospective single - center study . we included in this study patients with clinically significant prostate cancer proved on biopsy or prostatectomy . the patients with a history of positive prostate biopsy and the patients who were treated for prostate cancer were excluded . finally , 39 patients - mean age 68.02 years ( ranging from 51 to 78 years ) were included in this study . written informed consent was obtained from all the patients included in the study , in order to use their laboratory , imaging and histopathologic data . the study was carried out in agreement with the code of ethics of the world medical association ( helsinki declaration ) for experiments involving human subjects . all patients were examined by using a 1.5-tesla equipment ( magnetom avanto , siemens healthcare , erlangen , germany ) . the same mp - mri protocol was used for all patients : axial t2wi , sagittal t2wi , coronal t2wi , axial dwi with adc map , axial t2 fat - sat , coronal t1wi and axial dce - mri ( table i ) . for dce - mri a bolus injection of 0.1 mmol / kg body weight of gadolinium - based contrast agent followed by a saline flush of 20 ml was given . the examinations were read by a radiologist with 3 years of experience in prostate - mri . the images were analyzed using the syngo vb17 software with the commercially available applications ( siemens medical solutions , erlangen , germany ) and osirix viewer . suspected lesions were noticed in mri reports and were categorized according to the pi - rads 2 lexicon ; a pirads score was assigned for all mr abnormalities . for 17 patients examined between october 2013 and december 2014 , once the pi - rads version 2 scoring system was available , a revised pi - rads score was reported for these patients without modifying the initial mri report . the transition from pi - rads version 1 to pi - rads version 2 was made in order to use the same scoring system for all of the patients examined in the study . the mr findings were reported as positive if pirads 3 , pirads 4 or pirads 5 lesions were present and negative if only pirads 1 or pirads 2 findings were identified . a standardized multiparametric - mri reporting scheme - modified after rothke et al . - all 39 patients underwent a standard 12 core transrectal ultrasonography ( trus)guided biopsy ; additional targeted cores were picked up in 7 patients , from mr suspected lesions . during the study period , antibodies used were p63 ( clone 7jul , novocastra ) , high molecular weight cytokeratin ( clone 34betae12 , novocastra ) and also alpha - methylacyl - coa racemase ( amacr , p504s , clone epmu1 , novocastra ) . a pathologist with 8 years of experience in prostate pathology examined the slides , using an olympus bx43 microscope . the clinically significant cancer was defined , according to pi - rads version 2 system , as gleason score 7 , and/or volume 0.5 cc , and/or extraprostatic extension . when prostatectomy was not performed and only biopsy result was available , we defined a clinically significant prostate cancer core as gleason score 7 , and/or having a cancer length greater than 5 mm . the standard of reference was settled by the results of systematic trus - guided biopsy : a patient was considered true positive if biopsy specimens showed pathologically positive results and true negative if biopsy result was negative . numerical variables were descriptive presented . for testing normality distribution of the numerical variables we used the kolmogorov - smirnov test . for comparison of numerical variables , the student test , mann - whitney and kruskal - wallis ( depending on its distribution ) were used . the mean age of the examined patients was 68.026.38 years , ranging between 51 and 78 years . 10.3% of patients were between 5059 years , 43.6% ranged from 60 to 69 years and 46.2% were older than 70 table ii . the mean psa taken from blood samples was 22.6939.34 ng / ml , with a median psa of 12.95 ng / ml . patients were categorized based on psa value as follows : patients with psa < 10 ng / ml 38.5% ( 15 ) , patients with psa ranging from 10 to 20 ng / ml 28.2% ( 11 ) and with psa > 20 ng / ml 33.3% ( 13 ) . in our series , 8 patients ( 20.5% ) had prior negative biopsies and 31 patients ( 79.5% ) had no previous biopsies ; we found no statistically significant difference regarding the psa values between these two groups 25 patients had mr abnormalities , which were stratified according to the pi - rads 2 lexicon : focal abnormalities ( 1 ) , lesions ( 3 ) , masses ( 4 ) , nodules ( 5 ) , diffuse ( 4 ) , multifocal ( 4 ) , regional abnormalities ( 4 ) . pirads 5 was the dominant score ( 35.9 % ) in our mri reports ( figure 2 ) . we found a statistically significant difference regarding the distribution of pirads score between the psa groups ( p=0.011 ) table iv . all 39 patients underwent the standard transrectal ultrasonography ( trus)guided biopsy ; 7 patients had additional targeted cores on mr suspected lesions . 20 patients had positive biopsies and gleason 7 was the dominant score ( 35.9 % ) table v. there was no statistically significant difference regarding the gleason score distribution between the psa groups . we found a statistically significant difference between the mri reports in the psa groups ( p=0.016 ) table vi . the psa groups differed significantly regarding the biopsy results ( p=0.003 ) table vii . sensitivity , specificity , positive predictive value and negative predictive value were calculated among the whole group ( table viii ) . the highest incidence of prostate cancer was for males between 7079 years ( 46.2% ) . the result is consistent with data provided by the cancer report in north - western region of romania . in our study , the mri reports were positive for 25 ( 64.1% ) of 39 patients , of which 12 patients had high psa levels ( 20 ng / ml ) table vi . we found a statistically significant difference between the mri reports and the psa groups ( p=0.016 ) . pirads 1 and pirads 2 lesions were found more frequently in patients with psa value < 10 ng / ml ( 9 of 15 patients ) . in the group with psa20 ng / ml , pirads 4 and pirads 5 lesions were predominant ( 12 of 13 patients ) . a statistically significant difference regarding the distribution of pirads score between psa groups was found ( p=0.011 ) . . demonstrated that the benefit of mri and targeted biopsy increases with the increasing level of psa . recent studies that included patients with psa levels > 10 ng / ml , reported the sensitivity , specificity , accuracy , ppv and npv for the detection of prostate cancer using combined mri , between 6995% , 6396% , 6892% , 7586% and 8095% , respectively [ 914 ] . in our series of patients , trus - guided biopsies were positive in 20 patients ( 51.3% ) of which 11 had a psa 20 ng / ml . 19 patients ( 48.7% ) had negative biopsies , 12 of them having low psa levels ( < 10 ng / ml ) . we found a statistically significant difference between the biopsy results and the psa groups ( p=0.003 ) . our overall sensitivity and specificity for mp - mri detection of clinically significant prostate cancer detection were 100% and 73.68% , respectively . evaluated the clinical value of dwi and dce - mri in combination with t2wi for the detection of prostate cancer , in a series of 83 consecutive male patients , taking as reference the systematic biopsy results . they reported a sensitivity of 95% , a specificity of 74% and an accuracy of 86% . in a retrospective study of 2011 , tamada et al . reported a sensitivity of 83% and a specificity of 80% for combined mri techniques ( t2wi , dce - mri and dwi ) in prostate cancer detection , on a per - patient basis . their study evaluated a series of 50 patients and reference test was the12 cores trus - guided systematic biopsy . the accuracy , ppv and npv reported were 82% , 91% and 67% , respectively . . found great variations in the detection of clinically significant prostate cancer in a systematic review of the literature from 2015 . 12 studies ( 1981 patients ) were included in this meta - analysis of which 5 were prospective studies . six studies were performed at 3-t scanner , two studies used a 1.5-t unit , and four studies alternately used 1.5- and 3-tesla equipments . the selected studies performed prostate mri with at least two functional techniques ( dw - mri , dce - mri or mrsi ) in addition to anatomical t2wi . sensitivity ranged from 58 to 96% , specificity from 23 to 87% and accuracy from 44 to 87% . the npv and ppv for the detection of clinically significant disease ranged from 63 to 98% and from 34 to 68% , respectively . the authors have concluded that mp - mri is able to detect significant prostate cancer in biopsy - nave males and men with prior negative biopsies . the high npv of mp - mri is important to the clinician because mp - mri could be used to rule out significant disease . in our study , the positive predictive value and the negative predictive value were 80% and 100% , respectively . eight of 39 patients had prior negative biopsies . in 4 patients the mri report was positive and in 3 cases the prostate cancer was confirmed by trus - guided biopsy ( two patients had gleason 7 and one had gleason 6 ) . assessed the performance of multiparametric mri in patients with prior negative trus - guided biopsy and showed that mp - mri had good performance for detecting and ruling out clinically significant prostate cancer , with a se of 90% and a npv of 95% . they concluded that mp - mri can be used as a triage test in the population with persistently elevated psa levels following a negative biopsy and thereby identify patients who can avoid unnecessary prostate biopsy . a published study in 2014 by itatani et al . reported a clinical npv for mp - mri of 89.6% for clinically significant prostate cancer , over a longitudinal follow - up period of 5 years . therefore , the authors concluded that mp - mri can to rule out clinically significant prostate cancer before biopsy some potentially influential factors need to be discussed . a limit of our study is the lack of accurate correlation between the mr localization of suspicious lesions and the trus - guided biopsy . using the mr - in bore biopsy or the mri - ultrasound fusion techniques could exceed this limit . some recent studies have shown that the detection of more clinically significant cancers in the mri - guided biopsy compared with systematic biopsy , improve the biopsy performance and the diagnostic benefits [ 17,2023 ] . the positive mri was reported per patient and not per lesion and this might potentially influenced our data . another limit of this study was that we used only one reader for mp - mri examinations . therefore , studies on larger groups of patients , with multiple readers are needed to confirm our data . our results show that 1.5 t mp - mri has a high sensitivity for the detection of clinically significant prostate cancer and high negative predictive value in order to rule out significant disease .

background and aimmultiparametric - magnetic resonance imaging ( mp - mri ) is the main imaging modality used for prostate cancer detection . the aim of this study is to evaluate the diagnostic performance of mp - mri at 1.5-tesla ( 1.5-t ) for the detection of clinically significant prostate cancer.methodsin this ethical board approved prospective study , 39 patients with suspected prostate cancer were included . patients with a history of positive prostate biopsy and patients treated for prostate cancer were excluded . all patients were examined at 1.5-t mri , before standard transrectal ultrasonography guided biopsy.resultsthe overall sensitivity , specificity , positive predictive value and negative predictive value for mp - mri were 100% , 73.68% , 80% and 100% , respectively.conclusionour results showed that 1.5 t mp - mri has a high sensitivity for detection of clinically significant prostate cancer and high negative predictive value in order to rule out significant disease .

glutamate functions as the major excitatory neurotransmitter by binding to n - methyl - d - aspartate receptors ( nmdars ) that are widespread in the central nervous system . the nmdars constitute a major class of ionotropic glutamate receptors and play an essential role in synaptic transmission , plasticity , and memory . activation of nmdars results in cell membrane depolarization with an equilibrium potential near 0 mv , producing the excitatory postsynaptic potential ( epsp ) and leading to an increase of ca influx into the cell . the intracellular ca can in turn function as a second messenger , mediating a variety of signaling cascades . excessive activation of nmdars by glutamate mediates neuronal damage in many neurological disorders including ischemia and neurodegenerative diseases ( choi et al . the nmdars have long been considered the main target for the treatment of excitotoxicity - related neuronal injury , and a variety of antagonists or blockers of nmdars have been developed . unfortunately , the results of clinical trials have been disappointing because of the obvious side effects associated with blocking the physiological roles of nmdars ( chen and lipton , 2006 ) . therefore , a better understanding of the mechanism of how nmdars can be modulated by regulatory proteins should help in the development of new therapeutic agents to counteract overactive nmda receptor function , and may represent an alternative to treating nmdar - mediated excitotoxic injury . this short review focuses on the specific negative modulation of nmdars by a neuronal calcium sensor ( ncs ) protein , dream / calsenilin / kchip3 . nmdars are believed to be heterotetrameric complexes composed of combinations of the obligatory nr1 subunit and nr2 and/or nr3 subunits ( chazot and stephenson , 1997 ; laube et al . , 1998 ; schorge and colquhoun , 2003 ; furukawa et al . , 2005 ) . the nr1 subunit is encoded by a single gene but exists as eight functional splice variants , while the nr2 ( nr2a - b ) and nr3 ( nr3a - b ) subunits are encoded by four and two different genes , respectively . the nmdar subunits form a central ion conductance pathway selective for cations such as na , k , and ca , and share a common membrane topology , with each subunit consisting of four transmembrane ( tm ) domains ( m1m4 ) . the long extracellular n - terminal regions of nmdar subunits are organized as a tandem of two domains . the first domain , called the n - terminal domain ( ntd ) that includes the first 380 amino acids , is involved in tetrameric assembly ( mayer , 2006 ; paoletti and neyton , 2007 ; stroebel et al . , the second domain of about 300 amino acids is known as the agonist - binding domain ( abd ) that precedes the tm1 domain . the abd binds glycine ( or d - serine ) in the nr1 and nr3 subunits , whereas the nr2 abd binds glutamate ( furukawa et al . , 2005 ; yao and mayer , 2006 ) . the pore loop ( p loop ) , or the m2 region , forms the narrowest constriction of the channel ion conductance pathway and determines the permeation properties of nmdars . the nmdars feature an intracellular c - terminal tail of about 400600 residues that has a strong diversity in its amino acid sequence . the c - terminal tails of nmdar subunits contain a series of short motifs that interact with intracellular factors or binding partners involved in receptor trafficking , anchoring and signaling ( skeberdis et al . activation of nmdars requires a simultaneous binding of two co - agonists , glutamate , and glycine with different biophysical properties of ion permeation . the typical nmdars contain nr2 subunits with properties of high permeability to ca and extracellular mg block at hyperpolarized membrane potentials ( wrighton et al . different from conventional nr1/nr2 heterotetramers , nr3-containing nmdars have unique properties with a five to tenfold decrease of ca permeability , insensitivity to mg block , and reduced single - channel conductance and open probability , functioning as a negative modulator for nmda receptor channel function ( das et al . , 1998 ; 2009 ; cavara et al . , 2010 ; henson et al . , 2010 ) . nmda receptors are also regulated by other intracellular signals and proteins , including calcium , protein kinases , protein phosphatase calcineurin , and calcium - sensitive proteins such as calmodulin ( legendre et al . , 1993 ; vyklicky , 1993 ; lieberman and mody , 1994 ; tong et al . , 1995 ; ehlers et al . , 1996 ) . calcium - dependent nmda receptor desensitization and inactivation provides a feedback mechanism capable of regulating subsequent ca entry into the postsynaptic cell through nmda channels ( figure 1 ) . schematic representation for inhibitory effect of dream / calsenilin / kchip3 on nmdars in a ca - sensitive manner . upon activation of nmdars by glutamate binding , ca influx through nmdars increases the association between dream and nr1 subunits , resulting in reduced surface expression of nmdars , and subsequent inhibition of nmdars - mediated ca influx and excitotoxicity . dream functions as a ca - sensitive modulator for the negative feedback control of nmdar function . so far , a number of nr1 or nr2 subunit binding partners have been identified in the postsynaptic density . the nr1 binding proteins include calmodulin ( cam ) ( ehlers et al . , 1996 ; akyol et al . , 2004 ) , ca / cam - dependent protein kinase ii ( camkii ) ( leonard et al . , 2002 ) , -actinin ( wyszynski et al . , 1997 ; merrill et al . , 2007 ) , tubulin ( van rossum et al . , 1999 ) , spectrin ( wechsler and teichberg , 1998 ) , neurofilament ( ehlers et al . , calmodulin binding to the nr1 subunit is ca dependent and occurs with homomeric nr1 complexes , heteromeric nr1/nr2 subunit complexes from expression systems , and nmda receptors from the brain . calmodulin binding to nr1 causes a fourfold reduction in nmda channel open probability , mediating the negative modulation of nmdar function ( ehlers et al . , 1998 ) . the downstream regulatory element antagonist modulator ( dream ) protein , first identified in the nucleus as a ca - regulated transcriptional repressor through its binding to dna at specific regulatory elements , contains four ca - binding ef - hand domains and belongs to the ncs family ( carrion et al . , 1999 ; burgoyne , 2007 ) . dream was named for its ability to block gene expression in its ca - free form via direct binding with the downstream regulatory element ( dre ) sequence in target genes such as preprodynorphin ( ppd ) , c - fos , hrk , na , and ca exchanger ncx3 ( carrion et al . , 1999 ; sanz et al . , 2001 ; gomez - villafuertes et al . , dream was also named calsenilin or kv channel interacting protein 3 ( kchip3 ) ( buxbaum et al . , 1998 ; an et al . , 2000 ) , indicating that dream / calsenilin / kchip3 has multifunctional properties . in the nucleus the dream protein functions as a dimer , whereas outside the nucleus kchip3 is a monomer and regulates the surface expression and gating kinetics of kv4 channels ( an et al . , 2000 ; kim and sheng , 2004 ; scannevin et al . , 2004 ; pioletti et al . dream / calsenilin / kchip3 is preferentially expressed in the central nervous system , as well as in non - neuronal tissues ( link et al . dream / calsenilin / kchip3 knock - out mice display a hypoalgesic phenotype , suggesting a critical role of dream / calsenilin / kchip3 in pain modulation ( cheng et al . , in addition , emerging evidence reveals the role of dream / calsenilin / kchip3 in long - term potentiation ( ltp ) ( lilliehook et al . , 2003 ) and learning and memory ( alexander et al . , 2009 ; fontan - lozano et al . , 2009 ) , suggesting a possible connection between dream and nmda function . kchip13 were initially identified from a rat brain library in yeast two - hybrid ( yth ) screens using the cytoplasmic n - terminal domain ( amino acids 1180 ) of rat kv4.3 as a bait ( an et al . , 2000 ) . similarly , kchip4 from mouse and human was accidentally cloned using the c - terminal 43 amino acid residues of presenilin 2 ( ps2 , amino acids 406448 ) as a bait in the yth system ( morohashi et al . , 2002 ) . kchip4 , also known as calsenilin - like protein ( calp ) , binds to ps2 which is known to facilitate intramembranous -cleavage of -amyloid protein precursor ( app ) ( morohashi et al . , 2002 ) . kchip14 ( 216 256 amino acids ) can co - immunoprecipitate and co - localize with either kv4 from co - transfected cells or kv4 -subunits from tissues , and thus constitute integral components of native kv4 channel complexes ( wang , 2008 ) . kchip14 all share a conserved carboxy - terminal core region that contains four ef - hand - like calcium binding motifs , but have a variable amino - terminal region that causes diverse modulation of kv4 trafficking and channel function ( an et al . , 2000 ; holmqvist et al . , 2002 ; scannevin et al . , 2004 ; findings from co - immunoprecipitation experiments show that dream antibody can immunoprecipitate endogenous nr1 subunit and dream protein from rat hippocampal tissue ( zhang et al . , 2010 ) . in the reciprocal co - ip studies in hek 293 cells expressing dream and nr1 - 1a ( nr1a ) proteins , nr1 antibody can also immunoprecipitate dream along with the nr1 subunit . gst pull - down assays reveal that the n - terminus of dream directly interacts with the nr1a c - terminus , and that the dream - nr1 interaction is sensitive to ca and depends on the ef hand domains of dream ( zhang et al . , 2010 ) . psd-95 is a major scaffolding protein in the postsynaptic density , tethering nmdars to signaling proteins , and is critical for nmda receptor function ( kim and sheng , 2004 ) . wu et al . generated a line of transgenic mice ( tgdream ) overexpressing a dominant active dream mutant , and compared nmda receptor - mediated epscs in tgdream and wild - type mice under conditions of various stimulation intensities ( wu et al . they found that the amplitude of nmda receptor - mediated epscs in tgdream mice is significantly reduced compared to that in wild - type mice ( wu et al . , 2010 ) . in addition , ltd is significantly reduced in tgdream mice whereas ltp is not affected by dream , demonstrating that dream interacts with psd-95 , and that the interaction is negatively regulated by calcium ( wu et al . , 2010 ) . in xenopus oocytes expressing nr2b - containing nmdars alone or together with dream , two - electrode voltage clamp recordings show that , in the absence of dream , the peak currents of nmda channels activated by glutamate ( plus glycine ) are suppressed by dream , and the current decrease is caused by a reduction in the density of nmdars at the cell surface ( figure 1 ; zhang et al . , 2010 ) . fontan - lozano et al . recently provided another piece of evidence that dream negatively regulates the function of nmda receptors ( fontan - lozano et al . , 2011 ) . by taking advantage of mice lacking the dream protein , they demonstrated that the facilitated learning induced by decreased expression of kv4.2 in dream mice requires the activation of nmda receptors containing the nr2b subunit ( fontan - lozano et al . , 2011 ) . this study not only indicates the significance of the balance between kv4 channel function and nmdar activity , but also suggests the formation of a functional complex between dream / kv4.2/nmdars that regulates the synaptic efficacy mediating synaptic plasticity and learning . excitotoxicity is caused by overactivation of nmda receptor function , and inhibition of nmdars can reverse the neuronal toxicity . the data available so far support both the pro - apoptotic and anti - apoptotic roles of dream . in general , the pro - apoptotic role of dream closely correlates with its interaction with presenilins , the production of amyloid beta ( a ) and the modulation in ca signaling , whereas the anti - apoptotic role of dream is conferred by its transcriptional repressor activity on the apoptotic protein hrk . jo et al . reported that hela cells transiently transfected with dream exhibit the morphological and biochemical features of apoptosis and that expression of presenilin potentiates dream - induced apoptosis ( jo et al . , 2001 ) . jo et al . also reported that dream expression increases in either human neuroblastoma sk - n - be2(c ) cells or rat neuroblastoma b103 cells after exposure to a , but no other apoptotic inducers such as staurosporine , thapsigargin , and calcium ionophore a23187 . the pro - apoptotic role of dream is selectively induced during ab toxicity . because of the involvement of presenilins/-secretase in a formation and neuronal death , dream coordinates with presenilin activity to play a crucial role in these processes through binding with the c - terminus of presenilins ( jo et al . , 2003 , 2004 ) . stably expressed dream in h4 neuroglioma cells which showed no initiation of apoptosis in the absence of apoptosis triggers , and the apoptosis - associated caspase and calpain activities were not affected by dream ( lilliehook et al . on the other hand , binding of the transcriptional repressor dream to the hrk gene avoids inappropriate hrk expression and apoptosis in hematopoietic progenitor cell lines ( sanz et al . , 2001 , 2002 ) . nevertheless , the precise function of dream in pro - apoptosis and anti - apoptosis remains to be explored . from our previous observations , we noticed that cell viability is affected by the amount of exogenous dream gene and the method of transfection . however , we could not observe any obvious morphological changes associated with cell death after the transfection of dream . to prove the cytoprotective role of dream , we utilized cell lines and primary cultured hippocampal neurons with dream overexpression or sirna - mediated knockdown , and evaluated lactate dehydrogenase ( ldh ) leakage and propidium iodide ( pi ) uptake both in nmda and oxygen - glucose deprivation ( ogd ) -induced excitotoxic injury models ( zhang et al . , 2010 ) . administration of nmda markedly increases the number of pi - positive cells ( dead cells ) in nmdar - transfected cho cells , whereas co - expression of dream greatly reduces pi - positive cells ( zhang et al . , 2010 ) . ogd is commonly used in vitro to mimic ischemia - reperfusion insult to the brain , and ogd treatment induces a significant increase of ldh release ( dawson et al . , 1994 ) . with over - expression of dream , however , ogd treatment induces a smaller increase in ldh release . these results indicate that the over - expression of dream attenuates nmdar - mediated excitotoxicity ( zhang et al . , 2010 ) . we have previously tested the effect of dream sirna on nmda - induced current and excitotoxic injury in hippocampal neurons ( zhang et al . , 2010 ) . knockdown of endogenous dream with sirna results in an increased amplitude of nmda current recorded by whole - cell patch - clamp assays . after nmda exposure , pi - positive cells in the dream sirna group increase compared with control sirna , indicating the inhibitory effect of dream on nmdar - mediated current and excitotoxic injury ( zhang et al . , 2010 ) . the ncs protein dream / calsenilin / kchip3 acts as an auxiliary subunit and suppresses nmda receptor channel function . this negative modulation of nmda receptor function by dream likely provides a feedback mechanism by which overactive nmda receptors are inhibited . therefore , targeting regulatory proteins of nmdars may represent an alternative approach to treating nmdar - mediated excitotoxic damage and providing neuroprotection . the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest . the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest .

n - methyl - d - aspartate receptors ( nmdars ) are glutamate - gated ion channels highly permeable to calcium and essential to excitatory neurotransmission . the nmdars have attracted much attention because of their role in synaptic plasticity and excitotoxicity . evidence has recently accumulated that nmdars are negatively regulated by intracellular calcium binding proteins . the calcium - dependent suppression of nmdar function serves as a feedback mechanism capable of regulating subsequent ca2 + entry into the postsynaptic cell , and may offer an alternative approach to treating nmdar - mediated excitotoxic injury . this short review summarizes the recent progress made in understanding the negative modulation of nmdar function by dream / calsenilin / kchip3 , a neuronal calcium sensor ( ncs ) protein .

molecular simulations of biomolecules are expressions of their underlying force fields , sampling the interactions of chemically bonded atoms through newtonian approximations of the quantum systems . the typical model , given in eq 1 , is well - known . it approximates the energy of a molecular system as a thoroughly decomposable , easily differentiable sum of terms involving harmonic bonds , harmonic angles , lennard jones repulsion / dispersion terms , electrostatic interactions , and additional parameters guiding the dihedral preferences of the model . the additional parameters are both the least physically grounded and the most frequently edited part of most force fields ; numerous forms of the expressions modifying the dihedral potential energy include screening factors applied between the electrostatic and lennard jones interactions of atoms connected by chains of three bonds , fourier series in the dihedral angles made by such atoms , and coupling terms between fourier series of consecutive dihedral atom chains . changes in these parameters that have made considerable improvements in numerous force fields should be viewed in light of the fact that their adjusments to the system energy are small . the terms describe a regime between the high - frequency motions of bonded atoms and the low - frequency rearrangements of nonbonded chemical groups and appear to be a sort of keystone in numerous molecular models.1many molecular force fields can be classified into several lineages . while some force fields have been developed to reproduce bulk liquid properties , new models today tend to emerge as new levels of quantum mechanical theory become accessible and also as updates based on more extensive fitting data or inconsistencies with known biochemical data . within each lineage , the models tend to evolve while retaining a significant portion of their parameters from previous work . even comparatively minor changes can take years to gain acceptance , however , and any jump to parameter development based on a new level of quantum theory also requires significant effort to validate . the lineages of force fields are a natural consequence of the economics of force field development , particularly in the academic community where novelty and publications are essential . this environment has also driven the evolution of composite models where the various energy terms are shaped by different levels of quantum theory . , incorporated first into the amber ff94 force field , has remained in service for nearly 20 years . these charges were developed according to the kollmann resp method , using hf/6 - 31 g * quantum calculations to provide the target electrostatic potentials . numerous other force fields have adopted the charge model , most of them distinguished by new dihedral fourier series , typically based on quantum calculations at the mp2 level with either a cc - pvtz or 6 - 31++g basis set . while the mismatch in quantum targets implies that the torsion terms are effectively modifying the short - ranged behavior of the charge set to fit a different potential energy surface , the force fields derived in this way have led to impressively stable protein behavior . in 2003 , duan et al . derived an alternative charge model , based on b3lyp / cc - pvtz quantum calculations in a polarizable continuum ( pcm ) solvent intended to mimic the interior of a protein . backbone torsion fourier series were derived specifically for this new charge set at the mp2/cc - pvtz level of theory , also in the context of pcm solvent , to complete the amber ff03 force field . while the charges and single - point energies are derived with similar styles of qm theory , the standard torsion fitting procedure does not incorporate its own pcm solvent , and it is not trivial to extract the energy of the pcm - polarized wave functions in vacuum . molecular mechanics gas - phase energies computed with charges derived in the context of pcm solvent have accordingly been shown to double - count polarization effects , as the statically polarized charges are likely to be further affected by direct interaction with the solvent . it is unclear how this imbalance may have affected the quality of simulations performed with ff03 , but the force field has not become as widely used or further refined as ff94 and its derivatives . in recent work , we devised the implicitly polarized charge model ( ipolq ) , based on a new quantum mechanical method that integrates the condensed - phase environment due to explicit water molecules into quantum calculations at the mp2 level of theory . in this method , the appropriate atomic partial charges of a nonpolarizable model are estimated to be halfway between the charges of the system in vacuum and those of a system fully polarized in the presence of a condensed - phase reaction field potential . the averaging accounts for the polarization energy and instantaneous rearrangements of dipoles in otherwise nonpolarizable models . the choice of tip4p - ew to represent the solution - phase environment while developing the protein force field supports the use of this water model for later simulations . furthermore , the ipolq method provides a basis for understanding why essentially all fixed - charge water models carry a dipole moment of roughly 2.3 d : the dipole is halfway between the true condensed phase dipole of water , estimated to be 2.6 to 2.9 d , and the 1.85 d dipole of water in the gas phase . charges for solutes can therefore be derived under physical approximations that also obtain very similar results to existing water models . while the ipolq approach offers numerous benefits for creating a self - consistent force field , the process of deriving charges is laborious , and our previous work did not assign torsion fourier terms to complete the model . in this article , we will extend the ipolq method to facilitate derivation of complete models , and we will also describe automation of the procedure , which greatly reduces the human effort needed to create new force fields for proteins and drug - like molecules . the amber ff14ipq force field is intended to be a direct alternative to other nonpolarizable amber force fields and contains no new functional forms . however , ff14ipq is also designed to consistently adhere to a specific level of quantum theory , mp2/cc - pvtz . over the course of this study , we found two major obstacles to fitting a molecular mechanics ( mm ) expression to a quantum mechanical ( qm ) potential energy surface ( pes ) and devised our own solutions to each of them . the first concerns the means for adapting the charge set and torsion fourier series terms to work in concert . the second focuses on whether to make corrections for the inability of common mm models to reproduce certain high - energy features of the qm pes . the implicitly polarized charge method produces charges that , given certain assumptions detailed earlier , reflect the appropriate partial atomic charges of a nonpolarizable model intended for simulations in aqueous solution . the atomic partial charges of our ipolq amino acid charge set approximate electrostatic potentials around dipeptides ; these potentials are halfway between the potential calculated for an unpolarized dipeptide ( in numerous conformations ) in vacuo and the potential calculated for the dipeptide ( in the same conformations ) after its electron density has been polarized by a solvent reaction field potential due to the time - averaged tip4p - ew water density sampled around each conformation . the electrostatic potentials needed for fitting charges in the condensed phase are laborious but straightforward to compute . it is less obvious how to extract the internal potential energy of the dipeptides in the condensed phase or make an equivalent averaging to derive an appropriate pes for fitting torsion parameters . zgarbov and colleagues solved this problem by computing quantum mechanical energies of solutes at the hartree fock level of theory in the presence of the cosmo continuum solvent model , removing the solute solvent interaction energy and then comparing to molecular mechanics potential energies computed with a pb solvent model . in effect , they compared the internal energies of the qm and mm systems in the presence of equivalent continuum solvents . in our case , it is not as straightforward to decompose single - point energies at the mp2 level of theory , nor is it as certain whether a quantum - mechanical implicit solvent model could be substituted for tip4p - ew . we instead chose to extend the ipolq methodology and offer an alternative method for deriving torsion fourier series from gas - phase pess when the accompanying charge sets approximate polarization in a condensed - phase environment . it would be simple to derive torsion parameters for a set of charges derived strictly from the electrostatic potentials for solutes in vacuum : compute the single - point energies of many additional solute conformations at the same level of quantum theory , again in vacuum , and fit the mm energies of each conformation to match the qm results . furthermore , the role of the torsion fourier series is to artificially correct errors in the nonbonded electrostatic and van der waals interactions between atoms on either end of a dihedral ; the pes of a rotatable bond is mostly captured by the nonbonded parameters . we therefore sought a way to express the set of ipolq charges for a solute of interest , q , as a perturbation q of charges q derived for the solutes using only the vacuum quantum calculations:2it is simple to compute torsion parameters to match an mm pes q to a qm pes computed in vacuum . changes in the pes of a molecule s rotatable bond due to immersing it in water could then be assumed to be adequately represented by changes in local dipoles as expressed by q . this appears to be a safe assumption , given that q q and that the van der waals parameters , which also strongly influence the potential energy changes due to rotation about a bond , are constant across both phases . however , the linear least - squares fit from which our ipolq charges q and our proposed vacuum charges q are derived is able to obtain many different solutions with similar levels of accuracy , particularly for buried atoms whose charges are not well determined . in our earlier work , we used large numbers of conformations of each solute in order to guard against this behavior . however , because residual indeterminacy could exist in either q or q , the difference q could still be amplified . we therefore extended the ipolq least - squares fitting procedure to fit both q and q simultaneously , with additional restraint equations to keep q small . the results are solutions for each of q and q related by a minimal , smooth perturbation q . the original matrix equation constructed in the ipolq fitting procedure may be expressed in shorthand:3here , a is the fitting matrix whose elements are derived from the molecular coordinates and the kernel of coulomb s law . each row of a describes one linear equation through which the partial charges of a molecule in some conformation create an electrostatic potential at a nearby point in space . the electrostatic potential found by the target quantum method is stored in the vector u. each column of a solves for an independent charge variable ; if multiple atoms of a molecule are constrained to have the same charges , then they contribute to the same column . appended to a , the matrix v contains additional restraint equations that penalize the least - squares fit when particular charges stray from target values encoded in the vector vt appended to u. a more detailed statement of the linear least - squares problem is as follows:4here , the elements of the fitting matrix a(p , i)j pertain to the influence of all instances of the jth adjustable charge on the ith fitting point around the pth molecular conformation . the solution vector u on the right - hand side is filled with values of the electrostatic potential measured at many points rp , i . as was mentioned , there may be multiple instances of the same adjustable charge in each molecule . a general definition of the elements of a is then5where the summation runs over all atoms c bearing the fitted charge described by variable j. the various molecular conformations may be different poses of the same molecule or even a collection of poses of many different molecules . in the latter case ( which best describes the original ipolq fit we performed ) , the matrix a becomes a sparse matrix due to the fact that not all systems contain all charge variables , but it is not often sparse enough to warrant a special storage format . this can require a large amount of memory , but simultaneous solution of all charge variables permits multiple systems to share the same charges . in our ipolq fit , for instance , we chose to follow the cornell charge set convention of giving similar charges to backbone n , h , c , and o atoms involved in the peptide bonds for neutral , positively charged , and negatively charged amino acids , compressing what could have been more than 80 charge variables into just 12 , in the hope that this would expedite development of a common set of backbone torsion potentials . the restraint matrix v is appended to the fitting matrix a in order to keep the charges of certain atoms small and also to restrain the overall charges of certain groups of atoms : the fit is heavily penalized by vnc = 1.0 10 kcal / mole if the sum of atomic partial charges of neutral or ionic residues differs from the appropriate net charge ( nc ) , qk for the kth system . in eq 5 , p , j is 1 if the pth system contains the jth charge variable . to put all restraint constants vsa , j for specific atomic charges on the same scale , these constants were set proportional to the number of times nj the jth charge variable appeared in a6 in our ipolq fit , we set vsa0 to 1.0 10 kcal / mole . the extended least - squares fit for deriving ipolq charges as a perturbation of vacuum charges solves for twice as many variables based on the same molecular conformations . the extended problem to solve is simply7 in this system of equations , the original fitting matrix is replicated into several blocks of the extended system , and the solution vector contains electrostatic potentials computed from each molecular conformation in vacuum as well as the vacuum potential averaged with the electrostatic potential computed in the condensed phase u. the original restraint equations are still present , but they apply only to the vacuum charges q ; additional restraint equations are added to strongly force the sum of all perturbation charges in each system to zero . an additional block matrix loosely restrains perturbation charges individually to zero using stiffness constants vgp scaled by the number of instances of each ( perturbation ) charge variable nj ; numerical values of vgp are discussed below . in eq 7 , i is the identity matrix ; every perturbation charge is individually restrained . the extended system , which requires slightly more than four times the memory of the original problem and eight times the computation cost , yields q by applying eq 2 . the traditional least - squares approach for fitting torsion parameters is composed as shown in eq 8 . the fitting matrix is composed of the kernels t(p , i)j of the jth torsion term to be fitted for the ith conformation of the pth system , while other columns store energy adjustment constants cp applied to the pth chemical system present in the fitting data . the solution vector u in this equation contains the qm single - point energies of each conformation , less the average single - point energy of all systems with the same chemical composition , less the mm energy arising from other terms in the force field . the torsion terms are fitted so that the sum of their contributions compensates for inaccuracies in the molecular mechanics model to bring the relative energies estimated for different conformations of the same molecule into agreement with the qm pes.8 in principle , it would be feasible to leave the average single - point energy of each chemical system in u , but this would lead to a poorly conditioned matrix , as the energy adjustment constants cadj , here c1 ... ck , would need to take on very large values . it is also possible to compose u based on system conformational energies relative to the structure with the lowest quantum mechanical energy and to omit the constants cadj for each system . however , no quantum method is perfect , and omission of cadj would amplify the influence of a particular conformation over the fitted parameters . it is well - known to force field developers that the bond and angle terms that approximate high - frequency motions in molecular simulations are not consistent with the qm target models ; aside from the breakdown of the harmonic approximation with increasing strain , the equilibrium bond length or angle is dependent on the chemical context . the inconsistency elevates the mm energies for structures optimized by qm methods and is therefore a sort of contaminant when fitting lower - frequency degrees of freedom , in particular torsion fourier series terms . concern about introducing error by this method has led numerous groups to fit torsion fourier series by positing that a conformation of a molecule is defined strictly by its torsional degrees of freedom and making two slightly different variants of that conformation : relaxing all bond , angle , and nonbonded degrees of freedom by mm and qm approximations , respectively . the objective then becomes to fit torsion parameters such that the coordinates optimized by mm produce an energy most like the single - point energy found for the nuclear coordinates optimized according to qm . this method , hereafter the tandem optimization approach , relaxes much of the inconsistency arising from high - frequency degrees of freedom in the two pess , but it also introduces a new source of error in the nonbonded interactions between the different coordinate sets . while many investigators have accepted the trade - off , we performed an independent analysis , which led us to reject the approach in favor of a direct , one - to - one mapping between coordinates and energies . we analyzed the trade - off between error removed by relaxing high - frequency degrees of freedom and error introduced by changes in nonbonded interactions by computing mm energies for mm and qm optimized variants of 648 conformations in each of 15 amino acid dipeptides ( over 9000 pairs of energies ) . the conformations sampled 1 and 2 at 20 intervals while the backbone was held in an -helical or -sheet conformation ; 1 , 2 , , and dihedrals were held fixed during both optimizations . mm optimizations and all mm energy evaluations were performed with a new variant of the amber ff99sb force field provided to us courtesy of james maier and professor carlos simmerling s research group . qm optimizations , also performed for us by james maier , were performed at the mp2 level with the 6 - 311++g basis set . the mm and qm approximations are similar to those we have chosen for development of amber ff14ipq . first , we focused on the difference in the molecular mechanics energy computed for each variant of a given conformation ; by construction , the mm optimized variant was always lower in energy , as scored by amber ff99sb . table 1 gives the differences computed for 628 conformations of each dipeptide . by decomposing the mm energy , it is apparent that the mm and qm models do disagree about the optimal bond lengths and angles , and the energy differences in there terms are greater , sometimes much greater , than the energy differences arising from nonbonded terms . at face value , this result would appear to support the tandem optimization approach . each difference is given as an average standard deviation , in kcal / mol . includes lennard jones ( lj ) and electrostatic ( elec ) contributions . when fitting torsion fourier series to match a molecular mechanics pes to a qm pes , the objective is correct relative energies ; the mm pes is otherwise very far removed from the qm pes , which includes factors such as electron nuclei interactions and nuclear repulsion . the mean energy of the qm pes is therefore subtracted , and an additional constant cadj is included in the fit to arbitrarily adjust the energy of a particular molecule , regardless of conformation , up or down , to bring the two pess into agreement . because of this , the mean energy differences between the mm and qm optimized variants of each conformation will fall through the fit , absorbed into cadj . if the molecular mechanics and quantum models optimize bonds to different lengths , but the disagreement is consistent across all conformations of the molecule , then the mismatch in these high - frequency terms will have no effect on the fitted torsion parameters . standard deviations of the difference , the variability of the disagreement between the qm and mm pes , is a much better indicator of possible contamination in a parameter fit . table 1 shows that bond energy differences have a small deviation but angle energy differences carry a much higher deviation and therefore might contaminate the torsion parameter fit . comparing the standard deviations in the energy differences arising from the sum of bond and angle terms to those arising from all nonbonded terms suggests that the tandem optimization approach offers a marginal benefit in most cases but is detrimental in cases such as ser and thr . the total nonbonded energy differences appear to be smaller than those of bonds , but the nonbonded energy is a sum of many interactions . the major amber molecular dynamics engines conveniently break nonbonded interactions into 14 contributions between atoms at either end of a dihedral group and all other contributions . these two parts of the lennard jones interactions are uncorrelated , but the sum of electrostatic 14 interactions tends to be strongly anticorrelated with the sum of all other electrostatic contributions , as shown in table 2 . as a consequence , the standard deviations of the differences in the electrostatic 14 nonbonded terms are as large or larger than the deviations in total nonbonded energy differences . jones short - ranged interactions . ) these 14 interactions form the base of the torsional pes around each rotatable bond and , of all nonbonded interactions , are the most strongly connected to the torsion potentials . this suggests that the contaminant introduced by the tandem optimization method , a mismatch in the nonbonded interactions , is actually worse than the contaminant being removed , the mismatch in energies due to high - frequency modes . like table 1 , this table compares mm energies computed for each of two optimized variants of a dipeptide conformation . we conclude that it is at least as sane to accept errors that might be introduced by a mismatch between molecular mechanics bond and angle geometries and those of the quantum target as to accept errors arising from a mismatch in electrostatic or steric interactions . for this reason , we chose to submit the mm optimized coordinates , which reflect the states that the molecular mechanics model will actually explore in simulations , to single - point quantum calculations and demand that our fitted molecular mechanics model reproduce the qm single - point energies calculated for precisely the same set of nuclear positions ; details of our procedure for generating the actual fitting data for torsion fourier series can be found in the methods . due to the one - to - one mapping of coordinates and energies , the fitting data suffers some contamination from angle terms in the mm approximation being inadequate to describe the qm pes . these terms are roughly 1 order of magnitude higher in energy than the torsion terms or nonbonded interactions in the mm model ; as such , they could make erroneous contributions to the energy with the risk of torsion parameters becoming fitted against noise . however , the majority of angle strain is orthogonal to the torsional subspace of molecular motions . with adequate sampling of the torsional degrees of freedom in the fitting set , and with a consistent optimization of each set of coordinates relative to one model or the other we designed the amber ff14ipq force field to be similar to previous amber nonpolarizable force fields . bond and angle parameters were taken from the existing ff99sb force field , along with most lennard jones parameters . atomic partial charges were refitted according to the updated ipolq fitting procedure described in the theory section , using the same data produced in our previous study , but for future force field development , we automated the ipolq fitting cycle in the amber mdgx program . in our derivation of ipolq charges for amino acid side - chain analogues , we adjusted the lennard jones parameters of polar atoms in order to bring the computed hydration free energies into agreement with experiment . in this respect , the development philosophy of ff14ipq resembles that of the new gromos 54a8 force field . however , the interactions of these atoms with water were essentially the only changes that were of consequence during our hydration free energy calculations , and we found that larger radii fitted for most of the atom types intensified 14 nonbonded repulsion , making torsion parameters much more difficult to fit . to bring the lennard jones changes into ff14ipq , we made them applicable only between the polar atom types and the tip4p - ew oxygen type . the lennard jones lorentz berthelot mixing rule is broken for the interactions of these atom types with water , in the manner that charmm36 makes use of nbfix terms . with the bonded and nonbonded parameters established , the majority of the development in ff14 focused on torsion fourier series terms . a new module was added to the mdgx program to aid users in computing ipolq charges for arbitrary molecules . for a standard resp procedure , researchers must have a set of conformations of their molecule of interest ; the conformations serve as inputs to tools such as the red server , which manages the necessary quantum calculations and performs the restrained charge fit . with the new ipolq module , researchers must have a set of conformations of their molecule immersed in the solvent of interest . the mdgx program will read the solvated conformations as restart files along with an appropriate topology and begin dynamics with the solute molecule held in a fixed position . the mdgx program automates the process of collecting the solvent charge density , computing the solvent reaction field potential ( srfp ) , preparing inputs to a quantum program , and launching the calculations to ultimately produce grids of electrostatic potential computed for the molecule in vacuum and in the influence of the srfp . the mdgx ipolq module improves on the original protocol , always applying a shell of point charges around the average solvent charge density taken from the simulation . the charges in the shell are fitted to reproduce the srfp due to infinite electrostatics present in the simulation in the context of a quantum calculation on an isolated system . furthermore , users can specify up to three concentric charge shells to increase the accuracy of the srfp , evaluate the srfp at additional sites throughout the solute volume , and even specify an interior shell of charges to reproduce the srfp in and around the solute without including point charges nearer than an arbitrary distance from solute atoms . the final feature could be useful for researchers who are concerned about qm basis functions adversely interacting with solvent charges , although we did not notice any effects on the ipolq results for some systems we tested with mp2/cc - pvtz calculations used in the original ipolq protocol ( data not shown ) . in summary , mdgx manages a cycle of the ipolq procedure for a conformation of the molecule of interest ; the output of independent runs on multiple solute conformations can be pooled and sent to the mdgx charge fitting module to derive a new charge set , which can then be used to update the solvated system s topology and start another round of calculations with the ipolq module . in this manner , mdgx manages nearly all aspects of the ipolq procedure until the solute charge model converges . the module supports both gaussian and orca quantum chemistry packages , performs dynamics with modest parallelism and efficient cpu execution , and launches quantum chemistry programs for parallel execution in accord with the molecular dynamics run to avoid wasting cpu cycles . data for the fitting matrix a was derived from the same quantum data as the original ipolq charge set . the same protocol was followed for selecting fitting points from the electrostatic potentials evaluated around each conformation , but , due to memory constraints in the much larger matrix problem , only 3750 points per conformation were selected . ( our earlier work showed that anywhere from 3000 to 5000 points per conformation yielded convergent results in the fitted charges . ) this method implies two charge sets , one valid in vacuo and the other in solution . while the complete release version of ff14ipq takes the charges valid in solution , q + q in eq 7 , we also considered a variant based on q in some studies of systems in vacuo , hereafter named v - ff14 and not distributed for general use . the fitting set for amber ff14ipq was created by molecular simulations and energy optimizations performed with the amber ff99sb force field and new variants under development in the simmerling group . all of these mm models are based on the cornell charge set and have been edited over more than a decade . the most significant feature of the force fields , to us , was the similarity in form and some parameters to the proposed amber ff14ipq force field . while it is impossible to completely sample the available conformational space in the fitting data , we chose the most recent amber force fields to simulate each molecule in the hope that the these models would sample the unrestrained degrees of freedom in a manner that reveals their biases so that those biases could be eliminated in ff14ipq . nevertheless , critical dihedral angles near the center of each molecule were restrained to various values as we populated the fitting data set ; the exact details of the simulations , such as cutoff , time step , and solvent model , are therefore of secondary importance to the choice of protein - like molecules , the array of restraints , and the number of snapshots collected for single - point energy calculations . the most common structures included in our fitting data set were blocked dipeptides , ace - xx - nme , where xx is an amino acid , although we also included ace - ala - ala - ala - nme and ace - gly - gly - gly - nme tetrapeptides to sample backbone and angles and ace - xx - xx - nme tripeptides to further reduce the influence of the blocking groups on these backbone dihedrals . in all , the torsion fitting data set consisted of nearly 28 000 structures whose single - point energies were computed in vacuum by mp2/cc - pvtz calculations . to sample side chain conformations , we obtained a set of some 17 000 dipeptide conformations for neutral asp , asn , deprotonated asp , cys , -protonated his , -protonated his , ionic his , ile , leu , phe , ser , thr , trp , tyr , and val from james maier and the simmerling group . each amino acid was sampled in 628 conformations , exploring 1 and 2 angles at 20 intervals while holding the backbone in either an -helical or -sheet conformation . these amino acid conformations were among those used in our analysis of the effects of unrelaxed high - frequency degrees of freedom described in the theory section . to obtain a better sampling of backbone conformations and to reduce the risk of side chain dihedrals becoming coupled to particular backbone conformations , we generated another 180 conformations of each dipeptide by sampling and individually at 20 intervals with no restraints on any other degrees of freedom . to decorrelate the unrestrained degrees of freedom , we ran molecular dynamics simulations of each dipeptide while progressively advancing the and restraints . dynamics were run in baths of tip4p - ew water at 300 k , with 100 ps intervals between each snapshot . or were incremented with every snapshot , but each angle was completely rotated five times ; as a result , separate snapshots at the same or coordinate were spaced by 1.8 ns of dynamics . to sample backbone conformations , reduce the dependence of the fitted parameters on ace and nme blocking groups , and increase side - chain sampling , we included a set of over 7000 tripeptides . we sampled all 20 amino acids with ala , gly , or ser adjacent in either direction in the peptide chain , restraining and individually at 15 intervals with no other restraints and running dynamics on each system at 450 k to generate multiple conformations at each value of or . because there were so many of these systems , a generalized born solvent was used to generate the conformations . to further sample backbone conformations of two key amino acids , alanine and glycine tetrapeptides were sampled in 1296 conformations each , sampling the central residue s and space at 10 intervals on a two - dimensional grid . aside from simultaneous restraints on and , no other restraints were used as dynamics were performed for 100 ps between snapshots to decorrelate other degrees of freedom . snapshots of molecular dynamics trajectories are not suitable for direct incorporation into quantum calculations because of the potential for bond and angle high - frequency energy terms to contaminate the fitting data , as explained in the theory section . however , if the contributions from these terms are relaxed out with respect to either a mm or qm approximation , then the single - point qm energies calculated for each conformation then constitute an acceptable fitting set . we optimized each conformation with restraints on dihedrals involving four heavy atoms ( but not hydrogens ) in order to preserve the diversity of conformations created in our simulations while relaxing as much angle and bond strain as possible . we chose to optimize conformations with respect to the mm approximation because the calculations were cheap , allowing us to devote more time to evaluating single - point energies . also , we felt that it was more instructive to evaluate the local minima of an mm model with respect to the mp2/cc - pvtz target , to examine states that an mm model might have a propensity to populate and to verify their energies with qm in the fitting data . all torsion parameters for the amber ff14ipq force field were fitted simultaneously . while the length of each fourier series and also the phase angles were taken from ff99sb , new atom types added by the simmerling group as well as new atom types required by the ipolq charge set for amino acids were included , and the glycine c and proline backbone nitrogen atoms were given their own unique atom types distinct from other amino acids . including a new atom type implied replicating all bond , angle , and fourier series terms pertaining to the original type , thereby creating additional parameters to fit . all fourier series amplitudes were reoptimized by the standard linear least - squares approach in a single matrix equation to obtain the best overall fit for terms that appear in different contexts across multiple residues , as described in the theory section . another new module of the mdgx program was created to perform the atom type branching , set up the matrix equation , and perform the linear least - squares fit . while it was an advantage to have the extensive fitting set described in the preceding section , all torsion fourier terms were still restrained loosely toward zero by a penalty of 2.0 10 kcal / mol ( multiplied by the number of times each parameter appeared in the fitting matrix ) to keep the amplitudes small and avoid overfitting . torsion amplitudes were optimized to make molecular mechanics energies of the di- , tri- , and tetrapeptide systems computed in vacuo with charges from q agree with mp2/cc - pvtz single - point energies of the peptides also computed in vacuo . as explained in the theory section , we assume these torsion parameters to be transferable to describe the behavior of solvated peptides when paired with q. as a preliminary test of force field performance , we evaluated fidelity to the underlying mp2/cc - pvtz benchmark over the course of molecular mechanics energy minimization . the molecular conformations in the training set were already optimized with respect to a very similar mm model , but in the presence of one or more restraints on dihedral angles . energy minimization of each conformation of all systems found in the fitting set was performed in vacuo with v - ff14 , the variant of ff14ipq substituting q for the implicitly polarized charges found in the release version . new qm energy calculations were then performed on the resulting structures optimized by v - ff14 . these new conformations and energies were added to the training set , and new energy optimizations were performed in an iterative fashion until v - ff14 could score structures created by its own optimization consistently with those in its training set . building on the fitting data of small peptides , we computed potentials of mean force ( pmfs ) for pairs of dihedral angles in blocked dipeptides at 298 k with a standard two - dimensional umbrella sampling technique . we collected data in 1296 windows spaced by 10 in and or in 1 and 2 , depending on the amino acid and pmf . each window was seeded from a continuous , incrementally restrained simulation similar to that used to generate conformations for the corresponding tetrapeptides prior to mp2/cc - pvtz calculations . after seeding , each window dihedral angles were initially restrained by an 8 kcal / molrad harmonic penalty function , but if some of the 10 bins were left undersampled , then more windows were added on a 5 grid with 16.0 kcal / molrad dihedral restraints to completely fill out the pmf . we began with simulations of penta - alanine ( hereafter , ala(5 ) ) , the -helix k19 , and -hairpins chignolin ( starting structure pdb entry 1uao(32 ) ) and the gb1 hairpin from the c - terminal fragment of protein g. furthermore , we performed microsecond length simulations of the globular proteins gb3 ( starting structure pdb entry 1p7e , similar to protein g and containing a motif homologous to the gb1 hairpin ) and lysozyme ( 4lzt ) in solution . each system was equilibrated with protein backbone atoms held under progressively decreasing restraints for up to 7 ns , depending on the size of the system . all simulations were performed with tip4p - ew water ( the water model used to develop our solution phase charge set ) in sufficient quantity to enclose the peptide and solvated protein systems by at least 10.0 within octahedral boxes after equilibration under constant pressure dynamics . nonbonded interactions were calculated with a 10.0 cutoff on lennard jones interactions , a homogeneity approximation for long - ranged van der waals interactions , and smooth particle - mesh ewald electrostatics with direct and reciprocal space accuracies near 1 part in 200 000 ( this corresponds to a direct sum tolerance of 5.0 10 and a direct space cutoff of 9.0 with the default mesh grid spacing of up to 1 ) . a 2 fs time step was used in all simulations , along with the shake and settle algorithms to constrain the lengths of bonds to hydrogen . most of the small systems were simulated at 277 k , by a langevin thermostat with collision frequency 3/ps , to replicated nmr conditions . the gb1 hairpin system was simulated at 298 k to investigate -sheet stability at room temperature . the updated ipolq procedure described in the theory section uses the same fitting data as the original method to derived q , but it also derives q , a set of charges appropriate for modeling electrostatics in vacuo . these two charge sets would be independent , and the updated q would be the same as the original , but for the fact that the restraint equations used to temper the fit of the original q instead temper values in q , and the perturbation q that relates q to q by eq 2 is subject to its own set of restraints . in our protocol , the size of q is controlled by setting the parameter vgp in eq 7 . larger values of q could make it difficult to transfer torsion parameters developed for systems in vacuum to simulations in solution , but too small a q would imply q q , and neither set of charges would describe its intended environment well . we chose to set vgp to 0.005 kcal/(mole - instance ) , where e is the charge of a proton . this is half the stiffness of restraints holding charges of underdetermined sites to zero in this and earlier ipolq derivations . the fitting matrix is not well conditioned in either case : for main - chain amino acids , we found the ( 2-norm ) condition number of the matrix a in eq 3 to be 1.8 10 and that of the extended matrix in eq 7 to be 2.2 10 . the possibility of a worst - case loss of precision compels us to use double precision arithmetic for what is already a very large matrix , but the results are nonetheless consistent . the earlier ipolq protocol , for instance , judged convergence of the charge set by the point at which successive generations changed the fitted charges by less than the inclusion of one of the fitting conformations . ( these changes were on the order of 0.01e . ) as before , we are more concerned with the effect that a different set of constraints might affect the outcome . the value of vgp we chose seems to have minor effects on the overall accuracy of q and q. averaged over all systems and conformations , the original q reproduces the target electrostatic potential with 1.82 kcal / mole root - mean - squared error ( rmse ) , whereas q + q reproduces the same potentials with 1.89 kcal / mole rmse . the effects on selected systems are shown in the lower panel of figure 1 . over the range we tested vgp , the effects on electrostatic potential rmse appear to begin to approach an asymptotic limit ( half the difference between the electrostatic potentials computed in vacuo and in the condensed phase ) . if vgp is tuned even lower , then the rmse of q + q actually improves over the original q due to the fact that the perturbation charges are then much less restrained than the original charges . however , tuning vgp too low leads to undesirable effects such as ala c taking a charge of 0.21 at vgp = 0.0025 , as opposed to 0.04 in the original q. accuracies and charge perturbations for all amino acids at vgp = 0.005 are shown in table 3 . effects of the coupling constant vgp on q and the accuracy of electrostatic potentials for condensed - phase systems . vgp determines the strength of the harmonic penalty restraining all charges q toward zero . values of the root - mean - squared error ( rmse ) describe electrostatic potentials projected by each dipeptide s molecular mechanics charge set relative to the qm target . vgp = 0 corresponds to partial charges by the original ipolq method before the extension , which allows us to express them as a perturbation to charges appropriate for simulations in vacuo . errors and charge variations are expressed for a restraint of vgp = 0.005 kcal/(mole - instance ) applied to all perturbation charges . root - mean - squared error ( rmse ) of fitted mm charges in replicating the qm target electrostatic potential . the target is the average electrostatic potential of the solute s mp2/cc - pvtz wave function in vacuum and in the solvent reaction field potential due to tip4p - ew water . the target is the electrostatic potential of the solute s mp2/cc - pvtz wave function in vacuum . maximum absolute deviation in partial charges unique to this residue ; backbone atoms frequently showed q of 0.060.10 , as shown in the supporting information . at vgp = 0.005 , the majority of values in q are smaller than 0.05e , as shown by the top panel in figure 1 ; the larger values tend to be in buried methyl carbons , whose charges are small to begin with , and the atoms of polar head groups . neither of these changes are likely to make torsion parameters developed with q less transferable because the electrostatic potential energy surface will change only if atoms with large values of q can rotate around nearby atoms possessing large of charges of their own . the perturbation does not , in fact , bring q + q to the polarity of the original q : the carbonyl carbon atom becomes less positively charged by as much as 0.05e , the oxygen becomes less negatively charged by 0.03e , and the polarity of the n however , the perturbation does make the backbone significantly more polar than q alone would describe it . as a consequence , the ipolq protocol continues to model protein backbones with more polarity than the cornell and duan charge sets , maintaining a principal finding of our earlier study . torsion fourier series terms for all amino acids were fitted by a linear least - squares approach as described in the methods . the preliminary set of 28 000 structures and mp2/cc - pvtz single - point energies is , to our knowledge , the most extensive potential energy surface ever employed for this type of molecular mechanics parameter development . in addition to the atom types present in amber ff99sb and new atom types for c atoms introduced by the simmerling group , we included new types for each of the lennard jones modifications introduced to adjust hydration free energies of amino acid side - chain analogues in the initial ipolq derivation . we also included new types for the glycine c atom and proline backbone nitrogen because of the unique chemical and bonding structures around these atoms . when the electrostatics of each di- , tri- , and tetrapeptide are described by q , the fitted torsion fourier series terms complete a force field and describe the molecular mechanics energy of the system in vacuo . the rmse of these energy estimates relative to the mp2/cc - pvtz single - point energies for di- and tetrapeptides is given in table 4 . also given in this table are the molecular mechanics ( mm ) energies obtained by reoptimizing a much smaller set of torsion fourier series terms , the parameters found in amber ff99sb , in conjunction with our newly derived q and the mm energy estimates that would have been obtained if no torsion terms were used . the overall contributions from the torsion terms are often small , only reducing the rmse of mm energy estimates by 1.3 to 1.5 kcal / mol relative to a model that has no such terms . the overall size of the torsion terms contributions may understate their importance , given the number of published force field improvements based on changes in these terms . in all cases , the charge set q fitted to reproduce the electrostatic potentials of blocked dipeptides in vacuo was used to estimate the molecular mechanics energy of each blocked dipeptide in vacuum . all energies are given in kcal / mol . the v - ff14 force field : q has been substituted for the implicitly polarized charge set in the release version . the ff99 force field , with q as derived for v - ff14 ( identical to the force field in the first column , but with a smaller torsion parameter space ) . the new ff14ipq force field has many more torsion fourier series terms than the ff99sb force field : 427 to 67 . most of the new parameters were added by including the simmerling group s unique c atom types . while the total number of torsion parameters increases nearly 7-fold from ff99sb to ff14ipq , table 4 shows that the number of parameters expressed in any particular system doubles at most . new atom types c8 , 3c , and 2c for c have added new parameters for 1 and 2 , which distinguish the side - chain rotamer energetics for protonated his , arg , and lys , the amino acids ile , thr , and val , and other amino acids . this partitioning is just one of many possible approaches , but it seems to have achieved a similar effect to an earlier extension of the ff99sb force field , which improved the rotamer propensities of residues ile , leu , asp , and asn by adding new atom types and fitting the newly minted torsion parameters to mp2/(aug)cc - pvtz energy profiles . the ff14ipq parameter space makes notable improvements over the ff99sb parameter space for ile , asp , and asn ; it appears that providing only a single atom type for c forces the same set of torsion parameters to average between disparate potential energy surfaces and causes difficulty fitting the data . tg atom type for glycine c likewise decouples two distinct potential energy surfaces : the dihedral angles between a backbone -hydrogen and the backbone polar hydrogen , or the backbone carbonyl oxygen , are sampled twice ( roughly 120 apart ) in glycine and only once in all other amino acids . these dihedrals contribute directly to the protein and propensities , and the new tg atom type improves the fit for glycine tetrapeptide by roughly 0.5 kcal / mol rmse and the fit for all other dipeptides by up to 0.1 kcal / mol rmse ( data not shown ) . we also tried adding distinct atom types to the glycine -hydrogens , but this was not as effective as adding the tg atom type . in contrast , distinguishing the proline backbone nitrogen as tn has a negligible effect on the fit for any residues but the proline itself . given the inherent difficulty in sampling dihedrals related to this atom type , we considered whether to include it at all , but it does decouple the proline profile from other amino acids and serves as a handle for future parameter development . with the expanded parameter set , the torsion potentials contribute more and more to the total molecular mechanics energy ; their total contributions in ff14ipq are frequently double their contributions in the refitted ff99 parameter set . however , as shown by the final column of table 4 , a model with no torsion potentials is only perhaps 1 to 2 kcal / mol less accurate . the larger torsion potential contributions in ff14ipq are being counterbalanced by larger energy adjustment constants cadj , which never appear in the force field . the number of adjustable terms and the size of cadj are only weakly correlated ( pearson coefficient 0.40 ) , perhaps because many copies of the torsion terms can appear in the total mm energy and also because the amplitudes of the terms themselves are so variable . despite this , the sizes of cadj may be a useful indicator of whether a model is overfitted . the increases in cadj led us to track the sampling of each torsion parameter throughout the fitting set , as shown in the supporting information . with our exceptionally thorough data set , the torsion parameters related to rotatable bonds seem to be well - sampled , on the whole and in each system - dependent context in which they appear . even with this degree of coverage , however , these fitted torsion parameters are not the final settings distributed as ff14ipq , whether in combination with the partial charge set q or q ; they are , rather , a first draft . the following section presents results obtained when the draft model was allowed to guide geometry optimizations and thereby expose the ways in which its parameters could conspire to accumulate errors over the course of a simulation . energy optimization served as a preliminary test of our fitting program and also of the robustness of our fitting data . energy minimization of the fitting set s structures using the amber pmemd program and the preliminary v - ff14 force field confirmed that the mm energy computed for each initial structure matched that produced by the mdgx fitting module after solving eq 8 . after energy minimization in vacuo , some initial structures converged to the same final configuration , but a number of local minima were still produced for each di- , tri- , and tetrapeptide system . we computed mp2/cc - pvtz single - point energies for new structures in three tripeptide systems shown in figure 2 and compared them to the mm energy estimates to test whether the new model s 427 parameters were prone to overfitting . energies of dipeptide and tripeptide conformations before and after energy minimization with the preliminary v - ff14 force field . all molecular mechanical energies are adjusted according to the adjustment constants found while fitting torsion parameters ; quantum mechanical energies are normalized to a mean of zero . hence , the energies of conformations found in the fitting data ( black diamonds ) lie directly on the trendlines , and the energies of system conformations optimized according to the preliminary v - ff14 ( red , open squares ) may not track it . the results in figure 2 show that v - ff14 is often able to guide small peptides into configurations that mp2/cc - pvtz calculations agree have lower potential energy . however , after energy minimization , v - ff14 tends to estimate the energy of each configuration as being more favorable than the mp2/cc - pvtz single - point energy . the degree to which v - ff14 departs from the 1:1 trendline with its benchmark may be small , as shown in table 5 . however , geometry optimizations in systems with his , arg , and lys led to mm energies that departed severely from the qm benchmark , despite the agreement maintained in the training set . by adding the structures freely optimized by v - ff14 back into the training set , the second generation v - ff14 nearly eliminated departures from the qm benchmark in arg and his and also showed minor improvements in its ability to optimize the structures of most other residues . v - ff14 is able to guide unrestrained energy minimizations of structures in its own training set and reduce the internal potential energy by up to tens of kcal / mol . ( this may be realistic , as most training set structures were restrained in one or more torsional degrees of freedom . ) however , when re - evaluated at the mp2/cc - pvtz level , the resulting structures were often not as optimal as molecular mechanics depicted . negative numbers in the table indicate that v - ff14 strayed from its mp2 benchmark and estimated its optimizations to be too favorable . gaps in the table indicate that a system was omitted from one generation , due to compute cluster downtime or sufficiently low error in the previous generation . lysine presented more of a challenge : as shown in figure 3 , the second generation v - ff14 apparently contained a new artificial minimum that was subsequently found in all optimizations of the dipeptide . we examined the molecular mechanics energies of lysine conformations in the original training set , the second - generation training set , and the final training set with respect to each generation of torsion parameters . torsion parameters describing two of the rotatable bonds generate the severe departures from the benchmark seen in the first generation of the force field . first , a wildcard parameter with 3-fold periodicity describing rotation of the lysine side chain amino terminus takes on an amplitude of 4.99 . in the original training set , every conformation sampled the orientation of the terminus in a staggered conformation of the hydrogens at the crest of the cosine wave . the eclipsed conformation , favored by nearly 10 kcal / mol by this spurious parameter , was not sampled in the training set , and the result was six permutations of amino and aliphatic hydrogen interactions creating a nearly 60 kcal / mol fictitious energy release by adopting the unnatural , eclipsed conformation in every structure optimized by the first generation of the force field . a more serious problem occurred in the four - term fourier series describing interactions between the atom type c8 , coined by the simmerling group for lysine and arginine c atoms , and the backbone carbonyl carbons . because of its unique appearance in lysine and arginine , this fourier series strongly influences the backbone angles adopted by the residues . the original training set did not sample a 100 arc in for either residue , however , and the fourier series became fitted to produce an unnaturally low energy in the first generation , approximately 14 kcal / mol more favorable than any conformation of the angle should have allowed . only some of the lysine structures fell into this trap when optimized by steepest descent energy minimization under the first generation of the force field , and the result was the two striations , which can be seen in figure 3 . in the second - generation training set , sampling in these torsion parameters improved considerably by including the structures trapped in the first generation s spurious minima , as shown in table 6 . rotation of the lysine amino terminus needed to be sampled completely in order to get a transferable model , but this was accomplished by the third generation . energies of lysine dipeptide conformations estimated by three generations of the v - ff14 force field . each generation s fitting set contained all of the initial fitting set data plus conformations created by all previous generations . the torsion fourier series terms describing dihedral interactions of atom types c n cx c8 ( backbone carbonyl carbon of any residue n - terminal to lysine or arginine , backbone nitrogen , c , and c of lysine or arginine ) and x c8nl x ( generic torsion affecting amino terminal hydrogens ) evolve rapidly over three generations as sampling of the backbone angles and amino headgroup orientations becomes more complete . the set of torsion parameters optimized in the third generation of training was chosen for pairing with q and distributed as ff14ipq in ambertools14 ( see http://ambermd.org ) . blocked dipeptides , the simplest systems exhibiting protein - like backbone and side - chain dynamics , were studied extensively to characterize the effects of slight alterations in the charge set , q , which differentiate ff14ipq , our force field for simulations of proteins in water , from v - ff14 , a force field used as a parameter fitting apparatus that is otherwise appropriate only for simulations of proteins in vacuum . furthermore , while we found that torsion parameter refinement could eliminate catastrophic traps in the potential energy surfaces of some amino acids , the approach made modest improvements in the behavior of every other amino acid as well . we computed multiple two - dimensional potentials of mean force ( pmfs ) for alanine , glycine , and serine dipeptides to assess backbone propensities with either charge set and with alanine dipeptide to examine the impact of torsion refinement . as shown in figure 4 , the pmfs of simple , nonpolar amino acids are nearly identical for each charge set , particularly in the populated regions of the ramachandran plot . by inspection , the polarization of charges slightly increases the alanine propensity toward ( right - handed ) -helices , decreases its propensity toward -sheets , and leaves the model s strongest tendency , toward poly proline ii backbone conformations , unchanged . larger differences in all dipeptides appear near ( , ) = ( 0,0 ) , as the backbone n h and c = o dipoles become favorably aligned to create a more favorable free energy under the ff14ipq model , which strengthens both dipoles considerably . however , the ( , ) = ( 0,0 ) arrangement remains strongly disfavored by steric clashes . the pmfs for alanine and glycine amino acids are also very close to those of the ff99sb force field . the lowest row of panels in figure 4 suggests that solvent effects that make the side - chain charges more polar also facilitate transitions between poly proline ii and left - handed -helicies , but the relative weights of each major conformation are unchanged . while it is not certain what differences in protein folding or dynamics a g of 0.25 kcal / mol in a low - energy region of ( , ) space could lead to , these plots suggest that q changes protein folding transition states more than equilibria . potentials of mean force for blocked alanine , glycine , and serine dipeptides in water . the color scale for the preliminary versions of ff14ipq ( leftmost panels ) and v - ff14 ( middle panels ) measures g , the energy difference between any point in ( , ) space and the minimum free energy attainable in each model at 298 k. differences between models are shown on the rightmost panels in a separate color scheme . the importance of fitting torsion parameters in the context of an appropriate charge model is shown in figure 5 . in contrast to the changes introduced by fitting torsion parameters for v - ff14 to a gas - phase qm pes and then transferring them to work in ff14ipq , fitting torsion parameters in the context of ipolq charges , retroactively forcing this charge model to mimic a gas - phase qm pes , makes more substantial changes to the alanine pmf . the difference between this pmf and the ff14ipq pmf is much more frustrated than the difference between the ff14ipq and v - ff14 pmfs . the strong gradients evident in the difference map drive the -helical minimum approximately 15 to the south if the torsion parameters are fitted in the context of q and gas - phase quantum data . when working against an already polarized electrostatic model , the torsion parameters were also less effective at reproducing the gas - phase qm pes ( data not shown ) . difference plot of the alanine dipeptide pmf with torsion parameters derived for q rather than q. the color scheme is similar to difference plots in figure 4 : here , solid red implies that a hypothetical ( and incorrect ) model fitting gas - phase quantum data in the context of charges appropriate to the solution - phase estimates a point in / space more than 1 kcal / mol more favorably than a properly tuned model ; solid blue would imply that the incorrect model disfavors the conformation . refining the torsion parameters has its own effects on the alanine dipeptide pmf , as shown in figure 6 . even though the quantum mechanics target is unchanged and the initial set of torsion parameters had no serious problems optimizing the geometries of alanine ( tetra)peptide , the third - generation model produces noticeable differences in the pmf . notably , what was a saddle point between -helical and poly proline ii conformations in the first - generation ff14ipq becomes a local minimum in the third . transitions between poly proline ii , -helical , and l--helical conformations also tranverse lower barriers according to the third generation of torsion parameters , and in some of the high - energy regions of the pmf , the differences between the first and final generations are as great as those shown in figure 5 . while the magnitudes of the differences are comparable to those in figure 5 , however , the locations of all the major backbone conformational minima remain in the same places , and the most significant differences reside in high - energy regions of the pmf , which stands in contrast to the consequences of fitting torsion parameters with the wrong charge set . as before , the influence of these changes on protein folding is not discernible from the pmf alone , but the differences again appear mostly in highly strained configurations , suggesting that the refined torsion parameters depict more frequent transitions between major backbone conformations . potential of mean force for blocked alanine dipeptide in the release version of ff14ipq and comparison to the initial model . both plots refer to combinations of condensed - phase charges with torsion parameters fitted to reproduce gas - phase quantum data . peformance of the release version of ff14ipq , specifically the third generation of torsion parameters paired with the condensed - phase appropriate q , was evaluated on ala(5 ) , -hairpins chignolin and protein g c - terminal fragment , the -helical miniprotein k19 , two variants of the miniprotein trp cage , and globular proteins gb3 and lysozyme . sources of each protein structure , as well as sequences of the miniproteins , are given in table 7 . each of the miniproteins simulated in this study was selected to evaluate ff14ipq s performance on a particular secondary structure element . simulations combined third - generation torsion parameters with q. protein sequence ; blocking groups are indicated by ace- and -nme . the ala(5 ) system has recently become a standard diagnostic of a force field s ability to balance three major backbone configurations and reproduce nmr j - coupling results . best and colleagues made a comprehensive assessment of modern force fields with respect to nmr data from graf and co - workers , calculating mean values for each model s reproduction of 11 order parameters . the karplus relations used to calculate order parameters from the md simulations are sensitive to their own coefficients , but best and colleagues took three different sets of karplus coefficients and posited that a value of 2.25 or less under all three karplus relations indicated a high - quality force field . the results for ff14ipq using the same karplus coefficients and quadruplicate 375 ns simulations of the unblocked peptide with protonated c - terminus are shown in table 8 . the details of the simulated system were intended to match the acidic conditions of the nmr experiments ; new charges were derived specifically for the protonated c - terminal alanine and are given in the supporting information . the mean values obtained with the original karplus coefficients used by graf and two sets of dft - based karplus coefficients from case and colleagues were 1.3 0.0 , 2.6 0.1 , and 1.5 0.0 , respectively . ( the error bars are standard deviations of the from each of the four 375 ns simulations . ) the major drivers of the scores are disagreement with the j(c , c ) coupling for the second alanine residue and the j(hn , c ) coupling for the third residue . while ff14ipq does not meet best s standard by one of the dft results , it does score very well by the other two , and the j(hn , c ) coupling is known to be difficult for the karplus relation itself . by best s definitions of each secondary structure element , ff14ipq models the central residue of ala(5 ) in poly proline ii , -helical , and -sheet conformations for 56 , 18 , and 14% of the pooled trajectories , respectively . dft - based karplus coefficients from case . ff14ipq also stabilizes larger -sheet structures , as indicated by the plots in figure 7 . the gb1 hairpin from protein g appears to be challenging for other fixed - charge force fields to stabilize ( emilio gallicchio , personal communication ) but maintains its secondary structure throughout the 250 ns simulations whether simulated with blocking groups or without . while it is reassuring that ff14ipq stabilizes a -sheet structure , the hairpin should be only approximately 30% folded at 298 k. one element that appears to stabilize the system considerably is the ionic interaction between the termini : it remains intact for virtually the entire simulation with a mean and modal distance between the termini of 3.5 0.7 . we investigated the stability of the blocked peptide with quadruplicate 600 ns runs and found no significant unfolding of the antiparallel -sheet arrangement ( data not shown ; the 250 ns simulation reported in figure 7 is representative ) . given that the melting curves for this peptide and other -hairpins are well - established , it may be possible to chip away at the stability of these structures in ff14ipq , and this is probably easier than to try and obtain the right structure from a model that can not stabilize the peptide at all . backbone stability of the -hairpin from protein g over 250 ns . blocked and unblocked forms of the peptide were simulated , and backbone rmsd is plotted for both variants . the dssp chart below the rmsd plots refers to the blocked peptide system and indicates that the antiparallel -sheet is maintained throughout the simulation . the chignolin system could be simulated economically on equally long time scales and appears to fluctuate between two major backbone states . neither of them is very far from the structure obtained after equilibration ; figure 8 shows backbone rmsd relative to the first structure in the nmr ensemble , but if it is calculated relative to the first frame of the simulation after restrained equilibration , then the rmsd would fluctuate between 0.7 and 1.4 . in addition to calculating the standard , overall backbone positional root - mean - squared deviation ( rmsd ) relative the first nmr model , we computed the way in which individual residues deviate from the nmr ensemble as a whole . this was accomplished by making optimal alignments of the simulated backbones to each of the 18 chignolin nmr models found in the pdb and then computing the rmsd of each particular residue without further position alignment . the minimum residue rmsd to any of the models in the nmr ensemble was recorded for each frame of the trajectory to indicate how far from any of the plausible structures our simulations might have departed . the lower panel of figure 8 shows histograms of these per - residue backbone position rmsds for each residue over the course of the simulation . this confirms that the backbone adopts two major conformations , with larger fluctuations in the zwitterionic , unblocked terminal residues . the arrangement of the termini follows the gb1 hairpin system : the zwitterionic termini begin the simulation in contact with one another . in chignolin , the termini fluctuate in concert most of the time , although for 7% of the simulation the two ionic groups separate by more than 4.5 . the -hairpin fold is maintained throughout the simulation , but the state adopted in the middle of the simulation appears to be slightly outside the nmr structure ensemble . further simulations may be able to fold the peptide , test the pathway against the known mechanism , and suggest whether changes to the backbone backbone interactions would tighten up the equilibrium structural results . backbone rmsd in the top plot is calculated relative to the first nmr structure ; per - residue backbone rmsd reflects the deviation of each residue s backbone from the closest possible match out of the entire nmr ensemble . over the course of 1 s , residues 112 exhibit consistent helicity , while the c - terminus transiently explores alternative coil conformations and sometimes even forms a packed double - helical structure , as shown in figures 9 and 10 . the experimental data suggest that the protein s first 18 residues should consistently maintain an -helix , but the ff14ipq results are otherwise in qualitative agreement with experiment and previous simulations of this system . of particular interest was the degree to which highly solvent - exposed lysine residues in the c - terminal region might make hydrogen bonds with the backbone . jones radii in both the lysine amino headgroup and the backbone oxygen were altered when interacting with water for agreement with hydration free energies but allowed to maintain their original interaction with one another in the interest of keeping repulsion between nearby atoms from disrupting the internal potential energy surface . interactions between lysine and the backbone oxygen may not be well tuned : much has been done to reduce the backbone oxygen s affinity for water , but its affinity for amino groups is only balanced , if at all , by the fact that amino groups lennard jones radii were reduced to provide greater affinity for water . we calculated the minimum distance between each lysine headgroup on k19 with any backbone oxygen atom throughout the simulation . as shown in figure 11 , lysines 4 and 9 rarely make contact with backbone carbonyl groups , but lysines 14 and 19 are much more likely to form hydrogen bonds to the peptide backbone . defining an amino group to be hydrogen - bonded to a carbonyl if the nitrogen and oxygen atoms come within 3.2 of each other , lysines 4 , 9 , 14 , and 19 are bonded to the backbone in 4 , 7 , 17 , and 27% of the snapshots , respectively . while the interaction between some groups may be stronger than is realistic , their interactions remain transient . the nitrogen atom types in the lysine headgroup and the charged amino teminus of our -hairpin systems are the same , and the oxygen atom types in carbonyl and carboxylate groups were altered in a similar manner with respect to their precursors in ff99 . the artificial behaviors seen in each system are likely to have a common origin . the time axis applies to both the rmsd plot in the top panel and the dssp plot in the lower panel . the k19 peptide is predominantly -helical , with some instability at the c - terminus . residues 1619 begin to adopt a metastable 310 helical conformation near the middle of the simulation . all conformations have been aligned relative to the stable backbone of residues 214 . radial distributions of lysine head groups and backbone oxygen atoms in the k19 system .. as shown in figure 12 , both trp cage simulations showed very stable backbone configurations over the 500 ns simulations . the baseline rmsd of approximately 1.2 may indicate different preferences of the ipolq charge set compared to the parameters used in nmr refinement , but larger departures from the backbone configuration depicted in the nmr models were merely transient . figure 12 shows histograms of per - residue backbone position rmsds as were calculated for chignolin . most residues display low rmsds under this test , and most importantly the pro - pro - pro sequence near the c - terminus remains stable . some residues , in particular the asp - gly - gly - pro sequence in simulations of neidigh s trp cage ( pdb code 1l2y(45 ) ) , show a weakly bimodal distribution , suggesting that the simulations explore an alternate conformation not seen in the nmr ensemble . when simulating a hyperstable trp cage mutant ( pdb code 1rij(46 ) ) , the asp - gly - gly - pro sequence again departs from the nmr ensemble more significantly than other regions of the protein . the conformations of these residues are good candidates for future analysis and refinement of ff14ipq . backbone positional root - mean - squared deviations ( rmsds ) for trp cage miniprotein simulations . simulations of each of two trp cage proteins are indicated by their respective pdb codes . the top panel shows overall backbone rmsd to the first published nmr model for each system . lower panels show histograms of per - residue backbone rmsd to the closest possible match out of all published nmr models , darkened to indicate increasing occupancy at a particular deviation . even though the ipolq charge model is directed toward solvated molecules , ff14ipq is expected to have application to globular proteins . most residues on proteins of even a few hundred residues have some degree of solvent exposure , and the majority of buried residues will be nonpolar and therefore little different when represented by ipolq as opposed to other charge models . simulations of both gb3 and lysozyme showed that , overall , ff14ipq stabilizes the crystallographic backbone configurations of both proteins ( figure 13 ) . gb3 and lysozyme ( 56 and 129 residues , respectively ) were simulated with ff14ipq in baths of tip4p - ew water for 1 s . multiple simulations of gb3 are shown , each performed with a different generation of the ff14ipq torsion parameters . see figure 3 for the accuracy of each generation in predicting energetics of lysine dipeptide . because we began simulations of gb3 before realizing the importance of iterative torsion parameter refinement , the system provides an indication of the symptoms of underfitted parameters when simulating a complex biomolecule . the first generation of torsion parameters caused one of the lysine - containing loops ( residues 916 ) to take on alternative conformations that drove the overal rmsd significantly higher . application of the second - generation torsion parameters , which fixed an artificial minimum in the lysine backbone angle , greatly diminished these excursions , as shown in figure 14 . however , another very short loop of the protein , residues 3941 connecting -sheet to -helical structures , makes excursions from the x - ray backbone structure in any generation . it may be significant that this loop contains an aspartate residue : in the torsion fitting results ( table 4 ) , aspartate presented one of the most difficult potential energy surfaces for our torsion parameters to capture . while ff14ipq seems to provide a better fit than would be possible with ff99 , the greatly expanded parameter space that is almost certainly the basis for this improvement may have allowed some overfitting that we have not yet been able to eliminate with our iterative scheme . alternatively , aspartate polarization in solution may not be well captured by our approximations . ( a third possibility remains , whereby in solution this loop really does take on conformations not seen in the crystal lattice . ) we intend that future releases of ff14ipq will build on all of the existing quantum data with further refinements to improve the description of larger protein systems . per - residue backbone rmsd for gb3 , with three generations of the ff14ipq model . per - residue rmsd was calculated in the same manner as was done for trp cage , but the reference ensemble comprised only the one x - ray structure . loops that make significant departures from the x - ray structure in solution - phase simulations are emphasized on the x axis . the simulations performed to date with ff14ipq suggest that the force field is ready to guide much longer time scale experiments with proteins and solvated peptides . while some results indicate possible weaknesses in ff14ipq , it is straightforward to develop hypotheses for their origins in the parameter development . it is encouraging to get this level of performance from a new force field and to see the model improve over generations of new fitting data . before completing our molecular mechanics model based on the ipolq charge set , we needed a set of charges compatible with the gas - phase quantum calculations available to guide our torsion parameter optimization . this , in turn , required us to revisit the way in which we derived charges , decomposing the ipolq charges into foundational values describing the electrostatics of solutes in vacuo and perturbations describing the manner in which atoms polarize in aqueous solution . because of the iterative nature of the ipolq procedure , we must consider whether this latest change will require additional iterations of molecular simulations with fixed solutes followed by charge fitting . first , although the polarities of certain bonds have changed somewhat , the individual charges of polar side - chain atoms and overall dipoles of the molecules did not change very much . restraints on q seem to have the strongest effects on buried atoms or groups : increasing vgp reveals indeterminacy in the model fitting but is not likely to affect hydration free energies . indeed , we chose this approach to exploit the fact that there exist many partial charge models that reproduce a molecule s electrostatic potential with nearly the same accuracy : increasing vgp chooses two models that are most similar in terms of the mean - squared value of q . second , the objective of the ipolq procedure is a solvent reaction field potential ( srfp ) that is consistent with the way a solute polarizes ; the result is a set of charges that is consistent with the srfp . restraints on q can make minor changes to the result , but they should not change the objective . it would therefore be appropriate to apply the original ipolq procedure until convergence and then to make other stipulations about how the model s partial charges reproduce the fitting data . one surprising result of this analysis is that the electrostatic potentials of solutes in vacuo are harder to fit than their potentials in the condensed phase when the wave function has been computed in the presence of a polarizing charge density . this occurs in spite of the fact that the charges for polar groups , including the backbone , become significantly larger in the condensed phase to represent stronger electrostatic fields . while an analysis of why this occurs is beyond the scope of this work , it is a worthy subject for future studies and may inform the design of polarizable charge models . in another study , extension of the standard resp protocol , which is similar in spirit to our extended ipolq scheme if one takes the vacuum charges to be dresp s baseline charge model . dresp employs variable restraint stiffnesses on polar and nonpolar atoms to accommodate different polarizabilities , something that our arbitrary constant vgp does not support . we had thought that , with a complete charge parameter scheme and no obvious reasons to make further changes to bonded parameters or lennard jones terms , producing viable torsion parameters would be the simplest step in developing ff14ipq . beyond the aforementioned issues with double - counting solvent contributions , there were many hurdles in deriving a transferable torsion parameter set , and this aspect of the model development required as much effort as the charge derivation . we hope that our experience and the programs we designed to meet each challenge will benefit future ab initio force field development . we found inclusion of new atom types and the new fourier series terms they generated to be very helpful in fitting the vacuum - phase potential energy data . the new types introduced to accommodate the ipolq charge set did not introduce many new parameters , as the atom types they replaced were already found in unique bonded arrangements . however , introducing atom types specific to individual residues introduced many new terms that could be fitted to the unique potential energy surface of the residue and relieve the burden that would have been placed on generic terms . in this manner , including a new atom type for c made improvements in the rmse for nearly all dipeptide systems , as the and torsions of the uniquely achiral center of glycine could then be decoupled from those of all other , chiral amino acids . while we had wanted to include separate atom types for the c atoms of positively and negatively charged residues to match the unique charges derived for all backbone atoms in these residues , we were not confident in our ability to generate enough fitting data to sample the and torsion space for these individual residues . in particular , sampling and for consecutive arginine or lysine residues would require mp2/cc - pvtz calculations on large tripeptide systems at very high computational cost . expansion of the data set in this manner will likely be part of the next version of ff14ipq . our study also highlights the perils of adding new parameters without adequate sampling . while we believe it is correct to assign unique parameters to unique chemical environments , if the degrees of freedom governed by each parameter might take on values in simulations that are not present in the training set , then catastrophic excursions into artificial minima can occur . data collected with the first - generation ff14ipq showed unnatural backbone angles adopted by lysine residues in the gb3 protein , which disappeared in subsequent generations trained with complete sampling of this rotatable bond . while such cases might be apparent to an especially careful investigator , we programmed the mdgx fitting routines to produce a great deal of information on the sampling of each parameter , and we did not connect higher backbone fluctuations in the gb3 protein loops to undersampling in particular parameters until significant improvements were seen in the second generation of the force field and a complete breakdown of the molecular mechanics energy was determined for each of the hundreds of lysine structures in each training set . perhaps ore important is our finding that the descriptions of residues that do not exhibit such catastrophic artificial minima can still be improved by the simple approach of reintroducing the products of molecular simulations back into the model s training data . whatever artificial minima were removed from the other residues by the third generation of ff14ipq must have been shallow and probably involved combinations of several parameters , implying a very large conformational space to sample in order to obtain transferable parameters on the first attempt . if allowed to drive simulations , then any molecular model should be expected to overpopulate regions of the conformational space in which it scores too favorably . however , because all of the torsion parameters discussed in this work are based on cosines , an unrefined model s propensity to overstate the favorable nature of some conformations may conversely create a tendency to overestimate barrier heights . if this is true , then it may not be a coincidence that the third generation of ff14ipq models transition states in alanine dipeptide with lower barriers than the first generation . in our charge parameter development , we were forced to alter the radii of certain polar atoms , mostly by making them larger , to adjust hydration free energies of amino acid side - chain analogues into agreement with experiment . in this work , we were forced to break the combining rule when integrating these atoms into a complete force field because the larger radii of nearby atoms would clash and make the internal potential energy surfaces of polar amino acids very hard to model . we have covered the possible implications of this decision in the k19 system , where lysine head groups appear to interact too strongly with the backbone . the special combining rules for these polar atoms would also affect the stability of backbone backbone contacts , particularly -sheets and -helicies , and are likely the source of the overstabilization reported for the gb1 -hairpins . goetz and colleagues reported strong performance for the ipolq charge set when studying aggregation of ionic amino acids , but in that system the correct result was for the amino acids to aggregate . while a prototype version of ff14ipq was able to form these aggregates with the correct radial distributions , their study did not test whether the contacts formed might , in fact , be too strong . more recently , debiec , gronenborn , and chong reported that the ipolq charge set produced the correct association constants for charged amino acid side chains when the full amino acids were considered : positively charged side chains showed affinity for both negatively charged side chains and the backbone carbonyl group , and the competition drove the equilibrium of side chains contacting one another in the right direction . however , they did not rule out the possibility that the side chain to backbone contacts were themselves overstated , as our results on k19 lead us to believe . debiec and co - workers also did not have available to them the special combining rules we adopted for ff14ipq . when these rules are applied , they found that ff14ipq overstabilized salt bridge formation between charged amino acid side - chain analogues by about 1 kcal / mol ( personal communication ) . we are collaborating with debiec and others to find a pair - specific lennard jones matrix that will not create excessive 1:4 strain but will properly balance the strength of polar interactions . we also intend to add new residues to the palette , including nucleic acids and phospholipids . the folding and aggregation of these residues is dominated by electrostatic interactions , which will demand the proper balance between charged groups interacting with one another and also with water . for proteins jones interactions will still contain special combining rules but that the departures from a standard rule will be smaller than they are in ff14ipq . some of the lennard jones interactions in ipolq itself can be traced to limitations of the nuclear - centered charge model . for instance , the nuclear - centered charges can not portray the hydroxyl group with as strong a polarity as it should have without making larger errors elsewhere in the electrostatic field . the hydroxyl oxygen radius was reduced to correct alcohol hydration free energies , but this probably would not have been necessary with a virtual site scheme that could model the correct polarity . for lipids and nucleic acids , any new departures from a standard lennard jones combining rule may hold clues to the inherent limitations of a nonpolarizable , nuclear - centered charge model .

we present the ff14ipq force field , implementing the previously published ipolq charge set for simulations of complete proteins . minor modifications to the charge derivation scheme and van der waals interactions between polar atoms are introduced . torsion parameters are developed through a generational learning approach , based on gas - phase mp2/cc - pvtz single - point energies computed of structures optimized by the force field itself rather than the quantum benchmark . in this manner , we sacrifice information about the true quantum minima in order to ensure that the force field maintains optimal agreement with the mp2/cc - pvtz benchmark for the ensembles it will actually produce in simulations . a means of making the gas - phase torsion parameters compatible with solution - phase ipolq charges is presented . the ff14ipq model is an alternative to ff99sb and other amber force fields for protein simulations in programs that accommodate pair - specific lennard jones combining rules . the force field gives strong performance on -helical and -sheet oligopeptides as well as globular proteins over microsecond time scale simulations , although it has not yet been tested in conjunction with lipid and nucleic acid models . we show how our choices in parameter development influence the resulting force field and how other choices that may have appeared reasonable would actually have led to poorer results . the tools we developed may also aid in the development of future fixed - charge and even polarizable biomolecular force fields .

as an uncommon injury , laryngeal trauma accounts for less than 1% of blunt trauma in adult patients . possible sequelae of laryngeal trauma include soft - tissue edema , ruptures of ligaments , fractures of laryngeal cartilage , and dislocation of arytenoid cartilage . dislocation of the arytenoid cartilage results in reduced mobility of the vocal fold and incomplete glottic closure that mimic vocal fold paralysis . it has been rarely reported to be caused by external blunt forces [ 15 ] . diagnosis of arytenoid dislocation with indirect laryngoscopy , flexible fiberoptic laryngoscopy , videostrobolaryngoscopy , high - resolution ct , and lemg is generally accepted . lemg is recommended as a major diagnostic tool , although it still has some limitations in distinguishing arytenoid dislocation from laryngeal nerve injury . however , as lemg requires special equipment and skills as well as considerable experience , it is still rarely used in many clinics and hospitals . as such , we investigated whether it is possible to definitively diagnose and effectively treat arytenoid dislocation due to external laryngeal trauma without using lemg . this retrospective study was performed over a period of 15 years from january 1997 to december 2011 . from all patients who visited our department during this period , 97 patients in total were considered to have arytenoid cartilage dislocation , of which 85 ( 87.6% , 85/97 ) had received tracheal intubations . in the current study , we re - visited the records of the 12 patients who had a known histology of external neck trauma . written informed consent was obtained from each patient , and the study was approved by the institutional review board of second affiliated hospital , school of medicine , zhejiang university . they were divided into the satisfied group ( n=9 ) and dissatisfied group ( n=3 ) based on patients self - satisfied with voice qualities at 1 week after the last closed reduction manipulation . the 12 patients underwent a complete head and neck examination and a thorough voice evaluation using the vhi scale , indirect laryngoscope , flexible fiberoptic laryngoscope , video strobolaryngoscope , and/or high - resolution ct . the vhi scale , including functional ( f ) , physiological ( p ) , emotion ( e ) , and the sum denoted by t , was applied in self - assessment before initial closed reduction and at 1 week after the last closed reduction , and mpt was measured as well . the laryngoscopic examinations were selectively performed before initial closed reduction , immediately after the last closed reduction , and 1 week after the last closed reduction . laryngoscopic findings in arytenoid dislocation include impaired or absent vocal fold mobility and the asymmetric position and different length of vocal cords with poor glottic closure . in addition , arytenoid dislocation should be highly suspected if video strobolaryngoscopy further shows shortened mpt , normal vocal fold vibration , discrepancy between vocal cord heights , and absence of a jostle phenomenon ( a symptom of vocal cord paralysis characterized by lateral movement of the arytenoid on the immobile side , caused by contact with the mobile side during adduction ) . ct scan of the larynx was used to reveal arytenoid dislocation by interarytenoid asymmetry and clouding or obliteration of cricoarytenoid joint space . attempted closed reduction , as a diagnostic and therapeutic method , anterior dislocations were reduced with posterior - upward push on the arytenoids during phonation and posterior dislocations with anterior - upward push during inspiration . arytenoid dislocation was thought to be confirmed in patients who experienced immediate improvement in laryngeal symptoms such as hoarseness after attempted closed reduction . after each closed reduction manipulation , patients were encouraged to speak as much as they could , and taught to hold the larynx gently with fingers and shake it side to side while making a strong sound as if coughing up phlegm from the throat . attempted closed reduction the means were compared using paired t - test , wilcoxon match - pair signed - ranks test , or mann - whitney u - test . this retrospective study was performed over a period of 15 years from january 1997 to december 2011 . from all patients who visited our department during this period , 97 patients in total were considered to have arytenoid cartilage dislocation , of which 85 ( 87.6% , 85/97 ) had received tracheal intubations . in the current study , we re - visited the records of the 12 patients who had a known histology of external neck trauma . written informed consent was obtained from each patient , and the study was approved by the institutional review board of second affiliated hospital , school of medicine , zhejiang university . they were divided into the satisfied group ( n=9 ) and dissatisfied group ( n=3 ) based on patients self - satisfied with voice qualities at 1 week after the last closed reduction manipulation . the 12 patients underwent a complete head and neck examination and a thorough voice evaluation using the vhi scale , indirect laryngoscope , flexible fiberoptic laryngoscope , video strobolaryngoscope , and/or high - resolution ct . the vhi scale , including functional ( f ) , physiological ( p ) , emotion ( e ) , and the sum denoted by t , was applied in self - assessment before initial closed reduction and at 1 week after the last closed reduction , and mpt was measured as well . the laryngoscopic examinations were selectively performed before initial closed reduction , immediately after the last closed reduction , and 1 week after the last closed reduction . laryngoscopic findings in arytenoid dislocation include impaired or absent vocal fold mobility and the asymmetric position and different length of vocal cords with poor glottic closure . in addition , arytenoid dislocation should be highly suspected if video strobolaryngoscopy further shows shortened mpt , normal vocal fold vibration , discrepancy between vocal cord heights , and absence of a jostle phenomenon ( a symptom of vocal cord paralysis characterized by lateral movement of the arytenoid on the immobile side , caused by contact with the mobile side during adduction ) . ct scan of the larynx was used to reveal arytenoid dislocation by interarytenoid asymmetry and clouding or obliteration of cricoarytenoid joint space . attempted closed reduction , as a diagnostic and therapeutic method , was performed by indirect laryngoscope and laryngeal forceps under local anesthesia . anterior dislocations were reduced with posterior - upward push on the arytenoids during phonation and posterior dislocations with anterior - upward push during inspiration . arytenoid dislocation was thought to be confirmed in patients who experienced immediate improvement in laryngeal symptoms such as hoarseness after attempted closed reduction . after each closed reduction manipulation , patients were encouraged to speak as much as they could , and taught to hold the larynx gently with fingers and shake it side to side while making a strong sound as if coughing up phlegm from the throat . attempted closed reduction the means were compared using paired t - test , wilcoxon match - pair signed - ranks test , or mann - whitney u - test . all analyses were performed using spss 16.0 statistical software ( spss inc . , chicago , il ) . voice change occurred immediately after injury in 10 cases , and in the other cases at 4 and 7 days after injury . there were no obvious external signs of laryngeal trauma , neither subcutaneous emphysema nor signs of hematoma and fractures , in these patients . examination with indirect laryngoscope , flexible fiberoptic laryngoscope , and video strobolaryngoscope revealed immobility of vocal cords in 8 patients and impaired movement of vocal cords in 4 patients . the typical observations were the asymmetric position and different length of vocal cords with poor glottic closure ( figures 1a , 1b and 2a , 2b ) . arytenoid cartilage was anteriorly displaced in 11 patients ( 5 on the left side and 6 on the right side ) . one patient had both left - sided anterior and right - sided posterior displacement of the arytenoid cartilages ( table 1 ) . vocal cords were located at the intermediate , paramedian , and lateral positions in 7 , 1 , and 3 patients , respectively . in the patient with affected bilateral vocal cords , the left vocal cord was located at the intermediate position and the right one at the lateral position . video strobolaryngoscopic examination demonstrated that mpt was longer than 5 s in 8 patients with normal vocal fold vibration , and mpt for the other 4 patients was too short for us to observe vibration of vocal folds during phonation . the sensibility of the endolaryngeal mucosa was unaffected in all patients , indicating a normal function of the internal branch of the superior laryngeal nerves . jostle sign was absent , and discrepancy between vocal cord heights was noticeable in these patients ( table 2 ) . typically , the level of the affected vocal cord was lower than that of the normal one during phonation in 9 of the 11 patients with unilateral lesion . the different length between both vocal cords was observed in 12 patients . the affected vocal cord was shorter than the normal one in the cases with anterior malposition of arytenoid cartilage . seven patients underwent high - resolution ct scans of the larynx . among them , anterior displacement of the arytenoid cartilage was confirmed in 4 patients ( figure 3 ) . as illustrated in table 3 , vhi scores at 1 week after the last closed reduction were lower than those before initial reduction in the satisfied group ( p<0.05 ) , but these scores were not statistically significantly different from those in the dissatisfied group . among the 9 patients in the satisfied group , fiberoptic laryngoscopic and video strobolaryngoscopic examination demonstrated that 5 patients obtained normal movement of the vocal cord and the symmetric position and equal length of both vocal cords , with tight closure of the glottis during phonation instantly after closed reduction ( figure 1c , 1d ) . impaired movement of the vocal cord was still visible and vocal cords were located almost at the same level , with improved glottic closure in the other 4 patients ( figure 2c , 2d ) . mpt ( 20.116.11s ) in the satisfied group was significantly lengthened at 1 week after the last closed reduction ( p<0.05 ) . hoarseness , together with immobility of the vocal cord and incomplete glottic closure , remained in the dissatisfied group . the time interval between injury and closed reduction in this group was 157.6776.07 days , which was significantly longer than that in the satisfied group ( p<0.05 ) . laryngeal trauma caused by external forces is relatively rare , with an incidence of 1 in every 22 900 emergency room visits [ 911 ] . in this report , the finding of 12 out of 97 ( 12.4% ) patients with arytenoid dislocation caused by external trauma is similar to an earlier finding by rubin that 15.9% of their arytenoid dislocation cases were attributed to external neck trauma . it is worth noting that vocal cord immobility may originate either from neurological paralysis or joint abnormalities , or even from both . lemg could be useful in differentiating these 2 causes by indicating the presence of an innervated vocal cord . however , as pointed out by rubin et al . , lemg has its limitations , with 39.7% of their patients with arytenoid cartilage dislocation displaying abnormalities on lemg . this is possibly due to co - occurrence of denervation and a joint dislocation , nerve susceptibility to inflammatory mediators infiltrating after trauma , and/or hematoma and scarring in the posterior portion of the thyroarytenoid muscle , leading to the appearance of denervation on lemg . importantly , lemg alone may fail to establish the diagnosis of laryngeal paralysis , because cross - innervation may occur in a paralyzed vocal fold [ 1214 ] . the time interval between injury and initial medical examination in our patients was 14240 days ( median time 72.0067.59 days ) , which might be long enough for the occurrence of reinnervation . furthermore , lemg is unavailable in the majority of the hospitals in china , including our hospital . therefore , combined with our video strobolaryngoscopic observation showing good muscle tension with vibration of vocal folds in our patients , lemg was not included in the current study . except forin addition to lemg , flexible fiberoptic laryngoscopy , videostrobolaryngoscopy , and computed tomography are also helpful in the diagnosis of arytenoid cartilage dislocation . according to rubin et al . , the most useful examination is video strobolaryngoscopy , which allows evaluation of jostle sign , flickering of muscle activity associated with an intact nerve supply , and submucosal hematoma that can result in scarring and vocal cord stiffness . most of the studies investigating the difference in level between paralyzed and innervated vocal cords reported that the paralyzed vocal fold presented at a higher level than a normally innervated one [ 1519 ] . in our cases with arytenoid dislocation , conversely , the level of affected vocal cords was lower than that of normal ones in 9 of the 11 patients with a unilateral lesion . as suchtherefore , we would suggest thate an obvious discrepancy in height between vocal cords is an important feature in the differential diagnosis between arytenoid dislocation and laryngeal paralysis . during the 15-year period , we preferpreferred to perform attempted closed reduction by indirect laryngoscope and laryngeal forceps manipulation under local anesthesia . firstly , it is a feasible method to facilitate diagnosis when patients are highly suspected of arytenoid dislocation , especially in the medical institutions without lemg . the diagnosis of arytenoid dislocation can be confirmed in patients who experience instantaneous improvement in laryngeal symptoms such as hoarseness after attempted closed reduction . more importantly , it can shorten the time interval between the diagnosis and treatment of arytenoid dislocation , which is helpful to achieve a successful reduction . secondly , it prevents the larynx from being further injured by tracheal intubation under general anesthesia . thirdly , it is impossible to judge the position of arytenoid cartilage after reduction and to monitor the patient s voice under general anesthesia . fourthly , most of patients can tolerate the procedure by indirect laryngoscope and laryngeal forceps under local anesthesia , and high cure or improvement rate ( 78.4% , 76/97 ) was achieved during our 15-year experience . rubin et al . reported that 7 out of 10 patients ( 70% ) with arytenoid dislocation due to external trauma regained normal or improved voice only by closed reduction using direct laryngoscope under local anesthesia and/or voice therapy . in our series , the patients obtained similar outcomes ( 75% , 9/12 ) . therefore , closed reduction for patients with arytenoid cartilage dislocation under local anesthesia is practicable and effective . early treatment of arytenoid cartilage dislocation is crucial for good clinical recovery of voice quality . we have found that vhi scores and mpt were significantly improved in the satisfied group , who had a median time interval between injury and treatment of 43.4434.13 days , whereas the dissatisfied group showing no voice improvement had a median time interval of 157.6776.07 days . a critical difference between the 2 groups was immobility of the vocal cord in the latter , probably related to scarring and fixation of the cricoarytenoid joint that occurred due to the delayed treatment . similar results were also found by sataloff , who reported that the average time interval between injury and surgical treatment for patients who regained their normal voice was 10 weeks and an average time interval of 29 weeks for patients who had no response to treatment , although the causes of laryngeal trauma were different . nevertheless , it is obvious that recovery will become more difficult if clinical intervention is delayed , regardless of whether arytenoid cartilage dislocation is caused by internal or external forces . in addition , in our study , the 3 patients had no voice improvement by using a diagnostic reposition approach . there may be another possibility that arytenoid cartilage dislocation and associated vocal fold paralysis could occur among them . arytenoid dislocation after external blunt laryngeal trauma does occur , although rarely , and is often associated with impaired mobility of the vocal cord . attempted closed reduction should be considered as a diagnostic approach for highly suspected arytenoid dislocation , especially in the clinics or hospitals without lemg . furthermore , early closed reduction under local anesthesia is also a practicable and effective treatment option for this kind of disorders as well .

backgroundintubation trauma is the most common cause of arytenoid dislocation . the aim of this study was to investigate the diagnosis and treatment of arytenoid cartilage dislocation from external blunt laryngeal trauma in the absence of laryngeal electromyography ( lemg ) and to explore the role of early attempted closed reduction in arytenoids cartilage reposition.material/methodsthis 15-year retrospective study recruited 12 patients with suspected arytenoid dislocation from external blunt laryngeal trauma , who were evaluated through 7 approaches : detailed personal history , voice handicap index ( vhi ) test , indirect laryngoscope , flexible fiberoptic laryngoscope , video strobolaryngoscope , and/or high - resolution computed tomography ( ct ) , and , most importantly , the outcomes after attempted closed reduction under local anesthesia . they were divided into satisfied group ( n=9 ) and dissatisfied group ( n=3 ) based on their satisfied with voice qualities at 1 week after the last closed reduction manipulation.resultseach patient was diagnosed with arytenoid dislocation caused by external blunt laryngeal trauma . in the satisfied group , vhi scores and maximum phonation time ( mpt ) at 1 week after the last reduction were significantly improved compared with those before the procedure ( p<0.05 ) . normal or improved mobility and length of the affected vocal fold were also noted immediately after the end of the last closed reduction . the median time interval between injury and clinical intervention in satisfied group was 43.4434.13 days , much shorter than the median time of 157.6776.07 days in the dissatisfied group ( p<0.05).conclusionsmultimodality assessment protocols are essential for suspected arytenoid dislocation after external blunt laryngeal trauma . early attempted closed reduction should be widely recommended , especially in health facilities without lemg , mainly , because it could be helpful for early diagnosis and treatment of this disease . in addition , early closed reduction could also improve the success of arytenoid reduction .

the incidence of diabetes mellitus is increasing worldwide ; this trend is particularly strong for type 2 diabetes mellitus ( t2dm)1 . t2 dm is a chronic disease characterized by decreased insulin sensitivity and overall poor glucose control . the systematic review by boule et al . indicates structured exercise programs have a statistically and clinically significant beneficial effect on glycemic control in patients with t2dm3 . in addition , patients with t2 dm who use insulin , low - intensity exercise significantly reduces the prevalence of hyperglycemia4 . despite the benefits of physical activity , many people with t2 dm for some individuals , secondary diabetes - related complications such as lower - limb amputations , neuropathies , hypertension , nephropathies , and retinopathies can either contraindicate exercise or make it more difficult . in addition , many elderly people with t2 dm do not have sufficient physical ability to perform aerobic exercise and thus have problems maintaining euglycemia5 . passive static stretching occurs when sustained tension develops within a person s muscles through external forces . accordingly , blood glucose levels could decrease following a program of successive sustained muscle stretching . in addition , because passive stretching requires minimum effort by the person performing the stretch , people with t2 dm who are reluctant or unable to exercise may be willing to follow a stretching protocol6 . therefore , this study determined the effect of passive static stretching on blood glucose levels in people with t2 dm . fifteen in - patients with t2 dm at a hospital in busan , korea participated in this study . patients were eligible if they were sedentary ( i.e. , not participating in regular aerobic or strengthening exercises 6 months before the study ) and willing to commit to an 8-week supervised exercise program7 . all patients were diagnosed with t2 dm confirmed by a glycated hemoglobin ( hba1c ) level 6.5% or higher as a criterion for the diagnosis of diabetes8 . all patients meeting the inclusion criteria were given verbal and written information about this study . patients were randomized to the control group ( n = 7 ) or passive static stretching group ( pss , n = 8) . the control group was instructed to maintain their diet and medications for diabetes and not to perform any exercise during the experiment . meanwhile , patients in the pss group followed the same instructions as the control group but received a 40-minute intervention consisting of 6 lower - body and 4 upper - body static passive stretches . for each stretch , the muscle was held in the stretched position for 30 seconds and was repeated 4 times . each repetition was separated by a 15-second relaxation period , and different stretches were separated by a minimum of 1 minute . a description of stretch is provided in table 1table 1.descriptions of the stretches used in the interventionstretchdescriptionseated knee flexor ( bilateral)the patient sat on the floor with their legs extended and arms above their head . from this position , they lowered their head toward their knees while the experimenter pushed down on their back.seated knee flexor hip adductor ( bilateral)the patient sat on the floor in the cross - legged position . from this position , the patient lowered their head toward the floor while the experimenter pushed down on their back.seated shoulder lateral flexor ( bilateral)the patient sat in a chair with fingers interlaced behind their head . keeping their arms in this position , the experimenter stood behind the patient and pulled the elbows back toward the body s midline.supine hip flexor knee extensor ( unilateral)the patient lay on their back with their leg hanging over the edge of the table with the knee flexed at approximately 90. the hip was then hyperextended by the experimenter while pushing down on the thigh.seated hip external rotators , extensors ( unilateral)the patient sat on the floor with one leg extended . the opposite leg was flexed at the knee , and the foot was placed flat against the extended leg s inner thigh . the patient then lowered their head toward the extended knee while the experimenter pushed down on their back.seated shoulder extensors , adductors , retractors ( unilateral)while seated in a chair , the patient extended one arm and placed it horizontally across the front of the chest . the experimenter stood behind the patient , grabbed their wrist , and pulled their arm against the chest as much as possible while keeping the arm parallel to the floor.supine knee flexor plantar flexor ( unilateral)the patient lay on their back with the legs extended . the experimenter then raised one leg and simultaneously flexed the hip and dorsiflexed the ankle.prone hip flexor ( unilateral)the patient lay on their stomach and flexed one knee at approximately 60. keeping the knee in the flexed position , the experimenter lifted the thigh to hyperextend the hip.seated shoulder flexors , depressors ( bilateral)the patient sat on the floor with the legs extended . the experimenter then grabbed their wrists and hyperextended the shoulder by raising the arms behind the back and up toward the head while keeping the back and elbows straight.seated shoulder and elbow flexors ( unilateral)the patient sat on the floor with the legs extended , with one elbow flexed and brought up near the ear . from this position , the shoulder was hyperflexed by the experimenter by pushing the upper arm down toward the floor.6 . the pss group performed the stretches 3 times per week for 8 weeks . for outcome measurements , a 10-ml blood sample was collected from each patient to determine blood glucose levels using an hba1c analyzer ( variant turbo , bio - rad laboratories , inc . a paired t - test was used to determine whether there were significant changes in blood glucose levels before and after the intervention . meanwhile , an independent t - test was used to analyze differences between the 2 groups . the level of significance was set at p < 0.05 . the baseline characteristics of the patients are shown in table 2table 2.baseline characteristics of the patientscon ( n = 7)pss ( n = 8)page ( years)58.4 1.849.6 5.20.2duration of diabetes ( years ) 5.2 2.9 5.4 con : control group ; pss : passive static stretching group ; bmi : body mass index . there were no significant differences in the baseline characteristics between groups ( p > 0.05 ) . the results of outcome measures are summarized in table 3table 3.outcome measurescontrol group(n = 7)passive static stretching group(n = 8)pre - intervention post - interventionpre - interventionpost - interventionhba1c ( % ) 7.4 1.37.4 1.47.4 1.56.8 1.5*values are means sd . * p < 0.05 vs. post - intervention . hba1c : glycated hemoglobin a1c .. there was no significant difference in hba1c level after the intervention in the control group ( p > 0.05 ) . however , hba1c levels decreased significantly in the pss group after the intervention ( p < 0.05 ) and were significantly different between groups ( p < 0.05 ) . con : control group ; pss : passive static stretching group ; bmi : body mass index values are means sd . * p < 0.05 vs. post - intervention . as mentioned above , this study determined the effect of passive static stretching on blood glucose levels in patients with t2 dm . the results showed hba1c levels decreased significantly after an 8-week passive static stretching intervention . there are several possible mechanisms that could explain how passive stretching of skeletal muscles decreased blood glucose levels . according to a review by dohm9 , glucose transport into the skeletal muscles is primarily mediated by a glucose transport protein , glut-4 ; accordingly , exercise can increase glut-4 levels in the skeletal muscles . furthermore , increased metabolic activity accompanying passive muscle stretching is related to the glut-4 activation pathway9 , 10 . therefore , passive muscle stretching could induce the incorporation of glut-4 into the stretched skeletal muscles . other studies also support the possibility of stretching - induced incorporation of glut-4 into the skeletal muscles . first , the activity of protein kinase b controls glut-4 incorporation ; accordingly , protein kinase b is activated by passive stretching of isolated muscles11 . report that ischemia induces the translocation of glut-4 to the plasma membrane of cardiac myocytes12 ; accordingly , passive stretching of the skeletal muscles can cause ischemia13 . third , in an experimental study by roberts et al . , exercise - induced increases in nitric oxide levels resulted in increased glucose transport14 ; accordingly , passive stretching can increase nitric oxide release from excised soleus muscles by 20%15 . finally , mitogen - activated protein kinase activity stimulates glucose uptake in muscle cells16 ; martineau et al . report that the activity of mitogen - activated protein kinase directly reflects the magnitude of mechanical stress ( e.g. , actively or passively generated tension ) applied to the muscle17 . the sample size is insufficient to generalize the results to all patients with t2 dm . in addition , as hba1c reflects the average plasma glucose level over the preceding 23 months , the 8-week study period might have been too short to determine changes in blood glucose levels as a result of stretching18 . therefore , further studies are required to ascertain the long - term ( i.e. , more than 3 months ) effects of passive static stretching on blood glucose levels in a larger population of patients with t2 dm .

[ purpose ] this study determined the effects of passive static stretching on blood glucose levels in patients with type 2 diabetes . [ subjects ] fifteen patients ( 8 males and 7 females ) with type 2 diabetes were recruited and randomly assigned to the control group or passive static stretching group . [ methods ] glycated hemoglobin was measured before and after the 8-week training period . [ results ] glycated hemoglobin levels decreased significantly in the passive static stretching group , and there were significant differences in blood glucose levels between the 2 groups . [ conclusion ] passive static stretching of the skeletal muscles may be an alternative to exercise to help regulate blood glucose levels in diabetes patients .

the definition of congenital heart block for the purposes of this review will be the presence of conduction system disease of any form , which is diagnosed on or before 28 days of life . the incidence of congenital heart block has been estimated from several studies to be about 1 in 22,000 live births.1 although this is clearly an uncommon disorder , it may be associated with high mortality and morbidity and therefore requires a high index of suspicion for early diagnosis and aggressive therapy when appropriate . aggressive therapy can be defined as offering the prenatal use of dexamethasone or the other maternal drugs , fetal pacing , or early delivery . there are no data on the appropriateness of aggressive therapy , but our recent paper2 implies it may improve hydrops in the sickest fetuses . secondly , we will spend some time discussing the unique subtype of congenital heart block , that which occurs in the absence of major structural anomalies and which is associated with maternal autoimmune antibodies . the commonest forms of congenital heart disease associated with heart block include left atrial isomerism , often with an accompanying atrioventricular septal defect , as well as levo transposition of the great arteries . when diagnosed in the postnatal period , approximately one - third of cases of congenital conduction system disease have associated structural disease . in utero , diagnosis of congenital heart block is associated with structural heart disease in approximately one half of the cases.1 there is a higher association of congenital heart block occurring with congestive heart failure in utero , and thus a poorer prognosis . the outlook for patients with congenital heart block depends largely on the presence or absence of underlying structural heart disease , as well as the rate of ventricular activation and the presence or absence of congestive heart failure . if the heart block is diagnosed as a bradycardia during the fetal period , there is a very high rate of fetal and neonatal loss . prenatal risk factors for mortality prenatally depend on the presence of structural heart disease and a heart rate less than a critical value , frequently quoted as 55 bpm . the presence of hydrops fetalis or other signs of physiologic disturbance in cardiac function , are very poor prognostic signs . in severe cases , there has been as high as an 85% mortality rate in the neonatal period . according to the jaeggi paper3 , mortality in complete atrioventricular block in the fetus was 43% ( 13 out of 15 total deaths were fetal ) ; in the neonatal stage was 6% ; and in children there were none . in fetal hydrops if the fetal heart rate ( fhr ) was less than 55bpm , the majority died ( 9 out of 15 ) . according to the kertesz paper1 , in various series of fetal congenital complete atrioventricular block , , only 14% survived the neonatal period compared to 85% survival of the autoimmune isolated congenital complete atrioventricular block . if the congenital heart block is first diagnosed in the newborn period , presumably the higher risk fetuses have not survived , and therefore the prognosis is somewhat better . once again , the presence or absence of underlying structural heart disease often determines the outcome . the survival rate in newborns with congenital heart block and no structural heart disease is about 85% . if the congenital heart block first presents beyond the newborn period , the outlook for survival is improved . these patients are unlikely to have severe structural heart disease , and the survival rate is much higher than 85% . such children , however , still almost always require pacemaker implantation as well as treatment for any underlying structural heart disease . finally , some patients are first diagnosed with their presumably congenital conduction system disease in later childhood or adulthood . such patients are unlikely to have structural heart disease and they tend to have a good prognosis after pacemaker implantation . however , it must be remembered that they might present with severe life threatening events as their first manifestation of bradycardia , and they seem to have a late risk of developing left ventricular dilation and mitral insufficiency , presumably from longstanding bradycardia or immunological damage to the heart . several studies have attempted to elucidate the risk factors for the requirement of pacemaker implantation in patients with congenital heart block.1 it is fairly well accepted that a mean resting heart rate below a determined number for the age group could be an indication to place a pacemaker . this is frequently quoted as a 55 bpm in the newborn period and gradually decreases with advancing age . here we give some examples of electrocardiograms displaying varying degrees of heart block ( figures 14 ) . it is also well accepted that any symptomatic bradycardia requires pacemaker implantation , and it should be recognized that this may be either a sudden presentation or simply limited exercise capability . in addition , the presence of significant structural congenital heart disease is felt to be an indication to pace a patient with congenital heart block . some studies have suggested that a prolonged qtc interval or a wide qrs escape rhythm with complex ventricular ectopy may warrant the use of pacemaker therapy . electrocardiogram showing first degree atrioventricular block ( pr= 160 msec , heartrate= 170 bpm ) in a newborn . electrocardiogram showing second degree atrioventricular block ( mobitz type ii ) with progressive pr prolongation leading to dropped beats . electrocardiogram showing third degree heart block with atrioventricular dissociation and slow ventricular rate ( atrial rate is 150 , ventricular rate is 85 bpm ) . electrocardiogram showing third degree heart block ( atrioventricular dissociation with atrial rate of 170 bpm ) and ventricular pacemaker capturing at 125 bpm . it is sometimes difficult to determine if the child is having symptomatic bradycardia , because children will limit their exertion based on their symptomatology . echocardiograms may be helpful also to determine progressive loss of systolic function of the ventricle with increasing heart size and the development of mitral regurgitation . the congenital heart block associated with neonatal lupus is considered a form of passively acquired autoimmune disease in which maternal autoantibodies to the intracellular ribonucleoproteins ro ( ss - a ) and la ( ss - b ) , cross the placenta and injure the previously normal fetal heart . other manifestations of neonatal lupus may include the presence of skin rashes , liver abnormalities determined biochemically and abnormalities in the cellular elements of the blood including various cytopenias.2 while the non - cardiac manifestations of neonatal lupus are generally transient and resolve at approximately the time that the maternal antibodies are cleared from the infant 's circulation at several months of age , the conduction system disease is essentially irreversible . neonatal lupus is usually diagnosed in the presence of a slow heart rate discovered in a fetus or newborn in the absence of associated structural cardiac abnormalities . maternal serum testing subsequently reveals antibodies to ro and/or la , usually evaluated by elisa testing . while the mother may have systemic lupus or other autoimmune diseases such as sjogren 's syndrome , approximately half of the women at the time of diagnosis are asymptomatic . in utero , the peak onset of the diagnosis of bradycardia is between 18 and 24 weeks of gestation , corresponding to the window of opportunity about six weeks after effective placental transport of maternal igg antibodies begin . while the precise mechanism is unknown it is presumed that anti - ro / la antibodies directly or indirectly cause the cardiac damage . the degree of heart block may vary from first degree to third degree block , but most cases diagnosed in utero present with a least second degree or more advanced block . there is a high mortality rate , particularly in fetuses diagnosed in utero with hydrops , and it is approximately 20% . of all cases that have been recognized with congenital heart block , current data show that approximately two - thirds of these patients will have a pacemaker placed before reaching adulthood ( see table 1 ) . autoimmune congenital heart block statistics2 in those cases of autoimmune conduction system disease due to neonatal lupus , the bradycardia alone is not always the full extent of disease . recently , there has been the recognition of a relatively high incidence of the development of late cardiomyopathy leading to heart failure , death or transplantation despite successful pacemaker implantation ( table 1).34 as referenced in the moak paper4 , late cardiomyopathy is associated with immune - related congenital heart block in 5 - 11% of cases . clinical deterioration of cardiac function was seen up to 9.3 years . in our experience , other organ systems may be involved in the newborn as well , including the characteristic neonatal rash which appears generally as annular lesions , mostly on the face , particularly around the eyes and is photosensitive ( figure 5 ) . in addition , on serum testing , some of the newborns with maternal autoantibodies will have various low levels of red blood cell counts , white blood cell counts , and platelets . top shows 2d - directed m - mode echocardiogram of a newborn with a normal shortening fraction as the ventricle contracts in systole . the interventricular septum and the left ventricular posterior wall thicken toward each other during systole . bottom shows 2d - directed m - mode echocardiogram of a newborn showing a very poorly contractile , dilated ventricle . the occurrence rate of neonatal lupus has been estimated at approximately 2 to 3% in all pregnancies born to women with anti - ro or anti - la antibodies . the recurrence rate in a mother with antibodies who has a previous child who was affected , is approximately 18%.5 the mechanism of causation of neonatal lupus is not completely understood but evidence points to the fetus beginning life with a normal cardiac structure and conduction system . at approximately 12 weeks of gestation , maternal igg antibodies against ro and la intracellular ribonuclear proteins are actively transported across the placenta and are thought to bind specific cells of the fetal conduction system . there is also an element of maturation of the fetal immune system involved in the development of fetal immune disease . the majority of cases of congenital heart block , diagnosed in utero are detected by either auscultation or routine obstetrical ultrasound in low risk pregnancies . the diagnosis is confirmed by the performance of maternal fetal monitoring ( mfm ) and a fetal echocardiogram with doppler techniques ( figures 610 ) . in the past the purpose of the fetal echocardiogram is to determine the level of block and also to rule out major associated structural lesions of the heart , such as left atrial isomerism with or without atrioventricular septal defects , and ventricular inversion , which are structural diseases associated with the presence of heart block without antibodies . the fetal echocardiogram is also able to detect any associated myocarditis by looking for the presence of decreased contractility on fetal echocardiogram as well as any secondary changes of cardiac enlargement , tricuspid regurgitation , pericardial effusion , or the development of hydrops fetalis ( figure 11 ) . lower tracing is uterine contractions . note slow fetal heart rate ( fhr ) of 80 - 115 bpm . bottom shows fetal lvot doppler with measurement of mechanical pr interval , from onset of mitral a wave ( nadir of flow between the e wave and the a wave , when e and a are not distinctly separated ) to the onset of aortic flow . x axis= time in seconds ; y axis= velocity in meters / second . fetal doppler pr interval shows 1st degree heart block with the addition of profound sinus bradycardia ( fetal heart rate of 60 bpm ) . long pause between the onset of the atrial contraction and onset of ejection time ( pr interval ) . fetal doppler pr interval shows wenckebach mobitz type i , a type of 2nd degree heart block . the pr intervals become progressively longer ( top right and bottom left ) , with a non - conducted pr interval ( bottom right ) top shows fetal doppler pr interval with 3rd degree heart block . spectral doppler labeled a symbolizes a rapid atrial rate moving about 3 times as fast as ventricular rate seen below the baseline . bottom shows m - mode of ventricle and atrium in 3rd degree heart block with slow ventricular rate versus rapid atrial rate . . transverse section of fetal thorax displaying 4 chamber view of heart surrounded by pleural and pericardial effusions . with increasing prenatal care and use of ultrasound technology in pregnancy , increasing numbers of cases of autoimmune congenital block are being diagnosed between 18 and 24 weeks of gestation . unfortunately , although these babies are at high risk for morbidity and mortality , guidelines are not well established nor based on definite scientific evidence . based on the assumption that treatment for identified heart block in utero may be effective if it can reduce a generalized inflammatory insult and lower the titer of maternal autoantibodies , several prenatal therapeutic protocols have been utilized . these include the use of adrenocorticosteroids , which are not metabolized by the placenta , principally dexamethasone . some researchers have also attempted plasmapheresis and the use of maternal alpha adrenergic agents.2 our therapeutic approach to a fetal diagnosis of congenital heart block is as follows.2 if the heart block is already third degree and has been present for more than three weeks , we feel that an attempt at reversing this complete heart block is futile , and therefore we provide serial echocardiographic and obstetrical follow - up but no therapy is initiated . if , however , the third degree heart block has been recently diagnosed , we offer the patient a therapeutic course of dexamethasone 4 mg . if there has been no change in fetal status , we taper the course and discontinue it . on the other hand , if the fetus conduction system disease has improved to second degree block or better , then we continue dexamethasone until delivery and subsequently taper in the mother . if the fetus presents with alternating second and third degree block , we again offer dexamethasone at 4 mg orally daily for a six - week period of time . if the conduction system disease progresses to third degree block then we taper the drug and stop it . but if there has been improvement to second degree or better , we continue the steroids until delivery and taper thereafter . if the fetus is discovered to have only second degree or a simply prolonged mechanical pr interval ( first degree block),6 then we offer the mother dexamethasone 4 mg . orally daily until delivery and taper her dose after that . on the other hand , if this early block progresses to permanent third degree block , we will taper the steroid if third degree block has been present for six weeks or longer . occasionally , the fetal congenital heart block is associated with early signs of myocarditis and fetal hydrops . in such a case , we again offer dexamethasone at 4 mg orally daily until improvement of the hydrops fetalis per se , and then taper . some studies have suggested7 that in severely hydropic fetuses there may be some benefit to daily dexamethasone at 4 mg . other varied therapies in such cases of hydrops have included plasmapheresis , maternal terbutaline , digoxin , diuretics or direct fetal pacing . there has been no long - term survival from these desperate measures , and therefore if the lungs are mature at this point , we would advise early delivery . these include the glucocorticoid associated risks of increased infection , loss of bone density , diabetes , hypertension and cataracts . the fetal risks of maternal steroids include oligohydramnios , intrauterine growth retardation and adrenal suppression . there is also some suggestion of a risk to the developing fetal brain when exposed to steroids . some questions have arisen as to the appropriate use of prophylactic therapy in the pregnancy with a high - risk mother , such as those women with very high titers of the antibodies or a previous child with neonatal lupus . we feel that there is no support for the initiation of immune modulating treatment as a pre - emptive strike prior to the development of fetal conduction system disease . it is clearly advantageous to provide close fetal follow - up for monitoring the patient at risk for congenital heart block in the presence of maternal autoantibodies . we recommend that all women with anti - ro antibodies be evaluated by serial fetal echocardiograms . particularly high - risk groups appear to be those women with very high titers of anti - ro and anti - la antibodies , as well as those with previously affected pregnancies . we have recently developed a new technique in fetal echocardiography that allows us the possibility to detect the first possible changes of fetal conduction system disease , that is , the presence of first degree heart block in the fetus . in this case , the overall fetal heart rate will still be normal , but our new non - invasive doppler technique can measure the mechanical pr interval in the absence of an electrocardiogram from the left ventricular outflow tracing . this will allow the earlier diagnosis and the possibility of very early treatment , which may be able to reverse the disease.68 for this reason , we strongly suggest weekly fetal echocardiograms with doppler for pregnancies at risk . supportive treatment for low output or congestive heart failure can clearly be offered as well as pacemakers for babies with significant bradycardia , such as those with a heart rate less than 55 bpm . although we recognize that the newborn serum contains maternal antibody titers , we have no real data on immune modification of the newborn after birth . similarly , we can not comment on the fact that anti - ro and anti - la antibodies have been detected in maternal breast milk . we do know that neonates at risk for developing lupus rashes should be protected from sun exposure , but otherwise treatment is fairly conservative with the use of topical corticosteroids . the liver enzyme abnormalities and blood count irregularities are usually self - limited and require no specific treatment . the risk of a baby born with neonatal lupus syndromes developing active lupus in the future , is small and probably related to the genetic inheritance of the risk of developing rheumatic diseases rather than the maternal antibodies themselves . women with serum titers of anti - ro antibody carry a 3% risk of having a child with neonatal lupus syndrome . if she has a prior experience with affected fetuses , her risk rises to about 18%.5 therefore , we believe that all women at risk with antibodies present , should be closely followed during the pregnancy with serial echocardiograms , specifically looking for the earliest signs of conduction system disease such as pr interval prolongation by doppler . although prophylactic therapy is not indicated at the present time , if manifestations of congenital heart block develop , we have established empiric treatment guidelines . neonatal lupus congenital heart block has a fairly high morbidity and mortality but we believe that the outcome can be improved with early diagnosis and aggressive therapy . those patients whose congenital heart block is associated with structural heart disease have a higher morbidity and mortality , which is determined more by the underlying structural congenital heart disease than it is by the need for a pacemaker per se .

congenital heart block is a rare disorder . it has an incidence of about 1 in 22,000 live births . it may be associated with high mortality and morbidity . this should generate a high index of suspicion for early diagnosis and aggressive therapy when appropriate . the congenital heart block associated with neonatal lupus is considered a form of passively acquired autoimmune disease in which maternal autoantibodies to the intracellular ribonucleoproteins ro ( ss - a ) and la ( ss - b ) , cross the placenta and injure the previously normal fetal heart . women with serum titers of anti - ro antibody carry a 3% risk of having a child with neonatal lupus syndrome . recurrence rates are about 18% . we believe that serial echocardiograms should be acquired so that early diagnosis is made and aggressive therapy administered , if signs of conduction system disease such as pr interval prolongation by doppler are found , so as to optimize the outcome . establishment of guidelines for therapy have been set empirically , should signs of congenital heart block develop . those patients whose congenital heart block is associated with structural heart disease have a higher morbidity and mortality , which is determined more by the underlying structural congenital heart disease than it is by the need for a pacemaker per se .

malnutrition is one of the most important causes for improper physical and mental development of children . one in every five children in the developing world is malnourished , and poor nutrition is associated with half of all child deaths worldwide . malnutrition in children causes an increase in morbidity and mortality and has an adverse effect on intellectual ability . globally , acute malnutrition causes more than 50% of childhood mortality in children under 5 years old , which implies that about 3.5 million children die of malnutrition each year . weight - for - height ( wfh ) index ( figure 1 ) , height - for - age ( hfa ) index , weight - for - age ( wfa ) index ( figure 2 ) , body mass index , and skin fold thickness are to be used more frequently in the field . in april 2006 , the who released new global growth charts for infants and children as old as 5 years to replace the existing cdc / who international growth charts , which were based on the 1977 nchs growth charts . the worldwide malnutrition estimation rates indicate that 35.8% of preschool children in developing countries are underweight , 42.7% are stunted , and 9.2% are wasted . the increasing prevalence of childhood obesity has become a growing matter of public health concern worldwide . obesity has increased from 4.2% , in 1990 , to 6.7% , in 2010 , worldwide and is expected to reach 9.1% , in 2020 [ 9 , 10 ] . in iran , like many of the other developing countries , the prevalence of obesity in children has been moving on . according to a survey in west azerbaijan , 8.7% , 7.5% , and 4.3% of the children aged less than five years suffered from stunting , wasting , and underweight , respectively . the present study aimed at assessing the prevalence of malnutrition ( underweight , stunting , wasting , overweight , and obesity ) in under - five - year - old children in salmas district . the current study is a cross - sectional study which was conducted for assessing the nutritional status of children under 5 years old in salmas district on the basis of national guide and has been performed from 16 until 30 october , 2011 , with the cooperation of the office of community nutrition improvement and the united nations children 's fund ( unicef ) . using cluster sampling , the statistical population included 059-month - old children residing in the cities and villages of salmas and by ena software sample size was calculated with 5% confidence interval ; 902 children were determined . in this study , children being mentally and physically retarded and having problems in terms of anthropometry were removed from the study and replaced by other children . for medical ethics , the parental consent form must be completed to conduct completed design . in this study , first , the total number of households residing in rural and urban areas and the total number of children between 059 months in salmas city were cumulatively calculated . then , in the ena software items , in the planning part , the desired items were entered into sample size calculation as follows : the number of children under 5 years old : 19824 people , estimate of the prevalence number of malnutrition : 8.7 , widespread confidence interval : 2.3 , design effect : 1.5.after entering the above information , 841 were determined as the sample size and , with 5% confidence interval for a sample sufficiency , the number of children was calculated as 885 children and , finally , in the study , 902 questionnaires were completed . given that it was supposed to study 18 children in each cluster , 50 clusters were determined for this study . then , the ratio of urban and rural populations in salmas was calculated in this region ; the urban population was 49% and the rural population was 51% . therefore , 26 rural clusters and 24 urban clusters were determined and in the next stage the names of all villages and urban blocks were separately entered into the part of selecting clusters in ena software according to the number of clusters . the required data were collected through measuring the height and weight and arm circumference of the children in the study , completing the questionnaire and interviews with mothers or caregivers of children . the scale used in this study was a single pan balance with the maximum capacity of 150 kg and accuracy of 100 gr . if possible , the child was directly weighed . if the baby was too small or cried so hard , first , the mother was weighed alone and then hugged the child . the scale automatically calculated the weight of the child by subtracting . also , every day before starting work , to ensure the accuracy of the scale , the scale was tested using the control scale . the height of less - than - two - year children was measured in a supine and larger children were measured in standing position with an accuracy of one tenth of millimeter . the middle of the left arm circumference in children from 6 to 59 months was measured using a special band of measuring arm circumference based on the following steps and was recorded in millimeters . chi - square test was to be used for relationship independent variables ( sex and region ) with malnutrition . the number of children under 5 years old : 19824 people , estimate of the prevalence number of malnutrition : 8.7 , widespread confidence interval : 2.3 , this study was done on 902 children under 5 years old including 49.6% being boys and 50.4% being girls . most children were in the age group of 1829 months ( 24.7% ) and the lowest number was in the age group of 5459 months ( 8.8% ) ( table 1 ) . totally , the prevalence of malnutrition based on underweight , stunting , and wasting was estimated to be 2.3% , 7.3% , and 1.4% among children , respectively . the results of the study showed that underweight in girls and rural areas was more common . malnutrition under height for age in girls and boys in rural areas was more than that of the urban areas ; also , we found that wasting index was not different in both sex and area . current study showed that prevalence of overweight and obesity in girls and rural areas was less than in boys and urban areas but this difference was not significant ( table 3 ) . the relationship between gender and region with malnutrition showed that there was no statistical difference between sex and underweight , wasting , and stunting , but stunting was more common in rural areas and this different was significant ( p < 0.001 ) ( table 4 ) . the graph of the normal distribution of height for age in studied children in salmas shows that distribution of height for age was skewed to left comparing with who standard ( figure 3 ) . the aim of the current study was to assess the nutritional status of under - five - year - old children in urban and rural areas in salmas district . the prevalence of malnutrition based on underweight , stunting , and wasting was estimated to be 2.3% , 7.3% , and 1.4% among children , respectively , in salmas district . in a study in west azerbaijan province by farrokh - eslamlou , prevalence of underweight , stunting , and wasting was estimated to be 4.3% , 8.7% , and 7.5% , respectively . in another study , by veghari , malnutrition was observed in 3.20% , 4.93% , and 5.13% based on underweight , stunting , and wasting , respectively . in another study in khorasan province northeast of iran the rate of underweight , stunting , and wasting was reported to be 7.5% , 12.5% , and 4.4% , respectively . according to the unicef report , 11% , 15% , and 5% of under - five - year - old iranian children suffer from underweight , stunting , and wasting up , respectively . results of current study showed that underweight in girls and rural areas was common more which is consistent with other studies [ 7 , 13 ] . malnutrition based on height for age in both girls and boys in rural areas was more than urban areas which is due to the poor economic status , cultural status , income level , food behavior , and less health care in rural areas that are known as the risk factor for malnutrition . our findings show that there was no statistical difference between sex and underweight , wasting , and stunting , but we found statistically significant differences between stunting and region where stunting was more common in rural areas which is consistent with previous studies in iran and stunting is still highly prevalent in underdeveloped and developing countries . in current study , the prevalence of overweight and obesity is increasing worldwide and has become a public health challenge [ 10 , 15 ] . the tracking of childhood overweight and associated health consequences into adulthood is of concern ; several serious physical conditions are associated with overweight , especially obesity , among children including asthma , sleep problems , cardiovascular diseases , and type 2 diabetes . prevalence of obesity and overweight in boys and urban areas was more than girls and rural areas but this difference was not significant . in this area , stunting , overweight , and obesity are the most important priorities that health officials must pay more attention to . given the differences between various provinces and regions of the country which are a result of the differences between the levels of development in these areas , the necessity of designing and implementing targeted strategies it is worth noting that the present study has been conducted in a single period and in only one city of each province and using ena software ; therefore , the judgment about the whole province requires general investigation in all cites of the province and the obtained results are solely applied to these three cities . this study , also , showed that the ena software has a special ability to determine the samples and clusters and is a simple , rapid , and accurate method , especially in epidemiological studies compared to other methods that were used in studies in our country which can be a convenient tool and its use is suggested for the same studies . also , the quality control of the performed activities by the teams in the field is another distinctive feature of this software which is considered of high importance and emphasizes the use of this software .

introduction . malnutrition is one of the most important causes for improper physical and mental development of children . childhood obesity is a worldwide public health problem . the increasing prevalence of childhood obesity has become a growing matter of public health concern worldwide . the aim of the current study was to determine the prevalence of malnutrition and obesity in children under 5 years old in salmas district . methods . the current study is a cross - sectional study conducted on 902 of children under 5 years old to assess the nutritional status in salmas district and performed from 16 until 30 october , 2011 , with the cooperation of the office of community nutrition improvement and the united nations children 's fund . ena ( emergency nutrition assessment ) and spss software were used for data analysis . results . 49.6% of children were boys and 50.4% were girls . the prevalence of malnutrition based on underweight , stunting , and wasting was estimated to be 2.3% , 7.3% , and 1.4% among children , respectively . stunting was more common in rural areas and this difference was significant ( p < 0.001 ) . conclusion . in this area stunting , overweight and obesity were the most important priorities that health officials must pay more attention to . ena software has a special ability to determine the samples and clusters and is a simple , rapid , and accurate method , especially in epidemiological studies in the country , and can be a convenient tool and its use is suggested for the same studies .

the association of pancreatic disease with fat necrosis at distant foci was first described by chiari in 1883 ( 1 ) . the most common pancreatic disorders associated with pancreatic panniculitis are acute or chronic pancreatitis , especially the alcohol - related types , and pancreatic carcinoma . other pancreatic disorders have been infrequently reported to be associated with pancreatic panniculitis , and these include post - traumatic pancreatitis , pancreatic pseudocyst , pancreas divisum , and hemophagocytic syndrome ( 2 ) . to the best of our knowledge , one case was associated with pancreatic adenocarcinoma , and the other two were associated with acute and chronic pancreatitis , respectively ( 1 , 3 , 4 ) . we report here on a case of pancreatic panniculitis associated with acute pancreatitis that had a fatal outcome . physicians should be aware of this disease entity because this rare cutaneous manifestation may be associated with major morbidity and significant mortality . a 50-yr - old man with a history of alcohol abuse presented with increasing fatigue , generalized weakness , decreased appetite , and abdominal distension and discomfort for the past 2 weeks . he was admitted to the department of internal medicine under the impression of acute pancreatitis , and he was referred to the department of dermatology for the multiple painful subcutaneous nodules on his legs , which had suddenly developed 3 weeks before ( fig . his hemoglobin value was 11.4 g / dl ( reference range : 13 - 18 g / dl ) , the total count of white blood cells was 31,300/l ( reference range : 4,000 - 10,000/l ) with 90.7% segment neutrophils , but the coagulation profiles and platelet counts were normal . the serum amylase was 1,909 u / l ( reference range : 20 - 120 u / l ) , and the lipase was 2,306 u / l ( reference range : 5 - 51 u / l ) , an alanine aminotransferase level of 24 u / l ( reference range : 7 - 40 u / l ) , and a lactate dehydrogenase level of 665 u / l ( reference range : 200 - 400 the fasting glucose level was 133.9 mg / dl ( reference range of 70 - 110 mg / dl ) and the electrolytes were unbalanced . the calcium level was 7 mg / dl ( reference range : 8.6 - 10 mg / dl ) , and the sodium level was 125 the blood urea nitrogen and creatinine were 40.1 mg / dl and 2.1 mg / dl , respectively . after 48 hr , the blood urea nitrogen was increased to 62.7 mg / dl after intravenous fluid administration . as his leukocytosis , elevated serum ldh at admission , and hypocalcemia , hypoalbuminemia , increase in blood urea nitrogen during initial 48 hr were poor prognostic factors in ranson criteria , increased risk of complications was predicted . the chest radiograpy showed mild pleural effusion . on the abdominal computed tomography scan and magnetic resonance ( mr ) imaging taken on the second day of admission , a swollen pancreas with an dilated pancreatic duct , a loculated fluid collection in the left anterior perirenal space , multiple hepatic cysts , and massive ascites u / l , the serum - ascites albumin gradient was calculated to be 1.07 , and there was no evidence of malignancy . a skin biopsy performed on the 4th day of admission from the nodule on the left lower leg showed a diffuse subcutaneous fat necrosis and ghost - like cells with thick shadowy walls and no nuclei . there was a fine granular basophilic material deposited within and around the necrotic fat cells ( fig . after 10 days of intensive medical care for the pancreatic disease , the patient 's condition began to worsen ; he and his family began to refuse any further treatment . despite a strong warning by physicians the pathogenesis of pancreatic panniculitis is still unknown , but the released pancreatic enzymes such as trypsin may increase the permeability of the microcirculation . lipase or amylase is then involved in the process of fat degradation , which results in the liberation of free fatty acids that combine with calcium to form soap ( 2 ) . although elevation of the pancreatic enzymes is common in pancreatitis patients , pancreatic panniculitis is a very rare malady . mullin et al . ( 5 ) identified only one case in a retrospective review of 893 patients who had suffered with pancreatic disease from various causes . furthermore , well documented cases of fat necrosis with normal serum lipase levels have also been described ( 6 ) . these reports , suggest that there would be some other factors that allow the pancreatic enzymes to escape from the circulation and act on the subcutaneous fat . zellman ( 7 ) suggested that some damage to the blood vessels via inflammation , edema , or altered immunity may act as the initiating factor . as for the two cases in the korean literature , one case was associated with chronic pancreatisis , in which the cutaneous lesions had occurred 3 months later than the other systemic symptoms , and they involuted as the underlying pancreatitis was ameliorated ( 4 ) . the other case was associated with acute pancreatitis with pancreas divisum , in which his skin lesions had appeared 2 - 3 days after his admission due to acute pancreatitis ; the skin lesions also resolved as the underlying pancratitis subsided with conservative treatment ( 1 ) . on the contrary , in our patient , the skin lesions preceded the other systemic symptoms . although the underlying pancreatic pathologic conditions can vary , the clinical features of pancreatic panniculitis are similar . the legs were the most commonly affected area , but the lesions can also occur on the arms , thighs , and trunk . the lesions usually began as erythematous or red - brown subcutaneous nodules with a tendency to show central softening . in the mild form if the fat necrosis is severe , individual nodules may break down and extrude necrotic material ( 8) . although patients with other panniculitides such as erythema nodosum , erythema induratum , lupus panniculitis , weber - christian pannicultitis , or alpha-1 antitrypsin deficiency - associated panniculitis can have similar clinical lesions , the diagnosis of pancreatic panniculitis is suggested by the presence of pancreatic disease and this the pathognomonic findings are collections of ' ghost cells ' and anucleated adipocytes containing intracytoplasmic fine basophilic granular material from the saponification of fat . the resistance of the fat cell membrane to lipase produces the shadowy walls and the fatty acids combine with calcium to form calcium soap ( 10 ) . sometimes conservative care can ameliorate the pancreatitis , and this then results in the resolution of the skin lesions . in isolated cases , surgical correction of anatomic ductal anomaly or pancreatic pseudocyst , or the removal of gallstones has resulted in complete resolution ( 8) . however , disseminated fat necrosis is associated with major morbidity and mortality . in a review of 27 patients with pancreatic panniculitis , all 8 patients with pancreatic carcinoma and 42% of the 19 patients with pancreatitis died of their disease , as was seen in our case ( 9 ) . pancreatic panniculitis is a pathognomic finding of pancreatic disease , and as in our case , the cutaneous lesions may precede the usual manifestations of underlying pancreatic disease by several weeks to months ( 11 ) .

pancreatic panniculitis is a rare disease in which necrosis of fat in the panniculus and other distant foci occurs in the setting of pancreatic diseases ; these diseases include acute and chronic pancreatitis , pancreatic carcinoma , pseudocyst , and other pancreatic diseases . this malady is manifested as tender erythematous nodules on the legs , buttock , or trunk . histopathologically , it shows the pathognomonic findings of focal subcutaneous fat necrosis and ghost - like anucleated cells with a thick shadowy wall . we herein report a case of fatal pancreatic panniculitis that was associated with acute pancreatitis in a 50-yr - old man . he presented with a 3-week history of multiple tender skin nodules , abdominal pain and distension . laboratory and radiologic findings revealed acute pancreatitis , and skin biopsy showed pancreatic panniculitis . despite intensive medical care , he died of multi - organ failure 3 weeks after presentation .

a significant number of these admissions are attributable to a small number of complex patients with other comorbidities who do not engage well with mainstream services . assertive outreach teams have been used in the field of psychiatry to engage patients who are poorly compliant . this study examines whether an alcohol assertive outreach team ( aaot ) can engage with this group and reduce hospital admissions . the aaot is a multidisciplinary team with medical , psychiatric , substance misuse , psychology , nursing and social work specialists . the team worked with patients with the highest number of alcohol - related admissions and case managed in a community setting for 6 months . the admission and emergency department attendances of the cohort were compared for the 3-month period before and after the intervention . christo inventory for substance misuse services ( ciss ) scores were determined pre and post the intervention period . the total number of admissions in 3 months fell from 151 prior to the intervention period to 50 following the intervention . emergency department attendances also fell from 360 in 3 months to 146 following the intervention period . an aaot model appears to reduce hospital admissions and emergency department attendances in a complex group of patients that display high alcohol - related admissions . in the uk nhs , a continued rise in alcohol - related admissions is clearly seen . the current costs for alcohol - related admissions in england are estimated at 1.2 billion.1 within this general rise , there is a cohort of patients who frequently attend the emergency department and have high levels of repeated hospital admissions . this group have complex physical health , psychiatric and social needs , but the most common factor is high levels of alcohol use.2 within this group , there are a high proportion of dual diagnosis patients : those with both mental health and alcohol problems . unfortunately , they do not interact well with mainstream services and higher death rates are seen.3 previous studies have looked at strategies to impact patients who are frequent attenders at emergency departments . patient care plans have been used to reduce emergency department attendances with mixed results.46 these plans involve a specific management plan that is implemented when the patient attends the emergency department . case management strategies have also been used.7 8 this involves an ongoing clinical interaction which extends beyond the hospital into the community . case management involves multidisciplinary teams usually comprising psychiatric , social work , substance misuse , nursing and medical specialists . in mental health services , these are multidisciplinary teams which work with people with complex health needs , drug , alcohol , mental health and housing issues . they have been shown to be effective in patients with high levels of inpatient bed use.9 these teams are commonly targeted at people who engage poorly with services . assertive outreach teams are typically defined by workers with a low caseload , usually 10 patients per worker , assertive engagement mechanisms and a high proportion of contact within the community.10 recent work has demonstrated the use of assertive treatment methods to improve engagement in alcohol services and to improve rates of patients entering recovery.11 in view of the high levels of alcohol - related admissions seen in the north west of england , the north west chief executives challenge , led by david dalton and other experts , developed strategies to improve the quality of alcohol care . the two initiatives modelled were the development of an alcohol assertive outreach team ( aaot ) and a 7-day alcohol specialist nurse service.12 an aaot was developed to work with two separate cohorts of patients . the first group are patients with alcohol - related liver disease who are increasingly using hospital services and exceed the threshold of two admissions wholly attributable to alcohol . the aim of this study is to establish whether an aaot is an effective model for reducing hospital admissions and emergency department attendances in the top 30 most frequently admitted patients . the study was carried out between january 2011 and july 2012 at the salford royal nhs foundation trust . the study was a retrospective cohort analysis of the effects of assertive outreach methods on the admission rates pre and post the intervention period . the aaot comprised a consultant in emergency medicine , a consultant psychiatrist specialising in addiction disorders , an emergency department nurse , a social worker , a psychologist , an alcohol worker , a support worker and an administrator . in the uk , a national audit system exists to record the number of alcohol - related admissions to each hospital . a database was created to identify the adult patients with the highest number of alcohol - related unscheduled care admissions . patients were case managed for a period of 6 months ; after this time the database was refreshed . a new cohort of patients was then case managed for the subsequent 6 months . to case manage the patients , case profiles were initially formed by information gathered from the patient , the acute trust , mental health trust , community alcohol team , primary care services , council and criminal justice services . the face - to - face interaction as part of the case management was organised in the community away from the hospital . common strategies were fast access to alcohol detoxification ; appropriate referral to outpatient specialities and support in getting there ; alcohol and psychological support ; facilitating housing solutions ; and robust responses to violence and aggression . a part of the case management involved the production of individual care plans that would be implemented when a patient attended the emergency department . the plans were immediately available to the emergency department staff at the point of triage and helped produce a consistent coordinated response whenever patients interacted with the hospital . the primary outcome measure was the number of admissions per month in the 3-month period immediately before and after the 6 months of case management intervention . emergency department attendances were also recorded during these periods . at the start and end of the 6-month case management period , christo inventory for substance misuse services ( ciss ) scores were calculated.13 this is a subjective score that reflects a patients social functioning , psychological and general health , compliance , drug and alcohol use and criminality . the score is a 020 score , with lower scores reflecting better outcomes ( above 11 reflects high problem severity ) . the score has been previously validated.13 ethics approval was not required as this study fulfils the criteria for service evaluation . statistical analysis was performed with non - parametric statistical analysis as the ciss scores , admissions and attendance rates were not normally distributed . medians are quoted and the wilcoxon rank sum test was used for comparing effects between groups . the team was established and began to case manage patients in april 2011 . due to delays in fully establishing the team , only 24 patients were case managed in the initial cohort . a further 30 patients were case managed in the 6-month period starting in october 2011 . a significant reduction in hospital admissions was seen in the 3 months following case management as compared with the 3-month period immediately prior to the start of case management . the total number of hospital admissions were 151 prior to case management , reduced to 50 following the case management period ( w=2070 , p<0.001 ) . a similar significant reduction was also seen in the total number of emergency department attendances in the 3-month periods before and after intervention ( 360 reduced to 146 , w=2215 , p<0.001 ) . the white bar represents the 6-month period in which the team actively case managed 54 patients . there was a significant reduction in the ciss scores at the start as compared with the end of the case management period ( median 11 reduced to 8 , p<0.001 ) . there were no deaths in the study group during the period of case management and up to 3 months after . however , one patient left the local area and contact was not able to be maintained . there was loss of engagement with a second patient who did remain within the local area . this study demonstrated that case management using the alcohol assertive outreach model can reduce emergency department attendances and hospital admissions . this study is unique in that the focus is on patients with high level of alcohol admissions rather than frequent emergency department attendances alone . while these groups share similar complexity , this is a slightly different cohort than the traditional group of emergency department regular attenders . our pilot work demonstrated a lower level of homelessness in this group in contrast to our most frequent emergency department attenders . our high admissions group had more stable accommodation and we were able to remain in contact and engage in the outreach aspect of the team 's work . however , we had no exclusion criteria and homeless patients were included in the study . a second reason for addressing a high admissions group rather than frequent emergency department attenders was the perceived cost benefit of reducing admissions rather than emergency department attendances alone . based on national indicators and length of stay costs , an average alcohol - related admission costs a primary care organisation 1824 and an alcohol - related emergency department attendance costs 80.14 in the 3-month period after the end of case management alone , we demonstrated a reduction in admissions from 151 to 50 . taking into account the reductions in admissions and attendances seen during the first year of the team alone , the cost reductions cover the 300 000 required to establish the service . these initial cost reductions do not take into account any ongoing reduction in admissions and subsequent savings . previous studies using care plans and case management strategies have shown mixed outcomes in emergency department frequent attenders . studies from the uk , australia and the usa have all demonstrated reduction in emergency department attendances,2 4 5 8 15 but other teams have reported no benefit6 16 or even an increase in emergency department use.7 there is less evidence on the effects on admission rates . a uk study reported a good impact on admission rates,4 but this was not repeated in other healthcare systems . a us randomised control trial reported no impact on admissions with case management;8 however , increased admissions were seen in an australian trial.7 traditional clinical approaches to medical , alcohol and psychiatric disorders are effective when a single disorder is identified . unfortunately , when complex comorbidity is present , patients tend to fall between the gaps between services and commonly present and are admitted to inpatient hospital beds . we found that through effective multidisciplinary working , the aaot could more effectively support these complex individuals . the most important factor that increased the effectiveness of the team was the creation of an extensive case profile for each patient from a wide ranging variety of information sources . this permitted the team to plan effective interventions which supported the patient 's medical , psychiatric , substance misuse and social difficulties in a coordinated fashion . as with all projects that impact the frequent users of emergency departments , there has to be concern that patients are not simply being diverted to other emergency departments . this is true especially with homeless emergency department regular attenders who commonly move between areas . one undoubtedly had moved out of the area and was likely to have attended other emergency departments . the second patient remained within the local area but the team was not able to engage with him . for the rest of the patients , the ongoing community engagement allowed us to be confident that they remained within the locality of the hospital . indeed , the reduction in the ciss scores that are demonstrated point to improved functioning and a reduction in problem severity . the postintervention ciss score remains high in comparison with other settings such as abstinence based treatment centres or outpatient alcohol services , but this reflects the severity of the cohort that this group comprises . assertive outreach is a uk term to refer to the north american assertive community treatment for psychiatric care . it is more successful when hospital use is high.9 assertive approaches to engagement in uk community alcohol work have demonstrated to be beneficial . passetti et al11 set up a clinic which targeted patients with a history of disengagement . they used an assertive approach and saw more people completing withdrawal treatment and entering an aftercare placement . we needed to modify the original model to adapt to our local application , but maintained the fidelity of the assertive approach.10 this was defined by a small caseload with 10 patients per worker , backed by a multidisciplinary team with psychiatry , nursing and substance misuse workers , and uses assertive engagement mechanisms and focuses on community engagement . we used a preintervention and postintervention comparison whereby all patients were used as their own control . some of the effects could be attributed to a natural reduction in emergency department attendances and admissions over time . this effect has been previously noted in two us studies,6 8 but it is not a consistent effect with studies demonstrating no reduction16 or even an increase in emergency department attendance and admissions over time.7 a multicentre randomised controlled trial would be needed to adequately power a study to address this issue . we took a pragmatic approach in using a prestudy and poststudy to describe the effects of alcohol assertive outreach work in reducing admissions in this cohort . the main clinical findings of this study describe an aaot approach to the support and management of a cohort of patients who demonstrate high numbers of alcohol - related admissions to a uk inner city hospital . this approach appears to reduce admissions , emergency department attendances and improves social functioning . what is already known on this topica complex group of patients with high levels of repeat alcohol - related admissions are well recognised , but difficult to impact what this study addsan alcohol assertive outreach team can engage with this complex patient group and reduce hospital admissions how might it impact on clinical practice in the foreseeable futurealcohol assertive outreach teams provide a solution for reducing alcohol - related admissions in the highest users of inpatient services a complex group of patients with high levels of repeat alcohol - related admissions are well recognised , but difficult to impact an alcohol assertive outreach team can engage with this complex patient group and reduce hospital admissions alcohol assertive outreach teams provide a solution for reducing alcohol - related admissions in the highest users of inpatient services

objectivealcohol - related admissions are increasing . a significant number of these admissions are attributable to a small number of complex patients with other comorbidities who do not engage well with mainstream services . assertive outreach teams have been used in the field of psychiatry to engage patients who are poorly compliant . this study examines whether an alcohol assertive outreach team ( aaot ) can engage with this group and reduce hospital admissions.designthe aaot is a multidisciplinary team with medical , psychiatric , substance misuse , psychology , nursing and social work specialists . the team worked with patients with the highest number of alcohol - related admissions and case managed in a community setting for 6 months . the admission and emergency department attendances of the cohort were compared for the 3-month period before and after the intervention . christo inventory for substance misuse services ( ciss ) scores were determined pre and post the intervention period.results54 patients were case managed . the total number of admissions in 3 months fell from 151 prior to the intervention period to 50 following the intervention . emergency department attendances also fell from 360 in 3 months to 146 following the intervention period . ciss scores fell from 11 preintervention to eight postintervention.conclusionsan aaot model appears to reduce hospital admissions and emergency department attendances in a complex group of patients that display high alcohol - related admissions .

opces is a bioequivalent , randomized , double blind , multi - centric clinical trial enrolling six centers in five cities of iran . from march 2007 to november 2009 , 442 patients were included in this study . those centers that have the facility and requirements for stenting and also the tendency for research collaboration patients were randomized at least 12 hours before planned pci to receive a 300 mg loading dose of plavix or osvix ( 1:1 ) and followed by 75 mg / day for 30 days in bms and 6 months in des groups . all patients received daily aspirin therapy before pci and were continued until one and six months in bms and des groups , respectively . the study was approved by the ethical committee of the isfahan cardiovascular research center , a who collaborating center and was registered in the iranian registration clinical trial ( irct138712111723n1 ) . patients were eligible for enrollment if they were 18 years of age , had chronic stable angina and selected for stenting in a coronary artery with diameter 2.5 mm . exclusion criteria included previous treatment with a clopidogrel or warfarin for any reason , history of allergy or any contraindication to clopidogrel , high - risk patients ( e.g. , severe left ventricular dysfunction with ejection fraction less than 30% , stenting of saphenous vein grafts or internal mammary artery ) , total vessel occlusion without myocardial viability in des and large thrombus in angiography before pci . baseline and demo graphic information including sex , age , indications for stenting , morphology , length , and diameter of stent , previous cardiac interventions , and left ventricular ejection fraction based on echocardiography reports were recorded according to the patients medical charts . history of smoking , hypercholesterolemia , hypertriglyceridemia , high blood pressure , diabetes mellitus and family history of premature coronary heart disease ( cad ) in first relatives were also recorded . blood samples were drawn from each patient before the intervention to measure the platelet and neutrophil counts as well as liver enzymes . patients who were selected for stenting were randomly assigned to receive a 300 mg loading dose of plavix ( n = 221 ) or osvix ( n = 221 ) ( 1:1 randomization ratio ) 12 hours before the procedure followed by 75 mg / day for 30 days in bms and 6 months in des groups . all patients received daily aspirin therapy three days before pci and were continued until one and six months in bms and des groups , respectively . the laboratory assessment was performed for all patients before the study , then one month after pci for patients with bms and one and six months after pci for patients with des . blood samples were taken in one month and six months in bms and des groups to measure platelets , neutrophils , alanine ami - notransferase ( alt ) , and aspartate ami - notransferase ( ast ) . pci was considered successful when a residual diameter stenosis was less than 20% with thrombolysis in myocardial infarction ( timi ) flow less than iii . timi iii means contrast material flows briskly into and clears rapidly from the distal segment or good distal runoff.13 timi grade 3 flows were obtained . type a included lesions with the following characteristics : discrete ( < 10 mm length ) , concentric , readily accessible , non - angulated segment , smooth contour , little or no calcium , less than totally occlusive , not ostial in location , no major side branch involvement , and absence of thrombus . type b included lesions of tubular ( 10 - 20 mm length ) , eccentric , moderate tortuosity of proximal segment 45 , irregular contour , moderate to heavy circulation , total occlusion , ostial location , bifurcation lesion requiring double guide wire , and some thrombus present . type c involved diffuse ( > 20 mm length ) , excessive tortuosity of proximal segment , extremely angu - lated segments ( 90 ) , with total occlusion > 3 months old , inability to protect major side braches , and degenerated side branches with friable lesions . mace which is the primary endpoint of this study included death , q wave and non - q wave mi , stroke , target lesion revascularization ( tlr ) , target vascular revascularization ( tvr ) , in - stent thrombosis . cardiovascular death was considered as any death with a cardiovascular cause and included those deaths following a cardiovascular procedure ( for example , pci ) , cardiac arrest , mi , pulmonary embolus , stroke , hemorrhage or death due to unknown cause . non- cardiovascular death was defined as death due to a clearly documented non - cardiovascular cause ( e.g. , trauma , infection , malignancy ) . mi was defined by symptoms suggestive of infarction , electrocardiographic changes , and positive cardiac enzymes . in - stent thrombosis was classified in three groups ; definite : the presence of an acute coronary syndrome with angiographic or autopsy evidence of thrombus or occlusion ; probable : unexplained deaths within 30 days after procedure or acute myocardial infarction involving the target - vessel territory without angiographic confirmation ; possible : all unexplained deaths occurring at least 30 days after the procedure . stroke defined as a new focal neurological deficit thought to be vascular in origin with signs and symptoms lasting more than 24 hours . secondary endpoints included changes of platelet or neutrophil number or liver enzymes , hospitalization for angina , and major bleeding . all primary and secondary end points were assessed during hospitalization and after 30 days in bms and six months in des group . the academic research consortium definitions were used for stent thrombosis.14 results were reported as mean standard deviation ( sd ) for the quantitative variables and percentages for the categorical variables . the groups were compared using the chi - square test ( or fisher 's exact test if required ) for categorical variables and the t - test or mannwhitney u test for the continuous variables . all the statistical analyses were performed using spss version 16.0 ( spss inc . , chicago , il , usa ) for windows . opces is a bioequivalent , randomized , double blind , multi - centric clinical trial enrolling six centers in five cities of iran . from march 2007 to november 2009 , 442 patients were included in this study . those centers that have the facility and requirements for stenting and also the tendency for research collaboration patients were randomized at least 12 hours before planned pci to receive a 300 mg loading dose of plavix or osvix ( 1:1 ) and followed by 75 mg / day for 30 days in bms and 6 months in des groups . all patients received daily aspirin therapy before pci and were continued until one and six months in bms and des groups , respectively . the study was approved by the ethical committee of the isfahan cardiovascular research center , a who collaborating center and was registered in the iranian registration clinical trial ( irct138712111723n1 ) . patients were eligible for enrollment if they were 18 years of age , had chronic stable angina and selected for stenting in a coronary artery with diameter 2.5 mm . exclusion criteria included previous treatment with a clopidogrel or warfarin for any reason , history of allergy or any contraindication to clopidogrel , high - risk patients ( e.g. , severe left ventricular dysfunction with ejection fraction less than 30% , stenting of saphenous vein grafts or internal mammary artery ) , total vessel occlusion without myocardial viability in des and large thrombus in angiography before pci . baseline and demo graphic information including sex , age , indications for stenting , morphology , length , and diameter of stent , previous cardiac interventions , and left ventricular ejection fraction based on echocardiography reports were recorded according to the patients medical charts . history of smoking , hypercholesterolemia , hypertriglyceridemia , high blood pressure , diabetes mellitus and family history of premature coronary heart disease ( cad ) in first relatives were also recorded . blood samples were drawn from each patient before the intervention to measure the platelet and neutrophil counts as well as liver enzymes . patients who were selected for stenting were randomly assigned to receive a 300 mg loading dose of plavix ( n = 221 ) or osvix ( n = 221 ) ( 1:1 randomization ratio ) 12 hours before the procedure followed by 75 mg / day for 30 days in bms and 6 months in des groups . all patients received daily aspirin therapy three days before pci and were continued until one and six months in bms and des groups , respectively . the laboratory assessment was performed for all patients before the study , then one month after pci for patients with bms and one and six months after pci for patients with des . blood samples were taken in one month and six months in bms and des groups to measure platelets , neutrophils , alanine ami - notransferase ( alt ) , and aspartate ami - notransferase ( ast ) . pci was considered successful when a residual diameter stenosis was less than 20% with thrombolysis in myocardial infarction ( timi ) flow less than iii . timi iii means contrast material flows briskly into and clears rapidly from the distal segment or good distal runoff.13 timi grade 3 flows were obtained . type a included lesions with the following characteristics : discrete ( < 10 mm length ) , concentric , readily accessible , non - angulated segment , smooth contour , little or no calcium , less than totally occlusive , not ostial in location , no major side branch involvement , and absence of thrombus . type b included lesions of tubular ( 10 - 20 mm length ) , eccentric , moderate tortuosity of proximal segment 45 , irregular contour , moderate to heavy circulation , total occlusion , ostial location , bifurcation lesion requiring double guide wire , and some thrombus present . type c involved diffuse ( > 20 mm length ) , excessive tortuosity of proximal segment , extremely angu - lated segments ( 90 ) , with total occlusion > 3 months old , inability to protect major side braches , and degenerated side branches with friable lesions . mace which is the primary endpoint of this study included death , q wave and non - q wave mi , stroke , target lesion revascularization ( tlr ) , target vascular revascularization ( tvr ) , in - stent thrombosis . cardiovascular death was considered as any death with a cardiovascular cause and included those deaths following a cardiovascular procedure ( for example , pci ) , cardiac arrest , mi , pulmonary embolus , stroke , hemorrhage or death due to unknown cause . non- cardiovascular death was defined as death due to a clearly documented non - cardiovascular cause ( e.g. , trauma , infection , malignancy ) . mi was defined by symptoms suggestive of infarction , electrocardiographic changes , and positive cardiac enzymes . in - stent thrombosis was classified in three groups ; definite : the presence of an acute coronary syndrome with angiographic or autopsy evidence of thrombus or occlusion ; probable : unexplained deaths within 30 days after procedure or acute myocardial infarction involving the target - vessel territory without angiographic confirmation ; possible : all unexplained deaths occurring at least 30 days after the procedure . stroke defined as a new focal neurological deficit thought to be vascular in origin with signs and symptoms lasting more than 24 hours . secondary endpoints included changes of platelet or neutrophil number or liver enzymes , hospitalization for angina , and major bleeding . all primary and secondary end points were assessed during hospitalization and after 30 days in bms and six months in des group . the academic research consortium definitions were used for stent thrombosis.14 results were reported as mean standard deviation ( sd ) for the quantitative variables and percentages for the categorical variables . the groups were compared using the chi - square test ( or fisher 's exact test if required ) for categorical variables and the t - test or mannwhitney u test for the continuous variables . all the statistical analyses were performed using spss version 16.0 ( spss inc . , chicago , il , usa ) for windows . opces is a bioequivalent , randomized , double blind , multi - centric clinical trial enrolling six centers in five cities of iran . from march 2007 to november 2009 , 442 patients were included in this study . those centers that have the facility and requirements for stenting and also the tendency for research collaboration patients were randomized at least 12 hours before planned pci to receive a 300 mg loading dose of plavix or osvix ( 1:1 ) and followed by 75 mg / day for 30 days in bms and 6 months in des groups . all patients received daily aspirin therapy before pci and were continued until one and six months in bms and des groups , respectively . the study was approved by the ethical committee of the isfahan cardiovascular research center , a who collaborating center and was registered in the iranian registration clinical trial ( irct138712111723n1 ) . patients were eligible for enrollment if they were 18 years of age , had chronic stable angina and selected for stenting in a coronary artery with diameter 2.5 mm . exclusion criteria included previous treatment with a clopidogrel or warfarin for any reason , history of allergy or any contraindication to clopidogrel , high - risk patients ( e.g. , severe left ventricular dysfunction with ejection fraction less than 30% , stenting of saphenous vein grafts or internal mammary artery ) , total vessel occlusion without myocardial viability in des and large thrombus in angiography before pci . all the patients underwent complete history and physical examination . baseline and demo graphic information including sex , age , indications for stenting , morphology , length , and diameter of stent , previous cardiac interventions , and left ventricular ejection fraction based on echocardiography reports were recorded according to the patients medical charts . history of smoking , hypercholesterolemia , hypertriglyceridemia , high blood pressure , diabetes mellitus and family history of premature coronary heart disease ( cad ) in first relatives were also recorded . blood samples were drawn from each patient before the intervention to measure the platelet and neutrophil counts as well as liver enzymes . patients who were selected for stenting were randomly assigned to receive a 300 mg loading dose of plavix ( n = 221 ) or osvix ( n = 221 ) ( 1:1 randomization ratio ) 12 hours before the procedure followed by 75 mg / day for 30 days in bms and 6 months in des groups . all patients received daily aspirin therapy three days before pci and were continued until one and six months in bms and des groups , respectively . the laboratory assessment was performed for all patients before the study , then one month after pci for patients with bms and one and six months after pci for patients with des . blood samples were taken in one month and six months in bms and des groups to measure platelets , neutrophils , alanine ami - notransferase ( alt ) , and aspartate ami - notransferase ( ast ) . pci was considered successful when a residual diameter stenosis was less than 20% with thrombolysis in myocardial infarction ( timi ) flow less than iii . timi iii means contrast material flows briskly into and clears rapidly from the distal segment or good distal runoff.13 timi grade 3 flows were obtained . type a included lesions with the following characteristics : discrete ( < 10 mm length ) , concentric , readily accessible , non - angulated segment , smooth contour , little or no calcium , less than totally occlusive , not ostial in location , no major side branch involvement , and absence of thrombus . type b included lesions of tubular ( 10 - 20 mm length ) , eccentric , moderate tortuosity of proximal segment 45 , irregular contour , moderate to heavy circulation , total occlusion , ostial location , bifurcation lesion requiring double guide wire , and some thrombus present . type c involved diffuse ( > 20 mm length ) , excessive tortuosity of proximal segment , extremely angu - lated segments ( 90 ) , with total occlusion > 3 months old , inability to protect major side braches , and degenerated side branches with friable lesions . mace which is the primary endpoint of this study included death , q wave and non - q wave mi , stroke , target lesion revascularization ( tlr ) , target vascular revascularization ( tvr ) , in - stent thrombosis . cardiovascular death was considered as any death with a cardiovascular cause and included those deaths following a cardiovascular procedure ( for example , pci ) , cardiac arrest , mi , pulmonary embolus , stroke , hemorrhage or death due to unknown cause . non- cardiovascular death was defined as death due to a clearly documented non - cardiovascular cause ( e.g. , trauma , infection , malignancy ) . mi was defined by symptoms suggestive of infarction , electrocardiographic changes , and positive cardiac enzymes . in - stent thrombosis was classified in three groups ; definite : the presence of an acute coronary syndrome with angiographic or autopsy evidence of thrombus or occlusion ; probable : unexplained deaths within 30 days after procedure or acute myocardial infarction involving the target - vessel territory without angiographic confirmation ; possible : all unexplained deaths occurring at least 30 days after the procedure . stroke defined as a new focal neurological deficit thought to be vascular in origin with signs and symptoms lasting more than 24 hours . secondary endpoints included changes of platelet or neutrophil number or liver enzymes , hospitalization for angina , and major bleeding . all primary and secondary end points were assessed during hospitalization and after 30 days in bms and six months in des group . results were reported as mean standard deviation ( sd ) for the quantitative variables and percentages for the categorical variables . the groups were compared using the chi - square test ( or fisher 's exact test if required ) for categorical variables and the t - test or mannwhitney u test for the continuous variables . all the statistical analyses were performed using spss version 16.0 ( spss inc . , chicago , il , usa ) for windows . the total study population represents a group of 442 patients ( mean age 59 9.5 years ) with stable angina pectoris in whom pci was performed . among them , 225 participants implanted bms and the rest implanted des . a total of 224 patients treated with osvix ( 110 patients in bms and 114 patients in des ) and 218 subjects were treated with plavix ( 115 patients in bms and 103 patients in des ) . except male to female ratio that was higher in the osvix than the plavix group , the two drug groups were comparable with respect to baseline characteristics , clinical data especially left ventricular ejection fraction , and medical history . there were significantly more frequent hypertensive patients in the osvix than in the plavix group ( p < 0.01 ) , but no other significant differences were seen in patient characteristics between the two groups table 2 . besides , in both groups with des and bms , there was no significant difference in the type of coronary lesion , direct stenting , stent length and stent diameter ( table 3 ) . baseline characteristics and clinical data of all participants based on plavix or osvix use recorded characteristics and clinical data of all patients based on their stent type . coronary angiography and stent characteristics of patients and drug type used figure 1 shows the frequency and 95% confidence intervals of events such as death , instant thrombosis , mace rate , and mi following osvix or plavix placement in bare - metal or drug eluting stents patients . frequency and 95% confidence intervals of events following osvix or plavix placement in bare - metal or drug eluting stents patients there was no significant difference regarding mace rate in patients treated with osvix compared with plavix ( figure 1 ) . the study groups had the same frequency of mi , thrombosis , stroke and tvr ( figure 1 ) . regarding changes in laboratory variable , no significant changes were found concerning liver enzymes , platelet and neutrophil counts between the two drug regimens in both des and bms groups ( figures 2 and 3 ) . changes of serum platelet and neutrophil counts following osvix and plavix regimens in patients who underwent bare - metal or drug eluting stenting changes of serum aspartate aminotransferase and alanine aminotransferase levels following osvix and plavix regimens in patients who underwent bare - metal or drug eluting stenting opcess is the first randomized trial to compare the effect of a generic form of clopidogrel on mace after coronary stenting in iran . increased platelet activity has been shown to increase cardiovascular complications in acute coronary syndrome ( acs ) , and subsequent to pci.15 various studies have demonstrated that administration of antiplatelet drugs such as aspirin , ticlopidine and clopidogrel reduce short and long - term cardiovascular events in patients suffering acs or undergoing pci.16 we studied the short and long - term cardiovascular complications , including death , myocardial infarction , stroke , tlr and tvr in patients treated with osvix ( 300 mg bolus , and 75 mg / daily maintenance ) or plavix ( 300 mg bolus , and 75 mg / daily maintenance ) . assessment of secondary end - points such as angina , heart failure ( hf ) , major hemorrhagic episodes , and reduction of platelet or neutrophil counts and liver enzyme changes revealed no significant difference between the two treated groups . reduced systolic performance and ageing are among important factors underlying mace in these patients . however , the frequency distribution of age , sex and left ventricular systolic function were similar in osvix and plavix groups . tavazzi showed that concomitant disease and cvd risk factors can increase mace.17 whereas these risk factors were evenly distributed in the two groups in this randomized clinical trial ; a higher frequency of hypertension in the osvix group was seen ( p = 0.001 ) ( table 2 ) . kang and colleagues demonstrated that hypertension does not affect short and long - term cardiovascular complications of pci.18 patients in osvix group showed nonsignificant lower mace rate than the plavix group ( p < 0.05 ) . thus , it can be concluded that osvix has at least similar cvd complications as plavix . ali - doosti and colleagues showed that the type of vessel selected for pci did not affect the short and long term complications of the procedure.19 in our study , the frequency distribution according to the vessel elected for angioplasty was the same in both groups . various studies have demonstrated an increase in short and long term post - angioplasty complications in patients with complex lesions : the worse the anatomy and morphology of the lesion , the more difficult the procedure and the poorer the outcomes.2021 in our study , the frequency distribution of the type of coronary lesion elected for angioplasty was not significantly different in the osvix and plavix groups . in separate studies , singh and pompa showed that the length and diameter of stents are directly , and inversely proportional to tlr.2223 in our study , the stent length and diameter , as well as angioplasty technique had similar distribution in both groups , although the rate of tvr was low in our study as shown by results . this may have been due to the short follow - up period ( one month for non - pharmacological stents and six months for pharmacological stents ) . tvr was seen in two of our patients who underwent cabg a day after unsuccessful angioplasty . based on comparison of frequency distribution of patients according to mace in the preceding one and six months for bms and des stents , respectively , the incidence of mace including death was similar in the osvix and plavix groups . the incidence of mi was 0.4% in the osvix and 1.4% in the pla - vix groups with no significant difference . no significant difference was observed between the two groups in regard to episodes of angina , hf and bleeding which suggest no inferiority of osvix compared to plavix . although the occurrence of events was not significantly different in the two groups , the distribution of findings revealed an up - sloping trend in complications in the plavix group . thus , it may be concluded that osvix was not associated with more complications than plavix in patients who underwent pci . safety of antiplatelet drugs is of great importance and must be assessed against possible side - effects . gurbel demonstrated that including clopidogrel in the therapy of unstable angina patients , significantly reduced mortality from mace . although clopidogrel may significantly increase the risk of bleeding , its benefits have been shown to outweigh the risks.24 yusuf and colleagues showed that adding clopidogrel to aspirin reduced mace but increased the risk of bleeding . however , in our study , the rate of major bleeding in osvix and plavix groups who were treated with aspirin too , were not significant . during our follow - up period , mean platelet counts did not change significantly in either groups ( plavix , p = 0.80 and osvix , p = 0.99 ) or between them ( p = 0.87 ) . however , neutrophil counts were reduced significantly in the two groups ( p < 0.001 ) , but the trend of change in counts was not significantly different between the two groups . the above findings showed that adverse hematological effects ( thrombopenia or neutropenia ) did not increase in the osvix group as compared to the plavix group . we noted some limitations to our study , the most important of which was the small study population . however , the power of the study was enough to achieve the main and secondary objectives of the study . the other important limitation was that the angiographies were performed by different cardiologists in different centers . thus , interobserver variability was inevitable . in conclusion , our data demonstrated the safety of osvix in improving clinical outcome compared to plavix after coronary stenting in bms or des using patients . the frequency of mace , non - cardiac events or adverse hematological changes did not increase with osvix compared to plavix patients which suggest it 's safety . however , due to more cost advantages of osvix regimen over plavix , administration of osvix among iranian patients is preferable . akh designed the study and drafted this manuscript , mp designed the study , mo , gkm , mn , akh , mh , fr , shs , and jk carried the study , mgh drafted the manuscript and tables , hz helped in designing the study and carried the study , aat helped in designing the study , bs data analysis and drafted the tables , mgh helped in designing the study , mrkh and fn helped in statistical analysis , ekh and mj helped in data gathering , and ns helped in designing the study , revising the manuscript procedures , and drafting the manuscript .

background : to compare the early and late cardiovascular events as well as side effects of osvix , a generic form of clopidogrel versus plavix regimens in patients with chronic stable angina , undergoing bare metal stent ( bms ) or drug eluting stent ( des ) placement , this study was carried out.methods:a total of 442 patients with chronic stable angina who were scheduled for elective percutaneous coronary intervention ( pci ) were included in a randomized , double blind , multi - centric clinical trial being performed in 6 distinct university hospitals in 5 cities of iran from march 2007 to november 2009 . baseline , demographic and history of risk factors were recorded using the patients medical charts . stenting procedure was performed via transfemoral approach using low osmolar contrast agents . patients underwent bms or des placements based on the physician selection and were randomly assigned to osvix or plavix groups . patients were followed by telephone in 0 and 6 months intervals regarding the major adverse cardiovascular events ( mace ) including death , myocardial infarction , in - stent thrombosis , stroke , target lesion revascularization , and target vascular revascularization . angina episodes , bleeding , liver enzymes , neutrophils and platelets count were also assessed in these intervals.results:there was not any significant difference between these two groups regarding the baseline characteristics . in the des group , the 6-month mortality rate and the incidence of mace in osvix and plavix groups were 0.9% and 1.9% ( p = 0.61 ) and 1.8% and 4.9% ( p = 0.26 ) , respectively . during the follow up period after des or bms placement , there was nt any significant difference regarding neutrophil and platelet counts or liver enzymes between study groups.conclusions:using osvix and plavix are followed by similar major cardiovascular events and side - effect profile in patients undergoing pci .

in africa , pelvic inflammatory disease ( pid ) and its sequelae are a predominant cause of gynecologic morbidity [ 1 , 2 ] . these include tubal factor infertility , ectopic pregnancy , chronic pelvic pain , and recurrent pelvic infections [ 3 , 4 ] . hiv-1 seroprevalence in women with pid is consistently 27 times greater than measured in matched populations without pid [ 57 ] ; both infections are most commonly acquired through unprotected sexual activity . prompt diagnosis and treatment of women with upper genital tract infections is important in reducing morbidity , but it is complicated by lack of a sensitive and specific clinical and laboratory diagnostic test . laparoscopy is the gold standard for the diagnosis of salpingitis , but is not practical for routine clinical practice . kiviat et al . evaluated women with clinical pid ; evidence of endometritis as defined by 1 plasma cell ( pc ) and 5 polymorphonuclear lymphocytes ( pmn ) per high - powered field ( hpf ) was 92% sensitive and 87% specific compared with visual findings of salpingitis determined by laparoscopy . using the same diagnostic criteria in a study of acute salpingitis in kenya , plasma cell endometritis as defined by 1 pc / hpf was identified in 49% of women with salpingitis : this increased with disease severity and hiv - infection . studies on hiv-1-infected women have found an increased prevalence of plasma cell endometritis even in the absence of clinical disease [ 10 , 11 ] . thus , we conducted this analysis to determine the optimum endometrial histopathological criteria for predicting salpingitis in a population with a high hiv-1 seroprevalence . we anticipate that these data will help to plan future clinical trials , increase the understanding of the pathogenesis of upper genital tract infection among hiv-1 infected women , and in certain circumstances provide a tool to confirm the clinical diagnosis of pid . briefly , between april 2000 and july 2002 , women aged 1840 admitted to kenyatta national hospital ( knh ) acute gynecology ward with a complaint of lower abdominal / pelvic pain for 2 weeks or less plus one or more of the following signs or symptoms : temperature 38c , dysuria , and complaint of abnormal vaginal discharge were eligible for enrollment . after induction of anesthesia , an endometrial biopsy was obtained with a pipelle suction curette ( unimar , inc . , wilton , conn ) . at laparoscopy , samples from peritoneal fluid , tubal ostia , and pyosalpinx / tubo - ovarian abscess ( toa ) were obtained for n. gonorrhoeae and c. trachomatis pcr . using the jacobson and westrom criteria , the severity of acute salpingitis was graded as ( 1 ) mild ( tubal erythema or edema , mobile tubes , and with or without spontaneous exudate ) , ( 2 ) moderate ( marked tubal erythema and edema , limited tubal mobility , questionable or no tubal patency , and gross exudate ) , and ( 3 ) severe ( pyosalpinx or toa ) . women desiring permanent sterilization underwent laparoscopic tubal ligation preceded by an endometrial biopsy obtained using a pipelle suction curette . hiv-1-seropositive controls were enrolled from an hiv care and treatment clinic at the center for respiratory disease research at the kenya medical research institute . subjects had no clinical evidence of pid . enrollment and study procedures of the hiv-1-seropositive control group are detailed elsewhere . after informed consent was obtained , an endometrial pipelle biopsy was obtained in the research clinic . samples from the cervix , endometrium , fallopian tube , and abscess were examined by pcr ( roche molecular diagnostics , pleasanton , calif , usa ) for n. gonorrhoeae and c. trachomatis . endometrial specimens were fixed in 10% buffered formalin , processed , and stained with hematoxylin , eosin , and methyl green pyronin . pmns in glands and pcs in stroma were counted per high - power field . one pathologist ( nk ) who was blinded to the patients ' diagnosis read the slides . serum was tested for hiv antibodies by elisa ( detect hiv , biochem immunosystems , montreal , canada ) with positive results confirmed by a second elisa ( recombigen , cambridge biotech , ireland ) . univariate analyses used chi - square and fisher 's exact tests for categorical data and student 's t - test for continuous variables . logistic regression was done for multivariate analysis . one hundred and sixty women were enrolled with clinical pid , 140 ( 88% ) had laparoscopically confirmed salpingitis , 125 ( 89% ) of whom had an endometrial biopsy specimen : 56 ( 45% ) had mild , 31 ( 25% ) had moderate , and 38 ( 30% ) had severe disease based on laparoscopic criteria . nineteen women had other diagnoses at laparoscopy including appendicular abscess ( n = 2 ) , endometriosis ( n = 1 ) , ovarian cyst ( n = 12 ) , frozen pelvis ( n = 1 ) , pelvic tuberculosis ( n = 1 ) , cancer of the sigmoid volvulus with abscess ( n = 1 ) , and ovarian torsion ( n = 1 ) . asymptomatic women ( n = 20 ) desiring permanent sterilization underwent laparoscopic tubal ligation and served as hiv-1-negative controls . a single control subject had a sticky exudate emanating from the fallopian tubes and was excluded from the analysis leaving 19 hiv-1-seronegative controls . forty - five asymptomatic hiv-1-seropositive controls were enrolled from an hiv care clinic ; one woman had c. trachomatis detected . forty - eight ( 38% ) of the women with salpingitis were hiv - seropositive . women with salpingitis were younger , less likely to be married , and less likely to have ever used contraception ( table 1 ) . as expected , none of the hiv-1-seronegative controls had signs or symptoms consistent with pid , and none were infected with n. gonorrhoeae or c. trachomatis . however , t. vaginalis was detected in a similar proportion of salpingitis cases ( 23% ) and hiv - seronegative controls ( 21% ) ( table 1 ) . of the 125 women with salpingitis , endometrial biopsies from 107 ( 86% ) were evaluated histological . inadequate biopsies corresponded to endometrial specimens demonstrating sloughing , frank pus , and lack of tissue . in general , more severe disease as demonstrated by higher clinical severity score ( css ) ( 15.5 versus 13 , p < .03 ) and severity of salpingitis based on laparoscopic findings ( p - trend < .04 ) was associated with unevaluable endometrial biopsy results ( table 2 ) . similarly , history of depomedroxyprogesterone acetate ( dmpa ) was associated with an increased likelihood of obtaining an unevaluable biopsy ( p = .02 ) . hiv - infected women were more likely to have an unevaluable endometrial biopsy ( 57% versus 36% , p < .05 ) than hiv - uninfected women . although not significant , participants with an inadequate endometrial histological specimen had a higher prevalence of gonorrhea compared to those with an adequate biopsy ( 23% versus 12% p < 0.23 ) ( table 2 ) . in multivariate analysis , after controlling for factors found significant in univariate analysis , the use of dmpa at any time ( adjusted or = 3.1 , 95% ci 1.18.5 ) , hiv-1 infection for women with mild ( aor = 4.6 , 95% ci 1.118.3 ) but not moderate salpingitis ( aor = 0.89 , ci 0.155.3 ) , or severe salpingitis ( aor = 2.63 , ci 0.6810.2 ) was associated with an increased odds of an unevaluable endometrial biopsy . in addition , 12 ( 63% ) of 19 specimens from hiv-1-seronegative subjects were evaluable for histopathology . we reviewed the distribution of pmn and pc by hiv-1 serostatus and severity of salpingitis . women with severe salpingitis regardless of hiv-1 serostatus had the highest frequency of pmn and pc per high - power field . only two patients with hiv-1 infection and salpingitis did not have pmn found in the endometrium . although pmn density did not increase with severity of salpingitis among women with hiv-1 infection ( p - trend = .49 ) , this association was significant for hiv-1-uninfected women with salpingitis ( p - trend = .05 ) . in contrast , the frequency of pcs increased with severity of salpingitis among those with hiv-1 infection ( p - trend = .04 ) , but not among hiv-1 uninfected ( p - trend = .14 ) . furthermore , hiv-1 infection was associated with a higher frequency of pcs / hpf ( p - trend < .001 ) , and presence of lymphoid follicles ( p < .04 ) . only 2 ( 6% ) of 34 hiv-1-infected women with salpingitis did not have any plasma cells present in the endometrium versus 23 ( 41% ) of 56 hiv-1-uninfected women with salpingitis . we next set out to determine the sensitivity , specificity , and positive predictive value of four histopathologic criteria for diagnosis of endometritis in comparison to the laparoscopic diagnosis of salpingitis . the four rules evaluated included : ( a ) 3 pmn and 1 pc per high - power field , ( b ) 1 pmn and 1 pc per high - power field , ( c ) 1 pmn per high - power field , and ( d ) 1 pc per high - power field . women with moderate and severe disease were grouped together and compared to women with mild salpingitis and to the two control groups . table 3 outlines the comparison between the laparoscopic diagnosis for mild and moderate / severe salpingitis and the four histological rules stratified by hiv-1 serostatus . because the diagnosis of the moderate and severe disease requires more objective evidence of tubal inflammation ( e.g. , pus from tubes , pyosalpinx , abscess , and fresh adhesions ) than mild disease , we chose to gauge the sensitivity of each histological rule using laparoscopic evidence of moderate / severe salpingitis as the gold standard . rule a , although less sensitive than rules b through d for women with moderate / severe salpingitis ( hiv - seropositive = 74% versus 63% ; hiv - seronegative ; 93% versus 75% ) , was the most specific , demonstrating endometritis in 25% and 7% of hiv-1-seronegative and hiv-1-seropositive controls , respectively , in comparison to 58%67% and 38%62% for rules b through d . among the 19 women enrolled with a clinical diagnosis of pid , but who did not have salpingitis on laparoscopy , rule a had the least false positive , while rule d , at least one plasma cell , scored the highest false - positive rate . this study had three key findings : ( 1 ) 3 pmn and 1 pc per hpf as a histologic criteria for the diagnosis of moderate to severe salpingitis , while performing better than the other criteria , appears to have limited utility even more so for cases of mild salpingitis ; ( 2 ) endometrial specimens were often unevaluable for histopathology , and unevaluable specimens were more likely in subjects with severe salpingitis and hiv - infection , and thus may affect the utility of endometrial histopathology to confirm the clinical diagnosis of pid in similar settings ; ( 3 ) the pmn response increased with disease severity for hiv-1 seronegative but not hiv-1 seropositive women with salpingitis . since kiviat et al . published their paper , histologic endometritis has been used as a surrogate marker for salpingitis , especially in the study of mild to moderate pid . even though the criteria for histologic endometritis had never been validated in hiv-1-infected populations , several studies of pid were conducted in high hiv-1 seroprevalence settings [ 6 , 7 , 9 , 12 , 14 ] . 1990 , criteria for 5 pmns and 1 pc for the diagnosis of pid . in the kiviat et al . cohort , n. gonorrhoeae and/or c. trachomatis was found in 49% of the patient population ; in comparison , the cohort in our study had a high hiv-1 prevalence and a combined gonorrhea and/or chlamydia prevalence of 18% ( table 1 ) . similar to another report , only 72% of endometrial biopsies in our study were evaluable . the increased frequency of unevaluable endometrial biopsies in women with severe salpingitis , likely due to increased endometrial sloughing and presence of pus , and hiv-1 infection further limits the utility of endometrial histopathology as a diagnostic tool for studies of pid in similar populations . the low sensitivity of histologic endometritis for mild salpingitis amongst women symptomatic for pid was unexpected . studies of endometritis in populations of asymptomatic women have consistently demonstrated a relatively high prevalence of endometritis [ 10 , 15 , 16 ] which led authors to describe endometritis as an intermediate infection to pid . . studied hiv-1-infected women presenting to a family planning clinic and found endometritis in 38% of participants . this is a higher prevalence than what we found in hiv-1-negative women ( 16% ) and a lower prevalence than what we found in hiv-1-seropositive women ( 57% ) with mild salpingitis using less stringent criteria for endometritis . an alternative explanation may result from the subjectivity of the laparoscopic criteria for mild salpingitis that leads to misclassification of cases . the distribution of pmns and pcs in the endometrium of women with salpingitis was affected by hiv-1 serostatus and disease severity . pmns are only found in the healthy endometrium during menses , and form part of the endometrial immune response , they are also the first line immune defense against bacterial infections . the increased density of pmn with severe disease in hiv-1-uninfected but not in hiv-1-infected women with salpingitis is not well understood . consistent with other studies [ 7 , 9 , 10 , 14 ] , we found increased pc endometritis with hiv-1 infection . this could represent hiv-1 infection in the genital tract ; chronic plasma cell endometritis [ 11 , 14 ] ; or the presence of opportunistic infections . reported an association between hsv-2 seropositivity and plasma cell endometritis ; notably hsv-2 is extremely prevalent among hiv-1-infected persons ( kais 2007 . evaluated 20 endometrial biopsy samples from women with asymptomatic histologic endometritis and failed to detect herpes simplex virus by pcr , and cytomegalovirus was detected equally in women with and without histological endometritis . one limitation of this study is that hiv-1-infected controls did not undergo laparoscopic evaluation . therefore , unlikely we can not firmly exclude subclinical salpingitis from this population as we can for the hiv-1-seronegative controls . furthermore , we did not attempt to detect suspected etiologies of endometritis such as bacteria other than n. gonorrhoeae and c. trachomatis including m. genitalium and potential etiologies such as cytomegalovirus and herpes simplex virus infection . such data might help to elucidate the reason for the different findings among hiv-1-seropositive and hiv-1-seronegative women with salpingitis in regards to endometrial histopathology . this study raises some important questions regarding pid and its sequelae . with increased access to highly active antiretroviral therapy ( haart ) , hiv-1-infected population data from uganda plus others have demonstrated reduced fertility in hiv-1-infected women regardless of disease stage . further research is required to determine if women using haart return to normal fertility or not . lastly , although endometrial histopathology serves as a reasonable surrogate for salpingitis in hiv-1-uninfected populations , its utility in populations with a high hiv-1 seroprevalence appears to be limited . discovery of a sensitive and specific biomarker or set of biomarkers for salpingitis could facilitate further research on pid and its sequelae in such settings .

study objective . to identify sensitive and specific histological criteria for endometritis in women with laparoscopically - confirmed acute salpingitis . methods . women , age 1840 years of age presenting with complaints of lower abdominal pain 2 weeks and no antibiotics use in past two weeks , were enrolled . they underwent clinical examination , screening for hiv ; other sexually transmitted infections plus endometrial biopsy sampling for histopathology . diagnostic laparoscopy confirmed the diagnosis of acute salpingitis . controls were women undergoing tubal ligation and hiv-1 infected women asymptomatic for genital tract infection . results . of 125 women with laparoscopically - confirmed salpingitis , 38% were hiv-1 seropositive . nineteen hiv-1 negative controls were recruited . for the diagnosis of endometritis , 1 plasma cells ( pc ) and 3 polymorphonuclear lymphocytes ( pmn ) per hpf in the endometrium had a sensitivity of 74% for hiv-1-seropositive , 63% for hiv-1-seronegative women with a specificity of 75% and positive predictive value of 85% regardless of hiv-1-infection for predicting moderate to severe salpingitis . for hiv-1-seronegative women with mild salpingitis , 1 pc and 3 pmn had a sensitivity of 16% and a ppv of 57% . conclusion . endometrial histology , did not perform well as a surrogate marker for moderate to severe salpingitis , and failed as a surrogate marker for mild salpingitis .

muscle morphological characteristics , such as anatomical muscle cross - sectional area ( csa ) , muscle length , and muscle volume ( mv ) are important physiological variables for assessing the functional capacity of a muscle . although magnetic resonance imaging ( mri ) and computed tomography scans are the gold standard for measuring anatomical csa and mv , ultrasound is a utilizable technique that can be easily applied to clinical assessment and field surveys1 . in a small muscle , anatomical csa and mv can be measured using multiple images of individual muscles created by portable ultrasound . one study2 investigated the validity and reliability of mv measurements of the medial gastrocnemius using three - dimensional ultrasound , and reported that ultrasound overestimated mv by approximately 2 ml ( 1.1% ) and underestimated muscle length by 3 mm ( 1.3% ) across all joint angles compared to mri - measured values . in addition , the same study reported excellent reliability for repeated measures of mv ( intrarater correlation coefficient ( icc ) = 0.99 ) and muscle length ( icc = 0.97 ) . these results suggest that multiple ultrasound images of a small muscle can accurately measure muscle morphological variables . the foot is the point of direct contact between the body and ground surface during standing and walking . muscle forces generated by the toes and ankles may play an important role in maintaining balance because muscle strength is essential for posture and stability3 , 4 . a few studies have reported that toe flexor muscle strength is associated with postural control . for example , handa et al.5 reported significant positive correlations between toe flexor strength and one - leg standing balance with the eyes open ( r = 0.443 , p < 0.01 ) as well as functional reach ( r = 0.620 , p < 0.01 ) , in an analysis grouping men and women together . kurihara et al.6 examined the relationships between toe flexor muscle strength and intrinsic and extrinsic foot muscle sizes in young men and women and found significant correlations between toe flexor muscle strength and mri - measured anatomical csa in the medial parts ( r = 0.775 , p < 0.01 ) and lateral parts ( r = 0.739 , p < 0.01 ) of foot intrinsic muscles . recently , abe et al.7 reported a significant positive association between toe flexor muscle strength and accelerometer - determined light and moderate physical activities and average step counts . the results of these previous studies suggest that bigger intrinsic foot muscles in active individuals may be associated with greater toe flexor strength as well as good postural control . in a previous mri study6 , however , only one mri image was used to determine the anatomical csa of individual intrinsic foot muscles , even though the foot has several toe flexor muscles with differing distributions among the toes . therefore , the purpose of this study was to test the hypothesis that toe flexor muscle strength is related to the anatomical and physiological csa of intrinsic toe flexor muscles and that these morphological and functional variables associate with physical performance . thirty - four young adults aged 20 to 35 years ( 17 men and 17 women ) were recruited through printed advertisements and by word of mouth . before accepting their informed consent , a written description of the purpose and safety of the study was distributed to all of the potential subjects . all subjects were healthy and free of overt chronic disease ( e.g. , neuromuscular disorders , arthritic disorders , etc ) as assessed by self - report . the rate of regular sports activity ( at least twice a week ) , among the subjects was 65% ( 82% in men and 47% in women ) . the main types of sports activities were judo ( 29% ) , resistance exercise ( 21% ) , and soccer ( 21% ) for men , and jogging / running ( 50% ) and canoeing ( 25% ) for women . the study was conducted in accordance with the principles of the declaration of helsinki and was approved by the ethics committee for human experiments of the national institute of fitness and sports in kanoya , japan . anatomical csa was measured using b - mode ultrasound ( aloka prosound 6 , tokyo , japan ) with a 7.5 mhz linear array transducer ( 76 mm wide ) in two intrinsic toe flexor muscles , the flexor digitorum brevis ( fdb ) and abductor hallucis ( abh ) , as described previously8 . all subjects lay in the prone position during scanning of the two muscles . using anatomic landmarks described by crofts et al.8 , a linear transducer coated with water - soluble transmission gel was placed on the skin surface of the measurement sites , and cross sections of each muscle were imaged . ultrasound images of each site were stored on a personal computer , and anatomical csa was measured using image - j software . the mean values ( two images ) of each site were used for data analysis . all ultrasound measurements were performed on the left ( non - dominant ) foot , and dominance was ascertained by asking each subject which foot they used to perform well - learned skills using a questionnaire9 . the muscle volume and muscle length of the fdb were estimated using multiple ultrasound images from the sole of the foot . after foot length ( the distance between the tip of the great toe and the edge of the heel ) measurements , all measurement sites were marked at 50% of the foot length as well as at contiguous 1-cm intervals from the point of 50% of the foot length in both the proximal and distal directions . 1.before the start of ultrasound testing , foot length ( the distance between the tip of the great toe and the edge of the heel ) was measured and then all measurement sites were marked at 50% of the foot length as well as at contiguous 1-cm intervals from the point of 50% of the foot length in both the proximal and distal directions . typical ultrasound images ( young woman , 20 yr ) revealing transverse scans of the foot at contiguous 1-cm intervals from the point of 50% of the foot lengthfdb : flexor digitorum brevis ) and the anatomical csa of the fdb was measured using the procedure described above . muscle volume was calculated by multiplying anatomical csa by distance interval ( 1 cm ) . the distance between the most proximal image and the most distal image in which the fdb was visible was defined as the length of the fdb muscle . to calculate physiological csa , the fiber length of the fdb was estimated using the ratio of fiber length to muscle length ( average of the second , third , fourth toes ) as reported by kura et al10 . the physiological csa of the fdb was calculated by dividing muscle volume by fiber length . the test - retest reliability ( icc , sem and minimal difference ) was previously determined using the data of 7 young subjects ( 5 men and 2 women ) scanned twice within 7 days ( at least one day apart ) for anatomical csa of the fdb ( 0.924 , 0.13 cm , 0.36 cm ) and abh ( 0.949 , 0.21 cm , 0.58 cm ) and muscle volume of fdb ( 0.971 , 0.57 cm , 1.57 cm ) . before the start of ultrasound testing , foot length ( the distance between the tip of the great toe and the edge of the heel ) was measured and then all measurement sites were marked at 50% of the foot length as well as at contiguous 1-cm intervals from the point of 50% of the foot length in both the proximal and distal directions . typical ultrasound images ( young woman , 20 yr ) revealing transverse scans of the foot at contiguous 1-cm intervals from the point of 50% of the foot length fdb : flexor digitorum brevis toe flexor muscle strength ( tfs ) was measured using a toe - grasp dynamometer ( tkk3361 , takei , tokyo , japan ) , as described previously11 , 12 . while barefoot , subjects stood in front of a wall and the left foot then the subjects were instructed to lift the right foot and maintain a one - legged upright standing position on the dynamometer , with both hands on the wall in front of them , while holding the dynamometer grasp bar with their toes . the distance between the bar and the heel was adjusted to the foot size of the subjects so that the distal phalanges of the great toe and fifth toe and the middle phalanges of the second to fifth toes could be placed on the toe grasp bar . subjects were allowed to perform one test trial , followed by two maximum effort trials ( tfs-5-toes ) , and the best value of the left foot was used for the data analysis . the subjects also performed two maximum effort trials to measure toe flexor muscle strength without the contribution of the great toe ( tfs-4-toes ) . in these trials , a small metal plate was placed between the great toe and the toe grasp bar during these measurements for preventing the great toe from grasping the toe grasp bar of the dynamometer . maximal toe flexor strength divided by body weight was calculated to evaluate the relative toe flexor strength . the test - retest reliability of toe flexor strength ( tfs-5-toes and tfs-4-toes ) measurements using the icc , sem and minimal difference was previously determined using the data of 7 young subjects ( 5 men and 2 women ) tested twice within 7 days ( at least one day apart ) : 0.962 , 1.2 kg and 3.3 kg for tfs-5-toes and 0.883 , 1.1 kg and 3.1 kg for tfs-4-toes . maximum walking speed was measured by timing each subject as they walked along a 10-meter corridor with a ceramic floor surface . the total length of the marked corridor was 14 meters , allowing 2-meter acceleration and deceleration zones . the width of the corridor was constricted to 1 meter to encourage subjects to maintain a straight course . subjects used their own footwear and had to start 2 meters before the beginning of the start line , and to continue until the 2 meters past the goal line . after one practice trial , subjects performed two maximum speed timed trials . subjects were asked to walk down the corridor as fast as possible without running . times were measured with an electronic timing system ( nearest 0.01 s , brower timing system , draper , usa ) . the best time was converted to a maximum speed measurement ( unit , m / s ) and the best value was used as the maximum walking speed . the test - retest reliability of this measurement using the icc , sem and minimal difference was previously determined using the procedure described above : 0.930 , 0.14 the functional reach test was measured using the method described in a previously reported study4 . before the start of the test , subjects were instructed to stand with both feet touching a marked line and to maintain that foot position throughout testing . the subject then performed shoulder forward flexion with the right shoulder until an angle of 90 degrees was reached . next , the subjects tried to extend their middle finger as far forward as possible without moving their feet and keeping their arm parallel to the ground . the distance moved by the end of the middle finger between the starting position and maximum forward position subjects performed two trials , and the best value was used for the functional reach test . the test - retest reliability of this measurement using the icc , sem and minimal difference was previously determined fusing the procedure described above : 0.889 , 2 cm and 5 cm , respectively . before comparisons were made , the distributions of the dependent variables were tested for normality using the shapiro - wilk test . the difference between men and women was tested for significance using the unpaired student s t - test , and when variables were not normally distributed , the mann - whitney u test was used . pearson product correlations were performed to determine the relationships between toe flexor muscle strength and intrinsic toe flexor muscle size as measured by ultrasound and between the morphological and functional variables of toe flexor muscle and physical performance . men were taller and heavier than women . compared with women , men had higher anatomical csa in the fdb and abh , as well as muscle volume and physiological csa of the fdb . maximum tfs-5-toes and tfs-4-toes were higher in men than in women ; however , the specific strength ( tfs-4-toes per unit physiological csa ) of the fdb , walking speed and functional reach were similar for both men and women ( table 1table 1.maximum toe flexor muscle strength , physical performance and ultrasound measurements of intrinsic foot muscle sizes of young men and womenmenwomenoverall(n=17)(n=17)(n=34)age ( yrs)24 ( 4)24 ( 4)24 ( 4)height ( m)1.71 ( 0.05)*1.60 ( 0.05)1.66 ( 0.07)body mass ( kg)72.9 ( 11.4)*52.1 ( 5.1)62.5 ( 13.6)body mass index ( kg / m)24.8 ( 3.1)*20.4 ( 2.0)22.6 ( 3.4)foot length ( cm)25.2 ( 1.2)*22.6 ( 0.7)23.9 ( 1.6)flexor digitorum brevisacsa max ( cm)2.61 ( 0.38)*1.65 ( 0.33)2.13 ( 0.60)mv ( cm)11.95 ( 2.93)*6.66 ( 2.10)9.31 ( 3.67)pcsa ( cm)6.38 ( 1.04)*3.95 ( 0.97)5.17 ( 1.58)abductor hallucisacsa ( cm)2.89 ( 0.69)*2.02 ( 0.59)2.46 ( 0.77)tfs-5 toes ( kg)29.1 ( 5.3)*21.2 ( 4.7)25.1 ( 6.4)tfs-4 toes ( kg)10.4 ( 3.2)*6.4 ( 2.6)8.4 ( 3.5)tfs-4 toes / pcsa ( kg / cm)1.63 ( 0.40)1.62 ( 0.56)1.63 ( 0.48)walking speed ( m / s)3.19 ( 0.65)3.14 ( 0.66)3.17 ( 0.64)functional reach ( cm)38.1 ( 6.3)39.1 ( 4.0)38.6 ( 5.2)maximum toe flexor muscle strength with ( tfs-5 toes ) and without ( tfs-4 toes ) the contribution of the great toe ; acsa , anatomical cross - sectional area ; mv , muscle volume ; pcsa , physiological cross - sectional area . * maximum toe flexor muscle strength with ( tfs-5 toes ) and without ( tfs-4 toes ) the contribution of the great toe ; acsa , anatomical cross - sectional area ; mv , muscle volume ; pcsa , physiological cross - sectional area . * significant difference from women , p<0.001 there was a significant correlation between tfs-5-toes and tfs-4-toes of men ( r = 0.739 , p < 0.001 ) and women ( r = 0.731 , p < 0.001 ) . both tfs-5-toes and tfs-4-toes correlated positively with the maximum walking speed of men ( r = 0.584 , p = 0.014 and r = 0.553 , p = 0.021 , respectively ) , women ( r = 0.748 , p < 0.001 and r = 0.533 , p = 0.028 , respectively ) and the whole sample ( r = 0.535 , p = 0.001 and r = 0.459 , p = 0.006 , respectively ) . however , the correlations between tfs-5-toes and functional reach of both men ( r = 0.399 ) and women ( r = 0.166 ) were not significant ( p>0.05 ) . anatomical csa of the abh did not significantly correlate with tfs-5-toes of either men ( r = 0.034 , p = 0.896 ) or women ( r = 0.387 , p = 0.125 ) ; however , the correlation of the whole sample was significant ( r = 0.454 , p = 0.006 ) . for women , there were significant positive correlations between tfs-5-toes and anatomical csa max ( r = 0.713 , p = 0.001 ) , muscle volume ( r = 0.604 , p = 0.010 ) , and physiological csa ( r = 0.687 , p = 0.002 ) of the fdb . for men , however , the observed anatomical csa max ( r = 0.143 , p = 0.584 ) , muscle volume ( r = 0.332 , p = 0.192 ) , and physiological csa ( r = 0.333 , p = 0.191 ) of the fdb did not correlate significantly with tfs-5-toes . physiological csa of the fdb correlated positively with tfs-4-toes of men ( r = 0.541 , p = 0.025 ) , women ( r = 0.573 , p = 0.016 ) and the whole sample ( r = 0.720 , p < 0.001 ) ( fig . 2fig . 2.relationships between physiological cross - sectional area ( csa ) in the flexor digitorum brevis ( fdb ) and toe flexor muscle strength ( tfs-4 toes ) of young men and women . tfs-4 open circles are men and filled circles are women ) . relationships between physiological cross - sectional area ( csa ) in the flexor digitorum brevis ( fdb ) and toe flexor muscle strength ( tfs-4 toes ) of young men and women . tfs-4 the main findings of the present study were that physiological csa of the fdb was significantly correlated with tfs-4-toes of both men and women ; physiological csa of the fdb was significantly correlated with tfs-5-toes of women , but not that of men ; there was a significant correlation between toe flexor muscle strength and maximum walking speed in both genders . in the present study , the fdb was selected as representative of the intrinsic toe flexor muscles , because a more accurate estimation of muscle volume by ultrasound and calculation of physiological csa of the muscle can be achieved than of the other intrinsic toe flexor muscles . the fdb muscle volume reliability results ( icc of 0.971 and sem of 0.57 cm ) indicate that the ultrasound method of the present study is a good repeatable technique for measuring muscle volume . the fdb is located in the sole of the foot and separates into four tendons that insert onto the middle phalanges of the four lateral toes10 ( without the great toe ) . therefore , our results demonstrate that the physiological csa of the fdb is associated with tfs-4 toes of both men and women , even though the correlation was only moderate . toe flexor muscle strength is generated from a combination of the intrinsic and extrinsic foot muscles . in the hand , the grip strength decreases by approximately 50% after median and ulnar nerve ( intrinsic muscles ) blocks compared with the pre - block measurement13 . accordingly , the contribution of extrinsic toe flexor muscles may reflect the moderate correlations between tfs-4 toes and physiological csa of the fdb . in addition , individual differences in the moment arm ( located between the center of curvature of the metatarsal head and the center of the flexor tendon ) and/or differences in the dominant / non - dominant sides may also be unknown factors14 . our findings show that the anatomical and physiological csa of the fdb of women were significantly correlated with tfs-5 toes . for men only one study6 has examined the relationships between toe flexor muscle strength and intrinsic and extrinsic foot muscle sizes in young sedentary adults ( 14 men and 12 women ) and a pooled sample was used for data analysis . that study reported significant correlations between tfs-5 toes and mri - measured anatomical csa of the medial parts ( r=0.775 , p<0.01 ) and lateral parts ( r=0.739 , p<0.01 ) of the foot intrinsic muscles , although only one mri image was used in that investigation . in the present study , the reason for the lack of a significant correlation between tfs-5 toes and the fdb muscle size observed in men is unknown . approximately half of our young women were sedentary and the other half performed mainly jogging / running or canoeing . therefore , our women participants may have had relatively homogeneous morphological and functional properties of the toe flexor muscles , which might partially explain the significant correlations . on the other hand , most of our young men were physically active and performed different sports including judo , resistance exercise and/or soccer . a likely explanation is that physical activity in different sports may elicit non - homogeneous features among toe flexor muscles , especially between the great toe and the four other toes . together , the results of the present and previous studies suggest that differences in sports experience may be a factor underlying the poor correlation of tfs-5-toes and fdb muscle size in young men . additional research into these issues is needed . in the present study , both tfs-4-toes and tfs-5-toes this finding is consistent with the results of previous studies5 , 15 which found significant correlations between 10-m walking performance and tfs-5-toes ( r=0.459 , p<0.01)5 and improved 50-m dash time following 8 weeks of toe flexor strength training15 . thus , the results of the present and previous studies demonstrate that toe flexor muscle strength is an important factor determining maximum walking speed . on the other hand , no significant correlation of tfs-5-toes and functional reach of either young men or women was found . although a pooled sample with a wide age range ( 20 to 84 yrs ) was used , one study reported a significant positive correlation ( r=0.620 , p<0.01 ) between tfs-5-toes and functional reach5 . the discrepancy in the results of the present and that previous study is not known , but the difference in subject age ranges between the two studies may have played a role . in conclusion , although toe flexor muscle strength correlated positively with walking speed in both genders , its correlation with functional reach was not statistically significant . the lack of a significant relationship between tfs-5-toes and physiological csa of the fdb in men may be related to different experiences in sports .

[ purpose ] to investigate the relationships between toe flexor muscle strength with ( tfs-5-toes ) and without ( tfs-4-toes ) the contribution of the great toe , anatomical and physiological muscle cross - sectional areas ( csa ) of intrinsic toe flexor muscle and physical performance were measured . [ subjects ] seventeen men ( 82% sports - active ) and 17 women ( 47% sports - active ) , aged 20 to 35 years , volunteered . [ methods ] anatomical csa was measured in two intrinsic toe flexor muscles ( flexor digitorum brevis [ fdb ] and abductor hallucis ) by ultrasound . muscle volume and muscle length of the fdb were also estimated , and physiological csa was calculated . [ results ] both tfs-5-toes and tfs-4-toes correlated positively with walking speed in men ( r=0.584 and r=0.553 , respectively ) and women ( r=0.748 and r=0.533 , respectively ) . physiological csa of the fdb was significantly correlated with tfs-5-toes ( r=0.748 ) and tfs-4-toes ( r=0.573 ) in women . in men , physiological csa of the fdb correlated positively with tfs-4-toes ( r=0.536 ) , but not with tfs-5-toes ( r=0.333 ) . [ conclusion ] our results indicate that physiological csa of the fdb is moderately associated with tfs-4-toes while toe flexor strength correlates with walking performance .

why have we not been able to use neuroimaging in the clinical management of psychoses ? after 30 years of using this approach , we have established that structural brain alterations are present in individuals with psychoses . while this information has advanced academic knowledge on the pathophysiology of these disorders , it has had limited utility in clinical practice . the main reason can be found in the nature of brain alterations in psychoses : they are subtle and spatially distributed . still , the time may now have come when quick and noninvasive structural neuroimaging abandons the academic solitude in which it has been operating , and brings its novel methods and findings into the clinical arena . this paper aims to appraise this evidence , and discuss it in the context of potential future applications of magnetic resonance imaging ( mri ) in the clinical management of psychoses . the first part of this review will present evidence on the presence of neuroanatomical alterations in psychoses . it will then discuss studies that have examined the relationship between brain alterations and various medium- to long - term clinical and functional outcomes . in the third section , it will discuss more recent evidence on the relationship between brain alterations and early outcomes , with a particular focus on the relationship between symptomatic responses and initial treatment with antipsychotic drugs . the paper will then present a discussion of more novel approaches for the analysis of mri data , such as support vector machine . findings from these approaches have started to suggest that mri has the potential to provide clinically meaningful information . finally , it will discuss the implications of having strong and valid neuroimaging markers of psychosis outcomes in developing targeted , as well as novel , treatment interventions for these disorders . moving from the first evidence that individuals with established schizophrenia have subtle structural brain alterations , most notably an enlargement of the lateral ventricles , we have been studying individuals earlier and earlier along the disease course . more than 10 years ago , the seminal work by pantelis and colleagues showed that individuals at high risk of developing psychosis , but not ill yet , already show smaller volumes of the same brain areas that have been reported as smaller in individuals with an established psychosis , namely frontal and temporal cortices . even more novel , however , was their report that this is particularly the case for individuals who then actually develop the illness , and that some of these alterations could be specific to the type of psychosis that these subjects developed . for example , they found that subjects who develop an affective type of psychosis showed reductions in the subgenual anterior cingulate cortex , an area involved in the generation of affective symptoms . complementary to these gray - matter changes , ultra - high - risk individuals also have white matter alterations similar to , albeit less extensive than , first - episode patients . these include poorer integrity of the major associative fibers that connect fronto - parieto - temporal ( superior longitudinal fasciculus ) and fronto - parieto - occipital ( inferior fronto - occipital fasciculus ) regions , commissural fibers ( corpus callosum ) , and cortico - subcortical pathways ( corona radiata , corticospinal tract , and corticopontine tract ) . these findings , integrated with evidence from many reviews and meta - analyses , suggest that brain alterations become more extensive at the time of a first full psychotic episode , and then possibly even more marked over the years , when the illness becomes established . these are indeed two main factors that have limited our ability to use this information about brain alterations in the clinical management of the individual patient . therefore , many studies have tried to establish whether these distributed brain changes are more likely to occur in individuals with a more severe illness course or a worse clinical and functional outcome . for example , a poorer clinical outcome has been variously defined as response to early treatment , number of subsequent episodes , severity of symptoms , hospitalizations , or duration of remissions ; while functional outcome has been defined as the ability to live independently , maintain employment , or be in a relationship . nevertheless , many studies have tried to establish if structural brain alterations are related to illness characteristics by comparing brain structure in patients and healthy controls and then evaluating the relationship with outcomes at a single time point ; or conducting repeated evaluations of brain structure , and then investigating the relationship between longitudinal brain changes and outcomes . most of these studies have evaluated the relationship with medium to long - term outcomes , and only more recently has the attention shifted to the evaluation of the relationship between brain alterations and early outcomes such as response to the first treatment . studies that have evaluated brain structure at the time of illness onset and again following treatment , have often reported smaller volumes of gray matter in association with indicators of subsequent poorer clinical outcomes ( eg , higher number of admissions or worse symptom severity ) , and with worse social or occupational outcomes . for example , smaller volumes of prefrontal areas have been found to predict poorer functioning ( defined as a combination of social , occupational , and psychological functioning ) 1 year later in a small sample of first - episode psychosis patients . similarly , in a sample of patients with schizophrenia , drawn from an unselected general population sample , jskelinen and colleagues found that individuals showing higher density ( an indirect measure of volume ) of frontal and limbic areas had an overall better clinical ( defined as a lower number of hospitalizations ) and functional ( defined as not being on a disability pension ) outcome in the subsequent 16 years . wassink and colleagues reported an association between smaller volumes of another brain area , the cerebellum , and longer negative and psychotic symptom duration , as well as psychosocial impairment ( eg , quality of relationships , sexual activity , enjoyment of recreation , and work performance ) in individuals with schizophrenia spectrum disorders after a 7-year follow - up . still , others have not found an association between brain volumes at onset and subsequent outcomes . for example , the prospective study by van ilaren and colleagues found no relationship between brain volume measurements and symptom severity , level of functioning ( defined as the ability to maintain a variety of social roles ) , need for care , and illness course after 2 years . mitelman and colleagues examined white matter integrity using diffusion tensor imaging ( dti ) , in a group of 104 patients with an average 4-year schizophrenia duration . they classified them as having a poor or good outcome , based on both symptom severity and level of functioning at this time point . they found that although both patient groups showed reduced overall white matter integrity of the prefrontal and temporal areas ( as a measure of fractional anisotropy [ fa ] ) , these alterations were more extensive in patients with a poor outcome than in those with a good outcome . longitudinal studies that have evaluated brain structure at multiple time points after illness onset can help clarify if brain alterations , evident at illness onset , progress differently in individuals who then developed a poorer outcome . for example , a larger decrease in the volume of the lingual gyrus , insula , and cerebellum has been reported in patients with a worse functional outcome ( a combination of social , occupational , and psychological functioning ) 4 years after the first psychotic episode . with a similar duration of follow - up , van ilaren and colleagues also reported a greater gray matter volume reduction , and a greater thinning of temporal and frontal cortices in individuals who had poorer outcomes ( including a higher number of hospitalizations and a lower level of social and personal functioning ) 5 years after illness onset . similarly , in a sample of children and adolescents with early onset first - episode schizophrenia , arango and colleagues showed that more weeks of hospitalization and higher severity of negative symptoms were correlated with greater left frontal gray matter volume loss and greater cerebral spinal fluid increase , respectively . dynamic changes in relation to outcomes were also observed by our group in a 6-year follow - up study of hippocampal volume in first - episode psychosis patients . in this sample , we specifically found that individuals who had an increase in hippocampal volume over the first 6 years of illness were the ones who showed a less severe illness course and lower symptom severity , as well as a better functional outcome at follow - up , thus suggesting an important role for brain neuroplasticity . taken together , these findings suggest that brain alterations are present at illness onset and may be associated with worse medium- to long - term illness outcomes and ability to function . still , two important considerations need to be made in the interpretation of these findings . first , it is not possible to establish what the casual link is between brain structure and illness course . while brain alterations could be the substrate of a more severe illness , the more severe illness itself could affect brain plasticity by increasing exposure to substance abuse , worse living conditions , or a less stimulating environment . it is , therefore , possible that patients with a more severe illness are exposed to longer periods of treatment or higher doses of antipsychotics , which may themselves affect brain volumes . from a clinical perspective , it may be more informative to study the relationship between brain alterations at illness onset and outcome measures that occur earlier in the course of illness , which is the topic of the next section . if brain alterations could be used to predict , as early as possible , which patients are destined to have a poorer response to the first few weeks or months of treatment with antipsychotic medications ( the main stake for the treatment of psychosis ) , we could implement interventional strategies targeted at this specific subgroup . currently , we treat the first psychotic episode on a trialand - error basis , and we simply have no way of saying who will respond to the first antipsychotic drug and who might benefit from longer or different interventions . in fact , we know that only -55% of patients respond to antipsychotics in the first 12 months of treatment . yet , this early response is thought to be one of the strongest predictors of subsequent functional and clinical outcomes in psychosis . a pooled analysis , published in 1992 , examined the relationship between response to antipsychotics and brain measures , such as ventricular - brain ratio , sulcal prominence , third ventricles , and other brain measures . the authors found that the composite effect sizes were very small and did not reach statistical significance . however , recent studies have lent renewed support to the notion that alterations in both gray and white matter may be associated with a poorer response to antipsychotics , possibly because they used more homogeneous populations , or in some cases , better imaging acquisition approaches and more sophisticated methods of analysis . for example , zipursky and colleagues examined global tissue volumes in firstepisode psychosis patients , and found that subjects with a poorer response ( defined as having a lower reduction in symptom severity from baseline ) to the antipsychotic haloperidol in the first month of treatment had smaller cortical gray matter volume than those who showed symptomatic improvement . relatively consistent data have been reported on the relationship between response to clozapine and brain structure . cortical measures , such as sulcal enlargement , particularly in the prefrontal cortex , have been frequently related to poor response to clozapine in patients with chronic schizophrenia . in a double - blind , 10-week trial of clozapine and haloperidol , arango and colleagues found that a larger prefrontal gray matter volume was associated with better treatment response in clozapine - treated patients , but with a poor response in haloperidol- treated patients . they also found no relationship between response to these drugs and hippocampal and caudate nucleus volumes . the authors suggested that the presence of larger brain volumes might characterize patients who are more likely to benefit from clozapine . this finding is also consistent with evidence from a study in patients with treatment - resistant schizophrenia who were treated for with clozapine 6 months . patients with a larger baseline volume of the dorsolateral - prefrontal cortex were more likely to show improvement in their negative symptoms . interestingly , amelioration of positive symptoms was related to a larger temporal cortex volume , and amelioration of disorganization symptoms was inversely related to hippocampal volume . another temporal area has been reported as being related to early outcome the parahippocampal gyrus . using voxel - based morphometry , bodnar and colleagues found that the parahippocampal gyrus was significantly smaller in first - episode psychosis patients who , after 6 months of antipsychotic treatment , had not remitted , compared with those who had achieved remission . furthermore , by building a classification model using parahippocampal gray matter concentration , they were able to correctly classify remission status in 79% of the cases . in another study of patients with established schizophrenia treated with olanzapine or risperidone , 3-week response to these antipsychotics was associated with smaller volumes of other areas , such as the insula and rectal gyrus , and larger volumes of the basal ganglia . white matter has also been associated with treatment response , but the evidence is considerably more scant . response to antipsychotics was evaluated by garver and colleagues in a small sample of patients with schizophrenia after 28 days of treatment . here , somewhat counterintuitively , the 8 patients who responded to antipsychotics showed worse white matter microstructural integrity than the 5 patients who did not respond to medication . of note , individuals , in this study , had an established illness , and therefore , in contrast , studies in antipsychotic naive or minimally treated patients have the advantage that any structural brain alterations would be less likely to reflect the effect of antipsychotics on brain structure , and more likely to be a marker of illness . luck and colleagues analyzed a sample of first - episode psychosis patients using dti , and then reassessed them at 6 months , which is when they classified them as having either a poor or good treatment outcome . they specifically examined three white matter tracts connecting frontal and temporal regions : the cingulum , the superior longitudinal fasciculus , and the uncinate fasciculus . patients with a poor outcome showed greater alterations in white matter integrity in the superior longitudinal fasciculus and uncinate than patients with a good outcome , suggesting that abnormal fronto - temporal connectivity may represent an early marker of short - term clinical outcomes . our group has also recently focused on the evaluation of the relationship between neuroanatomical markers in white and gray matter at the time of presentation , and response to treatment at 12 weeks . this is a time when all patients would have received at least one full therapeutic course of antipsychotic medication . we examined white matter integrity , using dti and tractbased spatial statistics , to assess fa in a large sample of patients at their first episode of any psychosis . we operationalized response to treatment as having achieved symptomatic remission according to the criteria of the schizophrenia working group . we found that already at illness onset , patients who subsequently did not respond ( n=40 ) had lower fa than healthy controls in the uncinate , cingulum , and corpus callosum . furthermore , they also had lower fa in these regions than patients who responded to treatment ( n=40 ; figure 1 , tables i and ii ) . one of the most striking findings of this work was that the responders were indistinguishable from the healthy control subjects , suggesting that the original clinical sample was biologically rather heterogeneous . we rescanned these subjects at the 12-week clinical evaluation , and found that the same regional differences seen at baseline between the two patient groups were also present at 12 weeks , although the differences appeared less widespread . furthermore , although there was some increase in fa over time , this was in the same direction in the two patient groups , with no significant time vs group interaction . we interpreted these findings to indicate that white matter integrity and brain connectivity are important moderators of response to antipsychotics . it is intriguing to speculate whether these alterations are established early on , as a neurodevelopmental deviation . the presence of a neurodevelopmental alteration would find further support in our gray matter findings from this sample . in these individuals , we performed the first 3d evaluation of cortical gyrification , a marker potentially indicative of early neurodevelopmental disturbances , in relation to subsequent treatment response . gyrification defects have been associated with exposure to obstetric complications and with psychotic symptoms resistant to treatment . interestingly , the earlier neuroimaging studies in treatment - resistant patients that were mentioned in the previous section , found that sulcal enlargement ( which could lead to hypogyria ) was associated with a poorer response to clozapine . in our samples , we found that , already at illness onset , patients who subsequently did not respond to treatment had significant cortical folding defects ( hypogyria ) of several frontotemporal regions and the insula when compared with the responders . they also had widespread deficits in gyrification extending to the precuneus , angular gyrus , and lingual gyrus when compared with healthy controls ( figure 2 , table iii ) . in contrast , and similarly to what we saw for white matter , patients who subsequently responded were virtually indistinguishable from the healthy controls . these findings are very consistent with the evidence described earlier that only the nonresponders have reduced integrity in the white matter tracts that connect these cortical regions . it is also interesting that the alterations in gyrification and white matter were evident in nonresponders with both affective ( bipolar disorder or major depression with psychotic symptoms ) and nonaffective ( schizophrenia , schizophreniform disorder , schizoaffective disorder and psychosis not otherwise specified ) psychosis , suggesting that as a group , nonresponders are likely to have a more homogeneous pathophysiological process underlying psychoses than the responders . taken together , these findings suggest that individuals who are less likely to respond to treatment represent either a more severely affected group , or a group that has experienced a pathophysiological insult at an early neurodevelopmental stage than those who respond to treatment , possibly with a differential exposure to factors that determine axonal integrity ( genetic or molecular ) , which , in turn , influences brain connectivity and gyrification . although crucial to our understanding of psychosis , these group findings need to be extended if they are to inform clinical outcomes at the level of a single individual . structural imaging has strong potential for clinical applicability as it is : ( i ) noninvasive ; ( ii ) quick to acquire ; ( iii ) widely available ; and finally , ( iv ) cheap compared with other imaging methods such as positron emission tomography . therefore , what progress have we made toward its clinical applicability ? over the last few years , the application of novel approaches to the analysis of structural imaging data has made us feel closer to their potential use in clinical management . to this end , machine - learning methods , such as support vector machine , have shown promise in differentiating patients or ultra - high - risk individuals from healthy controls , based on structural mri data . obviously , an even more interesting translational question would be : can we use structural mri data at illness onset to predict which individuals will develop worse outcomes or poorer responses to treatment ? about 2 years ago , our group published the first paper showing that structural brain scans at the first episode could be used to predict , with significant accuracy , outcomes at 6 years . in this study , we found that the mri scan obtained when patients first presented to services could be used to predict which patients developed a continuous nonremitting illness course ; distinguishing them from both healthy controls ( sensitivity , 71 ; specificity , 61 ) and from patients who had an episodic , more benign illness ( sensitivity , 71 ; specificity , 68 ) . consistent with our recent data , discussed in the previous section , we were not able to distinguish the patients who went on to have an episodic course from the healthy controls . other papers have since confirmed , in different clinical populations , the potential of machine - learning approaches to discriminate individual patients in predicting the onset and subsequent severity of psychosis in ultra - high - risk individuals . while promising , these approaches need replication in larger samples of patients at the same illness stages and treated with the same pharmacological interventions , and also need validation for scans obtained using different scanners . these are just some of the aims of an ongoing , large , multicenter study funded by the fp7 programme of the european commission , optimise ( optimization of treatment and management of schizophrenia in europe , www.optimisetrial.eu ) . across various centers in europe , we are acquiring mri scans from a very large sample of patients with first - episode schizophrenia , who are then all treated with the same antipsychotic , amisulpride , for 4 weeks , when their symptomatic response is evaluated . this study will use both univariate and multivariate image analysis approaches , ie , support vector machine , to establish whether it is possible to predict the 4-week response based on the mri scan obtained at the first episode , alone or in combination with , other biological markers , including magnetic resonance spectroscopy . we know that the outcome and response to available treatments for schizophrenia , and for psychosis in general , is heterogeneous . still , we do not have strong biological markers that could help us disentangle this heterogeneity and be useful in clinical practice . there are , however , structural brain alterations that , if refined , have potential for use early in the stratification of patients with psychosis . for this to be achieved for example , validation with very large datasets could help establish a reference group ( a databank ) , which clinical imaging centers could access for an automated classification of their patients ' mris in order to obtain an estimate of the likelihood of a specific outcome . for this to be achieved , it would be essential to have standardized definitions of outcomes within the clinical academic community that are relevant to clinicians , patients , and their careers . such a system could then be used to assign a patient to targeted assertive case management at first presentation to services , in the optimization of pharmacological treatment , or in cognitive and family interventions . these have all been shown to improve treatment adherence and reduce relapse rates , eventually improving outcome . at the same time , they could help identify those patients most likely to have a good remitting illness after their first episode , who could then avoid long - term exposure to antipsychotic medication . furthermore , the identification of gray matter and connectivity markers that characterize poor response poses the question of whether these markers could be used in patient stratification in clinical trials of new treatment strategies , or even to inform drug development of agents that can enhance and restore brain connectivity , which may elicit a better response in those who currently do not respond to available antipsychotics .

studies that have used structural magnetic resonance imaging ( mri ) suggest that individuals with psychoses have brain alterations , particularly in frontal and temporal cortices , and in the white matter tracts that connect them . furthermore , these studies suggest that brain alterations may be particularly prominent , already at illness onset , in those individuals more likely to have poorer outcomes ( eg , higher number of hospital admissions , and poorer symptom remission , level of functioning , and response to the first treatment with antipsychotic drugs ) . the fact that , even when present , these brain alterations are subtle and distributed in nature , has limited , until now , the utility of mri in the clinical management of these disorders . more recently , mri approaches , such as machine learning , have suggested that these neuroanatomical biomarkers can be used for direct clinical benefits . for example , using support vector machine , mri data obtained at illness onset have been used to predict , with significant accuracy , whether a specific individual is likely to experience a remission of symptoms later on in the course of the illness . taken together , this evidence suggests that validated , strong neuroanatomical markers could be used not only to inform tailored intervention strategies in a single individual , but also to allow patient stratification in clinical trials for new treatments .

warfarin is the most commonly used oral anticoagulant both for prophylaxis as well as treatment for arterial and venous thromboembolic conditions . it has a narrow therapeutic index and is metabolized by hepatic cytochrome p450 2c9 enzyme ( cyp2c9 ) . various polymorphisms exist in the gene that code for cyp2c9 , of which * 1 is the wild form expressing normal activity . amongst the variants , * 2 and * 3 are more common than the rest and exhibit lesser enzymatic activity . the anticoagulant effect of warfarin is measured by prothrombin time - international normalized ratio ( pt - inr ) . warfarin exerts its anticoagulant effect by reducing the regeneration of active vitamin k through inhibition of vitamin k epoxide reductase , encoded by vitamin k epoxide reductase complex subunit 1 gene ( vkorc1 ) . increased risk of deranged inr or bleeding has been reported in patients harboring mutant form of either cyp2c9 or vkorc1 . this case report describes a patient on low - dose warfarin , who succumbed to a subdural hematoma and was found to have polymorphic cyp2c9 ( * 1/*3 ) . a 49-year - old male patient , who was a known case of superior sagittal sinus thrombosis for the past 8 years on tablet warfarin 2.5 mg every night , was brought to the hospital unconscious . the patient was non - compliant in not following up with his physician nor did he check his pt - inr levels regularly for the past 1 year . the patient 's relative , however , maintained that the patient was regular in taking the prescribed dose of warfarin ( 2.5 mg ) every night . on physical examination , he was found to be unconscious ( glasgow coma scale score of 3 ) , pulse was feeble and the blood pressure was not recordable . a plain computed tomography ( ct ) brain revealed 24.2 mm hyperdense cresentic subdural hematoma in left fronto - temporo - parieto - occipital region with subfalcine extension and midline shift [ figure 1 ] . his pt - inr was found to be 9 [ normal ( in the absence of anticoagulation therapy ) is 1 and it has to be maintained between 2 and 3 for an appropriate therapeutic effect for warfarin ] . fresh frozen plasma was initiated at a dose of 15 ml / kg / day . blood ( 5 ml ) was collected from the patient and genotyping was done for both cyp2c9 and vkorc1 . cyp2c9 was found to be polymorphic for * 3 ( * 1/*3 ) and vkorc1 was found to be wild - type ( normal ) . plain ct brain revealing 24.2 mm hyperdense cresentic subdural hematoma ( red arrow ) in left fronto - temporo - parieto - occipital region pharmacogenetics is the study of genetic influence on inter - individual variation in the response of a drug . pharmacogenetics has been shown to be of value in detecting an optimal response as well as in preventing adverse reactions following drugs such as warfarin , clopidogrel , and phenytoin . warfarin is metabolized by cyp2c9 and over 30 polymorphic alleles have been identified in the gene coding for cyp2c9 . the wild form of the gene is * 1 and the most common polymorphic forms include * 2 and * 3 . allelic frequency of cyp2c9 * 2 and * 3 in various populations including indians were found to be in the range of 0 - 20% . a recent systematic review concluded that in comparison to the patients with cyp2c9 * 1/*1 genotype , the cyp2c9 * 1/*2 , cyp2c9 * 1/*3 , cyp2c9 * 2/*2 , cyp2c9 * 2/*3 , and cyp2c9 * 3/*3 patients required warfarin doses that were 19.6 , 33.7 , 36.0 , 56.7 , and 78.1% lower , respectively . similarly , in the vkorc1 gene , two alleles have been identified in vkorc1 namely g and a. patients with gg ( wild type ) are resistant requiring a higher dose of the drug whereas aa are sensitive requiring a lesser dose and ga requires intermediate . a randomized clinical trial has also shown that pharmacogenetic - based dosing of warfarin was associated in maintaining the pt - inr levels within the acceptable range for a longer time than by the standard dosing . united states food and drug administration ( fda ) has recently modified the warfarin labeling by suggesting cyp2c9 and vkorc1 genotyping before initiating the drug to ensure warfarin safety . our patient had a fatal subdural hematoma despite low - dose ( 2.5 mg hs ) of warfarin . he had massively deranged inr of 9 , was brought in with glasgow coma scale of 3 . the * 1/*3 variant cyp2c9 explained this serious adverse event with low - dose warfarin . the * 3 variant is less than 5% as active as the wild type whereas * 2 retains around 12% of the activity . a recent indian review of oral anticoagulant use revealed lack of proper laboratory facilities , with irregular pt - inr monitoring in 25% patients and deranged inr values in a high proportion of patients . pharmacogenetic testing needs to be done only once and hence can be considered cost - effective . studies have shown that along with age , gender , body weight , cyp2c9 and vkorc1 polymorphisms account for almost 64% of the variability in the dose of warfarin required for an individual . hence , by doing these genetic testing a priori to the treatment initiation , there is a high possibility that the patients may receive an appropriate dose than just by routine practice . even , several international algorithms for predicting dose of warfarin have been developed with these predicting factors of which , gage 's and wadelius algorithms have been shown to be the most accurate . recently , an algorithm for indian patients has also been developed , which was found to be more accurate , sensitive and significantly reduced the risk of overestimation of dose of warfarin . this algorithm can be used for predicting the appropriate dose of warfarin in our population with the genotype details of cyp2c9 and vkorc1 . to conclude , this case highlights the necessity of adopting the practice of routine genetic testing for cyp2c9 and vkorc1 while initiating warfarin therapy .

warfarin is the most common and cheap oral anticoagulant currently used in clinical practice . a high inter - individual variation is seen in the response to warfarin . recently , pharmacogenetics has gained importance in managing patients on warfarin , both in predicting the optimum required dose as well as in decreasing the risk of bleeding . this case report is a description of a 49-year - old patient who had a lethal subdural hematoma with low - dose warfarin . he was subsequently found to have cyp2c9 gene polymorphism ( * 1/*3 ) . this case report stresses the importance of pre - prescription assessment of genetic analysis for those initiated on warfarin .

despite the advent of new microbials , meningococcal infection remains a leading cause of morbidity and mortality.[14 ] based on the sequence of pathophysiological events and the agent and host factors , a wide spectrum of presentations may be seen . patients with invasive meningococcal disease can present with meningitis alone or meningitis with shock.[14 ] the waterhouse friderichsen syndrome is a severe complication of meningococcal infection.[13 ] the patient presents with meningococcal disease along with shock due to adrenal hemorrhage.[14 ] here we report a fatal case of waterhouse - friedrichsen syndrome in an adult male patient with meningococcal disease . a 29-year - old male patient was admitted with a history of high - grade fever with chills and vomiting of 7 days duration , along with skin rash over the abdomen and trunk for the last 2 days . after admission he developed a hemorrhagic rash . there was no significant finding noted in the family history or the past history . on examination , his general condition was unstable and cyanosis was present . he was febrile , with a pulse rate of 90/min and systolic bp of 70 mm hg . a rash was observed over the whole of the body , but it was predominantly over the abdomen and trunk [ figure 1 ] . the cerebrospinal fluid ( csf ) tapped was collected under aseptic precautions and was processed according to standard bacteriological procedures . routine microscopy of the csf showed a cell count of 9400/mm , with 86% polymorphs and 14% lymphocytes . the csf protein was raised to 309 mg% and the sugar was reduced to 20 mg% . the gram stained smear of the csf showed pus cells along with gram - negative diplococci , some of which were intracellular and some extracellular [ figure 2 ] . the csf was cultured on chocolate agar , blood agar , and macconkey agar and the media were incubated at 37c in humid conditions in a candle jar . after overnight incubation , tiny translucent colonies were observed on chocolate agar and blood agar , which was later identified by standard laboratory procedures to be that of neisseria meningitidis . ct scan confirmed adrenal hemorrhage , which is supposed to be a diagnostic factor for waterhouse - friderichsen syndrome . antibiotic sensitivity tests showed that the isolate was sensitive to all the antibiotics tested , i.e. , ceftriaxone , chloramphenicol , penicillin , and trimethoprim + sulfamethoxazole . the patient meanwhile was started on ceftriaxone and steroids , but he went into shock and expired on the third day after admission . purpuric , hemorrhagic skin lesions associated with the waterhouse - friedrichsen syndrome gram stained smear showing pus cells along with gram - negative diplococci this case is being reported as waterhouse - friderichsen syndrome is comparatively rare in this antibiotic era , especially in adults . if early diagnosis and antibacterial treatment , along with steroids , is not administered then there is high associated mortality . asia has been the focus of meningococcal meningitis . many outbreaks of meningococcal meningitis were documented during 1966 and 1985 in delhi and adjoining areas . in early 2005 , a spurt in the number of cases of meningococcemia and meningitis due to n meningitidis was reported from india . the majority of the cases , and all the deaths , occurred in young adults between 1630 years of age . microscopically , these lesions are characterized by endothelial damage , which leads to hemorrhages , and microthrombi in small vessels . the lesions are the result of endotoxins- and cytokine - primed vasculitis that is mediated by the upregulation of adhesion molecules on endothelium and degranulation activated neutrophils . massive adrenal hemorrhage ( waterhouse - friderichsen syndrome ) although disseminated intravascular coagulation is a generalized phenomenon , it may affect any organ and when it causes adrenal hemorrhages the condition is called waterhouse - friderichsen syndrome . gram 's staining of the csf is still considered an important method for rapid diagnosis , but culture from csf or skin lesion biopsy is the gold standard.[16 ] in some cases , blood culture may yield the organism . in the present case only csf most of the studies showed that the isolates were sensitive to penicillin , ampicillin , and ceftriaxone ; in two - thirds of the cases the isolates were resistant to ciprofloxacin . our isolate was sensitive to all the drugs tested , but the patient expired despite adequate antibiotic therapy , probably due to the adrenal hemorrhage leading to shock . waterhouse - friderichsen syndrome , first reported in 1911 by rubert waterhouse , is characterized by fever , rash , purpura , coagulopathy , and shock . it has been suggested by many authors that this syndrome is more common than what literature reports indicate , many cases probably being missed due to lack of familiarity with the condition . the majority of the diseases caused by meningococci occur in children under 2 years of age , but it can occur at any age . though waterhouse - friderichsen syndrome is common with n meningitidis , other organisms are also associated with this syndrome , including streptococcus pneumoniae , -hemolytic streptococcus group a , staphylococcus aureus , neisseria gonorrhoeae , escherichia coli , hemophilus influenzae , klebsiella sp , and pasturella sp . although the condition is predominantly associated with meningococcal infection and with sepsis due to other organisms , there are also noninfectious causes of the waterhouse - friderichsen syndrome , such as anticoagulant treatment , antiphospholipid syndrome , trauma , and postoperative adrenal hemorrhages . though n meningitidis is susceptible to the commonly used antibiotics , the mortality in waterhouse - friderichsen syndrome is approximately 20% , rising to 50% if the patient is in shock as happened in the present case . if there are any signs and symptoms suggestive of waterhouse - friderichsen syndrome then 100 mg of hydrocortisone should also be given to the patient . if possible it should be given intravenously , but if a vein is not accessible the intramuscular route will suffice . studied have shown that even prophylactically administered steroid can be life saving . starting steroids like dexamethasone prior to antibacterial therapy may be useful to diminish the meningococcal inflammation brought about by bacterial cell death . thus early diagnosis and proper treatment is necessary to prevent death from this otherwise fatal condition .

waterhouse - friderichsen syndrome is one of the fatal complications of meningococcal infection . here we report a fatal case of this syndrome due to neisseria meningitidis in a 29-year - old male patient who was admitted with high - grade fever and chills and vomiting since 7 days , a skin rash over the abdomen and trunk , and altered sensorium since 2 days . on examination , the signs of meningitis were present along with the hemorrhagic rash . the diagnosis of adrenal hemorrhage was confirmed by computerized tomographic scan findings . the patient was started on intravenous ceftriaxone , and the cerebrospinal fluid was processed for bacterial culture , which yielded growth of n meningitidis . the patient 's condition deteriorated ; he developed purpura along with a fall in platelet count , and died due to shock . this case is being reported as such a complication is comparatively rare in this antibiotic era , especially in adults , and starting steroids like dexamethasone prior to antibacterial therapy may be useful to diminish the inflammation brought about by bacterial cell death and thus help in reducing the otherwise high mortality in these cases .

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

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

seasonal influenza epidemics impose a heavy burden on society , with 35 million cases and 250 000500 000 deaths worldwide every year.1 the resulting economic impact is large and includes both direct and indirect costs.2,3 traditionally , attention has been directed toward influenza a , which accounts for the majority of influenza cases in most seasons46 ; its subtypes are also responsible for influenza pandemics.7 during interpandemic periods , however , influenza b can represent a considerable proportion of total cases.8 since the 1970s , influenza b viruses have belonged to two antigenically distinct lineages called the victoria and yamagata lineages9 ; this has been a challenge for seasonal influenza vaccines as only one influenza b strain is included in the trivalent vaccine . studies in the united states have shown that the frequent influenza b vaccine mismatches of recent years have been associated with substantial increases in cases , hospitalizations and deaths ( up to annual 970 000 cases , with 8200 hospitalizations and 485 deaths , in the usa),10 as well as with large influenza - related medical costs , and costs associated with productivity loss.11 despite the important role of influenza b , much of the published scientific literature regarding the epidemiology of influenza has focused on influenza a , and we still have a relatively poor understanding of global epidemiology and burden of disease of influenza b , especially outside europe and the united states.8 several studies have reported on the burden of disease attributable to influenza b in a single season , or during consecutive seasons in a single country,12,13 but only one study thus far has looked at the global epidemiology of influenza b.4 in particular , it is very important to assess the epidemiology of influenza in the tropics , as this is where approximately 40% of the world s population live,14 and influenza activity there is quite different from other world regions15,16 : countries in the tropics may experience two annual peaks , and epidemics are not as short and intense as in the northern and southern hemispheres.1719 these differences can have important implications for effective and evidence - based decisions regarding the composition and period of administration of influenza vaccines . the global influenza b study ( gibs ) was launched in 2012 with the main aim of collecting information on the epidemiology and global burden of disease of influenza b during the past 1015 years , to support future prevention policies . gibs is a project of the global influenza initiative , an expert scientific forum established to address the ongoing problems related to influenza worldwide . to achieve this objective , we contacted countries around the world during the period june 2013 to february 2014 , requesting access to data from their national influenza surveillance systems . here , we compare four important epidemiological and virological characteristics of influenza a and b in 26 countries : the proportion of influenza b over all influenza cases ; the community impact of influenza b ; the frequency of influenza b vaccination mismatches ; and the age distribution of influenza a and b cases . the global influenza b study ( gibs ) was launched in 2012 with the main aim of collecting information on the epidemiology and global burden of disease of influenza b during the past 1015 years , to support future prevention policies . gibs is a project of the global influenza initiative , an expert scientific forum established to address the ongoing problems related to influenza worldwide . to achieve this objective , we contacted countries around the world during the period june 2013 to february 2014 , requesting access to data from their national influenza surveillance systems . here , we compare four important epidemiological and virological characteristics of influenza a and b in 26 countries : the proportion of influenza b over all influenza cases ; the community impact of influenza b ; the frequency of influenza b vaccination mismatches ; and the age distribution of influenza a and b cases . we contacted national influenza centers in 43 countries in the northern and southern hemispheres and the intertropical belt ; countries were selected to represent all world health organization ( who ) influenza transmission zones.20 all countries were asked to make available data originating from their national influenza surveillance system during recent years ( ideally from 20002013 ) . spreadsheet data reporting templates were provided , along with instructions on how to report data . each participating country was asked to provide the following : virological data : weekly number of influenza cases reported by the national surveillance system , broken down by age group ( 05 , 635 months , 34 , 517 , 1839 , 4064 and 65 years ) ; virus type ( a versus b ) ; and virus subtype [ a(h1n1 ) , a(h3n2 ) , a(h1n1)pdm2009 , a(h1n2 ) , a , unsubtyped ] or lineage ( b / victoria , b / yamagata , b , not characterized ) . epidemiological data : weekly influenza - like illness ( ili)/acute respiratory infection ( ari ) rates per 100 000 population or 100 consultations ( depending on what is routinely available within each national surveillance system ) . virological data : weekly number of influenza cases reported by the national surveillance system , broken down by age group ( 05 , 635 months , 34 , 517 , 1839 , 4064 and 65 years ) ; virus type ( a versus b ) ; and virus subtype [ a(h1n1 ) , a(h3n2 ) , a(h1n1)pdm2009 , a(h1n2 ) , a , unsubtyped ] or lineage ( b / victoria , b / yamagata , b , not characterized ) . epidemiological data : weekly influenza - like illness ( ili)/acute respiratory infection ( ari ) rates per 100 000 population or 100 consultations ( depending on what is routinely available within each national surveillance system ) . for countries that extend over large areas , especially when stretched across different climate zones ( such as china and brazil ) , we asked for data stratified by region / province , if it were available . all countries received a national feedback report shortly after providing the data , so that they had the opportunity to check the data they had sent . they were also all asked to complete a short questionnaire on the main features of their national influenza surveillance system ( see table s1 ) . the questionnaire included questions on the ili / ari case definition in use ; patients being sampled ; representativeness of data ; methods used for identification and characterization of influenza virus ; and the population denominator . influenza epidemics usually occur between october of a given year and april of the following year in countries in the northern hemisphere , and between april and october of a given year in the southern hemisphere , with greater variability observed for countries situated near the tropics.17 for the purposes of this study , we define a season as being the period between the first and last week of a given year ( for the tropics and the southern hemisphere ) or between the 27th week of a given year and the 26th week of the following year ( for the northern hemisphere ) , so that each season includes the whole period of increased influenza activity in each country . for conciseness , when looking at season 2005 , we refer to 20052006 for countries in the northern hemisphere , and year 2005 for all other countries . for each country , only the seasons with at least 50 influenza reported cases and at least 20 weeks of data reporting were included in the analysis . analyses were conducted for all countries and then separately for countries situated in the northern or southern hemisphere or in the intertropical belt ( defined as the country centroid , when available , or the largest city being located north of the tropic of cancer and south of the tropic of capricorn).21 for each country and season , we calculated the percentage of influenza cases that were due to influenza b virus and then worked out its median value for countries in the northern and southern hemispheres and in the intertropical belt . we calculated the number of seasons that were dominated by either lineage among those where there was a significant circulation of influenza b ( defined as the proportion of influenza b being 20% of all influenza cases reported during the season ) . for this analysis , we only considered seasons where the proportion of influenza b cases characterized was 10% . an influenza b vaccine mismatch was defined as a mismatch between the influenza b lineage included in the vaccine and the lineage that caused the majority ( > 50% ) of cases in a season with significant circulation of influenza b ; using this definition , we calculated the proportion of seasons where a vaccine mismatch was observed . the information on vaccine composition was obtained from the who website.22 we calculated the percentage of vaccine mismatch according to three alternative scenarios : ( i ) all tropical countries situated north of the equator adopting the who recommendations for the northern hemisphere , and vice versa ; ( ii ) all tropical countries using the northern hemisphere who recommendation ; or ( iii ) all tropical countries adopting the southern hemisphere who recommendation . to explore whether there was an association between the magnitude of the influenza season and the proportion of influenza cases due to b virus type , we obtained the country - specific z - score of the weekly ili / ari rate ( defined as the number of standard deviations above or below the country - specific average of the ili / ari rate ) and calculated the pearson s correlation coefficient between its maximum value and the proportion of influenza b cases during each season . we hypothesized that a moderate - to - mild inverse correlation would be seen between the proportion of influenza b and the maximum weekly ili / ari rate in the northern and southern hemispheres , as influenza a viruses cause most influenza cases and are responsible for short and intense epidemics especially influenza a(h3n2)compared with influenza b. we calculated the percentage of influenza a and b cases in each country in each of the following age categories : 04 , 517 , 1864 and 65 years ; we tested whether the percentage of influenza a versus b cases differed in each age group using a chi - square test . when the exact age was available , we also obtained virus type - specific median age and interquartile range ( iqr ) and used the wilcoxon rank sum test to detect any differences in median age of influenza a versus b cases . all analyses were performed using stata version 11 ( statacorp lp , college station , tx , usa ) and microsoft excel . all statistical tests were two - sided , and a p - value of < 005 was considered significant . we contacted national influenza centers in 43 countries in the northern and southern hemispheres and the intertropical belt ; countries were selected to represent all world health organization ( who ) influenza transmission zones.20 all countries were asked to make available data originating from their national influenza surveillance system during recent years ( ideally from 20002013 ) . spreadsheet data reporting templates were provided , along with instructions on how to report data . each participating country was asked to provide the following : virological data : weekly number of influenza cases reported by the national surveillance system , broken down by age group ( 05 , 635 months , 34 , 517 , 1839 , 4064 and 65 years ) ; virus type ( a versus b ) ; and virus subtype [ a(h1n1 ) , a(h3n2 ) , a(h1n1)pdm2009 , a(h1n2 ) , a , unsubtyped ] or lineage ( b / victoria , b / yamagata , b , not characterized ) . epidemiological data : weekly influenza - like illness ( ili)/acute respiratory infection ( ari ) rates per 100 000 population or 100 consultations ( depending on what is routinely available within each national surveillance system ) . virological data : weekly number of influenza cases reported by the national surveillance system , broken down by age group ( 05 , 635 months , 34 , 517 , 1839 , 4064 and 65 years ) ; virus type ( a versus b ) ; and virus subtype [ a(h1n1 ) , a(h3n2 ) , a(h1n1)pdm2009 , a(h1n2 ) , a , unsubtyped ] or lineage ( b / victoria , b / yamagata , b , not characterized ) . epidemiological data : weekly influenza - like illness ( ili)/acute respiratory infection ( ari ) rates per 100 000 population or 100 consultations ( depending on what is routinely available within each national surveillance system ) . for countries that extend over large areas , especially when stretched across different climate zones ( such as china and brazil ) , we asked for data stratified by region / province , if it were available . all countries received a national feedback report shortly after providing the data , so that they had the opportunity to check the data they had sent . they were also all asked to complete a short questionnaire on the main features of their national influenza surveillance system ( see table s1 ) . the questionnaire included questions on the ili / ari case definition in use ; patients being sampled ; representativeness of data ; methods used for identification and characterization of influenza virus ; and the population denominator . influenza epidemics usually occur between october of a given year and april of the following year in countries in the northern hemisphere , and between april and october of a given year in the southern hemisphere , with greater variability observed for countries situated near the tropics.17 for the purposes of this study , we define a season as being the period between the first and last week of a given year ( for the tropics and the southern hemisphere ) or between the 27th week of a given year and the 26th week of the following year ( for the northern hemisphere ) , so that each season includes the whole period of increased influenza activity in each country . for conciseness , when looking at season 2005 , we refer to 20052006 for countries in the northern hemisphere , and year 2005 for all other countries . for each country , only the seasons with at least 50 influenza reported cases and at least 20 weeks of data reporting were included in the analysis . analyses were conducted for all countries and then separately for countries situated in the northern or southern hemisphere or in the intertropical belt ( defined as the country centroid , when available , or the largest city being located north of the tropic of cancer and south of the tropic of capricorn).21 for each country and season , we calculated the percentage of influenza cases that were due to influenza b virus and then worked out its median value for countries in the northern and southern hemispheres and in the intertropical belt . we calculated the number of seasons that were dominated by either lineage among those where there was a significant circulation of influenza b ( defined as the proportion of influenza b being 20% of all influenza cases reported during the season ) . for this analysis , we only considered seasons where the proportion of influenza b cases characterized was 10% . an influenza b vaccine mismatch was defined as a mismatch between the influenza b lineage included in the vaccine and the lineage that caused the majority ( > 50% ) of cases in a season with significant circulation of influenza b ; using this definition , we calculated the proportion of seasons where a vaccine mismatch was observed . the information on vaccine composition was obtained from the who website.22 we calculated the percentage of vaccine mismatch according to three alternative scenarios : ( i ) all tropical countries situated north of the equator adopting the who recommendations for the northern hemisphere , and vice versa ; ( ii ) all tropical countries using the northern hemisphere who recommendation ; or ( iii ) all tropical countries adopting the southern hemisphere who recommendation . to explore whether there was an association between the magnitude of the influenza season and the proportion of influenza cases due to b virus type , we obtained the country - specific z - score of the weekly ili / ari rate ( defined as the number of standard deviations above or below the country - specific average of the ili / ari rate ) and calculated the pearson s correlation coefficient between its maximum value and the proportion of influenza b cases during each season . we hypothesized that a moderate - to - mild inverse correlation would be seen between the proportion of influenza b and the maximum weekly ili / ari rate in the northern and southern hemispheres , as influenza a viruses cause most influenza cases and are responsible for short and intense epidemics especially influenza a(h3n2)compared with influenza b. we calculated the percentage of influenza a and b cases in each country in each of the following age categories : 04 , 517 , 1864 and 65 years ; we tested whether the percentage of influenza a versus b cases differed in each age group using a chi - square test . when the exact age was available , we also obtained virus type - specific median age and interquartile range ( iqr ) and used the wilcoxon rank sum test to detect any differences in median age of influenza a versus b cases . all analyses were performed using stata version 11 ( statacorp lp , college station , tx , usa ) and microsoft excel . all statistical tests were two - sided , and a p - value of < 005 was considered significant . china provided separate data for the northern and southern parts of the country.23 brazil provided data stratified by its five administrative regions : north , north - east , central - west , south - east and south ; however , as results of the analyses did not differ across regions , results for the whole country are shown . participating countries are distributed in the northern ( n = 7 ) and southern ( n = 5 ) hemispheres and in the intertropical belt ( n = 14 ) , cover 16 of the 18 who influenza transmission zones,20 and account for around 37% of the world s population ( table1 ) . comparison of geography , demographics and main features of influenza surveillance systems of participating countries ( from southern- to northernmost ) . the global influenza b study ari , acute respiratory infection ; ili , influenza - like illness ; pcr , polymerase chain reaction ; sari , severe acute respiratory infection ; who , world health organization . the influenza surveillance systems of the participating countries differ from each other ( table1 ) ; in most cases , however , they cover the whole country , sample both outpatients and hospitalized patients , and send isolates to a who collaborating center for reference . most countries in the northern and southern hemispheres had data on ili rates ( ari rates for singapore ) , with a mixture of consultation and population denominators , but many countries in the tropics had no such data . overall , 935 673 influenza cases were reported to the national influenza centers during 200 seasons between 2000 and 2013 ( table2 ) . of these , 288 130 cases ( 308% ) were reported before the emergence of the 2009 a(h1n1 ) pandemic influenza . countries provided a median of seven seasons , ranging from four seasons for argentina , costa rica , and honduras , to 13 for new zealand . the proportion of influenza a cases subtyped was 651% , and 171% of influenza b cases were characterized . the information on age was available for 476% and 591% of influenza a and b cases , respectively . influenza cases reported to the national influenza surveillance system of each participating country ( from southern- to northernmost ) , and percentages of cases that were subtyped , by virus type . the global influenza b study seasons 20012002 and 20032004 in the ukraine were not included as the number of reported influenza cases was < 50 . the proportion of influenza cases due to type b virus was < 20% for 90 seasons , 2050% for 82 seasons and figure2 shows the distribution of seasons according to the proportion of influenza cases caused by type b virus , separately for countries situated in the northern or southern hemisphere or in the intertropical belt . the median proportion of influenza b over all influenza seasons was 178% in the southern hemisphere ( iqr 35304% ; 39 seasons ) , 243% in the intertropical belt ( iqr 102408% ; 94 seasons ) , and 214% in the northern hemisphere ( iqr 73380% ; 67 seasons ) . the p - value for the comparison of the median proportion of influenza b in the southern hemisphere versus the intertropical belt was borderline significant ( 007 ) and not significant for the other comparisons . distribution of influenza seasons by proportion of influenza b cases and geographical area ( southern hemisphere , intertropical belt and northern hemisphere ) . the spearman s rank correlation coefficient for the association between the proportion of influenza b during a given season and the maximum ili rate z - score during the same season was 031 in the southern hemisphere [ 95% confidence interval ( ci ) 064 to 012 ; 23 seasons ] , 009 in the intertropical belt ( 95% ci 043 to 026 ; 32 seasons ) , and 031 in the northern hemisphere ( 95% ci 054 to 004 ; 51 seasons ) ( figure3 ) . proportion of influenza b and maximum influenza - like illness ( ili ) rate ( z - score ) during each season , by geographical area ( southern hemisphere , intertropical belt and northern hemisphere ) . the proportion of influenza b cases that were characterized was 10% for 79 of the 200 seasons . if one assumes that all countries in the intertropical belt used the who recommendations for the hemisphere they are situated or the who recommendation for the southern hemisphere , an influenza b vaccine mismatch was seen in 19 of 79 seasons ( 2425% ) : 11 of 36 seasons in the northern hemisphere ( 31% ) , six of 22 seasons in the southern hemisphere ( 27% ) , and two of 21 seasons in the intertropical belt ( 10% or 14% ) ( table s2 ) . if one assumes the who recommendation for the northern hemisphere was followed , the results are very similar : there were a total of 20 mismatches and three mismatches for the intertropical belt countries . influenza b accounted for 20% or more of all influenza cases during 50 of these 79 seasons : victoria and yamagata lineages predominated in 32 ( 64% ) and 18 ( 36% ) seasons , respectively . also , the victoria and yamagata lineages often co - circulate in the same season : in 16 of 50 seasons , both lineages accounted for at least 20% of influenza b cases ( table s2 ) . table s3 presents the age distribution of a versus b influenza cases in each country . a consistent finding across most countries is a younger age for influenza b versus a cases . in particular , there was a consistently higher proportion of influenza b cases in the 517 years age group , and a cases in the 1864 years age group in southern and northern hemisphere countries ( except south africa , brazil , turkey , and ukraine ) and in some countries of the intertropical belt ( madagascar , indonesia , singapore , costa rica , nicaragua , guatemala , and vietnam ) . influenza b cases were also younger than a cases in panama , el salvador , and honduras , where there was a higher proportion of b cases among patients aged 04 years . no differences in age distribution were observed in kenya , cameroon , and the ivory coast ; these were the only countries where over 50% of all influenza cases were aged 4 years . finally , influenza a cases were older than b cases in south africa , brazil , turkey , and ukraine . the gibs was conceived and implemented to obtain a better understanding of the global epidemiology of influenza b , with a particular focus on the tropics , which have been relatively neglected by the research so far.8 our main finding was that influenza b is a common virus in the 21st century , representing roughly 20% of all cases reported to national influenza centers in 26 countries around the world during 20002013 . although the differences were not statistically significant , there is some evidence of geographical variability in the occurrence of influenza b around the world , with it being most common in the tropics ( median 243% ) and least common in the southern hemisphere ( 178% ) . we found that influenza b rarely represented over 50% of flu cases ( once every seven seasons ) and was generally associated with lower rates of ili in the northern and ( with borderline significance ) southern hemispheres . we also found that there was frequently a vaccine mismatch when influenza b circulated in a country ; this happened more often in the northern and southern hemispheres compared with the tropics . finally , influenza b generally affected younger persons than influenza a , with the former mainly affecting school - aged children ( aged 517 years ) and the latter adults ( aged 1864 years ) . it is not yet clear what causes the differences in influenza epidemiology ( including timing , periodicity , and patterns of transmission ) in the tropics compared with the southern and northern hemispheres.15 the non - significant higher proportion of influenza b in the tropics may simply reflect the relatively higher proportion of children , who are the most affected age group , in most countries of this region compared to the northern and southern hemispheres . it would , however , be necessary to calculate age - specific incidence rates of influenza to confirm or refute this hypothesis . future research should prioritize the study of influenza epidemiology in this very populous area of the world , to optimize prevention strategies and the composition and timing of administration of the influenza vaccine . the divergent results of the correlation between the proportion of influenza b and peak of ili rate mirror the differences in influenza epidemiology in temperate and tropical countries , with short epidemics and greater year - to - year fluctuations for influenza a than b in the former4 and a year - round influenza activity and higher average proportion of b in the latter.17 it is usually not possible to predict with reasonable accuracy the impact of the upcoming influenza season based on historical data,24 and we have shown that victoria and yamagata lineages often co - circulate in the same season ( table s2 ) . the frequency of vaccine mismatch has been high in recent years ( 2425% in the gibs database ) , but its potential consequences in terms of influenza cases , influenza - related deaths , and economic costs are difficult to estimate at the beginning of an influenza season . this has important implications for vaccination strategies , including the decision to adopt a quadrivalent influenza vaccine.10 differences in the age distribution of influenza a versus b patients across countries may be explained in a variety of ways , including differences in the age structure of the population and in the national influenza surveillance systems ( e.g. whether the latter is mainly outpatient or hospital based ) . inequalities in access to health care by age or the presence of comorbidities ( children and older patients are more likely to see a general practitioner or be taken to hospital , and therefore be sampled , compared with adults over 18 years of age ) may also affect the age distribution of influenza cases . in some countries ( e.g. cameroon and kenya ) , the lack of differences in age distribution may be due to the small number of influenza cases in those age categories where the differences are most frequently observed , that is , 517 and 1864 years . finally , the percentage of influenza a cases due to seasonal and pandemic a(h1n1 ) and a(h3n2 ) subtypes may differ across gibs countries ( especially as a consequence of each country providing data for different influenza seasons ) , so comparing b versus a as a whole may be suboptimal virus a subtypes may preferentially affect people of different age groups.25 a more in - depth analysis of age distribution across virus subtypes and lineages will be the topic of a future gibs publication . the major limitation of our study lies in the differing characteristics of the national influenza surveillance systems of participating countries . in particular , the different definitions of ili ( or ari ) that are in use and the differences in the sources of patients included in the national databases ( outpatients , hospitalized patients , severe ari patients ) may reduce the comparability of data across countries . some world regions ( such as northern africa and central asia ) are currently not represented in our database , which somewhat lessens the generalizability of our results . however , the gibs database already includes more than 900 000 influenza cases and is still growing ; in the future , therefore , it will be possible to address specific questions on subsets of cases with common characteristics . large countries may have very different epidemiological patterns at a regional level and climatic characteristics , and in those cases , the lack of stratified data may prevent the execution of analyses with the required level of detail . for some countries , regional data are available ( e.g. brazil and china ) , but in other countries , for example the united states and australia , it remains an issue this study indicates that it is important to take into consideration influenza b in the epidemiology of seasonal influenza , as it often co - circulates with influenza a and accounts for roughly 20% of total cases in all regions of the world , despite it rarely being the dominant strain . we believe that global data on the epidemiology of influenza b , as those produced by the gibs , are needed on a continuing basis to help optimize influenza prevention policies and determine the public health value of introducing influenza vaccines containing two b lineages . in particular , we recommend that future studies exploit the potential of the gibs database , with its age - specific data , to assess the benefits of adopting influenza vaccination in different regions of the world , and tailor the vaccination campaigns to each country s requirements . sc , fs , qsh , mac , spl , and jp participated in the design of the study . gk , ro , sw , cmph , rn , raf , hy , lf , mz , awc , hk , spu , hak , ge , j - mh , qsh , lwa , mv , mac , am , lb , and ltqm collected the data . sc , fs , qsh , mac , spl , and jp interpreted the data . other than some of the authors being gii members , and the gibs being supported by an unrestricted research grant from sanofi pasteur , the authors have no competing interests to declare . the global influenza b study includes the following members : juan manuel rudi , instituto nacional de enfermedades respiratorias dr . emilio coni , santa fe , argentina ; rhonda owen , influenza surveillance section , surveillance branch , office of health protection , department of health and ageing , woden , australia ; kunzang dorji , public health laboratory , department of public health , ministry of health , thimphu , bhutan ; jos ricardo pio marins and walquiria aparecida ferreira de almeida , ministry of health , braslia , df , brazil ; marie - astrid vernet and guy vernet , service de virologie , centre pasteur du cameroun , yaounde , cameroon ; winston andrade , seccin de virus respiratorios y exantemticos , instituto de salud pblica de chile , santiago de chile , chile ; juan yang and ming li , division of infectious disease , key laboratory of surveillance and early - warning on infectious disease , chinese center for disease control and prevention , beijing , china ; jenny lara , national influenza center , ministry of health , san jos , costa rica ; celina de lozano , national influenza center , ministry of health , san salvador , el salvador ; richard pebody , joanna ellis and helen green , respiratory diseases department , public health england , colindale , uk ; leticia castillo , national influenza center , ministry of health , guatemala city , guatemala ; maria luisa matute , national influenza center , ministry of health , tegucigalpa , honduras ; nurhayati , ministry of health , republic of indonesia , and us naval medical research unit no . 2 , jakarta indonesia ; isabella donatelli , national influenza center , istituto superiore sanit , rome , italy ; coulibaly daouda , national institute of public hygiene , abidjan , cte divoire ; joshua a. mott , us centers for disease control and prevention , nairobi , kenya ; norosoa harline razanajatovo , national influenza center , virology unit , institut pasteur of madagascar , antananarivo , madagascar ; laurence randrianasolo , epidemiology unit , institut pasteur of madagascar , antananarivo , madagascar ; liza lopez , institute of environmental science and research , wellington , new zealand ; angel balmaseda , national influenza center , ministry of health , managua , nicaragua ; brechla moreno , national influenza center , ic gorgas , panama city , panama ; jeffrey cutter , communicable diseases division , ministry of health , singapore , singapore ; vernon j. lee , communicable diseases division , ministry of health , singapore , and saw swee hock school of public health , national university of singapore , singapore ; cheryl cohen , centre for respiratory diseases and meningitis ( crdm ) , national institute for communicable diseases , johannesburg , south africa , and school of public health , faculty of health science , university of the witwatersrand , johannesburg , south africa ; selim badur , istanbul university , istanbul , turkey ; larysa radchenko , l.v . gromashevsky institute of epidemiology and infectious diseases national academy of medical science of ukraine , kiev , ukraine ; joseph bresee , epidemiology and prevention branch , influenza division , centers for disease control and prevention , atlanta , ga , usa . table s1 . questionnaire on national influenza surveillance systems used for the global influenza b study . proportion of influenza b cases due to victoria or yamagata lineage virus , and mismatch with recommended world health organization ( who ) influenza vaccine . seasons were only considered during which at least 20% of influenza cases were due to virus type b with at least 10% of these being characterized . the global influenza b study . proportion of influenza a versus b cases across age categories , median age and interquartile range ( iqr ) , in countries ( from southern- to northernmost ) participating in the global influenza b study .

introductionliterature on influenza focuses on influenza a , despite influenza b having a large public health impact . the global influenza b study aims to collect information on global epidemiology and burden of disease of influenza b since 2000.methodstwenty-six countries in the southern ( n = 5 ) and northern ( n = 7 ) hemispheres and intertropical belt ( n = 14 ) provided virological and epidemiological data . we calculated the proportion of influenza cases due to type b and victoria and yamagata lineages in each country and season ; tested the correlation between proportion of influenza b and maximum weekly influenza - like illness ( ili ) rate during the same season ; determined the frequency of vaccine mismatches ; and described the age distribution of cases by virus type.resultsthe database included 935 673 influenza cases ( 20002013 ) . overall median proportion of influenza b was 226% , with no statistically significant differences across seasons . during seasons where influenza b was dominant or co - circulated ( > 20% of total detections ) , victoria and yamagata lineages predominated during 64% and 36% of seasons , respectively , and a vaccine mismatch was observed in 25% of seasons . proportion of influenza b was inversely correlated with maximum ili rate in the same season in the northern and ( with borderline significance ) southern hemispheres . patients infected with influenza b were usually younger ( 517 years ) than patients infected with influenza a.conclusioninfluenza b is a common disease with some epidemiological differences from influenza a. this should be considered when optimizing control / prevention strategies in different regions and reducing the global burden of disease due to influenza .

cell culture tissue culture reagents were purchased from commercial sources ( invitrogen ) . the human pancreatic epithelial cancer cell lines , panc-1 , aspc-1 , capan-1 , capan-2 , bxpc-3 , l3.6 , miapaca-2 , and cfpac-1 , were obtained from american type culture collection and maintained according to the supplier 's suggestions . all cells were grown at 37 c in a humidified incubator under 5% co2 . plasmid construction standard molecular biology techniques were used to clone klf4 , klf5 , klf7 , klf9 , klf11 , klf14 , and klf15 into the pcdna3.1/his ( invitrogen ) and pcmvtag2 ( stratagene ) vectors for expression as his - tagged or flag - tagged proteins , respectively , as well as the truncated ( 263 bp ) tgfrii promoter into the pgl3-lux vector ( promega ) . the p3tp - lux reporter plasmid containing tgf-responsive elements anita roberts ( national institutes of health ) as a positive control for tgf1 stimulation experiments ( data not shown ) . semi - quantitative rt - pcr total rna was extracted from cells according to the manufacturer 's instructions using an rneasy kit ( qiagen ) , and 5 g was used for cdna synthesis using oligo(dt ) primer using the superscript iii first - strand synthesis system for rt - pcr ( invitrogen ) per the manufacturer 's protocol . rt - pcr was performed using la taqdna polymerase with a gc buffers kit ( takara ) per the manufacturer protocols . semiquantitative rt - pcr analysis was performed with the primer sets provided in supplemental table s1 . amplification of four human housekeeping genes , gapdh ( unigene hs.544577 ) , 2-microglobulin ( b2 m , hs.709313 ) , 2-tubulin ( tubb2 , hs.300701 ) , and hypoxanthine phosphoribosyltransferase 1 ( hprt1 , hs.412707 ) was used for all samples as an internal control . densitometric values were obtained and normalized to the average of the housekeeping cdnas for each individual sample using scion image beta 4.02 software ( scion corp . ) . to represent the tgf inducibility of individual klf transcripts , each tgf-treated sample was compared with an untreated control that was originally plated and ultimately collected at the same time to minimize compounding factors , which can often influence gene expression ( i.e. cell cycle stage , cell density , potential unknown paracrine , and/or autocrine stimuli ) . at each time point , values were determined by first normalizing the tgf-treated sample ( t , treated ) to the average of the four aforementioned housekeeping genes in the same sample ( tc , treated housekeeping gene control ) . this resultant value was divided by the corresponding untreated sample ( ut , untreated ) normalized in the same manner ( utc , untreated housekeeping gene control ) , to express the fold of tgf induction ( [ t / tc]/[ut / utc ] = fold tgf-induction ) . another housekeeping gene , -actin ( actb , hs.520640 ) , was not used in our analyses , because it showed significant differences with tgf treatment over control values ( data not shown ) . total protein extracts were prepared by lysing cells in radioimmune precipitation assay buffer supplemented with complete protease inhibitor mixture ( roche applied science ) . cellular lysates were subjected to 10% sds - page and then separated proteins are transferred to polyvinylidene difluoride membranes ( millipore ) . membranes were incubated overnight at 4 c in blocking solution ( tris - buffered saline solution containing 5% nonfat dried milk and 0.1% tween 20 ) . immune complexes were visualized by enhanced chemiluminescence ( pierce ) and exposed to x - ray film . transcriptional reporter assays cells were transfected with specified reporter constructs along with expression constructs and/or empty vector using electroporation ( 2 10 cells/0.4-cm microcuvette , 360 v , and 10 ms ) and subsequently serum - starved overnight . transfection was performed with equimolar concentrations of dna , and expression was quantified with western blotting directed against epitope - tagged proteins as described . cells were stimulated with tgf1 ( r&d systems ) as specified and assayed at various specified time points . at 24 or 48 h after transfection and treatment as noted , cells were lysed , and luciferase measurements were performed using a 20/20 luminometer ( turner designs ) according to manufacturer 's suggestions ( promega ) . data were normalized as relative light units and normalized to the protein concentration as the mean s.d . cells were transfected with flag - tagged constructs . at 24 h post - transfection , cells were washed and lysed in lysis buffer ( 150 mm nacl , 0.5% nonidet p-40 , 50 mm tris - hcl , ph 7.5 , 20 mm mgcl2 ) supplemented with complete protease inhibitor tablets ( roche applied science ) for 30 min at 4 c . immunoprecipitations were performed using anti - flag m2 agarose - conjugated antibodies ( sigma ) for 2 h at 4 c . to detect interaction with endogenous co - repressors , immunocomplexes were collected by centrifugation , washed with lysis buffer , and analyzed by western blot as described above using anti - msin3a and hdac2 antibodies ( santa cruz biotechnology ) . the following primer set for the 263-bp tgfrii promoter was used for pcr : 5-gca gat gtt ctg atc tac ta-3 ( forward ) ; 5-agc tgg gca gga cct ctc tc-3 ( reverse ) using takara la taq according to the manufacturer 's protocol ( mirus ) . site - directed mutagenesis was performed with quikchange ii site - directed mutagenesis kits per the manufacturer 's protocol ( stratagene ) . all constructs were sequenced by the mayo clinic molecular biology core facility . gst fusion the klf14 cdna fragment encoding amino acids 191323 corresponding to the dna - binding zinc finger region was cloned into the gst fusion vector pgex 5x-1 ( amersham biosciences ) using standard techniques . gst fusion protein expression was induced in bl21 cells ( stratagene ) by the addition of 1 mm isopropyl - d - thiogalactopyranoside and incubation for 2 h. cells were lysed and subsequently purified by using glutathione - sepharose 4b affinity chromatography as previously described ( 15 ) . briefly , 1.75 pmol of double - stranded oligonucleotides were end - labeled with [ -p]atp using 10 units of t4 polynucleotide kinase and appropriate buffer ( 700 mm tris - hcl , ph 7.6 , 100 mm mgcl2 , 50 mm dithiothreitol ) according to the manufacturer 's instructions ( promega ) . the reaction was incubated at 37 c for 10 min and halted with addition of te plus edta ( 0.5 m edta , 1 m tris , ph 8.0 , h2o ) . protein lysates included 0.5 g of purified gst - klf14/zf fusion protein and in some experiments rhsp1 ( promega ) at the indicated dilutions . a 5 zncl2 buffer was used in this reaction ( 100 mm hepes , ph 7.5 , 250 mm kcl , 25 mm mgcl2 , 50 m zncl2 , 30% glycerol , 1 mg / ml bovine serum albumin , 250 g / ml poly(di - dc ) , h2o ) for 10 min at room temperature . the -p - labeled oligonucleotides were added for 20 min . in some cases , an excess of cold probe , at the indicated dilutions , was added concomitant with the addition of radiolabeled probe in addition to anti - sp1 polyclonal rabbit antibody purchased from commercial sources ( millipore ) . the mixtures were electrophoresed in a 4% nondenaturing polyacrylamide gel in a hoeffer midi - gel using 0.5 tris borate - edta for 4 h at 160 v. gels were then transferred to blotting paper ( whatman 3 mm ) , covered in plastic wrap , and vacuum dried for 1.5 h at 65 c . dried gels were then analyzed using a storm scanner 860 phosphorimager ( amersham biosciences ) . sp1 consensus and sp1 mutant double - stranded oligonucleotides were obtained from commercial sources ( santa cruz biotechnology ) with the following sequences : sp1 consensus , 5-att cga tcg ggg cgg ggc gag c-3 ( forward ) ; 5-gct cgc ccc gcc ccg atc gaa t-3 ( reverse ) and sp1 mutant , 5-att cga tcg gtt cgg ggc gag c-3 ( forward ) ; 5-gct cgc ccc gaa ccg atc gaa t-3 ( reverse ) . figure 1.a , repression of the tgfrii promoter occurs after 24 h of tgf stimulation in panc1 cells . panc1 cells were transfected with a full - length and a truncated 263-bp tgfrii promoter luciferase reporter and then stimulated with tgf1 after overnight serum starvation . luciferase levels were obtained at specified time points after treatment and compared with untreated controls . data are the mean s.d . from three independent experiments , with triplicates for each experiment . binding sites . the diagram represents a schematic of the human tgfrii proximal promoter containing five gc - rich sp1-like elements . a , repression of the tgfrii promoter occurs after 24 h of tgf stimulation in panc1 cells . panc1 cells were transfected with a full - length and a truncated 263-bp tgfrii promoter luciferase reporter and then stimulated with tgf1 after overnight serum starvation . luciferase levels were obtained at specified time points after treatment and compared with untreated controls . data are the mean s.d . from three independent experiments , with triplicates for each experiment . the diagram represents a schematic of the human tgfrii proximal promoter containing five gc - rich sp1-like elements . a yet undefined sp / klf repressor protein plays a role in silencing of the type ii tgf-receptor promoter previous studies have described the regulation of the tgfrii by tgf ligands , primarily showing a bimodal response consisting of an sp1-dependent up - regulation ( 14 , 17 ) and a subsequent down - regulation of receptor transcript levels upon this stimulation ( 1820 ) . interestingly , although the activation of tgfrii promoter , in particular by sp1 , has received precise attention , how this receptor is repressed remains poorly understood . consequently , the major goal of the current study has been to characterize the role of a specific family of non - smad proteins ( sp / klf transcription factors ) , which may functionally explain the down - regulation tgfrii through gc - rich sp1-like sequences . our studies began with examination of the transcriptional activity of the tgfrii gene promoter in panc1 epithelial cells , a widely used model for studying tgf signaling . we have performed an initial series of reporter assays using full - length tgfrii and the previously described , tgf-sensitive , 263-bp core promoter , which is located 5 of the transcriptional start site ( 14 ) . treatment of panc1 cells with exogenous tgf1 leads to a marked reduction in activity of the full - length tgfrii reporter when compared with untreated control cells ( fig . this silencing effect is recapitulated in the 263-bp tgfrii core promoter , suggesting that both the previously described activation pathway ( 14 ) and the negative transcriptional regulatory mechanism characterized further here ( fig . 1a ) , are operational at this core promoter level . using bioinformatics analyses ( transfac public ) , we have identified five putative sp / klf binding sites within this tgf-sensitive , 263-bp core promoter region ( fig . four of these sites ( # 14 ) have been previously identified , although an additional site ( # 5 ) has not been previously reported ( 14 , 21 ) . some of these previously identified sites have been shown to be activated by sp1 . however , whether novel sp / klf silencer proteins can also bind to this sequence to reverse the activation by sp1 remained unknown . therefore , this analysis has led us to the hypothesis that a yet undefined sp / klf repressor protein plays a role in the silencing of this promoter . novel tgf-inducible non - smad , sp / klf proteins are identified as candidate regulators of the type ii tgf-receptor to test our hypothesis , we have developed a four - tier screening approach . this approach includes first , testing which of the 24 known sp / klf proteins are expressed in tgf-sensitive epithelial cells and , thus , can be considered initial candidates to target the tgfrii promoter . our experimental cell model , panc1 , a human epithelial cell line , is an optimal model for our studies , because they have adequate expression of tgfrii mrna and display growth inhibition to exogenous tgf1 stimulation ( 22 ) . in addition , as shown in fig . 2a , each of the 24 known sp / klf transcription factors are consistently expressed in these cells , which made it ideal for performing a comprehensive screen . second , we determine whether any sp / klf genes are tgf-inducible with a kinetic that is consistent with playing a role in the down - regulation of the tgfrii . third , by utilizing transfection studies combined with reporter assays , we test the potential of distinct members of this family to repress tgfrii promoter activity and then , by electromobility shift assays , determine which sites on the promoter are utilized by our candidate klf protein . finally , we examine whether the repressor that is isolated according to these criteria binds to the endogenous tgfrii gene and can remodel chromatin on this target , suggesting the bona fide target status of the candidate klf protein . thus , by applying this comprehensive screening , our study has been robust in evaluating the klf protein family in the tgf response and regulation of the tgfrii . the genomic axiom that functionally related genes follow a similar pattern of expression suggested that , hypothetically , the protein that represses the tgfrii may be expressed in a similar manner after tgf treatment , in particular , during the repression response to this cytokine . consequently , we have evaluated which , if any , of these sp / klf members are inducible by tgf1 treatment . thus , using rna from panc1 cells either untreated or treated with tgf1 , we have performed rt - pcr at various time points ( fig . 2b ) . as a positive control for tgf-mediated transcriptional induction , we monitor the expression of p21 , a known tgf-inducible gene within the pathway . of the 24 known sp / klf transcription factors , we have identified 7 that were markedly induced with exogenous tgf1 treatment , suggesting that these could be potential candidate transcriptional repressors of the tgfrii promoter ( fig . these results are not only consistent with our previous work , which identified klf11 as a tgf-inducible gene ( 16 , 23 ) , but , more importantly , it characterizes previously unidentified klf targets for this cascade , namely klf4 , -5 , -7 , -9 , -14 , and -15 . thus , based upon their expression patterns , these seven genes are good candidates to further investigate their regulation of the tgfrii promoter . figure 2.a , eight pancreatic cancer cell lines were screened by rt - pcr for expression of 24 members of the sp / klf family of transcription factors . the figure shows that all 24 sp / klf members are expressed in the tgf-sensitive panc1 cell line . panc1 cells were serum - starved overnight , treated with 10 ng / ml tgf1 , and screened for the expression of each klf gene by semi - quantitative rt - pcr . the expression levels for each klf gene from tgf1-treated samples were compared with untreated controls and normalized to housekeeping genes as described under material and methods . the expression of the p21 gene was used as a positive control for tgf1 treatment . four different housekeeping genes , namely gapdh , b2 m , tubb2 , and hprt1 , were used as internal standards for normalization . c , quantification of tgf induction of transcript levels over time . at each time point , values were determined by first normalizing the densitometric measurement of the tgf-treated sample to the average of four housekeeping genes ( gapdh , b2 m , tubb2 , and hprt1 ) in the same sample . this resulting value was divided by the corresponding value of the untreated sample normalized in the same manner in order to express the -fold of tgf induction . a , eight pancreatic cancer cell lines were screened by rt - pcr for expression of 24 members of the sp / klf family of transcription factors . the figure shows that all 24 sp / klf members are expressed in the tgf-sensitive panc1 cell line . panc1 cells were serum - starved overnight , treated with 10 ng / ml tgf1 , and screened for the expression of each klf gene by semi - quantitative rt - pcr . the expression levels for each klf gene from tgf1-treated samples were compared with untreated controls and normalized to housekeeping genes as described under material and methods . the expression of the p21 gene was used as a positive control for tgf1 treatment . four different housekeeping genes , namely gapdh , b2 m , tubb2 , and hprt1 , were used as internal standards for normalization . c , quantification of tgf induction of transcript levels over time . at each time point , values were determined by first normalizing the densitometric measurement of the tgf-treated sample to the average of four housekeeping genes ( gapdh , b2 m , tubb2 , and hprt1 ) in the same sample . this resulting value was divided by the corresponding value of the untreated sample normalized in the same manner in order to express the -fold of tgf induction . figure 3.a , panc1 cells were transfected with klf(flag / his ) epitope - tagged expression constructs or control empty vector to test tgfrii promoter activity . b , panc1 cells were transfected with indicated klf(flag / his ) epitope - tagged expression constructs or control vector and then stimulated with 10 ng / ml tgf1 after overnight serum starvation to test tgfrii promoter activity . c , panc1 cells were transfected with various concentrations of epitope - tagged klf14(flag ) expression construct or control vector and then stimulated with 10 ng / ml tgf1 after overnight serum starvation to observe the effect on tgfrii promoter activity . western controls ( flag / his ) were shown for epitope - tagged klf expression in all experiments . expression of flag - tagged klf4 , klf7 , klf14 , and klf15 was confirmed by an anti - flag antibody ( sigma ) , whereas expression of his - tagged klf5 , klf9 , and klf11 was verified by the omni d8 antibody ( santa cruz biotechnology ) . data are the mean s.d . from three independent experiments , with triplicates for each experiment . a , panc1 cells were transfected with klf(flag / his ) epitope - tagged expression constructs or control empty vector to test tgfrii promoter activity . b , panc1 cells were transfected with indicated klf(flag / his ) epitope - tagged expression constructs or control vector and then stimulated with 10 ng / ml tgf1 after overnight serum starvation to test tgfrii promoter activity . c , panc1 cells were transfected with various concentrations of epitope - tagged klf14(flag ) expression construct or control vector and then stimulated with 10 ng / ml tgf1 after overnight serum starvation to observe the effect on tgfrii promoter activity . western controls ( flag / his ) were shown for epitope - tagged klf expression in all experiments . expression of flag - tagged klf4 , klf7 , klf14 , and klf15 was confirmed by an anti - flag antibody ( sigma ) , whereas expression of his - tagged klf5 , klf9 , and klf11 was verified by the omni d8 antibody ( santa cruz biotechnology ) . subsequently , we have tested the transcriptional activity of these candidates on the tgfrii promoter using reporter assays by co - transfecting the 263-bp core tgfrii promoter - luciferase construct with cdnas encoding each of the seven tgf-inducible klf candidates . out of these , five candidates induced a marked decrease in tgfrii promoter activity , namely klf4 , -7 , -11 , -14 , and -15 ( fig . klf5 did not affect tgfrii promoter activity above control levels , whereas klf9 appeared to slightly activate this promoter , therefore these two proteins were not continued in subsequent experiments due to our objective of identifying tgfrii repressors . to confirm whether this observed repression of tgfrii promoter regulation is consistent with tgf pathway activation , these five proteins were further tested in panc1 cells treated with exogenous tgf1 treatment . interestingly , upon treatment , these klf proteins were capable of further repressing tgfrii promoter activity , with the largest repression achieved by klf14 ( fig . these data also indicate that a second tgf-dependent mechanism , directly ( signal - induced klf expression or post - translational modifications ) or indirectly ( induction of another transcription factor ) , cooperates with klf proteins to additionally repress this gene . upon increasing concentrations of klf14 cdna in transfection studies , we found a concentration - dependent repression of the core tgfrii promoter ( fig . together , these results strongly identify klf14 as a good candidate to be a regulator of tgfrii promoter activity and expression . klf14 , a novel non - smad protein , regulates the type ii tgf-receptor the gene encoding klf14 has been previously identified by our group and named bteb5 due to its sequence similarities to members of this subfamily of klf silencing proteins.5 although , recently , genetic studies have reported the genomic structure , intronless nature , and potential imprinted status of this gene ( 24 ) , a functional characterization of this protein at the cellular and biochemical level has never been performed . because our data indicate that klf14 appears to be a potent regulator of the tgfrii in promoter assays combined with this existing gap in knowledge on this klf family member and its targets , the choice to further investigate the role of this particular klf in tgfrii regulation would significantly expand the current knowledge on the functional properties of members of this family . noteworthy , we have validated these in vitro reporter results in vivo by assessing whether this protein has a regulatory effect on endogenous tgfrii levels in panc1 cells . initial correlative experiments demonstrate that the levels of tgfrii mrna levels decrease at a time in which the amount of klf14 increases ( repression phase of the bimodal expression pattern of tgfrii in response to tgf ) , raising the possibility that klf14 is induced to subsequently down - regulate the tgfrii ( fig . mechanistically support this correlation , we have performed rt - pcr on cells overexpressing klf14 to determine tgfrii levels in comparison to mock transfected cells in the presence or absence of exogenous tgf1 stimulation ( fig . 4b ) . klf14 overexpression alone is sufficient to decrease tgfrii mrna , interestingly to the same extent as tgf1 stimulation alone . furthermore , subsequent tgf1 treatment in cells transfected with klf14 leads to further down - regulation of tgfrii transcripts . because klf14 is a tgf-inducible gene , the further decrease in tgfrii mrna levels observed upon tgf1 treatment likely results from the induction of endogenous sp / klf transcription factors by this cytokine . these results suggest that tgf down - regulates tgfrii transcripts through klf14 expression with subsequent negative regulation of promoter activity . next , we have tested whether the effect of klf14 on tgfrii expression interfered with tgf-induced signals that target downstream genes , such as p21 ( 25 ) . indeed , as expected , tgf1 treatment increases p21 promoter activity , as observed via reporter assays , as well as its mrna levels ( fig . 4 , c and d ) , whereas klf14 reduces both p21 promoter activity and mrna levels upon tgf1 treatment ( fig . these results suggest that klf14 interferes with the activation of downstream tgfsignaling effects , at least in part , by its ability to repress the tgfrii . figure 4.a , tgfrii and klf14 transcript levels were analyzed by determination of density , and relative transcript levels were represented by the ratio tgfrii cdna / gapdh cdna and klf14 cdna / gapdh cdna . panc1 cells were transfected with klf14 or control empty vector and then stimulated with 10 ng / ml tgf1 for 24 h after overnight serum starvation . densitometry was performed after normalizing to the average of gapdh , b2 m , tubb2 , and hprt1 levels and untreated control cdna . c , panc1 cells were transfected with klf14 flag epitope - tagged constructs or control empty vector along with a p21 promoter reporter construct and then stimulated with 10 ng / ml tgf1 after overnight serum starvation . panc1 cells were transfected with flag - klf14 or control empty vector and then stimulated with 10 ng / ml tgf1 for 24 h after overnight serum starvation . densitometry was obtained after normalizing to the average of gapdh , b2 m , tubb2 , and hprt1 levels and untreated control cdna . a , tgfrii and klf14 transcript levels were analyzed by determination of density , and relative transcript levels were represented by the ratio tgfrii cdna / gapdh cdna and klf14 cdna / gapdh cdna . panc1 cells were transfected with klf14 or control empty vector and then stimulated with 10 ng / ml tgf1 for 24 h after overnight serum starvation . densitometry was performed after normalizing to the average of gapdh , b2 m , tubb2 , and hprt1 levels and untreated control cdna . c , panc1 cells were transfected with klf14 flag epitope - tagged constructs or control empty vector along with a p21 promoter reporter construct and then stimulated with 10 ng / ml tgf1 after overnight serum starvation . panc1 cells were transfected with flag - klf14 or control empty vector and then stimulated with 10 ng / ml tgf1 for 24 h after overnight serum starvation . densitometry was obtained after normalizing to the average of gapdh , b2 m , tubb2 , and hprt1 levels and untreated control cdna . klf14 represses the tgfrii promoter via distinct sp1-like gc - rich sequences and competition with sp1repressor klf proteins , such as klf14 , have been previously shown to compete with the canonical sp1 protein for overall transcriptional activity of a promoter , such as the cyp1a1 and ldlr promoters ( 26 , 27 ) . therefore , herein we test the ability of klf14 to repress transcription of the tgfrii promoter in the presence of exogenous sp1 expression . indeed , as shown in fig . 5a , the expression of exogenous sp1 relieves the repression mediated by klf14 in a dose - dependent manner . due to the direct , sp1-dependent up - regulation of the tgfrii promoter as part of its bimodal response during activation , we dissect whether the repression of the tgfrii promoter via klf14 is also acting through a direct mechanism on the promoter rather than indirect due to klf14 mediating a secondary effector . panc1 cells were transfected with klf14(flag ) and sp1(his ) epitope - tagged expression constructs or control empty vector as indicated to evaluate in vivo competition on tgfrii promoter activity . western control ( flag / his ) is shown for epitope - tagged expression . b , klf14 binds to four of the putative sp1 sites and competes with sp1 on the tgfrii promoter . electromobility shift assay was performed using klf14-zf gst fusion proteins and radiolabeled oligonucleotides for each of the five putative sp1 sites . lane 1 : control - sp1 consensus oligonucleotide ; lane 2 : control - sp1 mutant oligonucleotide ; lane 3 : sp1 consensus oligonucleotide and rhsp1 protein ; lane 4 : wt oligonucleotide ; lane 5 : mut#1 oligonucleotide ; lane 6 : mut#2 oligonucleotide ; lane 7 : wt oligonucleotide and 25 cold probe ; lane 8 : wt oligonucleotide and 100 cold probe ; lane 9 : wt oligonucleotide and anti - gst ; lane 10 : wt oligonucleotide and 1 rhsp1 protein ; lane 11 : wt oligonucleotide and 5 rhsp1 protein . c , klf14 utilizes four sp / klf sites to repress the tgfrii promoter in vivo . panc1 cells were transfected with tgfrii - luciferase with mutated sp1 sites as indicated and stimulated with 10 ng / ml tgf1 after overnight serum starvation , and transcriptional activity was measured and normalized to control empty vector . data are the mean s.d . from three independent experiments , with triplicates for each experiment . panc1 cells were transfected with klf14(flag ) and sp1(his ) epitope - tagged expression constructs or control empty vector as indicated to evaluate in vivo competition on tgfrii promoter activity . western control ( flag / his ) is shown for epitope - tagged expression . b , klf14 binds to four of the putative sp1 sites and competes with sp1 on the tgfrii promoter . electromobility shift assay was performed using klf14-zf gst fusion proteins and radiolabeled oligonucleotides for each of the five putative sp1 sites . lane 1 : control - sp1 consensus oligonucleotide ; lane 2 : control - sp1 mutant oligonucleotide ; lane 3 : sp1 consensus oligonucleotide and rhsp1 protein ; lane 4 : wt oligonucleotide ; lane 5 : mut#1 oligonucleotide ; lane 6 : mut#2 oligonucleotide ; lane 7 : wt oligonucleotide and 25 cold probe ; lane 8 : wt oligonucleotide and 100 cold probe ; lane 9 : wt oligonucleotide and anti - gst ; lane 10 : wt oligonucleotide and 1 rhsp1 protein ; lane 11 : wt oligonucleotide and 5 rhsp1 protein . c , klf14 utilizes four sp / klf sites to repress the tgfrii promoter in vivo . panc1 cells were transfected with tgfrii - luciferase with mutated sp1 sites as indicated and stimulated with 10 ng / ml tgf1 after overnight serum starvation , and transcriptional activity was measured and normalized to control empty vector . data are the mean s.d . from three independent experiments , with triplicates for each experiment . thus , to first delineate the dna binding properties of klf14 to the promoter of tgfrii in vitro , we characterize the five putative sp / klf sites ( shown in fig . radiolabeled oligonucleotides for each of the 5 sp1 sites have been created as well as mutants for each site containing either 2- or 4-nucleotide substitutions ( mut#1 or mut#2 , respectively ) within the gc - rich binding sequence . sp1-consensus oligonucleotides and respective mutants have been used as internal binding controls ( fig . the interaction between a recombinant klf14 zinc finger dna - binding domain and four of the sp / klf site probes ( # 13 and 5 ) is specific as indicated by the fact that an excess of unlabeled probe competes with the radiolabeled probes ( fig . furthermore , we were able to abolish this binding by using antibodies against the recombinant klf protein ( anti - gst antibody ) , thus confirming specificity of the complex ( fig . 5b , lane 9 ) . further specificity is evidenced by the impaired ability of recombinant klf14 protein to bind to mutated sp / klf probes ( fig . visible but reduced binding is noted with the 2-bp mutant probes ( mut#1 , lane 5 ) for sites # 13 , however a significant loss of binding was observed with the 4-bp mutant probes ( mut#2 , lane 6 ) at all sites . moreover , through addition of recombinant sp1 protein to the reaction , an increase in the concentration of sp1 diminished the binding of klf14 , demonstrating that klf14 and sp1 compete for these gc - rich binding sites ( fig . 5b , lanes 10 and 11 ) , complementing the competition results we observed in vivo ( fig . 5a ) . only a weak complex was visualized with site # 4 , indicating that this site was less specific for klf14 binding than the other four sites . to functionally complement these studies , we have performed tgfrii reporter assays with both wild - type and mutant promoter - luciferase constructs . site - directed mutagenesis has been utilized to create a 2-nucleotide substitution in each of the 5 gc - rich sp / klf sites ( fig . 5c ) . these assays resulted in a marked loss of the repression observed with the wild - type tgfrii promoter upon klf14 overexpression with mutation in sp / klf sites # 1 , 2 , 3 , and 5 , particularly with a significant gain of transcriptional activity upon mutating site # 1 . these findings demonstrate that these four are operational to silence the transcriptional activity of the tgfrii promoter by klf14 . furthermore , we did not observe a significant loss of repression with mutation of site # 4 with klf14 overexpression , which supports our finding of negligible binding of klf14 to this specific sp / klf site ( fig . 5b ) . all together , klf14 appears to bind and act on four of the gc - rich sites of the tgfrii promoter to repress its transcriptional activity , and this occurs through a mechanism that includes , at least in part , competition with sp1 . repression of the tgfrii gene by klf14 occurs via mechanisms involving chromatin - modifying enzymes careful examination of klf14 sequence reveals a 26-amino acid domain that is highly similar to repression domains of klf9 ( bteb1 ) , klf13 ( bteb3 ) , and klf16 ( bteb4 ) proteins ( fig . 6a ) , which we have previously shown to repress transcription by recruiting sin3a via its paired amphipathic helix 2 domain , as well as hdac ( 15 , 28 ) . interestingly , other studies have found that hdac inhibition leads to transcriptional activation of the tgfrii promoter in various cancer cell lines , raising the possibility that a repressor of this type may be operational to achieve this effect ( 29 , 30 ) . thus , we have tested whether klf14 does indeed interact with sin3a and hdac by immunoprecipitating full - length flag - tagged klf14 from panc1 cells . the presence of sin3a and hdac complexed to klf14 is detected by western - blot analysis ( fig . thus , these results indicate that klf14 interacts with sin3a and hdac in mammalian cells , implying that these proteins are part of a repressor complex . subsequently , we have investigated whether this repressor complex occupies the endogenous tgfrii promoter in vivo via chip assays . first , we find that klf14 indeed binds to the tgfrii promoter ( fig . 6c ) , treatment with tgf1 increases the amount of klf14 bound to the tgfrii promoter . in addition , the treatment with tgf1 coincides with the appearance of sin3a on the same region of the promoter as klf14 , further implicating this repressor complex in the mechanism of klf14 repression of the tgfrii promoter ( fig . 6d ) . to gain insight into the type of chromatin remodeling that accompanies klf14 and sin3a occupation , we also have performed chip using antibodies to various histone marks . interestingly , we find that , under native conditions , acetylated histones h3 and h4 are present on the tgfrii promoter ; however , upon tgf1 treatment , these fall to negligible levels , indicating a change from a transcriptionally active , acetylated state to a relatively inactive , non - acetylated state ( fig . moreover , we observe that , although methylated k20 h4 , a mark of repression ( 31 ) , is not present under native conditions , it occupies the promoter upon tgf1 treatment along with sin3a ( fig . these types of experiments are in agreement with the histone code hypothesis ( 32 ) , revealing that the state of chromatin in the tgfrii promoter appears to switch from active to repressed upon klf14 occupation after treatment with tgf1 , all consistent with the idea that klf14 binds to the tgfrii promoter to cause repression ( fig . therefore , both competition with sp1 and direct repression via the n - terminal domain , are likely to behave as a dual mechanism of repression that would make it more difficult to reverse than if one of them was operational . this redundancy would ensure that the promoter remains silent even under circumstances that inactivate either of the single mechanisms ( fig . figure 6.a , alignment displaying the 26-amino acid sin3-interacting domain of klf14 similar to other klf repressors , klf9 , klf13 , and klf16 . panc1 cells were transfected with flag - tagged klf14 or control empty vector constructs and flag - immunoprecipitated . panc1 cells were transfected with flag - tagged klf14 or control empty vector constructs and subject to chromatin immunoprecipitation . a 263-bp fragment of the tgfrii promoter was amplified by pcr from anti - flag or mock immunoprecipitated dna samples . note that the tgfrii promoter was amplified from anti - flag ( -flag ) but not mock immunoprecipitated samples ( mock ) transfected with klf14 . d , tgf1 leads to repressive chromatin modifications upon klf14 occupation of the tgfrii promoter . panc1 cells were transfected with flag - tagged klf14 and treated with tgf1 and chromatin landscape chip assay was performed using specified antibodies . a , alignment displaying the 26-amino acid sin3-interacting domain of klf14 similar to other klf repressors , klf9 , klf13 , and klf16 . panc1 cells were transfected with flag - tagged klf14 or control empty vector constructs and flag - immunoprecipitated . panc1 cells were transfected with flag - tagged klf14 or control empty vector constructs and subject to chromatin immunoprecipitation . a 263-bp fragment of the tgfrii promoter was amplified by pcr from anti - flag or mock immunoprecipitated dna samples . note that the tgfrii promoter was amplified from anti - flag ( -flag ) but not mock immunoprecipitated samples ( mock ) transfected with klf14 . d , tgf1 leads to repressive chromatin modifications upon klf14 occupation of the tgfrii promoter . panc1 cells were transfected with flag - tagged klf14 and treated with tgf1 and chromatin landscape chip assay was performed using specified antibodies . the current study provides us with several novel mechanistic insights on the regulation of the tgf pathway . for instance , the data reported here represent the first functional characterization of the klf14 protein , outline a novel pathway for the silencing of the tgfrii promoter , provide insight into the molecular mechanisms by which these phenomena are achieved , and report additional tgf-inducible , klf proteins that are good candidates to regulate this promoter . these findings are of significant relevance to the areas of klf proteins , tgf signaling , the maintenance of cell homeostasis , and their potential contribution to disease states . tgf1 ligand itself may play a role in regulating signaling by down - regulating the cell surface receptor and thus abrogating downstream messages . others have also shown that the tgf receptors are under autoregulation control by ligand stimulation ( 18 ) . it has been previously shown that treatment with tgf1 leads to down - regulation of tgfrii levels ( 19 ) . derynck and colleagues ( 20 ) have previously described a process in osteoblastic differentiation in which at first there is a marked up - regulation of tgf1 and sensitivity followed by marked down - regulation of receptors and insensitivity to tgf1 . furthermore , recent in vitro studies on the biosynthesis of the receptors indicate that the half - life of tgfrii is 60 min and is further reduced to 45 min in the presence of exogenous tgf1 ( 33 ) . therefore , understanding how this down - regulation of the tgfrii promoter occurs is of primary biological importance for better understanding tgf signaling . activation of the tgfrii would occur via tgf1 ligand initiating an up - regulation of the tgfrii promoter by sp1 . in our model , activation of the tgf pathway also causes an induction of klf14 expression ( a ) , which would , in turn , bind to the tgfrii promoter through its gc - rich sites ( b ) , competing away sp1 ( c ) , recruiting the sin3a / hdac complex ( d ) , and remodeling chromatin from a transcriptionally active , acetylated state to an inactive state with silencing methyl marks ( e ) . thus , klf14 mediates a negative feedback loop to down - regulate the type ii tgf receptor upon tgf stimulation . model of klf14-mediated tgfrii silencing . activation of the tgfrii would occur via tgf1 ligand initiating an up - regulation of the tgfrii promoter by sp1 . in our model , activation of the tgf pathway also causes an induction of klf14 expression ( a ) , which would , in turn , bind to the tgfrii promoter through its gc - rich sites ( b ) , competing away sp1 ( c ) , recruiting the sin3a / hdac complex ( d ) , and remodeling chromatin from a transcriptionally active , acetylated state to an inactive state with silencing methyl marks ( e ) . thus , klf14 mediates a negative feedback loop to down - regulate the type ii tgf receptor upon tgf stimulation . in this regard , the current study reports several novel observations of significant biochemical relevance for extending our knowledge of tgf signaling regulation by non - smad proteins . for instance , this study represents the first functional characterization of klf14 as a novel tgf-inducible repressor protein that mediates silencing of the tgfrii , and the mechanisms by which this phenomenon occurs , in particular the role of corepressors and chromatin modifications on the tgfrii promoter . previous promoter studies have primarily focused on the role of sp1 in the activation of this promoter . however , recent studies have uncovered a paradoxical behavior for this promoter , namely that histone deacetylase inhibitors are needed to activate this promoter ( 34 , 35 ) . these data point to the existence of a repressive state of this promoter , which is less likely to be regulated by sp1 but rather by sp / klf repressors of the type previously described by our laboratory ( 11 ) . however , in the current study , rather than looking for a candidate gene within the family of klf repressors , we have performed an unbiased screening for the tgf inducibility of each of the 24 klf transcription factors and find that klf14 is up - regulated upon tgf treatment and also can efficiently repress the tgfrii . interestingly , we demonstrate that there is a strong inverse correlation between expression patterns in a temporal manner of tgfrii mrna and klf14 mrna in human pancreatic cancer cells . expression of exogenous klf14 decreases the level of transcription from the tgfrii promoter , implying a repressive role for klf14 in tgfrii expression . further analysis reveals the mechanism by which this protein works , indicating that klf14 is a member of the sin3-dependent klf repressors . this is important in light of there being two types of repressors in this family as recently reviewed by us ( 11 ) , the sin3a - dependent proteins , which include klf9 , -10 , -11 , -16 , and now -14 , and the ctbp - dependent proteins , such as klf3 and -8 . this knowledge can now be very useful for performing rapid screening of the proteins that may be acting as repressors of particular genes , besides the traditional hdac inhibitor experiments . this screening , we propose , can utilize sirna to either target ctbp or sin3a . an abolition of the silencing activity would then rapidly focus the investigations on a reduced number of candidates belonging to each of these groups . in addition to the recruitment of a klf14-msin3a - hdac2 repressor complex to the tgfrii promoter , we observed a concurrent remodeling of chromatin , which involves loss of transcriptionally active acetylated histone marks and an increase in histone marks that associate with transcriptional silencing . finally , we defined binding sites involved in klf14-mediated repression of the tgfrii promoter , namely sites -143/138 ( # 1 ) , -101/-95 ( # 2 ) , -59/52 ( # 3 ) and -11/-5 ( # 5 ) , which are consistent with previously reported sp1 sites , and these same sites , when mutated , lose the repressive effect of klf14 . overall , however , the major relevance of this study is the fact that it contributes to organize our thoughts on how tgf signaling is regulated . it has recently been appropriately proposed to classify these mechanisms by whether they are directly mediated by smads ( the best studied to date ) or those less understood events regulated by non - smad proteins ( 4 ) . in fact , our laboratory has previously described a role for klf repressors , namely klf10 ( tieg1 ) and klf11 ( tieg2 ) , as non - smad effectors of the tgf response in cell growth ( 9 , 36 ) . now , due to the findings reported in the current study , we can propose a model wherein tgf cytokines activate and repress the tgfrii promoter using the very same gc - rich sites utilized by this family of proteins . activation would occur in an initial phase via sp1 , whereas inhibition would follow activation and require the action of klf14 , competition of sp1 , recruitment of sin3a , hdac , and distinct chromatin modifications on the promoter ( see model in fig . therefore , klf14 becomes another important non - smad protein , in which at least one of its functions is to silence the tgfrii . this novel transcriptional pathway thus becomes an important step for modulating the activity of tgf signaling .

the role of non - smad proteins in the regulation of transforming growth factor- ( tgf ) signaling is an emerging line of active investigation . here , we characterize the role of klf14 , as a tgf-inducible , non - smad protein that silences the tgf receptor ii ( tgfrii ) promoter . together with endocytosis , transcriptional silencing is a critical mechanism for down - regulating tgf receptors at the cell surface . however , the mechanisms underlying transcriptional repression of these receptors remain poorly understood . klf14 has been chosen from a comprehensive screen of 24 members of the sp / klf family due to its tgf inducibility , its ability to regulate the tgfrii promoter , and the fact that this protein had yet to be functionally characterized . we find that klf14 represses the tgfrii , a function that is augmented by tgf treatment . mapping of the tgfrii promoter , in combination with site - directed mutagenesis , electromobility shift , and chromatin immunoprecipitation assays , have identified distinct gc - rich sequences used by klf14 to regulate this promoter . mechanistically , klf14 represses the tgfrii promoter via a co - repressor complex containing msin3a and hdac2 . furthermore , the tgf pathway activation leads to recruitment of a klf14-msin3a - hdac2 repressor complex to the tgfrii promoter , as well as the remodeling of chromatin to increase histone marks that associate with transcriptional silencing . thus , these results describe a novel negative - feedback mechanism by which tgfrii activation at the cell surface induces the expression of klf14 to ultimately silence the tgfrii and further expand the network of non - smad transcription factors that participate in the tgf pathway .

unawareness , lack of insight , or anosognosia refers to impaired awareness in persons with dementia [ 17 ] . awareness is multifactorial and likely modular [ 4 , 810 ] , with each domain separable and potentially unique . most of the literature on awareness in persons with dementia describes the clinical correlates of one awareness domain ( reviews by [ 1 , 4 , 11 ] ) , but the few studies that have contrasted awareness for different domains have found differential patterns of clinical correlates [ 1215 ] . this paper provides further support for the modality specific nature of awareness in dementia by contrasting the clinical correlates for awareness of balance in addition to more commonly measured awareness of day - to - day function and memory . awareness quantification remains elusive , and there is no consensus method for measuring awareness ( e.g. , [ 4 , 9 ] ) . awareness has been measured with clinician ratings [ 16 , 17 ] ; or based on discrepancy between self - report versus clinicians ' impression or versus informant report assessed with interview [ 10 , 18 ] or questionnaires [ 7 , 12 , 14 , 1921 ] ; or discrepancy between self - report and objective performance [ 21 , 22 ] , which , depending on the task , measures self - monitoring or metacognitive abilities . detail difficulties with patient / informant discrepancies and the assumption that caregiver informants ' or clinicians ' reports are a better reflection of reality . moreover , worry , anxiety , defensiveness , denial , or focus on more important problems can influence reflection for persons with dementia . caregivers ' reports may be more highly correlated with objective measures of cognition than patient self - reports , suggesting there is value in using patient / informant discrepancies . nevertheless , caregiver / patient discrepancies may be a better measurement of patients ' awareness of function , whereas clinician / patient discrepancies may be a better measure of patients ' awareness of cognition . due , in part , to measurement challenges when assessing awareness , the literature on the clinical correlates of awareness in persons with dementia is contradictory . most , but not all , of the cumulative data suggest increasing dementia severity is associated with reduced awareness [ 1 , 16 , 18 ] . others have found few group - based differences , but high individual variability in awareness declines when studied over one year , potentially because severity and awareness are mediated by cognitive reserve . other important clinical correlates of awareness include depression [ 18 , 21 , 22 , 25 ] , neuropsychiatric status [ 1921 , 26 ] caregiver burden [ 10 , 11 , 14 , 26 ] , activities of daily living , and neuropsychological status [ 12 , 19 , 20 , 22 , 2729 ] which demonstrate variability in associations with awareness across domains and measurement methods . models of awareness suggest awareness is mediated by the frontal lobes [ 2 , 3 ] or the right frontal lobe [ 30 , 31 ] , and lack of awareness is associated with other behavioural indicators of frontal dysfunction , such as increased apathy . localized imaging has implicated the orbitofrontal cortex , but most have implicated medial structures [ 34 , 35 ] , including the anterior and posterior cingulate in awareness . despite converging evidence for prefrontal involvement in awareness , awareness may or may not be associated with tests of executive function ( see review by ) . some studies demonstrate strong relationships between awareness and executive functioning [ 12 , 19 , 20 , 27 ] , with others reporting nonsignificant and trivial associations [ 19 , 29 ] . although clearly composite measures that rely on more basic cognitive functions , many summary scores from traditional tests of executive function are associated with the dorsolateral prefrontal circuit , and awareness has been associated with more midline [ 3436 ] or orbitofrontal aspects of the prefrontal cortex . contradictory data on the clinical correlates of awareness is also due to the assumption that awareness is a unitary construct ( see reviews by [ 4 , 8 ] ) . differential patterns of clinical correlates have been demonstrated for awareness of cognitive deficits versus awareness of behaviours [ 1215 ] , suggesting that awareness is modality specific . the primary purpose of this paper is to describe the clinical correlates of awareness of balance in addition to the more commonly measured awareness of function in basic and instrumental activities of daily living ( badls and iadls , resp . ) and awareness of memory . awareness of balance is important in persons with dementia due to its relation with fall risk . we hypothesize that awareness of balance will be associated with physical variables , such as gait , falls , and objectively measured balance . based on the conceptualization of markova and colleagues and work suggesting differential clinical correlates for awareness of specific domains [ 13 , 15 ] , we hypothesize that awareness of functional abilities , memory , and balance will have differential patterns of clinical correlates . a secondary purpose of this paper is to contrast awareness for balance , function , and memory for those diagnosed with mild cognitive impairment ( mci ) . reduced awareness has been demonstrated for persons at high risk for dementia , and specifically for persons with amnestic mci ( amci ) . moreover , since awareness appears to differ based on dementia subtype , awareness for balance , function , and memory could be differentially affected for participants diagnosed with dementia due to ad versus non - ad dementias . therefore , the final comparison will explore awareness in groups of persons diagnosed with amci , non - amnestic mci , ad , and non - ad dementias . patients were from an interdisciplinary memory clinic established to provide early stage dementia differential diagnoses for rural persons . for this irb approved study , patients who were diagnosed with no cognitive impairment were excluded , and only patients who received a diagnosis of dementia or a variant of mci were included . diagnoses in this specialty clinic were consistent with the review of diagnostic guidelines provided by the canadian consensus on the diagnosis and treatment of dementias using recent comprehensive blood work , ct head scan , and interprofessional assessment data from neurology , neuropsychology , and physical therapy . the assessment procedures included standardized approaches ( e.g. , questionnaires , neuropsychological testing ) and also family interviews for clinical history and interviews with the informal caregiver who accompanied patients to the clinic ( families were strongly encouraged to attend the assessment and patients were asked to bring someone who knew them well . in the unusual circumstance when patients attended the clinic alone , telephone interviews were conducted with someone who knew them well , but questionnaire data were not collected ) . the sample consisted of 259 patients ( in the clinic 's 6th data release ) , and the vast majority ( 74% ) reported their ancestry as european , 9% were first nations or metis , and 17% chose other , rather than selecting one of the aforementioned categories , african or asian ancestry . table 1 includes descriptive information for the total clinical sample ( n = 259 patients ) and details the subgroups based on diagnosis . patients with dementia due to ad was the most common diagnosis ( n = 113 ) , and another heterogeneous subgroup ( n = 100 ) was created of patients with non - ad dementias ( i.e. , vascular dementias , mixed dementias , diffuse lewy body disease , dementia due to parkinsons ' disease , huntington 's dementia , variants of frontotemporal lobar degeneration , and dementias not otherwise specified ) . in addition , a third group included patients diagnosed with amnestic ( single or multiple domain ) mild cognitive impairment ( amci ; n = 23 ) and a fourth group included patients diagnosed with nonamnestic mci ( non - amci ; n = 23 ; single or multiple domains , which included those with diagnoses of vascular cognitive impairment , no dementia ) . although most of the clinic data were focused on patients for diagnostic purposes , informal caregivers ( n = 244 ) provided important collateral and personal information . caregivers ( m age = 61.40 , sd = 14.63 ) were typically family members and most of many were females ( 64% ) : 33% were wives , 20% were husbands , 31% were daughters , 10% were sons of the patient , with a remaining 7% whose relationship status included grandchildren , nieces , nephews , or friends . clinical correlates of awareness measures ( 1 ) assessment of severity . the clinical dementia rating ( cdr ) is a standardized and psychometrically sound clinician - based rating scale ( 0 to 3 ; no impairment to severe impairment ) , but summing the box scores of the six rating areas of the cdr ( cdr - sob ) provides a more detailed quantitative measure of global dementia severity and is more sensitive to detecting changes in dementia severity over time . patients rated their performance on the reliable and valid lawton instrumental activities of daily living ( iadl higher scores indicating independent functioning ) . patients ' caregivers rated patients ' performance of adls on two psychometrically strong scales : the functional assessment questionnaire ( faq ) and the bristol adl questionnaire where higher scores indicate impaired performance . the neuropsychiatric inventory ( npi ) is a well - researched and psychometrically strong caregiver rating of patients ' behaviours and associated caregiver distress . the centre for epidemiologic studies of depression ( cesd ) , a reliable and valid screen of depression , was self - rated by patients , with four factors : ( 1 ) depressed affect ( 2 ) lack of positive affect ( 3 ) somatic / vegetative and ( 4 ) interpersonal measuring social disconnectedness . self - report of caregiver burden was assessed with the short form of the zarit burden interview , which was shown to be psychometrically similar to the longer versions . the global severity index from the brief symptom inventory ( bsi ) measured caregiver self - report of overall psychological distress . ( 5 ) assessment of physical variables . a comprehensivephysical therapy assessment included the psychometrically strong berg balance scale ( bbs ) and the performance oriented mobility assessment ( poma , which is a measure of gait and balance ) . caregiver and patient reports of falls within the past 6 months were combined with bbs to estimate the probability of falling . patient self - report on the activities - specific balance confidence ( abc ) scale measured self - reported confidence in balance while doing a variety of day - to - day activities . each patient received a comprehensive neuropsychological assessment ( see for a review of the strong psychometric properties of these tests ) , and selected measures of executive function and working memory were analyzed . the ability to alternate attention was measured with the trail making test part b ( tmt b ) . the ability to inhibit an automatic response was measured with the stroop interference score . cognitive flexibility with speeded retrieval of language - based knowledge was measured using animal naming and phonemic fluency ( benton oral word association test ) . digit span backward subtest from the wechsler adult intelligence scale 3rd edition ( wais - iii ) measured working memory . finally , the index scores from the repeatable battery for the assessment of neuropsychological status ( rbans ) were analyzed . awareness of functional deficits ( af ) was operationalized using patient / caregiver congruence on reports of the patient 's ability to independently perform six iadls : management of finances , use of telephone , use of transportation , shopping , meal preparation , and performance of housework ( patient self - report version of the lawton iadl scale ; and caregiver report of patient 's function from the bristol adl scale ) . for each iadl , congruence was ranked on a 5-point scale ; the congruence ranking was summed across the six iadl items for a total of 30 possible points , with higher scores indicating greater awareness . data were available for 201 participants , and as can be seen in table 1 , most patients had good awareness of their functional abilities . af was significantly higher in the two mci diagnostic groups when compared with the two groups with dementia diagnoses . awareness of memory ( am ) was based on congruence between patient 's self - reports of memory on a standardized scale ( self - rating of memory scale ) and performance on a neuropsychological test of new learning ( repeatable battery for assessment of neuropsychological status ; rbans delayed memory index score ) . the delayed memory measure was chosen since it best captured consolidation difficulties asked about in the self - rating of memory scale . both the self rating of memory standardized scores and the rbans index scores were transformed into a linear ranked scale from 1 to 5 , with 1 indicating the lowest self - rating of memory and the lowest memory performance . the am score was created based on the congruence in ranking between self - reported and objectively measured memory , with 5 indicating perfect congruence . complete data were available for 192 participants . as can be seen in table 1 , most participants ' am was at the mid - point of the scale , but the ad and the amci subgroups reported significantly lower am than the non - ad dementia and non - amci groups . awareness of balance ( ab ) was based on congruence between patients ' ratings of balance confidence on the abc scale and the probability of falling . the abc and probability of falling were each transformed into ranked scores with 1 indicating low confidence or high probability of falling and 4 representing high confidence or low probability of falling . the ab score was created based on the congruence in ranking between self - reported balance confidence and objectively measured probability of falling , with 1 indicating low congruence and 4 indicating perfect congruence . perfect congruence between balance confidence and objective measurement may not be sufficient for stability ; rather underestimation of balance ( i.e. , less confidence than objective measurement would support ) has been shown to be associated with greater stability ( e.g. , [ 38 , 65 ] ) . consistent with this premise , ab ranking of 5 represented an underestimation of balance confidence relative to objectively measured balance . approximately one third of the sample ( 36% ) reported equivalent balance confidence to measured stability , 27% reported greater balance confidence than would be supported by objective measurement ( i.e. , reduced awareness ) , and 37% reported an underestimation of balance , which may be appropriate awareness for maximal stability ( e.g. , [ 38 , 65 ] ) . although the sample size was relatively small , ab was high for all groups and did not differ significantly for patients with diagnoses of amci , non - amci , ad , or non - ad dementias ( see table 1 ) . zero - order correlations were completed separately for af ( table 2 ) , am ( table 3 ) , and ab ( table 4 ) . only variables with significant correlations were used to minimize specification errors ( potential over- or undercorrection ) as predictors in simultaneous multiple regression equations . for each measure of awareness , analyses were conducted for the overall sample , but also for each of the four diagnostic groups . descriptors of magnitude of association were consistent with guidelines for small , medium , and large effect sizes provided by cohen . as can be seen in table 2 , af was highly correlated with caregiver report of adls ( faq and bristol adl each large magnitude associations ) . together these data suggest af was highly associated , but not redundant , with more comprehensive measures of day - to - day function . similarly , am was highly associated , but not redundant with the measures used to create it . as can be seen in table 3 , these associations were strong for the non - ad dementia and two mci groups but trivial for the ad group . finally , ab also demonstrated moderate , but nonredundant , associations with the variables used to create it . as can be seen in table 4 , the association with the abc scale was a moderate magnitude overall and for all diagnostic groups except the non - amci group . also seen in table 4 , ab was associated with both fall history in the past 6 months for the larger samples and overall . ab was associated with probability of falling only for specific diagnostic groups and the overall sample . the cell sizes for the poma were below 10 for the mci groups , but the small association was significant for the overall sample . overall , the correlations provide evidence for the convergent validity for each of the derived awareness measures . of interest , each measure of awareness appeared orthogonal : awareness of function was not associated with either awareness of memory ( rs = 0.026 , p > 0.05 , trivial magnitude ) or balance ( rs = 0.207 , p > 0.05 , small magnitude ) , and the latter two measures of awareness are similarly not well associated ( rs = 0.153 , p > 0.05 , small magnitude ) . for the overall sample , regression diagnostics suggested the bristol adl - caregiver , npi - distress , and rbans immediate and total scale indices were multicollinear , so these variables were excluded from the regression equation . the remaining predictors of cdr - sob , iadl - patient , faq - caregiver , npi - severity , zbi , probability of falling , bbs , clock drawing , rbans language , and visuospatial / constructional indices accounted for a large proportion of af variance ( r = 0.688 , p < 0.001 ) . not all predictors were equally predictive , however , and only the faq - caregiver ( t = 4.64 , p < 0.001 ) and zbi ( t = 3.06 , p = 0.003 ) were significant predictors of af . equivalent regression procedures were conducted separately for the four diagnostic subgroups , and across these analyses only measures of function were significant predictors of af ( ad group faq - caregiver t = 3.29 , p = 0.002 ; non - ad group faq - caregiver t = 3.03 , p = 0.004 ; amci group iadl - patient t = 2.30 , p = 0.035 ; and non - amci group badl - caregiver t = 2.37 , p = 0.029 ) . awareness of memory demonstrated a different pattern of zero - order correlations ( see table 3 ) , and the results of the regression analyses also suggested that the clinical correlates of am clearly differed from those of af . for the overall sample , the initial regression diagnostics resulted in removal of npi - severity and distress in addition to the rbans total scale index score due to multicollinearity . the overall model accounted for a large proportion of variance in am ( r = 0.736 , p < 0.001 ) , but of the predictors ( iadl - patient , cesd overall , cesd depressed affect , cesd somatic / vegetative , cesd interpersonal , bsi global severity , animal naming , and rbans immediate memory were excluded ) only the cesd - lack of positive affect ( t = 3.19 , p = 0.002 ) , the rbans visuospatial / constructional index ( t = 2.63 , p = 0.01 ) , and the rbans delayed memory index ( t = 8.65 , p < 0.001 ) remained significant . the regression equations for the diagnostic groups differed from the predictors for the overall group . for the non - amci group the overall model was nonsignificant ( likely due to only 9 participants having all variables complete ) . for the non - ad dementia group the rbans delayed memory index was the only significant predictor of am ( t = 2.32 , p = 0.028 ) , but for the few amci patients with all predictors complete the delayed memory was not significant ( t = 2.22 , p = 0.053 ) . perhaps most salient was the radically different associations between am and the clinical correlates for the ad group . here , only the iadl - patient ( t = 2.15 , p = 0.035 ) and cesd lack of positive affect ( t = 2.32 , p = 0.024 ) predicted am . the zero - order correlations that drove the regression equations for ab are shown in table 4 . the regression equations for the overall sample were less plagued by small sample size problems than the two mci groups . the clinical correlates of probability of falling , poma , and the bbs were excluded , however , due to multicollinearity . the remaining predictors were all significant : faq - caregiver ( t = 4.77 , p < 0.001 ) , the abc scale ( t = 6.61 , p < 0.01 ) , and fall history in last 6 months ( coded yes / no , t = 8.01 , p < 0.001 ) and together accounted for a large proportion of variance in ab ( r = 0.757 , p < 0.001 ) . when compared across the diagnostic groups , the two regression equations for the mci groups were not statistically significant , likely due to small sample sizes . for both dementia groups the abc scale was a significant predictor of ab ( ad group t = 4.61 , p = 0.038 , non - ad t = 2.34 , p = 0.014 ) but , in addition , for the non - ad group the fall history in last 6 months was significant ( t = 3.81 , p = 0.001 ) . these data support the hypothesis that awareness for different domains , specifically awareness of function , memory , and balance , would differentially relate to clinical correlates . this is in keeping with early work on awareness demonstrating differential clinical correlates for specific awareness domains [ 1215 ] and provides support for the assertion by markova and colleagues that awareness must be conceptualized as specific to the domain being measured , and that research on one domain can not be generalized to another domain . in addition to finding differential patterns of clinical correlates across domains of awareness , these data suggest that diagnostic group is also an important consideration in the clinical correlates of awareness . this was most evident in the clinical correlates for the ad group versus the non - ad and amci groups for awareness of memory . here , the relationship between specific symptoms of depression and awareness of memory was only evident for the ad group . in contrast , the clinical correlates for the other groups remain restricted to measures of memory . regarding caution in cross - domain generalization is not sufficient , and diagnostic grouping is another important consideration , at least for some domains of awareness . awareness of function was lower for the groups diagnosed with dementia than those with mci whereas awareness of memory was lower for the group with ad dementia and the group with amci , often considered a precursor to ad , than for the non - ad dementia or non - amci groups . our data suggest that awareness of specific domains was orthogonal : awareness of function was not associated with awareness of memory or awareness of balance . this was in contrast to the findings by ott and colleagues who found moderate correlations between awareness of memory and awareness of function . our method for measuring awareness of function was similar to that used by ott and colleagues ( namely , patient / caregiver discrepancy ) , but we used a discrepancy between performance and self - report to assess memory and balance awareness , which may account for these inconsistent findings . evidence for the modality specific nature of awareness is provided by the differential patterns of clinical correlates depending on domain measured . we found that the relation between caregiver burden and distress depended on the modality of awareness measured : reduced awareness of function was associated with increased caregiver reports of burden , which is consistent with findings from previous research [ 10 , 11 , 14 ] . balance awareness was the only awareness measure associated with physical variables such as past history of falls and self - reported balance confidence . the relation between balance awareness and falls is consistent with previous research demonstrating a strong relationship between proprioception and balance or falls . balance awareness was not related to any measure of neuropsychological functioning , despite previously reported relationships between risk of falls and measures of executive function . although none of the domains of awareness were associated with measures of executive function , awareness of memory was associated with neuropsychological variables of the delayed memory index and the visuospatial / constructional indices from the rbans . the associations with neuropsychological variables differed , however , when the diagnostic groups were considered separately . interestingly , awareness of memory was not associated with the delayed memory scores for the ad group . a floor effect in the delayed memory score appears to have created problems with heteroscedasticity in the bivariate memory awareness relationship , which may have attenuated any associations . the possible floor effect in memory measures did not , however , impact the association between depressive symptoms and awareness of memory , only for the ad group . the findings of differential clinical correlates for awareness of memory based on diagnostic group may speak to some of the most contradictory findings regarding correlates of awareness . neuropsychological function is inconsistently related to awareness [ 1 , 12 , 19 , 20 , 27 , 29 ] , and the relationship between awareness in dementia and depression is complicated , with clinical lore and empirical data supporting the notion that increased awareness is associated with more symptoms of depressed mood [ 18 , 25 ] , but increased awareness and depression may only be related to subclinical ( or dysthymia ) rather than major depression . our data suggest that in addition to modality of awareness being considered when measuring associations of neuropsychological and depressive symptoms with awareness , diagnostic group is an additional important consideration . patients with dementia due to ad appear to have differential clinical correlates for awareness of memory versus patients with non - ad dementia , for example . although the prediction of differential clinical correlates for the different domains of awareness was supported , some of our predictions regarding these clinical correlates were contrary to previous research . in zero - order associations , severity was associated with awareness of function , but with no other domain of awareness . moreover , severity did not account for sufficient unique variance in awareness of function and was , therefore , not a significant predictor . this finding is in contrast to the cumulative cross - sectional data demonstrating an association between severity of cognitive impairment and awareness ( see for a thorough review ) and is contrary to the more compelling longitudinal data demonstrating decreasing awareness with increasing cognitive impairment [ 16 , 18 ] . these contrasting findings likely speak to the orthogonal and domain - specific nature of the construct of awareness . aalten and colleagues ' and mcdaniel and colleagues ' prospective studies measured awareness by clinician ratings based on an interview with patient and caregiver regarding the patient 's history of cognitive deficits and their impact on function . finally , all measures of awareness were associated with some measure of independence in daily function , particularly instrumental activities of daily living which is evidence against modality specificity . assessment of functional abilities as a clinical correlate for measures of awareness is not often reported in the literature , but the few studies that do exist suggest that decreased awareness is associated with increased functional limitations , which is consistent with our data . despite adding to the converging research on the modality - specific nature of awareness [ 4 , 810 ] , these data are limited by the inconsistency in the measurement methods used for each modality of awareness . measurement of awareness of function was based on caregiver / patient discrepancy , whereas awareness of memory and of balance was based on discrepancy between self - report and objective measures of performance . if awareness is a true construct , it should not be highly dependent on how it is measured . if , for example , awareness of function differs greatly when measured with caregiver / patient discrepancy versus patient / observation discrepancy , then this would not be a construct at all and would be considered an artifact of measurement . if awareness is an artifact of measurement , future research measuring awareness of different modalities of awareness with different methods for measuring awareness will find markedly different clinical correlates than those presented here . another more problematic limitation of these data is the fact that this sample is derived from a specialty clinic . specialty clinic patients have been postulated to have higher awareness than the general dementia population due to the referral process for specialty clinics . it is unclear how having higher awareness may have impacted the differential patterns of clinical correlates for these multiple domains of awareness . although replication is required , these data demonstrate differential patterns of clinical correlates for awareness of function and memory . in addition , this study provides a novel contribution by describing the clinical correlates for awareness of balance . these data provide evidence for modality specific relations between awareness and clinical correlates in a database with a wide range of standardized measures of clinical correlates including severity , function , neuropsychiatric symptoms , depression , caregiver burden and distress , and a comprehensive assessment of neuropsychological status . differing patterns of clinical correlates for awareness of function , awareness of memory , and awareness of balance provide support for the modality specific uniqueness of awareness measures for each of these domains . data demonstrating different patterns of awareness based on diagnostic group ( namely , amci , non - amci , ad , and non - ad dementia diagnoses ) provides further evidence for the modality specific nature of awareness . modality specificity , if replicated in different populations , for example , stroke patients , could have implications for rehabilitation . these data would suggest that rehabilitation needs to be targeted to a domain of awareness since awareness is not a unitary construct . future research is needed on the clinical course and implications for day - to - day care associated with impairments in specific modalities of awareness for persons with dementia .

awareness in dementia is increasingly recognized not only as multifactorial , but also as domain specific . we demonstrate differential clinical correlates for awareness of daily function , awareness of memory , and the novel exploration of awareness of balance . awareness of function was higher for participants with mild cognitive impairment ( amci and non - amci ) than for those with dementia ( due to alzheimer disease ; ad and non - ad ) , whereas awareness of memory was higher for both non - amci and non - ad dementia patients than for those with amci or ad . balance awareness did not differ based on diagnostic subgroup . awareness of function was associated with instrumental activities of daily living and caregiver burden . in contrast , awareness of balance was associated with fall history , balance confidence , and instrumental activities of daily living . clinical correlates of awareness of memory depended on diagnostic group : associations held with neuropsychological variables for non - ad dementia , but for patients with ad dementia , depression and instrumental activities of daily living were clinical correlates of memory awareness . together , these data provide support for the hypothesis that awareness and dementia are not unitary and are , instead , modality specific .

although some recent studies have shown declines in the prevalence of hearing impairment in recent generations,1 2 according to world health organization global estimates , it still represents the most common sensory disorder in the world , with 328 million adults having disabling hearing loss ( hearing thresholds above 40 db ) , which can impair their quality of life and potentially result in feelings of isolation , loneliness , and frustration due to communication difficulties , economic participation , and lack of access to services.3 4 however , even in developed countries , it is estimated that less than 1 in 40 people who need hearing aids have access to them.3 in 2004 , with the establishment of the national hearing health care policy , the brazilian government , through the unified health system ( sus ) , via the organization of state hearing health networks , made it possible for the population to have access to the service levels of basic , medium , and high - complexity audiological care.5 in parallel , the processes of globalization and urbanization have resulted in changes in lifestyle . inactivity and unhealthy diet have resulted in an epidemic of excess weight,6 associated with stress , smoking , and alcohol abuse . in turn , these conditions have led to an increased prevalence of chronic diseases , the most common being cardiovascular , which includes hypertension and hypercholesterolemia ; osteoarticular ; psychiatric disorders ; chronic respiratory diseases ; diabetes ; kidney disease ; and cancer.6 7 8 9 all are significantly more prevalent in women.10 certain chronic degenerative diseases may contribute to the onset and worsening of hearing loss via different pathophysiological mechanisms.7 8 according to the national sampling policy of households ( pnad ) , conducted by the brazilian institute of geography and statistics in 2008 , hypertension is the most prevalent chronic disease in both sexes.10 11 12 it evolves from increased blood viscosity and microcirculatory failure that can compromise the auditory and vestibular systems at the peripheral and central levels . this is aggravated by hypercholesterolemia , which affects one - third of individuals over 45 years of age.6 7 13 diabetes , although the seventh most prevalent chronic disease in the same pnad statistics , showed the strongest growth , up 37% , compared with the prevalence described in the 2003 study.6 9 although there is still no consensus on the pathophysiology of hearing damage caused by diabetes , it is suggested that changes in the metabolism of lipids and glucides could be related to the development and/or aggravation of not only hearing loss but also tinnitus and balance disorders because of reduced blood flow and disability in the transport of nutrients , due to diabetic microangiopathy and secondary degenerative neuropathy affecting peripheral and central auditory pathways.7 14 15 such situations , besides contributing to the concomitant hypoacusis with other otologic complaints,16 17 18 increase the progression of hearing loss and are of the utmost importance when selecting and fitting hearing aids . several studies have shown the high concurrency of otologic symptoms in hearing aid users , especially tinnitus , dizziness , itching , and ear pain . although results vary , tinnitus symptoms have been reported by at least 70% of the subjects in most polls , followed by itching and/or dizziness in 50% of individuals.19 20 21 tenrio et al found that 100% of a sample for hearing aid wearers had at least one associated symptom.12 if on the one hand adaptation of the hearing aids has improved some of these symptoms,20 21 22 23 then on the other hand , hearing aid user may be related to a worsening clinical picture in cases of allergic contact dermatitis24 25 and even worse tinnitus if the device has no ventilation or is an open mold.22 such symptoms can compromise the benefits of hearing aid use . recently , andersson et al found that hearing aid users with tinnitus had worse responses than the group without tinnitus in unfavorable listening situations ( low signal - to - noise ratio ) , generating more complaints than improvements with the device.26 the impact of hearing loss in the world , the prevalence of systemic diseases and otologic symptoms in the adult population , in addition to the increase in number of sus users getting hearing aids show the importance of developing protocols for integrated assessment of candidates for fitting . greater attention should be paid to device selection and adaptation to better characterize and manage hearing loss and user expectations and otolaryngologic symptoms associated with systemic comorbidities to optimize the hearing aid fitting process . this study aims to compare the occurrence of otologic complaints , systemic diseases , and effective use of hearing aids in men and women with deafness . this is an exploratory , descriptive , cross - sectional study , approved by the research ethics committee under the number cep/027/2008 . all participants signed a consent form authorizing the use of the collected data . between march and september 2013 , during the development of this research , 278 patients using hearing aids were followed by the otolaryngology service at a hearing health service center accredited by sus in curitiba , brazil . for this study , individuals were selected who fit the following criteria : over 50 years old with a diagnosis of hearing loss , hearing aid use for at least the past 6 months , having complaints about the adaptation and/or functioning of the device . following this criteria the sample all subjects were evaluated by an ear , nose , and throat ( ent ) physician and audiologist . gender , age , otologic complaints , presence of associated systemic diseases , and otoscopy findings were variables taken into account for the ent assessment . in the clinical assessment , the type , degree , and configuration of hearing loss observed in pure tone audiometry were considered , as well as the type of hearing aid worn . for the audiometry , an itera audiometer ( madsen , denmark ) , calibrated in a soundproof booth according to the standards required by the federal council of speech - language pathology , the subjects were divided into two groups for purposes of comparison and analysis of results : group a , made up of women , and group b , by men . the results were statistically analyzed using the chi - square test ( to compare possible differences between observed and expected frequencies for an event ) and the difference in proportions test ( to compare differences between two populations ) , with 0.05 as the significance level . in the period that data were collected , 278 people presented for a follow - up visit at an accredited clinic . of these , 61 ( 21% ) subjects reported otologic or operational problems with their devices . the sample was composed of 61 individuals , 35 ( 57% ) women and 26 ( 43% ) men . the age in group a ( women ) ranged from 53 to 85 , with a mean of 72 , and in group b ( male ) , from 53 to 82 , with a mean of 69 . a family history of hearing loss was reported in 41% of subjects , 13 women and 12 men . there was a predominance of widows in group a ( 51% ) , followed by married ( 37% ) and single ( 11% ) , and group b was predominantly married ( 81% ) , followed by divorced ( 11% ) and widowers ( 8% ) . both genders were mostly made up of retirees or pensioners ( 71% of the women and 85% of the men ) . when talking about hearing aids , three individuals in group a used unilateral prosthesis , four used intracanal hearing aids , and 53 ( 86% ) used behind - the - ear ( bte ) prosthesis with silicone molds . in the group b , two individuals used prosthesis in one ear , six used intracanal hearing aids , and 48 ( 78% ) used bte prosthesis with silicone molds . the chi - square test predominantly showed mild to moderate hearing loss with descending slopes . about 80% of subjects had comorbidities , and the data are shown in table 2 . note : using chi - square test , there is significant difference in the degree of loss ( p = 0.0103 ) and configuration ( p = 0.0422 ) . abbreviation : n / a , not applicable . note : the number of citations is greater than the number of respondents because some individuals had more than one associated morbidity . using the difference of proportions test , there is significant difference between the number of morbidities for hypercholesterolemia and hypertension . the majority of the sample ( 92% ) had bilateral fitting of hearing aids , with the same percentage represented in both groups . the hearing aid use profile is shown in table 3 . considering only the totals of continuous and discontinuous use , the chi - square test at a significance level of 0.05 showed no significant dependence between usage type between the two genders . fourteen subjects ( 22% ) reported little use of hearing aids , either because of otologic problems associated with deafness or malfunction of the unit itself , and the results are shown in table 4 . of the 61 participants , 54% had otologic complaints related to deafness , and the data are presented in table 5 ; the correlation between morbidity and discontinuity of use for the hearing aid is shown in table 6 . note : the number of complaints is greater than the number of respondents because some individuals had more than one complaint associated with hearing loss . using the difference of proportions test , there was significant difference in the two genders for itching ( p = 0.0085 ) and tinnitus ( p = 0.0015 ) . note : the number of citations is greater than the number of respondents because some individuals had more than one associated morbidity . all 61 participants had hearing loss , and of these , 80.3% reported having systemic diseases , among which the most commonly reported were hypertension , hypothyroidism , hypercholesterolemia , and diabetes . the study population had an average age of 69 years for men and 72 years for women ; all were considered seniors . although the appearance of systemic diseases and hearing loss is natural , it can often be disabling and contribute negatively to quality of life , generating increasing demands for specialized health care services . to improve auditory perception , the elderly population has sought out hearing health services in sus for evaluation of hearing and fitting of hearing aids.27 28 in the present study , conducted within a hearing health clinic , most of the sample was elderly . a high incidence of morbidity was assessed in the sample , predominantly hypercholesterolemia in women and hypertension in men . investments are necessary so that medical care for hearing health services is not restricted to ent care . according to a published study,8 the body of an elderly person has peculiarities that must be evaluated when dealing with issues related to ent disorders . the physician should consider underlying diseases , medical history , and the drugs taken by patients . in the study group , 62% of women reported having no life partner ; only 19% of men did not have a partner . this coincides with work that notes the feminization of old age associated with greater longevity and independence of women compared with men.11 there was a balance between the two genders with regard to occupation , as most were retired or pensioners . a study with 320 subjects in curitiba revealed that more than half of the sample was made up of retirees and had a minimum wage income , which explains the use of the free , government - sponsored sus.27 this study ( table 1 ) was made up of patients with predominantly sensorineural hearing loss , from mild to moderate degree with a downward sloping audiometric curve , significant for both groups . as most subjects were elderly , the prevalence of hearing loss caused by aging ( i.e. , presbycusis ) as found in other studies had been expected.12 20 29 the majority of the sample ( table 3 ) had been wearing a hearing aid for over 2 years , and 14 subjects ( 22% ) reported not using the device continuously , either because of otologic factors or functional factors with the device . there was no significant difference between the two genders regarding continuous or nonuse of the device . studies point to the fact that it is common for users to give up on wearing hearing aids for several reasons , ranging from discomfort with the sound amplification to aesthetic considerations.28 29 in this study most respondents used bte hearing aids . of those who discontinued use of the device , half did so because of problems with the ear itself and half had problems with the device . ear pain and itching may compromise the use of the device , because they prevent the adaptation of molds,28 and failures in the devices are among the most common causes of abandonment of use.27 hearing loss is commonly accompanied by symptoms such as tinnitus , vertigo , and aural fullness.1 the most common otologic complaint in both groups was tinnitus , reported by 48 subjects , and the second most common was vertigo , reported by 17 subjects . tinnitus is an otologic problem that manifests itself frequently in patients with hearing loss.19 20 this problem , besides compromising the proper use of the device , may reduce its benefits and cause significant emotional and psychiatric sequelae . vertigo also compromises the user 's adequate adaptation for the hearing aid.21 contrary to the published literature18 , tinnitus was significant among women in this study.18 improvement in hearing symptoms was achieved , particularly for those complaining of tinnitus ( 16 women and 10 men ) , which confirms data from the literature.20 22 this information reinforces the thesis that the hearing aid constitutes an important resource for physicians and audiologists who engage in hearing rehabilitation . after investigating the causes of otologic symptoms , systemic diseases should be studied as they may be generating their own complaints and cause discontinuation of hearing aid use because of the important relationship between hearing , metabolic , and vascular issues.3 13 15 in this study , 14 respondents who do not wear the device appropriately , referring to otologic problems such as tinnitus , vertigo , ear pain and itching , mentioned the presence of comorbidities ( table 2 ) . the literature also reports that hypertension has been shown to be a common case in both genders , but in this study a predominance of males had this complaint.10 12 finally , as reported in the literature,20 22 use of the hearing aid was decisive for reducing some complaints , such as decreased sensation of dizziness and tinnitus ( table 3 ) for both women and men . this study showed that the incidence of systemic and otologic complaints is high in this population . during the data collection period , 278 people had follow - up visits ; of these subjects , 61 ( 21% ) reported otologic problems or operational problems with their devices . basocochlear hearing loss , a characteristic of presbycusis , was the most prevalent both in men and women , with the majority of the subjects studied being senior citizens . the most frequently reported comorbidities were hypercholesterolemia ( more significant in women ) and hypertension ( more significant in men ) . in 14 subjects , use of the device had been discontinued and there was no significant difference between genders . the reasons for discontinuation of use were itching and tinnitus , with women complaining more often . it is clear that this group of patients should be evaluated in its entirety , for actions of this nature can contribute to improving the quality of life and assisting in the process of hearing aid adaptation .

introduction optimization of the selection , adaptation , and benefit of hearing aids is necessary to characterize and manage hearing loss , user expectations , otolaryngologic symptoms , and systemic comorbidities . objective to compare the occurrence of otologic complaints , systemic diseases , and effective use of hearing aids in men and women with deafness . methods patients from a unified health system accredited hearing health service , who reported problems in adapting to their hearing aids , were evaluated by a physician and audiologist . an anamnesis , ent evaluation , and audiological evaluation were performed . results during the data collection period , 278 subjects came in for follow - up visits ; of these , 61 ( 21% ) reported otologic or operational problems with their equipment . the most prevalent type of hearing loss was basocochlear , a characteristic of presbycusis , in both men and women ; the most frequently reported comorbidities were hypercholesterolemia ( more significant in women ) and hypertension ( more significant in men ) . fourteen subjects reported using their device discontinuously , with no significant difference between genders ; the reasons for discontinuation of use were itching and ringing , with more complaints from women . conclusion the incidence of systemic and audiological complaints is high in this population . these patients should be evaluated thoroughly , as resolutions of these complaints can contribute to improving the quality of life and assist in the process of hearing aid fitting .

psoriasis is known to be associated with an increased risk of several comorbidities including inflammatory arthritis , metabolic syndrome , and atherosclerotic disease . the etiology of this autoimmune disorder is unknown but is believed to be an interplay between genetic predisposition and environmental factors such as smoking , high body mass index , and excessive alcohol consumption . the association between psoriasis and enteropathy has been recognized since 1971 when marks and shuster reported a small group of patients with severe psoriasis who presented with diarrhea / steatorrhea and were ultimately found to have enteropathy that was characterized by histological changes similar to celiac disease ( cd ) . however , subsequent epidemiologic studies attempting to characterize this association have yielded inconclusive results , primarily because of small sample size . therefore , to further investigate this possible relationship , we conducted a systematic review and meta - analysis of epidemiologic studies that compared the risk of cd in patients with psoriasis versus nonpsoriasis participants . the first two investigators independently searched published articles indexed in medline and embase database from inception to march 2016 using the search strategy that comprised the terms for psoriasis and cd as described in supplementary data 1 . the eligibility criteria included the following : ( 1 ) cohort study , cross - sectional study , or case control study reporting risk of cd among patients with psoriasis compared with participants without psoriasis ; ( 2 ) relative risk , hazard ratio , incidence ratio ( ir ) , or standardized ir with 95% confidence intervals ( cis ) or sufficient data to calculate those ratios were provided . the search and literature review process were overseen by the senior investigator who resolved any different decisions between the first two investigators . this scale assessed each study in three domains including the recruitment of cases and comparators , the comparability between the cohorts and the ascertainment of the outcomes of interest for a cohort study , and the ascertainment of exposure of interest for case control study . a standardized data collection form was used to extract the following information : first author 's name , title of the study , year of publication , year of study , country of origin , study design , study population , method used to identify cases and comparators , method used to diagnose psoriasis and cd , number of participants , and demographic data of participants and confounders that were adjusted and adjusted effect estimates with 95% ci . data analysis was performed using review manager 5.3 software from the cochrane collaboration ( london , united kingdom ) . we pooled the point estimates from each study using the generic inverse - variance method of dersimonian and laird which assigned a weight for each study based on its variance . in light of the high likelihood of between - study variance , we used a random effect model rather than a fixed effect model . cochran 's q test , which is complemented with the i statistic , was used to assess statistical heterogeneity . this i statistic quantifies the proportion of total variation across studies that is due to heterogeneity rather than chance . a value of i of 0%25% represents insignificant heterogeneity , > 25% but 50% low heterogeneity , > 50% but 75% moderate heterogeneity , and > 75% high heterogeneity . the first two investigators independently searched published articles indexed in medline and embase database from inception to march 2016 using the search strategy that comprised the terms for psoriasis and cd as described in supplementary data 1 . the eligibility criteria included the following : ( 1 ) cohort study , cross - sectional study , or case control study reporting risk of cd among patients with psoriasis compared with participants without psoriasis ; ( 2 ) relative risk , hazard ratio , incidence ratio ( ir ) , or standardized ir with 95% confidence intervals ( cis ) or sufficient data to calculate those ratios were provided . the search and literature review process were overseen by the senior investigator who resolved any different decisions between the first two investigators . this scale assessed each study in three domains including the recruitment of cases and comparators , the comparability between the cohorts and the ascertainment of the outcomes of interest for a cohort study , and the ascertainment of exposure of interest for case control study . a standardized data collection form was used to extract the following information : first author 's name , title of the study , year of publication , year of study , country of origin , study design , study population , method used to identify cases and comparators , method used to diagnose psoriasis and cd , number of participants , and demographic data of participants and confounders that were adjusted and adjusted effect estimates with 95% ci . data analysis was performed using review manager 5.3 software from the cochrane collaboration ( london , united kingdom ) . we pooled the point estimates from each study using the generic inverse - variance method of dersimonian and laird which assigned a weight for each study based on its variance . in light of the high likelihood of between - study variance , we used a random effect model rather than a fixed effect model . cochran 's q test , which is complemented with the i statistic , was used to assess statistical heterogeneity . this i statistic quantifies the proportion of total variation across studies that is due to heterogeneity rather than chance . a value of i of 0%25% represents insignificant heterogeneity , > 25% but 50% low heterogeneity , > 50% but 75% moderate heterogeneity , and > 75% high heterogeneity . our search strategy yielded 572 potentially relevant articles ( 428 articles from embase and 144 articles from medline ) . four hundred and two articles were excluded at this stage since they were not observational studies , did not report the outcome of interest , or were not conducted in patients with psoriasis , leaving 31 articles for full - length article review . ten studies were excluded as they were descriptive studies without comparators , whereas five of them were excluded since they compared the prevalence of cd - related antibodies between those with and without psoriasis but did not compare the prevalence of clinical disease . four retrospective cohort studies with 12,912 cases of psoriasis and 24,739 comparators met our inclusion criteria and were included in this meta - analysis . figure 1 outlines our study identification and literature review process . the clinical characteristics and newcastle ottawa scales of the studies included in this meta - analysis are described in table 1 . it should be noted that the inter - rater agreement for the quality assessment was high with the kappa statistics of 0.66 . search methodology and literature review process main characteristics of the studies included in this meta - analysis of the association between psoriasis and celiac disease all included studies revealed an increased risk of cd among patients with psoriasis even though did not always reach the statistical significance . the pooled analysis demonstrated a significantly higher risk of cd among patients with psoriasis compared with participants without psoriasis with the pooled odds ratio of 3.09 ( 95% ci , 1.924.97 ) . forest plot of this meta - analysis to confirm the robustness of our analysis , we performed jackknife sensitivity analysis by excluding one study at a time from the pooled analysis . the results of this sensitivity analysis ranged from 2.87 to 8.90 and remained statistically significant . since only four studies were included in this meta - analysis , evaluation for publication bias was not performed . since only four studies were included in this meta - analysis , evaluation for publication bias was not performed . this study is the first systematic review and meta - analysis to investigate the risk of cd among patients with psoriasis using comprehensive data from all available studies . we were able to demonstrate a statistically significant increased risk of incident atrial fibrillation among patients with psoriasis with approximately 3-fold increased risk of cd compared with participants without psoriasis . the pathophysiologic mechanisms behind this increased risk are not known , and further investigations are required . however , there are few possible explanations . first , the association between cd and several autoimmune diseases , such as type i diabetes mellitus and autoimmune thyroid disease , is well documented . it is believed that shared genes ( at - risk human leukocyte antigen [ hla ] haplotypes ) might be responsible for this association . for example , type i diabetes mellitus and cd share multiple common genetic loci such as hla - dr3 , hla - dq2 , and hla - dq8 . the shared genes might play a similar role in the association between psoriasis and cd . second , the hyperproliferated keratinocytes found in patients with psoriasis are known to produce an excessive amount of interleukin ( il)-1 and il-18 , the essential signals for the induction of th1 response . interestingly , mucosal inflammation in patients with cd is also caused by activation of th1 in response to dietary gluten . third , it is also possible that , in fact , cd increases the risk of psoriasis , but diagnosis of cd is often delayed or missed as its clinical manifestation could be subtle and nonspecific . intestinal barrier dysfunction associated with undiagnosed or untreated cd may allow increased passage of immune triggers resulting in increased risk of autoimmune diseases including psoriasis . given the possible mechanistic links between the two diseases , gluten - free diet , the cornerstone for the management of cd , may also have a role in the management of psoriasis . in fact , benefit of gluten - free diet among patients with psoriasis has been observed in nonrandomized studies . the major strength of this study was the advantage of systemic review and meta - analysis that comprehensively combined all available data . nevertheless , we acknowledge that the study has some limitations and the results should be interpreted with caution . second , we could not perform an evaluation for publication bias as only four studies were included in the analysis . therefore , we can not exclude the possibility of publication bias in favor of positive studies . third , the primary studies were conducted in only two countries which might limit the generalizability of our results to other populations . our meta - analysis demonstrated an approximately 3-fold increased risk of cd among patients with psoriasis . whether routine screening for cd is suggested in clinical practice patients with psoriasis are at approximately 3-fold increased risk of celiac disease compared with participants without psoriasis . patients with psoriasis are at approximately 3-fold increased risk of celiac disease compared with participants without psoriasis .

background and objectives : the possible association between psoriasis and celiac disease ( cd ) has long been observed , but epidemiologic studies attempting to characterize this association have yielded inconclusive results . this meta - analysis was conducted with the aims to summarize all available data.methods:we conducted a systematic review and meta - analysis of observational studies that reported relative risk , hazard ratio , odds ratio ( or ) , or standardized incidence ratio with 95% confidence interval ( ci ) comparing the risk of cd in patients with psoriasis versus participants without psoriasis . pooled risk ratio and 95% ci were calculated using random - effect , generic inverse - variance methods of dersimonian and laird.results:four retrospective cohort studies with 12,912 cases of psoriasis and 24,739 comparators were included in this meta - analysis . the pooled analysis demonstrated a significantly higher risk of cd among patients with psoriasis compared with participants without psoriasis with the pooled or of 3.09 ( 95% ci , 1.924.97).limitations : most primary studies reported unadjusted estimated effect , raising a concern over confounders.conclusions:our meta - analysis demonstrated an approximately 3-fold increased risk of cd among patients with psoriasis .

poly(vinyl alcohol ) ( pva ) is a biocompatible polymer with a great variety of biomedical and pharmaceutical applications . pva in aqueous solution is able to form chemical or physical gels under a variety of conditions . the physical gelation capacity of pva solutions has been well known for a long time ; however , the preparation of hydrogels by repeated freezingthawing cycles has attracted renewed interest . the high degree of swelling in water , the rubbery elasticity , the chemical and mechanical stability , the porous fibrilar network structure , and the lack of toxicity makes freezethaw pva hydrogels an attractive polymer matrix for biotechnological applications . to understand the origin of these properties and therefore the bioapplicability of this material , it is necessary to study the physical gelation process produced by the freezingthawing cycles as well as the formed network structure . komatsu el al.(13 ) investigated the phase diagram of the water / pva binary system . according to this diagram , gelation occurs with or without spinodal decomposition according to the polymer concentration and temperature . the process seems to start with a clustering of chains , which is primarily caused by the association of polar groups of the dissolved polymer , followed by polymer crystallization . this means that the physical cross - links , which are responsible for the network formation , could be formed by different processes , such as hydrogen bonding , polymer crystallization , and in some cases ( depending on the gelation conditions ) , phase separation.(14 ) in addition , the application of freezingthawing cycles to pva solutions leads to the formation of heterogeneous networks with different morphology and properties as compared with nonfrozen gel systems . were the pioneers in the development of this type of polymer cryogels.(7 ) in earlier work , they attribute the gel formation to a partial crystallization of chain segments to microcrystalline structures . however , the intensity of the diffraction maximum that corresponds to the 101 reflection is clear for only pva hydrogels with high polymer content . for hydrogels with 10 to 15% polymer , solid - state nmr was used to identify and quantify the crystalline phase , although x - ray analysis is also possible.(29 ) most recent works show that gelation by freezethaw processing forms heterogeneous networks of interconnected micro- and macropores . willcox et al.,(28 ) in an exhaustive study using transmission electron microscopy ( tem ) images and nmr , showed the formation of networks with rounded pore morphology , fibrillar network morphology , or both depending on the number of cycles as well as aging . they attribute the formation of cross - links to a kinetically frustrated crystallization in the first freezing cycle . after this process , subsequent cycles ( or aging processes ) lead to the creation of new ( secondary ) crystallites and the growing of the primary crystals ; however , the mesh spacing slightly changes . this fact seems to indicate that the average distance between the primary crystallites that are formed during the first thermal treatment constitutes the main controlling factor for the network structure . in addition , auriemma and coworkers in a series of works showed that the porous structure originates from freezing in the first step because of the incomplete crystallization of water . the polymer concentration in this unfrozen water phase is higher than that in the original solution , and the polymer network is formed within this microphase because of pva crystallization . the cross - link points are therefore constituted of polymer crystallites , and the formed gels could be described by a bicontinuous structure of polymer - rich and polymer - poor regions . on the basis of dsc measurements , also inferred the possibility of the existence of covalent cross - links between the polymer chains brought about by the formation of radicals due to strong local shear during water crystal growth.(33 ) in summary , the pva hydrogels obtained by freezingthawing cycles appear to be constituted by a very complicated network structure that is based on different phenomena ( i.e. , crystallization , hydrogen bonding , liquidliquid phase separation , and covalent bonds ) . therefore , a complete analysis of this structure requires the use of different characterization techniques . however , most of them require the manipulation of the hydrogel sample , provoking changes in the original structure . for example , the use of chromatographic techniques or scattering techniques to measure molecular weight or cluster size requires the dilution of the gel(24 ) ( although it was shown that it is possible ) . mechanical measurements have the limitation of water evaporation at higher temperatures.(9 ) the cryo - tem technique has the inconvenience of handling difficulties to avoid artifacts.(28 ) for the study of the crystallite content , very useful techniques such as differential scanning calorimetry ( dsc ) as well as x - ray diffraction are challenged by a sensitivity problem in dilute samples ; for this reason , measurements were sometimes made in dry samples.(34 ) in this vein , solid - state nmr is a powerful tool for studying polymer networks , but it has not been extensively used in this field . the focus of this work is the use of a low - field nmr spectrometer to analyze the structure of the pva gel in detail as a function of the freezingthawing cycles , checking all of the structural models suggested in the literature . in addition , a melting process that transforms these physical gels into isotropic solutions of pva in water was also studied . importantly , we focus on detecting protons so that sensitivity , even at the low concentrations under study , is not a serious issue . the use of an inexpensive low - field nmr spectrometer on as - prepared samples to study this complex matrix has the advantage that several of the cited inconveniences in sample characterization are avoided . by using different nmr methodologies , we are able to carry out a complete and quantitative study of the complex structure exhibited by the pva gels : multiple - quantum ( mq ) nmr allows us to gain direct access to residual dipolar coupling constants that persist because of the existence of cross - links and other topological constrains . therefore , this experimental procedure is a powerful tool for quantifying the gelation process(41 ) and gives us quantitative detail on not only the microstructure(42 ) but also the dynamics(43 ) of the gel , that is , a complete picture of the network structure evolution depending on , for example , the number of freezingthawing cycles . in addition , we used pulsed mixed magic - sandwich echo ( mse ) experiments that provide a near - quantitative refocusing of the rigid contribution to the initial part of the free induction decay ( fid)(44 ) and allow for an essentially quantitative determination of the crystallinity . to our knowledge , this is the first time that advanced nmr pulse sequences performed on a low - field spectrometer were applied to the study of the network structure and phase composition of physical hydrogels . the sample was > 99% hydrolyzed , had a weight - average molecular weight of 94 000 g / mol , and had syndio- , hetero- , and isotacticities of 17.2 , 54.1 , and 28.7% , respectively . pva was dissolved in deuterated water ( with polymer concentrations of 10 and 20% w / w ) at 80 c under continuous stirring to avoid inhomogeneities and local gelation . solutions were stored at 80 c overnight ; then , they were cooled to room temperature for 1 h. then , pva solutions were introduced to 10 mm od nmr tubes and were flame sealed to avoid variations in the water content . two technical considerations are important at this point : ( i ) the quantity of sample inside the tube has to be 8 mm in height and located in the center of the radio frequency ( rf ) coil to ensure good rf field ( b1 ) homogeneity . ( ii ) the length of the nmr tube should be as short as possible for good control of the temperature . this is important because in this way , the whole tube can be inserted in the probe head , thus avoiding possible temperature gradients due to heat conduction . pva hydrogels were obtained by repetition of freezingthawing cycles of solutions inside the sealed tubes that consisted of 1 h of cooling to 32 c and 1 h of thawing at room temperature . after this process , samples were immediately inserted in the nmr spectrometer and measured to avoid the effect of aging . in some cases ( specified below ) , the first freezing cycle was extended to 12 h ( overnight ) , and subsequent cycles were 3 h long . solid - state h nmr spectroscopy is a powerful and widely used experimental technique in the field of polymer science.(35 ) in recent years , the use of inexpensive and easy - to - use low - field spectrometers has gained the attraction of scientists because it certainly produces quantitative results on polymer structure and dynamics , albeit , of course , without chemical resolution . on the basis of different experiments , which will be extensively described in the next sections , applications include quantitative studies on polymer network structure(38 ) and polymer crystallization,(44 ) that is , the main topics of this study . in this work , experiments were carried out on a bruker minispec mq20 spectrometer operating at 0.5 t with 90 pulses of 1.7 s length and a dead time of 12 s . mq spectroscopy is one of the most versatile and robust quantitative techniques for investigating not only the structure but also the dynamics of polymer networks.(38 ) in solution , the fast segmental motion of the polymer chains through the different accessible conformations is isotropic and completely averages the dipolar coupling interaction that is typical for solid - state spectra . however , constraints , irrespective of their nature ( e.g. , cross - links , entanglements , or chain packing ) , lead to nonisotropic segmental fluctuations and therefore to the persistence of a weak residual dipolar coupling ( dres ) , which is our central nmr observable . in other words , the measured effect relies on the orientation dependence of the fluctuating dipolar coupling tensor with respect to the magnetic field , which can be described by an orientation autocorrelation function of the individual chain segments . fast segmental dynamics ( in the range of nanoseconds to microseconds ) lead to a loss of correlation to a plateau value that is related to the existence of preferential local orientation generated by the existence of cross - links . in fact , the measurable dres is directly proportional to a local dynamic order parameter of the polymer backbone,(38 ) which is defined as a time average over the fluctuations of the segment - fixed dipolar tensor over the time until the plateau region is reached . it connects the experimental observable with the network parameters : here dstat is the segmental averaged dipolar coupling constant in the static limit and k is a rescaling factor that takes into account the fast dynamics ( in the range of picoseconds ) inside the statistical ( kuhn ) segments . the nmr observable is related to r , that is , the ratio of the end - to - end vector to its average unperturbed melt state ( r = r/r0 ) , and to n , the number of statistical segments between constraints . a variety of nmr experiments was used to detect residual dipolar couplings to monitor gelation processes , and recently , h mq nmr has evolved as the most powerful tool for obtaining a direct measurement of dres and even its distribution . details of the pulse sequence as well as the data analysis are already published elsewhere . here temperature usually plays an important role in mq measurements(43 ) because as previously mentioned , the order parameter should be obtained as an average of the segmental fluctuations over all of the possible conformations on the experimental time scale ( less than microseconds ) . therefore , the temperature is required to be far above the polymer glass - transition temperature tg to ensure that the segmental dynamics is fast enough to sweep out the whole conformational space between topological constraints and to achieve full averaging . if this is not the case , then the obtained dres ( and therefore the number of junctions in the network ) will be overestimated.(41 ) in this work , the studied samples are hydrogels ; therefore , the dissolved state of the polymer chain ensures that the segmental dynamics is indeed fast enough to detect the order parameter in the plateau regime , even at room temperature . similar to the traditional transverse relaxation experiments , the analyzed result of the mq experiment is a time - dependent but normalized double - quantum ( dq ) signal function , indq , that has the advantage of being independent of any temperature - dependent true relaxation ( decay ) effect . it is dominated by the dipolar interactions and independent of the polymer dynamics . for this purpose , the directly measured experimental dq build - up curve ( idq ) must be relaxation - corrected by the use of the also measured reference intensity ( iref ) . initially , iref contains a signal from not only the dipolar - coupled network chains but also the uncoupled , isotropically mobile components such as sol , dangling chains , and so on . the total mq magnetization ( imq ) needed for normalization is the sum of idq and iref but only after subtracting the non - network contributions . this fraction has a slower relaxation and can be easily identified ( figure 1a ) . in the studied hydrogels , even though we used deuterated water to dissolve the polymer , we found a non - negligible water signal resulting from actual residual water and exchanged oh protons . therefore , we had to identify and subtract two exponential long - time contributions to imq ( i.e. , the noncoupled fraction of polymer chains ( b fraction in eq 2 ) and the solvent signal ( fraction c ) ) , which relax with longer relaxation times ( t2c * t2b * ) mq nmr build - up and decay data for 10% pva sample after one freezingthawing cycle . ( a ) as - acquired idq and imq and ( b ) imq ( after extraction of noncoupled contributions ) and normalized dq build - up curves ( idq ) . the solid line in a represents the exponential fit for water subtraction , and the dashed line is the exponential fit representing the subtracted noncoupled polymeric defects . in b , solid and dashed lines represent the fits assuming a gamma distribution of dres and a single residual dipolar constant , respectively . ( see figure 1b . ) finally , it is important to point out that in rubber networks the distribution effects of dres ( related to different end - to - end separations and polydispersities of network chains ) usually do not play any role.(56 ) therefore , the normalized dq build - up curve of a bulk rubber can be analyzed in the quasi - static limit in terms of a single dres(38 ) however , the complex and heterogeneous pva network structure , in addition to specific effects related to the swollen state of the sample ( which is proven to be heterogeneous in polymer networks(57 ) ) , leads to large deviations from the inverted gaussian shape in the studied pva gels . the actual coupling distribution ( related to a microstructurally heterogeneous distribution of constraints ) the insert in figure 1b shows the broad distribution of dipolar interactions in pva networks . it resembles a gamma distribution and is consistent with observations of very heterogeneous network structures proposed in the literature . all analyzed samples showed a similar distribution shape independently of the number of freezingthawing cycles . because regularization analysis is subject to some limitations(42 ) ( further work about this point is in progress ) , we have analyzed all build - up curves under the assumption of the gamma distribution of dipolar couplings that is predicted by the gaussian distribution of end - to - end separations ( but which is screened in bulk elastomers ) upon fitting , the integral is numerically evaluated within the curve fitting environment of the origin software . in using this distribution , we do not want to imply that this distribution is of any significance in the studied structure ; the resulting build - up curves merely describe all of our experimental build - up data very well and have the benefit of not introducing an additional fit parameter , yet they provide a well - defined average residual coupling davg . ( the width of the gamma distribution is directly related to the average . ) finally , note that the mq analysis pertains to the mobile fraction of the cryogels only ; this fraction always constitutes the major part of the sample ( > 80% ) ; the rest is rigid crystallites , and the signal of which rapidly relaxes and is not observable under the conditions of the mq experiment . according to previous papers , polymer crystallites act as junctions in pva networks obtained by freezingthawing cycles . solid - state nmr is a useful method for characterizing polymer crystallinity or phase composition in this type of system . the main concept is that the crystalline signal usually decays in the first 20 s of the free - induction decay ; therefore , the signal detected after the dead time ( 12 s in our case ) partially conceals this information . nevertheless , in some works , the fraction of protons in a glassy state in pva gels was estimated by monitoring the fid intensity during the first 20 s , which renders these studies qualitative . perhaps the most important is the use of solid echoes with different echo delays combined with back extrapolation(64 ) to correct for its inability to refocus the dipolar interactions in a multiple - spin system fully . certainly , the best approach is the use of a spectrometer with short ( 1 to 2 s ) dead time.(65 ) the use of an mse was recently proposed(44 ) as a more effective method for refocusing the multiple dipolar interactions that lead to the fast decay of the initial part of the fid , therefore removing the dead - time problem and obtaining a near - quantitative rigid fraction determination in polymer systems . under the same experimental conditions , differences of 40% in the quantity of rigid phase in pva compounds figure 2 shows the mse - fids for different total echo duration ( realized by increasing the interpulse spacings ) . the signal decay data are well represented by a gaussian function ( parabolic initial decay ) , and its extrapolation to t = 0 allows us to conclude that the signal loss is negligible . ( it could be estimated to be 2% on the absolute scale , which corresponds to 10% on the relative scale . ) parabolic ( gaussian ) extrapolation of the maximum mse - refocused fid intensities to t = 0 . it indicates only minor losses of rigid signal at the shortest echo time ( 10% on a scale relative to the total crystallinity ) . the inserted graph shows mse - refocused fids of 10% pva gel after six cycles measured at different echo times at room temperature ( 304 k ) . however , note that even for the mse the refocusing efficiency breaks when there is molecular motion on its time scale . in particular , this may be the case if the observed rigid component is glassy rather than crystalline and close to tg . under any condition irrespective of temperature , however , the typical decay time upon incrementing the echo was never significantly shorter than the ( relatively weak ) decay seen in figure 2 , which is ultimately associated with the breakdown of the pulse sequence efficiency because of higher - order imperfections . we thus conclude that all observed rigid signals are associated with crystal - like domains ; otherwise , a significant signal loss upon going through tg would have been observed . therefore , mse - fid curves were used to quantify the fraction of fast - decaying rigid polymer present in pva gels . the first fast decay ( caused by the rigid polymer fraction ) in the mse - fids ( insert in figure 2 ) has a quasi - gaussian decay , whereas the slower signal decay could be fitted with an exponential function . therefore , the first 140 s of the normalized mse - fid curves ( unity signal for zero time ) were fit according to eq 6 in this expression , a is the fraction of detectable rigid phase in the sample and b is an adjustable parameter for a better fit of the fast decay shape . in all of the cases , b 2 ( i.e. , the crystalline fraction is described by a gaussian function ) . in addition , to reference the hard polymer phase to the total amount of polymer ( instead of the sample fraction ) , we corrected the a parameter according to the actual fraction of polymer in the sample obtained from saturation recovery experiments ( note again that the samples contain residual water ) on the basis of the fact that water has a much longer t1 relaxation time . from such experiments , and assuming no significant noe transfer between the species on the same time scale , the water content can be obtained from a biexponential fit where d is the water fraction in the sample and t1w and t1p are the longitudinal relaxation time constant of water and polymer , respectively ( with t1w t1p ) . for example , at t = 304 k , the relaxation times were t1w = 1520 ms and t1p = 60 ms for a network with 10% pva after seven freezingthawing cycles . the fast - decaying rigid crystallite signal was not detected in these experiments ( the fid was evaluated at a time beyond 50 s ) such that d is determined relative to the mobile polymer fraction ( i.e. , ( 1 a ) in eq 6 ) . finally , the rigid polymer fraction discussed below was obtained according to obviously , for these corrections , it is essential to use a recycle delay that is long enough to observe the full equilibrium magnetization of the sample ( 5(t1w ) was used as a recycle delay in all experiments , i.e. , 720 s depending on the temperature ) . the aim of this work is to study the structure and phase composition of pva cryo - gels by using a low - field nmr spectrometer . to revise the structural models suggested in the literature , we will separately analyze the effects of three factors on the pva network structure : ( i ) the number of applied freezingthawing cycles , ( ii ) the temperature and melting process , and ( iii ) the aging process . to achieve this objective , in every point , dq experiments were performed to quantify the proportion of noncoupled network defects , and the molecular weight between cross - links and mse - fid pulse sequence were applied to determine the rigid fraction of the polymer . the starting point of our study is the characterization of two isotropic solutions of 10 and 20% ( w / v ) of pva in water . the 10% pva solution does not give any detectable dq signal , but for the decay ( iref ) data , two different processes can be identified ( i.e. , the decay of the polymer magnetization and the water relaxation ) . this result demonstrates that under these preparation and measurement conditions , the dilute solution can be considered to be nonentangled and above the gelation threshold . this is not a trivial result because dq experiments are , in principle , sensitive to transient links ( entanglements or hydrogen bonds ) , and this indeed appears indicated by the 20% pva solution ; under the same preparation conditions , dq experiments did yield a detectable build - up curve . however , this is in our case due to a nondissolved or partially gelled pva subphase . to avoid this problem , 20% pva solutions were prepared under more vigorous stirring as well as higher temperatures up to 90 c . in this way , a totally isotropic solution of polymer was obtained according the dq experiments . dq experiments were performed on pva samples subject to different numbers of freezingthawing cycles to determine the evolution of noncoupled network defects as well as the network mesh size . in figure 3 , we can see that after the first freezingthawing cycle , 50% of the polymer chains are coupled and are therefore part of the network , whereas the rest are not linked to it or at least belong to elastically inactive dangling chains or loops . the number of pva chains incorporated in the gel network increases with the successive cycles until it reaches a plateau after the sixth cycle , where the network has the minimum defect content . this important result shows that independent of the number of freezingthawing cycles applied to a pva solution 25% of the polymer is not coupled and therefore should not be elastically active . this is a very significant observation that is usually not taken into consideration when mechanical , dynamic - mechanical , or rheological properties of this material are analyzed . urushizaki et al.(66 ) have related the viscoelastic properties of this type of gel to the nonincorporated pva chains , and they have estimated their amount from the difference in weight between the polymer gel before and after immersion in distilled water . they found that 10% of the polymer was not incorporated after the first freezingthawing cycle . similar results were also indirectly inferred from the measure of intradiffusion coefficients of micellar aggregates of surfactant molecules dissolved in these types of gels.(67 ) obviously , our experimental values are much larger because we detect not only the non - cross - linked chains but also the dangling chains and other nonelastic network defects ( e.g. , loops ) . the effect of d2o in measured noncoupled network defects is another important point to take into consideration when this fraction of polymer is analyzed . pva monomers have an exchangeable proton ( oh ) , which , in presence of d2o , could be replaced by deuterium that is undetectable by h nmr , whereas the proton will become detectable as water ( hdo ) . free water signal ( h2o or hdo ) is subtracted from the long - time decay of the signal because it can be identified by its own longer t2 . however , the number of noncoupled network defects could be slightly overestimated because of the protondeuterium exchange . variation of noncoupled network defects ( i.e. , non - cross - linked polymer chains , dangling chains , and loops ) as a function of the number of freezingthawing cycles for the studied pva solutions . in addition , the decrease in the number of defects is correlated with an increase in dres that is proportional to the network chain order parameter ( eq 1 ) , see figure 4 . the formation of cross - links ( independent of their nature ) renders the polymer segmental motion nonisotropic , whereby the chain segments become ordered with respect to the end - to - end distance , and residual dipolar couplings arise . according to this basic principle , the increase in dres is directly related to the ( inverse ) length of the chains between the constraints . similar to the observation for the noncoupled network defects , the network chain length appears to approach a plateau after cycle number six , yet the tendency is not as clear as the former result . even after 16 cycles , the cross - link density still appears to increase . variation of the average residual dipolar couplings ( directly related to 1/n and therefore the cross - link density or the inverse mesh size ) extracted from dq experiments as a function of the number of freezingthawing cycles for the studied pva solutions . represents a sample containing 20% pva after 3 h of freezing , and is from the same sample after 16 h of freezing . with this observation , it is important to note that samples with different polymer concentration appear to exhibit the same behavior during gelation . a possible explanation could be that in the framework of a heterogeneous scenario cross - linking occurs via partial crystallization in the nonfrozen parts of the sample ; it simply means that in less concentrated samples more water freezes . this fact could explain the dependence of the dimension and shape of pores on polymer concentration and the regimes of cryogenic treatment.(20 ) in this context , it appears to be worthwhile to study the influence of the actual freezing temperature , which should affect the polymer concentration under the conditions at which the crystallites are formed . however , the freezing time ( especially in the first cycle when the network is formed ) seems to have some influence in the gel network structure . as is shown in figure 4 , the apparent dipolar coupling constant is very low ( 48 hz ) after 1 h of freezing at 32 c . if the freezing period is increased to 3 h , then the cross - link density of the gel almost doubles ( 82 hz ) , whereas longer freezing ( e.g. , 16 h ) does not lead to further variation this may indicate that 1 h of freezing is not long enough to complete the process , and for this reason , further analyses are based on samples with a first freezing cycle of 12 h and subsequent cycles of 3 h each . returning to the origin of the pva networks produced via freezingthawing cycles from homogeneous polymer solutions in water , it appears to be well demonstrated by now that during the first freezing cycle , freezing of some water(32 ) increases the concentration of pva in the still unfrozen phase.(31 ) crystallization of pva takes place in this concentrated microphase,(68 ) but it is kinetically frustrated by the gelation of the polymer solution . this fact could explain the small size of these primary crystallites ( 5 nm ) , which then act as physical junctions between the amorphous and mobile polymer chains . results extracted from the mse - fid curves of the pva gels after the first freezingthawing cycle ( figure 5 ) indicate that 8% of the polymer behaves like a rigid solid . this rigid fraction increases with the number of cycles until it reaches a plateau close to cycle number six , just as our other reported network properties . the maximum amount of rigid phase estimated by our nmr methodology ( 20% of the total polymer ) is substantially higher than the crystallinity of similar samples deduced by dsc as well as some other h nmr methods . variation of the rigid ( crystalline ) polymer fraction extracted from mse - fid curves as a function of the number of freezingthawing cycles for the studied pva solutions . in all cases , the crystallinity of pva cryo - gels obtained in dsc studies is estimated to be 5% ( not taking into account studies of dry networks(34 ) because the structure should be different ) . however , as was pointed out by willcox et al.,(28 ) this magnitude could very well be underestimated because the heat of fusion of the crystals in gels may be substantially lower than the assumed pva bulk value because of their small size and large surface effects ( hydrogen bonds to surrounding water , etc . ) . in addition , another important point is the difficulty in measuring small quantities of crystal phase in dilute gels via calorimetric methods in general ; for example , a crystallinity of 8% in a gel with a pva concentration of 20% represents only a sample fraction of 0.016 . nevertheless , this difficulty seems to be reduced by the use of microcalorimetry . in this way , similar results were obtained by performing this type of measurement on gels with low pva concentrations,(8 ) although the uncertainties related to the obtained results are still large . for these reasons , the use of solid - state nmr seems to be a better methodology for quantifying the crystallinity in dilute pva gels . however , there are still some discrepancies in the total amount of rigid phase measured by the use of different nmr methodologies . ricciardi et al.(18 ) estimated the crystallinity of pva gels after 11 freezingthawing cycles to be 8% by analyzing the first microsecond of the h fid . clearly , as was pointed out in the , this experimental procedure falls victim to the dead - time problem . depending on the spectrometer , the amount of rigid signal lost during this time could be more or less significant . ( ref ( 15 ) does not specify the dead time ; therefore , it is not possible to estimate the related error . ) for example , our low - field spectrometer has a dead time of 12 s ; therefore , the fid analysis would lead to an underestimation of the rigid polymer fraction by 50% . by using different c nmr experiments ( direct polarization as well as hc cross polarization ) , willcox et al.(28 ) established that after the first freezingthawing cycle , pva gel contains a rigid polymer phase of 5 2% , increasing up to 12 4% after cycle number 12 . as was pointed out by the authors,(28 ) the relative fraction comparing different samples could be estimated with a high accuracy , but the used methodology ( in particular , the use of the intrinsically nonquantitative cross polarization ) does not give a reliable absolute crystallinity . in conclusion , the key to obtaining the most accurate crystal fraction in these dilute polymer samples is to use a solid - state nmr method that allows us to detect the rigid part fully by refocusing the multispin dipolar interactions with the mse , thus solving the dead - time problem and minimizing the signal loss . of course , detecting the more - sensitive and 100% abundant protons instead of the naturally abundant c is an additional advantage , which more than compensates for the use of an intrinsically less - sensitive low - field spectrometer . summarizing all results discussed so far , the network structure of pva cryo - gels is very apparently based on the formation of rigid polymer areas ( most probably polymer crystallites ) during the cooling process . after the first cycle , almost half ( 8% ) of the final amount ( 20% ) of the rigid phase is already formed . in this stage , subsequent freezingthawing cycles increase the rigid polymer fraction , which has two consequences : first , the noncoupled polymer fraction is further reduced ( i.e. , more pva segments become elastically active ) , and second , the network mesh size is further reduced but only slightly during further freezingthawing cycles . therefore , we believe that subsequent freezingthawing cycles not only increase the size of the crystals formed during the first freezing process but also lead to the creation of other more imperfect crystals . independent of the polymer concentration of the gel , the melting of smaller and more imperfect secondary crystals takes place at lower temperature and over a large range , leading to a continuous decrease in the detected rigid polymer phase with the temperature ( figure 6 ) . at 7580 c ( in good agreement with the main endotherm observed in dsc measurements ) , this quantity drops below the detection limit . this should be correlated with the melting of the main ( primary ) crystals ( estimated to 5% of the polymer fraction ) that support the network structure of the gel . these results are consistent with dsc measurements because they show an increase in the melting endotherm that corresponds to the initial crystals as well as to the appearance of a second endotherm at lower temperatures when the number of freezingthawing cycles was increased for a given pva solution . variation of the rigid polymer fraction ( extracted from mse - fid experiments ) as a function of temperature for pva gels formed after seven freezingthawing cycles . a network with 10% pva was first heated until 324 k ( ) and then kept at room temperature for 12 h before the second heating procedure was performed ( ) . a solution with 20% pva was submitted to two different treatments , that is , one freezingthawing cycle and seven freezingthawing cycles , respectively . all samples were frozen for 12 h in the first cycle and 3 h in the subsequent cycles . the effect of the number of freezingthawing cycles on the crystallinity is easily understandable by comparing the evolution of the rigid polymer fraction of a pva gel obtained after seven cycles with that of a pva gel after only one freezingthawing cycle ( figure 6 ) . the onset of melting after only one freezing cycle is estimated to be 50 c , leaving the rigid polymer fraction constant at lower temperatures . therefore , subsequent cycles seem to create more , yet imperfect , secondary crystallite structures . above 50 c , all samples exhibit a similar behavior related to the partial melting of primary crystals or to the melting of less - perfect secondary structures . importantly , the temperature above which it is not possible to detect any more signal of rigid polymer remains invariant , yet the final fraction seems to increase slightly with the number of cycles . therefore , according to this result , freezingthawing cycles have a direct influence on not only the number of crystals formed during the first freezing process but also the creation of other more imperfect secondary crystallites . to ensure that the results shown in figure 6 are not artifacts produced by different magnetization relaxation at different temperatures that could possibly arise from regions that undergo a glass - to - liquid rather than a melting transition , the evolution of the signal at any given temperature was studied as a function of echo delay , see figure 7 . at all of the measured temperatures , similar behavior was found , with a loss of intensity estimated to between 0.7 and 1.8% for the two extreme cases , which corresponds to a constant 10% loss on a relative scale , as is usually found . importantly , the drop in overall signal corresponds , within the error margins , to the expected drop due to the curie factor ( temperature - dependent spin polarization ) . in addition , a direct check is the control of the total intensity , comparing the pva gels to the corresponding pva solutions ( insert in figure 7 ) . it is seen that no significant signal is lost / undetectable ( we observe almost all protons in the sample ) , and from this , it is clear that the loss of intensity caused by the mse in pva gels is at a minimum , with no other ( hidden ) phenomena that could be related to an intermediately mobile ( almost glassy ) rigid fraction . given this experimental evidence , the decay of the rigid h fraction with temperature can be related to only a melting of pva crystallites . variation of the initial signal intensity of mse - fids with increasing echo delay at different temperatures for a pva gel containing 20% polymer after one freezingthawing cycle ( 12 h of freezing and 1 h thawing ) . the insert presents the mse - fid of the pva gel measured at 304 k with the minimum echo delay ( 0.0022 ms ) as well as the estimated intensity at zero echo time in comparison with the mse - fid of the isotropic solution of the same sample measured under the same conditions after complete melting at 90 c . obviously , the temperature - dependent variation of the rigid polymer phase has a direct effect on the network structure , as shown in figure 8 . the melting of secondary crystals leads to an almost exponential increase in the number of network defects ( obviously , in a reversible pathway , comparing the phenomenon with the effect of successive freezingthawing cycles ) . however , the network mesh size ( as measured by the residual dipolar coupling ) remains almost constant . this means that secondary crystallization is very important in the network organization , reducing the number of network defects and therefore increasing the number of polymer segments that are elastically active . however , the molecular weight between the constraints appears to be dictated mostly by the primary crystallites formed during the first freezing cycles . for this reason , the network is completely destroyed at temperatures of 80 c when the primary crystal phase is molten , leaving no observable dq signal ( i.e. , an isotropic pva solution is formed ) . on the side , we note that the differences in the davg values given in figures 4 and 8b , which indicate a 50% decreased mesh size for the samples studied in figure 8 , are related to the increase in the freezing time ( 12 h in the first step and 3 h in the successive cycles instead of 1 h ) used in each cycle , as pointed out above . variation of ( a ) the network defect fraction and ( b ) the inverse mesh size ( given by davg measured by mq nmr ) with temperature for the 20% pva gel prepared after seven freezingthawing cycles ( 12 h first freezing cycle , 3 h subsequent cycles ) . error bars represent the fitting uncertainty , whereas lines are only guides to the eye . these nmr results on the mesh size perfectly agree with the trends observed in studies of mechanical properties reported in the literature . clearly , the elastic behavior of pva gels depends on the number of freezingthawing cycles . therefore , the main factor that determines the improvement of the mechanical properties upon further cycles should be associated with the number of elastically active chains . the polymer concentration in the original aqueous solution has an important effect on the elastic properties ; that is , the elastic modulus increases . however , in the view of our data , this does not appear to have important consequences for the actual network structure ; that is , there are no large variations in the network mesh size . therefore , the reported variations are simply related to differences in polymer concentration . in addition , different works demonstrate a slight decrease in the elastic modulus with the temperature , until a major drop occurs at 50 c.(65 ) this is obviously related to the partial melting of secondary and more imperfect crystallites that leads to an increasing amount of network defects ( elastically inactive chains ) . at temperatures close to 80 c , the complete loss of crystallinity essentially liberates all chains , and the system drops below the gel point . in view of the important role of the substantial polymer amount that is not elastically active ( free and dangling chains , loops ) , it may be worthwhile to reconsider detailed frequency - dependent mechanical measurements , where these components should contribute to an increased loss modulus over the frequency range covering their relaxation . the capacity of our investigated systems to be transformed from an isotropic aqueous solution to a physical cryo - gel by the application of freezingthawing cycles , including thermal reversibility back to an isotropic pva solution , is in clear contradiction with the assertion that covalent bonds could form between the polymer chains during gelation . if covalent cross - links were an important contribution to the final gel properties , then residual couplings should persist even above the melting temperature of the crystallites , which is not the case . however , the presence of ( rapidly tumbling ) microgels above the main melting point can not be ruled out and is in fact indicated by the relatively high viscosity ( as checked by a simple tilt test ) . however , we point out that even the pristine 20% pva solution exhibited some detectable dq signal , indicating extended gel - like structures , unless it was heated to > 90 c . therefore , an undetectably small fraction of crystallites ( or extended hydrogen - bonded aggregates ) may always be present , even at > 80 c , and hold the system close to the gel point , depending on the sample and its thermal history . from our perspective , permanent bonds can be ruled out as a major factor determining the cryo - gel structure . finally , we address aging phenomena that are a part of the data shown in figure 6 and were not commented on until now . after heating a sample containing 10% pva to 50 c and recording the loss of crystallinity , the sample was stored for 12 h , and another measurement was performed at room temperature . the amount of rigid polymer fraction after 12 h of storage was again considerably increased , as can be seen in figure 6 . yet , this new crystalline fraction melts at rather low temperatures , which again indicates the growth of secondary crystallites . similar prominent aging effects have been reported in the literature . for more clear evidence , a sealed sample containing 20% pva was submitted to seven freezingthawing cycles ( with 12 h of freezing in the first cycle ) and was characterized after different storage times at room temperature . according to figure 9 , aging clearly increases the secondary population of imperfect crystals . it also increases the amount of elastically active polymer segments but has no significant effect on the network mesh size . therefore , aging effects on pva gel properties are again mainly related to an increasing fraction of polymer incorporated into the network structure and not to a decrease in mesh size . effect of aging on ( a ) the rigid polymer fraction , ( b ) the noncoupled defects fraction , and ( c ) the average residual dipolar coupling of a gel with 10% pva obtained after seven freezingthawing cycles ( 12 h of freezing for the first cycle and 3 h for the following cycles ) . this physical gel is supported by rigid polymer areas ( pva crystallites ) that act as junctions between the remaining mobile and entropically elastic polymer chains and thus determine the network mesh size . in our study , the crystallites are quantitatively detected as a rigidlike fraction of quickly relaxing ( and fully refocused to overcome the dead - time problem ) magnetization , and the mesh size is deduced from mq experiments that measure residual dipolar couplings , the latter arising from constraints on the motion of the mobile chains ( i.e. , cross - links ) that render the segmental motion locally anisotropic . the most important crystal fraction forms in the first freezing cycle , and subsequent freezingthawing cycles produce only a slight increase in the initial crystal size as well as induce the formation of more imperfect , secondary crystallites . this secondary crystal fraction ( which constitutes around two - thirds of the total crystal phase in samples submitted to a large number of cycles , extended aging , or both ) plays an important role by increasing the number of elastically active polymer chains , but it does not appreciably affect the mesh size . pva cryo - hydrogels are shown to be totally thermoreversible ; that is , pva physical networks can be transformed into an isotropic aqueous solution by increasing the temperature . during this process , the more imperfect crystals are molten first , and the number of network defects increases exponentially . in contrast , the network mesh size does not undergo significant variations until the primary crystal phase is molten at temperatures of 80 c . our experiments did not reveal any evidence of the possible formation of covalent cross - links between pva chains during gelation ; no residual couplings were detected after the melting of this primary crystal fraction . independent of the number of cycles , the aging , or the polymer concentration in the original pva solution , a considerable fraction of polymer ( at least 25% ) is not elastically active , which is a result that is not yet not taken into account in the large body of literature addressing the formation and the properties of pva cryo - hydrogels . this finding is central to the interpretation and understanding of the elastic properties of this useful material and must be taken into account in a reinterpretation of the origin of the changes of the elastic moduli of pva cryogels . finally , it is important that the in - depth study of both processes ( i.e. , the pva gelation by the application of freezingthawing cycles ) as well as the destruction of the network structure by heating were , for the first time , entirely based on the use of an inexpensive low - field solid - state nmr spectrometer . we show that the combination of different advanced nmr strategies , that is , mq spectroscopy and component analysis of mse - refocused free - induction decays , is vital for obtaining quantitative information on network structure and phase composition ( crystallinity ) , respectively , to arrive at conclusions that can be matched with information extracted from other diverse techniques . in addition , the use of low - field solid - state nmr spectroscopy not only is useful for extracting structural information but also could be a powerful tool for investigating the polymer dynamics of these complex systems .

the network structure of poly(vinyl alcohol ) ( pva ) hydrogels obtained by freezingthawing cycles was investigated by solid - state 1h low - field nmr spectroscopy . by the application of multiple - quantum nmr experiments , we obtain information about the segmental order parameter , which is directly related to the restrictions on chain motion ( cross - links ) formed upon gelation . these measurements indicate that the network mesh size as well as the relative amount of nonelastic defects ( i.e. , non - cross - linked chains , dangling chains , loops ) decrease with the number of freezingthawing cycles but are independent of the polymer concentration . the formation of the pva network is accompanied by an increasing fraction of polymer with fast magnetization decay ( 20 s ) . the quantitative study of this rigid phase with a specific refocusing pulse sequence shows that it is composed of a primary crystalline polymer phase ( 5% ) , which constitutes the main support of the network structure and determines the mesh size , and a secondary population of more imperfect crystallites , which increase the number of elastic chain segments in the polymer gel but do not affect the average network mesh size appreciably . correspondingly , progressive melting of the secondary crystallites with increasing temperature does not affect the network mesh size but only the amount of network defects , and melting of the main pva crystallites at 80 c leads to the destruction of the network gel and the formation of an isotropic pva solution .

for many years , the global community has observed the calamitous effects of the human immunodeficiency virus ( hiv ) on the human population . hiv was historically viewed as an incurable disease that gradually progressed into acquired immune deficiency syndrome ( aids ) and led to one s untimely death . during his tenure ( 20012008 ) , united states president george w. bush recognized the devastating impact of hiv as a global health crisis that required immediate action . through bipartisan efforts between president bush and the 108 us congress , the passage of hr 1298 ( public law 10825 ) led to the creation of the president s emergency plan for aids relief , commonly referred to as pepfar . the objective of pepfar was to provide $ 15 billion in aid , targeting 12 focus countries in sub - saharan africa : botswana , cote divoire , ethiopia , kenya , mozambique , namibia , nigeria , rwanda , south africa , tanzania , uganda , and zambia . in an empirical research that was conducted on the impact of international humanitarian assistance on combating hiv / aids , azuine et al . discovered that the increase of donor assistance in developing countries led to an overall reduction of hiv infection rates . pepfar has illustrated the pertinence of an interconnected world and the urgency to embrace those connections during stages of global stress . according to the united nations , the united states has contributed more funding to the fight against hiv / aids than any other country . the research presented here provides a better understanding of how well pepfar advanced that fight and whether the allocation of a large amount of dollars for pepfar was crucial in saving lives and decreasing hiv infection rates in sub - saharan africa . the general policy research question for this research was to determine whether the pepfar funding allocated to the 12 resource - scarce countries in sub - saharan africa had any relevant effect in reducing the hiv infection rates of males and females between the ages of 15 and 49 . an analysis to determine whether the pepfar policy was effective and efficient may influence future global humanitarian assistance provided to other countries by the united states . researchers agree that the continent of africa is the focal point of the hiv pandemic and that hiv / aids is a detrimental problem for the global community . even though other countries such as guyana , haiti , and vietnam were also recipients of pepfar funding , the majority of the crisis , population affected , and funding allocated for pepfar was concentrated in the above - mentioned 12 african countries . the specific focus addressed herein is whether the pepfar funding allocation was effective in meeting its intended purpose of reducing hiv infection rates in the 12 african countries that received funding . to this day , africa prevails as the most hiv / aids affected continent in the world , in particular the sub - saharan areas . africans account for approximately two thirds of hiv infections and three quarters of deaths associated with hiv from around the world . the joint united nations programme on hiv / aids ( unaids ) , united nations children s fund ( unicef ) , and world health organization ( who ) determined that approximately 75% of people in sub - saharan africa between the ages of 15 and 49 are unaware of their hiv status . together , hiv and aids have devastating effects on society and the economy . together , hiv and aids create reproductive concerns , reduced revenues , increased healthcare expenses , decreased school enrollment , increased rates of absence at work , disruptions in the family environment , decreased human capital , increases in poverty rates , a lower gross domestic product ( gdp ) , and more orphaned children.[9 - 14 ] due to high levels of poverty , many developing countries in sub - saharan africa do not have the necessary resources or funding to combat the hiv / aids pandemic occurring in their nations . the methods that were used to treat and prevent further growth in cases of hiv / aids were education , counseling , hiv testing , antiretroviral medication , and building health care infrastructure . the treatment category of funding from pepfar focused on the amount of monetary support given to an african country for hiv treatment , the purchase of antiretroviral ( arv ) drugs , and the building of health infrastructure . one of the most essential components of the pepfar program was the increased access to generic arv medication and therapy . the purpose of the arv medication is to reduce the plasma level of hiv viral load.[19 , 20 ] the reduced viral load resulting from arv therapy not only improves the health of the infected individual , but also lowers the risk of passing the disease on to someone else . the availability of arv was critical in prolonging the lives of hiv infected individuals and reducing transmission rates . the prevention category of pepfar focused on the foundational strategy of preventing the proliferation of hiv . this foundation was known as the abc approach : ( a ) abstain , ( b ) be faithful , and ( c ) use condoms . there was strong emphasis on the abstinence from sex before marriage and be faithful aspects , which sometimes generated controversy because of the religious component in the program . safe - sex education was also a pertinent program in the fight against hiv / aids . the care category of pepfar focused on counseling , testing people for hiv / aids , taking care of orphan children , and palliative care for individuals with hiv / aids . since the majority of people in sub - saharan africa are unaware of their hiv status , this negligence could well lead to the rapid spread of the disease through sexual intercourse . the final category of pepfar efforts focused on training in health management , increasing health care staffing levels , and strengthening the healthcare management system . health management and effective management systems are important because one of the common theories is that the spread of hiv / aids in africa is worsened by high corruption rates and lack of accountability among government officials . the rampant level of government corruption may prevent government officials from being effective leaders in addressing the hiv / aids pandemic . to this day , africa prevails as the most hiv / aids affected continent in the world , in particular the sub - saharan areas . africans account for approximately two thirds of hiv infections and three quarters of deaths associated with hiv from around the world . the joint united nations programme on hiv / aids ( unaids ) , united nations children s fund ( unicef ) , and world health organization ( who ) determined that approximately 75% of people in sub - saharan africa between the ages of 15 and 49 are unaware of their hiv status . together , hiv and aids create reproductive concerns , reduced revenues , increased healthcare expenses , decreased school enrollment , increased rates of absence at work , disruptions in the family environment , decreased human capital , increases in poverty rates , a lower gross domestic product ( gdp ) , and more orphaned children.[9 - 14 ] due to high levels of poverty , many developing countries in sub - saharan africa do not have the necessary resources or funding to combat the hiv / aids pandemic occurring in their nations . the methods that were used to treat and prevent further growth in cases of hiv / aids were education , counseling , hiv testing , antiretroviral medication , and building health care infrastructure . the treatment category of funding from pepfar focused on the amount of monetary support given to an african country for hiv treatment , the purchase of antiretroviral ( arv ) drugs , and the building of health infrastructure . one of the most essential components of the pepfar program was the increased access to generic arv medication and therapy . the purpose of the arv medication is to reduce the plasma level of hiv viral load.[19 , 20 ] the reduced viral load resulting from arv therapy not only improves the health of the infected individual , but also lowers the risk of passing the disease on to someone else . the availability of arv was critical in prolonging the lives of hiv infected individuals and reducing transmission rates . the prevention category of pepfar focused on the foundational strategy of preventing the proliferation of hiv . this foundation was known as the abc approach : ( a ) abstain , ( b ) be faithful , and ( c ) use condoms . there was strong emphasis on the abstinence from sex before marriage and be faithful aspects , which sometimes generated controversy because of the religious component in the program . safe - sex education was also a pertinent program in the fight against hiv / aids . the care category of pepfar focused on counseling , testing people for hiv / aids , taking care of orphan children , and palliative care for individuals with hiv / aids . since the majority of people in sub - saharan africa are unaware of their hiv status , this negligence could well lead to the rapid spread of the disease through sexual intercourse . the final category of pepfar efforts focused on training in health management , increasing health care staffing levels , and strengthening the healthcare management system . health management and effective management systems are important because one of the common theories is that the spread of hiv / aids in africa is worsened by high corruption rates and lack of accountability among government officials . the rampant level of government corruption may prevent government officials from being effective leaders in addressing the hiv / aids pandemic . based on theoretical evidence , this study expected that the 12 focus countries in africa that received pepfar funding would see a decrease in the hiv infection rates . even though arv medications are able to prolong the lives of hiv infected individuals , the hiv infection rates that are calculated by the world bank take into consideration individuals who are taking the arv medication . the software used for the collection of hiv infection rates reduced the infectivity rates among people receiving arv treatments . table 1 lists the 12 countries in africa that received pepfar funding and table 2 provides the amount of pepfar funding that was received from 2004 through 2008 in millions of us dollars . table 3 shows the hiv infection rates of males and females between the ages of 15 and 49 in the 12 countries in africa that received pepfar funding from 2004 through 2008 . pepfar focus countries in sub - saharan africa source : united states president s emergency plan for aids relief countries that received pepfar funding ( millions of us dollars ) , 20042008 source : united states president s emergency plan for aids relief hiv infection rates ( % ) of males and females ages 1549 , 20042008 source : united states president s emergency plan for aids relief the following theoretical models were applied to this research : hivrateit= 1 + 2%pepfar per gdpit+ it hivrateit= 1 + 2%pepfar per gdpit + 3%health per gdpit + 4corruptionit + 5gdppcit+ it the dependent variable was hiv infection rates in africa . the control independent variables were healthcare expenditure per gdp , corruption rate of a country , and gdp per capita . where : 1 is the intercept of the models and it is the stochastic error term of the models.hivrateit was the hiv infection rates of males and females between the ages of 15 and 49 measured in percentage ( world bank).%health per gdpit was the percent of total public and private healthcare expenditures per gdp in us dollars ( world bank).gdppcit was the gdp per capita of each country in us dollars ( world bank).%pepfar per gdpit was the percent of pepfar funding received per gdp in us dollars ( pepfar).corruptionit was the score of perceptions on the level of corruption in the public sector , based on the corruption perceptions index , where 0=very corrupt and 10=not corrupt ( transparency international ) . 1 is the intercept of the models and it is the stochastic error term of the models . hivrateit was the hiv infection rates of males and females between the ages of 15 and 49 measured in percentage ( world bank ) . % health per gdpit was the percent of total public and private healthcare expenditures per gdp in us dollars ( world bank ) . gdppcit was the gdp per capita of each country in us dollars ( world bank ) . % pepfar per gdpit was the percent of pepfar funding received per gdp in us dollars ( pepfar ) . corruptionit was the score of perceptions on the level of corruption in the public sector , based on the corruption perceptions index , where 0=very corrupt and 10=not corrupt ( transparency international ) . this study used panel data from 2002 through 2010 covering the 12 countries that received pepfar funding in the continent of africa . even though pepfar was active from 2004 through 2008 , this research includes two years before ( 2002 and 2003 ) and two years after ( 2009 and 2010 ) in order to increase the ability to estimate the actual effect of receiving pepfar funding . in order to analyze the correlation between pepfar funding and hiv infection rates in africa , this research used stata software version 13 and conducted a fixed - effects panel regression analysis . the fixed - effects technique was chosen based on results from the hausman and sargan - hansen tests , which were performed through stata . there was the possibilities of both heteroskedasticity and autocorrelation in this study because this analysis included a combination of cross - sectional and time - series data . the use of panel data for this research required statistical tests to determine if the regression model did indeed encounter a problem with heteroskedasticity and autocorrelation . as expected , the results of the tests indicated the presence of these problems in the regression model . in order to correct for the heteroskedasticity and autocorrelation problems , this research used the robust standard errors for panel regression with cross - sectional dependence model . hivrateit= 1 + 2%pepfar per gdpit + 3%health per gdpit + 4corruptionit + 5gdppcit+ it the dependent variable was hiv infection rates in africa . the control independent variables were healthcare expenditure per gdp , corruption rate of a country , and gdp per capita . where : 1 is the intercept of the models and it is the stochastic error term of the models.hivrateit was the hiv infection rates of males and females between the ages of 15 and 49 measured in percentage ( world bank).%health per gdpit was the percent of total public and private healthcare expenditures per gdp in us dollars ( world bank).gdppcit was the gdp per capita of each country in us dollars ( world bank).%pepfar per gdpit was the percent of pepfar funding received per gdp in us dollars ( pepfar).corruptionit was the score of perceptions on the level of corruption in the public sector , based on the corruption perceptions index , where 0=very corrupt and 10=not corrupt ( transparency international ) . 1 is the intercept of the models and it is the stochastic error term of the models . hivrateit was the hiv infection rates of males and females between the ages of 15 and 49 measured in percentage ( world bank ) . % health per gdpit was the percent of total public and private healthcare expenditures per gdp in us dollars ( world bank ) . gdppcit was the gdp per capita of each country in us dollars ( world bank ) . % pepfar per gdpit was the percent of pepfar funding received per gdp in us dollars ( pepfar ) . corruptionit was the score of perceptions on the level of corruption in the public sector , based on the corruption perceptions index , where 0=very corrupt and 10=not corrupt ( transparency international ) . this study used panel data from 2002 through 2010 covering the 12 countries that received pepfar funding in the continent of africa . even though pepfar was active from 2004 through 2008 , this research includes two years before ( 2002 and 2003 ) and two years after ( 2009 and 2010 ) in order to increase the ability to estimate the actual effect of receiving pepfar funding . in order to analyze the correlation between pepfar funding and hiv infection rates in africa , this research used stata software version 13 and conducted a fixed - effects panel regression analysis . the fixed - effects technique was chosen based on results from the hausman and sargan - hansen tests , which were performed through stata . there was the possibilities of both heteroskedasticity and autocorrelation in this study because this analysis included a combination of cross - sectional and time - series data . the use of panel data for this research required statistical tests to determine if the regression model did indeed encounter a problem with heteroskedasticity and autocorrelation . as expected , the results of the tests indicated the presence of these problems in the regression model . in order to correct for the heteroskedasticity and autocorrelation problems , this research used the robust standard errors for panel regression with cross - sectional dependence model . although several components have likely contributed to the declining hiv rates in the pepfar focus countries , the main objective of this research was to determine whether pepfar was an effective factor in lowering the hiv / aids infection rates , and , if so , by how much . table 4 provides the results from the fixed - effects panel regression analysis for models 1 and 2 . in model 1 , pepfar funding per gdp was statistically significant at the 0.01 level . in model 2 , pepfar funding per gdp and gdp per capita were statistically significant at the 0.01 level . even though this research found correlation between hiv infection rates and pepfar funding , as the adage goes , correlation does not imply causation . thus , the results must be considered in light of this caveat , as is the case with all empirical analyses . in model 1 , the results illustrate that on average , ceteris paribus , for every 1 percentage point increase in pepfar funding per gdp a country received , the country s hiv infection rate decreased by 0.41 percentage points . the within r value for this model was 0.1684 , indicating that 16.84% of the variability in the dependent variable was explained by the independent variables . in model 2 , the results illustrated that on average , ceteris paribus , for every 1 percentage point increase in pepfar funding per gdp a country received , the country s hiv infection rate decreased by 0.355 percentage points . for every $ 1,000 increase in gdp per capita a country received , the hiv infection rate decreased by 0.37 percentage points . for every 1 percentage point increase in healthcare expenditure per gdp , the hiv infection rate decreased by 0.10 percentage points . the within r value for this model was 0.3733 , indicating that 37.33% of the variability in the dependent variable was explained by the independent variables . figure 1 provides graphs depicting a comparison between the amounts of pepfar funding received by the 12 countries in sub - saharan africa and the hiv rates from 2002 through 2010 . even though pepfar funding had steadily increased for each of the countries , most of the hiv infection rates decreased slightly , but remained relatively steady overall . comparison of hiv infection rates and pepfar funding the united states provided large monetary support toward the pepfar program . even though the results indicated that pepfar was statistically significant , the evaluation of whether pepfar was politically and economically significant remains subjective . some may argue that any decrease in hiv infection rates highlights the relevance of the continued efforts of pepfar , while others could argue that the large amount of money invested in pepfar should have generated larger decreases in the hiv infection rates in africa than the rates found in this study . based on this research , the overall hiv rate decreased , but the large amount of money invested to combat hiv infections did not drastically lower the hiv infection rates . the success of combating hiv / aids involves the collaboration of many public and humanitarian organizations . future research should include analysis on the impact of public sector agencies , universities , faith - based organizations , private contractors , and non - governmental organizations in this philanthropic mission . the center for global development released data that provided specific dollar amounts on how much pepfar funding was allocated to the different organizations from 2004 through 2006 in the categories of treatment , prevention , care , and other . although the data is sparse , analyzing the data supplied by the center for global development would provide a better indication of the role and impact each sector had in the pepfar program . a clear understanding of the global impact with the struggle against hiv / aids and the role that pepfar has played in that struggle is appropriate for future public policies , legislation , and programs . to the degree that it succeeded , the objectives and intentions of pepfar were met through an embrace of global connections and cooperation in order to reduce the increasing spread of hiv / aids in sub - saharan africa . the global community is becoming more interconnected and societies are becoming mutually dependent on one another to combat health crises . the increase of social , economic , and political globalization broadens the realization that hiv / aids does not recognize race , gender , ethnicity , sexual orientation , or geographic borders . pepfar had some impact on the hiv pandemic in the 12 focus countries in sub - saharan africa through increasing humanitarian funding , providing access to cost - effective arv medication , developing improved healthcare infrastructures , and implementing a number of prevention programs . even though hiv / aids can not be cured as of yet , there is intrinsic value in preventing the rapid spread of this malady . although the programs and allocated funding of pepfar accomplished most of the stated objectives , there remains even more that needs to be accomplished , for hiv / aids continues to persist as a global pandemic presenting significant challenges . despite a few limitations , this study was able to analyze and illustrate the relative effectiveness of pepfar funding in lowering the hiv infection rates on the continent of africa , and has hopefully provided valuable insight for future policy considerations . the objectives and intentions of pepfar were met through an embrace of global connections and cooperation in order to reduce the increasing spread of hiv / aids in sub - saharan africa.on average , ceteris paribus , for every 1 percentage point increase in pepfar funding per gdp a country received , the country s hiv infection rate decreased by 0.36 percentage points.for every $ 1,000 increase in gdp per capita a country received , the hiv infection rate decreased by 0.37 percentage points.for every 1 percentage point increase in healthcare expenditure per gdp , the hiv infection rate decreased by 0.10 percentage points . the objectives and intentions of pepfar were met through an embrace of global connections and cooperation in order to reduce the increasing spread of hiv / aids in sub - saharan africa . on average , ceteris paribus , for every 1 percentage point increase in pepfar funding per gdp a country received , for every $ 1,000 increase in gdp per capita a country received , the hiv infection rate decreased by 0.37 percentage points . for every 1 percentage point increase in healthcare expenditure per gdp , the hiv infection rate decreased by 0.10 percentage points .

background : hiv and aids continue to have a calamitous effect on individuals living on the continent of africa . u.s . president george w. bush implemented the president s emergency plan for aids relief ( pepfar ) with the objective of committing approximately $ 15 billion from 2004 through 2008 to assist with the reduction of the hiv pandemic worldwide . the majority of the pepfar policy and funding focused on 12 countries in sub - saharan africa : botswana , cote divoire , ethiopia , kenya , mozambique , namibia , nigeria , rwanda , south africa , tanzania , uganda , and zambia . the policy question this research paper seeks to analyze is whether the pepfar funding ( as a % of gross domestic product ( gdp ) ) allocated to the 12 countries in africa had any effect on the decrease of hiv infection rates of males and females between the ages of 15 and 49.methods:a fixed - effects panel regression analysis was conducted to determine if this association exists . this study examined the 12 african countries that received pepfar funding over the years 2002 to 2010 ; even though pepfar was only active from 2004 through 2008 , this research included two years prior and two years after this timeframe in order to better estimate the effect of pepfar funding on hiv reduction.results:the results illustrate that on average , ceteris paribus , for every 1 percentage point increase in pepfar funding per gdp a country received , the country s hiv infection rate decreased by 0.355 percentage points.conclusions and global health implications : while the empirical findings in this study suggested that the correlation between pepfar funding and hiv reduction is statistically significant , the practical significance is perhaps less obvious . arguably , the reduction rate should be higher given the extent of funding targeted to this project . the conclusion of this research provides suggestions on future research and the policy implications of pepfar .

the incidence of colorectal cancer is one of the highest malignancies . in netherlands alone , colorectal cancer is diagnosed in 12,000 patients annually and it is the second most frequent cause of death due to malignancies . in an effort to improve the standard of care for these patients , new techniques have been introduced over the years , such as laparoscopy and tme surgery [ 3 , 4 ] . recently , much attention has been given to patient volume of both the hospital and the individual surgeon . publications have shown that a high - volume surgeon operating in a high - volume hospital leads to an improved short - term outcome such as a lower number of adverse events , shorter hospital - stay , lower postoperative mortality , and cost reduction . furthermore , a number of studies have reported increased long - term survival when patients are treated in high - volume centers . however , the relationship between operative volume of the surgeon and long - term outcome remains unclear . we therefore conducted the current study to evaluate survival rates of patients with colorectal cancer following a procedure performed by high - volume surgeons compared to low - volume surgeons . the rijnland hospital is a teaching hospital in leiderdorp , netherlands , serving approximately 200,000 people . for the current study a retrospective analysis was conducted from our prospectively collected database including all colorectal cancer patients who underwent surgery in our hospital between 2004 and 2011 . eight hundred and twenty - four patients underwent a colorectal procedure between 2004 and 2011 . for our study we used the same inclusion criteria as the national web - based registry for the surgical treatment of colorectal cancer in netherlands : the dutch surgical colorectal audit ( dsca ) . patients were excluded in case the procedure was performed for metastatic disease following previous surgery ( n = 13 ) , in case the primary tumor could not be resected ( n = 9 ) , or when the pathology report showed a different type of tumor than an adenocarcinoma ( n = 28 ) . after applying these exclusion criteria our study population of 774 patients consisted of a homogenous cohort . in order to qualify as a high - volume surgeon a cut - off point of 25 colorectal resections per year , averaged over the study period , was chosen based on recent studies [ 6 , 913 ] . taking this criterion into account for our analysis , 13 low - volume surgeons operated on 453 patients and four high - volume surgeons operated on 321 patients . perioperatively , all patients received equal care using the colorectal enhanced recovery after surgery ( eras ) protocol [ 14 , 15 ] . the data collected in our database were the patient characteristics , including the american society of anesthesiology- ( asa- ) classification ; the intraoperative data ( high - volume surgeon versus low - volume surgeon ) ; and the postoperative data , including the tnm - stage , resection margins , length of hospital - stay , and adverse events . in case of an adverse event , the type ( surgical or nonsurgical ) and the severity were recorded according to netherlands ' society of surgery standard [ 18 , 19 ] . follow - up took place in our hospital according to netherlands ' society of surgery protocol . this protocol dictates that patients are seen in the hospital for follow - up by an attending surgeon every 4 months for the first 2 years and every 6 months for the years after , with a minimum of 5-year follow - up . in the current study patients were followed up for a minimum of 3 years . at each visit , an ultrasound and cea levels were performed . also the iknl ( integral cancer centre netherlands ) was consulted in case a patient deceased , which provided us with the date and cause of death . for some patients the follow - up did not take place in our hospital , mostly due to relocation of the patient . in those cases we consulted the general practitioner and the hospital where the follow - up was taking place for survival data . it was possible to evaluate the 5-year disease - free survival ( dfs ) of 761 patients ( 13 patients lost to follow - up : 8 low - volume and 5 high - volume ) and overall survival ( os ) of 772 patients ( 3 low - volume patients were lost to follow - up ) . for assistance with the statistical analysis , the department of statistics in our hospital and the leiden university medical center were consulted . comparisons were made between the high - volume and the low - volume group for all variables : perioperative characteristics , disease - free survival , and overall survival . the -test and the independent sample t - test were used to determine the association between perioperative characteristics and volume ( high - volume versus low - volume ) . multivariate poisson regression survival models were used to determine the effect of volume on dfs and os . variables in the univariate analysis that showed a significant association were then introduced into a cox regression multivariate model . preoperative clinicopathological characteristics of the 453 low - volume and the 321 high - volume patients are shown in table 1 . the groups were comparable except the fact that a greater number of the low - volume patients had a higher asa - classification ( p < 0.001 ) and laparoscopic surgery was more frequently performed in the high - volume group compared to the low - volume group , 78% ( n = 249 ) versus 59% ( n = 266 ) , respectively ( p < 0.001 ) . the type of resection also showed a difference ( p < 0.001 ) , largely caused by a higher number of abdominoperineal resections ( apr ) in the high - volume group . the significantly larger number of patients who received chemoradiotherapy as neoadjuvant regimen in this group can be explained by the higher number of rectal cancer cases ( p < 0.001 ) . the intraoperative data are listed in table 2 . in the high - volume group , significantly less blood loss was observed compared to the low - volume group , 308 ml versus 547 ml , respectively ( p < 0.001 ) . also the conversion rate in case of laparoscopic surgery was significantly lower in the high - volume group ( 18% versus 27% , p = 0.01 ) . the postoperative characteristics are listed in table 3 . a significantly more advanced tumor ( t ) stage ( p = 0.03 ) and metastatic ( m ) stage ( p < a larger median number of lymph nodes were harvested in the high - volume group , 15.3 versus 13.5 ( p < 0.001 ) . in the high - volume group the median postoperative hospital - stay was lower compared to the low - volume group : 10 versus 13 days , respectively ( p < 0.001 ) . no difference was seen between nodal ( n ) stages or resection margins and the number and neither did the severity of both surgical and nonsurgical adverse events show a difference in both groups . the 5-year dfs in the high - volume group was 66% compared to 48% in the low - volume group ( p < 0.001 , figure 1 ) . we performed a univariate analysis to estimate the effect of all variables on the dfs . the high - volume group showed a significantly increased dfs ( hazard ratio ( hr ) 0566 ; 95% ci 0.440.74 ; p < 0.001 ) . the other pre- , intra- , and postoperative variables that showed a statistical significance for dfs in the univariate analysis are listed in the left half of table 4 . we then incorporated the statistically significant variables of the univariate analysis into a cox multivariate regression model to determine which variables remained as prognostic factors for dfs . surgeons ' volume showed to be an independent prognostic factor for dfs in favor of the high - volume surgeon ( hr 0.739 ; 95% ci 0.560.99 ; p = 0.04 ) . other independent prognostic factors for a longer dfs were lower patient 's age ( p < 0.001 ) , lower asa - classification ( p = 0.05 ) , and a lower t ( p = 0.04 ) , n ( p < 0.001 ) , and m ( p < 0.001 ) stage . we also analyzed if the time periods ( 20042007 versus 20082011 ) had an influence on dfs ; however , neither in the univariate or in the multivariate analysis did this show significance . the patients in the high - volume group showed a 5-year os of 75% as compared to 54% for the low - volume group ( p < 0.001 , figure 2 ) . similarly to what is described above , we performed a univariate analysis for os . the high - volume surgeon was significantly associated with an increased os ( hr 0.495 ; 95%ci 0.350.69 ; p < 0.001 ) . the other pre- , intra- , and postoperative variables that showed a statistical significance for os in the univariate analysis are listed in the left half of table 5 . after incorporating the statistically significant variables of the univariate analysis into a cox multivariate regression model to determine which variables remained as prognostic factors for os , the high - volume surgeon did not remain significant ( hr 0.731 ; 95% ci 0.711.68 ; p = 0.09 ) . we also analyzed if the time periods ( 20042007 versus 20082011 ) had an influence on os ; however , neither in the univariate or in the multivariate analysis did this show significance . the factors that did prove to be an independent prognostic factor were advanced age ( p < 0.001 ) , higher asa - classification ( p = 0.01 ) , and higher n ( p < 0.001 ) and m ( p < 0.001 ) stage which showed to have an independent negative influence on os . laparoscopic surgery appeared to be a positive independent prognostic factor for os ( p < 0.001 ) . in the current analysis we found that a high - volume surgeon is an independent prognostic factor for increased dfs for colorectal cancer surgery when compared to a low - volume surgeon . however , high - volume surgery did not remain as an independent prognostic factor for os in the multivariate analysis . although increased dfs is an important outcome in research , ultimately a longer os is what is most desirable in medicine and what is important to the patient . possibly in a larger cohort of patients we may show an increased os in the future since os did show to be significantly increased in the high - volume surgery patients in the univariate analysis . previous studies have been performed to investigate possible variables of short - term and long - term outcomes following colorectal resection for malignancies . these outcomes depend on numerous patient- , surgeon- , and hospital - related variables [ 5 , 9 , 2227 ] . while the patient - related variables are difficult , if not impossible to adjust , efforts aimed at improving the perioperative care have been shown to have positive impact on the postoperative outcome . some of these efforts include the administration of preoperative antibiotics , maintaining normothermia during surgery , and implementing an eras protocol [ 15 , 16 , 2830 ] . another approach that has been shown to be effective is implementing high - volume surgery of colorectal procedures . recent studies have shown an improved short - term outcome , when a high - volume surgeon performed the procedure [ 9 , 23 , 24 , 31 ] . studies reporting long - term effects for high - volume colorectal surgery , however , have shown less unanimous results [ 12 , 26 , 27 , 32 , 33 ] . although high - volume surgery showed a significant relationship towards an increased os in the univariate analysis , it did not remain as an independent prognostic factor for os in the multivariate analysis . when looking at the difference in os between the high - volume group and the low - volume group ( figure 2 ) in the univariate analysis , it seems likely that , with either a larger patient population or an increased median follow - up time , this observed difference could also become statistically significant in the multivariate analysis . this would of course be an important outcome for our patients , as increased os is even more relevant than an increased dfs . our findings are in agreement with the outcomes of studies of low - volume surgical procedures , such as esophageal and pancreatic cancer surgery , in which it has been shown that the surgeon 's caseload is an important predictor for outcome [ 34 , 35 ] . also support for our assumption of an increased os can be found in the article by rogers jr . et al . who showed that , in a group of 26,644 patients with a median follow - up of 6 years , those who were operated upon by a high - volume surgeon had an increased os following colorectal cancer . the same is seen in the cochrane analysis in which an improved survival is reported for both the high - volume surgeon and the high - volume hospital . however , not all studies reporting survival after high - volume surgery for colorectal cancer are in agreement . [ 27 , 32 , 33 , 36 , 37 ] , showing that more research in this field is required before any definite statements can be made regarding the volume an individual surgeon should perform . recently published literature has increasingly published a similar observation showing an improved os following laparoscopic colorectal surgery when compared to open colorectal surgery [ 3841 ] . it has been suggested that this improved os observed in the recent years is mostly caused by the increased experience with the procedure together with technical and procedural advances . although this can not be attributed to the implementation of high - volume surgeons alone , the fact that dfs remained as an independent prognostic factor in the multivariate analysis shows that high - volume surgery is attributed to improved survival . in a previous report , renzulli et al . observed a similar increase in dfs for high - volume surgery . however , in the multivariate analysis this did not remain significant showing that the difference in dfs seen between both groups can not be explained by the timing of the surgery and the on - call surgeon . apart from the increased dfs in the multivariate and the increased os in the univariate analysis , a number of perioperative variables also showed statistical significance in favor of the high - volume surgeon . in case a high - volume surgeon performed a laparoscopic procedure , a significantly lower number of conversions were observed . furthermore , intraoperative blood loss was significantly less in the high - volume group and a greater number of lymph nodes were harvested leading to a more accurate staging . a decrease in postoperative adverse events has been reported in case the operation was performed by a high - volume surgeon [ 9 , 11 ] . possibly , underreporting of adverse events may have taken place in our study : the number of days a patient was admitted to the hospital following surgery by a high - volume surgeon was significantly lower , suggesting a quicker and uncomplicated recovery . therefore , in our hospital , high - volume surgery does not only improve the dfs and possibly os but also improve short - term outcome the low - volume group consisted of more patients with a higher asa - classification and a higher tnm - stage [ 16 , 17 ] . the early drop of dfs seen in the low - volume patients that is demonstrated in the kaplan - meier curve is most likely due to the more advanced disease in this group ( figure 1 ) . for this reason a multivariate analysis was conducted to correct for these differences in patient population . even after this correction high - volume surgery remained as an independent factor for dfs . rectal cancer resections were performed more frequently by the high - volume surgeons , which could have caused fewer postoperative complications due to increased experience . on the other hand , one could have expected more complications following the neoadjuvant radiotherapy and chemotherapy in this group , but this was not the case in the statistical analyses . in conclusion , the current study shows that in our hospital high - volume surgery is an independent prognostic factor for increased dfs following surgery for colorectal cancer . although high - volume surgery also significantly improved os in the univariate analysis , it did not remain statistically significant in the multivariate analysis . it is possible that , with either more patients included or a longer follow - up time , this observed difference will also become statistically significant for os . to our opinion , introducing high - volume surgeons will provide better perioperative care for patients suffering from colorectal cancer resulting in both improved short - term and long - term results .

background . surgery performed by a high - volume surgeon improves short - term outcomes . however , not much is known about long - term effects . therefore we performed the current study to evaluate the impact of high - volume colorectal surgeons on survival . methods . we conducted a retrospective analysis of our prospectively collected colorectal cancer database between 2004 and 2011 . patients were divided into two groups : operated on by a high - volume surgeon ( > 25 cases / year ) or by a low - volume surgeon ( < 25 cases / year ) . perioperative data were collected as well as follow - up , recurrence rates , and survival data . results . 774 patients underwent resection for colorectal malignancies . thirteen low - volume surgeons operated on 453 patients and 4 high - volume surgeons operated on 321 patients . groups showed an equal distribution for preoperative characteristics , except a higher asa - classification in the low - volume group . a high - volume surgeon proved to be an independent prognostic factor for disease - free survival in the multivariate analysis ( p = 0.04 ) . although overall survival did show a significant difference in the univariate analysis ( p < 0.001 ) it failed to reach statistical significance in the multivariate analysis ( p = 0.09 ) . conclusions . in our study , a higher number of colorectal cases performed per surgeon were associated with longer disease - free survival . implementing high - volume surgery results in improved long - term outcome following colorectal cancer .

angiomyolipoma ( aml ) is a benign mesenchymal tumour consisting of varying amounts of mature adipose tissue , smooth muscle , and thick walled blood vessels . it is relatively rare benign tumour appearing in about 0.3% of general population and accounts for 3% of solid renal masses . extra renal amls ( eramls ) are rare tumours that present as incidentalomas upon imaging for other conditions . case reports of extra renal aml are rare with < 60 reported cases since they were first described by friis and hjortrup in 1982 . our case described one of the largest ever reported myolipomas , which despite its size , can be rather asymptomatic . magnetic resonance imaging ( mri ) revealed 9.5 cm 8 cm 2 cm right adrenal mass [ figure 1 ] . the tumour was predominantly hyperintense with signal intensity similar to fat on t1/t2-weighted images and lost signal on the fat - suppressed sequences . a well - encapsulated , firm globular mass separate from the right kidney and no definable adrenal was found [ figures 2 and 3 ] . gross post - operative evaluation of the adrenal mass showed approximately 10 cm 8 cm 2 cm greyish white solid , smooth , and firm mass [ figure 4 ] . histological examination revealed benign adrenal cortical elements , with a predominance of mature benign adipose tissue interspersed with pockets of haemopoietic ( erythro - myeloid ) intermediates , and megakaryocytes which was confirmatory for the diagnosis of adrenal aml . angiomyolipoma is a rare clinical entity mostly involving kidney and is part of the group of tumour known as tumours of perivascular epithelioid cell origin . the most widely accepted theory of origin is adrenocortical cell metaplasia in response to stimuli , such as necrosis , inflammation , infection or stress ( meyer et al . ) . the presence of perivascular epithelioid cells is characteristic of aml since these cells show immunoreactivity for muscle markers ( epithelial membrane antigen , keratin , vimentin , desmin , and actin ) and hmb-45 . positive immunoreactivity for hmb-45 , a monoclonal antibody , is characteristic of amls and can be used to differentiate amls from other similar appearing lesions . one is isolated aml , and the other is that is associated with tuberous sclerosis . most patients of isolated cases are in the age group of 27 - 72 years , mean age being 43 years . although the majority of eramls are benign , two cases of metastatic and recurrent eramls have been reported . the demonstration of fat density ( hypodense ) within an adrenal mass by computed tomography is virtually diagnostic of aml . mri is sometimes required to demonstrate origin of the tumour , to define the tissue planes when the tumour is large and heterogeneous , and to distinguish benign from malignant lesions by comparing signal intensity ratios of adrenal to liver . a small , < 5 cm , asymptomatic myelolipoma could be followed - up over 1 - 2 year period with imaging controls . on the contrary , asymptomatic lesion or a large > 5 cm myelolipoma should be surgically excised , since there are reports of spontaneous rupture and haemorrhage of the mass presented with life - threatening cardiovascular shock . unstable patients may benefit from emergent tumour embolisation and a subsequently staged surgical resection . since it is a benign disease , its prognosis is good . nevertheless , follow - up is recommended because of atypical morphology . currently , there is no agreed protocol on follow - up but an ultrasound three to 6 months following the surgery with annual clinical examination for large tumours is recommended . the adrenal myolipoma is a rare urological entity , which seems to increase its frequency , probably due to causes affecting primarily the function and physiology of the adrenals . ever since its introduction by gagner in 1992 , laparoscopic adrenalectomy has become the standard of care for the treatment of functioning and non - functioning adrenal tumours . many authors have found a decrease in perioperative morbidity and convalescence after this procedure when compared with open surgery .

angiomyolipoma ( aml ) is a rare mesenchymal tumour arising from perivascular epithelioid cells . it is most commonly seen in kidney , but rarely aml can arise in extra renal sites . adrenal aml is a very rare clinical entity , and very few cases have been reported so far . we present our experience with a 43-year - old female , who presented with right flank pain . magnetic resonance imaging showed a right adrenal mass . laparoscopic adrenelectomy was performed , and the histopathology report confirmed the diagnosis of aml . patient was discharged uneventfully .

when patients present with acute occlusion of either the internal carotid artery ( ica ) or the proximal middle cerebral artery ( mca ) within the therapeutic time window for restoration of cerebral blood flow , rapid recanalization using intravenous / intra - arterial ( ia ) thrombolysis and ia mechanical embolectomy is the goal of acute stroke management.11 ) however , if the patient presents beyond the therapeutic time window , life - threatening brain swelling and herniation , known as malignant mca infarction , can manifest within one week after the onset of stroke symptoms , along with a further decrease of consciousness and pupillary dilatation , necessitating early application of a decompressive hemicraniectomy for achievement of a better clinical outcome.24 ) the present article reviews the pathophysiology , historical background in previous studies , operative timing , surgical technique and clinical outcomes of decompressive hemicraniectomy for malignant mca infarction involving the mca territory with or without anterior cerebral artery and posterior cerebral artery territories . acute occlusion of either the ica or the proximal mca and insufficient collateral blood flow result in cerebral infarction of the mca territory and associated severe brain edema . cytotoxic edema results from failure of sodium - potassium adenosine triphosphatase in brain cell membranes and is followed by vasogenic edema in association with disruption of the blood - brain barrier . the space - occupying lesion inside the cranial vault increases the intracranial pressure , thereby reducing the cerebral perfusion pressure and cerebral blood flow . in particular , a space - occupying lesion in a unilateral hemisphere causes pressure gradients between the supratentorial and infratentorial compartments and between the bilateral supratentorial compartments . the transtentorial uncal herniation and the resultant displacement of the subthalamic - upper brainstem structures will impair the consciousness of the patient . thus , the aim of decompressive surgery is external herniation of the swollen infarcted brain for relief of brainstem compression and to reduce intracranial pressure . the original purpose of decompressive hemicraniectomy was to help patients survive in cases of acute large hemispheric infarction.25 ) various case reports , retrospective studies and trials have suggested that this surgical treatment lowers mortality without increasing the incidence of severely disabled survivors.8)13)15)23)27)29 ) three european randomized , controlled clinical trials were conducted between 2001 and 2007.10)12)32 ) however , the decimal ( decompressive craniectomy in malignant middle cerebral artery infarction ) trial in france and destiny ( decompressive surgery for the treatment of malignant infarction of the middle cerebral artery ) trial in germany were stopped due to slow recruitment of cases and significant difference in mortality between groups.12)32 ) notwithstanding , the hamlet ( hemicraniectomy after middle cerebral artery infarction with life - threatening edema trial ) study , conducted in the netherlands , was completed and published in 2009.10 ) thirty two patients were randomly assigned to undergo surgical decompression and another 32 patients received the best medical treatment over a five - year period . according to the results , the use of hemicraniectomy resulted in a reduction in the number of case fatalities and poor outcomes ( modified rankin scale score 5 ) for patients with large hemispheric infarction who were treated within 48 hours of stroke onset . the clinical outcome for patients is considered to show improvement with early surgical decompression before or immediately after any neurological deterioration related to brain swelling.10)22)26)28)31)33)36 ) this clinical deterioration includes pupil asymmetry , an altered consciousness level and aggravated hemiplegia.6 ) patients commonly manifest such neurological deterioration within one week after the onset of stroke symptoms . in particular , one third of patients show deterioration within 24 hours , while another third show deterioration 24 - 48 hours after symptom onset.24 ) to date , many clinical and radiological data defining early predictors of malignant hemispheric infarction have been reported.6)7)9)14)16)18)20)30)34 ) malignant edema after acute infarction can be predicted based on the volume of the infarcted brain tissue , however , extension of the initial infarct territory , delayed spontaneous recanalization of the occluded vessel , hemorrhagic transformation of the infarcted brain and the fluid volume state of the patient can all make the prediction difficult . thus , the predictors lack a sufficient predictive value with regard to selection of candidates for a decompressive hemicraniectomy prior to neurological deterioration . in addition , in order to ensure timely decompressive surgery , all patients with acute large hemispheric infarction should be observed in an intensive care unit or stroke unit setting . thus , determination of strict cutoff criteria with a high specificity and positive predictive value for malignant infarction is needed.21 ) the predictive value of the infarct volume assessed by early ct scan after stroke onset has not been found to be satisfactory , as follows : ( 1 ) hypodensity covering > 50% of mca territory within five hours after symptom onset was predictive of a malignant course with a sensitivity of 61% and specificity of 94%;34 ) ( 2 ) hypodensity covering > 50% of mca territory within 12 hours was predictive with a sensitivity of 64% and specificity of 66%;17 ) ( 3 ) hypodensity covering > 50% and 67% of mca territory within 18 hours was predictive with a sensitivity of 58% and 45% , respectively and specificity of 94% and 100% , respectively.7 ) one useful predictor is the initial infarct volume assessed using diffusion - weighted magnetic resonance ( mr ) imaging > 145 cm within 14 hours after acute mca occlusion , which achieved a sensitivity of 100% and a specificity of 94% in the study by oppenheim et al.20 ) the cutoff criteria for lesion volume and associated midline brain shift for prediction of malignant edema should differ according to the timing after stroke onset.4)21 ) gerriets et al . reported a midline shift 2.5 , 3.5 , 4.0 and 5.0 mm in transcranial color - coded duplex sonography as a predictor of malignant edema after 16 , 24 , 32 and 40 hours , respectively , after stroke onset with a specificity of 100% and positive predictive value of 100%.4 ) meanwhile , assessment of the final infarct volume can be performed using perfusion ct or perfusion mr images on admission , however , such perfusion parameters are still not optimal.3)30 ) in the mr perfusion study reported by thomalla et al . , perfusion lesion volume > 162 ml on a time - to - peak ( ttp ) map with a ttp delay threshold of > 4 seconds was found to predict malignant infarction with a sensitivity of 83% and specificity of 75%.30 ) conceptually , decompressive craniectomy procedures include both internal and external decompression . in the case of external decompression , the frontal , temporal and parietal bones overlying the infarcted hemisphere are removed , allowing for external herniation of the swollen infarcted brain . with the patient in a state of general anesthesia , a skin incision is started just above the zygomatic arch 0.5 cm anterior to the tragus and then carried superiorly and posteriorly over the ear and around the parietal bone to the contralateral frontal midpupillary line . the hemicraniectomy then involves the removal of a large fronto - temporo - parietal bone flap , as large as possible . the bone flap is made anteriorly in order to avoid violation of the frontal sinus , except in the case of a huge frontal sinus . the medial limit is 2 cm from the midline , in order to minimize venous bleeding on the dura . the posterior limit of the bone flap is approximately 5 - 6 cm posterior to the external auditory canal , thereby covering the mca territory posteriorly and allowing for a neutral head position without compressing the brain . inferiorly , the temporal squama is removed to the level of the zygomatic arch . following a stellate - shaped dural incision , it is commonly recommended that the infarcted brain tissue not be removed due to the presence of a salvageable penumbra area or viable tissue.10)12)32 ) however , the internal decompression ( removal of the infracted brain tissue and/or an anterior temporal lobectomy ) can be performed for patients with whole hemispheric infarction.19 ) expansive duraplasty is then performed using a large flap of pericranial tissue or an artificial dura substitute . the dimensions of the expansive duraplasty should be extended in order to accommodate the subsequent aggravation of the brain edema . multiple dural tenting sutures , bipolar coagulation of bleeding points on the dural surface , application of commercial hemostatic materials and placement of a closed suction drain in the epidural / subgaleal space are all used . finally , the temporalis muscle and skin flap are reapproximated and sutured layer by layer . however , the temporalis muscle and fascia can be resected in order to maximize external herniation of the swollen brain . removal of the temporalis muscle does not cause problems with chewing , as the grinding phase of the closure stroke only requires one - third of the maximal bite force and only leads to a minimal decrease in the maximal bite force.1)5 ) resection of the temporalis muscle and fascia provides a two - fold volume expansion on average when compared with the conventional technique on postoperative day 3 ( fig . 1).22 ) technical obstacles to obtaining the best external decompression are an insufficient craniectomy size , epidural / subgaleal hematoma , thick and swollen temporalis muscle , tough and inelastic temporalis fascia and tight scalp.2)22)35 ) making hemicraniectomy as large as possible , meticulous hemostasis and resection of the temporalis muscle and fascia will all maximize external herniation of the infarcted brain . cranioplasty is then performed using the autogenous bone flap two to three months after the craniectomy . after decompressive surgery for malignant mca infarction , clinical outcomes at 6 - 12 months after stroke onset have been reported . for the criteria determining favorable and unfavorable outcomes , a dichotomization of the mrs score between 0 - 3 and 4 to death or between 0 - 4 and 5 to death has been used ; however , an mrs score of 0 - 3 is most appropriate for a favorable outcome as it includes independent functional outcomes . in the hamlet study , the patients were randomly assigned to undergo surgical decompression or to receive the best medical treatment within four days of stroke onset.10 ) the decompressive - surgery group had a lower incidence of mortality ( 22% versus 59% ) and mrs 5 - 6 ( 41% versus 59% ) than the group that received the best medical treatment . however , the incidence of patients with mrs of 0 - 3 did not differ between the two groups ( 25% versus 25% ) . results of a pooled analysis of the three european randomized controlled trials indicated a better clinical outcome than that of the hamlet study.31 ) performance of decompressive surgery within 48 hours of stroke onset resulted in reduced mortality and an increase in the number of patients who had a favorable functional outcome . in addition , more patients in the decompressive - surgery group had mrs 0 - 3 ( 43% versus 21% ) and mrs 0 - 4 ( 75% versus 24% ) compared with those in the control group who received the best medical treatment . in analysis of the three predefined subgroups of the decimal trial , in the decompressive - surgery group , younger age showed correlation with a favorable outcome and there was a trend toward a worse outcome in patients with higher infarct volumes.32 ) however , no significant difference in the clinical outcome with mrs scores was observed between surviving patients with dominant and non - dominant hemisphere infarction . in patients with malignant hemispheric infarction , decompressive surgery can reduce the number of cases of fatality and increase the number of favorable outcomes . in particular , if a decompressive hemicraniectomy is performed early , before irreversible cerebral herniation , using appropriate surgical techniques , favorable outcomes with functional independence can be achieved in a high proportion of patients .

in spite of the best medical treatment , large hemispheric infarction , resulting from acute occlusion of either the internal carotid or the proximal middle cerebral artery with insufficient collateral blood flow is associated with a high case fatality rate of approximately 60% . thus , a decompressive hemicraniectomy is considered a life - saving procedure for this devastating disease . findings of three recent randomized , controlled clinical trials and their meta - analysis showed that early surgical decompression not only reduced the number of case fatalities but also increased the incidence of favorable outcomes . the authors review the pathophysiology , historical background in previous studies , operative timing , surgical technique and clinical outcomes of surgical decompression for malignant hemispheric infarction .

approximately 95% occur in the neck and axilla , while the remaining 5% are found in the chest and abdomen . lymphangiomas of the abdomen are rare ( accounting for 1 per 100,000 hospital admissions ) , but have been reported in the mesentery , retroperitoneum , gastrointestinal tract and intra - abdominal solid viscera . the clinical presentation of lymphangiomas varies from incidental discovery on imaging to presenting with an acute abdomen . mesenteric lymphangiomas , in particular , can result in complications such as intestinal obstruction or volvulus leading to infarction . this case report documents a lymphangioma of the small bowel mesentery , incidentally discovered on pelvic ultrasound and initially thought to represent a complex cystic adnexal mass . a 42-year - old female presented with a history of chronic iron deficiency anemia and menorrhagia for several years . ultrasound was technically difficult , but demonstrated multiple fibroids [ figure 1 ] and a simple cyst within the left ovary . the right ovary was not visualized , but ultrasound demonstrated a complex cystic right adnexal mass measuring 9.7 5.2 6.3 cm that was presumed to originate from the right ovary [ figure 2 ] . 42-year - old female with a history of chronic iron deficiency anemia and menorrhagia for several years and later diagnosed with cystic lymphangioma of the small bowel mesentery . transabdominal ultrasound ( a ) sagittal and ( b ) transverse scans demonstrate a bulky uterus with a subserosal fibroid ( white arrows ) seen in both sagittal and transverse planes . 42-year - old female with a history of chronic iron deficiency anemia and menorrhagia for several years and later diagnosed with cystic lymphangioma of the small bowel mesentery underwent pelvic ultrasound for investigation of menorrhagia and was found to have a complex cystic right adnexal mass . transabdominal ultrasound demonstrates a cystic lesion with numerous thin septae ( curved arrows ) and good through transmission ( straight arrows ) . this confirmed multiple uterine fibroids , t1 bright foci within the left ovary suggestive of an ovarian endometrioma , as well as a dilated left fallopian tube with t1 high signal consistent with a hematosalpinx . was only partially visualized on the pelvic mri , but was deemed to originate from the abdomen rather than the ovary , which was tethered to the uterus [ figure 3 ] . 42-year - old female with a history of chronic iron deficiency anemia and menorrhagia for several years and later diagnosed with cystic lymphangioma of the small bowel mesentery . axial t2 images ( a and b ) demonstrate tethering of right ovary ( arrows ) to the uterus posteriorly . this study demonstrated a cystic mesenteric lesion with high t2 signal and low t1 signal and measuring 17.6 6.8 8.7 cm . the lesion insinuated around adjacent vessels within the small bowel mesentery and demonstrated multiple thin internal septations [ figure 4a ] . mass effect was notably absent , and the cisterna chyli and thoracic duct were not enlarged . the lesion was considered to represent a mesenteric lymphangioma . 42-year - old female with a history of chronic iron deficiency anemia and menorrhagia for several years and later diagnosed with cystic lymphangioma of the small bowel mesentery . ( a ) coronal fast imaging with steady - state precession ( fisp ) demonstrates a multiloculated cystic lesion with numerous septae ( straight arrows ) , lacking mass effect . ( b ) sagittal t2 ( 13 months later ) of lesion , shows increase in size of lesion with thin - walled septae ( straight arrows ) and insinuating around the mesenteric vessels ( curved arrow ) . a 13-month follow - up mri examination showed that the lymphangioma had increased in size compared to the initial examination , measuring 17.6 8.4 11.4 cm and extending from the uncinate process of the pancreas to the uterine fundus [ figure 4b ] . the increase in size prompted a surgical referral . furthermore , the cystic fluid of the lymphangioma demonstrated signal loss on the t1 out - of - phase sequence compared to the t1 in - phase sequence , consistent with microscopic lipid content . the signal intensity on t1 in - phase was 78.4 and on t1 out - of - phase was 56.2 , thus denoting a 28.3% signal intensity loss between phases . this finding is compatible with the presence of chylous fluid and is a very suggestive feature of a lymphangioma [ figure 5 ] . 42-year - old female with a history of chronic iron deficiency anemia and menorrhagia for several years and later diagnosed with cystic lymphangioma of the small bowel mesentery . corresponding axial t1 ( a ) in - phase and ( b ) out - of - phase mr images demonstrate the cystic lymphangioma ( straight arrows ) with signal intensity loss on the out - of - phase image ( b ) compared to the in - phase image , indicative of microscopic lipid content of chylous fluid . lymphangiomas are low - flow vascular malformations that develop as a result of failure of communication of lymph sacs with the venous drainage system . it is postulated that these lesions arise secondary to inflammatory processes , surgery , or radiotherapy that lead to lymphatic obstruction . lymphangiomas are histologically divided into several types : simple capillary , cavernous , and cystic lymphangiomas . cysts may contain serous , chylous , bloody fluid or even purulent fluid in the case of secondary infection . the presence of emulsified fats within chylous fluid most likely explains the signal loss on the t1 out - of - phase mr sequence compared to the t1 in - phase mr sequence in our study . intra - abdominal lymphangiomas mostly occur in the mesentery followed by the omentum , mesocolon , and retroperitoneum . involvement of solid viscera has been described in the literature in the liver , spleen , pancreas , adrenals , kidneys , bladder , and the ovaries . the clinical symptoms of a mesenteric lymphangioma are non - specific and include abdominal pain , vomiting , and constipation , making a specific diagnosis practically impossible on clinical grounds . the majority of lymphangiomas are discovered incidentally on imaging for the investigation of unrelated clinical indications . the differential diagnosis includes a wide range of cystic intra - abdominal lesions , ranging from pancreatic pseudocysts to abdominal tuberculosis , hydatid disease , or malignancies such as mucinous carcinomatosis . the patient did not have a history of previous pancreatitis to suggest pseudocysts or risk factors for pancreatitis such as gallstones or alcohol use . abdominal tuberculosis and malignancy were excluded largely on clinical grounds , as the patient was relatively healthy with no clinical complaints other than menorrhagia . hydatid disease was unlikely , given the absence of daughter cysts and calcifications , and the insinuating nature of the lesion lacking mass effect despite its size . the absence of aggressive features on imaging , such as invasion or solid component , made malignancy unlikely . a combination of imaging modalities may be helpful in confirming the diagnosis , with mri being the preferred examination of choice . typical imaging findings are those of a thin - walled , multiloculated cystic lesion lacking solid components or mural nodularity . lymphangiomas typically show t2 high signal fluid , while signal loss may be visualized on chemical shift t1-weighted imaging due to microscopic fat content from chylous fluid this is a very suggestive finding for a lymphangioma occurring in 2030% of cases , although rarely it may be found in other limited differentials such as lymphoceles , lymphoepithelial cysts , and pancreatic pseudocysts . they typically show little in the way of mass effect and generally insinuate around adjacent structures . the imaging algorithm for the work - up of lymphangiomas would depend on the local resources and availability of imaging modalities such as us , ct , and mri . in our institution , cystic lesions that are either indeterminate or suspicious for lymphangiomas on us or ct are referred to mri for further characterization . on mri , the presence of a thin - walled , multiloculated cystic lesion in the abdomen , without mass effect on adjacent structures , is suggestive of the diagnosis after relevant differential diagnoses have been excluded on clinical , laboratory , and imaging grounds . the presence of signal loss on chemical shift imaging is very suggestive of the diagnosis . if definitive confirmation is required , imaging - guided diagnostic aspiration of the cyst fluid could be performed . secondary infection , bowel obstruction , volvulus , and rupture with hemorrhage are a few recognized complications . so , to date , radical excision remains the definitive treatment , in particular , to prevent recurrence . in conclusion , intra - abdominal lymphangiomas are rare entities , which although are typically asymptomatic , can result in life - threatening complications . in our case , the lesion was discovered incidentally on imaging for an unrelated clinical indication and demonstrated interval growth at 1 year warranting a surgical referral . as of the present time , the surgical consultation has not yet been conducted and our diagnosis is based on imaging . our findings were those of a thin - walled multiloculated cystic lesion , lacking both mass effect and enhancement , and which insinuated around vessels within the small bowel mesentery . the cyst fluid showed signal loss on chemical shift t1-weighted mr imaging due to the microscopic fat content of chylous fluid a very suggestive finding for a lymphangioma that helps to differentiate it from other etiologies of cystic lesions in the abdomen .

lymphangiomas are benign lesions of mesenchymal origin . although more commonly encountered in the head and neck , intra - abdominal lymphangiomas are a rare entity that typically present as multiloculated intra - abdominal cystic lesions that are often incidentally discovered on imaging . this case report discusses such a case .

cysteine point mutations for residues were introduced via the quikchange mutagenesis kit ( stratagene ) using a human p2x2 receptor or rat p2x4-myc ( y378a ) plasmid as the template . human p2x2 receptor plasmid ( gift of dr . andrew powell , glaxosmithkline , united kingdom ) contained the sequence of the p2x2a isoform ( q9ubl9 ) with the nh2-terminal variant maaaqpkypagata mv as described.4 for the rat p2x4-myc ( gift of dr . francois rassendren , cnrs , montpellier , france ) , the mutant y378a was created and used as the template for more stable currents ( 21 ) . production of the correct mutations and absence of coding errors in the p2x2 and p2x4 mutant constructs was verified by dna sequencing ( automated abi sequencing service , university of leicester ) . expression in xenopus laevis oocytes wild type and mutant constructs were transcribed to produce sense strand crna ( mmessage mmachine , ambion , tx ) as described previously ( 22 ) . manually defoliculated stage v x. laevis oocytes were injected with 50 nl ( 50 ng ) of crna using an inject+matic microinjector ( j. alejandro gaby , genva , switzerland ) and stored at 18 c in nd96 buffer ( 96 mm nacl , 2 mm kcl , 1.8 mm cacl2 , 1 mm mgcl2 , 5 mm sodium pyruvate , 5 mm hepes , ph 7.6 ) . electrophysiological recordings two - electrode voltage clamp recordings ( at a holding potential of 60 mv ) were carried out on crna - injected oocytes using a geneclamp 500b amplifier with a digidata 1322 analog to digital converter and pclamp 8.2 acquisition software ( axon instruments ) as previously described ( 22 ) . native oocyte calcium - activated chloride currents in response to p2x receptor stimulation were reduced by replacing 1.8 mm cacl2 with 1.8 mm bacl2 in the nd96 bath solution . atp ( magnesium salt , sigma ) was applied via a u - tube perfusion system . atp ( 0.1 m to 10 mm ) was applied at 5-min intervals , using this regime reproducible atp - evoked responses were recorded . individual concentration - response curves were fitted with the hill equation : y = [ ( x ) m]/[(x ) + ( ec50 ) ] , where y is response , x is agonist concentration , h is the hill coefficient , m is maximum response , and ec50 is the concentration of agonist evoking 50% of the maximum response . mutants that had considerably shifted atp potency were tested with atp concentrations up to 10 mm . for the calculation of ec50 values individual concentration - response curves were generated for each experiment and statistical analysis carried out on the pec50 data generated . in the figures , characterization of the effects of methanethiosulfonate compounds to study the effect of mts compounds on atp activation at wild type and cysteine mutants , atp ( ec50 concentration ) was applied and either mtsea or sodium ( 2-sulfonatoethyl)methanethiosulfonate ( mtses ) ( toronto research chemicals , toronto , canada ) were bath - perfused ( for at least 5 min ; the recovery time required between application to see reproducible responses ) prior to coapplication with atp via the u - tube as described previously ( 15 , 16 ) . mts reagents ( 1 mm ) were made in nd96 solution immediately prior to use . mts reagents were applied for 10 min with the maximal , or near maximal response seen after 5 min . the effects of the mts reagents on the p2x2 receptor mutants were the same following washout of the compounds showing that they irreversibly modified the receptors . the effects of the mts compounds on the mutant p2x4 receptors were reversible following a 510 min washout . the p2x4 receptor has been shown to be subject to extensive constitutive and agonist induced trafficking ( 23 ) even for the y378a mutant used in this study ( 21 , 24 ) . to determine whether longer incubations could be used to irreversibly modify p2x4 mutant receptors we treated the oocytes for 3 h in the presence of mtses ( 1 mm ) and 3.2 units / ml of apyrase to break down any endogenous atp , or just apyrase . this was followed by washing and subsequent testing of responses to an ec50 concentration of atp ( in the absence of mtses ) . this treatment had no effect on the amplitude of wt p2x4 receptor currents , however , reductions of the mutants were equivalent to those where mtses was present during the recordings demonstrating that any changes in properties for the mutant receptors result from irreversible modification of introduced cysteine residues and that cysteine residues were available in the resting un - liganded state of the receptor . mts reagents have a reasonably short half - life and this suggests that the 1 mm concentration used is supramaximal and there was still sufficient reagent present during the 3-h incubation to irreversibly modify the receptors . these results suggest that the reversal of mts effects following washout of short applications is likely to result from trafficking of un - modified receptors to the cell surface and longer incubations are required to irreversibly modify the whole pool of p2x4 mutant receptors . we have therefore presented data for effects on an ec50 concentration of atp during the presence of the mts reagents as this allows us to analyze data from the same oocyte before and during application of the reagents . the effects of mts reagents on the concentration responses to atp for p2x2 receptors were investigated following washout of mtsea ( applied at 1 mm for 10 min in the absence of agonist ) , there was no effect on wt responses . for p2x4 receptors the effects of mtsea on concentration responses were investigated by application of atp following incubating the oocytes for 1 h in mtsea ( 1 mm ) and washout of mtsea . atp concentrations were applied via the u - tube with nd96 bathing solution ( no mts reagents present ) . western blotting expression levels of wild type and mutant receptors were estimated by western blot analysis of total cellular protein and cell surface proteins . total cellular protein samples were prepared from oocytes injected with wild type or mutant receptor crna homogenized in buffer h ( 100 mm nacl , 20 mm tris - cl , ph 7.4 , 1% triton x-100 , and 10 l / ml protease inhibitor mixture ) at 20 l / oocyte . sulfo - nhs - lc - biotin ( pierce ) labels cell surface proteins by reacting with primary amines and was used to estimate the level of wild type or mutant receptor trafficked to the cell surface membrane . sulfo - nhs - lc - biotin is impermeable to the cell membrane and can only biotinylate proteins available at the cell surface . oocytes injected with wild type or mutant receptor crna were treated with sulfo - nhs - lc - biotin ( 0.5 mg / ml ) in nd96 for 30 min and washed with nd96 . oocytes were homogenized in buffer h , and the spin - cleared supernatant was mixed with streptavidin - agarose beads ( sigma ) treated as described previously ( 25 ) . all samples were mixed with sds sample buffer and heated to 95 c for 5 min prior to loading . samples were run on a 10% sds - page gel , transferred to nitrocellulose , and screened for immunoreactivity for the anti - p2x2 or p2x4 antibody ( 1:500 ) ( alomone , israel ) significant differences between wt and mutant maximum current amplitudes , agonist potency , and mts reagent modification were calculated by one - way analysis of variance followed by dunnett 's test for comparisons of individual mutants against control using the graphpad prism package . differences in mutant concentration response curves for individual mutants , on treatment with mtsea , were tested with student 's t test . effects of cysteine mutants on atp responses at p2x2 receptors at wt human p2x2 receptors atp evoked concentration - dependent inward currents with an ec50 of 13 m ( fig . 2 and table 1 ) similar to that reported previously for this receptor ( 14 , 15 , 26 ) . robust currents were also recorded for f183c , t184c , and f289c mutants , however , they showed a modest 410-fold decrease in atp potency ( fig . the peak amplitude of currents to a maximal concentration of atp ( 10 mm ) were reduced by 90% for the mutant r290c and for this receptor the amplitude of responses continued to increase between 3 and 10 mm indicating the ec50 is > 3 mm atp . at mutants n288c and k307c 10 mm atp evoked very small currents suggesting that the ec50 for these receptors is > 3 mm . no currents were recorded in response to 10 mm atp for mutants k69c and k71c . analysis of the expression of both total and surface levels of p2x2 wt and mutant receptors with reduced peak current amplitudes showed that there was at most an 50% reduction in surface expression ( k307c ) , predicting a halving in peak current amplitude , however , there was a > 97% reduction in amplitudes for k69c , k71c , and n288c . this indicates that any reductions in current amplitude compared with wt were predominantly due to an effect on channel properties and not trafficking to the cell surface . overall these results are consistent with those from similar mutants for the p2x1 receptor ( 12 ) . table 1summary of the effects of cysteine substitution on p2x2 and p2x4peak current values taken on 1st application of a maximal concentration of atp ( a saturating concentration 300 m to 10 mm ) . mts data ( effect of atp applied in the presence of mts reagent ) calculated as a percentage of the control ( effect of atp applied in the absence of mts reagent ) which was set as 100%.na ( max)ec50pec50mtseamtseshuman p2x2 wt 4465 477 13 m 4.89 0.1 127 2% 101 3% k69c nfa nf nf no effect no effect k71c nf nf nf no effect no effect f183c 4661 1209 40 m 4.40 0.1b 128 2% 96 1% t184c 4515 1402 117 m 3.93 0.1c 195 11%d 20 3%c n288c 13 2b > 3 mm < 2.52 22 5%b 21 3%c f289c 2279 583 64 m 4.19 0.1c 123 11% 78 11%d r290c 525 70d > 3 mm < 2.52 1237 249%c 27 7%c k307c 49 20c > 3 mm < 2.52 27500 6660%c 15 1%crat p2x4-myc ( y378a ) wt 2513 187 13 m 4.89 0.02 103 11% 108 6% k67c 66 21d nde nd 2076 485%c 15 4%c k69c 821 146 > 3 mm < 2.52 350 50%c 1 1%c f185c 587 224 265 m 3.58 0.07c 93 7% 95 2% t186c 1706 425 650 m 3.19 0.04c 35 6%d 8 3%c n293c 521 164 > 3 mm < 2.52 6 1%c 2 1canf , nonfunctional.bp < 0.01 , n = 3-18.cp < 0.001 , n = 3-18.dp < 0.05 , n = 3-18.end , not determined . summary of the effects of cysteine substitution on p2x2 and p2x4 peak current values taken on 1st application of a maximal concentration of atp ( a saturating concentration 300 m to 10 mm ) . mts data ( effect of atp applied in the presence of mts reagent ) calculated as a percentage of the control ( effect of atp applied in the absence of mts reagent ) which was set as 100% . atp potency was tested on wt and p2x2 receptor mutants expressed in oocytes by two - electrode voltage clamp ( holding potential = 60 mv ) . a , trace data representative of currents observed for wild type and mutant receptors in response to atp applications ( indicated by the bar , concentrations in micromolar ) at 5-min intervals . b , concentration - response curves to atp for wt and mutants k69c , k71c , f183c , t184c , n288c , f289c , r290c , and k307c . k69c , k71c , n288c , and k307c with reduced peak currents in response to a maximal concentration of atp , are expressed as a percentage of the amplitude of the wt maximum response , whereas all others are expressed as a percentage of their own maximum response ( n = 310 ) . c , western blots of total and surface expression levels of wt and mutant p2x2 receptors with reduced peak current amplitudes . lower panel shows densitometric analysis of the blots expressed as % of wt levels . atp potency was tested on wt and p2x2 receptor mutants expressed in oocytes by two - electrode voltage clamp ( holding potential = 60 mv ) . a , trace data representative of currents observed for wild type and mutant receptors in response to atp applications ( indicated by the bar , concentrations in micromolar ) at 5-min intervals . b , concentration - response curves to atp for wt and mutants k69c , k71c , f183c , t184c , n288c , f289c , r290c , and k307c . k69c , k71c , n288c , and k307c with reduced peak currents in response to a maximal concentration of atp , are expressed as a percentage of the amplitude of the wt maximum response , whereas all others are expressed as a percentage of their own maximum response ( n = 310 ) . c , western blots of total and surface expression levels of wt and mutant p2x2 receptors with reduced peak current amplitudes . effects of mts reagents on cysteine mutant p2x2 receptors at p2x receptors the 10 conserved cysteine residues in the extracellular domain are thought to form five intrasubunit disulfide bonds ( 25 , 27 ) . the substituted cysteine accessibility method has been used to look at the contribution of introduced cysteine mutants to receptor function , where modification of properties following application of mts reagents indicates that the introduced cysteine residue is accessible in the aqueous environment and may play an important role in receptor function ( 28 ) . previously , charged mts reagents ( positively charged mtsea and negatively charged sodium mtses ) , have been used to probe residues involved in atp action at p2x receptors ( 15 , 16 ) as they may interact with the negative charge of the phosphate tail of atp or magnesium complexed with atp in physiological solutions giving rise to localized positive charge . at wt p2x2 receptors mtsea ( 1 mm ) produced a small increase ( 128 3% of control ) in the response to an ec50 concentration of atp ( table 1 , fig . mtsea had no effect on the potency of atp and current amplitudes returned to control levels on washout . mtsea had a similar effect at f183c and f289c mutant p2x2 receptors ( table 1 , fig . there was an increase in potentiation to 195 11% of control , however , on washout this was reduced to 141 11% of control indicating that it results from a combination of the reversible effect seen for wt and an additional irreversible modification . r290c and k307c mtsea produced a significantly larger increase in the response to an ec50 concentration of atp ( table 1 , fig . 3 ) that was sustained on washout indicating that it results from irreversible modification of the introduced cysteine . at r290c the amplitude of responses was still rising in response to increases from 3 to 10 mm atp , indicating a large decrease in atp sensitivity at this mutant . mtsea application for 10 min in the absence of agonist resulted in a large increase in amplitude of responses and a leftward shift in the concentration response to atp and now a fully saturating concentration response was generated following mtsea treatment ( pec50 values 2.8 0.1 , estimated from a free fit as responses did not saturate for control , to 3.3 0.2 , p < 0.01 , for mtsea , n = 34 ) ( fig . responses to a high concentration of atp ( 10 mm ) were small ( 50 na ) for the k307c mutant and responses were below the limit of detection for 1 mm atp , however , following a 10-min mtsea treatment ( and washout ) there was a very large increase in the amplitude of responses and the pec50 for atp in the presence of mtsea was 3.5 0.1 ( n = 7 ) . as mtsea was able to rescue the responses to mutants with small amplitude currents we tested whether mtsea had any effect on mutants where receptor protein could be detected at the cell surface but little or no current was detected in response to high concentrations of atp . at n288c the responses to atp ( 3 mm ) were reduced to 22 5% of control following mtsea treatment , this effect was sustained on mtsea washout . for mutants k69c and k71c , atp - evoked currents were still not detected following mtsea treatment . the negatively charged mts reagent mtses ( 1 mm ) had no effect on currents at wt p2x2 receptors ( table 1 , fig . similarly mtses had no effect on atp responses at f183c mutant p2x2 receptors ( table 1 , fig . mtses , however , reduced the amplitude of response to an ec50 concentration of atp by 80% for mutants t184c , n288c , r290c , and k307c , a more modest 20% decrease was recorded at f289c ( table 1 , fig . these effects were maintained following washout of mtses indicating that the reagent was acting irreversibly to modify the introduced cysteine residues . for non - functional mutants k69c and k71c in the presence of mtses , 3 mm atp still did not evoke a response . effects of cysteine mutants on atp responses at p2x4 receptors the myc - tagged rat p2x4 receptor with the y378a mutation was used as the background for these studies . the y378a mutation was introduced to improve surface expression of the receptor ( 21 ) and increased the peak amplitude of atp currents by > 2050-fold in the present study . atp evoked concentration - dependent responses at the rat p2x4-myc y378c receptor with an ec50 of 13 m ( table 1 , fig . 5 ) similar to that reported previously for rat p2x4 receptors ( 2931 ) and this construct will be referred to as p2x4 wt . for f185c , t186c , and f294c mutants full concentration - response curves were generated and the ec50 for atp was increased by 20- , 50- , and 4-fold , respectively ( table 1 , fig . 5 ) . for the remaining mutants ( k67c , k69c , n293c , r295c , and k313c ) atp - evoked responses did not saturate at the maximum concentration tested ( 10 mm ) and for these mutants the atp ec50 is reported as > 3 mm ( table 1 , fig . mutants k67c , r295c , and k313c had reduced maximal current amplitudes compared with wt , however , this was not reflected by a marked reduction in either total or surface expression of the receptors ( fig . 4c ) . for the mutant n293c the smaller peak current amplitudes may result in part from a reduction in trafficking of the receptor to the cell surface ( fig . effects of mts reagents on cysteine mutant p2x4 receptors positively charged mtsea ( 1 mm ) had no effect on atp - evoked currents at wt p2x4 receptors ( table 1 , fig . atp - evoked responses were also unaffected by mtsea for the mutants of the conserved aromatic phenylalanine residues f185c and f294c ( table 1 , fig . 6 ) . at t186c and r295c , responses evoked by an ec50 concentration of atp were reduced to 40% of control levels and at mutant n293c responses were reduced by 95% ( table 1 , fig . in contrast , at mutants where a positively charged lysine residue was mutated to cysteine ( lys , lys , and lys ) application of mtsea potentiated the amplitude of the atp response ( table 1 , fig . . a , trace data representative of currents observed before and during the addition of mtsea ( 1 mm ) . mtsea reagent was bath applied 5 min prior to u - tube atp coapplication with mtsea . b , graph representing the effect of mtsea on the p2x2 wt and mutant receptors ( n = 36 ) . c , trace data representative of currents observed with the addition of mtses ( 1 mm ) . mtses reagent applied in a similar manner to mtsea traces are shown for a given oocyte before and during mtses application . d , graph representing the effect of mtses on the p2x2 wt and mutant receptors ( n = 36 ) . * , p < 0.05 ; * * , p < 0.01 ; * * * , p < . a , trace data representative of currents observed before and during the addition of mtsea ( 1 mm ) . mtsea reagent was bath applied 5 min prior to u - tube atp coapplication with mtsea . b , graph representing the effect of mtsea on the p2x2 wt and mutant receptors ( n = 36 ) . c , trace data representative of currents observed with the addition of mtses ( 1 mm ) . atp application ( ec50 concentration ) is indicated by the bar . mtses reagent applied in a similar manner to mtsea traces are shown for a given oocyte before and during mtses application . d , graph representing the effect of mtses on the p2x2 wt and mutant receptors ( n = 36 ) . * , p < 0.05 ; * * , p < 0.01 ; * * * , p < 0.001 . a , atp concentration - response curve for r290c under control conditions or following washout of mtsea after a 10-min incubation with mtsea ( 1 mm)(n = 34 ) . b , atp concentration - response curve for k307c under control conditions or following washout of mtsea after a 10-min incubation with mtsea ( 1 mm)(n = 36 ) . the data are expressed as a percentage of the amplitude of the response to 3 mm atp under control conditions . a , atp concentration - response curve for r290c under control conditions or following washout of mtsea after a 10-min incubation with mtsea ( 1 mm)(n = 34 ) . b , atp concentration - response curve for k307c under control conditions or following washout of mtsea after a 10-min incubation with mtsea ( 1 mm)(n = 36 ) . the data are expressed as a percentage of the amplitude of the response to 3 mm atp under control conditions . the application of negatively charged mtses ( 1 mm ) had no effect on the amplitude of atp evoked responses at wt or f185c and f294c mutant p2x4 receptors ( table 1 , fig . 6 ) . at the remainder of the mutants ( k67c , k69c , t186c , n293c , r295c , and k313c ) mtses reduced the amplitude of atp evoked responses by 8095% . these results suggest that the negative charge at this position interferes with the action of atp possibly by repelling the negatively charged phosphate tail . these effects of mtsea and mtses are broadly consistent for both the p2x2 and p2x4 receptors . however , in contrast to the p2x2 receptor the effects of mts reagents were reversible on washout ( showing 50100% recovery following 5 min washout ) indicating that either the reagent was not irreversibly modifying the receptor or that un - modified receptors were being trafficked to the cell surface during the washout period . it is known that p2x4 receptors undergo cyclical trafficking to the cell surface ( 21 , 23 , 24 ) so we decided to see whether longer incubation with the mts reagents had an irreversible effect . in addition , as the mts reagents were applied before and during atp application the question arises as to whether the introduced cysteines are accessible in the un - liganded or ligand bound state . to address these questions we incubated mutant p2x4 receptors in membrane impermeant mtses ( 1 mm ) in the presence of apyrase ( 3.2 units / ml to break down any endogenous atp in the solutions ) for 3 h followed by washing . this treatment had no effect on the amplitude of wt p2x4 receptors compared with oocytes treated with just apyrase . for the mutants , however , there was a significant reduction in the amplitude of responses for the mutants k67c , k69c , t186c , n293c , r295c , and k313c ( 8 1 , 10 3 , 10 3 , 35 8 , 25 6 , and 17 4.7% of atp responses following apyrase treatment only and washing , with p < 0.01 , 0.001 , 0.005 , 0.005 , 0.005 , 0.01 , respectively , n = 47 ) . these results show that longer incubations with mts reagents are required to irreversibly modify the receptors , give equivalent results to bath - applied mtses , and suggest that washout following short applications of mts reagents ( 510 min ) results from trafficking of un - modified mutant p2x4 receptors to the cell surface . these results also demonstrate that these introduced cysteine residues are accessible in the absence of agonist . this is consistent with previous studies on the p2x2 receptor indicating that mts effects at the region around lys and lys are blocked by atp ( 15 ) and mtsea biotinylation studies on the p2x1 receptor showing that mutants n290c , f291c , r292c , and k309c are mtsea biotinylated in the absence of atp and this is reduced following application of atp ( 16 ) . taken together these results show that the mts reagents are acting at introduced cysteine residues that are accessible in the absence of ligand . atp potency was tested on wt and p2x4 receptor mutants expressed in oocytes by two - electrode voltage clamp ( holding potential = 60 mv ) . a , trace data representative of currents observed for wild type and mutant receptors in response to atp applications ( indicated by bar , concentration in micromolar ) at 5-min intervals . b , concentration - response curves to atp for wt and mutants k67c , k69c , f185c , t186c , n293c , f294c , r295c , and k313c . k67c , k69c , n293c , r295c , and k313c , with reduced peak currents in response to a maximal concentration of atp , are all expressed as a percentage of peak wt maximum response whereas , all others are expressed as a percentage of their own maximum response ( n = 34 ) . c , western blots of total and surface expression levels of wt and mutant p2x4 receptors with reduced peak current amplitudes . lower panel shows densitometric analysis of the blots expressed as % of wt levels . atp potency was tested on wt and p2x4 receptor mutants expressed in oocytes by two - electrode voltage clamp ( holding potential = 60 mv ) . a , trace data representative of currents observed for wild type and mutant receptors in response to atp applications ( indicated by bar , concentration in micromolar ) at 5-min intervals . b , concentration - response curves to atp for wt and mutants k67c , k69c , f185c , t186c , n293c , f294c , r295c , and k313c . k67c , k69c , n293c , r295c , and k313c , with reduced peak currents in response to a maximal concentration of atp , are all expressed as a percentage of peak wt maximum response whereas , all others are expressed as a percentage of their own maximum response ( n = 34 ) . c , western blots of total and surface expression levels of wt and mutant p2x4 receptors with reduced peak current amplitudes . lower panel shows densitometric analysis of the blots expressed as % of wt levels . for mutants k67c , k69c , and k313 , where mtsea resulted in an increase in the amplitude of the response to atp we constructed concentration - response curves following application of mtsea . we found that incubation with membrane - permeant mtsea for 1 h ( in the absence of agonist ) followed by washout and testing of atp sensitivity in the absence of mtsea resulted in an increase in atp potency at the receptors with pec50 values of 3.61 0.24 , 3.95 0.09 , and 3.63 0.16 for k67c , k69c , and k313c , respectively ( n = 4 , 3 , and 3 ) ( fig . 7 ) . these results suggest that positive charge at these positions plays an important role in determining atp potency . the aim of this study was to determine whether a model of the atp binding site based on systematic mutagenesis studies on the p2x1 receptor could be applied , using cysteine - based mutagenesis , to other p2x receptor family members , and to extend this to see if the actions of atp could be modified in a similar way by mts reagents . the data for the effects of cysteine mutants on atp potency at p2x2 and p2x4 receptors support previous work about residues involved in atp action . . a , trace data representative of currents observed on the addition of mtsea ( 1 mm ) . mtsea reagent was bath applied 5 min prior to u - tube atp coapplication with mtsea . traces are shown in response to atp for a given oocyte before and during the application of mtsea . b , graph representing the effect of mtsea on the p2x4 wt and mutant receptors ( n = 38 ) . c , trace data representative of currents observed on the addition of mtses ( 1 mm ) . traces are shown in response to atp for a given oocyte before and during the application of mtses . d , graph representing the effect of mtses on the p2x4 wt and mutant receptors ( n = 36 ) . * , p < 0.05 ; * * , p < 0.01 ; * * * , p < . a , trace data representative of currents observed on the addition of mtsea ( 1 mm ) . mtsea reagent was bath applied 5 min prior to u - tube atp coapplication with mtsea . traces are shown in response to atp for a given oocyte before and during the application of mtsea . b , graph representing the effect of mtsea on the p2x4 wt and mutant receptors ( n = 38 ) . c , trace data representative of currents observed on the addition of mtses ( 1 mm ) . traces are shown in response to atp for a given oocyte before and during the application of mtses . d , graph representing the effect of mtses on the p2x4 wt and mutant receptors ( n = 36 ) . * , p < 0.05 ; * * , p < 0.01 ; * * * , p < 0.001 . there are three conserved lysine residues in the extracellular domain that have been suggested to be involved in atp binding at p2x receptors . mutagenesis of these residues has shown a marked decrease in agonist potency at a variety of receptors with the majority of the mutations resulting in a shift in ec50 to > 3 mm and in some cases , e.g. p2x2 receptors , currents are below the limit of detection even at high agonist concentrations ( 15 ) . at p2x4 receptors atp sensitivity at k67c , k69c , and k313c was increased on treatment with mtsea , consistent with the fact that the incorporation of mtsea at cysteine produces a side chain of similar size and charge to lysine ( 17 ) . this clearly indicates that the localized charge at these three positions plays an important role in regulating atp action at the receptor . for p2x2 receptors a response to atp one feature that is common to the p2x1,2,4 receptors is that the decrease in atp potency at the cysteine mutant of the residue equivalent to lys in the p2x1 receptor can be rescued by treatment with mtsea , consistent with an important role of positive charge at this position . a recent study on the p2x2 receptor using single channel analysis has suggested the lysine residue 308 can play a role in regulation of gating of the channel . this was based on the fact that mutation of this residue could abolish spontaneous gating of the t339s mutant p2x2 channel and that mutation k308r reduced atp potency by 200-fold and decreased the amplitude of the maximal response compared with control ( 32 ) . in the current study the concentration responses to p2x2 k307c and p2x4 k313c did not saturate so it is not possible to determine whether there was a reduction in the amplitude of the maximal response . at the p2x1 receptor the 200-fold reduction in atp potency at k309r and k309c mutants did not result in a decrease in the amplitude of the maximal response ( 16 , 22 ) as would have been expected if an effect on gating of the receptor has been primarily responsible for the reduction in potency ( 33 ) . these results suggest that the effects of mutation of this conserved residue can be manifest as a principal effect on atp binding at the p2x1 receptor ( 16 ) and an additional effect on gating at the p2x2 receptor ( 32 ) . this may indicate that in the p2x2 receptor the lysine residue interacts with another amino acid that is not conserved at the p2x1 receptor to regulate gating . taken together these results support that the three positively charged lysine residues play an important role in atp action most likely contributing to the atp binding site . indeed it would be unusual to have an atp - binding protein without positive charge being important in coordinating agonist binding . a , mtsea significantly increased the potency of atp and amplitude of responses at k67c mutants . b , mtsea significantly increased the potency of atp and amplitude of responses at k69c mutants . c , mtsea significantly increased atp potency and peak response at k313c mutants ( n = 34 ) . the data are expressed as a percentage of the amplitude of the response to 3 mm atp . for mtsea treatment ( 1 mm , 1 h incubation , followed by washout and construction of the concentration response in the absence of mtsea ) these have been corrected with a scaling factor corresponding to the mean -fold increase , at 3 mm atp , in currents compared with untreated oocytes . a , mtsea significantly increased the potency of atp and amplitude of responses at k67c mutants . b , mtsea significantly increased the potency of atp and amplitude of responses at k69c mutants . c , mtsea significantly increased atp potency and peak response at k313c mutants ( n = 34 ) . the data are expressed as a percentage of the amplitude of the response to 3 mm atp . for mtsea treatment ( 1 mm , 1 h incubation , followed by washout and construction of the concentration response in the absence of mtsea ) these have been corrected with a scaling factor corresponding to the mean -fold increase , at 3 mm atp , in currents compared with untreated oocytes . the mutations of conserved aromatics f183c in p2x2 and f185c in the p2x4 receptor gave rise to a modest 3- and 20-fold decrease in atp potency at the receptors . this is consistent with previous studies on p2x1 and p2x4 receptors ( 34 , 35 ) and indicates that this conserved residue contributes to atp action . the mts reagents used are of similar size ( or smaller ) than atp and can gain access to the atp binding pocket . the effects of the mts compounds at cysteine mutants of the adjacent threonine residue ( t184c and t186c for p2x2 and p2x4 , respectively ) suggest that this region of the receptor is accessible to these reagents and the lack of effect at the cysteine - substituted phenylalanine residues is likely to reflect that this residue is either not very close to the atp binding pocket or is involved in interaction with other residues that could regulate gating . the former interpretation suggests that the phenylalanine may not play a direct role in binding of the adenine ring of atp as we have suggested previously for the p2x1 receptor ( 34 ) . however , cysteine substitution does give rise to changes in agonist action at the receptor . the introduction of a polar side chain ( cysteine ) to replace the non - polar phenylalanine could have an effect on bonding / electrostatic interactions or a structural change to the receptor . mutation of threonine in the conserved ft motif to cysteine resulted in a 10- and 40-fold decrease in atp potency at p2x2 and p2x4 receptors . previous studies on the p2x2 receptor have shown that alanine substitution at this residue deceases atp potency by 100-fold ( 15 ) . this is the first time a mutation has been made at this position for the p2x4 receptors and it supports a similar mode of agonist action as p2x1 and p2x2 receptors . at p2x2 and p2x4 receptors the response was inhibited by mtses . these results clearly indicate that modification of the side chain at this conserved threonine residue can regulate channel function . close to the ft motif is a nearby conserved lysine residue that has been shown to be involved in regulating atp potency ( lys in p2x1 and lys in p2x2 that when mutated to alanine resulted in 10- ( 22 ) and 100-fold decreases in atp potency ( 36 ) ) . thus this region in the middle of the extracellular loop of the channel plays a role in regulation of atp potency , however , whether this results from an effect on agonist binding and/or gating remains to be determined . what is clear is that residues involved in agonist action at p2x receptors are not just focused around regions close to the transmembrane segments and the pore of the channel . at the conserved nfr motif cysteine substitution at asn and arg reduced atp potency by > 300-fold and gave marked reductions in peak current amplitudes ( > 80% ) at p2x2 and p2x4 receptors . the asparagine to cysteine mutation had the same effect at p2x2 ( n288c ) and p2x4 ( n293c ) receptors with inhibition of currents by either mts reagent as reported previously for p2x1 receptors ( 16 ) . the reduction of atp responses at the mutants of the conserved arginine by mtses is the same for p2x1,2,4 receptors , however , mtsea inhibited p2x1 and p2x4 receptors , but potentiated the p2x2 receptor ( ref . 16 and this shows that the p2x1 and p2x4 receptors are similar in that the mts reagents interfere with the action of atp in a way that is charge independent . however , for p2x2 the results are consistent with the polarity of charge at this position being important . when the phenylalanine of the nfr motif was replaced with cysteine there was only a modest 4-fold reduction in atp potency , no effect on peak current amplitude , and mts reagents were ineffective . this suggests that at p2x2 and p2x4 receptors the conservation of the asparagine and arginine residues is more important in regulating the actions of atp than the phenylalanine of which they are adjacent . at p2x1 receptors and a non - desensitizing chimeric p2x1/2 receptor , marked effects of > 100-fold decrease in atp potency with no effect on the peak response were recorded with the equivalent mutations ( 16 , 18 ) . taken together these results show that whereas there are marked similarities between the receptors , there are also differences in regulation of atp potency between the p2x receptor subunits ( e.g. the effects of mtsea on cysteine substitution of the arginine of the nfr motif ) . this is consistent with variations in the pharmacological properties of p2x receptors and suggests that non - conserved amino acids are associated with agonist action . table 2summary of data for the effects of mts reagents at p2x receptor cysteine mutantsarrows correspond to significant effects of mts reagents on atp responses . shaded areas correspond to similar effects of the mts reagents at different p2x receptor subtype mutants . summary of data for the effects of mts reagents at p2x receptor cysteine mutants arrows correspond to significant effects of mts reagents on atp responses . shaded areas correspond to similar effects of the mts reagents at different p2x receptor subtype mutants . the present study shows that the nfr region plays an important role in regulation of atp action at p2x2 and p2x4 receptors . we have previously shown , for p2x1 receptors , that reductions in atp potency at cysteine mutants of the nfr motif are likely to result from effects on agonist binding at arg and effects on both binding and gating for asn and phe ( 16 ) . the contribution of this region to the agonist binding site is supported by a recent paper ( 18 ) showing that the phenylalanine is close to the conserved lys residue in the p2x1 receptor and cysteine mutants k68c and f291c can form an inter - subunit disulfide bond and this is inhibited by atp , suggesting that both residues are close to the atp binding site . similarly a recent single channel study on p2x2 receptors reported that the reduction of atp potency for the k69a mutant of the p2x2 receptor results predominantly from an effect on agonist binding ( 32 ) . previously we had suggested that the phenylalanine residues in the nfr motif were involved in binding of the adenine ring of atp . however , the present results from p2x2 and p2x4 receptors with modest changes in atp for cysteine mutants of these phenylalanines and the lack of effect of mts reagents on atp responses suggests that the conserved aromatic residue may not contribute directly to agonist recognition . the inhibition of cysteine - substituted mutants of the arginine of the nfr motif for p2x1 and p2x4 receptors by both positive and negatively charged mts reagents suggests that arginine is not directly involved in binding of the phosphate tail of atp . this suggests that asparagine and arginine are most likely involved in binding of the ribose and/or adenine ring of atp . portions of the extracellular loop adjacent to either the first ( in gray ) or second ( in black ) transmembrane domain are shown for two adjacent p2x receptor subunits ( numbering for the p2x4 receptor ) . the gray arrow represents the disulfide bond that can be formed between cysteine mutations of these residues at the p2x1 receptor ( 18 ) . residues that showed a decrease in atp potency when mutated to cysteine are shown , those in black correspond to those that were modified by mts reagents , whereas those in gray were unaffected by either mtsea or mtses . positively charged mtsea increased the amplitude and potency of responses at cysteine mutants of the p2x4 receptor at positions lys , lys and lys , this suggests a role of these residues in coordinating the binding of the negatively charged phosphate of atp , however , it is possible that they also contribute to the gating of the channel as has been suggested for the residue equivalent to lys at the p2x2 receptor ( 32 ) . the conserved ft region is shown as a dotted line as it has been shown to be involved in agonist potency and is unclear whether the effects result from changes in agonist binding and/or gating . portions of the extracellular loop adjacent to either the first ( in gray ) or second ( in black ) transmembrane domain are shown for two adjacent p2x receptor subunits ( numbering for the p2x4 receptor ) . the gray arrow represents the disulfide bond that can be formed between cysteine mutations of these residues at the p2x1 receptor ( 18 ) . residues that showed a decrease in atp potency when mutated to cysteine are shown , those in black correspond to those that were modified by mts reagents , whereas those in gray were unaffected by either mtsea or mtses . positively charged mtsea increased the amplitude and potency of responses at cysteine mutants of the p2x4 receptor at positions lys , lys and lys , this suggests a role of these residues in coordinating the binding of the negatively charged phosphate of atp , however , it is possible that they also contribute to the gating of the channel as has been suggested for the residue equivalent to lys at the p2x2 receptor ( 32 ) . the conserved ft region is shown as a dotted line as it has been shown to be involved in agonist potency and is unclear whether the effects result from changes in agonist binding and/or gating . our results support the idea that mammalian p2x receptors share a common agonist binding site with the negative charge of atp coordinated by positively charged lysine residues and the nfr motif involved in binding of the adenine / ribose component ( fig . the conserved ft motif also contributes to agonist action , however , whether this results from an effect on agonist binding and/or gating remains to be determined .

the agonist binding site of atp - gated p2x receptors is distinct from other atp - binding proteins . mutagenesis on p2x1 receptors of conserved residues in mammalian p2x receptors has established the paradigm that three lysine residues , as well as ft and nfr motifs , play an important role in mediating atp action . in this study we have determined whether cysteine substitution mutations of equivalent residues in p2x2 and p2x4 receptors have similar effects and if these mutant receptors can be regulated by charged methanethiosulfonate ( mts ) compounds . all the mutants ( except the p2x2 k69c and k71c that were expressed , but non - functional ) showed a significant decrease in atp potency , with > 300-fold decreases for mutants of the conserved asparagine , arginine , and lysine residues close to the end of the extracellular loop . mts reagents had no effect at the phenylalanine of the ft motif , in contrast , cysteine mutation of the threonine was sensitive to mts reagents and suggested a role of this residue in atp action . the lysine - substituted receptors were sensitive to the charge of the mts reagent consistent with the importance of positive charge at this position for coordination of the negatively charged phosphate of atp . at the nfr motif the asparagine and arginine residues were sensitive to mts reagents , whereas the phenylalanine was either unaffected or showed only a small decrease . these results support a common site of atp action at p2x receptors and suggest that non - conserved residues also play a regulatory role in agonist action .

it is well established that the transition of vascular smooth muscle cells ( vsmcs ) from a differentiated phenotype to a dedifferentiated state plays a critical role in the pathogenesis of atherosclerosis . the phenotypic modulation in vsmcs is accompanied by accelerated migration , proliferation and production of extracellular matrix components . eventually , these cellular events result in the formation of atherosclerotic lesions . however , the molecular mechanisms involved in phenotypic control are still unclear . micrornas ( mirnas ) are a class of endogenous , small , non - coding rnas that pair with sites can in 3 ' untranslated regions in mrnas of protein - coding genes to downregulate their expression . more importantly , one mirna is able to regulate the expression of multiple genes because it can bind to its mrna targets as either an imperfect or a perfect complement . thus , a mirna can be functionally as important as a transcription factor . as a group , mirnas it is therefore not surprising that mirnas are involved in the regulation of all major cellular functions . recently , the role of mirnas in cardiac cell differentiation has been described . in this study , kwon et al . demonstrated that mir-1 plays important roles in modulating cardiogenesis and in maintenance of muscle - gene expression by targeting transcripts encoding the notch ligand delta . the biological roles of mir-145 in diverse cancer cells have been recently identified ( reviewed in ) . in a recent article reported by xu et al . , mir-145 was found to be a critical switch for embryonic stem cell differentiation by repressing some core pluripotency factors . whether mirnas participate in the phenotypic control of vsmcs was unknown until recently . in this respect , three independent groups have reported exciting new discoveries regarding the critical role of the vsmc - enriched mirna , mir-145 , in vsmc phenotypic modulation [ 8 - 10 ] . they identified that mir-145 plays a role not only in the differentiation of multipotent neural crest stem cells into vsmcs , but also in the differentiation of adult vsmcs . other mirnas that may participate in the phenotypic modula tion of vsmcs are mir-143 and mir-221 [ 9 - 12 ] . this minireview summarizes the current research progress regarding the roles of mir-145 in the vsmc phenotype and the potential therapeutic opportunities of mirnas in atherosclerotic vascular disease . ji et al . demonstrated that mir-145 is the most abundant mirna in arteries . its expression is significantly downregulated in rat balloon - injured arteries with neointimal lesion growth . a recent nature article by cordes et al . showed that mir-145 expression was also decreased in mouse carotid arteries after ligation injury . more interestingly , transcripts of mir-145 were down regulated to nearly undetectable levels in atherosclerotic lesions containing neointimal hyperplasia . our own unpublished data also revealed that mir-145 is largely downregulated in atherosclerotic mouse and human arteries , although the downregulation is less pronounced compared with that from cordes ' study . if the selected atherosclerotic tissue had fewer vsmcs , the expression level of mir-145 could be lower . recently , cheng et al . found that mir-145 is the most abundant mirna in differentiated vsmcs . also , its expression is quickly downregulated in subcultured dedifferentiated vsmcs and in dedifferentiated vsmcs induced by stimulation with platelet - derived growth factor ( pdgf ) . our unpublished data also indicate that mir-145 expression in vsmcs isolated from balloon - injured rat carotid arteries and atherosclerotic apoe - knockout mouse aortas is significantly decreased compared with that in vsmcs isolated from normal control arteries . . found that , during the development of arteries , the expression of mir-145 is associated with the state of vsmc differentiation . mir-145 expression is notably absent in the aorta and pulmonary arteries during later cardiogenesis , during which vsmcs and arteries are developing . in contrast , high transcript levels of mir-145 in vsmcs of the arteries are demonstrated postnatally , after vsmcs and arteries have completed their development . cordes et al . demonstrated that mir-145 was necessary and sufficient to induce differentiation of multipotent neural crest stem cells into vsmcs . in addition , cheng et al . identified for the first time that mir-145 is a critical modulator for the vsmc phenotype both in vitro and in vivo . vsmc differentiation marker genes such as sm -actin , calponin , and sm - mhc were downregulated by mir-145 downregulation , and upregulated by mir-145 upregulation . the regulatory effect of mir-145 on the vsmc phenotype was further verified by cordes and colleagues . in contrast , another co - expressed mirna , mir-143 , had no significant effect on marker genes for vsmc differentiation , although it had a strong effect on vsmc proliferation . . demonstrated that the expres sion of the mir-143/145 cluster is confined to vsmcs during development . they found that mir-143 and mir-145 are required for vsmc acquisition of the contractile phenotype because the vsmcs from mir-143/145-deficient mice are locked in the synthetic state . the regulatory effects of mirnas on the vsmc phenotype are not limited to mir-143 and mir-145 . two recent studies revealed that mir-221 is also related to the vsmc phenotype that affects vsmc proliferation and migration . it is well known that phenotypic modulation of vsmcs is an initial cellular event in the development of atherosclerotic vascular disease . to determine the therapeutic potential of mir-145 in vascular disease , . demonstrated that restoration of mir-145 in rat balloon - injured carotid arteries via adenovirus - mediated gene transfer significantly inhibited neointimal lesion growth . in contrast , knockout of mir-143 and mir-145 resulted in the formation of neointima in mouse arteries . the roles of mir-145 in the vsmc phenotype and atherosclerotic vascular disease are summarized in figure 1 . expression modulation of mir-145 in the vascular walls and the potential roles of mir-145 in vascular smooth muscle cell phenotype and atherosclerosis . its expression is significantly downregulated in developing arteries and in adult arteries containing intimal hyperplasia . the downregulated mir-145 in adult arteries will increase vsmc dedifferentiation and result in the development of atherosclerotic lesions . mirna - based therapy may have some advantages compared with that for other molecular targets because one endogenous mirna can target its multiple target genes . although the recent studies have demonstrated that targeting mirnas , and mir-145 in particular , may represent a new therapy for atherosclerosis , there is still a long road before mirna - based technology can be translated to clinical therapy . first , the critical mirnas responsible for the development of atherosclerosis must be further identified , especially in human atherosclerotic arteries . second , the detailed cellular and molecular mechanisms of these critical mirnas in the prevention and treatment of atherosclerosis should be studied . third , in addition to the biological effects of these mirnas on vsmcs , their effects on other atherosclerosis - related cellular events should be identified . fourth , although methods are available to downregulate a mirna in vivo , technology for upregulating a mirna in the vascular walls in vivo requires development . finally , the potential side - effects of mirna - based therapy should be studied before application in the clinic . mirna : microrna ; mir-145 : microrna-145 ; pdgf : platelet - derived growth factor ; vsmc : vascular smooth muscle cell . this work was supported by a national institutes of health grant hl080133 and a grant from the american heart association 09grnt2250567 .

micrornas ( mirnas ) represent a class of small , non - coding rnas that negatively regulate gene expression via degradation or translational inhibition of their target mrnas . recent studies have identified that mir-145 is the most abundant mirna in normal arteries and vascular smooth muscle cells ( vsmcs ) , and its expression is significantly downregulated in dediffer - entiated vsmcs and atherosclerotic arteries . mir-145 plays a critical role in modulating vsmc phenotype . because phenotypic modulation of vsmcs is an initial cellular event in the development of atherosclerosis , mirnas , and mir-145 in particular , may represent new therapeutic targets for atherosclerosis .

the hundred trillions of cells which make up our bodies are continually exposed to various mechanical stimuli , including muscle contraction , ongoing blood flow , blood pressure , distension of visceral organs , etc . , which initiate a wide range of cellular responses . these responses include ca mobilization,1,2 protein phosphorylation,3 - 5 rearrangement of the cytoskeleton,6,7 transcriptional regulation,8 apoptotic cell death,9,10 and cell differentiation11 and so on . mechanical forces are sensed by mechanosensors that presumably undergo change in their enzymatic activity or interaction with signaling molecules in response to forces . however , the particular molecular entities and the underlying biophysical mechanisms of these mechanosensing molecules are largely unknown except for the mechanosensitive ( ms ) channels.12 - 14 a major reason for this slow progress is that mechanosensors , by their nature , do not possess the specific chemical ligands such as agonists , antagonists and inhibitors , which are used as biochemical tools to detect and purify receptor molecules . an alternative way toward the molecular identification of mechanosensors relies on an idea that putative mechanosensors are most likely localized at cellular sites of high concentrations of stress . the cell membrane is such a structure due to its high lateral elastic modulus , and is actually endowed with ms channels , although the correlation between the highly stressed membrane region and the ms channel localization has yet to be demonstrated . here we focus on adhesive structures , including focal adhesions , the actin cytoskeleton , and the molecular apparatus connecting these structures , where stress is presumably highly concentrated . generally focal adhesions comprise a high stress concentration site , linking extracellular matrices and the actin cytoskeleton.15 mechanical forces imposed from inside or outside of the cell are transmitted through the focal adhesions bidirectionally , i.e. , in an outside - in or inside - out direction.16 exogenous mechanical forces are exerted on integrins , an extracellular matrix receptor enriched in focal adhesions that activate a variety of intracellular signaling cascades.17 - 19 the activities of actin modulating proteins20 are also influenced by endogenous cell contractile force21 or exogenous mechanical stimuli.6,7 thus , mechanosensors22 and directly associated signaling molecules15 are thought to be involved in the focal adhesion , the actin cytoskeleton and/or cellular structures linking focal adhesions and the actin cytoskeleton . a recent in vitro biophysical study has shown that the apparent actin filament severing activity of cofilin and the rates of binding of cofilin to actin filaments are both affected by the tension present in the actin filaments,23 implying that the actin filament itself works as a mechanosensor . here , we review the recent progress in the study of tension - sensing by focal adhesion proteins and actin filaments , and evaluate the possible physiological roles of such tension - sensing by actin filaments . mounting evidence suggests that focal adhesion proteins are involved in the mechanically triggered activation of intracellular signaling molecules,15 including map kinases,24 akt,25,26 and pi3 kinase.27 direct manipulation and imaging of single protein molecules enables an application of mechanical forces to a target protein while monitoring its response , using these methods talin28 and possibly p130cas29 have been proposed to work as a mechanosensor . the focal adhesion protein talin has a head domain that binds to the cytoplasmic tail of the integrin subunit , while its rod domain contains actin - binding sites , each of which locates adjacent to a vinculin - binding site . when mechanical force was applied to talin through the manipulation of a small bead attached on a focal adhesion , integrin - cytoskeleton linkage at the focal adhesion was strengthened30 and vinculin was translocated to the focal adhesion underneath the bead in vivo,31 suggesting that talin acts as a mechanosensor in focal adhesions.30,32 a recent in vitro study demonstrated tension - sensing by the talin rod domain . the recombinant talin rod domain was extended approximately 100 nm by direct application of force ( 20 pn)28 with an afm tip ( fig . 1a ) , resulting in an increase in the number of vinculin head domains bound to the talin rod domain . combining molecular and cellular level studies , the authors have proposed that the force applied through the integrins in the focal adhesions extends the talin rod domain and exposes its binding sites for vinculin , which reinforces the actin - integrin linkage in vivo . ( a ) structure of the talin rod domain consisting of 12 helices ( upper panel ) . the rod domain which is unfolded under force28 ( lower panel ) is shown by the black arrows in the upper panel . ( b ) schematic drawing of the experimental setup to apply force to a single actin filament.23 one end of an actin filament ( red ) was tethered to a bead fixed on a coverslip , and the other end of the filament was tethered to a small bead manipulated by optical tweezers . ( c ) ( i ) , schematic drawing of the experimental setup to trace the torsional fluctuations of a single actin filament . an actin filament is tethered on the coverslip via gelsolin , and a bead was attached to the lower end of the actin filament . rotation of the bead is monitored using fluorescent small beads that are attached on the large bead . ( ii ) , rotational angular fluctuations of a bead attached to an actin filament during the time the large bead was trapped , but not stretched , by optical tweezers . the rotational angular fluctuations were decreased when the actin filament was stretched by moving the trapping point in downward direction . the data show the results at zero and ca . 5 pn stretch force ; one can change the applied force by increasing the laser power of the optical tweezers . panels a and c are based on studies ( 28 ) and ( 23 ) , respectively . p130cas , a substrate for p60src,29 is a scaffolding protein that localizes at focal adhesions . stretching of the cell substrate induces tyrosine phosphorylation of p130cas , followed by the activation of the p38 map kinase cascade via small gtpase rap133 in intact cells , suggesting that p130cas is involved in mechanically - induced signal transduction . a recent biophysical study using the purified p130cas substrate domain for p60src suggested that p130cas might work as a mechanosensor . mechanical extension of the elastic substrate , to which the purified recombinant p130cas substrate domain is adhered , induces tyrosine phosphorylation of the p130cas substrate domain by p60src , implying that the mechanical unfolding of the p130cas substrate domain increases the phosphorylation level of the p130cas not only in vitro but also in vivo.34 however , further studies are needed to confirm this phenomenon actually takes place in live cells . in intact spreading cells , the staining pattern made by an antibody against the extended p130cas substrate domain shows a similar staining pattern to that made by an antibody against phosphorylated p130cas in the cell periphery,34 suggesting that the p130cas substrate domain is extended by force , and tyrosine - phosphorylated by p60src.33 in these mechanosensing processes , the unfolding of the sensor molecules ( talin and p130cas ) is presumably required to sense forces and to transmit them to the downstream signaling molecules . titin , a giant elastic muscle protein connecting the z - disc and m - line in the sarcomere , is unfolded by force in its kinase domain.35 the improper unfolding of the kinase domain is thought to be involved in muscle disuse atrophy.36 fibronectin , an extracellular matrix protein , is mechanically unfolded by cell contractile forces,37 and the unfolding may be involved in accelerated fibronectin assembly , resulting in an enhancement of the fibronectin - integrin linkage.38,39 a recent in vitro study revealed that the actin filament itself functions as a mechanosensor.23 one end of a single actin filament was tethered to a myosin - coated bead fixed on a coverslip , while the other end was tethered to a small myosin coated bead manipulated with optical tweezers so as to tense the filament ( fig . when the filament was tensed ( ~30 pn ) , it was severed by cofilin with a larger delay compared with the filament when it was not tensed , or was not severed within the observation period ( ca . additionally , the binding of cofilin to the bundles of actin filaments was imaged with fluorescein labeled cofilin , which showed that the rate of the binding of cofilin to the actin bundles decreased when the bundles were tensed . approximately 2 pn of force is sufficient to decrease the apparent severing activity of cofilin,23 which is comparable to the force generated by a single myosin head . a single actin stress fiber is composed of 1030 actin filaments,40 and the contractile force in a single stress fiber is estimated to be on the order of nn,41 suggesting that the contractile force in stress fibers ( > 2 pn for each actin filament , assuming the stress in the stress fiber is evenly distributed among the actin filaments ) is high enough to prevent cofilin from binding to the actin filaments in vivo . there are enormous numbers of biochemical,42,43 structural,44,45 and computational46 studies on the binding of cofilin to actin filaments . electron microscopic analyses revealed that the twist of the actin filament is increased when the filament is fully decorated by cofilin.44,45 on the other hand , the rotational angular conformation of actin protomers in native actin filaments is variable47 ; the angle between neighboring actin protomers reportedly ranges from 156 to 170.45 based on these observations , it is hypothesized that cofilin preferentially binds to actin filaments in solution when the protomers of the actin filament are in the twisted state ; i.e. , large torsional fluctuations are required for the binding of cofilin to the filament . the torsional fluctuations of single actin filaments were visualized by monitoring the rotation of a bead attached on one end of the filament ( fig . application of a force of approximately 5 pn reduced the torsional fluctuations of the filament ( fig . 1c ) , indicating that the actin filament fluctuates less when the filaments are tensed , supporting the hypothesis that tension in the actin filament reduces torsional fluctuations of the actin filament , which decreases the effective number of cofilin binding sites so as to prevent the binding of cofilin , resulting in an inhibition of the cofilin severing of the filament . , the sensor molecule actin filament senses the applied force and transduces it into changes in the fiber fluctuation that in turn modulates the activity ( binding here ) of the signaling molecule cofilin , eventually regulating the fiber dynamics by itself . the tension - dependent local disassembly of actin filaments by cofilin presumably works under certain specific physiological conditions . the distribution of cofilin in living cells was examined using a gfp - cofilin fusion protein . gfp - cofilin translocated to the stress fibers within a period of one minute when tension in the stress fibers was reduced by relaxing the pre - stretched elastic cell substratum ( fig . 2a ) , followed by disassembly of the stress fibers,23 suggesting that cofilin mediates disassembly of the stress fibers with a decline in tension in living cells . bdm , a myosin atpase inhibitor , reduces tension in the stress fibers , and induces stress fiber disassembly in living cells . stress fiber disassembly by bdm is reportedly enhanced in cells overexpressing cofilin.21 by contrast , stress fiber disassembly was not detected in the cells without bdm , strongly supporting the proposal that cells make use of the tension - dependent local disassembly of actin filaments by cofilin . ( a ) when a certain amount of tension was generated in the stress fibers in adherent cells , the binding of cofilin to the stress fibers was reduced ( upper panel ) . when the tension was reduced by relaxing the cell substratum ( the direction is indicated by the black arrows ) , cofilin bound and started to disassemble the stress fibers ( lower panel ) . the prominent transverse stress fibers generating a large amount of contractile force are not disassembled . ( c ) schematic drawing of the actin cytoskeleton in an adherent cell during migration . the actin filaments are disassembled in the trailing region of the cell , where the tension in the stress fibers is low , while the stress fibers generating tension in the middle region of the cell are not disassembled . the double - headed arrows indicate the width of the lamellipodia . in accord with the above hypothesis , a tension decline in the actin stress fibers leads to the disassembly of the stress fibers , however , the possibility can not be excluded that it is the disassembly that leads to the decline in tension in the stress fibers . to resolve this problem , simultaneous measurement of the tension decline and the disassembly of the actin stress fibers is required . we recently examined the relationship between the tension decline and stress fiber disassembly in living cells.48 fibronectin - conjugated beads were adhered to the endothelial cells in that study . actin stress fibers were formed between focal adhesions underneath the bead and the focal adhesions at the cell bottom . the force ( 35 nn generated by 1 m displacement of the bead ) applied to the stress fibers and to the connected focal adhesions was estimated from the displacement of the beads embedded in the elastic substrate of the cells . when the attached bead was displaced , the force applied to the focal adhesions transiently rose , and then declined in less than a few seconds , indicating a tension decline in the stress fibers , probably due to a partial destruction of the linkage among the actin fibers within the stress fiber , which would decrease its elastic modulus . subsequently , the same stress fibers were gradually disassembled within a period of 10 min , indicating that the decrease in tension in a particular stress fiber is followed by its disassembly , though the involvement of cofilin with this arrangement has not been examined yet . the potential roles of the tension - dependent local disassembly of actin stress fibers in migrating cells can be discussed in cells such as keratocyte and osteocarcinoma cells , in which the magnitude and distribution of the intrinsic contractile force generated by the actin cytoskeletons were quantitatively analyzed.49 - 51 the traction forces in migrating keratocytes were estimated from the local distortion in the elastic substratum of the cells . the traction forces generated beneath the front area of a migrating keratinocyte cell are relatively small , while the forces beneath the lateral area of the cell are substantial.49 in locomotive keratocytes , actin filaments are polymerized near the leading edge of the cells and then disassembled within a few minutes , thereby maintaining a filament length of a few m , while , in contrast , the prominent transverse stress fibers that connect both the lateral sides of the cell are not disassembled.50 similar prominent stress fibers connecting the lateral sides of the cells are also found in migrating osteosarcoma cells , and are disassembled with a decline in tension by the myosin inhibitor , blebbistatin.51 the width of lamellipodia ( shown by the double - headed arrow in figure 2b ) is almost proportional to the length of the actin filaments extending at the leading edge , and knockdown of cofilin by rnai increases the width of lamellipodia,52 suggesting that cofilin mediates the actin filament disassembly near the leading edge . these findings fit the hypothesis that the disassembly of stress fibers by cofilin is inhibited when the filaments are tensed . the tension across vinculin , a focal adhesion protein , has been assessed in the migrating endothelial cells using a vinculin - fusion protein named a tension sensor module53 that was designed based on the fret mechanism . the fret signal is high at disassembling or sliding focal adhesions near the trailing edge of migrating cells , implying that the tension sensed by the sensor is low ( < 2.5 pn ) where stress fibers are disassembled . this also agrees with the above hypothesis , and suggests that a force as small as in a pn range can presumably be sensed by actin filaments , and , when decreased , initiates the stress fiber disassembly in living cells . proper disassembly of stress fibers is crucial for cell migration , because actin stabilization by phalloidin,54 and enhancement of stress fiber formation55 both inhibit cell migration . thus , the selective disassembly of non - tensed stress fibers is crucial for cell migration ; e.g. , cell migration is partly realized by the continual processing of actin fiber dynamics , including disassembly at the trailing edge and assembly at the leading edge of cells . the invasion of cancer cells is enhanced by mechanical stimulation ; the number of cancer cells that invaded the three dimensional matrix gel was increased when the gel was deformed by twisting small magnet beads embedded in the gel by an application of an external magnet field ( i.e. , mechanical stimulation to the cells in the gel ) . in addition , cofilin is involved in the mechanically stimulated invasion,56 suggesting that the mechanosensing by actin filaments plays an important role in cancer cell invasion . elucidating the role of the tension sensing performed by actin filaments in combination with cofilin binding / unbinding will ultimately provide great insights into cell behaviors under not only physiological , but also pathophysiological conditions .

mechanosensitive ion channels have long been the only established molecular class of cell mechanosensors with known molecular entities . however , recent advances in the state - of - the - art techniques , including single - molecule manipulation and imaging , have enabled an investigation of non - channel type cell mechanosensors and the underlying biophysical mechanisms of their activation . to date , two focal adhesion proteins , talin and p130cas , have been postulated to act as putative mechanosensors , acting through mechano - induced unfolding of their particular soft domain(s ) susceptible to phosphorylation . more recently , the actin filament has been demonstrated to act as a mechanosensor in the presence of the soluble actin - severing protein , cofilin . the cofilin severing activity negatively depends on the tension in the actin filament through tension - dependent binding / unbinding of cofilin to / from the actin filament . as a result , relaxed actin filaments are severed , while tensed ones are either not severed or severed after a long delay . here we review the latest progress in the mechanosensing by non - channel type proteins and discuss the possible physiological roles of the mechanosensing performed by actin filaments in the course of cell migration .

mammals are the only animals that secrete a complex fluid from an elaborated skin gland to provide both innate protection and nourishment for their newborn . there are more than 4,000 species of mammals with striking similarities in the structure and function of their mammary glands as well as in their unique milk components such as the caseins , -lactalbumin , lactoferrin , lactose , and milk fat . nevertheless , variations are exhibited in the arrangement and numbers of mammary gland , milk composition , and suckling strategies . mammary gland development begins at puberty and is maintained throughout pregnancy until lactation . during these last stages , development compromises numerous overlapping programs such as branching morphogenesis , inductive stromal - epithelial interactions , programmed cell death , extracellular matrix remodeling , and hormone action . current knowledge of the molecular regulation of mammary development and lactation has largely been derived from the dissection of signaling networks in cell culture systems and phenotypic characterization of genetically altered mice as well as genomewide approaches such as microarrays . nonetheless , to date , regulation of mammary gland development during pregnancy and lactation is incompletely understood . lactation is regarded as one of the most remarkable products of evolution whose processes include the development of mammary tissue as well as the synthesis and secretion of milk . consequently , despite the fact that the development of mammary tissue and the synthesis and secretion of milk are considered as complex dynamic physiological processes , both must preserve overall common characteristics among mammals . considering the underlying assumption that important biological functions are often conserved across species , genes expressed across multiple species are likely to have conserved functions . given the completion of the dna sequence of the human , mouse , and rat genomes , genes identified in microarray studies can be readily compared across species with respect to orthologous genes . therefore , a cross - species hybridization ( csh ) experiment could provide significant information concerning probable conserved gene networks among mammals . in a csh experiment , there is hybridization of rna from one or more ( target ) species to a microarray that contains dna ( cdna or oligomers ) from another ( reference ) species and represents a valuable tool for the identification of orthologous genes . thus , a csh microarray analysis offers the possibility of furthering our understanding of cross - species commonalities and differences that could lead to more effective use of animal models to understand the regulation of mammary gland development at the molecular level . dissection of unique patterns of expression of orthologous clusters of genes among species throughout distinct physiological time points along pregnancy and lactation could prove useful in the integrative analysis of the information available for discerning the molecular events underlying the regulation of mammary gland development and function that lead to milk synthesis . in this study , these data resulted from heterologous microarrays of target rna samples derived from rat mammary gland during distinct stages of pregnancy and lactation in order to extrapolate and enhance the understanding on transcriptional module networks or coregulated functional gene groups conserved in rodents and in the development of the mammary gland in humans . fifteen female sprague dawley rats were obtained from the animal care facility of centro mdico nacional siglo xxi of the mexican institute of social security ( imss ) in mexico city . animals were housed at 22 2c with a 12 h light / dark cycle with free access to water , and a purified diet was administered ad libitum during pregnancy and lactation . , rats were randomly assigned to five groups representing distinct time points in mammary gland development : virgin ( v ) , day 5 ( p5 ) and , day 14 ( p14 ) of pregnancy and day 1 ( l1 ) , and day 12 ( l12 ) of lactation . the day on which sperm was identified in vaginal smears was designated as day 1 of pregnancy and the day of parturition was designated as day 1 of lactation . rats were euthanized , and whole mammary tissue was removed from v , p5 , p14 , l1 , and l12 rats . tissue was immediately frozen in liquid nitrogen and stored at 70c for subsequent total rna isolation or histological analysis . total rna was isolated from tissue ( 0.1 - 0.2 g ) using trizol ( invitrogen , carlsbad , ca , usa ) following the method of chomczynski and sacchi . total rna from mammary tissue was isolated from three different animals of each physiological period ( v , p5 , p14 , l1 , and l12 ) , pooled , and kept in aliquots for later determination of purity and integrity . four microarray datasets generated using the custom - designed mus musculus oligonucleotide array containing 65-mer probe sets representing 22,000 transcripts ( microarray unit , cellular physiology institute , unam , mexico city ) were analyzed . each dataset represented distinct time points in mammary gland development such as p5 , p14 , l1 , and l12 . histologically , the mammary proliferative stage is represented by p5 , the secretory differentiation stage by p14 , early lactation by l1 , and full lactation by l12 . design of the microarray experiments is presented in table s1 in supplementary materials available online at http://dx.doi.org/10.1155/2013/624681 . ten g of total pooled rna was reverse transcribed into cdna incorporating dutp - cy3 or dutp - cy5 and using the cyscribe first - strand cdna labeling kit ( amersham biosciences , piscataway , nj , usa ) . sunnyvale , ca , usa ) , equal quantities of labeled cdna were hybridized to the m22 k_01 microarray for 14 h at 42c . four hybridization assays were carried out as follows : ( a ) the fluorophore used was dutp - cy3 for control nonpregnant virgin rats ( v ) and dutp - cy5 for p5 , ( b ) dutp - cy3 for v and dutp - cy5 for p14 , ( c ) dutp - cy3 for v and dutp - cy5 for l1 , and ( d ) dutp - cy3 for v and dutp - cy5 for l12 . data acquisition and analysis of array images were performed in scanarray 4000 with its accompanying software scanarray 4000 from packard biochips . microarray data analysis was performed with free software genarise , which was developed in the computing unit of the cellular physiology institute of the unam ( http://www.ifc.unam.mx/genarise/ ) . the goal of genarise is to identify which genes show good evidence of being differentially expressed . the software identifies differentially expressed genes by calculating an intensity - dependent z - score . elements with a z - score > 1.5 standard deviations are considered to be significantly and differentially expressed genes . the complete set of raw excel data files have been deposited at gene expression omnibus ( geo ) and are available on the geo website ( i d geo gse22545 ) . gene lists were generated by a set of multiple comparisons among the distinct developmental stages and intersection in venn diagrams . two - way hierarchical clustering with average linkage and a range of 5 to 15 k - means classifications were used to group our time series data using open source software cluster v3.0 . java treeview was used to display the clustering results as dendogram or heat map representations . we adopted the procedure as described in to code the mean expression of a cluster at each stage as flat , decreased , and increased and converted it to numerical representation . putative orthologous genes in rat , mouse , and human were identified from a genome comparative search with roundup ( http://rodeo.med.harvard.edu/tools/roundup ) . roundup is an ortholog and phylogenetic profile retrieval tool backed by a massive repository of orthologous and associated evolutionary distances that were built using the reciprocal smallest distance algorithm . the search was done with a stringent blast e - value threshold of 1.0 10 and a divergence threshold of 0.2 . the david 2.0 functional annotation tool ( http://david.abcc.ncifcrf.gov/summary.jsp ) was used to sort and arrange the similar , redundant , and heterogeneous annotation contents from a set of genes into defined functional groups . in the case of insufficient gene ontology information , published pathway mapping was accomplished using the kyoto encyclopedia of genes and genomes ( kegg ) database of biological systems that integrates genomic , chemical , and systemic functional information ( http://www.genome.jp/kegg/kegg2.html ) . gene lists were converted to human swissprot ids using tables from the ensembl database , release 42 . for each list of human swissprot ids , interactions between those gene products were obtained from online predicted human interaction database ( ophid ) and postprocessed using custom scripts to determine all linkages in the network and to generate a network file . this network file was then explored using navigator v2.0.15 , a program for large network analysis ( http://ophid.utoronto.ca/navigator/index.html ) . transcription - factor - binding site ( tfbs ) prediction was accomplished using core_tf ( conserved and over - represented transcription factor binding sites ) , a web - based tool that identifies overrepresented tfbs in promoters from coexpressed genes aided by the evaluation of cross - species conservation . we measured the relative transcript levels of 14 target genes , and five genes commonly used as references such as glyceraldehyde-3-phosphate dehydrogenase ( gapdh ) , beta - actin ( actb ) , and ribosomal large protein p0 ( rlp0 ) were used as high abundance internal controls as well as splicing factor arginine / serine - rich 1 ( sfrs1 ) and hypoxanthine guanine phosphoribosyl transferase 1 ( hprt1 ) as medium- and low - abundance internal controls , respectively ( table s2 ) . quantitative real - time pcr was performed in a 20 l reaction with 5.0 l from 1/4 reverse transcription dilution using the lightcycler probes master mix ( roche diagnostics , mannheim , germany ) containing 0.2 m of mrna - specific primers and 0.1 m corresponding upl probe into lightcycler microplate wells under reduced light conditions . the results are expressed as mean sem of at least three individual experimental observations . statistically significant differences among experimental groups ( between the mean values of each group ) were determined by an unpaired students t - test ( z - test ) , anova , or a modified fisher 's exact test . the rat mammary gland undergoes a series of dramatic phenotypic changes during pregnancy and lactation . in order to determine the integrity of the dissected inguinal mammary glands , a gross histological evaluation of the characteristic cytomorphological features four time points ( pregnancy days 5 and 14 ; lactation days 1 and 12 ) were selected to represent distinct periods in mammary gland development . histologically , the mammary proliferative stage is represented by p5 , the secretory differentiation stage by p14 , early lactation by l1 , and full lactation by l12 . as reported elsewhere , initial changes observed during pregnancy include an increase in ductal branching and the formation of alveolar buds ( figures 1(c ) and 1(d ) ) . the latter half of pregnancy is characterized by the expansion of alveolar buds to form clusters of lobuloalveolar units followed by the differentiation of these structures into presecretory structures . by day 14 of pregnancy , there is a readily apparent increase in the size of the epithelial compartment ( ep ) ( figures 1(e ) and 1(f ) ) , and expansion of the epithelium ( whereas the adipose compartment decreases ) continues until the epithelial compartment predominates by onset of lactation ( figures 1(g ) and 1(h ) ) . by day 12 of as expected , examination of the histology of the mammary gland at this stage reveals prominent luminal structures ( l ) and ducts and few adipocytes visible at this time ( figures 1(i ) and 1(j ) ) . in this study , we analyzed expression profiles of 22,000 transcripts using murine microarrays and rna samples of mg from virgin , pregnant , and lactating rats by cross - species hybridization . we first identified the total number of genes differentially expressed throughout distinct time points in mammary gland development such as p5 , p14 , l1 , and l12 . a total of 807 oligonucleotide probe sets representing 521 annotated genes showed differential expression in at least one of four physiological time points evaluated , taking into consideration a mean z - score cutoff value of 1.50 standard deviations using genarise . during early pregnancy ( day 5 ) , most of these transcripts ( 123 , 77.8% ) were upregulated , suggesting a feasible tendency in the direction of gain of function versus the virgin stage ( v ) . likewise , in mid - pregnancy ( day 14 ) , as opposed to the virgin stage , the number of transcripts with an altered expression maintained a similar value ( 133 transcripts ; 89.26% upregulated ) . during early and mid - lactation ( days 1 and 12 ) , 342 and 461 transcripts were differentially expressed , corresponding to a percentage of 64.0 and 38.4 overexpressed , respectively ( figure 2(a ) ) . to further illustrate the differences and commonalities among the four physiological time points , changes in gene expression were also interpreted with a venn diagram . as shown in figure 2(b ) , the descriptive table of the venn diagram denotes the number of genes showing upregulation ( ) or downregulation ( ) uniquely at pregnancy ( day 5 or 14 ) or lactation ( day 1 or 12 ) and differential expression at a combination of stages . venn diagram analysis indicated that 47.2% ( 381/807 ) of all the differentially expressed transcripts presented an average significant z - score fold change ( z > 1.5 ) exclusively during either or both time points of lactation . interestingly , among the 381 altered gene transcripts during lactation , 64.8% ( 247/381 ) were found downregulated , implying as previously stated by lemay et al . ( 2007 ) that mammary epithelial cells become biofactories not by gain of function but by a broad suppression of function to effectively push all cell resources towards a very few important tasks . all the gene sets that shared spatial and temporal distributions ( overlapping changes in expression ) are listed in additional data files ( table s3 ) . to determine global alterations in gene expression across developmental stages of the mammary gland from early pregnancy to mid - lactation , we performed a complete - linkage hierarchical clustering with an euclidean distance similarity metric on the expression profiles of the differentially expressed genes ( annotated and est ) across all four time points . the expression profiles of the 807 genetic elements resulted in six predominant clusters on a dendogram ( designated clusters 16 in figure 3 ) . this major trend is a decline in gene expression during mid - pregnancy that remains low during lactation . cluster 2 ( c2 ) describes 25.77% of the total data population and is characterized by a linear decrement in gene expression towards mid - lactation . however , positive z - score values are retained with respect to the reference stage ( virgin ) . the remainder of the clusters ( c3c6 ) appears to explain between 3.09 and 17.84% of the data variation . in c3 and c4 , gene expression rises exponentially from early pregnancy , reaching a plateau during mid - lactation . however , the slope of the curve is even steeper in c3 in comparison to c4 . in cluster 5 ( c5 ) , 65 elements matched the profile outline ( inverted sigmoid form ) of major trend c1 although the reduction tendency was less marked . in cluster 6 ( c6 ) , expression was roughly unchanged during pregnancy and lactation . even so , the relative abundance of transcripts remained in a higher proportion than the reference virgin stage as described for c2 . this transcriptional profile , involved in the mammary development program identified in rat , could be conserved in others mammals like mouse . consequently , in order to delineate potential groups of coregulated genes , final cluster membership was determined by a k - means analysis based on the preestimated number ( six ) of gene clusters . k - means clustering revealed six distinct clusters ( k16 ) that distinguished up from down , early from middle , and transient from sustained changes in expression ( figure 4 ; table s5 ) . each of the six clusters was designated with its unique trajectory expression profile signature across stages ( pregnancy days 5 and 14 , lactation days 1 and 12 ) as presented in figure 4 . there were two major groups of 245 under- and 175 overexpressed tags during lactation only ( table s5 , k1 : 1,1,0,0 and k3 : 1,1,2,2 according to the procedure of rudolph et al . , to code the mean expression , see section 2 ) . among the typical upregulated genes of lactation stage are the milk protein ( casein alpha ( csn1s1 , csn1s2a ) , casein beta ( csn2 ) , and whey acidic protein ( wap ) ) and biogenesis genes that mainly concern glucose and lipid metabolism ( akr1c6 , aldob , ugt2b1 , plb1 , apoe , and sult2b1 ) and transcriptional regulation ( stat5a , pou2af1 ) . among those genes found significantly downregulated only during lactation , several play an important role in the regulation of apoptosis , mediation of metastatic behavior ( epithelial - mesenchymal transition ) , or ubiquitin - mediated protein catabolism ( lysosome degradation ) in the mammary gland including igfbp5 , mmp2 , and ube2r2 [ 14 , 15 ] . one hundred forty - nine genes were upregulated exclusively during early pregnancy ( k2 : 2,1,1,1 ) such as esr1 , esr2 , tshr , and oxt . these participate in the transduction of hormonal status [ 2 , 16 ] involved in the modulation of important physiological processes such as carbohydrate metabolism ( creb3l4 , hk1 , and coasy ) ; glutathione metabolism ( ggt1 , mgst1 , and gstm6 ) ; cell differentiation ( foxa1 , mtap7 , gdf1 , twist2 , hey1 , dll4 , and pcaf ) [ 1923 ] , stromal - epithelial communication ( cell - cell junctions ) ( cldn10 , mpp5 , and epb4 ) , and cell adhesion ( matn1 , krt71 , mpzl2 , and dscaml1 ) . the smallest group of 24 genes were significantly upregulated exclusively at the onset of lactation ( k4 : 1,1,2,1 ) such as lpo , cd8a , and irs1 , important for lactogenesis , particularly in milk production capabilities and related immunotropic constituents ( antigen - specific cd8 + t cells ) found in colostrum [ 26 , 27 ] . for example , acta1 , flnc , and pax7 , which are either restricted to muscular tissues or involved in myogenic development and cellular differentiation [ 28 , 29 ] , are included in this group . according to the trajectory profile signature , 95 additional genes were found upregulated at all stages evaluated ( k6 : 2,2,2,2 ) . interestingly , most of the overexpressed genes in this group include general transcription and translation ( including spliceosome and protein folding ) machinery factors ( eif4a2 , eif2ak1 , etf1 , taf1 , ercc2 , sart1 , ppih , and dbr1 ) [ 30 , 31 ] as well as structural ( itga5 , actg1 , add2 , cldnd2 , rptn , and triobp ) and basal metabolic genes ( pank4 , agpat5 , cyp24a1 , and phyh ) . once the gene clusters were properly defined , identification of orthologous gene transcripts among the time course differentially expressed gene list subsets was critical for reliable comparison of gene function and subsequent determination of probably conserved transcriptional modules implicated in biological processes during the development of mammary tissue . according to genome comparative roundup orthologous database of a total of 448 transcripts upregulated and 371 downregulated , 213 ( upregulated ) and 183 ( downregulated ) genes were identified as orthologous to rat and/or human . the remainder of the genes was discarded or removed from subsequent analysis due to lack of similarity , insufficient information , or unknown identifiers . a complete list of orthologous genes from each dataset was compiled ( table s6 ) . among the upregulated orthologous genes to rat and/or human are those encoding to milk proteins , carbohydrate and lipid metabolism , transcriptional factors [ 17 , 34 ] , transduction of hormones , glutathione metabolism , and cell differentiation [ 19 , 20 ] . others are associated with stromal - epithelial communication , cell adhesion , lactogenesis , and general transcription and translation machinery factors as well as structural and basal metabolic genes [ 3335 ] . among those genes found significantly downregulated , several play an important role in the regulation of apoptosis , mediation of metastatic behavior ( epithelial - mesenchymal transition ) , or ubiquitin - mediated protein catabolism ( lysosome degradation ) [ 14 , 15 ] . also , genes restricted to muscular tissues or involved in myogenic development and cellular differentiation [ 28 , 36 ] are downregulated . taking into consideration their temporal expression profile signature and the fact that they represent different k - means cluster , 14 genes were selected for real - time pcr analysis ( table s2 ) . results show that the expression trends were consistent with the results from the microarray analysis . correlation analysis showed good agreement between real - time rt - pcr and microarray analysis . microarray results for all 14 genes tested were confirmed by real - time rt - pcr with regard to direction and significance of change ( figure 5 ) . conservation of functional blocks of genes is likely to be more important in a cross - species comparison . we found distinct blocks of significantly differentially expressed genes within different cytogenetic regions of the rat with homologous chromosomal segments in human and mouse . however , human , mouse , and rat have different chromosomal arrangements . genes in these blocks appear in contiguous cytogenetic regions , irrespective of species and chromosomal location . this finding is not surprising considering the close evolutionary distance between the species where 278 orthologous segments are reported to be shared between human and rat , and 280 segments are reported to be shared between human and mouse . it is proposed that these gene blocks may be significant for mammary gland development and maintenance and progression of lactation across human , rat , and mouse . for example , genes in the blocks may be coordinately expressed to share transcription programs as stated in previous studies . the argument may be made against the feasibility of using rodent data to draw inferences to human mammary gland gene expression . however , our objective in this study was to utilize the best available sources of information such as rat gene expression data during mammary development and mapping data to develop hypotheses on putative functional gene blocks conserved across species , underlying similarities despite reported differences in the architecture and hormonal control of mammary glands between rats and other rodents and between rats and humans [ 38 , 39 ] . in an effort to further characterize potential highly coregulated gene blocks , we combined transcription - factor binding - site prediction along the promoters of each gene member with the detection of expression profiles of annotated altered transcripts categorized as nucleic acid binding protein . several families of transcription factors were identified ( table s7 ) . for the most part , the presence of cis - elements found with core_tf ( http://www.lgtc.nl/core_tf ) in the promoters of the genes slc44a4 , ppt2 , and b3galt4 that compromises the conserved block d15 ( table s8 ) along with the cotranscription of mrnas that encoded for trans - regulator elements suggests that they are most likely modulated by transcription factors runx2 , creb3l4 , pou3f2 , and pou2af1 . correspondingly , the gene members of block u1 may possibly be coregulated by transcription factors stat5a , foxa1 , creb3l4 , and pou2af1 . in the same manner , other gene blocks ( u3 , u8 , u9 , d1 , d9 , and d14 , table s8 ) were found most likely co - regulated by a minor number of transcription factors ( foxa1 , creb3l4 , pou2af1 , or egr2 ) . hence , identification of conserved overrepresented upstream motifs unravels putative regulatory elements for transcription ( figure 6 ) in at least half of the gene block members reported in this study . consequently , these results strongly substantiate the maintenance of comparable transcriptional regulation programs among the predicted coexpressed modules . because cotranscription of genes in conserved blocks may allow concerted expression of gene products involved in the same response or pathway , integration of this type of analysis thus , the co - regulated clusters we proposed may indeed be conserved transcriptional modules through evolution , at least between rodents and primates . heterologous hybridization experiments on any microarray are of limited use for genes that have undergone rapid evolutionary change in coding regions , large rearrangements , and duplication . long oligonucleotide - based microarray platform may be more suitable for cross - species gene expression studies than a short oligonucleotide - based system . this comparative approach is based on the assumption that similar gene sequences in closely related species allow a reasonably reliable detection of many orthologous genes . for instance , according to several independent and unrelated studies carried out on comparable 50 to 60-mer oligonucleotide microarrays , cross - hybridization was observed only with genes with 50%75% overall sequence identity , respectively [ 44 , 45 ] . considering that orthologous genes between human and mouse and between human and rat both have a mean of ~85% sequence identity , validity of the results obtained in this study despite the problems encountered by csh in comparison to ssh seems upholding . in fact , the nucleotide sequence alignment confirmed an > 75.3% homology at least for the transcript members of the distinct gene blocks described , depending on the sequence evaluated among primates and rodents reinforcing the notion of attaining valid biological results . in addition , similar expression trends for distinct probe sets for one corresponding gene ( data not shown ) seem to largely substantiate the certainty and reproducibility of hybridization results obtained in this study . because of the challenges inherent to csh data , their confirmation by other techniques is essential . in addition to qrt - pcr , orthologous gene expression profiles with syntenic regions of rat , mouse , and human chromosomes reinforce another confirmation method that potentially substantiates the csh results obtained in this study . nonetheless , further validation of the results must be carried out by using csh of human rna to mouse oligonucleotide arrays . this study provides access to a prevalidated platform for analyzing transcriptional changes in rat mammary gland . this paper will hopefully spur an increase of mammary gland csh transcriptome analysis , thus adding to our knowledge base of this interesting evolutionary feature among mammals . however , although we acknowledge the multitude of aspects that can be elucidated by traditional ssh transcriptome analysis , we believe the biggest potential of the presented microarray lies in the multispecies - type studies described . we demonstrated that data analysis strategies such as the combination of orthologous gene expression profiles and chromosome mapping in conjunction with directed promoter transcription - factor binding - site prediction presented here can add strength to conclusions and help identify systems and responses that are conserved across the mammal taxa . the possibility of studying the evolutionary depth of transcriptional regulation adds a new dimension to comparative transcriptomic , particularly identification of differentially co - regulated gene blocks mapped to highly conserved syntenic chromosomal regions , which is important in mammary gland development using csh experiments among mammal species .

the mammary gland ( mg ) undergoes functional and metabolic changes during the transition from pregnancy to lactation , possibly by regulation of conserved genes . the objective was to elucidate orthologous genes , chromosome clusters and putative conserved transcriptional modules during mg development . we analyzed expression of 22,000 transcripts using murine microarrays and rna samples of mg from virgin , pregnant , and lactating rats by cross - species hybridization . we identified 521 transcripts differentially expressed ; upregulated in early ( 78% ) and midpregnancy ( 89% ) and early lactation ( 64% ) , but downregulated in mid - lactation ( 61% ) . putative orthologous genes were identified . we mapped the altered genes to orthologous chromosomal locations in human and mouse . eighteen sets of conserved genes associated with key cellular functions were revealed and conserved transcription factor binding site search entailed possible coregulation among all eight block sets of genes . this study demonstrates that the use of heterologous array hybridization for screening of orthologous gene expression from rat revealed sets of conserved genes arranged in chromosomal order implicated in signaling pathways and functional ontology . results demonstrate the utilization power of comparative genomics and prove the feasibility of using rodent microarrays to identification of putative coexpressed orthologous genes involved in the control of human mammary gland development .

gliomas are the most frequent primary tumors of the central nervous system ( cns ) and represent approximately 2% of all malignant diseases . their annual incidence is about 11.5 new cases per 100.000 persons per year [ 1 , 2 ] . the who classification of tumors of the central nervous system distinguishes the subtypes of glioma according to morphology and grade . high - grade gliomas ( hgg , who - grade 3 and 4 glioma ) are malignant tumors with a poor survival outcome . in a pivotal phase iii trial , where patients diagnosed with glioblastoma ( gb , who - grade iv glioma ) who were treated with postoperative radiation therapy ( rt ) and concomitant temozolomide ( tmz ) followed by six cycles of adjuvant tmz , the median survival was 14,6 months , while the overall survival ( os ) was 27.2% at 2 years , 16.0% at 3 years , 12.1% at 4 years , and 9.8% at 5 years . the prognosis of patients with who - grade iii glioma is superior to that of gb patients but much more heterogeneous and correlated with the histopathological and molecular - genetic subtype [ 5 , 6 ] . following initial resection and postoperative rt , anaplastic gliomas recur after a median of 2 - 3 years . most often , recurrent grade iii glioma will have transformed into a more aggressive tumor at recurrence ( a so - called secondary glioblastoma ) . the survival of patients with recurrent high - grade glioma following prior therapy with alkylating chemotherapy is grim and no treatment has demonstrated to improve the survival in a randomized clinical trial [ 6 , 7 ] . hgg are among the most angiogenic tumors and typically express high amounts of vascular endothelial growth factor ( vegf ) . vegf is a key molecular mediator of tumor - associated neoangiogenesis and its expression level has been correlated with tumor vascularisation , who - grade and prognosis [ 8 , 9 ] . hgg also express the vegf - receptors and frequently carry an amplicon of chromosome 4q12 comprising the vegf - receptor-2 ( vegfr2 ) , pdgfr - alfa , and ckit genes in 2330% of cases [ 1012 ] . coexpression and/or amplification of both the vegf and vegfr2 constitute an autocrine / paracrine loop . bevacizumab ( bev ; avastin , roche , basel , switzerland ) is a humanized immunoglobulin g1 monoclonal antibody that binds to and inhibits vegf . it has proven to be active in combination with cytotoxic agents and is registered by the fda and ema as part of a combination treatment regimen with chemotherapy for metastatic colorectal cancer , non - small - cell lung cancer , and breast cancer , and in combination with interferon - alfa in metastatic renal cell carcinoma [ 8 , 1319 ] . on may 5 , 2009 , the fda granted accelerated approval to bevacizumab as a single agent for the treatment of patients with recurrent glioblastoma . the approval was based on the results of two phase ii clinical trials ( avf3708 g and nci 06-c-0064e ) . the largest phase ii trial , involving a total of 167 patients with recurrent gb , randomized patients between treatment with bev ( at a biweekly dose of 10 mg / kg ) in one arm and the combination of bev ( at the same dose ) and irinotecan in a second arm . in the bevacizumab - alone and the bevacizumab - plus - irinotecan groups , the objective response rates were 28.2% and 37.8% , and the estimated 6-month progression - free survival rates were 42.6% and 50.3% , respectively . the number of adverse events in the bev plus irinotecan population was higher ( 65.8% versus 46.4% grade 3 adverse events ) , while median overall survival times was comparable between the two arms ( 9.2 months and 8.7 months , resp . , compared to 7.5 months in a historical population ) . in a single - arm phase ii trial , investigating the sequential use of bev and the combination of bev and irinotecan , no activity was found for the combination after failure of bev as a single agent . bev has also demonstrated antitumor activity as a single agent in patients with recurrent anaplastic glioma [ 22 , 23 ] ; in combination with a variety of cytotoxic agents [ 2426 ] , and when administered on a once every 3-week schedule ( at a dose of 15 mg / kg every 3 weeks ) . mri - based tumor evaluation in patients treated for recurrent glioma have been characterized by a rapid regression of tumor - associated edema and restoration of the blood - brain / tumor barrier . progression of disease by diffuse , non - gadolinium - enhancing infiltration of the brain ( = gliomatosis ) may occur in patients that respond to bev . notwithstanding these atypical patterns of progression , updated overall survival of the patients treated in the brain ( avf3708 g ) study indicated that 16% of patients remained alive at 30 months of followup , a percentage that compares favorably with historical controls . besides the effect of bev on glioma - associated vasculature , responses documented by positron emission tomography ( pet ) using fluorothymidine ( flt ) , an imaging marker of cell proliferation , were correlated with an improved overall survival in patients treated with irinotecan and bev [ 30 , 31 ] . on 19 november 2009 , the chmp ( ema ) refused to change the terms of the marketing authorization for bevacizumab in the eu to include recurrent glioblastoma . from april to november 2009 , the belgian riziv / inami provided partial ( 60% ) reimbursement for bevacizumab following an individual request to the bijzonder solidariteitsfonds / fonds spcial de solidarit . this paper reports the first experience with bevacizumab for recurrent glioma in patients treated at two brussels university hospitals . this observational ( noninterventional ) study was performed with the clinical data that were retrospectively retrieved from the medical files of all patients with recurrent high - grade glioma who initiated bev treatment between 9 january 2009 and 27 january 2010 . these patients represent the first patients treated with bev for recurrent glioma at two belgian university hospitals , the uz brussel and ulb erasme . we collected data regarding the general clinical and neurological evolution during bev treatment , the bev treatment disposition ( table 3 ) , as well as laboratory tests performed during bev therapy . adverse events were classified according to the common terminology criteria for adverse events v3.0 ( ctcae ) . , we made the sums of the maximal cross - sectional radii of the contrast enhancing tumor measured by consecutive contrast mr . a complete response ( cr ) was defined as a disappearance of all contrast enhancing tumor , with the patient neurologically improved or stable and off corticosteroids . a partial response ( pr ) was defined as a 50% or more decrease in the size of the contrast - enhancing tumor with the patient neurologically improved or stable and with the corticosteroid dose stable or decreased . progressive disease ( pd ) was defined as a 25% or more increase in the size of the contrast enhancing tumor or appearance of a separate tumor . stable disease ( sd ) was defined for all other situations . in addition , we assessed the abnormalities on sequential t2 and flair mri sequences in a similar fashion , and changes on pet scan of the brain for the subgroup of patients evaluated by this imaging modality . bev treatment disposition , bev - related adverse events , demographic and baseline patient , and disease characteristics were summarized using descriptive statistics . kaplan - meier statistics were used to estimate the probability of survival ( spss inc . , chicago , illinois 60606 , usa ) . nineteen patients ( 11 men and 8 women ) with recurrent supratentorial hgg were identified to have received treatment with bev for progressive disease following failure after prior treatment including surgery , radiation therapy , and chemotherapy . the median patient age at the initiation of bev treatment was 40 years ( range 28 to 70 ) . eighty - nine percent of the patients were younger than 50 years at the start of bev treatment . eight ( 53% ) patients had a pathological diagnosis of primary gb , 4 ( 21% ) of secondary gb , and 5 ( 26% ) of recurrent grade iii glioma . in six patients , initial treatment consisted of surgery followed by radiotherapy . in 13 patients , the initial treatment consisted of surgery , followed by rt with concomitant tmz and adjuvant tmz ( median number of adjuvant tmz cycles : 6 , range 1 to 12 ) . three patients had been treated with radiation therapy at recurrence ( one patient was administered fractionated radiotherapy at a dose of 55,5 gy , two patients were treated using -knife radiosurgery ) . twelve patients underwent additional modalities of salvage therapy for recurrent disease ( chemotherapy , dendritic cell vaccination ) before initiating bev therapy . three patients initiated bev at a dose of 5 mg / kg every 2 weeks ( in one of them , the dose was escalated to 10 mg / kg every 2 weeks after the first administration ) , 14 patients at a dose of 10 mg / kg every 2 weeks , and 2 patients at a dose of 15 mg / kg every 3 weeks , according to bev administration regimens published in the literature [ 20 , 21 , 24 , 27 , 3335 ] . a total number of 123 bev treatment cycles were analyzed in this study . a median number of 4 cycles were administered per individual patient ( range 1 to 16 ) . three patients were simultaneously treated with a cytotoxic drug ( hydroxyurea , tmz , or ccnu ) . fourteen patients ( 74% ) were treated with corticosteroids at the initiation of bev treatment . the dose of corticosteroids could be tapered in 4 patients and stopped in two of them . nine bev - related adverse events were encountered , of which none were grade 4 or 5 ( table 2 ) . two grade 3 adverse events ( ulceration of skin striae and an abdominal pain syndrome ) necessitated stopping bev administration in the absence of documented tumor progression . four patients ( 21% ) experienced a rapid increase in disease - related symptoms after the initiation of bev . their clinical condition prohibited an objective tumor evaluation with mri after the initiation of bev . in all 4 patients , fifteen ( 79% ) patients were assessable for tumor response on t1 gadolinium - enhanced mri - sequences ( gd - t1 ) . tumor regression was complete in 2 patients and more than 50% in an additional 3 patients . this correlates to an objective response rate of 33% according to the macdonald criteria for the 15 evaluable pts on mri and 26% for the intent to treat population . all of these patients had stabilization or improvement of disease - related symptoms and none of them had an increase in corticosteroid dose . seven patients ( 47% ) obtained a stable disease , and 3 ( 20% ) patients experienced immediate progression of disease during bev therapy ( figures 1 and 2 ) . assessment of tumor response by t2/flair mri - sequences was available for the same 15 patients who were evaluable by t1-weighted mri . complete disappearance of nonenhancing lesions was observed in 1 patient , and partial regression was observed in 6 additional patients ( 47% ; for an objective tumor response according to the rano criteria of 37% ) . no change was observed in four patients ( 27% ) , and an increase of abnormalities at the first evaluation was observed in 4 patients ( 27% ) ( figures 1 and 3 ) . four patients were evaluated by 2-(18f)-fluoromethyl - l - phenylalanine pet ( fmp - pet ) or 11c - methionine - pet of the brain before and during bev treatment . a reduced uptake of amino - acid tracer on pet - scan was documented in 3 out of 4 pts during bev treatment , in 2 patients with the most favorable progression - free survival , and a complete normalization of pet - tracer uptake was observed during bev therapy ( figure 4 ) . as of october 2010 ( the time of this analysis ) , two patients ( 10,5% ) remained free - from progression after 1 year of bev treatment . in one of these patients bev was stopped after 1-year of therapy in the absence of metabolic activity on methionine - pet and normalization of gadolinium enhancement on t1-mri . the second patient developed progression of disease following 18 months of remission on bev therapy . the six - month progression - free survival rate ( 6mpfs% ) was 21% ( 95% ci 2.739.5 ) , and the 6mos% was 47.4% ( 95% ci 24.869.9 ) . the median pfs and overall survival ( os ) were 10 weeks ( 95% ci 225 ) and 25 weeks ( 95% ci 1732 ) , respectively ( figure 5 ) . high - grade gliomas are highly aggressive and therapy resistant malignant tumors . with contemporary standard treatment options for patients diagnosed with gb , the prognosis remains grim and most patients do not survive for more than 2 years following the diagnosis . salvage therapies with cytotoxic agents are seldom successful ( < 10% orr ) [ 7 , 36 ] . uncontrolled clinical studies with the vegf targeted igg1 monoclonal antibody bevacizumab have shown unprecedented tumor response rates and survival outcomes that compare favorably with historical control series . within the context of these prospective clinical trials , bev - associated toxicities have been acceptable and reflect a typical spectrum of side effects that are associated with vegf(r ) targeted therapies . as most of the prospective trials have used quite stringent patient recruitment criteria , safety and activity of bev when used outside of a clinical trial merit consideration . we , therefore , retrospectively analyzed the clinical outcome of nineteen patients treated with bev for recurrent hgg during the first year that bev became available in this indication at two university hospitals in belgium . as expected , the baseline characteristics of the patients included in our analysis compared unfavorably with those of the patients treated in the pivotal phase ii trial . a larger proportion of patients treated in our series were treated at second or third relapse , and the baseline kps was less or equal to 60% in a significant proportion of patients . we , therefore , consider that the results from our study , although preliminary , indicate that the safety profile of bev for recurrent hgg outside the context of a prospective clinical trial is comparable to what has been reported in the literature . nevertheless , two patients needed to stop treatment because of bev - related side effects in the absence of documented progression . it , therefore , needs to be considered that frail patients might be at higher risk for bev - related adverse events . the objective tumor response , either analyzed by gadolinium - enhanced t1 mri and/or t2/flair imaging , was interestingly high in our patient population . reflecting the poor baseline prognostic characteristics of our population , both time to progression ( ttp ) and overall survival ( os ) , in contrast , were low when compared to published series . however , two patients in our series experienced a durable complete response and progression - free survival for over 1 year following bev therapy for recurrence , indicating the potential for a durable therapeutic effect in a subgroup of patients . patterns of tumor response and progression during antiangiogenic therapy are a matter of controversy in the recent literature . in our small series , the tumor response pattern to bev was heterogeneous and could be divided in three distinct patterns . a first group of patients demonstrated no evidence of response ( clinical or radiological ) to bevacizumab therapy . such was the case in four patients , who deteriorated rapidly and could not be evaluated by mri , and three ( 20% ) of the fifteen assessable patients on gadolinium - enhanced t1 mri . a second group of patients initially responded to therapy on gadolinium - enhanced t1 mri , but subsequently showed early ( < 6 months ) regrowth of the gadolinium - enhancing tumor mass ( n = 7 ; 47% ) or deteriorated clinically without characteristic increase in the diameter of gadolinium - enhancing t1 mri abnormities ( n = 3 , 20% ) . in these 3 patients , there was a marked progression of abnormalities on t2/flair mri , most likely representing vegfr - independent tumor cell infiltration of the brain . a small third group of patients ( 2 patients ; 13% ) experienced a very favorable and sustained tumor response to bev therapy , evident on both gd - t1 and t2/flair mri . further useful differentiation of response to bev may be obtained by metabolic tumor imaging using pet . pet - imaging has proven to be useful in assessing the response of recurrent glioma treated with a variety of modalities . likewise , flt - pet has been correlated with clinical outcome of patients treated with the combination of irinotecan and bevacizumab . in our series , normalization in pet tracer accumulation was observed in the 2 cases with the most favorable evolution on mri and survival . these case observations indicate that single agent bev can be associated with a reduction of pet - tracer uptake by the tumor , suggestive of a metabolic effect . these observations merit further study of pet as a tool for response assessment in patients with recurrent glioma treated by bev . pet response may be more predictive for survival as opposed to response assessment by mri . we conclude that our analysis of the first experience with bev for the treatment of patients with recurrent hgg is in line with the reported tolerability and activity of this new treatment from prospective clinical studies . our observations support the usefulness of bev as a new treatment option for patients with recurrent hgg taken into account the absence of alternative treatment options with proven activity . further observational study of the use of bev in this indication should be considered to optimize its use in daily practice . correlative studies between clinical , radiological , pet parameters with molecular - genetic features of the hgg should be conducted to provide predictive markers for response and survival benefit from bev .

background . bevacizumab ( bev ) , a humanized immunoglobulin g1 monoclonal antibody that inhibits vegf has demonstrated activity against recurrent high - grade gliomas ( hgg ) in phase ii clinical trials . patients and methods . data were collected from patients with recurrent hgg who initiated treatment with bev outside a clinical trial protocol at two belgian university hospitals . results . 19 patients ( 11 m/8 f ) were administered a total of 138 cycles of bev ( median 4 , range 131 ) . tumor response assessment by mri was available for 15 patients ; 2 complete responses and 3 partial responses for an objective response rate of 26% for the intent to treat population were observed on gadolinium - enhanced t1-weighted images ; significant regressions on t2/flair were documented in 10 out of 15 patients ( 67% ) . a reduced uptake on pet was documented in 3 out of 4 evaluable patients . the six - month progression - free survival was 21% ( 95% ci 2.739.5 ) . two patients had an ongoing tumor response and remained free from progression after 12 months of bev treatment . conclusions . the activity and tolerability of bev were comparable to results from previous prospective phase ii trials . reduced uptake on pet suggests a metabolic response in addition to an antiangiogenic effect in some cases with favorable clinical outcome .

the alpha omega trial is a multicenter , double - blind , placebo - controlled trial with a 2 2 factorial design , which has been described in detail elsewhere ( 6,7 ) . in collaboration with cardiologists from 32 hospitals in the netherlands , we recruited 4,837 patients aged 6080 years with a clinically diagnosed mi up to 10 years before randomization . for the current study , the results of 1,014 patients with diabetes were analyzed ( supplementary fig . these patients were randomly assigned to daily consumption of four different trial margarines for a period of 40 months . the patients were randomly allocated to four groups and received either no additional n-3 fatty acids or 400 mg / day epa+dha , 2 g / day ala , or the combination . the doses corresponded to recommended dietary allowances of those fatty acids . in trial margarines for active treatment groups , the margarines were developed by unilever research and development and could not be distinguished from each other in taste , odor , texture , and color . the trial was conducted in accordance with the helsinki declaration and approved by one central medical ethics committee and by the committees of all participating hospitals . written informed consent was obtained from all patients . for logistical reasons , all patients used placebo margarine during the first 46 weeks after randomization , after which actual treatment started . after randomization , the patients received eight blinded margarine tubs of 250 g every 12 weeks . based on this information , the daily intakes of margarine and n-3 fatty acids were calculated . an objective measure of compliance was obtained by determining fatty acids in plasma cholesteryl esters in random subgroups of patients at baseline and after 20 and 40 months of intervention . our definition of diabetes was based on a physician s diagnosis , use of antidiabetic drugs , and/or elevated blood glucose , defined as a casual plasma glucose level of 7.0 mmol / l ( 126 mg / dl ) for patients who had fasted > 4 h or a level 11.1 mmol / l ( 200.0 mg / dl ) in patients who did not fast . demographic factors , lifestyle , drug use , and medical history were assessed by questionnaire ( 6 ) . medication was coded according to the anatomical therapeutic chemical ( atc ) classification system ( world health organization , 2009 ) , including atc code a10 for antidiabetic medication . physical activity was assessed by the validated physical activity scale for the elderly ( pase ) questionnaire . a patient was called physically active when he or she had at least 5 days per week of > 30 min / day of physical activities of at least three metabolic equivalents of task . anthropometric measures , blood pressure , and heart rate were taken , and nonfasting blood was collected for determining serum lipids , plasma cholesteryl esters , and glucose ( 6 ) . examinations were repeated in a random sample of 159 ( 15.7% ) patients after 20 months and after 40 months in 476 patients ( 46.9% ) who completed the trial before 1 january 2009 . due to budget constraints , the remainder of the cohort filled out mailed questionnaires only . all patients were followed for clinical events , including those who discontinued margarine use during the trial . we monitored vital status of all patients using a computerized link with municipal registers with 100% coverage . for fatal cases , death certificates were obtained from statistics netherlands and general practitioners filled out a standard form on primary and contributing causes of death . additional information on fatal events was obtained from hospitals , nursing homes , and family members . three members of the endpoint adjudication committee who were blinded for patient identity , treating physician , and allocated treatment coded the fatal events independently according to the 10th revision of the international classification of diseases . in the cases of disagreement , information about the underlying causes of death was discussed until consensus was reached . in the original trial protocol , the primary end point was fatal chd , and one of the secondary end points was sudden death , a proxy for fatal arrhythmias ( 6 ) . due to the low mortality rate , the steering committee approved the switch from primary to secondary end point for fatal chd , and the extension with placement of cardioverter defibrillators for the definition of sudden death , as described in the statistical analysis plan ( 7 ) . fatal chd was defined as mortality from mi ( i20i25 ) , cardiac arrest ( i46 ) , and sudden death undefined ( r96 ) and the composite end - point ventricular arrhythmia related events as nonfatal or fatal cardiac arrest ( i46 ) , sudden death ( r96 ) , and implantable cardioverter defibrillator placement ( 6,7 ) . sudden death was extended with placement of cardioverter defibrillators because ventricular arrhythmias are the main cause of sudden death and cardioverter defibrillators are implanted in patients who are at high risk for developing ventricular arrhythmias ( 16,17 ) . self - reported data on implantable cardioverter defibrillator placement were verified against hospital records by trained research nurses or the research physician . all indications for implantable cardioverter defibrillator placement could be retrieved and verified from hospital discharge letters or by telephone with the departments of cardiology where the placements had taken place . the definitions of fatal chd and of the composite end - point ventricular arrhythmia related events include both fatal cardiac arrest ( i46 ) and sudden death undefined ( r96 ) ( 7 ) . to prevent overlap in these definitions , we limited in the present analysis fatal chd to death from mi ( i20i25 ) . baseline characteristics were compared among the four treatment groups of patients using anova or tests , when appropriate . time - to - event data were analyzed with the kaplan - meier method and the log - rank test . the three treatment groups ( i.e. , epa - dha , ala , and epa - dha plus ala groups ) were compared with the placebo group for each outcome . hazard ratios ( hrs ) and 95% cis were computed for ventricular arrhythmia related events , fatal mi , and the combination of these two end points , using cox proportional hazard models . no adjustments were made for multiple comparisons because only a priori formulated hypotheses were tested based on animal experiments of the effects of n-3 fatty acids on atherosclerosis and cardiac arrhythmias ( 5 ) . the alpha omega trial is a multicenter , double - blind , placebo - controlled trial with a 2 2 factorial design , which has been described in detail elsewhere ( 6,7 ) . in collaboration with cardiologists from 32 hospitals in the netherlands , we recruited 4,837 patients aged 6080 years with a clinically diagnosed mi up to 10 years before randomization . for the current study , the results of 1,014 patients with diabetes were analyzed ( supplementary fig . these patients were randomly assigned to daily consumption of four different trial margarines for a period of 40 months . the patients were randomly allocated to four groups and received either no additional n-3 fatty acids or 400 mg / day epa+dha , 2 g / day ala , or the combination . the doses corresponded to recommended dietary allowances of those fatty acids . in trial margarines for active treatment groups , the margarines were developed by unilever research and development and could not be distinguished from each other in taste , odor , texture , and color . the trial was conducted in accordance with the helsinki declaration and approved by one central medical ethics committee and by the committees of all participating hospitals . written informed consent was obtained from all patients . for logistical reasons , all patients used placebo margarine during the first 46 weeks after randomization , after which actual treatment started . after randomization , the patients received eight blinded margarine tubs of 250 g every 12 weeks . based on this information , the daily intakes of margarine and n-3 fatty acids were calculated . an objective measure of compliance was obtained by determining fatty acids in plasma cholesteryl esters in random subgroups of patients at baseline and after 20 and 40 months of intervention . our definition of diabetes was based on a physician s diagnosis , use of antidiabetic drugs , and/or elevated blood glucose , defined as a casual plasma glucose level of 7.0 mmol / l ( 126 mg / dl ) for patients who had fasted > 4 h or a level 11.1 mmol / l ( 200.0 mg / dl ) in patients who did not fast . demographic factors , lifestyle , drug use , and medical history were assessed by questionnaire ( 6 ) . medication was coded according to the anatomical therapeutic chemical ( atc ) classification system ( world health organization , 2009 ) , including atc code a10 for antidiabetic medication . physical activity was assessed by the validated physical activity scale for the elderly ( pase ) questionnaire . a patient was called physically active when he or she had at least 5 days per week of > 30 min / day of physical activities of at least three metabolic equivalents of task . anthropometric measures , blood pressure , and heart rate were taken , and nonfasting blood was collected for determining serum lipids , plasma cholesteryl esters , and glucose ( 6 ) . examinations were repeated in a random sample of 159 ( 15.7% ) patients after 20 months and after 40 months in 476 patients ( 46.9% ) who completed the trial before 1 january 2009 . due to budget constraints , all patients were followed for clinical events , including those who discontinued margarine use during the trial . we monitored vital status of all patients using a computerized link with municipal registers with 100% coverage . for fatal cases , death certificates were obtained from statistics netherlands and general practitioners filled out a standard form on primary and contributing causes of death . additional information on fatal events was obtained from hospitals , nursing homes , and family members . three members of the endpoint adjudication committee who were blinded for patient identity , treating physician , and allocated treatment coded the fatal events independently according to the 10th revision of the international classification of diseases . in the cases of disagreement , information about the underlying causes of death was discussed until consensus was reached . in the original trial protocol , the primary end point was fatal chd , and one of the secondary end points was sudden death , a proxy for fatal arrhythmias ( 6 ) . due to the low mortality rate , the steering committee approved the switch from primary to secondary end point for fatal chd , and the extension with placement of cardioverter defibrillators for the definition of sudden death , as described in the statistical analysis plan ( 7 ) . fatal chd was defined as mortality from mi ( i20i25 ) , cardiac arrest ( i46 ) , and sudden death undefined ( r96 ) and the composite end - point ventricular arrhythmia related events as nonfatal or fatal cardiac arrest ( i46 ) , sudden death ( r96 ) , and implantable cardioverter defibrillator placement ( 6,7 ) . sudden death was extended with placement of cardioverter defibrillators because ventricular arrhythmias are the main cause of sudden death and cardioverter defibrillators are implanted in patients who are at high risk for developing ventricular arrhythmias ( 16,17 ) . self - reported data on implantable cardioverter defibrillator placement were verified against hospital records by trained research nurses or the research physician . all indications for implantable cardioverter defibrillator placement could be retrieved and verified from hospital discharge letters or by telephone with the departments of cardiology where the placements had taken place . the definitions of fatal chd and of the composite end - point ventricular arrhythmia related events include both fatal cardiac arrest ( i46 ) and sudden death undefined ( r96 ) ( 7 ) . to prevent overlap in these definitions , we limited in the present analysis fatal chd to death from mi ( i20i25 ) . baseline characteristics were compared among the four treatment groups of patients using anova or tests , when appropriate . time - to - event data were analyzed with the kaplan - meier method and the log - rank test . the three treatment groups ( i.e. , epa - dha , ala , and epa - dha plus ala groups ) were compared with the placebo group for each outcome . hazard ratios ( hrs ) and 95% cis were computed for ventricular arrhythmia related events , fatal mi , and the combination of these two end points , using cox proportional hazard models . no adjustments were made for multiple comparisons because only a priori formulated hypotheses were tested based on animal experiments of the effects of n-3 fatty acids on atherosclerosis and cardiac arrhythmias ( 5 ) . data were analyzed with spss 17.0 statistical software ( spss inc . , chicago , il ) . the definition of diabetes was made in 72.4% on the combination of elevated blood glucose levels , physician - diagnosed self - report , and drug treatment , in 14.4% on elevated glucose levels only , in 9.9% on self - report only , and in 3.4% on drug treatment only . insulin was used by 273 ( 26.9% ) patients and 577 ( 56.9% ) used other antidiabetic medication , of whom 110 used both insulin and other antidiabetic medication . the proportion of women was lowest in the placebo group and highest in the ala group , and 5 ( 1.9% ) of the 267 women used hormone replacement therapy . the percentage of smokers was highest in the placebo group and lowest in the epa - dha plus ala group . baseline characteristics of the 1,014 patients with an mi and diabetes , * according to n-3 fatty acid supplementation group data are means sd unless otherwise indicated . as data for a number of patients were missing from several variables ( bmi , 4 patients ; alcohol use , 1 patient ; and physical activity , 9 patients ) , some percentages are based on a smaller number than the column total . * diabetes was considered to be present if a patient reported having received the diagnosis from a physician , was taking antidiabetic drugs , or had an elevated plasma glucose level ( 7.0 mmol / l [ 126 mg / dl ] in the case of patients who had fasted more than 4 h or 11.1 mmol / l [ 200.0 mg / dl ] in the case of nonfasting patients ) . to convert the values for cholesterol to milligrams per deciliter , divide by 0.02586 . to convert the values for triglycerides to milligrams per deciliter , divide by 0.01129 . the bmi is the weight in kilograms divided by the square of the height in meters . * * greater than or equal to three metabolic equivalent of task ( indicating at least moderate intensity for at least 30 min / day ) during 6 or 7 days / week . the mean intake ( sd ) of the trial margarine was 18.6 ( 5.2 ) g / day ; 86.7% of the patients adhered fully to the protocol and consumed a mean of 20.7 ( 4.2 ) g / day . patients in the two epa - dha groups received , on average , an additional amount of 223 ( 62 ) mg epa and 149 ( 42 ) mg of dha per day and those in the two groups of ala 1.9 ( 0.5 ) g / day . plasma cholesteryl esters were determined as a measure of compliance . at the final examination , the effect of n-3 fatty acid supplementation on fatty acids in serum cholesteryl esters was compared with placebo ( supplementary fig . 2 ) . ala supplementation increased ala by 64.2% and epa by 40.8% ; epa - dha supplementation increased plasma epa by 49.8% and dha by 30.9% ; and epa - dha plus ala supplementation increased plasma ala by 67.4% , epa by 118.4% , and dha by 44.0% . the median follow - up period was 40.7 months ( iqr 36.641.6 months ) , during which 3,195 person - years of follow - up were accumulated . during follow - up , 29 patients developed a ventricular arrhythmia related event , 2 died suddenly , 1 had a nonfatal cardiac arrest , 11 had a fatal cardiac arrest , and 15 had a cardioverter defibrillator implanted . indications for these implantable cardioverter defibrillator placements were a left ventricular ejection fraction 35% ( n = 13 ; 87% ) , resuscitated ventricular fibrillation ( n = 1 ) , and ventricular tachycardia with a left ventricular ejection fraction of 45% ( n = 1 ) . in addition , 27 patients died of mi , 23 from other vascular diseases , 34 from cancer , and 26 from other causes . the kaplan - meier curves showed that both epa - dha and ala reduced ventricular arrhythmia related events compared with placebo ( fig . the lowest incidence was observed in patients who were randomized to the combined supplementation of epa - dha plus ala . after adjustment for imbalances in age , sex , and current smoking , the combined supplementation of n-3 fatty acids reduced the ventricular arrhythmia related events by 84% compared with placebo ( cumulative incidence at median follow - up 0.9 vs. 5.6% , fig . similar effects were observed for the combined end - point cardiac arrest and sudden death ( 0.13 ; ci 0.021.09 ) and for placement of cardioverter defibrillators ( 0.19 : 0.021.55 ) . kaplan - meier curves of ventricular arrhythmia related events ( a ) , death from mi ( b ) , or both end points combined ( c ) . the cumulative incidence of end points is shown in 1,014 patients with an mi and diabetes . patients were randomly assigned to receive a margarine containing supplemental epa combined with dha , a margarine containing supplemental ala , a margarine containing both epa - dha and ala , or a placebo margarine . unadjusted and adjusted hrs of n-3 fatty acid supplementation on end points in 1,014 patients with diabetes , according to n-3 fatty acid supplementation group * data are hrs with 95% cis and p values , with the use of cox proportional hazards models . adjusted for age , sex , and smoking status . for fatal mi , the kaplan - meier curves did not differ significantly among the four groups ( fig . after adjustment for confounders , the strongest , although not significant , effect was observed for epa - dha plus ala ( hr 0.53 ; 95% ci 0.151.81 ; table 2 ) . the kaplan - meier curves for the composite end - point ventricular arrhythmia related events and fatal mi showed the strongest effect for epa - dha plus ala ( fig . after adjustment , these three n-3 fatty acids together reduced this combined end point by 72% ( cumulative incidence at median follow - up 2.6 vs. 8.5% , fig . the definition of diabetes was made in 72.4% on the combination of elevated blood glucose levels , physician - diagnosed self - report , and drug treatment , in 14.4% on elevated glucose levels only , in 9.9% on self - report only , and in 3.4% on drug treatment only . insulin was used by 273 ( 26.9% ) patients and 577 ( 56.9% ) used other antidiabetic medication , of whom 110 used both insulin and other antidiabetic medication . the proportion of women was lowest in the placebo group and highest in the ala group , and 5 ( 1.9% ) of the 267 women used hormone replacement therapy . the percentage of smokers was highest in the placebo group and lowest in the epa - dha plus ala group . baseline characteristics of the 1,014 patients with an mi and diabetes , * according to n-3 fatty acid supplementation group data are means sd unless otherwise indicated . as data for a number of patients were missing from several variables ( bmi , 4 patients ; alcohol use , 1 patient ; and physical activity , 9 patients ) , some percentages are based on a smaller number than the column total . * diabetes was considered to be present if a patient reported having received the diagnosis from a physician , was taking antidiabetic drugs , or had an elevated plasma glucose level ( 7.0 mmol / l [ 126 mg / dl ] in the case of patients who had fasted more than 4 h or 11.1 mmol / l [ 200.0 mg / dl ] in the case of nonfasting patients ) . to convert the values for cholesterol to milligrams per deciliter , divide by 0.02586 . to convert the values for triglycerides to milligrams per deciliter , divide by 0.01129 . the bmi is the weight in kilograms divided by the square of the height in meters . * * greater than or equal to three metabolic equivalent of task ( indicating at least moderate intensity for at least 30 min / day ) during 6 or 7 days / week . the mean intake ( sd ) of the trial margarine was 18.6 ( 5.2 ) g / day ; 86.7% of the patients adhered fully to the protocol and consumed a mean of 20.7 ( 4.2 ) g / day . patients in the two epa - dha groups received , on average , an additional amount of 223 ( 62 ) mg epa and 149 ( 42 ) mg of dha per day and those in the two groups of ala 1.9 ( 0.5 ) g / day . plasma cholesteryl esters were determined as a measure of compliance . at the final examination , the effect of n-3 fatty acid supplementation on fatty acids in serum cholesteryl esters was compared with placebo ( supplementary fig . 2 ) . ala supplementation increased ala by 64.2% and epa by 40.8% ; epa - dha supplementation increased plasma epa by 49.8% and dha by 30.9% ; and epa - dha plus ala supplementation increased plasma ala by 67.4% , epa by 118.4% , and dha by 44.0% . the median follow - up period was 40.7 months ( iqr 36.641.6 months ) , during which 3,195 person - years of follow - up were accumulated . during follow - up , 29 patients developed a ventricular arrhythmia related event , 2 died suddenly , 1 had a nonfatal cardiac arrest , 11 had a fatal cardiac arrest , and 15 had a cardioverter defibrillator implanted . indications for these implantable cardioverter defibrillator placements were a left ventricular ejection fraction 35% ( n = 13 ; 87% ) , resuscitated ventricular fibrillation ( n = 1 ) , and ventricular tachycardia with a left ventricular ejection fraction of 45% ( n = 1 ) . in addition , 27 patients died of mi , 23 from other vascular diseases , 34 from cancer , and 26 from other causes . the kaplan - meier curves showed that both epa - dha and ala reduced ventricular arrhythmia related events compared with placebo ( fig . the lowest incidence was observed in patients who were randomized to the combined supplementation of epa - dha plus ala . after adjustment for imbalances in age , sex , and current smoking , the combined supplementation of n-3 fatty acids reduced the ventricular arrhythmia related events by 84% compared with placebo ( cumulative incidence at median follow - up 0.9 vs. 5.6% , fig . similar effects were observed for the combined end - point cardiac arrest and sudden death ( 0.13 ; ci 0.021.09 ) and for placement of cardioverter defibrillators ( 0.19 : 0.021.55 ) . kaplan - meier curves of ventricular arrhythmia related events ( a ) , death from mi ( b ) , or both end points combined ( c ) . the cumulative incidence of end points is shown in 1,014 patients with an mi and diabetes . patients were randomly assigned to receive a margarine containing supplemental epa combined with dha , a margarine containing supplemental ala , a margarine containing both epa - dha and ala , or a placebo margarine . unadjusted and adjusted hrs of n-3 fatty acid supplementation on end points in 1,014 patients with diabetes , according to n-3 fatty acid supplementation group * data are hrs with 95% cis and p values , with the use of cox proportional hazards models . adjusted for age , sex , and smoking status . for fatal mi , the kaplan - meier curves did not differ significantly among the four groups ( fig . after adjustment for confounders , the strongest , although not significant , effect was observed for epa - dha plus ala ( hr 0.53 ; 95% ci 0.151.81 ; table 2 ) . the kaplan - meier curves for the composite end - point ventricular arrhythmia related events and fatal mi showed the strongest effect for epa - dha plus ala ( fig . after adjustment , these three n-3 fatty acids together reduced this combined end point by 72% ( cumulative incidence at median follow - up 2.6 vs. 8.5% , fig . low - dose supplementation of n-3 fatty acids epa - dha plus ala significantly reduced ventricular arrhythmia related events in post - mi patients with diabetes . the combined supplementation of these three fatty acids also reduced the composite end - point ventricular arrhythmia related events plus fatal mi but not fatal mi alone . a limitation of the current study is the small number of patients who developed ventricular arrhythmias ( n = 29 ) or died of mi ( n = 27 ) . the study had enough power to detect significant effects of combined supplementation of epa - dha and ala on the end - point ventricular arrhythmia related events whether or not in combination with fatal mi . significant effects on these end points were not observed for either epa - dha or ala supplementation . the combined effects of epa - dha and ala on the two composite end points are compatible with additive effects of either epa - dha or ala . assuming an additive model , an hr of 0.47 0.58 = 0.27 is expected for ventricular arrhythmia related events and of 0.81 0.60 = 0.49 for the combination of ventricular arrhythmias and fatal mi . these results fit well with the 95% ci of these end points and are compatible with the hypothesis that all three n-3 fatty acids reduce the risk of ventricular arrhythmias . this is also in accord with the results of animal experiments suggesting that ala , epa , and dha have similar antiarrhythmic effects by influencing the na and l - type ca channels of cardiomyocytes ( 5 ) . in the gissi - prevenzione trial , patients with a recent mi ( <3 months ago ) were supplemented with 0.9 g of epa - dha per day , which reduced their risk of sudden death by 45% ( 8) . in later trials , recent meta - analyses quantified the effect of epa - dha supplementation on sudden death at a 19% ( hr 0.81 ; 95% ci 0.521.25 ) ( 2 ) and 13% ( 0.87 ; 0.760.99 ) reduction ( 3 ) . an explanation for the smaller effect of epa - dha in the more recent trials could be improved drug treatment of cardiovascular risk factors ( 18 ) . this is supported by the recently published results of the omega trial in which patients were randomized 314 days after acute mi . in these high - risk patients , a supplement of 0.8 g epa - dha per day did not reduce sudden cardiac death ( 19 ) . in this trial and in the alpha omega trial , almost all patients received antithrombotic drugs , antihypertensive drugs , and statins ( 7 ) . the patients in the gissi - prevenzione trial were also well treated with antithrombotic and antihypertensive drugs but only 29% received statins ( 8) . the alpha omega trial started in 2002 , the omega trial in 2003 , and the gissi - prevenzione in 1993 . one of the secondary end points in the alpha omega trial was ventricular arrhythmia related events . ventricular arrhythmias are the major cause of sudden death and cardiac arrest and cardioverter defibrillators were generally implanted in post - mi patients with a low ejection fraction who are at high risk for these arrhythmias ( 16,17 ) . the protective effect of the combined supplementation of the three n-3 fatty acids on this end point ( hr 0.16 ; 95% ci 0.040.69 ) contrasts with the absence of an effect of epa - dha in cardiac patients with implanted cardioverter defibrillators ( who all had had a severe arrhythmic event before placement ) ( 2 ) . these discrepant results suggest that n-3 fatty acids may reduce primary arrhythmias in post - mi patients with diabetes with a low ejection fraction but not secondary arrhythmias in cardiac patients with cardioverter defibrillators . epa - dha supplementation reduced fatal mi by 47% but this reduction was not significant , possibly due to the small number of events . a possible protective effect of epa - dha on fatal mi would be in accord with the results of a prospective study carried out in postmenopausal women ( 20 ) . in that study , smaller increases in stenosis were observed in diabetic women who consumed greater than two servings of fish or greater than one serving of fatty fish compared with diabetic women who consumed less fish . an intervention study carried out in japan showed that diabetic patients who were supplemented with 1.8 g epa per day in contrast with the control group did not show an increase in intima - media thickness during 2.1 years of follow - up ( 21 ) . these results suggest that in diabetic patients , fish and epa supplementation may reduce progression of atherosclerosis and the eventual risk of fatal mi . diabetes alters smooth muscle function through atherosclerotic lesion formation , plaque instability , and cardiovascular events . basic research shows that peroxisome proliferator activated receptor ( ppar- ) plays a critical role in the regulation of insulin sensitivity . to become functional , ppar- should bind to an appropriate ligand . cell culture studies provide evidence that epa and dha are potential ligands for ppar- activation ( 22 ) . recently published results of an in vivo experiment suggested that fish oil increases the expression of glucose uptake genes , leading to reduced glucose levels ( 23 ) . experiments in obese mice showed that activation of the g protein coupled receptor 120 ( gpr120 ) by epa and dha inhibited multiple inflammation cascades and reversed insulin resistance caused by a high - fat diet ( 24 ) . these mechanistic findings support an important role for n-3 fatty acids in the etiology of diabetes , a major risk factor of fatal mi . in conclusion , low doses of n-3 fatty acids reduced the risk of ventricular arrhythmia related events in post - mi patients who also have diabetes . ongoing trials in diabetic patients , e.g. , origin ( outcome reduction with an initial glargine intervention ; clinical trial reg . nct00069784 ) ( 25 ) and ascend ( a study of cardiovascular events in diabetes ; nct00135226 ; http://www.ctsu.ox.ac.uk/ascend/ ) , evaluate the effect of epa - dha on sudden death and fatal chd . the results of more trials on the effect of different n-3 fatty acids are needed before definitive conclusions can be drawn on their role in the etiology of ventricular arrhythmias and fatal chd .

objectivewe carried out a secondary analysis in high - risk patients with a previous myocardial infarction ( mi ) and diabetes in the alpha omega trial . we tested the hypothesis that in these patients an increased intake of the n-3 fatty acids eicosapentaenoic acid ( epa ) , docosahexaenoic acid ( dha ) , and -linolenic acid ( ala ) will reduce the incidence of ventricular arrhythmias and fatal mi.research design and methodsa subgroup of 1,014 post - mi patients with diabetes aged 6080 years was randomly allocated to receive one of four trial margarines , three with an additional amount of n-3 fatty acids and one placebo for 40 months . the end points were ventricular arrhythmia related events and fatal mi . the data were analyzed according to the intention - to - treat principle , using multivariable cox proportional hazards models.resultsthe patients consumed on average 18.6 g of margarine per day , which resulted in an additional intake of 223 mg epa plus 149 mg dha and/or 1.9 g ala in the active treatment groups . during follow - up , 29 patients developed a ventricular arrhythmia related events and 27 had a fatal mi . compared with placebo patients , the epa - dha plus ala group experienced less ventricular arrhythmia related events ( hazard ratio 0.16 ; 95% ci 0.040.69 ) . these n-3 fatty acids also reduced the combined end - point ventricular arrhythmia related events and fatal mi ( 0.28 ; 0.110.71).conclusionsour results suggest that low - dose supplementation of n-3 fatty acids exerts a protective effect against ventricular arrhythmia related events in post - mi patients with diabetes .

this sticky , greenish - brown product has different compositions depending on the location of the bees and what trees and flowers they have access to . propolis from turkey or egypt will not have the same chemical properties as propolis from europe or brazil . this is because it is very difficult for researchers to come to general conclusions about its health benefits . caffeic acid phenethyl ester ( cape ) [ figure 1 ] is one of important compounds found in propolis that has antiviral , antioxidant , anti - inflammatory , antiproliferative , antitumor , and immunomodulatory effects . this marvelous compound has been used to prevent oxidative stress - based deterioration in cells / tissues / organs in both cell culture and experimental animals . lately , the protection of cape on central and peripheral nervous system as well as a reproductive system have been extensively reviewed [ 3 - 5 ] . it has extensively been used to treat a broad of malignancies including hodgkin s and non - hodgkin s lymphoma , burkitt s lymphoma , chronic lymphocytic leukemia , ewing s sarcoma , breast cancer , testicular cancer , etc . it has toxic effects in almost every system in the human body including the heart , liver , and kidney of which mostly due to its structural properties prone to induce oxidative stress in vitro and in vivo . the chemical illustration of caffeic acid phenethyl ester this study aimed to collect data and compare the protective effects of propolis and cape against cp - induced injury in animals . , ameliorative effect of propolis against cp - induced toxicity in mice was studied by el - naggar et al . . it throws light on the side effects of a common anticancer agent , cp , used in the treatment of various malignancies and possible remedies to prevent that type of side effects in vital organs such as liver and kidney . uysal et al . conducted an experimental animal study to determine protective role of cape on cp - induced hemorrhagic cystitis ( hc ) . while cp - induced hc lead to increase in superoxide dismutase , catalase , and malondialdehyde activities / levels , cape significantly reduced these parameters showing the protective effects . in addition to this biochemical effects , cape also ameliorates edema , hemorrhage , inflammation , and mucosal ulceration of cp - induced hc . we published a review article about toxicities of some therapeutic compounds and the protective effect of cape on chemotherapy- and radiotherapy - induced toxicity . we have shown that cape has protective effects on oxidative stress - induced toxicities by doxorubicin ( nephrotoxicity ) , cisplatin ( nephrotoxicity , ototoxicity , and hepatotoxicity ) [ 10 - 13 ] , and bleomycin ( lung fibrosis ) . currently , there is no medically recommended dose for propolis , since the mixture of propolis is subjected to change depending on its source . the most successful medical application field of propolis is beauty and skin care , especially in acne vulgaris because of its antibacterial , antiviral , antifungal , and anti - inflammatory properties . despite the fact that both water and ethanolic extractions of propolis have been used in the in vivo and in vitro experiments , water - soluble extracts of propolis exhibit higher antioxidant and inhibitory activities as compared ethanolic extract in vitro . in this perspective , even though the extraction method selection is dependent on the authors desire , it would be expected for authors to study propolis for their experiments comparatively by selecting propolis extracted by both extraction methods . cape is the most potent antioxidant agent of propolis mixture having free radical scavenging activity and potent inhibition of nf-b . so , the protective antioxidant effect of ethanol extract of propolis on organs depends mostly on cape rather than other polyphenolic compounds such as flavonoids , phenolic acids , and their esters [ figure 2 ] . cape was shown to completely block the production of reactive oxygen species in human neutrophils and in the xanthine / xanthine oxidase systems at 10 m concentration by its competent antioxidant capacity . indeed , cape has a regulatory effect on antioxidant enzyme activities such as catalase , superoxide dismutase , and glutathione peroxidase [ figure 2 ] . proposed mechanism of how cyclophosphamide - induced oxidative stress is blocked by antioxidant enzymes in several parts of hepatocytes and renal cells and how cape shows its protective effects against oxidative stress . aas : amino acids , cape : caffeic acid phenethyl ester , cat : catalase , fe : ferrous iron , gpx : glutathione peroxidase , gsh : reduced glutathione , gr : glutathione reductase , gssg : oxidized glutathione , h2o : water , nadp : oxidized nicotinamide adenine dinucleotide phosphate , nadph : reduced nicotinamide adenine dinucleotide phosphate , o2 : molecular oxygen , o2 : superoxide anion radical , oh : hydroxyl ion , oh : hydroxyl radical , onoo : peroxynitrite , no : nitric oxide , nos : nitric oxide synthase , pufa : polyunsaturated fatty acid , sod : superoxide dismutase it has been shown that cape application to the rats modifies the enzyme activity of cytochrome p450 ( cyp ) isoforms involved in the activation of diethylnitrosamine such as cyp1a1/2 and cyp2b12 . furthermore , treatment with cape of carbon tetrachloride - induced hepatotoxicity in mice blocks cyp2e1-mediated ccl4 bioactivation and protects against fas / fasl - mediated apoptosis . it will be very interesting to see the effect of cape on cyp2b6 , which constitutes 3 - 6% of total hepatic cyp content and metabolizes several pharmaceuticals including cp . to achieve this , further studies on the every single bioactive constituent of propolis such as cape and some other polyphenols are necessary to identify interactions mediating their biological effects on cyp2b6 , since there are roughly 150 different polyphenolic compounds within propolis . as a conclusion , studying propolis to prevent cp - induced oxidative stress in animals has several limitations since the proposed effect can not be specified to one or several molecules within the mixture . in that case , every single bioactive constituent of propolis needs to be studied to show the source of real effects and the molecular mechanisms of this effects .

propolis is a mixture having hundreds of polyphenols including caffeic acid phenethyl ester ( cape ) . they have been using in several medical conditions / diseases in both in vitro and in vivo experimental setup . cyclophosphamide ( cp ) has been used to treat a broad of malignancies including hodgkin s and non - hodgkin s lymphoma , burkitt s lymphoma , chronic lymphocytic leukemia , ewing s sarcoma , breast cancer , testicular cancer , etc . it may cause several side effects after treatment . in this mini review , the protective effects of propolis and cape were compared each other in terms of effectiveness against cp - induced injuries .

the many genetic studies performed in recent years showed that genes such as interleukin 23 receptor ( il23r ) and il12b and tumor necrosis factor alpha ( tnf ) are closely associated with psoriasis and related diseases such as rheumatoid arthritis , psoriatic arthritis , and crohn 's disease . human leukocyte antigen c ( hla - c)0602 is the allele most closely associated with this disease . the age at onset of psoriasis follows a bimodal distribution : type i psoriasis appears before the age of 40 years ( early - onset ) , with a peak at 1622 years ; type ii psoriasis appears after the age of 40 years ( late - onset ) , with a peak at 5760 years . type i psoriasis has been associated with several single - nucleotide polymorphisms ( snps ) in genes associated with the immune response ( table 1 ) . for example , hla - c0602 is more strongly associated with type i psoriasis than with type ii psoriasis . although several association studies have already been performed in psoriasis in both populations ( type i or type ii psoriasis patients ) , knowledge of age at onset remains limited and controversial ( table 1 ) . therefore , we performed a candidate gene study , where we evaluated genetic susceptibility to type i or type ii psoriasis in patients with moderate - to - severe chronic plaque psoriasis . this approach may help us to identify snps previously associated with psoriasis or other autoimmune diseases that are specific to type i or type ii psoriasis . furthermore , our genetic study could improve our understanding of psoriasis and of its etiology and pathogenesis . we recruited 198 caucasian patients with moderate - to - severe plaque type psoriasis ( psoriasis area and severity index > 10 ) who attended the department of dermatology in four university hospitals in madrid between 16/10/2007 and 17/12/2012 . five samples did not fulfill the quality criteria of the human genotyping unit - cegen ( cegen , spanish national cancer research centre , madrid , spain ) , and 2 samples had insufficient volume . we also included 197 healthy volunteers ( controls ) recruited between 10/01/2011 and 14/12/2012 from the clinical pharmacology service ( hospital universitario de la princesa , madrid , spain ) . all the volunteers were caucasian and had no personal or family history of psoriasis ( at least 2 generations ) . the protocol fulfilled spanish law on biomedical research and was approved by the ethics committee for clinical investigation of hospital universitario de la princesa . all controls and patients gave their written informed consent to donate a sample for investigation . we preselected 320 snps based on an extensive review of 449 articles describing the association between polymorphisms and psoriasis and response to biological drugs and psoriasis and related inflammatory diseases ( rheumatoid arthritis , psoriatic arthritis , and crohn 's disease ) . we finally selected 192 snps based on minor allele frequency ( 0.05 ) and on the results of studies performed in caucasians and psoriatic patients . information on the 173 snps analyzed can be found in supplementary table s1 , which is published in . dna was obtained from samples using an automatic dna extractor ( magna pure system , roche applied science , usa ) and its concentration was quantified in nanodrop nd-1000 spectrophotometer ( wilmington , usa ) . the extracted dna was stored at 80c in the clinical pharmacology service until use . a total of 196 samples from patients ( 2 samples of 198 cases had insufficient volume ) and 197 samples from controls were sent to the human genotyping unit - cegen to genotype 192 snps . the analysis was performed using the illumina veracode genotyping platform . if fluorescence was low or the genotype clusters were undifferentiated , the snps were removed . in addition , if the call rate was less than 95% of the average of the 192 snps analyzed , the samples were removed . since cegen quality criteria were not met in 19 snps and 5 patients , we finally analyzed 173 snps in 191 patients and 197 controls . the statistical analysis was performed to compare the following stratified populations : patients with type i psoriasis ( n = 155 ) or type ii psoriasis ( n = 36 ) versus controls ( n = 197 ) and patients with type i psoriasis versus cases with type ii psoriasis . hardy - weinberg equilibrium was tested for all the snps analyzed using the snpstats program . snps that were not in hardy - weinberg equilibrium in controls were removed from the subsequent analysis . we constructed various logistic regression models depending on the main types of inheritance ( codominant , dominant , recessive , and additive ) . in the additive model , the presence of 2 mutant alleles confers double the risk of 1 mutant allele . the results were adjusted for rs12191877 ( snp that is strongly associated with the hla - c0602 allele and is highly prevalent in our population ) [ 3 , 35 ] . subsequently , snps with p < 0.1 in the univariate analysis ( adjusted for rs12191877 ) were included in a multivariate logistic regression model to adjust for relevant confounding factors ( spss 15.0 ) . the results of the univariate analysis were adjusted for rs12191877 , except when we compared patients with type i psoriasis and patients with type ii psoriasis ( the influence of rs12191877 was not very relevant ) . we expressed the results as the odds ratio ( or ) , 95% confidence interval , and p value . the study population included 155 patients with moderate - to - severe chronic plaque type i psoriasis ( 92 men and 63 women ) , 36 patients with type ii psoriasis ( 19 men and 17 women ) , and 197 controls ( 98 men and 99 women ) . the mean age was 46.01 13.11 years in patients with type i psoriasis ( 45.72 11.69 in men and 46.43 15.04 in women ) , 67.72 11.85 years in patients with type ii psoriasis ( 65.95 11.18 in men and 69.71 12.59 in women ) , and 24.51 4.29 years in the controls ( 25.07 4.94 in men and 23.95 3.46 in women ) . the mean age at onset of psoriasis was 23.31 8.52 in patients with type i psoriasis and 52.58 10.45 in patients with type ii psoriasis . a total of 192 snps were analyzed ( see supplementary table s1 published in ) . one snp was monomorphic ( rs165161 in the junb gene ) and was excluded from the statistical analysis . all the minor allele frequencies were in hardy - weinberg equilibrium except 9 snps in the controls and 12 snps in the patients ( see supplementary table s1 published in ) . the 9 snps which were not in hardy - weinberg equilibrium in controls were removed from the statistical analysis . our findings showed an association between type i psoriasis and 10 snps ( n = 155 versus n = 197 controls ) : rs1634517 ( ccl4l ) , rs1975974 ( c17orf51 ) , rs12720356 ( tyk2 ) , rs1800925 ( il13 ) , and rs6908425 ( cdkal1 ) decreased the risk of psoriasis 2.94-fold , 2.08-fold , 10-fold , 100-fold , and 2.44-fold , respectively ; and rs2282276 ( clmn ) , rs10782001 ( fbxl19 ) , rs3792876 ( slc22a4 ) , rs12191877 ( hla - c ) , and rs13437088 ( hla - b / mica ) increased the risk of psoriasis 3.90-fold , 2.10-fold , 3.75-fold , 30.54-fold , and 2.52-fold , respectively ( table 2 ) . however , comparison of 36 patients with type ii psoriasis and 197 controls revealed no significant association ( results not shown ) . four snps were associated with significant decreases in the risk of type i psoriasis ( n = 155 ) compared with type ii psoriasis ( n = 36 ) , namely , rs191190 ( tnfr1 ; 126.08-fold ) , rs361525 ( tnf- ; 190.76-fold ) , and rs10499194 and rs6920220 ( tnfaip3 ; 155.02-fold and 19.14-fold , resp . ) . we also found 5 snps that were associated with a significant increase in the risk of type i psoriasis , namely , rs1801274 ( fcgr2a ; 5.26-fold ) , rs763361 ( cd226 ; 33.3-fold ) , rs12459358 ( psors6 ; 11.11-fold ) , rs12191877 ( hla - c ; 12.5-fold ) , and rs1576 ( cchcr1 ; 166.66-fold ) ( table 3 ) . about 75% of patients with chronic plaque psoriasis have type i psoriasis before age 40 , whereas a lower number of patients develop psoriasis at around 5060 years . our results are consistent with these findings , since 79.06% of our patients developed psoriasis before the age of 40 . when we compared patients with type i psoriasis and controls , we found 10 significant snps in clmn , fbxl19 , ccl4l , c17orf51 , tyk2 , il13 , slc22a4 , cdkal1 , hla - c , and hla - b / mica . the hla - c0602 allele is a risk factor for psoriasis and has been associated with both type i [ 69 ] and type ii psoriasis . in one study , 85.3% of patients with type i psoriasis had this allele , whereas only 14.7% of patients with type ii psoriasis were carriers . other authors found an association between rs10484554 ( hla - c ) and type i psoriasis compared with type ii psoriasis ( or = 3.24 in type i ) . the hla - c gene was associated with type i psoriasis ( p = 2.97e 18 for rs1265181 , p = 2.58e 15 for rs12191877 , p = 1.84e 15 for rs4406273 , and p = 1.10e 07 for rs2395029 ) , but not with type ii psoriasis after application of the bonferroni correction . in addition , our results showed significant differences in rs12191877 ( hla - c ) in patients with type i psoriasis ( p = 2.50e 19 ) . however , we did not find this association in patients with type ii psoriasis , probably owing to the small sample size in this group ( n = 36 ) . found an association between rs1295685 in the il13 gene and type i psoriasis ( p = 2.47e 03 ) . our results showed an association between another snp in il13 ( rs1800925 ) and type i psoriasis ( p = 0.034 ) . in addition , munir et al . did not obtain significant results when they compared controls with type ii psoriasis or type i psoriasis with type ii psoriasis . both snps in il13 have been associated with predisposition to psoriasis [ 36 , 37 ] . our comparison of patients with type i psoriasis and controls is the first to obtain significant results for a series of snps in type i psoriasis , although the snps have already been associated with the risk of psoriasis . rs10782001 in fbxl19 , rs1975974 in c17orf51 , rs12720356 in tyk2 [ 3 , 39 ] , rs3792876 in slc22a4 , rs6908425 in cdkal1 , and rs13437088 in hla - b / mica have previously been associated with psoriasis , but not with type i psoriasis . furthermore , snps in clmn ( rs2282276 ) and ccl4l ( rs1634517 ) have not been associated with psoriasis or age at onset . we found no significant differences between patients with type ii psoriasis and controls owing to the small sample size ( n = 36 ) . comparison between patients with type i psoriasis and patients with type ii psoriasis revealed significant associations for the following genes : fcgr2a , tnfr1 , cd226 , psors6 , tnfaip3 , hla - c , tnf- , and cchcr1 . polymorphisms in cchcr1 ( 386 and 404 , cchcr1ww allele ) have been associated with type i psoriasis [ 9 , 31 ] . we found significant differences between rs1576 in cchcr1 and age at onset . in a study comparing controls ( 54.8% ) and patients with psoriasis type ii ( 66.0% ) , allen et al analyzed rs763361 in cd226 in patients with early - onset psoriasis and patients with late - onset psoriasis , although they found no associations . rs12459358 in psors6 has been associated with type i psoriasis ( g risk allele , or = 1.47 and p = 0.005 ) . in contrast , our data showed an association between the t allele and type i psoriasis ( or = 11.11 ; p = 0.026 ) . rs361525 ( 238 ) in the tnf gene has been associated with susceptibility to psoriasis , and the a allele was more frequent in male patients with type i psoriasis ( p = 2e 07 ) [ 15 , 22 ] . we found significant results in rs361525 ( tnf- ) when we compared patients with type i psoriasis and patients with type ii psoriasis , although we found no gender differences . other authors confirmed our association with type i psoriasis in caucasian [ 20 , 23 ] and mongolian patients . baran et al . found no significant differences between rs1800629 in the 308 promoter ( tnf ) and type i or type ii psoriasis . likewise , rs12191877 in hla - c has been associated with increased risk of psoriasis . compared patients with type i psoriasis and patients with type ii psoriasis and obtained significant results for rs1265181 , rs4406273 , and rs12191877 in hla - c . we replicated these results in rs12191877 ( t allele risk ; p = 0.045 ) . rs191190 in tnfr1 and rs10499194 in tnfaip3 have been associated with psoriasis , but not with age of onset . moreover , rs1801274 in fcgr2a and rs6920220 in tnfaip3 have not been studied in patients with psoriasis according to age of onset . given the small sample size in the group with type ii psoriasis in our study , our results should be interpreted with caution . our results highlight the role of the immune system in psoriasis and enhance our understanding of pathogenic mechanisms . second , the sample size was limited by the number of study patients treated in the dermatology department , thus making it difficult to detect snps with a low probability of causing psoriasis . third , since the snps were selected based on a literature review , several major snps may not yet have been investigated . in conclusion , our study confirmed an association between rs12191877 ( hla - c ) and type i psoriasis and between type i and type ii psoriasis patients . ours is the first study to show an association between clmn , fbxl19 , ccl4l , c17orf51 , tyk2 , il13 , slc22a4 , cdkal1 , and hla - b / mica and type i psoriasis . in addition , psors6 and tnf have been described as more prevalent genes in type i psoriasis and we showed a significant association when we compared type i psoriasis and type ii psoriasis . ours is the first study to identify an association between fcgr2a , tnfr1 , cd226 , tnfaip3 , and cchcr1 and age at onset of psoriasis .

psoriasis is a chronic skin disease in which genetics play a major role . although many genome - wide association studies have been performed in psoriasis , knowledge of the age at onset remains limited . therefore , we analyzed 173 single - nucleotide polymorphisms in genes associated with psoriasis and other autoimmune diseases in patients with moderate - to - severe plaque psoriasis type i ( early - onset , < 40 years ) or type ii ( late - onset , 40 years ) and healthy controls . moreover , we performed a comparison between patients with type i psoriasis and patients with type ii psoriasis . our comparison of a stratified population with type i psoriasis ( n = 155 ) and healthy controls ( n = 197 ) is the first to reveal a relationship between the clmn , fbxl19 , ccl4l , c17orf51 , tyk2 , il13 , slc22a4 , cdkal1 , and hla - b / mica genes . when we compared type i psoriasis with type ii psoriasis ( n = 36 ) , we found a significant association between age at onset and the genes psors6 , tnf- , fcgr2a , tnfr1 , cd226 , hla - c , tnfaip3 , and cchcr1 . moreover , we replicated the association between rs12191877 ( hla - c ) and type i psoriasis and between type i and type ii psoriasis . our findings highlight the role of genetics in age of onset of psoriasis .

in fixed orthodontic treatment , brackets were used for transferring orthodontic forces to the teeth . at first , to attach the brackets to the tooth , orthodontic bands were used and after welding brackets to bands , they were cemented to the tooth . in 1955 , buonocore introduced the acid - etch technique that was gradually used in different dental treatments . in 1965 , newman used direct bonding of orthodontic brackets that was considered as the first step in application of appliances with the improvement of esthetic presentation . this technique was developed rapidly due to its simplicity , efficacy and providing more esthetic qualities . for achieving successful bonding , the bonding agent must penetrate to the enamel surface , have easy clinical use , dimensional stability and enough bond strength . the bond strength of orthodontic brackets should be enough to not cause bonding failure and delay in treatment and it also should have adequate resistance against chewing forces and stresses from archwires . on the other hand , easy debonding of the brackets without any damage to the teeth needs sufficient and safe bond strength . according to few stages for bonding of orthodontic brackets and related problems in the conventional system , other techniques such as application of self - etch primers or laser irradiation was suggested to simplify the bonding procedure . in the acid - etching technique , microporosity was produced on the enamel surface to provide micromechanical bonding . enamel etching with phosphoric acid results in loss of the superficial layer of the enamel and dissolution of the enamel subsurface . the amount of enamel loss depends on phosphoric acid concentration and the time of application . laser etching was performed by the erbium family with two different wavelengths ( 2940 and 2780 nm ) . this technique has some advantages such as having no vibration or heat and producing a surface which is acid resistant by altering the calcium to phosphor ratio and formation of less soluble compounds . there are some studies which have evaluated the effect of laser etching on bond strength of orthodontic brackets with controversial results . so , the purpose of this study was to compare shear bond strength ( sbs ) of orthodontic brackets bonded to enamel prepared by er : yag laser with two different powers and conventional acid - etching . forty - five human premolars extracted for orthodontic purposes were selected for this study . in transillumination examination , the teeth showed healthy enamel on the buccal surface , without attrition , fracture , restoration , congenital anomalies and structural defects . there was no history of chemical substance application such as hydrogen peroxide for these teeth . after rinsing the teeth , they were placed in 0.5% chloramine t for inhibiting bacterial growth for 2 hours . the teeth were divided into three groups according to conditioning method : group 1 : conventional etching with 37% phosphoric acid ; group 2 : laser irradiation by er : yag laser with output power of 1w ; and finally group 3 : laser irradiation with er : yag laser with output power of 1.5w . in group 1 , the samples were etched with 37% phosphoric acid gel ( 3 m , dental products , st.poul ) for 15 sec , then rinsed for 15 sec with water spray and dried with air spray for 10 sec in a 2 cm distance above the surface of the enamel . laser irradiation in group 2 and 3 was carried out by er : yag laser ( us20d , deka , italy ) with a 2940 nm wavelength . the area was marked before irradiation . in group 2 , laser was used with an output power of 1w , energy of 100 mj and frequency of 10 hz . these parameters were 1.5 w , 150 mj and 10 hz , respectively for group 3 . the handpiece of laser was used 5 mm above the surface in non - contact mode and sweeping motion . subsequently , the adhesive kit ( transbond xt , 3 m , unitek ) was used . the adhesive paste was placed on the bracket base and the brackets were placed on the enamel with a 300 gr compressive force with gauge for 10 sec to produce uniform thickness . the resin was polymerized by led ( mectron , starlight pro gac , italy ) with a 440480 nm wavelength and 400 mw / cm intensity for 40 sec . consequently , the samples were thermocycled for 200 cycles between 5c and 55c water baths with 30 sec dwell time for each . the specimens were mounted in auto - cure acrylic resin and the shear bond strength was measured by using a universal testing machine with a crosshead speed of 0.5 mm per second . after debonding , the amount of resin remaining on the teeth was determined using the adhesive remnant index ( ari ) scored 1 to 5 ( table 1 ) by stereomicroscope ( nikon d - cs , japan ) with 10x magnification . one - way analysis of variance was used to compare shear bond strengths and the kruskal - wallis test was performed to evaluate differences in the ari for different etching types . the mean and standard deviation of the conventional acid - etch group , laser group ( 1w ) and laser group ( 1.5w ) was 3.82 1.16 , 6.97 3.64 and 6.93 4.87 , respectively . there was no significant difference between laser group ( 1.5w ) and laser group ( 1w ) ( p=1.000 ) and conventional group ( p=0.085 ) , but there was a significant difference between laser group ( 1w ) and conventional group ( p=0.016 ) . according to graph 1 , the variances of values of the laser samples bond strengths was higher than the acid - etch group . table 2 shows the frequency distribution of ari degrees in the three groups . according to the kruskal - wallis test there are some studies which evaluate the enamel preparation by laser irradiation for orthodontic brackets . the aim of this study was to assess the shear bond strength of orthodontic brackets bonded to enamel prepared by er : yag laser or acid - etch . the bond strength of light curing composites may be influenced by thermal changes of the oral cavity and the quality of polymerization . thermocycling is a common method for stimulating this condition ; therefore , we used this technique in this research . the result of this study showed that both laser groups had higher bond strengths than the acid - etch group . although this difference was significant between the laser group with an output power of 1w and the acid - etch group , the laser group with an output power of 1.5w showed no significant difference with the acid - etch group . according to usumez s study , laser irradiation with a power of 2w in comparison with the acid - etch technique showed similar shear bond strengths , but application of laser with a power of 1w showed a lower bond strength . on the other hand , gokcelik et al.s study which assessed the shear bond strength of samples prepared by er : yag laser and acid - etch found no significant difference between these two groups . controversial results were obtained from different studies which evaluated the effect of laser irradiation compared to conventional methods due to different study designs and various parameters used in these studies . morphological changes of enamel produced after laser irradiation depends on the energy density of the laser , the time of exposure , the distance of the laser handpiece from the surface and percentage of water irrigation . samples irradiated with 1.5w power showed no significant difference compared to laser group with 1w power which is in agreement with the results obtained from basaran s study . in the present study , the laser groups showed higher bond strengths with higher standard deviations compared to the acid - etch group . this finding reduced the credibility of laser application for enamel preparation , considered as an unfavorable characteristic . in similarity , usumez et al . reported higher distribution coefficient for shear bond strength of orthodontic brackets in laser prepared surfaces . the reason may be related to the irregular etching pattern of surfaces irradiated by laser . sasaki et al . found that preparation of enamel surfaces by er : yag laser can not be done homogeneously . surfaces irradiated by laser showed some areas which were similar to unlased enamel surfaces but surface preparation by acid etch technique showed more homogeneous patterns which was like honey comb pattern that is favorable structure for adhesion process . higher standard deviations in the laser groups may be associated with intrinsic nature differences of the teeth collected from different people , time of storage and environmental effects on the tooth after extraction . in order to control these problems , animal teeth can be used because numerous tooth samples can be provided from an animal . among different animals maijer and smith stated that bond strength of 8 mpa is essential for orthodontic treatment . in this study , the mean of shear bond strength in the three groups was below the suggested value . cerekja and cakirer showed that that thermocycling process reduced the shear bond strength of orthodontic brackets . in addition , daub confirmed that this condition was due to differences in thermal expansion coefficients of the adhesive , brackets and enamel . two thirds of the samples showed an ari degree of 4 or 5 which showed that the highest debonding happened in resin to teeth contact surface which needs less cleaning of debonded enamel leading to reduction of abrasion risk to the enamel , but it is better to have debonding in resin - bracket contact or inside the resin because the less adhesive remaining on the tooth , the more stress affecting the enamel surface in clinical condition , this kind of debonding is rare because providing favorable etching in enamel surface is difficult due to lack of controlling humidity , time and cooperation of patients in preparing the surfaces . in addition , the structural pattern of the bracket base makes debonding in the resin bracket contact surface uncommon . in contrast to these results , lee in the evaluation of bonded brackets observed that samples prepared by acid - etch technique or er : yag laser irradiation showed more fracture pattern in resin - bracket contact surface . these different results may be contributed to the debonding test procedure , which was tensile bond strength in lee s study . valletta reported that debonding happened in bracket resin surface in tensile bond strength and in resin tooth contact surface in shear bond strength . : yag laser with an output power of 2 w and frequency of 2 hz showed no significant increase in shear bond strength compared to the control group . in the present study , ari degree among the two laser groups and between laser groups and in contrast , gokcelik showed higher ari degrees in er : yag laser compared to the acid - etch group . in laboratory conditions , loading forces to brackets were different from clinical conditions . in clinical conditions , besides , in the oral cavity , there are different kinds of stresses such as thermal changes , humidity and microbial plaque that make the simulation condition in laboratory difficult . although bond strength tests are still far from ideal , attempts should be made to standardize these tests to make comparisons easier . the shear bond strength of bracket to laser - prepared enamel with two different powers of 1 and 1.5 w was similar and laser groups showed higher bond strengths than the acid etch group . however , high variances of values in bond strength of irradiated enamel should be considered to find the appropriate parameters for applying er : yag laser as a favorable alternative for surface conditioning .

introduction : the purpose of this study was to compare shear bond strength ( sbs ) of orthodontic brackets bonded to enamel prepared by er : yag laser with two different powers and conventional acid-etching.materials and methods : forty - five human premolars extracted for orthodontic purposes were randomly assigned to three groups based on conditioning method : group 1- conventional etching with 37% phosphoric acid ; group 2- irradiation with er : yag laser at 1 w ; and group 3- irradiation with er : yag laser at 1.5 w. metal brackets were bonded on prepared enamel using a light - cured composite . all groups were subjected to thermocycling process . then , the specimens mounted in auto - cure acryle and shear bond strength were measured using a universal testing machine with a crosshead speed of 0.5 mm per second . after debonding , the amount of resin remaining on the teeth was determined using the adhesive remnant index ( ari ) scored 1 to 5 . one - way analysis of variance was used to compare shear bond strengths and the kruskal - wallis test was performed to evaluate differences in the ari for different etching types.results:the mean and standard deviation of conventional acid - etch group , 1w laser group and 1.5w laser group was 3.82 1.16 , 6.97 3.64 and 6.93 4.87 , respectively.conclusion:the mean sbs obtained with an er : yag laser operated at 1w or 1.5w is approximately similar to that of conventional etching . however , the high variability of values in bond strength of irradiated enamel should be considered to find the appropriate parameters for applying er : yag laser as a favorable alternative for surface conditioning .

the interaction of ultraviolet or visible light of a specific wavelength with certain molecules or photosensitizing chemicals leads to a delayed type hypersensitivity reaction that causes photocontact dermatitis . the primary investigation for the detection of photodermatitis is the photopatch test , which helps in the investigation and detection of specific allergens that cause photodermatitis in a susceptible individual . it involves exposure of the skin to appropriate amounts of an allergen implicated in causing photoallergic contact dermatitis and recording the subsequent response with and without light exposure . the primary indication for the test would be dermatitis predominantly limited to sun exposed sites of uncertain aetiology . the antigens used in the photopatch series include sunscreens , nonsteroidal anti - inflammatory drugs ( nsaids ) , and fragrances ; specific antigens are added based of information provided by the patient . although various differences exist in the procedure , irradiation doses , interpretation , and antigens of photopatch test , only 4%20% of patients undergoing photopatch tests show clinically positive relevant results . common photosensitizing agents include chemicals present in sunscreens , antiseptic agents , fragrances , and nonsteroidal anti - inflammatory drugs . in india , parthenium hysterophorus is probably the most common cause of photoallergic contact dermatitis and airborne contact dermatitis . the diagnosis of a photodermatitis is based on the history and clinical examination and is confirmed by photopatch testing . it is important to rule out other causes of photoexposed site reactions such as connective tissue disease , drugs , allergic contact dermatitis , and pophyrias while investigating a patient of suspected photoallergy . there is no indian standard photopatch test series available , and studies carried out in india have used european or scandinavian photopatch test trays , which may not be relevant for indian patients . hence we have used a combination of photopatch test and uv - irradiated indian standard series ( iss ) in our study , to detect light - induced antigens in patients with photosensitive dermatitis . this study was done as a hospital - based , descriptive , observer blinded study . a total of 35 patients were included in the study during a one year period between september 2012 and october 2013 . clearance from the ethical committee was obtained and written informed consent was taken from all patients involved in the study . patch and photopatch testing was performed on all the patients using the iss and photopatch series . 1- control , 2- potassium dichromate , 3- neomycin sulfate , 4- cobalt chloride , 5- benzocaine , 6- formaldehyde , 7- paraphenylenediamine ( ppd ) , 8- parabens , 9- nickel sulfate , 10- colophony , 11- gentamicin , 12- mercapto mix , 13- epoxy resin , 14- fragrance mix , 15- mercaptobenzothiazole , 16- nitrofurazone , 17- polyehyleneglycol-400 , 18- chlorocresol , 19- wool alcohols , 20- balsam peru , 21- thiruram mix , 22- chinoform , 23- black rubber mix , and 24- p - tbp f resin . the photopatch series consisted of : 1- benzophenone-3 , 2- benzophenone-4 , 3- eusolex 232 , 4- butylmethoxydibenzoyl methane ( parsol 1789 ) , 5- para amino benzoic acid , 6- 3(4 methyl benzyliden ) camphor , 7- octyltriazone , 8- octyl methoxycinnamate , 9- ibuprofen 5% , 10- piroxicam 5% , 11- ketoprofen 2.5% , 12- isomyl 4-methoxycinnamate 10% , 13- fragrance mix 8% , and 14- parthenium . the patients included in the study presented with dermatitis predominantly affecting sun exposed areas or with a history of photosensitivity . clinically suspected cases of phototoxic or photoallergic contact dermatitis secondary to drugs , chemicals , or airborne antigens were also included in the study . patients on systemic immunosuppressants , oral steroids , exfoliative and active dermatitis , pregnant or lactating women , and those below 18 years of age were excluded from the study . those with photosensitivity due to conditions such as connective tissue disease and genetic disorders with photosensitivity were also excluded . a detailed history was taken with regard to onset , duration and progression of disease , type and distribution of lesions , and presence of any comorbid conditions . all patients that fit the criteria for inclusion were subjected to a thorough clinical examination . photographs of the patch test sites were taken at each reading to document positive reactions . both sets of patches were applied in duplicate on the patient 's back , on either side of the spine by the standard method . after 24 h , the tapes were carefully removed and squares representing each chamber were marked using a marker pen . readings were recorded after a gap of half an hour , into the respective proformas . after noting relevant readings , one side was closed with an opaque black cloth and the other side was irradiated with 14 j / cmsq of uva . the selection of side to be irradiated was done in a randomized , observer - blinded manner . a distance of 15 cm was kept between the patient 's back and irradiation source . readings were then recorded after 48 h. at the end of the protocol , two sets of readings were obtained considering the day of patch application as day 0 . first reading was at 24 h after application of patches , followed by uva irradiation ( day 1 ) . the patch test results were evaluated using the international contact dermatitis research group ( icdrg ) grading . photopatch test was interpreted according to the standard photopatch criteria . according to the criteria , if only the irradiated side shows a positive reaction , it is labeled as a photoallergic reaction . if both sides show a positive reaction with the irradiated side showing greater than 1 + positivity , it is termed as a contact dermatitis with photoaggravation . if both sides show equal reaction after irradiation , a contact allergy is the result . out of the 35 patients included in the study , 10 ( 29% ) were females and 25 ( 71% ) were males and all patients were outdoor workers . majority ( 66% ) of patients belonged to the age group ranging from 35 to 65 years . a total of 24 ( 69% ) patients had positive patch or photopatch test results . majority of these patients 21 ( 60% ) had features of chronic dermatitis confined mainly to the sun - exposed areas with a history of photosensitivity . the remaining 14 ( 40% ) patients presented with a history of photosensitivity with dermatititis present both in sun - exposed and covered areas . twelve patients among the total study population had a positive history of atopy with ige levels above 1000 iu / ml . the highest number of photopatch reactions was noted with parthenium , with 18 ( 51% ) patients showing positive results . the detailed results of the 18 patients with a positive photopatch test suggestive of photodermatitis are shown in table 1 . out of the 18 patients , 9 ( 50% ) showed contact allergy , 4 ( 22% ) had photoallergy , and 5 ( 28% ) had contact dermatitis with photoaggravation to p. hysterophorus . five patients among the 18 patients had coexistent contact dermatitis to other antigens in the iss . detailed results of the 18 patients who showed positive photopatch test results six patients had contact dermatitis without any photoaggravation or photoallergy to varied antigens such as potassium dichromate , chinoform , fragrance , para - phenylenediamine , nickel , and cobalt . in our study , p. hysterophorus was the leading allergen with 51% of the study population showing a positive reaction . this is expected due to the widespread presence of parthenium in the locality and surrounding areas . in a similar study carried out by jindal et al . , 30 patients were subjected to photopatch testing along with some antigens obtained from the standard series . fourteen positive tests to several allergens were obtained with fragrance mix being the leading antigen ( 30% ) , followed by para - phenylenediamine ( 20% ) and p. hysterophorus ( 17% ) . sharma and kaur found 78% of patients with airborne contact dermatitis to have parthenium sensitivity . in another study done by sharma et al . three patients showed photoallergic reaction and another 3 showed photoaggravation out of 19 patients . in our study 4 patients showed photoallergy and 5 patients showed contact dermatitis with photoaggravation . the major antigens in p. hysterophorus are sesquiterpene lactones . some of the identified lactones are parthenin , hymenin , ambrosin , and coronopilin . parthenium is established to cause both photoallergy and contact dermatitis with photoaggravation . in a study done by kar et al . , it was observed that parthenium plays a significant role in the initiation and spread of air borne contact dermatitis and chronic actinic dermatitis ( cad ) . a 20-year analysis of antigens causing photoallergic contact dermatitis done in new york showed 11.6% positivity to plant derivatives including sesquiterpene lactone mix . the coexistence of allergic contact dermatitis ( acd ) with cad is well known . in most of these cases about 75% of patients with cad show a positive patch test response to one or more allergens . among the various plant antigens , sesquiterpene lactones obtained from plants of the compositae family are the most common causative antigens apart from fragrance , rubber , metals , colophony , chromates , and sunscreens . the second most common allergens in our study were fragrance mix and potassium dichromate . in our study , photoallergy due to fragrance can be due to the perfume compound itself of the fixative agent such as musk ambrette . in a study done by panja et al . , fragrance mix was the leading photosensitizer . metals such as nickel , cobalt , and dichromates are common sensitizing agents . three patients ( 30% ) showed positive patch test reactions , to nickel and cobalt in our study . these sensitizers are found in jewelry , watches , cement , leather , and dyes . chronic exposure to these allergens in the presence of ultraviolet radiation facilitates increased immune recognition and aggravation of pre - existing dermatitis . various differences exist in the pattern of antigen positivity depending on the area and population under study . the positivities obtained in western literature are to certain antigens not so frequently encountered in the indian scenario . in a recent study from the united states , sunscreens and anti - microbial agents were the predominant antigens and a decreased incidence in fragrance induced photoallergic contact dermatitis was found . the most common positive antigens in photopatch tests in western studies were sunscreens and drugs such as nsaids . no positivity to sunscreens was detected in our study , probably due to infrequent use in the given population . the leading allergens in the scandinavian multicentric photopatch study were musk ambrette and para amino benzoic acid . one patient showed positive reaction to ppd . in a study done by jindal et al . 6 out of 20 patients showed contact allergy to ppd with two patients having photoaggravation . although ppd is known to cause photoallergic reactions , our patient did not exhibit the same . in our study , six patients had contact dermatitis to various antigens such as nickel , cobalt dichromates , and ppd . although these sensitizers have been implicated in photoaggravated contact dermatitis , none of our patients showed such a response pattern . photodermatitis is prevalent in india , and prompt identification of the causative antigens will alleviate the morbidity associated with this condition . photoallergic contact dermatitis is largely underdiagnosed in our country due to lack of availability of proper photopatch protocols . parthenium was found to be the leading cause of photodermatitis in our study causing photoallergy , contact dermatitis with photoaggravation , and contact allergy . among the 18 parthenium - positive patients , five had coexistent contact dermatitis to other antigens in the iss . in this context , patch testing with both iss and photopatch series could lead to clinically relevant results . it is advantageous as it is noninvasive , simple to carry out , and can be performed on an outpatient basis . thus , patch testing with combined iss and photopatch series can be efficacious in the detection of antigens causing photosensitive dermatitis . a 24- and 48-h photopatch test reading was taken . our inability to take delayed readings at 72/96 h or even one week may have resulted in false - negative results.minimal erythema dose or med is the least amount of uv radiation required to produce perceptible erythema on light exposed skin . 48-h photopatch test reading was taken . our inability to take delayed readings at 72/96 h or even one week minimal erythema dose or med is the least amount of uv radiation required to produce perceptible erythema on light exposed skin . a 24- and 48-h photopatch test reading was taken . our inability to take delayed readings at 72/96 h or even one week may have resulted in false - negative results.minimal erythema dose or med is the least amount of uv radiation required to produce perceptible erythema on light exposed skin . 48-h photopatch test reading was taken . our inability to take delayed readings at 72/96 h or even one week minimal erythema dose or med is the least amount of uv radiation required to produce perceptible erythema on light exposed skin .

background : the photopatch test is used to detect photoallergic reactions to various antigens such as sunscreens and drugs . photosensitive dermatitis can be caused due to antigens like parthenium , fragrances , rubbers and metals . the photopatch test does not contain these antigens . therefore , the indian standard series ( iss ) along with the standard photopatch series from chemotechnique diagnostics , sweden was used to detect light induced antigens.aim:to detect light induced antigens in patients with photosensitive dermatitis.methods:this study was done in a descriptive , observer blinded manner . photopatch test and iss were applied in duplicate on the patient 's back by the standard method . after 24 hours , readings were recorded according to icdrg criteria . one side was closed and other side irradiated with 14 j / cm2 of uva and a second set of readings were recorded after 48 hrs.result:the highest positivity was obtained with parthenium , with 18 out of 35 ( 51% ) patients showing a positive patch test reaction with both photoallergic contact dermatitis and photoaggravation . four patients ( 11% ) showed positive patch test reaction suggestive of contact dermatitis to potassium dichromate and fragrance mix . six patients had contact dermatitis to numerous antigens such as nickel , cobalt , chinoform and para - phenylenediamine . none of these patients showed photoaggravation on patch testing.conclusion:parthenium was found to cause photoallergy , contact dermatitis with photoaggravation and contact allergy . hence , photopatch test and uv irradiated patch test can be an important tool to detect light induced antigens in patients with photosensitive dermatitis .

pycnodysostosis was first reported in 1923 by montanari , and he called it as atypical achondroplasia . the main characteristics are short stature , cranial dysplasia , increased bone density and fragility . other clinical features include open cranial sutures , hypoplastic paranasal sinuses , dysplastic lateral clavicle , shortened terminal phalanges , proptosis , blue sclera and frontal or occipital bossing . oral manifestations include obtuse gonial angle , grooved palate , anterior cross - bite , malpositioned teeth associated with increased incidence of dental caries and periodontitis , hypoplastic maxilla , receded chin , delayed eruption of permanent teeth , delayed exfoliation of deciduous teeth and hypoplasia of root - obliterated pulp spaces . a 47-year - old man reported to the department with a complaint of deformed lower jaw for the past 10 years . history revealed that the patient had undergone extraction of his teeth that was uneventful , following which there was fracture of jaw at the extraction site . subsequently , there was frequent exfoliation of teeth with fracture at different sites in the lower jaw . his medical history revealed that he had multiple fractures of the upper and lower limbs and a history of diabetes mellitus and hypertension . general examination revealed that the patient 's height was 127 cm and weight was around 49 kg , with proportionate dwarfism [ figure 1 ] . the hand and feet had short digits with overlying cutaneous wrinkles that tapered off with large overriding nails [ figure 2 ] . a 47-year - old man , 127 cm in height shortened digits and cutaneous wrinkles on extraoral examination , there was facial dysmorphia with prominent forehead ( frontal bossing ) , proptosis , beaked nose , deep nasolabial skin folds , micrognathia and obtuse mandibular angle on the right side [ figure 3 ] . mild proptosis and facial dysmorphia intraoral examination revealed multiple clinically missing teeth , chronic periodontitis , narrow and grooved palate , no features of enamel hypoplasia , malposed teeth [ figure 4 ] and evidence of sequestrum in relation to the tooth 46 [ figure 5 ] . based on the history and clinical presentation , a provisional diagnosis of a bone dysplasia , probably pycnodysostosis , was made and differential diagnosis of cleidocranial dysplasia and osteopetrosis was included . grooved palate and clinically missing maxillary anteriors sequestrum in relation to tooth 46 laboratory findings were within normal limits , including hemoglobin conc . , differential count calcium , phosphate , alkaline and acid phophatase level . computed tomography of the bone window of the skull showed open sutures and fontenalles with nonaerated paranasal sinuses , flattening of the mandibular angle on the right side with evidence of fracture and loss of bone architecture on the left side involving the ramus of the body of the mandible and hypoplastic maxilla [ figures 68 ] . orthopantomograph revealed generalized bone loss , multiple missing teeth and obtuse gonial angle with loss of bone structure on the left side of the mandible involving the body and ramus [ figure 9 ] . 3d - reconstructed computed tomography showing open sutures and fontanelles 3d - reconstructed computed tomography showing fractured body of the mandible and obtuse gonial angle 3d - reconstructed computed tomography showing fractured body of the mandible opg showing generalized bone loss , multiple missing teeth , and obtuse gonial angle lateral skull showing open fontanelles with nonaerated paranasal sinus and fractured body of the mandible the patient was surgically managed for osteomyelitis by removal of the sequestrum and curettage , and further mandibular reconstruction was performed . the patient presents with characteristic facies , dwarfism , beaked nose , prominent head and generalized increase in the density of bones not sufficient to obliterate medullary canals or cranial orifices . frequent fractures due to trauma can aid in diagnosing this condition . in our case , the patient was negligent about the condition and reported to us with a fractured jaw . intraoral clinical presentation included altered pattern of exfoliation of deciduous teeth and eruption of the permanent dentition . the disease is diagnosed at an early age , wherein the main reasons for consultation are generally short stature and open anterior fontanelles . in later stages , consultation is usually for fracture resulting from slight or moderate trauma , given the severe bone fragility . symptoms include dental abnormalities , with hypoplasia of the enamel , obliterated pulp chambers and hypercementosis . protrusion of the incisors with anterior open bite may be found , and dental crowding associated with extensive caries and periodontitis is frequent . in our case , multiple clinically missing teeth , chronic periodontitis , narrow and grooved palate , features of enamel hypoplasia and malposed teeth these conditions cause the premature loss of dentition that may already be complete by the fourth decade of life , similar to our patients . our patient showed evidence of sequestrum in relation to the tooth 46 as a result of osteomyelitis . orofacial infections are commonly encountered by the dentist and there are wide ranges of modalities that can be implemented in managing them . this is caused by the increased bone volume of the sella turcica that , on compressing the pituitary gland , causes its hypoplasia and a deficient production of the growth hormone . our patient 's height was 127 cm and weight was around 49 kg , with proportionate dwarfism . the hand and feet had short digits with overlying cutaneous wrinkles that tapered off with large overriding nails . diagnosis of pycnodysostosis is based on the clinical presentation , and medical treatment for the condition is symptomatic . the differential diagnosis of pycnodysostosis includes osteopetrosis , acroosteolysis , mandibular acral dysplasia and cleidocranial dysplasia . unlike osteoporosis , hepatosplenomegaly and difference between pycnodysostosis and cleidocranial dysplasia is that dense and brittle bones are found in pycnodysostosis but not in cleidocranial dysplasia . new treatment modalities like gene therapy and bone marrow transplant can be expected to be the mainstay in the future , now that the abnormal expression of cathepsin k and the gene defect has been located . now - a - days , symptomatic treatment is provided for patients with pycnodysostosis , with the main intention of prevention of fractures . as pycnodysostosis is associated with inappropriate bone remodeling , it can pose a challenge for a dental health care professional to provide treatment as there can be serious complications , such as osteomyelitis arising as a result of dental infections . tooth extractions in these patients demand certain special care , such as carrying out the surgery atraumatically with proper asepsis . oral hygiene practices and frequent visits to

pycnodysostosis , a sclerosing bone dysplasia , is a rare autosomal recessive disorder with an estimated prevalence rate of one in one million . patients with pycnodyostosis usually have normal intelligence , sexual development and life span . this condition is characterized by increased bone density and fragility along with oral manifestations like malposition teeth , hypoplastic maxilla , receded chin and delayed eruption of permanent teeth with discharging sinuses in the jaws because of poor blood supply . this is one such rare case report of a 47-year - old patient presenting with a complaint of fractured jaw and reviewing the clinical and radiographic characteristics of pycnodysostosis .

because steroid hormones play an important role in a wide range of physiological processes , the potential to disturb endocrine effects is a major concern in the development of novel pharmaceutical drugs such as etomidate and aminoglutethimide . the adrenal gland is the most common target for toxicity in the endocrine system in vivo , because steroid hormones are primarily synthesized through enzymatic reactions in the adrenal cortex [ 25 ] . indeed , in these studies based on chemically induced endocrine lesions observed in vivo , the most frequent site of reported effects was the adrenal gland . therefore , the prediction of human adrenal toxicity based on the mechanism of on- or off - target actions in the early stages of drug development is important . the nci - h295r human adrenocortical carcinoma cell line has been used to elucidate mechanisms of adrenal steroidogenic disrupting compounds [ 1 , 6 ] . the h295r cell line was established by gazder and his collaborators in 1990 , which expresses all key steroidogenic enzymes and steroidogenesis - related proteins [ 79 ] . h295r cells have the physiological characteristics of zonally undifferentiated human fetal adrenal cells and the ability to produce steroid hormones found in the adult adrenal cortex [ 1 , 7 , 9 ] . in vitro bioassays using the h295r human cell line have been able to evaluate the effects of chemicals on steroid hormone production [ 1015 ] , steroidogenic enzyme activities [ 11 , 16 , 17 ] , and the expression of steroidogenic genes [ 11 , 18 ] . in transcriptome studies , the mechanisms of action of many steroidogenic disrupting compounds have been qualitatively assessed in terms of adrenal toxicity . furthermore , measuring a few specific steroid hormones may not be a useful approach to study the mechanisms of steroidogenic disrupting effects in complex pathways such as adrenal steroidogenesis . to systematically understand how exogenous compounds affect adrenal steroidogenesis , simultaneous determination of all detectable steroid hormones and integrative analysis of these complex data would be important . as an exploratory approach to analyze complex data , toxclust developed by zhang and colleagues in 2009 is able to visualize concentration - dependent response relationships in the characteristics of chemically induced toxicological effects . however , this exploratory approach is unable to provide a quantitative understanding of the mechanism of action of adrenal toxicants or reveal systematic information about the effect of each enzymatic reaction , interactions , and feedback in the adrenal steroidogenesis pathway . systems biology based on computational models of biological processes and the comprehensive measurement of biological molecules is the most powerful approach to quantitatively understand the influence of each factor in complex biological pathways . in recent studies by our collaborators , a computational model of adrenal steroidogenesis has been developed in nci - h295r cells , including the steroidogenic disrupting effects of metyrapone to inhibit enzymatic reactions of cyp11b1 [ 21 , 22 ] . the model reproduces the dynamics of adrenal steroidogenesis in nci - h295r cells and the influence of metyrapone . a current computational model of adrenal steroidogenesis was incorporated with a reaction of oxysterol synthesis as a bypass to consume cellular cholesterol . in addition , all reactions in this model are described by a kinetic equation of the first - order reaction . it is difficult to quantitatively evaluate the influence of each protein in the complicated system of adrenal steroidogenesis using the reported models , because it is simple and any biochemical and cellular biological information is not sufficient . for example , to clearly understand the cause of the change from the differentially dynamic patterns of steroid hormones , it is necessary to consider the substrate inhibition of steroidogenic enzyme because most of steroidogenic enzymes recognize multiple steroids as the enzymatic substrate . however , the substrate inhibition of steroidogenic enzyme can not be described by the mathematical model based on kinetic equations of first - order reaction that does not consider michaelis constant km expressing the affinity of the substrate . to quantitatively estimate the mechanism of steroidogenic disrupting compounds from comprehensive experimental data of adrenal steroidogenesis in nci - h295r cells , the reported model should be improved according to the following two points . first , the kinetic equation of enzymatic reactions should be exchanged from the first - order equation to a steady - state kinetic equation based on the mechanism of the enzymatic reaction . because a mathematical model organized by first - order equations operates in a simple structure - dependent manner , it does not show complex behavior based on molecular interactions , feedback , or regulation . second , intracellular localization processes of cholesterol should be incorporated as a considerable mechanism . because intracellular cholesterol molecules are stored as cholesterol esters or widely distributed as membrane components , only a few cholesterol molecules localized on the mitochondrial inner membrane are available for the adrenal steroidogenesis pathway [ 23 , 24 ] moreover , cholesterol - trafficking processes from the outer to inner mitochondrial membranes , which are regulated by steroidogenic acute regulatory ( star ) protein , are one of the rate - limiting steps in adrenal steroidogenesis . by overcoming these limitations in the reported steroidogenesis model , systems analysis of adrenal steroidogenesis in h295r cells may be able to quantitatively estimate the mechanism of action of steroidogenic disrupting compounds . in the present study , to quantitatively estimate the toxicological mechanism of endocrine - active compounds in adrenal steroidogenesis and to predict human adrenal toxicity of novel pharmaceutical drugs in the drug discovery phase , we developed a novel computational model of steroidogenesis in nci - h295r cells . it includes cholesterol transport into intracellular regions from the extracellular space , the cholesterol translocation system in intracellular regions , including oxysterol synthesis , the metabolic pathway of adrenal steroidogenesis , and transport of steroid hormones . global sensitivity analysis of this adrenal steroidogenesis model is able to evaluate the influence of each steroidogenic enzyme and related protein for each steroid hormone observed in an in vitro steroidogenesis assay of nci - h295r cells . furthermore , the mechanisms of action of steroidogenesis disrupting compounds for steroidogenic enzymes can be estimated by the optimization method to solve the reverse problem from the concentration changes of 12 steroid hormones measured by liquid chromatography / mass spectrometry in the steroidogenesis assay of nci - h295r cells in vitro . using this developed model of adrenal steroidogenesis and the analytical approach , the in vitro steroidogenesis assay of nci - h295r cells can assess the human adrenal toxicity of a novel pharmaceutical drug based on quantitative understanding of its toxicological mechanism in adrenal steroidogenesis . nci - h295r human adrenocortical carcinoma cells were purchased from the american type culture collection ( cat # crl-2128 , manassas , va ) and cultured at 37c in a humidified atmosphere with 5% co2 . the cells were maintained in a 1 : 1 mixture of dulbecco 's modified eagle 's medium ( dmem , gibco , life technologies , carlsbad , ca ) and f-12 medium ( mp biomedicals inc . , irvine , ca ) supplemented with 15 mm hepes ( dojindo laboratories , kumamoto , japan ) , 0.00625 mg / ml insulin ( sigma - aldrich , inc . , st . louis , mo ) , 0.00625 mg / ml transferrin ( sigma - aldrich inc . , st . louis , mo ) , 30 nm sodium selenite ( wako pure chemical industries ltd . , osaka , japan ) , 1.25 mg / ml bovine serum albumin ( bsa , sigma - aldrich inc . , st . louis , mo ) , 0.00535 mg / ml linoleic acid ( sigma - aldrich inc . , st . louis , mo ) , 2.5% nu serum ( becton , dickinson and company , franklin lakes , nj ) , 100 u / ml penicillin ( meiji seika pharma , tokyo , japan ) , and 100 mg nci - h295r cells were stimulated with adrenocorticotrophic hormone ( acth ) , forskolin , and angiotensin ii to initiate steroidogenesis . changes in steroid concentrations over time were measured after stimulation in both cells and culture medium to construct a simulation model . the cells were seeded at 6 10 cells / well in 6-well plates . after 3 days of culture , the culture medium was changed to stimulation medium consisting of dmem / f-12 ( 1 : 1 ) medium supplemented with 0.00625 mg / ml insulin , 0.00625 mg / ml transferrin , 30 nm sodium selenite , 1.25 mg / ml bsa , 0.00535 mg / ml linoleic acid , 10% fetal bovine serum ( gibco , life technologies , carlsbad , ca ) , 100 u / ml penicillin , 100 mg / l streptomycin , 50 nm acth ( sigma - aldrich inc . , louis , mo ) , 20 m forskolin ( sigma - aldrich inc . , st . louis , mo ) , and 100 nm angiotensin ii ( calbiochem , merck millipore , darmstadt , germany ) . culture media and cells were collected at 0 , 8 , 24 , 48 , and 72 h after stimulation . the cells were collected in 100 l distilled water and sonicated to produce a cell lysate . the cultures were conducted in four wells / time point ( n = 4 ) . the concentrations of 12 steroids , pregnenolone ( preg ) , 17-hydroxypregnenolone ( hpreg ) , dehydroepiandrosterone ( dhea ) , progesterone ( prog ) , 17-hydroxyprogesterone ( hprog ) , androstenedione ( dione ) , testosterone ( testo ) , 11-deoxycorticosterone ( dcortico ) , 11-deoxycortisol ( dcort ) , corticosterone ( cortico ) , cortisol ( cort ) , and aldosterone ( aldo ) , in the medium and cell lysate were measured by lc / ms . concentrations of estrone ( e1 ) and 17-estradiol ( e2 ) were measured by enzyme - linked immunosorbent assays ( wako pure chemical industries ltd . in addition , the concentration of cholesterol was measured using a commercial kit ( wako pure chemical industries ltd . , osaka , japan ) based on the cholesterol oxidase method . a lc - vp series ( shimadzu , kyoto , japan ) consisting of an sil - htc autosampler , lc-10advp pump , cto-10acvp column oven , and dgu-14am degasser was used to set the reverse - phase liquid chromatographic conditions . the column was a cadenza cd - c18 column ( 100 2 mm i.d . , 3 m , imtakt corp . , kyoto , japan ) used at 45c . the mobile phase included water / acetonitrile / formic acid 95/5/0.05 ( v / v / v , solvent a ) and water / acetonitrile / formic acid 35/65/0.05 ( v / v / v , solvent b ) . the gradient elution programs were 0% b ( 0 - 1 min with an isocratic gradient ) , 040% b ( 1 - 2 min with a linear gradient ) , 40% b ( 27 min with an isocratic gradient ) , 40100% b ( 712 min with a linear gradient ) , 100% b ( 1214 min with an isocratic gradient ) , 1000% b ( 14 - 15 min with a linear gradient ) , and 0% b ( 15 - 16 min with an isocratic gradient ) at a flow rate of 0.3 ml / min . the autosampler tray was cooled to 45c and the injection volume was 5 l . a triple quadrupole mass spectrometer api4000 ( applied biosystems / mds sciex , concord , canada ) coupled with an electrospray ionization source was operated in the positive ion mode . the optimized ion source conditions were as follows : collision gas , 6 psi ; curtain gas , 40 psi ; ion source gas 1 , 50 psi ; ion source gas 2 , 80 psi ; ion source voltage , 5500 v ; ion source temperature , 600c . nitrogen was used as the collision gas in the multiple reaction monitoring ( mrm ) mode . the conditions of declustering potential , collision energy , and collision cell exit potential were optimized by every steroid . the transitions in mrm were as follows : preg m / z 317 299 , hpreg m / z 315 297 , dhea m / z 289 271 , prog m / z 315 109 , hprog m / z 331 109 , dione m / z 287 97 , dcort m / z 331 123 , dcortico m / z 347 161 , cortico m / z 347 100 , cort m / z 363 309 , aldo m / z 361 343 , and testo m / z 289 109 . mass spectroscopic data were acquired and quantified using the analyst 1.4.2 software package ( applied biosystems / mds sciex , concord , canada ) . cell volume was estimated from the number of cells in the well and the average diameter of the cells . cells were detached from the well using 0.025% trypsin ( mp biomedicals , inc . , irvine , ca ) in a 0.02% edta solution ( dojindo laboratories , kumamoto , japan ) at the start of preculture , start of stimulation , and at 24 , 48 , and 72 h after stimulation . the numbers and diameters of the cells were measured by a cell counter vi - cell xr 2.01 ( beckman coulter , krefeld , germany ) after trypan blue staining . parameters of the cell volume and number of cells were estimated to fit experimental time - course data using exponential curves . nci - h295r cells were exposed to seven well - characterized inhibitors of steroidogenesis , and then the concentrations of the steroids in the culture medium were measured to estimate the enzyme inhibition to evaluate the performance of the simulation model . the adrenal steroidogenic inhibitors included aminoglutethimide ( agt , bachem ag , bubendorf , switzerland ) , o , p-ddd ( ddd , sigma - aldrich inc . , st . louis , mo ) , spironolactone ( sp , sigma - aldrich inc . , st . louis , mo ) , metyrapone ( mp , sigma - aldrich inc . , st . louis , mo ) , ketoconazole ( kc , wako pure chemical industries , ltd . , osaka , japan ) , miconazole ( mc , wako pure chemical industries , ltd . , osaka , japan ) , and daidzein ( dz , sigma - aldrich inc . , st . the cells were also exposed to four adrenal toxicants whose adrenal toxicity is not mediated through steroidogenesis inhibition . the toxicants were acrylonitrile ( an , wako pure chemical industries , ltd . , osaka , japan ) , salinomycin ( sm , sigma - aldrich , inc . , st . louis , mo ) , thioguanine ( tg , tokyo chemical industry co. , ltd . , tokyo , japan ) , and fumaronitrile ( fn , wako pure chemical industries , ltd . , osaka , japan ) and added to the culture medium at 1 : 1000 dilutions . nci - h295r cells were cultured for 3 days in 6-well plates and then stimulated with the above - mentioned compounds . upon the start of stimulation , various concentrations of test chemicals were added to the cultures . after a further 3 days of culture with the chemicals , the concentrations of 12 steroids ( preg , hpreg , dhea , prog , hprog , dione , dcortico , dcort , cortico , cort , aldo , and testo ) in the culture medium were measured by lc / ms / ms . the cytotoxicity assay was conducted in 96-well plates using atp content in cells as an endpoint ( celltiter - glo luminescent cell viability assay , promega ) . the test concentrations of adrenal steroidogenesis inhibitors and other compounds are shown in table 1 . comparisons were performed by the two - sample welch 's t - test with bonferroni multiple testing correction for each steroid hormone species . steroid hormones secreted from human adrenal corticocarcinoma nci - h295r cells are synthesized from cholesterol through the c21-steroid hormone biosynthesis pathway . a mathematical model of adrenal steroidogenesis in nci - h295r cells was constructed with cholesterol transport and the intracellular localization pathway , the oxysterol synthesis pathway as a bypass of steroidogenesis , the c21-steroid hormone biosynthesis pathway as the main steroidogenesis pathway , passive transport of steroid hormones , and cell proliferation ( figure 1 ) . in this model , two compartments , the intracellular space and culture medium , were incorporated as the available region . equations and parameters of the cell proliferation and diffusional transport of steroid hormones have been proposed by previous studies [ 21 , 22 ] . cholesterol transport and the intracellular localization pathway including the oxysterol bypass were integrated using a part of the acth - stimulated cortisol secretion model described by dempsher and colleagues . the c21-steroid hormone biosynthesis pathway includes 14 steroid hormones , preg , hpreg , dhea , prog , hprog , dione , testo , dcortico , dcort , cortico , cort , aldo , e1 , and e2 , and 17 enzymatic reactions catalyzed by nine steroidogenic enzymes , cholesterol side chain cleavage enzyme ( cyp11a1 ) , 17-hydroxylase ( cyp17h ) , c17,20-lyase ( cyp17l ) , 3-hydroxysteroid dehydrogenase ( hsd3b2 ) , 21-hydroxylase ( cyp21a2 ) , 11-hydroxylase ( cyp11b1 ) , 18-hydroxylase ( cyp11b2 ) , 17-hydroxysteroid dehydrogenase ( hsd17b3 ) , and aromatase ( cyp19a1 ) . in this mathematical model of adrenal steroidogenesis in nci - h295r cells , the flux velocities of molecular transportation and enzymatic reaction rates of steroidogenic enzymes were defined based on the first - order reaction and rapid - equilibrium enzyme kinetics , respectively . all equations in the mathematical model of adrenal steroidogenesis of nci - h295r cells were described in a supplementary document ( see supplementary material available online at http://dx.doi.org/10.1155/2016/4041827 ) . the rate constants and the maximum activities were estimated by fitting to experimental time - course data of the concentrations of cholesterol and all steroids . initial values of cholesterol and the 14 steroid concentrations were used in each experimentally measured value , and every steroid concentration was assumed to rapidly reach the equilibrium state between the culture medium and intracellular space . all fixed values of static parameters and initial values of variable parameters in this model were described in tables s1 and s2 in a supplementary document , respectively . this computational model of adrenal steroidogenesis in nci - h295r cells was developed on the simbio platform which is a general environment of biological dynamic simulation and computational model development . odes were solved by the fourth - order runge - kutta method with a variable time step . the time step ( dt ) was adjusted to refer to the maximum absolute value of flux velocities or enzymatic reaction rates at each time point , and the range of the time step was from 1 10 to 10 . to confirm whether the range of the time step was suitable , the numerical error ratio was calculated by certain fixed time steps in the range of the time step , which was under 1 10 in every time step . the duration time of computational simulation of adrenal steroidogenesis in nci - h295r cells was set at 72 h. to reconstruct experimental time - course patterns of the concentrations of cholesterol and the 14 steroids in the culture medium and intracellular space , we optimized every rate constant and maximum velocity of the steroidogenic enzymes . this parameter optimization problem was solved by the levenberg - marquardt method which is one of the nonlinear least squares methods [ 4951 ] . the objective function of optimization was used as the following normalized least squares distance ( nlsd):(1)nlsd=h i jxh , i , jexpxh , i , jsim2xh , imax2,where h is the compartment ( culture medium or intracellular space ) , i is the molecular species ( cholesterol and the 14 steroids ) , j is the time point ( 0 , 8 , 24 , 48 , and 72 h ) , xh , i , j is the experimentally measured concentration of molecule i in compartment h at time point j , xh , i , j is the simulated concentration of molecule i in compartment h at time point j , and xh , i is the maximum concentration of molecule i in compartment h over all time points . data points under the lower quantitation limit were excluded from the evaluation by the objective function . effects of every static model parameter for parameter optimization were calculated from differences of fitting the objective function using sensitivity analysis . metabolic steroid profiling and differential patterns of the adrenal steroid hormones by chemical perturbation were reconstructed to optimize the relative activities of the steroidogenic enzymes . the input data for the quantitative mechanistic analysis of adrenal toxic compounds was a fold change ( ratio ) of the measured 12 steroid concentrations induced by drug exposure for 72 h. the two - step optimization method of the real - coded genetic algorithm ( rcga ) was adopted as a global optimization method in the quantitative mechanistic analysis of adrenal toxic compounds . the operations of the crossover and generation alteration model in rcga were used for the real - coded ensemble crossover ( rex ) and just generation gap ( jgg ) [ 5255 ] . as the initial parameters of rcga , maximum generation , population size , selection size of parent individuals , population size of child individuals , and termination criteria were 1000 , 100 , 6 , 25 , and under 0.1 of nlsd , respectively . the search space for the relative activities of the steroidogenic enzymes was from 1/100 to 100 . to evaluate the fitness of each individual , the sum of squared residuals for fold changes of measured 12 steroid concentrations was used as the objective function . nonlinear least squares optimization by the levenberg - marquardt method was used as a local search [ 4951 ] . as the estimated mechanisms of actions of the adrenal toxic compounds , the relative activities of eight steroidogenic enzymes ( cyp11a1 , cyp17h , cyp17l , hsd3b2 , cyp21a2 , cyp11b1 , cyp11b2 , and hsd17b3 ) were optimized by the above - mentioned 2-step optimization method . the property of every kinetic parameter in this computational model of steroidogenesis in nci - h295r cells was evaluated by dynamic sensitivity analysis . the sensitivity ( sx , y ) of kinetic parameter x for variable parameter y was defined by the following equation:(2)sx , yt=yt / ytx / x , where variable parameter y was the concentration of a steroid hormone in the cytosolic space of nci - h295r cells . the perturbation for kinetic parameters was + 10% ( x / x = 0.1 ) . all steroid hormones in the culture medium were significantly increased after 72 h of stimulation with 50 nm acth , 20 m forskolin , and 100 nm angiotensin ii ( figure 2(a ) ) . mass balances in steroidogenesis of nci - h295r cells under nontreatment and control ( stimulated ) conditions are shown in figures 2(b ) and 2(c ) , respectively . under stimulation , the dynamics of net mass in these experiments were unchanged , and accumulated cholesterol was converted to adrenal steroids . viabilities of cells treated with each compound were expressed as a relative value to the atp level of the control . effects of an , sm tg , fn , agt , ddd , sp , mp , kc , mc , and dz on cell viability were determined to be valid under 80% of the relative atp level at 7 days after treatment . an , sm , tg , and fn showed cytotoxicity at over 100 , 1 , 10 , and 10 m , respectively . agt , mp , and dz did not affect cell viability at up to 100 m . ddd , sp , kc , and mc induced less than 80% of cell viability at over 100 , 50 , 100 , and 25 m , respectively . after nci - h295r cells were exposed to each test compound during three days , the concentrations of 12 steroid hormones in the culture medium were simultaneously measured by lc / ms / ms . all effects of the compounds on adrenal steroidogenesis were evaluated at the concentration without any overt cytotoxicity . the differential metabolic steroid profiles of 11 adrenal toxic compounds were classified and visualized by using hierarchical clustering analysis ( figure 3 ) . four adrenal toxicants without steroidogenic inhibition , an , sm , tg , and fn , did not change the medium concentrations of all steroid hormones by more than 2-fold . above - mentioned 4 compounds at every condition and 7 adrenal steroidogenic inhibitors at the low exposure concentration were gathered into a big cluster as nonchange group . the 7 steroidogenic inhibitors at the maximum exposure concentration showed the characteristic steroid profiles each , but 100 m dz and 10 m sp were classified as a cluster . agt drastically decreased the medium concentrations of preg , hpreg , dhea , prog , dcortico , cortico , and aldo at 100 m . ddd dose - dependently decreased the medium concentrations of prog , dcortico , cortico , cort , and aldo at > 10 m and decreased preg , hpreg , dhea , prog , hprog , dione , and dcort at the maximum exposure concentration of 25 m . sp increased preg , hpreg , and dhea and decreased prog , dione , dcortico , dcort , cortico , aldo , and testo at 10 m . mp dose - dependently decreased cortico , cort , and aldo and decreased dhea , hprog , dione , and testo at the maximum exposure concentration of 100 m . kc drastically decreased the medium concentrations of preg , hpreg , dhea , hprog , dione , dcortico , dcort , cortico , corto , aldo , and testo at 10 m . mc increased the medium concentrations of prog and decreased dione , dcort , cort , and testo at 10 m . dz increased preg , hpreg , and dhea and decreased dione , dcortico , dcort , cortico , cort , aldo , and testo at 100 m . the mathematical model of adrenal steroidogenesis in nci - h295r cells was optimized for several kinetic parameters of cholesterol transport , intracellular localization , the oxysterol pathway , and maximum velocity of steroidogenic enzymes to fit the experimental time - course data . the optimized mathematical model was reconstructed with the experimental dynamic patterns of cholesterol and the 14 steroid hormones in the intracellular space and culture medium . optimized kinetic parameters were calculated sensitivities for the nlsd value as the fitting score and are shown in table s1 in a supplementary document . the highly sensitive parameters for fitting the nlsd score were the extracted nine kinetic parameters , kcholesterol transport , kf , kf , kb , vmax , km , vmaxa , vmaxa , and vmaxa , which had higher than 3.0 fitting sensitivity . effects of adrenal toxic compounds on steroidogenic enzymes were quantitatively predicted from the change in the ratio of the measured medium concentrations of the 12 steroid hormones at 72 h after drug exposure using the mathematical model of adrenal steroidogenesis in nci - h295r cells . the estimated effects of 11 adrenal toxic compounds on eight steroidogenic enzymes are shown in figure 5 . the adrenal toxic compounds without steroidogenic inhibition , such as vasculotoxic agents ( an , sm , tg , and fn ) , were not estimated for the target steroidogenic enzymes under noncytotoxic conditions . every fitness values were under 0.05 of nlsd values used as the fitting objective function ( figures 5(a)5(d ) ) . other steroidogenic inhibitors ( agt , ddd , sp , mp , kc , mc , and dz ) are described in detail below . the mechanism of action of agt in adrenal steroidogenesis was estimated by inhibition of cyp11a1 , hsd3b2 , cyp21a2 , and cyp11b1 at 100 m ( estimated inhibitions were 77.0% , 78.0% , 81.1% , and 59.8% , resp . ) ( figure 5(e ) ) . agt has been reported to inhibit cyp11a1 , cyp21a2 , cyp11b1 , and cyp11b2 [ 6 , 2730 ] . in particular , cyp11a1 appeared to be inhibited strongly by agt . in our study , hsd3b2 inhibition of agt was shown by mechanistic analysis based on systems biology approaches as a novel mechanism of action of agt . however , the concentration of aldo in the culture medium decreased to 3.8% of the normal stimulated condition . inhibition of agt by cyp11b2 has been shown using sheep adrenal homogenates as well as a human adrenal homogenate from a patient with cushing 's syndrome . the activity of 18-hydroxylase induced by cyp11b2 was determined as the conversion of corticosterone to 18-hydroxycorticosterone in the previous study . the cause of the discrepancy regarding the effect of agt on cyp11b2 was suggested to be substrate inhibition , because the intracellular concentration of cortico was increased by over 10 times of that in the culture medium to reach 50 m . another possibility was poor quantitative reliability of the experimental data , because the aldo concentration was under the lower limit of quantification at 100 m agt . hecker and colleagues reported that 3 m agt decreases preg and prog concentrations and increases the testo concentration . one possibility is that the concentration of testo was already enhanced by about 3.3-fold through stimulation with acth , forskolin , and angiotensin ii . the mechanism of action of ddd in adrenal steroidogenesis was estimated by dose - dependent inhibition of cyp11a1 , hsd3b2 , cyp21a2 , and cyp11b1 ( estimated inhibitions at 25 m were 87.0% , 86.9% , 76.9% , and 84.9% , resp . ) ddd has been reported to inhibit cyp11a1 , hsd3b2 , cyp21a2 , cyp11b1 , and cyp11b2 [ 29 , 31 , 32 ] . inhibition of ddd by cyp11b2 was not estimated in our study . however , the concentration of aldo in the culture medium decreased to 3% of that in the normal stimulated condition . inhibition of ddd by cyp11b2 has been shown using mitochondrial and microsomal fractions prepared by standard centrifugation procedures from a bovine adrenal cortex homogenate . the cause of the discrepancy regarding the inhibition of ddd by cyp11b2 could not be explained by same effect in the case of agt . the mechanism of action of sp in adrenal steroidogenesis was estimated by inhibition of hsd3b2 , cyp21a2 , and hsd17b3 ( estimated inhibitions at 10 m were 70.2% , 59.5% , and 59.3% , resp . ) sp has been reported to inhibit cyp17h , cyp17l , cyp11b1 , and cyp11b2 [ 6 , 3335 ] . the inhibitory effect of sp on the hsd3b2 enzyme was a novel mechanism of action . sp has also been reported to exert some off - target effects by binding to androgen , glucocorticoid , and progesterone receptors [ 5658 ] . sp has been shown to inhibit the production of aldo and cort from preg induced by angiotensin ii in h295r cells , but the specific mr antagonist eplerenone did not show the inhibitory effects . therefore , hsd3b2 inhibition by sp is not mediated via mr , and the action might be direct inhibition of hsd3b2 enzymes or a part of known off - target effects mediated through other nuclear hormone receptors . regarding cyp17h and cyp17l , our results were consistent with previous reports [ 33 , 34 ] . the fact that there were no inhibitions of cyp17h or cyp17l in our study suggests that sp might not be deacetylated to 7-thiospironolactone in nci - h295r cells . it has been shown that 30 m sp inhibits cyp11b1 and cyp11b2 in human and bovine adrenal mitochondria . the cause of cyp11b1 and cyp11b2 inhibition by sp could not be determined in our study , which might be due to the lower maximum exposure concentration of sp than that in the previous report . we could not examine sp concentrations over 10 m because these concentrations were cytotoxic in nci - h295r cells . the mechanism of action of mp in adrenal steroidogenesis was estimated by dose - dependent inhibition of cyp11b1 ( estimated inhibitions at 1 , 10 , and 100 m were 57.1% , 82.7% , and 98.2% , resp . ) mp has been reported to inhibit cyp11b1 as its major effect and cyp11a1 and cyp11b2 as a weak effect [ 6 , 29 , 3639 ] . the results were able to show that mp is a selective inhibitor of cyp11b1 in the previous report . however , the estimated effect of mp at a high concentration , 100 m as the maximum exposure concentration , was unclear . according to the previous report , selectivity of mp for cyp11b1/cyp11b2 is about five times . in addition , 20 m mp has a slight inhibition effect on cyp11a1 in h295r cells . the mechanism of action of kc in adrenal steroidogenesis was estimated by inhibition of cyp11a1 , cyp17h , cyp17l , hsd3b2 , cyp21a2 , cyp11b1 , and cyp11b2 ( estimated inhibitions at 10 m were 92.6% , 94.3% , 51.8% , 83.0% , 88.2% , 97.4% , and 79.8% , resp . ) kc has been reported to inhibit cyp11a1 , cyp17h , cyp17l , hsd3b2 , cyp21a2 , and cyp11b1 [ 6 , 29 , 4043 ] . kc inhibits cyp11a1 , cyp17h , cyp21a2 , and cyp11b1 in nci - h295r cells at 10 m and cyp17h , cyp17l , cyp21a2 , and cyp11b1 in human adrenal mitochondria and leydig cell microsomes at 25 m [ 60 , 61 ] . however , kc has shown only weak inhibition of hsd3b2 and hsd17b3 in leydig cells at the millimolar level [ 60 , 61 ] . regarding cyp11b2 and hsd17b3 , we considered that these estimated inhibitions of kc did have sufficient reliability in terms of quantitative prediction precision , because aldo and testo concentrations were less than the lower limit of quantification at 10 m kc . the mechanism of action of mc in adrenal steroidogenesis was estimated by inhibition of cyp17h , cyp17l , cyp11b1 , and hsd17b3 ( estimated inhibitions at 10 m were 69.1% , 53.0% , 76.4% , and 57.1% , resp . ) ( figure 5(j ) ) . mc has been reported to inhibit not only cyp17h and cyp17l but also cyp11a1 , cyp21a2 , and cyp11b1 [ 41 , 44 , 45 ] . the results in the previous reports were able to estimate that mc is a cyp17 inhibitor . however , cyp11a1 inhibition by mc , probably instead of a reduction in star expression , was not clearly detected in our study using nci - h295r cells , because there were no decreases in the concentrations of preg and prog in the culture medium . indirect inhibition of cyp11a1 via the peripheral - type benzodiazepine receptor has been reported in mouse adrenocortical y-1 cells treated with mc in the absence of stimuli by measuring preg production . on the other hand , reductions of star protein expression and/or transport activity without affecting total steroid synthesis or cyp11a1 and hsd3b2 enzyme expression or activities have been reported in ( bu)2camp - stimulated ma-10 leydig tumor cells treated with mc by measuring prog production . therefore , the effect of mc on the initial reaction in adrenal steroidogenesis from cholesterol should be different according to the cell type and stimulation condition . inhibition of cyp21a2 and cyp11b1 by mc has been reported as decreases in the consumption of prog and dcortico , respectively . inhibition of cyp11b1 was estimated by the action of mc in this study , but that of cyp21a2 was not detected . in the previous experimental report , inhibitory sites by mc might have been reflected by inhibition of cyp17h activity , because cyp21a2 activity was measured as a decrease in labeled prog . the mechanism of action of dz in adrenal steroidogenesis was estimated by inhibition of cyp11a1 , hsd3b2 , cyp21a2 , cyp11b1 , and hsd17b3 ( estimated inhibitions at 100 m were 58.6% , 94.1% , 96.5% , 87.2% , and 98.1% , resp . ) ( figure 5(k ) ) . these estimated effects of dz on cyp11b1 and hsd17b3 were unclear , because the concentrations of aldo and testo were less than the lower limit of quantification at 100 m . in addition , these enzymes act downstream of the strong action points of dz , such as hsd3b2 and cyp21a2 . to comprehensively understand the dynamics of adrenal steroidogenesis , dynamic sensitivities were calculated for steroid concentrations secreted by nci - h295r cells using our constructed mathematical model of steroidogenesis . the results of dynamic sensitivity analysis at 72 h of duration and 6 h of interval time are presented as a heat - map in figure 6 . the top 10 parameters of the total area under the curve of dynamic sensitivity for cholesterol and the 14 steroids in culture medium were vmaxa , vmax , vmaxa , kf , kb , km , vmaxa , kcholesterol transport , kf , and vmaxb in order from the top . cholesterol uptake ( kcholesterol transport ) , star protein ( kf ) , and cyp11a1 ( vmax ) , which are determining factors of the capacity for steroidogenesis , promoted the production of mineralocorticoids ( dcortico , cortico , and aldo ) and restrained the synthesis of glucocorticoids ( dcort and cort ) and sex steroids ( dione , testo , and e1 ) because of the accumulation of intermediate molecules in steroidogenesis ( preg , hpreg , dhea , prog , and hprog ) only by self - activation . the dynamic patterns of the intermediate molecules in steroidogenesis were mainly dependent on the activity of cyp17h and hsd3b2 with preg as the substrate of these enzymes , in which the dynamic sensitivities of vmaxa for hpreg , and hprog and vmaxa for prog , hprog , and dcortico reversed the direction of sensitivity at 4966 h after stimulation . the dynamic sensitivities of the maximum activities of hsd3b2 for preg ( vmaxa ) and cyp21a2 for prog ( vmaxa ) were related to all steroids at 72 h. almost all model parameters had positive sensitivity for downstream steroids in the adrenal steroidogenic pathway and negative sensitivity for direct - binding steroids as substrates of the steroidogenic enzyme . the sensitivity of vmax in all steroidogenic enzymes was relatively higher than km for the same steroid substrate . to clearly show the property of the metabolic shift between mineralocorticoid and glucocorticoid biosynthesis , we performed two - dimensional parameter scanning of the enzymatic activities of cyp17h and hsd3b2 ( figure 7 ) . nci - h295r cells lost the ability to produce all steroid hormones when enzymatic activities of cyp17h and hsd3b2 were changed by over 60% and 30% , respectively . activation of cyp17h and/or hsd3b2 induced the metabolic shift that enhanced the glucocorticoid biosynthesis and deviated from the mineralocorticoid biosynthesis . on the other hand , inhibition of cyp17h and/or hsd3b2 induced the metabolic shift that enhanced the mineralocorticoid biosynthesis and deviated from the glucocorticoid biosynthesis . moreover , the enzymatic activity of hsd3b2 regulated the metabolic balance of sex steroids and the precursors on adrenal steroidogenesis of nci - h295r cells . e1 , testo , and dione were produced by nci - h295r cells when activating the enzymatic activity of hsd3b2 . conversely , e2 and dhea were produced by nci - h295r cells when suppressing the enzymatic activity of hsd3b2 . the biosynthesis of downstream steroids in adrenal steroidogenesis pathway , such as mineralocorticoids and glucocorticoids , was almost completely terminated when the enzymatic activity of hsd3b2 was decreased by over 80% . our systematic analysis using the mathematical model of adrenal steroidogenesis in nci - h295r cells revealed that the enzymatic activity of 3-hsd controls the dynamics of adrenal steroidogenesis . the activity of the star protein controls the net capacity of steroidogenesis in steroidogenic cells , which is the transport of cholesterol from the outer to inner mitochondrial membranes . both the expression levels of star protein and mrna are rapidly elevated in response to stimulation by tropic hormones such as acth [ 62 , 63 ] . another important factor in adrenal steroidogenesisis is the cholesterol side chain cleavage enzyme cyp11a1 , the first rate - limiting and hormonally regulated step in the synthesis of all steroid hormones , which is conversion of cholesterol to pregnenolone in mitochondria . according to our results of global sensitivity analysis ( supplementary table 1 and figure 6(d ) ) , in addition to cyp11a1 and star proteins , 3-hsd was one of the key regulators in adrenal steroidogenesis of nci - h295r cells . and also , this result suggests that a significant regulatory mechanism in steroidogenesis pathway is very reasonable . star , cyp11a1 , and 3-hsd ( isoforms 1 or 2 in humans ) proteins generally respond to the same hormones that stimulate steroid production through common pathways such as camp signaling in adrenal glands and testes [ 65 , 66 ] . moreover , our data also support recent experimental evidence from clinical and in vivo studies , suggesting that the enzymatic activity of 3-hsd plays an important role in the regulation of mineralocorticoid synthesis in adrenal steroidogenesis and contributes to hypertension caused by abnormal overproduction of aldosterone [ 6770 ] . circadian clock - deficient cry - null mice show salt - sensitive hypertension due to abnormally high synthesis of aldosterone , which is caused by constitutively high expression of hsd3b6 mrna and protein in the adrenal cortex [ 67 , 68 ] . recent clinical observations have revealed predominant expression of hsd3b2 mrna and protein in tumor cells of aldosterone - producing adenoma ( apa ) , and hsd3b1 mrna significantly correlated with cyp11b2 mrna levels and plasma aldosterone concentrations in apa patients [ 69 , 70 ] . however , the relationship is unclear and disputed in a small - scale clinical study indicating that genetic variation in hsd3b1 affects blood pressure and hypertension in apa patients . the results of our simulation study suggest that 3-hsd protein ( human genes are hsd3b1 and hsd3b2 ) is one of the determination factors for the dynamic property of adrenal steroidogenesis . our results support the clinical evidence of doi and colleagues , and we believe that the hsd3b1 enzyme has a promising potential as novel drug target for endocrine hypertension . the metabolic properties of adrenal steroidogenesis in nci - h295r cells were revealed by dynamic sensitivity analysis using the mathematical model ( figure 6 ) . mineralocorticoids , such as dcortico , cortico , and aldo , and intermediate steroids upstream of the adrenal steroidogenesis pathway , such as preg , hpreg , dhea , prog , and hprog , were accelerated by reactions of cholesterol import ( kcholesterol transport ) , star protein ( kf and kf ) , and cyp11a1 ( vmax ) . on the other hand , glucocorticoids , such as dcort and cort , and sex hormones , such as dione , testo , and e1 , were suppressed by these model parameters . therefore , enhancement of the net adrenal steroidogenesis capacity , which supplies preg precursor to the pathway , causes a production shift from glucocorticoids to mineralocorticoids by substrate inhibitions of cyp17h , hsd3b2 , and cyp21a2 caused by accumulation of initial intermediate steroids such as preg and prog . sensitivities of cyp17h ( vmaxa ) and hsd3b2 ( vmaxa ) for the products were dynamically changed and these parameters determined the metabolic balance of downstream steroids in the adrenal steroidogenesis pathway . according to these results of dynamic sensitivity analysis of star , cyp11a1 , cyp17h , and hsd3b2 , we suggest that the enhancement of cyp17h and hsd3b2 activity during acth stimulation was important to shift the steroidogenic output away from aldo biosynthesis towards cort biosynthesis , as well as adrenal androgen production . this suggestion partially supports a comparative animal study in which molecular and cellular variations in cyp17h activity dramatically affect acute cortisol production , resulting in distinct physiological and behavioral responses . results of two - dimensional parameter scanning of the enzymatic activities of cyp17h and hsd3b2 quantitatively showed the detail of the metabolic relationship between mineralocorticoid and glucocorticoid biosynthesis ( figure 7 ) . particularly , the results showed that the balance of these enzymatic activities was very important for the typical function of nci - h295r cells , namely , the ability to produce all steroid hormones . nci - h295r cells lost this function when enzymatic activities of cyp17h and hsd3b2 were changed by over 60% and 30% , respectively . in addition , they became mineralocorticoid ( aldo ) secreting cells when the enzymatic activity of cyp17h or hsd3b2 was inhibited by over 50% or glucocorticoid ( dcort and cort ) secreting cells when these enzymes were activated by over 50% . in particular , this analysis also showed that hsd3b2 was a key player in the adrenal steroidogenesis of nci - h295r cells , because hsd3b2 inhibition by over 80% almost completely inhibited the biosynthesis of downstream steroids . the ratio of cyp17a1 to hsd3b2 mrna expression levels has been related to several endocrine diseases with a low level in apas and high level in cortisol - producing adenomas . furthermore , the expression levels or enzymatic activities of cyp17a1 and hsd3b1 have been related to androgen production in polycystic ovary syndrome [ 75 , 76 ] . these clinical studies support our simulation results indicating that the balance of enzymatic activity of cyp17h and hsd3b2 determines the shift in steroidogenic output to mineralocorticoids , glucocorticoids , or androgens . according to our results obtained using the mathematical model of steroidogenesis in nci - h295r cells , such as sensitivity analysis , comprehensive analysis based on systems biology is available to quantitatively estimate the mechanism of action of steroidogenic disrupting compounds from differential profiling of adrenal steroid hormones , because dynamic patterns of steroid hormones in adrenal steroidogenesis pathway are highly complex . our proposed method of quantitative mechanistic analysis of steroidogenic inhibitors was able to predict known action sites in the adrenal steroidogenesis pathway at only one time point ( 72 h after drug exposure ) . moreover , according to the results of sensitivity analysis ( figure 6 ) , vmax of all steroidogenic enzymes was more sensitive than the km , because the intracellular concentrations of steroid hormones were almost maintained at sufficiently high levels compared with km values of steroidogenic enzymes . these results suggested that estimation of the mechanism of action of drugs is more effective and detectable when using the influences of vmax as the searching parameters such as our proposed method . our data showed that the proposed method based on a systems biology model is a very powerful tool for exploratory screening of steroidogenic disrupting compounds . rcga as a solver of parameter estimation problems in systems biology has been applied to biological network identification of gene regulatory networks and metabolic pathways and optimization of biological processes using experimentally observed time - course data [ 7782 ] . in this study , rcga was useful to estimate the mechanism of action of novel pharmaceutical drug candidates for adrenal steroidogenesis as a new application of rcga in systems biology . we had two issues when applying rcga to the quantitative mechanistic analysis of drug actions . these issues were the vast calculation cost and multimodality of quasi - optimum solutions in solving the optimization problem , because the mathematical model in systems biology consists of many equations and parameters . a proposed optimization strategy using rcga based on rex / jgg was a highly stable and efficient calculation method for a better quasi - optimum solution than the unimodal normal distribution crossover ( undx)/minimum generation gap ( mgg ) method that is well applied in the engineering field . in addition , we expanded the rcga optimization program based on rex / jgg to a hybrid method of ga and then applied a local search as recommended by harada and kobayashi [ 28 , 83 ] . a final optimal solution was obtained with a good convergence property . because these problems are general in systems biology studies , we suggest that the proposed hybrid method based on rex / jgg is a very useful tool for quantitative mechanistic analysis of novel pharmaceutical drugs , not limited to steroidogenic disrupting compounds . the novel mathematical model of adrenal steroidogenesis was constructed in this study , including cholesterol transport and distribution , the c21-steroid hormone pathway , steroid transport , and cell proliferation , which could reproduce adrenal steroidogenesis in nci - h295r cells . according to the results of dynamic sensitivity analysis using the new model , hsd3b2 plays the most important role in the metabolic balance of adrenal steroidogenesis in nci - h295r cells . moreover , to quantitatively estimate mechanisms of action of adrenal toxic compounds , we analyzed differential metabolic profiles of 12 steroid hormones at 3 days after exposure to 11 adrenal toxic compounds , by using the new mathematical model and a hybrid optimization method of the rcga and a local search ( nonlinear least squares ) . we could estimate which steroidogenic enzymes were affected by these compounds using the hybrid optimization method . vasculotoxic agents were estimated to have no effect according to the results obtained by our method . in terms of adrenal steroidogenic inhibitors , the predicted action sites were approximately matched to the target enzymes as reported in the literature . thus , our computer - aided method based on a systems biology approach may be useful to analyze the mechanism of action of endocrine - disrupting compounds and to assess the human adrenal toxicity of novel pharmaceutical drugs based on steroid hormone profiling .

adrenal toxicity is one of the major concerns in drug development . to quantitatively understand the effect of endocrine - active compounds on adrenal steroidogenesis and to assess the human adrenal toxicity of novel pharmaceutical drugs , we developed a mathematical model of steroidogenesis in human adrenocortical carcinoma nci - h295r cells . the model includes cellular proliferation , intracellular cholesterol translocation , diffusional transport of steroids , and metabolic pathways of adrenal steroidogenesis , which serially involve steroidogenic proteins and enzymes such as star , cyp11a1 , cyp17a1 , hsd3b2 , cyp21a2 , cyp11b1 , cyp11b2 , hsd17b3 , and cyp19a1 . it was reconstructed in an experimental dynamics of cholesterol and 14 steroids from an in vitro steroidogenesis assay using nci - h295r cells . results of dynamic sensitivity analysis suggested that hsd3b2 plays the most important role in the metabolic balance of adrenal steroidogenesis . based on differential metabolic profiling of 12 steroid hormones and 11 adrenal toxic compounds , we could estimate which steroidogenic enzymes were affected in this mathematical model . in terms of adrenal steroidogenic inhibitors , the predicted action sites were approximately matched to reported target enzymes . thus , our computer - aided system based on systems biological approach may be useful to understand the mechanism of action of endocrine - active compounds and to assess the human adrenal toxicity of novel pharmaceutical drugs .

bladder cancer is the second most common urological malignancy in humans , and it was estimated in 2012 in the united states that 73,510 new cases of cancer of the urinary bladder were diagnosed along with 14,680 deaths . in korea also , bladder cancer is the second most common genitourinary tumor and is about five times as common in men as in women . bladder cancer is classified into two large groups of non - muscle - invasive bladder cancer ( nmibc ) and muscle - invasive bladder cancer ( mibc ) according to pathology and clinical features . although only 20% of bladder cancer is confirmed as mibc at first diagnosis , mibc accounts for the majority of cancer - specific deaths . more than three - quarters of all bladder cancer cases are nmibc that can be treated by transurethral resection ( tur ) . unfortunately , about 70% of patients with nmibc who have undergone tur experience a tumor recurrence within 2 years . furthermore , 20% to 30% of patients who have had a complete tur and intravesical therapy performed with bacillus calmette - gurin ( bcg ) have experienced progression to mibc . thus , many diagnostic tools and biomarkers have been proposed and developed to predict the recurrence and progression of nmibc [ 8 - 10 ] , but most of these have proven to be inadequate in terms of efficacy and accuracy because of the heterogeneous behavior of bladder cancer . micrornas ( mirnas ) are small , noncoding rnas that are 18 to 22 nucleotides in length in their mature form . micrornas act as posttranscriptional gene modulators and play an important role in cell proliferation , differentiation , survival , programmed cell death , and oncogenesis of cells and organisms , usually by inhibiting translation . micrornas are aberrantly expressed in human cancer and may function as a novel class of oncogenes or tumor suppressor genes . actually , numerous studies have detected mirna deregulation in human malignancies , including chronic lymphocytic leukemia , breast cancer , primary glioblastoma , lung cancer , thyroid cancer , colon cancer , and pancreatic cancer . also , when histologically matched with normal urothelium , various types of mi - rnas are aberrantly expressed in bladder cancer . accordingly , it has been suggested that mirnas play roles as oncogenes or tumor suppressors in the tumorigenesis and progression of bladder cancer . however , although many mirnas , their targets , and their mechanism of action as well as their clinical value have recently been described , few studies have focused on cell - free mirnas isolated from urine , particularly in bladder cancer . in the present study , we measured the expression levels of urinary cell - free mir-214 in nmimc patients and then investigated whether urinary cell - free mir-214 could be a prognostic biomarker for nmibc . a total of 138 patients with primary urothelial carcinoma of the urinary bladder and 144 healthy controls were enrolled in the study . controls were selected to match the age and gender proportions of the bladder cancer patients , and subjects were screened to ensure that they were within the normal range of all laboratory results with no history of malignant tumors . urine samples were collected in the morning and stored at 4 , then centrifuged at 25,000 rpm for 15 minutes . each supernatant and sediment was portioned into eppendorf tubes and stored at -20 until use . all primary tumor samples were obtained from patients who underwent tur and were histologically verified to have urothelial carcinoma . to reduce confounding factors affecting the analyses , and to delineate a more homogeneous study population , any patients diagnosed with a concomitant carcinoma in situ or for whom data collection was incomplete were excluded . the collection and analysis of all samples was approved by the institutional review board of our institute , and written informed consent was obtained from each subject ( irb approval number 2006 - 01 - 001 ) . patients who had a t1 tumor , multiple tumors , large tumors ( > 3 cm in diameter ) , or high - grade ta nmibc received one cycle of intravesical treatment ( bcg or mitomycin - c ) . patients who were free of disease within 3 months after treatment were assessed every 3 months for the first 2 years , and then every 6 months thereafter . patients who refused or did not complete an imaging work - up such as a computed tomographic scan or an magnetic resonance imaging at least once every 3 months to evaluate their response tumors were staged and graded according to the 2002 tnm classification and the 1973 world health organization grading system , respectively . recurrence was defined as a recurrence of primary nmibc at either a lower or the same pathological stage , and progression was defined as disease with a higher tnm stage upon relapse of nmibc . a genolution urine mirna purification kit ( genolution pharmaceuticals inc . , seoul , korea ) urine sample supernatant ( 500 l ) was added to each tube containing genolution proprietary mirna separation solution , which was then vortexed for 20 seconds . next , 200 l of chloroform was added and the samples were vortexed for 10 seconds , after which they were centrifuged at 13,000 rpm for 10 minutes at 4. a 600-l fraction from the top aqueous phase was taken and transferred into a new 1.5 ml tube , and 0.8 ml of isopropanol was added , followed by centrifugation for 5 minutes at 13,000 rpm and 4. after removing the aqueous solution , 1 ml of 70% ethanol was added and the sample was again centrifuged for 2 minutes at 13,000 rpm and 4. after removing the ethanol , the pellet was dissolved in 30 l rnase - free water and stored at -80 until use . a fixed concentration of microrna ( 5 ng/l ) from a given volume of starting urine was used as the input into the reverse transcription reaction . the miscript reverse transcription kit ( qiagen korea , seoul , south korea ) was used for reverse transcription of the mirnas . after mixing with template rna , 5x miscript buffer , miscript reverse transcriptase mix , and rnase - free water in a final volume of 20 l , the mixture was centrifuged briefly and incubated for 60 minutes at 37. to inactivate the miscript reverse transcriptase mix , for quantification of the mirna expression , real - time amplification was performed with a rotor - gene 6000 . real - time pcr assays were performed in microreaction tubes ( corbett research , mortlake , australia ) using the miscript pcr starter kit ( qiagen korea , seoul , korea ) . to amplify target mirnas , forward primers were designed for the mir-214 ( 5'-aca gca ggc aca gac agg ca gt-3 ' ) . after thawing the reagents and template cdna , the pcr reaction was carried out in a final volume of 20 l containing 10 l 2x quantitect sybr green pcr master mix , 2 l 10x miscript universal primer , 2 l 10 pmol forward primer , 2 l template cdna , and rnase - free water . real - time pcr conditions were as follows : one cycle of initial activation for 15 minutes at 95 , followed by 50 cycles of 15 seconds at 94 for denaturation , annealing for 30 seconds at 55 , and extension for 30 seconds at 70. the melting program was performed at 70 to 99 at a heating rate of 1 per 5 seconds . spectral data were captured and analyzed by using rotor - gene real - time analysis software 6.0 , build 14 . all samples were run in triplicate . for accurate and reproducible results by real - time pcr , it was necessary to normalize the amount of target mirna by using a suitable endogenous reference rna . was used as a control for the normalization of real - time pcr results in mirna quantification studies using the miscript pcr system . u6 was analyzed in parallel as an endogenous rna reference gene and all data were normalized to u6 expression . a student t - test was applied to assess the association of the mirna expression level with the clinical variables . to compare the levels of mirna-214 in enrolled patients and controls , the prognostic value of the levels of mir-214 in nmibc was studied by using multivariate cox proportional hazards regression models . statistical analysis was performed by using ibm spss ver . 19.0 ( ibm co. , armonk , ny , usa ) , and a p - value of < 0.05 was considered statistically significant . a total of 138 patients with primary urothelial carcinoma of the urinary bladder and 144 healthy controls were enrolled in the study . controls were selected to match the age and gender proportions of the bladder cancer patients , and subjects were screened to ensure that they were within the normal range of all laboratory results with no history of malignant tumors . urine samples were collected in the morning and stored at 4 , then centrifuged at 25,000 rpm for 15 minutes . each supernatant and sediment was portioned into eppendorf tubes and stored at -20 until use . all primary tumor samples were obtained from patients who underwent tur and were histologically verified to have urothelial carcinoma . to reduce confounding factors affecting the analyses , and to delineate a more homogeneous study population , any patients diagnosed with a concomitant carcinoma in situ or for whom data collection was incomplete were excluded . the collection and analysis of all samples was approved by the institutional review board of our institute , and written informed consent was obtained from each subject ( irb approval number 2006 - 01 - 001 ) . patients who had a t1 tumor , multiple tumors , large tumors ( > 3 cm in diameter ) , or high - grade ta nmibc received one cycle of intravesical treatment ( bcg or mitomycin - c ) . patients who were free of disease within 3 months after treatment were assessed every 3 months for the first 2 years , and then every 6 months thereafter . patients who refused or did not complete an imaging work - up such as a computed tomographic scan or an magnetic resonance imaging at least once every 3 months to evaluate their response tumors were staged and graded according to the 2002 tnm classification and the 1973 world health organization grading system , respectively . recurrence was defined as a recurrence of primary nmibc at either a lower or the same pathological stage , and progression was defined as disease with a higher tnm stage upon relapse of nmibc . a genolution urine mirna purification kit ( genolution pharmaceuticals inc . , seoul , korea ) was used to purify the urine samples . urine sample supernatant ( 500 l ) was added to each tube containing genolution proprietary mirna separation solution , which was then vortexed for 20 seconds . next , 200 l of chloroform was added and the samples were vortexed for 10 seconds , after which they were centrifuged at 13,000 rpm for 10 minutes at 4. a 600-l fraction from the top aqueous phase was taken and transferred into a new 1.5 ml tube , and 0.8 ml of isopropanol was added , followed by centrifugation for 5 minutes at 13,000 rpm and 4. after removing the aqueous solution , 1 ml of 70% ethanol was added and the sample was again centrifuged for 2 minutes at 13,000 rpm and 4. after removing the ethanol , the pellet was dissolved in 30 l rnase - free water and stored at -80 until use . a fixed concentration of microrna ( 5 ng/l ) from a given volume of starting urine was used as the input into the reverse transcription reaction . the miscript reverse transcription kit ( qiagen korea , seoul , south korea ) was used for reverse transcription of the mirnas . after mixing with template rna , 5x miscript buffer , miscript reverse transcriptase mix , and rnase - free water in a final volume of 20 l , the mixture was centrifuged briefly and incubated for 60 minutes at 37. to inactivate the miscript reverse transcriptase mix , the samples were incubated for 5 minutes at 95 and placed on ice . for quantification of the mirna expression , real - time amplification was performed with a rotor - gene 6000 . real - time pcr assays were performed in microreaction tubes ( corbett research , mortlake , australia ) using the miscript pcr starter kit ( qiagen korea , seoul , korea ) . to amplify target mirnas , forward primers were designed for the mir-214 ( 5'-aca gca ggc aca gac agg ca gt-3 ' ) . after thawing the reagents and template cdna , the pcr reaction was carried out in a final volume of 20 l containing 10 l 2x quantitect sybr green pcr master mix , 2 l 10x miscript universal primer , 2 l 10 pmol forward primer , 2 l template cdna , and rnase - free water . real - time pcr conditions were as follows : one cycle of initial activation for 15 minutes at 95 , followed by 50 cycles of 15 seconds at 94 for denaturation , annealing for 30 seconds at 55 , and extension for 30 seconds at 70. the melting program was performed at 70 to 99 at a heating rate of 1 per 5 seconds . spectral data were captured and analyzed by using rotor - gene real - time analysis software 6.0 , build 14 . all samples were run in triplicate . for accurate and reproducible results by real - time pcr , it was necessary to normalize the amount of target mirna by using a suitable endogenous reference rna . was used as a control for the normalization of real - time pcr results in mirna quantification studies using the miscript pcr system . u6 was analyzed in parallel as an endogenous rna reference gene and all data were normalized to u6 expression . a student t - test was applied to assess the association of the mirna expression level with the clinical variables . to compare the levels of mirna-214 in enrolled patients and controls , the prognostic value of the levels of mir-214 in nmibc was studied by using multivariate cox proportional hazards regression models . statistical analysis was performed by using ibm spss ver . 19.0 ( ibm co. , armonk , ny , usa ) , and a p - value of < 0.05 was considered statistically significant . the mean age of the patients was 62.0813.38 years for 110 males and 28 females ; the mean age of the controls was 63.7912.65 years for 114 males and 30 females . the number of patients in grades g1 , g2 , and g3 was 53 , 64 , and 21 , respectively . the number of patients in stages ta and t1 was 64 and 74 , respectively . table 2 summarizes the association between urinary levels of mirna-214 and the clinicopathological parameters of bladder cancer patients . the levels of mir-214 were significantly higher in the nmibc patients than in the controls ( 20.083.21 vs. 18.962.68 , p=0.002 ) . however , the urinary levels of mir-214 were not significantly associated with either the grade or the stage of bladder cancer ( p>0.05 , each ) . when we compared the urinary levels of mir-214 according to clinical outcomes , we found that the urinary levels of mir-214 were significantly different between the recurrence and the nonrecurrence groups ( table 3 ) . patients with recurrence of nmibc had lower levels of mir-214 than did those who had no recurrence ( 19.242.67 vs. 20.413.41 , p=0.023 ) . there was no significant difference in the levels of mir-214 between the progression and the nonprogression groups ( p=0.919 ) . in the univariate cox regression analysis , the number of tumors ( especially 8 ) and the levels of mir-214 were influential factors in the recurrence of nmibc ( hazard ratio [ hr ] , 2.636 ; 95% confidence interval [ ci ] , 1.220 to 5.699 ; p=0.014 ; hr , 2.231 ; 95% ci , 1.175 to 4.238 ; p=0.014 , respectively ) . multivariate cox regression analysis revealed that a low level of urinary mir-214 was the only independent predictor of nmibc recurrence ( hr , 2.011 ; 95% ci , 1.027 to 3.937 ; p=0.041 ) ( table 4 ) . the patients with low levels of mir-214 had a significantly longer recurrence - free survival time than did the patients with high level of mir-214 ( p=0.012 ) ( fig . the mean age of the patients was 62.0813.38 years for 110 males and 28 females ; the mean age of the controls was 63.7912.65 years for 114 males and 30 females . the number of patients in grades g1 , g2 , and g3 was 53 , 64 , and 21 , respectively . the number of patients in stages ta and t1 was 64 and 74 , respectively . table 2 summarizes the association between urinary levels of mirna-214 and the clinicopathological parameters of bladder cancer patients . the levels of mir-214 were significantly higher in the nmibc patients than in the controls ( 20.083.21 vs. 18.962.68 , p=0.002 ) . however , the urinary levels of mir-214 were not significantly associated with either the grade or the stage of bladder cancer ( p>0.05 , each ) . when we compared the urinary levels of mir-214 according to clinical outcomes , we found that the urinary levels of mir-214 were significantly different between the recurrence and the nonrecurrence groups ( table 3 ) . patients with recurrence of nmibc had lower levels of mir-214 than did those who had no recurrence ( 19.242.67 vs. 20.413.41 , p=0.023 ) . there was no significant difference in the levels of mir-214 between the progression and the nonprogression groups ( p=0.919 ) . in the univariate cox regression analysis , the number of tumors ( especially 8 ) and the levels of mir-214 were influential factors in the recurrence of nmibc ( hazard ratio [ hr ] , 2.636 ; 95% confidence interval [ ci ] , 1.220 to 5.699 ; p=0.014 ; hr , 2.231 ; 95% ci , 1.175 to 4.238 ; p=0.014 , respectively ) . multivariate cox regression analysis revealed that a low level of urinary mir-214 was the only independent predictor of nmibc recurrence ( hr , 2.011 ; 95% ci , 1.027 to 3.937 ; p=0.041 ) ( table 4 ) . the patients with low levels of mir-214 had a significantly longer recurrence - free survival time than did the patients with high level of mir-214 ( p=0.012 ) ( fig . the current study showed that urinary cell - free mirnas can serve as noninvasive prognostic markers for the recurrence of nmibc . micrornas have been identified in all types of bodily fluids , including cerebrospinal fluid , pleural fluid , peritoneal fluid , and even tears , saliva , and urine . numerous studies have recently shown that mirnas are emerging as a new class of cancer biomarkers . in the case of urothelial carcinoma , urine is a particularly desirable source of such biomarkers . an ideal biomarker must be accessible by noninvasive protocols , be inexpensive to quantify , be specific to the disease of interest , and be a reliable indicator of disease before the appearance of clinical symptoms . urine is more convenient and less invasive to collect than blood ; in addition , mirnas exist in a stable form in urine even after seven cycles of freezing and thawing or 72 hours at room temperature . cell - free mirnas in urine could be direct indicators of urological conditions including injury , malignancy , and so on . the exact mechanisms of release and the roles of cell free circulating mirnas are unclear . recent studies have suggested , however , that the interaction between cells via mrna and mirna is accomplished by microvesicle transfer . circulating mirnas might be released from cancer cells and communicate with recipient cells in the surrounding microenvironment by microvesicles . for this reason , it is certain that urine would have the highest exposure to microvesicles from urothelial cancer tissue . a number of studies have established either the over - expression or under - expression of mirnas in different types of human malignancy [ 21 - 27 ] . accumulated evidence has shown that mirna expression signatures correlate well with the specific characteristics of each malignancy and can be used to classify normal and malignant tissues as well as the subtype of malignancy . these observations suggest that mirnas can function either as tumor suppressors or oncogenes , regulating different cellular processes by targeting hundreds of genes and conferring a predictive diagnostic value to mirna expression . in solid tumors , such as stomach , pancreatic , and prostate cancer , alteration of the levels of a small number of mirnas including mir-214 has been identified as a signature . in breast cancer , mir-214 expression is reduced , and in pancreatic and ovarian cancers , mir-214 has a relationship with chemoresistance . however , studies that have evaluated the relationship between the level of mir-214 and bladder cancer are rare , which , of course , includes urinary mir-214 . in the present study , we confirmed that urinary mir-214 can serve as a noninvasive prognostic biomarker of bladder cancer . the levels of mir-214 capably distinguished nmibc patients from the noncancerous controls , but could show no correlation with either the grade or the stage of nmibc . we suggest that the nmibc patients had relatively high levels of mir-214 compared with the control patients owing to the tumor - suppressive effects of mir-214 . in nmibc patients , low levels of urinary mir-214 could be associated with a recurrence of nmibc owing to relatively low tumor - suppressive effects . on the basis of these results , we inferred that mir-214 could be related to the inhibition of angiogenesis , to cell proliferation , and to tumor recurrence . as far as we could ascertain , only one study has evaluated urinary cell - free mirnas as diagnostic and prognostic biomarkers of bladder cancer . the current study is the first to link urinary mirna-214 with bladder cancer . the results of this study indicated that mir-214 in urine might serve as a noninvasive biomarker for predicting the prognosis of bladder cancer . low levels of urinary mir-214 can be used as an independent prognostic parameter for the recurrence of nmibc . thus , cell - free urinary microrna-214 might be a useful noninvasive biomarker for the recurrence of nmibc .

purposemicrornas are small noncoding rnas and microrna-214 ( mir-214 ) has been associated with the inhibition of cancer cell growth , migration , and invasion . the aim of this study was to investigate whether cell - free mir-214 isolated from urine could be used as a biomarker for non - muscle - invasive bladder cancer ( nmibc).materials and methodsa total of 138 patients with primary nmibc and 144 healthy normal controls were enrolled in this study . by use of quantitative polymerase chain reaction ( pcr ) , the urinary levels of cell - free mir-214 were measured and the clinicopathological parameters of patients with nmibc were compared with those of the controls.resultsthe urinary levels of cell - free mir-214 were significantly higher in the nmibc patients than in the controls ( 20.083.21 vs. 18.962.68 , p=0.002 ) . however , the urinary levels of cell - free mir-214 were neither graded nor staged for the nmibc patients ( p>0.05 , each ) . when we compared the urinary levels of cell - free mir-214 according to clinical outcomes , patients with recurrence had lower levels of mir-214 than did those with no recurrence ( 19.242.67 vs. 20.413.41 , p=0.023 ) . by contrast , there were no significant differences in the urinary level of cell - free mir-214 between the nmibc patients showing progression and those showing no progression ( p=0.919 ) . multivariate cox regression analysis revealed that urinary levels of cell - free mir-214 were an independent predictor of nmibc recurrence ( hazard ratio , 2.011 ; 95% confidence interval , 1.027 to 3.937 ; p=0.041).conclusionsurinary levels of cell - free mir-214 could be an independent prognostic parameter for nmibc recurrence . thus , urinary cell - free microrna-214 might be a useful prognostic marker for nmi bc .

neuroblastoma ( nb ) is the most common extracranial solid tumor in children , with an incidence of 1 case per 100.000 children per year , and causes 15% of cancer deaths in pediatric age . nb originates from the sympathetic nervous system , most frequently in the adrenal medulla or the paraspinal ganglia . the causes are unknown , although 1 - 2% of nb may have a hereditary basis . different genetic alterations have been characterized in nb , that is , gain - of - function of alk gene , losses of 11q and 1p , gain of 17q , and amplification of the mycn gene . nb is heterogeneous , as it may undergo spontaneous remission or evolve to progressive metastatic disease , with dissemination to lymph nodes , bone , bone marrow , liver , skin , and other organs . in particular the international neuroblastoma risk group staging system takes into account genetic alterations , dna ploidy , histological features , and clinical data , as criteria for defining the risk classes . the prognosis of low / intermediate risk nb patients is favorable , and tumors can be cured by surgery alone or minimal chemotherapy . in contrast , high - risk nb patients ' prognosis is poor , in spite of aggressive treatment based on surgery , chemotherapy , radiation therapy , hematopoietic stem cell transplantation , and adjuvant therapy with retinoic acid . in fact , survival rates of these patients at 5 years are less than 50% . in the last years , the role of hla - class ib molecules in the progression of nb has been characterized by our group [ 47 ] and by others . hla - ib family includes hla - g , hla - e , hla - f , and hla - h . in contrast with high polymorphic hla - ia molecules ( hla - a , hla - b , and hla - c ) all these molecules display a limited polymorphism , with few alleles encoding a limited number of functional proteins . moreover , although hla - class ib molecules can bind small peptides and present them to specific cd8 t cell subsets ( similarly to hla - class ia counterparts ) , their main function is the modulation of the immune response in both physiological and pathological conditions . hla - g and hla - e are the best characterized among hla - ib molecules . hla - g has seven different isoforms , four membrane bound ( namely , hla - g1 , hla - g2 , hla - g3 , and hla - g4 ) and three soluble ( namely , hla - g5 , hla - g6 , and hla - g7 ) , that are generated by alternative splicing from the same primary transcript . hla - g can interact with at least four receptors , namely , immunoglobulin - like transcript ( ilt)2 , ilt4 , kir2dl4 , and cd160 , thus affecting the function of different immune effector cells ( t and b lymphocytes , natural killer nk cells , dendritic cells , granulocytes , and monocytes ) . in contrast , hla - e can be expressed as membrane bound or soluble isoform ( generated through metalloproteases cleavage ) and can inhibit cd8 t cells or nk cells though interaction with cd94/nkg2a heterodimeric receptor . however , hla - e can also interact with the activating receptor cd94/nkg2c , thus leading to nk cell activation . these interactions are crucial during trophoblast implantation to abrogate nk cell lysis of semiallogeneic fetal tissue and , on the other hand , to activate nk cell functions in the process of tissue remodeling . we have previously demonstrated that soluble ( s)hla - g concentration is higher in plasma samples from nb patients than in controls , and shla - g can be released by nb cells themselves , or by monocytes ( stimulated by soluble factors secreted by tumor cells ) . moreover , high shla - g plasma levels correlated with nb patients ' relapse . finally , we have assessed that hla - g is expressed by metastatic nb cells in the bone marrow from nb patients . also soluble hla - e levels are higher in nb patients than in healthy controls . however , we have demonstrated that high plasma levels of shla - e at diagnosis correlated with a better overall survival ( os ) of nb patients at follow - up , in contrast with shla - g . here , we demonstrated for the first time that shla - g and shla - e are present also in bm plasma samples derived from either nb patients at diagnosis or healthy donors . moreover , we have assessed that shla - g and shla - e levels in bm plasma samples are related to the stage of the disease . analysis of these patients at follow - up will reveal whether shla - g and shla - e concentration in bm plasma may predict the clinical outcome of nb patients . the study was approved by the ethics committee of the g. gaslini institute , genoa , italy . bone marrow ( bm ) samples were collected at diagnosis and centralized at istituto giannina gaslini in genoa , italy . patients ' characteristics , that is , age at diagnosis , sex , mycn amplification ( single copy or amplified ) , bm infiltration , and stage , are summarized in table 1 . as controls , bm aspirates were obtained from 13 healthy donors , selected according to the transplant unit clinical protocol of ematologia 2 at the irccs san martino - ist in genoa , following a written informed consent at the time of donation . samples were processed as described in , and an aliquot was taken at the end of processing to perform quality control tests , such as cd34 cell count , in vitro progenitors ' cell growth , and sterility . the remaining bm blood sample from this aliquot was subjected to centrifugation ( 3000 g 10 ) to obtain bm plasma . enzyme - linked immunosorbent assay ( elisa ) for shla - g and shla - e was performed as previously described . briefly , maxisorp nunc - immuno 96-microwell plates ( nunc a / s , roskilde , denmark ) were coated overnight at 4c with 1 g / ml of mem - g9 , specific for hla - g hc ( exbio , prague ) , that recognizes shla - g1/g5 , or 3d12 mab , specific for hla - e hc ( ebioscience , science center drive , san diego , ca , usa ) . after three washes with pbs 0.05% tween 20 ( washing buffer ) , plates were saturated with 200 l / w of pbs 2% bsa ( sigma , st . one hundred l of bm plasma samples and serial dilutions of 721.221.g1 cell line supernatant ( for hla - g ) or total extract from normal peripheral blood mononuclear cells ( standard ) were added to each well and incubated at rt for 1 hour . after three washes , 100 l of detection reagent ( hrp - conjugated anti-2 microglobulin mab , exbio , vestec , cz ) was added , and plates were incubated for 1 hour at rt . after three washes , 100 l of tmb ( substrate for hrp , sigma ) was added , and reaction was stopped after approximately 30 minutes by adding h2so4 5 n. absorbance at 450 nm was measured using infinite 200 pro spectrometer ( tecan group ltd . , results are expressed as ng / ml shla - g and arbitrary units / ml shla - e ( 1 unit = quantity of shla - e in 1 g of total extract ) . normal distribution of data was tested using kolmogorov - smirnov test , using prism software ( graphpad software inc . , la jolla , ca ) . since data distribution was not normal , differences in plasma levels between ( i ) patients and controls or ( ii ) different groups of patients were compared by mann - whitney test , using prism software . correlations between plasma levels of shla - g and shla - e were calculated by spearman 's test using prism software . a p value 0.05 significance ranges are the following : p < 0.05 ; p < 0.01 ; and p < 0.001 . first , we have tested shla - g and shla - e concentration in bm plasma samples from nb patients and healthy donors . as shown in figure 1(a ) , shla - g concentration was similar between nb patients ( median se : 24.69 8.45 ng / ml ) and controls ( 25.16 7.38 ng / ml ) . in contrast , shla - e levels were lower in nb patients ( 3.72 7.89 u / ml ) than in controls ( 48.01 10.93 u / ml ) . however , such difference was not statistically significant , likely due to the wide distribution of the results in both groups ( figure 1(b ) ) . finally , shla - g and shla - e levels in bm plasma samples from nb patients ( r = 0.96 , p < 0.0001 , figure 1(c ) ) and healthy donors ( r = 0.92 , p < 0.0001 , figure 1(d ) ) strongly correlated with each other . we have next analyzed possible correlation between shla - g and shla - e levels in bm plasma samples and patient 's characteristics or clinical parameters . accordingly , nb patients were divided into two groups on the basis of ( i ) mycn amplification ( single copy versus amplified ) , ( ii ) bm infiltration ( not infiltrated versus infiltrated ) , ( iii ) age at diagnosis ( < 18 months versus > 18 months ) , ( iv ) stage of the disease ( stages 1 - 2 versus stages 3 - 4 ) , and ( v ) sex ( male versus female ) . next , differences in shla - g and shla - e levels between these groups of nb patients have been evaluated . no significant differences in shla - g levels have been detected between nb patients ( i ) carrying amplified ( 29.96 13.57 ng / ml ) or single - copy ( 23.65 9.6 ng / ml ) mycn gene ( figure 2(a ) ) and ( ii ) presenting ( 31.17 11.99 ng / ml ) or not ( 21.05 9.91 ng / ml ) nb cells infiltrating the bm ( figure 2(b ) ) . in contrast , shla - e levels were higher in ( i ) patients with single - copy mycn ( 7.56 9.17 u / ml ) than in those with amplified mycn ( 1.03 16.2 u / ml ) ( figure 2(a ) ) and in ( ii ) patients with infiltrated bm ( 6.45 10.29 u / ml ) than in those without bm infiltration ( 1.86 12.43 u / ml ) ( figure 2(b ) ) . however , such differences were not statistically significant . furthermore , both shla - g and shla - e levels were similar between patients with an age below ( 21.05 9.92 ng / ml shla - g and 6.45 10.29 u / ml shla - e ) or above ( 28.77 11.36 ng / ml shla - g and 2.37 12.81 u / ml shla - e ) 18 months at diagnosis ( figure 3(a ) ) . notably , no correlation was found between age and shla - g or shla - e levels in bm plasma samples in healthy donors ( data not shown ) . both shla - g and shla - e levels were significantly higher in patients with disease stages 3 - 4 ( 32.34 8.08 ng / ml shla - g and 13.87 9.42 u / ml shla - e ) than in those with disease stages 1 - 2 ( 0 4.32 ng / ml shla - g and 0 3.27 u / ml shla - e , p = 0.01 and 0.03 , resp . ) surprisingly , both shla - g and shla - e levels were found to be higher in male ( 45.87 12.5 ng / ml shla - g and 34.19 14.83 u / ml shla - e ) than in female ( 2.52 8.81 ng / ml shla - g and 0 8.18 u / ml shla - e , p = 0.05 and 0.03 , resp . ) in contrast , healthy donors showed higher levels of shla - g and shla - e in female ( 50.74 14.1 ng / ml shla - g and 52.25 14.8 u / ml shla - e ) than in male ( 12.35 10.98 ng / ml shla - g and 11.33 13.25 u / ml shla - e ) subjects . however , such differences were not statistically significant ( figure 3(d ) ) . to the best of our knowledge , this is the first demonstration of the presence of shla - class ib molecules hla - g and hla - e in bm plasma samples . previous studies have demonstrated that shla - g can be released by some cell populations that are present in the bm environment , such as erythroblasts and mesenchymal stromal cells [ 1619 ] . in contrast , no information is available regarding hla - e expression and release in the bm . the strong correlation observed between the levels of these two molecules in bm samples either from nb patients or controls suggested that both molecules may be released by the same cell populations , or at least induced by similar stimuli . we have previously demonstrated that metastatic nb cells in the bm expressed high levels of hla - g on their surface , in contrast with primary tumors , that tested negative for hla - g . here , we have demonstrated that both shla - g and shla - e are present at similar levels in nb patients and healthy donors , thus suggesting that malignant metastatic nb cells are unlikely involved in their release . this observation is further confirmed by the finding that bm infiltration by metastatic nb cells did not affect shla - g or shla - e levels in bm plasma samples . moreover , mycn amplification and age at diagnosis that represent important prognostic factors were not related to shla - g and shla - e levels in bm , thus further suggesting that these molecules might be released by bm stromal cells or bm resident cell populations instead of nb cells themselves , and may be present in the bm environment in physiological conditions . however , the increased tumor burden might be correlated to a higher release of tumor - derived factor(s ) that , in turn , can upregulate hla - g and hla - e production by bm stromal cells . the finding that shla - g and shla - e bm plasma levels are higher in male than in female patients is in line with a previous study on multiple sclerosis , where the authors demonstrated that shla - g levels in plasma samples were higher in male than in female patients . however , this study has been carried out using peripheral blood plasma samples , and this is the first demonstration of this difference between male and female subjects in bone marrow plasma samples . notably , such difference may be a prerogative of nb patients , since shla - g and shla - e levels were higher in female than in male normal subjects . the most important finding of our study is the demonstration that shla - g and shla - e levels were significantly higher in bm plasma samples from patients with metastatic disease than in patients with localized nb . this data may suggest that the levels of these molecules in the bm at diagnosis might be associated with disease progression and might be predictive of the clinical course of nb patients . however , this hypothesis can be confirmed only by analyzing the clinical parameters of these patients at follow - up . in conclusion , we demonstrated for the first time that soluble hla - ib molecules hla - g and hla - e are present in bm plasma samples in physiological and pathological conditions , and their concentration correlated with stage disease in nb patients . the prognostic value of shla - g and shla - e concentration in bm plasma samples from nb patients at diagnosis has to be confirmed in future studies .

the role of nonclassical hla - class ib molecules hla - g and hla - e in the progression of neuroblastoma ( nb ) , the most common pediatric extracranial solid tumor , has been characterized in the last years . since bm infiltration by nb cells is an adverse prognostic factor , we have here analyzed for the first time the concentration of soluble ( s)hla - g and hla - e in bone marrow ( bm ) plasma samples from nb patients at diagnosis and healthy donors . shla - g and shla - e are present in bm plasma samples , and their levels were similar between nb patients and controls , thus suggesting that these molecules are physiologically released by resident or stromal bm cell populations . this hypothesis was supported by the finding that shla - g and shla - e levels did not correlate with bm infiltration and other adverse prognostic factors ( mycn amplification and age at diagnosis ) . in contrast , bm plasma levels of both molecules were higher in patients with metastatic disease than in patients with localized nb , thus suggesting that concentration of these molecules might be correlated with disease progression . the prognostic role of shla - g and shla - e concentration in the bm plasma for nb patients will be evaluated in future studies , by analyzing the clinical outcome of the same nb patients at follow - up .

diabetes mellitus is a global health problem and an important cause of mortality and morbidity in many countries . the trend of increasing diabetes prevalence seems to prevail among developing countries . in brazil , diabetes affected 11.3 million people in 2011 , and this number is likely to triple by 2030 . estimates suggest that the diabetes rate in less developed countries will increase by 69% between 2010 and 2030 . diabetes imposes a burden for society such as high socioeconomic costs that have an impact on productivity as well as life and health quality . this situation seems to be worse in developing countries , where the healthcare system often fails to meet demand . studies have concluded that a western dietary pattern , sedentary lifestyle , and genetic factors play a central role in diabetes development . the brazilian ministry of health has followed the world health organization 's recommendations and has taken some actions to monitor diabetes such as an annual telephone - based survey . socioeconomic disparities might contribute to some degree of heterogeneity in measures of prevalence between regions . a study demonstrated that diabetes prevalence across the brazilian states ranged from 11% to 25% , with an overall rate of 16% in 2001 . brasilia , the capital of brazil , is located in the central - west region of the country . the city has the highest human development index in brazil , but it has one of the highest levels of social inequality compared with other brazilian regions [ 11 , 12 ] . these characteristics of brasilia warrant further investigation in many aspects , including the health status of its population . thus , the goal of this study was to estimate the prevalence of diabetes and its associated risk factors in adults of brasilia , brazil . the present cross - sectional population - based study was conducted in brasilia , brazil , from february to may 2012 . the sample size was calculated based on an estimation of 16% of self - reported diabetes cases . considering a 95% confidence interval ( ci ) , precision of 2.25% , and a design effect of 1.8 , we added 10% of the sample size to compensate for any eventual attrition , which resulted in a final sample of 2,019 individuals . participants were selected by a two - stage probability sampling process by cluster and were stratified by sex and age . a total of 220 census tracts were randomly selected from 3,886 urban tracts with more than 200 inhabitants . up to 10 households were selected from each census tract . in total , one adult per household was selected following the predefined quotas of sex and age to answer the interview . trained professionals surveyed all of the participants in their homes using a semistructured questionnaire . to ensure reliability , 20% of the interviews were audited by telephone . to test the understanding and acceptability of the questionnaire , 150 pilot interviews were held prior to data collection . the dependent variable was self - reported diabetes . independent variables included demographic characteristics ( age group , sex , marital status , living arrangements , and household location ) , socioeconomic characteristics ( level of education , occupation , and social class ) , chronic health conditions ( self - reported hypertension , depression , respiratory diseases , cardiovascular diseases , and other chronic diseases ) , access to healthcare ( health insurance , medical consultation , and hospitalization ) , and perceived health status ( mobility , self - care , usual activities , pain / discomfort , and anxiety / depression ) . the stratification was based on the brazilian criterion of economic classification , which defines five classes , with a being the wealthiest group and e being the poorest . in all of the analyses , self - reported diabetes prevalence in the population was then calculated at a 95% ci . to identify factors related to diabetes prevalence , we calculated prevalence ratios ( pr ) using bivariate analysis and calculated the adjusted pr by a poisson regression model with robust variance . in this model , we preferred to use this more conservative model that included all of the variables to allow for better confounding adjustment . other models that included only the most significant variables were tested and did not change the significance of the variables . associations were considered to be statistically significant when p < 0.05 . the stata software version 10.1 was used for all of the calculations . approximately 60% of the participants were women , and 57% were aged between 35 and 60 years . most of the participants belonged to economic class c , had completed high school , were married or cohabitating , lived with at least one more person in the household , and dwelled in a satellite town . diabetes was self - reported by 10.1% ( 95% ci : 8.5%11.6% ) of the adult population in brasilia . table 1 depicts diabetes prevalence and prevalence ratios ( pr ) before and after adjustment by poisson regression . the age group of 3565 years , hypertension , respiratory disease , cardiovascular disease , and pain / discomfort were significantly associated with diabetes . sex , marital status , living arrangements , social class , education level , employability , living location , health insurance , medical consultation , hospitalization , physical mobility , self - care , usual activities , and anxiety / depression revealed no significant association . figure 2 illustrates differences in diabetes prevalence between all persons and the population with comorbidities . diabetes prevalence in the age range 3065 years is higher among individuals with cardiovascular disease , followed by those with hypertension and those with respiratory diseases . this result suggests that the likelihood of diabetes increases with age and is greater in persons with comorbidities . an age of 35 years and over , presence of pain or discomfort , cardiovascular disease , hypertension , and respiratory disease were positively associated with diabetes in the adult population of brasilia . the main limitations of our study were the self - reported assessments of the primary outcome and independent variables . self - reported diabetes might be a source of bias because individuals need to be aware of the diagnosis prior to answering , which could result in disease underestimation . however , performing a clinical test for diagnosing diabetes is not always possible in population - based studies . thus , self - reported answers regarding diabetes have been a common practice according to the literature [ 17 , 18 ] . another shortcoming was the cross - sectional design of the study , which hampers a causal relationship between diabetes and the significantly associated factors identified herein . a previous population - based study developed in brazil in 2008 used telephone interviews to investigate self - reported diabetes prevalence and found low prevalence rates in brasilia . another study found that brasilia was the region with the highest diabetes prevalence compared with other brazilian regions from 2002 to 2007 . research identified a significant increase in self - reported diabetes in the brazilian population because it ascended from 3.3% in 1998 to 5.3% in 2008 . in south and central america , the estimated diabetes prevalence in 2013 was 8.0% ; brazil demonstrated the highest prevalence , followed by colombia and argentina . the variability of diabetes prevalence may be due to a poorer diet and a lack of physical activity , or it could be related to better access to diagnostic testing . as expected , our results demonstrated that the likelihood of having diabetes increases with age . from a healthcare policy perspective , diabetes prevention and management programs should target young people and not only the elderly population . diabetes prevalence was higher among individuals with cardiovascular disease , hypertension , and respiratory disease compared with the general population . there is convincing evidence of the association between diabetes and hypertension , which increases the risk of a cardiovascular event . a 2003 study conducted in so luis , a city located in one of the poorest areas of brazil , observed a positive association between diabetes and hypertension . a cross - sectional study conducted between 2004 and 2005 in so jose do rio preto , a city in the brazilian southeast region , revealed that the diabetes prevalence was almost threefold higher in a population of hypertensive individuals compared with the general population . a cohort study performed in women between 1988 and 1996 throughout 11 states in the united states found that chronic obstructive pulmonary disease was a diabetes risk factor . a retrospective cohort study conducted in northern california reported that individuals with diabetes are at a greater risk of developing asthma , chronic obstructive pulmonary disease , fibrosis , and pneumonia . in contrast , a systematic review of 10 studies suggests that growing up in a socioeconomically disadvantaged environment may contribute to diabetes in later life . an australian study also described a positive association between socioeconomic variables and diabetes in adults aged 45 years and over . the perceived health dimensions physical mobility , self - care , usual activities , and anxiety / depression were not associated with diabetes in our sample . in 2012 , a literature review found that diabetes was considered a potential risk factor for the poor performance of daily life activities among individuals aged 50 years and over . a study conducted with older adult other than depression , this finding might depict an association between diabetes and activities of daily living , which may be developed at older ages . diabetes is a common health condition in adults living in brasilia and is positively associated with older age , cardiovascular disease , hypertension , respiratory disease , and presence of pain or discomfort .

aim . the aim of this study was to estimate the prevalence of diabetes and its associated risk factors in adults from brasilia , brazil . methods . the present cross - sectional population - based study consisted of interviews with individuals aged 1865 years . participants were selected through two - stage probability sampling by clusters and stratified by sex and age . demographic and clinical data were collected directly with participants from february to may 2012 . self - reported diabetes prevalence was calculated at a 95% confidence interval ( ci ) . prevalence ratios ( pr ) were adjusted by poisson regression with robust variance . results . in all , 1,820 individuals were interviewed . diabetes prevalence in the adult population of brasilia was 10.1% ( 95% ci , 8.5%11.6% ) . variables associated with diabetes were an age between 35 and 49 years ( pr = 1.83 ; 95% ci , 1.192.82 ) or 50 and 65 years ( pr = 1.95 ; 95% ci , 1.173.23 ) , hypertension ( pr = 4.04 ; 95% ci , 2.666.13 ) , respiratory disease ( pr = 1.67 ; 95% ci , 1.112.50 ) , cardiovascular disease ( pr = 1.74 ; 95% ci , 1.152.63 ) , and pain / discomfort ( pr = 1.71 ; 95% ci , 1.212.41 ) . conclusion . diabetes is a prevalent condition in adults living in brasilia , and disease risk increases with age and comorbidities . future health policies should focus on screening programs and prevention for the more vulnerable groups .

musculoskeletal disorders of the upper back , shoulders , and neck are common in many occupations that require physical exertion , repetitive movements , static muscle contraction , prolonged sitting before a computer , or maintaining fixed or extreme postures.16 in some instances , muscle fatigue subsequent to overwork or to poor posture results in tension or overactivation of the muscles of the neck.2,5,7,8 work - related neck , shoulder , and upper back musculoskeletal disorders include tension neck syndrome , rotator cuff syndrome , upper back pain , and shoulder tendonitis or tenosynovitis.912 these conditions may cause deterioration of joints , muscles , and soft tissue , persistent pain in related body areas , and disability.6 workplace interventions ( eg , resistance training , stretching exercise programs ) may help to prevent and manage mus - culoskeletal disorders.13 in addition , pharmacologic options , such as acetaminophen and nonsteroidal anti - inflammatory drugs ( nsaids ) , are used to manage musculoskeletal pain , and muscle relaxants may be used to manage muscle pain , stiffness , and spasm.1417 although there are many skeletal muscle relaxants , systemically active products are generally available with prescription and are associated with well - documented adverse effects such as somnolence , dizziness , weakness , and dry mouth.17 guaifenesin , an over - the - counter ( otc ) expectorant , has exhibited a muscle relaxant effect in preclinical studies and in patients with cerebral palsy when used in doses higher than those currently available for otc use.18,19 in other preclinical investigations , guaifenesin has shown modest but significant analgesic and anti - inflammatory effects.20 little is known about the potential mechanism by which guaifenesin produces these effects , but a chemically similar muscle relaxant , mephenesin , has been found to be an antagonist of the excitatory amino acids n - methyl - d - aspartate ( nmda ) and -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid ( ampa).18,21 whether a true muscle relaxant effect occurs at otc doses of guaifenesin has not been studied previously . the primary objective of this proof - of - principle study was to explore the efficacy and safety of 2 otc dose strengths of guaifenesin compared with matched placebos for symptom relief in subjects with acute upper back , neck , or shoulder muscle spasm and pain . this multicenter , randomized , placebo - controlled , repeat - dose , 4-arm , parallel study was conducted at 4 investigational sites in the united states ( clinicaltrials.gov identifier : nct01562548 ) . eligible subjects with upper back , neck , or shoulder muscle spasm and pain were randomly assigned in a 2:2:1:1 ratio to receive guaifenesin 600 mg ( a single 600 mg mucinex extended release bi - layer tablet ; reckitt benckiser , parsippany , nj , usa ) , guaifenesin 1200 mg ( two 600 mg mucinex extended release tablets ) , placebo matched to guaifenesin 600 mg ( ie , 1 placebo tablet , hereafter referred to as placebo-600 ) , or placebo matched to guaifenesin 1200 mg ( ie , 2 placebo tablets , hereafter referred to as placebo-1200 ) . the randomization schedule was generated by the biostatistics department of glaxo - smithkline consumer healthcare ( parsippany , nj , usa ) . all doses were self - administered twice daily ( bid ) for 7 consecutive days . subjects recorded the intensity of upper back , neck , or shoulder muscle spasm , stiffness , tension , pain , and discomfort twice daily ( 8 am and 8 pm ) on a diary card , using an 11-point numeric rating scale ( nrs ) for each symptom , with anchors of 0=symptom not present and 10=unbearable symptoms . on days 4 and 7 , investigators telephoned subjects to evaluate muscle relaxation and to perform a global assessment of treatment helpfulness ( gath ) , sleep disturbance ( gasd ) , and headache frequency / intensity ( gahf / gahi ) . at that time muscle relaxation was scored on a 5-point categorical scale of 0=no relaxation , 1=a little relaxation , 2= fair relaxation , 3=good relaxation , and 4=complete relaxation . the gath was scored on a scale of 0=poor , 1=fair , 2=good , 3=very good , and 4=excellent . the gasd , gahf , and gahi recorded whether the symptom had increased , decreased , or remained the same . the vernon mior disability questionnaire was originally designed to assess how neck pain affects everyday life with regard to pain intensity , personal care , lifting , reading , headaches , concentration , work , driving , sleeping , and recreation , with each item rated on a 6-point scale , with 1 being no disability and 6 being complete disability . here , the same questionnaire was used to measure disability resulting from neck , upper back , and shoulder pain . this study , which was conducted from february 2012 through may 2013 , conformed to the principles of the declaration of helsinki and was approved by an institutional review board ( quorum review , inc , seattle , wa , usa ) . subjects enrolled in the study were healthy men and women aged 1865 years with a new episode of acute upper back , neck , or shoulder muscle spasm and pain . inclusion criteria were muscle pain and/or spasm with an onset within 48 hours of randomization and at least 30 days since a previous episode ; symptoms consistent with a clinical diagnosis ; and subject - rated muscle spasm and pain scores of at least 40 mm on a 100 mm visual analog scale . subjects had to have a normal neurologic evaluation , physical examination , and clinical laboratory test results . exclusion criteria were current or recent history of neck , back , and/or shoulder injuries ; spinal disk disease ; osteoporosis ; liver and/or kidney disease ; myocardial infarction within 12 months of randomization ; history of upper back pain with active hypersensitive spots ; history of other chronic pain ( eg , headache , osteoarthritis , or lower back pain ) ; and known or suspected intolerance or hypersensitivity to guaifenesin or methocarbamol ( or closely related compounds ) or any other study medication ingredient . individuals who had used muscle relaxants , narcotics , monoamine oxidase inhibitors , or selegiline within 2 weeks of randomization or who were currently using tricyclic antidepressants , lithium , anticoagulants , or tramadol were ineligible . additional exclusion criteria were current pregnancy or breastfeeding , drug or alcohol abuse within the previous 2 years , use of an investigational drug or participation in another clinical trial within 30 days prior to randomization , and involvement in a worker s compensation case . subjects had to agree to refrain from use of analgesics ( including nsaids , with the exception of aspirin 325 mg / day ) , muscle relaxants , physical or massage therapy , acupuncture , and heat and spa treatments for the duration of the study . the primary efficacy end point was the change in muscle spasm over the 7-day study period , measured as mean change from baseline for both morning and evening spasms in the 11-point nrs scores recorded in the subjects diary cards and averaged over the 7-day treatment period . secondary efficacy end points included change from baseline in muscle stiffness , tension , pain , discomfort , and relaxation over the 7-day period , measured as mean change from baseline for both morning and evening symptoms on the 11-point nrs scores . other secondary end points included muscle relaxation scores , as well as scores for gath and the individual vernon mior disability questionnaire components on days 4 and 7 . safety assessments included treatment - emergent adverse events ( teaes ) and serious adverse events ( saes ) . safety was monitored from the start of treatment until 7 days after the last treatment administration , and treatment - related saes were reported from the time of informed consent through all follow - up contact . severity and relationship to treatment were assessed for all teaes . given the proof - of - concept objective , no formal sample size calculations were performed . a total of 78 subjects ( 26 in each active treatment randomization group and 13 in each placebo randomization group ) were considered sufficient to visualize any directional change in increased efficacy from placebo to lower- and upper - dose guaifenesin groups for muscle spasm , stiffness , tension , pain , discomfort , and relaxation scores . directional changes in efficacy were based primarily on comparisons of absolute values of means of guaifenesin 1200 mg bid with placebo-1200 and with guaifenesin 600 mg bid . placebo-600 was not included in these comparisons , as its only purpose was to test the effect of guaifenesin 600 mg bid . statistical testing to detect significant differences between treatments was exploratory , as no formal sample size calculations were done for this study . efficacy analyses were conducted on the intent - to - treat ( itt ) population , which was defined as subjects who had received at least 1 dose of study medication and who had at least 1 post - baseline efficacy assessment . all primary and secondary end points were summarized using descriptive statistics ( mean , standard deviation [ sd ] , minimum , maximum ) in order to provide an absolute comparison between treatments for the purpose of identifying a directional change in efficacy . in addition , a mixed - model analysis of covariance ( ancova ; proc mixed of sas ) was used to analyze changes from baseline in subject nrs ratings over the 7-day period ( ie , mean of each morning s and evening s changes from baseline on days 17 ) . a similar mixed model was used to assess mean muscle relaxation and gath scores on days 4 and 7 ( incorporating morning and evening scores ) . both ancova models included factors for treatment ( fixed effect ) , site ( random effect ) , and baseline assessment of the end point being analyzed as a covariate . treatment differences were presented with 95% confidence intervals and associated p - values . global assessments ( gasd , gahf , and gahi ) were summarized by percentage of subjects recording that symptoms had increased , decreased , or stayed the same . no imputation was performed for missing values for any of the assessments , and subjects who dropped out or were excluded were included in all analyses for which they were evaluable . adverse events were classified using the medical dictionary for drug regulatory activities version 15.1 and summarized using descriptive statistics . this multicenter , randomized , placebo - controlled , repeat - dose , 4-arm , parallel study was conducted at 4 investigational sites in the united states ( clinicaltrials.gov identifier : nct01562548 ) . eligible subjects with upper back , neck , or shoulder muscle spasm and pain were randomly assigned in a 2:2:1:1 ratio to receive guaifenesin 600 mg ( a single 600 mg mucinex extended release bi - layer tablet ; reckitt benckiser , parsippany , nj , usa ) , guaifenesin 1200 mg ( two 600 mg mucinex extended release tablets ) , placebo matched to guaifenesin 600 mg ( ie , 1 placebo tablet , hereafter referred to as placebo-600 ) , or placebo matched to guaifenesin 1200 mg ( ie , 2 placebo tablets , hereafter referred to as placebo-1200 ) . the randomization schedule was generated by the biostatistics department of glaxo - smithkline consumer healthcare ( parsippany , nj , usa ) . all doses were self - administered twice daily ( bid ) for 7 consecutive days . subjects recorded the intensity of upper back , neck , or shoulder muscle spasm , stiffness , tension , pain , and discomfort twice daily ( 8 am and 8 pm ) on a diary card , using an 11-point numeric rating scale ( nrs ) for each symptom , with anchors of 0=symptom not present and 10=unbearable symptoms . on days 4 and 7 , investigators telephoned subjects to evaluate muscle relaxation and to perform a global assessment of treatment helpfulness ( gath ) , sleep disturbance ( gasd ) , and headache frequency / intensity ( gahf / gahi ) . at that time muscle relaxation was scored on a 5-point categorical scale of 0=no relaxation , 1=a little relaxation , 2= fair relaxation , 3=good relaxation , and 4=complete relaxation . the gath was scored on a scale of 0=poor , 1=fair , 2=good , 3=very good , and 4=excellent . the gasd , gahf , and gahi recorded whether the symptom had increased , decreased , or remained the same . the vernon mior disability questionnaire was originally designed to assess how neck pain affects everyday life with regard to pain intensity , personal care , lifting , reading , headaches , concentration , work , driving , sleeping , and recreation , with each item rated on a 6-point scale , with 1 being no disability and 6 being complete disability . here , the same questionnaire was used to measure disability resulting from neck , upper back , and shoulder pain . this study , which was conducted from february 2012 through may 2013 , conformed to the principles of the declaration of helsinki and was approved by an institutional review board ( quorum review , inc , seattle , wa , usa ) . subjects enrolled in the study were healthy men and women aged 1865 years with a new episode of acute upper back , neck , or shoulder muscle spasm and pain . inclusion criteria were muscle pain and/or spasm with an onset within 48 hours of randomization and at least 30 days since a previous episode ; symptoms consistent with a clinical diagnosis ; and subject - rated muscle spasm and pain scores of at least 40 mm on a 100 mm visual analog scale . subjects had to have a normal neurologic evaluation , physical examination , and clinical laboratory test results . exclusion criteria were current or recent history of neck , back , and/or shoulder injuries ; spinal disk disease ; osteoporosis ; liver and/or kidney disease ; myocardial infarction within 12 months of randomization ; history of upper back pain with active hypersensitive spots ; history of other chronic pain ( eg , headache , osteoarthritis , or lower back pain ) ; and known or suspected intolerance or hypersensitivity to guaifenesin or methocarbamol ( or closely related compounds ) or any other study medication ingredient . individuals who had used muscle relaxants , narcotics , monoamine oxidase inhibitors , or selegiline within 2 weeks of randomization or who were currently using tricyclic antidepressants , lithium , anticoagulants , or tramadol were ineligible . additional exclusion criteria were current pregnancy or breastfeeding , drug or alcohol abuse within the previous 2 years , use of an investigational drug or participation in another clinical trial within 30 days prior to randomization , and involvement in a worker s compensation case . subjects had to agree to refrain from use of analgesics ( including nsaids , with the exception of aspirin 325 mg / day ) , muscle relaxants , physical or massage therapy , acupuncture , and heat and spa treatments for the duration of the study . the primary efficacy end point was the change in muscle spasm over the 7-day study period , measured as mean change from baseline for both morning and evening spasms in the 11-point nrs scores recorded in the subjects diary cards and averaged over the 7-day treatment period . secondary efficacy end points included change from baseline in muscle stiffness , tension , pain , discomfort , and relaxation over the 7-day period , measured as mean change from baseline for both morning and evening symptoms on the 11-point nrs scores . other secondary end points included muscle relaxation scores , as well as scores for gath and the individual vernon mior disability questionnaire components on days 4 and 7 . safety assessments included treatment - emergent adverse events ( teaes ) and serious adverse events ( saes ) . safety was monitored from the start of treatment until 7 days after the last treatment administration , and treatment - related saes were reported from the time of informed consent through all follow - up contact . severity and relationship to treatment were assessed for all teaes . given the proof - of - concept objective , no formal sample size calculations were performed . a total of 78 subjects ( 26 in each active treatment randomization group and 13 in each placebo randomization group ) were considered sufficient to visualize any directional change in increased efficacy from placebo to lower- and upper - dose guaifenesin groups for muscle spasm , stiffness , tension , pain , discomfort , and relaxation scores . directional changes in efficacy were based primarily on comparisons of absolute values of means of guaifenesin 1200 mg bid with placebo-1200 and with guaifenesin 600 mg bid . placebo-600 was not included in these comparisons , as its only purpose was to test the effect of guaifenesin 600 mg bid . statistical testing to detect significant differences between treatments was exploratory , as no formal sample size calculations were done for this study . efficacy analyses were conducted on the intent - to - treat ( itt ) population , which was defined as subjects who had received at least 1 dose of study medication and who had at least 1 post - baseline efficacy assessment . all primary and secondary end points were summarized using descriptive statistics ( mean , standard deviation [ sd ] , minimum , maximum ) in order to provide an absolute comparison between treatments for the purpose of identifying a directional change in efficacy . in addition , a mixed - model analysis of covariance ( ancova ; proc mixed of sas ) was used to analyze changes from baseline in subject nrs ratings over the 7-day period ( ie , mean of each morning s and evening s changes from baseline on days 17 ) . a similar mixed model was used to assess mean muscle relaxation and gath scores on days 4 and 7 ( incorporating morning and evening scores ) . both ancova models included factors for treatment ( fixed effect ) , site ( random effect ) , and baseline assessment of the end point being analyzed as a covariate . global assessments ( gasd , gahf , and gahi ) were summarized by percentage of subjects recording that symptoms had increased , decreased , or stayed the same . no imputation was performed for missing values for any of the assessments , and subjects who dropped out or were excluded were included in all analyses for which they were evaluable . adverse events were classified using the medical dictionary for drug regulatory activities version 15.1 and summarized using descriptive statistics . ninety - five people were screened , of whom 79 met the enrollment criteria and were randomly assigned to receive guaifenesin 600 mg bid ( n=25 ) , guaifenesin 1200 mg bid ( n=25 ) , placebo-600 ( n=15 ) , and placebo-1200 ( n=14 ) . one subject in the guaifenesin 1200 mg bid group was lost to follow - up and did not receive treatment and was excluded from the safety and itt populations . one subject in the placebo-1200 group was excluded from the itt population because the number of pills returned at the end of the study indicated a much lower adherence to study procedures than that indicated in the subject s diary records . thus , 78 subjects were included in the safety population and 77 in the itt population . the overall mean ( sd ) age of randomized subjects was 45.9 ( 11.5 ) years , and slightly more than half of all subjects were males ( 55.1% ) . most study subjects were white ( 66.7% ) ; the remaining subjects were primarily black ( 32.1% ) . the demographics of the subjects in the individual treatment arms are shown in table 1 . over the 7-day study period , the guaifenesin 1200 mg bid group showed a potential directional change for a greater effect on decreasing muscle spasm compared with its matched placebo and guaifenesin 600 mg bid ( figure 1a ) . reduction of muscle spasm in subjects treated with guaifenesin 1200 mg bid was 25% greater than that of subjects in the placebo-1200 group and 16% greater than that of subjects in the guaifenesin 600 mg bid group . guaifenesin 600 mg bid did not show a clear effect on decreasing muscle spasm compared with its matched placebo ( mean change score , 1.53 vs 1.74 ; table 2 ) . over the 7-day study period , treatment with guaifenesin 1200 mg bid provided numerically greater improvements than its matched placebo and guaifenesin 600 mg bid on most other subject - reported symptoms , including pain ( figure 1b ) , tension ( figure 1c ) , and discomfort . subjects in the guaifenesin 1200 mg bid group experienced greater mean reductions of 52% and 31% in pain , 28% and 13% in tension , and 108% and 20% in discomfort compared with subjects in the placebo-1200 and guaifenesin 600 mg bid groups , respectively ( table 2 ) . guaifenesin 600 mg bid did not show a clear effect toward improvement on these outcomes compared with its matched placebo . improvement in muscle discomfort with guaifenesin 1200 mg bid was significantly greater than placebo-1200 ( p=0.0358 ) . all other differences among treatments for the above - mentioned end points were not significant ( p>0.05 ; table 2 ) . there was a directional change suggesting increasing effects on muscle relaxation from placebo-1200 to guaifenesin 600 mg bid to guaifenesin 1200 mg bid ( table 3 ) . mean muscle relaxation on day 4 for subjects in the guaifenesin 1200 mg bid group was 39% and 3% greater than mean muscle relaxation for subjects in the placebo-1200 and guaifenesin 600 mg bid groups , respectively . on day 7 , this directional change continued with a 28% and 17% greater effect of guaifenesin 1200 mg bid over placebo-1200 and guaifenesin 600 mg bid groups , respectively . guaifenesin 600 mg bid did not produce greater muscle relaxation compared with its matched placebo on both days 4 and 7 ( table 3 ) . similar results to muscle relaxation were observed for gath ( table 3 ) . on day 4 , subjects rated guaifenesin 1200 mg bid 94% and 10% higher than subjects in the placebo-1200 and guaifenesin 600 mg bid groups , respectively . on day 7 , these values were 23% and 10% , respectively . the greatest treatment effect for gath and muscle relaxation was observed on day 4 ( table 3 ) . there was a significant difference between guaifenesin 1200 mg bid and placebo-1200 ( p=0.0211 ) for gath and a borderline significant difference between these 2 groups for muscle relaxation ( p=0.0523 ) . for example , the effect of guaifenesin 1200 mg bid on muscle relaxation on day 7 was 22% higher than on day 4 . there were no directional changes in effect with regard to effects on sleep disturbance , headache frequency , or headache intensity based on gasd , gahf , and gahi scores , respectively ( table s1 for day 7 data ) . on day 4 , subjects in the guaifenesin 1200 mg bid group had overall better ( lower ) mean vernon mior index scores than subjects in the guaifenesin 600 mg bid group , and subjects in the guaifenesin 600 mg bid group had overall better ( lower ) scores than subjects in the placebo-1200 group ( figure 2 ) . there was a directional change on day 4 of placebo-1200 ( 2.39 ) > guaifenesin 600 mg bid ( 2.31 ) > guaifenesin 1200 mg bid ( 2.01 ) . there was a reduction of 16% and 13% in the overall mean vernon mior index for subjects in the guaifenesin 1200 mg bid group compared with subjects in the placebo-1200 and guaifenesin 600 mg bid groups . a similar directional change was observed on day 7 , where there was a reduction of 10% and 20% in the overall vernon mior index of subjects in the guaifenesin 1200 mg bid group compared with the placebo-1200 and guaifenesin 600 mg bid groups , respectively . within each treatment group , vernon mior disability scores improved noticeably from day 4 to day 7 ( figure 2 ) . a summary of results for upper back / neck / shoulder pain disability end points is presented in table 4 . twenty - one teaes were reported by 17 ( 21.8% ) subjects in the study . eight teaes occurred in 6 subjects ( 25.0% ) in the guaifenesin 1200 mg bid group , 4 teaes in 3 subjects ( 21.4% ) in the placebo-1200 group , 8 teaes in 7 subjects ( 28.0% ) in the guaifenesin 600 mg bid group , and 1 teae in 1 subject ( 6.7% ) in the placebo-600 group . the most common categories of teaes were nervous system disorders ( eg , headache , dizziness , dysgeusia , and somnolence ) and infections ( eg , upper respiratory infections and nasopharyngitis ) . nearly all teaes were mild ; there were 2 moderate teaes ( upper respiratory tract infection and hyperhidrosis ) in the guaifenesin 1200 mg bid group , 3 moderate teaes ( headache , sinusitis , and sleep disorder ) in the placebo-1200 group , 2 moderate teaes ( headache and arthralgia ) in the guaifenesin 600 mg bid group , and no moderate teaes in the placebo-600 group . seven teaes in 7 subjects were considered to be treatment - related , including 1 case of moderate hyperhidrosis in the guaifenesin 1200 mg bid group , 1 moderate headache and 1 moderate sleep disorder in the placebo-1200 group , 1 mild case each of dizziness , dysgeusia , and fatigue in the guaifenesin 600 mg bid group , and 1 case of mild abdominal discomfort in the placebo-600 group . ninety - five people were screened , of whom 79 met the enrollment criteria and were randomly assigned to receive guaifenesin 600 mg bid ( n=25 ) , guaifenesin 1200 mg bid ( n=25 ) , placebo-600 ( n=15 ) , and placebo-1200 ( n=14 ) . one subject in the guaifenesin 1200 mg bid group was lost to follow - up and did not receive treatment and was excluded from the safety and itt populations . one subject in the placebo-1200 group was excluded from the itt population because the number of pills returned at the end of the study indicated a much lower adherence to study procedures than that indicated in the subject s diary records . thus , 78 subjects were included in the safety population and 77 in the itt population . the overall mean ( sd ) age of randomized subjects was 45.9 ( 11.5 ) years , and slightly more than half of all subjects were males ( 55.1% ) . most study subjects were white ( 66.7% ) ; the remaining subjects were primarily black ( 32.1% ) . the demographics of the subjects in the individual treatment arms are shown in table 1 . over the 7-day study period , the guaifenesin 1200 mg bid group showed a potential directional change for a greater effect on decreasing muscle spasm compared with its matched placebo and guaifenesin 600 mg bid ( figure 1a ) . reduction of muscle spasm in subjects treated with guaifenesin 1200 mg bid was 25% greater than that of subjects in the placebo-1200 group and 16% greater than that of subjects in the guaifenesin 600 mg bid group . guaifenesin 600 mg bid did not show a clear effect on decreasing muscle spasm compared with its matched placebo ( mean change score , 1.53 vs 1.74 ; table 2 ) . over the 7-day study period , treatment with guaifenesin 1200 mg bid provided numerically greater improvements than its matched placebo and guaifenesin 600 mg bid on most other subject - reported symptoms , including pain ( figure 1b ) , tension ( figure 1c ) , and discomfort . subjects in the guaifenesin 1200 mg bid group experienced greater mean reductions of 52% and 31% in pain , 28% and 13% in tension , and 108% and 20% in discomfort compared with subjects in the placebo-1200 and guaifenesin 600 mg bid groups , respectively ( table 2 ) . guaifenesin 600 mg bid did not show a clear effect toward improvement on these outcomes compared with its matched placebo . improvement in muscle discomfort with guaifenesin 1200 mg bid was significantly greater than placebo-1200 ( p=0.0358 ) . all other differences among treatments for the above - mentioned end points were not significant ( p>0.05 ; table 2 ) . there was a directional change suggesting increasing effects on muscle relaxation from placebo-1200 to guaifenesin 600 mg bid to guaifenesin 1200 mg bid ( table 3 ) . mean muscle relaxation on day 4 for subjects in the guaifenesin 1200 mg bid group was 39% and 3% greater than mean muscle relaxation for subjects in the placebo-1200 and guaifenesin 600 mg bid groups , respectively . on day 7 , this directional change continued with a 28% and 17% greater effect of guaifenesin 1200 mg bid over placebo-1200 and guaifenesin 600 mg bid groups , respectively . guaifenesin 600 mg bid did not produce greater muscle relaxation compared with its matched placebo on both days 4 and 7 ( table 3 ) . similar results to muscle relaxation were observed for gath ( table 3 ) . on day 4 , subjects rated guaifenesin 1200 mg bid 94% and 10% higher than subjects in the placebo-1200 and guaifenesin 600 mg bid groups , respectively . on day 7 , these values were 23% and 10% , respectively . the greatest treatment effect for gath and muscle relaxation there was a significant difference between guaifenesin 1200 mg bid and placebo-1200 ( p=0.0211 ) for gath and a borderline significant difference between these 2 groups for muscle relaxation ( p=0.0523 ) . on day 7 , treatment effect increased compared with day 4 . for example , the effect of guaifenesin 1200 mg bid on muscle relaxation on day 7 was 22% higher than on day 4 . there were no directional changes in effect with regard to effects on sleep disturbance , headache frequency , or headache intensity based on gasd , gahf , and gahi scores , respectively ( table s1 for day 7 data ) . on day 4 , subjects in the guaifenesin 1200 mg bid group had overall better ( lower ) mean vernon mior index scores than subjects in the guaifenesin 600 mg bid group , and subjects in the guaifenesin 600 mg bid group had overall better ( lower ) scores than subjects in the placebo-1200 group ( figure 2 ) . there was a directional change on day 4 of placebo-1200 ( 2.39 ) > guaifenesin 600 mg bid ( 2.31 ) > guaifenesin 1200 mg bid ( 2.01 ) . there was a reduction of 16% and 13% in the overall mean vernon mior index for subjects in the guaifenesin 1200 mg bid group compared with subjects in the placebo-1200 and guaifenesin 600 mg bid groups . a similar directional change was observed on day 7 , where there was a reduction of 10% and 20% in the overall vernon mior index of subjects in the guaifenesin 1200 mg bid group compared with the placebo-1200 and guaifenesin 600 mg bid groups , respectively . within each treatment group , vernon mior disability scores improved noticeably from day 4 to day 7 ( figure 2 ) . a summary of results for upper back / neck / shoulder pain disability end points is presented in table 4 . twenty - one teaes were reported by 17 ( 21.8% ) subjects in the study . eight teaes occurred in 6 subjects ( 25.0% ) in the guaifenesin 1200 mg bid group , 4 teaes in 3 subjects ( 21.4% ) in the placebo-1200 group , 8 teaes in 7 subjects ( 28.0% ) in the guaifenesin 600 mg bid group , and 1 teae in 1 subject ( 6.7% ) in the placebo-600 group . the most common categories of teaes were nervous system disorders ( eg , headache , dizziness , dysgeusia , and somnolence ) and infections ( eg , upper respiratory infections and nasopharyngitis ) . nearly all teaes were mild ; there were 2 moderate teaes ( upper respiratory tract infection and hyperhidrosis ) in the guaifenesin 1200 mg bid group , 3 moderate teaes ( headache , sinusitis , and sleep disorder ) in the placebo-1200 group , 2 moderate teaes ( headache and arthralgia ) in the guaifenesin 600 mg bid group , and no moderate teaes in the placebo-600 group . seven teaes in 7 subjects were considered to be treatment - related , including 1 case of moderate hyperhidrosis in the guaifenesin 1200 mg bid group , 1 moderate headache and 1 moderate sleep disorder in the placebo-1200 group , 1 mild case each of dizziness , dysgeusia , and fatigue in the guaifenesin 600 mg bid group , and 1 case of mild abdominal discomfort in the placebo-600 group . this is the first study to evaluate guaifenesin for managing symptoms of upper back , neck , and shoulder pain . the results suggest further investigation of the potential of guaifenesin 1200 mg bid to provide some relief in muscle spasm , pain , tension , and discomfort of the upper back , neck , and shoulders is warranted . the reduction in muscle discomfort with guaifenesin 1200 mg compared with placebo-1200 was statistically significant ( p=0.0358 ) ; the rest of these improvements were not significant ( p>0.05 ) ; it is noted that in this small proof - of - principle study , analyses were not powered for pre - specified statistical testing between treatments . the 1200 mg bid dose of guaifenesin suggested a potential for therapeutic muscle relaxant effect . however , any observed differences between treatments diminished over the course of the study , possibly due to the natural course of upper back pain / muscle spasm , which is generally a self - limiting disorder such that patients may have recovered at day 7 . this suggests that future studies should focus on earlier ( eg , day 4 ) outcomes . the directional change in effect observed for upper back / neck / shoulder pain disability on the vernon mior questionnaire end points was consistent with that of most of the other end points in the study . subjects in the guaifenesin 1200 mg bid group had better ( lower ) overall scores than subjects in the guaifenesin 600 mg bid group , and subjects in the guaifenesin 600 mg bid group had better ( lower ) scores than subjects in the placebo-1200 group . this study adds support to the limited evidence suggesting that guaifenesin has muscle relaxant activity.19 in an earlier small , exploratory clinical study , guaifenesin at doses up to 220.5 mg / kg ( 100 mg / lb ) of body weight showed muscle relaxant and antispasmodic effects in children with athetoid or tonic spasticity ( n=18).19 methocarbamol , a car - bamate derivative of guaifenesin , also has muscle relaxant properties22,23 and is prescribed for the relief of discomfort associated with acute , painful musculoskeletal conditions.24 based on these prior studies , muscle spasm was selected as the primary outcome for our investigation ; however , results of the secondary end points showed the largest treatment effect for guaifenesin 1200 mg bid vs placebo-1200 on muscle pain and discomfort . therefore , pain and discomfort may be more appropriate primary end points in future studies . acute , non - traumatic back , shoulder , and neck pain may be self - limiting and , for most patients , may improve or resolve within several days of onset without intervention ; however , the pain sometimes persists or recurs and converts to a chronic condition lasting months or even years.2527 the availability of an otc muscle relaxant may provide timely access to an effective remedy for relief of acute symptoms . while none of the directional changes in effect observed in the primary and secondary outcomes achieved statistical significance , it should be noted that this was a small , proof - of - principle study for which sample size was not powered to show statistical difference between active treatments and their matched placebos . furthermore , the 2:2:1:1 randomization ratio led to only 15 subjects assigned to placebo-600 and 14 assigned to placebo-1200 ; the small numbers of subjects may have contributed to the higher than expected placebo response in the placebo-600 group due to sampling bias and the possibility that the subjects were not representative of the general population experiencing upper back , neck , and shoulder pain . nonetheless , this study has provided interesting preliminary evidence on another potential therapeutic use for otc guaifen - esin . therefore , a larger , adequately powered study is warranted to further investigate the potential muscle relaxant and pain reduction effects of guaifenesin 1200 mg bid . results from this proof - of - principle study showed a directional change in benefit from placebo to lower and upper doses of guaifenesin for muscle spasm , pain , tension , discomfort , and relaxation , suggesting that the otc dose of guaifenesin 1200 mg bid may potentially provide symptom relief from upper back , neck , and shoulder musculoskeletal pain and discomfort . gasd , gahf , and gahi at day 7 ( itt population ) notes : bid , twice daily ; placebo-600 , placebo matched to guaifenesin 600 mg bid ; placebo-1200 , placebo matched to guaifenesin 1200 mg bid . abbreviations : gasd , global assessment of sleep disturbance ; gahf , global assessment of headache frequency ; gahi , global assessment of headache intensity ; itt , intent - to - treat .

background / objectiveguaifenesin , an over - the - counter ( otc ) expectorant , has exhibited muscle relaxant effects preclinically and clinically . this proof - of - principle study explored whether otc doses of guaifenesin can provide relief from acute upper back , neck , or shoulder muscle spasm and pain.methodsthis multicenter , placebo - controlled , repeat - dose , parallel study randomly assigned adults experiencing acute pain and muscle spasm in their upper back , neck , or shoulder to guaifenesin 600 or 1200 mg or matched placebo twice daily ( bid ) in a 2:2:1:1 ratio for 7 days . the primary end point was the change from baseline in muscle spasm relief , measured using an 11-point numeric rating scale ( 0=not present to 10=unbearable ) recorded twice daily and averaged over the 7-day treatment period . analyses were performed using a linear mixed model that included treatment as a fixed effect and site as a random effect.resultsa total of 77 subjects were included in the 4 treatment groups . least squares mean muscle spasm score over 7 days was 1.77 with guaifenesin 1200 mg , 1.42 with its matched placebo , 1.53 with guaifenesin 600 mg , and 1.74 with its matched placebo . treatment with guaifenesin 1200 mg bid provided 25% greater reduction in mean muscle spasm over its matched placebo and 16% greater reduction than guaifenesin 600 mg bid . these differences were not statistically significant . based on comparisons of absolute mean values , a consistent directional change in effect was observed , suggesting some benefit from placebo to lower - to - upper doses of guaifenesin with regard to muscle spasm , tension , pain , discomfort , and relaxation . no severe or serious adverse events were reported.conclusionresults suggest the potential for otc dose of guaifenesin 1200 mg bid to provide symptomatic relief of upper back musculoskeletal pain and spasm . confirmation of this preliminary result in a larger , adequately powered study is needed .

left ventricular thrombus ( lvt ) is one of the most common complications of myocardial infarction ( mi ) . the incidence of lvt in patients with acute anterior mis in the prethrombolytic era is ranged from 20% to 40% and with a non anterior acute myocardial infarction ( ami ) and the risk of lvt was < 5% . although controversial , in the contemporary era of routine early revascularization and more aggressive anticoagulation , the incidence of lvt complicating as an anterior ami is likely reduced and is currently estimated at 5 - 15% . the risk factors for the development of lvt are consistently irrespective of infarct treatment and include large infarct size , severe apical akinesia , or dyskinesia left ventricular ( lv ) aneurysm , and anterior mi . two - dimensional transthoracic echocardiography is the imaging modality used most often for assessing the presence , shape , and size of an lv mural thrombus with an excellent specificity of 85 - 90% and sensitivity of 95% . here , we report a case of 40-year - old male patient who is presented in emergency department with the complaint of substernal chest pain radiating to left arm and back associated with sweating for the last 1 day , there was no history of dyspnea , palpitation , any limb weakness , or any history suggestive of transient ischemic attack and peripheral embolization . his 12-lead electrocardiogram was suggestive of inferior wall mi [ figure 1 ] . his total leukocyte and eosinophil counts were normal , prothrombin time / international normalized ratio and activated partial thromboplastin time were normal , and other tests for hypercoagulable states were planned for the follow - up evaluation . a transthoracic echocardiogram was performed bedside which revealed hypokinesia of basal , mid inferior , and inferoseptal wall [ figure 2 and video 1 ] . surprisingly , it also revealed a mobile mass which is the most probably thrombus attached to hypokinetic inferobasal septum just near the lv outflow tract ( lvot ) [ figure 3 and video 2 ] . considering the possibility that this mass was high risk for embolism , we planned for coronary angiogram followed by the surgical extraction of possible thrombus . however , unfortunately , despite the proper counseling and explanation of the risk of embolism , the patient refused for further intervention and got discharged against medical advice . small arrows showing hypokinesia of basal inferior septum , large arrow indicates left ventricle probable thrombus apical four chambers view thrombi formation at basal interventricular septum near lvot is extremely rare as it is a region of high - velocity blood flow . traditionally , the causes of lvt formation after acute st - segment elevation mi include segmental dysfunction of the infracted myocardium resulting in the stasis of blood , endocardial tissue inflammation that provides a thrombogenic surface and a hypercoagulable state . the higher mortality has been reported in patients with lv thrombi after infarction , especially when these develop within the first 48 h after infarction . the treatment for cardiac mass is a prompt surgical resection of the mass with the patients placed on cardiopulmonary bypass . therefore , better understanding of the circumstances in which lv thrombosis occurs may influence the patient management . in our case , thrombus formed at the very unusual site and could lead to dreaded complication for the patient . the possible etiology of thrombus formation , in our case , is hypokinesia of basal septum due to mi , but the presence of a subtle septal rupture which is not detectable on echocardiography could not be ruled out .

left ventricular ( lv ) thrombus formation is a frequent complication in patients with acute anterior myocardial infarction ( mi ) . its incidence is lower with inferior wall mi . risk factors for the development of lv thrombus are consistently irrespective of infarct treatment and include large infarct size , severe apical akinesia or dyskinesia lv aneurysm , and anterior mi .

the number of cardiovascular implantable electronic device ( cied ) implantation has increased over the past few years . the conventional access of lead implantation is the subclavian vein , which is often accompanied by high complication rate , including pneumothorax , hemopneumothorax , inadvertent artery puncture , local hematoma , subclavian crush syndrome , and difficulty in lead operating . thus , the axillary vein puncture has been proposed as an alternative technique to the conventional subclavian vein access . however , the comparison between subclavian and axillary vein access through large - size sampled and randomized clinical trial is still limited . here , we proposed a randomized , two - armed , open - label study to analyze the efficacy and safety of optimized axillary vein puncture versus conventional subclavian puncture in cied implantation . patients aged 18 years or older with the indication of permanent pacemakers , cardiac resynchronization therapy pacemakers / defibrillators ( crt - p / crt - d ) , and implantable cardioverter defibrillators ( icd ) were included in the study . all eligible patients were asked to sign and date an informed consent document in person before randomized into different groups . patients undergoing lead replacement were excluded from the study . the study was reviewed and approved by the clinical research ethics board at china - japan friendship hospital and was registered to www.clinicaltrials.gov with trial number nct02358551 . according to a computer - generated randomization list , patients were randomized to the optimized axillary vein puncture group or the conventional subclavian vein puncture group at a ratio of 1:1 using an 8-sized block . the pulse generators were implanted on the surface of pectoralis major on the same side . operated chest walls were locally strapped for 24 h and pressed with sandbags for 4 h. operations in both groups were performed consistently by one skilled electrophysiology physician . is usually fixed , we used the 21-g needle to search for the axillary vein with x - ray fluoroscopy at the point where the first rib meets the clavicle . the direction of needle was toward the first anterior rib , with an angle of about 4560 between the needle and the skin . after the venous blood was drawn , a 0.018 inch j - shaped guidewire was put into the 21-g needle , and the 5 fr sheath ( cook group incorporated , bloomington , indiana , usa ) was inserted into the vein [ figure 1 ] . subsequently , both the 0.018 inch guidewire and 5 fr sheath were replaced by the standard 0.035 inch j - shaped guidewire and the peel - away electrode lead sheath ( biotronik , berlin , germany ) . if five attempts of puncture failed , an ipsilateral cubital vein angiography will be performed to observe the passage of axillary vein [ figure 2 ] and subsequently the same method of vein puncture should be used as stated above . the left arrow indicates the axillary vein and the right arrow indicates the subclavian vein . subclavian vein puncture ipsilateral cubital vein trocar was retained before the procedure . pacemaker pocket was created below the clavicle . we used the 18-g needle to search for the subclavian vein with x - ray fluoroscopy at the point of clavicle . the entry point of the needle was a one - third point to the sternoclavicular joint , and direction of the needle was toward the suprasternal fossa , with an angle of about 1530 between the needle and the skin . after the venous blood was drawn , a standard 0.035 inch j - shaped guidewire and the peel - away electrode lead sheath were inserted into the vein . if five attempts of puncture failed , an ipsilateral cubital vein angiography will be performed to observe the passage of subclavian vein and the same method of vein puncture should be used as stated above . success of puncture was defined as drawing of venous blood and insertion of guidewire and sheath . one - time success was defined as minor adjustment of needle direction and point of puncture , without withdrawing the needle . number of puncture needed , length of time in puncture and x - ray exposure , parameters of pacemakers including threshold , impedance , and p / r wave amplitude were recorded . patients were followed up at 1 , 3 , 6 , and 12 months after the operation and for every 12 months afterward . continuous variables were used to compare between groups by t - test for normally distributed values ; otherwise , mann - whitney u - test should be used . patients aged 18 years or older with the indication of permanent pacemakers , cardiac resynchronization therapy pacemakers / defibrillators ( crt - p / crt - d ) , and implantable cardioverter defibrillators ( icd ) were included in the study . all eligible patients were asked to sign and date an informed consent document in person before randomized into different groups . patients undergoing lead replacement were excluded from the study . the study was reviewed and approved by the clinical research ethics board at china - japan friendship hospital and was registered to www.clinicaltrials.gov with trial number nct02358551 . according to a computer - generated randomization list , patients were randomized to the optimized axillary vein puncture group or the conventional subclavian vein puncture group at a ratio of 1:1 using an 8-sized block . the pulse generators were implanted on the surface of pectoralis major on the same side . operated chest walls were locally strapped for 24 h and pressed with sandbags for 4 h. operations in both groups were performed consistently by one skilled electrophysiology physician . is usually fixed , we used the 21-g needle to search for the axillary vein with x - ray fluoroscopy at the point where the first rib meets the clavicle . the direction of needle was toward the first anterior rib , with an angle of about 4560 between the needle and the skin . after the venous blood was drawn , a 0.018 inch j - shaped guidewire was put into the 21-g needle , and the 5 fr sheath ( cook group incorporated , bloomington , indiana , usa ) was inserted into the vein [ figure 1 ] . subsequently , both the 0.018 inch guidewire and 5 fr sheath were replaced by the standard 0.035 inch j - shaped guidewire and the peel - away electrode lead sheath ( biotronik , berlin , germany ) . if five attempts of puncture failed , an ipsilateral cubital vein angiography will be performed to observe the passage of axillary vein [ figure 2 ] and subsequently the same method of vein puncture should be used as stated above . the left arrow indicates the axillary vein and the right arrow indicates the subclavian vein . subclavian vein puncture ipsilateral cubital vein trocar was retained before the procedure . pacemaker pocket was created below the clavicle . we used the 18-g needle to search for the subclavian vein with x - ray fluoroscopy at the point of clavicle . the entry point of the needle was a one - third point to the sternoclavicular joint , and direction of the needle was toward the suprasternal fossa , with an angle of about 1530 between the needle and the skin . after the venous blood was drawn , a standard 0.035 inch j - shaped guidewire and the peel - away electrode lead sheath were inserted into the vein . if five attempts of puncture failed , an ipsilateral cubital vein angiography will be performed to observe the passage of subclavian vein and the same method of vein puncture should be used as stated above . success of puncture was defined as drawing of venous blood and insertion of guidewire and sheath . one - time success was defined as minor adjustment of needle direction and point of puncture , without withdrawing the needle . number of puncture needed , length of time in puncture and x - ray exposure , parameters of pacemakers including threshold , impedance , and p / r wave amplitude were recorded . patients were followed up at 1 , 3 , 6 , and 12 months after the operation and for every 12 months afterward . continuous variables were used to compare between groups by t - test for normally distributed values ; otherwise , mann - whitney u - test should be used . a total of 247 patients were enrolled in this study between january 2013 and november 2015 , with no loss during follow - ups . among all the patients , 125 were randomized into the axillary vein group and 122 were randomized into the subclavian vein group . the sex and age distribution , body mass index , and types of cied were similar between the two groups . baseline characteristics of the study population * normally distributed continuous variables are presented as mean sd . : value ; : t value . cied : cardiovascular implantable electronic device ; crt - p / crt - d : cardiac resynchronization therapy pacemakers / defibrillators ; icd : implantable cardioverter defibrillator ; sd : standard deviation . patients in the axillary vein group were implanted with 231 pacing / defibrillation electrode leads , of which 221 punctures succeeded . patients in the subclavian vein group were implanted with 227 pacing / defibrillation electrode leads , of which 218 punctures succeeded . one - time success rate of both groups was 68.4% ( 158/231 ) and 66.1% ( 150/227 ) , respectively . the mean time spent in puncture of the axillary vein group was 45.9 14.1 s , whereas in the subclavian vein group , it was 28.7 13.9 s ( t = 9.679 , p < 0.001 ) . the duration of x - ray exposure was 150.1 33.6 s and 145.5 34.9 s , respectively [ table 2 ] . success rate and duration of the procedure * normally distributed continuous variables are presented as mean sd . the vein punctures failed in 11 patients , who all received cubital vein angiographies to observe the passage of axillary veins or subclavian veins . among them , three patients in the axillary vein group had total occlusion of veins and two had shifting veins ; two patients in the subclavian vein group had total occlusion of veins , one had vein stenosis , and three had shifting veins . we successfully performed the puncture under the guidance of angiography or performed the procedure on the opposite side . in the subclavian vein group , pneumothorax occurred in three patients . three patients subclavian gaps were too tight to allow operation of the electrode lead ( all of them had puncture repeated once to complete the operation ) . there were no puncture - associated complications in the axillary vein group . in either group , none had pocket hematoma or other severe complications . in a mean follow - up of 24.1 7.4 months , lead dislocations occurred in one patient in both groups , both of whom underwent lead replacements . pocket infection occurred in one patient in both groups , both of whom underwent pocket debridement and later were implanted with the cieds in the opposite side . two patients in the subclavian vein group had subclavian crush syndrome , both of whom underwent lead replacements . the remaining patients had no complications , and threshold , p / r wave amplitude , and impedance of their cieds were all in the normal range during the follow - ups . the complication rate in perioperative period and follow - ups of the axillary and subclavian vein group was 1.6% ( 2/125 ) and 8.2% ( 10/122 ) , respectively ( = 5.813 , p = 0.016 ) [ table 3 ] . complications in perioperative period and follow - ups a total of 247 patients were enrolled in this study between january 2013 and november 2015 , with no loss during follow - ups . among all the patients , 125 were randomized into the axillary vein group and 122 were randomized into the subclavian vein group . the sex and age distribution , body mass index , and types of cied were similar between the two groups . baseline characteristics of the study population * normally distributed continuous variables are presented as mean sd . : value ; : t value . cied : cardiovascular implantable electronic device ; crt - p / crt - d : cardiac resynchronization therapy pacemakers / defibrillators ; icd : implantable cardioverter defibrillator ; sd : standard deviation . patients in the axillary vein group were implanted with 231 pacing / defibrillation electrode leads , of which 221 punctures succeeded . the overall success rate was 95.7% . patients in the subclavian vein group were implanted with 227 pacing / defibrillation electrode leads , of which 218 punctures succeeded . one - time success rate of both groups was 68.4% ( 158/231 ) and 66.1% ( 150/227 ) , respectively . the mean time spent in puncture of the axillary vein group was 45.9 14.1 s , whereas in the subclavian vein group , it was 28.7 13.9 s ( t = 9.679 , p < 0.001 ) . the duration of x - ray exposure was 150.1 33.6 s and 145.5 34.9 s , respectively [ table 2 ] . success rate and duration of the procedure * normally distributed continuous variables are presented as mean sd . : the vein punctures failed in 11 patients , who all received cubital vein angiographies to observe the passage of axillary veins or subclavian veins . among them , three patients in the axillary vein group had total occlusion of veins and two had shifting veins ; two patients in the subclavian vein group had total occlusion of veins , one had vein stenosis , and three had shifting veins . we successfully performed the puncture under the guidance of angiography or performed the procedure on the opposite side . three patients subclavian gaps were too tight to allow operation of the electrode lead ( all of them had puncture repeated once to complete the operation ) . group , none had pocket hematoma or other severe complications . in a mean follow - up of 24.1 7.4 months , lead dislocations occurred in one patient in both groups , both of whom underwent lead replacements . pocket infection occurred in one patient in both groups , both of whom underwent pocket debridement and later were implanted with the cieds in the opposite side . two patients in the subclavian vein group had subclavian crush syndrome , both of whom underwent lead replacements . the remaining patients had no complications , and threshold , p / r wave amplitude , and impedance of their cieds were all in the normal range during the follow - ups . the complication rate in perioperative period and follow - ups of the axillary and subclavian vein group was 1.6% ( 2/125 ) and 8.2% ( 10/122 ) , respectively ( = 5.813 , p = 0.016 ) [ table 3 ] . common vein access for cied lead implantation was cephalic , subclavian , and axillary veins . subclavian vein puncture has faster learning curve , and the success rate is generally high . however , due to its anatomic characteristics , complications are relatively common , including pneumothorax , hemopneumothorax , inadvertent subclavian artery puncture , brachial nerve plexus injury , subclavian crush syndrome , and electrode lead fracture . a large - scale retrospective study found that cephalic vein access was related with lower rate of lead failure . however , it requires vein incision which renders the operation more complicated and time consuming . moreover , the size of the axillary vein is relatively small , thus it often suffers from a high failure rate , especially with multiple leads . the number of implantations of icds and crt - ps / crt - ds has increased in the past few years , and cephalic vein puncture may not be an optimal procedure . anatomically , the axillary vein terminates at the lateral margin of the first rib and becomes the subclavian vein . its passage is outside the clavicle and far from the cupula pleurae , and the diameter is larger compared with the cephalic vein with little variation . therefore , the axillary vein puncture avoids nerve or pleura injury and subclavian crush syndrome , which ensures a high success rate . however , these maneuvers are complicated and have steep learning curves , which limit the clinical application . belott reported an approach based on anatomic landmark on body surface and radiograph , where the axillary vein terminates and becomes the subclavian vein with little variation . overall , axillary vein punctures guided by fluoroscopic landmark , contrast venography , or ultrasound have shown high success rate and low complication rate . in this trial , furthermore , we used the 21-g needle instead of the routine 18-g needle , which reduced tissue injury as well as the risk of artery injury and pneumothorax . in this study , the overall success rate and one - time success rate of axillary and subclavian vein puncture were similar . no pneumothorax or difficulty on lead operating occurred in the operation and no subclavian crush syndrome occurred in the follow - ups , which confirmed the efficacy and safety of this technique . no pocket hematoma or other severe complications occurred in either group , probably due to the operator 's experience and the use of electrotome . the duration of procedure in the axillary vein group is longer than the subclavian group , probably because the axillary vein puncture requires more fluoroscopy guidance and searching in local area . in addition , the axillary vein puncture included an extra procedure : exchange of guidewire and sheath , which was time consuming . the relatively steep learning curve of axillary vein puncture is an issue , and innovations in the axillary vein puncture have been proposed recently , such as using the cephalic vein as anatomic landmark , introducing guidewire retrogradely from the femoral vein up to the left axillary vein as roadmap , and utilizing the caudal fluoroscopic view . first , the relatively short follow - up period is not sufficient to evaluate long - term efficacy . second , it was a single - centered trial and all the procedures were performed by one operator , thus interpretation and extrapolation of the results should be cautious . a multi - centered trial with larger scale and longer follow - ups is necessary to present more evidence of the advantage of the axillary vein puncture technique . in conclusion , optimized axillary venous approach may be superior to conventional subclavian vein approach for cied lead placement . this work was supported by grants from fund for clinical study of capital characteristic ( no . this work was supported by grants from fund for clinical study of capital characteristic ( no .

background : the conventional venous access for cardiovascular implantable electronic device ( cied ) is the subclavian vein , which is often accompanied by high complication rate . the aim of this study was to assess the efficacy and safety of optimized axillary vein technique.methods:a total of 247 patients undergoing cied implantation were included and assigned to the axillary vein group or the subclavian vein group randomly . success rate of puncture and complications in the perioperative period and follow - ups were recorded.results:the overall success rate ( 95.7% vs. 96.0% ) and one - time success rate ( 68.4% vs. 66.1% ) of punctures were similar between the two groups . in the subclavian vein group , pneumothorax occurred in three patients . the subclavian gaps of three patients were too tight to allow operation of the electrode lead . in contrast , there were no puncture - associated complications in the axillary vein group . in the patient follow - ups , two patients in the subclavian vein group had subclavian crush syndrome and both of them received lead replacement . the incidence of complications during the perioperative period and follow - ups of the axillary vein group and the subclavian vein group was 1.6% ( 2/125 ) and 8.2% ( 10/122 ) , respectively ( 2 = 5.813 , p = 0.016).conclusion : optimized axillary vein technique may be superior to the conventional subclavian vein technique for cied lead placement.trial registration : www.clinicaltrials.gov , nct02358551 ; https://clinicaltrials.gov/ct2/show/nct02358551?term=nct02358551 & rank=1 .

in view of growing awareness of the side - effects of some orthodox medicines , people are turning more towards alternative medicines with fewer and less - toxic side - effects , and homeopathy has become a major complementary and alternative medicine ( cam ) in many countries today . in homeopathy , microdoses of very high dilutions ( potentized ) of natural substances are generally preferred over mother tinctures ( crude extracts ) [ 2 - 4 ] for stronger and longer - effects . the initial drug substance is generally dissolved in an aqueous solution of ethanol ( mostly 70% ) and is potentized in gradual steps of dilution with agitation or succussion . on a centesimal scale , when 1 ml of mother tincture is diluted with 99 ml of an aqueous solution of ethanol ( vehicle of drug ) and given 10 mechanical jerks , potency 1c is produced . when 1 ml of 1c is again diluted with 99 ml of an aqueous solution of ethanol and given 10 jerks , the potency 2c is produced , and so on . therefore , when the drug has attained potency 12c , it has been diluted to 10 ( beyond avogadro s limit ) , and the existence of even a single molecule of the original drug substance becomes highly improbable . although some researchers have demonstrated the existence of nanoparticles of the original drug in such ultra - highly diluted homeopathic drugs [ 5 , 6 ] , the efficacy is often questioned by rationalists , as the precise mechanism of drug action has still not been firmly established . therefore , we became interested in the study of any perceivable differences in the actions between dilutions below avogadro s limit ( 6c , diluted 10 times ) and above avogadro s limit ( 30c , diluted 10 times ) in living cells , in vitro , and we attempted to understand the possible signalling pathway of their action . non - small cell lung cancer ( nsclc ) is the most prevalent accounting for - 80% of all lung cancer cases . in addition , benzo[a]pyrene is one of the major polycyclic aromatic hydrocarbons found in cigarette smoke and is responsible for inducing lung tumours in smokers . patients with lung cancer are commonly treated with conventional modalities including chemotherapy , radiation therapy , etc . which also affect normal cells . , are now gaining importance in the cure / amelioration of many difficult- to - cure diseases including cancer . first , do the two potentized homeopathic drugs , condurango 6c and 30c show any ability to induce apoptosis in nsclc , and do they act via reactive oxygen species ( ros ) generation and mitochondrial membrane potential ( mmp)-depolarization . second , dose if condurango 30c have more apoptosis - inducing ability than condurango 6c . nci - h460 ( h460 ) human nsclc cells were procured from national centre for cell science ( nccs ) , pune , india and cultured in rpmi-1640 media , supplemented with 10%-fetal bovine serum ( fbs ) and 1%-antibiotic - antimicotic solution . h460 cells ( 110 /well ) were treated with different concentrations of condurango 6c and 30c ( 0.5l/100l media- 5l/100l media ) and with placebos ( successed 70% alcohol - vehicle for the drugs ) as a control for 24 hours and 48 hours . the concentrations at which both the drugs showed nearly 50% cell death were determined by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide ( mtt ) assays . h460 cells were also treated with half maximal inhibitory concentration ( ic50 ) doses of condurango 6c and 30c and were compared against placebo - treated and untreated cells . after 24 hours and 48 hours of treatment , the cells were observed and photographed under an inverted phase - contrast microscope ( axiscope plus 2 , zeiss , germany ) . for further confirmation of morphological changes , if any , a scanning electron microscopy ( sem ) study was done using an s530-hitachi scanning electron microscope . reactive oxygen species ( ros)-accumulation is generally known to occur at early hours of apoptosis . cells were treated with condurango 6c and 30c ( ic50 doses ) and with their respective placebos for 2 hours , 6 hours , 12 hours , 18 hours , and 24 hours , to estimate the specific time - point at which maximum ros accumulated . after treatment , cells were incubated with 2,7-dichlorodihydrofluorescein diacetate ( h2 dcfda ) ( 5 mm ) and ros was estimated by using fluorimetry ( perkinelmer , usa ) . fluorescence microscopy was done at the specific time - point(s ) when ros - generation was maximum . the changes in mmp were recorded at 2 hours , 6 hours , 12 hours , 18 hours , and 24 hours of treatment with ic50 doses of both drugs were compared against drug - untreated cells by using rhodamine-123 and the cells were analyzed by using fluorescence microscope ( leica , dmls ) . changes in mmp were also measured using flowcytometry ( facs , aria iii , bd bioscience ) at the specific time - point(s ) at which the mmp showed the maximum decrease . cells were stained separately with 4,6-diamidino-2-phenylindole ( dapi ) ( 10gml ) and acridine orange / ethidium bromide ( ao / eb ) ( 1mg / ml ) to visualize changes in nuclear morphology . after 48 hours of treatment , stained cells were observed and photographed under a fluorescence microscope ( leica , dmls ) . dna - fragmentation was assayed using the conventional phenol / chloroform method and was visualized under a uv - transilluminator ( ultracam digital imaging , genei , india ) . dna strand breakage was analysed flowcytometrically by labeling the treated and the untreated cells with 5-bromo- 2-deoxyuridine 5-triphosphate ( br - dutp ) by using bd facs verse . the percent of cells in each phase ( subg0/g1 , g0/g1 , s , and g2/m ) was quantified flowcytometrically using bd facs verse . rt - pcr analysis was done using primers of bax , bcl2 , cytochrome - c , caspase-3 and gapdh . the cells were treated with ic50 dose of both drugs for 18 hours in the case of bax - bcl2 , 24 hours for cytochrome - c , and 48 hours for caspase-3 and poly ( adp - ribose ) polymerase ( parp ) . the protein activities were measured by using indirect- enzyme linked immunosorbent assay ( elisa ) and western blot analyses . quantifications of developed proteins after the western blot analyses were done densitometrically by using image j software . localization of caspase-3 was done by using an immunofluorescence study and was photographed under fluorescence microscope . the observers were blinded during observation as to whether they were observing the control and/or drug - treated materials . data were analyzed , and the signicance of differences between the mean values was determined by using a one - way analysis of variance ( anova ) with fisher s least significant difference ( lsd ) post hoc tests by using spss 14-software ( spss inc , chicago , il , usa ) . ic50 values for 48 hours treatment with condurango 6c ( 3.57l/100l ) and condurango 30c ( 2.43l/100l ) against h460 were selected for the entire study ( fig 1 ) the cell viability of both placebo - treated cells at maximum dose ( 5l/100l ) was found to be very close to that of the untreated ( control ) ones . cells were exposed to condurango 6c and 30c for 24 hours and 48 hours ( 0.5l/100l-5l/100l dose ) with respective placebos ( 5l/100l ) . results are expressed as mean percent of cell viability standard deviation ( sd , n = 6 ) . signicance levels are presented as,*p < 0.05 vs. untreated cells andp < 0.05 vs. both placebo - treated cells . the light microscopy ( fig . 3 ) studies revealed that cell morphologies remained unaltered with intact cell membrane and cellular extensions in both untreated and placebo - treated cells . however , condurango 6c - treated cells showed gradual deformation with cellular shrinkage ; condurango 30c - treated cells became gradually smaller and rounded with distorted nuclei and damaged cellular extensions , depicting the great apoptosis - inducing potential of condurango 30c . the fluorescence intensity in optical density ( od ) was too low in untreated and placebo - treated cells at different hour intervals . a significant increase in the fluorescence intensity was observed at 18 hours in both condurango 6c and 30c - treated cells ( fig . fluorescence microscopy showed a greater h2 dcfda - intensity in drug - treated cells at 18 h ( fig . signicance levels are presented as * p < 0.05 and * * * p < 0.001 between untreated and condurango 6c and 30c - treated cells and between untreated and placebo - treated cells . results showed up - regulation and downregulation of bax and bcl2 , respectively , at 18 hours in response to both drug treatments as compared to drug - untreated cells ( fig . condurango 30c - treated sample showed significantly increased bax and decreased bcl2-expressions than condurango 6c and placebo - treated samples . signicance * p < 0.05 untreated ( ut ) vs condurango 6c and 30c , p < 0.05 placebo ( 6c ) vs condurango 6c andp < 0.05 placebo ( 30c ) vs condurango 30c . figure 6a shows bright green fluorescence in drug - un - treated and cells at different time - points while condurango 6c and 30c - treated cells showed a gradual decrease in the fluorescence intensity with increasing time , but a marked decrease occurred at 24 hours , suggesting that mmp depolarizes the maximum at 24h that trigger cytochrome- c release from mitochondria . an increased expression of cytochrome - c was found in response to drugs at 24 hour of treatment ( fig . condurango 30c - treated cells showed more significant upregulation of cytochrome - c than condurango 6c - treated ones . signicance levels are presented as * p < 0.05 untreated ( ut ) vs condurango 6c and 30c , p < 0.05 placebo ( 6c ) vs condurango 6c andp < 0.05 placebo ( 30c ) vs condurango 30c . a gradual increase in the dapi - fluorescence intensity was observed in condurango 6c and 30c - treated cells at 48 hours of treatment ( fig . further , ao / eb- staining showed both a change in the fluorescence pattern from green ( normal - cellular dna ) to orange ( nicked - cellular dna ) and an increase in the fluorescence of eb in drug - treated cells ( fig . figure8c shows the formation of dna - laddering in both drug - treated samples , especially in condurango 30c - treated samples , compared to the dna in the drug - untreated groups . a gradual increase in the number of dutp - nicks was noted in drug - treated cells compared to drug - untreated cells ( fig . this strongly suggests the abilities of both drugs to generate dna - nicks that induce apoptosis . quantitative evaluation revealed the percent of tunel - positive nuclei was greater in condurango 30c - treated cells . the sub - diploid cell population at subg0/g1 was more highly increased in the condurango 6c and 30c - treated cells than in the placebo - treated and untreated cells ( fig . rt - pcr and elisa data showed upregulation of caspase-3 after condurango 6c and 30c treatment as compared to untreated samples ( fig . the western blot analysis revealed that condurango 30c had more capacity to increase caspase-3 expression significantly by forming cleaved fragments ( 20kda and 12kda ) . p < 0.05 untreated ( ut ) vs condurango 6c and 30c , p < 0.05 placebo ( 6c ) vs condurango 6c andp < 0.05 placebo ( 30c ) vs condurango 30c . the immunofluorescence study demonstrated a gradual increase in the localization of caspase-3 within cell - cytosol in drug - treated cells , especially in condurango 30c - treated cells ( fig . parp activation , the end process of the caspase-3-mediated pathway is designated by the formation of two cleaved fragments of 116kda ( inactive ) and 89kda ( active ) . the western blot analysis showed the formation of two fragments in condurango 6c and 30c - treated cells at 48 h of treatment , which was nearly absent in drug - untreated samples ( fig . band - intensities confirmed gradual down - regulation of the 116kda fragment and significant up - regulation of the 89kda fragment , especially in the condurango 30c - treated cells . present findings would demonstrate that exposure of nsclc- h460 cells to ic50 doses of both condurango 6c and 30c for 48 hours resulted in apoptotic cell - death . further , the effects of condurango 30c , which actually was highly- diluted , caused relatively more palpable alterations in all parameters of this study than did condurango 6c . this phenomenon did not apparently follow the general pharmacological rule that the effect of a drug increases linearly with its concentration , but was in line with the claim of the higher the dilution , the stronger the effect as per homeopathic doctrine . incidentally , apparent evidence for discernible effects produced by two dilutions , one below and one above avogadro s limit an enigma to many rationalists who believe in the accepted laws of physical sciences , as well as pharmacological sciences . however , claims are accumulating that homeopathic potencies beyond avogadro s limit show demonstrable beneficial/ curative effects against different diseases including cancer [ 20 - 22 ] . in fact , our earlier in vivo findings on benzo[a ] pyrene - induced lung cancer in rats also convincingly demonstrated the ameliorative effect of condurango 30c , for which we tried to verify that the potentized remedy showed anti - cancer potential in nsclc cells . apoptosis induction in cancerous cells is often targeted as one of the key events of cancer chemotherapy . thus , one approach was to look at the generation of ros and to ascertain if it had a specific role in the induction of apoptosis . the time - course studies showed elevation of ros , mmp - depolarization and cytochrome - c release at 18 hours through 24 hours of treatment , indicating that these events were earlier than apoptotic execution at 48 hours . our result for cytochrome - c release at 24 hours of treatment further suggested that these events occurred due to an alteration in the mitochondrial structure that could trigger apoptosis . along with mitochondrial dysfunctions , ros generation may be initiated at early hours of drug exposure via bax- bcl2 modulation . in this study , we found increased and decreased expression of bax and bcl2 , respectively , at 18 hours in the drug - treated series . however , surprisingly bax - bcl2 expressions were more significant in the condurango 30c - treated samples , which is a good indication of the higher efficacy of a more highly diluted homeopathic drug . another indication of apoptosis is internucleosomal dna breakdown . results of dapi and ao / eb - staining would suggest the formation of dna nicks , as further confirmed by dna - laddering in drug - treated cells . tunel - positivity in both drug - treated cells as compared to placebo- treated cells would further strengthen ros - dependent dna - breakage - mediated apoptotic events . interestingly quantitatively more dna damage was found in condurango 30c - treated cells , than was found in condurango 6c . increased ratio of bax - bcl2 is well known to stimulate the release of cytochrome - c from mitochondria to promote activation of caspase-9 that binds to apaf-1 to lead to caspase-3 and parp - activation . results provide clear indication of caspase-3 activation in drug - treated cells . increased expression and formation of cleavage of caspase-3 and parp after drug exposure confirmed apoptosis induction via caspase-3-mediated- intrinsic pathway . however , noticeably , condurango 30c - treated cell showed more significant modulation of caspase-3 and parp cleavage than did condurango 6c - treated cells . these strongly support again the idea that more potentized condurango 30c had greater ability to induce apoptosis in nsclc - h460 . if the similar results are taken into consideration , one possible conclusion is that homeopathic drugs may act by interacting with certain high affinity receptors that regulate expressions of specific genes , but more work is necessary to identify some missing links . the present study has gain significance because it attempts to determine if potentized forms of condurango , below ( 6c ) and above ( 30c ) avogadro s limit , which are occasionally used to treat digestive problems and stomach cancer [ 22 , 24 ] , had abilities to inhibit lung cancer progression , in vitro . the various changes noted in this study can only be brought about by the activities of certain genes and by epigenetic modifications , which would support the hypothesis [ 3 , 23 ] that ultra - highly - diluted drugs might somehow manage to correct expressions of relevant genes , the regulation of which had failed in cancer cells . interestingly , many factors , including the presence of nanoparticles [ 5 , 25 ] , interaction between containers ( e.g. , silica from glass or a polymer ) and drug molecules , etc . , are now under scrutiny and are implicated in modifications of the structural orientation , the size , and the physico - chemical properties of resultant homeopathic drugs even in absence of any original molecule [ 6 , 26 ] . the overall results of this study suggest that condurango 30c has more apoptosis - inducing ability than condurango 6c , which is consistent with the claim made in the homeopathic doctrine .

objectives : in homeopathy , it is claimed that more homeopathically - diluted potencies render more protective / curative effects against any disease condition . potentized forms of condurango are used successfully to treat digestive problems , as well as esophageal and stomach cancers . however , the comparative efficacies of condurango 6c and 30c , one diluted below and one above avogadro s limit ( lacking original drug molecule ) , respectively , have not been critically analyzed for their cell - killing ( apoptosis ) efficacy against lung cancer cells in vitro , and signalling cascades have not been studied . hence , the present study was undertaken.methods:3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylte-trazolium bromide ( mtt ) assays were conducted on h460-non - small - cell lung cancer ( nsclc ) cells by using a succussed ethyl alcohol vehicle ( placebo ) as a control . studies on cellular morphology , cell cycle regulation , generation of reactive oxygen species ( ros ) , changes in mitochondrial membrane potential ( mmp ) , and dna - damage were made , and expressions of related signaling markers were studied . the observations were done in a blinded manner.results:both condurango 6c and 30c induced apoptosis via cell cycle arrest at subg0/g1 and altered expressions of certain apoptotic markers significantly in h460 cells . the drugs induced oxidative stress through ros elevation and mmp depolarization at 18 - 24 hours . these events presumably activated a caspase-3-mediated signalling cascade , as evidenced by reverse transcriptase- polymerase chain reaction ( rt - pcr ) , western blot and immunofluorescence studies at a late phase ( 48 hours ) in which cells were pushed towards apoptosis.conclusion:condurango 30c had greater apoptotic effect than condurango 6c as claimed in the homeopathic doctrine .

crestal incision placed about 1.5 mm palatal to the greatest bony height with an angulation of 45 with no . 15 bard parker blade [ figure 1 ] a vertical releasing incision placed mesially and distally to the crestal incision without disturbing the adjacent teeth papilla and extending about 3 mm labially [ figure 2 ] soft tissue reflected to expose the cover screw [ figure 3 ] palatal extension of the incision followed closer to the adjacent teeth on both sides , and a release incision made around the palatal portion of the exposed cover screw [ figure 4 ] then healing abutment is placed after thoroughly cleaning the soft tissue around internal hex of the implant [ figure 5 ] soft tissue approximated with 40 suture material ( vicryl)sutures removal has to be done after 10 daysafter 2 weeks , second stage prosthetic procedure should be carried out . crestal incision placed about 1.5 mm palatal to the greatest bony height with an angulation of 45 with no . 15 bard parker blade [ figure 1 ] a vertical releasing incision placed mesially and distally to the crestal incision without disturbing the adjacent teeth papilla and extending about 3 mm labially [ figure 2 ] soft tissue reflected to expose the cover screw [ figure 3 ] palatal extension of the incision followed closer to the adjacent teeth on both sides , and a release incision made around the palatal portion of the exposed cover screw [ figure 4 ] then healing abutment is placed after thoroughly cleaning the soft tissue around internal hex of the implant [ figure 5 ] soft tissue approximated with 40 suture material ( vicryl ) sutures removal has to be done after 10 days after 2 weeks , second stage prosthetic procedure should be carried out . incision and flap outlined ( after reflection ) more importantly it plays an important and crucial role in an esthetic component of a natural dentition . hence , it is very vital to maintain the integrity of the papilla during second stage implant surgery . several methods with unique incisions and suturing designs have been proposed to regenerate interdental papilla around implants . palacci suggested that a full thickness flap be raised from the buccal and palatal side of the implant on the ridge and rotated 90 to accommodate the interproximal space of the implant . palatal sliding strip flap to form papilla between implants and natural dentition in the anterior maxilla . nemcovsky et al . suggested an u- shaped incision , with opening toward the buccal aspect of the implant site with divergent incisions . tinti and benfenati reported a ramp mattress suture design , which pulled the buccal flap coronally , to obtain a papilla between two implants . suggested a split finger surgical method in which three interlacing finger like incisions were made and each of the fingers was sutured over the desired interdental papillary position . proposed a method for papilla regeneration wherein the healing abutment is buried beneath the gingival flap to produce a dead space with expectation of the space being filled by the soft tissue . even though these procedures provided papillary height , the results were not predictable . various factors were involved in the success of any papilla regeneration procedures , such as preservation of blood supply , prevention of scar formation , and more importantly ideal placement of the implant into the bone . this new and innovative surgical technique provided significantly excellent results compared to the conventional mid - crestal technique [ figures 6 and 7 ] . in this case report , in order to overcome the limitations of above - mentioned techniques a novel window technique was followed to regenerate papilla around implants during second stage surgery prior to prosthetic restoration . unlike most other procedures mentioned above , this technique avoided the labial papilla on the adjacent teeth and involved wholly the palatal papilla , which is considered to be the key element in the success of this technique . moreover , there is no cross incisions on to the papilla , which may lead to , disturbed wound healing and scar formation . above all avoiding more invasive surgery is considered one of the best methods of soft tissue regeneration , which is being incorporated in this technique . the major advantages of this technique , when compared with earlier ones , are minimal invasive incision , less postoperative discomfort , and excellent esthetic and emergence profile obtained . the aim of any soft tissue procedure should be to obtain a resilient and scar less soft tissue . this can be obtained with atraumatic tissue handling , minimal tension during approximation , adequate vascularity , adequate hydration , and expedient surgical procedures that do not compromise blood supply . this case report shows the excellent results following the above requisites to obtain esthetic soft tissue profile . the patients were followed for 2 years at regular intervals and still the esthetic results obtained during the surgery and after 2 years were the same [ figure 8 ] . with above considerations with respect to follow - up and esthetic component , this technique provided the required and desired results . in order to obtain esthetic papillary height and tissue integrity around implants proper and meticulous implant positioning is needed . being a long term follow - up report this technique can be considered as a simple , minimal invasive procedure , which can be followed during 2 stage surgery .

reconstructing predictable and esthetic papilla is the most complex and challenging aspect of implant dentistry . to obtain an esthetic and predictable gingival architecture and implant restoration , interdental papilla plays an important role . the main objective of the surgeon during the second stage of implant treatment should be the creation of interdental papilla prior to prosthetic restoration . the aim of this case report was to demonstrate a novel window technique for developing predictable and esthetic papilla around dental implants , which was followed for 2 years with excellent esthetic results .

periprosthetic joint infection ( pji ) is a devastating complication seen in total joint arthroplasty ( tja ) patients . it could lead to prolonged hospitalization , repeated surgical intervention , significant permanent deformity , or even definitive loss of the implant . the symptoms of pji are often nonspecific , which makes the diagnosis of pji quite challenging . in caring for a painful joint arthroplasty , the ability to distinguish between septic and aseptic failures of the prosthesis is critical as the treatment for pji necessitates unique surgical strategies that aim to eradicate the infecting organism(s ) . traditionally , the hematological diagnosis of pji is performed by measuring inflammatory factors of white blood cell ( wbc ) levels , erythrocyte sedimentation rate ( esr ) , and serum c - reactive protein ( crp ) . in addition , microbiology analysis of synovial fluid and periprosthetic tissue using histology and synovial fluid culture and imaging tests such as enhanced computed tomography bone scanning , magnetic resonance imaging , and positron emission tomography are also used . however , some of these results are nonspecific for pji , and the test results have to be combined with the clinical history and symptoms ; thus , a more specific and sensitive routine for pji diagnosis is required . to address the inconsistency of different tests , the american academy of orthopedic surgeons ( aaos ) published their first guideline in 2010 as a reference for the diagnosis of pji , in which esr and crp were used as screening tests and joint aspiration should be performed when the level of these markers are elevated . then , in 2012 , the musculoskeletal infection society ( msis ) renewed with a consensus statement providing a concise definition of a pji . although the msis definition provides a standard for definitive retrospective diagnosis and research , its complexity makes it difficult to use in daily clinical practice . the ideal method of pji diagnosis would be a single test or panel that is highly sensitive , specific , and simple to interpret . in recent years , researches on pji diagnosis have started to focus on synovial fluid instead of serum , since synovial fluid is the site of primary infection , and the diagnosis should be more sensitive than that of serum theoretically . studies have revealed that number of antimicrobial peptides and inflammatory cytokines including crp , interleukin ( il)-1 , il-6 , il-17a , interferon- , tumor necrosis factor- , defensin , cathelicidin ll-37 , and so on in synovial fluid could be used as biomarkers for diagnosis . crp is a protein that has long been measured in the serum as an indicator of infection , and it has already become a well - known protein in the orthopedic community . until now , several studies have suggested that the crp levels in synovial fluid may serve as a simple and cost - effective means for improving the diagnosis of pji as the local crp is thought to enhance complement activation and phagocytosis . however , there are also studies concluded that crp from synovial fluid does not offer a diagnostic advantage in the detection of pjis after comparing the value of synovial fluid crp with serum crp . to address this controversy , we believe that establishing a prompt , precise , and convenient diagnostic guideline based on current evidence , consensus , expert opinions , and reviews is necessary . therefore , in this study , we aim to conduct a meta - analysis to investigate the diagnostic accuracy of synovial fluid crp for diagnosing pji . ultimately , this will improve the management of patients with pji as an effective treatment of pji requires an accurate and quick diagnosis . to the best of our knowledge , our study is the first meta - analysis that evaluates the clinical utility of synovial fluid crp in the diagnosis of pji . in our study , we compared the diagnostic performance of synovial fluid crp with the consensus - based guidelines . the methodological approach to evidence searching and synthesis described in this protocol was based on the cochrane collaboration 's diagnostic test accuracy methods . in our study , we performed a literature search , screened the studies identified , and selected the studies that meet the eligibility criteria . we then extracted the data from the selected studies and assessed the eligible studies by means of the revised quality assessment of diagnostic accuracy studies ( quadas-2 ) criteria . statistical analysis , evidence synthesis , and report compilation were carried out as the steps below . we strictly adhered to standards of the preferred reporting items for systematic reviews and meta - analyses in reporting the findings of this review . we searched the electronic databases including pubmed , embase , web of science , the cochrane library , and science direct for entries recorded from the time of database inception to december 2015 . prosthesis - related infections to represent the disease , synovial fluid or fluid , synovial to represent the source of our target biomarker , and c - reactive protein or protein , c - reactive as our target index . studies that were related with patients suffering from the hip , knee , and shoulder joint arthroplasties or investigated our target biomarker were included . therefore , animal - only studies and studies that do not report data on the diagnostic performance of our target index were excluded . screening was performed in a two - step process : title / abstract screening and full - text screening . two researchers independently reviewed the title and abstract of each assay to select those that were likely for further screening . in the initial stage of the screening , 1015 articles should be used to reach acceptable levels of agreement among the researchers . when confronted with disagreements , two researchers had to come to a consensus about the screening methods . following full - text screening , a list of excluded studies with reasons for exclusion was presented . inclusion criteria were as follows : patients who have undergone knee , hip , or shoulder joint replacements ; sufficient synovial fluid had to be aspirated for study method and crp of synovial fluid was detected ; the diagnosis of pji was confirmed by msis or aaos ; sufficient data can be extracted for the construction of a 2 2 contingency table . exclusion criteria were as follows : unrelated biomarkers , crp of the serum , not synovial fluid ; insufficient data to calculate sensitivity and specificity ; case reports , commentaries , expert opinion , and narrative reviews ; duplicates . the methodological quality of the included studies was appraised by an adapted version of the quadas-2 , which consisted of four key domains that discussed patient selection , index test , reference standard , and flow and timing . risk of bias assessment of the four domains and clinical applicability of the first three domains were assessed with signaling questions . questions were answered as yes for low risk of bias / concerns , no for high risk of bias / concerns , or the following information was extracted : ( 1 ) study characteristics including author , year of publication , country , design , sample size , and number analyzed for each study ; ( 2 ) population characteristics including patients mean age , sex ; ( 3 ) intervention characteristics including method of sampling , method of measuring , and threshold ; ( 4 ) gold standard including msis or aaos ; ( 5 ) outcomes including false / true positive , false / true negative from 2 2 table for diagnostic studies , sensitivity and specificity , positive likelihood ratio ( plr ) , and negative likelihood ratio ( nlr ) . data were extracted by a single reviewer with all outcomes and then verified by the other reviewers . for all the studies from which we constructed the 2 2 table , pooled sensitivity , specificity , plr , nlr , and the diagnostic odds ratio ( dor ) were calculated using the bivariate model . the summarized receiver operating characteristic ( sroc ) curve was constructed . in diagnostic test , if there were more than one threshold in an article , the threshold with the largest youden index was chosen . the percentage of the total variation across studies was described by the i statistic , which indicated the existence of significant heterogeneity when the value exceeded 50% . the value of i ranges from 0 to 100% , with 0 implying no observed heterogeneity , and larger values indicating increasing heterogeneity . the random effects model was chosen due to the expected clinical and statistical heterogeneity among the studies . all analyses were conducted using meta - disc software ( version 14.0 , unit of clinical biostatistics team , madrid , spain ) . the methodological approach to evidence searching and synthesis described in this protocol was based on the cochrane collaboration 's diagnostic test accuracy methods . in our study , we performed a literature search , screened the studies identified , and selected the studies that meet the eligibility criteria . we then extracted the data from the selected studies and assessed the eligible studies by means of the revised quality assessment of diagnostic accuracy studies ( quadas-2 ) criteria . statistical analysis , evidence synthesis , and report compilation were carried out as the steps below . we strictly adhered to standards of the preferred reporting items for systematic reviews and meta - analyses in reporting the findings of this review . we searched the electronic databases including pubmed , embase , web of science , the cochrane library , and science direct for entries recorded from the time of database inception to december 2015 . prosthesis - related infections to represent the disease , synovial fluid or fluid , synovial to represent the source of our target biomarker , and c - reactive protein or protein , c - reactive as our target index . studies that were related with patients suffering from the hip , knee , and shoulder joint arthroplasties or investigated our target biomarker were included . therefore , animal - only studies and studies that do not report data on the diagnostic performance of our target index were excluded . screening was performed in a two - step process : title / abstract screening and full - text screening . two researchers independently reviewed the title and abstract of each assay to select those that were likely for further screening . in the initial stage of the screening , 1015 articles should be used to reach acceptable levels of agreement among the researchers . when confronted with disagreements , two researchers had to come to a consensus about the screening methods . following full - text screening , a list of excluded studies with reasons for exclusion was presented . inclusion criteria were as follows : patients who have undergone knee , hip , or shoulder joint replacements ; sufficient synovial fluid had to be aspirated for study method and crp of synovial fluid was detected ; the diagnosis of pji was confirmed by msis or aaos ; sufficient data can be extracted for the construction of a 2 2 contingency table . exclusion criteria were as follows : unrelated biomarkers , crp of the serum , not synovial fluid ; insufficient data to calculate sensitivity and specificity ; case reports , commentaries , expert opinion , and narrative reviews ; duplicates . the methodological quality of the included studies was appraised by an adapted version of the quadas-2 , which consisted of four key domains that discussed patient selection , index test , reference standard , and flow and timing . risk of bias assessment of the four domains and clinical applicability of the first three domains were assessed with signaling questions . questions were answered as yes for low risk of bias / concerns , no for high risk of bias / concerns , or the following information was extracted : ( 1 ) study characteristics including author , year of publication , country , design , sample size , and number analyzed for each study ; ( 2 ) population characteristics including patients mean age , sex ; ( 3 ) intervention characteristics including method of sampling , method of measuring , and threshold ; ( 4 ) gold standard including msis or aaos ; ( 5 ) outcomes including false / true positive , false / true negative from 2 2 table for diagnostic studies , sensitivity and specificity , positive likelihood ratio ( plr ) , and negative likelihood ratio ( nlr ) . data were extracted by a single reviewer with all outcomes and then verified by the other reviewers . for all the studies from which we constructed the 2 2 table , pooled sensitivity , specificity , plr , nlr , and the diagnostic odds ratio ( dor ) were calculated using the bivariate model . , heterogeneity was commonly caused by threshold effect . when threshold effect existed , there was a negative correlation between sensitivity and specificity . if there were more than one threshold in an article , the threshold with the largest youden index was chosen . the percentage of the total variation across studies was described by the i statistic , which indicated the existence of significant heterogeneity when the value exceeded 50% . the value of i ranges from 0 to 100% , with 0 implying no observed heterogeneity , and larger values indicating increasing heterogeneity . the random effects model was chosen due to the expected clinical and statistical heterogeneity among the studies . all analyses were conducted using meta - disc software ( version 14.0 , unit of clinical biostatistics team , madrid , spain ) . of the identified 237 articles , 186 of which were excluded with the reasons of duplicates . among the left 51 articles , 40 were excluded after reading the title and abstract , reasons including the unqualified source of crp detected and inappropriate article type ( reviews , comments , or letters ) . after reading the whole 11 articles included , 4 were unqualified due to insufficient data , and 7 of which were considered suitable for systematic review . among these articles , one used improper cutoff value and was excluded , leaving 6 further analyzed for meta - analysis . graphical summary of the methodological assessment based on quadas-2 quality assessment for the 6 studies of meta - analysis is shown in figure 2 . . a total of 456 samples from patients who had undergone hip or knee joint replacement were included in the meta - analysis . all studies were conducted prospectively , and five of the studies took the synovial fluid samples before any clinical treatment while one did not mention . however , the cutoff value of synovial fluid crp varied in each study : 2.8 , 3.65 , 6.6 , 9.5 , and 12.2 mg / l , respectively . in addition , to determine synovial fluid crp , enzyme - linked immunosorbent assay ( elisa ) was used in two studies while turbid metric immunoassay or kinetic infrared immunoassay was used in the other four . as for the standard diagnosis , msis and aaos were both included since the details had an overlap with each other . detailed characteristics of individual study are summarized in table 1 , and detailed number of patients involved in each study and their diagnosis results are illustrated in table 2 . characteristics of studies included for meta - analysis ua : unavailable ; p : prospective study ; elisa : enzyme - linked immunosorbent assay ; rbm : rules - based medicine s human inflammation multianalyte profiling ; msis : musculoskeletal infection society ; aaos : academy of orthopedic surgeon s ; crp : c - reactive protein . data extracted for the construction of 2 2 table tp : true positive ; fp : false positive ; fn : false negative ; tn : true negative . for the included studies , the overall pooled sensitivity was 0.92 ( 95% confidence interval [ ci ] : 0.860.96 ) , and the pooled specificity was 0.90 [ 95% ci : 0.870.93 , figure 3a and 3b ] . the pooled plr and nlr were 9.00 ( 95% ci : 6.1513.16 ) and 0.10 [ 95% ci : 0.060.18 , figure 4a and 4b ] , respectively . the area under the sroc ( ausroc ) was 0.9663 [ standard error 0.0113 , figure 5 ] and the dor was 101.40 ( 95% ci : 48.07213.93 ) . spearman 's correlation coefficient ( 0.40 ) and the p value ( 0.60 ) , which represent threshold effect were tested for the between - study variability ( heterogeneity ) . we could also come to the same conclusion since there was no shoulder - like roc plane curve . the heterogeneity for sensitivity and specificity was tested through i range ( 0 and 26.8% , respectively ) . pooled sensitivity and specificity of crp in the diagnosis of pji . lr : likelihood ratio ; crp : c - reactive protein ; pji : periprosthetic joint infection . summary of sroc of crp in the diagnosis of pji . sroc : summarized receiver operating characteristics curve ; crp : c - reactive protein ; pji : periprosthetic joint infection ; se : standard error . summary results of bivariate model analysis sen : sensitivity ; ci : confidence interval ; spe : specificity ; plr : positive likelihood ratio ; nlr : negative likelihood ratio ; dor : diagnostic odds ratio ; sroc : summarized receiver - operating curve ; se : standard error . pji is currently one of the most common complications associated with tja and difficult to diagnose . the major reasons for this difficulty are the absence of specific clinical signs and symptoms , the relative lack of accurate laboratory tests , and low culture rate in isolation of pathogens due to prior therapy and formation of biofilms . the accurate definition of what constitutes pji is still controversial ; therefore , several orthopedic associations have established clinical guidelines for diagnosing pji . the msis recently responded to this diagnostic difficulty by developing a definition for pji . according to the msis , the diagnosis of pji definition requires positive result in either one of two major criteria ( sinus tract communication with a prosthesis or pathogen isolated by culture from two separate fluid samples ) or four of six minor criteria ( elevated esr , elevated crp , elevated wbc count , elevated percentage of polymorphonuclear neutrophils ( pmn ) , presence of purulence , and greater than five neutrophils per high - power field on frozen section ) . by comparison , aaos guideline is similar to msis , including the following four thresholds : esr > 30 mm / h , serum crp value > 10 mg / l , synovial wbc count > 1760 cells/l for chronic infection or 10,700 cells/l for acute infection , and synovial pmn differential percentage > 73% for chronic infection or greater than 89% for acute infection . although clinically useful , these definitions are complex and time - consuming , with the subjective interpretation of the frozen section histology and the delay in diagnosis of several independent culture results . on the contrary , synovial fluid aspirated from patients with joint replacement may provide researchers with a perfect source of pji diagnosis since host proteins with direct antimicrobial activity may play an important role in response to pathogen elimination . the promise of synovial fluid biomarkers to diagnose pji has been reported during the past few years ; however , the reference standard in some of these studies is not based on msis or aaos , which makes the comprehensive analysis of these studies more challenging . according to our search results , none of the articles so far have carried out a systematic review or meta - analysis about synovial fluid biomarkers in the diagnosis of pji and we consider it necessary to fill this gap . at the initial stage of our systematic study , we used the keywords such as biological biomarkers , inflammatory cytokines , and antimicrobial peptides in the research strategy to obtain as many highly correlated articles as possible . after reviewing searched studies , only two out of five qualified studies used synovial fluid il-6 as a biomarker for diagnosis of pji ; hence , the meta - analysis would not be accurate due to the limited numbers . as for -defensin , all qualified five studies came to the same conclusion that it was a biomarker with high sensitivity and specificity for the diagnosis of pji . based on the studies we have searched , opinion toward the diagnostic value of synovial fluid crp is still in debate ; thus , we focused on this biomarker . crp release is induced by the recognition of pathogenic patterns , playing several mechanistic roles in the innate immune response and is currently assayed in the serum as a common and inexpensive test to screen for the presence of pji in msis . however , elevated concentration of serum crp is nonspecific for the diagnosis of localized infection since crp is an acute - phase reactant in numerous noninfectious diseases . after quality assessment , six articles were highly qualified for our meta - analysis , four of which used msis as the reference standard and the rest used aaos as the reference standard . in our meta - analysis , we found that synovial fluid crp showed high sensitivity and specificity for the diagnosis of pji . pooled estimates of sensitivity and specificity were 0.92 and 0.90 , respectively . based on the low i ( 0 and 26.8% , respectively ) , spearman 's correlation coefficient with p > 0.05 , and inexistence of shoulder - like curve , we believe that the heterogeneity among studies is low . as for the diagnostic performance estimated by the summary roc , synovial fluid crp had a high ( area under the curve [ auc ] > 0.9 ) diagnostic ability to identify pji patients based on the suggested guidelines for the interpretation for the ausroc . the dor of our pooled analysis is 101.40 , indicating a high diagnostic value of synovial fluid crp in pji diagnosis . of seven articles screened for systematic review , the one carried out by deirmengian et al . was not included in meta - analysis due to the use of an improper cutoff value . in the article however , crp was used solely as a complementary biomarker and evaluated through roc analysis with the purpose of improving the specificity of -defensin assays used . as part of the combined algorithm , after the detection of both -defensin and crp , the false positive -defensin results could be reversed to true negative , which meant this crp cutoff value was decided only for the false positive samples . therefore , the crp cutoff value used was relatively low and the generated 2 2 table was not suitable for the meta - analysis in this study . two studies conducted by parvizi et al . came to the conclusion that future investigations are needed to confirm their findings in a larger cohort . we performed this meta - analysis with the primary aim of enlarging the number of samples , which is in accordance with the studies of parvizi et al . and vanderstappen et al . , who found that intra - articular crp level could also reflect the severity of pji in their studies . admitted that synovial fluid crp was easier to obtain , less expensive , and less dependent on the technique of obtaining and interpreting the frozen section . found that measurement of crp in synovial fluid rather than serum using readily available assays does not offer a diagnostic advantage in the detection of pjis . however , based on our meta - analysis , the diagnostic value of synovial fluid crp is higher than that of serum crp . the pooled estimates for sensitivity , specificity , and the auc for the serum crp of the 25 included studies were 0.82 ( 95% ci : 0.800.84 ) , 0.77 ( 95% ci : 0.760.78 ) , and 0.877 0.016 , respectively . first , despite an in - depth search of several electronic databases , there were only six articles qualified for our meta - analysis and it was impossible to further analyze and divide the studies into subgroups to explore other potential factors that may affect the heterogeneity and perform meta - regression analysis . therefore , characteristics of patients included in each study including age , basic condition before surgery , and the existence of systematic diseases could not be fully analyzed in the meta - analysis . second , the ideal cutoff value for the synovial fluid crp test could not be determined since the raw data were not provided in the published articles . it is hard to come to a consistent cutoff value since different laboratories used different methods to detect synovial fluid crp , for example , elisa and turbidimetric immunoassay , both of which performed antibody response with the target protein ( crp ) . as a matter of fact , there is still no standard cutoff value for the diagnosis worldwide currently even for the same method . thus , our meta - analysis also indicates that large - scale , prospective , randomized trials with standardized reference and detecting method , strict included , and excluded criteria are in urgent requirement to generate a more precise cutoff value for clinicians . to the best of our knowledge , our study is the first meta - analysis that evaluates the clinical utility of synovial fluid crp in the diagnosis of pji . although the number of studies included in our meta - analysis is limited , all the included studies are highly qualified and illustrate the high sensitivity and specificity of synovial fluid crp in discriminating pji patients from those who had undergone joint replacement and showed similar symptoms . this systematic review has constituted a primary foundation for evidence - based guides on the diagnostic performance of synovial fluid , which can provide recommendations to clinicians for diagnosing pji accurately and efficiently . meanwhile , prospective studies are in urgent need to further validate our findings , and more synovial fluid biomarkers of high sensitivity and specificity are required in clinical practice for the diagnosis of pji .

background : periprosthetic joint infection ( pji ) is the main cause of failure following total joint arthroplasty . until now , the diagnosis of pji is still confronted with technical limitations , and the question of whether synovial fluid biomarker , c - reactive protein ( crp ) , can provide high value in the diagnosis of pji remains unanswered and , therefore , was the aim of the study.methods:first , we conducted a systematic review on crp in the diagnosis of pji by searching online databases using keywords such as periprosthetic joint infection , synovial fluid , and c - reactive protein . eligible studies providing sufficient data to construct 2 2 contingency tables were then selected based on the list of criteria and the quality of included studies was assessed subsequently . finally , the reported sensitivity , specificity , diagnostic odds ratio ( dor ) , summary receiver operating characteristic ( sroc ) curve , and the area under the sroc ( ausroc ) were pooled together and used to evaluate overall diagnostic performance.results:seven studies were included in our review , six of which comprising a total of 456 participants were further investigated in our meta - analysis . the pooled sensitivity , specificity , and dor were 0.92 ( 95% confidence interval [ ci ] : 0.860.96 ) , 0.90 ( 95% ci : 0.870.93 ) , and 101.40 ( 95% ci : 48.07213.93 ) , respectively . the ausroc was 0.9663 ( standard error , 0.0113).conclusions : synovial fluid crp is a good biomarker for the diagnosis of pji with high sensitivity and specificity .

pelvic exenteration ( pe ) describes a radical surgery involving the en bloc resection of the pelvic organs , including the internal reproductive organs , bladder , and rectosigmoid . indications include advanced primary or recurrent pelvic malignancies , most commonly centrally recurrent cervical carcinoma , but also other gynecologic tumors and urologic and rectal cancers . as the best chance for disease - free survival is surgical resection of regional disease , this procedure is an opportunity to cure advanced and recurrent cancers confined to the pelvis . pe has also been used for palliation of symptoms related to radiation necrosis or extensive tumor burden . both total and partial pe require extensive reconstruction and surgical recovery with significant associated morbidity and mortality . careful patient selection is required to balance the potential goal of cure or symptom palliation with surgical risk . the first cases of total pe were described by brunschwig in 1948 as a palliative procedure for symptoms caused by locally advanced gynecologic cancers . this demonstrated proof of concept for pe , with a postoperative survival of up to 8 months , and a 23% surgical mortality rate . subsequent data demonstrated that the technique could offer a chance of cure for centrally located tumors , not just palliation , and the focus of the surgery shifted to one of curative intent . various surgical approaches both for sparing uninvolved pelvic organs and removing extraperitoneal structures such as the sacrum were attempted . major breakthroughs included separate stomata for urine and fecal diversion and the use of omentum to protect the empty and denuded pelvic space and reduce abscess formation and intestinal obstruction [ 2 , 3 ] . more recently , techniques to resect tumor involving the pelvic sidewall , previously a contraindication to pe , have been described offering more patients a chance at curative surgery . pe may also be combined with intra - operative radiation therapy for improved disease control at the pelvic sidewall or possible positive margins [ 5 , 6 ] . since 1948 several developments in perioperative care and surgical technique have improved survival , morbidity , and mortality , with recent mortality rates quoted < 5% . development of continent urinary conduits and orthotopic neobladders , as well as low rectal anastomoses has led to the completion of pe without formation of stomata . various techniques for functional neovaginas have been described , allowing patients to maintain sexual function if they desire . advances in laparoscopic and robotic assisted technology applied to pe have improved operative recovery . despite these significant advances and five - year survival rates of approximately 50% , pe remains a radical procedure with significant complications ( 3192% ; see table 1 ) , both physical and psychological . traditionally pe has been used for centrally recurrent cervical carcinoma , both squamous and adenocarcinoma , with well - documented salvage potential . up to 25% of women with figo stage ib - iia cervical cancer may recur after initial therapy . frequently , these recurrences may be treated with radiotherapy ; however , radical surgery may offer an alternative for curative treatment . survival rates ranging from 16 to 60% are reported for these patients [ 10 , 11 ] . long - term survival is directly correlated with complete tumor resection [ 12 , 13 ] , so establishing resectability is a key aspect of preoperative planning . time from primary treatment , with radiation or chemoradiation , to time of pe has also been shown to be related to survival and disease - free interval , with women requiring pe for recurrence less than 2 years following primary therapy demonstrating an 8-month survival versus 33 months in women who recurred more than 2 years following initial treatment in one study , though this has not been shown in all series . pe has also been utilized as a potentially curative primary treatment for locally advanced cervical cancer ( figo stage iva ) , a practice exercised more frequently in germany than the united states . for example , in their series , marnitz et al . reported a 52.5% five - year survival . cases of pe for a variety of histologic types of uterine cancer have been reported , with outcomes similar to pe for other indications . most recurrent uterine cancers spread beyond the pelvis , given their propensity for diffuse abdominal or heterogenous spread , making pe appropriate intervention for only a select group of patients with recurrent uterine malignancies . women with only loco - regional recurrence , however , may be candidates for pe with curative intent . described a series of 21 women with recurrent uterine cancers who underwent pe and demonstrated a five - year survival of 40% . the study also noted varying outcomes dependent on histology , with endometrioid adenocarcinoma ( 50% five - year survival rate ) and sarcoma ( 66% five - year survival rate ) demonstrating improved survival over a group of women with tumors with serous , mixed , and carcinosarcoma - histology ( 14% five - year survival rate ) . morris et al . reported a five - year survival rate of 45% following pe for recurrent endometrial cancer . given the similarity of complication rates ( 4860% ) and survival to pe for cervical cancer , patients with locally recurrent uterine cancer may be considered candidates for the procedure . vulvar cancer has a propensity for regional metastases . for patients with advanced primary or recurrent vulvar cancer who do not have the option of treatment with radiation therapy , pe may be appropriate . the authors demonstrated results similar to other gynecologic malignancies , with a five - year survival of 62% . complete resection with no evidence of residual disease was associated with improved outcomes , a five - year survival rate of 74% , compared to 21% in patients without complete resection . absence of tumor lymph node invasion was also associated with an improved five - year survival rate ( 83% versus 36% ) . in contrast , combination therapy with vulvectomy and radiotherapy has been described for locally advanced vulvar cancer with the goal to spare the pelvic organs , with five - year survival in two series of 45% and 72% , and sparing of the pelvic organs in 62.5% and 89% of patients [ 19 , 20 ] . given the propensity of ovarian cancer to spread throughout the abdomen , women with this disease are rarely candidates for pe with curative intent . supralevator pe has been reported when needed for optimal cytoreduction , combined with standard staging procedures and for recurrent disease . two series of modified posterior pe for ovarian cancer demonstrated median survival 33 and 37.4 months after initial surgery . optimal cytoreduction was achieved in 46% and 58% of patients in the series [ 21 , 22 ] , demonstrating this technique may be used to achieve optimal cytoreduction in patients with disease requiring rectosigmoid resection . as vaginal cancer is rare , this review could not identify any literature specifically addressing pe for this indication . several cases of vaginal cancer , both primary and recurrent , undergoing pe have been included in larger studies , most frequently including the results for these patients combined with results for cervical cancer [ 10 , 23 ] . it may be hypothesized , that results following pe for vaginal carcinoma would be similar to those for cervical cancer provided the same other parameters for patient selection apply . pe has been described for palliation rather than for curative intent , most frequently in the setting of severe radiation necrosis . both morbidity and mortality were shown to be higher in this group of patients as opposed to those undergoing pe with curative intent , though improvements in quality of life are reported [ 24 , 25 ] . pe is thus only considered for palliation if there is no reasonable alternative , though with the development of minimally invasive surgical technology , pe may become a more feasible option . pe is a major surgery with significant morbidity , and as such selecting appropriate patients is essential . if the surgery is undertaken with curative intent , the tumor should be fully resectable with negative margins . classically , disease burden was required to be limited to the central pelvis , but with new surgical developments candidates for curative pe may now also include patients with positive lymph nodes , pelvic sidewall involvement , and local bone invasion . regardless of the indication , patients undergoing pe must be in otherwise good medical health to be able to tolerate a long surgical procedure with extensive fluid shifts and prolonged hospital stay . preoperative evaluation includes a complete history , physical exam , and , if necessary , an exam under anesthesia , biopsy of any suspicious lesion such as an enlarged lymph node , evaluation of specific patient concerns suggesting metastatic spread , such as unilateral leg pain , chest radiograph or computed tomography ( ct ) . in general , cystoscopy and sigmoidoscopy are not necessary unless the bladder or rectum is to be spared . in this case , laboratory tests should include a complete blood count , platelet count , comprehensive metabolic panel , including hepatic and renal function , as well as clotting factors and urinalysis . patients should be offered testing for human immunodeficiency virus , which may be a contraindication to pe . given the nature of the surgery , patients should be counseled about changes in body image and function . specifically , patients should have an understanding of anatomical changes involving creation of colostomy and urinary conduit and need to be accepting of major changes in body image even in the setting of reconstructive surgery . patients require significant family support , intact mental capacity and access to continued and long - term medical care . we recommend sharing printed literature and illustrations depicting ostomies and conduits , as well as offering patients the opportunity to speak with other women who have undergone the procedure . patients should meet with ostomy nursing staff to begin the education process preoperatively and gain confidence with management of the ostomy and conduits . during these visit part of the counseling sessions should focus on sexual function and how this will change for both patients choosing to have creation of a neovagina , as well as for those declining this part of the reconstruction . patients should be informed of all possible perioperative complications , including infectious , thromboembolic , gastrointestinal , urinary , psychiatric , readmission , and reoperation . women being considered for pe should be informed of a 35% risk of operative mortality . some of these complications occur more frequently in the remote postoperative period ( days 3190 ) than in the immediate one ( 030 days ) . patients should expect frequent visits to the hospital during this time given the risk not only of immediate but also delayed complications . of note , part of the preoperative counseling should include the impact aborting the operation for unexpected surgical findings may have on the patient . that is , patients should be informed that even with the use of state - of - the art , preoperative imaging , the possibility of finding metastatic disease continues to exist , and a minority of exenterative procedures are aborted at the time of surgical exploration . it is critical that patients have sufficient medical and emotional support to manage the physical and psychological challenges central to the operation . therefore , the goal of diagnostic techniques is to find evidence of unresectable or metastatic disease ; thus , making the woman an unsuitable candidate for pe . a number of diagnostic techniques can aid in assessing unresectable disease in a patient who is believed to have central pelvic disease . computed tomography ( ct ) and magnetic resonance imaging ( mri ) can be helpful in assessing the presence of lateral pelvic wall invasion or liver metastasis . however , major limitations of ct and mri lie in their inability to assess minimally enlarged nodes to detect microscopic peritoneal disease and to distinguish fibrosis from tumor in recurrent disease , and the fact that most patients have usually received extensive radiation makes distinguishing radiation fibrosis from malignant tumor extremely difficult . f - fluorodeoxyglucose positron emission tomography ( fdg pet ) has been shown to perform better in this population . the only prospective study to date in which all patients underwent surgical exploration with curative intent and in which almost all pet positive sites were biopsied showed that sensitivity and specificity of pet imaging in metastatic disease in patients being considered for pe was 100 and 73% , respectively . despite a negative predictive value of 100% , the high false positive rate found in this study makes surgery obligatory for all pe candidates . bone scans are usually not indicated unless there is a history of recent bone pain and concern for bony metastases . the procedure begins with the patient in low lithotomy position to allow for abdominal and perineal portions of the surgery . pe is traditionally performed as an open abdominal procedure , but recent developments in laparoscopy and robotics have allowed for the minimally - invasive adaptation of the technique . the abdomen is opened with a vertical midline incision to allow for maximum ability to explore the upper abdomen as well as the pelvis . any suspicious lesion is biopsied and sent for frozen section to exclude the possibility of distant metastatic disease that would preclude complete resection or alter the surgical plan . for recurrent cervical cancer , low para - aortic and pelvic lymph node dissection once the disease has been confirmed to be resectable , the operation may proceed [ 8 , 30 , 31 ] . the round ligaments are divided , and the paravesical and pararectal spaces are developed . at this time the pelvic sidewalls may again be examined . at this point , the extent of pe must be determined . total pe includes removal of the bladder and distal ureters , portions of rectum and sigmoid colon , internal reproductive organs ( if still present ) , and vagina . in well - selected patients , occasionally , if the anatomic location of the recurrence is only the anterior or posterior compartment of the pelvis , the colon or bladder may be spared and only an anterior exenteration or posterior exenteration may be necessary . removal of the specimen begins with the ligation and division of the fibrovascular pedicle containing the uterine vessels , cardinal ligament , and the ureter bilaterally . the uterine artery is ligated at is origin from the hypogastric , lateral to the ureter . the sigmoid is then mobilized and transected with a gastrointestinal anastomotic stapler ( gia ) , and the sigmoid vessels are identified and ligated . care must be taken to preserve blood flow to the remaining colon usually the sigmoid artery is left intact and the superior hemorrhoidal artery is ligated . the avascular plane between the sigmoid and the sacrum is developed to the level of the levator ani muscles . the prevesical space is extended bluntly . at this point , the specimen should be freely mobile in the pelvis . the perineal portion of the procedure is then performed ( or may be performed synchronously with an additional surgeon ) . an incision is marked to include the urethra , vaginal opening , anus , and possibly the vulva . the pubococcygeal and anococcygeal ligaments are identified and divided . upon completion of the dissection , the entire specimen is free to be removed . anterior pe involves the removal of the bladder and internal reproductive organs but spares the gastrointestinal tract . the rectosigmoid , anus , and lower portion of the posterior vagina are left intact . after division of the cardinal ligaments , uterine vessels , and ureters , the rectum is separated from the upper vagina . the rectum is retracted posteriorly by rectovaginal bimanual exam to ensure the space is clear of tumor and resectable . an incision is made into the peritoneum of the cul - de - sac , and the rectum is dissected sharply off the upper vagina . posterior pe removes the internal reproductive organs and the rectosigmoid but spares the anterior vagina , urinary bladder , and ureters . in previously irradiated pelves , it is important to consider the possibility of urinary fistulae developing following a posterior pe given the possibility for devascularization . the ureters are identified and dissected as in a radical hysterectomy , and the uterine arteries and cardinal ligaments ligated . modified posterior pe , as for cytoreduction in ovarian cancer , is a supralevator dissection . there is no perineal phase to the operation . if enough rectum remains ( more than 6 centimeters ) , a low rectal anastomosis may be made , sparing the patient a stoma . if the tumor does not involve the vulva or lower third of the vagina , the patient may be a candidate for a supralevator pe . after the specimen is mobilized as in a total pe , an incision is made into the posterior vaginal wall below the tumor , ensuring an adequate margin . the rectum is isolated and divided with a stapling device , leaving an anorectal stump and possibility for low rectal anastomosis . brunschwig initially designed reconstruction after pe with an ureterosigmoidostomy , known as a wet colostomy , with urine and feces emptying through one stoma . in current practice , both incontinent ileal and colonic conduits are used , as well as a variety of continent urinary reservoirs , most commonly the miami pouch [ 30 , 31 ] . the standard ileal conduit is formed by an isolated segment of distal ileum with its vasculature . the ureters are anastomosed directly to the ileum at one end , and the other end is brought to the skin as a stoma . the miami pouch was first reported by bejany and politano in 1988 , a modification of prior continent colonic pouches designed to reduce incontinence . the ileum is transected 10 to 15 centimeters proximal to the ileocecal valve , and the transverse colon is transected distal to the middle colic artery . an appendectomy is performed . to form the bulk of the pouch , the colon is opened along the tenia , and the open edges are approximated by folding the colon segment into a u - shape conduit and the edges closed with a stapling device . this formation of the colon creates a reservoir and interrupts the ability of the bowel to peristalse and increase the pouch pressure . for the ureteral anastomoses , the distal ends of the ureters attention is then turned to the ileum , which is tapered distally to support the ileocolic valve and prevent reflux . the free end of the ileum is brought to the skin surface as a stoma . the patient will be required to self - catheterize this stoma , but she is spared a drainage device if the procedure is successful . in a follow - up study of the miami pouch reported 92% continence and reservoir volume of average 650 ml , allowing for a reasonable catheterization interval . for patients whose disease requires infralevator dissection posteriorly , permanent end colostomy will be required because the anal sphincter is compromised or excised . if the sphincter and enough rectum may be spared without compromising the chance at complete disease resection , low rectal anastomosis may be considered to restore continence . direct end to end anastomosis with circular staplers is a reasonable option if enough healthy tissue remains . to improve frequency of stooling by improving the reservoir of the rectum , a colonic j - pouch may be used , particularly in patients with very little rectum remaining ( less than 5 centimeters ) . some authors , however , cite the frequency of recurrence of disease near the site of rectal anastomosis ( 45% ) as a reason to perform complete resection and end colostomy in all patients . other authors strongly support low rectal anastomosis for a chance at preserved function and avoidance of undesirable colostomy for the patient . after vaginectomy , construction of a neovagina for restoration of sexual function should be offered to patients undergoing pe . several options exist for the creation of a neovagina , including split - thickness skin grafts , myocutaneous grafts , and colon . rectus abdominus myocutaneous ( ram ) flaps have been reported routinely in the literature , with 93% viability in the series from ucla . the ram flap may be harvested from the same midline vertical incision used for the pe , improving cosmetic outcomes for the patient . consideration must be made in the selection of the flap , such as previous maylard incision or other compromise to the inferior epigastric artery . a transverse or vertical flap may be constructed , at least 10 to 12 centimeters in length , maintaining the blood supply from the inferior epigastric artery . the flap is freed , elevated , and sutured into a tube with the skin at the interior , which will serve as the neovagina . the tube is then secured in the pelvis at the vaginal introitus . a mold with estrogen cream is left in the vagina to maintain the lumen for 5 to 7 days . patient satisfaction and coitus rates are quoted as 5878% [ 34 , 35 ] . if a neovagina has been created with a myocutaneous flap , such as a ram flap , this graft is usually sufficient to fill the pelvic dead space and ensure adequate vascularity . if no such procedure has been performed , it minimizes complications such as bowel obstruction and maximizes hemostasis to close the pelvic dead space . the omentum is detached at the greater curvature of the stomach while preserving its origin containing the left gastroepiploic artery , which will supply the flap . other options , such as mesh and pelvic packing , were attempted with poor outcomes . the indications for its use have widened , and the superseding of open surgery seems inevitable in many areas of surgery . this revolution in surgery is in part associated with the technological advancement and a concomitant acquisition of advanced minimally invasive surgical skills by many gynecologic oncologists . laparoscopy is now a well - accepted tool in the armamentarium of the treatment of gynecological cancer , and data have been published by various centers [ 3639 ] . minimal invasive surgery is generally associated with less intraoperative blood loss , postoperative pain , and shorter hospital stay . [ 40 , 41 ] were the first group to report two cases of laparoscopic pe for gynecological cancer . both patients enjoyed the other well - known advantages of laparoscopy including minimal blood loss and quick ambulation , all contributing to a better postoperative quality of life . subsequently , lin et al . reported a case of laparoscopy - assisted transvaginal total pe . in addition , ferron et al . published a series of five patients that underwent a laparoscopic assisted vaginal pe . their series reports the first application of a rational combination of laparoscopic , perineal , and hand - assisted surgery , with the goal of limiting the potentially long laparoscopic time to a strict minimum . of note , the authors elected to perform a hand - assisted miami pouch through a minilaparotomy ( 5 cm ) in order to reduce the operative time , safely perform the ureteral anastomosis , restore bowel continuity and , in addition , build the omental cylinder for vaginal reconstruction . the use of a perineal or vaginal approach allowed to quickly and safely free the specimen well above the pelvic floor . in a subsequent report by the same authors , with a mean follow - up of 14 months , four patients died of the disease ( three were metastatic ) , one patient presented a local recurrence , and two patients are disease free . reported in a series of 16 consecutive patients , the technique , feasibility , and safety of laparoscopic anterior pe as primary treatment for locally advanced pelvic cancers . thirteen patients underwent anterior pe with ureterosigmoidostomy , while two patients required total pe with wet colostomy . of note , after a mean follow up of 15 months , all patients were disease free . also described the feasibility of doing a laparoscopic total pe for palliation in advanced cervical cancer . of the 7 patients included in their series , no patients required conversion to open surgery . the mean followup was 11 ( 424 ) months and mean symptom free period was 8 ( 324 ) months . the mean followup of the patients was 11 months ( range 4 to 24 months ) ; and the mean symptom free survival period was 8 months ( range 3 to 24 months ) . three patients are now disease - free for more than a year . the development of robotic technology has facilitated the application of minimally invasive techniques for the treatment and evaluation of patients with gynecological cancers . robotic surgery offers several advantages over laparoscopy : a three - dimensional vision system , wristed instrumentation , and ergonomic positioning for the surgeon while performing surgical procedures . the enhanced visualization gives the gynecologic surgeon an improved ability to identify tissue planes , blood vessels , and nerves while performing the surgical procedure [ 4749 ] . since the first report of robotic - assisted radical hysterectomy by sert and abeler in 2006 for cervical cancer , there have been some reports of robotic - assisted laparoscopic pe [ 5052 ] . the first cases of robotic - assisted laparoscopic pe were described by pruthi et al . in 12 women for clinically localized bladder cancer . urinary diversion was performed extracorporeally ( 9 ileal conduit diversion , 3 orthotopic neobladder ) . lim reported the first case report of robotic assisted total pe with an ileal loop urinary diversion and an end colostomy for treatment of recurrent cervical cancer . the authors reported that concerning hospital stay , there was no benefit comparing to laparotomy , essentially due to urinary diversion management ( catheterization ) and to self catheterization patient 's autonomy . despite the apparent encouraging early results suggesting an advantage of minimally invasive surgery for pe , questions remain about the surgical effectiveness of this approach . further study of minimally invasive techniques to perform a pe is needed prior to widespread clinical application of these techniques . as it is a radical surgery performed in the setting of advanced tumor growth and frequently on irradiated tissue , pe is associated with a significant rate of complications , quoted about 4050% for major complications and about 80% for minor complications . mortality is quoted from 116% , with disparate causes including sepsis , thromboembolic disease , and cardiopulmonary failure . despite significant advances in the last fifty years , the extensive nature of the surgery , including blood loss , fluid shifts , and operative time , have led to unavoidable risks . infection is the most frequent morbidity ( 1986% ) , with urinary infections and wound infections most commonly reported . anastomotic leaks and fistulae from either diverting system are also relatively frequent , cited at 836% . most of these complications can be managed conservatively , but significant numbers of patients require operative revision [ 10 , 11 , 55 ] . death in the perioperative period occurs in fewer than 5 percent of patients , with women over the age of 65 at highest risk . given the radical and prolonged nature of this procedure , patients and providers must be prepared for a long and potentially complicated hospital course . many patients require a stay in the intensive care unit immediately postoperatively for close monitoring , particularly in the setting of potentially dramatic fluid shifts . special attention to thromboembolism prophylaxis , respiratory care , and nutrition is required . while no longer routine , some patients will require total parenteral nutrition due to prolonged inability to eat postoperatively , as ileus is relatively common . a team - based approach , including case managers , dedicated nurses , and social workers , may help patients as they heal both mentally and physically postoperatively . as a portion of preoperative counseling and postoperative support , the changes in a woman 's body image following pe must be reviewed . some patients , particularly those undergoing this surgery for palliative management of pain or fistulae , do report improved quality of life following surgery , with decreased narcotic requirements and malodorous discharge . most women , however , note a decline in specific areas of quality of life . notably , body image , physical ability , and social function have all be reported decreased in questionnaires compared to patients ' preoperative answers . these changes are more pronounced in younger patients and those who do not undergoing vaginal reconstruction . interestingly , overall function and mental and emotional quality of life are comparable [ 5759 ] . pe is a radical operation , involving en bloc resection of pelvic organs , including reproductive structures , bladder , and rectosigmoid . in gynecologic oncology , it is most commonly indicated for the treatment of advanced primary or locally recurrent cancer . patients need to be carefully selected and counseled about risks and long - term issues related to the surgery . total pe is associated with significant surgical morbidity , a fact that underscores the importance of careful patient selection and counseling . the emergence of minimally invasive surgery and application of this technology to radical pelvic surgery including pe may result in a reduction operative morbidity and mortality . further studies are necessary prior to a widespread adoption of this technology to exenterative procedures .

total pelvic exenteration ( pe ) is a radical operation , involving en bloc resection of pelvic organs , including reproductive structures , bladder , and rectosigmoid . in gynecologic oncology , it is most commonly indicated for the treatment of advanced primary or locally recurrent cancer . careful patient selection and counseling are of paramount importance when considering someone for pe . part of the evaluation process includes comprehensive assessment to exclude unresectable or metastatic disease . pe can be curative for carefully selected patients with gynecologic cancers . major complications can be seen in as many as 50% of patients undergoing pe , underscoring the need to carefully discuss risks and benefits of this procedure with patients considering exenterative surgery .

it is well recognized that the mealtime insulin requirement in type 1 diabetes ( t1 dm ) patients is driven mostly by carbohydrate content and that monitoring it can improve glucose levels . the american diabetes association recommends meal plans based on carbohydrate counting as a key strategy to achieve glycemic control . an impact of protein containing food on glycemia in diabetes has been studied for many decades ; however , it is unclear whether or not the calculation of other food components can also be used to optimize glucose levels [ 3 , 4 ] . some studies have suggested that counting the amount of fat and protein ( using fat / protein exchanges ) may be beneficial for reaching glycemic control in t1 dm children . such an approach could be particularly feasible in t1 dm patients treated with continuous subcutaneous insulin infusion ( csii ) via insulin pump . one of the features implemented in some personal insulin pumps is the dual - wave bolus ( dwb ) option , which delivers the combination of an instant standard premeal insulin bolus followed by a square bolus ( sb ) infused over several hours , helping to tailor prandial insulin delivery to the composition of a meal . this pump option may be particularly useful for mixed , fat-/protein - rich meals since such food seems to modify postmeal glucose patterns with a less rapid and more prolonged increase in plasma glucose concentration . it has recently been shown among a cohort of pediatric patients with long - lasting t1 dm , using pump therapy , that for mixed meals insulin dosing based on both carbohydrate and fat / protein counting leads to lower postprandial glycemic levels than the conventional counting of carbohydrates only . . however , the studies performed so far estimating the glycemic effect , mostly on postprandial glucose levels , of fat and protein ingestion had some serious limitations . for example , they evaluated either the effect of a meal containing both fat and protein [ 5 , 8 ] or the effect of just fat added to a mixed meal . to our knowledge , until now no study has examined the effect of sole protein ingestion . the aim of this study was to estimate the impact of ingestion of a pure protein load on glucose levels in t1 dm patients treated with personal insulin pumps . we examined 10 t1 dm patients ( 6 females , 4 males ) treated with insulin pumps ( medtronic paradigm 722 or veo ) equipped with a continuous glucose monitoring system ( cgms ) option . during the study , enlite sensors ( medtronic ) all of the patients were white caucasians under the medical care of the department of metabolic diseases , university hospital , krakow , poland . the selection of patients was based on good prestudy compliance as assessed by good glycemic control ( hba1c level less than 7.5% , 58 mmol / mol ) , the patient 's usage of the bolus wizard ( bolus calculator ) option for more than 90% of boluses , changing infusion sets according to manufacturer 's recommendation , and using at least one electrode for cgms per month during the preceding year . all patients gave informed consent , and the study was approved by the bioethical committee of the jagiellonian university . before study entry ( one to two weeks preceding day 1 ) , the patients ' baseline insulin infusion was optimized to minimize the differences in fasting glucose levels to less than 30 mg / dl between any two time points between 9 a.m. and 3 p.m. the procedure of optimization was based on a retrospective analysis of cgms records and involved increasing or decreasing the rate of basal insulin infusion two hours before the observed rise or fall in glucose level . subsequently , the new settings of the basal infusion rate were rechecked with cgms to meet the study criteria . in phase i ( day 1 ) , a fasting test ( between 9 a.m. and 3 p.m. ) was performed to confirm the stable glucose patterns ( as defined above ) on current basal insulin infusion settings . in phase ii ( day 2 ) , the patients were exposed to a single pure protein load by ingesting a commercially available preparation ( protifar , nutricia , milk derived proteins ) . in its 100 ml volume , the product contained 88.5 grams of pure protein , 1.6 grams of fat , and less than 1.5 grams of carbohydrates ( lactose ) , as well as minerals and micronutrients . all the patients were exposed to a dose of 0.3 g of pure protein ( 0.34 ml of protifar / kg dissolved in 200 ml of water ) for each kg of body weight ; the load was administered at 9 a.m. such a dose of protein is the equivalent of the usual protein portion in a medium - size meal , based on the dietary recommendations for patients with diabetes ( 1520% of total daily energy intake ) . both phases were performed in home settings ; the patients were given precise instructions concerning the procedure . the rate of basal insulin infusion during the protein load test was the same as that defined initially in phase i ; no modifications or extra insulin boluses were permitted . cgms record was performed in both phases , during which the patients avoided physical activity . glucose patterns were recorded during 6 hours of phase i ( fasting ) and 6 hours of phase ii ( protein load ) . there was no consistency in the age of cgm sensors during phase i and phase ii ; however , we strictly avoided performing tests during the first or last ( 6th ) day after sensor insertion . the difference between the two phases was assessed by using the dependent t - test or paired sample wilcoxon signed rank test , when applicable . the patients ' mean age was 32.3 years , mean t1 dm duration was 11.7 years , and mean hba1c was 6.85% ( 51 mmol / mol ) . mean baseline glucose levels were 119.8 and 117.6 mg / dl for phase i and phase ii , respectively ( p = 0.68 ) . mean maximal glucose levels were 146.4 and 145.2 mg / dl for phase i and phase ii , respectively ( p = 0.85 ) . mean maximal glucose level increment was similar for the entire 6-hour fasting and protein load test ( 26.6 mg / dl versus 27.6 mg / dl , resp . there was only a borderline difference between the change in baseline versus 6th hour glucose levels for the fasting state versus protein load test ( 12.5 mg / dl and 19.0 mg / dl , resp . , p = 0.04 ) . the glucose variability assessed by cgms - based standard deviation of mean glucose levels was 36.4 and 38.9 mg / dl for phase i and phase ii , respectively ( p = 0.01 ) . there were no episodes of infusion set malfunctions during phase i or phase ii of the study , and no hypoglycemia was recorded during fasting or after loading . here , for the first time , we evaluated the impact of the ingestion of a pure protein load on glucose levels in adult t1 dm subjects treated with insulin pumps . in this short - term clinical experiment , we found that protein consumption had very little effect on the glycemia of the examined cohort of patients . contradictory to our study results , there have been several earlier papers reporting a substantial rise in glucose levels in t1 dm patients as an effect of the consumption of noncarbohydrate food components [ 5 , 8 , 10 ] . however , these reports involved either fat only or fat and protein combined , because , in real - life settings , they are often consumed together in products chosen by diabetic individuals , such as meat , poultry , fish , and dairy products . thus , the discrepancy between earlier studies and our results may be explained by the variable short - term impact of fat and protein versus protein only consumption on glucose homeostasis through different mechanisms of hepatic gluconeogenesis , postmeal insulin resistance , release of glucagon and gut hormones , gastric emptying , and rate of absorption of other food components like carbohydrates and other factors [ 1113 ] . there are some earlier reports from different healthy and diabetic populations suggesting variable glycemic effects of high protein meals ; for example , they promote gluconeogenesis and slow gastric emptying [ 13 , 14 ] . in a recently published study in t1 dm , it was shown that a mixed meal consisting of 30 g of carbohydrate and 40 g of protein has a greater glycemic effect than a pure carbohydrate load . the study by smart was performed in a pediatric population , while ours involved adult individuals . the differences in outcomes of both studies may be associated with various physiological reactions to protein load in the examined age groups . furthermore , there may be discrepancies in the proportion of basal versus prandial insulin doses depending on the age of patients , which could affect study outcomes . moreover , the effect of protein when mixed with carbohydrates might be different compared to the effect of protein alone . finally , the impact of protein may be dose - dependent , because , in the earlier study , 40 g of protein per meal was used , while the protein dose in our study was much lower . it is also important to underline that factors other than meal size or composition may influence the rate of food absorption and postprandial glucose patterns . the list of such factors includes the method of preparation ( e.g. , cooking , frying , or grilling ) , rapidity of consumption , accompanying beverages , time elapsed since the previous meal ( second - meal phenomenon ) , and variability in individual rates of gut absorption , as well as some other factors . moreover , various types of protein consumed as a meal component or during the load test might have affected the postmeal / load glucose patterns . the outcomes of our study may have practical clinical consequences as there are some small protein - based snacks available , such as protein bars or shakes , which are small meal options for t1 dm patients . so , it is important to provide them with information about their impact on glucose level . this is particularly important as , through their satiating effect , they may be helpful in weight management . an important advantage of our study was the carefully optimized rate of basal insulin infusion based on the earlier fasting phase of the experiment . consequently , we were able to exclude the potential bias related to an excess or deficit of basal insulin on postload glucose patterns . additionally , our subjects were highly preselected ; they were adult t1 dm patients characterized by very good compliance prior to the study , frequent cgms use , and willingness to follow a challenging study protocol . the direct clinical implication of our study is the recommendation that small protein - based snacks may be consumed by adult t1 dm csii - treated patients without insulin bolusing . obviously , the general guidelines concerning daily protein consumption for patients with diabetes should be followed . the results of this study can not be generalized to other noncarbohydrate food components , especially fat . additionally , we examined glucose patterns for 6 hours ; thus , it is not possible to exclude the protein load effect on glycemia beyond this time , for example , during subsequent meal . finally one has to underline , that we have used cgm systems that are still characterized with limited accuracy and reliability . in conclusion , the ingestion of a pure protein load does not seem to have a clinically significant impact on glucose levels in adult t1 dm patients treated with insulin pumps .

we aimed to estimate the impact of ingestion of a pure protein load on the glucose levels in t1 dm patients treated with insulin pumps . we examined 10 t1 dm patients ( 6 females , mean age32.3 years , mean hba1c6.8% ) treated with insulin pumps equipped with a continuous glucose monitoring system ( cgms ) . in phase i , baseline insulin infusion was optimized to minimize the differences in fasting glucose levels to less than 30 mg / dl between any two time points between 9 a.m. and 3 p.m. in phase ii , the patients were exposed to single pure protein load . cgms record was performed and the glucose pattern was defined for 6 hours of each phase . the maximal glucose level increment was similar for the entire duration of the fasting and the protein load test ( 26.6 versus 27.6 mg / dl , resp . , p < 0.78 ) . there was only a borderline difference in change between baseline versus 6th hour glucose ( 12.5 and 19.0 mg / dl , p = 0.04 ) . glucose variability , assessed by standard deviation of mean glucose levels , was 36.4 and 37.9 mg / dl , respectively ( p = 0.01 ) . the administration of a pure protein load does not seem to have a clinically significant impact on glucose levels in t1 dm patients treated with insulin pumps .

paul ehrlich described mast cells for the first time more than 100 years ago , through their characteristic metachromatic staining with aniline dyes . he noticed that these cells had abundant and dense granules ; he assumed that the granules were caused by excessive feeding . these cells were abundant in areas near tumors , so ehrlich postulated that they played a role in the protection against cancer . since then , many functions were proposed for mast cells in the regulation of organism homeostasis , but experimental evidence to support these assumptions was lacking . the discovery of mice with a diminished number of mast cells , and their reconstitution with bone marrow - derived mast cells , provided a tool to test these hypotheses and to extend our knowledge about the functions of mast cells in the body . mast cells derive from hematopoietic precursors that are released from the bone marrow into the bloodstream and are recruited to connective tissues , where they complete their differentiation . mast cells can live for months in the tissues , where they proliferate in response to different signals . the tissues that usually contain abundant mast cells are those that interact with the external environment ( the skin and the gastrointestinal , urogenital and respiratory tracts ) , where mast cells are located near lymphatics and blood vessels , or close to nerve terminals or smooth muscle cells . the morphology of mast cells depends on their anatomical location , ranging from rounded - shaped to elongated forms , when they are associated with blood vessels . their size ranges from 5 to 20 m and their cytoplasm contains abundant granules , with approximately 1,000 granules per cell . mast cell granules contain several chemical mediators , including histamine , proteoglycans , cytokines like tnf- and il-4 , and neutral proteases like tryptase , chymase , carboxypeptidase and cathepsin g. granule composition is not identical in all mast cells : human mast cells that reside in the skin have all the aforementioned neutral proteases , while epithelial lung mast cells have significant amounts of tryptase , but lack chymase , carboxypeptidase and cathepsin g. accordingly , human mast cells are classified as either mct ( skin ) or mt ( lung ) . in addition to these pre - formed mediators that are stored in granules , mast cells are efficient producers of lipid - derived mediators , such as platelet - activating factor ( paf ) and the arachidonic acid - derived prostaglandins , thromboxanes and leukotrienes . mast cells are also efficient producers of many soluble protein mediators , such as cytokines , chemokines and antimicrobial peptides [ 6 , 7 ] . activation of mast cells induces the release of one group of these mediators or of all of them , depending on the triggering signal . however , not all the mediators are released at the same time : chemical mediators from the granules are secreted in seconds after stimulation , while lipid - derived mediators are produced minutes after activation and protein soluble mediators are detected hours after the initial trigger . mast cells produce abundant histamine , and since the discovery that histamine is a crucial element for the development of type i hypersensitivity , the relevance of mast cells for the initiation of allergic reactions was underscored . by far , the most studied mast cell - activating signal is the cross - linking of fcri by ige and allergen , an event that leads to mast cell degranulation and to the release of several chemical mediators that cause the immediate inflammatory response characteristic of type i hypersensitivity . as a consequence , much of our knowledge of mast cell biology is biased towards their participation in this pathological process . type i hypersensitivity , or allergic inflammation , is an acute inflammation produced in sensitized individuals when they are exposed to the corresponding allergen . sensitization occurs when dendritic cells capture an allergen in the airways , migrate to the regional lymph nodes and activate nave t cells . in the presence of il-4 ( derived from mast cells , eosinophils or innate lymphoid cells ) , these cytokines promote class - switch recombination in b cells , which then produce allergen - specific ige . ige binds to fcri on mast cells , which are then primed for activation upon re - exposure to the allergen . during this subsequent exposure , the allergen cross - links fcri - bound ige , which leads to mast cell activation and degranulation . the mediators derived from activated mast cells cause bronchoconstriction , vasodilation , increased vascular permeability , mucus production and recruitment of eosinophils and neutrophils . repetitive or persistent exposure to the allergen leads to a chronic allergic inflammation , which causes tissue injury and remodeling ; this last process includes the severe narrowing of the airway lumen observed in asthmatic patients . however , mast cells are conserved in different groups of vertebrates , and from an evolutionary point of view , it makes no sense that humans should have conserved a cell population that causes a harmful and even deadly response if it conferred no evolutionary advantage . so , in the last years , the study of mast cells has been redirected to investigate their participation in processes such as innate immune response to pathogens , regulation of inflammation , wound healing and regulation of tumoral growth . in this review , we focus on the mechanisms used by mast cells in the lung to keep the proper function of this vital organ . mast cells have a vast collection of cell membrane receptors that allow them to sense the external environment . a set of these receptors are pattern recognition receptors ( prr ) that recognize pathogen associated molecular patterns ( pamp ) . these receptors are crucial for the initiation of the innate immune response , and they are classified in five groups : toll - like receptors ( tlr ) , nod - like receptors ( nlr ) , c - type lectin receptor ( clr ) , rig - i - like receptor ( rir ) and scavenger receptors . mast cells express at least one receptor of each group , allowing them to recognize a multitude of microorganisms , including viruses , bacteria , protozoa , fungi and multicellular parasites [ 13 - 23 ] ( fig . 1 ) . the first evidence that mast cells are crucial elements of the immune response to pathogens came from a study of the infection of the rat gastrointestinal tract with multicellular parasites . there are few studies that investigate if mast cells play a role in the containment of parasitic diseases in the lungs . one of the firs studies that established a role for mast cells in fungal infections in vivo reported that , in a mouse model of infection with sporothrix schenckii , mast cell depletion decreased the severity of cutaneous lesions . whether mast cells in the lungs plays a role in fungal infections needs to be evaluated , but in vitro experiments suggest that they do have a role , because they are activated by aspergillus fumigates . identified for the first time that mast cells are essential for the early containment of bacterial infections in the lungs ; klebsiella pneumonie , a gram - negative bacteria that causes community - acquired pneumonia , was among the bacteria tested in this study . the authors observed that the lung bacterial load was higher in mice deficient in mast cells , compared with wild - type mice , and this correlated with a defective recruitment of neutrophils to the lung . these results indicated that mast cells did not have a direct antimicrobial activity , but they induced inflammation through tnf- , which allowed the recruitment of neutrophils . mast cells also participate in the immune response to mycoplasma pneumoniae , a bacteria that is among the smallest free - living organisms and lacks a classical bacterial cell wall . airway infection of mice deficient in mast cells induced faster weight loss , more severe pneumonia , higher mortality and increased bacterial burden in the lungs , compared with wild - type mice . this work showed that mast cells play an important role in innate immune response against bacteria that infect lungs . tularemia is a life - threatening disease caused by francisella tularensis , a gram - negative coccobacillus that can be spread through aerosols . intranasal infection of wild - type mice with f. tularensis induces an increased accumulation of mast cells in the lungs . accordingly , mast cell - deficient mice showed impaired bacterial control and a significant decrease in il-4 production . in fact , mast cells confer protection by inhibiting bacterial entry and replication in macrophages , thus showing a different mechanism by which mast cells confer protection against bacterial infection in the lungs . it is caused by mycobacterium tuberculosis ( mtb ) , a bacteria that has a cell wall unique among bacteria , and that infects and usually persists in the lungs . studies with humans have shown an increase of mast cells in the lymph nodes of patients with tuberculous lymphadenitis . in vitro studies demonstrated that mtb activates mast cells , inducing degranulation , production of il-6 and tnf- and even bacterial internalization through lipid rafts [ 36 , 37 ] . moreover , in vivo experiments have suggested a role of mast cells in the containment of mtb infection . in one of the first studies , mice received c48/80 , a compound that induces mast cell degranulation , before infection with mtb . these mice showed an altered production of cytokines and chemokines , which induced a decrease in lung inflammation and an increase in mtb load , compared with untreated mice . a second study used tlr-2 knockout mice , which are highly susceptible to mtb infection . when these mice received mast cells that express tlr-2 before infection with mtb , they had an improved inflammatory response , with myeloid cell recruitment and granuloma formation that correlated with increased protection against the bacilli . these studies reinforce the notion of mast cells as crucial players of the lung innate immune response against bacterial pathogens . cytomegalovirus is a beta - herpes virus that causes interstitial pneumonia in immunocompromised patients and is a serious problem in patients with an organ transplant . infection of mast cell - deficient mice with cytomegalovirus is followed by high viral replication and delayed virus clearance from the lungs , compared with wild - type mice . interestingly , this phenomenon was associated with a defective production of the chemokine ccl5 ( also known as rantes ) and a decreased recruitment of cd8 + t cells into the infected lungs , providing evidence that mast cells can recognize viral infection and induce the recruitment of essential effector cells to the site of infection in the lung . influenza is a frequent infectious disease of the human respiratory tract , which is caused by a group of viruses from the orthomyxoviridae family ; the most common of these viruses is influenza a. these viruses have attracted attention because they are associated with pandemics that have severely affected human health , like the spanish flu of 1918 . interestingly , studies with this strain have reported alterations in lung architecture , which are caused by an excessive inflammatory response promoted by an uncontrolled production of inflammatory cytokines and chemokines , also known as cytokine storm . in a mouse model of infection with a pathogenic strain of h5n1 virus , lung lesions were reduced if mice were treated with ketotifen , a well - known mast cell degranulation inhibitor . moreover , mice survival was dramatically increased if mice received a combined treatment of ketotifen and oseltamivir , an antiviral drug . these results suggest that mast cell activation correlated with pathology development during h5n1 infection , and that inhibition of mast cell activation improved host health . recently showed that the pathology associated with influenza a virus infection is significantly decreased in mast cell - deficient mice , compared with wild - type mice . this event is associated with a lower production of the inflammatory cytokine tnf- and the chemokines ccl2 , ccl3 , ccl4 , cxcl2 and cxcl10 in the lungs , suggesting a critical role of mast cells during the induction of the cytokine storm . the studies in mice described above show that mast cells play a pro - inflammatory role against bacterial and viral infections , with both beneficial and detrimental effects . on the one hand , mast cells are critical for the innate immune response because they recruit effector cells that are fundamental for the containment of the pathogens during the early stages of infection ; this event has been observed in other organs besides the lungs . on the other hand , excessive mast cell activation by pathogens can lead to damage and loss of lung function , as evinced by influenza studies . this excessive mast cell activation has also been observed in calves with paroxystic respiratory distress syndrome caused by bovine respiratory syncitial virus . recent evidence indicates that mast cells also play a predominant role in the development of pathology during dengue virus infection . st john a , et al . observed a direct correlation between the serum levels of chymase and the severity of dengue disease in humans ; interestingly , treatment of mice with drugs that block mast cell degranulation or with an antibody that blocks leukotriene receptors improved the vasculopathy associated with dengue virus infection . these studies point to mast cells as a new therapeutic target to limit the damage provoked by excessive inflammation during infectious diseases , such as influenza and dengue . in contrast with their classical pro - inflammatory function , it has been reported that mast cells can produce significant amounts of anti - inflammatory cytokines , like il-10 . mast cell - produced il-10 plays a crucial role in limiting the sterile inflammation provoked by allergic contact dermatitis or uv radiation . moreover , mast cell - produced il-10 is essential for the induction of immune suppression by uv exposure [ 49 , 50 ] . recently demonstrated that mast cells produce il-10 during in vivo bladder infection with uropathogenic escherichia coli , and this event was necessary to maintain the tolerance that allowed bacterial persistence . this study demonstrated that microorganisms can tune mast cells into a pro - inflammatory or an anti - inflammatory program in order to subvert the immune response . choi et al , recently showed that salmonella typhimurium injects a potent phosphatase ( sptp ) into the cytosol of mast cells ; sptp interferes with mast cell activation , even with a very strong signal ( fcri cross - linking ) . this strategy is also exploited by yersinia pestis , which uses a phosphatase ( yoph ) with high homology to sptp to dampen mast cell activation . pathogenic bacteria are not the only ones that effectively suppress mast cell activation , symbiotic bacteria have established a delicate balance with the immune response in order to colonize and survive in different anatomical locations of the host . breakdown of such balance usually results in the development of different pathologies . so , it is not surprising that bacteria that usually colonize the gastrointestinal tract can regulate mast cell activation . one of the first evidences of this strategy came from a study in which murine mast cells were stimulated with non - pathogenic e. coli from human gut . unexpectedly , mast cells did not degranulate , even after incubation with a high bacterial load , which contrast with the swift degranulation in response to pathogenic e. coli . furthermore , mast cells incubated with non - pathogenic e. coli lost the ability to degranulate in response to strong activators , such as a23187 and fcri cross - linking . a similar phenomenon is also observed with lactobacillus and bifidobacterium , other probiotic and symbiotic bacteria that reside in the human gut [ 56 , 57 ] . these results are of great interest in view of the association that exists between the use of probiotics and the prevention and treatment of several atopic diseases . however , how this prevention and treatment are achieved is still a matter of discussion , and several mechanisms have been proposed to explain this phenomenon . recent evidence derived from a mouse model of allergic inflammation points out that probiotics have the ability to decrease the effector phase in allergic diseases , even in the presence of specific ige antibodies . this effect is related to the inhibition of mast cell activation through the alteration of the signaling pathways triggered by fcri . the probiotics bifidobacterium breve and lactobacillus rhamnosus have been proposed as treatments for atopic diseases because of their potent effect in suppressing mucosal mast cell degranulation during chronic asthma . as described in the previous section , mast cells play an essential role in the initiation of the innate immune response to pathogens . in fact , all the cells that participate in type i hypersensitivity also participate in type 2 immune responses , which protect against infections with bacteria , viruses and multicellular parasites , and participate in the clearance of venoms , xenobiotics and irritants .type 2 innate lymphoid cells and th2 lymphocytes produce il-5 , which induces eosinophil activation and recruitment ; they also produce il-9 , which causes basophil and mast cell proliferation , and il-13 , which increases mucus production and causes fibrosis . these cell populations constantly interact with each other : for example , type 2 innate lymphoid cells produce il-13 in response to mast cell - derived prostaglandin d2 , for which they have a specific receptor , crth2 . since mast cells play a fundamental role during the early stages of type i hypersensitivity and also during the initiation of the innate immune response to pathogens , two important questions arise : 1 . is the response of mast cells to pathogens affected in an allergic environment ? 2 . can pathogens exacerbate type i hypersensitivity through mast cell activation ? in relation to the first question , there is considerable epidemiological evidence that asthma is a risk factor for viral and bacterial infections in the lungs , but what promotes this increased susceptibility to infections is poorly understood [ 63 - 67 ] . a deficient immune response could explain this increased susceptibility ; for example , jung j et al . observed that asthmatic patients have decreased titers of specific pneumococcal antibodies when compared to not - asthmatic individuals . other authors suggest a defective innate immune response as the cause of this increase in the susceptibility to infections . several mechanisms of the innate immune response are altered in allergic patients , including a defective epithelial barrier , an excessive mucus production and a decreased production of cytokines . however , this defective innate immune response apparently does not affect the potential of mast cell to combat infections , as is demonstrated in a mouse model of infection with mycoplasma pneumonie in an allergic setting . in relation to the second question , infectious diseases , especially those caused by bacteria and viruses , are considered important environmental elements that exacerbate atopic diseases . several reports indicate that infections with bacteria and viruses that affect the respiratory tract induce the production of ige antibodies specific for bacterial or viral components . infection of newborn mice with respiratory syncital virus ( rsv ) induces the production of virus - specific ige , and reinfection of these mice caused the airway symptoms classically associated with an allergic response . moreover , in a mouse model of acute infection with influenza a virus , an enhanced susceptibility of the host to develop a severe allergic response to house dust - mite was described . these studies suggest that the induction and the exacerbation of allergic responses by pathogens are associated with a modification of the environment that favors ige production ; however , another mechanism could be the exacerbation of mast cell responses . as we discussed earlier , several bacterial and viral pathogens can induce mast cell activation , inducing a pro - inflammatory environment that recruits effector immune cells . but how does a mast cell respond if it is simultaneously activated through fcri and through receptors that sense infection ? early studies suggested that this double stimuli results in an increased activation of mast cells . for example , mast cell lines activated through fcri and infected with rhinovirus release more histamine and produce more cytokines than mast cell lines activated with a single stimuli . patients with allergic rhinitis that were experimentally infected with rhinovirus showed increased levels of histamine and eosinphil recruitment in bronchoalveolar lavages , compared with allergic individuals that were not infected with rhinovirus , suggesting an altered mast cell response . several reports also associate infection with pathogenic bacteria with the development of an environment that facilitates or exacerbates the development of type i hypersensitivity . for example , the pathogenic bacteria mycoplasma pneumoniae , streptococcus pneumoniae , haemophilus influenza and chlamydia pneumoniae induce the production of ige specific for bacterial components , suggesting that infection promotes an environment favorable for the development of allergies [ 76 - 78 ] . however , little information exists about how these pathogenic bacteria could affect mast cell activation through fcri . as mentioned above , the only bacteria that have been analyzed in this respect are symbiotic bacteria from the gastrointestinal tract ( e. coli , lactobacillus spp and bifidobacterium spp ) , and these bacteria ameliorated fcri - mediated activation of mast cells , showing a therapeutic potential of these bacteria for allergic reactions in the gut . however , other symbiotic bacteria that colonize a different environment , the vagina , apparently do not mimic this effect . interestingly , new evidence has demonstrated that the human respiratory tract is home to several symbiotic bacteria , and environments that were thought to be sterile , like the bronchial tree , in fact contain about 2,000 bacterial genomes per cm . moreover , asthmatic patients showed altered bacterial communities in their airways , when compared with healthy individuals , with an increase in haemophillus spp and a decrease in prevotella spp . how these changes in microbiota airway affect the progress of type i hypersensitivity and how they alter mast cell function are fields that need to be clarified in the future . this process develops in an orderly fashion , involving hemostasis , inflammation , cell proliferation and remodeling of the affected tissue . alterations in the sequence , duration or intensity of any of these stages result in an impaired tissue repair that is usually reflected in the development of different pathologies . several cell populations are involved in this process , and cells of the immune system have been implied in different steps of the process . first , they tend to accumulate and degranulate in the wounded tissue , favoring the recruitment of effector cells , mainly neutrophils and macrophages . the neutral proteases chymase and tryptase favor the degradation of the extracellular matrix [ 83 , 84 ] ; mast cell production of arachidonic acid - derivatives induces the production of type i collagen by fibroblasts , and mast cell production of fibroblast growth factor ( fgf ) , vascular endothelial growth factor ( vegf ) and transforming - growth factor 1 ( tgf-1 ) is considered important for fibroblast proliferation and for the formation of new blood vessels . however , much of the knowledge about the role of mast cells during wound healing comes from studies in the skin , and little work has been performed in the lung , perhaps because of the lack of an appropriate model of lung injury , making this an interesting field that needs more research to clarify the role of mast cells in this environment . paul ehrlich described that mast cells were abundant in areas near tumors , and assumed that these cells played an important role in the regulation of tumor growth more than 100 years ago . since then , several reports have confirmed that mast cells usually accumulate in areas near tumors , and that this accumulation is associated with a poor prognosis in breast cancer , melanoma , hodgkin s lymphoma , oral squamous cell carcinoma , renal carcinoma , pancreatic cancer , endometrial cancer , cervical cancer , etc . in 2000 , imada et al . observed that patients with stage i non - small cell lung adenocarcinoma had an increased number of mast cells in tumor areas around blood vessels , and those patients with higher mast cells counts had a worse prognosis . it has been suggested that tumor cells produce chemotactic mediators that attract mast cells to the tumor , but few of these chemotactic mediators have been identified . one example is stem cell factor ( scf ) , which is produced by several tumor - derived cell lines , including a human alveolar adenocarcinoma cell line . scf interacts with the receptor c - kit , which is expressed on mast cells , and directs their migration to the tumor . how mast cells contribute to tumor growth is still matter of research , but three main mechanisms have been proposed : 1 ) production of tumor growth factors , 2 ) facilitation of tumor angiogenesis and 3 ) modification of the interaction between the epithelial cells and the stroma . a mast cell product that leads to the proliferation of tumor cells is histamine , which promotes the proliferation of alveolar basal carcinoma cells . adrenomedullin - stimulated mast cells produce cytokines , such as fibroblast growth factor ( fgf ) and vascular endothelial growth factor ( vegf ) , which are associated with increased angiogenesis in vivo . observed an increase in the number of mast cells that produce vegf in patients with lung adenocarcinoma . in contrast with previous reports by several authors , welsh et al . reported that , in non - small cell lung adenocarcinoma , the number of mast cells in the tumor stroma had no correlation with tumor progression , while an increased number of mast cells in the tumor cell islets correlated with an improved prognosis . these results suggested an anti - tumor role for the infiltrating mast cells , but the mechanism was not investigated , and further studies are needed to clarify this issue . mast cells can trigger different mechanisms that contribute to the homeostasis and adequate function of the lungs . disequilibrium in the function of mast cells can lead to pathological states , of which asthma is the most recognized . mast cells play an essential role in type i hypersensitivity ( allergic inflammation ) and also during the early stages of the innate immune response to pathogens . several causes , from infections to tumor growth , alter the function of mast cells ( fig . the regulation of mast cell activation by conventional drugs or microorganisms , either pathogenic or symbiotic , opens a new window of opportunities to develop new treatments that could preserve and restore lung health .

lungs are indispensable organs for the respiratory process , and maintaining their homeostasis is essential for human health and survival . however , during the lifetime of an individual , the lungs suffer countless insults that put at risk their delicate organization and function . many cells of the immune system participate to maintain this equilibrium and to keep functional lungs . among these cells , mast cells have recently attracted attention because of their ability to rapidly secrete many chemical and biological mediators that modulate different processes like inflammation , angiogenesis , cell proliferation , etc . in this review , we focus on recent advances in the understanding of the role that mast cells play in lung protection during infections , and of the relation of mast cell responses to type i hypersensitivity - associated pathologies . furthermore , we discuss the potential role of mast cells during wound healing in the lung and its association with lung cancer , and how mast cells could be exploited as therapeutic targets in some diseases

this study aims to model the financial benefits of implementing an electronic medical record ( emr ) system for hospitals in low - income settings . to date the limited attention given to analyzing financial soundness has been couched solely in high - income settings . we quantify three areas of significant potential cost savings of an emr system based on non - emr data from a hospital in lilongwe , malawi : ( 1 ) length of stay ; ( 2 ) transcription time ; and ( 3 ) laboratory use . advocates of the use of emr in low - income settings have primarily emphasized their potential to improve quality of care,1 benefits that mainly accrue to patients and the community . however , these benefits are realized at a significant cost to healthcare organizations ; these costs are cited as a primary barrier to adoption of emr in the usa , and represent a more significant obstacle in low - income settings.2 so far , there has been little consideration of the financial benefits of emr in the literature , much less their potential cost savings in low - income settings . in this paper we present a prospective analysis of some of the main categories in which a hospital - wide emr system could generate financial benefits in a low - income setting , modeled on an emr system in development in a hospital in lilongwe , malawi . while there is interplay between clinical and financial outcomes , analyses of emr in low - income settings emr in these settings have been shown to reduce wait times and medication order errors.3 financial analyses of emr systems in high - income countries have identified cost savings due to reductions in transcription and chart pulling , drug expenditures and adverse drug events , more efficient laboratory and radiology testing , and more accurate billing.2 49 these savings can be classified as either non - personnel related , such as meals , which will generate immediate savings , or personnel costs , such as transcription time , which will not be realized immediately . the distinction between high - income and low - income settings is important because the impact of an emr system is a function of factors such as resource availability , scale , and existing inefficiencies in the paper - based workflow . for example , an emr in lilongwe must adapt to low computer literacy rates , limited healthcare budgets , inadequate staffing , electric power disruptions , and inadequate security , none of which are significant challenges in the usa.10 these differences are all likely to contribute to a very different financial scenario for an emr in a low - income setting . for example , financial benefits due to reductions in malpractice premiums and insurance reimbursement are more relevant in the usa , while reductions in drug stockouts are a more significant consideration in malawi . this paper adds to the emr valuation literature by considering the financial , rather than clinical , benefits of an emr system in a low - income , rather than a high - income , setting . baseline information about the paper - based system in the lilongwe hospital was paired with estimated cost savings from analyses of emr in western hospital settings . in the absence of comparable data from low - income settings when multiple studies have been found we have used the most modest improvements to maintain the conservative nature of the model . throughout , there is an emphasis on the economies of scale of this emr system ; each component of the emr generates benefits beyond the immediate department . kamuzu central hospital ( kch ) , located in lilongwe , malawi , is a government - operated tertiary referral hospital with 710 beds . , it treated 275 880 patients , with an average bed occupancy of 83%.11 kch employs 176 physicians and clinical officers ( similar to nurse practitioners ) and 262 nurses . the hospital has long - standing shortages of both staff and supplies,12 factors that affect how fully the benefits of an emr system are realized . resource constraints are relevant to this analysis because they affect the ability of the hospital to capture the financial benefits ; non - personnel savings are naturally more easily realized than personnel - related savings , and this difference is compounded by the existing staff shortages . for example , the hospital complement is supposed to have 532 nurses , indicating a shortage of over 50% in 2010.13 these issues are compounded by similar shortages in surrounding health centers , which increase the number of patients seeking treatment at kch and the amount of primary care that is provided . in 2001 the non - governmental organization baobab health trust implemented a prototype of the emr in operation at kch.14 over the following decade the emr was iteratively developed , beginning with a pediatric module and expanding to include patient registration , radiology , laboratory specimen management , inpatient admission / discharge , and chronic disease management for hiv / aids and non - communicable diseases.15 16 additional modules currently under development include a laboratory management system , and computerized provider order entry for ordering medications . the kch emr has approximately 70 touchscreen clinical workstation appliances ( workstations ) deployed at the point of care in the ambulatory setting that enable health workers to retrieve medical records using barcode scanners.16 two central servers within kch store the emr data on a local network supported by a centralized power backup system . we estimated a one - time cost for the full implementation of all modules of the emr that included initial software configuration to accommodate clinical workflow , followed by routine operating costs such as electricity and consumables . a 2010 internal analysis identified 13 potential areas of cost savings from the emr system at kch.11 we attempted to re - analyze in greater detail the four areas with the largest potential benefit : length of stay ; transcription time ; adverse drug events ; and laboratory use . we collected data from several departments to quantify a baseline under the paper - based system . the hospital administration provided data on patient volume and length of stay , the laboratory supplied data on test volume and supply orders , and interviews with staff such as nurses and clinical officers gave insight into the daily workflow . we chose to exclude the category of adverse drug events from this analysis , as we did not have the data to support this initial study ; we are currently independently studying this category in more detail . costs and projected savings are modeled in us$. we chose the hospital as the unit of analysis to capitalize on the returns to scale of the emr system and account for the relationships among the different hospital departments . furthermore , because the decision to invest in an emr system was made at the hospital level , and its sustainability is the responsibility of the ministry of health , it seemed relevant to analyze the effect of the emr system on the hospital 's budget . however , it is also possible to extend the theory that economies of scale are important in realizing the financial benefit of emr and analyze potential financial benefits at a higher level of organization , such as at the district , zonal , and national levels . for example , one of the natural consequences of an emr is the collection of large amounts of data . these data can easily be aggregated to generate routine reports required by the ministry of health , and thus additional cost savings may also be realized at higher levels . in the usa , a business case analysis conducted by kaiser permanente estimated that approximately 35% of net benefits identified from the introduction of an emr are attributable to a reduction in the average length of stay resulting from efficiency gains.17 electronic communication of orders and results between clinical and ancillary departments ( laboratory , radiology , pharmacy ) are cited as a driver of efficiency gains.18 evidence of the impact of emr on hospital length of stay varies widely . an early study conducted on an internal medicine service in a large urban hospital in the usa showed a 0.89 day ( 10.5% ) reduction in length of stay resulting from electronic ordering.19 the government accountability office benchmark for length of stay reduction associated with an emr is 30%.18 thus far , we have found no study measuring the impact of emr on length of stay in a low - income setting . length of stay at kch averaged 4.82 days in 201011 , but varied significantly by ward . for example , the male surgical ward averaged 18.6 days per stay , while it was only 2.18 days in the maternity ward . our analysis includes all non - paying inpatient wards at kch . while there are challenges that contribute to length of stay that the emr system may not directly benefit , such as pharmaceutical and blood shortages , communication lapses also contribute to longer stays in the hospital ; for example , if a patient 's laboratory results are ready but have not been picked up in time for rounds , the patient stays admitted until the next opportunity for rounds , which may be 23 days due to limited rounding at the weekend ( b. gondwe , personal communication , 2012 ) . to maintain the conservative nature of this model , we assumed a 10.5% reduction in length of stay for inpatients and consider only cost savings associated with meal provision and time saved for clinical staff . the hospital provides meals to a patient at a cost of malawi kwacha 350 , or approximately us$1.75 , per day . in calculating a reduction in nurse and clinician time required we assume that approximately 60% of their time is spent managing inpatients ( t. bui , personal communication , 2012 ) . transcription activities are those that involve transferring information from one place to another . in the usa , emr systems have been found to reduce transcription time by 2850% , largely through the partial elimination of dictation.2 18 dictation is not a relevant part of the kch workflow , but medical staff spend a significant amount of time engaged in indirect care , defined as discussing cases with colleagues , reading and writing patient charts , and prescribing medications.20 time is also spent in the compilation of mandatory reports for the ministry of health , admission registration , and chart creation . a study of three hiv clinics in uganda found that providers spent approximately 34% less time during the workday engaging in indirect patient care after the implementation of an emr system.20 a similar study at a rural health center in kenya found a two - thirds reductions in inter - clinic personnel contact time as a result of the implementation of the mosoriot medical record system.21 anecdotal evidence of the time burden of indirect care under a paper - based system is provided for hospital antiretroviral therapy clinics in ethiopia , where providers cite the excessive time spent in activities such as error checking with pharmacists and consultations with laboratory technicians.22 based on interviews conducted in 2010 , we estimated that doctors , laboratory technicians , nurses and some administrative staff spend approximately 1 h per working day on transcription.11 the integration of barcodes and label printing into the emr system reduces transcription time by eliminating the need to write information that has previously been entered elsewhere in the system . we attribute a reduction of 17 min per employee per working day ( 28% ) in transcription time to the emr system . laboratory data are involved in 70% of medical diagnoses and can significantly affect the success and cost of patient treatment.23 in the usa , emr have been found to reduce test orders by 715%.2 24 some of this reduction is attributed to improved access to health records ; it has been estimated that 13.7% of laboratory tests are ordered because of lack of access to earlier results.25 the hiv / aids pandemic has triggered investment in laboratory capacity building with some emphasis on information systems to improve laboratory workflow in several low and middle - income countries , including cte d'ivoire , haiti , malawi , peru , rwanda , south africa , vietnam and zambia.2629 however , for the benefits of information systems in this setting to be fully realized the management of laboratory information must extend beyond the walls of the laboratory , starting at the point the clinician requests the laboratory investigation and finishing at the time the clinician makes a decision based on the test result.27 smaller health centers must send specimens to centralized laboratories for tests to be completed , further complicating the process and creating additional delays . the increasing dependence on expensive pcr testing in hiv management in malawi means that more laboratory testing needs to be centralized.30 the 11-step process and associated information management challenges of centralized laboratory testing for early infant diagnosis of hiv has been described in this context.26 benefits resulting from the use of information systems to support the management of laboratory information have been demonstrated in low - income countries such as zambia and rwanda , commonly finding a reduction in turnaround time and fewer results that do not reach clinicians.28 31 a 65% reduction in the delay of accessing tuberculosis results as well as the detection of duplication of laboratory testing has been demonstrated in peru.32 in this model we limit cost savings from the emr system to benefits realized in two areas : fewer samples redrawn due to better labeling , and fewer tests ordered due to improved access to past test results . a study examined 3549 samples sent to the kch laboratory for testing during a 4-week period , identifying whether they were correct and complete or needed to be redrawn ; if the latter , the reason(s ) the sample was inadmissible were recorded.33 overall , 18% of samples sent to the laboratory were discarded due to paperwork and labeling errors . similar challenges associated with incomplete laboratory request forms and associated wastage and delays in processing laboratory specimens have been described in south africa.34 while an emr system should completely eliminate documentation and labeling errors , the model assumed a 50% reduction in wasted samples due to documentation and labeling problems . improving clinicians access to medical history , specifically recent laboratory tests , should reduce the number of duplicate tests ordered . we make the conservative assumption that an emr would reduce the number of tests conducted at kch by 7% . based on information provided by the kch laboratory , we estimated the cost of a sample to be us$1 , and the cost of the test to be us$3 ; both figures account for employee time and materials . the investment cost of the emr system is calculated at us$337 847 , and includes hardware , software configuration , project management , installation and training ; these costs are outlined in table 1 . in representing the costs of the comprehensive emr system implementation , we excluded software development costs because the software is freely available and open source.16 while the level of emr deployment at kch is significant ( approximately 70 workstations ) , costs described here are not incremental and assume no existing infrastructure . costs are extrapolated from the actual installation of the emr system at kch , and include 151 workstations and two servers . each workstation included a touchscreen computer , label printer , and barcode scanner . the hardware , network electronics and power backup system are described in detail in a previous publication.16 project management cost is an estimated 1 year salary for a local project manager who coordinates the system implementation . training includes the costs for providing half - day training sessions for clinical staff who will use the emr , and salary for local software support staff who provide training full time for an estimated 6 months during emr implementation . installation includes the cost of a team of three local hardware technicians who install all system hardware over a period of 4 weeks . software configuration includes 1 month of salary for one local software support staff and one software developer who configure the software modules to support the clinical workflow within each point - of - care setting . the yearly operating costs are estimated to be us$29 824 , and include consumables ( eg , labels ) ( us$9258 ) , electricity ( us$9656 ) , hardware and software maintenance ( us$5910 ) , and recurrent training ( us$5000 ) . in year 3 , the operating cost is higher ( us$47 424 ) due to the cost of replacing the batteries ( us$17 600 ) . investment costs for the emr system at kch emr , electronic medical record ; kch , kamuzu central hospital . the benefits and costs outlined here are summarized over time in table 2 , and the 5-year financial outlook is shown in table 3 . to maintain the conservative nature of this analysis , we assume that no benefits are realized in the first 6 months of use . this choice recognizes that benefits of the emr will accrue gradually , and that any initial efficiency improvements are likely to be offset by productivity losses . costs and benefits are discounted on a yearly basis at a 5% annual percentage rate , the assumed cost of capital for the hospital . five - year return on emr implementation * discounted yearly at 5% to reflect the cost of capital . the estimated reductions in the various categories are determined based on a mixed - methods approach , using the existing ( western ) literature , data from kch , and interviews with kch administration and staff . in table 4 we present variations on the assumptions in table 3 to examine how the financial outlook changes when the benefits vary . first , we examine how the outlook changes when we assume only half of western the reductions identified in emr in the model . second , because savings associated with consumables are more quickly realized than those associated with personnel , we distinguish between personnel and non - personnel cost savings by removing personnel cost savings from our original analysis . in the usa , a business case analysis conducted by kaiser permanente estimated that approximately 35% of net benefits identified from the introduction of an emr are attributable to a reduction in the average length of stay resulting from efficiency gains.17 electronic communication of orders and results between clinical and ancillary departments ( laboratory , radiology , pharmacy ) are cited as a driver of efficiency gains.18 evidence of the impact of emr on hospital length of stay varies widely . an early study conducted on an internal medicine service in a large urban hospital in the usa showed a 0.89 day ( 10.5% ) reduction in length of stay resulting from electronic ordering.19 the government accountability office benchmark for length of stay reduction associated with an emr is 30%.18 thus far , we have found no study measuring the impact of emr on length of stay in a low - income setting . length of stay at kch averaged 4.82 days in 201011 , but varied significantly by ward . for example , the male surgical ward averaged 18.6 days per stay , while it was only 2.18 days in the maternity ward . our analysis includes all non - paying inpatient wards at kch . while there are challenges that contribute to length of stay that the emr system may not directly benefit , such as pharmaceutical and blood shortages , communication lapses also contribute to longer stays in the hospital ; for example , if a patient 's laboratory results are ready but have not been picked up in time for rounds , the patient stays admitted until the next opportunity for rounds , which may be 23 days due to limited rounding at the weekend ( b. gondwe , personal communication , 2012 ) . to maintain the conservative nature of this model , we assumed a 10.5% reduction in length of stay for inpatients and consider only cost savings associated with meal provision and time saved for clinical staff . the hospital provides meals to a patient at a cost of malawi kwacha 350 , or approximately us$1.75 , per day . in calculating a reduction in nurse and clinician time required we assume that approximately 60% of their time is spent managing inpatients ( t. bui , personal communication , 2012 ) . transcription activities are those that involve transferring information from one place to another . in the usa , emr systems have been found to reduce transcription time by 2850% , largely through the partial elimination of dictation.2 18 dictation is not a relevant part of the kch workflow , but medical staff spend a significant amount of time engaged in indirect care , defined as discussing cases with colleagues , reading and writing patient charts , and prescribing medications.20 time is also spent in the compilation of mandatory reports for the ministry of health , admission registration , and chart creation . a study of three hiv clinics in uganda found that providers spent approximately 34% less time during the workday engaging in indirect patient care after the implementation of an emr system.20 a similar study at a rural health center in kenya found a two - thirds reductions in inter - clinic personnel contact time as a result of the implementation of the mosoriot medical record system.21 anecdotal evidence of the time burden of indirect care under a paper - based system is provided for hospital antiretroviral therapy clinics in ethiopia , where providers cite the excessive time spent in activities such as error checking with pharmacists and consultations with laboratory technicians.22 based on interviews conducted in 2010 , we estimated that doctors , laboratory technicians , nurses and some administrative staff spend approximately 1 h per working day on transcription.11 the integration of barcodes and label printing into the emr system reduces transcription time by eliminating the need to write information that has previously been entered elsewhere in the system . we attribute a reduction of 17 min per employee per working day ( 28% ) in transcription time to the emr system . laboratory data are involved in 70% of medical diagnoses and can significantly affect the success and cost of patient treatment.23 in the usa , emr have been found to reduce test orders by 715%.2 24 some of this reduction is attributed to improved access to health records ; it has been estimated that 13.7% of laboratory tests are ordered because of lack of access to earlier results.25 the hiv / aids pandemic has triggered investment in laboratory capacity building with some emphasis on information systems to improve laboratory workflow in several low and middle - income countries , including cte d'ivoire , haiti , malawi , peru , rwanda , south africa , vietnam and zambia.2629 however , for the benefits of information systems in this setting to be fully realized the management of laboratory information must extend beyond the walls of the laboratory , starting at the point the clinician requests the laboratory investigation and finishing at the time the clinician makes a decision based on the test result.27 smaller health centers must send specimens to centralized laboratories for tests to be completed , further complicating the process and creating additional delays . the increasing dependence on expensive pcr testing in hiv management in malawi means that more laboratory testing needs to be centralized.30 the 11-step process and associated information management challenges of centralized laboratory testing for early infant diagnosis of hiv has been described in this context.26 benefits resulting from the use of information systems to support the management of laboratory information have been demonstrated in low - income countries such as zambia and rwanda , commonly finding a reduction in turnaround time and fewer results that do not reach clinicians.28 31 a 65% reduction in the delay of accessing tuberculosis results as well as the detection of duplication of laboratory testing has been demonstrated in peru.32 in this model we limit cost savings from the emr system to benefits realized in two areas : fewer samples redrawn due to better labeling , and fewer tests ordered due to improved access to past test results . a study examined 3549 samples sent to the kch laboratory for testing during a 4-week period , identifying whether they were correct and complete or needed to be redrawn ; if the latter , the reason(s ) the sample was inadmissible were recorded.33 overall , 18% of samples sent to the laboratory were discarded due to paperwork and labeling errors . similar challenges associated with incomplete laboratory request forms and associated wastage and delays in processing laboratory specimens have been described in south africa.34 while an emr system should completely eliminate documentation and labeling errors , the model assumed a 50% reduction in wasted samples due to documentation and labeling problems . improving clinicians access to medical history , specifically recent laboratory tests , should reduce the number of duplicate tests ordered . we make the conservative assumption that an emr would reduce the number of tests conducted at kch by 7% . based on information provided by the kch laboratory , we estimated the cost of a sample to be us$1 , and the cost of the test to be us$3 ; both figures account for employee time and materials . the investment cost of the emr system is calculated at us$337 847 , and includes hardware , software configuration , project management , installation and training ; these costs are outlined in table 1 . in representing the costs of the comprehensive emr system implementation , we excluded software development costs because the software is freely available and open source.16 while the level of emr deployment at kch is significant ( approximately 70 workstations ) , costs described here are not incremental and assume no existing infrastructure . costs are extrapolated from the actual installation of the emr system at kch , and include 151 workstations and two servers . the hardware , network electronics and power backup system are described in detail in a previous publication.16 project management cost is an estimated 1 year salary for a local project manager who coordinates the system implementation . training includes the costs for providing half - day training sessions for clinical staff who will use the emr , and salary for local software support staff who provide training full time for an estimated 6 months during emr implementation . installation includes the cost of a team of three local hardware technicians who install all system hardware over a period of 4 weeks . software configuration includes 1 month of salary for one local software support staff and one software developer who configure the software modules to support the clinical workflow within each point - of - care setting . the yearly operating costs are estimated to be us$29 824 , and include consumables ( eg , labels ) ( us$9258 ) , electricity ( us$9656 ) , hardware and software maintenance ( us$5910 ) , and recurrent training ( us$5000 ) . in year 3 , the operating cost is higher ( us$47 424 ) due to the cost of replacing the batteries ( us$17 600 ) . investment costs for the emr system at kch emr , electronic medical record ; kch , kamuzu central hospital . the benefits and costs outlined here are summarized over time in table 2 , and the 5-year financial outlook is shown in table 3 . to maintain the conservative nature of this analysis , we assume that no benefits are realized in the first 6 months of use . this choice recognizes that benefits of the emr will accrue gradually , and that any initial efficiency improvements are likely to be offset by productivity losses . costs and benefits are discounted on a yearly basis at a 5% annual percentage rate , the assumed cost of capital for the hospital . five - year return on emr implementation * discounted yearly at 5% to reflect the cost of capital . the estimated reductions in the various categories are determined based on a mixed - methods approach , using the existing ( western ) literature , data from kch , and interviews with kch administration and staff . in table 4 we present variations on the assumptions in table 3 to examine how the financial outlook changes when the benefits vary . first , we examine how the outlook changes when we assume only half of western the reductions identified in emr in the model . second , because savings associated with consumables are more quickly realized than those associated with personnel , we distinguish between personnel and non - personnel cost savings by removing personnel cost savings from our original analysis . the calculations presented compare the known costs of deploying an emr system with a conservative estimate of its cost savings . a breakdown of the annual cost savings is shown in table 2 . given 43 484 inpatients with an average length of stay of 4.82 days , a 10.5% reduction in length of stay would save us$128 645 in food and personnel costs per year . reducing transcription time by 17 min ( 28% ) each workday for all doctors , laboratory technicians , nurses and some administrative employees the reductions in laboratory samples and duplicate tests would save the hospital us$91 187 each year . the benefits from reductions in length of stay , transcription , and laboratory samples are estimated at us$284 395 per year . the yearly financial return for the first 5 years of use is shown in table 3 . , the emr pays for itself in less than 3 years , and over a 5-year period the hospital saves us$613 681 . this model serves as a proof of concept that emr can have financial , in addition to clinical , benefits in low - income settings . these results highlight the coupled nature of clinical and financial outcomes , as well as the more general relationships among various hospital departments . despite initially juxtaposing the clinical and financial impacts of emr in motivating this research for example , improvements in length of stay due to an emr , while generating cost savings for the hospital , should raise the quality of care . the financial benefits build upon the natural economies of scale associated with expanding an emr across all departments of the hospital . for example , installing the emr laboratory system reduces length of stay in the inpatient wards by improving diagnostic efficiency and the communication of pre - surgical laboratory tests . the significant correlation among these areas , due to their tight relationships in providing care , is why this analysis focused only on small pieces of each category . for example , food and personnel costs were the only considerations in calculating length of stay savings , ignoring the contributions of other departments such as laboratory , radiology and pharmacy . the goal of this paper was to examine the financial impact of an emr system in a low - income setting . even with discount rates as high as 31% , the break - even point would be realized in the third year of use , which is important given that discount rates in low - income settings tend to be higher than those in high - income areas.35 the list of financial benefits modeled is not comprehensive , suggesting that this estimate represents a lower bound on the financial return of this emr system . furthermore , this analysis does not consider factors such as the residual value of the equipment . other previously cited benefits , such as chart handling and more efficient drug procurement , are also likely to be realized at kch , but adequate data did not exist to explore these categories . to avoid overlap and maintain the conservative nature of this analysis , the results suggest that over a relatively short time horizon the emr system generates cost savings for the hospital , the equivalent of the annual salaries of over 70 physicians . the next natural step is to analyze each of these areas in more detail , acknowledging the relationships among them and collecting more data to quantify these benefits better . these results can be aggregated to the level of the hospital , and can take advantage of the implementation of the emr modules currently in development . by acknowledging the complexity of categories such as length of stay and laboratory testing , the benefits of an emr system in a low - income setting such as kch will be more clearly articulated . while we believe the role and the effects of emr systems are different in low - income and high - income country settings , table 4 examines the robustness of our findings to other sets of assumptions . to permit better comparison , the break - even month is calculated , assuming that the annual savings and costs are realized in equal monthly increments . over 5 years scenario 1 presents the financial analysis conducted earlier in this paper . in acknowledgement that the assumed reductions may not be realized at kch , scenario 2 assumes that kch realizes only half of the reductions ; a net financial gain from the emr system is realized during the fourth year after implementation . finally , in scenario 3 we distinguish between personnel cost savings and non - personnel cost savings by focusing on the latter . non - personnel cost savings , such as a reduction in food expenses due to shortened length of stay , are more easily realized than personnel cost savings , such as a reduction in transcription , which imply a reduction in the work force necessary to provide the same level of service . kch , like other health facilities in the area , faces a long - standing staff shortage . therefore , while it may not be feasible to reduce the existing staff level , or hire additional staff , the emr would allow employees to be repositioned into critical , currently unfilled roles within the ministry of health . in this way , the savings in length of stay , transcription time , and laboratory use can be thought of as a reduction in the staff time required to perform the same level of care , thus allowing that saved time to be redirected to other tasks currently affected by the staff shortage . ignoring personnel costs significantly lowers the 5-year value of the emr system , and profit is not realized until the fifth year of implementation . the results of this prospective analysis suggest that emr can be financially sound investments in low - income settings . it strove to be conservative enough in its assumptions to serve as a lower bound estimate of the financial impact of this system and required simplification of the contributing factors to avoid overlap ; more comprehensive analyses of each component are planned . this paper provides a framework for these further studies , which can improve the model by substituting its assumptions for evidence ; this model can also be applied to cost savings analyses of emr in other low - income hospital settings . understanding the benefits of emr in low - income settings is a burgeoning area of research that so far has focused on the clinical effects , but the adoption of emr in any setting also depends on articulating the financial soundness of the initial investment . although this means that implementing an extensive emr system would require a long - term approach to budgeting , the timing of such an undertaking may be right ; the objectives of the 2011 malawi health sector strategic plan reflect a similarly long time - horizon and articulate goals that overlap well with the known strengths of emr . these aims included interest in systems for laboratory information , revenue management , continuous monitoring and evaluation , and improved vital registration.36 these results suggest that the dialogue surrounding emr in low - income settings should focus on how , rather than whether , to make these investments .

objectiveto model the financial effects of implementing a hospital - wide electronic medical record ( emr ) system in a tertiary facility in malawi.materials and methodswe evaluated three areas of impact : length of stay , transcription time , and laboratory use . we collected data on expenditures in these categories under the paper - based ( pre - emr ) system , and then estimated reductions in each category based on findings from emr systems in the usa and backed by ambulatory data from low - income settings . we compared these potential savings accrued over a period of 5 years with the costs of implementing the touchscreen point - of - care emr system at that site.resultsestimated cost savings in length of stay , transcription time , and laboratory use totaled us$284 395 annually . when compared with the costs of installing and sustaining the emr system , there is a net financial gain by the third year of operation . over 5 years the estimated net benefit was us$613 681.discussiondespite considering only three categories of savings , this analysis demonstrates the potential financial benefits of emr systems in low - income settings . the results are robust to higher discount rates , and a net benefit is realized even under more conservative assumptions.conclusionsthis model demonstrates that financial benefits could be realized with an emr system in a low - income setting . further studies will examine these and other categories in greater detail , study the financial effects at different levels of organization , and benefit from post - implementation data . this model will be further improved by substituting its assumptions for evidence as we conduct more detailed studies .

in today 's cost - conscious health care environment , equity and efficiency are becoming increasingly important considerations in managing and evaluating health care systems , especially as pressure to contain further growth in health care expenditures increases . equity and efficiency are an integral and essential part of a well functioning health care system . equitable and efficient health care systems will identify those in need of care , allow timely access to appropriate providers for adequate level of care , and protect service users against the risk of high expenditures . an efficient health care system will also induce proper and cost - effective provider and consumer behaviors through appropriate payment and benefit design . among many sectors of health care , the mh / sa care system has been particularly challenged with problems in equity and efficiency . studies have documented that access in mh / sa remains a serious problem . not only is mh / sa care manpower concentrated in certain areas , but also the geographic distribution of mh / sa care facilities is very uneven in the united states . in terms of service use , many studies raise concerns over an inefficient and inequitable pattern ; underutilization of services among the severely ill and vulnerable population and potential overutilization of care among the less serious may indicate problems in the system ( frank and lave , 1986 , lave and frank , 1988 ) . faced with many challenges , players in the mh / sa care system have searched , and continue to search , for ways to enhance the system 's equity and efficiency . significant legislative progress toward a more equitable provision of mh / sa care recently took place at the federal level through the mental health parity act of 1996 ( public law 104 - 204 ) . taking effect on january 1998 , the act requires that mental benefit provisions be no more restrictive than medical / surgical benefits in group health plans with 50 or more employees . at the state level , 13 states had already enacted a statewide mental illness parity laws , while three additional states enacted a parity law applicable only to state employees ( american managed behavioral healthcare association , 1997a ) . another notable activity in the current mh / sa market is the proliferation of new delivery and payment models , i.e. , managed behavioral health care . nearly absent a couple of decades ago , the managed behavioral health industry is estimated to cover approximately 170 million americans for their mh / sa care in 1997 ( american managed behavioral healthcare association , 1997b ) . and growth is expected to continue as more state medicaid programs adopt a managed behavioral care model . in such a dynamic and evolving market , we need to appraise the impact of new developments on access , use , and financing of mh / sa care . concomitant with the health care financing review 's interest in mh / sa care research , this issue disseminates research findings on access , use , and financing of mh / sa care for vulnerable populations . from 1980 - 85 , a comprehensive mh / sa epidemiological survey was conducted by the national institute of mental health ( nimh ) . known as the epidemiological catchment area ( eca ) study , the survey revealed a surprisingly high prevalence of mh / sa disorders in america . in a given month , about one in six adults are found to have a mh / sa disorder , and approximately one in four adult americans meet the criteria for mh / sa disorder during a year . equally surprising was that , of the population with mh / sa disorders , only one - half of them received mh / sa care at all , and less than one in three receive care within 1 year ( reiger et al . , 1993 ) . mental disorders encompass a broad range of conditions with different degrees of debilitating effects . as such , prevalence alone does not adequately depict the problems in mh / sa care need in america . based on the eca study conducted between 1980 and 1985 , the nimh estimates some five million adults are suffering from often incapacitating severe mental disorders , such as schizophrenia , manic - depressive illness , major depression , panic disorder , and obsessive - compulsive disorder . it was reported that about 60 percent of the persons with severe mh / sa disorders received care ( goodwin et al . , 1993 ) . a recent nationally representative survey , the national comorbidity survey reconfirmed this figure ( kesseler et al . prevalence of severe mh / sa disorders among children are not as well understood . however , according to a recent survey , mental disorders in child and adolescent populations ( meca ) , 3.2 percent of the sample adolescent population had a severe mh / sa disorder ( national institute for mental health , 1993 ) . accurate assessment of the need for hm / sa care is an essential guide in formulating a policy that allocates resources efficiently and equitably . especially in today 's changing health care market , we have to know what the current need is and to what extent these vulnerable population receive care . changing population characteristics and family and social structures necessitate continuous tracking of trends in need for mental health care . in an effort to assess the need and treatment in substance abuse care , this issue features an article by woodward and colleagues . this article presents new estimates of the numbers of persons in this country who need and receive substance abuse treatment . additionally , a summary of mental health data from the medicare current beneficiary survey ( mcbs ) round 13 is featured . as previously discussed , the epidemiological evidence clearly indicates we have serious problems with unmet needs among vulnerable populations . more than any other factors , the unequal provisions of mental health benefits in health insurance seems to have perpetuated this problem . rightfully fearing that generous mh / sa benefit would draw unfavorable selection , many private health insurance plans limited their mh / sa care benefits over time ( schuttinga , falik , and steinwald , 1985 ) . if the mental health parity act can restore equity in the provision of mh / sa benefits , it would considerably alleviate the inequity problem . with the rising concern over the health care expenditure inflation , we need to be more resourceful in planning and allocating resources . ( 1990 ) estimated the total expenditure for mh / sa in this country to be as much as $ 67 billion in 1990 . this amounts to approximately one - tenth of the total national expenditure on health care for the same year . studies indicate evidence of an inefficient pattern of service utilization in mh / sa care : underutilization of services among the severely ill and potential overutilization of care among the less serious . to enhance equity and efficiency in the system , it is important to monitor patterns of service utilization . in this issue of the review , there are a number of articles related to mh / sa service utilization in medicare and medicaid . rosenbach and ammering analyze medicare 's impact of mental health outpatient coverage expansion on utilization and expenditure between 1987 and 1992 . ettner and hermann examine the pattern of mental health specialist use in medicare , and report the factors that influence specialist use . an article by slifkin and colleagues proposes a reimbursement system for the care of the mentally retarded in intermediate care facilities . an article by cano , hennessy , warren , and lubitz presents information on demographic , diagnostic , utilization , and expenditure characteristics associated with inpatient psychiatric care among medicare beneficiaries in 1995 . the most sweeping change in the recent mh / sa system is the proliferation of managed mental health care . survey data show that the industry nearly doubled in covered lives between 1993 and 1997 ; the latest estimate puts the enrollment figure at approximately 170 million in 1997 ( american association for marriage and family therapy , 1996,american managed behavioral healthcare association , 1997b ) . this means approximately two - thirds ( 68 percent ) of the insured population are enrolled in a managed mh / sa care plan . this industry 's phenomenal expansion was driven , in part , by implementation of medicaid waivers in many states , a trend which seems certain to persist for the next several years . a total of 28 states and the district of columbia have implemented some form of managed mh / sa care waiver . nine states already received approval on their waiver requests , and ten additional states have pending waiver requests with various managed mh / sa care provisions . the managed mh / sa care contracts typically feature such characteristics as carved - out mh / sa benefits , person - level capitated budgets , risk sharing arrangements , and case management . compared with the incentives in indemnity insurance , each feature of the managed care contract can differently impact the utilization , financing , and outcome of the mh / sa care . at a time when the managed behavioral health system is emerging as the dominant mh / sa care delivery model , timely evaluation of its performance the third group of articles in this issue address this issue both empirically and theoretically . an article by stoner and colleagues evaluates a capitation payment demonstration in the community mental health centers ( cmhcs ) in the utah medicaid program . the authors report how capitation affected utilization and expenditures in three cmhcs . using afdc carve - out experience from massachusetts , norton , lindrooth , and dickey investigate whether the managed mh / sa care vendor shifted costs to the medical care sector . the findings from this relatively mature managed public program will be of great interest to other states that are at an early stage of managed behavioral care contracting . based on economic theory , frank and colleagues address the problem of adverse selection in the context of behavioral health care , and offers an explanation of why carve - out and cost sharing two key features of managed behavioral contracts offer a solution to this problem . the mh / sa care system in america is undergoing a sweeping transformation . while shifting demographics and social and family structure affect the need for mh / sa care , new financing and delivery models are emerging in both the private and public sector . in this highly dynamic and often uncertain era the articles presented in this issue of the review describe , analyze , and evaluate the transformations that are taking place in the mh / sa care system . it is our hope that such research activities ultimately help shape a more equitable and efficient mh / sa care system .

this overview presents an introduction to the articles published in this issue of the health care financing review , entitled mental health services and vulnerable populations . this article discusses the challenges the mental health and substance abuse ( mh / sa ) care system is confronted with in terms of equity and efficiency and how the system is responding to these challenges . it further addresses research issues in assessing the need and use of mental health services and summarizes recent activities in the research and evaluation of new delivery and payment systems .

inflammatory bowel disease ( ibd ) is a common diagnostic and therapeutic problem , affecting patients at increasingly young ages . a standard diagnostic method includes macroscopic assessment of the intestinal mucosa during colonoscopy and histopathological evaluation of the obtained biopsies . according to porto criteria , the diagnosis of ibd is based on the clinical presentation , intestinal endoscopic and histological features , laboratory tests , and radiological examination . recently , reports on the usefulness of a noninvasive examination , that is , faecal calprotectin measurement in ibd diagnosis , have been published . evaluation of faecal calprotectin ( fc ) seems to be a screening test selecting patients requiring further invasive diagnostics . furthermore , there have been reports on using calprotectin assays in monitoring the treatment of crohn 's disease and ulcerative colitis in adults and children [ 14 ] . to our knowledge , there are no published data on the usefulness of faecal calprotectin assays in the diagnosis of other atypical forms of bowel inflammation , such as eosinophilic , lymphocytic , or nonspecific colitis . the aim of the study is to assess the usefulness of faecal calprotectin measurement in children with various types of ibd and to evaluate fc concentration in children with crohn 's disease and ulcerative colitis in relation to disease activity . 91 patients were included in the analysis , including 49 boys ( 53.85% ) and 42 girls ( 46.15% ) , ranging from 6 to 18 years of age ( the mean age was 13.38 years ) . the study group comprised 71 children with various types of ibd , who were subsequently divided into six subgroups : b124 ( 33.8% ) children with crohn 's disease ( cd ) , b216 ( 22.5% ) with ulcerative colitis ( uc ) , b37 ( 9.8% ) with eosinophilic colitis ( ec ) , b48 ( 11.26% ) with lymphocytic colitis ( lc ) , and b516 ( 22.5% ) with nonspecific colitis ( nc , colitis indeterminata the control group ( k ) comprised 20 healthy , age- and sex - matched subjects . patients with ibd underwent following procedures : anamnesis , physical examination , laboratory tests ( inflammatory state markers , biochemical parameters of liver , pancreas , and kidney function , sweat test , coprological tests , and immunoassays ) , diagnostic imaging ( abdominal ultrasound ) , and endoscopy with histopathological evaluation . in all patients , faecal calprotectin was measured by means of elisa method , using phical test ( calpro ) . calprotectin concentrations ranging from 0 to 50 mg / kg were considered to be normal reference values . the results were evaluated by using the following tests : komogorow - smirnow , t - student , u mann - whitney , fisher , and yates ; the analysis of correlation was based on the spearman 's rank correlation coefficient . a p value of < 0.05 was considered statistically significant . all patients and their caregivers gave informed consent to participate in the study , which was approved by the bioethics committee of the medical university of silesia in katowice ( consent no . a statistically significant increase in the mean concentrations of faecal calprotectin was observed in the group of children with cd and uc , as compared to the control group . concentrations of fc were also higher in children with uc than in patients with cd . faecal calprotectin concentrations were within the normal limits in patients with eosinophilic , lymphocytic , and nonspecific colitis , similarly to the healthy subjects ( table 1 , figure 1 ) in children with cd , faecal calprotectin concentrations positively correlated with the disease severity assessed according to the pcdai scale . in patients suffering from uc , faecal calprotectin also positively correlated with the truelove - witts scale and the rachmilewitz endoscopic index ( figures 2 , 3 , and 4 ) . a significant increase in faecal calprotectin concentrations was observed in children suffering from cd , with lesions located in both small and large intestine , and in patients presenting with inflammatory changes in 5 or more sections of the intestine ( figure 5 ) . faecal calprotectin is a promising , noninvasive screening method for diagnosing patients suffering from gastrointestinal disorders , such as abdominal pain or diarrhea , which are also typical for ibd [ 57 ] . so far , many authors have considered calprotectin as a useful marker in differential diagnosis of ibd and functional gastrointestinal disorders ( e.g. , irritable bowel syndrome ) [ 8 , 9 ] . many authors evaluated faecal calprotectin concentrations in patients with suspected inflammatory process of the large intestine . demonstrated in the group of paediatric patients that this assay is characterised by 95% sensitivity and 93% specificity , and high calprotectin concentrations show strong positive correlation with the presence of inflammatory lesions in the large intestine . according to these authors so far , only limited studies , based on very small groups of patients , evaluated faecal calprotectin concentrations in patients with other types of bowel inflammations less common than cd and uc , such as lymphocytic , eosinophilic , and nonspecific colitis , which seem to be a considerable clinical problem in everyday pediatric practice . in studies conducted by bunn et al . , two children with nonspecific colitis and 3 children with allergic colitis were included in the analysis . in both cases , calprotectin concentrations were found to be within the normal ranges . in our study involving 31 children : 7 with eosinophilic colitis , 8 with lymphocytic colitis , and 16 with nonspecific colitis , respectively , these results support the hypothesis that in the aforementioned types of bowel inflammation , histopathological examination does not reveal infiltrations of neutrophil cells , whose cytosols contain calprotectin . therefore , its concentration in faeces is directly related to the number of neutrophils in the large intestine lumen [ 11 , 12 ] . in paediatric patients , the diagnosis of nonspecific colitis ( indeterminata colitis ) remains unchanged in approximately 36% . over time in some patients , the diagnosis may be changed into ulcerative colitis ( in approximately 3372.5% ) or crohn 's disease ( in approximately 1727.5% ) . in our group of 6 cd patients , previously diagnosed conditions included single cases of ulcerative colitis , lymphocytic colitis , and nonspecific colitis , whereas eosinophilic colitis was found in 3 subjects . increased calprotectin concentrations may be useful when making a decision on extending diagnostic procedures in patients with less frequent types of bowel inflammation . in our study , the mean calprotectin concentration in the examined patients with ibd was higher than it was observed by bremner et al . ; however , patients in remission were also enrolled in the latter research . in the presented material , a significant correlation was demonstrated between calprotectin activity and the disease severity assessed by the modified pcdai scale for cd and the modified truelove - witts scale and the rachmilewitz endoscopic index for uc . , demonstrating a strong positive correlation between calprotectin concentrations and the disease severity according to the modified truelove - witts scale only in uc . analyses performed in children assessed a correlation between fc concentrations and intensity of macroscopic and microscopic inflammatory lesions in the large intestine , observed in the course of ibd . studies carried out by fagerberg et al . included 39 children with ibd , in whom calprotectin concentrations strongly correlated with intensity and extent of micro- and macroscopic abnormalities . norwegian researchers also confirmed such a correlation and , moreover , suggested that intensity of inflammatory lesions rather than their extent influences faecal calprotectin concentration . this suggestion can be supported by data obtained from our study , indicating that in patients with uc calprotectin concentration depends on disease activity and not on the extent of lesions , in contrast to patients suffering from cd . in the latter group , calprotectin concentrations correlated with the disease activity and were significantly higher in children with inflammatory lesions present in both small and large intestine and located in 5 or more sections of the large intestine . these results support conclusions from the studies on clinical expression of the disease , which demonstrated that in children and adolescents the disease is more severe and cd lesions are located in the small intestine [ 17 , 18 ] . so far faecal calprotectin measurement seems a promising test to evaluate disease activity and a tool for monitoring ibd treatment . in everyday practice , a disadvantage of this method is its low specificity , which is connected with the fact that in patients with increased calprotectin concentration many organic diseases should be excluded . it seems that measurement of faecal calprotectin concentration can be a useful , safe , and noninvasive test in children suspected for ibd , since it is found to be increased in children with cd and uc as compared to patients with other inflammatory diseases ( eosinophilic , lymphocytic , and nonspecific colitis ) and also in the reference to healthy subjects . when the faecal calprotectin concentration is increased in children with less common types of bowel inflammation , a further follow - up of such patients faecal calprotectin concentration correlates positively with the disease severity in cd and uc patients ; thus , it may be useful when choosing or modifying the appropriate treatment regimen .

introduction . the aim of the study was to assess the usefulness of the fc measurement in children with various types of ibd and relation to the disease activity . patients and methods . 91 patients ( 49 boys : 53.85% and 42 girls : 46.15% , mean age : 13.38 years , range 618 years ) were included in the analysis . patients were divided into the groups : b124 children with cd , b216 patients with uc , and a group comprising 31 children with other types of colitis ; the control group ( k ) comprised 20 healthy children . fc was assayed by elisa method , using phical test ( calpro ) . results . the mean faecal calprotectin concentrations were higher in children with cd and uc as compared to healthy controls , patients with eosinophilic , lymphocytic , and nonspecific colitis . a positive correlation was observed between fc concentrations and the disease activity ( the pcdai scale , the truelove - witts scale , and the endoscopic rachmilewitz index ) . conclusion . it seems that the fc concentrations can be a useful , safe , and noninvasive test in children suspected for ibd , since fc concentration is higher in children with cd and uc than in patients with other inflammatory diseases .

phytoremediation technology using floating macrophytes ( eichhornia crassipes ) performed very well in remediation eutrophic water body since e. crassipes is capable of assimilating large amount of nutrients efficiently [ 13 ] . during 20102012 , large - scale confined growth of e. crassipes was used to remove pollutants ( mainly n and p ) from dianchi lake as well as the rivers connecting to the lake . there are more than 31 rivers , which carried wastewater discharged from different types of sewage treatment plants ( stps ) , agriculture , and domestic source , flowing into the lake . the macrophytes significantly improved water quality in both inflow rivers and dianchi lake . to evaluate the contributions of water hyacinth to the removal of nitrogen from the lake , both assimilation and stimulated denitrification by the macrophyte are important since n-15 tracing experiment in labs indicated that the values of n-15 at.% excess of n2-n production were significantly ( p < 0.05 ) higher with the growth of e. crassipes than that without [ 5 , 6 ] . the presence of e. crassipes roots has positive effect on stimulating the activity , abundance , and diversity of denitrifiers . previous studies also suggested that the root system of floating macrophyte could support the attachment of microorganism and enhance the growth and activity of bacteria for removing organic matter and nutrients . environmental conditions are also critically important in mediating the activity , abundance , and diversity of bacteria [ 9 , 10 ] . the abundance and diversity of denitrifiers on e. crassipes roots grown in the rivers receiving wastewater with different water properties may vary with the variation of environmental factors . the abundance of the functional genes and community compositions of denitrifiers can be affected by many factors , such as water temperature , ph , do , and nutrient concentrations . in the rivers with different sources of condensed pollutants and diverse physiochemical properties , the abundance and diversity of denitrifiers on the e. crassipes roots may be modified in various patterns . hence , in the present study , we investigated the abundance and diversity of denitrifying bacteria on e. crassipes roots in 11 rivers with different pollution sources in the north side of dianchi lake . it put an emphasis on understanding the interactions between the changes of environmental factors and the abundance and diversity of denitrifying bacteria attached to e. crassipes roots . it was expected that the results would shed some insight on how environmental factors and cultivation of e. crassipes mediate denitrification process in different eutrophic rivers flowing into the dianchi lake . a total of 11 rivers around dianchi lake located from 249 to 250 latitude and 1026 to 1027 longitude were investigated ( figure 1 ) . as shown in figure 1 , xinbaoxiang ( xbx ) , daguan ( dg ) , chuanfang ( cf ) , and panlongjiang ( plj ) rivers receive effluents from sewage treatment plants ( stps ) . haihe ( h ) , guangpugou ( gpg ) , jinjia ( jj ) , xibahe ( xbh ) , and xinyunliang ( xyl ) rivers receive raw sewage from industrial , domestic , and agricultural sources . xiaba ( xb ) and yaoan ( ya ) rivers represent the same sewage origin from the same upstream ( not sampled due to water hyacinth not being grown ) but different tributaries separated at water treatment wetland named wujia ( not sampled due to water hyacinth not grown ) . water temperature , ph , and dissolved oxygen ( do ) were measured in situ using portable meter ( ysi proplus , usa ) . one - liter water samples were collected from each site with three replicates at 00.5 m of the water column of the eleven rivers using cylinder sampler in september 25 , 2012 . total nitrogen ( tn ) , ammonium ( nh4 ) , nitrate ( no3 ) , nitrite ( no2 ) , and total soluble nitrogen ( tsn ) were analyzed using a seal autoanalyzer 3 ( seal analytical co. , hampshire , uk ) . mixed root samples were collected randomly with three replicates at each sampling site using sterile scissors and forceps and then stored in an ice box and taken back to the laboratory . fresh roots ( 2 g ) of e. crassipes were transferred into 200 ml sterile water . bacteria attached to e. crassipes roots were detached by vigorous shaking for 30 min ( 18.3 hz , thermomixer eppendorf ) and filtered through a 0.45 m sterile filter . the resultant filtrates were filtered through 0.22 m millipore membrane filters using a vacuum air pump and the membranes stored at 80c for dna extraction . all the abovementioned membranes were cut into pieces with sterile scissors and used immediately for dna extraction , which was performed using an e.z.n.a . water dna kit ( omega bio - tek inc . , real - time quantitative pcr was performed to estimate the denitrifying bacteria abundance using the primers listed in table 1 . real - time polymerase chain reaction ( qpcr ) was performed on abi 7500 real - time system ( life technologies , usa ) . amplification was performed in triplicate in a total volume of 20 l reaction mixtures by using sybr premix ex taqtm ( tiirnaseh plus ) qpcr kit as described by the suppliers ( takara bio , dalian , china ) . for each assay , three different pcr conditions were performed separately for the same sample by varying annealing temperature at either 54c ( nirs- cd3af / nirs - r3cd and nosz - f / nosz-1622r ) or 58c ( nirk - f1acu / nirk - r3cu ) . the qpcr amplification was performed as follows : initial denaturation at 95c for 2 min , followed by 35 cycles consisting of denaturation step at 95c for 5 s , varying annealing temperature for 30 s , and elongation at 72c for 30 s. the data were collected during the 72c for 30 s step . data was analyzed using the abi 7500 software ( version 2.0.6 , life technologies , usa ) . the parameter ct ( threshold cycle ) was determined as the cycle number at which a statistically significant increase in the reporter fluorescence was detected . the standard curves for real - time pcr assays were developed as previously described . for denaturing gradient gel electrophoresis ( dgge ) analysis , the pcr was performed in reaction mixtures including 1 l of template dna , 5 l of 10 pcr buffer , 1 l of dntps ( 10 mm each ) , 1 l of each primer ( 20 m ) ( table 1 ) , and 2 u of taq polymerase ( takara bio , dalian , china ) and adjusted to a final volume of 50 l with sterile deionized water . the reaction was performed in a bio - rad c1000 thermal cycler ( bio - rad , usa ) using different cycling conditions . the nirk gene ( f1acu / r3cu - gc ) pcr program was carried out with an initial denaturation at 94c for 3 min , followed by 32 cycles of 94c for 30 s , 58c for 30 s , and 72c for 45 s , followed by 72c for 10 min , and ended at 10c . the touchdown pcr amplification of nirs ( cd3af / r3cd - gc ) and nosz ( nosz - f / nosz1622r - gc ) was performed as follows : 94c for 2 min , followed by 10 cycles , 94c for 30 s , and 57c for 30 s in the initial cycle and at decreasing temperatures by 0.5c / cycle until a temperature of 52c was reached in the subsequent cycles . after the touchdown program , 30 cycles at 94c for 30 s , 53c for 30 s , and 72c for 1 min , followed by 72c for 10 min , and ended at 10c . the amplified products were pooled and resolved on dgge gels using a dcode system ( bio - rad laboratories , hercules , usa ) . the purified pcr products ( 30 l ) of nirs , nirk , and nosz containing approximately equal amounts of pcr amplicons were loaded onto the 1 mm - thich 6% ( w / v ) polyacrylamide ( 37.5 : 1 , acrylamide : bisacrylamide ) gels with denaturing gradients of 5075% for 15 h ( nirs ) , 5070% for 12 h ( nirk ) , and 5070% for 15 h ( nosz ) ( 100% denaturant contains 7 mol / l urea and 40% ( v / v ) formamide ) . the gels were run in 1 tae ( 40 mm tris - acetate and 1 mm edta ) at 100 v and 60c . polaroid pictures of the dgge gels were scanned using an epson perfection v700 photo scanner ( seiko epson corporation , nagano , japan ) . dgge profiles were digitized after average background subtraction for the entire gel using quantity one software ( version 4.5 , bio - rad , usa ) as previously described . digitized information from the dgge banding profiles was used to calculate the diversity indices such as richness ( s ) , which was determined from the number of bands in each lane , and shannon - wiener index ( h ) , which was calculated from h = pi lnpi , where pi is the importance probability of the bands in a gel lane , calculated as pi = ni / n , where ni is the intensity of a band and data presented as mean values sd . one way analysis of variance ( anova ) followed by s - n - k - test was performed to check for quantitative differences between samples ; p < 0.005 was considered to be statistically significant . the relative intensity of a specific band was transformed according to the sum of intensities of all bands in a pattern . redundancy analysis ( rda ) for community ordination was conducted using canoco ( version 4.5 , centre for biometry , wageningen , netherlands ) for windows using relative band intensity data obtained from the quantity one analysis . eight environmental parameters , including water temperature , ph , do , ammonia , nitrate , nitrite , total nitrogen , and total soluble nitrogen , were selected to perform rda - based variance inflation factor ( vif ) analysis with 499 unrestricted permutations to statistically evaluate the significance of the first canonical axis and of all canonical axes together . the corresponding environmental parameters ( table 2 ) of the eleven rivers represented their own properties of different pollution sources . the water from stp sites was characterized by relatively high concentrations of nitrate ( 4.7912 mg l ) and organic matter , which had contrary properties comparing to those rivers receiving raw sewage from industrial , domestic , and agricultural sources . the xb and ya rivers had similar characteristics to those rivers receiving water from stp but lower dissolved oxygen and higher ammonia nitrogen . the results showed that the abundance of nirk , nirs , and nosz gene copies per gram fresh root ranged from 4.13 10 to 6.11 10 , 1.45 10 to 1.99 10 , and 2.20 10 to 2.20 10 , respectively ( figure 2 ) . the nirk and nirs abundance on the roots of e. crassipes in ya river and xb river were significantly higher than those in other rivers ( p < 0.05 ) . the lowest abundance of nirk and nosz type denitrifiers were determined on the root sample from dg river . the nirk , nirs , and nosz copy abundance varied between sites indicated that different pollution source would influence the abundance of denitrifiers in rivers . the highest abundance ratio ( 125.34 ) of nosz/(nirk + nirs ) occurred in jj river , followed by gpg ( 39.75 ) , while the lowest ratio was in xyl river ( 1.43 ) , and the ratios in other rivers were similar , ranging from 3.26 to 8.38 . however , the nirk / nirs ratio in all samples ranged from 1.70 to 6.60 . to explain the relationship between environmental factors and the abundance of nirk , nirs , and nosz , the gene copy numbers of three denitrifiers and eight parameters were explored by redundancy analysis ( figure 3 ) . the gray circle area implies a positive correlation and the white circle area implies a negative correlation . the larger the circle area , the greater the impact corresponding to the changes in environmental factors that would have influenced the denitrifiers . denitrifier lines at the end in the gray circle had positive regression coefficients for that environmental variable with the corresponding t - value larger than 2.0 . the results showed that the temperature , ph , and nitrate circle areas were larger than other environmental factors , which indicated that temperature , ph , and nitrate circle greatly affected the nirs , nirk , and nosz abundance than other factors . the abundance of nirk and nirs was positively correlated with water temperature , nitrate , and nitrite concentrations and was negatively correlated with the other factors ( ph , do , dtn , tn , and ammonium ) . the abundance of nosz was negatively correlated with water temperature and was positively correlated with the ph and do , while there were no significant correlations with other factors . only one of the three replicates of dgge profiles was listed for each gene type to illustrate resolution . however , all the three replicates of the profiles were digitized and were used in statistics analysis . the shannon indices ( h ) calculated from dgge gels ranged from 2.23 to 2.90 for nirk , 2.08 to 2.69 for nirs , and 2.11 to 2.73 for nosz , which showed that high diversity of denitrifier ( nirk , nirs , and nosz ) genes on the root of e. crassipes ( table 3 ) . the significant differences among them were observed statistically ( p < 0.05 ) . with respect to the richness and diversity of denitrifier communities in all sites , similar trends emerged with low richness and diversity of nirk and nirs genes in xbx and dg rivers , which mainly received effluent from stps . next trends were in the h , jj , and xbh rivers with relatively higher richness and diversity of nirk and nirs genes , which were less impacted by the effluent from stps . the highest richness and diversity of nirk showed in the xb and ya rivers , which received wastewater after flowing through a wetland incubation on the water way . the richness and diversity of nosz , which was mainly impacted by temperature , gave a similar trend for all rivers due to the fact that temperature did not vary too much in all sites . to determine to what extent the eight environmental properties affected the three types of denitrifier community compositions , nirk , nirs , and nosz dgge fingerprints were evaluated by redundancy analysis ( table 4 ) . the first axis explained 26.9% of the nirk - type denitrifier diversity , and the second axis explained 21.8% of the diversity . for nirs - type denitrifier , the first two canonical axes explained 29.5% and 11.5% of the variation , respectively . for nosz - type denitrifier , 37.8% and 13.7% of the variation were explained by the first two canonical axes ( table 4 ) . of the parameters , total n , do , ph , and water temperature appeared to be the relatively important environmental factors for denitrifiers ( table 5 ) . for nirk - type denitrifier , water temperature , do , and total n explained 46% variations of microbial communities , leaving 54% of the variation unexplained . variation partitioning analysis showed that water temperature , do , and total n separately explained 19% ( p = 0.020 ) , 13% ( p = 0.054 ) , and 14% ( p = 0.240 ) of the variation , respectively . for nirs - type denitrifier , water temperature ( 18% , p = 0.066 ) , ph ( 10% , p = 0.304 ) , and do ( 8% , p = 0.038 ) explained 36% variations of microbial communities , leaving 64% of the variation unexplained . compared to nirs , the total n rather than do was relatively important for nosz - type denitrifier ( table 5 ) . the relationships of microbial patterns to environmental variables were summarized in rda ordination plots ( figures 4(b ) , 5(b ) , and 6(b ) ) . the rda charts ( figure 4(b ) ) of nirk gene showed four rivers ( plj , xyl , dg , and cf ) grouped into one type , while other four rivers ( xbh , xbx , h , and ya ) clustered together . for nirs gene , plj , jj , and cf rivers were similar and grouped into one type , while other three rivers ( xbh , xyl , and xbx ) clustered together . other rivers were not similar and did not belong to either group ( figure 5(b ) ) . according to the rda chart ( figure 6(b ) ) of nosz gene , dg , h , gpg , and xbh rivers were located independently and did not cluster with any group . however , plj , xyl , jj , and cf rivers clustered into one group , while other three rivers ( xb , xbx , and ya ) clustered together . the activity of denitrifying microorganisms leads to significant net removal of dissolved nitrogen from the water , resulting in considerable improvement of water quality in aquatic ecosystem . denitrifiers play an important role in buffering of the excessive load of nitrogen from upstream to downstream . in aquatic ecosystems , mats of macrophytes are important sites for microbial mediated biogeochemical processes , as accrual of biomass and increases in mat density reduce the degree of external factors to influence internal processes . the suspended root system of e. crassipes could provide a large surface area , approximately 2.5 to 8.0 mkg on a dry weight basis , for microbial attachment [ 5 , 18 ] . releasing of oxygen and dissolved organic carbon from roots of e. crassipes would support an appropriate microenvironment for nitrification and/or denitrification [ 19 , 20 ] . process of denitrification is driven by the denitrifying microorganisms under the influence of environmental conditions . water properties in different rivers in the present study were shown altering the abundance and diversity of denitrifiers on e. crassipes roots . the abundance of nirk , nirs , and nosz denitrifiers on the root of macrophytes varied with the variation of environmental parameters in different rivers , which seemed depending on the nitrogen concentrations , water temperature , water turbulence , and pretreatment of wastewater using wetland . the abundance of nirk , nirs , and nosz denitrifiers on root samples from xbx , dg , cf , and plj rivers was relatively stable and low . these rivers were larger than other rivers around dianchi lake , which were important sites receiving effluent from the stps . the fast - flowing water and irregular discharge of effluent of these rivers may prevent development of the stable environment properties from microbial attachment and propagate . contrarily , the abundance of denitrifiers genes on roots samples in xb and ya rivers ( xiaba and yaoan rivers ) was higher than that in other rivers . the xb river received both the wastewater from industrial and residential areas and the tidal water from dianchi lake , when water level increased in rainy seasons ( may to october ) . the water merged at wujia wetland , and then part of it was pumped into ya river after 45 days of retention in wujia wetland . this implies that a combination of wetland and growth of water hyacinth may further promote denitrification processes in eutrophic water . the abundance of nirk gene was always greater than that of nirs gene on the roots of e. crassipes , suggesting that the fresh water of dianchi lake was more suitable for the growth of nirk - type denitrifying bacteria . the nirs gene of cytochrome cd1 type has also been found more often in anoxic locations , where do levels were consistently low . in contrast , nirk genes of copper containing type have been found where diurnal do swings are greater [ 24 , 25 ] . this finding was of coincidence with that e. crassipes releases oxygen from roots , which facilitates the creation of aerobic microsites on the roots . even though the nirk and nirs are functionally equivalent , denitrifying bacteria harboring either nitrite reductase seems to be likely not under the same community assembly rules . philippot et al . suggested that the existence of the two types of nitrite reductase ( nir - gene ) was due to differential niche preferences . this speculation was consistent with previously identified habitat preferences of nirs - gene and nirk - gene bearing organisms . jones and hallin found that most nirk sequences were derived from soil but that most nirs sequences were prominently derived from marine and estuarine environment . bacteria suspended in water and attached to the root of e. crassipes may originate from many different sources . autochthonous bacterioplankton populations that developed in the water column were likely to be mixed with allochthonous populations from forest soils , urbanized land , farm fields , and wetlands as well as hyporheic sediments in the rivers . this mixed origination , impacted by varied environmental parameters , seemed to be the main cause of the discrepancy of denitrifiers found in the eleven rivers . this , however , did not necessarily indicate that nirk - type denitrifiers contributed more or less in denitrification than nirs - type ones ; rather it may only imply that the root of e. crassipes could provide a broad support for different kinds of microorganisms . dianchi lake together with surrounding rivers comprised a plateau water catchment to provide ecological services and fresh water supply for more than seven million people in the area . its geochemical characteristics have made the water ph relatively high ( 7.568.03 ) and its geophysical characteristics have made the water temperature moderate with winter months ( december to march next year ) around 12c . its heavy load of organic matters have made the do level relatively low ( 0.203.80 mg these environmental properties dominated the community assembly processes of the genetic makeup of the denitrifiers in the rivers . nevertheless , specific environmental conditions in different rivers favored the variation in richness and diversity of different denitrifying genes . the dgge profiles for denitrification genes encoding nitrite and nitrous oxide reductase ( nirk , nirs , and nosz ) on the root of e. crassipes growing in 11 rivers around dianchi lake supported our hypothesis of profound differences in community composition , although a complex picture of denitrifier community similarity emerged depending on which functional denitrification gene was evaluated . the correlations of denitrifying microbial community compositions with abiotic environmental factors , using redundancy analysis ( rda ) , confirmed that water temperature ( temp ) , dissolved oxygen ( do ) , and ph appeared to be the most important factors to alter the denitrifier community structures significantly by serving as essential conditions for the growth of microorganisms on the roots of e. crassipes ( table 5 ) . the results of this study indicated that the development of denitrifier communities on roots corresponded to different origins of rivers . the physiochemical characteristics of water from the river inlet varied with water origin and pollution sources [ 22 , 23 , 30 ] , resulting in the variation in do , ph , pollutant species and concentrations , and organic carbons in rivers . these environmental factors , including do , carbon content , water temperature , and ph , influenced denitrification rates in rivers and as a consequence they might also affect the denitrifier community composition [ 32 , 33 ] . . found that the change of temperature resulted in gradually changed denitrification activity but also in abundance mutative of nitrate reducers and in different denitrifier community compositions . there are some indications that temperature and ph may directly or indirectly influence the abundance and communities composition of denitrifiers [ 35 , 36 ] . the excess o2 resulted in reduced denitrifying bacterial growth and a smaller bacterial density versus nitrate reducing bacteria ration , which indicated that the development of the denitrifying bacteria was influenced by the do concentration . many investigators had found that the ph , temperature , and do generally affect diversity and richness of denitrifier community [ 32 , 38 ] , and microbial community assembly was more dependent on local - scale environmental factors . on the other hand , different microorganisms may have their physiological constraints for growth and reproduction within narrow ph ranges , specific do , and nutrient availability , which affect the community structures directly [ 40 , 41 ] . activities of microorganisms could change the environmental properties that differed in the concentrations of enzymes and nutrients or do , the form and amount of dissolved carbon present , and ph hence to affect denitrifier community structure . previous studies have found that growth of e. crassipes could regulate water at neutralize ph significantly , as a result of increase in the rate of denitrification in aquatic ecosystems . the variation in abundance of denitrifier communities on e. crassipes roots , grown in rivers flowing into dianchi lake , corresponded to different water properties of rivers . the ratio of nirk / nirs gene copies abundance was always greater than 1 , indicating that the surface of e. crassipes roots was more suitable for the growth of nirk - type denitrifying bacteria . the temperature of water , nitrate concentration , and ph greatly affected the nirs , nirk , and nosz abundance than other factors . meanwhile , the temperature of water , do , and ph appeared to be the most important factors to alter the community structures of denitrifiers on the roots of e. crassipes . as process of denitrification is driven by denitrifies under the influence of environmental conditions , a variation of denitrification capability in different rivers would be expected .

to evaluate effects of environmental conditions on the abundance and communities of three denitrifying genes coding for nitrite ( nirk , nirs ) reductase and nitrous oxide ( nosz ) reductase on the roots of eichhornia crassipes from 11 rivers flowing into the northern part of dianchi lake . the results showed that the abundance and community composition of denitrifying genes on e. crassipes root varied with different rivers . the nirk gene copies abundance was always greater than that of nirs gene on the roots of e. crassipes , suggesting that the surface of e. crassipes roots growth in dianchi lake was more suitable for the growth of nirk - type denitrifying bacteria . the dgge results showed significant differences in diversity of denitrifying genes on the roots of e. crassipes among the 11 rivers . using redundancy analysis ( rda ) , the correlations of denitrifying microbial community compositions with environmental factors revealed that water temperature ( t ) , dissolved oxygen ( do ) , and ph were relatively important environmental factors to modifying the community structure of the denitrifying genes attached to the root of e. crassipes . the results indicated that the specific environmental conditions related to different source of rivers would have a stronger impact on the development of denitrifier communities on e. crassipes roots .

to report a case of recurrent idiopathic frosted branch angiitis ( fba ) successfully treated with adalimumab . a 14-year - old otherwise healthy boy was referred to the uveitis clinic for bilateral panuveitis with diffuse retinal vascular sheathing and severe macular edema . two years ago , a similar episode had been treated with oral prednisolone , however it was complicated by adverse psychiatric effects . the progressive course of the condition mandated considering other therapeutic measures ; adalimumab was chosen based on its purported efficacy for treatment of childhood uveitis and a favorable safety profile . the patient responded dramatically to a single subcutaneous injection of adalimumab without any side effect during and after injection . to the best of our knowledge , this is the first case of idiopathic fba treated successfully with adalimumab without adjunctive steroid therapy . frosted branch angiitis ( fba ) is a rare disease characterised by visual disturbance associated with anterior chamber and vitreous inflammation and severe sheathing of retinal vessels resembling the appearance of frosted branches of a tree . it was originally described in a six - year - old child by ito in 1976.1 fba can be associated with ocular and systemic diseases . cytomegalovirus retinitis , acquired immunodeficiency syndrome , and toxoplasma chorioretinitis are frequent ocular associations , while systemic lupus erythematosus , crohn 's disease , behcet s disease , large cell lymphoma , and acute lymphoblastic leukemia have been described as systemic associations.2 the idiopathic type typically occurs in young and healthy persons and is associated with good recovery of vision . a 14-year - old boy , who was otherwise healthy , was referred to the uveitis clinic for progressive blurring of vision in both eyes since 1 month before . on examination , anterior segment examination revealed mild ciliary injection and anterior chamber reaction ( 2 + cells ) but no keratic precipitates or posterior synechiae . both eyes had inflammatory reaction in the vitreous ( 2 + cells ) but there were no snow balls or snow bank formation . fundus examination disclosed extensive retinal vascular sheathing and scattered intraretinal hemorrhages with severe macular edema in both eyes ( fig . 1 ) . systemic work - up and investigations , including a complete blood count ( cbc ) , erythrocyte sedimentation rate ( esr ) , c - reactive protein ( crp ) titer , angiotensin converting enzyme ( ace ) titer , tuberculin skin test ( tst ) , urine analysis and chest x - ray were unremarkable . rheumatologic tests including antinuclear antibodies ( ana ) , anti double strand dna antibody ( anti ds - dna ) , rheumatic factor ( rf ) , antiphospholipid antibody , anti - neutrophil cytoplasmic antibodies ( ancas ) , and cryoglobulin assay were all negative . blood cd4 and cd8 counts were within normal range and serology for the human immunodeficiency virus was also negative . blood serology for possible infectious causes including herpes family viruses , i.e. herpes simplex virus ( hsv ) type 1 and 2 , varicella zoster virus ( vzv ) , cytomegalovirus ( cmv ) , and epstein - barr virus ( ebv ) , hepatitis b and c viruses , treponema , borrelia , and toxoplasma were checked . immunoglobulin g ( igg ) levels against hsv type 2 and cmv ( but not igm ) were elevated . we tested the aqueous specimen after an anterior chamber paracentesis to investigate the presence of deoxyribonucleic acid ( dna ) of hsv type 1 and cmv viruses . the result of polymerase chain reaction ( pcr ) on aqueous samples was negative for both viruses . until the results of blood tests and aqueous pcr became ready , vision continued to deteriorate down to 20/1000 and 20/800 in the right and left eyes , respectively . based on the inconclusive work - up a diagnosis of idiopathic fba the patient s parents mentioned that two years ago , for treatment of a similar condition , oral prednisolone 1 mg / kg had been commenced but soon after initiation , the patient developed severe mood changes which persisted long after discontinuation of treatment . the progressive nature of the condition mandated considering other therapeutic measures to prevent macular scarring . due to the safety profile of anti - tumor necrosis factor ( tnf ) agents and their rapid onset of effect , adalimumab was considered as an alternative to conventional treatment for which the parents provided written informed consent . a single dose of 40 mg pre - filled adalimumab ( humira , abbott laboratories , north chicago , il , usa ) was injected subcutaneously in the patient s right thigh . no local or systemic adverse effect was noted at the time of injection or later on . three weeks after injection , visual acuity improved to 20/200 and 20/120 in the right and left eyes , respectively . anterior chamber and vitreous reaction decreased to 1 + cell , and vascular sheathing and macular edema resolved dramatically . repeat fluorescein angiography and optical coherence tomography ( oct ) confirmed the improvement ( figs . 2 and 3 ) . 10 weeks after injection , visual acuity improved to 20/40 and 20/30 in the right and left eyes , respectively . in both eyes , clinical improvement continued up to the last visit ( 6 months after the injection ) when visual acuity reached 20/25 in both eyes with no sign of active inflammation . fba is basically divided into three different subgroups.3 the first comprises patients affected by lymphoma and leukemia presenting with a frosted branch - like appearance in the fundus . the second group includes patients with associated autoimmune or viral disease which can present with fba in association with the underlying disease . the characteristic feature of this latter type is its occurrence in otherwise healthy young individuals with good recovery of vision . clinically , there is severe sheathing of retinal vessels appearing like the frosted branches of a tree , and acute visual disturbance associated with anterior chamber and vitreous inflammation ; fluorescein angiography shows leakage from sheathed vessels without signs of occlusion or stasis.4,5 systemic steroids are the main and only suggested treatment for this condition . in our patient however , prior adverse psychiatric effects of steroid treatment encouraged us to employ another therapeutic option : adalimumab , a fully humanized monoclonal anti - tnf antibody , was chosen based on its suggested effect for treatment of refractory childhood uveitis including juvenile idiopathic arthritis.6 - 8 our patient responded dramatically to a single subcutaneous injection of adalimumab . the improvement was rapid and long lasting and the patient did not report any side effect during follow up . review of the literature revealed no other therapeutic option for treatment of idiopathic fba except systemic steroids . to our best knowledge , this is the first case of idiopathic fba treated successfully with an anti - tnf agent without adjunctive steroid therapy .

purposeto report a case of recurrent idiopathic frosted branch angiitis ( fba ) successfully treated with adalimumab.case reporta 14-year - old otherwise healthy boy was referred to the uveitis clinic for bilateral panuveitis with diffuse retinal vascular sheathing and severe macular edema . extensive work - up including aqueous sampling for detection of viral causes was inconclusive . two years ago , a similar episode had been treated with oral prednisolone , however it was complicated by adverse psychiatric effects . the progressive course of the condition mandated considering other therapeutic measures ; adalimumab was chosen based on its purported efficacy for treatment of childhood uveitis and a favorable safety profile . the patient responded dramatically to a single subcutaneous injection of adalimumab without any side effect during and after injection . the therapeutic effect was rapid and relatively long-lasting.conclusionto the best of our knowledge , this is the first case of idiopathic fba treated successfully with adalimumab without adjunctive steroid therapy .

although liver resections have been associated with high mortality and morbidity rates , recent advances in anesthetic and surgical management have significantly reduced the operative risk . the techniques of vascular control during hepatectomy are highly demanding and should be performed under special anesthetic considerations . hepatic vascular control methods can be categorized as those involving occlusion of liver inflow and those involving occlusion of both liver inflow and outflow . hepatic pedicle occlusion : continuous pringle maneuver ( cpm),intermittent pringle maneuver ( ipm ) . selective inflow occlusion . hepatic pedicle occlusion : continuous pringle maneuver ( cpm),intermittent pringle maneuver ( ipm ) . continuous pringle maneuver ( cpm ) , intermittent pringle maneuver ( ipm ) . inflow and outflow vascular exclusion total hepatic vascular exclusion ( thve ) , inflow occlusion with extraparenchymal control of the major hepatic veins : with selective hepatic vascular exclusion ( shve ) . total hepatic vascular exclusion ( thve ) , inflow occlusion with extraparenchymal control of the major hepatic veins : with selective hepatic vascular exclusion ( shve ) . when performing these techniques , the conduct of anesthesia should take into account hemodynamic management , risks of vascular air embolism , ischemia reperfusion liver injury , intraoperative blood loss , and the need for transfusion , factors which usually complicate hepatic vascular control methods . special attention should also be paid to the preoperative assessment and induction of anesthesia , as patients undergoing liver resection usually have a compromised health status . careful selection of the anesthetic drugs can minimize the effects of hepatic blood flow decrease induced by the surgical technique adopted . our objective was to identify the anesthetic considerations in techniques of hepatic vascular control methods . all prospective randomized studies were thoroughly evaluated and presented , as they are the most important source of information on the outcomes of surgical and anesthetic manipulations . few case reports and smaller studies are mentioned , given the fact that they highlight special anesthetic aspects . healthy patients undergo a routine preoperative assessment including a full blood count and a standard biochemical and coagulation test . preexisting hepatic impairment is a risk factor , even for nonhepatic surgery , with higher blood transfusion requirements , a longer hospital stay , a higher number of complications , and increased mortality rates of 16.3% in cirrhotic patients as compared to 3.5% in controls . estimating the health status of patients presenting for hepatectomy is quite challenging : coagulopathy , volume and electrolyte disturbances , viral infections ( hep c ) , hepatorenal [ 24 ] and hepatopulmonary syndrome , portopulmonary hypertension , and low cardiovascular reserve capacity can occur in patients with chronic liver disease . the identification of patients at risk to develop postoperative hepatic or renal failure is important and , ideally , involves many related disciplines such as surgery , anesthesia , and intensive care . although vascular occlusion techniques have minimized hepatic bleeding , the risk for postoperative liver and/or renal failure remains high for patients of advanced age and those with steatosis and cirrhosis , on preoperative chemotherapy and with small remnant liver volumes . have developed and validated a prediction score for postoperative acute liver failure following liver resection based on the preoperative parameters of cardiovascular disease , chronic liver failure , diabetes , and alt levels , which seems to be an easily applicable and attractive tool in clinical practice . hepatic ischemia and reperfusion on subsequent liver dysfunction is associated with unexpected responses to surgical stress [ 79 ] and poor prognosis . patients with end - stage liver disease have a characteristic hemodynamic profile : increased cardiac output with blunted response to painful stimuli , splanchnic vasodilatation and central hypovolemia . as a result , silent moderate - to - severe coronary artery disease can not be easily recognized . currently , there are no specific guidelines for the identification of coronary artery disease in patients with advanced liver disease [ 11 , 12 ] . preoperative invasive assessment of preexisting cardiovascular dysfunction is indicated only for high risk patients , provided that any coagulopathy is corrected . in the noninvasive assessment of coronary artery disease in patients with cirrhosis , beta blockade discontinuation in order to permit adequate cardiac function assessment may be hazardous in patients with advanced liver disease . beta blockers reduce portal hypertension , decrease cardiac workload , and their use seems to be beneficial to both the liver and the heart in the setting of hepatectomy . in general , the preoperative assessment needs to be adapted to the individual patient to minimize the perioperative liver insults of hepatic vascular control . cis - atracurium is the nondepolarizing muscle relaxant of choice in patients with liver disease as it is hydrolyzed by hoffman elimination . atracurium can provide stable neuromuscular blockade , as its requirements remained unchanged during exclusion of the liver from the circulation . an intravenous hypnotic is used for induction and a halogenated volatile agent in air - oxygen mixture is used for maintenance . hepatic vascular control techniques depress cardiovascular function in addition to the depression caused by general anesthesia . careful selection of the volatile agent is required . most commonly used volatile anesthetics for maintenance are isoflurane and sevoflurane . isoflurane has mild cardiodepressive effects but maintains hepatic oxygen supply , due to vasodilatation in the hepatic artery and portal vein . both isoflurane and sevoflurane upregulate heme - oxygenase-1 , release iron and carbon monoxide , and thus decrease portal vascular resistance in rats . in humans , sevoflurane decreases portal vein blood flow but increases hepatic artery blood flow . , in a randomized controlled trial on patients undergoing liver surgery , showed that ischemic preconditioning with sevoflurane before inflow occlusion limited postoperative liver injury , even in patients with steatosis . although various inhalational anesthetics are used in liver surgery , no optimal anesthetic technique has been established for the maintenance of anesthesia . additionally , desflurane undergoes only minor biodegradation ( it is metabolized at a ratio of 0.02% ) and in fact it may cause less hepatocellular damage due to its reduced metabolism . ko et al . , comparing the effects of desflurane and sevoflurane on hepatic and renal functions after right hepatectomy in living donors reported better postoperative hepatic and renal function tests with desflurane as compared to sevoflurane at equivalent doses of 1 mac without , however , being able to validate the clinical importance of their study . arslan et al . comparing the effects of anesthesia with desflurane and enflurane on liver function , showed that during anesthesia with desflurane , liver function was well preserved ; glutathione - s - transferase and aspartate aminotransferase levels were significantly lower in the desflurane group . on the other hand , laviolle et al . suggested that propofol has an early protective effect against hepatic injury compared with desflurane after partial hepatectomy under inflow occlusion . however , few studies on the effects of general anesthesia during hepatectomies under vascular control techniques are available in patients with significant comorbidities . portal triad clamping increases systematic vascular resistance by up to 40% and reduces cardiac output by 10% . . however , the systemic circulation in patients with cirrhosis is hyperdynamic and dysfunctional , with increased heart rate and cardiac output , decreased systemic vascular resistance , and low or normal arterial blood pressure . thus , maintaining adequate organ perfusion may be difficult to achieve and preoperative optimization of the patient is required . the anesthetic management is dictated by the surgical approach and the patient 's health status . invasive monitoring provided by a central venous line or pulmonary catheterization is reserved for patients with poor cardiovascular status or when prolonged vascular occlusions are performed . a low cvp ( between 2 and 5 mmhg ) , while aiming at euvolemia , reduces blood loss during liver surgery and improves survival [ 30 , 31 ] . a low cvp can be achieved by limitation of intravenous fluids administration pre- and intraoperatively . maintenance fluids and crystalloids to stabilize blood pressure > 90 mmhg and ensure diuresis of at least 0.5 ml / kg / h can be used safely with minor hemodynamic disturbance . if fluid restriction is ineffective to keep a low cvp , vasoactive agents are used . nitroglycerin reduces cvp to the desired level during the resection phase or when excessive oozing is observed from the resected surface [ 13 , 16 ] . cpm with a cvp of 5 mmhg or less is associated with minor blood loss and a shorter hospital stay . if , however , it is applied under a low cvp during transection , blood loss and hemodynamic changes are minimal [ 3437 ] . in an experimental animal study , sivelestat , a neutrophil elastase inhibitor , reduced hepatic injury and stabilized hemodynamics after ischemia - reperfusion following ipm . the advantages of a low cvp must be weighed against inadequate perfusion of the vital organs and loss of volemic reserve in case of bleeding and/or air embolism . support that in low cvp anesthesia during liver resection , the incidence of perioperative renal failure does not increase significantly . ( 1 ) total hepatic vascular exclusion ( thve)in thve , rapid hemodynamic changes ( table 1 ) are frequent due to surgical events such as caval clamping , sudden blood loss , and hepatic reperfusion . cross - clamping of the inferior vena cava and portal vein result in a 4060% reduction of venous return and cardiac output , with a compensatory 80% increase in systemic vascular resistance and a 50% increase in heart rate . although systemic vascular resistance and heart rate increase , the cardiac index is reduced by half , secondary to a preload reduction . unclamping is followed by an increase in cardiac index and a significant reduction in systemic vascular resistance .the anesthetist should take prompt steps to manage the preload reduction and the sudden decrease in cardiac output evoked by the inferior vena cava and portal vein clamping . intraoperative monitoring includes ecg , pulse oximetry , etco2 tension , invasive blood pressure monitoring through an arterial line , and cvp monitoring through a large bore central venous line . in addition , the presence of a pulmonary artery catheter allows the tailored administration of vasopressors in case of massive hemorrhage due to vena cava injury . the vigileo , an uncalibrated arterial pulse contour cardiac output monitoring system , has been proved to be unreliable in cirrhotic patients with hyperdynamic circulation undergoing major liver surgery .before colloids , beyond correcting volume deficits , improve splanchnic circulation , displace fluid into the blood compartment , and reduce bowel edema . blood pressure and circulatory support vasopressin or norepinephrine are administered if volume loading is inadequate to maintain blood pressure following clamping of the vena cava .there is no standard approach to the use of vasoactive agents in thve . vasoactive agents should be used carefully , as they improve cardiac output at the expense of microcirculatory blood flow . during vascular isolation of the liver in eight pigs , norepinephrine infusion ( 0.7 g / kg / min ) decreased hepatic vascular capacitance by activation . in a recent study in septic patients , krejci et al . showed that norepinephrine increased systemic blood flow but reduced microcirculatory blood flow on liver 's surface.vasopressin on the other hand , is known to rapidly restore blood pressure during septic shock . however , in an experimental study , vasopressin proved to be inferior to norepinephrine in terms of improving hepatosplanchnic blood flow . the response to both norepinephrine and vasopressin is blunted in patients with cirrhosis [ 44 , 45 ] . renal autoregulation ceases below a renal perfusion pressure of 70 to 75 mmhg , below which , flow becomes pressure dependent . perioperative fluid shifts , intravascular hypovolemia , and sympathetic activation during thve result in a reduction of renal blood flow . low dose dopamine have been used with the aim of preventing intraoperative renal injury without evidence of substantial benefit . fenoldopam had beneficial effects on postoperative creatinine levels and creatinine clearance of critically ill patients . recently , terlipressin along with volume expansion have been shown to improve renal function , without , however , improving survival .hemodynamic intolerance to thve or ischemia under thve exceeding 30 or 60 minutes , require venovenous bypass [ 50 , 51 ] . thve should be limited to selected cases , as hemodynamic intolerance has been observed in 1020% of patients , as well as increased morbidity and hospital stays ( table 2 ) . in thve , rapid hemodynamic changes ( table 1 ) are frequent due to surgical events such as caval clamping , sudden blood loss , and hepatic reperfusion . cross - clamping of the inferior vena cava and portal vein result in a 4060% reduction of venous return and cardiac output , with a compensatory 80% increase in systemic vascular resistance and a 50% increase in heart rate . although systemic vascular resistance and heart rate increase , the cardiac index is reduced by half , secondary to a preload reduction . unclamping is followed by an increase in cardiac index and a significant reduction in systemic vascular resistance . the anesthetist should take prompt steps to manage the preload reduction and the sudden decrease in cardiac output evoked by the inferior vena cava and portal vein clamping . intraoperative monitoring includes ecg , pulse oximetry , etco2 tension , invasive blood pressure monitoring through an arterial line , and cvp monitoring through a large bore central venous line . in addition , the presence of a pulmonary artery catheter allows the tailored administration of vasopressors in case of massive hemorrhage due to vena cava injury . the vigileo , an uncalibrated arterial pulse contour cardiac output monitoring system , has been proved to be unreliable in cirrhotic patients with hyperdynamic circulation undergoing major liver surgery . before thve , colloids can be administered to prevent the abrupt decrease in cardiac output . colloids , beyond correcting volume deficits , improve splanchnic circulation , displace fluid into the blood compartment , and reduce bowel edema . blood pressure and circulatory support vasopressin or norepinephrine are administered if volume loading is inadequate to maintain blood pressure following clamping of the vena cava . vasoactive agents should be used carefully , as they improve cardiac output at the expense of microcirculatory blood flow . during vascular isolation of the liver in eight pigs , norepinephrine infusion ( 0.7 g / kg / min ) decreased hepatic vascular capacitance by activation . in a recent study in septic patients , krejci et al . showed that norepinephrine increased systemic blood flow but reduced microcirculatory blood flow on liver 's surface . vasopressin on the other hand , is known to rapidly restore blood pressure during septic shock . however , in an experimental study , vasopressin proved to be inferior to norepinephrine in terms of improving hepatosplanchnic blood flow . the response to both norepinephrine and vasopressin is blunted in patients with cirrhosis [ 44 , 45 ] . renal autoregulation ceases below a renal perfusion pressure of 70 to 75 mmhg , below which , flow becomes pressure dependent . perioperative fluid shifts , intravascular hypovolemia , and sympathetic activation during thve result in a reduction of renal blood flow . low dose dopamine have been used with the aim of preventing intraoperative renal injury without evidence of substantial benefit . fenoldopam had beneficial effects on postoperative creatinine levels and creatinine clearance of critically ill patients . recently , terlipressin along with volume expansion have been shown to improve renal function , without , however , improving survival . hemodynamic intolerance to thve or ischemia under thve exceeding 30 or 60 minutes , require venovenous bypass [ 50 , 51 ] . thve should be limited to selected cases , as hemodynamic intolerance has been observed in 1020% of patients , as well as increased morbidity and hospital stays ( table 2 ) . ( 2 ) selective hepatic vascular exclusion ( shve)shve is a flexible technique that can be applied in a continuous or intermittent manner . should accidental tears of major hepatic veins occur , rapid conversion to thve must be undertaken . the literature suggests that many institutions favor shve as one of the standard methods of vascular control because it provides a bloodless surgical field and it is tolerated by most patients . no special anesthetic considerations regarding the hemodynamic management of shve are referred , as this method diminishes blood pressure and heart rate fluctuations during liver resection ( table 1).in a cohort study among 246 patients , hemodynamic tolerance to shve was excellent with only a slight increase in systemic and pulmonary resistance during clamping . no deaths were reported and the mean hospital stay was 9.6 days.shve is the method of choice in cases when cvp can not be lowered ( i.e. , right heart failure , poor cardiovascular status ) [ 5356 ] . in a retrospective study on 102 patients , shve was shown to be unaffected by cvp levels and the authors concluded that it should be used whenever cvp remains high despite adequate anesthetic management . although the performance of shve requires significant surgical expertise , it is tolerated by most patients and has a hemodynamic profile similar to that of cpm [ 53 , 54 ] . furthermore , it controls backflow bleeding of the hepatic veins . in a large clinical study , shve proved to be more effective than cpm in controlling intraoperative bleeding , preventing blood loss , and reducing postoperative complications and mortality rates ( table 2 ) . combined shve and perioperative fluid restriction has also been suggested as a liver and renal protective procedure in partial hepatectomy . . demonstrated that active preoperative dehydration of the patient , low cvp anesthesia and shve resulted in minimal blood loss , low morbidity , and zero mortality in patients undergoing partial liver resection . in conclusion , shve which is not associated with cardiorespiratory and hemodynamic alterations is well tolerated by the majority of patients and requires shorter hospitalization times . shve is a flexible technique that can be applied in a continuous or intermittent manner . should accidental tears of major hepatic veins occur , rapid conversion to thve must be undertaken . the literature suggests that many institutions favor shve as one of the standard methods of vascular control because it provides a bloodless surgical field and it is tolerated by most patients . no special anesthetic considerations regarding the hemodynamic management of shve are referred , as this method diminishes blood pressure and heart rate fluctuations during liver resection ( table 1 ) . in a cohort study among 246 patients , hemodynamic tolerance to shve was excellent with only a slight increase in systemic and pulmonary resistance during clamping . shve is the method of choice in cases when cvp can not be lowered ( i.e. , right heart failure , poor cardiovascular status ) [ 5356 ] . in a retrospective study on 102 patients , shve was shown to be unaffected by cvp levels and the authors concluded that it should be used whenever cvp remains high despite adequate anesthetic management . although the performance of shve requires significant surgical expertise , it is tolerated by most patients and has a hemodynamic profile similar to that of cpm [ 53 , 54 ] . furthermore , it controls backflow bleeding of the hepatic veins . in a large clinical study , shve proved to be more effective than cpm in controlling intraoperative bleeding , preventing blood loss , and reducing postoperative complications and mortality rates ( table 2 ) . combined shve and perioperative fluid restriction has also been suggested as a liver and renal protective procedure in partial hepatectomy . . demonstrated that active preoperative dehydration of the patient , low cvp anesthesia and shve resulted in minimal blood loss , low morbidity , and zero mortality in patients undergoing partial liver resection . in conclusion , shve which is not associated with cardiorespiratory and hemodynamic alterations is well tolerated by the majority of patients and requires shorter hospitalization times . although the relative risk of air embolism in hepatic surgery is low ( < 5% ) , factors predisposing to vascular air embolism during liver resections include : ( a ) surgical technique , ( b ) size and place of the tumor , ( c ) blood loss , and ( d ) low cvp anesthesia . clinical signs of vascular air embolism during anesthesia with respiratory monitoring are : a decrease in end - tidal carbon dioxide and decreases in both arterial oxygen saturation ( sao2 ) and tension ( po2 ) , along with hypercapnia . from the cardiovascular system monitoring , tachyarrhythmias , electromechanical dissociation , major hemodynamic manifestations such as sudden hypotension may occur before hypoxemia becomes present . when performing techniques of inflow vascular occlusion ( cpm , ipm , selective inflow occlusion ) , air embolism may be observed during parenchymal transection under low cvp anesthesia or during reperfusion , due to mobilization of air bubbles trapped in opened veins . resection of large tumors situated in the right lobe , close to the inferior vena cava or the cavohepatic junction , put the patient at risk of venous air embolism . recent clinical trials assessing the efficacy of shve and pringle maneuver in preventing vascular air embolism showed that embolism occurred in three out of 2100 patients or in one out of 29 patients of the pringle group , following massive blood loss during tumor resection . air embolism did not occur in any case of the shve group [ 6264 ] . massive bleeding ( > 5000 ml ) and subsequent air embolism can even result in intraoperative death in patients undergoing major liver resections . the morbidity and mortality of air embolism depend on the volume and rate of air accumulation . from case reports of accidental intravascular delivery of air , the adult lethal volume has been described as between 200 and 300 ml or 35 ml / kg [ 67 , 68 ] . low cvp further enhances the negative pressure gradient at the surgical field compared to the right atrium and increases the possibility of air embolism . currently , the most sensitive monitoring devices for vascular air embolism are transesophageal echocardiography and precordial doppler ultrasonography , detecting as little as 0.02 ml / kg and 0.05 ml / kg of air , respectively [ 6971 ] . the consequences of air embolism can be minimized by placing the patient in a 15 degree trendelenburg position [ 7274 ] . furthermore , moulton et al . in a small study among ten patients , showed that patient positioning alone during liver surgery does not affect the risk of venous air embolism . thus , the beneficial effects of low cvp in liver resections must be carefully weighed against adequate hydration and volume status optimization . additionally , cirrhotic patients undergoing hepatectomy have pulmonary abnormalities including intrapulmonary shunting , pulmonary vascular dilatation , and arteriovenous communications . in these patients , air can pass into the systemic circulation ( paradoxical air embolism ) , even if cardiac abnormalities ( patent foramen ovale ) are not present , evoking fatal consequences . recent literature suggests that shve prevents vascular air embolism and provides operative tolerance . however , recognizing the risk for vascular air embolism and planning the appropriate level of monitoring and treatment is the key to patient safety . liver resections may result in significant blood loss and subsequent transfusion of rbc ( red blood cells ) in about 25%30% of patients . the two main sources of bleeding during a liver resection are ( a ) the inflow system ( hepatic artery and portal vein ) and ( b ) the outflow system ( backflow bleeding from the hepatic veins ) . bleeding may also occur during liver mobilization , hepatic transection , and dissection of biliary structures . blood loss has been linked to morbidity and mortality since 1989 , whereas rbc transfusions are associated with multiple disadvantages , risks , and side effects . furthermore , operative blood loss independently predicts recurrence and survival after resection of hepatocellular carcinomas . operative mortality in patients refusing blood transfusions was 7.1% for patients with hemoglobin levels > 10 g / dl and 61.5% for patients with hemoglobin levels < 6 g / dl [ 79 , 80 ] . the refinement of inflow and outflow occlusive techniques as well as the appropriate anesthetic management has reduced intraoperative bleeding and the need for blood transfusions . study of the literature reveals the following results regarding bleeding with different vascular occlusion techniques : pringle maneuver has been shown to be effective in reducing blood loss during parenchyma transection . portal triad clamping is associated with less bleeding compared with no clamping . in procedures of liver ischemia time belghiti et al . , in a prospective study of ipm versus cpm , found no difference in total blood loss or the volume of blood transfused between the two groups , despite higher blood loss during parenchyma transection . , in two prospective studies of ipm versus no use of vascular control at all , showed lower total blood loss and fewer transfusions in the ipm group [ 8385 ] . hemihepatic vascular clamping was shown superior to ipm and to no application of vascular control , with reduced both blood loss and transfusion requirements . shve provides a bloodless surgical field similar to thve , but is better tolerated by patients . many authors favor shve as one of the standard methods of vascular control , as it substantially prevents massive blood loss and diminishes transfusion needs . from an anesthetic standpoint , a low cvp level plays an important role in reducing intraoperative blood loss and transfusion rates [ 30 , 57 , 87 ] . maintaining a cvp < 5 mmhg by volume restriction and intravenous infusion of nitroglycerine and a systolic blood pressure above 90 mmhg by intravenous infusion of dopamine ( 46 g / kg ) the anesthetist should also provide normothermic conditions to the patient undergoing liver resection , because hypothermia reduces blood coagulation , especially platelet function , and increases intraoperative blood loss . of the pharmacological methods , desmopressin , although used in treating hemophilia , was not effective in reducing blood loss and transfusion needs in patients undergoing liver resection . in a randomized clinical trial , the use of recombinant factor viia in major liver resections failed to reduce the number of units transfused . a significant reduction in blood transfusion needs in liver resections has been shown with the use of aprotinin . aprotinin was found to reduce intraoperative blood loss by 25% and transfusion requirements by 50% . used half dose aprotinin ( 10 kiu followed by 2.5 10 kiu / hour infusion ) during hepatic transplantation in patients who have a significant coagulopathy or portal hypertension and in those who had previous abdominal surgery . however , lentschner et al . cautioned against the routine use of aprotinin due to the incidence of life threatening allergic reactions , thrombotic potential , and renal failure . currently , there is no scientific support for the routine use of aprotinin in patients undergoing partial hepatectomy , whereas its efficacy in liver transplantation is well established . tranexamic acid has also been shown to reduce blood requirements in liver resection surgery but safety concerns have been raised and require further investigation [ 92 , 93 ] . in the future , two artificial oxygen carriers ( hemoglobin solutions and perfluorocarbons ) may become essential in reducing the need for allogeneic rbc transfusions [ 9496 ] . artificial oxygen carriers improve o2 delivery and tissue oxygenation as well as the function of organs with marginal o2 supply . more studies examining their efficacy in ischemic liver during hepatectomy need to be performed . undoubtedly , the improvement of vascular control techniques during hepatectomy has permitted an aggressive approach for liver resections with low mortality rates ( 4% ) . in addition , anesthesia orientated towards an almost transfusion free setting has also improved mortality and morbidity following liver surgery . to this direction , a transfusion risk score , including variables of : ( a ) preoperative hemoglobin concentrations below 12.5 g / dl , ( b ) largest tumor more than 4 cm , ( c ) need for exposure of the vena cava , ( d ) need for an associate procedure , and ( e ) cirrhosis , accurately predicted the likelihood of blood transfusions in liver resections . recently , cescon et al . , in a retrospective review assessing the outcome of 1500 consecutive patients who underwent hepatic resection , estimated overall mortality and morbidity at 3% and 22.5% , respectively . their multivariate analysis revealed that blood transfusions , primary liver tumors , and additional procedures were associated with an increased risk of postoperative complications , whereas blood transfusions , cirrhosis , biliary malignancies , and extended hepatectomy were associated with an increased risk of postoperative mortality . , evaluating the long - term outcomes of liver resection for hepatocellular carcinoma , estimated that 86.9% of the patients did not require perioperative blood transfusion and that pringle maneuver and rbc transfusions are independent prognostic factors influencing survival . blood transfusions are well known to carry the risk of transmitted infections , acute or delayed reactions and wrong blood incidents . in liver resections , blood transfusions there is strong evidence that blood transfusions have an impact on tumor recurrence for patients with early stages of hepatocellular carcinoma . however , no such effect could be demonstrated for patients undergoing partial liver resection for late stages of hepatocellular carcinoma , colorectal metastasis , or cholangiocarcinoma . transfusion evoked immunosuppression is also responsible for trali ( transfusion - related acute lung injury ) . dyspnea , hypotension , fever , and bilateral noncardiogenic pulmonary edema , present within 6 h of transfusion and complicate the postoperative outcome of patients following major liver surgery . patients with chronic liver disease have the greatest risk of developing trali , in comparison to other populations [ 101 , 102 ] . although all blood products can lead to this life - threatening situation , plasma - containing products were responsible for the majority of cases in patients undergoing liver transplantation . recent studies suggest that trali fatalities followed plasma transfusion components were linked to multiparous female donors with leukocyte antibodies [ 103 , 104 ] . therefore , the establishment of new strategies in blood donation excluding multiparous women as donors , as potential carriers of trali - inducing antibodies , is expected to eliminate this entity . in conclusion , given the influence of blood loss and transfusions on the surgical outcome , techniques of liver vascular control and anesthetic management should be adjusted to the individual patient . the tumor location , the underlying liver disease and the patient 's cardiovascular status should therefore be taken into account , in order to minimize blood loss and transfusion requirements . ischemia / reperfusion ( i / r ) injury is a serious complication of liver surgery , especially after extended hepatectomies . it causes a local and systemic inflammation response and its clinical manifestations may vary from transient arrhythmias to multiorgan dysfunction and death . reperfusion injury is mediated via reactive oxygen species which damage cellular membranes , stimulate leukocyte activation and endothelial adhesion , and activate the complement . hepatic i / r injury affects patient recovery after major surgery and bears a risk of poor postoperative outcome . in liver surgery , ischemic preconditioning ( ip ) and intermittent clamping are the only established methods to provide protection against tissue damage due to ischemia during inflow occlusion [ 98 , 108 ] . ip is defined as a process in which a short period of ischemia , separated by intermittent reperfusion , renders an organ more tolerant to subsequent episodes of ischemia [ 107 , 109 ] . it was initially described for a canine heart by murry et al . in 1986 . as far as the liver is concerned , the beneficial effect of ip was first demonstrated in a rodent model by lloris - carsi et al . . it leads to improvement of hepatic microcirculation , reduction in tissue apoptosis , and improvement of survival . experimental data suggest that generation of adenosine , activation of adenosine a2 receptors with subsequent generation of no and release of no cause vasodilation and prevent the increase in endothelins , thus protecting the liver from reperfusion injury . ip stimulates adenosine receptors on kupffer cells in nonischemic lobes to produce oxygen radicals , leading to the promotion of liver regeneration after partial hepatectomy . in a clinical study of 61 patients undergoing liver surgery performed by heizmann et al . , the absence of preconditioning was found to be an independent risk factor for postoperative complications . it has been stated that ip might also be less beneficial during extended liver resections , due to hyperperfusion - induced derangement in hepatic microcirculation . similarly , the effect of preconditioning was lost in patients undergoing tissue loss above 50% . in small liver remnants of about 30% , it may in fact have detrimental effects . this is because the small remaining tissue suffers from shear stress - associated microvascular injury . ischemic preconditioning seems to attenuate the apoptotic response of hepatic cells in major hepatectomies performed under shve . on the other hand , azoulay et al . found that ip failed to protect human liver against ir injury after major hepatectomy under continuous vascular occlusion with preservation of caval flow . ip and salvialonic acid - b have been shown to possess synergistically protective effects in rats , mediated through reduction of postischemic oxidative stress , higher atp levels and reduction in hepatocellular apoptosis . the preconditioning effect fades away when the ischemic time is prolonged . in this case , intermittent vascular occlusion , although more complex surgically , seems to be the method of choice . van wagensveld et al . demonstrated that prolonged intermittent vascular inflow occlusion in pig liver surgery caused less microcirculation impairment and hepatocellular necrosis compared with continuous occlusion and recommend it when a prolonged period of vascular inflow occlusion is expected . it has been found that when ischemia persists for more than 40 minutes , intermittent vascular occlusion offers better protection of liver cells , demonstrated by lower ast values , lower apoptotic activity and reduced capsase-3 activation . in several animal models , pharmacological preconditioning with a volatile anesthetic has been proven to provide protection against ischemic injury . beck - schimmer et al . evaluated the effects of sevoflurane preconditioning before liver ischemia and concluded that this particular volatile anesthetic limited the postoperative increase of serum transaminase levels by 261 u / l for the alt and by 239 u / l for the ast . the sevoflurane group had less major complications ( such as sepsis , bilioma , bleeding , and infection ) than the control ( propofol ) group . the protective effects were more pronounced in patients with liver steatosis . however , according to wang et al . , propofol also seems to have the ability to protect human hepatic l02 cells from h2o2-induced apoptosis . intraportal administration of l - arginine , a precursor of no , has been recently studied in pigs and appears to reduce cell damage during the early phase of reperfusion , by downregulating capsase-3 activty and by preserving mitochondrial structure . clinically , it resulted in a reduction of ast and an increase in bile production . in another animal study , simvastatin ( 5 mg / kg ) protected the rat liver from i / r injury by regulating the inflammatory response and by improving microvascular flow . prostaglandins have also been found to have protective effects on i / r - injured livers by inhibiting the generation of reactive oxygen species , preventing leucocyte migration , improving hepatic insulin and lipid metabolism and regulating the production of inflammatory cytokines . finally , ramalho et al . reported that angiotensin ii type i receptor ( at1r ) antagonist increased regeneration in nonsteatotic livers , while in the presence of steatosis both at1r and at2r antagonists increased liver regeneration . intolerance to thve is not unusual and this method should be reserved for patients in need for extensive reconstruction of the inferior vena cava . inflow occlusion techniques , although simple and effective , require specific anesthetic manipulations to reduce liver injury and prevent backflow bleeding . every method of hepatic vascular control applied under a carefully selected anesthetic plan can improve the outcome of patients undergoing hepatectomy . anesthetic vigilance along with thorough knowledge of the surgical manipulations promotes team - based health care in the operative room .

background . hazards of liver surgery have been attenuated by the evolution in methods of hepatic vascular control and the anesthetic management . in this paper , the anesthetic considerations during hepatic vascular occlusion techniques were reviewed . methods . a medline literature search using the terms anesthetic , anesthesia , liver , hepatectomy , inflow , outflow occlusion , pringle , hemodynamic , air embolism , blood loss , transfusion , ischemia - reperfusion , preconditioning , was performed . results . task - orientated anesthetic management , according to the performed method of hepatic vascular occlusion , ameliorates the surgical outcome and improves the morbidity and mortality rates , following liver surgery . conclusions . hepatic vascular occlusion techniques share common anesthetic considerations in terms of preoperative assessment , monitoring , induction , and maintenance of anesthesia . on the other hand , the hemodynamic management , the prevention of vascular air embolism , blood transfusion , and liver injury are plausible when the anesthetic plan is scheduled according to the method of hepatic vascular occlusion performed .

as a consequence of the availability of whole - genome expression methodologies , regulation of gene expression is at the core of current post - genomic studies . once a set of genes is clustered on the basis of similar expression profiles , a logical next step is that of searching their upstream regions for potential binding sites for transcriptional regulators . the predicted binding sites in dna can then be mutated or used to fish out the dna - binding regulatory protein . different methods exist for finding binding sites , with a recent rapid increase in different methods with small variations and improvements . however , as the computational biology community has long been aware , a common limitation of such methods is the high rate of false - positives that they generate as a result of the low degree of conservation of the dna sequences of binding sites . this work is a contribution towards a more detailed evaluation of the performance of these methods , with the aim of finding the best selection of thresholds to provide reliable predictions . on the basis of our evaluations , we suggest improved methods to search for novel binding sites that give a much lower rate of false positives . we use information gathered in regulondb , a database on regulation of transcription in escherichia coli compiled from the literature . the database contains data on regulons - sets of genes in transcription units whose expression is regulated by the same regulatory proteins - with different types of evidence and different levels of description . for instance , at the time of writing , the database contains information on 112 regulatory proteins , but binding sites in dna are only described for 60 of these . the data for 26 of the regulatory proteins includes information on at least three regulated genes , with at least one binding site per gene ( table 1 ) . as explained below , we distinguish between pattern discovery and pattern search and evaluate each separately . one is dyad - analysis , a program developed to find over - represented small words separated by a given distance . we also describe and evaluate an elaboration of this method that aims to search for probable binding sites using the dyads generated ( dyad sweeping ) . the other method uses consensus , a program that generates optimized ungapped multiple alignments for sets of known or suspected regulatory sequences and builds matrices representing the frequency of each base at each position of the aligned sequences . its companion program ' patser ' uses the matrices generated to scan for similar new sequences . the evaluations take into account the interest in minimizing the false - positive rate , as even a very small false - positive rate can overshadow true positives because of the small number of genes expected to be part of each regulon ( see below ) . as most regulatory sites for dna - binding proteins are found 200 to 400 base - pairs ( bp ) upstream of the regulated genes , we built two sets of upstream regions . one contained 200 bp of the region upstream of the genes ' start sites plus 50 bp downstream ( 200 + 50 set ) ; the other contained 400 bp upstream plus 50 bp downstream of the start sites ( 400 + 50 set ) . repressor sites are located near the promoter site , whereas activators tend to occupy a larger region upstream of the promoter . it is therefore potentially useful to evaluate the performance of the methods with these two different ranges of sequence . additional information can also influence the decision of the experimentalist to select the length of upstream region to analyze . for instance , some proteins tend to have a single binding site per promoter , which has to be proximal to the promoter ( for example lexa ) , whereas other proteins tend to have several binding sites per upstream region , with some of them farther upstream of the promoter ( for example arac , lrp and metj ) . another factor that influences the size of region to analyze is whether the precise site of transcription initiation ( the + 1 position ) is known . when the promoter is known , the search can be limited to 200 bp upstream from the + 1 position . if it is not known , then the reference point has to be the start codon and the 400 bp upstream of this are used - which assumes an average of 50 to 100 bp between the promoter and the beginning of the gene . we used the total set of upstream regions containing at least one reported binding site in regulondb as the basic data for evaluation . in each case , upstream regions of genes regulated by the same protein ( regulons ) were separated from the collection and constituted the ' training sets ' . for each set , the remaining upstream regions , known to be regulated by other proteins , are assumed to be the collection of ' known negatives ' . though there is still a risk that the known negatives contain genes that also pertain to the regulon we are contrasting them with , the fact that they have been the subject of experimental work allows us to think that this risk is minute . because of the small amount of data for each protein , we could not leave out a set of known positives to evaluate the rate of true positives , except in the case of the regulatory protein crp . for those families having at least five upstream regions we were able to apply a ' leave one out ' procedure as described below . we also have information , in some cases , on genes regulated by a given protein in the regulons analyzed , but with no reported binding site . the upstream regions of these genes were used to search for binding sites and provide further evaluation . a more detailed analysis was performed for lexa , comparing our predictions with a recent report in the literature . depending on the information available , there are basically two computational approaches to predicting binding sites for transcription initiation factors in dna . in the best cases , there is information on experimentally determined examples of binding sites for a given regulatory protein . in such cases , the search programs can be trained using the sequences corresponding to the binding sites , and the information obtained ( dyads , weight matrices ) can then be used to find similar sequences , and thus other genes that might be under the control of the same regulatory protein . on the other hand , a common scenario at present is that a set of apparently co - regulated genes is identified from transcriptome experiments . in this case , a program would be trained with a collection of upstream regions from these genes with the goal of identifying probable shared regulatory sites . if the data come from transcriptome experiments , the collection of co - regulated genes might not be complete . because of the noise inherent to such experiments , and/or to the limitations of clustering algorithms , a researcher might wish to try to find other genes likely to be under the control of the same protein . however , other genes regulated by the same protein might display a different pattern of expression as a result of complications such as regulation by more than one regulatory protein . on the basis of these considerations , the analyses we present contemplate the use of experimentally determined binding sites as training sets to study pattern search , and the use of upstream regions of co - regulated genes to study pattern discovery . more precisely , we use the set of binding sites in dna for each regulatory protein reported in regulondb to try to find additional genes in the genome with similar sites . we also use the data on known co - regulated genes to try to find the binding site within the genes ' upstream regions . as training sets , we ran the dyad or matrix search programs on the sequences of known regulatory binding sites and on upstream regions of 200 + 50 and 400 + 50 bp from genes regulated by a given regulatory protein . families corresponding to a given regulatory protein were evaluated only if there were at least three sequences in the corresponding training set ( 40 in the collection of binding sites ; 26 in the 200 + 50 and the 400 + 50 datasets ) . subsequently , the dyads and matrices were evaluated against the complete collections of 200 + 50 and 400 + 50 upstream regions . this gives a total of 3 2 = 6 evaluations for each regulon analyzed . the evaluations included regions 200 + 50 or 400 + 50 only if there was at least one reported binding site within that range ; thus , the total set of 200 + 50 regions contained 172 sequences , and the 400 + 50 set contained 189 . we used the dyad - analysis program to find dyads within each training set . the options used were to find dyads of 3 bp long separated by distances of 0 to 16 bp , with any kind of dyad ( direct repeat , inverted repeat , asymmetric ) , searching in both dna strands . further analyses were limited to the training sets where the program found at least one dyad with a significance equal to or above 1.0 ( see for a detailed description of significance ) . this left 19 families from the binding - sites training sets , 11 from the 200 + 50 regions , and 14 from the 400 + 50 regions ( the program dyad - analysis did not find any dyad in about 75% of the rejected families , and found just one in most of the rest of them ) . the program consensus was run to obtain alignments and matrices 20 bp long - the most frequent size among binding sites for regulatory proteins . to assign match scores , we used an ' alphabet ' based on the frequency of each base at upstream regions of 200 + 50 and 400 + 50 of all genes in e. coli . the search was done in a single strand . although we also ran the program to find symmetric patterns , we first present results of pattern discovery , then concentrate on the selection of the best thresholds , analyzing their performance on the basis of the evaluation criteria described above . as most regulatory sites for dna - binding proteins are found 200 to 400 base - pairs ( bp ) upstream of the regulated genes , we built two sets of upstream regions . one contained 200 bp of the region upstream of the genes ' start sites plus 50 bp downstream ( 200 + 50 set ) ; the other contained 400 bp upstream plus 50 bp downstream of the start sites ( 400 + 50 set ) . repressor sites are located near the promoter site , whereas activators tend to occupy a larger region upstream of the promoter . it is therefore potentially useful to evaluate the performance of the methods with these two different ranges of sequence . additional information can also influence the decision of the experimentalist to select the length of upstream region to analyze . for instance , some proteins tend to have a single binding site per promoter , which has to be proximal to the promoter ( for example lexa ) , whereas other proteins tend to have several binding sites per upstream region , with some of them farther upstream of the promoter ( for example arac , lrp and metj ) . another factor that influences the size of region to analyze is whether the precise site of transcription initiation ( the + 1 position ) is known . when the promoter is known , the search can be limited to 200 bp upstream from the + 1 position . if it is not known , then the reference point has to be the start codon and the 400 bp upstream of this are used - which assumes an average of 50 to 100 bp between the promoter and the beginning of the gene . we used the total set of upstream regions containing at least one reported binding site in regulondb as the basic data for evaluation . in each case , upstream regions of genes regulated by the same protein ( regulons ) were separated from the collection and constituted the ' training sets ' . for each set , the remaining upstream regions , known to be regulated by other proteins , are assumed to be the collection of ' known negatives ' . though there is still a risk that the known negatives contain genes that also pertain to the regulon we are contrasting them with , the fact that they have been the subject of experimental work allows us to think that this risk is minute . because of the small amount of data for each protein , we could not leave out a set of known positives to evaluate the rate of true positives , except in the case of the regulatory protein crp . for those families having at least five upstream regions we were able to apply a ' leave one out ' procedure as described below . we also have information , in some cases , on genes regulated by a given protein in the regulons analyzed , but with no reported binding site . the upstream regions of these genes were used to search for binding sites and provide further evaluation . a more detailed analysis was performed for lexa , comparing our predictions with a recent report in the literature . depending on the information available , there are basically two computational approaches to predicting binding sites for transcription initiation factors in dna . in the best cases , there is information on experimentally determined examples of binding sites for a given regulatory protein . in such cases , the search programs can be trained using the sequences corresponding to the binding sites , and the information obtained ( dyads , weight matrices ) can then be used to find similar sequences , and thus other genes that might be under the control of the same regulatory protein . is that a set of apparently co - regulated genes is identified from transcriptome experiments . in this case , a program would be trained with a collection of upstream regions from these genes with the goal of identifying probable shared regulatory sites . if the data come from transcriptome experiments , the collection of co - regulated genes might not be complete . because of the noise inherent to such experiments , and/or to the limitations of clustering algorithms , a researcher might wish to try to find other genes likely to be under the control of the same protein . however , other genes regulated by the same protein might display a different pattern of expression as a result of complications such as regulation by more than one regulatory protein . on the basis of these considerations , the analyses we present contemplate the use of experimentally determined binding sites as training sets to study pattern search , and the use of upstream regions of co - regulated genes to study pattern discovery . more precisely , we use the set of binding sites in dna for each regulatory protein reported in regulondb to try to find additional genes in the genome with similar sites . we also use the data on known co - regulated genes to try to find the binding site within the genes ' upstream regions . as training sets , we ran the dyad or matrix search programs on the sequences of known regulatory binding sites and on upstream regions of 200 + 50 and 400 + 50 bp from genes regulated by a given regulatory protein . families corresponding to a given regulatory protein were evaluated only if there were at least three sequences in the corresponding training set ( 40 in the collection of binding sites ; 26 in the 200 + 50 and the 400 + 50 datasets ) . subsequently , the dyads and matrices were evaluated against the complete collections of 200 + 50 and 400 + 50 upstream regions . this gives a total of 3 2 = 6 evaluations for each regulon analyzed . the evaluations included regions 200 + 50 or 400 + 50 only if there was at least one reported binding site within that range ; thus , the total set of 200 + 50 regions contained 172 sequences , and the 400 + 50 set contained 189 . we used the dyad - analysis program to find dyads within each training set . the options used were to find dyads of 3 bp long separated by distances of 0 to 16 bp , with any kind of dyad ( direct repeat , inverted repeat , asymmetric ) , searching in both dna strands . further analyses were limited to the training sets where the program found at least one dyad with a significance equal to or above 1.0 ( see for a detailed description of significance ) . this left 19 families from the binding - sites training sets , 11 from the 200 + 50 regions , and 14 from the 400 + 50 regions ( the program dyad - analysis did not find any dyad in about 75% of the rejected families , and found just one in most of the rest of them ) . the program consensus was run to obtain alignments and matrices 20 bp long - the most frequent size among binding sites for regulatory proteins . to assign match scores , we used an ' alphabet ' based on the frequency of each base at upstream regions of 200 + 50 and 400 + 50 of all genes in e. coli . the search was done in a single strand . although we also ran the program to find symmetric patterns , no clear improvement was observed . in the results section , we first present results of pattern discovery , then concentrate on the selection of the best thresholds , analyzing their performance on the basis of the evaluation criteria described above . finally , we present some specific predictions . pattern discovery starts with a collection of co - regulated genes for which no binding sites are yet known . to evaluate the methodology , we counted the number of times a sensor can locate a known binding site in a collection of 200 + 50 or 400 + 50 regions . over - represented words would be expected to occur at the binding sites , and thus the first step was to determine if the resulting dyads match the binding sites . we found that there are significant dyads all along the sequences analyzed , with most of them matching at or near the known binding sites . figure 1 shows , using the purr family , that most dyads were found at distances very close to or overlapping the true binding sites . we thus decided to search for stretches of contiguous matches , which we call ' regions of overlapping matches ' ( roms ) , in the upstream sequences being analyzed by counting ( sweeping ) , base by base , the number of matching dyads . as seen in figure 2 , the roms with the highest number of matching dyads overlap the true known binding sites in the dna . this result motivated us to use the highest number of matches within a rom as the score . we call this method dyad sweeping . as the highest - scoring roms frequently overlap reported binding sites ( figure 2 , table 2 ) , we decided to keep , for subsequent analyses , the dyads found within the highest - scoring roms of each upstream region , as long as the rom contained at least two dyads . in table 3 it can be seen that , except in a few of the regulons , the fraction of regions with known binding sites found is quite high . in other words , the set of dyads that result after keeping only those that contribute to the highest rom in each family is able to recover a large fraction of all the known binding sites in the family . it is important to keep in mind that a given dyad can match several positions - and therefore sites - in a single region or family . thus , selecting only those dyads appearing in the highest peak does not restrict their ability to find more than one site per region . the number of dyads that describe the set of known binding sites in a given regulatory family is quite variable . for instance , if we use the known binding sites as training sets , the tyrr family involves 14 different dyads whereas arca has 65 . there is no clear correlation between the number of dyads per site and the total number of sites in the training set for any given family , or any other property of the regulatory site , such as its size . consensus is a program designed to find and align shared stretches of sequence among a given set of sequences . the searching method based on the results of consensus is already available ; the weight matrix generated can be used to search , with the companion program patser , for sites in other upstream regions . the search using patser was made using the first matrix ( highest informational content ) obtained in the final cycle of consensus . this cycle requires all regions to contribute at least one sequence to the matrix . using patser , we searched for the highest - scoring sequence in each region in the training set . the lowest value among these results was set as the minimal score and a second search was performed with this threshold in order to find new sites above this limit within each upstream region in e. coli for further searches and analyses . the capacity for pattern discovery of the two methods can be estimated by calculating the fraction of binding sites found when the training sets were the 200 + 50 or 400 + 50 bp regions , as shown in table 4 . a site was considered found when the predicted pattern overlaps 20% of the binding site . we also show the results of using the sequences of the binding sites with 10 bp extensions on each side as training sets , so we could distinguish between pattern discovery and pattern abstraction or identification . in the case of dyad - analysis / sweeping we evaluated whether the filtered dyads overlap the set of true sites . in the case of consensus / patser we evaluated whether the set of sites selected by consensus / patser overlaps the set of known sites . consensus / patser is able to abstract a pattern for each of the 25 families , whereas dyad - analysis / sweeping can only do it for 19 of the families . in 11 of these 19 families consensus / patser finds more sites , in two families dyad - analysis / sweeping finds more sites , and in the remaining six both methods perform equally well . the real pattern discovery situation is that of the 450/sites cases ( see legend to table 5 for definition ) , where consensus generates matrices for 24 of the families and dyad - analysis finds significant dyads for 11 of them . dyad sweeping finds on average more than 70% of the binding sites ( when dyad - analysis obtains significant dyads ) as compared to around 60% with patser . note that using shorter regions to search for dna binding sites ( 200 + 50 ) , improves the performance of both methods by about 5 - 7% . once table 4 was generated , we estimated the fraction of upstream regions recovered ( table 3 ) . a region is considered found when at least one site in that region is found . therefore , the results differ from those in table 4 because of the occurrence of multiple sites in some upstream regions . a clear case of this is the argr regulon , where each of the six regions has two binding sites . the methods detect from 17% to 58% of the sites , but find from 33 to 100% of the regions . detection of new members of regulons requires the selection of an optimal threshold to accept a sequence as a predicted binding site , and the genes downstream of such sequences as new members of the regulon family . the selection of the best threshold requires the evaluation of the following parameters : sensitivity ( rate of true positives ) , specificity ( rate of true negatives ) , accuracy ( overall rate of true results ) , and , very important in this case , the positive predictive value ( rate of true positives among the total number of positives , true and false ) . we used a leave one out ( loo ) procedure to evaluate the true - positive and false - negative rates with families containing at least five reported genes with binding sites . the loo method consists of leaving one gene at a time out of the training set ; then , with the matrix or dyads built with the remaining sites , a search is made for a probable binding site within the upstream region of the gene that was left out . we combined the results of the left - out regions to build the total set of known positives for evaluation of true positives and false negatives . the evaluation of true negatives and false positives was carried out using the whole set of known positives as training sets and all the remaining regions known to be regulated by any other protein , as known negatives . instead of calculating an average of the scores , and defining the threshold on the basis of standard deviations , we scanned the scores scale form the minimum score obtained in the collection of positive , to the maximum one , calculating the evaluation parameters noted above at each point of the scale . there is no point in searching at lower scores as there is no effect on sensitivity at such values . in figure 3 we show the results of the analyses of the purr regulon using dyad sweeping . here , the minimum number of matches evaluated was one . note that , as the dataset of known negatives exceeds that of known positives , high accuracy coexists with a large number of true negatives . nevertheless , at the threshold of 10 matches , despite a very low false - positive value ( less than 10% ) , and a very high accuracy ( approximately 95% ) and sensitivity ( 90% ) , the positive predictive value ( ppv ) shows that the total true positives in the whole ' predicted ' set is about 60% . as most regulatory proteins regulate just a few genes in comparison with the whole set of genes in a given organism , such a difference means that false positives might dilute reliable predictions even at very low false - positive rates . the ppv alone would leave results with very little recovery of true binding sites . therefore , calculating an optimal point for prediction requires the use of a balanced evaluation criterion . after examining several graphs , we noticed that the average between accuracy and ppv ( which we call the overall performance or op ) would be a good criterion . this makes sense , as op represents a trade - off between those two statistical measures . other criteria , such as the product of accuracy and ppv , might be used instead , but op worked well for our purposes . in a few cases , the point of highest op leaves a very small sensitivity value ( around 50% in purr , for instance ) . if the sensitivity value was less than 60% , we used the last point where the sensitivity was above 60% . in figure 4 we show the results of sensitivity and false - positive rate for all regulons at their best op value using dyad sweeping . the use of weight matrices derived from consensus ( with patser ) is not illustrated , as the selection of the best threshold is the same as in dyad sweeping . in figure 5 we show the results of sensitivity and false - positive rates of each regulon at the best overall performance point of each regulon analyzed using patser . in table 6 we give the fraction of sites found per family in regions of 400 + 50 bp when starting from different training sets using the threshold chosen as described above . dyad - detection / sweeping still performs better at finding the sites within an upstream region , while consensus / patser trained with binding sites finds the sites at an average of almost 77% . an interesting finding here was that , when trained with all the upstream 400 + 50 sequences , consensus finds an alignment and matrix that clearly discriminates between the sequences used in the training set , or regulon , from any other upstream sequence in e. coli . however , in some families , the matrix matches at sites different from the experimentally determined dna binding site of the regulon under analysis ( figure 6 ) , and such sites do not correspond to any known site , motif or region annotated in regulondb in the upstream sequence . we also verified that they do not match conserved regions in between pairs of sites . it will be indeed interesting to find out if these sequences have any biological meaning . once the optimal threshold was obtained , we proceeded to predict other members of each regulon using the complete collection of upstream regions ( 200 + 50 and 400 + 50 ) of the e. coli genome . in order to further evaluate the predictions obtained , we used the recent annotations of cellular functions assigned by monica riley and her group to known e. coli genes . about 30% of the genes in e. coli have no function assigned , and each gene or gene product can be assigned to more than a single cellular role . in table 7 we show the consistency between the functional annotations of genes experimentally demonstrated to belong to each regulon as compared with the functional annotations of the set of predicted genes . in the cases of predictions of high confidence ( for example , argr , crp and purr - all with correspondences above 90% ) , a putative function can be reliably assigned to genes of unknown function . for instance , in the case of the purr family , the genes without functional annotations might be assigned to macromolecule ( dna / rna ) biosynthesis . this is an example of functional gene prediction based on analysis of its regulatory elements . annotations like ' active transporter ' would require other kinds of evidence ( see additional data files ) . functional annotations might be quite helpful in cleaning up wrong predictions , or adjusting the proposed thresholds , although limited by the genomic coverage of the functional assignments . regulondb contains information on a few genes belonging to some of the regulons studied but with no mapped binding site for the relevant regulatory protein . as further evaluation , we show the results of dyad sweeping and patser , trained with the known binding sites of each regulon , for all of these genes ( tables 8,9 ) . in the tables we indicate whether the gene would be included in the corresponding predictions , the highest scoring rom ( dyad sweeping , table 8) or pattern match ( patser , table 9 ) found in the 400 + 50 region of the gene , and the actual sequence suggested as part of the possible binding site . some genes would be rejected as predictions , but the small amount of data makes it impossible to appropriately evaluate this problem . a researcher might choose to use a different , perhaps lower , threshold if the intention is to find every gene for a given regulon experimentally , and such a decision would depend on how many confirmatory experiments it is possible to perform ( an example is shown in the next section ) . lower thresholds can also be used if the intention is to confirm new members suggested by other data , like clustering of a gene or genes with known members of a regulon . the latter case is exemplified by the results with those regulon members lacking a mapped binding site . most contain roms or patterns scoring above the minimal score obtained for a known member of the regulon ( no search is performed below this lower limit ) , often just below our suggested threshold . thus , if there is additional evidence that a gene belongs to a given regulon , the roms found can be proposed as the putative binding sites . a recent attempt has been made by fernandez de henestrosa et al . to locate all the members of the lexa regulon by a combined strategy that included prediction of probable binding sites and experimental confirmation . experimental confirmation showed that only 10 of the 49 predicted new members responded to lexa . the authors also give a table of previously found members of the lexa regulon , which includes a few genes not annotated in regulondb . we could analyze only five of their experimentally confirmed genes and 31 of their wrong predictions ( predictions they later found experimentally not to be regulated by lexa ) because of the lack of updating of the e. coli k12 genome annotations . in table 10 we present our results for the genes noted as previously determined members of the lexa regulon in , plus the five new members found by this study . in table 11 , we find 20 out of the 23 confirmed members of the lexa regulon , whereas we would reject 20 out of their 31 wrong predictions . with consensus , we detect 18 of the 23 confirmed members of the regulon , while rejecting 19 of their wrong predictions . pattern discovery starts with a collection of co - regulated genes for which no binding sites are yet known . to evaluate the methodology , we counted the number of times a sensor can locate a known binding site in a collection of 200 + 50 or 400 + 50 regions . over - represented words would be expected to occur at the binding sites , and thus the first step was to determine if the resulting dyads match the binding sites . we found that there are significant dyads all along the sequences analyzed , with most of them matching at or near the known binding sites . figure 1 shows , using the purr family , that most dyads were found at distances very close to or overlapping the true binding sites . we thus decided to search for stretches of contiguous matches , which we call ' regions of overlapping matches ' ( roms ) , in the upstream sequences being analyzed by counting ( sweeping ) , base by base , the number of matching dyads . as seen in figure 2 , the roms with the highest number of matching dyads overlap the true known binding sites in the dna . this result motivated us to use the highest number of matches within a rom as the score . we call this method dyad sweeping . as the highest - scoring roms frequently overlap reported binding sites ( figure 2 , table 2 ) , we decided to keep , for subsequent analyses , the dyads found within the highest - scoring roms of each upstream region , as long as the rom contained at least two dyads . in table 3 it can be seen that , except in a few of the regulons , the fraction of regions with known binding sites found is quite high . in other words , the set of dyads that result after keeping only those that contribute to the highest rom in each family is able to recover a large fraction of all the known binding sites in the family . it is important to keep in mind that a given dyad can match several positions - and therefore sites - in a single region or family . thus , selecting only those dyads appearing in the highest peak does not restrict their ability to find more than one site per region . the number of dyads that describe the set of known binding sites in a given regulatory family is quite variable . for instance , if we use the known binding sites as training sets , the tyrr family involves 14 different dyads whereas arca has 65 . there is no clear correlation between the number of dyads per site and the total number of sites in the training set for any given family , or any other property of the regulatory site , such as its size . consensus is a program designed to find and align shared stretches of sequence among a given set of sequences . the searching method based on the results of consensus is already available ; the weight matrix generated can be used to search , with the companion program patser , for sites in other upstream regions . the search using patser was made using the first matrix ( highest informational content ) obtained in the final cycle of consensus . this cycle requires all regions to contribute at least one sequence to the matrix . using patser , we searched for the highest - scoring sequence in each region in the training set . the lowest value among these results was set as the minimal score and a second search was performed with this threshold in order to find new sites above this limit within each upstream region in e. coli for further searches and analyses . the capacity for pattern discovery of the two methods can be estimated by calculating the fraction of binding sites found when the training sets were the 200 + 50 or 400 + 50 bp regions , as shown in table 4 . a site was considered found when the predicted pattern overlaps 20% of the binding site . we also show the results of using the sequences of the binding sites with 10 bp extensions on each side as training sets , so we could distinguish between pattern discovery and pattern abstraction or identification . in the case of dyad - analysis / sweeping we evaluated whether the filtered dyads overlap the set of true sites . in the case of consensus / patser we evaluated whether the set of sites selected by consensus / patser overlaps the set of known sites . consensus / patser is able to abstract a pattern for each of the 25 families , whereas dyad - analysis / sweeping can only do it for 19 of the families . in 11 of these 19 families consensus / patser finds more sites , in two families dyad - analysis / sweeping finds more sites , and in the remaining six both methods perform equally well . the real pattern discovery situation is that of the 450/sites cases ( see legend to table 5 for definition ) , where consensus generates matrices for 24 of the families and dyad - analysis finds significant dyads for 11 of them . dyad sweeping finds on average more than 70% of the binding sites ( when dyad - analysis obtains significant dyads ) as compared to around 60% with patser . note that using shorter regions to search for dna binding sites ( 200 + 50 ) , improves the performance of both methods by about 5 - 7% . once table 4 was generated , we estimated the fraction of upstream regions recovered ( table 3 ) . a region is considered found when at least one site in that region is found . therefore , the results differ from those in table 4 because of the occurrence of multiple sites in some upstream regions . a clear case of this is the argr regulon , where each of the six regions has two binding sites . the methods detect from 17% to 58% of the sites , but find from 33 to 100% of the regions . detection of new members of regulons requires the selection of an optimal threshold to accept a sequence as a predicted binding site , and the genes downstream of such sequences as new members of the regulon family . the selection of the best threshold requires the evaluation of the following parameters : sensitivity ( rate of true positives ) , specificity ( rate of true negatives ) , accuracy ( overall rate of true results ) , and , very important in this case , the positive predictive value ( rate of true positives among the total number of positives , true and false ) . definitions of these terms are given in the legend to table 5 . we used a leave one out ( loo ) procedure to evaluate the true - positive and false - negative rates with families containing at least five reported genes with binding sites . the loo method consists of leaving one gene at a time out of the training set ; then , with the matrix or dyads built with the remaining sites , a search is made for a probable binding site within the upstream region of the gene that was left out . we combined the results of the left - out regions to build the total set of known positives for evaluation of true positives and false negatives . the evaluation of true negatives and false positives was carried out using the whole set of known positives as training sets and all the remaining regions known to be regulated by any other protein , as known negatives . instead of calculating an average of the scores , and defining the threshold on the basis of standard deviations , we scanned the scores scale form the minimum score obtained in the collection of positive , to the maximum one , calculating the evaluation parameters noted above at each point of the scale . there is no point in searching at lower scores as there is no effect on sensitivity at such values . in figure 3 we show the results of the analyses of the purr regulon using dyad sweeping . here , the minimum number of matches evaluated was one . note that , as the dataset of known negatives exceeds that of known positives , high accuracy coexists with a large number of true negatives . nevertheless , at the threshold of 10 matches , despite a very low false - positive value ( less than 10% ) , and a very high accuracy ( approximately 95% ) and sensitivity ( 90% ) , the positive predictive value ( ppv ) shows that the total true positives in the whole ' predicted ' set is about 60% . as most regulatory proteins regulate just a few genes in comparison with the whole set of genes in a given organism , such a difference means that false positives might dilute reliable predictions even at very low false - positive rates . the ppv alone would leave results with very little recovery of true binding sites . therefore , calculating an optimal point for prediction requires the use of a balanced evaluation criterion . after examining several graphs , we noticed that the average between accuracy and ppv ( which we call the overall performance or op ) would be a good criterion . this makes sense , as op represents a trade - off between those two statistical measures . other criteria , such as the product of accuracy and ppv , might be used instead , but op worked well for our purposes . in a few cases , the point of highest op leaves a very small sensitivity value ( around 50% in purr , for instance ) . if the sensitivity value was less than 60% , we used the last point where the sensitivity was above 60% . in figure 4 we show the results of sensitivity and false - positive rate for all regulons at their best op value using dyad sweeping . the use of weight matrices derived from consensus ( with patser ) is not illustrated , as the selection of the best threshold is the same as in dyad sweeping . in figure 5 we show the results of sensitivity and false - positive rates of each regulon at the best overall performance point of each regulon analyzed using patser . in table 6 we give the fraction of sites found per family in regions of 400 + 50 bp when starting from different training sets using the threshold chosen as described above . dyad - detection / sweeping still performs better at finding the sites within an upstream region , while consensus / patser trained with binding sites finds the sites at an average of almost 77% . an interesting finding here was that , when trained with all the upstream 400 + 50 sequences , consensus finds an alignment and matrix that clearly discriminates between the sequences used in the training set , or regulon , from any other upstream sequence in e. coli . however , in some families , the matrix matches at sites different from the experimentally determined dna binding site of the regulon under analysis ( figure 6 ) , and such sites do not correspond to any known site , motif or region annotated in regulondb in the upstream sequence . we also verified that they do not match conserved regions in between pairs of sites . it will be indeed interesting to find out if these sequences have any biological meaning . once the optimal threshold was obtained , we proceeded to predict other members of each regulon using the complete collection of upstream regions ( 200 + 50 and 400 + 50 ) of the e. coli genome . in order to further evaluate the predictions obtained , we used the recent annotations of cellular functions assigned by monica riley and her group to known e. coli genes . about 30% of the genes in e. coli have no function assigned , and each gene or gene product can be assigned to more than a single cellular role . in table 7 we show the consistency between the functional annotations of genes experimentally demonstrated to belong to each regulon as compared with the functional annotations of the set of predicted genes . in the cases of predictions of high confidence ( for example , argr , crp and purr - all with correspondences above 90% ) for instance , in the case of the purr family , the genes without functional annotations might be assigned to macromolecule ( dna / rna ) biosynthesis . this is an example of functional gene prediction based on analysis of its regulatory elements . annotations like ' active transporter ' would require other kinds of evidence ( see additional data files ) . functional annotations might be quite helpful in cleaning up wrong predictions , or adjusting the proposed thresholds , although limited by the genomic coverage of the functional assignments . regulondb contains information on a few genes belonging to some of the regulons studied but with no mapped binding site for the relevant regulatory protein . as further evaluation , we show the results of dyad sweeping and patser , trained with the known binding sites of each regulon , for all of these genes ( tables 8,9 ) . in the tables we indicate whether the gene would be included in the corresponding predictions , the highest scoring rom ( dyad sweeping , table 8) or pattern match ( patser , table 9 ) found in the 400 + 50 region of the gene , and the actual sequence suggested as part of the possible binding site . some genes would be rejected as predictions , but the small amount of data makes it impossible to appropriately evaluate this problem . a researcher might choose to use a different , perhaps lower , threshold if the intention is to find every gene for a given regulon experimentally , and such a decision would depend on how many confirmatory experiments it is possible to perform ( an example is shown in the next section ) . lower thresholds can also be used if the intention is to confirm new members suggested by other data , like clustering of a gene or genes with known members of a regulon . the latter case is exemplified by the results with those regulon members lacking a mapped binding site . most contain roms or patterns scoring above the minimal score obtained for a known member of the regulon ( no search is performed below this lower limit ) , often just below our suggested threshold . thus , if there is additional evidence that a gene belongs to a given regulon , the roms found can be proposed as the putative binding sites . a recent attempt has been made by fernandez de henestrosa et al . to locate all the members of the lexa regulon by a combined strategy that included prediction of probable binding sites and experimental confirmation . experimental confirmation showed that only 10 of the 49 predicted new members responded to lexa . the authors also give a table of previously found members of the lexa regulon , which includes a few genes not annotated in regulondb . we could analyze only five of their experimentally confirmed genes and 31 of their wrong predictions ( predictions they later found experimentally not to be regulated by lexa ) because of the lack of updating of the e. coli k12 genome annotations . in table 10 we present our results for the genes noted as previously determined members of the lexa regulon in , plus the five new members found by this study . in table 11 we show our results with their wrong predictions . using dyad sweeping , we find 20 out of the 23 confirmed members of the lexa regulon , whereas we would reject 20 out of their 31 wrong predictions . with consensus , we detect 18 of the 23 confirmed members of the regulon , while rejecting 19 of their wrong predictions . stringent evaluations of pattern discovery and pattern searching methods should be carried out to establish the confidence of a given prediction . here we take advantage of the availability of reasonable negative samples - all other known regulons described in regulondb , except the one under study - in order to use standard statistical measurements of performance such as specificity and ppv . the ppv allowed us to stress how important even low rates of false positives might become in a large population . the small proportion of genes expected to be regulated by a given regulatory protein makes it important to emphasize the need for a stringent threshold to admit new members of regulons , as the true positives might be diluted in a high number of false positives . nevertheless , if additional independent evidence is available , thresholds can be relaxed to include as many predictions as the confirmation procedure ( genetic evidence of the regulatory effect , for instance ) would allow . for instance , if the two computational methods were combined , only one of the genes known to be regulated by lexa ( see previous section ) would be rejected by both methods ( ybfe in table 10 ) , while 16 of the wrong predictions are rejected by both methods ( table 11 ) . a very striking observation that deserves experimental analysis the program discovers patterns that discriminate , very specifically , the upstream regions used as training sets from the other regions . these results imply the existence of new sequence elements specific to each family , different from those reported in the literature . we have not yet found ( data not shown ) any additional property that could suggest their function ; their distance from the start site of transcription to known binding sites is not conserved ; in some cases the predicted motif occurs upstream of the known sites in some promoters and downstream in other promoters . we have , of course , verified these observations twice , and find no additional property to associate with such families . in the comparison of the two methods we have not found that one of them performs better in all the evaluations and scenarios considered ( pattern search , pattern abstraction and pattern discovery ) . this implies that one could consider combining the different methods to make the best use of their respective strengths . for instance , if there is evidence of co - regulation only , we would suggest using dyad - analysis / sweeping first to find the binding sites . if dyad - analysis finds significant dyads , the dyad sweeping methodology can be used to extract possible binding sites . after that , the predicted sites can be used to train consensus and search for further co - regulated genes . in cases where the dna binding sites are known , consensus / patser , which are both very fast and simple to use , can give very reliable results in a short time . the combination of computationally more confident predictions , together with additional independent evidence - for example , functional classes or operon organization - is an intelligent strategy for making more robust predictions . these more robust upstream regulatory analyses can be used to assign function to unknown genes , as illustrated here with the argr , crp and purr regulons . one can envisage highly relevant genomic applications of these predictions , such as distinguishing orthologs within families of paralogous genes , based on their differential regulation , or identifying non - orthologous gene displacement on the basis of regulatory comparisons . the goal in computational biology is twofold : to provide , on the one hand , methods that generate useful and evaluated predictions , and , on the other hand , to use such methods as models of the biology under study . this latter virtue could generate new ways of understanding fundamental processes in gene regulation , along with , as suggested here , new properties of gene regulation at the genomic level . each algorithm should be tested on well - defined problems in order to find their strengths . thus it should be possible to choose which method , or combination of methods , is best suited for the problem at hand . additional data files containing the functional annotations ( using dyad analysis and consensus analysis , respectively ) associated to the genes within each regulons , and of those genes downstream of predicted binding sites are available . functional annotations using dyad analysis click here for additional data file functional annotations using consensus analysis click here for additional data file this research was supported by grants from conacyt no . 0028 and from dgapa to j.c.v . position of dyads found by the dyad - analysis program in relation to the binding sites in dna for the whole purr family . the graph shows the distances between all the dyads found in relation to the known binding sites of the purr regulon . contiguous regions of overlapping matching dyads ( roms ) frequently overlap with the known binding sites . this example shows results after finding significant dyads in the 200 + 50 regions of the purr regulon , and finding the roms within the same regions . the two roms with the highest peaks completely overlap with the two reported regulatory binding sites in this region ( sites lie at positions -59 to -43 and at 29 to 45 ) . it can be seen , for instance , that blue dyads occur only in the two true binding sites . different thresholds , defined as number of overlapping matches , were evaluated for all regulons . this graph shows the case of the purr regulon when the dyads are obtained from the known binding sites and the evaluation is carried out on the 400 + 50 regions . the only dyads used in the search were those found at the roms with the highest value per region in the purr regulon . the statistical parameters ( see table 5 ) are plotted as percentages instead of fractions . the arrow indicates the point of maximum overall performance ( op ) ( see text ) . performance of dyad - analysis / dyad sweeping at the best threshold defined for each family . sensitivity and false - positive rate ( expressed as percentages ) at the highest overall performance for each regulon are shown , using the binding sites as training sets , and the 400 + 50 regions as evaluation sets . sensitivity , and false - positive rate ( expressed as percentages ) at the highest overall performance for each regulon are shown , using the binding sites as training sets , and the 400 + 50 regions as evaluation sets . the positions found by consensus / patser . if consensus is run to find an alignment within the 400 + 50 regions , the resulting matrix finds sites within each region ( indicated here by the sites labeled ' matrix ' ) that do not always match the binding sites for the relevant regulatory protein ( arac in the case illustrated here ) , but are very specific to the gene family . the sequence found does not correspond to known binding sites for other regulatory proteins ( for example crp ) within the regions nor to the promoter . summary of the datasets in regulondb regulondb contains information for the 86 regulons shown in this table . of these , only 60 have at least three known binding sites for their corresponding regulatory protein . the second column indicates the total number of known sites , which are distributed in upstream regions ( fourth column ) . the last column indicates the number of upstream regions for which there is experimental evidence suggesting regulation , but no direct proof of binding of the regulator to the upstream site is yet available . for instance , there are 12 known sites for argr located in only six regions ( with two sites per region ) , plus one region for a different gene for which there is evidence of regulation by argr . pattern discovery using roms ( regions of overlapping matches ) with maximal score to find binding sites in dna the total number of genes in the regulon with a known binding site ( in the 400 + 50 upstream regions ) . the number of regions where a rom ( region of overlapping matches ) with the highest number of matches ( max rom ) touches a known binding site . number of regions where either a rom or dyad touches a known binding site , but the max rom does not . the percentage of all upstream regions in which any rom touches a binding site . number of regions with dyads , but no match between known binding sites and roms . pattern discovery at the level of upstream regions for each family , we show the results with dyad - analysis / sweeping and with consensus / patser . the data shown are obtained using different training sets - the 200 + 50 and 400 + 50 regions ( 250 and 450 ) and a comparison with training sets of known binding sites ( sites ) as a reference standard . results are given as the number of regions where at least one binding site was found divided by the total number of regions , and expressed as percentages . note that only the dyads extracted from the max roms within each region are used here . in each column heading , the first word refers to the training set and the second refers to the regions where the patterns were searched . for instance , columns headed 450/sites show the results of pattern discovery when consensus or dyad - analysis has as input the 450 + 50 bp regions , and the sensor is evaluated with the files of known sites . we counted only those regions containing known binding sites within the range covered ( that is , if a known binding site is present more than 200 bp upstream of the gene start site , the corresponding 200 + 50 region is not counted ) . dashes mean that either there was no binding site within the region , or the programs failed to provide a matrix ( consensus ) or significant dyads ( dyad - analysis ) . a region is considered found if at least one of its binding sites is matched . pattern discovery at the level of binding sites for each family , we show the results of applying dyad - analysis / sweeping and consensus / patser to the problem of discovering binding sites . the results contain pattern discovery data similar to those in table 3 , but this time counting the number of binding sites found per total number of sites . definitions of parameters used in evaluating the predictions fn , false negative ; fp , false positive ; tn , true negatives ; tp , true positives . binding sites remaining at best threshold for each family , we show the results with dyad - analysis / sweeping and with consensus / patser accepting a match only if its score exceeds the defined best threshold . a comparison between the functional annotations of genes known to be regulated by a given protein and the functional annotations of the predicted set of genes . the percentage with related function is calculated against all the genes with functional annotations , while the percentage without functional annotations is calculated against the whole set of predicted genes . the number in parentheses excludes genes known to be part of the corresponding regulon . in cases with high correlation of functional annotations we can propose a related function for genes without functional annotations , as in the consensus / patser predictions of argr , crp and purr ( all with correspondences above 90% ) . . dyad - analysis / sweeping predictions in regions without binding sites reported in regulondb sequences and positions of binding sites predicted by dyad sweeping in genes with experimental evidence for co - regulation in regulondb , but with no binding site experimentally identified . genes follow the alphabetic order of the regulatory proteins , with the name of the protein separating each group . the number in parentheses after the regulator is the value of the threshold - derived from requesting best overall performance . the site coordinates are ' i ' for initial base , ' f ' for final position relative to the start codon . the score is given as the maximum number of matching ( ' m ' ) dyads within a rom . the number of families used was the same for any method , but we only show families where the methods provided significant results . consensus / patser prediction in regions without binding sites reported in regulondb data and analysis as described in table 8 . sc , the number of families used was the same for any method , but we only show families where the methods provided significant results . the table shows our binding - site predictions with dyad sweeping and with patser , using their corresponding best overall performance thresholds . sc , score as obtained by patser ; m , maximum number of matching dyads . note that most genes clearly have roms with 10 or more matches and with scores of patser above 10 . contrasting predictions : regions known to lack lexa sites after experiment , fernandez de henestrosa et al . rejected this set of genes in which they had predicted lexa sites using other computational methods . we tested the capacity of dyad sweeping and patser to also reject these false positives . sc , score as obtained by patser ; m , maximum number of matching dyads . note that for both methods , most of the genes here show much smaller scores than genes belonging to the lexa regulon ( see table 10 ) .

backgroundsites in dna that bind regulatory proteins can be detected computationally in various ways . pattern discovery methods analyze collections of genes suspected to be co - regulated on the evidence , for example , of clustering of transcriptome data . pattern searching methods use sequences with known binding sites to find other genes regulated by a given protein . such computational methods are important strategies in the discovery and elaboration of regulatory networks and can provide the experimental biologist with a precise prediction of a binding site or identify a gene as a member of a set of co - regulated genes ( a regulon ) . as more variations on such methods are published , however , thorough evaluation is necessary , as performance may differ depending on the conditions of use . detailed evaluation also helps to improve and understand the behavior of the different methods and computational strategies.resultswe used a collection of 86 regulons from escherichia coli as datasets to evaluate two methods for pattern discovery and pattern searching : dyad analysis / dyad sweeping using the program dyad - analysis , and multiple alignment using the programs consensus / patser . clearly defined statistical parameters are used to evaluate the two methods in different situations . we placed particular emphasis on minimizing the rate of false positives.conclusionsas a general rule , sensors obtained from experimentally reported binding sites in dna frequently locate true sites as the highest - scoring sequences within a given upstream region , especially using consensus / patser . pattern discovery is still an unsolved problem , although in the cases where dyad - analysis finds significant dyads ( around 50% ) , these frequently correspond to true binding sites . with more robust methods , regulatory predictions could help identify the function of unknown genes .

physical activity during preschool age has significant effects on physical , social and psychological health of children . fundamental movement skills ( fms ) including locomotor ( e.g. run , gallop , hop , leap , horizontal jump and slid ) , object control ( e.g. catch , kick , overhand throw and dribble ) and body management ( e.g. balance , climb and forward roll ) provide a base for more advanced physical skills . fms develop during early childhood and are essential for complex activities at adulthood . some studies have shown that the level of locomotor skills of children is positively correlated with the levels of participation in physical activities at adulthood[34 ] . d'hondt et al reported that the levels of gross motor skills in obese children are lower than the levels of these skills in normal - weight children . therefore it seems likely that more proficient children in fms are more physically active , have more self confidence , are less obese and would be healthier in their adulthood . fms would not be developed naturally as a result of growth and maturation in all children . it has been suggested that the optimal ages for fms learning are between 2 to 7 years[2 , 6 ] . brian w et al have recommended that physical activity promotional programs for preschool children should be on base of children 's natural activities such as being spontaneous and intermittent . also preschool children activities should be enjoyable and contain gross motor plays and locomotor activities . therefore , it seems that an effective physical activity program for preschool children needs to be developmentally appropriate for children in the listed age range . furthermore , physical , emotional and psychosocial needs of children should be considered in development of appropriate physical activity program for these children . ideally physical activity programs in nursery schools also a well developed training course for these instructors is essential in efficient teaching of movement skills to children in nursery schools[1 , 9 ] . in addition to the role of fms in long - term health , fms play a cardinal role in physical development of preschool children . to our knowledge there is no national curriculum for preschool children physical activity education in iranian nursery schools . the objectives of this study were a ) to develop a physical activity program for nursery schools in iran , and b ) to evaluate the effects of this program on improvement of fundamental movement skills of preschool children in selected nursery schools in iran . this quasi - experimental study evaluated the effect of a 10 weeks physical activity program on fundamental movement skills levels of nursery children in iran . the participants were 147 children with age range between 4 to 6 years that were selected by convenience sampling . children selected form five available nursery schools in five different cities in iran . a manual for nursery physical activity instructors was developed by a panel of 20 experts in different related fields in iran including sports medicine specialist , pediatrician , expert nursery teachers , psychologist , nutritionist , physical education specialist , phd in sports physiology , and phd in movement and behavior . this manual contained two parts ; a ) instructions for nursery physical activity instructors about goals and structure of the program , and materials on children 's growth and development , children 's nutrition , physical education , psychology of children , and health and safety , b ) twenty four sections which cover different lessens containing physical activity and games aimed at developing locomotor and object control skills of children aged 4 to 6 years . some sections of this package were developed based on the iranian traditional games and plays that were appropriate for preschool ages . five nursery schools were selected in five different cities ( tehran , isfahan , shahrood , neishaboor and gorgan ) and volunteer nursery teachers of these nursery schools were enrolled in an educational course . this one week course was designed to train nursery physical activity instructors to be able to physically train children in nursery based on our designed physical activity program . the aim of this short - term educational program was to ensure that all physical activity instructors are alert to their duties about implementing of the developed physical activity manual . also physical activity instructors were educated to test children using the test of gross motor development - edition 2 ( tgmd-2 ) before and after intervention . persian version of this test has been employed by akbari h , et al . and bakhtiari s , et al . in iran[9 , the levels of gross motor development of all subjects were measured before intervention and after 10 weeks physical activity program employing tgmd-2 . tgmd-2 is a valid and reliable ( test - retest reliability = 0.88 - 0.96 ) criterion - referenced instrument designed to assess gross motor development among children . each subtest consists of 6 locomotor items ( run , hop , gallop , leap , horizontal jump , and slide ) , and 6 object control items ( throw , catch , kick , strike , dribble , and roll ) . subtest raw score was defined by sum of 6 items of each subtests of locomotor or object control ( scored between 0 - 48 ) . standard scores of each subtest ( scored between 0 - 20 ) and also gross motor quotient were calculated in accordance with tgmd-2 manual . ( gmq ) for each subject was reported as very superior ( gmq > 130 ) , superior ( gmq = 121130 ) , above average ( gmq = 111120 ) , average ( gmq = 90110 ) , below average ( gmq = 8089 ) , poor ( gmq = 7079 ) , and very poor ( gmq < 70 ) according to the suggestions of the tgmd-2 manual . according to developed physical activity manual , physical activity program was conducted 5 days a week for 10 weeks by trained nursery physical activity instructors . paired - samples t test was used to determine whether there were significant differences between the levels of locomotor and object control of participants before and after intervention . independent samples t - test was used to compare differences between boys and girls on variables . in this quasi - experimental study 147 children with mean ( sd ) age of 4.95 ( 0.8 ) ( range 3 - 6 ) year from five nursery schools in 5 cities in iran were included . of all subjects 49% ( 72 ) were girls and 51% ( 75 ) were boys . the base line and post intervention tgmd-2 scores of subjects are presented in tables 1 and 2 . in the baseline , there were no statistically significant differences between the locomotor and object control raw scores of boys and girls ( p=0.49 and p=0.9 ) respectively . base line and post intervention tgmd-2 scores of all subjects sd : standard deviation means ( sd ) of base line and after intervention tgmd-2 raw scores of all subjects by age and gender sd : standard deviation after intervention , differences between the locomotor raw scores of boys and girls were not statistically significant ( p=0.5 ) . however , there was a statistically significant difference between boys and girls in the object control raw scores ( p=0.048 ) . the gmq of all subjects which is the most reliable score of tgmd-2 was statistically significantly increased after 10 weeks of intervention . both subtests of tgmd-2 including locomotor raw score and object control raw score in both genders were statistically significantly increased after 10 weeks of intervention ( table 1 ) . descriptive rating of gmq for subjects before and after intervention is shown in table 3 . before intervention only 11.5% of all subjects were rated superior / very superior in gmq scores ( i.e. gmq > 120 ) , however this rate increased to 49.7% of all subjects after 10 weeks of intervention . before the intervention , 26.6% of all subjects were rated as poor / very poor , this rate decreased to 2% of subjects after 10 weeks intervention . age equivalents of subjects on the locomotor and object control subtests before and after intervention are shown in table 4 . age equivalents or developmental age defined as developmental level or age that corresponds to a raw score made by an individual . descriptive rating of the gross motor quotient of all subjects before and after intervention age equivalents for locomotor and object control raw scores according to age groups of all subjects before and after intervention the main finding of this study was that the developed physical activity intervention program that was focused on gross motor skills development had a significant positive effect on proficiency in fundamental movement skills in preschool children from selected nursery schools in five cities in iran . differences in tgmd-2 results before and after the intervention were significant in locomotor , object control , sum of standard cores and gross motor quotient in all subjects . to our knowledge there has been no published data on the levels of physical activity of iranian preschool children . however , base line tgmd-2 subtests scores of our subjects in this study were in the range of reported data from the united states ( table 5 ) . comparison of tgmd-2 standard scores of subjects in this study with reported data from the united states , mean ( sd ) sd : standard deviation our data suggested that a supervised physical activity program could increase these scores to be better than normative reported data . there was no significant difference in the base line mean of the locomotor and object control raw scores between girls and boys ( 30 vs 29.5 ) and ( 24.8 vs 26 ) , respectively . however after the intervention the mean of both object control raw scores ( p=0.048 ) and object control standard scores ( p=0.02 ) in girls were higher than these scores in boys . cliff et al have reported that locomotor raw score in preschool children was higher in girls compared with boys but there was no significant difference between girls and boys in the object control raw score . in this study , base line means of standard scores of the locomotor ( 9.3 vs 8.9 ) , object control ( 9.1 vs 8.1 ) and gmq ( 95.5 vs 91.2 ) were not significantly different between girls and boys . in contrast cliff et al have reported significant differences between girls and boys in the mean of locomotor standard scores ( 9.9 vs 7.9 ) , object control standard scores ( 10.1 vs 8.6 ) and gmq ( 99.7 vs 88.2 ) . one study has reported that locomotor skills proficiency is higher in girls and in contrast boys are more proficient in object control skills . some studies have suggested that the levels of moderate and vigorous physical activities in children with better motor performance are significantly higher than the levels of these activities in children with less developed skills[4 , 19 ] . therefore , conduction of a physical activity program such as the program used in this study may help children to improve their fms which may help to have a higher physical activity in their future . first , same person conducted both training sessions and outcomes measurement in each nursery school . furthermore , to our knowledge there was no normative data of tgmd-2 for iranian children to be used for comparison with our data . further studies are needed to evaluate the long - term effects of physical activity intervention on fms in iran . it seems that our developed physical activity program conducted by trained nursery physical activity instructors could be an effective and practical way of improving gross motor skills of preschool children in short term in iran . conduction of this program in nursery schools could indirectly help with increasing health levels and levels of physical activities in the society . we recommend using of this kind of physical activity programs in all nursery schools in iran and similar counties .

objectivethe objectives of this study were a ) to develop a physical activity program for nursery schools , and b ) to evaluate the effects of this program on fundamental movement skills of preschool age children in iran.methodsin this quasi - experimental study 147 children from five nursery schools in five different cities in iran were enrolled . a physical activity program was developed for nursery children . trained nursery physical activity instructors conducted the program for 10 weeks for all subjects . the levels of gross motor development of all subjects were measured before intervention and after 10 weeks physical activity program employing the test of gross motor development - edition 2 ( tgmd-2).findingsthe participants in this study had a mean ( sd ) age of 4.95 ( 0.83 ) years . at the end of the study , scores of subjects at all components of tgmd-2 ( including locomotor , object control , sum of standard scores and gross motor quotient ) were significantly improved compared to the baseline scores ( p<0.001 ) . based on descriptive rating of the " gross motor quotient " in the base line , 11.5% of subjects were superior / very superior ( gmq > 120 ) and after 10 weeks intervention this rate was increased to 49.7% of all subjects.conclusionit seems that the developed physical activity program conducted by trained nursery physical activity instructors could be an effective and practical way of increasing levels of fundamental movement skills of preschool children in iran .

prostatic acid phosphatase ( pap ) , a glycoprotein synthesized by the prostate gland , is traditionally used to diagnose prostate cancer . pap has two isoforms , secreted and transmembrane isoforms , and , more than in the prostate gland , is also extensively expressed in nonprostate tissue such as nervous tissues .. recent studies have identified pap is molecular identity to thiamine monophosphatase ( tmpase ) and , historically , tmpase , with the acid phosphatase characteristics , has been widely used for labeling nociceptive dorsal root ganglia ( drg ) neurons . pap hydrolyzes adenine monophosphate ( amp ) , particularly in acidic environments , to adenosine that acts as an analgesic effector . moreover , small nociceptive drg neurons , including peptidergic and nonpeptidergic neurons , coexpress pap , suggesting that pap profiles correlate with pain modulation . for example , a growing body of evidence has shown that intrathecal injection of recombinant pap counteracts inflammatory pain and enhances neuropathic pain in pap - knockout mice . previously , we have established a mouse model of small - fiber neuropathy , in which small sensory nerves were specifically depleted by resiniferatoxin ( rtx ) , a capsaicin analog acting on the transient receptor potential vanilloid receptor 1 ( trpv1 ) . this rtx - induced neuropathy model mimics human small - fiber neuropathy ; that is , the model exhibits the degeneration of cutaneous sensory nerves and neuropathic pain . furthermore , rtx - induced neuropathy with skin denervation markedly increases the expression of activating transcription factor 3 ( atf3 ) , a marker of neuronal injury , in nonpeptidergic p2x3 neurons , which is correlated with neuropathic pain . whether the expression profiles of this subpopulation of pap neurons correlate with injury - induced neuropathic pain should be determined , and the mechanism responsible for modulating the analgesic potential in injury - induced neuropathic pain should be investigated . the compound 4-methylcatechol ( 4mc ) enhances the synthesis of neurotrophins such as nerve growth factor ( ngf ) and promotes skin reinnervation . cutaneous sensory nerves function as the peripheral receptors of small drg neurons ; thus , inducing atf3 expression in these neurons caused skin denervation and neuropathic pain in our rtx - induced neuropathy model . ngf is trophic to small drg neurons and modulates neuropathic pain through its high - affinity receptor , trka ; a reduction in ngf and trka expression is accompanied by various peripheral neuropathies . whether 4mc modulates pap expression profiles through ngf - trka signaling is unclear . rtx - induced neuropathy is an excellent model for determining how the loss of the analgesic effect of pap leads to injury - induced neuropathic pain . in the present study , we investigated the intervention of pap profiles to neuropathic pain and the effect of 4mc on reversing neuropathic pain through ngf - trka signaling . this study investigated pap - trka signaling - mediated pain behavior in rtx - induced neuropathy by investigating whether pap neuropathology is correlated with pain development and whether 4mc reverses pain and pap profiles in rtx - induced neuropathy in an ngf - trka signaling - dependent manner . administration of a single dose of rtx ( 50 g / kg , sigma , st . louis , mo ) ( the rtx group ) . injections of 4mc ( 10 g / kg , wako , osaka , japan ) immediately after rtx treatment for seven consecutive days ( the 4mc group ) , or an equal volume of vehicle as a control ( the vehicle group ) . to confirm the effect of 4mc , . administered . briefly , 2.5s ngf ( alomone labs , jerusalem , israel ) was dissolved in dulbecco s modified eagle medium , and mice received ngf ( 1 g/10 g ) immediately after rtx treatment ( the ngf group ) . some mice in the 4mc group received additional anti - ngf antisera ( 30 g / kg , sigma ) to neutralize ngf induced by 4mc ( the abngf group ) . to compare each experimental approach , 2.5s ngf and anti after treatment , the mice were housed in plastic cages in a 12-h light / dark cycle and were given access to water and food ad libitum . all procedures were conducted in accordance with ethical guidelines for laboratory animals , and the protocol was approved by the institutional animal care and use committee of kaohsiung medical university . all experimental procedures were performed carefully , and all efforts were made to minimize animal suffering . the changes in the mechanical threshold of each group were assessed using the up - and - down method with different calibers of von frey monofilaments ( somedic sales ab , hrby , sweden ) in accordance with our established protocol . briefly , a series of monofilaments was applied to the plantar region of the hindpaw . if paw withdrawal occurred , a monofilament of a smaller caliber was applied . in the absence of paw withdrawal four additional stimuli with monofilaments of various calibers were applied on the basis of the preceding responses , and the mechanical thresholds were calculated using a formula published in a previous study . animals were sacrificed by intracardiac perfusion with 0.1 m phosphate buffer ( pb ) followed by 4% paraformaldehyde ( 4 p ) in 0.1 m pb . after perfusion , the 4th and 5th ( l4 and l5 , respectively ) lumbar drgs were removed carefully and postfixed in 4p for another 6 h. the drgs were cryoprotected with 30% sucrose in 0.1 m pb overnight , and 8 -m - thick cryosections were obtained using a cryostat ( cm1850 , leica , wetzlar , germany ) . for adequate sampling , two ganglia ( l4/l5 ) per mouse and 5 to 8 sections per drg tissue ( at 80-m intervals ) were immunostained . the following primary antisera were used in this study : anti - pap ( chicken , 1:600 , aves labs , tigard , or ) , anti - atf3 ( rabbit , 1:100 , santa cruz biotechnology , santa cruz , ca ) , anti - peripherin ( rabbit , 1:800 , chemicon , temecula , ca ) , anti - neurofilament smi32 ( mouse , 1:600 , covance , emeryville , ca ) , anti - p2x3 ( rabbit , 1:400 , neuromics , edina , mn ) , and anti - trka ( rabbit , 1:200 , santa cruz ) . briefly , sections were incubated overnight at 4 with one of the primary antiserum combinations : p2x3/pap , pap / peripherin , pap / smi32 , atf3/pap , and pap / trka . thereafter , the sections were incubated with texas red or fluorescein isothiocyanate - conjugated secondary antisera ( 1:100 , jackson immunoresearch , west grove , pa ) corresponding to the appropriate primary antisera for 1 h. sections were mounted with vectashield ( vector , burlingame , ca ) for quantification . to quantify the various phenotypic drg neurons , each drg section was systematically photographed at 200 under a fluorescence microscope ( axiophot microscope , zeiss , oberkochen , germany ) to produce a montage of the entire drg section according to an established procedure . to avoid density bias , only neurons with a clear nuclear profile were counted , and only sections containing neuronal ganglia were measured using imagej version 1.44d ( national institutes of health [ nih ] , bethesda , md ) . for morphometric analysis of pap neuropathology , the diameters of pap(+ ) neurons and atf3(+)/pap(+ ) neurons were measured using image pro - plus ( media cybernetics , bethesda , md ) and plotted as a histogram . to minimize individual bias , five to seven mice were included in each group , and the coding information was masked during the behavioral tests and quantification procedures . all data are expressed as mean standard derivation ( sd ) of the mean . a t test was performed to analyze the data following a gaussian distribution . for data which did not follow a gaussian distribution , a nonparametric mann this study investigated pap - trka signaling - mediated pain behavior in rtx - induced neuropathy by investigating whether pap neuropathology is correlated with pain development and whether 4mc reverses pain and pap profiles in rtx - induced neuropathy in an ngf - trka signaling - dependent manner . administration of a single dose of rtx ( 50 g / kg , sigma , st . louis , mo ) ( the rtx group ) . injections of 4mc ( 10 g / kg , wako , osaka , japan ) immediately after rtx treatment for seven consecutive days ( the 4mc group ) , or an equal volume of vehicle as a control ( the vehicle group ) . to confirm the effect of 4mc , . administered . briefly , 2.5s ngf ( alomone labs , jerusalem , israel ) was dissolved in dulbecco s modified eagle medium , and mice received ngf ( 1 g/10 g ) immediately after rtx treatment ( the ngf group ) . some mice in the 4mc group received additional anti - ngf antisera ( 30 g / kg , sigma ) to neutralize ngf induced by 4mc ( the abngf group ) . to compare each experimental approach , 2.5s ngf and anti after treatment , the mice were housed in plastic cages in a 12-h light / dark cycle and were given access to water and food ad libitum . all procedures were conducted in accordance with ethical guidelines for laboratory animals , and the protocol was approved by the institutional animal care and use committee of kaohsiung medical university . all experimental procedures were performed carefully , and all efforts were made to minimize animal suffering . the changes in the mechanical threshold of each group were assessed using the up - and - down method with different calibers of von frey monofilaments ( somedic sales ab , hrby , sweden ) in accordance with our established protocol . briefly , a series of monofilaments was applied to the plantar region of the hindpaw . if paw withdrawal occurred , a monofilament of a smaller caliber was applied . in the absence of paw withdrawal four additional stimuli with monofilaments of various calibers were applied on the basis of the preceding responses , and the mechanical thresholds were calculated using a formula published in a previous study . animals were sacrificed by intracardiac perfusion with 0.1 m phosphate buffer ( pb ) followed by 4% paraformaldehyde ( 4 p ) in 0.1 m pb . after perfusion , the 4th and 5th ( l4 and l5 , respectively ) lumbar drgs were removed carefully and postfixed in 4p for another 6 h. the drgs were cryoprotected with 30% sucrose in 0.1 m pb overnight , and 8 -m - thick cryosections were obtained using a cryostat ( cm1850 , leica , wetzlar , germany ) . for adequate sampling , two ganglia ( l4/l5 ) per mouse and 5 to 8 sections per drg tissue ( at 80-m intervals ) were immunostained . the following primary antisera were used in this study : anti - pap ( chicken , 1:600 , aves labs , tigard , or ) , anti - atf3 ( rabbit , 1:100 , santa cruz biotechnology , santa cruz , ca ) , anti - peripherin ( rabbit , 1:800 , chemicon , temecula , ca ) , anti - neurofilament smi32 ( mouse , 1:600 , covance , emeryville , ca ) , anti - p2x3 ( rabbit , 1:400 , neuromics , edina , mn ) , and anti - trka ( rabbit , 1:200 , santa cruz ) . briefly , sections were incubated overnight at 4 with one of the primary antiserum combinations : p2x3/pap , pap / peripherin , pap / smi32 , atf3/pap , and pap / trka . thereafter , the sections were incubated with texas red or fluorescein isothiocyanate - conjugated secondary antisera ( 1:100 , jackson immunoresearch , west grove , pa ) corresponding to the appropriate primary antisera for 1 h. sections were mounted with vectashield ( vector , burlingame , ca ) for quantification . to quantify the various phenotypic drg neurons , each drg section was systematically photographed at 200 under a fluorescence microscope ( axiophot microscope , zeiss , oberkochen , germany ) to produce a montage of the entire drg section according to an established procedure . to avoid density bias , only neurons with a clear nuclear profile were counted , and only sections containing neuronal ganglia were measured using imagej version 1.44d ( national institutes of health [ nih ] , bethesda , md ) . for morphometric analysis of pap neuropathology , the diameters of pap(+ ) neurons and atf3(+)/pap(+ ) neurons were measured using image pro - plus ( media cybernetics , bethesda , md ) and plotted as a histogram . to minimize individual bias , five to seven mice were included in each group , and the coding information was masked during the behavioral tests and quantification procedures . all data are expressed as mean standard derivation ( sd ) of the mean . a t test was performed to analyze the data following a gaussian distribution . for data which did not follow a gaussian distribution , a nonparametric mann in a previous study , we developed an rtx - induced small - fiber neuropathy mouse model , in which sensitized p2x3 purinociceptors - mediated mechanical allodynia . in the present study , rtx affected pap expression , as demonstrated by the coexpression of p2x3 and pap ( figure 1 ) . notably , pap and p2x3 showed inverse patterns after neuropathy ; specifically , there was an increase in p2x3(+ ) and decrease in pap(+ ) neurons , resulting in an increased p2x3/pap ratio ( 67.4% 2.1% vs. 44.7% 3.4% , p < 0.0001 ) and decreased pap / p2x3 ratio ( 87.1% 5.4% vs. 76.8% 2.3% , p = 0.001 ) ( figure 1(e ) ) . furthermore , pap expression was higher in peripherin(+ ) neurons than in neurofilament ( smi32)(+ ) neurons ( figure 2 ) . the diameter histograms confirmed that the pap(+ ) neurons were small to medium drg neurons ( 25th75th percentile : 17.123.7 m ) ( figure 2(f ) ) . figure 1.colocalization of prostatic acid phosphatase ( pap)(+ ) and p2x3(+ ) dorsal root ganglion neurons in resiniferatoxin ( rtx)-induced neuropathy . ( a d ) double immunofluorescence staining was performed with the following primary antiserum combinations : anti - pap ( a , c ) and anti - p2x3 ( b , d ) antisera in the vehicle ( a , b ) and rtx ( c , d ) groups . ( e ) the graph indicates the changes of colocalized ratios of p2x3(+)/pap(+ ) and pap(+)/p2x3(+ ) neurons in the vehicle ( opened bar ) and rtx ( filled bar ) group according to panels ( a ) to ( d).bar , 50 m . * * p < 0.01 , * * * p < 0.001 . figure 2.expression of prostatic acid phosphatase ( pap)(+ ) by the peripherin(+ ) and neurofilament ( sim32)(+ ) dorsal root ganglion ( drg ) neurons . double immunofluorescence staining of the drg sections was performed with the following two primary antisera combinations : ( a , b ) pap / smi32 and pap / peripherin ( c , d ) . ( a , b ) the micrographs show extremely low colocalization of pap ( a ) and smi32 ( b ) . ( c , d ) the micrographs show high colocalization of pap ( c ) and peripherin ( d ) . ( e ) the graph quantifies the colocalized ratios of pap(+)/smi32(+ ) ( open bar ) and pap(+)/peripherin(+ ) neurons ( filled bar ) according to panels ( a ) to ( d ) . ( f ) the graph shows the diameter histogram of pap(+ ) neurons , indicating that pap(+ ) neurons are small to medium neurons.bar , 50 m . * * p < 0.01 colocalization of prostatic acid phosphatase ( pap)(+ ) and p2x3(+ ) dorsal root ganglion neurons in resiniferatoxin ( rtx)-induced neuropathy . ( a d ) double immunofluorescence staining was performed with the following primary antiserum combinations : anti - pap ( a , c ) and anti - p2x3 ( b , d ) antisera in the vehicle ( a , b ) and rtx ( c , d ) groups . ( e ) the graph indicates the changes of colocalized ratios of p2x3(+)/pap(+ ) and pap(+)/p2x3(+ ) neurons in the vehicle ( opened bar ) and rtx ( filled bar ) group according to panels ( a ) to ( d ) . bar , 50 m . * * p < 0.01 , * * * p < 0.001 . expression of prostatic acid phosphatase ( pap)(+ ) by the peripherin(+ ) and neurofilament ( sim32)(+ ) dorsal root ganglion ( drg ) neurons . double immunofluorescence staining of the drg sections was performed with the following two primary antisera combinations : ( a , b ) pap / smi32 and pap / peripherin ( c , d ) . ( a , b ) the micrographs show extremely low colocalization of pap ( a ) and smi32 ( b ) . ( c , d ) the micrographs show high colocalization of pap ( c ) and peripherin ( d ) . ( e ) the graph quantifies the colocalized ratios of pap(+)/smi32(+ ) ( open bar ) and pap(+)/peripherin(+ ) neurons ( filled bar ) according to panels ( a ) to ( d ) . ( f ) the graph shows the diameter histogram of pap(+ ) neurons , indicating that pap(+ ) neurons are small to medium neurons . bar , 50 m . * * p < 0.01 to understand the expression of pap and its relationship with atf3 , an injury marker , we performed double immunofluorescence staining of drg neuronal sections in rtx - induced neuropathy ( figure 3(a ) to ( e ) ) . pap(+ ) neurons were mildly lower in the rtx group than in the vehicle group ( 190.6 12.2 vs. 269.5 6.9 neurons / mm , p = 0.0003 ) ( figure 3(f ) ) . by contrast , atf3(+ ) neurons were markedly higher in the rtx group than in the vehicle group ( 192.3 18.0 vs. 9.0 13.1 neurons / mm , p < moreover , atf3 was induced preferentially in pap(+ ) neurons ( figure 3(a ) to ( e ) ) , resulting in an increased ratio of atf3(+)/pap(+ ) neurons in the rtx group ( 29.0% 5.6% vs. 0.2% 0.2% , p = 0.0043 ) ( figure 3(h ) ) . figure 3.phenotypic changes in prostatic acid phosphatase ( pap)(+ ) dorsal root ganglion neurons in resiniferatoxin ( rtx)-induced neuropathy . ( a e ) double immunofluorescence staining was performed with anti - activating transcription factor 3 ( atf3 ; a e in red ) and anti - pap ( a e in green ) antisera in the vehicle ( a ) , rtx ( b ) , 4-methylcatechol ( 4mc ) ( c ) , ngf ( rtx + 2.5s ngf ; d ) , and ( e ) abngf ( 4mc + anti - ngf antisera ) groups . ( f h ) the graphs quantify the density changes in ( f ) pap(+ ) and ( g ) atf3(+ ) neurons and ( h ) the ratio changes in atf3(+)/pap(+ ) neurons according to panels ( a ) to ( e ) . ( i ) the mechanical thresholds were assessed using the up - and - down method with von frey monofilaments , as described in the materials and methods . the graph indicates the changes in the mechanical thresholds in the vehicle ( open bar ) , rtx ( filled bar ) , 4mc ( grey bar ) , ngf ( slashed bar ) , and abngf ( dotted bar ) groups . ( j , k ) the graphs indicate that the mechanical thresholds correlated with pap expression , that is , a linear correlation with ( j ) pap densities and ( k ) an inverse correlation with the ratio of atf3(+)/pap(+ ) neurons . ( l , m ) the graphs show the diameter histogram of atf3(+)/pap(+ ) neurons in the rtx ( l ) and 4mc ( m ) groups . the diameter histogram shows no difference between the rtx and 4mc groups , but higher numbers of atf3(+)/pap(+ ) neurons in the rtx group.bar , 50 m . * p < 0.05 , * * p < 0.01 , * * * p < 0.001 . phenotypic changes in prostatic acid phosphatase ( pap)(+ ) dorsal root ganglion neurons in resiniferatoxin ( rtx)-induced neuropathy . ( a e ) double immunofluorescence staining was performed with anti - activating transcription factor 3 ( atf3 ; a e in red ) and anti - pap ( a e in green ) antisera in the vehicle ( a ) , rtx ( b ) , 4-methylcatechol ( 4mc ) ( c ) , ngf ( rtx + 2.5s ngf ; d ) , and ( e ) abngf ( 4mc + anti - ngf antisera ) groups . ( f h ) the graphs quantify the density changes in ( f ) pap(+ ) and ( g ) atf3(+ ) neurons and ( h ) the ratio changes in atf3(+)/pap(+ ) neurons according to panels ( a ) to ( e ) . ( i ) the mechanical thresholds were assessed using the up - and - down method with von frey monofilaments , as described in the materials and methods . the graph indicates the changes in the mechanical thresholds in the vehicle ( open bar ) , rtx ( filled bar ) , 4mc ( grey bar ) , ngf ( slashed bar ) , and abngf ( dotted bar ) groups . ( j , k ) the graphs indicate that the mechanical thresholds correlated with pap expression , that is , a linear correlation with ( j ) pap densities and ( k ) an inverse correlation with the ratio of atf3(+)/pap(+ ) neurons . ( l , m ) the graphs show the diameter histogram of atf3(+)/pap(+ ) neurons in the rtx ( l ) and 4mc ( m ) groups . the diameter histogram shows no difference between the rtx and 4mc groups , but higher numbers of atf3(+)/pap(+ ) neurons in the rtx group . p < 0.05 , * * p < 0.01 , * * * p < 0.001 . mice in the rtx group showed mechanical allodynia compared with the vehicle group ( 747.1 156.0 vs. 299.6 70.6 mg , p = 0.0025 ) ( figure 3(i ) ) . this behavioral change correlated with the pap profiles ; that is , the densities of pap(+ ) neurons ( r = 0.75 , p < 0.0001 ; figure 3(j ) ) and the ratios of atf3(+)/pap(+ ) neurons ( r = 0.60 , p = 0.003 ; figure 3(k ) ) were inversely linear to the mechanical thresholds . collectively , these findings indicate that pap expression had a strong influence in mediating pain development . after neuropathy was induced using rtx , 4mc normalized pap expression and relieved mechanical allodynia ; specifically , the pap(+ ) neuronal density ( 269.7 48.8 neurons / mm , p = 0.61 ) ( figure 3(c ) and ( f ) ) and mechanical allodynia ( 693.8 146.0 mg , p = 0.54 ) ( figure 3(i ) ) in the 4mc group were comparable with those in the vehicle group . furthermore , although 4mc reduced the atf3(+ ) neuronal density ( 102.4 27.8 neurons / mm , p < 0.0001 ) and ratio of atf3(+)/pap(+ ) neurons ( 15.8% 5.9% , p = 0.0095 ) , the ratios of atf3(+)/pap(+ ) neurons were still higher in the 4mc group than in the vehicle group ( p = 0.016 ; figure 3(c ) , ( g ) , ( h ) ) . morphometric analysis demonstrated that 4mc reduced the absolute number of atf3(+)/pap(+ ) neurons but did not have a significant effect on their mean diameter ( 15.5 2.2 vs. 16.5 2.8 m , p > 0.05 ) ( figure 3(l ) vs. ( m ) ) . to understand the putative mechanisms of pain relief by 4mc , we examined the coexpression of pap(+)/trka(+ ) neurons ( figure 4 ) . for each group , pap(+ ) neuronal density was similar to trka(+ ) neuronal density ( figure 3(f ) vs. 4(k ) ) . the pap / trka ratios correlated linearly with the mechanical threshold ( r = 0.62 , p = 0.0062 ; figure 4(m ) ) . to confirm the effect of 4mc , the mice in the ngf group were systemically ( i.p . ) administered 2.5s ngf . the ngf group exhibited the same effect as the 4mc group ; that is , pap and trka expression profiles were reversed , atf3 induction was normalized , and mechanical allodynia relieved . those findings , however , were not observed in the abngf group ( administered anti - ngf antisera to neutralize ngf ) ( figures 3 and 4 ) . figure 4.colocalization of prostatic acid phosphatase ( pap ) and high - affinity nerve growth factor ( trka ) receptor after resiniferatoxin ( rtx)-induced neuropathy . ( a j ) double immunofluorescence staining of dorsal root ganglia sections was performed with anti - pap ( a e ) and trka ( f - j ) in the vehicle ( a , f ) , rtx ( b , g ) , 4-methylcatechol ( 4mc ; c , h ) , ( d , i ) ngf ( rtx + 2.5s ngf ) , and ( e , j ) abngf ( 4mc + anti - ngf antisera ) groups . ( k , l ) the graphs show the changes in ( k ) trka density and ( l ) the colocalized ratios of pap(+)/trka(+ ) neurons in the vehicle ( open bar ) , rtx ( filled bar ) , 4mc ( grey bar ) , ngf ( slashed bar ) , and abngf groups ( dotted bar ) according to panels ( a ) to ( j ) . ( m ) the graph shows that the mechanical thresholds correlate linearly with the ratios of pap(+)/trka(+ ) neurons.bar , 50 m . * p < 0.05 , * * p < 0.01 . colocalization of prostatic acid phosphatase ( pap ) and high - affinity nerve growth factor ( trka ) receptor after resiniferatoxin ( rtx)-induced neuropathy . ( a j ) double immunofluorescence staining of dorsal root ganglia sections was performed with anti - pap ( a e ) and trka ( f - j ) in the vehicle ( a , f ) , rtx ( b , g ) , 4-methylcatechol ( 4mc ; c , h ) , ( d , i ) ngf ( rtx + 2.5s ngf ) , and ( e , j ) abngf ( 4mc + anti - ngf antisera ) groups . ( k , l ) the graphs show the changes in ( k ) trka density and ( l ) the colocalized ratios of pap(+)/trka(+ ) neurons in the vehicle ( open bar ) , rtx ( filled bar ) , 4mc ( grey bar ) , ngf ( slashed bar ) , and abngf groups ( dotted bar ) according to panels ( a ) to ( j ) . ( m ) the graph shows that the mechanical thresholds correlate linearly with the ratios of pap(+)/trka(+ ) neurons . in a previous study , we developed an rtx - induced small - fiber neuropathy mouse model , in which sensitized p2x3 purinociceptors - mediated mechanical allodynia . in the present study , rtx affected pap expression , as demonstrated by the coexpression of p2x3 and pap ( figure 1 ) . notably , pap and p2x3 showed inverse patterns after neuropathy ; specifically , there was an increase in p2x3(+ ) and decrease in pap(+ ) neurons , resulting in an increased p2x3/pap ratio ( 67.4% 2.1% vs. 44.7% 3.4% , p < 0.0001 ) and decreased pap / p2x3 ratio ( 87.1% 5.4% vs. 76.8% 2.3% , p = 0.001 ) ( figure 1(e ) ) . furthermore , pap expression was higher in peripherin(+ ) neurons than in neurofilament ( smi32)(+ ) neurons ( figure 2 ) . the diameter histograms confirmed that the pap(+ ) neurons were small to medium drg neurons ( 25th75th percentile : 17.123.7 m ) ( figure 2(f ) ) . figure 1.colocalization of prostatic acid phosphatase ( pap)(+ ) and p2x3(+ ) dorsal root ganglion neurons in resiniferatoxin ( rtx)-induced neuropathy . ( a d ) double immunofluorescence staining was performed with the following primary antiserum combinations : anti - pap ( a , c ) and anti - p2x3 ( b , d ) antisera in the vehicle ( a , b ) and rtx ( c , d ) groups . ( e ) the graph indicates the changes of colocalized ratios of p2x3(+)/pap(+ ) and pap(+)/p2x3(+ ) neurons in the vehicle ( opened bar ) and rtx ( filled bar ) group according to panels ( a ) to ( d).bar , 50 m . * * p < 0.01 , * * * p < 0.001 . figure 2.expression of prostatic acid phosphatase ( pap)(+ ) by the peripherin(+ ) and neurofilament ( sim32)(+ ) dorsal root ganglion ( drg ) neurons . double immunofluorescence staining of the drg sections was performed with the following two primary antisera combinations : ( a , b ) pap / smi32 and pap / peripherin ( c , d ) . ( a , b ) the micrographs show extremely low colocalization of pap ( a ) and smi32 ( b ) . ( c , d ) the micrographs show high colocalization of pap ( c ) and peripherin ( d ) . ( e ) the graph quantifies the colocalized ratios of pap(+)/smi32(+ ) ( open bar ) and pap(+)/peripherin(+ ) neurons ( filled bar ) according to panels ( a ) to ( d ) . ( f ) the graph shows the diameter histogram of pap(+ ) neurons , indicating that pap(+ ) neurons are small to medium neurons.bar , 50 m . * * p < 0.01 colocalization of prostatic acid phosphatase ( pap)(+ ) and p2x3(+ ) dorsal root ganglion neurons in resiniferatoxin ( rtx)-induced neuropathy . ( a d ) double immunofluorescence staining was performed with the following primary antiserum combinations : anti - pap ( a , c ) and anti - p2x3 ( b , d ) antisera in the vehicle ( a , b ) and rtx ( c , d ) groups . ( e ) the graph indicates the changes of colocalized ratios of p2x3(+)/pap(+ ) and pap(+)/p2x3(+ ) neurons in the vehicle ( opened bar ) and rtx ( filled bar ) group according to panels ( a ) to ( d ) . bar , 50 m . * * p < 0.01 , * * * p < 0.001 . expression of prostatic acid phosphatase ( pap)(+ ) by the peripherin(+ ) and neurofilament ( sim32)(+ ) dorsal root ganglion ( drg ) neurons . double immunofluorescence staining of the drg sections was performed with the following two primary antisera combinations : ( a , b ) pap / smi32 and pap / peripherin ( c , d ) . ( a , b ) the micrographs show extremely low colocalization of pap ( a ) and smi32 ( b ) . ( c , d ) the micrographs show high colocalization of pap ( c ) and peripherin ( d ) . ( e ) the graph quantifies the colocalized ratios of pap(+)/smi32(+ ) ( open bar ) and pap(+)/peripherin(+ ) neurons ( filled bar ) according to panels ( a ) to ( d ) . ( f ) the graph shows the diameter histogram of pap(+ ) neurons , indicating that pap(+ ) neurons are small to medium neurons . to understand the expression of pap and its relationship with atf3 , an injury marker , we performed double immunofluorescence staining of drg neuronal sections in rtx - induced neuropathy ( figure 3(a ) to ( e ) ) . pap(+ ) neurons were mildly lower in the rtx group than in the vehicle group ( 190.6 12.2 vs. 269.5 6.9 neurons / mm , p = 0.0003 ) ( figure 3(f ) ) . by contrast , atf3(+ ) neurons were markedly higher in the rtx group than in the vehicle group ( 192.3 18.0 vs. 9.0 13.1 neurons / mm , p < moreover , atf3 was induced preferentially in pap(+ ) neurons ( figure 3(a ) to ( e ) ) , resulting in an increased ratio of atf3(+)/pap(+ ) neurons in the rtx group ( 29.0% 5.6% vs. 0.2% 0.2% , p = 0.0043 ) ( figure 3(h ) ) . figure 3.phenotypic changes in prostatic acid phosphatase ( pap)(+ ) dorsal root ganglion neurons in resiniferatoxin ( rtx)-induced neuropathy . ( a e ) double immunofluorescence staining was performed with anti - activating transcription factor 3 ( atf3 ; a e in red ) and anti - pap ( a e in green ) antisera in the vehicle ( a ) , rtx ( b ) , 4-methylcatechol ( 4mc ) ( c ) , ngf ( rtx + 2.5s ngf ; d ) , and ( e ) abngf ( 4mc + anti - ngf antisera ) groups . ( f h ) the graphs quantify the density changes in ( f ) pap(+ ) and ( g ) atf3(+ ) neurons and ( h ) the ratio changes in atf3(+)/pap(+ ) neurons according to panels ( a ) to ( e ) . ( i ) the mechanical thresholds were assessed using the up - and - down method with von frey monofilaments , as described in the materials and methods . the graph indicates the changes in the mechanical thresholds in the vehicle ( open bar ) , rtx ( filled bar ) , 4mc ( grey bar ) , ngf ( slashed bar ) , and abngf ( dotted bar ) groups . ( j , k ) the graphs indicate that the mechanical thresholds correlated with pap expression , that is , a linear correlation with ( j ) pap densities and ( k ) an inverse correlation with the ratio of atf3(+)/pap(+ ) neurons . ( l , m ) the graphs show the diameter histogram of atf3(+)/pap(+ ) neurons in the rtx ( l ) and 4mc ( m ) groups . the diameter histogram shows no difference between the rtx and 4mc groups , but higher numbers of atf3(+)/pap(+ ) neurons in the rtx group.bar , 50 m . * p < 0.05 , * * p < 0.01 , * * * p < 0.001 . phenotypic changes in prostatic acid phosphatase ( pap)(+ ) dorsal root ganglion neurons in resiniferatoxin ( rtx)-induced neuropathy . ( a e ) double immunofluorescence staining was performed with anti - activating transcription factor 3 ( atf3 ; a e in red ) and anti - pap ( a e in green ) antisera in the vehicle ( a ) , rtx ( b ) , 4-methylcatechol ( 4mc ) ( c ) , ngf ( rtx + 2.5s ngf ; d ) , and ( e ) abngf ( 4mc + anti - ngf antisera ) groups . ( f h ) the graphs quantify the density changes in ( f ) pap(+ ) and ( g ) atf3(+ ) neurons and ( h ) the ratio changes in atf3(+)/pap(+ ) neurons according to panels ( a ) to ( e ) . ( i ) the mechanical thresholds were assessed using the up - and - down method with von frey monofilaments , as described in the materials and methods . the graph indicates the changes in the mechanical thresholds in the vehicle ( open bar ) , rtx ( filled bar ) , 4mc ( grey bar ) , ngf ( slashed bar ) , and abngf ( dotted bar ) groups . ( j , k ) the graphs indicate that the mechanical thresholds correlated with pap expression , that is , a linear correlation with ( j ) pap densities and ( k ) an inverse correlation with the ratio of atf3(+)/pap(+ ) neurons . ( l , m ) the graphs show the diameter histogram of atf3(+)/pap(+ ) neurons in the rtx ( l ) and 4mc ( m ) groups . the diameter histogram shows no difference between the rtx and 4mc groups , but higher numbers of atf3(+)/pap(+ ) neurons in the rtx group . bar , 50 m . * p < 0.05 , * * p < 0.01 , * * * p < 0.001 . mice in the rtx group showed mechanical allodynia compared with the vehicle group ( 747.1 156.0 vs. 299.6 70.6 mg , p = 0.0025 ) ( figure 3(i ) ) . this behavioral change correlated with the pap profiles ; that is , the densities of pap(+ ) neurons ( r = 0.75 , p < 0.0001 ; figure 3(j ) ) and the ratios of atf3(+)/pap(+ ) neurons ( r = 0.60 , p = 0.003 ; figure 3(k ) ) were inversely linear to the mechanical thresholds . collectively , these findings indicate that pap expression had a strong influence in mediating pain development . after neuropathy was induced using rtx , 4mc normalized pap expression and relieved mechanical allodynia ; specifically , the pap(+ ) neuronal density ( 269.7 48.8 neurons / mm , p = 0.61 ) ( figure 3(c ) and ( f ) ) and mechanical allodynia ( 693.8 146.0 mg , p = 0.54 ) ( figure 3(i ) ) in the 4mc group were comparable with those in the vehicle group . furthermore , although 4mc reduced the atf3(+ ) neuronal density ( 102.4 27.8 neurons / mm , p < 0.0001 ) and ratio of atf3(+)/pap(+ ) neurons ( 15.8% 5.9% , p = 0.0095 ) , the ratios of atf3(+)/pap(+ ) neurons were still higher in the 4mc group than in the vehicle group ( p = 0.016 ; figure 3(c ) , ( g ) , ( h ) ) . morphometric analysis demonstrated that 4mc reduced the absolute number of atf3(+)/pap(+ ) neurons but did not have a significant effect on their mean diameter ( 15.5 2.2 vs. 16.5 2.8 m , p > 0.05 ) ( figure 3(l ) vs. ( m ) ) . to understand the putative mechanisms of pain relief by 4mc , we examined the coexpression of pap(+)/trka(+ ) neurons ( figure 4 ) . for each group , pap(+ ) neuronal density was similar to trka(+ ) neuronal density ( figure 3(f ) vs. 4(k ) ) . the pap / trka ratios correlated linearly with the mechanical threshold ( r = 0.62 , p = 0.0062 ; figure 4(m ) ) . to confirm the effect of 4mc , the mice in the ngf group were systemically ( i.p . ) administered 2.5s ngf . the ngf group exhibited the same effect as the 4mc group ; that is , pap and trka expression profiles were reversed , atf3 induction was normalized , and mechanical allodynia relieved . those findings , however , were not observed in the abngf group ( administered anti - ngf antisera to neutralize ngf ) ( figures 3 and 4 ) . figure 4.colocalization of prostatic acid phosphatase ( pap ) and high - affinity nerve growth factor ( trka ) receptor after resiniferatoxin ( rtx)-induced neuropathy . ( a j ) double immunofluorescence staining of dorsal root ganglia sections was performed with anti - pap ( a e ) and trka ( f - j ) in the vehicle ( a , f ) , rtx ( b , g ) , 4-methylcatechol ( 4mc ; c , h ) , ( d , i ) ngf ( rtx + 2.5s ngf ) , and ( e , j ) abngf ( 4mc + anti - ngf antisera ) groups . ( k , l ) the graphs show the changes in ( k ) trka density and ( l ) the colocalized ratios of pap(+)/trka(+ ) neurons in the vehicle ( open bar ) , rtx ( filled bar ) , 4mc ( grey bar ) , ngf ( slashed bar ) , and abngf groups ( dotted bar ) according to panels ( a ) to ( j ) . ( m ) the graph shows that the mechanical thresholds correlate linearly with the ratios of pap(+)/trka(+ ) neurons.bar , 50 m . * p < 0.05 , * * p < 0.01 . colocalization of prostatic acid phosphatase ( pap ) and high - affinity nerve growth factor ( trka ) receptor after resiniferatoxin ( rtx)-induced neuropathy . ( a j ) double immunofluorescence staining of dorsal root ganglia sections was performed with anti - pap ( a e ) and trka ( f - j ) in the vehicle ( a , f ) , rtx ( b , g ) , 4-methylcatechol ( 4mc ; c , h ) , ( d , i ) ngf ( rtx + 2.5s ngf ) , and ( e , j ) abngf ( 4mc + anti - ngf antisera ) groups . ( k , l ) the graphs show the changes in ( k ) trka density and ( l ) the colocalized ratios of pap(+)/trka(+ ) neurons in the vehicle ( open bar ) , rtx ( filled bar ) , 4mc ( grey bar ) , ngf ( slashed bar ) , and abngf groups ( dotted bar ) according to panels ( a ) to ( j ) . ( m ) the graph shows that the mechanical thresholds correlate linearly with the ratios of pap(+)/trka(+ ) neurons . this is the first report documenting the intervention of pap and ngf - trka signaling , which mediates neuropathic pain . we speculate one possible mechanism : neuropathic pain in rtx - induced small - fiber neuropathy develops because of pap neuropathology and the decrease in ngf - trka signaling . rtx is specifically neurotoxic to trpv1(+ ) neurons ; it also induces small - fiber neuropathy , such as skin denervation , which results in thermal hypoalgesia , small - diameter neuron injury , and peripheral sensitization of p2x3 purinoceptors , leading to mechanical allodynia . atf3 is both an injury marker and a marker of pain ; for example , the ratios of p2x3(+)/atf3(+ ) neurons correlate linearly with the degree of neuropathic pain . pap is susceptible to rtx because of the high ratios of pap(+)/p2x3(+ ) and pap(+)/peripherin(+ ) neurons . thus , injured pap neurons are critical for modulating neuropathic pain ; that is , the ratios of atf3(+)/pap(+ ) neurons are inversely linear to the mechanical threshold . the balance of extracellular purine nucleotides such as adenine nucleotides is maintained by functional membrane - bound ectonucleotidase activity , which responded for the cellular pathophysiological homeostasis . the ectonucleotidase activity of pap may facilitate the hydrolysis of amp to adenosine , which acts as an analgesic ligand . pap downregulation leads to the imbalance of amp / adenosine ratios , inhibiting the analgesic effect of adenosine . taken together , these findings suggest the neuronal soma underlies the irritation and/or injury coincident with the metabolic imbalance of extracellular nucleotides , resulting in the loss of the analgesic effect of pap . additional studies should investigate the effects of altered purine metabolism to clarify the cellular responses to neuronal soma irritation . a previous study demonstrated that pap - mediated neuropathic pain was trpv1-dependent . in our rtx - induced neuropathy model , trpv1 was completely depleted , and the present study provides evidence of an alternative nociceptive pathway to the trpv1-dependent nociceptive pathway ; the loss of the analgesic effect of pap is mediated by neuronal soma injury and/or irritation . furthermore , pain development correlated with trka profiles ; for example , the ratios of pap(+)/trka(+ ) neurons were linearly correlated with the degree of mechanical allodynia . this report demonstrates the amelioration of neuropathic pain by 4mc through the intervention of pap and ngf - trka signaling . in addition to previous studies demonstrating that 4mc promotes skin reinnervation , the present study demonstrated that 4mc could facilitate the recovery irritated and/or injured small - diameter neuronal soma , as indicated by the reduced ratios of atf3(+)/pap(+ ) neurons . conversely , ngf - sensitized sensory nerves resulted in pain perception and targeting of ngf - trka signaling is a new direction for pain therapy . coincidentally , several studies have suggested that the interaction of ngf and trpv1 is critical for pain transmission . the present study shows contrary outcomes ; for example , 4mc - promoted ngf played the role of a modulator in the recovery of pap and pap(+)/trka(+ ) neuronal profiles coinciding with the normalization of mechanical allodynia . 4mc appears to have a pleiotropic effect in modulating neuronal activity ; for example , it also increases the synthesis of the brain - derived neurotrophic factor ( bdnf ) and enhances the phosphorylation of the trk family and activation of the mapk / erk cascade . regarding the targeted signaling pathway of 4mc in rtx - induced neuropathy , bdnf modulates the function of the motor and myelinated sensory neurons , but no effect was observed in this current neuropathic model because ( 1 ) rtx specifically affects small - diameter sensory neurons with no pathologic alternations in the motor and myelinated sensory neurons , and ( 2 ) 4mc has no effect on trkb and/or trkc signaling because there are no changes in bdnf and neurotrophic 3 ( nt3 ) expression in the peripheral tissues , as determined in our previous study . regarding the possible mechanism of the effect of 4mc on pain - relief in rtx - induced neuropathy , this report provides immunohistochemical evidence of the potential effect of 4mc , as demonstrated by the pap / trka ratios of approximately 70% . collectively , these data reinforce our speculations : 4mc regulates pap pathology through ngf - trka signaling , which was confirmed through alternative ngf administration and ngf neutralization experiments . combining with the 4mc reduced the atf3(+)/pap(+ ) neurons ; this study suggests that 4mc has a pleiotropic effect on both the recovery of irritated and/or injured neuronal soma and restores ngf - trka signaling . in summary , atf3 might be an upstream molecule initiating the loss of the analgesic effect of pap , and pap is a downstream molecule of ngf - trka signaling , restoring the analgesic effect of pap in a trka - dependent manner ( figure 5 ) . figure 5.diagram of prostatic acid phosphatase ( pap ) pathology modulates the pain perception in resiniferatoxin ( rtx ) neuropathy . this diagram illustrates the pap pathology mediating pain development follows a ngf - trka - dependent manner , as described in the following steps : ( 1 ) rtx sensitizes the transient receptor potential vanilloid subtype 1 ( trpv1 ) . ( 2 ) rtx then induces activating transcription factor 3 ( atf3 ) upregulation on pap(+ ) neurons , indicating the pap pathology . ( 3 ) the pap pathology reduces the amp hydrolysis . ( 4 ) this reduction evokes pain perception because of the decrease in the adenosine analgesic effect . ( 5 ) nerve growth factor ( ngf ) binds to high - affinity ngf receptor ( trka ) and ( 6 ) reverses the induction of the atf3 and pap pathology . ( 7 ) this recovers the pap - mediated analgesic effect by returning the ability of amp hydrolysis . diagram of prostatic acid phosphatase ( pap ) pathology modulates the pain perception in resiniferatoxin ( rtx ) neuropathy . this diagram illustrates the pap pathology mediating pain development follows a ngf - trka - dependent manner , as described in the following steps : ( 1 ) rtx sensitizes the transient receptor potential vanilloid subtype 1 ( trpv1 ) . ( 2 ) rtx then induces activating transcription factor 3 ( atf3 ) upregulation on pap(+ ) neurons , indicating the pap pathology . ( 3 ) the pap pathology reduces the amp hydrolysis . ( 4 ) this reduction evokes pain perception because of the decrease in the adenosine analgesic effect . ( 5 ) nerve growth factor ( ngf ) binds to high - affinity ngf receptor ( trka ) and ( 6 ) reverses the induction of the atf3 and pap pathology . ( 7 ) this recovers the pap - mediated analgesic effect by returning the ability of amp hydrolysis . rtx is specifically neurotoxic to trpv1(+ ) neurons ; it also induces small - fiber neuropathy , such as skin denervation , which results in thermal hypoalgesia , small - diameter neuron injury , and peripheral sensitization of p2x3 purinoceptors , leading to mechanical allodynia . atf3 is both an injury marker and a marker of pain ; for example , the ratios of p2x3(+)/atf3(+ ) neurons correlate linearly with the degree of neuropathic pain . pap is susceptible to rtx because of the high ratios of pap(+)/p2x3(+ ) and pap(+)/peripherin(+ ) neurons . thus , injured pap neurons are critical for modulating neuropathic pain ; that is , the ratios of atf3(+)/pap(+ ) neurons are inversely linear to the mechanical threshold . the balance of extracellular purine nucleotides such as adenine nucleotides is maintained by functional membrane - bound ectonucleotidase activity , which responded for the cellular pathophysiological homeostasis . the ectonucleotidase activity of pap may facilitate the hydrolysis of amp to adenosine , which acts as an analgesic ligand . pap downregulation leads to the imbalance of amp / adenosine ratios , inhibiting the analgesic effect of adenosine . taken together , these findings suggest the neuronal soma underlies the irritation and/or injury coincident with the metabolic imbalance of extracellular nucleotides , resulting in the loss of the analgesic effect of pap . additional studies should investigate the effects of altered purine metabolism to clarify the cellular responses to neuronal soma irritation . a previous study demonstrated that pap - mediated neuropathic pain was trpv1-dependent . in our rtx - induced neuropathy model , trpv1 was completely depleted , and the present study provides evidence of an alternative nociceptive pathway to the trpv1-dependent nociceptive pathway ; the loss of the analgesic effect of pap is mediated by neuronal soma injury and/or irritation . furthermore , pain development correlated with trka profiles ; for example , the ratios of pap(+)/trka(+ ) neurons were linearly correlated with the degree of mechanical allodynia . this report demonstrates the amelioration of neuropathic pain by 4mc through the intervention of pap and ngf - trka signaling . in addition to previous studies demonstrating that 4mc promotes skin reinnervation , the present study demonstrated that 4mc could facilitate the recovery irritated and/or injured small - diameter neuronal soma , as indicated by the reduced ratios of atf3(+)/pap(+ ) neurons . conversely , ngf - sensitized sensory nerves resulted in pain perception and targeting of ngf - trka signaling is a new direction for pain therapy . coincidentally , several studies have suggested that the interaction of ngf and trpv1 is critical for pain transmission . the present study shows contrary outcomes ; for example , 4mc - promoted ngf played the role of a modulator in the recovery of pap and pap(+)/trka(+ ) neuronal profiles coinciding with the normalization of mechanical allodynia . 4mc appears to have a pleiotropic effect in modulating neuronal activity ; for example , it also increases the synthesis of the brain - derived neurotrophic factor ( bdnf ) and enhances the phosphorylation of the trk family and activation of the mapk / erk cascade . regarding the targeted signaling pathway of 4mc in rtx - induced neuropathy , bdnf modulates the function of the motor and myelinated sensory neurons , but no effect was observed in this current neuropathic model because ( 1 ) rtx specifically affects small - diameter sensory neurons with no pathologic alternations in the motor and myelinated sensory neurons , and ( 2 ) 4mc has no effect on trkb and/or trkc signaling because there are no changes in bdnf and neurotrophic 3 ( nt3 ) expression in the peripheral tissues , as determined in our previous study . regarding the possible mechanism of the effect of 4mc on pain - relief in rtx - induced neuropathy , this report provides immunohistochemical evidence of the potential effect of 4mc , as demonstrated by the pap / trka ratios of approximately 70% . collectively , these data reinforce our speculations : 4mc regulates pap pathology through ngf - trka signaling , which was confirmed through alternative ngf administration and ngf neutralization experiments . combining with the 4mc reduced the atf3(+)/pap(+ ) neurons ; this study suggests that 4mc has a pleiotropic effect on both the recovery of irritated and/or injured neuronal soma and restores ngf - trka signaling . in summary , atf3 might be an upstream molecule initiating the loss of the analgesic effect of pap , and pap is a downstream molecule of ngf - trka signaling , restoring the analgesic effect of pap in a trka - dependent manner ( figure 5 ) . figure 5.diagram of prostatic acid phosphatase ( pap ) pathology modulates the pain perception in resiniferatoxin ( rtx ) neuropathy . this diagram illustrates the pap pathology mediating pain development follows a ngf - trka - dependent manner , as described in the following steps : ( 1 ) rtx sensitizes the transient receptor potential vanilloid subtype 1 ( trpv1 ) . ( 2 ) rtx then induces activating transcription factor 3 ( atf3 ) upregulation on pap(+ ) neurons , indicating the pap pathology . ( 3 ) the pap pathology reduces the amp hydrolysis . ( 4 ) this reduction evokes pain perception because of the decrease in the adenosine analgesic effect . ( 5 ) nerve growth factor ( ngf ) binds to high - affinity ngf receptor ( trka ) and ( 6 ) reverses the induction of the atf3 and pap pathology . ( 7 ) this recovers the pap - mediated analgesic effect by returning the ability of amp hydrolysis . diagram of prostatic acid phosphatase ( pap ) pathology modulates the pain perception in resiniferatoxin ( rtx ) neuropathy . this diagram illustrates the pap pathology mediating pain development follows a ngf - trka - dependent manner , as described in the following steps : ( 1 ) rtx sensitizes the transient receptor potential vanilloid subtype 1 ( trpv1 ) . ( 2 ) rtx then induces activating transcription factor 3 ( atf3 ) upregulation on pap(+ ) neurons , indicating the pap pathology . ( 3 ) the pap pathology reduces the amp hydrolysis . ( 4 ) this reduction evokes pain perception because of the decrease in the adenosine analgesic effect . ( 5 ) nerve growth factor ( ngf ) binds to high - affinity ngf receptor ( trka ) and ( 6 ) reverses the induction of the atf3 and pap pathology . ( 7 ) this recovers the pap - mediated analgesic effect by returning the ability of amp hydrolysis . for pain development , this study provides evidence of an alternative pathway to conventional small nociceptor sensitization - induced neuropathic pain ; the loss of analgesic effect of pap is mediated by neuronal soma irritation and/or injury . alternatively , restoring the analgesic ligand may be a new direction for targeting painful neuropathy , that is , maintenance of steady - static adenosine content and functional pap ectonucleotidase activity at the neuronal soma level . the author(s ) declared no potential conflicts of interest with respect to the research , authorship , and/or publication of this article . the author(s ) disclosed receipt of the following financial support for the research , authorship , and/or publication of this article : this work was supported by grants from the national science council ( nsc100 - 2320-b-037 - 018 and nsc102 - 2320-b-037 - 009 ) ; ministry of science and technology ( most103 - 2320-b-037 - 015-my3 ) , taiwan ; and kaohsiung medical university hospital ( kmuh104 - 4m19 ) .

backgroundneuropathic pain in small - fiber neuropathy results from injury to and sensitization of nociceptors . functional prostatic acid phosphatase ( pap ) acts as an analgesic effector . however , the mechanism responsible for the modulation of pap neuropathology , which leads to loss of the analgesic effect after small - fiber neuropathy , remains unclear.resultswe used a resiniferatoxin ( rtx)-induced small - fiber neuropathy model to examine whether functional pap(+ ) neurons are essential to maintain the analgesic effect . pap(+ ) neurons were categorized into small to medium neurons ( 25th75th percentile : 17.123.7 m ) ; these neurons were slightly reduced by rtx ( p = 0.0003 ) . by contrast , rtx - induced activating transcription factor 3 ( atf3 ) , an injury marker , in pap(+ ) neurons ( 29.0% 5.6% vs. 0.2% 0.2% , p = 0.0043 ) , indicating pap neuropathology . moreover , the high - affinity nerve growth factor ( ngf ) receptor ( trka ) colocalized with pap and showed similar profiles after rtx - induced neuropathy , and the pap / trka ratios correlated with the degree of mechanical allodynia ( r = 0.62 , p = 0.0062 ) . the ngf inducer 4-methylcatechol ( 4mc ) normalized the analgesic effects of pap ; specifically , it reversed the pap and trka profiles and relieved mechanical allodynia . administering 2.5s ngf showed similar results to those of administering 4mc . this finding suggests that the analgesic effect of functional pap is mediated by ngf - trka signaling , which was confirmed by ngf neutralization.conclusionsthis study revealed that functional pap(+ ) neurons are essential for the analgesic effect , which is mediated by ngf - trka signaling .

however , the iron overload in various organs leads to serious problems such as metabolic , endocrine , and growth abnormalities in such patients despite the expanded use of iron - chelator drugs ( 3 - 5 ) . patients with thalassemia intermedia ( ti ) although the rate of iron loading is slower in ti than in tm , patients with ti can eventually develop complications similar to those of the patients with tm , including hepatic , endocrine and cardiac dysfunction ( 6 ) . moreover , in patients with transfusion independent thalassemia , serum ferritin was a poor predictor of liver iron concentration ; therefore , chelation therapy may be postponed in patients with ti ( 7 ) . hepcidin is a small peptid hormone secreted by hepatocytes to regulate plasma iron concentration and distribution in different tissues ( 8 , 9 ) . chronic excess of hepcidin causes iron restricted anemia ( 9 ) , while the hepcidin deficiency leads to iron overload with iron deposition in the liver and parenchyma ( 10 ) . increased plasma and stored iron stimulate hepcidin production , which in turn blocks dietary iron absorption and consequently iron loading .conversely , hepcidin is suppressed in iron deficiency ( 11 ) . hepcidin concentration is significantly higher in chronically transfused patients than in non - transfused ones , presumably due to iron overload ( 12 - 16 ) . despite using iron chelatorin patients with thalassemia , iron overload is one of the main mortality factors in such patients and hepcidin targeted therapeutics can help with better management of iron overload in patients with thalassemia ( 17 ) . the current study aimed to determine the correlation between serum ferritin and hepcidin levels in patients with tm and ti , also compare serum hepcidin and ferritin levels between the two groups of patients . the current cross - sectional study was conducted in hematology research center of shiraz university of medical sciences , shiraz , iran , in 2013 . the study subjects included 90 patients , 50 tm and 40 ti , selected by systematic random sampling from the 500 tm and 280 ti patients registered at the thalassemia clinic of tertiary state hospital affiliated to shiraz university of medical sciences . this is a referral center with 45 beds for patients with thalassemia in southern iran . this study was approved by medical ethics committee of shiraz university of medical sciences ( grant number : 3652 , approval date : 21.03.2011 ) . the diagnosis of thalassemia was based on complete blood count ( cbc ) , hb electrophoresis and clinical history . in the tm group , blood transfusion had been started before two years old and all of them were on regular blood transfusion every - two to four weeks . in the tm group , inclusion criteria were age above 10 years old , under regular follow up and regular blood transfusion , and iron chelation therapy . in the ti group , inclusion criteria were transfusion independence and taking hydroxyurea ( hu ) 10 - 15 mg / kg / day for a duration of at least ten years . patients with the history of any recent infection or surgery , congestive heart and liver failures as well as positive viral hepatitis were excluded from the study . a 2.5 ml sample of venous blood withdrawn from each participant was added to the tubes containing clot activator . plasma centrifuge was done immediately and 2 ml serum was separated and preserved at -20c , then carried by cold box to the laboratory to check serum hepcidin and ferritin levels . also , 2 ml sample of venous blood was withdrawn from each patient and added to the tubes of cbc containing ethylenediamine tetraacetic acid edta anti - coagulant . complete blood count test was performed immediately with peripheral blood smears and then stained with wright stain and the rate of nucleated red blood cells ( nrbc ) per 100 white blood cells ( wbc ) was counted . fasting serum ferritin was checked by enzyme linked fluorescent assay elfa method , using mini vidas ( biomerievx s.a , france ) machine , and hepcidin was measured by enzyme linked immunosorbent assay elisa method ( competitive assay ) , using elisa reader dynex ( usa ) machine with drg hepcidin kit ( eia 4 705 ) . according to the direction of hepcidin kit manufacturer , the normal range of hepcidin in 5% - 95% of people with the age range of 23 to 67 years is from 13.3 to 54.4 ng / ml . the normal range of ferritin was considered as 20 - 300 g / lin males 10 - 100 g / l in females before menopause , 20 - 200 g / l in females after menopause and 150 - 500 test of normality was done by shapiro - wilk test . among all the evaluated variables , only age had normal distribution . descriptive data were summarized as mean and standard deviation , as well as median and interquartile range ( iqr ) , where appropriate . correlation between serum hepcidin and quantitative variables in each of the two groups of patients with tm and ti was evaluated by spearman correlation test . quantitative data between the two groups of patients were compared by student t - test and mann - whitney test . considering the correlation coefficient of 0.68 for urinary hepcidin and serum ferritin levels reported by origa et al . ( 14 ) in 44 patients with thalassemia including 22 tm and 22 ti , = 0.05 and = 0.01 , the sample size was estimated as 29 patients in each group . for higher accuracy , sample size test of normality was done by shapiro - wilk test . among all the evaluated variables , only age had normal distribution . descriptive data were summarized as mean and standard deviation , as well as median and interquartile range ( iqr ) , where appropriate . correlation between serum hepcidin and quantitative variables in each of the two groups of patients with tm and ti was evaluated by spearman correlation test . quantitative data between the two groups of patients were compared by student t - test and mann - whitney test . considering the correlation coefficient of 0.68 for urinary hepcidin and serum ferritin levels reported by origa et al . ( 14 ) in 44 patients with thalassemia including 22 tm and 22 ti , = 0.05 and = 0.01 , the sample size was estimated as 29 patients in each group . for higher accuracy , the mean age of the patients with tm and ti were 21.4 6.1(from 11 to 33 years ) and 23.7 6.1 years ( from 8 to 38 years old ) respectively , and there was no statistically significant difference between the two groups ( p = 0.076 ) . considering gender distributions , 21 ( 43.8% ) and 22 ( 55% ) of the subjects in the tm and ti groups were female , respectively ; there was no statistically significant difference ( p = 0.392 ) . the median iqr for serum hepcidin levels were significantly higher in the tm compared to ti group ( 87.6 , 43.9 vs. 51.8 , 23.4 , p < 0.001 ) . in addition , the median iqr for serum ferritin levels were 2208 ( 3761 ) , and 465 ( 632 ) in the tm and ti groups respectively ; the difference was statistically significant ( p < 0.001 ) ( table 1 ) . table 2 indicates the results of correlation between serum hepcidin levels and the evaluated parameters for the subjects with tm and ti . no statistically significant correlations were found between serum hepcidin with any of the evaluated variables in the two groups . also there were no statistically significant differences in serum hepcidin levels between males and females in patients with tm and ti ( p = 0.107 and p = 0.861 respectively ) . considering the cut - off point of 54.4 ng / ml for serum hepcidin levels , 44 ( 91.7% ) , and 18 ( 45% ) subjects had hepcidin levels higher than normal range in the tm and ti groups , respectively ( p < 0.001 ) . abbreviations : iqr , interquartile range ; sd , standard deviation ; nrbc , nucleated red blood cells ; wbc , white blood cell . data are presented as mean sd , median ( iqr ) or male / female . data are presented for n = 40 . statistically significant . in thalassemia major , it was pre - transfusion hemoglobin abbreviations : nrbc , nucleated red blood cells ; rs , spearman s rho correlation coefficient ; ti , thalassemia intermedia ; tm , thalassemia major ; wbc , white blood cell . in thalassemia major , it was pre - transfusion hemoglobin . determining hepcidin concentrations in subjects with iron - loading anemias may be useful to identify the patients at higher risk of iron toxicity due to severely decreased hepcidin levels ( 17 ) . based on the current study results , 91.7% of the subjects with tm had serum hepcidin levels above normal compared to the patients with ti , 45% . also , the median of serum hepcidin levels were significantly higher in the tm compared to ti group that was similar to those of the previous studies ( 14 ) . in iron loading anemias not treated by frequent transfusions , such as beta thalassemia intermedia and congenital dyserythropoietic anemias , urinary and serum hepcidin decrease due to high erythroid signals . hepcidin deficiency in turn allows excessive iron absorption and development of systemic iron overload ( 12 - 14 , 18 ) . also , it could be due to more iron overload resulted from regular blood transfusion in the patients with tm compared to ti . as observed in the current study , ferritin levels were significantly higher in the subjects with tm . hemoglobin levels were significantly lower in the subjects with tm than in ti , which may be due to the time of blood sampling in the subjects with tm , immediately before transfusion . during transfusion intervals , it is known that in patients with ti , there is also increased risk of iron over load due to increased intestinal iron absorption triggered by chronic anemia , ineffective erythropoiesis and decreased serum hepcidin ( 19 , 20 ) . the current study showed higher erythropoietic activity in the subjects with ti than tm , since the median value for nrbc was significantly higher in ti compared to tm group . in ti group , since all subjects were on hu therapy for a long time , erythropoietic activity was expected to be suppressed and consequently hepcidin level was expected to be higher . but it did not occur and it seems that hu at a dose of 10 - 15 mg / kg / day was not enough to suppress bone marrow activity and make erythropoiesis ineffective . however , previous reports documented that hu with the same dose was effective in decrease or cessation of the need for regular blood transfusion , as well as increasing hb levels in the patients with thalassemia ( 21 - 24 ) . the current study showed no significant correlation between serum hepcidin and ferritin levels as a marker of iron overload in any of the tm or ti groups . this result was in accordance with the previous reports that demonstrated the dominant role of erythropoiesis compared to iron overload in regulation of hepcidin in patients with thalassemia ( 25 - 27 ) . ( 15 ) , the current study found no statistically significant correlation between hepcidin and pre - transfusion hemoglobin levels in tm patients . in a longitudinal study on 31 patients with tm . the strong point of the current study was large sample size compared to those of the previous reports ; however , there were some limitations in the study , since molecular erythropoietic activity was nit evaluated . in conclusion , based on the current study results , there was insignificant correlation between serum hepcidin and ferritin levels as a marker of iron overload in any of the two groups of patients with tm and ti , which supported the results of previous studies that regulation of hepcidin in patients with thalassemia is more affected by erythropoeitic activity than by iron overload . also serum hepcidin levels were significantly higher in the tm than in ti group , which could mainly result from higher erythropoeitic activity in ti . in the subjects with ti , it is believed that low dose hu ( 10 - 15 mg / kg / day ) therapy for a long time could increase hb levels and lead to transfusion - independence , but probably not high enough to suppress bone marrow activity and make erythropoiesisin effective . increased awareness of hepcidin levels in the patients with thalassemia could be helpful to diagnose and manage iron burden in such patients .

background : hepcidin is a key regulator of iron absorption in humans . it is mainly affected by hypoxia and iron stores.objectives:the current study aimed to determine the correlation between serum hepcidin and ferritin levels in patients with thalassemia major ( tm ) and thalassemia intermedia ( ti).patients and methods : the current cross - sectional study investigated 88 randomly selected patients with thalassemia , 48 tm and 40 ti , registered at the thalassemia clinic of shiraz university of medical sciences , a referral center for thalassemia in southern iran in 2013 . all patients with ti were receiving hydroxyurea ( hu ) 10 - 15 mg / kg / day for at least 10 years . the serum hepcidin , ferritin levels , hemoglobin ( hb ) and nucleated red blood cell ( rbc ) of the two groups were measured.results:no statistically significant correlation was observed between serum hepcidin and ferritin levels in any of the two groups of patients with tm ( rs = 0.02 , p = 0.892 ) or ti ( rs = 0.055 , p = 0.734 ) . the median interquartile range ( iqr ) for serum hepcidin and ferritin levels were significantly higher in tm compared to ti group , ( hepcidin : 87.6 ( 43.9 ) vs. 51.8 ( 23.4 ) , p < 0.001 ; ferritin : 2208 ( 3761 ) vs. 465 ( 632 ) , p < 0.001).conclusions : there was insignificant correlation between serum hepcidin and ferritin levels in the two groups of patients with tm and ti . it seems that regulation of hepcidin in patients with thalassemia is more affected by erythropoeitic activity than iron stores . also , hepcidin levels were significantly higher in patients with tm than ti , possibly due to higher erythropoeitic activity in ti . in ti , it seems that low dose hu increases hb levels and leads to transfusion - independence , but it is not high enough to suppress bone marrow activity and ineffective erythropoiesis . consequently , serum hepcidin level decreases .

though the pathogens causing aris vary geographically and by season , globally viruses play a major role [ 13 ] . in a recent systematic review , the most common respiratory viruses causing acute lower respiratory tract infection ( lri ) in children under five years of age were respiratory syncytial virus ( rsv ) , influenza virus ( ifv ) , parainfluenza virus ( piv ) human metapneumovirus ( hmpv ) , and rhinovirus ( rv ) . besides this , 10%50% of children affected with ari develop secondary bacterial infections , namely , acute otitis media , sinusitis , or pneumonia . moreover , viruses are the most common pathogen associated with severe respiratory diseases ( e.g. , bronchiolitis ) , exacerbation of asthma , or pneumonia in early life and are leading cause of hospitalization in children under two [ 57 ] . although viral etiology of aris and their impact on health care are much studied in developed countries , there is a gap in knowledge regarding the same in developing countries including india . from the public health point of view , it is important to know the most common viral agents causing aris , their manifestations , how often they cause severe disease , and how severe aris can be prevented . in this study , we aimed to characterize the viral spectrum and pattern of upper and lower aris in children under five from eastern part of india . the present study was conducted in the pediatrics department of svppgip , scb medical college , a tertiary care teaching hospital in eastern india over 2-year period ( october 2011 to september 2013 ) . children aged 2 to 60 months with symptoms and signs of acute respiratory tract infections suggested by who were included . who classification of acute respiratory tract infection in children presenting with cough , difficult breathing , or both is as follows : pneumonia respiratory rate per minute > 50 breaths ( 211 months of age ) or > 40 breaths ( 1259 months of age ) ; no lower chest indrawing ; severe pneumonia symptoms of pneumonia , and lower chest indrawing with or without rapid breathing ; very severe disease symptoms of severe pneumonia , inability to drink , convulsions , central cyanosis , being abnormally sleepy or difficulty to wake , stridor in calm child , or clinically severe malnutrition . those with suspected bacterial etiology ( e.g. , streptococcal sore throat , lobar pneumonia , pneumatocele , and empyema ) , underlying chronic conditions , hiv , illness lasting more than a week , severe malnutrition , and those hospitalized in last one month were excluded . nasal and throat swabs were collected and pooled into tubes containing 2 ml of virus transport medium ( vtm , copan , brescia , italy ) and kept in refrigerator at 2 to 8c . then the samples were transported over ice to the laboratory ( regional medical research centre , icmr ) situated at bhubaneswar , a grade 1 equipped virological laboratory . total nucleic acids ( including dna and rna ) were extracted from 200 l of each specimen using a qiaamp minelute virus spin kit ( qiagen , mississauga , on , canada ) according to the manufacturer 's instructions . for all collected specimens , pcr or rt - pcrs were performed to detect infection with rsv , ifv , piv , hmpv , adenovirus ( adv ) , human bocavirus ( hbov ) , human corona virus ( hcov ) , enterovirus ( ev ) , and rv . the method used for each virus is described in the following references : pivs 14 , ev , rv , ifv ( a , b , and c ) , and rsv ( a and b ) were detected by two multiplex nested rt - pcrs ; hcov and hmpv by two - step rt - pcr ; adv by single - step pcr ; and hbov by touchdown pcr . rt - pcr was performed using a superscript ii one - step rt - pcr platinum taq kit ( invitrogen , carlsbad , ca , usa ) . typing for ifv , piv , and rsv was performed according to pcr product size . a total of 358 children of 260 mo age and acute respiratory infections were screened and finally 300 patients were included after meeting the eligibility criteria . following were the reasons for exclusion in 58 children : chest x - ray suggestive of bacterial pneumonia ( n = 19 ) , streptococcal sore throat ( n = 15 ) , underlying chronic conditions ( n = 10 ) , severe malnutrition ( n = 7 ) , illness lasting more than a week ( n = 3 ) , complicated pneumonia ( n = 2 ) , and being hospitalized in last one month ( n = 2 ) . the m : f ratio was 1.7 : 1 ( uri = 1.6 : 1 ; lri = 1.9 : 1 ) . out of 300 , 174 ( 58% ) were lri and 126 ( 42% ) were uri cases . month - wise recruitment of uri and lri cases has been shown in figure 1 . most lri cases occurred in the month of january , august , november , march , and july . most children with lri were in the age range of 212 mo ( 54% ) and with uri were in the age range of > 1260 mo ( 45.2% ) . chest x - ray findings were hyperinflation ( 93% ) , peribronchial cuffing ( 60% ) , interstitial infiltrates ( 48% ) , and patchy opacity ( 46% ) . of the 300 nasal and throat swabs , 248 cases were positive for viruses [ lri = 142/174 ( 81.6% ) , and uri = 106/126 ( 84.1% ) ] . month - wise recruitment of ari cases and samples positive for viruses has been shown in figure 2 . the maximum number of cases recruited and samples positive for viruses were in the months of january , december , and august . the virus positivity in ari was as follows : uri , 65 ( 61.3% ) samples were positive for single virus and 41 ( 38.7% ) were positive for more than one virus , and lri , 86 ( 61% ) samples were positive for single virus and 56 ( 39% ) were positive for more than one virus . the coinfection pattern differed between acute uri and lri , and this has been detailed in table 2 . the most common viruses isolated from uri cases were rv ( 31.1% ) , adv ( 18.9% ) , rsv ( 17% ) , and ifv ( 17% ) . the most common viruses isolated from lri cases were rsv ( 30.3% ) , ifv ( 17.6% ) , rv ( 14.8% ) , and adv ( 13.4% ) . rsv , adv , rv , and ifv were the most prevalent viruses isolated from all the ari cases . ifv - a was predominant over ifv - b , and piv-3 was predominant over other subtypes of piv . rsv - a subtype dominated over rsv - b in uri cases , whereas rsv - b dominated over rsv - a in lri cases . the detection rates of viruses corresponding to different age groups are shown in table 1 . in case of uris , rv was the most prevalent virus ( 26 mo age = 2.8% ; > 612 mo age = 13.3% ; > 1260 mo age = 14.2% ) . rsv was the second most common virus detected in age groups of 26 mo and > 612 mo . adv was the second most common virus detected in age group of > 1260 mo . the least common viruses in all age groups were piv , hbov , and hmpv . hcov and ev were not detected in any age group . in case of lris , rsv was the most prevalent virus ( 26 mo age = 13.4% ; > 612 mo age = 11.3% ; > 1260 mo age = 5.6% ) . hmpv , hbov , and hcov were not detected up to 6 mo age . the least common viruses in all age groups were hbov , hcov , ev , and hmpv . rv was the most common virus detected in uris , but it was fourth most prevalent in lris . it was detected throughout the year , the maximum rate being in december , january , march , and august . rsv was the most common virus detected in lris , but it was third most prevalent in uris . it was detected throughout the year , the maximum rate being in december , january , and february . ifv was second most common virus detected in lris , but it was third most prevalent in uris ; maximum positivity rate was in august followed by in july , and minimum positivity rate was in november . piv had a higher prevalence in april to july , and a low prevalence from september to march . there detection rate was lower in lris . like hmpv , hbov was present throughout the year in lris . the clinical features , demographics , and risk factors of children among viral positive and virus negative group with lris were compared ( table 3 ) . it was observed that significantly higher number of children below 12 mo were virus positive ( p = 0.01 ) . children presenting with preceding bronchiolitis were significantly associated with total viral infections ( p = 0.003 ) . rhinorrhea was significantly present in the virus positive group ( p = 0.02 ) . among risk factors , ari in family was significantly associated with virus positivity ( p = 0.04 ) . in lris , out of 174 cases ( lri = 101 ; severe lri = 51 ; very severe lri = 22 ) , 161 were cured and 13 died . of 13 cases who died , 10 had coinfection with more than 1 respiratory virus ( ifv + rsv = 5 ; rsv + rv = 3 ; ifv + hcov43 + rv = 2 ) . in uris , in the present study , respiratory viruses were detected in 248 cases ( lri = 142 ; uri = 106 ) . rsv ( 25.8% ) , rv ( 21.9% ) , ifv ( 21% ) , and adv ( 12.5% ) were the most prevalent single viruses isolated from all the ari cases . the most common viruses isolated from acute uri cases were rv ( 32.6% ) , adv ( 19.6% ) , rsv ( 17.4% ) , and ifv - a ( 15.2 ) . the most common viruses isolated from acute lri cases were rsv ( 32.4% ) , ifv - a ( 19.6% ) , adv ( 18.6% ) , and rv ( 15.7% ) . most cases occurred in the month of january , december , and august . in uris , the most common age group affected was > 1260 mo , and in lris , the most common age group affected was 212 mo . present study results are similar to the previous study results from india . in the study by broor et al . , rsv , ifv - a , and piv-3 were important causes of ari in children under five in the rural indian community . in the study by singh et al . , rsv was the most frequently detected virus ( 21.3% ) from hospitalised children presenting as alri followed by ifv ( 9.0% ) , measles ( 8.5% ) , and adv ( 5.3% ) . in the study by bharaj et al . , rsv was the most frequently detected virus ( 58.1% ) from hospitalised children presenting as alri followed by piv ( 22% ) , hmpv ( 10.5% ) , and ifv - a ( 9.3% ) . in the study by maitreyi et al . , rsv was the most frequently detected virus ( 17% ) from hospitalised children presenting as alri followed by ifv ( 14.5% ) , piv ( 11.5% ) , and adv ( 1.5% ) . in the study by yeolekar et al . , rsv was the most frequently detected virus ( 26% ) from hospitalised children presenting as alri followed by ifv ( 5.4% ) , piv ( 2.07% ) , and adv ( 0.8% ) . panda et al . recently found rv as the most frequently detected pathogen ( 24.7% ) followed by rsv ( 4.22% ) , piv ( 2.11% ) , and hmpv ( 2.11% ) in children with ari from eastern india . in the present study , we found rv as the second most important agent to rsv causing ari in young infants and children . the present study findings are more or less similar to the recently published studies from other parts of the globe , though the most prevalent virus may be different [ 2127 ] . the viral uri and lri prevalence varied with age , uris being common in children of > 1260 mo and lris being common in infants of < 12 mo age . infants < 12 mo age were most commonly positive for rsv and less commonly for ifv , the latter occurring commonly beyond infancy in older children . this is because maternal antibody protection against rsv during infancy remains questionable , and as the child grows , the immunity builds up against rsv due to repeated exposures . ifv due to marked antigenic diversity evades the herd immunity and does not give long lasting immunity . therefore , the susceptibility to ifv increases as the child grows . adv is uncommon during first six months , where maternal antibody confers protection . previous indian studies [ 16 , 17 ] have compared the clinical , demographic , and risk factors for alri testing positive for respiratory viruses with those testing negative for respiratory viruses . one study observed that significantly higher number of children below 12 mo of age had rsv infection , those presenting with preceding bronchiolitis were significantly associated with virus positivity , and ari in family was significantly associated with virus positivity . in another study , patients testing positive for viruses showed significantly high percentage of cough , nasal discharge , and diarrhea . we found a significantly higher number of children below 12 mo being virus positive , those presenting with preceding bronchiolitis being significantly associated with virus positivity , rhinorrhea significantly presenting in the virus positive group , and ari in family being significantly associated with virus positivity . the respiratory illness exhibits a clear - cut seasonality , and all the published studies till date support this . most of the studies report increased incidence of acute respiratory infection ( both uri and lri ) during winter and fall season [ 2027 ] . this is because of the following sequence of events : decrease in air temperature causing decrease in the nasal airway temperature ( compromised cooling of the nasal airway ) leading to poor defence against infection ( because of decrease mucociliary clearance and phagocytosis ) . in the present study , the incidence of both uri and lri was higher in winter season ( as upper airway acts as an entry point to lower airway ) . though this can not be solely explained based on the above hypothesis , it may be because of an increase in the birth cohort during this period . in acute lris , severe clinical phenotypes ( severe and very severe pneumonia , acute respiratory distress syndrome ) the present study findings throw lights on the management of an individual case as well as the community . first , good hand hygiene , maintenance of warm temperature in the living place / avoidance of exposure to sudden cold temperature , avoidance of taking of freeze dried / cold food items , and wearing of the mask to prevent spread of the infection should be practiced . second , the nutritional status of the children in the community should be improved as malnutrition predisposes to increase risk of infection and severe illness . third , vaccines should be developed against respiratory viruses ( including against the new viruses ) to decrease the morbidity and mortality from respiratory illness . fourth , any child with ari and viral coinfection should be closely monitored for development of complications / severe illness . strengths of present study are adequate sample size and children being recruited over 2 consecutive years including all the seasons . limitations include children presenting to the health facility only were recruited , reinfection rate by the viruses were not considered , and simultaneous / secondary bacterial infections were not studied . rsv , adv , rv , and ifv were the most prevalent viruses isolated from the ari cases . the epidemiology of respiratory viruses needs to be studied further for vaccine development and implementation purpose . any child with ari and viral coinfection should be monitored for development of complications / severe illness .

background . acute respiratory infections ( aris ) are important cause of mortality and morbidity in children under five in developing country . methods . this observational study was conducted over two - year period in a tertiary care teaching hospital of eastern india . nasal and throat swabs were collected , transported to the laboratory at 28c in viral transport media , and then processed for detection of viruses using mono / multiplex real - time polymerase chain reaction . results . a total of 300 children aged 260 months with aris were included . the most common age group affected with lri was 212 mo and with uri was > 1260 mo . viruses were detected in 248 cases . in uri , 77 were positive for single virus and 19 were positive for more than one virus ; in lri , 113 were positive for single virus and 12 were positive for more than one virus . the most common viruses isolated from uri cases were rhinovirus and adenovirus . the most common viruses isolated from lri cases were respiratory syncytial virus and influenza virus . most cases occurred in the months of january , december , and august . conclusion . viruses constitute a significant cause of ari in children under five . rsv , adv , rv , and ifv were the most prevalent viruses isolated .

these include the intrinsic flexor pollicis brevis , flexor pollicis longus , lumbricalis , flexor brevis , and flexor longus muscles1 . toe - gripping strength is measured in the sitting position with the trunk vertical , the hip and knee joints at 90 , and the ankle joint in the neutral position1,2,3 . low toe - gripping strength is a risk factor of falls for the elderly , and several studies have reported that toe - gripping strength is lower in subjects with a history of falls than in those with no history of falls1,2,3,4 . furthermore , toe - gripping strength can be increased by training5 , which can decrease the risk of falls1 . souma et al.6 studied the % iemg of several crural muscles ( the soleus muscle , medial head of the gastrocnemius muscle , and tibialis anterior ) during toe - gripping in 3 different ankle joint positions10 of plantar flexion and 0 and 10 of dorsiflexion and reported that the crural muscles help the ankle joint by co - contracting during toe - gripping . in another study , nakae et al.7 calculated the % iemg of the same muscles during toe - gripping with subjects seated on the edge of a seat or standing , and reported that the % iemg of the medial gastrocnemius muscle was significantly lower when subjects were seated than when they were standing . however , as both studies focused on the activity of the crural muscles , the activity of the femoral muscles during toe - gripping remains unclear . we believe it is important to elucidate the contribution of femoral muscles to toe - gripping strength . in the present study , we investigated femoral muscle activity during toe - gripping to examine the role of the femoral muscles in toe - gripping strength . their average ( mean sd ) age , height , and body weight were 20.6 1.0 years , 159.4 6.3 cm , and 51.8 5.8 kg , respectively . this study was approved by the ethics committee for human research of tohoku fukushi university , and written informed consent was received from all of the subjects after the purpose of the study had been explained to them . toe - gripping strength of the dominant toe and emg activity of the ipsilateral thigh were synchronously recorded to assess the activity of the rectus femoris and biceps femoris muscles . toe - gripping strength was measured using a toe - gripping dynamometer ( t.k.k.3360 ; takei co , ltd , niigata , japan ) . as described by uritani8 , the subjects were instructed to sit with their trunk in the vertical position , hip and knee joints at 90 , and ankle joints in the neutral position . after a sufficient number of training trials and adequate rest , toe - gripping strength was measured twice and the maximal force was used in the analysis . for all subjects , the right toe was dominant ( defined as the toe used to kick a ball ) . to measure the maximum voluntary contraction ( mvc ) activity of the rectus femoris muscles , as described by kai9 , each subject was instructed to sit on a chair with the hip and knee joints at 90 , and to exert maximal isometric force of knee extension while resisting an opposing force applied by the examiner . the emg was recorded for 3 seconds while each subject exerted maximal force of knee extension . to measure the mvc of the biceps femoris muscle , each subject was instructed to generate maximal isometric force of knee flexion while resisting an opposing force applied by the examiner . the emg was recorded for 3 seconds while each subject exerted maximal force of knee flexion . after confirmation of adequate skin preparation ( skin resistance of less than 5 k ) , three bipolar lead electrodes ( de-2.1 , delsys , inc . , boston , usa ) were attached to the skin over the rectus femoris and the long head of the biceps femoris , as described by peroto10 . for measurement of the rectus femoris muscle , the electrode was attached at the midpoint between the superior edge of the patella and the anterior superior iliac spine . for measurement of the long head of the biceps femoris , the electrode was attached at the midpoint between the head of the fibula and ischial tuberosity . the emg signal was collected using ml846 power lab 4/26 ( sample : 1,000 hz ; ad instruments co. , ltd . ) and transferred to a personal computer . the bandwidth was 20500 hz . the emg signal segment selected and integrated ( integrated electromyogram : iemg ) for analysis was the middle 1 s of the entire 3-s duration of continuous maximal toe - gripping strength . the iemg was analyzed using lab chart pro v7.3.5 ( ad instruments co. , ltd . ) and normalized to the iemg of each muscle s mvc . repeated mann - whitney u tests were used to compare the % iemg of the rectus and biceps femoris muscles . relationships between the % iemg of the rectus and biceps femoris muscles were statistically analyzed using spearman s correlation coefficient . the average ( mean sd ) toe - gripping strength was 20.9 3.6 kg . the average ( mean sd ) % iemg values of the rectus and biceps femoris muscles were 7.0% 6.2% and 25.6% 15.9% , respectively . the % iemg of the biceps femoris was significantly higher than that of the rectus femoris ( p < 0.01 , table 1table 1.comparison of the % iemg values of the rectus femoris and biceps femoris muscles during toe - gripping actionrectus femorisbiceps femorismedian ( range : min max)median ( range : min max)%iemg4.8 * ( 1.521.1)27.2 ( 3.068.1)mann - whitney u test * : p < 0.05 ) . mann - whitney u test * : p < 0.05 using spearman s correlation coefficient analysis , a significant positive correlation was found between the % iemg of the rectus femoris and that of the biceps femoris ( r = 0.548 , p in the present study , we found that the % iemg of the biceps femoris was significantly higher than that of the rectus femoris . moreover , a significant positive correlation was found between the % iemg of these two muscles . these results suggest that femoral muscles co - contract during toe - gripping action , and thus possibly contribute to knee joint stability . we found that toe - gripping action stimulates femoral muscle activity and that the % iemg of the biceps femoris was significantly higher than that of the rectus femoris . sato et al.11 reported that during toe - gripping , the tibialis anterior and soleus muscles are activated first , followed by the gradual activation of the medial head of the gastrocnemius muscle . the biceps femoris initiates knee flexion , and the medial head of the gastrocnemius is a synergist for knee flexion . additionally , active tension of the biceps femoris decreases during toe - gripping in knee flexion . this may be because the length of the biceps femoris is shorter during active tension than during resting tension . we think that the activation of the biceps femoris during toe - gripping was caused by increasing motor unit recruitment and synchronization of the impulse to compensate for decreasing activity of the biceps femoris . in the present study , we found a significant positive correlation between the % iemg of the rectus femoris and that of the biceps femoris . this suggests that the % iemg of the rectus femoris increases with increases in the % iemg of the biceps femoris . toe - gripping strength was measured using a closed kinetic chain movement , and therefore , the likelihood of movement in the directions of flexion or extension of the knee joint is less . however , because the gastrocnemius also acts on knee joint flexion during exertion of toe - gripping strength , the knee joint is readily displaced in the direction of flexion . to prevent this , it is thought that the knee joint is immobilized by contraction of the rectus femoris , that is , we think the rectus femoris during exertion of toe - gripping strength acts in order to control the action of knee joint flexion by the gastrocnemius . first , we were unable to avoid the common problems that negatively affect surface emg recording , such as skin resistance , artifacts , and the effects of proximal muscles . second , as only healthy young women participated in this study , it is difficult to extend our findings to the general population . in future studies , we need to consider this aspect , and involve healthy young men and individuals of different age groups .

[ purpose ] in the present study , we investigated femoral muscle activity during toe - gripping , and the role of the femoral muscles in toe - gripping strength . [ subjects ] fourteen healthy young women were selected . [ methods ] we measured the maximum voluntary contraction of the rectus femoris and long head of the biceps femoris muscles . we then calculated the percent integrated emg ( % iemg ) during the toe - gripping action . [ results ] we found that the % iemg of the biceps femoris was significantly higher than that of the rectus femoris . moreover , a significant positive correlation was found between the % iemg of the rectus femoris and that of the biceps femoris . [ conclusion ] these results suggest that femoral muscles co - contract during the toe - gripping action , and thus possibly contribute to knee joint stability .

this study was performed as a part of the prospective ongoing research on the positioning of chronic kidney disease in specific health check and guidance in japan project . a new annual health check program , the specific health check and guidance in japan , was started by the japanese government in 2008 , targeting early diagnosis and intervention for metabolic syndrome . the target population comprises japanese citizens between the ages of 40 and 74 years . in japan , there are 47 administrative divisions ( prefectures ) , and 13 of these prefectures ( yamagata , miyagi , fukushima , and niigata from the tohoku region in northeastern japan ; tokyo , kanagawa , and ibaraki from the kanto region in central japan ; osaka , okayama , and kochi from the kansai , tyugoku , or shikoku region in western japan ; and fukuoka , miyazaki , and okinawa from the kyushu region in southern japan ) , which were randomly distributed across japan , agreed with the aims of this study and performed data collection prospectively from 2008 to 2009 . data were sent to an independent data center , the nonprofit organization japan clinical research support unit after anonymization in a linkable fashion , and verified by trained staff ( k.i . and y.o . ) . after that , the database was locked with a security password , which contained the participant s information managed by a research i d number but did not contain the participant s name , and was sent to each investigator on a recordable compact disc . there were a total of 346,942 subjects ( mean age , 63.4 years ; 41% [ n = 141,938 ] men ) for whom information on age , sex , bp , bmi , habitual smoking or drinking , use of antihypertensive drugs , and previous history of cardiovascular diseases ( i.e. , stroke and cardiac diseases such as angina and myocardial infarction ) were available , as well as data on the serum creatinine level and dipstick urine test for proteinuria ( 19 ) . some of the regions participating in our project ( i.e. , okinawa and osaka ) concomitantly performed regular health checkups for employees as legally mandated in japan ; as a result , the database used in the present analysis also included subjects aged 2039 years ( n = 2,025 ) . among the 346,942 subjects , 29,820 subjects with a previous history of cardiovascular disease , 243 subjects with chronic kidney disease stage 5 ( estimated glomerular filtration rate [ egfr ] < 15 ml / min/1.73 m ) , and 47 subjects with both were excluded from the present analysis . moreover , 88,101 subjects with insufficient blood sampling data of glucose and lipid parameters were excluded . supplementary table 1 shows the differences in clinical characteristics between subjects who were included in the present analysis ( n = 228,778 ) and those who had missing data ( n = 88,101 ) . the study was conducted according to the guidelines of the declaration of helsinki and ethical guidelines for epidemiological research ( 1 november 2007 , ministry of education , culture , sports , science , and technology and ministry of health , labor , and welfare of japan ) . all subjects completed a self - administered questionnaire to document their medical history , current medications , smoking habits ( current smoker or not ) , and alcohol intake ( daily drinker or not ) . the study physicians performed a physical examination of each subject and rechecked their medical history to improve the precision of the information . body height and weight were measured in light clothing without shoes , and the bmi was calculated ( kg / m ) . bp measurement and blood and urine sampling were performed at each local medical institution to cooperate with the nationwide medical checkup . according to the recommendations of the japanese ministry of health , labor , and welfare ( http://www.mhlw.go.jp/bunya/shakaihosho/iryouseido01/info03a.html ) , bp was measured by medical staff using a standard sphygmomanometer or an automated device on the right arm after the subject had rested for 5 min in a seated position with the legs not crossed . pulse pressure was calculated as systolic bp diastolic bp , and mean bp was calculated as diastolic bp + ( pulse pressure/3 ) . blood samples were collected after an overnight fast and were assayed within 24 h with an automatic clinical chemical analyzer . all measurements were conducted locally rather than at a central laboratory without calibration among different laboratories , despite the fact that beginning several years ago , standardized methods to measure laboratory data were recommended and widely adopted by the activity of the japan society of clinical chemistry . the value for hemoglobin a1c ( hba1c ) was estimated as a national glycohemoglobin standardization program equivalent value calculated with the following equation ( 20 ) : hba1c ( % ) = hba1c ( japan diabetes society ) ( % ) + 0.4% . diabetes was defined in accordance with american diabetes association guidelines ( 17 ) as a fasting glucose concentration of 126 mg / dl or higher , hba1c 6.5% or higher , or self - reported use of antihyperglycemic drugs . diagnosis of prediabetes was based on the new american diabetes association criterion of impaired fasting glucose ( fasting plasma glucose 100125 mg / dl ) or hba1c 5764% , or both ( 17 ) . urinalysis by the dipstick method was performed on a single spot urine specimen collected in the early morning after overnight fasting . urine dipstick results are interpreted by the medical staff in each local medical institution and recorded as , , 1 + , 2 + , and 3 + . in japan , it is recommended and widely adopted by the activity of the japanese committee for clinical laboratory standards ( http://jccls.org/ ) that all urine dipstick tests be manufactured so that a urine dipstick result of 1 + will correspond to a urinary protein level of 30 mg / dl . in the current study , egfr was derived using the following equation ( 21 ) : egfr ( ml / min/1.73 m ) = 194 age ( years ) serum creatinine ( mg / dl ) ( if women 0.739 ) . all statistical analyses were performed with spss version 18.0 j software ( spss , chicago , il ) . data were expressed as the means sd ( age , bmi , egfr , and bp values ) or median and interquartile range ( glucose and lipid parameters ) . clinical parameters and bp or metabolic values according to the presence of diabetes or prediabetes were compared using anova , and categorical parameters were compared with the test . we subdivided the study population according to the quintiles of pulse pressure , and the prevalence of proteinuria ( 1 + ) was compared by test among each group of the quintiles of pulse pressure separately in subjects with diabetes , prediabetes , or normal glucose tolerance , respectively . the highest quintile of pulse pressure ( 63 mmhg , n = 40,511 ) was defined as the high pulse pressure group in the present analysis . next , we used a multivariable logistic regression analysis to examine the independent association of high pulse pressure with proteinuria ( 1 + ) separately in subjects with diabetes , prediabetes , or normal glucose tolerance , respectively . in the initial model ( model 1 ) , these associations were assessed with adjustment for age , sex , bmi , current smoking and daily drinking , the presence of antihypertensive medications , and egfr . extended models were used to assess whether the association of high pulse pressure with proteinuria ( 1 + ) was attenuated by the potential confounding effects of glucose and lipid parameters ( model 2 ) and systolic bp ( model 3 ) . in addition , to minimize the influence of systolic bp in the association between pulse pressure and proteinuria , we examined the association only in patients with diabetes whose systolic bp was within the normal bp range ( i.e. , < 130 mmhg ) ( 22 ) . finally , we examined the association of a + 1 sd increase of pulse pressure ( + 13 mmhg ) , rather than pulse pressure as a dichotomous variable , with proteinuria in patients with diabetes by a multivariable logistic regression analysis . this study was performed as a part of the prospective ongoing research on the positioning of chronic kidney disease in specific health check and guidance in japan project . a new annual health check program , the specific health check and guidance in japan , was started by the japanese government in 2008 , targeting early diagnosis and intervention for metabolic syndrome . the target population comprises japanese citizens between the ages of 40 and 74 years . in japan , there are 47 administrative divisions ( prefectures ) , and 13 of these prefectures ( yamagata , miyagi , fukushima , and niigata from the tohoku region in northeastern japan ; tokyo , kanagawa , and ibaraki from the kanto region in central japan ; osaka , okayama , and kochi from the kansai , tyugoku , or shikoku region in western japan ; and fukuoka , miyazaki , and okinawa from the kyushu region in southern japan ) , which were randomly distributed across japan , agreed with the aims of this study and performed data collection prospectively from 2008 to 2009 . data were sent to an independent data center , the nonprofit organization japan clinical research support unit after anonymization in a linkable fashion , and verified by trained staff ( k.i . and y.o . ) . after that , the database was locked with a security password , which contained the participant s information managed by a research i d number but did not contain the participant s name , and was sent to each investigator on a recordable compact disc . there were a total of 346,942 subjects ( mean age , 63.4 years ; 41% [ n = 141,938 ] men ) for whom information on age , sex , bp , bmi , habitual smoking or drinking , use of antihypertensive drugs , and previous history of cardiovascular diseases ( i.e. , stroke and cardiac diseases such as angina and myocardial infarction ) were available , as well as data on the serum creatinine level and dipstick urine test for proteinuria ( 19 ) . some of the regions participating in our project ( i.e. , okinawa and osaka ) concomitantly performed regular health checkups for employees as legally mandated in japan ; as a result , the database used in the present analysis also included subjects aged 2039 years ( n = 2,025 ) . among the 346,942 subjects , 29,820 subjects with a previous history of cardiovascular disease , 243 subjects with chronic kidney disease stage 5 ( estimated glomerular filtration rate [ egfr ] < 15 ml / min/1.73 m ) , and 47 subjects with both were excluded from the present analysis . moreover , 88,101 subjects with insufficient blood sampling data of glucose and lipid parameters were excluded . supplementary table 1 shows the differences in clinical characteristics between subjects who were included in the present analysis ( n = 228,778 ) and those who had missing data ( n = 88,101 ) . the study was conducted according to the guidelines of the declaration of helsinki and ethical guidelines for epidemiological research ( 1 november 2007 , ministry of education , culture , sports , science , and technology and ministry of health , labor , and welfare of japan ) . all subjects completed a self - administered questionnaire to document their medical history , current medications , smoking habits ( current smoker or not ) , and alcohol intake ( daily drinker or not ) . the study physicians performed a physical examination of each subject and rechecked their medical history to improve the precision of the information . body height and weight were measured in light clothing without shoes , and the bmi was calculated ( kg / m ) . bp measurement and blood and urine sampling were performed at each local medical institution to cooperate with the nationwide medical checkup . according to the recommendations of the japanese ministry of health , labor , and welfare ( http://www.mhlw.go.jp/bunya/shakaihosho/iryouseido01/info03a.html ) , bp was measured by medical staff using a standard sphygmomanometer or an automated device on the right arm after the subject had rested for 5 min in a seated position with the legs not crossed . pulse pressure was calculated as systolic bp diastolic bp , and mean bp was calculated as diastolic bp + ( pulse pressure/3 ) . blood samples were collected after an overnight fast and were assayed within 24 h with an automatic clinical chemical analyzer . all measurements were conducted locally rather than at a central laboratory without calibration among different laboratories , despite the fact that beginning several years ago , standardized methods to measure laboratory data were recommended and widely adopted by the activity of the japan society of clinical chemistry . the value for hemoglobin a1c ( hba1c ) was estimated as a national glycohemoglobin standardization program equivalent value calculated with the following equation ( 20 ) : hba1c ( % ) = hba1c ( japan diabetes society ) ( % ) + 0.4% . diabetes was defined in accordance with american diabetes association guidelines ( 17 ) as a fasting glucose concentration of 126 mg / dl or higher , hba1c 6.5% or higher , or self - reported use of antihyperglycemic drugs . diagnosis of prediabetes was based on the new american diabetes association criterion of impaired fasting glucose ( fasting plasma glucose 100125 mg / dl ) or hba1c 5764% , or both ( 17 ) . urinalysis by the dipstick method was performed on a single spot urine specimen collected in the early morning after overnight fasting . urine dipstick results are interpreted by the medical staff in each local medical institution and recorded as , , 1 + , 2 + , and 3 + . in japan , it is recommended and widely adopted by the activity of the japanese committee for clinical laboratory standards ( http://jccls.org/ ) that all urine dipstick tests be manufactured so that a urine dipstick result of 1 + will correspond to a urinary protein level of 30 mg / dl . in the current study , proteinuria was defined as 1 + or more . egfr was derived using the following equation ( 21 ) : egfr ( ml / min/1.73 m ) = 194 age ( years ) serum creatinine ( mg / dl ) ( if women 0.739 ) . all statistical analyses were performed with spss version 18.0 j software ( spss , chicago , il ) . data were expressed as the means sd ( age , bmi , egfr , and bp values ) or median and interquartile range ( glucose and lipid parameters ) . clinical parameters and bp or metabolic values according to the presence of diabetes or prediabetes were compared using anova , and categorical parameters were compared with the test . we subdivided the study population according to the quintiles of pulse pressure , and the prevalence of proteinuria ( 1 + ) was compared by test among each group of the quintiles of pulse pressure separately in subjects with diabetes , prediabetes , or normal glucose tolerance , respectively . the highest quintile of pulse pressure ( 63 mmhg , n = 40,511 ) was defined as the high pulse pressure group in the present analysis . next , we used a multivariable logistic regression analysis to examine the independent association of high pulse pressure with proteinuria ( 1 + ) separately in subjects with diabetes , prediabetes , or normal glucose tolerance , respectively . in the initial model ( model 1 ) , these associations were assessed with adjustment for age , sex , bmi , current smoking and daily drinking , the presence of antihypertensive medications , and egfr . extended models were used to assess whether the association of high pulse pressure with proteinuria ( 1 + ) was attenuated by the potential confounding effects of glucose and lipid parameters ( model 2 ) and systolic bp ( model 3 ) . in addition , to minimize the influence of systolic bp in the association between pulse pressure and proteinuria , we examined the association only in patients with diabetes whose systolic bp was within the normal bp range ( i.e. , < 130 mmhg ) ( 22 ) . finally , we examined the association of a + 1 sd increase of pulse pressure ( + 13 mmhg ) , rather than pulse pressure as a dichotomous variable , with proteinuria in patients with diabetes by a multivariable logistic regression analysis . the mean age sd of the 228,778 subjects was 63.2 8.9 years , and 89,877 of the subjects ( 39.3% ) were men . there were 27,913 subjects ( 12.2% of the total subject population ) with diabetes , of whom 10,980 subjects ( 39.1% ) were taking antihyperglycemic medications . the clinical characteristics according to the presence of diabetes or prediabetes are shown in table 1 . compared with subjects with normal glucose tolerance ( as a reference ) , the odds ratio ( or ) for the increased risk of proteinuria ( 1 + ) in diabetes itself was 2.14 ( 95% ci 2.032.25 ) , and that in prediabetes was 1.10 ( 1.051.14 ) , even after adjustment for significant covariates , such as age , sex , bmi , current smoking and daily drinking , the presence of antihypertensive medications , and systolic bp level ( both p < 0.001 ) . characteristics of the study population according to the presence of diabetes or prediabetes clinical characteristics and metabolic or bp parameters according to the quintile of pulse pressure are shown in supplementary table 2 . the increasing prevalence of proteinuria ( 1 + ) in accordance with the increasing pulse pressure was more prominent in subjects with diabetes than those without diabetes ( fig . supplementary table 3 shows the prevalence of proteinuria subdivided by the dipstick positive scale according to the quintile of pulse pressure with or without diabetes . prevalence of proteinuria according to the quintile of pulse pressure in subjects with diabetes , prediabetes , or normal glucose tolerance . the prevalence of proteinuria ( 1 + ) was calculated among each group of the quintiles of pulse pressure separately in subjects with diabetes , prediabetes , or normal glucose tolerance , respectively . the p value was obtained by a test among each group of the quintiles of pulse pressure . next , a multivariable logistic regression analysis was performed to examine the independent association between the highest quintile of pulse pressure and proteinuria , separately in subjects with diabetes , prediabetes , and normal glucose tolerance . in patients with diabetes , the highest quintile of pulse pressure ( 63 mmhg ) was positively associated with proteinuria , independently of significant covariates , including systolic bp ( models 13 in table 2 ) . when we examined the association between pulse pressure and proteinuria only in patients with diabetes whose systolic bp was within the normal range ( i.e. , < 130 mmhg , n = 11,074 [ 39.7% ] ) , the highest quintile of pulse pressure still remained significantly associated with proteinuria ( or 1.46 [ 95% ci 1.032.08 ] ; p = 0.04 , respectively ) , even after adjustment for significant covariates , as shown in model 2 in table 2 . when diastolic bp or mean bp was entered into model 3 in table 3 in place of systolic bp , the association between the highest quintile of pulse pressure and proteinuria still remained significant ( 1.61 [ 1.491.75 ] and 1.42 [ 1.311.55 ] ; both p < 0.001 , respectively ) . in contrast , the highest quintile of pulse pressure in subjects with prediabetes or normal glucose tolerance was not associated with proteinuria independently of systolic bp ( model 3 in table 2 ) . when we examined the risk of the highest quintile of pulse pressure on proteinuria among subjects without antihypertensive medications ( n = 167,110 ) , the conclusion remained unchanged ( model 4 in table 2 ) . use of antihyperglycemic or antihyperlipidemic drugs did not influence any of the above results ( data not shown ) . in contrast , systolic bp , used as an adjusted factor in model 3 in table 2 , showed significant associations with proteinuria in subjects with diabetes , prediabetes , and normal glucose tolerance ( data not shown ) . or for the highest quintile of pulse pressure in the association of proteinuria ( 1 + ) according to the presence of diabetes or prediabetes or ( 95% ci ) for proteinuria in diabetes ( n = 27,913 ) finally , we analyzed the association of a + 1 sd increase of pulse pressure ( + 13 mmhg ) , rather than pulse pressure as a dichotomous variable , with proteinuria in patients with diabetes . we found that a + 1 sd increase of pulse pressure was associated with proteinuria independently of significant covariates , including systolic bp ( table 3 ) , diastolic bp , or mean bp ( data not shown ) . the mean age sd of the 228,778 subjects was 63.2 8.9 years , and 89,877 of the subjects ( 39.3% ) were men . there were 27,913 subjects ( 12.2% of the total subject population ) with diabetes , of whom 10,980 subjects ( 39.1% ) were taking antihyperglycemic medications . the clinical characteristics according to the presence of diabetes or prediabetes are shown in table 1 . compared with subjects with normal glucose tolerance ( as a reference ) , the odds ratio ( or ) for the increased risk of proteinuria ( 1 + ) in diabetes itself was 2.14 ( 95% ci 2.032.25 ) , and that in prediabetes was 1.10 ( 1.051.14 ) , even after adjustment for significant covariates , such as age , sex , bmi , current smoking and daily drinking , the presence of antihypertensive medications , and systolic bp level ( both p < 0.001 ) . characteristics of the study population according to the presence of diabetes or prediabetes clinical characteristics and metabolic or bp parameters according to the quintile of pulse pressure are shown in supplementary table 2 . the increasing prevalence of proteinuria ( 1 + ) in accordance with the increasing pulse pressure was more prominent in subjects with diabetes than those without diabetes ( fig . 1 ) . supplementary table 3 shows the prevalence of proteinuria subdivided by the dipstick positive scale according to the quintile of pulse pressure with or without diabetes . prevalence of proteinuria according to the quintile of pulse pressure in subjects with diabetes , prediabetes , or normal glucose tolerance . the prevalence of proteinuria ( 1 + ) was calculated among each group of the quintiles of pulse pressure separately in subjects with diabetes , prediabetes , or normal glucose tolerance , respectively . the p value was obtained by a test among each group of the quintiles of pulse pressure . next , a multivariable logistic regression analysis was performed to examine the independent association between the highest quintile of pulse pressure and proteinuria , separately in subjects with diabetes , prediabetes , and normal glucose tolerance . in patients with diabetes , the highest quintile of pulse pressure ( 63 mmhg ) was positively associated with proteinuria , independently of significant covariates , including systolic bp ( models 13 in table 2 ) . when we examined the association between pulse pressure and proteinuria only in patients with diabetes whose systolic bp was within the normal range ( i.e. , < 130 mmhg , n = 11,074 [ 39.7% ] ) , the highest quintile of pulse pressure still remained significantly associated with proteinuria ( or 1.46 [ 95% ci 1.032.08 ] ; p = 0.04 , respectively ) , even after adjustment for significant covariates , as shown in model 2 in table 2 . when diastolic bp or mean bp was entered into model 3 in table 3 in place of systolic bp , the association between the highest quintile of pulse pressure and proteinuria still remained significant ( 1.61 [ 1.491.75 ] and 1.42 [ 1.311.55 ] ; both p < 0.001 , respectively ) . in contrast , the highest quintile of pulse pressure in subjects with prediabetes or normal glucose tolerance was not associated with proteinuria independently of systolic bp ( model 3 in table 2 ) . when we examined the risk of the highest quintile of pulse pressure on proteinuria among subjects without antihypertensive medications ( n = 167,110 ) , the conclusion remained unchanged ( model 4 in table 2 ) . use of antihyperglycemic or antihyperlipidemic drugs did not influence any of the above results ( data not shown ) . in contrast , systolic bp , used as an adjusted factor in model 3 in table 2 , showed significant associations with proteinuria in subjects with diabetes , prediabetes , and normal glucose tolerance ( data not shown ) . or for the highest quintile of pulse pressure in the association of proteinuria ( 1 + ) according to the presence of diabetes or prediabetes or ( 95% ci ) for proteinuria in diabetes ( n = 27,913 ) finally , we analyzed the association of a + 1 sd increase of pulse pressure ( + 13 mmhg ) , rather than pulse pressure as a dichotomous variable , with proteinuria in patients with diabetes . we found that a + 1 sd increase of pulse pressure was associated with proteinuria independently of significant covariates , including systolic bp ( table 3 ) , diastolic bp , or mean bp ( data not shown ) . in this nationwide study of 228,778 japanese people ( mean age 63.2 years ) who had no known cardiovascular disease , we demonstrated for the first time that there was a significant difference in the association between the highest quintile of pulse pressure ( 63 mmhg ) and proteinuria ( 1 + on dipstick ) among subjects with diabetes , prediabetes , and normal glucose tolerance . the cross - sectional design of the current study did not allow us to elucidate the pathophysiological pathway linking high pulse pressure and proteinuria ( 1 + ) . however , there are some possible explanations for the observed association . since the glomerular afferent arterioles provide relatively low resistance , the glomerulus is susceptible to barotrauma if the pulse pressure is elevated ( 16 ) . in fact , prior studies have demonstrated an association of high pulse pressure with microalbuminuria even in subjects without diabetes ( 7,8 ) . in the current study , we examined the possible association of high pulse pressure and proteinuria ( 1 + ) , i.e. , macroalbuminuria , and found that this association was not significant independently of systolic bp in subjects without diabetes . in contrast , systolic bp was significantly associated with proteinuria in these subjects . although the usefulness of the urine dipstick test for risk stratification of renal and cardiovascular disease has been recognized , this method is a less sensitive measure of albuminuria compared with the measurement of urinary albumin excretion ( 2326 ) . accordingly , we can not deny the possibility of an association between high pulse pressure and microalbuminuria in subjects without diabetes . in spite of the strict collinearity between systolic bp and pulse pressure , the or of high pulse pressure to proteinuria was reduced but remained significant even after adjustment for systolic bp in patients with diabetes ( table 2 ) . table 3 also shows that a + 1 sd increase of systolic bp and a + 1 sd increase of pulse pressure were associated with proteinuria independently of each other , with the or of the systolic bp increase on proteinuria being higher than that of the pulse pressure increase . these findings indicate that high systolic bp showed a confirmed association with proteinuria and is an important confounder explaining the association between high pulse pressure and proteinuria ; however , even after adjustment for systolic bp , the pulsatile component of bp itself was still significantly associated with proteinuria in patients with diabetes . intriguingly , even in the patients with diabetes who were within the normal range of systolic bp values , high pulse pressure was associated with proteinuria . first , since renal autoregulation is impaired in diabetes ( 13,1113 ) , it may be possible that when pulse pressure is elevated , more barotrauma - induced glomerular ultrastructural changes leading to albuminuria occur in subjects with diabetes than in those without diabetes ( 15 ) . second , much as in the previous reports ( 27,28 ) , higher pulse pressure was observed in diabetes than nondiabetes ( table 1 ) , suggesting the possibility that diabetes accelerates aortic and large arterial stiffness ( 29 ) . aortic stiffness itself has a potential etiologic role in the causation and progression of renal dysfunction ( 3032 ) , because loss of the damping of ventricular ejection in the stiffened aortae could lead to an increase in the transmission of these pressure changes to the renal microcirculation . in the current study , however , we did not use any measure of vascular stiffness more direct than pulse pressure , such as pulse wave velocity , and thus the potential efficacy of such measures will need to be investigated in the future . third , overt proteinuria in patients with diabetes , which is observed in long - standing diabetes , together with hypertension and increased arterial stiffness , is a surrogate marker not only for renal structural damages but also generalized vascular damages ( 3,6,24,25 ) . therefore , we speculate that patients with diabetes with proteinuria are likely to have systemic vasculopathy , and as a consequence , they have high pulse pressure . lastly , since the current study is a cross - sectional analysis , we have to pay attention to another possibility that diabetic renal disease indicated by greater proteinuria raises systolic bp as well as pulse pressure rather than the reverse in patients with diabetes . the current study provided the first examination of the association of pulse pressure with proteinuria in prediabetes using a large sample size . understanding such risk estimates is important , given the increases in the prevalence of prediabetes that have occurred in many populations in conjunction with the increasing prevalence of obesity , particularly in asian populations ( 33,34 ) . in the current study , another japanese study performed in healthy japanese people ( n = 6,636 , mean age 50 years ) demonstrated that the prevalence of prediabetes was 32% ( 35 ) . this survey was performed between 1997 and 2003 , and since the prevalence of diabetes in asian populations has increased rapidly in recent years ( 33,34 ) , the high prevalence of prediabetes in the current study was not entirely unexpected . several limitations of our study should be mentioned . first , single - measurement readings of bp , fasting glucose or hbalc , and proteinuria can not be considered fully accurate . in particular , some of the dipstick - positive proteinuria could have been transient , and thus could not be taken as definitive evidence of the presence of persisting proteinuria . these factors may introduce a source of variability that could have led to a tendency to underestimate the true association between pulse pressure and proteinuria . however , the incidence of type 1 diabetes is extremely low ( approximately two cases / year/100,000 individuals ) , and japan has one of the lowest incidence rates of type 1 diabetes in the world ( 36 ) . third , we could not assess the diabetes- and atherosclerosis - related information , such as the duration of diabetes and the presence of diabetes complications ( e.g. , neuropathy ) , which would be informative and extend the knowledge achieved in the current study . lastly , we could not assess what kinds of antihypertensive drugs had been prescribed in treated hypertensive subjects . some antihypertensive drugs ( e.g. , angiotensin receptor blockers or angiotensin enzyme converting inhibitors ) have more favorable effects on vascular and renal protection ( 37 ) . therefore , their use was potentially confounding , although our conclusions remained unchanged when we analyzed our data while excluding the subjects with antihypertensive medications . in conclusion , among the japanese general population , high pulse pressure , particularly in individuals with diabetes , was associated with proteinuria , and this information has the potential to supplement other bp indices . to confirm our findings , a prospective study as well as interventions that examine whether or not reduction of pulse pressure can enhance nephron - protective benefits in diabetes will be required . since the glomerular afferent arterioles provide relatively low resistance , the glomerulus is susceptible to barotrauma if the pulse pressure is elevated ( 16 ) . in fact , prior studies have demonstrated an association of high pulse pressure with microalbuminuria even in subjects without diabetes ( 7,8 ) . in the current study , we examined the possible association of high pulse pressure and proteinuria ( 1 + ) , i.e. , macroalbuminuria , and found that this association was not significant independently of systolic bp in subjects without diabetes . although the usefulness of the urine dipstick test for risk stratification of renal and cardiovascular disease has been recognized , this method is a less sensitive measure of albuminuria compared with the measurement of urinary albumin excretion ( 2326 ) . accordingly , we can not deny the possibility of an association between high pulse pressure and microalbuminuria in subjects without diabetes . in spite of the strict collinearity between systolic bp and pulse pressure , the or of high pulse pressure to proteinuria was reduced but remained significant even after adjustment for systolic bp in patients with diabetes ( table 2 ) . table 3 also shows that a + 1 sd increase of systolic bp and a + 1 sd increase of pulse pressure were associated with proteinuria independently of each other , with the or of the systolic bp increase on proteinuria being higher than that of the pulse pressure increase . these findings indicate that high systolic bp showed a confirmed association with proteinuria and is an important confounder explaining the association between high pulse pressure and proteinuria ; however , even after adjustment for systolic bp , the pulsatile component of bp itself was still significantly associated with proteinuria in patients with diabetes . intriguingly , even in the patients with diabetes who were within the normal range of systolic bp values , high pulse pressure was associated with proteinuria . first , since renal autoregulation is impaired in diabetes ( 13,1113 ) , it may be possible that when pulse pressure is elevated , more barotrauma - induced glomerular ultrastructural changes leading to albuminuria occur in subjects with diabetes than in those without diabetes ( 15 ) . second , much as in the previous reports ( 27,28 ) , higher pulse pressure was observed in diabetes than nondiabetes ( table 1 ) , suggesting the possibility that diabetes accelerates aortic and large arterial stiffness ( 29 ) . aortic stiffness itself has a potential etiologic role in the causation and progression of renal dysfunction ( 3032 ) , because loss of the damping of ventricular ejection in the stiffened aortae could lead to an increase in the transmission of these pressure changes to the renal microcirculation . in the current study , however , we did not use any measure of vascular stiffness more direct than pulse pressure , such as pulse wave velocity , and thus the potential efficacy of such measures will need to be investigated in the future . third , overt proteinuria in patients with diabetes , which is observed in long - standing diabetes , together with hypertension and increased arterial stiffness , is a surrogate marker not only for renal structural damages but also generalized vascular damages ( 3,6,24,25 ) . therefore , we speculate that patients with diabetes with proteinuria are likely to have systemic vasculopathy , and as a consequence , they have high pulse pressure . lastly , since the current study is a cross - sectional analysis , we have to pay attention to another possibility that diabetic renal disease indicated by greater proteinuria raises systolic bp as well as pulse pressure rather than the reverse in patients with diabetes . the current study provided the first examination of the association of pulse pressure with proteinuria in prediabetes using a large sample size . understanding such risk estimates is important , given the increases in the prevalence of prediabetes that have occurred in many populations in conjunction with the increasing prevalence of obesity , particularly in asian populations ( 33,34 ) . in the current study , another japanese study performed in healthy japanese people ( n = 6,636 , mean age 50 years ) demonstrated that the prevalence of prediabetes was 32% ( 35 ) . this survey was performed between 1997 and 2003 , and since the prevalence of diabetes in asian populations has increased rapidly in recent years ( 33,34 ) , the high prevalence of prediabetes in the current study was not entirely unexpected . first , single - measurement readings of bp , fasting glucose or hbalc , and proteinuria can not be considered fully accurate . in particular , some of the dipstick - positive proteinuria could have been transient , and thus could not be taken as definitive evidence of the presence of persisting proteinuria . these factors may introduce a source of variability that could have led to a tendency to underestimate the true association between pulse pressure and proteinuria . however , the incidence of type 1 diabetes is extremely low ( approximately two cases / year/100,000 individuals ) , and japan has one of the lowest incidence rates of type 1 diabetes in the world ( 36 ) . third , we could not assess the diabetes- and atherosclerosis - related information , such as the duration of diabetes and the presence of diabetes complications ( e.g. , neuropathy ) , which would be informative and extend the knowledge achieved in the current study . lastly , we could not assess what kinds of antihypertensive drugs had been prescribed in treated hypertensive subjects . some antihypertensive drugs ( e.g. , angiotensin receptor blockers or angiotensin enzyme converting inhibitors ) have more favorable effects on vascular and renal protection ( 37 ) . therefore , their use was potentially confounding , although our conclusions remained unchanged when we analyzed our data while excluding the subjects with antihypertensive medications . in conclusion , among the japanese general population , high pulse pressure , particularly in individuals with diabetes , was associated with proteinuria , and this information has the potential to supplement other bp indices . to confirm our findings , a prospective study as well as interventions that examine whether or not reduction of pulse pressure can enhance nephron - protective benefits in diabetes will be required .

objectiveto examine whether there is a difference in the association between high pulse pressure and proteinuria , independent of other blood pressure ( bp ) indices , such as systolic or diastolic bp , among subjects with diabetes , prediabetes , or normal glucose tolerance.research design and methodsusing a nationwide health checkup database of 228,778 japanese aged 20 years ( mean 63.2 years ; 39.3% men ; none had pre - existing cardiovascular disease ) , we examined the association between high pulse pressure , defined as the highest quintile of pulse pressure ( 63 mmhg , n = 40,511 ) , and proteinuria ( 1 + on dipstick , n = 12,090 ) separately in subjects with diabetes ( n = 27,913 ) , prediabetes ( n = 100,214 ) , and normal glucose tolerance ( n = 100,651).resultsthe prevalence of proteinuria was different among subjects with diabetes , prediabetes , and normal glucose tolerance ( 11.3 vs. 5.0 vs. 3.9% , respectively ; p < 0.001 ) . in subjects with diabetes , but not those with prediabetes or normal glucose tolerance , high pulse pressure was associated with proteinuria independently of significant covariates , including systolic bp ( odds ratio 1.15 [ 95% ci 1.041.28 ] ) or diastolic or mean bp ( all p < 0.01 ) . in patients with diabetes , a + 1 sd increase of pulse pressure ( + 13 mmhg ) was associated with proteinuria , even after adjustment for systolic bp ( 1.07 [ 1.001.13 ] ) or diastolic or mean bp ( all p < 0.05).conclusionsamong the japanese general population , there was a significant difference in the association between high pulse pressure and proteinuria among subjects with diabetes , prediabetes , and normal glucose tolerance . only in diabetes was high pulse pressure associated with proteinuria independent of systolic , diastolic , or mean bp levels .

toll - like receptor ( tlr ) agonists such as endotoxins initiate the synthesis of the inactive il-1 precursor ( fig . although most of the il-1 precursor is in the cytosol , a fraction moves into specialized secretory lysosomes ( 1 ) . the next step is the conversion of the inactive procaspase-1 to active caspase-1 by a complex of proteins termed the current thinking is that in resting cells procaspase-1 is bound to a large inhibitor molecule , which prevents its activation . during initiation of il-1 synthesis , there is activation of caspase-1 , which then processes the il-1 precursor into a mature form ready for secretion . processing and release are closely linked ( fig . 1 , c and d ) . activation of the nucleotide p2x7 receptor triggers the efflux of potassium ions out of the cell , and within minutes the secretory lysosomes begin releasing their contents of processed il-1 into the extracellular milieu . in support of the role of p2x7 , overexpression of the receptor increases the secretion of il-1 ( 3 ) and the small peptide ll37 released from activated neutrophils and epithelial cells also stimulates the release of processed il-1 via the p2x7 receptor ( 5 ) . the efflux of potassium ions signals the influx of calcium ions ( 3 ) , which in turn activate phospholipases ( 6 ) . it appears that calcium - independent phospholipase a2 is required for caspase-1 processing in the specialized lysosomes , whereas phosphatidylcholine - specific phospholipase c is required for lysosomal exocytosis and release ( 6 ) . dysregulation in any of these steps might account for increased secretion of il-1 and for il-1mediated diseases . steps in the processing and secretion of il-1. ( a ) tlr ligands such as endotoxin trigger gene expression and synthesis of the il-1 precursor , which remains diffusely in the cytosol . in the same cell , inactive procaspase-1 is bound to components of the il-1 inflammasome , which contains the products of the nalp-3 gene . the il-1 inflammasome is kept in an inactive state by binding to a large molecular weight putative inhibitor . ( b ) after tlr signals , there is a transient uncoupling of the inhibitor and nalp-3 gene products from procaspase-1 , which then colocalizes with the il-1 in secretory lysosomes . ( c ) activation of the nucleotide receptor p2x7 by atp or ll37 initiates the efflux of potassium from the cell via a potassium channel . ( d ) the efflux of potassium ions results in the influx of calcium ions , which in turn activate phospholipases . phosphatidylcholine - specific phospholipase c ( pc - pla-2 ) facilitates lysosomal exocytosis and secretion of il-1 . sojia ( also known as systemic juvenile rheumatoid arthritis ) is a devastating , systemic inflammatory disease that affects growing children for which there are few treatment options other than high dose corticosteroid treatment . in this issue , pascual and colleagues show that blocking il-1 activity with an il-1 receptor antagonist ( il-1ra , anakinra ) resulted in convincing clinical and hematological responses in nine patients with sojia ( 7 ) . resolution of clinical symptoms including fever , marked leukocytosis , thrombocytosis , elevated erythrocyte sedimentation , anemia , and arthritis were rapid and sustained . the efficacy of il-1ra in these children contrasts sharply to that of blocking tnf in sojia . neutralization of tnf , a successful treatment in some patients with rheumatoid arthritis , is now discredited in sojia since the tnf inhibitors etanercept and infliximab are associated with treatment failures , worsening of disease and/or exacerbations of other autoimmune diseases in these patients . based on the present study and a similar observation ( 8) , blocking il-1 may become the standard of therapy for sojia . at present , only il-1ra is approved for use in humans , but other agents such as anti il-1 monoclonal antibodies or the il-1 trap molecule ( 9 ) reduce il-1 activity and are likely to be effective . the rapid resolution of clinical , hematological , and biochemical manifestations of sojia after a few days of il-1ra treatment is reminiscent of the treatment of refractory adult onset still 's disease , a systemic inflammatory disease of adults characterized by similar manifestations of disease seen in sojia . reduction or complete withdrawal of long - term steroid treatment was achieved without a rebound in disease activity , as is also the case in adult onset still 's disease patients treated with il-1ra . for growing children , in addition , il-1ra therapy in rheumatoid arthritis patients does not interfere with tetanus immunization , suggesting that this treatment will not interfere with childhood immunizations . pascual and coworkers took their investigation of disease mechanisms a step further than most studies . they added 20% serum from four sojia patients or from healthy controls to peripheral blood mononuclear cells ( pbmcs ) of healthy donors , and changes in gene expression were assessed by microarray analysis ( 7 ) . why these four sera of the 16 patients available to the authors were selected is not specified , nor is the reason for using 20% serum . nevertheless , increases in gene expression of cytokines , cytokine receptors , cell adhesion molecules , and other markers of inflammation were observed in pbmcs incubated with patient sera but not control sera . the authors found that incubating pbmcs with 20% serum from sojia patients increased secretion of il-1 compared with autologous sera . furthermore , it appeared that the sera from patients with systemic disease induced more il-1 secretion compared with patients with only active arthritis . it may be concluded that 20% serum from patients with sojia contains enough stimulant(s ) to increase a portfolio of proinflammatory genes as well as induce secretion of il-1 from healthy pbmcs , and that autologous sera does not contain such stimulants . in my opinion , however , such methods do not support the concept that disease is caused by a circulating factor(s ) ; rather , the effects of sera on cultured cells may be an epiphenomenon . presence of tlr ligands , such as bacterial lipopolysaccharide , in sojia sera would yield the same results , and a combination of particular serum acute phase reactants could also contribute to the observation . if the authors had added il-1ra to the cultures , they could have at least observed whether the serum stimulant(s ) was il-1 itself . the authors also examined steady - state gene expression in pbmcs from 16 sojia patients with active disease and compared the results to pbmcs from 12 healthy children . many of the same genes induced by sojia sera were spontaneously increased in the pbmcs of these 16 patients . most notable and significant were the genes encoding il-1 , the il-1 decoy receptor , cyclooxygenase-2 , tlr-2 , and the complement receptor c1q . pentraxin-3 , an il-1inducible gene ( 11 ) , was also highly overexpressed , and being an acute phase protein , it likely contributes to the high sedimentation rate of red blook cells in sojia . some genes that were up - regulated by the sojia sera were not increased in steady - state mrna from pbmcs of the 16 patients , including several chemokines and fibronectin , casting doubt on the relevance of the high expression level these genes have in serum studies . another potentially relevant gene overexpressed in sojia pbmcs is a potassium channel gene kcnj15 . as discussed above , influx of potassium ions is a trigger for activation of caspase-1 and secretion of il-1 in response to activation of the nucleotide receptor p2x7 . measurement of circulating il-1 is not a reliable indicator of a role for this cytokine in disease , nor does it provide rationale for selection of a therapeutic intervention such as il-1ra . il-1 is a highly active cytokine in humans ; injecting a few nanograms per kilogram results in fever , neutrophilia , thrombocytosis , acute phase proteins , and circulating il-6 ( for review see reference 12 ) . thus , circulating levels of il-1 in the picomolar range may easily escape detection by routine elisas or similar methods . although there are numerous reports that circulating cytokine levels correlate with severity in a variety of diseases , it is only specific blockade or neutralization of a cytokine that provides a convincing case for causation . for this reason , it is a general concept that il-1mediated disease severity is regulated at the level of ligand production and activity , and not at the receptor level . for example , il-1 type i receptors are expressed on all cells in healthy individuals and increases of only two- or threefold occur in disease . on the other hand , in circulating monocytes and bone marrow macrophages from healthy individuals , il-1 gene expression is absent but increases at least 100-fold when stimulated with microbial products or inflammatory molecules , including products of activated t cells . the total amount of il-1 precursor that is synthesized , however , does not necessarily equate to the amount of active il-1 that is produced , as the caspase-1dependent conversion of il-1 precursor to an active secreted cytokine is a tightly controlled event , despite the presence of constitutive procaspase-1 in the same cell . hence , the increase in il-1 secretion from pbmcs of sojia patients is a highly relevant observation . the study by pascual and coworkers provides evidence for increased secretion of il-1 by freshly cultured pbmcs from these patients compared with pbmcs from healthy subjects ( 7 ) . in the absence of exogenous stimulation , cultured pbmcs from healthy subjects do not release il-1 , but upon stimulation synthesize the il-1 precursor and release the processed form of il-1 into the supernatant . however , more than 50% of the total il-1 precursor synthesized remains inside monocytes from healthy donors . the amount of il-1 released from pbmcs of five sojia patients was more than 10-fold greater than five healthy controls ( 7 ) . in the same cultures , the release of tnf and il-6 was similar for healthy and affected subjects , suggesting that the elevated release of il-1 was not due to increased monocyte numbers or increased activation of sojia pbmcs . unfortunately , il-1 secretion was induced using the unorthodox combination of pma plus ionomycin , whereas most studies of dysfunctional il-1 release use tlr agonists . nevertheless , the disease - related increase in il-1 secretion may explain the role for il-1 and the responses to il-1ra in these patients who failed to respond to conventional treatments . increased secretion of il-1 from cultured pbmcs has also been reported for a growing number of inherited , chronic autoinflammatory syndromes , each of which responds to il-1ra ( 1316 ) . in these syndromes , increased secretion of il-1 is due to a single amino acid mutation in the nalp-3 gene , which controls the activation of caspase-1 found in the il-1 inflammasome ( 17 ) . similar to the situation in sojia , circulating levels of il-1 are not detected in these patients ( 13 ) but increased secretion of il-1 is observed in vitro . the single point mutation in the nalp-3 gene causes the loss of tight control of il-1 processing . as a result , relatively minor stresses such as exposure to cold results in increased secretion of il-1 with consequent systemic disease ( 13 ) . it appears that the nalp-3 gene provides an important roadblock to control the secretion of il-1 and raises the issue of a defective roadblock in sojia . although the reason for the increased secretion of il-1 in the sojia patients remains unclear , both sojia patients and patients with nalp-3 mutations share the phenotype of systemic disease and increased secretion of processed il-1 in vitro . there are chronic inflammatory syndromes without mutations in the nalp-3 gene but nevertheless elevated il-1 release in vitro and also respond to il-1ra therapy ; presently , these are mutation - negative neonatal onset multisystem inflammatory disease ( nomid ; reference 18 ) , pyogenic arthritis , pyoderma gangrenosum , acne syndrome ( papa ; reference 19 ) , and familial mediterranean fever ( fmf ; reference 20 ) . both papa and fmf are genetic diseases associated with the intracellular protein pyrin , which participates in maintaining procaspase-1 as an inactive enzyme . mutations of the pyrin gene in mice , similar to those found in humans with fmf , result in increased caspase-1 activity and increased secretion of il-1 ( 21 ) . although these systemic , multisystem syndromes are not common diseases , they reveal a fundamental role for il-1 in systemic inflammation regardless of the cause . as shown in fig . these are recurrent fevers , neutrophilia , thrombocytosis , elevated serum amyloid a and c reactive protein , and anemia . hearing loss , developmental delay , and aseptic meningitis can also be observed in childhood . the endothelium is a prime target for il-1mediated inflammation as il-1 receptors on the endothelium trigger prostaglandin e production , cause bone marrow release of neutrophils , and induce the production of il-6 . in fact , il-1 induction of il-6 accounts for hepatic acute phase protein synthesis and the thrombocytosis . the finetuning of il-1mediated inflammation is revealed upon stopping and restarting il-1ra . upon cessation of il-1ra therapy , clinical signs and symptoms of disease as well as biochemical and hematological abnormalities rebound within days and resolve upon resumption of il-1 receptor blockade ( 10 , 1416 ) . in the pascual study , il-1ra therapy was temporarily withdrawn in two patients due to viral - like illness , during which time the erythrocyte sedimentation rate increased , only to fall upon restarting therapy ( 7 ) . systemic manifestations of il-1. active il-1 is secreted by many cell types including monocytes and macrophages ( center ) . il-1 enters the circulation and triggers il-1 receptors on the hypothalamic vascular network resulting in synthesis of cyclooxygenase-2 , which causes brain levels of prostaglandin e2 to rise , thus activating the thermoregulatory center for fever production ( reference 27 ) . in the periphery , il-1 activates il-1 receptors on the endothelium resulting in rashes and the production of il-6 . circulating il-6 stimulates liver hepatocytes to synthesize several acute phase proteins , which accounts for the increase in erythrocyte sedimentation rate in sojia . il-1 also acts on the bone marrow to increase mobilization of granulocyte progenitors and mature neutrophils , resulting in peripheral neutrophilia although sojia clearly fits with the clinical , hematological , and biochemical manifestations of the autosomal dominant autoinflammatory syndromes , and although sojia shares with these inherited diseases increased secretion of il-1 , no genetic mutations have yet been described in sojia patients that relate to il-1 secretion or any other genetic marker . however , to our knowledge no systematic examination of gene defects in the components of the il-1 inflammasome has been made in sojia . there is no clear pattern of inheritance in sojia , and like other autoimmune diseases , the incidence is greater in families but without identification of a specific gene or mutation . it is likely that other agents that prevent il-1mediated disease will be effective in sojia . these are the il-1 trap , il-1specific monoclonal antibodies , il-1 receptor type i specific monoclonal antibodies , and the caspase-1 inhibitor ( 22 ) . it is also possible that agents that inhibit the nucleotide receptor p2x7 will reduce il-1mediated disease ( 23 ) . if sojia is due to dysfunctional control of caspase-1 activity or the p2x7 receptor , treatment options may be targeted to the mechanism of il-1 secretion . it is a general principal in therapeutics to target the most distal mechanism of a disease process . several levels of control of the synthesis , processing , and secretion of il-1 have evolved , and one may assume that these function to limit inflammation . moreover , once released , il-1 must contend with competition for receptor occupancy with the naturally occurring il-1ra , the binding and neutralization by the il-1 type ii decoy receptor ( 24 ) , and the formation of inactive complexes with constitutively secreted soluble il-1 receptor accessory protein ( 25 , 26 ) , each of which also limit il-1 responses . although increased secretion of il-1 may account for il-1 activity in sojia , loss of control of these additional mechanisms may also be disrupted in sojia .

a growing number of systemic inflammatory diseases characterized in part by recurrent fevers , leukocytosis , anemia , and elevated acute phase proteins are linked to interleukin ( il)-1 activity since rapid and sustained resolution is observed upon specific blockade of il-1 receptors . rapid resolution of systemic and local inflammation is now also reported in systemic onset juvenile idiopathic arthritis ( sojia ) . loss of control of the secretion of il-1 might be a common mechanism explaining the aberrant activity of il-1 in these diseases .

hormones , paracrines , autocrines and other mediators define the permeability of the endothelial barrier , the anti - thrombotic nature of the endothelial cell surface and endothelium - dependent blood pressure regulation ( reviewed in current special issue ; and in libby 2002 ; luscher 1990 ; landmesser and drexler 2007 ; vierhapper et al . 1990 ; wojciak - stothard and ridley 2002 ; palmer et al . endothelial mechanobiology is a young field of research and little is known about mechanics - dependent signaling pathways . this is mainly due to the lack of proper techniques to quantify mechanics in living cells . over the last decade , however , considerable progress has been made in various techniques , such as atomic force microscopy , laser tweezers , optical trap , pipette aspiration and microrheology . experimental science is now equipped with a full tool kit facilitating the investigation of cellular mechanics and its physiological relevance ( lee and lim 2007 ; van vliet et al . . this review will highlight recent advances in the field of endothelial nanomechanics and its impact in endothelial physiology . mechanobiology of the vascular system can be separated into cell mechanics and mechanical stimuli . on the one hand , external forces like fluid shear stress ( fss ) , vessel wall tension , vascular hydrostatic pressure and cell cell contacts determine the mechanical stimuli in the cardiovascular system . these stresses affect endothelial function via mechanotransduction , i.e. , activation of mechanosensitive pathways ( tzima 2006 ; ando and yamamoto 2009 ; shyu 2009 ; johnson et al . the corresponding mechanosensors exhibit various elements , including mechanosensitive ion channels , adhesion proteins , tyrosine kinase receptors , or caveolae ( liu et al . 2013 ) . cell mechanics , on the other hand , describes the dynamics of cell ( and tissue ) elasticity , measured as mechanical stiffness and its impact on endothelial physiology . in more detail , the four most prominent and mechanically distinct compartments in the endothelium are ( 1 ) the glycocalyx , ( 2 ) the cell cortex , ( 3 ) the cytoplasm and ( 4 ) the nucleus ( kasas et al . 2005 ; dahl et al . 2008 ; oberleithner et al . 2009 , 2011 ; martins et al . 2012 ; weinbaum et al . 2007 ) . recently , nanomechanics has come into the focus of research as it turned out that the stiffness of the single cellular compartments has a crucial impact on endothelial cell function . to understand the exact meaning of cell mechanics and its impact upon physiological mechanisms , it is important to define the molecular basis of the nanomechanical properties and to characterize their influence on cellular signaling processes . the endothelial glycocalyx ( egc ) is a thick carbohydrate - rich layer , lining the luminal side of the endothelial surface that consists of proteoglycans and glycoproteins . the proteoglycans are decorated with long carbohydrate side chains , the glycosaminoglycans , among which heparan sulfate is the most prominent in the egc . this mesh serves as a host for specific plasma proteins , soluble proteoglycans and hyaluronic acid . together , they form a dynamic and complex interface between blood and tissue ( fig . 1 ) . the total volume of the egc in the human body is about 1.7 l and its thickness varies from a few hundreds of nanometers in capillaries to a few micrometers in arteries ( van den berg et al . 2006 , 2008 ) . due to its high water content and the loose network , the egc is several times softer than the underlying subcellular structures ( oberleithner et al . glycocalyx and cytoskeletal organization of endothelial cells determine the mechanical characteristics of the endothelium cellular nanomechanics . glycocalyx and cytoskeletal organization of endothelial cells determine the mechanical characteristics of the endothelium one hallmark function of the egc is the transmission of biochemical and biomechanical signals from the blood into endothelial cells . different processes are known that alter the nanomechanical properties of the egc . as a polyanionic bio - gel , its volume and mechanics are regulated by the respective electrolyte concentration ( wolf and gingell 1983 ; peters et al . it has been shown that an extracellular sodium concentration in the upper physiological range leads to a compact egc ( = collapse ; oberleithner et al . in contrast , treatment of endothelial cells with the polyphenol - rich compound ws1442 induces an increase in volume ( = swelling ) of the egc ( peters et al . . the specific mechanisms of collapse and swelling depend on dominant interactions in the system ( hydrogen bondings , ionic interactions , hydrophobic / hydrophilic properties , etc . ) ( quesada - perez et al . 2011 ) . other processes , which modulate the nanomechanics of the egc , are shedding and biosynthesis . both can be induced by biochemical factors like hormones and enzymes ( reitsma et al . 2007 ) or by fss ( gouverneur et al . 2006 ; zeng and tarbell 2014 ) . enzymatic digestion of heparan sulfate as well as treatment of endothelial cells with thrombin , lipopolysaccharides , or tumor necrosis factor compromises the structural integrity of the egc leading to reduced egc volume and stiffness ( peters et al . in contrast , the biosynthesis of the egc after enzymatic degradation leads to an increase in volume and a decrease in stiffness , as has been shown in vitro ( bai and wang 2012 ) . the egc is the very first layer of the endothelium that comes into contact with blood . thus , alterations of egc nanomechanics lead to a change in the mechanical interaction between blood cells and the egc constituents . it is likely that a collapsed egc can be less deformed by fss and thereby becomes unable to transmit signals into the cell adequately , a condition that can promote cardiovascular disease . additional to their function as fss transmitters , the proteoglycan - associated heparin sulfate residues serve as attachment points for sodium ions and substances like albumin and other blood - borne proteins , hormones and enzymes ( siegel et al . 1996 ; reitsma et al . 2007 ; quinsey et al . 2004 ; kato 2002 ; li et al . 1998 ; ballinger et al . 2004 for instance , increased plasma sodium concentration leads to a stiffening of the glycocalyx and simultaneously increases cellular sodium uptake ( korte et al . 2a ) is supposed to stand for a fully - functional endothelium , whereas a shedded or collapsed egc most likely exerts adverse effects on the vascular system . 2b ) , facilitates edema formation ( salmon and satchell 2012 ; strunden et al . 2010 ) , cell vessel wall interactions ( constantinescu et al . 2003 ; henry and duling 2000 ; mulivor and lipowsky 2004 ) , loss of fss perception ( thi et al . 2004 ; mochizuki et al . 2003 ) and endothelial dysfunction ( nieuwdorp et al . 2005 , 2006 ; drake - holland and noble 2012 ) . 2c ) has been discussed as being a promoter of vascular diseases ( peters et al . c acute collapse leads to similar functional changes as described in b but egc nanomechanics are different egc stiffness in endothelial function . c acute collapse leads to similar functional changes as described in b but egc nanomechanics are different crossing the cell membrane , the first significant mechanical compartment inside the cell is , directly underneath the plasma membrane , the cell cortex . due to the three - dimensional cytoskeletal organization , the cell cortex as well as the cytoplasm and the nucleus , can be characterized by their distinct cytoskeleton - dependent nanomechanical properties ( oberleithner et al . 2009 ; kasas et al . 2005 ; dahl et al . 2008 ; martins et al . 2012 ) . directly beneath the plasma membrane ( 50200 nm ) , actin is organized in form of a dynamic network ( fig . 1 ) ( miranda et al . 1974 ; koning et al . 2008 ) . the cortical web , also known as peripheral actin , is made of cross - linked actin filaments ( f - actin ) that provide a supportive structure to the plasma membrane and its embedded proteins ( pollard and cooper 2009 ) . the organization of the cortical cytoskeleton is highly dynamic as the actin filaments are regulated by a variety of actin binding proteins ( dos remedios et al . additionally , actin polymerization is stimulated by cdc42 and rac1 , both members of the rho gtpase family . cortactin and other proteins , such as filamin or fascin , stabilize the actin web by cross - linking filaments . destabilization of the cortical actin network due to filament disassembly is induced by cofilin , gelsolin , or rhoa . finally , motor proteins are able to cross - link actin filaments and simultaneously facilitate the force administration to the cortical web ( dos remedios et al . . a high rate of actin polymerization and a dense filament organization go along with a relatively stiff cortex , whereas a depolymerization of f - actin results in cortical softening ( kasas et al . 2005 ) . filamin a and/or -actinin cross - link actin filaments and subsequently lead to an increased stiffness of the actin web ( esue et al . 2009 ; kasza et al . furthermore , motor proteins ( e.g. , non - muscle myosin ii ) contribute to cortical stiffness as they generate contractile forces within the filament network . by this , motor proteins induce a lateral tension within the cortical network leading to an inward directed tension ( paluch et al . there is evidence that nanomechanics of the cell cortex has significant influence on endothelial physiology ( hoffman and crocker 2009 ; paluch et al . in particular , nitric oxide ( no ) release and barrier function , both hallmarks of endothelial function , appear to be influenced by cortical nanomechanics . in the vascular endothelium , a softening of the cell cortex induces no synthesis and thereby is likely to facilitate vasodilation followed by an increase in tissue perfusion and decrease of blood pressure ( oberleithner et al . 2007 , 2009 ; szczygiel et al . 2011 ) . under certain physiological conditions , plasma potassium concentration can increase locally up to 12 mm , e.g. , due to muscle or neuronal activity ( nordsborg et al . 2003 ; kofuji and newman 2004 ) , which induces a rapid decrease in cortical stiffness ( oberleithner et al . 2009 ) . this softening of the cell cortex is driven by a membrane potential - dependent depolymerization of the submembranous actin web ( callies et al . a decrease in cortical stiffness is generally caused by a destabilization of the cortical actin web , which verifies the physiological link of elasticity , actin organization and endothelial function ( fels et al . 2012 ) . simultaneously , the activity of the endothelial no synthase ( enos ) increases ( fig . firstly , it has been shown that enos activity is stimulated by an association with g - actin while it is inhibited by an association with f - actin ( kondrikov et al . a decrease in cortical stiffness due to f - actin depolymerization may increase the association of enos with g - actin and therefore directly stimulates no release ( fels et al . mechanosensitive calcium channels in a flexible membrane are supposed to be readily activated by shear stress and subsequently increase intracellular calcium levels ( knudsen and frangos 1997 ; kuchan and frangos 1994 ; galan et al . 2011 ) . as enos is activated by the calcium - binding protein calmodulin , a soft cortex is likely to promote no release . since no is a vasodilating gas , softening - induced enos activity will lead to increased tissue perfusion furthermore , blood pressure may decrease in case of systemic softening of the endothelial cortex.fig . softening of the cell cortex , induced by cortical actin depolymerization , decreases membrane abundance of enac and endothelial sodium uptake and increases enos activity and increases endothelial barrier function cortical stiffness influences endothelial function . softening of the cell cortex , induced by cortical actin depolymerization , decreases membrane abundance of enac and endothelial sodium uptake and increases enos activity and increases endothelial barrier function interestingly , these modulations of cortical stiffness and enos activity are independent of the bulk nanomechanics of the endothelial cells ( oberleithner et al . 2009 ; fels et al . 2012 ) . while , under physiological conditions , potassium concentrations may rise to a larger extent only locally , other mediators potentially act more systemically , i.e. , on the whole vasculature . the mineralocorticoid hormone aldosterone , as well as the cytokine tumor necrosis factor alpha ( tnf ) , induce transient cortical softening associated with an increase in no release ( fels et al . interestingly , sustained exposure to aldosterone or tnf results in an opposite effect , described in detail in the subsequent section . in addition to the regulation of enos activity , other vascular mechanisms such as endothelial permeability are influenced by cortical nanomechanics . since the link between no synthesis and cortical stiffness is the dynamic reorganization of the cortical actin cytoskeleton , it was hypothesized that the stiffness - dependent barrier function is again based on cortical actin dynamics . this hypothesis is verified by the finding that exposure to sphingosine-1-phosphate increases peripheral ( cortical ) stiffness in pulmonary endothelial cells and thereby increases barrier function . the barrier - enhancing effect is most likely mediated via a signaling cascade including cortactin activation and subsequent actin filament formation in the cell cortex ( arce et al . in contrast , thrombin acts as the counterpart in regulation of endothelial permeability as it decreases cortactin in the cortex and at the same time increases permeability ( arce et al . stimulation of myosin activity , as a second important determinant of cortical stiffness , leads to an increase in barrier function ( dudek et al . hence , it may be concluded that a soft cell cortex indicates a physiological function of the endothelium . in analogy to cortical softening , increased polymerization of cortical actin and concomitant stiffening of the endothelial cell cortex has ( patho-)physiological consequences in the control of endothelial permeability and the response to hormone action ( hall 1984 ; birukova et al . endothelial stiffening by the c - reactive protein and the cathelicidin ll-37 peptide is found to have anti - inflammatory effects , possibly due to a decrease in endothelial permeability ( kusche - vihrog et al . 2011 ; byfield et al . these effects contribute to maintaining tissue fluid homeostasis and hence counteract the increased no production and subsequent drop in blood pressure often accompanying acute inflammatory processes and septic shock . 3b ) , as a cell with a stiff cortex produces reduced amounts of no ( oberleithner et al . 2007 , 2009 ; kidoaki and matsuda 2007 ; fels et al . 2010 ) . the phenomenon increased endothelial cortical stiffness / reduced no release was recently termed stiff endothelial cell syndrome ( secs ) ( lang 2011 ) . diminished no release and thus a shift of endothelial action towards increased vasoconstriction , is also the hallmark of endothelial dysfunction , a clinical predictor for expecting cardiovascular diseases later in life ( endemann and schiffrin 2004 ; schachinger et al . one of the crucial prerequisites leading to an increase in endothelial stiffness and a reduced no production is a rather high plasma aldosterone level , a major risk factor for vasculopathies . upon prolonged treatment ( > 20 min ) with aldosterone , endothelial cells swell ( oberleithner et al . both of these effects are blocked either by the specific epithelial sodium channel ( enac ) blocker amiloride or by the aldosterone antagonist spironolactone . both prevent a ( further ) augmentation in endothelial stiffness upon raising extracellular sodium concentrations from 135 to 145 mm in the presence of aldosterone ( oberleithner et al . incidentally , these two factors , aldosterone and high sodium , also cause an increase in enac surface expression ( kusche - vihrog et al . 2008 ; korte et al . 2012 ) , indicating a key role of aldosterone in controlling enac activity by increasing the channel s abundance at the endothelial cell surface ( alvarez et al . 2002 ; snyder 2002 ) . enac is widely abundant in various tissues throughout the human body , including epithelia tissues where this sodium channel mediates the rate - limiting step of sodium transport ( garty and palmer 1997 ; golestaneh et al . 2001 ) . in vascular endothelium , enac is different , as the major portion of sodium exits the blood capillary system through a more or less leaky paracellular pathway ( mehta and malik 2006 ) . also , the enac expression level is clearly lower as compared to epithelial tissues ( kusche - vihrog et al . recently , a direct link between enac expression and no release has been established , suggesting the functional role of enac in the vascular endothelium ( jeggle et al . 2013 ) . cells with elevated enac expression exhibit an increase in mechanical cortical stiffness in vitro and ex vivo . taken together , enac determines cortical endothelial stiffness and plays a major role in endothelial ( dys)function contributing to the control of vascular tone . the mechanistic basis of the link between enac and endothelial stiffness most likely relies on the direct interaction of enac with f - actin located in the subapical pool underneath the plasma membrane ( mazzochi et al . alterations in cortex formation upon changes in the enac surface expression could thus also be ascribed to this interaction , as it might increase actin polymerization in this compartment and hence increase cortical stiffness . the sequence of events , whether enac membrane insertion promotes actin polymerization or vice versa , has not yet been elucidated . in addition to chemical mediators , mechanical stimuli affect actin organization ( most likely influencing cortical stiffness ) and endothelial function . an in vitro increase in hydrostatic pressure , mimicking blood pressure in vivo , induces actin reorganization and affects endothelial permeability ( shin et al . fluid shear stress influences actin reorganization ( seebach et al . 2007 ) and alters the endothelial barrier ( tarbell 2010 ; katoh et al . finally , substrate stiffness was shown to induce actin polymerization , modulating barrier function in a dose - dependent manner ; substrate stiffness simulating physiological conditions improves barrier function while stiffer and softer substrates disrupt barrier function ( birukova et al . the elasticity of the cell cortex can be seen as a parameter that determines endothelial physiology in a more general way . for instance , aging cells lose their elasticity due to an increased cytoskeletal organization ( sokolov et al . 2006 ; schulze et al . 2010 ) . even basic processes that usually occur in cellular life , such as mitosis , differentiation and development , can be related to changes in cortical stiffness ( stewart et al . 2011 ; patel et al . 2012 ; kidoaki and matsuda 2007 ; hoffman and crocker 2009 ) . fountain of youth and guarantees a reasonably low blood pressure and a healthy organism . apparently , the physiological impact of cortical stiffness on physiological mechanisms is highly tissue - specific . in contrast to the endothelium , a soft cortex can even indicate a pathophysiological state of a cell . in ventricular myocytes , for instance , the relationship between elasticity and no release appears to be different . there , an inhibition of myosin by blebbistatin decreases cell stiffness and simultaneous inhibits nos activity ( walsh and cole 2013 ; dedkova et al . furthermore , it has been shown that the metastatic behavior of cancer cells directly correlates with the cell s elasticity . cancer cells with a low elasticity ( soft cells ) are more likely to spread than stiffer ones ( ketene et al . additionally , breast cancer cells induce softening of the endothelium to ease extravasation , leading to facilitated metastasis formation ( mierke 2011 ) . while single actin filaments are predominantly found in the cortex , actin stress fibers ( bundles of 1030 filaments ) span the whole cytoplasm . they also contribute to focal adhesions and thus are involved in the mechanisms of cell motility ( pellegrin and mellor 2007 ; fletcher and mullins 2010 ; prasain and stevens 2009 ) . microtubules are tube - like polymerized protein ( tubulin ) filaments facilitating the transport of organelles and vesicles . both microtubules and intermediate filaments determine cytoplasmic ( bulk ) stiffness ( wang 1998 ; janmey et al . 1991 ; kasas et al . 2005 ; herrmann et al . the molecular nature of the contribution of tubulin to cell nanomechanics has been investigated in detail ( gardel et al . so far , the microtubule network is supposed to represent a compressive load - bearing component that counteracts the tensile forces generated by the cortical actimyosin web ( ingber 2003 ) . the physiological relevance in stiffness - dependent signaling pathways in endothelial cells is , however , still unclear . what is known so far is that microtubule - associated proteins ( e.g. , map65 ) directly influence the flexibility of single microtubules , most likely resulting in a change in cellular elasticity ( portran et al . furthermore , in the developing organ of corti , the fibroblast growth factor induces a microtubule - dependent decrease in cell stiffness leading to hearing loss ( szarama et al . intermediate filaments , of which vimentin is the dominant network - forming member in the endothelium , support the three - dimensional organization of the cell and its organelles ( kamei 1994 ) . their mechanical properties as well as their contribution to cell mechanics have been reviewed by herrmann et al . intermediate filaments play a key role in mechanotransduction as mutation or deletion of several intermediate filament proteins leads to cell fragility , heart failure and muscle dystrophies . even leucocyte diapedesis and endothelial nitric oxide release depend on proper intermediate filament formation ( herrmann et al . although the role of intermediate filaments in ( endothelial ) physiology is well documented and the molecular contributions to cell mechanics are known , evidence for a direct link between intermediate filament - dependent mechanics and endothelial function is still missing . data on intermediate filament and microtubule mechanics and its impact on endothelial physiology indicate that several different nanomechanical signaling pathways exist that await future investigation . each individual component of the cellular cytoskeleton described in the previous sections is directly linked to the cell nucleus . plectin ( nesprin-3 ) provides a link to the cytoplasmic network of intermediate filaments ( wilhelmsen et al . 2005 ) , whereas nesprin-1/2 mediates binding to microtubules and the actin network ( padmakumar et al . nesprins connect cytoskeletal networks with the intranuclear lamin network via sun 1/2 proteins ( haque et al . it is therefore conceivable that all of the mechanical stimuli perceived by the cell through actin , microtubule , or intermediate filament networks are integrated on the level of the nuclear lamina . similar to the cortical mechanics , the nucleus of an eukaryotic cell could also be an important contributor to the overall mechanics of the cell . nuclei of several cell types have been shown to be two- to ten - fold stiffer than the respective cytosol ( martins et al . , the cell nucleus could be envisioned as being a supramolecular shock absorber capable of withstanding considerable stress ( dahl et al . a connection between cytoskeleton and the nuclear envelope further strengthens the notion of a direct involvement of the nucleus in mechanosensing and mechanotransduction ( maniotis et al . recently , the pioneering work from the laboratory of dennis discher demonstrated that the nucleus can serve as an intracellular mechanostat ( swift et al . 2013 ) , a structure that is able to sense and respond to changes in the mechanical properties of the cellular environment by changing its own stiffness ( fig . elasticity of the cell nucleus regulates gene expression and endothelial function nuclear elasticity in endothelial function . elasticity of the cell nucleus regulates gene expression and endothelial function in the light of recent advances in the field of nuclear mechanics , it is tempting to speculate that nuclei of endothelial cells could be directly involved in mediating physiological functions in response to mechanical stimuli . nuclei of endothelial cells are particularly large in comparison to those of other cell types . nuclei bulge into the lumina of blood vessels , thus being directly exposed to the shear strain exerted by the blood flow . consequently , any change in nuclear volume and/or stiffness could have a strong influence on blood flow , in particular in vessels of small inner diameters . according to hagen - poisseulle s law thus , a small change in nuclear volume , as shown and quantified in vascular endothelium in response to aldosterone ( oberleithner et al . bulging nuclei could be a significant hindrance for blood flow , in particular when the nuclei stiffen at the same time . in addition , nuclei of the cells exposed to shear stress stiffen ( fig . apparently , flattening and stiffening of the nuclei could be the consequence of a shape change induced by shear forces . however , the recovery in shape and , at the same time , the maintenance of nuclear stiffness upon release of the shear forces , point towards a more complex nuclear remodeling mechanism . the question , whether the expression of the lamin a network at the nucleoplasmic side a major determinant in the control of the nuclear envelope plasticity ( lammerding et al . 2006)is responsible for the nuclear mechanostat function , remains open ( swift et al . there is another reason indicating a potential role of lamin a in the regulation of nuclear plasticity . a mutation of lamin a , which results in an irreversible ( permanent ) anchoring of the mutated product in the nuclear envelope , results in a severe clinical manifestation termed hutchison - gilford progeria syndrome ( hgps ) ( merideth et al . affected individuals undergo drastically accelerated aging and die in their teens of cardiovascular - related diseases such as stroke or myocardial infarction ( gerhard - herman et al . 2012 ) . at tissue level , major arteries of hgps patients demonstrate a severe degeneration of the smooth muscle cell layer ( stehbens et al . the role of endothelial cells in the development of the syndrome has not been fully assessed to date . however , at the level of individual cell nuclei , the presence of a mutated rigid lamin a network at the inner side of the nuclear envelope has been linked to significant stiffening of such nuclei ( philip and dahl 2008 ) . it is therefore tempting to speculate that stiff nuclei cause an overall stiffening of the endothelial cell . this could result in a severe form of the stiff endothelial cell syndrome ( lang 2011 ) . in this case , the ability of the endothelial cell of sensing and responding to mechanical stimuli might be disrupted . as a result , the downstream signaling directed towards vascular smooth muscle is expected to be impaired , possibly explaining the degradation of vascular smooth muscle cell layer in hgps . in summary , the question , whether nuclear mechanics follows mechanical alterations evoked from the exterior environment as postulated by the nuclear mechanostat theory ( swift et al . 2013 ) and/or whether any changes in nuclear elasticity could primarily alter endothelial function , will be an exciting area of cell research in the near future . in conclusion , the elastic properties of the four compartments , ( 1 ) glycocalyx , ( 2 ) cortex , ( 3 ) cytoplasm and ( 4 ) nucleus , are mainly determined by the composition of the respective structural elements . the dynamic interactions between those elements with cross - linker and motor proteins determine the mechanical properties of the respective region . nanomechanics provides information on the physiological state of the endothelial cell in terms of nitric oxide release and barrier function . a soft cell cortex , combined with a soft , well - hydrated glycocalyx , increases no formation , which is a prerequisite for a functionally intact vasculature . any changes in elasticity at the level of the egc glycocalyx , the cell cortex and the cell nucleus , can have a significant influence on endothelial function in terms of local blood flow , tissue perfusion and , finally , arterial blood pressure .

the mechanical characteristics of endothelial cells reveal four distinct compartments , namely glycocalyx , cell cortex , cytoplasm and nucleus . there is accumulating evidence that endothelial nanomechanics of these individual compartments control vascular physiology . depending on protein composition , filament formation and interaction with cross - linker proteins , these four compartments determine endothelial stiffness . structural organization and mechanical properties directly influence physiological processes such as endothelial barrier function , nitric oxide release and gene expression . this review will focus on endothelial nanomechanics and its impact on vascular function .

voluntary euthanasia ( ve ) is the act of requesting deadly medical practices in order to terminate one s life ( 1 ) . the process of decision making ( like that for ve ) is composed of two interwoven brain based components ( 2 ) . one is the conscious power of analyzing the condition and utilizing algorithmic like strategies , which is known as cognition ( 2 , 3 ) . this part is important since it affects the person s ability of seeking in the condition ( 4 ) , estimating the parameters of a situation ( 5 ) and finally deciding ( 2 ) . chronic pain patients ( cpps ) develop disappointing emotional backgrounds , which affect their mental power of decision - making ( 6 ) . this article argues about the mental incompetence relating decision - making that accompanies chronic pain in such patients . this can be considered as equal to the historically known conditions of torturing a person and wanting him / her to accept or confess something in return for relieving pain ( 7 ) . in case of ve , the agent is the internal mental burden of a disease and the thing wanted in return for reliving the pain is dying acceptance . therefore , in this condition the stimulator of accepting death is the emotion of escaping from pain in return for anything . this is the exaggerated form of the normal emotion of fearing and escaping from painful situations ( 8) . in fact , a cpp fears from experiencing excess pain . according to this outlook and having in the mind that emotions - in the way it is described in the literature ( 9 ) - are the most important determinants of the alternative to be chosen ( 10 ) , here it is proposed that cpps are emotionally and hence mentally incompetent for making critical decisions . studies using mri technology have shown brain deficits in cpps ( 1114 ) . more specifically in the state of chronic pain it is stated that the orbitofrontal part of the brain cortex is preferentially involved . this part functions as a link between multiple brain regions with distinct roles in the emotional assessment . these regions include lateral prefrontal areas which handle memory , effector components of the brainstem which are responsible for producing emotional responses and those parts that are considered as the brain substrates of feelings like insula and amygdale ( 6 ) . according to these findings a cpp does not have a biologically healthy brain for providing the accuracy of the act of choice - making especially when he / she wants to do so for emotion - laden alternatives like that of whether to escape or cope with the pain . this can be considered as equal to the historically known conditions of torturing a person and wanting him / her to accept or confess something in return for relieving pain ( 7 ) . in case of ve , the agent is the internal mental burden of a disease and the thing wanted in return for reliving the pain is dying acceptance . therefore , in this condition the stimulator of accepting death is the emotion of escaping from pain in return for anything . this is the exaggerated form of the normal emotion of fearing and escaping from painful situations ( 8) . in fact , a cpp fears from experiencing excess pain . according to this outlook and having in the mind that emotions - in the way it is described in the literature ( 9 ) - are the most important determinants of the alternative to be chosen ( 10 ) , here it is proposed that cpps are emotionally and hence mentally incompetent for making critical decisions . studies using mri technology have shown brain deficits in cpps ( 1114 ) . more specifically in the state of chronic pain it is stated that the orbitofrontal part of the brain cortex is preferentially involved . this part functions as a link between multiple brain regions with distinct roles in the emotional assessment . these regions include lateral prefrontal areas which handle memory , effector components of the brainstem which are responsible for producing emotional responses and those parts that are considered as the brain substrates of feelings like insula and amygdale ( 6 ) . according to these findings a cpp does not have a biologically healthy brain for providing the accuracy of the act of choice - making especially when he / she wants to do so for emotion - laden alternatives like that of whether to escape or cope with the pain . the central concept of this article is that a person who is under the mental pressures is not capable of making critical decisions . the same concept is already accepted for the other categories of people who can not think well such as drunken individuals ( 15 ) . the fact that mentally under pressure patients can not think well is also indirectly accepted by all societies that refuse to obey a mentally ill patient s will in the light of labeling the patient unqualified for making a will according to the judgment of court . with the same bases , this article s concept can be accepted by the court and become a law in such societies . therefore , the patient s will of terminating his / her life must not be accepted like that of a drunken person . deciding to undergo euthanasia is the most critical decision of a person ( not only a patient ) since this decision leads to the end of making other decisions . in this respect , deciding to die is different from deciding to buy a car because the latter is reversible or can be compensated in the future and does not violate the right of making other decisions , something that the former intrudes in . therefore the ability of making the decision of undergoing euthanasia must be considered as something different from just a right . in this mental incompetence is the common quality in both conditions of chronic pain and will rejection in the court . therefore the accepted rules for both of these two conditions must be the same . this means that any nation that rejects the will of a mentally incompetent individual must do the same about the request of a cpp for ve .

emotional status and its brain bases change in a chronic pain patient and this change affects his / her decision making ability . moreover , it is accepted that a mentally disturbed individual is not competent to make critical decisions . according to these bases , this article demonstrates that such patients are not entitled to request voluntary euthanasia .

epilepsy is the second most prevalent neurological disorder ( \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } \(\sim 1\ ) \end{document}% prevalence ) affecting approximately 67 million people worldwide with up to 75% from developing countries . eeg is important in developing countries for various clinical uses , including evaluation of suitability for epilepsy surgery and treatment of status epilepticus . epilepsy diagnosis is complicated by the fact that electrical potentials recorded from scalp electroencephalogram ( eeg ) have poor sensitivity to localized epileptiform activity , especially high frequency oscillations ( hfos ) which now attract a lot of attention as a promising marker of epileptogenesis and ictogenesis . misdiagnosis of epilepsy is very common in patients of all ages and occurs in up to 50% of the patients , . the hfos occupy the gamma ( 30100 hz ) , ripple ( 100200 hz ) and fast ripple ( 200500 hz ) bands along with delta , theta , alpha and beta bands making up the remainder of the spectral content of eeg . appearance of fast eeg rhythms at the onset of seizures in humans and animal models of epilepsy has been described in many studies and first reported in humans by fisher et al . and allen et al . . fast rhythms are especially evident in intracranial recordings in patients considered for surgical treatment of their drug - resistant epilepsy . in recent years , there has been a surge in the number of publications on hfos and their role in epilepsy ( for reviews , see , . numerous studies on animal models and epilepsy patients have revealed that : 1 ) hfos are one of the most common early manifestations of seizures recorded within minutes before seizure onset ; 2 ) hfos appear to be the most likely eeg correlate of a seizure onset zone ( soz ) ; and 3 ) the removal of hfo - generating areas correlates with good surgical outcome , , , . thus , hfos appear to be an excellent marker for the epileptogenic zone but their recording from scalp with conventional disc electrodes is difficult . eeg has been a very useful research and clinical instrument for almost a century since its first human use by hans berger in 1920s . the distance to the brain and the relatively large electrical impedance of the skull significantly reduce small electrical signals generated by individual neurons . to be detectable at the scalp , the signal should be produced by a relatively large population of neurons acting more or less synchronously at scales of at least several centimeters . only in this case does the sum of individual neuronal signals become large enough to be detected at the scalp . synchronous activity within neuronal assemblies is now considered an important mechanism of information processing , and this synchrony is expressed through neuronal oscillations occurring at different frequencies spanning several orders of magnitude from infraslow ( \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } \(\sim 0.01\ ) \end{document } hz ) up to ultrafast ( \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } \(\sim 1000\ ) \end{document } hz ) ( for a comprehensive review of brain oscillations , see . there is an intricate relationship between temporal and spatial scales of brain oscillations in that higher frequency oscillations ( e.g. , high gamma band > 60 hz ) usually occur at smaller distances between sources and involve smaller neuronal populations . this is because more precise synchronization dictated by shorter oscillatory periods can be achieved only through shorter neuronal connections with shorter propagation times or non - synaptic communication such as ephaptic interactions and gap junctions . long distance connections have longer propagation times and therefore synchronization at larger distances is less precise and preferentially occurs at lower frequencies ( e.g. , within delta , theta , alpha and beta bands ) . as a result , high frequency oscillations > 60 hz usually reflect activity within well localized neuronal assemblies at spatial scales of a few mm , which are not typically visible from the scalp . thus , conventional eeg has lower sensitivity to high frequency components of brain activity , . conventional scalp eeg is also contaminated by noise represented by non - brain electrical activity such as ocular artifacts , scalp muscle potentials , and the electrocardiogram . additional noise is occasionally produced by abnormally large electrode impedance , electrode movement , amplifier drifts , etc . the noise can be as large as , or even larger than , the brain potential of interest and represents a significant problem for electroencephalographers and neurologists . this noise shows high spatial coherence due to smearing effects of the head volume conductor . conventional disc eeg recordings also have reference electrode problems . a common average reference and concentric electrodes have been proposed to resolve the reference electrode problems . however , in the common average reference recordings , it is possible that components present in most of the electrodes but absent or minimal in the electrode of interest may appear as ghost potentials . thus , there is a strong need to develop new types of electrodes beyond conventional disc electrodes . to overcome the poor signal - to - noise ratio and reference problems of the disc electrodes besio et al . have developed the tripolar concentric ring electrode ( tcre ) , a transformative electrode configuration . the tcre consists of three electrode elements - outer ring , middle ring , and the central disc ( fig . 1 , b ) . it is distinctively different from the disc electrode which has a single element ( fig . the novelty of the tcre lies in a new principle of recording electrical activity of the brain from the scalp through its three closely spaced elements . it can provide three separate signals from the three electrode elements used to record two bipolar differential signals for the tripolar laplacian derivation first described in as a weighted sum { 16*(m - d)-(o - d ) } where o , m , and d are the potentials on the outer ring , middle ring , and central disc , respectively . the tripolar signal is a ' hardware realization ' of the laplacian i.e. , the second spatial derivative of the scalp signal . unlike other software methods ( ' laplacian montages ' in conventional eeg systems ) which approximate the laplacian by taking differences between disc electrodes placed , at best , > 1 cm apart , the tcre performs the laplacian automatically and takes bipolar differences of the surface potentials from closely spaced ( \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } \(\sim 1\ ) \end{document } mm ) concentric electrode elements with our custom preamplifier . with the electrode elements so close to each other , globally originating sources such as eye blinks , muscle or motion - related artifacts contribute nearly equally and are attenuated sharply when bipolar differences are performed by the preamplifier . compared with disc signals , eeg recorded with the tcre ( teeg as described by ) has about a 4-fold ( 374% ) increase in signal - to - noise ratio ( snr ) and less than one - tenth ( 8.27% ) the mutual information between signals recorded from two adjacent tcres ( which suggests a significant attenuation of volume conductance effects and , as a result , higher spatial resolution ) , . the tcre also has strong attenuation of various artifacts , 100 db at one radius from the electrode . figure 1.conventional disc electrode ( a ) and tripolar concentric ring electrode ( b ) . conventional disc electrode ( a ) and tripolar concentric ring electrode ( b ) . the current study presents preliminary data from patients with epilepsy in whom teeg was recorded in parallel with clinical conventional scalp eeg . the goal is to demonstrate that tripolar electrodes may provide a unique opportunity to record hfos from scalp and thus improve diagnosis of epilepsy and localization of the seizure onset and the irritative zones . their implementation into clinical practice may eventually help determine where to place and reduce the number of intracranial grids and depth electrodes to be implanted during presurgical evaluation of patients . patients for this study were recruited from the national institute of neurology and neurosurgery ( ninn ; ten patients ) and rhode island hospital ( rih ; two patients ) . patients were referred by the epilepsy clinic in each institution with the diagnosis of drug resistant epilepsy using the international league against epilepsy criteria . diagnosis of epilepsy and epileptic seizures was based on the international classification of seizures 1981 and epileptic syndromes 1989 . ictal and non - ictal recordings were obtained ; for non - ictal recordings lateralization and localization of the irritative zone ( iz ) was determined whereas in ictal recordings the soz or iz was identified with the appearance of behavioral changes associated with focal low voltage , fast activity , flattening or slow wave interruption . to verify that tcres can be used to record physiological signals we recorded from three healthy subjects , one at stanford school of medicine and two at the university of rhode island ( uri ) placing conventional disc electrodes over the occipital lobe ( at the o1 and o2 locations of the 1020 international electrode system with reference at a1 and ground electrode placed on the right collar bone ) . two tcres were placed right next to the conventional electrodes at the o1 and o2 locations ( o1 ' and o2 ' positions , respectively ) . the tcre eeg signals ( teeg ) were first preconditioned with our custom made preamplifiers ( gain \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } \(=20\ ) \end{document } ) and then passed to the bipolar inputs of the ( stanford - eeg-1200 , nihon kohden corporation , tokyo , japan ; uri - grass technologies aura ltm-64 , grass technologies , west warwick , ri ) clinical eeg recording system . all signals were recorded with 1 to 120 hz band - width at 500 s / s and stored to the hard drive for post processing . the subjects were asked to close and open their eyes repeatedly to record the presence or absence of alpha rhythms , respectively . the study 's recording protocol was designed in such a way as to avoid any interference with clinical eeg recording and evaluation . at both the ninn and rih during the attachment of clinical conventional disc electrodes ( referred to as ' eeg electrodes ' or ' eeg signals ' in the subsequent text ) , the patient 's scalp was cleaned with nuprep and then eeg electrodes were placed at the 1020 international electrode system locations using ten-20 paste . collodion was also used to help hold all electrodes in place at the ninn . to obtain teeg recordings in parallel to the clinical eeg , the tcres were placed just behind the disc electrodes in locations close to the 1010 sites and attached to the scalp with ten-20 paste ( fig . the ground was placed on the forehead ( rih , ninn ) and the reference electrode was placed on the forehead at rih and on the oz location at ninn . clinical eeg was recorded with the comet as40 system ( grass technologies , west warwick , ri ) and stored separately for further clinical evaluation . the eeg sampling rate was 200 samples per second ( s / s ) and the low - pass filter was 70 hz ( ninn and rih ) . the teeg data were pre - amplified with the gain equal to either 6 ( for eight patients ) or 100 ( for four patients ) and amplified and digitized with an aura ltm-64 system ( grass technologies , west warwick , ri ) at different sampling frequencies for different patients . for four patients the data were filtered 1100 hz and digitized at 200 s / s , another six were filtered 1200 hz and digitized at 400 s / s and for the remaining two patients the data were filtered 1250 hz and 1500 hz respectively and digitized at 1600 s / s . the 60 hz notch filter was used for all patients . the recording sessions at the ninn usually lasted for six hours , from around 7 am to 1 pm . for the two patients at rih , the recording was stopped shortly after the patient had a seizure ( 130 minutes total ) and for the other the recording lasted 66 minutes . the ninn recording protocol included requests to patients to be sleep deprived the night before coming for a video - eeg monitoring and all patients signed an additional consent form as antiepileptic drugs dosage was reduced by half the previous day of the recording . recorded data were reviewed by board certified neurologists and seizure onset time and duration were determined for each seizure . seizure onset time was defined as the beginning of the first observable seizure pattern in either eeg or teeg . note : t7/p7 and t8/p8 are the same as t3/t5 and t4/t6 in 1020 nomenclature . the blue rings are for standard 1020 electrode locations . the 105 montage with tcres ( red ) placed near the 1020 locations . note : t7/p7 and t8/p8 are the same as t3/t5 and t4/t6 in 1020 nomenclature . the data were stored on the hard drive and exported from twin ( grass technologies , west warwick , ri ) into ascii text files that were then imported to matlab ( mathworks , natick , ma ) and converted to .mat files . for the teeg , next we used a modified version of the algorithm reported by gardner et al . for detection of hfos combined with the visual inspection to rule out high frequency artifacts . the algorithm performed a continuous short - time fourier transform to calculate the power within a particular frequency band over consecutive and half - overlapping one - second epochs using a hamming tapering window . as a result simple thresholding was also used to find events which had power significantly higher than the interictal background eeg . then the time periods when the hfo power was exceeding its average plus two standard deviations were found . frequency bands starting at high gamma band ( \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } \(\sim 60\ ) \end{document } hz ) and up to the upper bound of the band - pass filter that varied for individual patients were searched for hfos . we selected segments up to an hour prior to the onset of the seizures and generated spectrograms to follow the temporal dynamics of pre - seizure hfos . for the patients who had epileptiform activity only but no seizures we calculated spectrograms on 10 minute long segments centered on the periods of epileptiform activity . for the patients who did not have either epileptiform activity or seizures we performed spectral analysis on multiple 10 minute long segments at various times during the recording . to assess the relationship between teeg channels containing hfos preceding seizures and the soz the ratio between the number of teeg channels with hfos in the soz and the total number of teeg channels with hfos was calculated for each patient whose seizure was recorded . we also calculated the ratio between the total number of teeg channels with hfos and the total number of teeg channels to assess how widespread the hfos were for each patient . both ratios were averaged for the five patients with seizures included in this study . only the channels containing hfos preceding the seizures were assessed . soz or iz was determined for each patient independently by three epileptologists ( iemj , jng , and rsf ) based on eeg data and videos only . the epileptologists did not have access to teeg data and were not aware of the hfo detection results . in one patient the third soz was determined by the location of resection by the surgeon . a disjunctive ( logical or ) that is all channels determined as part of the soz ( iz ) by either one of the epileptologists were considered to be a part of the soz ( iz ) . first , we demonstrate the suitability of tcres to provide a sound eeg signal sensitive to physiological rhythms such as alpha activity as well as their insensitivity to myogenic activity which often corrupts scalp eeg recordings especially in the high frequency range > 30 hz . if the tcre attenuates volume conductance effects and has a greater sensitivity to local sources , how good is it for recording global brain rhythms such as alpha activity ? figure 3 , ( a / c ) shows the signals that were recorded with eyes closed / open and their corresponding power spectral densities are presented in fig . 3 , ( b / d ) . the alpha waves were present in teeg ( top trace , blue ) coinciding with the corresponding alpha rhythm recorded by conventional disc electrodes ( middle and bottom traces , red and green respectively ) ( fig . the alpha waves were blocked in both teeg and eeg when the eyes were open ( fig . 3 , c , d ) . thus , this result demonstrates the ability of tcres to record physiological rhythms . figure 3.global physiological rhythms such as alpha activity observable with eyes closed can be recorded by tcres . panel a : the top trace ( blue ) is teeg from a tcre placed next to the conventional disc electrode at o1 . the middle trace ( red ) both the top and middle traces are recorded with respect to the reference on the right collar bone ( rcb ) . the bottom trace ( green ) is from the o1 electrode with respect to the a1 reference electrode . panel b : the power spectral densities for the signals of panel a. panels c and d are the signals and power spectral densities when the eyes are open . it is evident that the alpha waves are present in both teeg and eeg records , specifically between 4 and 5 seconds ( panel a ) , which is confirmed by the spectra displaying a peak just below 10 hz ( panel b ) but are not seen in panels c and d. global physiological rhythms such as alpha activity observable with eyes closed can be recorded by tcres . panel a : the top trace ( blue ) is teeg from a tcre placed next to the conventional disc electrode at o1 . both the top and middle traces are recorded with respect to the reference on the right collar bone ( rcb ) . the bottom trace ( green ) is from the o1 electrode with respect to the a1 reference electrode . panel b : the power spectral densities for the signals of panel a. panels c and d are the signals and power spectral densities when the eyes are open . it is evident that the alpha waves are present in both teeg and eeg records , specifically between 4 and 5 seconds ( panel a ) , which is confirmed by the spectra displaying a peak just below 10 hz ( panel b ) but are not seen in panels c and d. a relative insensitivity of tcres to myogenic activity is demonstrated by teeg and eeg records obtained from the same subject during head movements ( fig . the bottom trace ( green ) it is evident that from about 3 to 6 seconds there is a high - frequency contamination in the bipolar disc recordings that is significantly attenuated in the teeg ( top trace , blue ) . this high - frequency interference coincided with head movements and is most likely caused by muscle artifacts . 4 shows the power spectral densities for the traces in panel a. the teeg power ( blue ) has the lowest power in the higher frequencies range where the muscle artifact is prominent in the records from the conventional bipolar disc signals . from our experience , such attenuation of myogenic artifacts panel a : the top trace ( blue ) is teeg from a tcre placed next to the conventional disc electrode at o1 . both the top and middle traces are recorded with respect to the reference on the right collar bone ( rcb ) . the bottom trace ( green ) is from the o1 disc electrode with respect to the a1 reference electrode . conventional eeg is heavily corrupted by muscle artifacts which are evident from approximately 3 to 6 seconds of the record ( middle [ red ] and bottom [ green ] traces ) coinciding with head movements by the subject . these artifacts are nearly unnoticeable in the teeg ( top trace [ blue ] ) . panel b : the power spectral densities for the signals shown in panel a. the power spectrum for the teeg ( blue ) has much less high frequency power ( > 30 hz ) due to myogenic activity . panel a : the top trace ( blue ) is teeg from a tcre placed next to the conventional disc electrode at o1 . both the top and middle traces are recorded with respect to the reference on the right collar bone ( rcb ) . the bottom trace ( green ) is from the o1 disc electrode with respect to the a1 reference electrode . conventional eeg is heavily corrupted by muscle artifacts which are evident from approximately 3 to 6 seconds of the record ( middle [ red ] and bottom [ green ] traces ) coinciding with head movements by the subject . these artifacts are nearly unnoticeable in the teeg ( top trace [ blue ] ) . panel b : the power spectral densities for the signals shown in panel a. the power spectrum for the teeg ( blue ) has much less high frequency power ( > 30 hz ) due to myogenic activity . we separated the patients into 3 groups : ( 1 ) patients who had clinical seizures ( n\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } \(=5\ ) \end{document } ) , ( 2 ) patients with epileptiform activity but no seizures ( n\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } \(=3\ ) \end{document } ) , and ( 3 ) patients with neither epileptiform activity nor seizures ( n\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } \(=4\ ) \end{document } ) . simultaneously recorded eeg and teeg and the corresponding spectrograms from representative patients are shown in fig . 5 and 6 . 5 the patient was a 42-year - old woman with seizure onset at 9 years . she had been diagnosed with right temporal lobe epilepsy and had undergone a right temporal lobectomy but seizures persisted . she was on clonazepam ( cnz ) , lamotrigine ( ltg ) and phenytoin ( pht ) at the time of the study . in figure 5 conventional eeg data are on panels a , b , e , f , and i and teeg data are on panels c , d , g , h , and j. the conventional eeg data were obtained from the bipolar montage ( channel f8-f4 ) . this patient had a generalized seizure ( onset at approximately 610 seconds in the time scale of panels a - d in fig . the seizure activity is evident by large increases in signal amplitude and power at all frequencies . panels e , f , g , and h are eleven - second eeg and teeg segments ( marked by the black line in panel c ) shown at higher sequential resolution . note the series of high gamma - band hfos between approximately 60 to 80 hz ( highlighted by ellipse in panel c ) occurring about every two seconds which are clearly evident in the teeg but not eeg , starting approximately 10 min prior to the generalized seizure activity ( compare panels a and c as well as e and g ) . with further zoom in panels i and j showing two - second segments , one can see a high frequency burst in the teeg ( black horizontal line in the panel j ) which is not present in the eeg ( panel i ) . conventional eeg ( bipolar montage , channel f8-f4 , panels b , f and i , 170 hz , 200 s / s ) , teeg ( from a tripolar electrode placed right behind the f8 position ( f8 ' ) , panels d , h and j , 1100 hz , 200 s / s , ) and their spectrograms ( panels a , e and panels c , g for eeg and teeg , respectively ) starting \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } \(\sim 10\ ) \end{document } min before and continuing during a generalized seizure with onset marked with the dashed vertical black line . panels e - h : eleven - second eeg and teeg segments ( marked by the black line in panel c ) are shown at higher temporal resolution . note a series of high gamma - band bursts hfos at 6080 hz ( highlighted by ellipse in c ) occurring about every 2 s , which are clearly seen in the teeg spectrogram only . panels i , j : further zoom - in of two - second segment shows one hfo in teeg ( black horizontal line in the panel j ) while this hfo is absent in eeg ( panel i ) . figure 6.panel b shows 12 minutes of bipolar eeg from fp2-f4 ( 170 hz , 200 s / s ) . panel e shows 30 seconds of eeg from panel b at the onset of the generalized seizure ( dashed line ) . note the high gamma - band burst hfos just prior to the partial seizure ( highlighted by ellipse in panel c ) . conventional eeg ( bipolar montage , channel f8-f4 , panels b , f and i , 170 hz , 200 s / s ) , teeg ( from a tripolar electrode placed right behind the f8 position ( f8 ' ) , panels d , h and j , 1100 hz , 200 s / s , ) and their spectrograms ( panels a , e and panels c , g for eeg and teeg , respectively ) starting \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } \(\sim 10\ ) \end{document } min before and continuing during a generalized seizure with onset marked with the dashed vertical black line . panels e - h : eleven - second eeg and teeg segments ( marked by the black line in panel c ) are shown at higher temporal resolution . note a series of high gamma - band bursts hfos at 6080 hz ( highlighted by ellipse in c ) occurring about every 2 s , which are clearly seen in the teeg spectrogram only . panels i , j : further zoom - in of two - second segment shows one hfo in teeg ( black horizontal line in the panel j ) while this hfo is absent in eeg ( panel i ) . panel b shows 12 minutes of bipolar eeg from fp2-f4 ( 170 hz , 200 s / s ) . panel e shows 30 seconds of eeg from panel b at the onset of the generalized seizure ( dashed line ) . note the high gamma - band burst hfos just prior to the partial seizure ( highlighted by ellipse in panel c ) . this patient was a 45 year old male , with onset of epilepsy at 22 years . his seizures consisted of simple partial seizures followed by complex partial with or without secondarily generalization . he was on carbamazepine ( cbz ) and ltg at the time of the recording . his magnetic resonance imaging showed left mesial temporal sclerosis and a right frontal venous angioma . panel d shows teeg recorded from location fp2 ' directly behind the fp2 disc electrode . , we also found gamma band bursts ( \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } \(\sim 70\ ) \end{document } hz ) in the teeg , approximately three minutes before the seizures . 6(c ) highlights the high gamma - band burst hfos in the teeg at location fp2 ' approximately 4 min prior to the partial seizure . the hfos are present throughout the pre - seizure spectrogram but became more consolidated around 70 hz about 3 min prior to the seizure . it is also important that the teeg during the tonic seizure was less contaminated with muscle and movement artifacts than the eeg ( compare panels e and f ) . also note the higher power in eeg ( compare panels a and c ) from approx . 400 to 650 s when the patient was still anxious / disoriented and moving while recovering from the seizure . during that same period overall , hfos preceding seizures were present in teeg data from all five patients whose seizures were recorded . for these five patients , out of all the teeg channels that contained hfos an average of 78.2% were also in the soz ( iz ) ( patient minimum : 42.9% ; maximum : 100% ) . the average percentage of the total number of teeg channels recorded that contained hfos is equal to 35.5% ( patient minimum : 5.3% ; maximum : 73.7% ) . figure 7 portrays the relationship between the clinical soz ( iz ) and hfo - containing channels for two patients that we present the spectrograms for in fig . 5 ( panel a ) and fig . 6 ( panel b ) respectively . figure 7.panel a shows the relationship between the clinical seizure onset zone ( soz ) and hfo - containing channels for the patient from fig . panel b shows the relationship between the clinical irritative zone ( iz ) and hfo - containing channels for the patient from fig . soz or iz was determined for each patient independently by three epileptologists ( iemj , jng , and rsf ) based on eeg data and videos only . panel a shows the relationship between the clinical seizure onset zone ( soz ) and hfo - containing channels for the patient from fig . panel b shows the relationship between the clinical irritative zone ( iz ) and hfo - containing channels for the patient from fig . soz or iz was determined for each patient independently by three epileptologists ( iemj , jng , and rsf ) based on eeg data and videos only . it has been shown that eeg is frequently contaminated by artifacts originating from various sources such as scalp muscles , eye blinks , eye movements , or patient movement . state what is intuitively obvious ; artifacts obscuring the eeg at the time of seizure onset can greatly hinder the interpretation of the recorded seizures . during a tonic or tonic - clonic seizure , muscle activity is very prominent and is reflected in the eeg by random , high frequency signals . . showed that when eegs were moderately contaminated with muscle artifacts blind source separation techniques based on canonical correlation provides signals that are more reliable for source estimation than raw eeg . they also found that in cases of severe muscle contamination of the eeg or when the muscle artifact had different spatial information than the spatial information of the epileptiform event , the blind source separation techniques based on canonical correlation did not work well . in general , a major reason why eeg has not reached its full potential in epilepsy diagnosis is due to artifact contamination . we have found that the tcre and the teeg automatically attenuate myogenic activity and movement artifacts . in contrast to the typical examples of artifact contaminated conventional bipolar eeg , the teeg is much less contaminated by the muscle activity . it should be noted that conventional approaches to remove muscle artifacts by digital signal processing ( such as filtering ) lead to significant loss of information since the gamma band activity and hfos in general are within the bandwidth of the electromyogram ( emg ) . in other words , removing the emg will inevitably remove high frequency components of the brain activity , which is not a desirable result . the teeg , although not as obvious , may also be contaminated with emg ( fig . 4 , a , top blue trace ) but from a much more local source . the emg from more distant sources have nearly equal contributions on the elements of the tcre and are cancelled out when the potentials on the elements are subtracted from each other with bipolar differences . in this study , we show , in a limited number of patients , that high - power hfo activity is detected at specific locations on the scalp surface in the teeg records of patients with epilepsy . this hfo activity was apparent prior to seizures . although there may have been hfo activity at various times in the teeg during the recordings the bandwidth of the hfo activity narrowed sharply and the power increased just prior to the seizure . we did not see the hfo activity in the conventional eeg , even when using bipolar montages which somewhat attenuates global artifacts . in some instances this may partially be due to the low - pass filter used to record the eeg . the eeg low - pass filter was set to 70 hz and this cutoff frequency may have partially filtered out gamma band activity . however , the gamma band activity detected in the teeg was often well below 70 hz , in one case as low as 63 hz , and in these cases the gamma band activity was still undetected in the conventional eeg . when a low - pass digital filter was applied to the teeg at 70 hz the hfos were still present in the teeg for patients whose hfos were below 70 hz . we do not know what the upper limit is for detecting hfos on the scalp . in rats we have found an increase in power up to 300 hz during ptz - induced seizure electrographic activity recorded with teeg . in two patients having seizures for whom the data were filtered 1250 hz and 1500 hz respectively and digitized at 1600 s / s we have detected hfo preceding seizures in gamma , ripple , and fast ripple bands at frequencies up to 425 hz . a potential disadvantage of recording with tcre is deemphasis of non - artifactual signals with wide spatial distribution , for example , spike - waves . we intend to develop the circuitry to record both teeg and an eeg approximation from the same sensor in the near future . despite a limited sample size of patients having seizures in this study ( 5 patients ) we were able to investigate a possible correlation between the clinically determined soz ( iz ) and the location of hfos detected by tcres . hfos preceding seizures were present in teeg data from all five patients whose seizures were recorded in an average of 35.5% of the patient 's teeg channels . out of those channels containing hfos an average of 78.2% were within soz ( iz ) determined independently by three epileptologists based on eeg data and videos . in all five patients who had recorded seizures , at least one channel in the soz exhibited hfos ( in one out of five patients hfos were present in a single teeg channel ) . in two patients no specific soz was determined but rather an iz . for those two patients hfos were present in 7 and 4 teeg channels respectively out of 20 ( 35% ) and 19 ( 21% ) channels total respectively which is on par with the population average of 35.5% . examples of the relationship between the soz ( iz ) and hfo - containing channels are presented in fig . the third epileptologist ( jng ) mentioned 4 hz activity over central leads but no clear electrographic onset . soz definitions from all three epileptologists ( iemj , jng , and rsf ) overlapped for two channels ( fp2 and f4 ) over the right frontal lobe . these preliminary results support that teeg is capable of detecting hfos from the scalp , which are not usually seen in conventional eeg records . further studies with more patients are needed to more accurately correlate locations of ( presumably abnormal ) hfo activity with clinically determined soz as well as the resected tissue in surgical cases . due to the improved signal quality of teeg we hope that in the future we will be able to use teeg to detect seizures and trigger stimulation to attenuate seizures . we have shown that transcranial focal electrical stimulation ( tfs ) via tcres in rat models was effective in reducing penicillin - induced myoclonic jerks , , pilocarpine - induced status epilepticus ( an extreme form of continuing seizures ) and in a third model , we applied tfs in rats treated by pentylenetetrazole ( ptz ) . tfs significantly reduced ptz - induced hypersynchrony at the beta and gamma frequencies , most recently we developed an automatic non - invasive seizure control system based on tfs and tested it successfully on rats .

epilepsy is the second most prevalent neurological disorder ( \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } \(\sim 1\ ) \end{document}% prevalence ) affecting \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } \(\sim 67\ ) \end{document } million people worldwide with up to 75% from developing countries . the conventional electroencephalogram is plagued with artifacts from movements , muscles , and other sources . tripolar concentric ring electrodes automatically attenuate muscle artifacts and provide improved signal quality . we performed basic experiments in healthy humans to show that tripolar concentric ring electrodes can indeed record the physiological alpha waves while eyes are closed . we then conducted concurrent recordings with conventional disc electrodes and tripolar concentric ring electrodes from patients with epilepsy . we found that we could detect high frequency oscillations , a marker for early seizure development and epileptogenic zone , on the scalp surface that appeared to become more narrow - band just prior to seizures . high frequency oscillations preceding seizures were present in an average of 35.5% of tripolar concentric ring electrode data channels for all the patients with epilepsy whose seizures were recorded and absent in the corresponding conventional disc electrode data . an average of 78.2% of channels that contained high frequency oscillations were within the seizure onset or irritative zones determined independently by three epileptologists based on conventional disc electrode data and videos .

hepatitis c virus ( hcv ) infection is responsible for considerable morbidity and mortality worldwide . moreover the hcv genotype has been shown to have an important role in clinical and histological features as well as the response to antiviral treatment . most studies suggest that subtype 1b isolate possibly represent more aggressive variants of the virus and better responses to ifn are found in patients infected with either hcv genotypes 2 or 3 in comparison with those infected with genotypes 1 and 4 . considering that approximately 1% of iran s population is infected , chronic hepatitis c remains a serious medical problem with considerable burden on iran s health care system . these observations have led us to determine hcv genotypes , particularly in the region of khuzestan province , southwest iran , which has no available data on hcv genotypes . herein , we report the results of our cross - sectional analyses of hcv genotype distribution and its association with certain clinical features in patients with chronic hcv infection in khuzestan province conducted from september 2006 to september 2009 . during a three year period ( between september 2006 and september 2009 ) , we conducted a cross - sectional study on 223 consecutive hcv positive individuals who referred to the ahwaz jundishapour university hospitals ( ajsuh ) and hepatitis clinic . the presence of anti - hcv antibody was determined by an elisa test ( ortho hcv 3.0 diagnostics , raritan , nj , usa ) . the presence of hcv rna in anti - hcv positive and indeterminate samples was detected by rt - pcr with the qualitative amplicor hcv test v.2.0 ( roche molecular systems , branchburg , nj , usa ) . hcv genotyping / sub - typing was performed by two rt - pcr assays , restriction length polymorphism analysis ( rflp ) of the 5noncoding region ( 5ncr ) and nested pcr with type specific primers following primary rt - pcr with consensus primers designed for the core region of the hcv genome . hcv genotyping by rflp was carried out using the restriction enzymes avaii and rsai on pcr - amplified from 5ncr as previously described . type - specific pcr assay was accomplished by means of the nested pcr assay originally developed by okamoto et al . for sub typing genotypes 1 and 2 , and sequences of the hcv core region were detected after synthesis of cdna and nested pcr with conserved primers covering positions 439 to 751 of the hcv genome . rank - sum and kruskal - wallis tests compared continuous variables such as age between the groups . fisher exact test assessed associations . , 1989 - 2002 , chicago , il , usa ) for windows in addition to univariate analysis and multivariate logistic regression . all 223 patients had documented positive anti - hcv antibodies for at least six months . there were 170 males ( mean age : 34.6 years ) and 53 females ( mean age : 49.1 years ) with an age range of 13 - 70 years . samples comprised 42 patients with unknown risk factors and 179 with parenteral risk of infection [ 11 dialyzed , 10 hemophiliacs , 95 intravenous drug users ( ivdu ) , with tattoos , 54 polytransfused , and needle sticks in 2 cases ] . possible routes of infection were recognized in 181 ( 81.2% ) patients . among these , patients with a history of ivdu , blood transfusion recipients , undergoing chronic hemodialysis , history of tattooing , accidental inoculation and non - marital sexual contact comprised 42.6% , 23.3 % , 4.93% , 3.15% , 0.9% and 0.9% of samples , respectively ( table 1 ) . rflp identified hcv genotypes in 181 of 223 samples , whereas 42 cases could not be sub - typed with this method . patients ages were analyzed as a categorical variable ( < 40 and > 40 years ) . genotypic distribution was as follows : 1a ( 41.7% ) ; 1b ( 2.7% ) ; 2 ( 4.1% ) ; 3a ( 31.4% ) and 4 ( 1.8% ) . there were 42 samples ( 18.84% ) not classified into any of the known hcv subtypes . univariate analysis demonstrated an independent association between ivdu and genotypes 1 ( 60.7% 1a ) and 3a ( 30% ) . statistical analysis did not show a difference in the frequency distribution of genotypes according to gender and age ( p > 0.56 ) . in this study , 64 ( 28.7% ) of 223 patients received blood transfusions before 1995 , whereas 5 ( 2.25% ) of 64 patients were positive for hcv rna who received transfusions after 1995 . hepatitis c infection is currently the most common cause of end - stage liver disease in many countries , however the whole population , prevalence is less than 1% in iran . a comparison of hcv genome sequences from various geographical regions of the world has shown substantial heterogeneity of nucleotide sequences within several regions of the viral genome . based on these genomic differences , six major genotypes with several subtypes have been identified and a nomenclature for these was given following a consensus proposal . in the first report on iranian patients , zali et al . in tehran studied the prevalence of specific genotypes in 15 samples with the following results : type i /1a ( 7 ) , type ii/1b ( 3 ) , type v/3a ( 4 ) and type 4 ( 1 ) . a recently published article by samimi - rad et al . in iranian patients with anti - hcv ab positive from tehran and five cities from different locations of iran showed a predominance of genotype 1a ( 47% ) , followed by 3a ( 36% ) , 1b ( 8% ) and 4 ( 7% ) , respectively . the current study has shown that in khuzestan province , southwest iran , genotype 1a accounted for the majority of hcv infections ( 41.7% ) . this is the first study of the genotype distribution in this area which suggests a pattern similar to that reported in other parts of iran , where most cases studied thus far belonged to genotype 1 . however the results of our study differed from other middle - east countries such as the republic of yemen , kuwait , iraq and saudi arabia where genotype 4 has been identified as the most prevalent . a study of saudi patients detected hcv genotype 4 in 50% of cases and genotype 1b in nearly 40% . from other parts of the world , studies have revealed that genotype 3 is prevalent in south east asia whereas genotype 1 is common in the usa and western europe . patients with risk factors for hcv infection enrolled in the present study were very few and therefore we were unable to reach any conclusion of genotype distribution within this group . the distribution of the two most common hcv viral types ( 1a and 3a , ) were not statistically different in terms of mean age or gender . the relative influence of hcv genotype , risk factors for infection , duration of infection , and host factors on progression of liver disease seemed to be important . our study indicated that ivdu and blood transfusions were the leading risk factors for hcv acquisition ( 71.3% ) , of which ivdu was the most frequent risk factor ( 42.6 % , ) . all hcv infected ivdu patients were male , which was consistent with other reports . the route of hcv transmission mainly causes this epidemiological pattern in genotype distribution such that ivdu associated with genotype 1a and 3a is currently the primary risk factor of hcv infection . screening for hcv is routine for all blood donors since 1995 in iran but 5 ( 2.25% ) out of 64 patients who received transfusions after 1995 were positive for hcv rna . therefore , more careful pre transfusion screening of blood for anti - hcv must be introduced in our blood banks . there were 42 cases ( 18.8% ) unable to be classified for hcv genotype by rflp technique . this may be due to multiple infecting genotypes in these patients , difficulties in sample handling or unusual hcv genotypes in this patient group which could not be classified by this method . we can conclude that genotype 1 , particularly the subtype 1a and genotype 3 , subtype 3a are predominate in this geographic part of iran . knowledge on the distribution of various genotypes in our area is essential for its prognostic implications in chronic hepatitis c infection . further investigations with larger sample numbers are necessary to determine the major genotypes that cause hcv infection in various clinical conditions and potential associations with disease severity . this work was supported by the tropical and infection research center , ahwaz jundishapour university of medical sciences , ahwaz , iran . we are grateful to the research deputy of ahwaz jundishapour university of medical sciences for his encouragement and constructive advice during the preparation of the manuscript . we thank the subjects for their compliance in participating in this study , and danesh lab center for genetic studies , sequence alignment and immunoassays .

background hepatitis c virus ( hcv ) infection is responsible for considerable morbidity and mortality worldwide . the hcv genotype has a geographic distribution and an important role in clinical and histological outcomes . this study determined hcv genotypes and their related risk factors among patients from khuzestan province , southwest iran . methods in a cross - sectional study , 223 patients infected with hcv who referred to ahwaz jundishapour university hospitals ( ajsuh ) and hepatitis clinic were enrolled . specific and nested polymerase chain reaction ( pcr ) and restriction fragment length polymorphisms ( rflps ) were performed to determine viral infection and genotype analysis . liver enzymes including alt and ast and the correlated risk factors were also determined . results the hcv genotype distribution was as follows : genotype 1a ( 41.7% ) ; genotype 1b ( 2.7% ) ; genotype 2 ( 4.1% ) ; genotype 3a ( 31.4% ) ; and genotype 4 ( 1.8% ) . there were 42 samples ( 18.84% ) not classified into any of the known hcv subtypes . no patient was infected with more than one genotype . hiv was found in four ( 1.8% ) cases , of which all were intravenous drug users . univariate analysis demonstrated an independent association of intravenous drug use ( ivdu ) and genotypes 1a ( 60.7% ) and 3a ( 30% ) . conclusion our findings suggest that genotypes 1a and 3a are the most common ones among iranian patients with chronic hepatitis c infection in khuzestan province , southwest , iran .

structural deficiencies and functional abnormalities of heart valves are conditions that need cardiovascular surgery . these abnormalities may be caused by congenital diseases or by a variety of acquired diseases that result in valvular stenosis , valvular insufficiency , or both . the rheumatic heart disease ( rhd ) , the most common heart valve disease in underdeveloped countries , is a condition that causes damage to the valve function , due to an abnormal immune response to group a streptococcal infection , especially during infancy . patients with rhd have valvular lesion caused by the rheumatic valve involvement ( stenosis and/or regurgitation ) or secondary to ventricular dilatation , leading to mitral or tricuspid insufficiency . the aortic stenosis , the most common valve disease in industrialized countries , had been considered , for many years , a degenerative disease that would appear with aging , and that was caused by the passive accumulation of calcium on the surface of the valve leaflet . recent studies , however , have demonstrated that this disease represents an active process that may be divided into 2 distinct phases : an early initiation phase , similar to atherosclerosis , and a later progression phase that involves pro - calcifying and pro - osteogenic factors . in the last few decades , these conditions have been the object of great attention in the cardiology field , mainly due to changes in their presentation profile and treatment . these changes came as a result of a significant reduction in the incidence and sequelae of rheumatic fever , increase in life expectancy , technological advancement , and the discovery of new causes for valve diseases , including the alleged role of infectious agents in their pathogenesis . infectious agents are well known to cause infective endocarditis , another major cause of valve replacement , but there are recent evidence also connecting these pathogens to valvular stenosis and/or regurgitation . in this context , an association between chlamydophila pneumonia infection and aortic stenosis has been suggested by nystrm - rosander et al and turgeman et al . oral micro - organisms have already been indirectly associated with other cardiovascular diseases ( cvd ) , such as atherosclerosis . oral bacteria involved in the etiopathogenesis of chronic periodontitis are known to cause an immune and moderate systemic inflammatory response , elevating the serum concentration of multiple cytokines and inflammatory markers that are abundantly produced in pathological periodontal tissues and strongly associated with the pathogenesis of some cvds . the presence of oral bacteria in valvular tissue with or without clinical endocarditis have been investigated through sensitive molecular exams , such as the polymerase chain reaction ( pcr ) , in order to investigate the mechanisms that can link oral infections to cvd . in addition to identifying oral bacteria dna in valvular tissue , nomura et al have reported the mechanisms that led cariogenic bacteria to colonize heart valves . considering the recent findings , the aim of this study is to identify cariogenic and periodontopathogenic micro - organisms in the dental plaque , saliva , and cardiac valves of patients undergoing valve replacement surgery , regardless of the disease , in order to contribute with epidemiologic data regarding the presence of oral bacteria in cardiac valves . detailed evaluation of oral health status of all patients , including presentation of caries and periodontal disease history , by dental examination , will also be reported . this data could provide evidence of a connection between oral diseases and the frequency of oral micro - organisms in valve samples . carlos alberto studart gomes ( fortaleza , cear , brazil ) who were consecutively scheduled for cardiovascular surgery of valve replacement between march 2012 and september 2012 were enrolled in this study , which accounted for a convenience sample of 42 patients . all participants gave their informed consent and this study was approved by the ethics committee of the hospitals where the research was performed . two examiners ( f.a.f.o and c.p.f ) were calibrated to evaluate teeth condition and current periodontal status of the patients , using the indexes dmft ( decayed , missing , and filled teeth ) and psr ( periodontal screening and recording ) , respectively . for inter - examiner calibration , both examiners evaluated the same 10 volunteers that were not enrolled in the study . after 1 week , the same patients were re - evaluated to assess intra - examiner calibration . the kappa values ranged from 0.80 to 0.97 . before the cardiovascular surgery , the previously calibrated examiners performed bedside oral examination . information regarding smoking history , hypertension , diabetes mellitus , dyslipidemia , and other comorbidities were obtained through medical charts and anamnesis . only 1 patient could not undergo the periodontal examination due to his poor physical condition . for dentate patients , during oral examination , supragingival and subgingival dental plaque were collected . only for edentulous patients , saliva samples were collected by rubbing sterilized absorbent paper points in the oral mucosa and on the alveolar ridge , palate , and tongue . the oral samples were stored in a sterile vial containing phosphate - buffered saline ( pbs ) at 20c for subsequent molecular analysis . a total of 47 cardiac valves were collected , aseptically , during valve replacement surgeries . a fragment of each sample was stored in a sterile container containing pbs at 20c for further analysis using real - time pcr . dna extraction and molecular analysis through real - time pcr were carried out , respectively , at the laboratory of human cytogenetics and laboratory of human and medical genetics of the federal university of par ( belm , brazil ) . a total of 114 samples of supragingival and subgingival dental plaque , saliva , and cardiac valves ( 30 mg ) were transferred to sterile plastic tubes of 2 ml containing a buffer , maintaining aseptic handling , and posteriorly homogenized . dna extraction proceeded according to a standard protocol that used proteinase k and cetyltrimethylammonium bromide ( ctab ) to remove complex polysaccharide . the samples of extracted dna were subjected to real - time pcr for the detection of dna from 4 different bacterial species : s. mutans , p. gingivalis , p. intermedia , and t. denticola . taqman probes to 16s bacterial ribosomal dna ( table 1 ) were specifically designed for this study ( life technologies ) . for real - time pcr , 1 l of genomic dna ( 5 ng ) was added to 8 l of taqmanuniversal pcr master mix ( applied biosystems ) , 0.35 l of probe , and 3.15 l of water . the final volume of 14 l was obtained by adding ipc ( 1 l ) and ipc dna ( 0.5 l ) , which was used as an internal control for the reaction . amplification was carried out in a thermal cycler 7500 real - time pcr system ( applied biosystems ) . bacterial dna was replaced by water as a negative control for the reaction . the real - time pcr protocol consisted of an initial step of denaturation at 95c for 10 min , followed by 40 cycles at 95c for 15 s , and 60c for 1 min . list of primers and probes designed for the identification of different cariogenic and periodontopathogenic micro - organisms the distribution pattern of the quantitative data of the sample was evaluated through the shapiro wilk test . posteriorly the quantitative data were submitted to the student t test and expressed as mean sem . qualitative nominal variables were expressed as absolute frequency ( relative frequency ) and analyzed by the fisher exact test or the chi - square test with bonferroni correction . when possible , the prevalence ratio expressed as the prevalence ratio ( confidence interval minimum maximum ) was calculated . ( cdc atlanta ) was used and a significance level of p < 0.05 was established for all analyses . carlos alberto studart gomes ( fortaleza , cear , brazil ) who were consecutively scheduled for cardiovascular surgery of valve replacement between march 2012 and september 2012 were enrolled in this study , which accounted for a convenience sample of 42 patients . all participants gave their informed consent and this study was approved by the ethics committee of the hospitals where the research was performed . two examiners ( f.a.f.o and c.p.f ) were calibrated to evaluate teeth condition and current periodontal status of the patients , using the indexes dmft ( decayed , missing , and filled teeth ) and psr ( periodontal screening and recording ) , respectively . for inter - examiner calibration , both examiners evaluated the same 10 volunteers that were not enrolled in the study . after 1 week , the same patients were re - evaluated to assess intra - examiner calibration . the kappa values ranged from 0.80 to 0.97 . before the cardiovascular surgery , the previously calibrated examiners performed bedside oral examination . information regarding smoking history , hypertension , diabetes mellitus , dyslipidemia , and other comorbidities were obtained through medical charts and anamnesis . only 1 patient could not undergo the periodontal examination due to his poor physical condition . for dentate patients , during oral examination , supragingival and subgingival dental plaque were collected . only for edentulous patients , saliva samples were collected by rubbing sterilized absorbent paper points in the oral mucosa and on the alveolar ridge , palate , and tongue . the oral samples were stored in a sterile vial containing phosphate - buffered saline ( pbs ) at 20c for subsequent molecular analysis . a total of 47 cardiac valves were collected , aseptically , during valve replacement surgeries . a fragment of each sample was stored in a sterile container containing pbs at 20c for further analysis using real - time pcr . dna extraction and molecular analysis through real - time pcr were carried out , respectively , at the laboratory of human cytogenetics and laboratory of human and medical genetics of the federal university of par ( belm , brazil ) . a total of 114 samples of supragingival and subgingival dental plaque , saliva , and cardiac valves ( 30 mg ) were transferred to sterile plastic tubes of 2 ml containing a buffer , maintaining aseptic handling , and posteriorly homogenized . dna extraction proceeded according to a standard protocol that used proteinase k and cetyltrimethylammonium bromide ( ctab ) to remove complex polysaccharide . the samples of extracted dna were subjected to real - time pcr for the detection of dna from 4 different bacterial species : s. mutans , p. gingivalis , p. intermedia , and t. denticola . taqman probes to 16s bacterial ribosomal dna ( table 1 ) were specifically designed for this study ( life technologies ) . for real - time pcr , 1 l of genomic dna ( 5 ng ) was added to 8 l of taqmanuniversal pcr master mix ( applied biosystems ) , 0.35 l of probe , and 3.15 l of water . the final volume of 14 l was obtained by adding ipc ( 1 l ) and ipc dna ( 0.5 l ) , which was used as an internal control for the reaction . amplification was carried out in a thermal cycler 7500 real - time pcr system ( applied biosystems ) . the real - time pcr protocol consisted of an initial step of denaturation at 95c for 10 min , followed by 40 cycles at 95c for 15 s , and 60c for 1 min . list of primers and probes designed for the identification of different cariogenic and periodontopathogenic micro - organisms the distribution pattern of the quantitative data of the sample was evaluated through the shapiro wilk test . posteriorly the quantitative data were submitted to the student t test and expressed as mean sem . qualitative nominal variables were expressed as absolute frequency ( relative frequency ) and analyzed by the fisher exact test or the chi - square test with bonferroni correction . when possible , the prevalence ratio expressed as the prevalence ratio ( confidence interval minimum maximum ) the statistical software epiinfo 3.5.1 for windows ( cdc atlanta ) was used and a significance level of p < 0.05 was established for all analyses . for the present study , a total of 114 oral samples ( supragingival plaque , subgingival plaque , and saliva ) and cardiovascular samples ( heart valves ) were collected from 42 patients with a mean age of 55.6 13.8 years . regarding the medical conditions that led to valve replacement surgery , mitral regurgitation ( 23.4% ) and aortic stenosis ( 21.2% ) were the most common ( table 2 ) . demographic , clinic , and dental characteristics of patients with heart valve diseases detailed oral examination for the investigation of dental caries and periodontitis was carried out in all 42 individuals . the mean number of teeth missing due to caries was 23.52 9.41 per patient , and all patients have already had a previous experience of caries resulting in tooth loss . according to the highest degree of periodontal disease observed in the individual , excluding edentulous patients ( 44.0% ) , periodontal pockets > 4 mm ( 43.4% ) , and dental calculus ( 34.7% ) were present in a greater number of patients . other demographic , medical , and dental characteristics of the patients are summarized in table 2 . molecular analysis of oral samples revealed high frequency of s. mutans and p. intermedia in supragingival plaque , saliva , and subgingival plaque of dentate and edentulous patients ( ranging from 60.0% to 100.0% ) , whereas p. gingivalis and t. denticola were present in fewer oral samples ( ranging from 17.6% to 64.0% ) ( fig . 1 ) . distribution profile of oral bacteria between dentate and edentulous patients revealed significant differences for p. gingivalis ( p = 0.024 ) and t. denticola ( p = 0.037 ) . according to the probing depth , p. gingivalis ( p = 0.002 ) and t. denticola ( p = 0.044 ) were found with a higher frequency in patients with periodontal pockets > 4 mm , with a rate > 75.0% . percentage distribution of cariogenic and periodontopathic bacteria in dental plaque , saliva , and heart valve samples . p < 0.05 versus valve sample of pi , pg , and td . p < 0.05 versus supragingival dental plaque , subgingival dental plaque , and saliva sample for each oral bacteria . p < 0.05 oral sample of dentate patients versus the oral sample of edentulous patients ( saliva ) for each oral bacteria . regarding the presence of oral bacteria in the heart valves , all 4 micro - organisms were found in at least 1 sample , and only 5 valves ( 10.6% ) were not infected with cariogenic or periodontopathic bacteria . the micro - organism most frequently found in the valve samples was the s. mutans ( 89.3% ) , followed by p. intermedia ( 19.1% ) , p. gingivalis ( 4.2% ) , and t. denticola ( 2.1% ) ( fig . significant difference was observed between the frequency of s. mutans and the studied periodontopathic bacteria in the valve tissue ( p < 0.001 ) ( fig . 1 ) . likewise , p. intermedia was significantly more present in the valve tissue compared to p. gingivalis ( p = 0.025 ) and t. denticola ( p = 0.007 ) . gingivalis , p < 0.001 ; t. denticola , p < 0.001 ) , there was no significant difference between the presence of s. mutans in heart valve and dental plaque or saliva ( p = 0.060 ) ( fig . 1 ) . there was no significant difference between the frequency of oral bacteria in the heart valves regarding dental condition ( dentate , edentulous ) ( p = 0.504 ) , anatomical location ( aortic or mitral ) ( p = 0.596 ) , and clinical diagnosis ( stenosis , insufficiency , or both ) ( p = 0.256 ) . for many years , studies have been developed with an aim to investigate the possible connection between periodontal disease and cardiovascular disease , through the evaluation of inflammatory markers that are common to both pathologies . however , recent studies , such as the study of nakano et al , attempted to elucidate the direct mechanisms that link oral diseases to cvds , and , according to this author , the presence of oral bacteria in the bloodstream ( bacteremia ) is probably one of the initiators of biological events that justify this association . the involvement of cariogenic bacteria in the pathogenesis of some cvds has been studied , and the reported detection rate of s. mutans in cardiovascular samples has been superior to the detection rate of periodontopathic bacteria . in the present study , a high detection rate of s. mutans in the heart valve samples , as well as dental plaque and saliva samples was observed . the higher frequency of s. mutans in the heart valves compared to other studies may be related to the great previous experience of dental caries among the participants of the present study . it was also observed a high percentage of tooth loss due to caries , and epidemiological studies have demonstrated that the absence of teeth may be a risk factor associated with many cardiovascular diseases , including aortic stenosis . in our research , the high detection rate of s. mutans in dental plaque samples and saliva samples may suggest that the s. mutans found in the valve samples was originated from the oral cavity . in the present study , edentulous patients also presented a high frequency of the cariogenic bacterium in saliva and cardiac valve samples . these bacteria can be found in the oral cavity even after complete tooth loss , adhered to soft tissue or dentures . the colonization of s. mutans in the cardiac tissue of these patients may have happened previously to complete tooth loss . another theory is that these bacteria may have entered the bloodstream after complete tooth loss through soft tissue trauma . statistically significant difference was found between the frequency of s. mutans and the other investigated micro - organisms in the heart valves . in the past few years , in vitro and in vivo experimental studies have reported that s. mutans has the ability to modify the expression of certain genes influenced by plasma components , in order to obtain advantages while inside the bloodstream . these findings may justify this bacterium 's high ability to settle in the heart valve tissue . jung et al reported the role of atla , a recent discovered fibronectin binding protein , in the s. mutans resistance to fagocitosis and in its ability to survive in the bloodstream . fibronectin , elastin , laminin , and collagen are considered the most common extracellular matrix components and may serve as receptors for s. mutans . the discovery of bacterial surface structures such as the proteins p1 , wapa , gtfb , gtfc , gtfd , cnm , and cbm , has been important to understand the mechanisms of attachment of these pathogens to the cardiovascular tissue , with subsequent invasion of endothelial cells , induction of inflammation with the production of cytokines , platelet aggregation , and foam cell formation . jung et al and matsumoto - nakano et al have already reported the important interaction between this bacterium and the platelets in heart valve tissue through experimental studies , where they show that platelet aggregation is stimulated by this micro - organism . pariodontophatic bacteria were found less frequently in heart valve tissue , when compared to the gram - positive bacterium s. mutans , corroborating the findings of nakano et al . great variability is reported in the literature regarding the prevalence of these bacteria in heart valve tissues and atherosclerotic plaques , and the reported frequency is either superior or inferior to that observed in the present study . many authors that found a higher frequency of these periodontal bacteria in cardiovascular tissues , compared to the present study , have included in their studies only dentate patients with moderate to severe periodontitis . in the present study , the majority of the dentate patients presented periodontal disease , exhibiting dental calculus and periodontal pockets > 3.5 mm for > 4 mm . this fact was determinant for the periodontal bacteria of the orange complex ( p. intermedia ) and red complex ( p. gingivalis , t. denticola ) to be detected at a high frequency in the dental plaque , specially subgingival plaque removed from periodontal pockets > 6 mm , corroborating previously reported findings . among the gram - negative micro - organisms , p. intermedia was significantly more detected in the heart valve tissue ( p < 0.05 ) . the high detection rate of this micro - organism in the oral samples of dentate ( supragingival and subgingival dental plaque ) and edentulous patients ( saliva ) probably explains its higher frequency in the heart valve samples , compared to the other periodontopathic bacteria . the hypothesis that periodontopathic bacteria can infect cardiovascular samples prior to complete tooth loss of a patient that had periodontal disease may be considered , as suggested by some authors , such as zaremba et al . the frequency of periodontopathic bacteria in the heart valve samples was considered low , when compared to their frequency in the oral samples , which may suggest that these micro - organisms have greater difficulty in surviving inside the bloodstream and adhering to heart valve tissue , compared to s. mutans . p. gingivalis has received special attention among the periodontopathic bacteria regarding its association with cvds , although it was found at a low frequency in the present study . this bacterium is detected at a high frequency in studies that investigate its presence in atherosclerotic plaques , and in vitro and in vivo experimental studies have demonstrated that p. gingivalis accelerates the process of atherosclerotic plaque formation through different mechanisms . in the present study , real - time pcr was used because it has previously demonstrated high sensitivity and efficacy in detecting bacterial dna in valve tissue and oral samples , even at low levels . nevertheless , the polymerase chain reaction may also detect dna of dead bacteria , which poses a doubt regarding the role of the detected bacteria in the pathogenesis of the disease . it is unlikely that the bacteria present in the heart valve tissue are innocuous , as recent laboratorial studies have given support to the idea that these bacteria may have a direct influence in the initiation and progression of the disease . . the evaluation of the expression of different biomarkers ( inflammatory , thrombotic , and osteogenic ) in human heart valves affected with chronic diseases and its correlation with the presence and frequency of oral bacteria in those samples may provide a positive association between the infection and the intensity of the local inflammatory process . this would be an important finding to confirm an active role of the oral bacteria in the progression of these diseases . in summary , cariogenic and periodontopathic bacteria were found in dental plaque , saliva , and cardiac valve samples of patients with cardiovascular disease . the detection rate of s. mutans in cardiovascular samples was superior to the detection rate of periodontopathic bacteria . this finding may be related to the great previous experience of dental caries among the participants of the present study and s. mutans possible ability to survive in the bloodstream and attach to extracellular matrix components .

abstractstructural deficiencies and functional abnormalities of heart valves represent an important cause of cardiovascular morbidity and mortality , and a number of diseases , such as aortic stenosis , have been recently associated with infectious agents . this study aimed to analyze oral bacteria in dental plaque , saliva , and cardiac valves of patients with cardiovascular disease . samples of supragingival plaque , subgingival plaque , saliva , and cardiac valve tissue were collected from 42 patients with heart valve disease . molecular analysis of streptococcus mutans , prevotella intermedia , porphyromonas gingivalis , and treponema denticola was performed through real - time pcr . the micro - organism most frequently detected in heart valve samples was the s. mutans ( 89.3% ) , followed by p. intermedia ( 19.1% ) , p. gingivalis ( 4.2% ) , and t. denticola ( 2.1% ) . the mean decayed , missing , filled teeth ( dmft ) was 26.4 6.9 ( mean sd ) , and according to the highest score of periodontal disease observed for each patient , periodontal pockets > 4 mm and dental calculus were detected in 43.4% and 34.7% of patients , respectively . in conclusion , oral bacteria , especially s. mutans , were found in the cardiac valve samples of patients with a high rate of caries and gingivitis / periodontitis .

in most teleost fishes head kidney , pronephros plays an important role as main hematopoietic organ and blood cell reservoir ( fange 1994 ; houston et al . 1996 ; fijan 2002a , b ; romano et al . 2004 ; rombout et al . 2005 ; gangopadhyay and homechaudhuri 2011 ) . according to wendelaar bonga ( 1997 ) and weyts et al . ( 1999 ) , pronephros shows not only hematopoietic activity but also is a lymphoid and endocrine organ . however , head kidney is not the only site of hematopoiesis in fish . according to various authors ( glomski et al . 1996 ; kobayashi et al . 2006 , 2007 , 2008 ; zapata et al . 2006 2007 and santos et al . 2011 ) , other organs such as spleen , gut - associated lymphoid tissue ( galt ) , mucosa - associated lymphoid tissue ( malt ) , and intertubular tissue of trunk kidney ( mesonephros ) may also show hematopoietic activity . in some fish species , several hematopoietic organs are active , while in the others only one of them ( liu et al . morphology of hematopoietic tissue in fish is quite well known ( e.g. , fange 1994 ; 2002a , b ) , but little information is available on the effects of environmental factors on its structure and activity , and studies of hematopoietic tissue are seldom included in evaluation of physiological effects of toxic agents on fish . hematopoietic activity of head kidney tissue involves proliferation of stem cells and early precursors of all cell lines , differentiation and maturation , as well as apoptosis , and the rate of all these processes is a key factor that determines efficiency of hematopoiesis . stem cell renewal and precursor cell proliferation are counterbalanced by apoptosis in functionally inactive or terminally differentiated cells ( mckenna and cotter 1997 ) . apoptosis plays an important role in regulating early progenitor and stem cells , and particularly for the development and function of lymphoid cells ( domen 2000 ) . the marker protein of apoptosis is caspase 3an enzyme participating in degradation of nuclear and cytoplasmic proteins during this process . this caspase is commonly defined as effector caspase ( migliarini et al . 2005 ) . evaluation of both precursor cell proliferation and apoptosis rate is applied in hematological studies to evaluate the rate of cell turnover and hematopoietic activity ( thiele et al . have been proved to react with mammalian ( mouse or rabbit ) monoclonal antibodies , and these antibodies were successfully used for evaluation of proliferation and apoptosis of various cells in thalassoma pavo ( monteiro et al . 2009 ) , oreochromis niloticus ( brunelli et al . 2011 ) , and salmo salar ( yousaf et al . cadmium and copper are well known to induce hematotoxicity in fish , often resulting in anemia and immunosuppression ( e.g. , svobodova et al . 2004 ; seong - gil et al . 2004 ; ates et al . 2008 ; witeska et al . sometimes the values of hematological parameters of intoxicated fish fluctuate , and their changes are not always directly related to metal concentrations and time of exposure or time post - exposure ( ruparelia et al . these fluctuations may result from translocation of cadmium and copper within the organism , and their toxic action on various functions at different time . cadmium and copper probably affect not only circulating blood cells , but also newly developing ones in hematopoietic tissue . very scarce data concerning hematopoietic effects of heavy metals in fish ( garofano and hirshfield 1982 ; ghosh et al . 2007 ; som et al . 2009 ) and mammals ( lutton et al . 1984 ; mitsumori et al . 1998 ; van den heuvel et al . 1999 ; 2001 ; celik et al . 2005 , 2009 ) indicate that they are cytotoxic to precursor cells , and various cell lineages show different sensitivity to metal toxicity . the aim of present study was to evaluate the effects of copper and cadmium under various exposure conditions ( concentration and time ) on hematopoietic potential of common carp head kidney . common carp ( cyprinus carpio l. ) 6-month - old juveniles of body mass 21.5 8.3 g were harvested in october from the rearing ponds of the inland fisheries institute in abieniec and brought to the department of animal physiology , university of natural sciences and humanities in siedlce in plastic bags with water and supplied with pure oxygen . before the experiment , the fish were acclimated for 4 weeks to the laboratory conditions in the flow - through tank , at 1718 c . water was constantly aerated , and o2 concentration was 6.18.0 mg / dm of o2 ( 6687 % saturation ) , while concentration of no2 0.020.06 mg / dm , and nh4 4.67.1 mg / dm . the fish were fed aller aqua classic 4 mm feed at the rate of 2 % of stock mass per day . prior to the experiment survival tests were performed , and 96hlc50 values were calculated using the probit method for both metals . fish were exposed for 3 h to cd and cu concentrations equal to 100 % of 96hlc50 ( 6.50 and 0.75 mg / dm for cd and cu , respectively)groups cd - s and cu - s or for 4 weeks to 10 % of 96hlc50 ( 0.65 and 0.075 mg / dm , respectively)groups cd - l and cu - l . control group ( c ) was kept in clean tap water ( < 0.31 g / dm of cd , 233 g / dm of cu ) . experimental solutions were made using cdcl2 2 h2o and cuso4 5h2o , and 3/4 was renewed everyday without disturbing fish . the fish were kept in 100 dm aerated tanks ( 10 fish in each ) , and fed aller aqua classic 4 mm ( 1 % of stock mass , once a day before water renewal ) . five fish were sampled weekly for 4 weeks from each experimental group and killed for head kidney isolation . water quality parameters were measured everyday ( table 1 ) using portable do meter ( hanna instruments hi 9143 ) , ph meter ( elwro prl tn 5123 ) , and kits for nitrogen metabolites ( visocolor ammonium 0,210 mg / dm and visocolor nitrite 0,052 mg / dm , machery nagel ) . water hardness values were provided by the water supplier ( www.pwik.siedlce.pl).table 1water quality parameters during the experimentparametergroupccu - s + cu - lcd - s + cd - ltemperature ( c)17.0 0.517.1 0.517.2 0.6hardness ( mg / dm caco3)179198ph6.87.0o2 ( mg / dm)8.9 0.69.0 0.28.8 0.5no2 ( mg / dm)0.03 0.010.06 0.020.04 0.02nh4 ( mg / dm)3.9 0.65.2 0.86.1 1.6 water quality parameters during the experiment five fish were sampled weekly for 4 weeks from each experimental group and killed for head kidney isolation . the kidneys were sliced and smeared on degreased slides and dried at room temperature for 24 h. two preparations from each fish were stained giemsa and may - grunwald for cytological analysis . hematopoietic precursor cells were identified ( 22 types of cells , according to fijan 2002a , b , and kondera 2011 ) . immunocytochemical staining was also performed to identify proliferating cells ( showing pcna ) , and cells undergoing apoptosis ( showing caspase 3 ) using monoclonal mouse antibodies anti - proliferating cell nuclear antigen clone pc 10 ( dako cytomation ) selectively binding to the pcna antigen , and anti - caspase 3 ( active ) antibody produced in rabbit , ( sigma ) binding to active caspase 3 . dried preparations were hydrated with deionized water , and then peroxidase activity was blocked using 3 % h2o2 ( trace pur , merck ) . the antibody solution diluted 1:300 was applied and left on slides for 1 h at room temperature . cells with antibody protein complexes were visualized using dako cytomation en vision + system - hrp for use with mouse primary antibodies , and dako cytomation en vision + system - hrp for use with rabbit primary antibodies , respectively , according to the producer s instruction . the pcna - positive and caspase 3-positive cells stained brownish and were easily distinguishable from other cells ( stained light blue with hematoxylin ) . negative control staining was also performed ( the preparations were treated the same way except for incubation with antibodies ) and resulted in no color reaction . the cells were counted in at least 6 fields , and percentage of proliferating and apoptotic cells was calculated per 300 cells in each preparation . all preparations were preserved with histokitt , glaswarenfabrik karl hecht gmbh germany , covered with cover glass , and viewed using nikon eclipse e600 microscope at 1,000 magnification . eclipse e600 microscope connected with digital nikon coolpix camera and computer image analysis system coolview ( precoptic , poland ) . the results were subjected to statistical evaluation of significance of differences in values of all parameters between the control and metal - exposed groups using mann the study obtained agreement of the iii local ethical committee at the warsaw university of life sciences ( no 41/2008 ) . both metals induced significant changes in frequency of blast cells , as well as proliferating and apoptotic precursors . short - term exposures to cd and cu resulted in an increase in the frequency of early blast cells in head kidney of carp in 1 week after the treatments ( figs . 1 , 2 ) . the level of blasts in cd - s fish remained significantly elevated until the 3-week post - exposure , while in cu - s group decreased already in the 2 weeks . on the contrary , during long - term exposure , cd induced only transient increase in blast frequency ( 3 weeks ) , while the fish from cu - l group showed significantly elevated level of early blast cells beginning from the 2 weeks of exposure until the end of the experiment.fig . 1the effects of cd and cu on the frequency of early blast cells in head kidney hematopoietic tissue of common carp ( arithmetic mean s.e . , * different from the value before metal exposure ( time 0 ) , p 0.05 , u mann whitney test ) . cd - s , cu - s and cd - l , cu - l groups subjected to short- or long - term exposures , respectivelyfig . 2early blast cells the effects of cd and cu on the frequency of early blast cells in head kidney hematopoietic tissue of common carp ( arithmetic mean s.e . , * different from the value before metal exposure ( time 0 ) , p 0.05 , u mann whitney test ) . cd - s , cu - s and cd - l , cu - l groups subjected to short- or long - term exposures , respectively similarly , also the frequency of proliferating ( pcna - positive ) cells significantly increased in 1 week after short - term exposures ( fig . 3 , 4 ) . in cd - s group , the level of cells undergoing mitosis remained elevated until the 2-week post - exposure , while in cu - s proliferation rate quickly decreased to the control level . during long - term cd exposure , a significant increase in precursor cell proliferation took place in 2 weeks , and their level remained elevated until the end of the experiment . the reaction of fish from cu - l group was different : a significant increase in frequency of proliferating cells took place at the end of the experiment ( in 4 weeks of exposure).fig . 3the effects of cd and cu on the frequency of proliferating cells in common carp head kidney hematopoietic tissue ( arithmetic mean s.e . , * different from the value before metal exposure ( time 0 ) , p 0.05 , u mann whitney test ) . cd - s , cu - s and cd - l , cu - l groups subjected to short- or long - term exposures , respectivelyfig . 4pcna - positive cells the effects of cd and cu on the frequency of proliferating cells in common carp head kidney hematopoietic tissue ( arithmetic mean s.e . , * different from the value before metal exposure ( time 0 ) , p 0.05 , u mann whitney test ) . cu - l groups subjected to short- or long - term exposures , respectively both metals induced a significant increase in the frequency of apoptosis of hematopoietic precursor cells ( fig . 5 , 6 ) . both short - term and long - term exposures caused similar effects , but in case of cd exposures , the reaction was more pronounced.fig . 5the effects of cd and cu on the frequency of apoptotic cells in common carp head kidney hematopoietic tissue ( arithmetic mean s.e . , * different from the value before metal exposure ( time 0 ) , p 0.05 , u mann whitney test ) . cd - s , cu - s and cd - l , cu - l groups subjected to short- or long - term exposures , respectivelyfig . 6caspase 3-positive cells the effects of cd and cu on the frequency of apoptotic cells in common carp head kidney hematopoietic tissue ( arithmetic mean s.e . , * different from the value before metal exposure ( time 0 ) , p 0.05 , u mann whitney test ) . cd - s , cu - s and cd - l , cu - l groups subjected to short- or long - term exposures , respectively caspase 3-positive cells these changes resulted in a reduction of hematopoietic potential in all metal - exposed fish , measured as the ratio of frequencies of precursor cell proliferation and apoptosis ( fig . 7 ) . it is noteworthy that in case of short - term copper exposure , reduction in hematopoietic activity at the end of the experiment was deeper than in group subjected to short - term cadmium intoxication or to fish subjected to a long - term copper treatment . however , in all cases , hematopoietic activity was over than 1 showing that the frequency of proliferating cells was all the higher than percentage of precursors undergoing apoptosis.fig . 7the effects of cd and cu on cell turnover rate in common carp head kidney hematopoietic tissue ( arithmetic mean s.e . , * different from the value before metal exposure ( time 0 ) , p 0.05 , u mann whitney test ) . cd - s , cu - s and cd - l , cu - l groups subjected to short- or long - term exposures , respectively the effects of cd and cu on cell turnover rate in common carp head kidney hematopoietic tissue ( arithmetic mean s.e . , * different from the value before metal exposure ( time 0 ) , p 0.05 , u mann whitney test ) . cd - s , cu - s and cd - l , cu - l groups subjected to short- or long - term exposures , respectively the obtained results show that cadmium and copper disturbed hematopoiesis in carp but on the other hand indicate a considerable compensatory potential of carp hematopoietic system . the pattern of changes after short - term exposures ( a rapid increase in cell proliferation rate and early blast frequency , accompanied by an increase in apoptotic rate ) and during long - term treatments ( gradual increase in the values of these parameters during the exposures ) was different but the final effect reduction in cell turnover rate was very similar . the increase in apoptotic rate was higher when compared to acceleration of precursor cell proliferation . no anemia was observed in peripheral blood or a significant reduction in leukocyte count , and the most pronounced effect of metal exposures was significantly reduced frequency of peripheral phagocytes ( neutrophils and monocytes ) , accompanied by reduction in their metabolic activity ( kondera and witeska 2012 , and unpublished data of the same study ) . these results indicate that hematopoietic potential of carp head kidney tissue was reduced due to the increase in apoptotic cell destruction since no necrosis or other visible cell damage was observed . hematological effects of heavy metal intoxication of fish were extensively studied , but very little data concerning metal - induced alterations in hematopoietic system are available . according to garofano and hirshfield ( 1982 ) , 2-h exposure of ictalurus nebulosus at 61.3 mg / dm of cd resulted in complete destruction and elimination of all hematopoietic precursor cells in head kidney hematopoietic tissue over 24-h post - exposure , and the only cells present after intoxication were mature erythrocytes . also , saxena et al . considerable hematopoietic disturbances were also observed in labeo rohita subjected to cu treatment : 50 % 72hlc5025 mg / dm cu or 100 % 72hlc5050 mg / dm cu ( som et al . 2009 ) . sublethal exposure resulted in an increase of abundance of both erythrocyte and leukocyte precursors in head kidney hematopoietic tissue , while under lethal conditions , their number initially decreased , and then increased until the end of the experiment . the authors also observed an increase in blast cell abundance in cu - exposed fish . studies of the effects of metals on hematopoietic tissue of other vertebrates also show their hematotoxic potential . reduction of hematopoietic potential that resulted in anemia was observed in cadmium - treated rat ( mitsumori et al . the results of in vitro study revealed that cadmium , lead , and silver adversely affected erythropoiesis in rat bone marrow ( lutton et al . ( 1999 , 2001 ) observed that cadmium was more toxic to human and murine hematopoietic progenitor cells than lead , erythroid cell being more sensitive than myeloid precursors . ( 2005 , 2009 ) reported genotoxic and cytotoxic action of cadmium upon erythroid cells in rat bone marrow . almost no data are available on the effects of heavy metals on proliferative and apoptotic activity of fish hematopoietic tissue . ( 2009 ) who observed an increase in labeo rohita blast cell apoptosis during both lethal and sublethal cu exposures , while their proliferation rate increased only under sublethal conditions . there are , however , some data showing the effects of cadmium and copper on proliferative and apoptotic activity in other fish tissues . according to brunelli et al . ( 2011 ) , cadmium induced increase in proliferation and apoptosis rates in gill epithelium of thalassoma pavo which was followed by a decrease . ( 1999 ) reported a significant increase in proliferation and apoptosis rate of intestine epithelial cells of salmo salar fed cd - containing feed . activation of both cell proliferation and apoptosis in the intestine of liza aurata from metal - polluted natural environment was also reported by ferrando et al . ( 2011 ) , an increase in cell proliferation rate in cadmium - exposed s. salar and sparus aurata may be a protective mechanism reducing adverse effect of metal on fish tissues . according to hernandez et al . ( 2011 ) , danio rerio larvae subjected to 19.5 mg / dm of cuso4 for 2 h showed an increase in frequency of apoptosis of various types of cells , head kidney cells being more sensitive than brain neurons and gill epithelial cells . monteiro et al . ( 2009 ) reported an increase in proliferation rate of gill epithelial cells in cu - exposed oreochromis niloticus which was not accompanied by activation of apoptosis . massive mitotic activity was detected using pcna immunostaining in olfactory epithelium of tilapia mariae following 4-day exposure to 20100 g / dm of cu ( bettini et al . these observations confirm that cell proliferation is involved in tissue regeneration after metal - induced damage . metal - induced activation of cell proliferation and apoptosis was reported also in mammals . according to habeebu et al . ( 1998 ) , cadmium induced a dose- and time - dependent activation of murine hepatocyte proliferation and apoptosis . tsangaris and tzortzatou - stathoupoulou ( 1998 ) observed that cadmium induced apoptosis of human immune cells , b lymphocytes being more sensitive than t cells and lymphoblasts . the data obtained in the present study indicate that cadmium and copper may affect hematological and immune status of fish organism by disturbing the process of hematopoiesis . hematopoietic precursor cells are sensitive to intoxication and heavy metals enhance the rate of their apoptotic destruction . on the other hand , hematopoietic system of carp shows high homeostatic potential and tends to compensate cell loss by activation of mitotic divisions . the results showed that toxic effects of both metals were persistent and hematopoietic activity reduced after short - term exposures to cadmium and copper did not recover in 4-week post - treatment . they also showed that low sublethal concentrations of cadmium , and particularly of copper may significantly disturb hematopoietic processes in fish that do not show any other symptoms of intoxication . in conclusion , anemia and immunosuppression often observed in fish intoxicated with copper and cadmium may result from toxic effect of metals on hematopoietic system . additionally , cellular parameters of hematopoietic tissue : frequency of blast cells and the rate of proliferation and apoptosis are sensitive indicators of sublethal intoxication .

the effects of cadmium and copper on activity of common carp head kidney hematopoietic tissue were evaluated . the fish were subjected to short - term ( 3 h , cd - s and cu - s ) or long - term ( 4 weeks , cd - l and cu - l ) exposures to 100 % 96hlc50 or 10 % 96hlc50 , respectively . head kidneys were isolated weekly from 5 fish of each group for 4 weeks ( post - short - term exposure and during long - term exposure ) . percentage of early blast cells among the hematopoietic precursors was calculated . proliferative and apoptotic activity were evaluated using immunocytochemical staining for proliferating cell nuclear antigen ( pcna ) and caspase 3 , respectively . hematopoietic activity was calculated as the ratio of proliferating to apoptotic cells . all metal exposures induced an increase in frequency of early blast cells . the frequency of proliferating ( pcna - positive ) cells also significantly increased . a considerable and significant increase in the frequency of apoptotic cells was the most pronounced effect of metal exposures . both short - term and long - term treatments caused similar effects , but in case of cd exposures , the reaction was more pronounced . all metal exposures reduced hematopoietic potential of fish measured as the ratio of proliferating to apoptotic precursor cell frequency . however , in all cases , hematopoietic activity was higher than 1 showing that the rate of repair of hematopoietic tissue prevailed over destruction .

in india , 23.9 lakh people were living with hiv / aids ( plha ) in 2009 and children under 15 years accounted for 3.5% of all infections . according to who / unaids estimates , 450,000 children were receiving antiretroviral therapy ( art ) and more than 2 million children still needed art in 2010 . zidovudine ( azt ) , being an important component of standard regimen for first - line art in children , is widely used for managing pediatric hiv . as more and more children are exposed to this drug , the range of adverse reactions can also be expected to expand . nail pigmentation with azt is a well - documented occurrence in adults , especially dark - skinned individuals . , we report a case of zidovudine - induced nail pigmentation in a 12-year - old boy . in december 2011 , a 12-year - old boy was brought by guardians to dermatology opd for nail pigmentation involving all fingernails . about 4 years back , art was started for him with stavudine - based regimen ( 12 mg stavudine + 60 lamivudine + 100 mg nevirapine ) because of too low hemoglobin level . but after improvement in hemoglobin level , he has been shifted to zidovudine - based regimen ( zidovudine 300 mg + lamivudine 150 mg + nevirapine 200 mg ) since last 1 year . after 3 months of initiating the new regimen , he noticed bluish - brown discoloration of thumbnails that gradually spread to other fingernails [ figure 1a and b ] and grew in intensity over time [ figure 1c and d ] . there was no history of trauma , use of coloring agents , any triggering factor , or abnormal skin or mucosal pigmentation . ( a ) pigmentation involving all nails ( december 2011 ) ; ( b ) thumbnail involvement ( december 2011 ) ; ( c ) increased pigmentation in all nails ( march 2012 ) ; ( d ) increased pigmentation in thumbnails ( march 2012 ) on examination , he was averagely built , nourished , and fully immunized . local examination revealed diffuse bluish - brown discoloration of all fingernails , but it was more marked in thumbnails . nails were brittle with loss of lunulae and periungual changes [ figure 1c and d ] . the patient and guardians were counseled about harmless nature of this adverse drug reaction ( adr ) and he is receiving the same regimen at present . nail pigmentation with zidovudine was first described by furth and kazakis in two black patients in whom progressive hyperpigmentation of all fingernails developed . since then , several such case reports have been published from various parts of the world.[68 ] but all these patients were adults , and we did not come across any case report on pediatric patients . though our patient was receiving three antiretroviral drugs , zidovudine was thought to be the most likely cause of nail pigmentation , as pigmentation developed after 3 months of initiating zidovudine - based regimen . the causality assessment by naranjo algorithm showed that this adr was probable with zidovudine . in previous studies , patients taking zidovudine had noticed nail changes in 48 weeks or even after 56 months.[1011 ] dark - skinned individuals are at higher risk of this adverse effect and it appears to be reversible and dose dependant . nail pigmentation occurs in variety of medical conditions and is also associated with use of several drugs , especially chemotherapeutic agents . the pattern of nail pigmentation may be transverse bands , longitudinal bands , or diffuse discoloration . histopathologic findings of nail biopsy show deposits of brown pigmented granules containing melanin throughout the epidermis . as more patients receive zidovudine , it is important to alert patients about nail pigmentation . though it is harmless and reversible , psychological aspects of this noticeable side effect may hamper adherence and efficacy of therapy . this may also lead to unnecessary investigations and treatment for misdiagnosis such as cyanosis and melanoma . physicians should also focus on preventing adverse effects and distinguishing serious adrs from self - limiting adrs in order to manage prejudiced and fearful patients with available art .

zidovudine is an important component of first - line antiretroviral treatment ( art ) regimens used to manage pediatric hiv . nail pigmentation with zidovudine is a well - documented occurrence in adults , especially dark - skinned individuals . but it has so far not been reported in children . here , we report a pediatric case of zidovudine - induced nail pigmentation . a 12-year - old boy receiving art with zidovudine , lamivudine , and nevirapine presented to dermatology opd with complaint of diffuse bluish - brown discoloration of all fingernails . the pigmentation was noticed by the patient after 3 months of initiating zidovudine - based regimen . it first appeared in thumb nails , gradually involved all fingernails , and increased in intensity over time . though harmless and reversible , psychological aspects of this noticeable side effect may hamper adherence to therapy and may lead to unnecessary investigations and treatment for misdiagnosis such as cyanosis or melanoma .

parental drug misuse and its effects on children and families are policy priorities in the uk and elsewhere ( flavin & paltrow , 2010 ; hm government , 2010 ) . polydrug use is the norm among drug users in the uk , and benzodiazepine use is common ( bird & robertson , 2011 ; department of health ( england ) , 2007 ; hay , gannon , casey , & mckeganey , 2009 ; jones , mogali , & comer , 2012 ) . increased risks and harm , and a range of adverse effects are associated with long - term benzodiazepine use ( lader , 2011 , 2014 ; obrien , 2005 ) . however , existing research on drug use and parenting has tended to focus on opioids , opioid substitution therapy ( ost ) , or crack cocaine ( radcliffe , 2009 , 2011 ; rhodes et al . , 2010 ) , and in the case of pregnancy , also alcohol , nicotine , marijuana and amphetamines ( behnke & smith , 2013 ) . in this paper , we examine accounts provided by polydrug - dependent parents about their use of , and dependence on , benzodiazepines as compared to ost . benzodiazepine use is highly prevalent among drug users worldwide ( jones et al . , 2012 ) . opioid - dependent and ost patients use benzodiazepines to prolong and enhance the effects of opiates ( jaffe et al . , 2004 ) , and increased rates of dependence are associated with co - occurring psychosocial problems ( jones et al . , 2012 ) . weizman , gelkopf , melamed , adelson , and bleich ( 2003 ) estimated a lifetime prevalence of benzodiazepine dependence in the opiate - dependent population ( in or out of treatment ) ranging from 6194% . benzodiazepines were originally marketed in the 1960s for the relief of anxiety , stress and insomnia ( who , 1996 ) and remain one of the most widely used psychoactive drugs ( lader , 2011 ) . their use within the general population is common , particularly among females ( obrien , 2005 ) . the gendered cultural meanings of diazepam ( valium ) , a well - known benzodiazepine , was cemented in the 1966 rolling stones song mother s little helper . benzodiazepines are recommended for the short - term treatment of anxiety and insomnia ( baldwin et al . , 2013 ) . tolerance and withdrawal effects can develop after only a few weeks of treatment ( lader , 2011 ) . adverse effects include sedation , impaired cognitive and psychomotor functioning , and increased likelihood of accidents and injuries ( lader , 2011 ; obrien , 2005 ) . withdrawal effects vary in severity and duration , and include anxiety , insomnia , depression , impaired memory , muscle spasms and tension , and rarely , seizures and psychosis ( lader , 2011 ) . combined benzodiazepine and opioid use is associated with increased sedation , decreased psychomotor performance ( lintzeris , mitchell , bond , nestor , & strang , 2007 ; lintzeris & nielsen , 2010 ) , poorer psychosocial adjustment and general health , heightened risk - taking behaviour , and increased risk of overdose ( bird & robertson , 2011 ) . however , evidence is limited ( jones et al . , 2012 ) , and few studies have focused on the combined effects of opioid and benzodiazepine dependence on pregnancy or parenting ( acmd , 2003 ) , despite evidence to suggest that both drugs are problematic in these contexts . whilst there is evidence that parental opioid use can compromise parenting capacity and pregnancy outcomes ( acmd , 2003 ) , the health and wellbeing of children and families can also be negatively affected by social and structural factors such as poverty , poor housing , unemployment , parental stress , domestic violence and criminal justice involvement ( hepburn , 2004 ; klee , jackson , & lewis , 2002 ) . however research indicates that despite this complexity , there is a tendency among service users and service providers to focus on drug use alone ( banwell & bammer , 2006 ; chandler et al . , 2013 ) , and in most cases the primary drug of misuse ( winklbaur et al . , 2008 ) . while this focus may relate to concerns regarding the effects of specific drugs per se on parenting capacity and the immediate safety of children , it could also result in a limited view of drug dependence that neglects to consider polydrug use and the impact of socio - economic and structural inequalities . while the use of opioids and ost is stigmatising , especially in the context of pregnancy and parenthood ( holland , forrester , williams , & copello , 2013 ) , use of benzodiazepines in the context of mothering has historically been characterised in a less problematic ( though not entirely positive ) manner ( metzl , 2003 ) . we found no published literature which compared benzodiazepine and opioid dependence , from the perspective of parents , or expectant parents . therefore the aim of our study was to address this gap in the evidence and to undertake a comparative thematic analysis of the ways in which parents accounted for their use of opioids , ost and benzodiazepines during and after pregnancy . this was a longitudinal , qualitative study involving 19 opioid - dependent service users over the course of approximately one year in order to examine changes over time in participants experiences with healthcare services , drug use and parenting . the truth , but rather as a resource through which to explore how parenting and drug use are understood , negotiated and mutually constructed ( bury , 2001 ; rhodes , bernays , & houmoller , 2010 ) . the study was carried out in scotland in an area with a mixed socio - economic profile , containing areas of significant deprivation . ost services for drug - dependent patients are delivered via primary care and specialist community - based substance misuse services . there are local guidelines both for the management of substance misuse in pregnancy ( whittaker , 2003 ) , and parental substance use ( elbeg , 2013 ) . notably , a large number of patients in the area are prescribed benzodiazepines in addition to ost . participants , recruited via healthcare practitioners , were interviewed at around 2832 weeks gestation ; 23 months postnatal ; and 69 months postnatal . inclusion criteria were opioid - dependence , being an expectant or recent parent , and > 18 years . all were unemployed and white , reflecting the population of opioid - dependent adults in the study area . five participants were first - time parents , fourteen other participants had between one and three children , aged 219 . all were receiving ost at the first interview , primarily methadone , with a minority prescribed buprenorphine or dihydrocodeine . eleven reported heroin use in the 12 months prior to the first interview and thirteen reported benzodiazepine use during the study period . in five cases , diazepam was prescribed long - term alongside ost . this reflects the pattern of poly - drug use and prescribing practices in this geographical setting ( bird & robertson , 2011 ) . in total , semi - structured interviews , lasting between 45120 minutes , addressed participants social background , drug use , experiences with parenting , and involvement with services . this was supplemented by close reading of transcripts and thematic coding by authors ac and aw ( ritchie , spencer , & oconnor , 2003 ) . the present paper is based on analysis of two content codes : benzodiazepine use and methadone practice . accounts were compared and contrasted and analytic themes were generated and agreed by the research team . participants gave informed consent and were provided with a 20 voucher for each interview completed . a common theme across parents accounts of all drug use was that their substance use helped to support their attempts to engage in normal daily life , providing a better context in which to undertake parenting . positive meanings were sometimes ascribed to ost which was portrayed as enabling parents to manage their opioid - dependence in a legal , safer and relatively more socially acceptable manner ( chandler et al . , 2013 ) . this was particularly the case for parents who had only recently ceased use of illicit opioids:[t]he only way to have a normal life [ ] and keep a normal life to normality , to being able to live again is a methadone prescription . ( alison , 3 months postnatal ) [ t]he only way to have a normal life [ ] and keep a normal life to normality , to being able to live again is a methadone prescription . ( alison , 3 months postnatal ) however , parents accounts more often emphasised the barriers and problems ost posed . particularly in the post - natal period , the structures around ost prescribing ( e.g. dispensing requirements ) were highlighted as having a negative impact on parents attempts to engage in a normal life or recovery - orientated activities such as training and employment : im no wanting to stay on daily supervised , especially to try and go back to work , to go back to college . ( laura , 9 months postnatal ) i m no wanting to stay on daily supervised , especially to try and go back to work , to go back to college . ( laura , 9 months postnatal ) this was particularly the case for those participants who were required to have their use of ost supervised where methadone was consumed in the pharmacy under the surveillance of the dispensing pharmacist ( and any onlookers ) . such practices were framed as especially problematic for parents : sometimes when i see ones in the chemist wi their weans [ children ] and i think they should nt be bringing their wean oot they re standing waiting for their meth and i try and sit in a seat away fae them . ( melanie , antenatal ) when you re being on supervised and that , because at the chemist i m at , they ve no got a bit that s blocked off , so you re standing at [ the locality ] swallowing your methadone in the middle of the shop flair . so you get folk looking at you [ ] whether i ve got [ daughter ] wi me or no , so it s just , there s the junkie , look at her . ( caitlin , 9 months postnatal ) sometimes when i see ones in the chemist wi their weans [ children ] and i think they should nt be bringing their wean oot they re standing waiting for their meth and i try and sit in a seat away fae them . ( melanie , antenatal ) when you re being on supervised and that , because at the chemist i m at , they ve no got a bit that s blocked off , so you re standing at [ the locality ] swallowing your methadone in the middle of the shop flair . so you get folk looking at you [ ] whether i ve got [ daughter ] wi me or no , so it s just , ( caitlin , 9 months postnatal ) both melanie and caitlin s accounts highlight the role of stigma in shaping the experiences of parents in ost programmes . in contrast , accounts of benzodiazepine use and dependence tended to be framed more positively , and crucially without reference to stigma . most participants described using benzodiazepines in constructive ways : treating anxiety states , mood enhancers , providing practical and emotional support for parenting , enabling them to engage in daily routines and household tasks , and ultimately providing a context in which carol , for instance , argued that her use of illicit diazepam was controlled , and helped her manage household chores.yes , like that song , mother s little helper , that s what i like to think of it as , just sort of gets me going , puts me in a good mood . ( carol , 6 months postnatal)similar justifications for illicit use were given by two fathers in the study . here , paul s account refers to his ( unsuccessful ) attempts to acquire a prescription for benzodiazepines : i said to him [ general practitioner ] i m not taking them to get out of my face [ intoxicated ] . i said i just take the two at night to help me get to sleep . ( paul , antenatal ) yes , like that song , mother s little helper , that s what i like to think of it as , just sort of gets me going , puts me in a good mood . ( carol , 6 months postnatal ) i said to him [ general practitioner ] i m not taking them to get out of my face [ intoxicated ] . i said i just take the two at night to help me get to sleep . ( paul , antenatal ) participant s explanations for their continued use of benzodiazepines often centred on the supposed functional benefits of the drug in helping parent s cope with day - to - day life . in contrast , accounts about ost were more likely to be framed around emphasising reduction and abstinence , narratives which were largely absent when it came to benzodiazepines . this is most evident in the accounts of parents who were dependent on both benzodiazepines and ost . in nicola s final interview she reported that she was committed to reducing her methadone , contrasting starkly with her account of her benzodiazepine prescription : im fed up being stuck on something , where i wake up with my daughter , feeling like crap , and the first thing i dae is have to swallow medicine and have a cup of tea , to feel normal [ ] aye , i m not ready for [ reducing ] diazepam . that s like mother s little helper [ ] they help me , to want to get up in time to do stuff and everything . ( nicola , 10 months postnatal ) i m fed up being stuck on something , where i wake up with my daughter , feeling like crap , and the first thing i dae is have to swallow medicine and have a cup of tea , to feel normal [ ] aye , i m not ready for [ reducing ] diazepam . that s like mother s little helper [ ] they help me , to want to get up in time to do stuff and everything . ( nicola , 10 months postnatal ) divergent orientations towards methadone and benzodiazepine reduction were also discussed with tricia . in earlier interviews , tricia had rejected the idea of reducing any of her prescriptions , however , like nicola , at 10 months postnatal she reported reducing her methadone , but not her benzodiazepine dosage : i feel like i m still a junkie because i need this green liquid [ methadone ] every day , it s crap , but i mean i dinnae feel like that with my valium [ ] aye i need to stay there [ on the same dose of benzodiazepine ] the now , i dinnae ken [ do nt know ] i would get all nervous . i do need my valium and if they said to me they were going to start cutting me i would panic [ ] but they re not , they re just happy at me being stable just now . ( tricia , 10 months postnatal ) i feel like i m still a junkie because i need this green liquid [ methadone ] every day , it s crap , but i mean i dinnae feel like that with my valium [ ] aye i need to stay there [ on the same dose of benzodiazepine ] the now , i dinnae ken [ do nt know ] i would get all nervous . i do need my valium and if they said to me they were going to start cutting me i would panic [ ] but they re not , they re just happy at me being stable just now . ( tricia , 10 months postnatal ) rhetoric about stability , abstinence and dependence , therefore , were employed by participants differently according to the particular substance they were discussing . accounts of substance use during pregnancy were for women especially more similar . in the majority of cases , women indicated that they wanted to reduce their dosage of both ost and benzodiazepine . each of these substances were framed as problematic during pregnancy , largely centring on potential harm to the unborn baby.ive forgotten the other one , stop smoking , come off that , like the valium , and the methadone [ ] and i said it would be a bit much for me doing all of that [ ] but it s no too much for me coming down from the methadone and the valium [ ] . because it s going to be helping the baby and me . ( iona , antenatal ) i ve forgotten the other one , stop smoking , come off that , like the valium , and the methadone [ ] and i said it would be a bit much for me doing all of that [ ] but it s no too much for me coming down from the methadone and the valium [ ] . because it s going to be helping the baby and me . ( iona , antenatal ) women stressed a keen awareness that use of a range of substances might have negative effects on their babies . those women dependent on multiple substances ( the majority ) described a careful balance of trying to stabilise or reduce one or more prescriptions , whilst avoiding relapse to illicit drug use . a clear contrast between parents accounts of opioid and benzodiazepine use and dependence was that the latter were often framed as a legitimate method of managing anxiety , negative emotions and past trauma . as such , while women described reducing their benzodiazepine use during pregnancy , as with nicola s account here , once the pregnancy ended , the need to reduce ( based on perceived potential harm to the baby ) also ceased.ive been on them maist of my life , and that s to stop paranoia , it s to stop me going ootside , and feel like everybody s looking at me , and agitated , anxiety , everything , so they know i ll no be coming off them . i ve already cut doon to ten milligrams , when i was pregnant , and nae mair , that s it . i ll sort the meth [ ost ] , and then see aboot that [ diazepam ] in the later future . ( nicola , 10 months post - natal ) i ve been on them maist of my life , and that s to stop paranoia , it s to stop me going ootside , and feel like everybody s looking at me , and agitated , anxiety , everything , so they know i ll no be coming off them . i ve already cut doon to ten milligrams , when i was pregnant , and nae mair , that s it . i ll sort the meth [ ost ] , and then see aboot that [ diazepam ] in the later future . ( nicola , 10 months post - natal ) while nicola reported recommencing her reduction of ost at 10 months postnatal , she maintained that she would not be doing the same with her benzodiazepine prescription . carrie described regular use of illicit benzodiazepines to manage anxiety states , and support her parenting . as with tricia and nicola above , she justified her use of benzodiazepines , and need for a prescription of benzodiazepines , on therapeutic grounds , whilst simultaneously affirming a desire to reduce her methadone prescription : ive been taking valium , but like the drug worker says , instead of getting it fae the streets , cos you do nt know what s in it , she s going to give me a prescription [ ] so i m not sitting all thingy [ pulls agitated face ] around the kids and stuff . cos i m a bit , still a bit , like anxious i think that s the word . but i m down to 19ml [ methadone ] noo , so i ve been coming doon a ml a week . ( carrie , 4 months postnatal ) i ve been taking valium , but like the drug worker says , instead of getting it fae the streets , cos you do nt know what s in it , she s going to give me a prescription [ ] so i m not sitting all thingy [ pulls agitated face ] around the kids and stuff . cos i m a bit , still a bit , like anxious i think that s the word . but i m down to 19ml [ methadone ] noo , so i ve been coming doon a ml a week . ( carrie , 4 months postnatal ) when participants described benzodiazepine use as therapeutic , their accounts framed this as reasonable , responsible and more importantly , acceptable . carrie emphasises that she was seeking , and expected to be given , a prescription for benzodiazepine , indicating that her dependence was validated by healthcare staff . another participant , michael , maintained that the illicit benzodiazepines he bought were obtained from a reputable source and were trustworthy in terms of pharmaceutical content . these narratives supported participants arguments that their benzodiazepine use , whether illicit or prescribed , was controlled and unproblematic . thus , participants constructed benzodiazepine use and dependence as a legitimate means to help them regulate problem emotional states which might otherwise interfere with their ability to live a normal life and effectively parent their children . benzodiazepine use and dependence for women was frequently normalised : women described using prescribed and illicit benzodiazepines to manage anxiety , perform household chores or regulate sleep patterns . in contrast , while men in the study described using benzodiazepines for similar reasons , their accounts indicated that this was seen as problematic . one participant framed this as explicitly related to gender : ive got all these people , telling me what s right , and what s wrong , and i take valium now and again , so , therefore , i m not a good parent . how many bloody mothers oot there , do you ken that take valium ? does that stop them from being good parents [ ] ? where s their just cause [ ] ? where s the argument , like , because i chose to be on valium when i was young , he must be f***ed up . to me , i would like to think i m reasonably intelligent . but , fae their angle , i m no , they must think i m a total madman , which i m no. ( stuart , 8 months postnatal ) i ve got all these people , telling me what s right , and what s wrong , and i take valium now and again , so , therefore , i m not a good parent . how many bloody mothers oot there , do you ken that take valium ? does that stop them from being good parents [ ] ? where s their just cause [ ] ? where s the argument , like , because i chose to be on valium when i was young , he must be f***ed up . to me but , fae their angle , i m no , they must think i m a total madman , which i m no. ( stuart , 8 months postnatal ) stuart alludes to the existence of gendered stereotypes about benzodiazepine dependence suggesting that use among women and mothers is accepted , but that among men and fathers is seen as dangerous or evidence of severe personality dysfunction . while the other men in the study did not make this gendered link explicit , the four male participants who reported illicit use of benzodiazepines provided similar accounts in other ways . all asserted the therapeutic benefits of benzodiazepines , described unsuccessful attempts to acquire prescriptions and subsequently affirmed a desire or need to cease their illicit use of benzodiazepines . while it is impossible to say based on this limited sample , these accounts allude to the existence of gendered patterns of prescribing , with healthcare practitioners seeming more ready to legitimise women s use of , and dependence on , benzodiazepines . benzodiazepine use among men was also portrayed as problematic by three women in the study who described illicit use of benzodiazepines by their partners . one participant suggested that her partner s benzodiazepine use was associated with aggression and domestic abuse , while the others clearly indicated that benzodiazepine use for their partners was more concerning than their use of other substances , particularly when combined with alcohol . this parallels findings of another study with young offenders ( forsyth , khan , & mckinlay , 2011).what i m doing , i m just pottering about the house quite happy , tidying up , sorting things out , and they [ benzodiazepines ] make me feel happy , and i go to the shops and chat away to people , and it gives me a bit more confidence , but with him they make him angry , like i do nt know , it s weird . ( carol , 9 months postnatal ) what i m doing , i m just pottering about the house quite happy , tidying up , sorting things out , and they [ benzodiazepines ] make me feel happy , and i go to the shops and chat away to people , and it gives me a bit more confidence , but with him they make him angry , like i do nt know , it s weird . ( carol , 9 months postnatal ) in contrast , while women occasionally noted adverse effects of benzodiazepine use , this was generally focused on pregnancy and risks to the unborn baby . the potential harms associated with benzodiazepine use , including the potential for cognitive impairment and acute withdrawal symptoms , appeared little understood or acknowledged in most participant accounts . this was a longitudinal , qualitative study involving 19 opioid - dependent service users over the course of approximately one year in order to examine changes over time in participants experiences with healthcare services , drug use and parenting . the truth , but rather as a resource through which to explore how parenting and drug use are understood , negotiated and mutually constructed ( bury , 2001 ; rhodes , bernays , & houmoller , 2010 ) . the study was carried out in scotland in an area with a mixed socio - economic profile , containing areas of significant deprivation . ost services for drug - dependent patients are delivered via primary care and specialist community - based substance misuse services . there are local guidelines both for the management of substance misuse in pregnancy ( whittaker , 2003 ) , and parental substance use ( elbeg , 2013 ) . notably , a large number of patients in the area are prescribed benzodiazepines in addition to ost . participants , recruited via healthcare practitioners , were interviewed at around 2832 weeks gestation ; 23 months postnatal ; and 69 months postnatal . inclusion criteria were opioid - dependence , being an expectant or recent parent , and > 18 years . all were unemployed and white , reflecting the population of opioid - dependent adults in the study area . five participants were first - time parents , fourteen other participants had between one and three children , aged 219 . all were receiving ost at the first interview , primarily methadone , with a minority prescribed buprenorphine or dihydrocodeine . eleven reported heroin use in the 12 months prior to the first interview and thirteen reported benzodiazepine use during the study period . in five cases , diazepam was prescribed long - term alongside ost . this reflects the pattern of poly - drug use and prescribing practices in this geographical setting ( bird & robertson , 2011 ) . in total , semi - structured interviews , lasting between 45120 minutes , addressed participants social background , drug use , experiences with parenting , and involvement with services . this was supplemented by close reading of transcripts and thematic coding by authors ac and aw ( ritchie , spencer , & oconnor , 2003 ) . the present paper is based on analysis of two content codes : benzodiazepine use and methadone practice . accounts were compared and contrasted and analytic themes were generated and agreed by the research team . participants gave informed consent and were provided with a 20 voucher for each interview completed . a common theme across parents accounts of all drug use was that their substance use helped to support their attempts to engage in normal daily life , providing a better context in which to undertake parenting . positive meanings were sometimes ascribed to ost which was portrayed as enabling parents to manage their opioid - dependence in a legal , safer and relatively more socially acceptable manner ( chandler et al . , 2013 ) . this was particularly the case for parents who had only recently ceased use of illicit opioids:[t]he only way to have a normal life [ ] and keep a normal life to normality , to being able to live again is a methadone prescription . ( alison , 3 months postnatal ) [ t]he only way to have a normal life [ ] and keep a normal life to normality , to being able to live again is a methadone prescription . ( alison , 3 months postnatal ) however , parents accounts more often emphasised the barriers and problems ost posed . particularly in the post - natal period , the structures around ost prescribing ( e.g. dispensing requirements ) were highlighted as having a negative impact on parents attempts to engage in a normal life or recovery - orientated activities such as training and employment : im no wanting to stay on daily supervised , especially to try and go back to work , to go back to college . ( laura , 9 months postnatal ) i m no wanting to stay on daily supervised , especially to try and go back to work , to go back to college . ( laura , 9 months postnatal ) this was particularly the case for those participants who were required to have their use of ost supervised where methadone was consumed in the pharmacy under the surveillance of the dispensing pharmacist ( and any onlookers ) . such practices were framed as especially problematic for parents : sometimes when i see ones in the chemist wi their weans [ children ] and i think they should nt be bringing their wean oot they re standing waiting for their meth and i try and sit in a seat away fae them . ( melanie , antenatal ) when you re being on supervised and that , because at the chemist i m at , they ve no got a bit that s blocked off , so you re standing at [ the locality ] swallowing your methadone in the middle of the shop flair . so you get folk looking at you [ ] whether i ve got [ daughter ] wi me or no , so it s just , there s the junkie , look at her . ( caitlin , 9 months postnatal ) sometimes when i see ones in the chemist wi their weans [ children ] and i think they should nt be bringing their wean oot they re standing waiting for their meth and i try and sit in a seat away fae them . ( melanie , antenatal ) when you re being on supervised and that , because at the chemist i m at , they ve no got a bit that s blocked off , so you re standing at [ the locality ] swallowing your methadone in the middle of the shop flair . so you get folk looking at you [ ] whether i ve got [ daughter ] wi me or no , so it s just , there s the junkie , look at her . ( caitlin , 9 months postnatal ) both melanie and caitlin s accounts highlight the role of stigma in shaping the experiences of parents in ost programmes . in contrast , accounts of benzodiazepine use and dependence tended to be framed more positively , and crucially without reference to stigma . most participants described using benzodiazepines in constructive ways : treating anxiety states , mood enhancers , providing practical and emotional support for parenting , enabling them to engage in daily routines and household tasks , and ultimately providing a context in which carol , for instance , argued that her use of illicit diazepam was controlled , and helped her manage household chores.yes , like that song , mother s little helper , that s what i like to think of it as , just sort of gets me going , puts me in a good mood . ( carol , 6 months postnatal)similar justifications for illicit use were given by two fathers in the study . here , paul s account refers to his ( unsuccessful ) attempts to acquire a prescription for benzodiazepines : i said to him [ general practitioner ] i m not taking them to get out of my face [ intoxicated ] . i said i just take the two at night to help me get to sleep . ( paul , antenatal ) yes , like that song , mother s little helper , that s what i like to think of it as , just sort of gets me going , puts me in a good mood . ( carol , 6 months postnatal ) i said to him [ general practitioner ] i m not taking them to get out of my face [ intoxicated ] . i said i just take the two at night to help me get to sleep . ( paul , antenatal ) participant s explanations for their continued use of benzodiazepines often centred on the supposed functional benefits of the drug in helping parent s cope with day - to - day life . in contrast , accounts about ost were more likely to be framed around emphasising reduction and abstinence , narratives which were largely absent when it came to benzodiazepines . this is most evident in the accounts of parents who were dependent on both benzodiazepines and ost . in nicola s final interview she reported that she was committed to reducing her methadone , contrasting starkly with her account of her benzodiazepine prescription : im fed up being stuck on something , where i wake up with my daughter , feeling like crap , and the first thing i dae is have to swallow medicine and have a cup of tea , to feel normal [ ] aye , i m not ready for [ reducing ] diazepam . that s like mother s little helper [ ] they help me , to want to get up in time to do stuff and everything . ( nicola , 10 months postnatal ) i m fed up being stuck on something , where i wake up with my daughter , feeling like crap , and the first thing i dae is have to swallow medicine and have a cup of tea , to feel normal [ ] aye , i m not ready for [ reducing ] diazepam . that s like mother s little helper [ ] they help me , to want to get up in time to do stuff and everything . ( nicola , 10 months postnatal ) divergent orientations towards methadone and benzodiazepine reduction were also discussed with tricia . in earlier interviews , tricia had rejected the idea of reducing any of her prescriptions , however , like nicola , at 10 months postnatal she reported reducing her methadone , but not her benzodiazepine dosage : i feel like i m still a junkie because i need this green liquid [ methadone ] every day , it s crap , but i mean i dinnae feel like that with my valium [ ] aye i need to stay there [ on the same dose of benzodiazepine ] the now , i dinnae ken [ do nt know ] i would get all nervous . i do need my valium and if they said to me they were going to start cutting me i would panic [ ] but they re not , they re just happy at me being stable just now . ( tricia , 10 months postnatal ) i feel like i m still a junkie because i need this green liquid [ methadone ] every day , it s crap , but i mean i dinnae feel like that with my valium [ ] aye i need to stay there [ on the same dose of benzodiazepine ] the now , i dinnae ken [ do nt know ] i would get all nervous . i do need my valium and if they said to me they were going to start cutting me i would panic [ ] but they re not , they re just happy at me being stable just now . ( tricia , 10 months postnatal ) rhetoric about stability , abstinence and dependence , therefore , were employed by participants differently according to the particular substance they were discussing . accounts of substance use during pregnancy were for women especially more similar . in the majority of cases , women indicated that they wanted to reduce their dosage of both ost and benzodiazepine . each of these substances were framed as problematic during pregnancy , largely centring on potential harm to the unborn baby.ive forgotten the other one , stop smoking , come off that , like the valium , and the methadone [ ] and i said it would be a bit much for me doing all of that [ ] but it s no too much for me coming down from the methadone and the valium [ ] . because it s going to be helping the baby and me . ( iona , antenatal ) i ve forgotten the other one , stop smoking , come off that , like the valium , and the methadone [ ] and i said it would be a bit much for me doing all of that [ ] but it s no too much for me coming down from the methadone and the valium [ ] . because it s going to be helping the baby and me . ( iona , antenatal ) women stressed a keen awareness that use of a range of substances might have negative effects on their babies . those women dependent on multiple substances ( the majority ) described a careful balance of trying to stabilise or reduce one or more prescriptions , whilst avoiding relapse to illicit drug use . a common theme across parents accounts of all drug use was that their substance use helped to support their attempts to engage in normal daily life , providing a better context in which to undertake parenting . positive meanings were sometimes ascribed to ost which was portrayed as enabling parents to manage their opioid - dependence in a legal , safer and relatively more socially acceptable manner ( chandler et al . , 2013 ) . this was particularly the case for parents who had only recently ceased use of illicit opioids:[t]he only way to have a normal life [ ] and keep a normal life to normality , to being able to live again is a methadone prescription . ( alison , 3 months postnatal ) [ t]he only way to have a normal life [ ] and keep a normal life to normality , to being able to live again is a methadone prescription . ( alison , 3 months postnatal ) however , parents accounts more often emphasised the barriers and problems ost posed . particularly in the post - natal period , the structures around ost prescribing ( e.g. dispensing requirements ) were highlighted as having a negative impact on parents attempts to engage in a normal life or recovery - orientated activities such as training and employment : im no wanting to stay on daily supervised , especially to try and go back to work , to go back to college . ( laura , 9 months postnatal ) i m no wanting to stay on daily supervised , especially to try and go back to work , to go back to college . ( laura , 9 months postnatal ) this was particularly the case for those participants who were required to have their use of ost supervised where methadone was consumed in the pharmacy under the surveillance of the dispensing pharmacist ( and any onlookers ) . such practices were framed as especially problematic for parents : sometimes when i see ones in the chemist wi their weans [ children ] and i think they should nt be bringing their wean oot they re standing waiting for their meth and i try and sit in a seat away fae them . ( melanie , antenatal ) when you re being on supervised and that , because at the chemist i m at , they ve no got a bit that s blocked off , so you re standing at [ the locality ] swallowing your methadone in the middle of the shop flair . so you get folk looking at you [ ] whether i ve got [ daughter ] wi me or no , so it s just , there s the junkie , look at her . ( caitlin , 9 months postnatal ) sometimes when i see ones in the chemist wi their weans [ children ] and i think they should nt be bringing their wean oot they re standing waiting for their meth and i try and sit in a seat away fae them . ( melanie , antenatal ) when you re being on supervised and that , because at the chemist i m at , they ve no got a bit that s blocked off , so you re standing at [ the locality ] swallowing your methadone in the middle of the shop flair . so you get folk looking at you [ ] whether i ve got [ daughter ] wi me or no , so it s just , there s the junkie , look at her . ( caitlin , 9 months postnatal ) both melanie and caitlin s accounts highlight the role of stigma in shaping the experiences of parents in ost programmes . in contrast , accounts of benzodiazepine use and dependence tended to be framed more positively , and crucially without reference to stigma . most participants described using benzodiazepines in constructive ways : treating anxiety states , mood enhancers , providing practical and emotional support for parenting , enabling them to engage in daily routines and household tasks , and ultimately providing a context in which carol , for instance , argued that her use of illicit diazepam was controlled , and helped her manage household chores.yes , like that song , mother s little helper , that s what i like to think of it as , just sort of gets me going , puts me in a good mood . ( carol , 6 months postnatal)similar justifications for illicit use were given by two fathers in the study . here , paul s account refers to his ( unsuccessful ) attempts to acquire a prescription for benzodiazepines : i said to him [ general practitioner ] i m not taking them to get out of my face [ intoxicated ] . i said i just take the two at night to help me get to sleep . ( paul , antenatal ) yes , like that song , mother s little helper , that s what i like to think of it as , just sort of gets me going , puts me in a good mood . ( carol , 6 months postnatal ) i said to him [ general practitioner ] i m not taking them to get out of my face [ intoxicated ] . i said i just take the two at night to help me get to sleep . ( paul , antenatal ) participant s explanations for their continued use of benzodiazepines often centred on the supposed functional benefits of the drug in helping parent s cope with day - to - day life . in contrast , accounts about ost were more likely to be framed around emphasising reduction and abstinence , narratives which were largely absent when it came to benzodiazepines . this is most evident in the accounts of parents who were dependent on both benzodiazepines and ost . in nicola s final interview she reported that she was committed to reducing her methadone , contrasting starkly with her account of her benzodiazepine prescription : im fed up being stuck on something , where i wake up with my daughter , feeling like crap , and the first thing i dae is have to swallow medicine and have a cup of tea , to feel normal [ ] aye , i m not ready for [ reducing ] diazepam . that s like mother s little helper [ ] they help me , to want to get up in time to do stuff and everything . ( nicola , 10 months postnatal ) i m fed up being stuck on something , where i wake up with my daughter , feeling like crap , and the first thing i dae is have to swallow medicine and have a cup of tea , to feel normal [ ] aye , i m not ready for [ reducing ] diazepam . that s like mother s little helper [ ] they help me , to want to get up in time to do stuff and everything . ( nicola , 10 months postnatal ) divergent orientations towards methadone and benzodiazepine reduction were also discussed with tricia . in earlier interviews , tricia had rejected the idea of reducing any of her prescriptions , however , like nicola , at 10 months postnatal she reported reducing her methadone , but not her benzodiazepine dosage : i feel like i m still a junkie because i need this green liquid [ methadone ] every day , it s crap , but i mean i dinnae feel like that with my valium [ ] aye i need to stay there [ on the same dose of benzodiazepine ] the now , i dinnae ken [ do nt know ] i would get all nervous . i do need my valium and if they said to me they were going to start cutting me i would panic [ ] but they re not , they re just happy at me being stable just now . ( tricia , 10 months postnatal ) i feel like i m still a junkie because i need this green liquid [ methadone ] every day , it s crap , but i mean i dinnae feel like that with my valium [ ] aye i need to stay there [ on the same dose of benzodiazepine ] the now , i dinnae ken [ do nt know ] i would get all nervous . i do need my valium and if they said to me they were going to start cutting me i would panic [ ] but they re not , they re just happy at me being stable just now . ( tricia , 10 months postnatal ) rhetoric about stability , abstinence and dependence , therefore , were employed by participants differently according to the particular substance they were discussing . accounts of substance use during pregnancy were for women especially more similar . in the majority of cases , women indicated that they wanted to reduce their dosage of both ost and benzodiazepine . each of these substances were framed as problematic during pregnancy , largely centring on potential harm to the unborn baby.ive forgotten the other one , stop smoking , come off that , like the valium , and the methadone [ ] and i said it would be a bit much for me doing all of that [ ] but it s no too much for me coming down from the methadone and the valium [ ] . because it s going to be helping the baby and me . ( iona , antenatal ) i ve forgotten the other one , stop smoking , come off that , like the valium , and the methadone [ ] and i said it would be a bit much for me doing all of that [ ] but it s no too much for me coming down from the methadone and the valium [ ] . because it s going to be helping the baby and me . ( iona , antenatal ) women stressed a keen awareness that use of a range of substances might have negative effects on their babies . those women dependent on multiple substances ( the majority ) described a careful balance of trying to stabilise or reduce one or more prescriptions , whilst avoiding relapse to illicit drug use . a clear contrast between parents accounts of opioid and benzodiazepine use and dependence was that the latter were often framed as a legitimate method of managing anxiety , negative emotions and past trauma . as such , while women described reducing their benzodiazepine use during pregnancy , as with nicola s account here , once the pregnancy ended , the need to reduce ( based on perceived potential harm to the baby ) also ceased.ive been on them maist of my life , and that s to stop paranoia , it s to stop me going ootside , and feel like everybody s looking at me , and agitated , anxiety , everything , so they know i ll no be coming off them . i ve already cut doon to ten milligrams , when i was pregnant , and nae mair , that s it . i ll sort the meth [ ost ] , and then see aboot that [ diazepam ] in the later future . ( nicola , 10 months post - natal ) i ve been on them maist of my life , and that s to stop paranoia , it s to stop me going ootside , and feel like everybody s looking at me , and agitated , anxiety , everything , so they know i ll no be coming off them . i ve already cut doon to ten milligrams , when i was pregnant , and nae mair , that s it . i ll sort the meth [ ost ] , and then see aboot that [ diazepam ] in the later future . ( nicola , 10 months post - natal ) while nicola reported recommencing her reduction of ost at 10 months postnatal , she maintained that she would not be doing the same with her benzodiazepine prescription . carrie described regular use of illicit benzodiazepines to manage anxiety states , and support her parenting . as with tricia and nicola above , she justified her use of benzodiazepines , and need for a prescription of benzodiazepines , on therapeutic grounds , whilst simultaneously affirming a desire to reduce her methadone prescription : ive been taking valium , but like the drug worker says , instead of getting it fae the streets , cos you do nt know what s in it , she s going to give me a prescription [ ] so i m not sitting all thingy [ pulls agitated face ] around the kids and stuff . just to calm me cos i m a bit , still a bit , like anxious i think that s the word . but i m down to 19ml [ methadone ] noo , so i ve been coming doon a ml a week . ( carrie , 4 months postnatal ) i ve been taking valium , but like the drug worker says , instead of getting it fae the streets , cos you do nt know what s in it , she s going to give me a prescription [ ] so i m not sitting all thingy [ pulls agitated face ] around the kids and stuff . just to calm me cos i m a bit , still a bit , like anxious i think that s the word . but i m down to 19ml [ methadone ] noo , so i ve been coming doon a ml a week . ( carrie , 4 months postnatal ) when participants described benzodiazepine use as therapeutic , their accounts framed this as reasonable , responsible and more importantly , acceptable . carrie emphasises that she was seeking , and expected to be given , a prescription for benzodiazepine , indicating that her dependence was validated by healthcare staff . another participant , michael , maintained that the illicit benzodiazepines he bought were obtained from a reputable source and were trustworthy in terms of pharmaceutical content . these narratives supported participants arguments that their benzodiazepine use , whether illicit or prescribed , was thus , participants constructed benzodiazepine use and dependence as a legitimate means to help them regulate problem emotional states which might otherwise interfere with their ability to live a normal life and effectively parent their children . benzodiazepine use and dependence for women was frequently normalised : women described using prescribed and illicit benzodiazepines to manage anxiety , perform household chores or regulate sleep patterns . in contrast , while men in the study described using benzodiazepines for similar reasons , their accounts indicated that this was seen as problematic . one participant framed this as explicitly related to gender : ive got all these people , telling me what s right , and what s wrong , and i take valium now and again , so , therefore , i m not a good parent . how many bloody mothers oot there , do you ken that take valium ? does that stop them from being good parents [ ] ? where s their just cause [ ] ? where s the argument , like , because i chose to be on valium when i was young , he must be f***ed up . to me but , fae their angle , i m no , they must think i m a total madman , which i m no. ( stuart , 8 months postnatal ) i ve got all these people , telling me what s right , and what s wrong , and i take valium now and again , so , therefore , i m not a good parent . how many bloody mothers oot there , do you ken that take valium ? does that stop them from being good parents [ ] ? where s their just cause [ ] ? where s the argument , like , because i chose to be on valium when i was young , he must be f***ed up . to me but , fae their angle , i m no , they must think i m a total madman , which i m no. ( stuart , 8 months postnatal ) stuart alludes to the existence of gendered stereotypes about benzodiazepine dependence suggesting that use among women and mothers is accepted , but that among men and fathers is seen as dangerous or evidence of severe personality dysfunction . while the other men in the study did not make this gendered link explicit , the four male participants who reported illicit use of benzodiazepines provided similar accounts in other ways . all asserted the therapeutic benefits of benzodiazepines , described unsuccessful attempts to acquire prescriptions and subsequently affirmed a desire or need to cease their illicit use of benzodiazepines . while it is impossible to say based on this limited sample , these accounts allude to the existence of gendered patterns of prescribing , with healthcare practitioners seeming more ready to legitimise women s use of , and dependence on , benzodiazepines . benzodiazepine use among men was also portrayed as problematic by three women in the study who described illicit use of benzodiazepines by their partners . one participant suggested that her partner s benzodiazepine use was associated with aggression and domestic abuse , while the others clearly indicated that benzodiazepine use for their partners was more concerning than their use of other substances , particularly when combined with alcohol . this parallels findings of another study with young offenders ( forsyth , khan , & mckinlay , 2011).what i m doing , i m just pottering about the house quite happy , tidying up , sorting things out , and they [ benzodiazepines ] make me feel happy , and i go to the shops and chat away to people , and it gives me a bit more confidence , but with him they make him angry , like i do nt know , it s weird . ( carol , 9 months postnatal ) what i m doing , i m just pottering about the house quite happy , tidying up , sorting things out , and they [ benzodiazepines ] make me feel happy , and i go to the shops and chat away to people , and it gives me a bit more confidence , but with him they make him angry , like i do nt know , it s weird . ( carol , 9 months postnatal ) in contrast , while women occasionally noted adverse effects of benzodiazepine use , this was generally focused on pregnancy and risks to the unborn baby . the potential harms associated with benzodiazepine use , including the potential for cognitive impairment and acute withdrawal symptoms , appeared little understood or acknowledged in most participant accounts . benzodiazepine use and dependence for women was frequently normalised : women described using prescribed and illicit benzodiazepines to manage anxiety , perform household chores or regulate sleep patterns . in contrast , while men in the study described using benzodiazepines for similar reasons , their accounts indicated that this was seen as problematic . one participant framed this as explicitly related to gender : ive got all these people , telling me what s right , and what s wrong , and i take valium now and again , so , therefore , i m not a good parent . how many bloody mothers oot there , do you ken that take valium ? does that stop them from being good parents [ ] ? where s their just cause [ ] ? where s the argument , like , because i chose to be on valium when i was young , he must be f***ed up . to me , i would like to think i m reasonably intelligent . but , fae their angle , i m no , they must think i m a total madman , which i m no. ( stuart , 8 months postnatal ) i ve got all these people , telling me what s right , and what s wrong , and i take valium now and again , so , therefore , i m not a good parent . how many bloody mothers oot there , do you ken that take valium ? does that stop them from being good parents [ ] ? where s their just cause [ ] ? where s the argument , like , because i chose to be on valium when i was young , he must be f***ed up . to me , i would like to think i m reasonably intelligent . but , fae their angle , i m no , they must think i m a total madman , which i m no. ( stuart , 8 months postnatal ) stuart alludes to the existence of gendered stereotypes about benzodiazepine dependence suggesting that use among women and mothers is accepted , but that among men and fathers is seen as dangerous or evidence of severe personality dysfunction . while the other men in the study did not make this gendered link explicit , the four male participants who reported illicit use of benzodiazepines provided similar accounts in other ways . all asserted the therapeutic benefits of benzodiazepines , described unsuccessful attempts to acquire prescriptions and subsequently affirmed a desire or need to cease their illicit use of benzodiazepines . while it is impossible to say based on this limited sample , these accounts allude to the existence of gendered patterns of prescribing , with healthcare practitioners seeming more ready to legitimise women s use of , and dependence on , benzodiazepines . benzodiazepine use among men was also portrayed as problematic by three women in the study who described illicit use of benzodiazepines by their partners . one participant suggested that her partner s benzodiazepine use was associated with aggression and domestic abuse , while the others clearly indicated that benzodiazepine use for their partners was more concerning than their use of other substances , particularly when combined with alcohol . this parallels findings of another study with young offenders ( forsyth , khan , & mckinlay , 2011).what i m doing , i m just pottering about the house quite happy , tidying up , sorting things out , and they [ benzodiazepines ] make me feel happy , and i go to the shops and chat away to people , and it gives me a bit more confidence , but with him they make him angry , like i do nt know , it s weird . ( carol , 9 months postnatal ) what i m doing , i m just pottering about the house quite happy , tidying up , sorting things out , and they [ benzodiazepines ] make me feel happy , and i go to the shops and chat away to people , and it gives me a bit more confidence , but with him they make him angry , like i do nt know , it s weird . ( carol , 9 months postnatal ) in contrast , while women occasionally noted adverse effects of benzodiazepine use , this was generally focused on pregnancy and risks to the unborn baby . the potential harms associated with benzodiazepine use , including the potential for cognitive impairment and acute withdrawal symptoms , appeared little understood or acknowledged in most participant accounts . this longitudinal qualitative study explored accounts of poly - drug use within the context of pregnancy and parenting , highlighting differential understandings and practices surrounding benzodiazepine and opioid use . our findings illuminate a number of issues with implications for theory , policy , prevention and education . the accounts in our study resonate with others ( klee , 1998 ; radcliffe , 2011 ) in demonstrating the morally difficult nature of being a drug - using parent , and the extent to which stigma can play an important role in the day - to - day lives of parents . our findings suggest that while drug use , in general , is seen as incompatible with parenting , there are clear indications that some drugs , and types of addiction , are more acceptable than others . crucially , our analysis indicates important differences in the way opioid and benzodiazepine use are understood and narrated within the context of parenthood . unlike benzodiazepine use , participants in this study often described ost in negative terms even when they acknowledged that ost was helpful in treating opioid dependence . this dominant negative discourse on ost by drug users themselves , has been described elsewhere ( harris & mcelrath , 2012 ; lloyd , 2013 ; radcliffe & stevens , 2008 ) , and highlights the unique stigma attached to ost for the treatment of opioid dependence ( keane , 2013 ) . participants described using benzodiazepines in constructive ways to enhance social and emotional functioning and engagement in normal life appealing to a self - medicating hypothesis of addiction to explain and justify continued use ( jones , et al . , 2012 ) . these meanings served to legitimise benzodiazepine dependence , contrasting sharply with participants alignment of ost with junkie identities , as documented elsewhere ( keane , 2013 ; radcliffe & stevens , 2008 ) . in the context of parenthood , parents were more likely to describe a desire to reduce opioid rather than benzodiazepine use , and there was evidence that illicit benzodiazepine use was considered less harmful than opioid use . this is noteworthy , because alongside this complacency were occasional notes of concern , particularly in relation to benzodiazepine use by men , and its link with increased aggression and domestic abuse ( reported by some women participants ) . accounts from this study provide a tentative indication of the way gender may shape understandings of benzodiazepine use . the long - term use of benzodiazepines by mothers was framed as being therapeutic and unproblematic . in contrast , while men reported using benzodiazepine for similar reasons , their accounts indicated that practitioners did not legitimise this and prescriptions were denied . these findings sit uneasily with the dominant recovery agenda in uk drug policy ( valentine & treloar , 2013 ; wardle , 2012 ) , where abstinence from all drug use is assumed to be both desirable and necessary for social reintegration . it may be that in practice recovery refers to particular categories of drugs , with the hierarchy of acceptability identified among service users in this study being apparently mirrored in prescribing practices and approaches taken to treating addiction by clinicians ( anthierens , habraken , petrovic , & christiaens , 2007 ) . thus , while opioid - dependent individuals may express a desire to reduce their opioid prescriptions , this is not necessarily reflected in their orientation towards other drug use , particularly benzodiazepines . as such , the claim that opioid - dependent individuals would prefer abstinence - based treatment approaches may result from the unique stigma attached to opioids and may not apply to all drug use , or all drugs of dependence ( lloyd , 2013 ; mckeganey , morris , neale , & robertson , 2004 ; neale , nettleton , & pickering , 2011 ) . our findings suggest a need for further preventative measures and targeted education to address benzodiazepine use and dependence within the context of pregnancy and parenting , especially in populations of parents engaged in ost programmes where stability on opioids may detract from issues associated with benzodiazepine use and dependence . reviews of benzodiazepine prescribing ( sirdifield et al . , 2013 ) , identify a variety of reasons for inconsistent practice , and conclude that improved education and training , provision of non - pharmacological interventions , and enhanced communication with patients is required . our findings support these recommendations , especially the need for enhanced education and training of both clinicians and patients , and extra provision of non - pharmacological interventions to promote the psychological and social functioning of parents on ost . this was an in - depth , qualitative exploration of the accounts of nineteen drug - dependent parents on ost in a specific geographical area . as such , our findings and conclusions should be read as indicative and suggestive rather than definitive . further qualitative exploration in different geographical areas and populations is needed , as it is likely that local or cultural contexts may shape the types of narratives expressed in respect of the range of substances consumed . our findings may have limited applicability to opioid dependent parents who are not engaged in ost programmes . however , the contrasting accounts depicted here underline the importance of qualitative research in illuminating understandings about the meanings that drugs of addiction have for those who use them ( neale , allen , & coombes , 2005 ) . although long - term benzodiazepine use and dependence among opioid - dependent populations has been a topic of concern for some decades ( who , 1996 ) and is associated with increased risks and harm ( lader , 2011 , 2014 ) , research regarding the impact on pregnancy , parenting and family life has been limited ( acmd , 2003 ) . our findings suggest that parents frame benzodiazepine use and dependence as largely unproblematic , less stigmatising and more legitimate than ost . we suggest these divergent accounts can be understood as drawing on differing cultural meanings attached to ost and benzodiazepine use , which are reflected in policy and practice . as such , prevention , education and policy initiatives should pay greater attention to the kind of narratives and strategies that are employed in addressing benzodiazepine use and dependence within opioid dependent populations , especially in respect of the parenting and child welfare agenda .

aimsto explore the ways in which opioid - dependent parents accounted for their use of opioids and benzodiazepines during and after pregnancy.methodslongitudinal qualitative interviews [ n = 45 ] with 19 opioid - dependent adults recruited in scotland , uk , were held during the antenatal and post - natal period . interviews focused on parenting and parenting support within the context of problem drug use and were analysed using a narrative informed , thematic analysis . findingsthe majority of participants described using benzodiazepines in addition to opioids . almost all indicated a desire to stop or reduce opioid use , whereas cessation or reduction of benzodiazepines was rarely prioritised . in stark contrast to opioid dependence , benzodiazepine dependence was portrayed as unproblematic , therapeutic and acceptable in the context of family life . whereas opioid dependence was framed as stigmatising , benzodiazepine use and dependence was normalised . an exception was benzodiazepine use by men which was occasionally associated with aggression and domestic abuse.conclusionsdrug-dependent parents attach different meanings to opioid and benzodiazepine use and dependence in the context of parenthood . divergent meanings , and stigma , may impact on stated commitment to stability or recovery from dependent drug - use . attention should be paid to the way in which policy and practice regarding ost and benzodiazepines reflects this divergence .

the incidence of bladder forming a part of an inguinal hernia is 14% . with correct knowledge of anatomy and careful dissection , injuries to the bladder during hernia repair we hereby report a case where the patient first presented with a scrotal abscess and vesicocutaneous fistula after surgical repair of sliding hernia . a 65-year - old morbidly obese man underwent mesh hernioplasty for large right inguinal hernia . after removing the urethral catheter , he developed gradually increasing right scrotal swelling with fever . thereafter , he developed continuous urine leakage from the site of incision and drainage , figure 1 . we carried out a cystogram via the urethral catheter that revealed a fistulous communication between bladder and scrotal skin , figure 1 . , cystoscopy revealed normal anterior and posterior urethra , non - obstructing prostatic lobes and a defect in the anterior bladder wall with no evidence of mesh erosion . almost the whole of the bladder was lying in the right scrotum and densely adherent to the right testis and cord structures and mesh . there was a fistulous opening at the dome of the bladder wall well away from the mesh . our main concerns were inguinal hernia repair and creation of extraperitoneal space to reposition the bladder in the normal position , which was not possible without performing right high inguinal orchiectomy . hence , we performed right high inguinal orchiectomy and removal of mesh and extraperitoneal space was made to reposition the urinary bladder to its normal position . fistula opening was repaired in two layers and the bladder was put on continuous drainage via 20 french urethral catheter , figure 2 . post - operatively at 2 weeks , there was no urinary leak on cystogram and the urethral catheter was removed and normal voiding was restored . scars of previous surgery with vesicocutaneous fistula and cystogram showing contrast in the left hemiscrotum the entire urinary bladder lying in the scrotum , with the bladder re - positioned into the normal position levine coined the term scrotal cystocoele in 1951 for inguinoscrotal herniation of the bladder . urinary bladder herniations are usually diagnosed at the time of inguinal herniorraphy and are commonly repaired through the same incision . they are sometimes found incidentally during the evaluation of a patient with lower urinary tract symptoms and associated inguinal hernias . two - stage micturition is the classical symptom , with the second stage facilitated by some form of external pressure on the bladder . the para - peritoneal type is the most common type and the extra - peritoneal type is the least common . because imaging all patients with large hernias may not be cost - effective , imaging studies are performed only when bladder herniation is suspected . the diagnostic triad of lateral displacement of the distal one - third of the ureter , small asymmetric bladder and incomplete visualization of the bladder base on an intravenous urogram has been described by reardon and lowman . iatrogenic injury to the bladder during hernia repair can be due to multiple factors , such as an inexperienced surgeon in the early part of the learning curve or an obese patient with large hernial sac with unrecognized bladder component . in our patient , there could have been an injury to the bladder that was not recognized at the time of hernia repair , which led to subsequent scrotal abscess formation resulting in a vesicocutaneous fistula . if unrecognized , these usually present immediately after catheter removal , but presentation can sometimes be delayed in case the fistula is very small and there is no infravesical obstruction . management includes immediate repair in case it is recognized intraoperatively . in case of unrecognized injury and with delayed presentation , the first step is to put a wide caliber per urethral catheter followed by thorough evaluation with urine culture examination and cystogram . a small fistula can be healed with only continuous bladder drainage with per urethral catheter or preferably suprapubic cystostomy , provided lower tract infravesical obstruction has been ruled out . a larger fistula needs open surgical management . careful dissection is needed in the extraperitoneal space while separating the sac from cord structures as the bladder forms a part of the posterior wall of the sac . after completing bladder dissection , the fistula is repaired in two to three layers and an adequate space is created in the extraperitoneal plane to reposition the bladder . sometimes , large hernias could be treated by resection of the herniated bladder as described by thomas and gomella . in our case , the whole bladder was lying in the scrotum and extensive adhesions were present between the cord structures and the bladder . high inguinal orchiectomy was performed to create space for the bladder and for proper closure of the inguinal canal . urinary bladder rarely forms a part of an inguinal hernia and , with the correct knowledge of anatomy and careful dissection injuries to the bladder during hernia repair , surgery can be prevented . surgically creation of an extraperitoneal space for bladder repositioning is of paramount importance , sometimes needing inguinal orchiectomy in the elderly .

sliding inguinal hernias are usually direct inguinal hernias containing various abdominal viscera . the incidence of bladder forming a part of an inguinal hernia , called as scrotal cystocele , is 14% . the risk of bladder injury is as high as 12% when repairing this type of hernia . this case report emphasizes this aspect in a 65-year - old man who presented with urinary leak through the scrotal wound following right inguinal hernia repair .

seven able - bodied male subjects ( age 30.7 4.2 yrs ; height 178.9 10.2 cm ; mass 73.9 12.2 kg , mean sd ) participated in this study the study was approved by the local ethics committee ( ethics committee of the swiss canton of bern , kek bern ) . the knee dynamometer is a custom made measurement device , which moves one leg at a constant velocity and measures the isokinetic torque produced during stimulated knee extension . the lower leg is fixed with a brace to an aluminium load cell ( lcb130 , me - mesysteme gmbh , germany ) , which moves , via a lever arm , a chain drive system connected to a magnetostrictive torque sensor ( s-2220 - 75 , ncte ag germany ) . the torque sensor and the load cell are used to bi - directionally measure the effective torque on the gauge bar in real time . a brushless motor ( ec45 , 250 w , maxon motor ag , switzerland ) is used with a planetary gear head ( gear ratio : 156:1 , gp42 , maxon motor ag , switzerland ) . the actuator can generate a maximum continuous torque of 90 nm . a position sensor ( vert - x 28 , contelec gmbh , switzerland ) is used for angle measurement with a resolution of 0.648 deg to control the motor torque . the measurements were performed at a constant angular velocity of ~110 /s , which is equivalent to a cycling cadence of 50 rpm , and the range of motion was set from 45 to 130 knee extension ( where 180 is full extension ) . the electrical pulses were generated with an eight channel stimulator ( rehastim , hasomed gmbh , germany ) . co. , ltd , usa ) were placed on the motor points of the m. quadriceps lateralis and medialis and dispersive electrodes were placed 10 15 cm proximal to the corresponding muscle motor point ( fig . 1 ) . the skin was cleaned and the body hair shaved at the position of the electrodes . muscle motor points were detected for each stimulated muscle prior to measurement with a stimulation pen ( motor point pen , compex , switzerland ) . subjects were stimulated with rectangular bi - phasic pulses at a constant amplitude of 40 ma . current was applied using an sdss electrode setup , which consists of four small electrodes with a surface area of 4.5 2.5 cm each and one large electrode ( 9 5 cm ) with the same total area . each of the four small electrodes used a frequency of 8.75 hz and a phase shift of 90 , which corresponds to an overall stimulation frequency of 35 hz . two different sdss electrode placements were investigated : distal versus proximal sdss electrodes . in both cases the active part the mean pulse widths were 73.3 14.2 s for the proximal and 73.3 14.4 s for the distal sdss setup . each subject participated in two sessions with only one electrode placement tested for each leg within each session . between the two measurements in each session , subjects had a break of 15 minutes . stimulation setup order and the leg were chosen randomly . before each measurement subjects were placed on the dynamometer and individual adjustments to body proportions were made . then a two - minute phase was started in which the measured leg was moved by the device without stimulation ( non - stimulation [ ns ] phase ) . then the pulse width was manually increased after every third extension , starting at 0 s . pulse width was increased up to the subjects pain threshold or up to the point they were no longer able to stay relaxed . 80 % of this maximal pulse width ( pwmax ) was then used for the test measurements . after a rest period of 10 minutes , the measurement started with an ns - phase of two minutes followed by a stimulation phase ( st - phase ) of 6 minutes . each session was conducted on a different day with at least one day of rest in between . electrode positions were marked to ensure identical placement each day only the extension phase of joint motion was evaluated , as the setup was to simulate cycling motion . the measured torque ( ) , together with the angular speed was used to calculate the instantaneous output power ( pm ) . the power used to move the leg during the ns - phase was denoted as pns . the net effective power output of one stimulation cycle is thus pstim = pns pm . for every knee extension the following outcomes were calculated : ( a ) mean power output during one extension ( pmean ) , ( b ) peak power output ( ppeak ) and ( c ) the time from onset of the stimulation to 80% of ppeak ( tpeak80 ) . to compare the different stimulation setups , pstim was scaled using a reference pulse width of 100 s ( pstim , s ) , e.g. subject a had a pulse width of 80 s , so the scaled mean power output pmean , s of that subject is pmean ( 100/80 ) and the scaled peak power output ppeak , s is ppeak ( 100/80 ) . all outcomes were calculated for the initial 15 knee extensions and for the final 15 knee extensions . a fatigue index ( fi ) describes the percentage reduction in pmean from the initial phase ( pinit ) to the final phase ( pfinal ) : fi=1-(pinit - pfinal)/pinit . the higher the value , the higher the fatigue resistance ; fi=1 means no fatigue . the data were tested for normality using the shapiro - wilk - test and then a paired t - test for normally distributed data and a wilcoxon test for non - normal data was applied to test differences of means . seven able - bodied male subjects ( age 30.7 4.2 yrs ; height 178.9 10.2 cm ; mass 73.9 12.2 kg , mean sd ) participated in this study the study was approved by the local ethics committee ( ethics committee of the swiss canton of bern , kek bern ) . the knee dynamometer is a custom made measurement device , which moves one leg at a constant velocity and measures the isokinetic torque produced during stimulated knee extension . the lower leg is fixed with a brace to an aluminium load cell ( lcb130 , me - mesysteme gmbh , germany ) , which moves , via a lever arm , a chain drive system connected to a magnetostrictive torque sensor ( s-2220 - 75 , ncte ag germany ) . the torque sensor and the load cell are used to bi - directionally measure the effective torque on the gauge bar in real time . a brushless motor ( ec45 , 250 w , maxon motor ag , switzerland ) is used with a planetary gear head ( gear ratio : 156:1 , gp42 , maxon motor ag , switzerland ) . a position sensor ( vert - x 28 , contelec gmbh , switzerland ) is used for angle measurement with a resolution of 0.648 deg to control the motor torque . the measurements were performed at a constant angular velocity of ~110 /s , which is equivalent to a cycling cadence of 50 rpm , and the range of motion was set from 45 to 130 knee extension ( where 180 is full extension ) . the electrical pulses were generated with an eight channel stimulator ( rehastim , hasomed gmbh , germany ) . co. , ltd , usa ) were placed on the motor points of the m. quadriceps lateralis and medialis and dispersive electrodes were placed 10 15 cm proximal to the corresponding muscle motor point ( fig . 1 ) . the skin was cleaned and the body hair shaved at the position of the electrodes . muscle motor points were detected for each stimulated muscle prior to measurement with a stimulation pen ( motor point pen , compex , switzerland ) . subjects were stimulated with rectangular bi - phasic pulses at a constant amplitude of 40 ma . current was applied using an sdss electrode setup , which consists of four small electrodes with a surface area of 4.5 2.5 cm each and one large electrode ( 9 5 cm ) with the same total area . each of the four small electrodes used a frequency of 8.75 hz and a phase shift of 90 , which corresponds to an overall stimulation frequency of 35 hz . two different sdss electrode placements were investigated : distal versus proximal sdss electrodes . in both cases the active part was placed on the previously detected motor point . the mean pulse widths were 73.3 14.2 s for the proximal and 73.3 14.4 s for the distal sdss setup . each subject participated in two sessions with only one electrode placement tested for each leg within each session . between the two measurements in each session , subjects had a break of 15 minutes . stimulation setup order and the leg subjects were placed on the dynamometer and individual adjustments to body proportions were made . then a two - minute phase was started in which the measured leg was moved by the device without stimulation ( non - stimulation [ ns ] phase ) . then the pulse width was manually increased after every third extension , starting at 0 s . pulse width was increased up to the subjects pain threshold or up to the point they were no longer able to stay relaxed . 80 % of this maximal pulse width ( pwmax ) was then used for the test measurements . after a rest period of 10 minutes , the measurement started with an ns - phase of two minutes followed by a stimulation phase ( st - phase ) of 6 minutes . each session was conducted on a different day with at least one day of rest in between . only the extension phase of joint motion was evaluated , as the setup was to simulate cycling motion . the measured torque ( ) , together with the angular speed was used to calculate the instantaneous output power ( pm ) . the power used to move the leg during the ns - phase was denoted as pns . the net effective power output of one stimulation cycle is thus pstim = pns pm . for every knee extension the following outcomes were calculated : ( a ) mean power output during one extension ( pmean ) , ( b ) peak power output ( ppeak ) and ( c ) the time from onset of the stimulation to 80% of ppeak ( tpeak80 ) . to compare the different stimulation setups , pstim was scaled using a reference pulse width of 100 s ( pstim , s ) , e.g. subject a had a pulse width of 80 s , so the scaled mean power output pmean , s of that subject is pmean ( 100/80 ) and the scaled peak power output ppeak , s is ppeak ( 100/80 ) . all outcomes were calculated for the initial 15 knee extensions and for the final 15 knee extensions . a fatigue index ( fi ) describes the percentage reduction in pmean from the initial phase ( pinit ) to the final phase ( pfinal ) : fi=1-(pinit - pfinal)/pinit . the higher the value , the higher the fatigue resistance ; fi=1 means no fatigue . the data were tested for normality using the shapiro - wilk - test and then a paired t - test for normally distributed data and a wilcoxon test for non - normal data was applied to test differences of means . figure 2 shows the development of pmean , s , ppeak , s and tpeak80 over the 6-minute stimulation phase . the corresponding outcome measures for the initial and final stimulation phases are summarised in tab . 1 . no significant differences between distal and proximal electrode placement were found for any outcome measures during the initial stimulation phase . in the final stimulation phase , ppeak and ppeak , s showed significantly higher values for the distal sdss setup : 25.4 8.1 w vs. 28.2 6.2 w , p=0.0062 and 34.8 9.5 w vs. 38.9 6.7 w , p=0.021 , respectively . in the final phase 3 ) and pmean , s with the distal sdss placement ( 11.8 3.8 w vs. 12.7 3.3 w , p=0.071 and 16.2 4.5 w vs. 17.4 3.4 w , p=0.14 ) , and of longer tpeak80 for distal sdss ( 347.6 29.2 ms vs. 359.4 38.2 ms , p=0.096 ) . the modestly higher mean power output in the final stage with distal sdss , and a lower dispersion of power values , can be conveniently visualised ( fig . fatigue resistance was not different between the two stimulation setups ( fi 0.61 0.14 vs. 0.64 0.9 , p=0.38 ) . the aim of this study was to compare the power output , fatigue and activation properties of proximally versus distally placed sdss electrodes in an isokinetic knee extension task simulating knee movement during recumbent cycling . overall , especially in the final phase of stimulation , the distal sdss setup showed higher power outputs ( the only exception was pmean , s in the initial phase , which was minimally lower for distal sdss ) . this might be regarded as surprising , since the active electrode was placed exactly on the motor point in the proximal sdss setup . splitting one large electrode into four small ones of the same overall size , and using a sequential stimulation strategy , was previously shown to give increased power output and better fatigue resistance compared to a standard electrode setup . the temporal and spatial shift had a significant impact on muscle activation in a dynamic knee extension task . using a standard electrode setup ( ses ) , it should not make a difference which electrode ( active , dispersive ) is positioned at the motor point : the electrical field is the same and the current direction should not activate the muscle fibres differently or change the number of recruited motor units . on the other hand , the electrical field changes with the size of the electrodes , and the distance between the active and dispersive electrodes has a substantial influence on the torque . to control for these factors in the two setups investigated in the present study , the distance was not changed and the active electrode was placed distally . that no substantial or significant difference was observed for any power output parameter in the initial phase of the task may indicate that both setups recruited and activated a similar number of motor units . the similar development over time of pmean and ppeak , as well as the different development of tpeak80 ( fig . 2a ) between setups , shows that the muscle fibre recruitment and the power curve of a single extension is not the same over time . although fi was not significantly different , the examination of power development over time ( fig . 2b , c ) shows that distal placement seems to have a slower power decrease and a higher power output in the final stimulation phase . smaller electrodes increase the current density compared to larger electrodes using the same amplitude and pulse width . thus , the non - equal size of the electrodes ( active , dispersive ) leads to an asymmetric electrical field , which seems to influence muscle activation . in contrast to the distal placement , where four small active electrodes are placed around the motor point , in the proximal setup one large active electrode is placed exactly on the motor point . in consequence , the change of the electrical field at this sensitive position is lower in this setup . less change over time in the pattern of activation has been shown to favourably affect fatigue and power output development ( 13 , 14 ) . activation time , i.e. the time from stimulation onset to 80% of peak power , plays a crucial role when electrical stimulation is used to produce a functional movement . usually , more than just one muscle group is involved , so that both the coordination of the force and the activation of the different muscles are of importance . in fes - cycling , often only three major muscle groups are involved ( m. quadriceps , m. hamstrings and m. gluteus ) and the coordination of these muscles is one factor for achievement of high power output . during repetitive activation , muscles not only fatigue , but tpeak80 for the proximal placement starts to flatten out after about 30 extensions , whereas tpeak80 for the distal placement is still increasing at this time and only begins to flatten out after about 65 extensions ( fig . the mean difference for tpeak80 of 11.8 ms in the final phase corresponds to a phase shift of 6 when cycling 50 rpm , which might have an influence on the overall cycling performance . by positioning the electrodes more precisely in relation to the motor points ( proximal setup ) , the activation becomes more efficient and the muscle activation time is less affected by the duration of the task . in conclusion , the sdss approach to muscle stimulation seems to provide substantial performance benefits , but the placement of the electrodes is still a crucial factor . distal placement of the sdss electrodes showed higher power output values in the final stimulation phase but also a slightly increased activation time . the development of new array electrodes , specifically for sdss , where the initial pulse is applied directly on the motor point and the following pulses are randomly distributed , may combine the positive effects of the proximal and distal electrode placements . based on the evidence presented here , for practical fes applications , distal placement of the sdss electrodes appears to be preferable .

spatially distributed sequential stimulation ( sdss ) has demonstrated substantial power output and fatigue benefits compared to single electrode stimulation ( ses ) in the application of functional electrical stimulation ( fes ) . this asymmetric electrode setup brings new possibilities but also new questions since precise placement of the electrodes is one critical factor for good muscle activation . the aim of this study was to compare the power output , fatigue and activation properties of proximally versus distally placed sdss electrodes in an isokinetic knee extension task simulating knee movement during recumbent cycling . m. vastus lateralis and medialis of seven able - bodied subjects were stimulated with rectangular bi - phasic pulses of constant amplitude of 40 ma and at an sdss frequency of 35 hz for 6 min on both legs with both setups ( i.e. n=14 ) . torque was measured during knee - extension movement by a dynamometer at an angular velocity of 110 deg / s . mean power , peak power and activation time were calculated and compared for the initial and final stimulation phases , together with an overall fatigue index . power output values ( pmean , ppeak ) were scaled to a standardised reference input pulse width of 100 s ( pmean , s , ppeak , s ) . the initial evaluation phase showed no significant differences between the two setups for all outcome measures . ppeak and ppeak , s were both significantly higher in the final phase for the distal setup ( 25.4 8.1 w vs. 28.2 6.2 w , p=0.0062 and 34.8 9.5 w vs. 38.9 6.7 w , p=0.021 , respectively ) . with distal sdss , there was modest evidence of higher pmean and pmean , s ( p=0.071 , p=0.14 , respectively ) but of longer activation time ( p=0.096 ) . the rate of fatigue was similar for both setups . for practical fes applications , distal placement of the sdss electrodes is preferable .

the first and second vertebrae ( atlas and axis , respectively ) are different from others with their function and anatomy . normally , the intervertebral disc is located between two vertebrae , but not located between atlas and axis . the most common cervical vertebrae anomalies ( cvas ) are fusion and posterior arch deficiency ( pad ) . the second ( c2 ) and third ( c3 ) cervical vertebrae are most commonly influenced by fusion . posterior arch anomalies occur probably the result of a locally decreased blood supply during fetal development . skeletal deviations in maxillofacial region , head and neck posture deformities , cervical inclination , and orthopedic findings can be associated with malocclusion . several studies declared the relation between the cervical vertebrae morphology and position of the mandible . cva is often asymptomatic , for this reason , patients may notice anomalies with decrease age or an injury the pathologic condition may reveal with the radiographic examination . although orthodontists do not have to be experts in cva these patients should be directed to the experts early as possible and prevents progressively degenerative neurologic defects . the present study is the first report to determine the frequency of cvas in orthodontic patients with dental malocclusions . the aims of this study were to investigate the distribution of cvas among angle class i , ii , and iii malocclusions in a turkish patient population and whether a correlation between cva and dental malocclusion ; thus , being the first series of cvas in our population described in the english literature . a power analysis conducted using the g*power version 3.1.7 . software ( franz faul , universitt kiel , germany ) indicated that a total sample size of 150 patients would give more than 80% power ( actual power = 0.8229 ) to detect significant differences with an effect size of 0.30 at an = 0.05 level of significance ( critical = 11.0705 ; noncentrality parameter = 13.5000 ) . the study was performed on 318 patients clinical records ( case histories , lateral cephalometric radiographs , and study models ) which were taken in archive at the department of orthodontics , faculty of dentistry , kirikkale university . if an accurate diagnosis of the cva could not be made from these records , the subject was excluded from the study . exclusion criteria included the patients , who were less than 18 years of age , had records with poor quality radiographs , had craniofacial anomaly and systemic muscle or joint disorder and , wound , burns , or scarring in the head and neck . subjects were selected according to the following criteria : turkish with turkish grandparents , no history of trauma , and previous orthodontic treatment , study models including the first molars and standardized lateral cephalometric radiographs with the first five cervical vertebrae visible . a total of 341 subjects were included in the study , but 23 subjects with developmental anomalies were excluded . the radiographs of the patients were obtained with the teeth in occlusion , the lips in a relaxed position in a standardized head posture ( the frankfort plane parallel to the horizontal ) . the radiographs were taken by an experienced x - ray technician using an orthopantograph ( planmeca proline cc 2002 , helsinki , finland ) with a film - to - focus distance of 165 cm and a film - to - median plane distance of 15 cm . dental malocclusion groups observed through molar relationship in plaster models were divided into angle class i , ii , and iii according to angle 's classification . angle class i : normal relationship of the molars ( mesiobuccal cusp of the upper molar occludes in buccal groove of the lower molar ) , but a line of occlusion incorrect because of malposed teeth , rotation , or other causes . angle class ii : lower molar distally positioned relative to upper molar , a line of occlusion not specified . angle class iii : lower molar mesially positioned relative to upper molar , a line of occlusion not specified . cvas were categorized and recorded for all patients : fusion was defined as no intradiscal radiolucency or osseous continuities without complete separation between two cervical vertebrae . pad was defined as a uniform radio - opacity without an internal cortical outline at the posterior arch of the cervical vertebra . the lateral cephalograms were examined by an orthodontist ( h.k . ) and a dentomaxillofacial radiologist ( e.y . ) simultaneously . to determine errors in the methods , 20% of the subjects with or without cva were selected randomly.first , each radiograph was separately evaluated by authors , but a final examination was done together , so a sentence was made to decide . in addition , the same examiners twice reevaluated all of the radiographs after 4 weeks of the first evaluation . a paired t - test was applied to both the first set and a second set of measurements , and no significant difference was found between the two sets . intra - examiner reproducibility was found to be 90 and 92% , respectively , and the agreement between both investigators was 95% . the chi - square and fisher 's exact test were used to determine the potential differences in the distribution of dental malocclusions , and genders when stratified by cvas . pearson 's correlation test was used to determine the correlation between cvas and different parameters ( dental malocclusions and genders ) . all of the statistical analyzes were performed with the spss software package ( spss version 16.0 , spss inc . , 233 south wacker drive , 11 floor , chicago , il , usa ) . a p < 0.05 was considered statistically significant . the final sample of 318 patients was examined ( 170 females and 148 males , mean age ; 20 0.9 years from 18 and 29 years ) . of these 318 patients , 98 ( 30.81% ) had angle class i , 124 ( 38.99% ) had angle class ii , and 96 ( 30.18% ) had angle class iii malocclusion . cva was observed in 42 individuals ( of which 26 [ 8.1% ] had fusion and 16 [ 5.0% ] had pad ) , with a frequency of 13.2% . of the 26 fusion defect , 8 ( 30.8% ) had angle class i , 8 ( 30.8% ) had angle class ii , and 10 ( 38.4% ) had angle class iii malocclusion . of the 16 pad , 8 ( 50% ) had angle class i , 8 ( 50% ) had angle class ii but no patients with angle class iii malocclusion was observed . the distribution of dental malocclusions regarding cvas was not statistically significant ( p = 0.076 ) [ table 1 ] . the distribution of cvas according to dental malocclusions of these 42 individuals with cva , 52.3% ( 15 fusions and 7 pad ) were females and 47.7% ( 11 fusions and 9 pad ) were males . the cvas and the gender were also compared [ table 2 ] and no statistically significant differences were found ( p = 0.339 ) . as shown in table 3 , statistically significant negative correlation ( p = 0.025 ) was found among cvas and dental malocclusions , but no statistically significant correlation was found between cvas and genders . according to the sample size calculation for a power of 0.80 at = 0.05 significant level , 150 subjects would be sufficient . at baseline , there were 341 subjects for this study group ; twenty - three subjects in the study group have been excluded from the study . at the end of the study , there were 318 patients for the study group . correlation between cva and vertebrae morphology , craniofacial malformations , and skeletal malocclusions were reported in the previous studies . the aims of our study were to identify the prevalence of cva in turkish population and also whether there is a correlation between cva and dental malocclusion . in our study , the prevalence of fusion and pad were found 8.1% and 5.0% , respectively [ table 1 ] . besides , a statistically significant correlation ( p = 0.025 ) was found among cva and dental malocclusions [ table 3 ] . the prevalence of cva was reported with a wide range of 0 - 61.4% in the literature . sonnesen and kjaer reported that the incidence of fusion ( 61.4% ) more common in skeletal class iii patients and skeletal horizontal overjet when compared with control group ( 14.3% ) . they revealed that if a skeletal horizontal overjet was caused by maxillary retrognathia , the decreased prevalence of fusion more likely seen . reported that the descending distribution of cva were determined in skeletal class i , ii , and iii malocclusion , respectively ( 17 ) . in addition , a higher prevalence of c1 level partial cleft and occipitalization was reported in their study . our study was designed to investigate dental malocclusion , but the previous studies were planned to examine skeletal anomalies . to the best of our knowledge , this is the first study evaluate the frequency and distribution of cvas in orthodontic patients with dental malocclusions . had stated that the angle of the cranial base and the head posture deviations were sexually dimorphic , females showing larger cervicohorizontal , and cranial base angles than males . in addition , they reported that the relation between the cervical column fusion and cranial base angle , the inclination of the upper cervical spine and cervical lordosis were determined in females . hence , it could be hypothesized that cervical column fusion has a dimorphic pattern in their occurrence . in contrast , some researchers affirmed that there was no significant relation between gender and the occurrence of cva in their studies . so that , our findings supports the studies showing no gender dimorphism [ table 2 ] . it is not clear why vertebral anomalies occur and why these anomalies occur in different craniofacial morphology groups and skeletal malocclusion traits . the genetic studies and insight of the early embryogenesis might be essential to understand the etiology of the cva . the recent studies affirmed that the notochord may be responsible to these anomalies . because the vertebral bodies were formed around the notochord in the prenatal period . besides , the jaws develop from the migration of the neural crest cells to the craniofacial area before the notochord is surrounded by bone tissue . however , the association between the precise signaling of notochord to the neural crest and the migration of the neural crest cells to the craniofacial area is still unknown . lateral cephalograms are usually used in orthodontic clinics to planning pretreatment and examined the postorthodontic treatment results . early diagnose of these pathologies on cephalograms can provide essential documentation to the patient due to symptoms , injury , aging , and progression of the degenerative process . however , two - dimensional radiographs may not be valid in the diagnosis of cva , because of the superimposition of the spine inclination and radiographic overlapping of the facets . for this reason , the suspectable sign of cva on cephalogram is reevaluated with three - dimensional imaging systems like cone beam computed tomography to prevent misdiagnose . the prevalence of fusion and pad were found 8.1% and 5.0% in turkish population , respectivelystatistically significant differences were found among the dental malocclusions , and no statistically significant correlation between cva and angle class i , ii , and iii malocclusionno statistically significant differences were also found between the genders , and no statistically significant correlation between cva and genders were found . the prevalence of fusion and pad were found 8.1% and 5.0% in turkish population , respectively statistically significant differences were found among the dental malocclusions , and no statistically significant correlation between cva and angle class i , ii , and iii malocclusion no statistically significant differences were also found between the genders , and no statistically significant correlation between cva and genders were found .

aims : the aims of our study were to investigate the distribution of cervical vertebrae anomalies ( cvas ) among dental angle class i , ii , and iii malocclusions in turkish population and whether a correlation between cva and dental malocclusion.materials and methods : the study was performed on lateral cephalometric radiographs which were taken at the department of orthodontics , faculty of dentistry , kirikkale university . the final sample of 318 orthodontic patients was included in the study . dental malocclusions were performed according to angle classification . cvas were categorized : ( 1 ) fusion and ( 2 ) posterior arch deficiency ( pad ) . the chi - square test was used to the analysis of the potential differences among dental malocclusions.results:the final sample of 318 patients was examined . cva was observed in 42 individuals ( of 26 [ 8.17% ] had fusion and 16 [ 5.03% ] had pad ) , with a frequency of 13.2% . of the 26 fusion defect , 8 ( 30.7% ) had angle class i , 8 ( 30.7% ) had angle class ii , and 10 ( 38.4% ) had angle class iii malocclusion . of the 16 pad , 8 ( 50% ) had angle class i , 8 ( 50% ) had angle class ii but no patients with angle class iii malocclusion was observed . the distribution of dental malocclusions regarding cva was not statistically significant ( p = 0.076 ) . of these 42 individuals with cva , 52.3% ( 15 fusions and 7 pad ) were females and 47.7% ( 11 fusions and 9 pad ) were males.conclusion:in our study , the prevalence of fusion and pad were found 8.1% and 5.0% in turkish population , respectively . besides , no statistically significant correlation between cva and angle class i , ii , and iii malocclusions were found . our findings support the studies showing no gender dimorphism .

animal and treatment : one week after the artificial insemination , second parity landrace yorkshire crossbred sows were randomly divided into two groups . sows in control group ( n = 6 ) were fed with standard diet ( table 1table 1.composition and nutrient content of the experimental dietcontrolbetaineingredient , g / kgcorn370370wheat300300bran8080soybean meal170170lignocelluloses3030cahpo42020soybean oil88premix*2020betaine03digestible energy , mj / kg13.113.1calculated compositioncrude protein , % 1515crude fiber , % 4.54.5calcium , % 0.840.84phosphorous , % 0.650.65 * the premix contains ( per kg ) : vitamin a : 240,000 iu ; vitamin d3 : 60,000 iu ; vitamin e : 720 iu ; vitamin k3 : 30 mg ; vitamin b1 : 30 mg ; vitamin b2 : 120 mg ; vitamin b6 : 60 mg ; vitamin b12 : 360 mg ; niacin : 600 mg ; pantothenic acid : 300 mg ; folic acid : 6 mg ; manganese sulphate : 1.0 g ; zinc oxide : 2.5 g ; iron sulphate : 4.0 g ; copper sulphate : 4.0 g ; sodium selenite : 6 mg ; calcium : 150 g ; phosphorus : 15 g ; sodium chloride : 40 g. ) , and those in betaine group ( n = 6 ) were fed with diet supplemented with 3 g / kg betaine hydrochloride of 98% purity ( skystone feed co. , ltd . , all animals were treated in the same way of insemination and reared under the same housing condition . the hippocami of male offspring ( one per litter ) were sampled at delivery , immediately frozen in liquid nitrogen and stored at 80c . * the premix contains ( per kg ) : vitamin a : 240,000 iu ; vitamin d3 : 60,000 iu ; vitamin e : 720 iu ; vitamin k3 : 30 mg ; vitamin b1 : 30 mg ; vitamin b2 : 120 mg ; vitamin b6 : 60 mg ; vitamin b12 : 360 mg ; niacin : 600 mg ; pantothenic acid : 300 mg ; folic acid : 6 mg ; manganese sulphate : 1.0 g ; zinc oxide : 2.5 g ; iron sulphate : 4.0 g ; copper sulphate : 4.0 g ; sodium selenite : 6 mg ; calcium : 150 g ; phosphorus : 15 g ; sodium chloride : 40 g. operating procedures for animal breeding and husbandry were in accordance with technical regulations for commercial pig production for intensive pig farms ( gb / t 17824.2 - 2008 ) . the experimental protocol was approved by the animal ethics committee of nanjing agricultural university , with the project number 2012cb124703 . the slaughter and sampling procedures complied with the guidelines on ethical treatment of experimental animals ( 2006 ) no . 398 after pretreated with rnase - free dnase , 2 g of total rna was reverse - transcribed to cdna in obedience to the protocol provided in the random hexamer primers kit ( promega , madison , wi , u.s.a . ) . two l of diluted cdna ( 1:25 , vol / vol ) was used for real - time pcr which was performed with a mx3000p real - time pcr system ( stratagene , santa clara , ca , u.s.a . ) . all the primers , synthesized by generay biotech , for determining gr total mrna and its variants and internal control ( -actin ) are listed in table 2table 2.primers for real - time pcr amplification of gr mrna and its variants , segments of gr promotertarget geneproduct lengthprimer sequencereference(bp)(f : forward , r : reverse)gr108f : 5-ccaaactctgccttgtgtgttc-3ay779185r : 5- tgtgctgtccttccactgct-3gr 1 - 4161f : 5-cacacagcacaacctttc-3r : 5-aaccttcacaggagttcc-3gr 1 - 5207f : 5-gcgtgcaacttccttcaa-3r : 5-cttggagtctggctgaga-3gr 1 - 6189f : 5-gagtgggccgcccagacgat-3r : 5-ccccccctcaggcttttat-3gr 1 - 7185f : 5-gcgaagagaaactagagaaa-3r : 5-aaccttcacaggagttcc-3gr 1 - 8144f : 5-tgcccagcgtcgccaaca-3r : 5-ccgcccctcaggcttttat-3gr 1 - 9,10177f : 5-cctgctttcacacgctaa-3r : 5-atcacatgggctctctcc-3gr 1 - 11163f : 5-ctggtggaagtgggcgtgtc-3r : 5-ttcctcccctcaggcttttat-3-actin201f : 5-cccacggaatcgagaaagag-3af057040r : 5-ttgacggaagggcacca-3gr segment1f : 5-cggcgaaggtctaggtacg-3r : 5- gaaggctgccccgtgt-3gr segment2f : 5- tctttgaaccccgcactt-3r : 5- ctcccagcgacaaaccag-3negative controlf : 5- ctgggcatcagaacctgt-3(actb promoter)r : 5- gagcaatcccctgaagaa-3. data analysis conformed to the method of 2 . the abundance of gr variants mrna was presented as the percentage of total gr mrna in control group . protein samples , after being measured concentrations with a pierce bca protein assay kit ( no . 23225 , thermo , rockford , il , u.s.a . ) , were denatured in waterbath for 5 min at 100c and seperated in a 7.5% or 10% sds - page . after protein transfer , nitrocellulose membranes ( biotrace , pall co. , ann arbor , mi , u.s.a . ) were blocked with 4% bsa and incubated respectively with anti - gr ( sc-1004 , santa cruz biotechnology , dallas , tx , u.s.a . , 1:500 ) and anti - actb ( ap0060 , bioworld , atlanta , ga , u.s.a . , 1:10,000 ) antibodies overnight at 4c . bands were visualized by enhanced chemiluminescence with the lumiglo substrate ( super signal west pico trial kit , pierce , rockford , il , u.s.a . ) and captured by versadoc 4000mp system ( bio - rad , hercules , ca , u.s.a . ) to calculate the value of band density using quantity one software ( bio - rad ) automatically . methylated dna immunoprecipitation analysis : hippocampal genomic dna was sonicated into fragements approximately 500 bp in size . two g of sonicated dna were denatured and incubated with the antibody against 5-methyl cytosine ( ab10805 , abcam , cambridge , ma , u.s.a . ) to immunoprecipitate the methylated dna fragments . then , protein g agarose beads were used to capture the dna - antibody complex which was washed and treated with proteinase k to release the precipitated dna . the medip dna was then extracted , purified and used to amplify the fragment of gr promoter by real - time pcr , following the procedure described previously . the promoter region of actb gene containing no cpg sites was amplified to serve as a negative control in medip analysis . data were normalized against the negative control and presented as the fold change relative to the average value of the control group . prediction and determination of mirnas : the potential mirnas targeting the 3 utr of gr mrna were predicted with the probability of interaction by target accessibility ( pita ) algorithm based on the online database of pig micrornas ( http://www.mirbase.org/ ) , using microrna prediction tool ( http://genie.weizmann.ac.il/pubs/mir07/mir07_prediction.html ) . two g of total rna from each piglet were polyadenylated by poly(a ) polymerase ( pap ) at 37c for 1 hr using a poly(a ) tailing kit ( am1350 ; ambion , rockford , il , u.s.a . ) , according to the manufacturer s instructions . after phenol / chloroform extraction and ethanol precipitation , treated rna was then dissolved and reverse - transcribed using poly(t ) adapter and m - mlv ( promega ) . real - time pcr was performed in an mx3000p ( stratagene ) with sybr_premix ex taqtm ii ( takara , otsu , japan ) using a mirna - specific forward primer and a universal reverse primer complementary to part of the poly ( t ) adapter sequence . the sequences for all the primers and the poly ( t ) adapter are listed in table 3table 3.primers for real - time pcr amplification of mirnasprimerprimer sequencemir-18b5-taaggtgcatctagtgcagttag-3mir-22 - 3p5-aagctgccagttgaagaactgt-3mir-22 - 5p5-agttcttcagtggcaagcttta-3mir-30a-3p5-ctttcagtcggatgtttgcagc-3mir-30a-5p5-tgtaaacatcctcgactggaag-3mir-130b5-cagtgcaatgatgaaagggcat-3mir-1385-agctggtgttgtgaatcaggc-3mir-181a5-aacattcaacgctgtcggtgagtt-3mir-1835-tatggcactggtagaattcactg-3mir-181d5-cccaccgagggatgaatgtcac-3mir-181c5-aacattcaacctgtcggtgagt-3universal reverse primer 5-tagagtgagtgtagcgagca-3u65-ggcaaggatgacacgcaaat-3poly ( t ) adapter 5-tagagtgagtgtagcgagcacagaattaatacg actcactataggttttttttttttttttvn-3. the abundance of mirnas was presented as the fold change relative to the average value of the control group . functional validation of mir-130b and mir-181d : dual luciferase activity assay was used to verify the function of mir-130b and mir-181d targeting gr 3utr in vitro , as previously described . briefly , fragments of mir-130b and mir-181d precursor and the scramble control ( sc ) sequences ( table 4table 4.nucleotides sequences used in functional validation of mirnasnamesequences ( f , forward ; r , reverse)mir-130b precursorf:5-gatccccttggcataacgtagcagcacataatggtttgtgggttttgaaaaggtgcaggccatattgtgctgcctcaaaaatacaaggttttttggaaa-3r:5-agcttttccaaaaaagcctgactgatgccctttcatcattgcactgcttcccagtggcccacagtagtgcaacagggaaagagtgtcaggcaggcg-3mir-181d precursorf:5-tgctgcccaccgagggatgaatgtcacgttttggccactgactgacgtgacatttccctcggtggg-3r:5-cctgcccaccgagggaaatgtcacgtcagtcagtggccaaaacgtgacattcatccctcggtgggc-3mir - scf:5-gatccgacttacagccagttcctagtatagtgaagcagcagatggtatactaggaactggctgtaagctttttttggaaa-3r:5-agcttttccaaaaaaagcttacagccagttcctagtataccatctgctgcttcactatactaggaactggctgtaagtcg-3gr 3utr primerf:5-tctagactttcgttggtgtat-3r:5- tctagagcaaacccattggg-3 ) were subcloned to construct plasmids named psilence - mir-130b , psilence - mir-181d and psilence - mir - sc , respectively ( fig . 3afig . 1.rt-pcr demonstrated that hippocampal expression of gr exon 1 - 4 and 1 - 9,10 mrna was higher in betaine - treated group , associated with the higher total gr mrna . meanwhile , western blotting analysis showed that total gr protein content was not changed so much . values are means , with their standard errors , and * indicates significant difference between groups at p<0.05 , * * means at p<0.01.fig . 2.cpg sites are underlined in detectable segment1 ( 4861 ~ 4722 ) and segment2 ( 1650 ~ 1515 ) from translation start codon ( atg ) , which were marked as block in gr exon 1 structure diagram ( blank arrow ) . medip results are presented in column figure , showing dna methylation status of cpgs in segment2 was increased . values are means , with their standard errors , and * indicates significant difference between groups at p<0.05.fig . ( c ) statistic results of the data from dual luciferase activity assay system . * * indicate significant difference between groups at p<0.05 and p<0.01 , respectively . ) . gr 3 utr sequence was amplified by pcr using the specific primers ( table 4 ) , and the product was then subcloned to pgl3-control using xbai ( invitrogen , rockford , il , u.s.a . ) to construct the pgl3-gr - utr plasmid ( fig . hela cells were transfected with the plasmids with an electroporation device , and prl - tk was used to normalize the transformation efficiency . after 24 hr incubation at 5% co2 and 37c , firefly and renilla luciferase activities were measured , and the targeting efficacy of mirnas in post - transcriptional repression of reporter protein was presented as the fold change relative to the average value of the psilence - mir - sc . rt - pcr demonstrated that hippocampal expression of gr exon 1 - 4 and 1 - 9,10 mrna was higher in betaine - treated group , associated with the higher total gr mrna . meanwhile , western blotting analysis showed that total gr protein content was not changed so much . values are means , with their standard errors , and * indicates significant difference between groups at p<0.05 , * * means at p<0.01 . cpg sites are underlined in detectable segment1 ( 4861 ~ 4722 ) and segment2 ( 1650 ~ 1515 ) from translation start codon ( atg ) , which were marked as block in gr exon 1 structure diagram ( blank arrow ) . predicted transcription factor binding sites among segment2 were framed . medip results are presented in column figure , showing dna methylation status of cpgs in segment2 was increased . values are means , with their standard errors , and * indicates significant difference between groups at p<0.05 . ( b ) plasmid structure containing gr 3utr sequences . cloning region is indicated in dot line with gr 3utr sequences ( 1,384 bp ) . ( c ) statistic results of the data from dual luciferase activity assay system . statistical analysis : all data were expressed as means sem and analyzed with one - way anova for independent samples with statistical packages for the social sciences ( spss ) 11.0 for windows . expression of total gr mrna and the alternative exon 1 mrna variants , as well as the total protein content of gr : as shown in fig . 1 , the hippocampal expression of total gr mrna was significantly higher ( p<0.01 ) in offspring piglets born to betaine - treated sows . seven alternative gr exon 1 mrna variants were detected in the hippocampus of newborn piglets . but , only gr 1 - 4 ( p<0.05 ) and gr 1 - 9,10 ( p<0.01 ) mrna variants were significantly upregulated in betaine group . nevertheless , no significant alteration was observed in total gr protein content as revealed by western - blot analysis ( fig . 1 ) . methylation level of gr promoter sequences : two segments of gr promoter , 4861 ~ 4722 and 1650 ~ 1515 from translation start codon ( atg ) , were analyzed using medip assay . the sequences of these two segments and the position relative to gr exon 1 variants are shown in fig . 2 . data from medip analysis indicated that the methylation level of segment 1 ( 4861 ~ 4722 ) was not significantly changed , but segment 2 ( 1650 ~ 1515 ) was hypermethylated ( p<0.05 ) in betaine group ( fig . the transcription factor prediction analysis with patch ( http://www.gene-regulation.com/cgi-bin/pub/programs/patch/bin/patch.cgi ) showed many potential transcription factor binding sites within this segment ( fig . 2 ) . expression of mirnas targeting gr : the abundance of mir-181a ( p<0.05 ) , mir-181d ( p<0.01 ) and mir-130b ( p<0.05 ) was significantly higher in the hippocampus of betaine - treated piglets compared to control counterparts ( table 5table 5.expression of mirnas targeting porcine gr 3utrpredicted mirnascontrol ( n = 5)betaine ( n = 5)p - valuemir-130b1.00 0.091.35 0.090.03 mir-1381.00 0.061.10 0.060.26 mir-18b1.00 0.091.09 0.100.50 mir-181a1.00 0.061.22 0.060.04 mir-181c1.00 0.081.07 0.090.61 mir-181d1.00 0.413.08 0.140.00 mir-1831.00 0.131.29 0.140.16 mir-22 - 3p1.00 0.061.07 0.090.51 mir-22 - 5p1.00 0.101.10 0.110.54 mir-30a-3p1.00 0.091.04 0.050.71 mir-30a-5p1.00 no significant difference was detected for the expression of mir-18b , 22 - 3p/5p , 30a-3p/5p , 138 , 183 or 181c between control and betaine - treated groups . functional validation of mir-130b and mir-181d : the schematic maps of the ssc - mir-130b and ssc - mir-181d over - expression plasmids , as well as the luciferase reporter plasmid containing 1384 bp of pig gr 3 utr , are shown in fig . 3a and 3b , respectively . overexpression of mir-130b ( p<0.05 ) or mir-181d ( p<0.01 ) was able to significantly reduce the luciferase activity of hela cells transfected with the pig gr 3utr reporter plasmid , as compared with the scramble control ( fig . it is well - documented that gr expression can be modulated by maternal nutritional interventions , such as protein restriction in rats [ 4 , 21 ] and undernutrition in sheep , as well as dietary supplementation of methyl donors in rats and in humans . moreover , infusion of l - methionine diminished hippocampal gr expression in the adult rat offspring of high licking / grooming and arched - back nursing ( lg - abn ) mothers . similarly , maternal dietary supplementation with methyl donor mixture inhibited hippocampal gr expression in three - month - old tame rats . in the present study , however , maternal betaine supplementation increased total gr mrna expression in the hippocampus of newborn piglets . the diverse responses of hippocampal gr expression to maternal methyl donor supplementation may attribute to multiple factors , such as the type and the dose of the substances , the timing and the duration of supplementation , the species , genotype and the age of the animals investigated . moreover , the distribution patterns of gr exon 1 mrna variants differ significantly between species . in pigs , gr mrna variants , 1 - 4 , 1 - 6 and 1 - 9,10 , are abundantly expressed and tightly regulated in the hippocampus [ 29 , 31 ] , while in rats , gr 1 - 7 appears to be the most important mrna variant of functional significance in hippocampus . therefore , it is presumed that the transcriptional regulation of gr expression in hippocampus is complex and species - specific . in our experiment , significant change of hippocampal gr expression in mrna level was observed with maternal betaine treatment , but not in protein level . probably , potential participant of transcript regulation , such as dna methylation and mirnas , was also induced by maternal betaine to maintain gr protein homeostasis in the hippocampi of these piglets . the effects of maternal methyl donors on the regulation of gr transcription often involve changes of the methylation status in gr promoter regions . higher choline intake led to hypermethylation of 5 untranslated exon 1f ( homologue of exon 1 - 7 in rats ) of gr promoter in human placenta , while methionine treatment reversed the hypomethylation of the exon 1 - 7 gr promoter in the hippocampus of adult rat offspring of high lg - abn mothers , as revealed with sodium bisulfite sequencing technique . nevertheless , it was also reported that feeding methyl - supplemented diet or methyl donor deficiency diet to pregnant dams did not alter the methylation level of gr exon 1 - 7 promoter in the hippocampus of rat offspring . these results suggest that the maternal or prenatal supplementation of methyl donors has minor , if any , effects on methylation status of gr exon 1 - 7 promoter in the hippocampus of rat offspring . in the present study , only two segments in the region of porcine gr promoter were investigated , and the segment ( 1650 ~ 1515 ) was found to be hypermethylated in the hippocampus of maternal betaine - treated piglets . the other regions can not be amplified due to extremely high enrichment of cpgs . certainly , more detailed and higher resolution analysis of methylation pattern across the whole region of gr promoter is required , by using more powerful techniques , to elucidate the role of cpg methylation in the transcription regulation of different gr mrna variants . it is well known that dna hypermethylation in gene promoter is functioned for suppression of mrna expression when positive transcript factors can not bind to the corresponding sequences . otherwise , binding loss of negative transcript factor would contributed to promoting mrna expression , as we previously reported that hypermethylation of differentially methylated regions in igf2 gene promoter was associated with high expression of igf2 mrna . here , we found that the segment ( 1650 ~ 1515 ) of gr gene promoter was hypermethylated , which might co - work with negative transcript factors to improve gr mrna expression . the segment ( 1650 ~ 1515 ) is located upstream of the translation start codon ( atg ) in exon 2 , which contains multiple transcription factor binding sites ( fig . 2 ) , predicted to be involved in the transcriptional regulation of gr expression . therefore , feeding betaine - supplemented diet to pregnant sows modulated hippocampal gr transcription in neonatal offspring piglets through , at least partly , alterations of methylation status in gr promoter . gr expression is also regulated at the post - transcriptional level . in this study , the increased abundance of total gr , as well as its 1 - 4 , and 1 - 9,10 mrna variants , did not lead to significant change in the level of gr protein in the hippocampus of betaine - treated piglets . we presumed that there might be mirna - mediated post - transcriptional regulation involved . methyl donor deficiency ( mdd ) in utero caused growth retardation of rat fetus at embryonic day 20 associated with upregulated expression of the stat3 regulator mir-124 . we reported previously that feeding betaine - supplemented diet to pregnant sows significantly altered hepatic expression of mir-497 and mir-181 in neonatal offspring piglets . in this study , hippocampal expression of mir-130b , mir-181a and mir-181d was significantly up - regulated in neonatal piglets of betaine group . overexpression of mir-130b was reported to decrease the expression of endogenous gr protein and the activity of the luciferase reporter containing the 3utr of human gr mrna in gc - sensitive mm.1s cells . in the present study , the suppressive function of ssc - mir-130b and ssc - mir-181d on gr expression was validated in hela cells transfected with a luciferase reporter plasmid containing pig gr 3utr sequence . mir-181d was previously reported to target methyl - guanine - methyl - transferase gene in human and was implicated to be a potential target for glioma therapy . furthermore , mir-181d was found to be one of the stress - responsive mirnas which may target a number of stress and metabolic signaling pathways in thymocytes . however , direct evidences to support the suppressive effect of mir-181d on gr expression are lacking . to our knowledge , here , we provide the first evidence that ssc - mir-181d is able to target porcine gr 3utr and suppress the luciferase reporter activity in hela cells . in conclusion , we demonstrate , for the first time , that maternal betaine supplementation during gestation enhances the expression of total gr mrna , as well as its exon 1 - 4 and 1 - 9,10 mrna variants , in the hippocampus of neonatal piglets , without affecting the total cellular gr protein content . alterations of cpg methylation in the proximal region of gr promoter and modified expression of mirnas targeting 3utr of porcine gr mrna appear to be involved in the regulation of hippocampal gr expression . long - term follow - up studies are required to evaluate the possible consequences of maternal betaine supplementation regarding the health and performance of pigs in later life .

methyl donor nutrients are critical for embryonic development of brain . hippocampus is the most susceptible brain region to various factors including prenatal supply of methyl donors . glucocorticoid receptor ( gr ) expressed in hippocampus is involved in the regulation of energy homeostasis and stress sensitivity . hippocampal gr expression is highly susceptible to epigenetic regulation , yet the effect of maternal methyl donor supplementation on epigenetic regulation of gr transcription in offspring hippocampus remains unclear . in this study , we fed sows with betaine ( 3 g / kg ) throughout the gestation and analyzed the hippocampal expression of gr mrna and its variants , as well as the cpg methylation status of the promoter and the micrornas predicted to target 3 utr of porcine gr gene in neonatal piglets . total gr mrna ( p<0.01 ) and its variants gr 1 - 4 ( p<0.05 ) and 1 - 9,10 ( p<0.01 ) , were significantly higher in the hippocampus of betaine - treated piglets , while the content of gr protein was not significantly changed . the cpgs located in the 1650 ~ 1515 segment of gr gene were hypermethylated ( p<0.05 ) . the hippocampal expression of mir-130b ( p<0.05 ) , mir-181a ( p<0.05 ) and mir-181d ( p<0.01 ) was significantly up - regulated . the targeting efficacy of mir-130b and mir-181d was validated in vitro using dual - luciferase reporter assay system . our results demonstrate that maternal betaine supplementation during gestation enhances gr mrna expression in offspring hippocampus , which involves alterations in mirnas expression .

in recent years , nanoparticles have been used increasingly for biomedical applications , including drug or gene delivery , imaging , sensing , or photothermal therapy . in particular , gold nanoparticles ( nps ) have been suggested as highly useful sensitizing agents in phototherapy due to their unique size and shape - dependent optical properties , high absorption coefficients , ease of synthesis , biocompatibility , and their ability to hold a variety of functional ligands . it is well - known that citrate - stabilized gold nps are endocytosed by cells and remain in intracellular vesicles . moreover , targeting of specific sites inside the cell by functionalization of the surface with cell penetrating peptides or peptides containing nuclear localization sequences has been reported . alternatively , to selectively target specific types of cells , gold nps can be modified with suitable antibodies . this approach has been studied for potential use in photothermal cancer therapy , where cancer cells overexpressing human epidermal growth factor receptor 2 ( her2 ) or epithelial growth factor receptor ( egfr ) were incubated with gold nps conjugated to anti - her2 or anti - egfr antibodies , respectively . the nps were then irradiated with light within their plasmon resonance absorbance band to heat the cells to temperatures leading to cell death . most interestingly , it has also been demonstrated that endocytosis of gold nps by cancer cells and subsequent irradiation of such intracellular nps can lead to cell death even at irradiation levels that are not high enough to cause significant heating . this nonthermal route to laser - induced cell death has been ascribed to an as yet not fully characterized photochemical reaction , although irradiation of endocytosed nps was reported to be accompanied by increased levels of reactive oxygen species . the use of such a photochemical mechanism could be of great advantage in situations where different types of cells coexist in close vicinity , since it would allow for more selective targeting of particular cells , whereas due to the fast diffusion of heat over the relevant length scales photothermally induced cell death will affect all cells within the irradiated volume more or less indiscriminately , as long as some of them contain nps . irradiation of gold nps with continuous wave ( cw ) lamp or laser light has been shown to lead to photogeneration of singlet oxygen ( o2 ) in vitro , suggesting that this highly reactive species , which is widely used in photodynamic therapy , may be involved in the photochemical pathway of cell killing by gold nps . in vitroo2 photogeneration by irradiation of spherical gold nps with short laser pulses or cw laser light at comparable powers and intensities has also been reported . in this study , it was suggested that the mechanism of singlet oxygen photogeneration may involve hot electrons , i.e. , the highly excited conduction band electrons which upon absorption of a short laser pulse by a np can reach quasi - equilibrated energy distributions corresponding to temperatures of several thousand degrees . this seems somewhat surprising , since cw light at the intensities used does not yield hot here we present new experimental results on the photogeneration of singlet oxygen by irradiation of gold nanoparticles with continuous or pulsed laser light , as well as theoretical work pertaining to the underlying mechanism(s ) , which so far had not been addressed . we show that electron temperatures in excess of 2000 c are easily achieved in pulsed laser irradiation experiments , whereas cw light under similar conditions yields electronic temperatures of at most 10 c above room temperature . thus , the photogeneration of o2 by gold nps proceeds by different mechanisms under different irradiation conditions ; the implications for the further development of medical applications of the effect are discussed in detail . furthermore , we also found that even a moderately thick , but dense , ligand layer significantly reduces the efficiency of o2 photogeneration at the np surface , which also has important consequences for practical applications . citrate - stabilized spherical gold nps with 15 and 46 nm diameter were prepared according to the turkevich frens and a seeded growth method , respectively . gold nanorods ( nrods ) were synthesized using the seeded - growth method reported by dickerson et al . with slight modifications . more details of the np preparation are provided in the supporting information . nps were characterized using uv vis spectroscopy ( genesys 10 uv ) , differential centrifugal sedimentation ( cps instruments dc24000 ) , and transmission electron microscopy ( tem , fei tecnai spirit microscope at 120 kv ) ( see figures s1 and s2 in the supporting information ) . the uv vis absorbance spectrum of nrods showed a transverse plasmon resonance band at 522 nm , and a longitudinal plasmon resonance band at 798 nm and tem revealed nrods to have a length of 40 nm and a diameter of 12 nm . functionalization with thiolated peg ligands or peptides was achieved by overnight incubation with excess ligand , followed by repeated centrifugation for excess ligand removal . the capping ligands used here were peg - oh ( hs-(ch2)11-(eg)4-oh ) , mpeg5000 ( hs-(ch2)2-(eg)n - o - ch3 , average mw 5000 g mol ) and peptide c - tat ( primary sequence calnnagrkkrrqrrr ) ; see figure s3 for the structures of the peg ligands . o2 was detected via the bleaching of 1,3-diphenylisobenzofuran ( dpbf ) , which is widely used for this purpose . all experiments involving dpbf were carried out in the dark . because dpbf is not soluble in neat water , all experiments were conducted in 50/50 ( v / v ) mixtures of water and ethanol . a fresh solution of dpbf ( 3.1 mg , 0.115 mm , a412 nm = 2 ) in etoh ( 100 ml ) was kept stirring in the dark . a 10 mm quartz cuvette and a 3 mm stirrer bar were left in aqua regia ( 1:3 hno3:hcl ) for 15 min and thoroughly rinsed multiple times with milli - q water ( mq h2o ) and etoh . in the clean cuvette with stirrer bar , either mq h2o ( 600 l ) or np solution ( 600 l ) was mixed with the ethanolic dye solution ( 600 l ) . where appropriate , the np concentration was adjusted prior to mixing to yield an absorbance of 0.4 at 532 nm in the final solution . the cuvette was sealed with an airtight lid and parafilm , and the uv vis absorbance spectrum was recorded ( genesys 10 uv ) . the sample was then placed on a stirring plate ( in the dark ) , and its absorbance spectrum measured every 10 min for 30 min to ensure the solution was stable . for the irradiation experiments , the cuvette containing 1200 l of sample solution was placed on a stirring plate in front of the laser . for experiments with nps , the cuvette was fitted with heat fins using non - silicone heat transfer paste and cooled using a fan . no significant increase of the cuvette temperature beyond a slight warming was observed . for most experiments , the sample was irradiated at 1000 mw ( unless stated otherwise ) using a 532 nm continuous - wave diode pumped solid state laser ( laser quantum opus 532 ) with a 1/e beam diameter of 1.85 mm ; for some experiments the beam was expanded to 8 mm diameter using a lens , as stated explicitly . the sample was irradiated for 10 min and then removed from the laser setup to record the absorbance spectrum ; this was repeated until the sample had been irradiated for 60 min in total . irradiation of gold nrods in the longitudinal plasmon resonance band at 800 nm was performed in the same setup , but using a titanium : sapphire laser ( coherent mira 900 ) , aligned on the auxiliary cavity , which prevents mode - locking and thus provides continuous - wave laser operation at 800 nm with 1000 mw power ; the beam was expanded to 3.4 mm using two lenses . pulsed laser irradiation was performed in the same setup , but using the second harmonic of a q - switched nd : yag laser ( quantel brilliant ) ( 532 nm , 5 ns pulse length , 10 hz repetition rate , 3.5 mm beam size , 15 mj pulse energy ) . for experiments that included nps , the absorbance of dpbf at 412 nm was calculated by subtracting the np absorbance at 412 nm ( obtained from neat np samples ) from the measured sample absorbance . the concentration of dpbf in the samples always yielded an initial absorbance very close to 1 at 412 nm ; for comparative data analysis , the irradiation - time - dependent dpbf absorbance at 412 nm was therefore normalized to 1 at zero irradiation time . 1,3-diphenylisobenzofuran ( dpbf ) readily undergoes a 1,4-cycloaddition on reaction with o2 to form endoperoxides which irreversibly yield 1,2-dibenzoylbenzene . dpbf strongly absorbs light at 412 nm ( figure 1a ) , but due to the loss of the -system of isobenzofuran , the product does not absorb light at this wavelength ; it is this loss of absorbance that is used to detect the presence of singlet oxygen . here , dpbf was chosen as singlet oxygen sensor as it has no absorbance at 532 or 800 nm , the wavelengths used for laser irradiation of gold nanoparticles in their plasmon resonance bands , and no photobleaching of dpbf was expected to occur upon irradiation in the absence of nps . contrary to this expectation , some bleaching was observed under our cw irradiation conditions , with the dpbf absorbance decreasing by ca . 10% upon irradiation at 1 w ( 37 w cm ) for 60 min ( figure 1a ) . as shown in figure 2a , and described in more detail in the supporting information , this photobleaching of dpbf has two phases : ( i ) a rapid phase , extending over the first 1020 min of irradiation under the conditions used here , and ( ii ) a slower phase , which on the time scale investigated appears linear with time . we found that phase i depends on laser power , beam size , and the presence of oxygen , whereas the slope of the time dependent bleaching after 20 min , phase ii , is essentially independent of the presence of oxygen and of the beam power / intensity in the range used here ( see figure s4 for details ) . it should be noted that similar effects have been reported previously , with cw irradiation at 514 nm for 60 min at significantly lower powers ( 40 mw ) than employed here , leading to a ca . 5% decrease of the absorbance of dpbf in benzene , although no explanation was suggested in that report . the oxygen dependence of phase i is in agreement with direct photogeneration of singlet oxygen by visible light , which has been suggested to be the reason for dpbf photobleaching upon irradiation with light at wavelengths above 470 nm . however , the slower phase ii is not affected by removing oxygen from the solution ( see figure s4b and figure 3 ) and hence can not be ascribed to singlet oxygen formation . at the present moment , the mechanism of both phases of dpbf photobleaching remains unclear , although both effects are highly reproducible and bleaching does not occur when the sample is not exposed to light ( see figure s4a ) . photobleaching of the dpbf absorbance upon cw irradiation at 532 nm , 1 w ( 37 w cm ) , in a 50/50 ( v / v ) mixture of water and ethanol : ( a ) in the absence of nps and ( b ) in the presence of 15 nm citrate - stabilized spherical gold nps . shown are absorbance spectra taken at intervals of 10 min from before the irradiation up to a maximum irradiation time of 60 min ; the arrows indicate the direction of change . ( a ) time dependence of the photobleaching of the dpbf absorbance at 412 nm , a412(dpbf ) , upon cw irradiation at 532 nm , 1 w ( 37 w cm ) , in the absence and presence of citrate - stabilized spherical nps with 15 and 46 nm diameter ; shown here are the results from several individual experiments ( dashed lines ) and the average ( solid lines ) , after subtraction of the np absorbance and normalization to 1 at time zero ; see the experimental section for details of data treatment and analysis . ( b ) effect of the nps alone , calculated by subtracting the photobleaching effect of dpbf in the absence of nps from the results obtained in the presence of nps ; the solid lines in ( b ) are linear fits of the data in the range 2060 min . gradient of the time - dependent dpbf absorbance photobleaching in the irradiation time window 2060 min for different samples under cw irradiation at 532 nm , 1 w ( 37 w cm ) . for experiments in the presence of nps , the np concentration was adjusted to yield an absorbance of 0.4 at 532 nm . experiments for dpbf in the absence of nps and in the presence of 15 nm spherical nps were also undertaken after bubbling the sample with nitrogen for 10 min , as indicated ( + n2 ) . the error bars correspond to the standard deviation of several repeat experiments , and indicates statistically significant differences with respect to the experiment on dpbf only , as determined by the anova f - test at p < 0.001 ; it should be noted that the results for 15 or 46 nm spherical nps without nitrogen bubbling or a peg capping layer were found to be different to all other results at this statistical significance level . no repeat experiment was undertaken for irradiation of nanorods at 532 nm , but the same result ( no additional bleaching in the presence of nanorods ) was obtained for irradiation at 800 nm ( figure s5 ) . in the presence of citrate - stabilized spherical gold nps , the photobleaching of dpbf upon irradiation at 532 nm , i.e. , within the nanoparticle plasmon resonance band , is significantly increased ( figure 1b ) . the time dependence of the dpbf photobleaching in the absence of nps and in the presence of nps with 15 and 46 nm diameter is shown in figure 2a . it can be clearly seen that the presence of nps leads to a significant increase of the dpbf photobleaching effect and that larger nps yield a larger effect although the np absorbance was adjusted to be the same for all samples , so that the same amount of light was absorbed . the additional photobleaching of dpbf caused by the presence of nps can be clearly ascribed to the generation of reactive oxygen species ( ros ) , since purging of the samples with nitrogen removes this effect ( see below ) . moreover , previous experiments had shown that light irradiation of citrate - stabilized spherical gold nps leads to the characteristic luminescence of o2 at 1280 nm and does not result in the generation of superoxide , o2 , or hydroxyl , oh , radicals , which leads us to conclude that the predominant ros species produced here is singlet oxygen , o2 . the fact that significant dpbf photobleaching occurs even in the absence of nps requires careful consideration for the analysis of these data . like dpbf photobleaching in the absence of nps , the effect in the presence of nps shows two phases . however , subtraction of the effect observed when only the dye is present yields an essentially linear time dependence for the additional effect ascribed to the nps on the time scale of the experiment ( figure 2b ) , indicating that the np - induced photogeneration of o2 is essentially constant over our experimental time interval . moreover , the second phase of direct dpbf photobleaching is largely independent of the laser power , at least down to 0.1 w , i.e. , significantly lower powers than those used for most of the experiments reported here , as shown in detail in the supporting information ( figure s4a ) , and therefore should be independent of the presence of nps whose absorbance leads to a decrease of the laser power along the beam path , allowing a direct comparison of the results obtained in the time frame of 2060 min . moreover , this slower phase of direct dpbf photobleaching is also independent of the presence of oxygen . for these reasons , only data starting at 20 min irradiation will be used for quantitative comparisons . figure 3 summarizes the main results obtained here after np photoexcitation at 532 nm . it is very obvious that citrate - stabilized spherical gold nps induce significantly faster dpbf photobleaching than is observed in their absence ( black ) ; this is found for 15 nm gold nanoparticles ( red ) but is even more pronounced for larger nps with 46 nm diameter ( blue ) . increasing the size of the spherical nps from 15 to 46 nm increases the np - induced dpbf photobleaching ( i.e. , the additional effect , after subtracting the direct dpbf photobleaching effect ) by ca . it is interesting to note that unlike the direct dpbf photobleaching , which is not affected by nitrogen purging , the np - induced additional bleaching is significantly reduced by nitrogen purging , almost down to the level of the direct dpbf photobleaching effect . this strongly supports the conclusion that the np - induced effect is caused by the formation of o2 which then leads to dpbf photobleaching . furthermore , capping the 15 nm gold nps with peg - oh , a moderately large ligand ( figure s3 ) , essentially removes the np - induced bleaching and reduces the observed effect to the level of the direct dpbf photobleaching . gold nanorods ( nrods ) with a length of 40 nm and a diameter of 12 nm , which have two plasmon resonance bands , namely the transverse band at 522 nm and the longitudinal one at 798 nm ( figure s1a ) , stabilized with a capping layer consisting of a mixture of a peg polymer ( mpeg5000 ) and a peptide ( c - tat ) ( see the experimental section ) , were also investigated . irradiation at 532 nm , i.e. , in the transverse plasmon resonance band , showed no additional dpbf bleaching above the direct dpbf effect ( figure 3 ) . irradiation with 1 w ( 11 w cm ) cw laser power at 800 nm , i.e. , in the longitudinal plasmon resonance band ( figure s5 ) , showed reduced direct dpbf photobleaching compared to irradiation at 532 nm , but again no additional photobleaching was observed in the presence of nrods . these results show that the nrods used here , which have a ligand layer consisting of a mixture of a peg polymer and a peptide , do not induce the formation of o2 upon laser irradiation , independent of the plasmon resonance which is photoexcited . irradiation at 532 nm in the presence and absence of citrate - stabilized spherical gold nps with 15 nm diameter was also investigated using laser pulses with a pulse duration of 5 ns ( figure 4 ) . in this case , the uv vis spectra show a slight broadening of the np plasmon resonance band at 520 nm during the first 5 min of irradiation , suggesting that some aggregation occurs ; after the initial 5 min , however , the nps remain stable . analysis of the spectra shows that photobleaching of dpbf in the absence of gold nps is similar to the results obtained using cw laser irradiation at comparable average powers ( figure s4a ) . in the presence of gold nps with 15 nm diameter , on the other hand , the np - induced bleaching effect ( i.e. , the effect remaining after subtracting the direct dpbf photobleaching effect ) which is induced by pulsed laser irradiation is larger than that caused by cw irradiation by almost 1 order of magnitude , in spite of the significantly lower laser power employed during the pulsed irradiation experiments ( 0.15 w vs 1 w ) , which leads to a correspondingly lower number of absorbed photons ; compare figure 4b ( pulsed irradiation ) with figure 3 ( cw irradiation ) . photobleaching of the dpbf absorbance upon laser irradiation with 5 ns laser pulses at 532 nm , 0.15 w , 10 hz repetition rate ( corresponding to a power density of 1.5 w cm and a pulse energy density of 0.15 j cm ) . ( a ) absorbance spectra in the presence of citrate - stabilized spherical gold nps with 15 nm diameter , taken at intervals of 5 min from before the irradiation ( gray ) up to a maximum irradiation time of 30 min ; the arrow indicates the direction of change . ( b ) gradient of the time - dependent dpbf photobleaching ( measured at 412 nm ) in the irradiation time window of 2030 min in the absence and presence of nps . it is straightforward to estimate the quantum yield of np - induced dpbf photobleaching from the absorbed laser power and the observed rate of absorbance bleach . for the citrate - stabilized spherical nps with 15 and 46 nm diameter under cw irradiation at 532 nm , this yields values of 5 10 and 8 10 , respectively . thus , less than one of each 1 million photons absorbed by a np leads to the photobleaching of a dpbf molecule under cw irradiation . for irradiation of 15 nm nps with 5 ns laser pulses , on the other hand , the quantum yield of np - induced dpbf photobleaching is 3.5 10 , i.e. , almost 2 orders of magnitude larger than for cw irradiation , but still very small . diffusion of oxygen over the lifetime of o2 ( approximately 6 s in 50/50 water / ethanol ) covers a distance larger than the average distance between dpbf molecules at the concentrations used here , and hence it can be concluded that a significant fraction of the photogenerated o2 which escapes from the np surface should be detected . 10 m , which is close to the concentration used here , is sufficient to detect 50% of photogenerated o2 . thus , the observed low quantum yield of dpbf photobleaching indicates a very low quantum yield of o2 photogeneration by gold nps ( i.e. , number of o2 generated for each photon absorbed ) , having values of the order of ca . 10 for cw irradiation and ca . 10 for irradiation with nanosecond laser pulses . our results show that irradiation of citrate - stabilized spherical gold nps at 532 nm , i.e. , in their surface plasmon band , with pulsed or cw laser light leads to the production of o2 , detected here by monitoring the bleaching of dpbf absorbance . however , the rate of o2 production is much larger when using short laser pulses than when using cw light of comparable intensity . whereas ca . 24% of the dye is bleached after only 10 min of irradiation of 15 nm nps with 5 ns laser pulses at an average power of 150 mw ( figure 4 ) , only 12% of the dye is bleached over this time by irradiation of the same nps with cw light at significantly higher power ( 1 w ) ( figure 2 ) . this difference allows one to draw important conclusions on the mechanism of o2 production by irradiation of gold nanoparticles . irradiation of spherical gold nps at 532 nm leads to the excitation of their plasmon resonance , which can be described as a coherent oscillatory motion of the conduction band electrons ; this oscillation dephases and decays on the sub-100 fs time scale , with only a very small radiative contribution , so that most of the excitation energy is retained as electronic excitation in the form of electron hole pairs . since the photon energy is close to the minimum energy required for direct excitation of d - band electrons into the conduction band of gold , a minor contribution of this excitation mechanism can not be ruled out ; however , d - band holes are filled by conduction band electrons on the 10 fs time scale , yielding essentially the same outcome as excitation of the plasmon resonance band . the excited electrons initially have a nonthermal energy distribution , and often are referred to as primary hot electrons , although the concept of temperature does not strictly apply to such a distribution . they rapidly ( within less than 500 fs ) equilibrate by electron electron scattering to yield a fermi distribution corresponding to an elevated temperature and can then be referred to as hot electrons . thiol bond dissociation at the surface of gold nps and have been suggested to be responsible for the creation of o2 by irradiation of gold nps with nanosecond laser pulses . they lose their energy on the time scale of a few picoseconds by interaction with the lattice ( electron phonon scattering ) with coupling times that are essentially size - independent for nps above 10 nm in diameter , although they strongly depend on the amount of energy deposited due to the temperature - dependent electronic heat capacity . because the lattice heat capacity is much larger than the electronic heat capacity , this leaves the electrons and the lattice in equilibrium at a temperature which is significantly lower than the initial electronic temperature ; finally , cooling occurs by heat transfer to the solvent and heat diffusion on the time scale of 10 to a few 100 ps , strongly depending on np size . since the dissipation of the absorbed energy proceeds on the picosecond time scale , excitation with a nanosecond laser pulse yields a highly nonequilibrium situation during the duration of the laser pulse where energy is continuously deposited into the electronic system and at the same time flows through the lattice into the surrounding solvent . it is therefore not straightforward to predict the temperature of the hot electrons achieved in such experiments . we used the two - temperature model for the electron and phonon heat baths coupled to finite - element heat transfer and diffusion simulations in the surrounding solvent to estimate this temperature ; details of these simulations are given in the supporting information . as shown in figure 5 , under our conditions the electrons are expected to reach a temperature of 2100 c , whereas the lattice reaches temperatures of the order of 1400 c and the solvent in the immediate vicinity of the np a temperature of about 900 c . time - dependent temperatures of the conduction band electrons ( red ) , lattice ( black ) , and first solvent layer ( blue ) , calculated for our experiments using nanosecond - laser pulse excitation ( 15 nm spherical nps in 50/50 etoh / water , 5 ns laser pulses with 0.15 j cm intensity , solid lines ) and for the experiments described in ref ( 24 ) ( 40 nm spherical nps in 80/20 etoh / water , 7 ns laser pulses with 0.03 j cm intensity , dashed lines ) using the two - temperature model for the electron and phonon heat baths , coupled to finite - element heat transfer and diffusion simulations in the surrounding solvent ( see supporting information for details ) ; time zero corresponds to the center of the laser pulse . a lattice temperature of 1400 c , which is above the melting temperature for solid gold , albeit present for only a few nanoseconds , might be sufficient to cause temporary melting of the nanoparticle , although the pulse intensities used here are still below the reported threshold for size reduction of gold nps of 15 nm diameter by nanosecond laser pulses . nevertheless , a minor effect on the shape and/or size of the nps , especially at the upper end of the size distribution , can not be ruled out and may be related to the minor np spectral changes observed upon irradiation which suggest some aggregation to occur ( see above ) . because of the slower heat dissipation around larger nps , these are heated to higher temperatures and hence are more likely to fragment , in agreement with experimental results on nanosecond - laser pulse induced fragmentation , which also show that fragmentation is finished after 5 min under conditions similar to the ones used here . since our nps were prepared without excess citrate , the resulting increase in the ratio of surface area to volume may indeed cause some aggregation . it also can not be ruled out that the solvent near the np surface temporarily forms bubbles , although the pulse intensities used here are still below the reported threshold for bubble formation by nanosecond pulses for 15 nm nps . such bubbles could lead to better thermal insulation , thus potentially increasing the maximum temperatures , but on the other hand , they might prevent oxygen from reaching the np surface , thus reducing the chance of o2 formation . the formation of o2 from the triplet ground state by interaction with a photoexcited sensitizer requires a change of the electron spin and hence can not result from dipole dipole ( frster ) interaction , but only from dexter - type electron exchange coupling ; the latter can be described as simultaneous transfers of an electron from one of the 2 * molecular orbitals on oxygen to a photogenerated hole on the sensitizer and of an electron with opposite spin from a high - energy excited sensitizer level to the same or the other 2 * orbital , resulting in the formation of the or singlet oxygen state , respectively . this mechanism requires significant overlap of the relevant electronic wave functions and hence only occurs at short distances of at most 10 . since the photoexcited hot electrons have such a short lifetime , this reaction can only occur if an oxygen molecule happens to be in the vicinity of the np or is temporarily adsorbed to its surface at the moment of excitation . a similar reaction occurs on photoexcited si nanocrystals , although in this case the reaction is more efficient than for au nps due to the much longer lifetime of the photoexcited excitons in si . thus , the short lifetime of hot electron excitation in gold nanoparticles easily explains the low quantum yield of o2 photogeneration which is observed here . the excitation of electrons to temperatures exceeding 2000 c means that a significant number have sufficient energy to excite an oxygen molecule to the state , which has an energy of 0.98 ev above the ground state ( see figure 6 ) . the number of hot electrons available at the higher energy and the number of holes available at the lower energy involved in this two - electron exchange reaction can be estimated from the density of states of gold and the fermi distribution , as described in more detail in the supporting information . for example , for a spherical np with 15 nm diameter at an electron temperature of 2100 c , there are 260 electrons within an energy interval of 0.1 ev around the state . ( it should be noted that the relevant energy interval is the width of this state for an oxygen in the vicinity or temporarily adsorbed onto a np , which is not known , so only relative numbers will be used here . ) thus , the electron temperatures achieved in our experiment are sufficient for a significant population of hot electrons and holes at the relevant levels . schematic diagram showing the population probability f(e ) for a np electron state at energy e near the fermi level , ef , under different conditions : ( a ) in equilibrium at room temperature , ( b ) at an electron temperature of te = 2100 c after electron electron equilibration ( hot electrons ) , and ( c ) immediately after the absorption of photons by single electrons ( primary hot electrons , with population changes highly exaggerated to make them visible ) . also shown are the energies of the ground - state triplet ( ) and lowest - excited singlet state ( ) of oxygen as well as the next singlet state ( ) under the assumption that ef is equidistant from the and energies . excitation of an oxygen molecule to o2 requires the simultaneous transfer of an electron from the oxygen to a hole at the energy of the state and of a hot electron with the opposite spin and an energy at the ( or ) level to the oxygen molecule . the large number of equilibrated hot electrons available during a nanosecond - laser pulse also rules out the primary hot electrons as the main source of o2 photogeneration . the number of primary hot electrons available during the pulse duration can be estimated from the number of photons which are absorbed per nanosecond , multiplied by their lifetime , which is less than 500 fs . however , these primary hot electrons populate np states at energies from the fermi level ef to ef + 2.34 ev ( the energy of a photon at 532 nm ) ( see figure 6c ) . for 15 nm nps and excitation with 5 ns laser pulses with 0.15 j cm intensity , this predicts that not more than three primary hot electrons are available within an energy interval of 0.1 ev around the level at any time during the laser pulse , assuming that all levels are equally populated and taking into account that the density of states for gold has an essentially constant value in the relevant energy range around ef . this is significantly less than the number of hot ( equilibrated ) electrons available at the maximum electron temperature , which was estimated to be on the order of 260 for the same energy interval ( see above ) . thus , we can conclude that it is indeed the hot electrons which are responsible for the observed o2 photogeneration when using short laser pulses for excitation . similar results to those obtained here using pulsed irradiation have recently been reported for spherical gold nps with 40 nm diameter . although a significantly lower laser pulse energy density ( 0.03 j cm , compared to 0.15 j cm here ) was used under otherwise similar experimental conditions , slightly faster o2 photogeneration was observed in this study , with the dpbf absorbance decreasing by ca . 24% decrease observed here in the first 10 min ( figure 4a ) . in this context , it is interesting to note that in spite of the lower laser pulse energy density the nps are heated to almost the same electron temperature as in our experiments ( see figure 5 ) . this is largely due to ( i ) heat dissipation from larger nps being slower and ( ii ) the use of 80/20 etoh / water as solvent in ref ( 24 ) , which has slower heat transport than a 50/50 etoh / water mixture . an explicit calculation of the number of hot electrons available at the energy of the oxygen state for the two experiments is given in the supporting information ; together with a detailed consideration of all other experimental differences , these numbers yield very good agreement between the expected and the observed relative o2 photogeneration rates ( see the supporting information for details ) . this provides further support for the conclusion that o2 photogeneration is mediated by the equilibrated hot electrons of gold nanoparticles under nanosecond - pulsed laser irradiation . compared to the effect of pulsed irradiation , the rate of o2 photogeneration is much smaller when using cw light of comparable intensity . whereas ca . 24% of the dye is bleached after only 10 min of irradiation of spherical nps with 15 nm diameter with 5 ns laser pulses at an average power of 150 mw ( figure 4 ) , only 12% of the dye is bleached over this time by irradiation of the same nps with cw light at significantly higher power ( 1 w ) ( figure 2 ) . in a previous publication , significantly higher rates of dpbf photobleaching had been reported under conditions which appear to be similar to the ones used here . in this context , we note that we also observed such significantly higher rates of bleaching , but only when the sample preparation protocol described in the experimental section was not followed accurately ; for example , the use of dye solution that had not been freshly prepared or of a cuvette that had not been cleaned thoroughly and rinsed multiple times with mq water or the use of a cell that was not sealed during irradiation , leading to some loss of ethanol from the solution , all resulted in larger and highly irreproducible bleaching of dpbf under cw irradiation , up to levels comparable to those reported in ref ( 24 ) , even in the absence of nps . in the following , we will show that photogeneration of o2 under cw irradiation , unlike pulsed irradiation , is mediated by the initially created primary hot electrons ; i.e. , it occurs during the short time during which the excited electrons have not yet relaxed to a thermal distribution ( see figure 6c ) . under the cw irradiation conditions used here ( 1 w , 1.85 mm beam diameter ) , a spherical np with 15 nm diameter absorbs photons at an average rate of 1.3 10 s , as estimated from the absorption cross section and the beam intensity . this means that after absorption of a photon there is enough time for full relaxation and transfer of the photon energy into the solvent , which occurs in less than 100 ps , before absorption of the next photon . absorption of one photon by a 15 nm np yields hot electrons at a temperature of 10 k above the surrounding after electron electron equilibration ; these hot electrons lose their energy by electron phonon scattering within a few picoseconds to yield a np whose temperature is only 80 mk above the surrounding . neither of these effects is expected to yield any significant photochemical effects ; estimates analogous to those described above predict that for a spherical 15 nm np with an electron temperature of 35 c there are ca . 2.6 10 electrons within an energy interval of 0.1 ev around the oxygen state and the same number of holes around the state energy . this means that there are on the order of 10 times less hot electrons and 10 times less holes available for the photoreaction than under our pulsed laser irradiation conditions , which rules out any significant reaction ; this is also confirmed by the fact that these population numbers are less than a factor 2 larger than those for room temperature , where no o2 is generated in the absence of light . this leaves only the primary hot electrons , i.e. , those electrons that are excited upon absorption of a photon but have not yet equilibrated by electron electron scattering , as potential cause for photogeneration of o2 . absorption of a single photon can potentially excite electrons to energies of up to 2.34 ev above the fermi level . if one assumes excitation of only one electron by each photon and equal excitation probability for all available electrons , as shown in figure 6c , on average there will be 0.043 electrons within an energy interval of 0.1 ev around the oxygen state , which is approximately 5000 times less than during a single laser pulse in the pulsed experiments , see above . taking into account that the same factor also applies to the holes required for the dexter mechanism , but correcting for the lifetime of the excitation ( 500 fs for primary hot electrons created during cw irradiation , ca . 3 ns for the hot electron distribution induced by a single laser pulse ; see figure s6 ) and the repetition rates ( 1.3 10 s for single photon absorption during cw irradiation , 10 s for the pulsed laser irradiation ) , one would predict a rate of o2 photogeneration under our cw irradiation conditions which is smaller than that expected for our pulsed irradiation conditions by a factor on the order of 10 . this is significantly closer to the experimental results ( ratio of o2 photogeneration rates under pulsed vs cw irradiation of 1020 ) than any estimate based on the equilibrated hot electrons after the absorption of a single photon . the main discrepancy between the predicted and observed cw results arises from the assumption of direct excitation of single electrons in the above estimate , which is not valid for irradiation at 532 nm , i.e. , in the gold np plasmon resonance band , since this leads to the coherent excitation of many electrons which rapidly dephases without the electrons exchanging energy . thus , absorption of a single photon yields more than one primary hot electron , with the photon s energy distributed over all of them . consequently , the energy distribution even of the primary hot electrons will not extend up to 2.34 ev above ef but will be shifted toward the states nearer the fermi level , thus increasing the population of states around the oxygen energy and hence the yield of o2 . a more quantitative estimate of this effect is beyond the scope of this paper . an alternative possibility for the mechanism of o2 photogeneration by gold nps could be envisaged , which is based on increased direct photoexcitation of oxygen due to the well - known local electric field enhancement in the vicinity of metal nps by the plasmon electrons . however , significant field enhancement extends to distances comparable to the dimensions of the nanoparticle , so that this mechanism is in disagreement with our observation that a peg - oh capping layer , which has a thickness of only 2 nm , completely inhibits o2 photogeneration ( figure 3 ) . dexter - type electron exchange coupling , on the other hand , is known to be of significance only over distances of less than 1 nm , as discussed above , and thus is further supported by this observation . in conclusion , cw irradiation is less efficient than pulsed laser irradiation in photogenerating o2 since the ( equilibrated ) hot electrons , which are the main mediator of the photochemistry in the case of pulsed irradiation , do not have sufficient energy / temperature to drive o2 photogeneration . nevertheless , our results confirm that cw irradiation of spherical gold nps produces detectable amounts of o2 . the absence of significant amounts of ( equilibrated ) hot electrons means that o2 photogeneration proceeds via a different mechanism under cw irradiation compared to pulsed laser generation ; the above estimates indicate that it is the primary hot electrons , i.e. , the directly photoexcited electrons , which are responsible for the photochemistry here and that the photochemical reaction must occur before these equilibrate by electron electron thermalization . photodynamic therapy holds great promises for medical applications , such as the treatment of cancer , because of the ability to selectively affect diseased tissue only . however , wider use of photodynamic cancer therapy is currently prevented by several limitations imposed by the available photosensitizers . these limitations include toxicity , poor stability and photostability , poor selectivity for cancer tissue , and the need of using visible light with poor tissue penetration . all of these limitations , in principle , can be overcome by the use of gold nanoparticles , which are nontoxic , have excellent stability even under irradiation , can target cancer tissue either passively by the enhanced penetration and retention ( epr ) effect or by active targeting , have extinction coefficients that are larger than those of dye molecules by several orders of magnitude , and can be tuned to absorb in the near - infrared spectral region for maximum tissue penetration . gold nanoparticles have been reported to have three potential modes of operation for inducing cell death by irradiation , namely ( i ) hyperthermia , which is based on the rapid conversion of the absorbed light energy into heat , ( ii ) np - assisted photodynamic therapy , in which the efficiency of a standard sensitizer is amplified by the np plasmon field enhancement effect , or ( iii ) a direct photochemical mechanism without involvement of a photosensitizer . the feasibility of photothermal therapy has been clearly established by careful experiments in vitro(1113,15,54 ) and has been shown to work in vivo . however , it should be noted that most studies reporting successful photothermal therapy made no attempt to either confirm significant heating or rule out photochemical effects , which means that some of these reported results could in fact arise from photochemical rather than photothermal effects or from a synergistic combination of photochemical and photothermal effects . as has been pointed out recently , another problem with many reports on photoinduced hyperthermia using nps is the relatively high light intensity required to reach sufficient temperatures , which often were well above the generally accepted skin tolerance threshold . the presence of gold nps can also lead to increased photogeneration of o2 by traditional photodynamic sensitizers , which can be ascribed to the local electric field enhancement in the vicinity of metal nps by the plasmon electrons , similar to the well - studied sers ( surface - enhanced raman spectroscopy ) effect . this approach might help to alleviate some of the drawbacks of traditional photodynamic therapy but does not directly overcome them , since it still requires the presence of a sensitizer . in fact , the requirement for two active components , gold np and sensitizer , which must be colocalized , introduces an additional complication , and care must be taken to avoid a reduction of the photosensitizer effect due to quenching of its excited state by the metal np . in an alternative approach , cell death has been demonstrated to occur following irradiation of intracellular ( endocytosed ) gold nps even at irradiation levels that are not high enough to cause significant heating . this photochemical effect has been related to the observation that irradiation of nps in vitro results in the formation of singlet oxygen , which is the active species in traditional photodynamic therapy . because of the short lifetime of o2 ( 3.4 s in water ) , its action is highly localized an oxygen molecule only diffuses over the length scale of 100 nm in this time . for this reason , only intracellular nps are expected to trigger cell death by the photochemical route , although they may initially be located inside endosomes which are known to be broken up by nps under cw irradiation . on the other hand , the localization of the photochemical effects within individual cells also means that in situations where different types of cells coexist in close vicinity , selective targeting of particular cells and minimization of collateral damage should be achievable . this is different than photothermally induced cell death , which affects all cells within the irradiated volume more or less indiscriminately , as long as some of them contain nps , because of the fast diffusion of heat over the relevant length scales . the results of the experiments described here provide more insight into the direct photochemical mechanism and allow some important conclusions to be made for the further development of practical applications . they show that detectable amounts of o2 are generated by irradiation of nps with short laser pulses or cw light even in the absence of a photodynamic sensitizer , albeit with low quantum yield . short laser pulses are significantly more efficient at this process , since they can heat a significant fraction of the np conduction band electrons to high enough temperatures to excite oxygen to the singlet state . however , this requires pulse energy densities that are well above generally accepted safe levels for the irradiation of skin with pulsed laser light . since the effect requires the absorption of many photons by a np during one laser pulse , it is highly nonlinear with respect to irradiation intensity , and thus it will not be possible to compensate lower irradiation levels by longer irradiation times . furthermore , pulsed irradiation at the required intensities also can cause other effects , such as np fragmentation or bubble formation , and it is not clear what consequences these effects may have when occurring in tissue . although the use of femtosecond laser pulses , as compared to the nanosecond pulses used here , might alleviate some of these problems to some extent , they would require even more sophisticated equipment which may not be suitable for a clinical environment . the use of cw light , on the other hand , is straightforward and does not even require a laser but can be achieved with simple lamps . in spite of the lower quantum yield of o2 photogeneration by nps under cw irradiation which is reported here , cell death induced by cw light in the presence of nps has been reported in vitro and in vivo . although the experiments described here , which did not involve any biological material , were undertaken at light intensities that are above safe irradiation levels , the mechanism of o2 photogeneration by nps under cw illumination is shown to be based on the absorption of single photons . this means that lower light intensities can be compensated for by longer irradiation times , allowing one to reduce the intensity to safe levels for biological or medical applications without affecting the amount of o2 generated . o2 photogeneration and cancer cell destruction in vitro and in vivo have indeed been observed upon irradiation on the minute time scale at intensities below safe levels and using nonlaser light sources . the results presented here indicate another design criterion which needs to be fulfilled for successful implementation of the photochemical route of inducing cell death by irradiation of nps , namely that the nps must not possess a dense capping layer . even the thin capping layer formed by peg - oh , with a thickness of only 2 nm , is sufficient to completely inhibit o2 photogeneration ( figure 3 ) . this is in full agreement with the suggestion that energy exchange between the np and the oxygen molecule occurs via the dexter ( two electron exchange ) mechanism , which is limited to distances of less than 1 nm . similar effects have been reported for o2 photogeneration by si nanocrystals , which is significantly affected by a thin oxide layer . it is important to point out that the assay used here for o2 detection ( bleaching of dpbf ) only reports on singlet oxygen found outside the np capping layer ; thus , it can not be ruled out that even on nps with a peg - oh capping layer some o2 is photogenerated at the np surface but reacts with the np ligands and hence is quenched . on the other hand , such singlet oxygen would not be of any direct use for practical applications , such as the induction of cell death , so that the assay results in fact report the effects relevant for such applications . uncapped ( citrate ) nps are rapidly covered by a protein corona after they have been taken up into live cells , but it appears that this corona is permeable enough for oxygen to not completely prevent singlet oxygen formation , as evidenced by fact that cell death by the photochemical route has been observed with such nps ; it may be that because of their size proteins are not able to form a capping layer ( corona ) of similar density as the smaller ligands used here . similarly , a lipid bilayer appears to allow oxygen access to the surface of gold nanorods , whereas a dense pentapeptide ( calnn ) capping layer on spherical gold nanoparticles is sufficient to suppress the photochemical mechanism of cell death . it also seems likely that the thick peg capping layer present on the nanorods used here is the main reason for the absence of o2 photogeneration by nanorods upon irradiation in either the transversal or the longitudinal plasmon resonance band that was observed here . because of their longitudinal plasmon resonance band , which is in the near - ir spectral region with high tissue penetration , nanorods are more suitable for practical photodynamic applications in tissue . however , standard synthesis protocols yield nanorods within a bilayer of cytotoxic ctab , so that ligand exchange is required before any biological or medical application , and care will need to be taken to choose suitable ligands to allow access of oxygen to the nanorod surface . potential examples for these are poly(vinylpyrrolidone ) , lipid bilayers , or mesoporous silica , all of which have been used successfully in experiments showing photochemically induced cell death using gold nanorods . dpbf has been used successfully to detect singlet oxygen that is formed when spherical nps are irradiated at 532 nm , either with cw or pulsed laser irradiation . singlet oxygen generation by pulsed laser irradiation has been shown to act via the equilibrated hot electrons that can reach temperatures of several thousand degrees during the laser pulse ; cw irradiation , on the other hand , can act only via the directly excited primary hot electrons , which rapidly lose their energy by electron electron equilibration , and hence is significantly less efficient for the formation of singlet oxygen . nevertheless , even cw irradiation can produce enough singlet oxygen for photodynamic therapy applications and will allow practical applications of the effect at safe irradiation levels . photodynamic therapy using gold nanoparticles will also require careful design of the nanoparticles with respect to size , shape , and capping layer and will require internalization of the nps , not just attachment to the cell surface , which is sufficient for photothermal therapy .

the formation of singlet oxygen by irradiation of gold nanoparticles in their plasmon resonance band with continuous or pulsed laser light has been investigated . citrate - stabilized nanoparticles were found to facilitate the photogeneration of singlet oxygen , albeit with low quantum yield . the reaction caused by pulsed laser irradiation makes use of the equilibrated hot electrons that can reach temperatures of several thousand degrees during the laser pulse . although less efficient , continuous irradiation , which acts via the short - lived directly excited primary hot electrons only , can produce enough singlet oxygen for photodynamic cancer therapy and has significant advantages for practical applications . however , careful design of the nanoparticles is needed , since even a moderately thick capping layer can completely inhibit singlet oxygen formation . moreover , the efficiency of the process also depends on the nanoparticle size .

alzheimer 's disease ( ad ) is a central nervous degenerative disease with memory impairment , aphasia , agnosia , and executive dysfunction , as well as personality and behavior changes . pathological features of ad include amyloid ( a ) deposition and neurofibrillary tangles , leading to progressive neuronal damage , and ultimately atrophy of the cortex and subcortical structures . magnetic resonance imaging ( mri ) however , relatively few studies are available assessing changes in other brain regions in ad patients , especially the frontal lobe . interestingly , evaluating 41 patients with ad , harwood et al . found that the insight and cognitive impairment as well as functional deficits in ad are associated with the glucose metabolic rate in the frontal cortex . we previously demonstrated that treatment with electroacupuncture could effectively improve the spatial learning and memory ability as well as glucose metabolism in the hippocampus of animals with ad . other studies suggested that music therapy could contribute to a supplementary treatment of ad . in china , an innovative therapy combined electroacupuncture and music therapy , and the term musical electroacupuncture ( mea ) addition of musical therapy helps overcome acupuncture intolerability ; therefore , the mea therapy in a way is superior to traditional electroacupuncture . in the current study , we aimed to address two questions : ( 1 ) what happens in the frontal lobe during mea ? do these changes differ from those observed in other brain regions ? ( 2 ) are there differences between the two therapies for ad treatment ? to this end , senescence - accelerated mouse prone 8 ( samp8 ) mice were selected as an ad animal model , and the differences between techniques were assessed . first , morris ' water maze test was used to evaluate behavioral changes in the model animals . then , micro - pet assessment of a region of interest ( roi ) and glucose metabolism evaluation in different brain regions were performed . finally , immunohistochemistry ( ihc ) was used to assess the changes of amyloid-1 - 42 deposition in the frontal lobe after the treatments . senescence - accelerated mouse prone 8 ( samp8 ) and cognate normal senescence - accelerated mouse - r1 ( samr1 ) breeding pairs were kindly provided by professor takeda at kyoto university , japan . all animals were male and specific pathogen - free ( spf ) , weighing 30 2 g. they were housed in a barrier facility at the experimental animal centre of first teaching hospital of beijing university of traditional chinese medicine , under controlled temperature ( 24 2c ) and 12 h/12 h dark - light cycle , with sterile drinking water and standard pellet diet ad libitum . all experiments were performed according to the national institute of health guide for the care and use of laboratory animals ( nih publications number 80 - 23 ) . thirty 7.5-month - old male samp8 mice were divided into three groups ( n = 10 per group ) , including samp8 alzheimer 's disease control ( ad ) , electroacupuncture ( ea ) , and musical electroacupuncture ( mea ) groups . ten 7.5-month - old male samr1 mice composed the normal control ( n ) group . electroacupuncture and musical electroacupuncture treatments were performed 20 minutes per day , once daily for 15 days ( no treatment on day 8) . prescription of acupuncture points included du20 baihui , du 26 shuigou , and ex - hn3 yintang ( significant extra points ) ; the locations of these points were according to the national acupuncture society for experimental research developed by the laboratory animal acupuncture atlas . the pricking method was used for du 26 shuigou and the flat thorn method for du20 baihui and ex - hn3 yintang . , the needle handle was connected to the hans - lh202 electroacupuncture device ( peking university institute of science nerve and beijing hua wei industrial development company ) , with sparse wave at 2 hz , 2 v , and 0.6 ma . in the mea group , the needle handle was connected to the zj-12h musical electroacupuncture device ( developed by chinese acupuncture society and manufactured by harbin zhihou medical devices co. , ltd . ) . clear rhythm , moderate speed , and music prescription intensity ( curing dementia prescription ) were selected ; music intensity was adjusted so that the animals remained quiet during the treatment . in the n and ad groups , no treatment was carried out , with grabbing and fixing the mice in order to ensure the same treatment conditions , once daily for 15 days . morris ' water maze consisted of a circular tank ( diameter , 90 cm ; height , 50 cm ) , filled with water to a depth of 29 cm , maintained at 24 1c , and rendered opaque with blue - black ink . a removable circular platform ( diameter , 9.5 cm ; height , 28 cm ) with the top surface 1 cm below the water was located inside the pool . the pool area was conceptually divided into four quadrants ( ne , nw , sw , and se ) of equal size . data were collected by a video camera ( tota-450d , japan ) , which was fixed to the ceiling and connected to a video recorder with an automated tracking system ( china daheng group , beijing , china ) . in the behavioral test , mice were placed in the pool of water containing a platform just below the surface of the water . distal visual cues are arrayed around the room , and , in general , mice are able to find the location of the hidden platform based on these cues . this test assesses the ability of mice to find the platform under conditions where they can not directly see the latter but must either remember it based on external cues or perform a search . the platform was placed 1 cm under the water surface ; the water was rendered opaque by a suspension of dark blue , nontoxic tempera paint . the platform was placed in a different location from that used in the visible platform testing . each mouse was released from one of the 4 locations and had 60 s to search for the hidden platform . at the end of each trial , the mouse was placed on the platform or allowed to stay there for 15 s. prominent spatial cues were arrayed around the room . the investigator also constituted a powerful spatial cue and always sat in the same location during each trial after releasing the mouse . six trials per day for 5 consecutive days were performed with the platform location kept constant . the time that the mouse took to find the platform was recorded and represented escape latency . the day after completion of the hidden platform test , the platform was removed ; each mouse was placed in the pool once for 60 s , starting from the same starting location used first in the hidden platform test . the time spent in the platform quadrant was recorded , and the percentage of total time spent swimming to the platform quadrant was derived . the f - fdg pet tracer was provided by the chinese medicine research institute pet room ; pet imaging was carried out on a siemens inveon pet / ct imaging system . before the experiments , mice ( 7.5 months , 28~32 g ) were submitted to blood glucose monitoring and showed levels in the normal range ( 7.0~10.1 mmol / l ) . the animals were placed in the suction chamber , inhaling oxygen mixed with 1.5% isoflurane for anesthesia . after complete anesthesia , approximately 14.8~16.5 mbqf - fdg pet were injected via the tail vein . after f - fdg pet tracer uptake for 60 min , the mice were placed in the prone position , parallel to the scanner long axis , with the head located within the scanner field of view . the mice were anesthetized by inhalation of oxygen mixed with 1.5% isoflurane ( 1 l / min ) . filtered back projection ( fbp ) and ct photon attenuation correction were used for image reconstruction . three - dimensional regions of interest were selected in the hippocampus , in transverse , coronal , and sagittal planes . after the morris water maze test , the remaining six mice in each group ( four were used in micro - pet ) were anesthetized by intraperitoneal injection of 10% chloral hydrate at 0.35 ml/100 g body weight . three minutes later , the chest was opened and the heart exposed ; intubation was performed from the left ventricle to the ascending aorta with quick injection of 100 ml saline . then , the right atrial appendage was cut , and 4% paraformaldehyde was injected until the liver turned white with clear fluid flowing out from the right atrial appendage . after the perfusion , the mouse was decapitated and the whole brain extracted and placed on ice . , paraffin embedded brain tissue sections were deparaffinized with xylene and hydrated with graded alcohol . then , the sections were treated with citric acid antigen repair buffer and washed with pbs ( ph 7.4 ) every 5 min three times with shaking . after incubation with 3% hydrogen peroxide for 20 min in the dark to quench endogenous peroxidase , the sections were incubated with anti - a1 - 42 antibody ( 1 : 50 , ab10148 , abcam ) overnight . then , secondary antibodies were added for 30 min at room temperature , and detection was performed with dab . micrographs of brain tissue samples were obtained at 400x magnification , and integral optical density ( iod ) values were calculated using image - pro plus 6.0 software . data are mean sd for each group . for the morris water maze test , the escape latency time of the hidden platform trial was analyzed by the huynh - feldt test , while one - way anova was conducted on probe trial , micro - pet test , and immunohistochemical data . all statistical analyses were performed with the spss software v.17.0 ( spss , usa ) . the effects of electroacupuncture and musical electroacupuncture in spatial location ability of samp8 mice in the wmw test are shown in figure 1(a ) . with training time extension , the ad group showed a marked retardation in escape latency compared with the n group ( p = 0.00 ) , probably due to memory deficits resulting from the rapid aging process impairing learning and memory . compared with the ad group , escape latency in the ea ( p = 0.031 ) and mea ( p = 0.023 ) groups was significantly reduced ( p < 0.05 ) . as shown in figures 1(a ) and 1(b ) , the mea group performed better than the ea group , although there was no statistically significant difference ( p = 0.895 ) . to assess therapeutic effects on spatial memory ability , performance on day 6 was examined by analyzing the percentages of swimming time in the expected platform position . a higher percentage of time spent in the platform quadrant is interpreted as a higher level of memory retention . in this trial , compared with the ad group , the ea ( p = 0.045 ) and mea ( p = 0.035 ) groups showed increased time spent in the platform quadrant ( p < 0.05 ) . what is more , percentages of time spent in the platform quadrant were similar between the ea and mea groups ( p = 0.907 , figure 2 ) . besides the escape time , the average animal swimming speed is also associated with spatial learning and memory ability . in some cases , swimming speed in the normal control group was slower , with the escape time longer than in the alzheimer 's disease group . thus , escapes times can not serve as the only indicator of learning ability and memory . the average swimming speed before getting on the platform is an important indicator of exercise ability and can reflect individual differences in experimental animals . compared with the n group , the swimming speed in ad group was significantly reduced ( p = 0.00 ) , indicating lower exercise ability for samp8 mice compared with samr1 animals . compared with the ad group , electroacupuncture and musical electroacupuncture both increased the swimming speed of samp8 mice ( ea , p = 0.023 ; mea , p = 0.009 ) . what is more , the mea group performed better than the ea group ( figure 3 ) . micro - pet images were obtained from the hippocampus , frontal lobe , and cerebral cortex of each mouse . the same color standard and code were used from top to bottom to display the metabolic rate of glucose . after treatment with electroacupuncture and musical electroacupuncture , f - fdg levels in each brain region assessed were higher than values obtained for the nontherapy group ( figures 4(a ) , 4(b ) , and 4(c ) ) . to obtain acute differences in glucose metabolism among groups , the uptake rate of f after treatment with electroacupuncture and musical electroacupuncture , uptake rates of f - fdg per gram in the hippocampus ( ea , p = 0.039 ; mea , p = 048 ) , cerebral cortex ( ea , p = 0.14 ; mea , p = 0.047 ) , and frontal lobe ( ea , p = 0.045 ; mea , p = 0.031 ) were higher than those obtained for the alzheimer 's disease group ( figures 5(a ) , 5(b ) , and 5(c ) ) . in the frontal lobe , the uptake rate of f - fdg for the mea group was higher than that of the ea group , while , in the other two regions , the ea group showed higher values . after the behavioral tests and imaging , brain tissue samples were analyzed by immunohistochemistry to assess the effects of the two therapeutic variants on amyloid-1 - 42 accumulation due to neuronal damage and memory impairment . compared with the n group , iod of amyloid-1 - 42 in the frontal lobe was significantly higher in the ad group ( p = 0.00 ) , ea ( p = 0.00 ) , and mea ( p = 0.007 ) groups . meanwhile , iod of amyloid-1 - 42 in frontal lobe samples from the ad group was significantly higher than those of the ea ( p = 0.00 ) and mea ( p = 0.00 ) groups . interestingly , iod of amyloid-1 - 42 in frontal lobe samples from the mea group was significantly lower than that of the ea group ( p = 0.01 < 0.05 ) ( figures 6 and 7 ) . the senescence - accelerated mouse ( sam ) is an accelerated aging model that was established through phenotypic selection from a common genetic pool of the akr / j mouse strain . it was shown that senescence - accelerated mouse ( samp8 ) , as a model of aging , displays many features known to occur in the early stage of ad such as increased oxidative stress , amyloid - beta level alteration , and tau phosphorylation . what is more , published data and our previous research demonstrated that samp8 could undergo acupuncture therapy to improve learning and memory ability . in this study , therefore , samp8 mice were selected as an ideal animal model for alzheimer 's disease . music therapy is a nonpharmacological treatment for the behavioral and psychological symptoms of alzheimer 's disease . appropriate music formulation could provide a form of relief to the ad patient and may stimulate cognitive activities so that areas subject to progressive degeneration are maintained . in the recent ten years , increasing evidence suggests that proper music formulation could improve ad symptoms [ 1621 ] , especially for mild cases . besides , since music therapy is nonpharmacological , it is often used in combination with other therapies . musical electro - acupuncture ( mea ) combines music therapy and electro - acupuncture ; during symptomatic selection of music , the sound wave could be turned into a pulse current . therefore , the effects of mea on the human auditory organ and acupoints were separately assessed . specifically , the fundamental characteristics and advantages included two aspects : music therapy and irregular pulse current . electroacupuncture ( ea ) is widely used in clinical practice and research and in experimental investigations into the mechanisms of acupuncture . this therapy has been applied for ad treatment and could improve the cognitive function and brain energy metabolism [ 5 , 27 ] . the mechanism of ea tolerance is that the central nervous system releases analgesic substances ( including brain norepinephrine and endogenous antiopioid substances ) as well as large amounts of endogenous monoamine . among them , cholecystokinin octapeptide ( cck-8 ) is by far the most recognized antiopioid contributing to ea tolerance , which is caused by long - term use of the same frequency in ea . since ad treatment requires a long - term course , it likely results in ea tolerance . it transformed musical rhythms into constantly changing frequencies and waveforms to overcome the shortcomings of human body 's tolerance of general electroacupuncture . meanwhile , patients were allowed to listen to relieving music , which plays a role of music therapy . it was shown that such therapy performs better than traditional electroacupuncture in relieving pains , improving the symptoms of dermatosis ( urticarial disease , chloasma ) , alleviating nervous system diseases such as depression [ 3638 ] , insomnia , and anxiety [ 40 , 41 ] . however , there was seldom research on dementia . studies assessing the pathogenesis of ad are currently more focused on the temporal lobe , parietal cortex , and hippocampus , with few analyzing changes in the frontal lobe . recent studies found that transgenic mice with alzheimer 's disease show early cognitive decline related to frontal atrophy , with the changes taking place even earlier than in the hippocampus . as shown above , although the mea therapy performed better than ea in the morris water maze test , differences were not statistically significant . in micro - pet , ea therapy showed higher glucose metabolism improvement in the hippocampus and cerebral cortex compared with mea . only in the frontal lobe , these findings prompted the question whether mea therapy was more inclined to play a role in the frontal lobe . to address this , amyloid - beta levels were assessed in the frontal lobe of samp8 mice , and the results confirmed the above notion . comparing the different therapies in iod of amyloid - beta in the frontal lobe , mea therapy performed significantly better than ea ( p < 0.05 ) . therefore , mea indeed is inclined to play a role in the frontal lobe . using behavioral tests , in vivo imaging , and protein detection , the differences between mea and ea therapy for ad treatment were assessed in samp8 mice . interestingly , both ea and mea could improve spatial learning and memory ability , improving glucose metabolism in different brain regions and amyloid - beta expression in the frontal lobe . what is more , the mea therapy performed better than ea in decreasing amyloid - beta amounts in the frontal lobe .

objectives . to compare musical electroacupuncture and electroacupuncture in a mouse model of alzheimer 's disease . methods . in this study , 7.5-month - old male senescence - accelerated mouse prone 8 ( samp8 ) mice were used as an alzheimer 's disease animal model . in the normal control paradigm , 7.5-month - old male samr1 mice were used as the blank control group ( n group ) . after 15 days of treatment , using morris water maze test , micro - pet , and immunohistochemistry , the differences among the musical electroacupuncture ( mea ) , electroacupuncture ( ea ) , alzheimer 's disease ( ad ) , and normal ( n ) groups were assessed . results . the morris water maze test , micro - pet , and immunohistochemistry revealed that mea and ea therapies could improve spatial learning and memory ability , glucose metabolism level in the brain , and a amyloid content in the frontal lobe , compared with the ad group ( p < 0.05 ) . moreover , mea therapy performed better than ea treatment in decreasing amyloid - beta levels in the frontal lobe of mice with ad . conclusion . mea therapy may be superior to ea in treating alzheimer 's disease as demonstrated in samp8 mice .

microleakage around resin composite restorations results from the formation of gaps where the restoration and the cavosurface margin of the natural tooth structure are joined . gap formation may be related to shrinkage of the resin during polymerization and/or poor adhesion of dentin bonding agents between the dentin and composite material which leads to consequences , such as discoloration of the restoration , marginal break down , recurrent caries , pulpal inflammation and post operative sensitivity which may affect the longevity of restoration and ultimately the vitality of the dental pulp . when we cut the tooth structure with rotary instruments such as burs , an amorphous layer of organic and non - organic debris named the smear layer covers the cavity surface . now it is clear that the quality and quantity of the smear layer severely depends on how it is created and in different conditions it has various properties [ 35 ] . many variations in the smear layer of the prepared teeth with different dental instruments have been reported that can affect microleakage of composite resin restorations . the use of different burs to increase the surface roughness of preparations was evaluated as early as 1987 . mowery , parker and davis used four different grits of sandpaper to prepare the dentin surface . they found that increasing the surface roughness may increase bond strength primarily because of the increase in the total surface area . in addition , surface irregularities were created ; subsequently , increasing the mechanical locking of resin into these irregularities . while most of these studies concentrated on improving the bond strength of resin composites to the tooth structure , microleakage does not always decrease in direct proportion to increased bond strength . many studies indicate that the quality and quantity of the smear layer is different when we use high speed or low speed cavity preparation devices or when we use different cutting instruments [ 35 ] formation of the smear layer by various diamond burs with different grits was evaluated by tani et al . he understood that the smear layer which covered the dentin when preparing with 50150 grit diamond bur is thicker than the smear layer formed with 1530 grit diamond bur . he also revealed that using different adhesive systems have affected the microleakage of composite restorations . studies have evaluated different dentin bonding agents designed to improve the bond between the tooth structure and restorative materials . bonded enamel is generally reliable in decreasing microleakage ; however , bonded dentin is not as predictable at reducing microleakeage of the gingival margins at or near the cement - enamel junction . dentin bonding agents have substantially reduced microleakage in the gingival margin but have not eliminated it completely . deliperi and others compared the degree of microleakage in self etch and total etch adhesive systems . they reported that i - bond , the one step self etch adhesive system , has more dye penetration in both gingival and occlusal margins and there was no signifcant difference between occlusal and gingival margins in xenoiii ( one step self etch adhesive ) , nt ( total etch adhesive ) and i - bond ( one step self etch adhesive ) . on the other hand clearfil se bond had more dye penetration in the occlusal margin than the gingival margin . in different studies , it has been revealed that the cutting efficacy of cutting instruments is reduced while applying and it may affect the quality and quantity of the underlying smear layer . therefore , this study evaluated the effects of cutting efficacy of different diamond burs on the microleakage of resin composite restorations using total etch and self etch adhesive systems , and it also determined whether or not bur cutting efficacy had an impact on the results . ninety non - carious extracted human third molars , stored in 0/2% thymol solution after extraction , were cleaned of calculus , soft tissue and debris with hand instrumentation . in order to omit the inter - operator bias , each sample was assigned randomly in equal numbers ( n=15 ) to one of six groups . conservative class v composite preparations were made using one of three different burs ; a coarse new diamond bur , a coarse used diamond bur and a fine diamond bur in an air / water cooled high speed headpiece ( ch-4t5nsk b2/b3 , japan a1101800 ) . the cavity preparations were standardized with a width of 3 mm and a height of 2.5 mm and a depth of 1.5 mm . the occlusal wall of the cavity was limited in the enamel wall and the gingival wall of the cavity was extended beyond the cej onto the cementum . first group : teeth were cut with used coarse diamond bur ( tizkavan - iran ) and were conditioned with clearfil se bond ( kurary medical inc . japan pef 1975-wd batch no : 1-primer : lot 00670a2-bond : 00957a ) adhesive system . second group : teeth were cut with new coarse diamond bur ( tizkavan - iran ) and were conditioned with se bond adhesive system . third group : teeth were cut with fine diamond bur ( komet brasseler germany ) and were conditioned with se bond adhesive system . fourth group : teeth were cut with fine diamond bur and were conditioned with single bond ( 3 m dental product st . fifth group : teeth were cut with new coarse diamond bur and were conditioned with single bond adhesive system . sixth group : teeth were cut with used coarse diamond bur and were conditioned with single bond adhesive system . all prepared cavities were washed for 15 seconds with an air / water spray and the excessive water was removed with a gentle air spray , leaving the preparation slightly moist . se bond adhesive system was applied for the cavities of group 1 , 2 and 3 , according to the manufacturer s instruction the primer was applied in the cavity , 10 seconds air dried gently , then applied a single layer bond in the cavity , the bonding agent was thinned with intermittent one - to - two second air blasts , which was followed by 20-second light polymerization by a led light curing unit ( led turbo light cure- taiwan ) with 600 mw / cm light intensity . single bond adhesive system was applied for the cavities of group 4,5 and 6 , according to the manufacturer s instruction , 20 seconds total etch time of the enamel , dentin and cementum with ultra etch 37% phosphoric acid ( ultraetch 505 west 10200 south south jordan , utah 84095 ultradent , usa ) , 15 seconds rinse , then a light one - to - two seconds stream of air leaving the surface slightly moist . this was followed by applying single bond into the preparation and rubbing the bonding resin into the dentin enamel and cementum with the applicator brush tip . single bond was thinned with intermittent one - to - two second air blasts to the point of not losing its glossy appearance . this was followed by 20 second light polymerization using an led light curing unit ( led turbo light cure - taiwan ) with 600 mw / cm light intensity . in all groups , the composite restorative material ( z100 - 3 m , shade a2 usa ) was placed and condensed incrementally until the preparations were completely filled . each increment of restorative material attempted to involve only two walls of the preparation to reduce shrinkage and direct stress strain away from the internal walls . each increment was light polymerized for 20 seconds prior to placement of the subsequent increment . all specimens were then subjected to 500 thermocycles at 5c , 55c with a 20 second dwell time . all specimens were then subjected to 500 thermocycles at 5c and 55c with a 20- second dwell time ( vafaei - iran ) . after 24 hours all the restorations were finished , although the required finishing was minimal . after cycling , the apices of all root surfaces were sealed with adhesive wax and two coats of finger nail were applied to within approximately 1 mm of the tooth - composite interface . after sealing , the teeth were immersed in a 5% solution of methylene blue dye for 12 hours . upon retrieval from the dye , the teeth were washed under running water and left to dry for dye fixation . the embedded samples were then sectioned once vertically approximately midway through the facial surface using a diamond coated cutting disk and nonstop section machine ( bego nonstop - germany ) . dye penetration was evaluated using a 10x stereomicroscope ( m6c- 10- germany ) at the occlusal and gingival margins . microleakage scores were based on the degree of dye penetration according to the criteria described in table 1 . microleakage scores were recorded for both the occlusal and gingival margins as shown in table 2 . the kruskal - wallis for non parametric data was used to analyze inter group comparisons of microleakage , while mann - whitney u and wilcoxon w tests with bonferoni s correction were used to test for differences in microleakage between pairs of groups in dentin and enamel margins . microleakage raw data scores by examiners are presented in table 2 . mean rank and means of dye penetration for the experimental groups at occlusal and gingival margins are presented in table 3 . kruskal - wallis test showed that new / se group has the most and used / sb group has the least microleakage in the occlusal margin ( p - value=0.05 ) . in the gingival margin new / sb group has the least and used / sb group has the most microleakage as shown in table 3 . the kruskal - wallis test revealed that using the same adhesive system in gingival margins , significant difference was seen between bur types . the se bond adhesive system had the most leakage in the used diamond bur and the least leakage in new diamond bur ( p - value=0.05 ) . the single bond adhesive system had the most leakage in the new diamond bur and the least leakage in the used diamond bur ( p - value=0.001 ) . the kruskal - wallis test revealed that using the single bond adhesive system in occlusal margins , significant difference was seen between bur types ( p - value=0.029 ) . the results showed that using the se bond adhesive system in occlusal margins there was no significant difference between bur types ( p - value=0.127 ) . the mann - whitney test was used to test for differences in microleakage between pairs of groups in gingival and occlusal margins . the results have been shown in table 4 . the mann - whitney test with bonferoni s correction showed that using the same bur type in occlusal margins , there was a significant difference ( p - value<0.0055 ) between used se and used sb groups . there was no significant difference using the same bur type in gingival margins between two types of adhesive system . the mann - whitney test with bonferoni s correction was used to compare microleakage at the occlusal and gingival margins of the samples for each group . the results showed that the gingival margins leaked significantly more than the occlusal margins in the used sb group ( p - value=0.000 ) . reviewing the previous literature makes it clear that the efficiency of the instrument used for cavity preparation has great effect on quantity and quality of the smear layer and the amount of leakage in cavities prepared with burs having different efficiency is not the same . in 2005 , von fraunhofer evaluated the effect of re - using of disposable diamond burs on restoration leakage . he showed that leakage of the first and third uses of the bur were similar to each other , but was much greater for the fifth use . he said that disposable diamond burs can cut preparations in up to three teeth before adversely affecting leakage behavior . oliveria reported that the roughness of the surface varied strongly with the degree of coarseness . the surface roughness and also the thickness of the smear layer increased significantly with the coarseness of the abrasive but did not differ significantly with the abrasive type . evaluating the smear layer modification ; they found a significant inverse association between the degree of coarseness and the tubule openness . the sem study carried out by sanitini et al suggested that the smear layer produced by rough diamond burs were consistently thicker than those produced by tungsten carbide fissure burs , which in turn were thicker than those produced by fine diamond burs . using the inverted cone at speeds of 6000 rpm without water spray , the research says that an understanding and recognition of appropriate treatment of smear layers is crucial to the development of improved dentin bonding systems . the research results showed that gingival margins leaked more than occlusal margins for all bur types . as well as organic in its nature , dentin is a complex structure of collagen and dentinal tubules . depending on the location of the preparation , the size , number and direction of dentinal tubules are different . dentin tubules may be absent if the preparation ends at the cej or below ; this will affect the bond and also microleakage . in the se bond group , we found that the used diamond bur had more leakage than the new diamond bur , which may be the result of the quality of the smear layer , which is unlike in different bur types and high ph of self etch primer in this system . vonfraunhofer related the higher restoration leakage in multiple uses of the disposable bur to the effect of greater smearing of the surface of cavity preparation together with some redeposition of cutting debris on the surface from the repeatedly used bur . we also found that when the same adhesive systems were used , there was significant difference between bur types in dentin margins , so when the single bond adhesive system was used , the used diamond bur had the most and a new diamond bur had the least microleakage and it had the same results when we used the se bond adhesive system . this might suggest that the smear layer which is produced by used diamond bur is denser and stickier , because of the low rate of efficiency , in order to prepare the cavity , more pressure of the hand is used by the dentist unintentionally . but when the bur types were the same in dentin margins , there was no significant difference between different adhesive systems . the results of this study revealed that using the same adhesive system in enamel margins caused no significant difference between bur types . but when the same bur type was used , there was significant difference between the types of adhesive systems . this might show that the phosphoric acid etchant in single bond system can totally remove the smear layer and resin can easily infiltrate into the demineralized space . but as the ph value of self - etching / priming solution in se bond system is generally low enough to demineralize the smear layer and the underlying dentinal surface , we have more leakage in se bond system than the single bond system . pashley and carvalho suggested that the smear layer interferes with the self - etching primer adhesion . in conclusion , the cutting efficiency of bur had a great increasing effect on microleakage of composite resin restorations . so long term use of burs may result in an increased microleakage of composite resin restorations . on the other hand ,

objective : in this study , the effect of bur cutting efficacy was evaluated on microleakage of class v composite restorations with two adhesive systems.materials and methods : class v cavities were produced on sound extracted human teeth , which had been assigned randomly to one of six groups ( n=15 ) as follows : groups 1 and 6 were prepared using used rough diamond bur ; group 2 and 5 were prepared using new rough diamond bur ; group 3 and 4 were prepared using soft diamond bur . after application of single bond ( 3 m dental product , usa ) in groups 4,5 and 6 and clearfil se bond [ kurary medical inc . japan ] in groups 1,2 and 3 , all cavities were restored with composite resin . the teeth were thermocycled and microleakage was evaluated by dye penetration . kruskal - wallis and mann - whitney tests with bonferroni s correction were used for statistical analysis.results:the results showed that gingival margins significantly leaked more than occlusal margins for all bur types and bonding systems . using the same adhesive system in gingival margins , significant difference was seen between bur types and using the same bur type in occlusal margins , there was a significant difference between the two types of adhesive systems.conclusion:cutting efficiency of bur had a great effect on microleakage of resin composite restorations . so long term use of burs may result in an increase in microleakage of composite resin restorations .

considerable disparities in cancer survival rates exist between african americans ( aas ) and white americans ( was ) . various factors such as socioeconomic status ( ses ) , cancer stage at time of diagnosis , and treatment have accounted for many of these disparities . other factors include insurance status , social determinants in general and genetics . however , even when analyses of cancer survival data include all known or suspected factors affecting survival , aas still tend to have a lower survival rate than that of was , possibly because of unmodeled factors such as biological differences , and perhaps as a consequence of educational level and access to health care as several authors have noted . discussions of cancer survival disparities generally overlook the role of vitamin d. for 20012004 , aas older than 60 y had a population mean serum 25-hydroxyvitamin d [ 25(oh)d ] concentration of 17 ng / ml compared with 25 ng / ml for was . prevalence of hypovitaminosis d [ ( 25(oh)d < 15 ng / ml ] in the south was 45% among blacks and 11% among whites . in patients participating in a randomized controlled trial of chemotherapy , serum 25(oh)d concentrations were lower in black patients than in white patients and patients of other race ( median , 10.7 vs 21.1 vs. 19.3 ng / ml , respectively ; p < 0.001 ) , as well as in females compared with males ( median , 18.3 vs 21.7 ng / ml , respectively ; p = 0.0005 ) . solar ultraviolet - b ( uvb ) irradiance is the primary source of vitamin d for most americans , accounting for 8090% of vitamin d. aas , with darker skin , are less efficient at producing vitamin d from uvb irradiance . in addition , aas are less likely to have as much vitamin d from oral intake . a large body of literature supports a beneficial effect of vitamin d in reducing the risk of cancer incidence and mortality rates . many ecological studies have supported this hypothesis , as have observational studies of breast and colorectal cancer . two ecological studies found stronger inverse correlations between solar uvb doses and cancer mortality rates than incidence rates . several reviews of the uvb - vitamin d - cancer hypothesis have also been published . the dose response relation for vitamin d has been derived from observational studies for breast and colorectal cancer . for the values for cancer incidence are not necessarily the same for cancer survival , but they do suggest the magnitude of the effect . this vitamin d - cancer dose response relation might underestimate the effect of lower serum 25(oh)d concentrations for aas since 20% of the black population is older than 60 y , in contrast to only 6% of whites ; also , the risk of cancer increases more rapidly for changes of serum 25(oh)d concentration at lower concentrations . a recent paper addressed vitamin d s role in explaining some of the cancer survival disparities . data from the third national health and nutrition examination survey ( nhanes iii ) were used to investigate the role of racial disparity from colorectal cancer ; adding vitamin d deficiency to the model attenuated the mortality risk associated with being black by a statistically significant 40% . grant and peiris investigated vitamin d s role in explaining disease disparities between aas and was in general . this paper surveys the literature on cancer disparities for aas and was as well as the literature on epidemiological studies on vitamin d and cancer to see whether differences in serum 25(oh)d concentrations might explain many of the otherwise - unaccounted - for residual disparities . table 1 presents the findings regarding cancer survival with respect to serum 25(oh)d concentrations at the time of diagnosis . significant inverse correlations between 25(oh)d and cancer survival were found for all - cancer , breast , colon , colorectal , lung , prostate cancer , chronic lymphocytic leukemia / chronic lyphocytic lymphoma , hodgkin lymphoma , and non - hodgkin lymphoma . studies also reported no significant correlation between serum 25(oh)d and survival for bladder , lung and ovarian cancer . ac , all cause ; cs , cancer specific ; hr , hazard ratio ; nhl , non - hodgkin lymphoma ; rr , relative risk . table 2 presents the multifactor - adjusted hazard ratios for survival for aas vs. was for cancer - specific survival . inclusion of ses , stage at diagnosis , and treatment in the analyses is indicated . the results for cancer - specific survival rates are a stronger indication of the effects of vitamin d than are all - cause survival rates because some of the all - cause deaths could be due to non - vitamin d - related diseases or to factors such as smoking . statistically significant disparities emerged for cancer - specific survival rates for 13 types of cancer : bladder , breast , colon , endometrial , lung ( non - small cell , stage iii , iv ) , ovarian ( advanced ) , pancreatic , prostate , rectal , testicular , vaginal cancer , hodgkin lymphoma , stage ii and melanoma . our analysis also found statistically significant disparities for cancer - specific survival rates for two types of cancer , endometrial and ovarian cancer . there were no statistically significant findings for gastric adenocarcinoma , or head and neck , and oral cancer and leukemia . aas lower serum 25(oh)d concentrations ( mainly from reduced vitamin d photoproduction owing to darker pigmentation ) may account for much of the unexplained survival disparity after consideration of such factors as ses , stage at diagnosis , and treatment . all cancers for which a disparity in cancer - specific survival was reported also have evidence for a beneficial role of vitamin d , as do most of those for which we found disparities for all - cause survival . one reason ecological studies are strong include that vitamin d plays an important role in reducing risk of cancer initiation and angiogenesis around tumors and metastases . since cancer can take years to decades to reach the stage of detection or death , continued high serum 25(oh)d concentrations over much of the lifetime is required for greatest risk reduction . also , ecological studies include many cases , thereby reducing the uncertainty of the values . among 45-y - old british citizens , casual solar uvb irradiance in summer increased serum 25(oh)d concentrations by about 15 ng / ml , enough to have an important impact on cancer risk . for example , breast cancer incidence rates are highest in spring and fall . the reasons for the seasonal variations given were increased production of vitamin d in summer and melatonin in winter . breast cancer has several subtypes , and rate of progression can vary widely , with some being very rapid . for slower growing cancers , serum 25(oh)d concentrations in summer may be sufficient to retard or reverse the growth . once cancer reaches the point where it can be diagnosed , vitamin d improves cancer - specific survival by several mechanisms , including antiangiogenesis and antimetastases . the disparities for hematopoietic cancers may be weak or nonexistent because angiogenesis and metastases are less important for blood cell - related tumors than for solid tumors . higher serum 25(oh)d concentrations also affect all - cause mortality rates since vitamin d protects against several major life - threatening conditions for the elderly , including diabetes and cardiovascular disease , influenza and pneumonia , and falls and fractures . secondary hyperparathyroidism due to osteoblastic metastases and hungry bone syndrome has been described with advanced prostate and breast cancer , it is likely that a vitamin d replete state may minimize such occurrences . coleman and mccloskey suggest that bisphosphonates may prevent metastases and reduce the risk of disease recurrence . based on animal data , a vitamin d replete state may be helpful in reducing bisphosphonate induced osteonecrosis of the jaw . factors other than ses , stage at diagnosis , treatment , and vitamin d status might also explain the cancer survival disparities . for example , the lack of survival disparities for lung cancer may be due to a stronger effect from smoking than from vitamin d. smoking cessation improves lung cancer survival rates associated with early - stage lung cancer . obesity is significantly correlated with cancer risk for nearly all types of cancer listed in tables 1 and 2 . one reason is that obesity is linked to poverty in the united states because of energy - dense but nutrient - poor foods are cheaper due to subsidies . a second reason is that aas have about twice the prevalence of apolipoprotein e 4 ( apoe4 ) than was . apoe4 increases production of cholesterol in the liver and of insulin in the pancreas to store excess food as fat for those with sporadic food supplies , such as hunter - gatherers . interestingly , overweight and obesity rates for white and black men differ little , whereas aa women are much heavier than wa women ( http://www.cdc.gov/nchs/data/hestat/obesity_adult_07_08/obesity_adult_07_08.pdf ) . thus , obesity does not seem to be a likely explanation for cancer disparities among men but could be for women . on the other hand , serum 25(oh)d concentrations are inversely correlated with body mass index , which has implications for cancer risk . interestingly , for pancreatic cancer incidence , higher body mass index was significantly associated with risk for aa and wa men and wa women but with only insignificantly reduced risk for aa women . cancer survival studies with respect to serum 25(oh)d concentrations at time of diagnosis offer strong evidence for a beneficial effect of vitamin d. all cancers with a beneficial effect of vitamin d on survival have been found inversely correlated with solar uvb doses , with the possible exception of chronic lymphocytic leukemia . there are also studies from norway indicating improved survival for those diagnosed with breast , colon , prostate cancer and hodgkin lymphoma in summer compared with winter . the uvb - vitamin d - cancer hypothesis receives its strongest support from ecological studies . observational studies also provide good support if the various studies are examined carefully and a good reason is found for why many observational studies have not found a beneficial effect of vitamin d in reducing the risk of cancer . nested case - control studies have a reduced strength since only a single serum 25(oh)d concentration measurement or oral intake assessment is made at time of enrollment , with follow - up periods lasting between 3 and 28 y. as the follow - up time increases beyond about 37 y , the single measurement is less meaningful . case - control studies , on the other hand , use serum 25(oh)d concentration or vitamin d oral intake values at the time of diagnosis . a review of observational studies of breast and colorectal cancer incidence with respect to serum 25(oh)d concentration found statistically significant inverse correlations for breast cancer out to 3 y and for colorectal cancer out to 12 y of follow - up . thus , the recently reported results from the vitamin d pooling project study of rarer cancer types ( endometrial , esophageal , gastric , ovarian , pancreatic , and renal cancer and non - hodgkin lymphoma ) probably failed to find an inverse correlation between incidence of these cancers and prediagnostic serum 25(oh)d concentrations because the mean follow - up period was 6.63 y and because there were so few cases that the 95% confidence intervals were about 50% . the correlation between serum 25(oh)d concentrations measured at different times decreases with time , dropping to a regression coefficient of 0.40 after 14 y. several ways exist to test the uvb - vitamin d - cancer hypothesis as an additional contributing factor for cancer survival disparities . one would be to measure serum 25(oh)d concentrations of newly diagnosed cancer patients and at several intervals during the course of the cancer . another would be to supplement newly diagnosed cancer patients with sufficient vitamin d to bring serum 25(oh)d concentrations up to 4080 ng / ml and compare results for those not supplemented , perhaps from previous patients in the same practice . a recent publication described the rationale for vitamin d supplementation , which is being done in some cancer treatment centers . increasing serum 25(oh)d concentrations would also reduce the risk of severe sepsis associated with cancer surgery as well as many other comorbid diseases . we acknowledge that while it appears very likely that vitamin d is an important and often ignored factor in the biology of cancer , the issue of cancer etiology is complex and is clearly multifactorial . moreover , outcomes studies may have skewed results since aa men are less likely to participate in cancer screening trials . an inverse relationship between physical activity and breast cancer in aa women has been reported . reported that aa patients are less likely to receive resection in non - metastatic rectal cancer . demonstrated that aa colorectal cancer survivors are less likely to receive post - treatment colorectal surveillance . these may not necessarily reflect racism in that physicians may make recommendations based on a patient s access to health care , presence of insurance , etc . in addition , poor health literacy in aa women may also impact access to available health care strategies . cultural differences may also play a role with cultural insensitivity among providers compounding the issue . margolis et al . demonstrated significant racial differences in belief prior to lung cancer surgery . some of these differences result in refusal of surgery on the part of aa patients . aas have less trust in their health providers and may not accept physicians assertions regarding treatment . van ness et al . indicates that lack of religiousness maybe associated with poor cancer survival in aa women . church attendance may be associated with greater emotional and social support , which is linked to better outcomes in breast cancer . we must also consider the possibility that apart from direct cellular benefits of vitamin d on cancer that vitamin d deficiency has indirect effects which are hard to quantify but may have a significant impact on cancer outcomes . vitamin d deficiency is also associated with a higher prevalence of depression and neurocognitive symptoms , which makes patients intrinsically less likely to seek medical attention . treating vitamin d deficiency some risk factors such as diet can be modified and increased consumption of vegetables may decrease the risk of breast cancer in aas , possibly by altering estrogen / progesterone receptor status . fortunately , it does appear that tumors are not intrinsically more aggressive in aas . in veterans with equal access to health care , dignam reported that black women , diagnosed at comparable disease stage as white women and treated appropriately , tend to experience similar breast cancer prognoses and survival . some of the residual disparity for prostate cancer may be due to the higher prevalence of the apoe4 allele among aas than was , which is related to increased cholesterol production . increased low - density lipoprotein concentrations increased the risk of prostate cancer for aas but not was . we acknowledge that while it appears very likely that vitamin d is an important and often ignored factor in the biology of cancer , the issue of cancer etiology is complex and is clearly multifactorial . moreover , outcomes studies may have skewed results since aa men are less likely to participate in cancer screening trials . an inverse relationship between physical activity and breast cancer in aa women has been reported . reported that aa patients are less likely to receive resection in non - metastatic rectal cancer . demonstrated that aa colorectal cancer survivors are less likely to receive post - treatment colorectal surveillance . these may not necessarily reflect racism in that physicians may make recommendations based on a patient s access to health care , presence of insurance , etc . in addition , poor health literacy in aa women may also impact access to available health care strategies . cultural differences may also play a role with cultural insensitivity among providers compounding the issue . some of these differences result in refusal of surgery on the part of aa patients . aas have less trust in their health providers and may not accept physicians assertions regarding treatment . van ness et al . indicates that lack of religiousness maybe associated with poor cancer survival in aa women . church attendance may be associated with greater emotional and social support , which is linked to better outcomes in breast cancer . we must also consider the possibility that apart from direct cellular benefits of vitamin d on cancer that vitamin d deficiency has indirect effects which are hard to quantify but may have a significant impact on cancer outcomes . vitamin d deficiency is also associated with a higher prevalence of depression and neurocognitive symptoms , which makes patients intrinsically less likely to seek medical attention . treating vitamin d deficiency some risk factors such as diet can be modified and increased consumption of vegetables may decrease the risk of breast cancer in aas , possibly by altering estrogen / progesterone receptor status . fortunately , it does appear that tumors are not intrinsically more aggressive in aas . in veterans with equal access to health care , dignam reported that black women , diagnosed at comparable disease stage as white women and treated appropriately , tend to experience similar breast cancer prognoses and survival . some of the residual disparity for prostate cancer may be due to the higher prevalence of the apoe4 allele among aas than was , which is related to increased cholesterol production . increased low - density lipoprotein concentrations increased the risk of prostate cancer for aas but not was . lower serum 25(oh)d concentrations among aas than was may explain many of the cancer survival disparities after consideration of ses , stage at time of diagnosis , and treatment . if substantially correct , programs to increase serum 25(oh)d concentrations among aas could reduce the cancer disparities . this approach would work not only for those of the ages where cancer is more likely but also for those younger . vitamin d can reduce the risk of cancer at the initiation stage and in the advanced stages , as well as raising serum 25(oh)d concentrations to over 40 ng / ml shortly after cancer diagnosis . given the biologic plausibility , the currently available evidence of beneficence , and the lack of harm with moderate vitamin d replacement , we recommend oncologists consider a more proactive stance on this issue pending additional studies . papers cited in this study came from the national library of medicine s pubmed database ( http://www.pubmed.gov ) . as of december 28 , 2011 , a search for cancer disparity papers ( search terms cancer disparities survival , african - american ) identified 457 articles . we examined in more detail those reporting hazard ratios for aas vs. was for survival . the tables in this review do not include the component papers of meta - analyses cited . we inspected the results in the papers , preferring those with disparities in survival that included all three factors ses , stage at diagnosis , and treatment , in addition to race over those including fewer or none of these factors . we examined the papers to see whether survival was cancer - specific or all - cause . in addition , papers reporting cancer survival with respect to serum 25(oh)d concentration were also sought .

considerable disparities in cancer survival rates exist between african americans ( aas ) and white americans ( was ) . various factors such as differences in socioeconomic status ( ses ) , cancer stage at time of diagnosis , and treatment which this analysis considers primary explanatory factors have accounted for many of these differences . an additional factor not usually considered is vitamin d. previous studies have inversely correlated higher solar ultraviolet - b ( uvb ) doses and serum 25-hydroxyvitamin d ( 25(oh)d ) concentrations with incidence and/or mortality rates for about 20 types of cancer and improved survival rates for eight types of cancer . because of darker skin pigmentation , aas have 40% lower serum 25(oh)d concentrations than was . this study reviews the literature on disparities in cancer survival between aas and was . the journal literature indicates that there are disparities for 13 types of cancer after consideration of ses , stage at diagnosis and treatment : bladder , breast , colon , endometrial , lung , ovarian , pancreatic , prostate , rectal , testicular , and vaginal cancer ; hodgkin lymphoma and melanoma . solar uvb doses and/or serum 25(oh)d concentrations have been reported inversely correlated with incidence and/or mortality rates for all of these cancers . this finding suggests that future studies should consider serum 25(oh)d concentrations in addressing cancer survival disparities through both measurements of serum 25(oh)d concentrations and increasing serum 25(oh)d concentrations of those diagnosed with cancer , leading to improved survival rates and reduced disparities .

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

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

wound healing defects and associated soft tissue damages are becoming more important in the orthopedic practice . this is due to the significant increase of prosthesis implantations , including knee prosthesis implantations and the demographic development of society . the demands on a long lifetime of the prostheses are consistently increasing due to the increasing life expectancy of the patients receiving prostheses . standardizations in endoprosthetics , increased experience of surgeons and the further development of prostheses has led to the consistent improvement of long - term results and lifetime of artificial joints , so that prostheses are implanted and replaced in younger patients as well . a secure and stable coverage with soft tissue is a basic requirement for good short - term and long - term results regarding the prosthesis . without a sufficient coverage with vital soft tissue with a good blood supply , the risks of infection of the prosthesis and reduced capacity of the new joint exist . this is especially important for the knee because there is little soft tissue coverage in general which contributes to the increased risk for wound healing defects due to the surgical wound leading to a reduced blood supply . transcutaneous measurements of oxygenation have shown that the level of oxygenation near the surgical scar necessary for wound healing is only reached after two to three days [ . besides the negative impact of the surgery itself on the primary wound healing , various other exogenous factors play a role in wound healing . the most important factors are rheumatoid arthritis , diabetes , long - term cortisone intake , malnutrition , nicotine use , hypothyroidism and existing multiple scars in the area of the surgery , , . laing et al . have described five stages of soft tissue defects after knee endoprosthetics 1992 : stage 0 : redness near the wound without wound dehiscence or development of necrosis stage 1 : only superficial skin necrosis or tension blisters , deeper layers not affected ; no fistula stage 2 : extensive skin necrosis with fistula towards the knee , deeper layers of tissue not affected stage 3 : joint fistula with dehiscence of deeper layers ; a small part of the joint prosthesis is visible stage 4 : widespread tissue necrosis with wound dehiscence and visible prosthesis stage 0 : redness near the wound without wound dehiscence or development of necrosis stage 1 : only superficial skin necrosis or tension blisters , deeper layers not affected ; no fistula stage 2 : extensive skin necrosis with fistula towards the knee , deeper layers of tissue not affected stage 3 : joint fistula with dehiscence of deeper layers ; a small part of the joint prosthesis is visible stage 4 : widespread tissue necrosis with wound dehiscence and visible prosthesis for stages 0 and 1 , a conservative treatment with fixation of the knee and bed rest for the patient is favoured and the soft tissue damage usually heals well without surgical treatment . for stage 2 and above , surgical measures are preferred . in stage 2 , the superficial skin necrosis should be removed and temporary soft tissue coverage and vac - therapy should occur . permanent soft tissue coverage can occur in the case of asepsis . depending on size , a split - skin ( mesh - graft ) or full - skin graft can be chosen . 1 ) ) , a muscle flap is necessary , because the deeper damages can not be treated with mere skin transplants . the benefits of muscle flaps , e.g. a gastrocnemius rotation flap , are evident : because the muscle flap is supplied with blood , it can be placed on infected wounds ; this leads to a significant improvement of wound healing . this is due to secure soft tissue coverage of possibly exposed bone and prosthetic material . it is also due to the transportation of immunocompetent cells to the site of infection with the normal blood supply which leads to an improvement of wound healing and defence against infection . for this stage , the elevation and use of one gastrocnemius muscle belly , medial or lateral , is sufficient ( figure 2 ( fig . the lateral gastrocnemius muscle flap plasty was first described by barford and pers and the medial gastrocnemius muscle flap plasty was first described by ger . the medial head is often longer and wider than the lateral head which is why it is usually used . surales medialis and laterialis , the elevation of an isolated flap and the coverage of almost the entire knee ( figure 3 ( fig . the muscle belly should be lifted without a skin graft because this could cause problems with the closure of the skin near the location of extraction . for the dermal closure of the implanted muscle flap , split - skin ( mesh - graft ) with a thickness of 0.3 to 0.5 mm the muscular aponeurosis and parts of the fascia of the muscle should be removed , so that it can heal well ( figure 4 ( fig . an area of up to 60 square centimetres can be covered this way . the muscle coverage with split - skin ( mesh - graft ) is also indicated ( figure 5 ( fig . if there is an intact suralis muscle , the expected muscular deficit regarding plantar flexion is remarkably small , and there is little aesthetic defect involved with muscle flap plasty . 16 patients were treated for stage 3 soft tissue damage and seven patients were treated for stage 4 soft tissue damage . accordingly , 16 patients received one - headed muscle flaps and seven patients received two - headed muscle - flaps with split - skin grafts from the ipsilateral thigh . out of 23 patients , dehiscence or necrosis at the split - skin graft occurred in three patients , and this required repeated split - skin grafts . one patient experienced dehiscence near the extraction point of the muscle which was successfully treated with split - skin graft after vac - therapy . four patients experienced recurring infection of the prosthesis with development of a fistula and large soft tissue destruction which was successfully treated with elaborate surgical measures in one case . the extremity could not be saved in three cases , and this resulted in above - knee amputation . due to the increasing number of implantation of knee endoprostheses and the associated number of soft tissue damages , a standardized procedure for the treatment of these complications is necessary . in the case of deep soft tissue damages with fistulas reaching the prosthesis or exposing the prosthesis , the gastrocnemius muscle flap is a good method for secure coverage of the prosthesis with well - perfused tissue . despite the high rate of complications of almost 35% which required at least another surgery , this method of soft tissue reconstruction is a necessary skill for surgeons involved with the implantation of knee endoprostheses .

with the increase of endoprosthetic knee replacements , there is also an increase of critical wounds to the knee due to a high incidence of soft tissue problems ( ranging from wound healing defects to severe wound infections ) . the literature describes a general rate of soft tissue complications of up to 20% [ 1 ] , [ 2 ] , with 5% [ 3 ] involving exposed bone . these complications are an increasingly important problem for surgeons . since sufficient coverage of bones , tendons and prosthetic material with soft tissue is a necessity , the use of a pediculated muscle flap is the only solution in some cases . the gastrocnemius muscle is very useful for this purpose . it is an elaborate procedure which is associated with a high rate of complications . however , this procedure can establish a secure coverage with soft tissue , and the function of the prosthesis and the patient s extremity can be saved . we have treated 23 patients with a gastrocnemius rotation flap after knee prosthesis or knee arthrodesis infection with consecutive soft tissue damage at our hospital from 8/2004 through 3/2011 . the overall rate of healing of the knee infections with stable soft tissue status is almost 87% . the revision rate with lifting of the flap and revision of the sutures at the point of insertion as well as the point of extraction was about 35% with long - term conservative or additional surgical treatments .

a 64-year - old man with a 15-year long history of hypertension was presented to the emergency room with a 24-hour history with symptoms of cyanosis and pain on the right upper extremity , consistent with acute ischemia . the major physical findings revealed that the right upper extremity was pale , cold , and exhibited reduced sensation and power . blood pressure was 130/80 mmhg and heart rate was regular at 64 beats per minute . laboratory values indicated an increase in white blood cell count of 11,400/ul , normal hemoglobin value of 15.5 g / dl and elevated creatininc kinasl mb ( ck - mb ) value of 33.2 ng / ml ( normal , 0 - 5 ng / ml ) . the increased levels of bun ( 31.9 mg / dl ) ( blood urea nitrogen ) and serum creatinine ( 1.8 mg / dl ) returned back to baseline value within three days . iu / l ) and alanine aminotransferase ( range , 28 - 79 ) were increased . a right transfemoral approach was performed with placement of a 6-f sheath . a 5-f angiographic catheter ( h1 angiogram obtained after injection of contrast medium in right axillary artery revealed a smooth , tapered narrowing of the brachial artery without opacification of radial and ulnar artery ( figs . 1a , b ) . at the time of angiography , passing of guide wire across the narrowing and occlusive segment was attempted , but these efforts were unsuccessful . the diagnosis of unusual pattern of raynaud 's disease was initially made , eventually incorrect . a fear of gangrenous changes in right upper extremity , he had undergone intraarterial papaverine injection of 30 mg and a right axillary - radial bypass surgery . over a period of approximately two hours , there was a dramatic improvement in the color of the patient 's upper extremity and radial pulses became palpable , but cyanosis and pain persisted at phalangeal area . in addition to intravenous infusion of prostaglandin e1 and heparin , two days later , the patient underwent ipsilateral sympathectomy with the subsequent disappearance of cyanosis and pain on phalangeal area . arteriography examination demonstrated normal arterial anatomy of the right arm as well as the patent graft ( figs . the patient 's postoperative recovery was uneventful , and he was discharged in a good condition . eight days later , the patient returned for the same ischemic symptoms of the right upper extremity . repeated angiography revealed the similar findings which had been demonstrated during first angiography and also occlusion of graft was noticed . the diagnosis of ergotamine - induced vasospasm was made only after that and treatment was initiated by discontinuing the intake of offending drug and an intravenous infusion of nitroprusside was initiated at a rate of 4 g / kg / minute . the drug infusion was continued over four days until there was relief from pain and cyanosis . we noticed that for over a period of 10 years the patient had been prescribed ergotamine for the treatment of migraine . people who consumed the fungus contaminated grain suffered from limb ischemia and discoloration of extremities . most of the cases of ergotamine toxicity are due to medication ingestion , either acutely or chronically ( 2 ) . this particular alkaloid can cause intense peripheral vasoconstriction which can lead to gangrene and amputation . ergotamine - induced vascular ischemia is rare , but potentially serious complication may be induced through two mechanisms : vasospasm and thrombus formation . the latter may be caused by stasis and a postulated direct endothelial damage may be caused by ergotamine . toxicity can occur from chronic use of therapeutic doses , acute ingestion of excessive amounts , and acute ingestion of normal doses in hypersensitive patients . a number of conditions are known to potentiate the vasospastic effects of ergotamine like , fever , sepsis , malnutrition , thyrotoxicosis , pregnancy , liver and renal insufficiency , coronary artery disease , and peripheral vascular disease ( 3 ) . the triggering factor for the development of ergotamine toxicity in the present case is still uncertain , but a possible reason could be hepatitis ( elevated liver enzyme ) with transient renal dysfunction . drugs may also be responsible for an increase in the side effects of ergotamine ( 4 , 5 ) : oral contraceptives , xanthine derivatives , antiviral agents , antibiotics interfering with the liver metabolism of ergotamine ( i.e. clarithromycin , ampicillin , erythromycin , and troleandomycin ) . recently , baldwin et al . described a case of ergotamine toxicity in an human immunodeficiency virus infected patient treated with antiviral protease inhibitor ( 6 ) . ischemia caused by ergotamine intoxication affects the lower extremities more commonly than the upper extremities . the external carotid arteries are often involved with a rare involvement of coronary , mesenteric , renal , and retinal arteries . the diagnosis of this rare condition requires a high index of suspicion in patients with migraine and a careful inquiry about their medication history . the angiographic findings of the affected vessels reveal thin , threadlike and smooth tapered narrowing of the arteries with or without collateral formation ( 7 ) . thrombus results from stasis distal to arterial spasm with either bilateral or symmetrical involvement , and the areas of stenosis may be diffused or localized . many therapeutic modalities can be used to reverse the vasoconstriction ( 8 , 9 ) . the first step of the treatment is discontinuation from the offending drug . when vascular thrombosis is suspected , heparin , a low molecular weight dextran and streptokinase has been suggested to reduce tissue loss . nitroprusside , a direct acting vasodilator , is effective but can be used only for shorter periods of time and prompt recurrence of symptoms have been reported after discontinuation . nifedipine is the most potent peripheral vasodilator of the group and has been successfully used to treat ergotamine toxicity . in severe forms of ischemia refractory to pharmacologic treatment , surgical sympathectomy or intraarterial balloon dilatation can be effective ( 10 ) , and the management depends on early diagnosis of the symptoms . in our case , uninterrupted medication for migraine is thought to play a significant role in the development of recurrent symptoms . the present case emphasizes the importance of acquiring a detailed medication history . combination of heparin , prostaglandin e1 , and sympathectomy did not prevent the progression of cyanosis in our case , but intravenous injections of nitroprusside could relieve the symptoms . an understanding of the clinical features and angiographic findings of ergotamine - induced ischemia is essential in early diagnosis and treatment to prevent irreversible complications .

ergotamine - induced limb ischemia is an extremely rare case . we present a case of a 64-year - old man , who developed ischemia on the right upper extremity due to long - term use of ergot for migraine headache . angiography revealed diffused , smooth , and tapered narrowing of the brachial artery . the patient was successfully treated with intravenous nitroprusside .

lumbar spine instability has been reported to be the leading cause of chronic back pain1 . an a abnormal vertebral joint creates mechanical lesions and pains in the lumbar spine , decreasing stability and increasing the range of motion , resulting in functional degeneration2 . these negative patterns have a variety of forms such as aging , medical history , and exercise deficiency and present serious problems in the age group of 2040 , due to developments of modern society and recent changes in various working forms of work3 , 4 . herniated nucleus pulposus ( hnp ) is a representative disease of the functional vertebral unit that occurs when the pulposus nucleus is exposed by rupture of the annulus fibrosus and it is a disease that causes chronic back pains5 , 6 . chronic back pain patients not only have weak deep muscles of the lumbar region and muscle imbalance compared to normal subjects , but also show reduction in the re - positioning ability which leads to problem with the stability of the spine , due to the reduction in proprioceptive sense , which causes lumbar pain and recurrence7 , 8 . they include exercises for increasing muscle strength , improving functions , and maintaining posture as well as preventing excessive movements of the lumbar spine9 . exercise rehabilitation approaches for strength and stability are important even after surgical treatments , due to degeneration and restricted activities10 , 11 and conservative methods of treatment by exercise have been recommended for the purpose of preventing recurrence of pain and improving functions12,13,14,15,16 . thus , exercise therapies based on the stabilization exercises are considered to be necessary for preventing chronic pain and reductions in functional capabilities by the reduction of functional instability in various of type and the improvement of motor functional capabilities of the lumbar joints . therefore , this study aims to help adult women with lumbar disc herniation recover muscle strength and stability through 8-week lumber extension strength exercise programs , and to provide data for constructing an integrated exercise program . in this study , an 8-week exercise program for lumbar muscle extension strength and stabilization was performed by 26 females older than 20 with lumbar disc herniation . this study was approved by the hankuk university of foreign studies and the institution where it was performed , and it complied with the ethical principles of the declaration of helsinki . the purposes and process of the study were fully explained to the subjects and their consent was obtained before participation . the subjects mean sd was 27.2 4.4 years old their mean sd height was 162.3 5.1 cm and their mean sd weight was 55.4 7.1 kg . the programs was conducted for a total of 8 weeks , 60 minutes a session , twice a week , to improve strength and stabilize lumbar extension . the exercise program was divided into warm - up exercises , main exercises and cool - down exercises . the warm - up exercises and cool - down exercises were conducted focusing on the range of joint motion ( rom ) without pain , utilizing cycles and steppers for 10 minutes , alternately . for the purpose of improving the lumbar stability and resistance muscles , the main exercises were designed to be individually conducted in the form of a circuit training in which the number of repetitions increased gradually , utilizing sling and weight exercise equipment . a lumbar extension strength machine ( medx , medx inc . , ocala , fl , usa ) was utilized for the evaluation of lumbar extensor strength . the maximum static muscular strength of lumbar extension was measured at 7 different angles of lumbar flexion : 0 , 12 , 24 , 36 , 48 , 60 , and 72 degrees . passive tests were conducted to determine whether to limit the range of motion before measurement . the tests were conducted 12 times at 45 lbs in accordance with the medx exercise measurement guidelines . the measurement was performed after fixing the pelvis and thigh of the subjects on the support . then , the motion of the lumbar spine was limited during measurement by adjusting the footrest . the subjects were asked to increasingly extend the lumbar spine by sufficiently considering the limited angular range of motion of the joints . the measurements were performed in the same manner at every angle by maintaining maximum muscle contraction for about 2 seconds . for the physical stability measurements , the total weight distribution index ( wdi ) was calculated by assessing the degree of interaction and coordination of the lower body through body away while maintaining a standing posture . for data processing , the statistical program spss ( spss inc . , the mean and standard deviation of the measurements was calculated for all metrics . to compare the difference between the pre - exercise and post - exercise state of the 8-week exercise program , the paired t - test was used and statistical significance was accepted for values of p < 0.05 . significant differences were observed in lumbar extension strength ( les ) between pre - exercise and post - exercise at every angle of lumbar flexion , but no significant difference was found in wdi ( % ) ( table 1table 1.changes of the les and wdivariableanglepre - exercisepost - exerciseles ( ft - lbs)061.8 33.880.1 31.6**1278.7 33.898.8 33.6**2487.6 36.5116.5 32.7***3698.6 35.9129.7 32.3***48104.7 35.0137.6 30.2***60113.1 36.9146.5 30.5***72122.3 40.1158.7 34.7***wdi ( % ) 5.9 3.35.0 2.8values are mean sd , les : lumbar extension strength , wdi : weight distribution index . the post - exercise les were found to be very significantly ( p<0.01 ) higher than pre - exercise at 0 degree and 12 degree , and extremity significantly ( p<0.001 ) higher than pre - exercise at 24 , 36 , 48 , and 72 degrees of lumbar flexion . values are mean sd , les : lumbar extension strength , wdi : weight distribution index . this study attempted to identify the positive impacts that were exerted on extension muscle strength and stability by an 8-week extension strength exercise program which was performed by 26 females with lumbar disc herniation . hnp is caused by the annulus fibrosus that is squeezed into the spinal canal when the pulposus nucleus is torn due to a degenerative intervertebral disc . it has been reported that approximately 29% of the projected and escaped intervertebral discs are associated with the pulposus nucleus17 . chronic back pains resulting from hnp aggravate the instability of the lumbar spine , causing degenerative changes , atrophy of muscle strength , and reduce flexibility and joint range of motion due to trunk damage and instability18 , 19 . lumbar herniated nucleus pulposus does not occur due to lumbar pain or disability , but more frequently occurs due to deterioration of the related muscles or functions interacting with them adjunctively8 . in addition , it is said that the joint range of motion is limited due to the loss of muscle strength and flexibility caused by occupational factors , mode of action , specific changes in posture , or degenerative disease18 . these musculoskeletal diseases can be generally improved by utilizing conservative treatments such as exercise9 , 18 . rehabilitation exercise therapies for muscular strength and stability are important even after surgery10 , 16 . various types of composite exercises and core exercises in addition to exercises for muscular strength and stability are currently being utilized8 , 12 . these exercise therapies help to stabilize the lumbar spine through lumbar and trunk dynamic stabilization and exercise modulation , and increase of muscle strength20 . similar to the present study , a study analyzing the impacts of an 8-week functional exercise program for lumbar muscle strength , that was performed by 26 females with degenerative disc findings , found that there were significant differences between pre - exercise and post - exercise15 in lumbar muscle extension strength at all 7 angles of lumbar flexion , proving that exercise participation develops stability of the muscles around the lumbar spine . in addition , a study analyzing the effects of decompression therapy by 4-week joint mobilization that was performed for patients with lumbar herniated nucleus pulposus , reported improvements in joint range of motion in flexion and extension21 , proving its effectiveness . similarly , a study of13 lumbar traction for patients with lumbar herniated nucleus pulposus , and a study11 of lumbar discectomy and stabilization exercises for patients with lumbar herniated nucleus pulposus , both reported improvements in flexion and extension . the improvements in functions pursuant to improved muscle strength and stability were statistically significant , as were the results of the studies cited above . therefore , prevention of excessive movement of the lumbar spine and trunk , in the patients with chronic back pains or lumbar herniated nucleus pulposus can be helpful in their daily life because they secure the joint range of motion , thereby improving strength and stability9 . thus , the development of muscular strength for stabilization and integrated exercise for pain reduction and rehabilitation help to maintain the range of joint motion , muscle strength , and balance21,22,23 . for the improvement and rehabilitation of inpaired capacity in daily life due to back pain - associated injuries experienced by about 80% of the population a higher occurrence rate8 , 10 when recurrence is included muscle weakness , and loss15 of balance the development of an integrated exercise program is required . the above results indicate that improving muscle extension strength to enhance and improve the functions the patients with chronic back pain or lumbar herniated nucleus pulposus can help improve muscle functions and increase the range of joint motion thereby exerting a positive impact on physical stability .

[ purpose ] the purpose of this study was to provide the data for constructing an integrated exercise program to help restore muscle strength and stability through extension strength exercise in adult females with lumbar disc herniation . [ subjects and methods ] an 8-week exercise program for lumbar muscle extension strength and stabilization was performed by 26 females older than 20 with lumbar disc herniation findings . [ results ] significant differences were found in lumbar extension muscle strength at every angle of lumbar flexion after participation in the 8-week stabilization exercise program ; but there was no significant difference in the weight distribution index . [ conclusion ] an integrated exercise program aiming to strengthen lumbar spine muscles , reduce pain and stabilize the trunk can help to maintain muscle strength and balance . in addition , improvement in extension strength is expected to be helpful in daily life by securing the range of joint motion and improving the strength and stability .

a common cause of brain injury during the perinatal period is hypoxic - ischemic injury , which frequently results in the chronic handicapping conditions such as cerebral palsy , mental retardation , learning disability , and epilepsy ( 1 ) . predicting the outcome of neonates with hypoxic - ischemic injury , however , is difficult , because some methods that predict outcome have not reliably or consistently predicted long term neurologic outcomes . recently , h - magnetic resonance spectroscopy ( mrs ) has been used as a quantitative noninvasive tool to assess the biomechanical changes associated with central nervous system injury . h - mrs , which provides objective extent of hypoxic - ischemic insults , enhances predictability and provides a method to assess treatment effect ( 2 - 5 ) . the exact mechanism by which hypoxic - ischemic brain injury occurs in neonates is not clear , although increasing evidences indicate that hypoxic - ischemic induced neuronal death includes both necrosis and apoptosis . necrosis may predominate in acute damage , whereas apoptotic injury may take time to develop . therefore , blocking the apoptotic cascade may prolong the therapeutic window after hypoxic - ischemic injury ( 6 - 8 ) . there is evidence for involvement of multiple caspases in hypoxic - ischemic brain injury . and caspase inhibitors which were developed as antiapoptotic agents , are believed to play a key role in the delayed neuronal cell death observed after hypoxic - ischemic injury ( 7 , 9 ) . the effects of growth hormone ( gh ) on the central nervous system have become more apparent in the past decade . not only it is involved in brain growth and development , but its qualities as a neuroprotective factor against injury are now appreciated . recent studies have demonstrated that gh is involved in neuroprotection during hypoxic - ischemic brain injury ( 10 , 11 ) . these protective roles are supported by the ability of gh to accelerate glial cell division and myelinogenesis , and gh is also thought to have neuroprotective roles in neurogenesis ( 11 ) . although this neuroprotective mechanism is not completely known , is probably achieved by inhibiting of caspase activities ( 12 , 13 ) . the lipid peak in the h - mr spectrum has been reported to be a marker for apoptosis during hypoxic - ischemic injury ( 14 ) . we have therefore used h - mrs to evaluate the effects of gh as a caspase inhibitor on hypoxic - ischemic injury in neonatal rat brains . the right common carotid arteries of 7-day old sprague - dawley rats ( mean weight=13.3 g ) were ligated under halothane anesthesia . after a recovery period of 3 hr gh ( eutrophin , lgphd , korea ) was administered just prior to hypoxic - ischemic insult . the rats were divided into four groups : control ( 10 l distilled water , n=29 ) , intracerebroventricular ( icv , 10 l gh in 10 l distilled water , n=23 ) , intracerebroventricular / intraperitoneal ( icv+ip , n=21 ) , and intraperitoneal ( ip , 10 mg / kg gh in distilled water , n=23 ) . localized in vivo h - mrs was performed on a bruker biospec 4.7 t mri / mrs system equipped with active shielded gradients and aspect3000 computer with tomikon hardware and software ( bruker , fallanden , switzerland ) . spectra were acquired in the right cerbral hemisphere of rats 24 hr after the onset of hypoxic - ischemic insult . water suppressed h - mr spectra were acquired using a vosy sequence with detection of the double - refocused spin echo signal from the selected voxel ( 322 l , 12 l ) using the following acquisition parameters : sw=5,000 hz , si=4,096 pts , ns=128 , tr / te=3,000/30 and 135 msec . to identify the peak at 1.3 ppm , the spectra were acquired at echo times of 30 and 135 msec in order to differentiate the lactate peak from the lipid peak . peak areas were measured and the lipid / n - acetyl aspartate ( naa ) and lipid / creatine ( cr ) ratios were used as apoptotic markers . after the h - mrs examinations on the 1st day , 6 brains from each group were perfused with 0.9% saline solution mixed with 2 units / ml of heparin , followed by perfusion with 4% paraformaldehyde in pbs solution . each brain was isolated , and tunel staining was performed using an in situ cell death detection kit , pod ( boehringer mannheim , germany ) , as described . apoptotic cells were counted 3 times in the parietal area of the brain using a 200 lens , and the mean apoptotic cell numbers were calculated using image analyzer software . gross morphologic changes were scored at 2 weeks using a 5 point grading system as method by palmer et al . ( 15 ) , where 0 indicates no change and 4 indicates the most severe injury . significance was assessed by unpaired t - test and anova followed by kruskall wallis test . spearman correlation was used to investigate relationships between lipid / naa and lipid / cr ratio and morphologic score . the right common carotid arteries of 7-day old sprague - dawley rats ( mean weight=13.3 g ) were ligated under halothane anesthesia . after a recovery period of 3 hr gh ( eutrophin , lgphd , korea ) was administered just prior to hypoxic - ischemic insult . the rats were divided into four groups : control ( 10 l distilled water , n=29 ) , intracerebroventricular ( icv , 10 l gh in 10 l distilled water , n=23 ) , intracerebroventricular / intraperitoneal ( icv+ip , n=21 ) , and intraperitoneal ( ip , 10 mg / kg gh in distilled water , n=23 ) . localized in vivo h - mrs was performed on a bruker biospec 4.7 t mri / mrs system equipped with active shielded gradients and aspect3000 computer with tomikon hardware and software ( bruker , fallanden , switzerland ) . spectra were acquired in the right cerbral hemisphere of rats 24 hr after the onset of hypoxic - ischemic insult . water suppressed h - mr spectra were acquired using a vosy sequence with detection of the double - refocused spin echo signal from the selected voxel ( 322 l , 12 l ) using the following acquisition parameters : sw=5,000 hz , si=4,096 pts , ns=128 , tr / te=3,000/30 and 135 msec . to identify the peak at 1.3 ppm , the spectra were acquired at echo times of 30 and 135 msec in order to differentiate the lactate peak from the lipid peak . peak areas were measured and the lipid / n - acetyl aspartate ( naa ) and lipid / creatine ( cr ) ratios were used as apoptotic markers . after the h - mrs examinations on the 1st day , 6 brains from each group were perfused with 0.9% saline solution mixed with 2 units / ml of heparin , followed by perfusion with 4% paraformaldehyde in pbs solution . each brain was isolated , and tunel staining was performed using an in situ cell death detection kit , pod ( boehringer mannheim , germany ) , as described . apoptotic cells were counted 3 times in the parietal area of the brain using a 200 lens , and the mean apoptotic cell numbers were calculated using image analyzer software . gross morphologic changes were scored at 2 weeks using a 5 point grading system as method by palmer et al . ( 15 ) , where 0 indicates no change and 4 indicates the most severe injury . significance was assessed by unpaired t - test and anova followed by kruskall wallis test . spearman correlation was used to investigate relationships between lipid / naa and lipid / cr ratio and morphologic score . the lipid / naa ratio was significantly lower in the icv ( 8.53.1 ) and icv / ip ( 9.22.5 ) groups than in the control group ( 12.24.6 ) ; although the lipid / naa ratio in the ip group ( 11.43.7 ) was lower than in the control group , this difference was not significant ( fig . the lipid / cr ratio was also significantly lower in the icv group ( 8.13.5 ) than in the control group ( 10.94.4 ) ; the lipid / cr ratio was lower in the icv / ip ( 9.23.4 ) and ip ( 9.63.5 ) groups than in the control group , but these differences were not statistically significant ( fig . although the number of tunel positive cells did not differ between the control and ip groups , there were fewer in the icv and icv / ip groups ( fig . we found that the morphologic scores were significantly lower in the icv group ( 1.41.3 ) and somewhat lower in the icv / ip group ( 1.81.4 ) than in the control group ( 2.21.4 ) , but not in the ip group ( 2.21.2 ) ( fig . morphologic scores significantly correlated with the lipid / naa and lipid / cr ratios ( fig . 5 ) . recently h - mrs has been used as a quantitative noninvasive assessment tool in monitoring of brain development and in the diagnosis of neurologically damaged infants ( 3 - 5 ) . h - mrs can detect metabolites such as naa and other acetyl compounds , which serve as primarily neuronal markers ; cr , including phosphocreatine and cr , which are bioenergetic markers ; choline - containing compounds ( cho ) , which are released during membrane disruption ; and lactate ( lac ) , which accumulates in response to anaerobic tissue metabolism ( 2 , 16 ) . decreased naa / cho and naa / cr ratio and increased cho / cr ratio have were in asphyxiated neonates with poor neurologic outcomes after 1 yr ( 4 , 17 ) . h - mrs in asphyxiated neonates has also shown increased lactate and decreased naa in thalamus , as well as increased lac and decreased cr in basal ganglia ( 18 , 19 ) . during hypoxic - ischemic injury , there is a significant increase in the lipid peak , which correlates with apoptotic cell death , as well as in the intensity of the lipid peak , which is directly related to the apoptotic cell count ( 14 ) . we therefore used the lipid / naa and lipid / cr ratios as apoptotic markers . newborn infants subjected to transient hypoxic - ischemic injury during birth asphyxia are apparently relatively normal soon after resuscitation but show evidence of delayed cerebral injury hours later , the magnitude of which predicts the severity of later neurodevelopmental impairment ( 20 , 21 ) . the mechanism of delayed injury is unclear , but apoptotic cells are detected in brains of infants who died after birth asphyxia , suggesting that inappropriate activation of the apoptotic pathway accounts , at least in part , for the delayed cell death ( 6 , 8 , 21 ) . apoptosis was first described as a type of cell death distinct from necrosis , with no swelling or loss of membrane integrity , and no inflammatory response from the host tissue ( 22 ) . apoptotic cells undergo a ubiquitous physiologic process that is essential to the development and survival of multicellular organisms . this process takes place during embryologic development , turnover of gastrointestinal epithelium , and the regulation of the immune system . many pathological events that cause necrosis , including hypoxic - ischemic injury , can also induce apoptosis ( 2 , 4 , 6 , 23 ) . necrosis may predominate in more intense ischemic damage , whereas apoptosis may occur during milder ischemic damage and may take time to develop ( 24 ) . immature cortical neurons have been shown to be more susceptible to apoptosis than mature neurons , perhaps because cells of younger animals more readily undergo apoptosis than cells of more mature animals ( 21 , 25 ) . thus , blocking the apoptotic cascade may prolong the therapeutic window after hypoxic - ischemic events , especially in the developing brain ( 6 - 8 ) . the ability of specific therapeutic agents to reduce caspase inhibitors , which have antiapoptotic activity and are believed to play a key role in the delayed neuronal cell death after hypoxic - ischemic injury ( 7 , 9 ) . caspase-3 is a terminal enzyme in the caspase family that activates an endonuclease ( caspase - activated dnase ) , resulting in dna fragmentation ( 26 ) . caspase inhibitors , including inhibitors of caspase-3 , may prolong the therapeutic window after hypoxic - ischemic injury ( 7 , 27 , 28 ) . recently , gh administration has been reported to inhibit neuronal death during hypoxic - ischemic injury , and to have a neuroprotective effect in the cerebral cortex , hippocampus , and thalamus ( 10 , 11 ) . although the mechanism is not completely known , several reports suggest that hypoxic - ischemic injury induces neuronal death by downregulating bcl-2 protein levels , followed by sequential activation of the caspases , and that gh protects neuronal cells by inhibiting alterations in bcl-2 protein levels and caspase activities ( 12 , 13 ) . we found that the lipid / naa ratio was significantly lower in rats administered gh by the icv and icv / ip routes , that lipid / cr ratio was significantly lower in rats administered gh by the icv , and that the degree of morphologic changes in the brain was significantly correlated with the lipid / naa and lipid / cr ratios . in our results , lipid / naa ratio and lipid / cr ratio were not significantly changed by ip administration of gh . because gh does not usually cross the blood - brain barrier , ip administration of gh taken together , these findings suggest that gh exerts neuroprotective effects in cerebral hypoxic - ischemic injury by inhibiting apoptosis , especially in the early stage after insult . our results also suggest that gh , as a caspase inhibitor , can have therapeutic value in neuroprotective effect of hypoxic - ischemic brain injury .

using 1h - mrs , we evaluated the effects of growth hormone ( gh ) as a caspase inhibitor on hypoxic - ischemic injury in neonatal rat brains . the right common carotid arteries of rats were ligated , allowed to recover for 3 hr , and exposed to 8% oxygen for 2 hr . gh was given just prior to hi insult and animals were divided into four groups : control , intracerebroventricular ( icv ) , intracerebroventricular / intraperitoneal ( icv / ip ) , and intraperitoneal ( ip ) . localized in vivo 1h - mrs and tunel staining were performed 24 hr after hi injury . lipid / n - acetyl aspartate ( naa ) and lipid / creatine ( cr ) ratios were used as apoptotic markers . gross morphologic changes at 2 weeks were used to evaluate the effects of gh . the lipid / naa ratio was lower in the icv and icv / ip groups than in the control , and the lipid / cr ratio was lower in the icv group than in the control . the number of tunel positive cells was decreased in the icv and icv / ip groups , and the degree of morphologic change indicative of brain injury was lower in the icv group and somewhat lower in the icv / ip group . the degree of morphologic change correlated with the lipid / naa and lipid / cr ratios . these findings suggest that gh exerts neuroprotective effects in cerebral hypoxic - ischemic injury .

myofascial pain syndrome ( mps ) can be caused by frequent muscle or fascial stiffness as a result of prolonged tension and muscle fatigue due to repetitive stress of muscles or overuse of particular muscles1 , 2 . mps activates trigger points in 54% of women and 45% of men , and is regarded as the most common cause of pain in the musculoskeletal system3 . in particular , the upper trapezius requires head movement according to the direction of view , but it can be vulnerable to damage when the hands and arms are used repetitively in work that requires precise control4,5,6 . once myofascial trigger points are activated , changes in the structural characteristics and contraction function of the muscle will occur . the most distinctive changes are taut bands , tender nodules , referred pain , local twitch response , muscle weakness , and restricted range of motion3 . to evaluate the contraction function of skeletal muscle , various methods can be used . among them , surface electromyography ( emg ) analyzes functional changes in muscle by measuring quantitative changes in motor unit action potential that is activated by muscle contraction7 , 8 . surface emg is widely used in kinetic analysis to diagnose normal or abnormal function in muscles and nerves according to amplitude and frequency9 . it has several advantages , as it is noninvasive and convenient , and can perform measurement even during dynamic motion10 , 11 . although a number of clinical reports on the pathologic mechanisms or clinical diagnosis and treatment of mps can be found , few studies have been conducted on the changes in contraction function in myofascial trigger point areas or on the electrophysiological characteristics and neuromuscular physiological information . this study aimed to compare the electrophysiological characteristics of normal muscles versus muscles with latent or active myofascial trigger points , and to identify their neuromuscular physiological characteristics , in order to provide a quantitative evaluation of mps and clinical foundational data for its diagnosis . this study was approved by the research agency , and all participants provided written informed consent . subjects were selected among those with no known neurologic disease , no regular exercise habit , and no drug use that could affect the experimental result . subjects in the active myofascial trigger point group were those diagnosed with mps by a physician and whose clinical characteristics were pain in the upper trapezius during rest , taut bands , tender nodules , referred pain , local twitch response , and muscle weakness . subjects in the latent myofascial trigger point group were those not diagnosed with mps but who felt tenderness when the upper trapezius was stimulated . mean age , height , and weight , respectively , were 23.27 4.21 years , 172.32 8.44 cm , and 64.14 6.54 kg in the active trigger point group ; 23.74 6.47 years , 174.76 6.22 cm , and 66.74 5.74 kg in the latent trigger point group ; and 24.17 3.14 years , 171.21 9.64 cm , and 63.44 7.51 kg in the control group . to measure maximum voluntary isometric contraction ( mvic ) , the shoulders and heads of the subjects were firmly fixed and pulled as the dynamometer as maximum power , thereby measuring mvic against the upper trapezius . endurance time was measured from the start of mvic to a 50% reduction in contraction force . median frequency ( mdf ) was measured by using emg at the muscle belly of the upper trapezius during mvic . muscle fatigue index was calculated by obtaining the mdf at a section where the mvic force was reduced from 100% to 50% , and remove the 50% mdf from 100% mdf was divided into 100% mdf . the sampling rate for the surface emg signal was set at 1,000 hz , and the frequency band filter was set at 20 to 450 hz , using one channel . storage and analysis of the emg signals was performed using acqknowledge 3.8.1 ( biopac systems , goleta , ca , usa ) . the average of three measurements was calculated and used in the analyses ; 10 minutes of rest was given between measurements to prevent muscle fatigue . statistical analyses were performed using spss version 18.0 for windows ( spss , inc . one - way analysis of variance was conducted to examine differences among groups with respect to the measured items , while the tukey test was conducted for post hoc analysis . there were no significant differences in mvic or endurance time among the three groups . however , mdf was significantly different among all three groups ( p < 0.05 ) ( table 1table 1.comparison of mvic , et , mdf , and fi among the three groupsgroup a ( n = 30)group b ( n = 30)group c ( n = 30)post hocmvic , kg19.811.5 * 22.19.423.510.2/et , s48.59.749.510.251.28.8/mdf , hz102.521.488.523.184.517.4a < cmfi0.150.070.080.040.060.04a < cdata are presented as mean sd . * significant difference among the three groups ( p < 0.05 ) . group a : active myofascial trigger points ; group b : latent myofascial trigger points ; group c : control . et : endurance time ; mdf : median frequency ; mfi : muscle fatigue index ; mvic : maximal voluntary isometric contraction ) . tukey post hoc results showed that the active myofascial trigger point group was significantly different from the control group . muscle fatigue index also was significantly different among all three groups ( p < 0.05 ) ( table 1 ) . tukey post hoc results showed that the active myofascial trigger point group was significantly different from the control group . data are presented as mean sd . * significant difference among the three groups ( p < 0.05 ) . group a : active myofascial trigger points ; group b : latent myofascial trigger points ; group c : control . et : endurance time ; mdf : median frequency ; mfi : muscle fatigue index ; mvic : maximal voluntary isometric contraction this study aimed to measure mvic , endurance , mdf , and muscle fatigue index of normal muscles versus muscles with latent or active myofascial trigger points , thereby identifying the neuromuscular physiological characteristics of muscles with active myofascial trigger points and providing clinical foundational data that are applicable for the quantitative evaluation and diagnosis of mps . surface emg , which was used to evaluate the functionality of trigger points in this study , is nonintrusive and convenient ; thus , it is widely used in studies on the functional characteristics of muscle , by analyzing the electrical activity of muscle . the results of mdf according to surface emg show changes in recruitment of fast - twitch muscle fibers and conduction velocity of motor unit action potential12 . in addition , increases in mdf reflect recruitment of type ii fibers , and muscle fatigue index can be analyzed according to the relationship between type ii fibers and mdf13 , 14 . in the present experiment , mdf was significantly higher in the active trigger point muscles than in normal muscles . the above results indicate that as trigger points were activated , recruitment of motor unit action potential of type ii fibers also increased . in addition , muscle fatigue index also increased due to the increased distribution of type ii fibers . however , mvic was lower in active trigger point muscles than in normal muscles . although a high correlation between muscle strength and mdf has been reported15 , 16 , the same correlation was not found in this study . this result was due to increased recruitment of motor unit action potential of type ii fibers in active trigger point muscles , which more easily induced muscle fatigue as a physiological phenomenon , thereby reducing muscle strength . in other words , the greater the fatigue , the weaker the muscle strength17 ; endurance also was decreased due to muscle weakening . therefore , this study s results will be effective in complementing the physical therapy diagnosis of mps which , until now , has focused on physical examination only by understanding not only the usefulness of electrophysiological analysis in mps diagnosis , but also the neuromuscular physiological characteristics of active trigger point muscles .

[ purpose ] this study compared the differences in electrophysiological characteristics of normal muscles versus muscles with latent or active myofascial trigger points , and identified the neuromuscular physiological characteristics of muscles with active myofascial trigger points , thereby providing a quantitative evaluation of myofascial pain syndrome and clinical foundational data for its diagnosis . [ subjects ] ninety adults in their 20s participated in this study . subjects were equally divided into three groups : the active myofascial trigger point group , the latent myofascial trigger point group , and the control group . [ methods ] maximum voluntary isometric contraction ( mvic ) , endurance , median frequency ( mdf ) , and muscle fatigue index were measured in all subjects . [ results ] no significant differences in mvic or endurance were revealed among the three groups . however , the active trigger point group had significantly different mdf and muscle fatigue index compared with the control group . [ conclusion ] given that muscles with active myofascial trigger points had an increased mdf and suffered muscle fatigue more easily , increased recruitment of motor unit action potential of type ii fibers was evident . therefore , electrophysiological analysis of these myofascial trigger points can be applied to evaluate the effect of physical therapy and provide a quantitative diagnosis of myofascial pain syndrome .

a 3-year - old girl was admitted to our hospital with unexplained fever and paroxysmal abdominal pain that had persisted for three days . her body temperature at the time of admission was 39.5. physical examination showed abdominal tension , though there was no obvious tenderness and rebound tenderness . the blood platelets count ( 24010/l ) was normal , and the c - reactive protein ( > 1.6 mg / dl ) , the white blood cell count ( 13.910/l ) and eosinophil count ( 4% , 0.5810/l ) were elevated , while hepatic glutamic oxalacetic transaminase ( 24 u / l ) and glutamate pyruvate transaminase ( 21 u / l ) were normal . however , the level of cholinesterase ( 2,395 the abdominal non - contrast ct obtained on the fifth hospital day revealed diffusely hypodense nodular lesions in the hepatic parenchyma with marked hepatomegaly , retroperitoneal lymphadenopathy and ascites in the pelvic cavity . eight days after admission , an abdominal mri revealed marked hepatomegaly with diffuse high intensity of the homogeneous nodular lesions ( 2 - 4 mm diameter ) throughout the hepatic parenchyma and retroperitoneal lymphadenopathy . in addition , disseminated miliary nodules of high signal intensity were located on the peritoneum on the t2-weighted images , as well as a group of diffuse , low intensity nodular lesions were seen throughout the hepatic parenchyma on the axial t1-weighted images ( fig . , a chest ct showed multiple small hyperdense nodules ( 2 - 4 mm in diameter ) in both lungs ( fig . after the small intestinal wall and peritoneal biopsy , histological examination revealed parenchymal tubercles containing several larvae of pentastomids and a large amount of inflammatory cell infiltration surrounding them ( fig . at this point , it was revealed by the patient 's parents that the patient had consumed snake meat several months prior to her admission . following the diagnosis , she was administered praziquantel ( 0.5 g , tid , per day ) and albendazole ( 0.75 g , bid , per day ) in order to expel the intestinal worms . the patient 's temperature began to slowly decrease two days after the start of the therapy , and after 10 days of the therapy the fever had disappeared and the abdominal pain was diminished . on examination , pentastomid worms were observed in a stool ( fig . the patient made an uneventful recovery after a half a month of therapy and she was discharged from our hospital . human pentastomiasis is divided into two types : the more common visceral pentastomiasis ( which is the main portion of the cases of pentastomiasis ) and nasopharyngeal pentastomiasis ( 1 ) . visceral pentastomiasis is an unusual parasitic zoonosis that is caused by the larval stages ( nymphs ) of several species of pentastomes ( ' tongue worms ' ) , and these are parasites that form a unique phylum with characteristics of both arthropods and annelids ( 2 ) . at present , there are ten known pathogenic species of pentastomid parasites and six known kinds of human pentastomiasis . the latter include armilliferosis , linguatulosis , porocephalosis , raillietiellosis , leiperiosis and sebekiosis ( 3 ) . the species that affect humans belong to the families linguatulidae , armilliferidae and porocephalidae , and all of which have different geographic distributions . more than 90% of the human cases are caused by the nymphs of only two species , l. serrata and a. armillatus ( 4 ) . the infection develops in humans when parasite ova are ingested from the respiratory secretions or feces from the final hosts ( dogs and other carnivores for linguatula , several species of large snakes for armillifer and porocephalus ) . in the digestive tract of the human host , the minute four - legged primary larvae hatch and invade the viscera of the digestive tract in the human host ( 5 , 6 ) . the nymphs involve many organs , including the liver , esophagus , stomach , duodenum , jejunum , ileum , colon , appendix , rectum , mesentery , gallbladder , lung , pleura , broad ligament , pancreas , omentum , bladder , adrenal gland , heart ( pericardium ) , lymph nodes , skin , epididymis , eye , brain , inguinal hernia sac , etc ; however , the liver is the most common site ( 7 ) . visceral pentastomiasis causes the majority of asymptomatic infections in humans , but a small number of cases are fatal , and the severity of symptoms is related to the organs that are infected and the degree of infection by the nymph ( 8) . the patients with a heavy infestation showed chronic fever , ascites , abdominal pain , diarrhea , mild anemia , hepatosplenomegaly , eosinophilia in the bone marrow and blood , and multiple polyps in the whole colon . the pathologic features of the liver biopsy are degeneration and necrosis of hepatocytes , along with obvious infiltration of eosinophils . as was demonstrated in this study , praziquantel or mebendazole is a medical treatment for this disease ( 9 ) . only a few cases of pentastomiasis infections have been reported in humans around the world , and there is limited literature about the imaging features of pentastomiasis infection . we report here a case where the patient had a history of eating snake meat several months prior to her hospital admission , and her main symptoms were a high fever and abdominal pain . an abdominal mri demonstrated diffuse nodules throughout the liver , an enlarged liver , a little ascites , diffuse retroperitoneal lymphadenopathy and disseminated miliary nodules of high signal intensity on the peritoneum . the laparoscopy revealed that the liver was diffusely enlarged , as well as there were diffusely scattered micro - cyst nodules of varying sizes on the surface of the liver and the peritoneum . microscopically , the small intestinal wall and peritoneal biopsy showed several larvae of pentastomids in the cyst nodules . the parasite worms were surrounded by a group of macrophages , inflammatory cells and fibrosis . when liver enlargement and diffuse hepatic hypodense nodular lesion is seen on a t1-weighted image , it is necessary to differentiate pentastomiasis hepatic involvement from other diseases that present with diffuse liver enlargement , high fever and abdominal pain ( 10 , 11 ) such as diffuse liver metastasis , diffuse hepatocellular carcinoma , lymphoma and diffuse granulomatosis ( 12 ) . biopsy or autopsy is necessary to determine the cause of the symptoms . in this report we studied the patient with magnetic resonance imaging . due to the spatial resolution of the current t2-weighted mr imagers , the detection of these microcysts and larvae within the microcysts was well accomplished in tandem with the clinical features and epidemiology , which aided in making the differential diagnosis ( 13 ) . in summary , we have presented a rare case of pentastomiasis infection that involved many organs , including the liver , small intestine , peritoneum , lung and retroperitoneal lymph nodes . the patient 's symptoms consisted of high fever , abdominal pain , abdominal tension and anemia . the imaging features of the abdominal mri revealed marked hepatomegaly with diffuse homogeneous nodular lesions throughout the hepatic parenchyma , diffuse retroperitoneal lymphadenopathy and disseminated miliary nodules on the peritoneum . the chest ct showed scattered small nodules in both lungs . when liver enlargement and hepatic disseminated miliary nodules are identified , it is necessary to differentiate pentastomiasis hepatic involvement from other diseases . pentastomiasis infections should be considered in the diagnoses of patients with a history of such abdominal symptoms and who have consumed snake meat , though the definite diagnosis depends on the pathology report .

we report here a case of pentastomiasis infection in a 3-year - old girl who had high fever , abdominal pain , abdominal tension and anemia . ultrasound scanning of the abdomen revealed disseminated hyperechoic nodules in the liver and a small amount of ascites . abdominal mri showed marked hepatomegaly with disseminated miliary nodules of high signal intensity throughout the hepatic parenchyma on t2-weighted images ; retroperitoneal lymphadenopathy and disseminated miliary nodules on the peritoneum were also noted . chest ct showed scattered small hyperdense nodules on both sides of the lungs . the laparoscopy demonstrated diffuse white nodules on the liver surface and the peritoneum . after the small intestinal wall and peritoneal biopsy , histological examination revealed parenchymal tubercles containing several larvae of pentastomids and a large amount of inflammatory cell infiltration around them . the pathological diagnosis was parasitic granuloma from pentastomiasis infection .

the so - called feminization of medicine is likely to have important implications in patient - physician relationship , local and societal delivery of care , and the medical profession itself . when specialty choices are examined , women are proportionately over represented in the primary care fields . they are underrepresented in most surgical fields , with the exception of obstetrics and gynecology , where women now comprise the majority of practicing physicians . why anesthesiology is not a more popular specialty choice among women is difficult to determine . lifestyle issues are often cited as an important consideration in the selection of a specialty for residency training , and anesthesiology is often included in lists of specialties said to be associated with favorable lifestyles . however , data indicate that anesthesiologists rank among the busiest specialists , with an average of 61 h / week committed to professional activities and an average of 59 h / week dedicated to patient care . income expectations and personality factors gender discrimination and sexual harassment may be important factors . some factors that may influence women on the choice of anesthesiology as a career is sparse or even no contact with anesthesiologists during the preclerkship medical school curriculum . careers in medicine historically demanded a selfless emphasis on caring for one 's patients , sometimes at the expense of one 's marriage , children , and personal life . as women entering the medical field is increasing in numbers , the conflicts between career and family became more prominent . thus , many challenges must confront woman physicians in balancing their multiple roles as physician , mother , and spouse . women are underrepresented in anesthesia and with an effort to equalize the numbers of men and women within the medical school , one might expect the number of women entering anesthesiology to increase concomitantly mansoura is the capital city of dakahlia which is one of the most important egyptian provinces and is famous with many great medical centers . the total number of anesthesiologists in mansoura city at the time of our study was 224 and the proportion of women among them was 25.9% , and the proportion of women among the doctors in general was nearly 51% . the aim of our work was to evaluate the implications of anesthesiology as a profession on personal and family life of women anesthesiologists . we also wished to elicit the differences between academic and nonacademic women anesthesiologists with respect to these effects and the effect of women anesthesiologists on the profession . a descriptive comparative study was conducted by surveying women anesthesiologists in mansoura , egypt during the period between january and may 2013 . the survey involved woman anesthesiologists affiliated with mansoura university hospital ( academic anesthesiologists ) and others working in hospitals related to ministry of health at mansoura ( nonacademic anesthesiologists ) . during the study period , a convenience sample of 51 women were recruited , and 46 agreed to participate in the study with a nearly 90% response rate . nonparticipation was due to lack of interest in the study , absence during the study period and incomplete questionnaires . approval to conduct our study was granted by our institution 's review board and from the management of the involved hospitals . the researchers introduced themselves to participants and informed them about the aims of the study . the content validity was determined by consulting a panel of experts . to ensure reliability , the questionnaire was pretested through a pilot study on a group of doctors who were not included in the final analysis and cronbach alpha coefficient of internal consistency was 0.87 . the constructed survey instrument was finally edited in english version and consisted of four sections : the first section elicited some personal information of the respondents ( sociodemographic characteristics ) . the second part consists of 6 items exploring the potential reflection of anesthesiology on family life of women ( implications on family life ) , the third contains 12 items asking about the possible reflection of anesthesiology on woman personality ( implications on personality and personal life ) and the final section elicits 11 items reflecting the perceived impact of women on anesthesiology as a profession ( job implications ) . the responses were coded using the variables in the responses to determine the coding guide . to facilitate quantification and analysis of data , respondents had options of strongly agree scored as five ; agree scored as four ; undecided scored as three ; disagree scored as two and strongly disagree scored as one on a five point likert scale . data were analyzed using spss program ( statistical package for social sciences ) version 13 windows ( ibm , inc . , chicago , illinois , usa ) . descriptive statistics ( i.e. , mean , frequency , percentage , and standard deviation ) and inferential statistics ( i.e. , chi - square test , fisher 's exact , and t - test ) were used . for comparison in order to evaluate the agreement among academic and nonacademic , items with multiple response levels were collapsed into binomial variables of agree and disagree . a score of three ( undecided ) on likert scale was considered as disagree . the age of the anesthesiologists with an academic career was not significantly different from those without academic career ( 33.1 vs. 31.8 years ) . nearly , one - fifth of our woman anesthesiologists were single ( 21.7% ) . the number of children in the two groups was not significantly different [ table 1 ] . sociodemographic characteristics of the studied woman anesthesiologists the implications on family life were significantly much more afflicted by an academic career compared to nonacademic career in the form of : delayed marriage ( 87% vs. 44% ) , delayed first baby ( 78% vs. 30% ) , child rearing ( 96% vs. 30% ) , maternity rights ( 91% vs. 26% ) and poor fulfillment of family demand ( 83% vs. 44% ) respectively [ table 2 ] . implications on family life among the studied woman anesthesiologists the majority of studied women reported personality changes . the positive implications on personality and personal life of anesthesiology as independence , empowerment and positive social interaction were not significantly different between the two groups . furthermore , most of anesthesiology drawbacks on personality ( aggression , nervousness , depression , stressful attitude , and drug experimentation ) were significantly more cited by women with academic careers . however , the financial limitation was significantly more complained by the noncareer anesthesiologists ( p = 0.032 ) . the other negative effects observed in a large number of women in both groups were affection of feminine attitude , sense of discrimination , and poor social acceptance [ table 3 ] . implications on personality and personal life of among the studied woman anesthesiologists anesthesiology was perceived privileged due to women 's employment in the field by soothing the work environment , more emotional reaction to patient complaints and increased perfectionism ( perceived by about 91% , 89% and 76% respectively ) . on the other hand , findings revealed that increased conflict with the surgeons ( 98% ) , poor surgeon acceptance ( 87% ) , poor patient acceptance and recurrent change of work schedule ( 80% each ) were the most common items perceived by women as negative implications on the field of anesthesiology as a result of women 's employment . difficult to manage night shifts was more significantly mentioned by academic career anesthesiologists compared to the others ( p < 0.001 ) [ table 4 ] . we can not deny that rearing and caring at home are the primary role for any woman whatever her level of education , especially in arab countries . biologically , the most appropriate time for the woman to have children coincides with the period when career demands are most intense , making the balancing of career and family demands particularly difficult during this period . this critical period usually overlaps with the medical study and training , particularly with financial limitations and high work demands . the implications on family life were significantly much more expressed by women anesthesiologists with academic career , and more than 50% of both women groups perceived that their family life was exposed to negative implications . although only 9% of women with delayed marriage blamed anesthesiology work as a predisposing factor for their pregnancy problems , however this needs consideration . these findings were concomitant with a previous study which reported that most doctors recommended postponing pregnancy until after the completion of training . in contrast , another survey study conducted on pediatricians found that they gave birth to their first child during residency . our results could be explained by the fact that most of the academic career anesthesiologists have an intense schedule with teaching , research and regular or duties . the present findings were also coinciding with the study that found woman resident physicians experienced more pre - eclampsia and preterm labor than other women . unfortunately , our results emphasized that the interest of female doctors in academic medicine is reduced . this could be explained by many concerns that may range from balancing their multiple roles to the worry of physician - mother on the child 's safety while she is away from home . furthermore , the view of some colleagues to a woman doctor 's pregnancy and family commitments as evidence of a diminished dedication to medicine and career can be a contributory factor . , support some of the previous concerns by concluding that decreased academic progress was related to childbearing . the present work showed that the maternity rights were affected for more than 58% of our women anesthesiologists , and this was significantly higher among career group . in egypt , even with the presence of a law that allows 3 months of full paid maternity leave , some women - doctors due to fear of demotion in their career , are pushed to sacrifice their maternity rights . it was surprising to know that many american physicians do not take the entire 6 weeks of maternity leave to which they are entitled . this action was partly explained by the concern of their colleagues attitudes toward their staying home with their infant or by their feeling guilty about being away from their patients and work . regarding implications on personality , the present study established that our ladies were privileged by their choosing anesthesia as a career , where most of them felt independent and empowered besides developing positive social interaction . previous studies support our finding by demonstrating that woman had greater adaptability to clinical situations , better vigilance , perceptual speed and associative memory that might give them an edge over males to recall more details . university staff in general has a better income compared to nonacademic which is a privilege granted by higher education committee in egypt to encourage the graduated physician to be a university staff . this can partly explain the significant financial problems encountered by nonacademic anesthesiologists in comparison to those in academic careers . the sense of discrimination due to gender is considered as a major factor that has a great negative impact on our woman anesthesiologists and represents 78% in this study . gardner et al . , have reported that although woman anesthesiologists in south africa have higher career satisfaction ; gender discrimination and harassment were found to be more likely among women . in contrast , a canadian study revealed an absence of gender discrimination . regarding the implications on profession the current study showed that around 98% of woman anesthesiologists complained from increased conflict with surgeons and this may give an explanation , not only for the high stressful attitude of two - third of our sample but also for the women perversion from anesthesia specialty . regardless of gender bias , conflicts with surgeons could be explained by the facts that surgeons have often been perceived by other physicians with negative features such as aggressive , dominating , cold , impatient , and selfish . these conflicts were commonly attributed to poor communication , and limited contact between males and females in our culture , especially in a society like mansoura . in this study , alternative change of work schedule , delayed response to on call and difficult to manage night shifts were reported by 80% , 54% and 52% of the studied groups , respectively . one study concluded that 85% of women were more prone to modify their job tasks or alter their career for the benefit of their families and children compared to only 35% of male physicians . job stress and its implications were so clear in our study , and this was supported by a previous egyptian study which observed more fatigue and psychological distress among anesthesiologists . kinzl et al . , have reported that woman anesthesiologists showed higher concentration and limited possibilities to control work environments as compared to their male colleagues . it is not surprising , that our findings revealed high significant domination of an academic career over noncareer woman anesthesiologists regarding most of the anesthesiology drawbacks reflected on women , which were discussed in this study . hence , we can use our findings to explain the results of another study , which reported that the percentage of women faculty members in all specialties who reach the designation of full professor insulates well - behind that of men , and this is definitely true for anesthesiology , the findings of our study are discouraging as literature has revealed that the healthy development of anesthesiology requires a vital increase in the number of women affiliate to academic anesthesiology . gender disparities in academic promotion leading to less likelihood of external grant funding and fewer publications than men could be a contributory factor . furthermore , the junior partners are assigned to more work pressures , less desirable pay and they are frequently scheduled for later hours , on weekends and during holidays . one of the major limitation is the cross - sectional design of the study that was based on self - reported questionnaires provided by women . furthermore , the small sample size may be another limitation to detect the accurate gender associations . also the exclusion of male 's opinions from the survey ; and the sociocultural factors of this study that make the number of woman anesthesiologists limited may interfere with the generalizability of our results to anesthesiologists in other societies . however , as we try to recruit most women anesthesiologists working in mansoura hospitals , the results have external validity of women working as anesthesiologists in mansoura , egypt . the study delineated the implications on the personality and family life of women who selected academic anesthesiology as a specialty and career . furthermore , anesthesiology as a profession is significantly benefited by women joining in the academic ranks .

background and aims : we hypothesized that feminization of medicine has an impact on the choice of anesthesiology as a specialty . however anesthesiology is still not a more popular choice among women in medicine . we aimed to evaluate the implications of anesthesiology as a profession on personal and family life of women anesthesiologists ; the differences between academic and nonacademic women anesthesiologists with respect to these effects and the effect of women anesthesiologists on the profession.materials and methods : this study surveyed a total of 46 woman anesthesiologists ; both academic and nonacademic institutions in our country between january and may 2013 . a convenient sampling method was used . a constructed self - reported questionnaire was developed to explore the 29 items of woman - anesthesiology - related implications ( family , personal , and job ) , in the form of close - ended questions.results:negative implications of anesthesiology on personal and family life of woman were common . with the exception of financial limitation , academic group was significantly more affected . increased surgeons conflict ( 98% ) , poor surgeon acceptance ( 87% ) , poor patient acceptance and recurrent change of work schedule ( 80% each ) were the most common perceived negative impacts of women in the field of anesthesiology . some positive implications were perceived as independent , positive social interaction , empowerment , soothing work environment , emotional reaction to patient complaint , and increased perfectionism ( nearly 94% , 98% , 87% , 91% , 89% , and 76% , respectively).conclusion : serious implications exist for the personality and family life of women who chose anesthesiology as a specialty and career , and most of these implications were significantly more prevalent among women with an academic career . furthermore , anesthesiology as a profession was significantly affected by women joining the department .

obesity is a major public health problem with over two - thirds of americans overweight and greater than one - third obese . while new data have suggested a leveling off of the prevalence of childhood obesity , 12.4% of kindergarteners are obese and overweight five - year olds are four times as likely as normal weight five - year olds to become obese [ 2 , 3 ] . it has been well documented that obesity leads to cardiac , metabolic , and other systemic health derangements . it has also been shown that these unfavorable changes may start as early as three years of age . well - child checks have long been recognized as opportunities to foster healthy growth and development . these regular visits , which are performed with a health care provider , traditionally occur during the newborn period , at one , two , four , six , nine , twelve , fifteen , eighteen , and twenty - four months and every year thereafter . the goal is to provide continuity of care and allow for anticipatory guidance to be given . recognizing that diet and nutrition are linked with obesity , it is important to address these topics . however , guidelines on nutrition counseling remain vague , and visit time is constrained by a multitude of other encouraged and essential components . yet studies show that when it comes to counseling during well - child checks , less is more and focusing on fewer topics is more effective . however , these are notably imprecise with regard to early and targeted interventions to prevent and treat obesity in pediatric populations . regarding nutrition counseling during well - child care within the first year of life , the american academy of pediatrics ( aap ) bright future guidelines contains goals for temporal introduction of foods rather than composition of diet . even at the one - year visit , surprisingly , there is no mention of nutrition guidance at the fifteen- or eighteen - month well - child checks and it is not until the two - year visit that the subject of obesity is first addressed in the guidelines . meanwhile , research shows that a rapid transition occurs between the one- and two - year well - child checks , when diet habits seemingly shift to favor fast foods , with the french fry as the number one consumed vegetable consumed by two - year olds . the world health organization ( who ) strongly advocates for exclusive breastfeeding in the first six months of life and offers recommendations on complimentary feeding habits . these include continuing on - demand breastfeeding until two years of age and starting at 6 months of age , gradually increasing the number of feedings , consistency , and variety of other foods . for a nonbreastfed child , who further recommends providing four to five meals per day , with one or two healthy snacks aimed to meet the child 's nutritional needs . these who guidelines are appropriately aimed toward a global audience , with an emphasis on preventing malnutrition and ensuring adequate growth . current literature on prevention and treatment for obesity has overwhelmingly focused on adolescent and school aged populations . the aap recommends screening for obesity starting at two years , while united states preventive services task force ( uspstf ) suggests waiting until age six [ 7 , 10 ] . these recommendations bypass a likely window of opportunity for primary prevention prior to the second year of life , and in the case of the uspstf prior to six years . given these vague universal recommendations , we hypothesize that significant variability exists in clinical practice . it has been demonstrated that there is variability among pediatric primary care practices regarding obesity counseling , attitudes , and perceptions . some prior studies have focused on family medicine primary care providers ' ( pcps ) beliefs and practices pertaining to childhood obesity . however , many of these studies have placed emphasis more specifically on evaluation of treatment modalities , perception of obesity as a disease , and physician training [ 1318 ] . further , much of the previous work on childhood obesity appears directed beyond the age of five years , with few interventions studied between two and six years of age . this study builds upon the existing literature by offering a family medicine perspective and focusing specifically on perceived barriers and anticipatory guidance discussed at the early well - child checks particularly those prior to the second year of life . this is further warranted as much of the current literature occurred prior to the updated 2008 aap bright futures guidelines [ 7 , 20 ] . given the recent emphasis on primary care and the likely influx of pediatric patients via the accountable care act , family physicians will continue to provide a substantial amount of care for pediatric populations . the goal of the present study is to assess the perception of family medicine pcps in a university based family medicine network surrounding the barriers of preventing and treating obesity in the young child and to analyze pcps reported behaviors at well - child checks . in addition , the present study allows for a comparison of attitudes and practice between family medicine pcps and pediatric pcps who participated in an earlier study using a similar survey . the sample included all family medicine physicians , physician assistants ( pas ) , and nurse practitioners ( nps ) at eleven family medicine duke primary care sites in duke university health systems . the survey used in the present study was developed based on a similar study performed in pediatric practices , current epidemiological literature , and recommendations from the american academy of pediatrics ( aap ) and the united states preventive services task force ( uspstf ) [ 6 , 10 , 11 ] . previous authors were contacted for permission to utilize a similar survey tool . the survey contained three independent sections , each with a brief introduction . the sections were as follows : ( i ) perceived barriers in treating obesity , ( ii ) current pcp practices at well - child checks , and ( iii ) demographic information . introductory phone calls with follow - up emails were sent in january 2012 , prior to the in - person survey administration . an agreed upon morning or lunch hour time was arranged to include a ten- minute presentation explaining the goals of the project , with subsequent survey administration to all pcps who attended the meetings . an agreement was made to present the study findings to the participating sites at the conclusion of the study . data collection was performed during january and february 2012 and the data were analyzed in march 2012 . demographic data are presented as percentages / proportions . for questions related to perceived barriers , we calculated the percentage of respondents who indicated how important each issue was on a likert scale 15 ( not important , slightly important , moderately important , very important , and critically important ) , as well as the combined percentage of those who responded either very important ( 4 ) or critically important ( 5 ) . for pcp behaviors assessing anticipatory guidance , multiple responses were accepted ( i.e. , mark all well - child checks that apply ) . this study was reviewed by the research advisory board of the primary care research committee and the institutional review board of duke university and was found to be an exempt study ( pro00034169 ) . surveys were completed by 56 of the 78 family medicine pcps ( 41 family medicine physicians , 8 physician assistants , and 7 nurse practitioners ) at the 11 participating clinics , for a 72% response rate . approximately 1/3 ( 35% ) were 4049 years old , 1/3 ( 33% ) were 3039 , 27% were 50 + years old , and 5% were twenty through twenty - nine years old . the majority of pcps were medical doctors or doctors of osteopathic medicine ( md / do ) ( 73% ) , with physician assistants and nurse practitioners making up 14% and 13% , respectively . the average reported body mass index ( bmi ) of respondents was 24.71 kg / m ( 7 of the 54 respondents did not complete the bmi measures ) . approximately 1/3 ( 33% ) of the pcps were classified as overweight and 0.6% were in the obese category . reported bmi closely mirrored pcps perception of their weight , as 30% indicated they were overweight and 2% believed they were obese . pcps were asked to rate the relative importance of specific barriers to preventing or treating overweight or obese children . these questions related to children of all ages and focused on many factors including the child , parents , family unit , influence of society , and pcp factors . the five barriers that were most often rated as either very important or critically important were as follows : ( i ) families do not get enough exercise ( 93% ) ; ( ii ) families often have fast food meals ( 86% ) ; ( iii ) parent is not motivated to change diet or lifestyle ( 81% ) ; ( iv ) families watch too much tv ( 79% ) ; and ( v ) child is not motivated to change diet or lifestyle ( 75% ) ( table 1 ) . the following barriers were rated as very important or critically important by 6072% of pcps : parent is unaware that child is overweight , parents are overweight so they are not concerned that child is overweight , families are too busy to eat home cooked meals , healthy foods are too expensive , tv advertisements promote unhealthy foods , pcps have limited time to discuss nutrition , and pcps are frustrated with the low success rate of treating overweight children . barriers that pcps were less likely to rate as very important or critically important were as follows : overweight child does not act sick , overweight child is a good eater , parents do not have time to shop for healthier foods , families are too busy to eat meals together , healthy lifestyle habits are too complicated to follow , published reports about diet and nutrition are often confusing , school lunches promote unhealthy eating habits , pcps ' time constraints , lack of training to treat overweight children , compensation for obesity treatment , access to nutritionists , and pcps ' weight status or body mass index ( table 1 ) . pcps were asked to mark all of the well - child checks in which they discuss a variety of health topics with patients . analyzing the barriers perceived by pcps as most important to preventing and treating obesity revealed trends regarding fast food consumption and physical inactivity . as shown in table 2 , even by their own report , most pcps did not discuss fast foods at or prior to the twelve - month visit . at the eighteen - month visit fast foods are discussed by 32% of pcps . meanwhile , the two - year visit is the first well - child visit at which the majority of pcps ( 68% ) discuss fast food . while the majority of pcps ultimately discuss this topic , the discussion is not undertaken by a majority of pcps at a specific encounter until the two- through five - year visits . fruit and vegetable discussion increases in frequency as the child ages , with a peak of 63% of pcps discussing this at the twelve - month visit , before dropping to 51% at the eighteen - month visit . between 22% and 26% of pcps discuss having 3 meals per day by the twelve - month visit , whereas 65% are discussing meal frequency at the two- through five - year visits . physical inactivity / exercise was another area of concern with 93% of pcps recognizing this as a contribution to obesity , yet this topic was discussed by at most 23% of pcps at and/or before the twelve - month visit . by the eighteen - month visit , at most 48% of pcps had ever discussed the topic with their patients or families . the two- through five - year visits are the first time at which the majority of pcps ( 68% ) discuss this topic . the percent of well - child visits where screen time is discussed closely mirrors physical activity / exercise , and a similar trend is seen regarding the discussion of fast foods ( table 2 ) . a major finding of the current study involved the relative importance pcps ascribe to perceived barriers in treating obesity . our results reproduced those of a previous study utilizing a similar survey but performed within a pediatric setting . our results confirm that family medicine pcps share the same top six concerns when dealing with perceptions surrounding obesity . these concerns center around physical inactivity , fast food consumption , and motivation to change . the barriers identified were in areas that were inconsistently addressed in practice , specifically prior to the two - year well - child check . despite the apparent lack of congruence between the identified barriers and the actions by pcps in clinical practice , furthermore , due to the self - reported nature , pcps may overestimate how often they address certain issues . a second major finding of the current study was the inconsistency pcps demonstrated concerning when discussion took place for physical activity , fruit and vegetable selection , screen time , juice , and other beverage choices . this study suggests that primary prevention interventions targeting obesity in practice are either misplaced or missed altogether in some cases , which is consistent with other recent studies demonstrating missed opportunity for primary prevention of obesity [ 2124 ] . as this study is representative of an academic practice population with close geographic proximity , further study on a larger scale and in other practice populations the aap recommendations make no mention of fruits and vegetables until the five- and six - year well - child visits . in another case , the aap guidelines suggest the discussion of having 3 meals per day at the nine- and twelve - month well - child checks . however , in this study , only 26% of pcps have this discussion specifically at or before the twelve - month well - child visit . interestingly 65% of pcps have this discussion at the two- through five - year well - child visits despite lack of specific recommendation to do so . pcps seem to be aware of existing guidelines but , in this example , delay the delivery . other than this specific recommendation at the five - year well - child visit , all of the earlier well - child visit nutrition and diet guidelines are relatively nonspecific regarding diet composition . this likely contributes toward the variability observed in this study , as many pcps chose to have discussions , such as fruits and vegetables , at differing well - child visits . therefore , we believe that it would be advantageous to the goals of obesity prevention and treatment to have early and targeted interventions that precede adoption of adverse lifestyle choices [ 2527 ] . although it is beyond the scope of this paper , innovative approaches in obesity treatment have been identified over the past decade [ 2830 ] . comparing this study of family medicine pcps with a similar study done with pediatric providers revealed some similarities in practice behavior . pcps behavior in both studies reflected the general aap anticipatory guidance guidelines pertaining to obesity [ 7 , 11 ] . however , the anticipatory guidance discussions were not consistently performed at specific visits in either study , with greater variability observed in this family practice study ( see table 2 ) . different methodologies were used in data collection , as the study for family medicine pcps was measured using a cumulative approach , whereas the pediatric study was measured at point of first intervention . in comparing these studies , we chose to use the most conservative estimates by summating the percentages of anticipatory guidance being discussed . for example , a single pcp may have indicated that they discussed fast food at both the four- and six - month visits . our study would count both of these as unique interventions being done by different pcps . consequently , these findings may offer a realistic representation or , otherwise , an overestimation of how often topics were discussed at well - child checks . the present study identifies discrepancies in pcps adherence to counseling guidelines for nutrition , exercise , and screen time . in regard to exercise and screen time , this study demonstrates similar increases in the percentage of pcps discussing these topics , with the majority doing so at the two- through five - year well - child checks , and a peak between the six- through eleven - year well - child checks . however , the aap guidelines suggest that a majority of pcps address each of these topics much earlier . it would be beyond the scope of this discussion to address all elements of the survey . therefore , the remainder of this discussion will focus on the results relevant to fast food counseling and nutrition and their role in obesity prevention . a surprising result from this study was that there is no clear consensus as to when pcps are having discussions about fast food . while a prior study indicated that 62% of pediatricians addressed fast foods at or prior to twelve - month well - child visits , this study shows that at most 39% of family physicians did so ( table 2 ) . these estimates suggest that there is still a large percentage of the population receiving no counseling on fast food during the entire first year of life . it is not until the two - year well - child visit that a majority of pcps discuss fast food . meanwhile , previous research has shown that there is a profound shift in dietary habits toward fast foods , such as french fries , that occurs between the one- and two - year well - child checks . to address fast food consumption , pcps could consider integrating a universal french fry discussion regularly at the twelve - month well - child care visit . given the recognition that food transitioning towards fast foods such as french fries occurs within the subsequent window from twelve months to twenty - four months , it might be beneficial to offer a specific intervention at this visit . furthermore , prior research has shown that a dedicated intervention targeting two - year olds and their families can significantly reduce bmi . the french fry discussion could take the form of a purposeful talk with family members about the importance of avoiding fast foods , fried foods , and sweetened beverages . for example , using motivational interviewing techniques has been shown to be effective in changing behavior relating to obesity , and this approach may address such a complex behavior [ 32 , 33 ] . this visit could additionally include handouts or printouts about alternative foods and snacks that are affordable and can be prepared quickly . rather , this should be an individualized discussion , in which family members are encouraged to voice their concerns and devise solutions that meet their unique situation . as such , we would not anticipate a discussion of this relevance requiring anything less than ten minutes of dedicated visit time . the goal of this discussion would be to leverage the patient - physician - family relationship to positively impact lifestyle choices for both the child and the family . further study is needed to examine actual obesity prevention and treatment guidelines in clinical practice and to discern the specifics , feasibility , and benefits of incorporating additional counseling into routine well - child care .

childhood obesity is a complex problem that warrants early intervention . general recommendations for obesity prevention and nutrition counseling exist . however , these are notably imprecise with regard to early and targeted interventions to prevent and treat obesity in pediatric populations . this study examines family medicine primary care providers ' ( pcps ) perceived barriers for preventing and treating pediatric obesity and their related practice behavior during well - child visits . methods . a written survey addressing perceived barriers and current practices addressing obesity at well - child visits were administered to pcps at eleven family medicine clinics in the duke university health system . results . the most common perceived barriers identified by pcps to prevention or treatment of obesity in children were families not getting enough exercise ( 93% ) and families too often having fast food meals ( 86% ) . most pcps do not discuss fast foods at or prior to the twelve - month well - child visit . the two - year visit is the first well - child visit at which a majority of pcps ( 68% ) discuss fast food . conclusion . no clear consensus exists as to when pcps should discuss fast food in early well - child checks . previous research has shown a profound shift in children 's dietary habits toward fast foods , such as french fries , that occurs between the one- and two - year well - child checks . consideration should be given to having a french fry discussion at every twelve - month well - child care visit .

diabetes is the most frequent cause of end - stage renal disease in industrialised countries [ 1 , 2 ] . clinically , diabetic nephropathy is characterized by the development of albuminuria and a subsequent decline in glomerular filtration rate . this severe complication significantly influences the risk of cardiovascular disease as well as mortality and quality of life [ 3 , 4 ] . at present the most important identified risk factors are diabetes duration , arterial blood pressure , and glycaemic regulation . as evident from an increasing incidence of affected patients , however , there is still a great need for new strategies in the treatment and prevention of diabetic nephropathy . clear evidence indicates that the pathogenesis of diabetic nephropathy is multifactorial and triggered by a complex series of pathophysiological events . the inflammatory response in diabetes is highly complex involving proinflammatory cytokines and chemokines , for example , il1 the impact of complement activation on the diabetic kidney may well , in part , be mediated through induction of cytokine response and inflammation [ 911 ] . several studies have linked diabetic late - complications to the complement system of the innate immune system [ 12 , 13 ] . the complement system plays a crucial role in recognition and clearance of infectious microbes and the system forms a link between innate and adaptive immunity . the activation of the complement system results in the release of multiple inflammatory signaling molecules . ultimately complement activation leads to the formation of pore - forming membrane attack complexes ( macs ) that are inserted in the cell membranes to mediate lyses of the cell through osmotic stress . however , in mammalian cells , it has been shown that sublytic amounts of mac can increase production of il-8 and monocyte chemoattractant protein 1 dependent on nfb nuclear translocation . furthermore , macs are shown to have a mitogenic effect and cause release of basic fibroblast growth factor and platelet - derived growth factor from endothelial cells leading to fibrosis in neighboring cells including glomerular mesangial cells [ 16 , 17 ] . the latter effects of mac may explain the link between complement and diabetic kidney damage . three activations pathways exist : the classical , the alternative , and the lectin pathway . the present paper focuses on the lectin pathway , in which at least five soluble pattern - recognition molecules are characterized that may activate the complement system , that is , mannan - binding lectin ( mbl ) , h - ficolin , l - ficolin , m - ficolin , and collectin - k1 . the three ficolins utilize a fibrinogen - like domain that binds , for example , n - acetylglucosamine , n - acetylgalactosamine , and n - acetyl - neuraminic acid , whereas mbl and cl - k1 have a carbohydrate recognition domain and through this bind specifically to patterns of monosaccharides . a very recent publication reports a close association between ficolin and diabetic nephropathy in patients with type 1 diabetes . the observational design of the study , however , limits its ability to study a cause - effect relationship . when ficolins bind they all initiate activation of associated serine proteases ( mbl associated serine proteases , masps ) , which subsequently cleave the complement factors , c2 and c4 , leading to further complement activation [ 20 , 21 ] . eventually , complement activation leads to the formation of macs causing cell lyses or induction of fibrosis . the balance between activation and inhibition of the complement cascade is tightly controlled by regulatory proteins in order to prevent damage of healthy host cells . in diabetes , inappropriate effects of the complement system may be present as glycation - induced dysfunction of the complement inhibitory mechanism and consequently overactivation of the system is indicated [ 2325 ] . it is speculated that diabetic patients are exposed to uncontrolled complement attack partly due to altered molecular patterns on the cell surfaces as a consequence of high blood glucose [ 24 , 25 ] . most significantly , an association is seen between diabetic nephropathy and the lectin pathway [ 2628 ] . we have previously demonstrated direct cause - effect relationship between presence of mbl and worsening of kidney injury in a mouse model of diabetic nephropathy [ 29 , 30 ] . we speculate that ficolins also exert detrimental effects in diabetes similar to mbl through activation of the lectin pathway as indicated in patients with type 1 diabetes . this study aimed to investigate the impact of ficolin b ( the orthologue to human m - ficolin ) on the development of diabetic nephropathy in a mouse model of type 1 diabetes . we used 11-week - old , female ficolin b knockout mice and age - matched , female c57bl/6j bomtac wild - type mice ( taconic , ry , denmark ) . the knockout ficolin b model was backcrossed more than 10 generations to a c57bl/6j bomtac genetic background ( own breeding ) . in each cage there were three to eight mice and they had free access to tap water and standard chow ( altromin number 1324 ; lage , germany ) . the environment was stable with a 12-hour light - dark cycle , temperature at 21 1c , and humidity of 55 5% . the ficolin b knockout mice and the wild - type mice were randomized into a diabetic and nondiabetic group ; thus four groups were made : ( 1 ) diabetic knockout mice ( n = 6 ) , ( 2 ) nondiabetic knockout mice ( n = 7 ) , ( 3 ) diabetic wild - type mice ( n = 11 ) , and ( 4 ) nondiabetic wild - type mice ( n = 11 ) . diabetes was induced by intraperitoneal injections of streptozotocin ( stz ) dissolved in a cold 10 mm citrate buffer ( doses of 55 mg / kg body weight , sigma aldrich , st louis , mo , usa ) on five consecutive days . the 18-week experiment was initiated when the mice were classified as diabetic ( blood glucose > 15 mm ) . animals with more than 15% sustained weight loss , signs of illness , or persistent ketonuria were excluded from the study . blood glucose was measured from tail vein by contour ( bayer diabetes care , kgs . lyngby , denmark ) . with combur test d strip ( roche diagnostics gmbh , mannheim , germany ) two mice from each diabetic group were excluded because of insufficient increase in blood glucose levels . furthermore , two mice from the diabetic knockout group were excluded because of weight loss > 15% of body weight . the excluded mice were not included in the number of animals per group indicated above . spot urine was collected in eppendorf tubes on five consecutive days prior to sacrifice of the animals . the blood samples were drawn from under the tongue at baseline and from the retroorbital venous plexus at study end and collected in potassium edta tubes ( sarstedt , nmbrecht , germany ) . the animals were anesthetized by an intraperitoneal dose of ketamine at 0.5 mg / g body weight and xylazine at 0.2 mg / g body weight ( ketaminol 4 vet and narcoxyl vet , resp . , urinary albumin excretion was determined by mouse albumin elisa quantification kit ( bethyl laboratories , inc . , urine creatinine was measured by isocratic high - performance liquid chromatography ( hplc ) on a zorbax scx300 column ( agilent , usa ) using a slight modification of a method first reported by yuen et al . . in brief , 5 l urine was added to 100 l acetonitrile containing 0.5% acetic acid and vortexed for 15 seconds to extract the creatinine . after 15 min of 20c storage and centrifugation the supernatants were evaporated and then reconstituted with 25 l 5 mm sodium acetate , ph 4.1 . duplicate samples ( 10 ul each ) were fractionated on a 50 mm 2.1 mm zorbax scx300 column with an in - front scx guard column . isocratic hplc was performed at a flow rate of 1 ml / min , and uv absorbance was monitored at 225 nm . a standard curve was created by including a 2-fold dilution series of creatinine anhydrous ( sigma aldrich ) . this study was designed with two independent factors ; diabetes / nondiabetes and knockout / wild - type and thus analysed by two - way anova for normal distributed variable with equal variance . the main focus of interest was the interaction between the diabetic factor and the knockout factor ; that is , does ficolin b modify the effects of diabetes on the effect parameters ? if no interaction was found , the independent effects of diabetes and ficolin b on the kidney were estimated . for pairwise comparison , normal distributed data was tested with student 's t - test , whereas otherwise the wilcoxon mann - whitney rank sum test was used . data are given as mean ( 95% confidence interval ( ci ) ) unless else is stated . at baseline , the knockout mice on average weighed 20.0 g , which was slightly less than the wild type mice , 20.8 g ( p = 0.04 ) . no difference was found between the two diabetic groups or between the two nondiabetic groups ( table 1 ) . after 18 weeks an expected difference in body weight was observed between the diabetic and the nondiabetic mice independently of knockout status ( p < 0.001 ) . the nondiabetic mice weighed 3.2 g ( ci : 2.0 g4.3 g ) more than the diabetic mice . furthermore the diabetic knockout mice were significantly smaller than the diabetic wild type ( p < 0.05 ) . as presented in table 1 , blood glucose , estimated as area under the curve ( auc ) , did not differ between the two diabetic groups ( p = 0.69 ) or between the two nondiabetic groups ( p = 0.13 ) . the kidney weight was equally increased in diabetic wild - type mice , 24% ( ci : 13%36% ) , and in the diabetic knockout mice , 29% ( ci : 12%47% ) , compared to the respective control groups ( figure 2(a ) ) . no interaction between knockout and diabetes was found ( p = 0.60 ) , indicating that wild - type and knockout mice develop the same degree of diabetes - induced renal hypertrophy . the considerable body weight difference between the two diabetic groups at study end indicated that the kidney weight was to be normalised to the body weight . ficolin b did not modify the diabetes - induced increase in kidney weight when testing for interaction ( p = 0.11 ) . furthermore , no significant statistical difference was found in kidney weight per body weight between the diabetic wild - type , 1.95 mg / g , and the diabetic knockout , 2.89 mg / g ( p = 0.09 ) . the albumin - to - creatinine ration ( acr ) was higher among the diabetic wild - type mice , 76 mg / g ( ci : 50103 mg / g ) , compared to the nondiabetic wild - type mice , 44 mg / g ( ci : 2563 mg / g ) , p = 0.07 . similarly , the acr of diabetic knockout mice was 96 mg / g ( ci : 71122 mg / g ) compared to the nondiabetic knockout group , 34 mg / g ( ci : 2344 mg / g ) , p < 0.001 . as depicted in figure 3 no interaction was observed between diabetes and ficolin b knockout , p = 0.21 . in the present study we found no association between diabetes - induced kidney changes and the presence of ficolin b. we conclude that ficolin b is not responsible for , or a crucial contributory factor in , the pathophysiology of diabetic nephropathy . in our study , the kidney weight and to some extent the acr were altered by diabetes as expected . the diabetes - induced increase in kidney weight , measured by comparing the diabetic mice with the nondiabetic mice , was not statistically different between the wild - type and ficolin b knockout mice . the diabetes - induced increase in kidney weight was 24% in the wild - type mice and 29% in the ficolin b knockout mice . taking the lower body weight of the knockout mice into account , the difference in renal hypertrophy was still insignificant when comparing the wild - type mice and the ficolin b knockout mice . similarly , the diabetic change seen in acr was not altered in the absence of ficolin b. the experimental setup including four groups matched on age , body weight , and genetical background was a strength to the study , as the diabetes factor and the knockout factor were the only modulators of the outcome . most importantly both diabetic groups did reach and sustain blood glucose levels of above 15 mm . at study end , the body weight differed among groups , which impeded the analyses of the diabetic kidney damage , because the knockout mice appear to be more vulnerable to type 1 diabetes mellitus . our study provides important new information on the association between the lectin pathway and diabetic kidney damage . we are the first to investigate the role of ficolin b ( which corresponds to ficolin m in human ) in the inflammatory response of diabetic nephropathy . in mice with deficiency of mbl , the classical functional and physical renal changes normally seen in this experimental model of type 1 diabetes were modified [ 29 , 30 ] . the fact that ficolin b does not appear to modulate diabetic effects on the kidney emphasizes the importance of mbl compared with ficolin b. both mbl and ficolin b activate the lectin pathway of the complement system , but only deficiency of mbl has been shown to protect against diabetic kidney damage . this indicates that the role of the lectin pathway in the development of diabetic nephropathy is complex and may depend on the specific carbohydrate - binding properties of mbl as previously described . the function of other complement factors in the first parts of the lectin pathway ( e.g. , ficolin a and masps ) in the pathology of diabetic kidney disease remains unknown and must be explored in further studies . one study indicates that ficolin a and ficolin b exert a cooperatively defensive role in destroying streptococcus pneumoniae , suggesting a synergetic immunological effect . this emphasises the need for further investigations involving both mouse ficolins . in order to fully understand the involvement of the lectin pathway in the development of diabetic nephropathy , an additional parallel experiment with masps is of particular interest given that they represent the limiting downward step in the complement activation in conclusion , this study demonstrates that ficolin b does not modify the kidney weight and acr in a type 1 diabetes mouse model . this indicates that the role of the lectin pathway in the development of diabetic nephropathy is specific and that hyperglycaemia - induced glycations on renal cells may be more prone to bind mbl than ficolin b.

background . the innate immune system may have adverse effects in diabetes and cardiovascular disease . the complement system seems to play a key role through erroneous complement activation via hyperglycaemia - induced neoepitopes . recently mannan - binding lectin ( mbl ) was shown to worsen diabetic kidney changes . we hypothesize that mouse ficolin b exerts detrimental effects in the diabetic kidney as seen for mbl . methods . we induced diabetes with streptozotocin in female wild - type mice and ficolin b knockout mice and included two similar nondiabetic groups . renal hypertrophy and excretion of urinary albumin and creatinine were quantified to assess diabetic kidney damage . results . in the wild - type groups , the kidney weighed 24% more in the diabetic mice compared to the controls . the diabetes - induced increase in kidney weight was 29% in the ficolin b knockout mice , that is , equal to wild - type animals ( two - way anova , p = 0.60 ) . in the wild - type mice the albumin - to - creatinine ratio ( acr ) was 32.5 mg / g higher in the diabetic mice compared to the controls . the difference was 62.5 mg / g in the ficolin b knockout mice , but this was not significantly different from the wild - type animals ( two - way anova , p = 0.21 ) . conclusions . in conclusion , the diabetes - induced effects on kidney weight and acr were not modified by the presence or absence of ficolin b.

dendritic cells ( dcs ) have been identified as a key component in manipulating and stimulating the immune system . activated dcs are potent antigen presenting cells that express both major histocompatibility complex ( mhc ) class i and ii molecules ( signal 1 ) and costimulatory molecules ( signal 2 ) and secrete immune modulating cytokines ( signal 3 ) resulting in activation of t lymphocytes . depending on the cytokine environment , dcs may elicit either a th ( t helper ) 1 or th2 cd4 t - cell response . for tumor immunotherapy , induction of a th1 t - cell response is pivotal , and secretion of il-12 ( interleukin 12 ) by dcs is of critical importance for differentiation of naive t cells into th1 cells . furthermore , il-12 stimulates the production of interferon - gamma ( ifn- ) and tumor necrosis factor - alpha ( tnf- ) from t cells and natural killer cells . in contrast , th2 responses , associated with cytokines il-4 , il-5 , il-6 , and il-10 , suppress th1 activity and may anergize effector t cells to tumor antigens . one of these strategies involves fusing dcs with tumor cells using electrical currents in a method called electrofusion , hence combining the antigen presenting properties of dcs with the full repertoire of antigens present within a tumor cell in order to stimulate effector t cells [ 6 , 7 ] . while dc - tumor hybrids alone are insufficient to elicit significant immune responses in vivo and are critically dependent upon exogenously administered 3rd signal adjuvants , murine studies using dc - tumor hybrids for vaccination given concomitantly with an adjuvant third signal , such as il-12 , ox-40- , 4 - 1bb - monoclonal antibody , or toll - like receptor agonists , showed regression of tumor metastases after a single vaccination in several tumor types including melanoma , breast , sarcoma , and squamous cell carcinoma [ 811 ] . however , systemic delivery of 3rd signal along with a dc - tumor fusion vaccine is clinically problematic due to 3rd signal toxicity and/or availability . therefore , a better understanding of the mechanisms affecting the dependence of dc - tumor fusions on 3rd signal adjuvants is of paramount importance for optimizing this immunotherapeutic approach . in this study , we show that production of the th1 skewing cytokine il-12 was dramatically downregulated in dc - tumor fusion cells . in addition , gene products that are involved in signaling pathways including nfb ( nuclear factor kappa - light - chain - enhancer of activated b - cells ) , pi3k / akt / mtor ( phosphatidylinositol 3-kinase / akt , protein kinase b / mammalian target of rapamycin ) , wnt ( wingless - related integration site ) , and mapk ( mitogen - activated protein kinase ) were differentially expressed in fusion cells . inhibitor studies revealed that interruption of the canonical wnt pathway did not affect il-12 production by dc - tumor fusion cells and that inhibition of mek ( mitogen extracellular signal - regulated kinase ) only increased il-12 production marginally . in contrast , il-12 production could significantly be enhanced by treatment of dc - tumor hybrids with inhibitors of the pi3k and mtor . given the critical role of the pi3k / akt / mtor signaling pathway in cancer biology and the immunostimulatory effect of pi3k / akt / mtor inhibitors on dc - tumor hybrids , combination therapy may represent a promising and novel cancer vaccine with enhanced clinical impact . animals were housed in a specific pathogen - free environment at the animal facility of the durham veteran affairs medical center . all mice used in this study were cared for in accordance with the guide for humane care and use of laboratory animals published by the national institutes of health . all the animal experimental protocols were approved by the duke university medical center institutional animal care and use committee . d5lacz is a -galactosidase expressing derivative of the b16 f10.9 melanoma cell line and has been shown to be poorly immunogenic . cells were cultured in complete media ( cm ) composed of rpmi 1640 media supplemented with 10% fetal bovine serum , 2 mm l - glutamine , 0.1 mm nonessential amino acids , 1 mm sodium pyruvate , 100 u / ml penicillin , 100 g / ml streptomycin , 0.5 g / ml fungizone , 50 g / ml gentamicin , and 5 10 m 2-mercaptoethanol ( invitrogen , carlsbad , ca ) . these cells were maintained at 37c with 5% co2 , harvested following a short incubation period with 0.05% trypsin with edta , and irradiated at 100 gy prior to use . b- and t - lymphocytes were depleted using antibody - coated magnetic beads ( dynal biotech , carlsbad , ca ) . the dc - enriched cell fraction was then cultured in cm supplemented with 10 ng / ml gm - csf and 10 ng / ml il-4 ( peprotech , rocky hill , nj ) at a concentration of 0.5 10 cells / ml at 37c with 5% co2 . on day 6 , cells were harvested , resuspended in fresh cm + gm - csf / il-4 media at 1 10 cells / ml , and incubated at 37c with 5% co2 for 24 hours . then , lps ( lipopolysaccharide , 100 ng / ml , sigma - aldrich , saint louis , mo ) was added to stimulate dc maturation . after 24 hours after 24 hours , dcs were stained intracellularly with cfse prior to use ( molecular probes , eugene , or ) . irradiated tumor cells and cfse stained dc were mixed in a 1 : 1 ratio and washed in prefusion media , followed by resuspension in fusion media at a concentration of 20 10 cells / ml . for electrofusion , the pulse generator ( model ecm 2001 generator , btx instruments , san diego , ca ) was used . cells were exposed to two consecutive , independent electrical currents : ( 1 ) a low voltage alternating current of 120 v / cm for 10 seconds to achieve alignment and chain formation , and ( 2 ) a high voltage direct current of 1100 v / cm for 25 microseconds to cause a reversible breakdown of cell membranes . the multinucleated hybrid cells were allowed to stand for at least 5 minutes before incubation in culture media overnight at 37c with 5% co2 . to separate unfused tumor cells ( t ) from t - t hybrids and unfused dcs from dc - dc hybrids facs sorting by size on forward scatter ( fsc ) and side scatter ( ssc ) dc - t hybrids were purified using a combination of mechanical and facs sorting techniques , based on their plastic adherence characteristics as well as cfse staining . therefore , after electrofusion and overnight culture , the nonadherent cell population representing unfused dcs and dc - dc hybrids was discarded . facs was then performed only on the adherent cell population containing unfused tumor cells , t - t hybrids , and dc - t hybrids . since only dcs were stained with cfse , facs sorting was used to separate cfse positive cells from the cfse negative populations ( unfused tumor cells and t - t hybrids ) . all cell samples were analyzed using the facs aria ii ( bd biosciences , san jose , ca ) . 24 hours after electrofusion , total rna was isolated using the rneasy plus mini kit protocol ( qiagen , valencia , ca ) . the cdna template was synthesized from 0.51.0 ug of total rna using the rt first strand kit protocol ( sabiosciences , frederick , md ) . each template was then combined with rt sybr green qpcr master mix ( sa biosciences ) and aliquoted into a 96-well mouse common cytokine plate array ( sa biosciences ) . the pcr cycling program was 95c for 10 minutes , followed by 40 cycles of 95c for 15 seconds , and then 60c for 1 minute on a stratagene mx3005p qpcr machine . briefly , all threshold values ( ct ) reported as greater than 35 indicated no detectable gene expression . genomic dna ( gdna ) contamination was detected if the gdna control ct value was below 35 . a reverse transcription control ( rtc ) detected impurities in the rna sample that affect the reverse transcription of the template and was considered positive if the ct was greater than 5 . gene expression associated with th1 ( ifn- , il-2 , il-12p40 , il-15 , il-18 , and tnf- ) and th2 ( il-4 , il-10 , il-13 , and il-25 ) immune responses was analyzed . total rna was isolated from tumor cells , dcs , and dc - t fusion cells using the rneasy plus mini kit protocol ( qiagen , valencia , ca ) . rna isolation for tumor and dendritic cells was done in triplicate . for the dc - t fusion cells , triplicate samples of d5lacz tumor cells , dcs , and dc - t fusion cells were each run through a microarray chip ( affymetrix ) by the duke dna microarray core facility . multiway anova was performed and fold change was determined to select target genes that were differentially expressed between fusion cells and dcs , or fusions cells and tumors cells , respectively . top differentially expressed genes were selected with p value cutoff of 0.01 based on anova test and fold change cutoff of > 5 . hierarchical clustering was performed on differentially expressed genes based on average linkage with pearson 's dissimilarity . the murine il-4 elisa kit ( ebioscience , san diego , ca ) and the murine il-12p70 elisa kit ( bd biosciences , san jose , ca ) were used according to the manual provided by the manufacturer . to determine cytokine secretion by dcs or dt - tumor fusion cells , 2 10 cells in 1 ml of aimv media ( invitrogen , carlsbad , ca ) were incubated in the presence of 100 ng / ml of lps for 24 hours at 37c , 5% co2 . where indicated , lps stimulation was performed in the presence of the following inhibitors ( purchased from sigma - aldrich , saint louis , mo ) : u0126 ( 1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthio ] butadiene ) is a highly selective inhibitor of both mek1 and mek2 and was used at a concentration of 100 nm , jw 74 ( 4-[4-(4-methoxyphenyl)-5-[[[3-(4-methylphenyl)-1,2,4-oxadiazol-5-yl]methyl]thio]-4h-1,2,4-triazol-3-yl]-pyridine ) an inhibitor of the canonical wnt pathway was used at a concentration of 10 m , rapamycin ( 23,27-epoxy-3h - pyrido[2,1-c]oxaazacyclohentriacontine ) forms a complex with fkbp12 ( fk506 binding protein 12 ) that binds to and inhibits mtor which was used at 0.5 m , and wortmannin which inhibits the pi3k / akt signal transduction cascade was used at 100 nm . experiments were performed in duplicate and error bars represent the sem ( standard error of the mean ) . in a first set of experiments , d5lacz tumor - tumor ( t - t ) cell hybrids , dc - dc hybrids , and dc - t hybrids were generated by electrofusion . fusion cells were purified by facs and rna isolated from hybrid cells was analyzed by quantitative real - time pcr ( qpcr ) for expression levels of mrnas encoding the th1 cytokines ifn- , tnf- , il-2 , il-12p40 ( the -subunit of bioactive il-12p70 ) , il-15 , and il-18 or the th2 cytokines il-4 , il-10 , il-13 , and il-25 . figure 1 shows the results of our qpcr analyses . comparison of dc - t hybrids with dc - dc fusion cells ( white bars ) reveals that cytokines associated with a th1 response including il-12p40 and il-15 were downregulated by more than 100- and 15-fold , respectively . in contrast , the th2 cytokine il-4 was dramatically upregulated by 115-fold . among all cytokines analyzed , only tnf- and il-12p40 exhibited higher expression levels in dc - t fusion cells when compared to t - t fusions ( figure 1 , black bars ) . in another series of experiments , the th1 and th2 cytokine expression profile of cells exposed to electrofusion was compared to unexposed cells . however , no significant changes in cytokine gene expression between tumor cells and t - t fusion cells or dcs and dc - dc fusion cells were observed ( data not shown ) . for this reason , we focused on the comparison of gene expression levels between dc - t hybrid cells and dcs in the subsequent analyses presented in this study . we next sought to determine changes in the expression of genes that may negatively impact the immunologic properties of dc - t fusion cells . in order to do so , rnas were isolated from facs - isolated dc - t hybrids cells , dcs , or d5lacz tumor cells , and microarray assays were performed . consistent with our qpcr data , expression of il12p40 and il-15 by dc - t fusion cells was markedly downregulated ( 13.2- and 8-fold ) when compared to dcs , albeit to a lesser degree than observed in pcr analyses ( figure 2(a ) ) . also , il-4 was upregulated 59.4-fold in dc - t fusions . the proinflammatory cytokines il-1 and il-1 were downregulated 5.5- and 8.2-fold , respectively , while tgf3 was upregulated 8.8-fold . furthermore , we observed a downregulation of receptors for colony - stimulating factor ( csfr1 ) , tnf- ( tnfr2 ) , and il-7 ( il-7r ) . in contrast the receptors for tweak ( tnf - like weak inducer of apoptosis , tweakr ) and for il-17 ( il-17rc ) were upregulated 9.5- and 6.5-fold . while overexpression of il-17rc has been implicated in bcl-2- and bcl - xl - independent protection of cancer cell lines from tnf-induced apoptosis , tweakr signaling has been shown to enhance the expression of nfb ( nuclear factor kappa - light - chain - enhancer of activated b - cells)-regulated genes including il-6 , il-8 , rantes , and icam-1 ( cd54 ) . however , upregulation of none of these gene products was observed in our study ( figures 2(a ) , 2(c ) , and 3(a ) ) . in addition to cytokine gene and cytokine receptor expression , there were also significant changes in the expression level of gene products that are involved in cytokine signaling ( figure 2(b ) ) . expression of tgfi ( transforming growth factor beta - induced ) , a protein that is induced by tgf and that acts to inhibit cell adhesion , was downregulated 14.1-fold . downregulation of this gene product was unexpected given that tgf3 was upregulated in dc - t hybrids ( figure 2(a ) ) and implies that the tgf-signaling pathway may not be hyperactive in dc - t hybrid cells . there were no differences in expression levels of tgf-receptors between dcs and dc - t hybrid cells . however , nedd4l ( neural precursor cell expressed developmentally downregulated gene 4-like ) was upregulated 13.8-fold in dc - t hybrid cells . nedd4l negatively regulates tgf signaling by ubiquitination - mediated degradation of tgf- receptor 1 and receptor - regulated smad2 ( mothers against decapentaplegic homolog 2 ) . as such , it is reasonable to assume that neddl4 overexpression suppressed transcriptional activity induced by tgf. expression of il-1ra , the interleukin-1 receptor antagonist , which modulates a variety of il-1 related immune and inflammatory responses , was downregulated 7.5-fold . moreover , expression of irfs ( interferon regulatory factors ) 4 , 7 , and 9 , which are involved in transcriptional regulation of type i interferon genes , interferon signaling , and hence the janus kinase- ( jak- ) signal transducer and activator of transcription ( stat ) pathway , was downregulated 7- , 6- , and 10.5-fold , respectively . also , jak-2 and stat-4 , known to be involved in il-12 receptor signaling , are downregulated 6.5- and 11.3-fold . last , three gene products that are associated with nf-b signaling were found to be downregulated in dc - t hybrid cells , namely , relb ( reticuloendotheliosis viral oncogene homolog b , 5.3-fold ) , traf-1 ( tnf receptor associated factor-1 , 11.3-fold ) , and the nf-b inhibitor ib ( nuclear factor of kappa light polypeptide gene enhancer in b - cells inhibitor alpha , 7.3-fold ) . relb is known to form heterodimers with nf-b p50 or p52 , and traf-1 forms a heterodimeric complex with traf2 , which is required for tnf--mediated activation of mapk8/jnk ( jun kinase ) and nf-b . on the other hand , these results are somewhat contradictory and argue that nf-b activity in dc - tumor fusion cells is not regulated at the transcriptional level . as shown in figure 2(c ) , expression of chemokines or their receptors which are involved in chemotaxis of neutrophils , monocytes , dcs , t cells , and nk cells were generally downregulated in dc - tumor fusions , with the exception of cxcl-10 ( ip-10 , interferon - gamma - induced protein 10 ) . surprisingly , even chemokines involved in chemotaxis of th2 cells and regulatory t cells ( ccl-17 and ccl-22 ) were downregulated while ip-10 which is implicated in the induction of th1 responses and chemotaxis of th1 cells was significantly upregulated [ 23 , 24 ] . we therefore hypothesize that the chemokine expression profile of dc - tumor hybrids does not have a major impact on the th - polarizing capacity of dc - tumor hybrid cells . it has been demonstrated that the expression of matrix metalloproteinases mt-1 ( mmp-14 ) and mmp-9 is a major contributing factor to the migratory capacity of dcs to lymph nodes through the degradation of extracellular matrix components . in this context , mmp-9 activity is of particular importance since it cleaves collagen iv , a major component of basement membranes . furthermore , it has been shown that the balance of mmp-9 and timp ( tissue inhibitor of mmps ) expression is crucial for dc migration in vivo . our data reveal that timp-2 was upregulated 11.8-fold in dc - t fusion cells , while mmp-9 is downregulated 7-fold ( figure 2(d ) ) . as such , these results suggest that the migratory capacity of dc - t hybrids toward lymph - node derived chemokines , namely , ccl-19 and ccl-21 , may be impaired . as shown in figure 3(a ) , expression of genes involved in antigen presentation in the context of mhc classes i and ii or cd1d was downregulated 5.7- , 16.5- , and 6-fold in fusion cells . furthermore , the expression of all well - established costimulatory molecules , including cd40 , cd54 , cd80 , cd83 , cd86 , 4 - 1bb , gitr ( glucocorticoid - induced tnfr - related protein ) , ox40l , and slam ( signaling lymphocytic activation molecule ) , was downregulated in dc - tumor fusion cells . these data explain to some degree why targeting of costimulatory molecules with agonistic antibodies can enhance the potency of dc - tumor fusion - based vaccines , as has been described previously . last , expression of pd - l2 ( programmed death ligand 2 ) , an inhibitory immune checkpoint molecule , was suppressed 7.8-fold in dc - fusion cells . no differences in pd - l1 expression between dcs and dc - t hybrid cells were observed . the development of melanocytes is highly dependent on the action of the microphthalmia - associated transcription factor ( mitf ) which has been shown to regulate a broad variety of genes , whose functions range from pigment production to cell - cycle regulation , migration , and survival . mitf was upregulated in dc - tumor fusion cells ( figure 3(b ) ) . concomitantly , also mitf - regulated mrnas encoding melanoma antigens , including tyr ( tyrosinase ) , trp-1 and trp-2 ( tyrosinase - related protein ) , gp100 ( silver ) , melan , melanophilin , m - cam ( melanoma cell adhesion molecule ) , and matp ( membrane - associated transporter protein also known as solute carrier family 45 member 2 ( slc45a2 ) or melanoma antigen aim1 ) , were also highly upregulated . moreover , expression of mitf - regulated mcr1 ( melanocortin 1 receptor ) , trpm1 ( transient receptor potential cation channel subfamily m member 1 ) , gpr143 ( g protein - coupled receptor 143 ) , and mbp ( myelin basic protein ) was highly upregulated in fusion cells . expression of mbp by melanoma cells is somewhat surprising , but it has been shown that b16f10 cells undergo differentiation to a myelinating glial phenotype characterized by induction of the transcriptional activity of the mbp promoter . last , osteonectin ( secreted protein acidic and rich in cysteine ( sparc ) ) , which has been implicated in metastasis of melanoma to the lungs , and plagl1 ( pleomorphic adenoma gene - like 1 ) , a potential tumor suppressor gene , were also overexpressed in dc - tumor fusions . these results suggest that the entire antigenic repertoire of melanoma cells is indeed strongly expressed in dc - tumor hybrid cells , as has been hypothesized . next , we analyzed expression levels of genes that are involved in signaling pathways known to be aberrantly regulated in cancer cells . the transcription factor tcf7l1 ( transcription factor 7-like 1 ) which is activated by -catenin and thus mediated wnt signaling was upregulated 6.2-fold ( figure 4(a ) ) . also , expression of frzb ( frisbee ) , a wnt - binding protein and competitor for the cell - surface receptor frizzled , and expression of wntless ( g protein - coupled receptor 177 ) , another receptor for wnt proteins , were increased 12.2- and 61.3-fold . furthermore , target genes of the canonical wnt pathway , wisp-1 ( wnt1-inducible - signaling pathway protein 1 ) and nrcam ( neuronal cell adhesion molecule ) , were upregulated 13.7- and 148.7-fold , indicating activation of the wnt pathway in dc - tumor fusion cells . expression of the fk506 binding proteins fkbp4 , fkbp6 , and fkbp9 , immunophilins known to interact with mtor , was upregulated in dc - tumor fusions 5.3- , 6.7- , and 28-fold ( figure 4(b ) ) . furthermore , expression of pik3r1 ( phosphatidylinositol 3-kinase regulatory subunit alpha , p85 ) was downregulated 6.5-fold , which may indicate aberrant activity of pi3k in dc - tumor fusion cells . nedd4 directly binds to and poly - ubiquitinates pten ( phosphatase and tensin homolog ) , targeting it for proteasomal degradation . therefore , posttranslational suppression of its expression level may lead to hyperactivation of the pi3k / akt signaling pathway . the lps - inducible mitogen - activated protein kinase 12 ( mapk12 ) , also known as extracellular signal - regulated kinase 6 ( erk6 ) or p38- , was upregulated 11.7-fold in dc - tumor fusions ( figure 4(c ) ) . in contrast , the lps - inducible gadd45 ( growth arrest and dna damage - inducible 45 ) was downregulated 10.8-fold . gadd45 is an nf-b target gene which , in combination with mekk4 , activates p38mapk . furthermore , mitogen - activated protein kinase kinase kinase 14 ( map3k14 ) also known as nf - kappa - b - inducing kinase was downregulated 7.25-fold in fusion cells . lastly , expression of c - jun amino - terminal kinase interacting protein 1 ( mapk8ip1 ) , a negative regulator of mapk8 ( c - jun amino - terminal kinase ) , was upregulated 6.9-fold in dc - tumor hybrid cells . lipid mediators such as prostaglandins have been implicated in tumor - mediated immunosuppression [ 37 , 38 ] . as presented in figure 4(d ) , several genes involved in eicosanoid biosynthesis and signaling were differentially expressed in dc - tumor fusion cells . additionally , cyclooxygenase-2 was downregulated 16.2-fold and the cysteinyl - leukotriene c4 synthase ( ltc4s ) was downregulated 12.1-fold . in contrast , phospholipase a2 ( pla2 ) and prostaglandin d2 synthase ( pgds ) were upregulated 47.2- and 61.3-fold , respectively . we next sought to determine whether inhibition of signaling pathways , for which inhibitors are available clinically , could restore secretion of bioactive il-12p70 by dc - tumor fusion cells . we chose wortmannin as an inhibitor of pi3k upstream of akt ( pkb ) , u0126 as an inhibitor of mek1 and mek2 , rapamycin as an inhibitor of mtor , and jw74 as an inhibitor of the canonical wnt pathway . admittedly , our data provide several lines of evidence that nf-b - signaling is impaired in dc - tumor fusion cells , but , even though nf-b - inhibitors are starting to emerge in the clinic , it would obviously not make sense to administer nf-b - agonists to cancer patients . dcs and dc - tumor fusion cells were stimulated with lps in the presence or absence of inhibitors as indicated in figure 5(a ) and supernatants were analyzed for il-12p70 secretion by elisa . as expected , dc - tumor fusions did not produce il-12p70 in response , while dcs responded to lps stimulation . u0126 led to a modest increase of il-12p70 by both dc - tumor fusions and dcs . inhibition of pi3k with wortmannin and inhibition of mtor with rapamycin increased secretion of il12-p70 significantly ( 11 - 13-fold ) . inhibition of the canonical wnt pathway with jw74 did not have any impact on il-12p70 production by dcs or dc - tumor fusion cells . we next asked whether combined inhibition of pi3k and of mtor could further enhance il-12p70 secretion by dc - tumor fusion cells . as shown in figure 5(b ) , combining wortmannin and rapamycin to inhibit pi3k and mtor did not significantly enhance il-12p70 secretion by dc - tumor hybrid cells , hence excluding a synergistic or additive effect of these inhibitors . this study is the first to investigate the mechanisms responsible for the dependence of dc - tumor hybrid vaccines on exogenously provided 3rd signal adjuvants . these include the induction of apoptosis of immune cells via expression of fas ligand , trail ( tnf - related apoptosis - inducing ligand ) [ 39 , 40 ] , or pd - l1 and pd - l2 ( programmed death ligand ) . furthermore , induction of tolerance through cytokines such as tgf- , il-6 , and il-10 or lipid mediators [ 37 , 38 ] has been described . lastly , activation of the mapk pathway by melanoma cells has been described as a mechanism to inhibit il-12 production by dcs in a paracrine manner . our results do not provide evidence for overexpression of apoptosis - inducing ligands by dc - tumor cell hybrids , nor did we observe an enhanced production of tolerance - inducing cytokine il-6 or il-10 by these cells . tgf-3 was upregulated 8.8-fold in dc - tumor fusions , but the observed downregulation of the tgf-induced protein in combination with upregulation of nedd4l argues against a major impact of this cytokine on fusion cells . surprisingly , despite a profound upregulation of mrna encoding il-4 in dc - tumor hybrids , there was no evidence of il-4 signaling in these cells . we did not observe any upregulation of target genes of the il-4 receptor i or ii , including socs-1 , il-4 receptor , ccl11 ( eotaxin 1 ) , or fc receptor ii . in addition , there were no changes in expression of gene products that are components of the il-4 receptors , namely , il-4 receptor , il13 receptor , and cd25 . even though the expression levels of phospholipases a2 , which release arachidonic acid from phospholipids , and of prostaglandin d2 synthase were upregulated in dc - tumor fusions , expression of cyclooxygenase , which catalyzes the downstream conversion of arachidonic acid into eicosanoids , was downregulated . furthermore , while pgd-2 has been shown to upregulate cd80 and to downregulate ip-10 in lps - matured dcs , the exact opposite was observed in our experiments ( figures 2(c ) and 3(a ) ) . accordingly , we conclude that pgd-2 may not be the main culprit for the dramatic downregulation of il-12 production in dc - tumor hybrids . however , it has been described that , in the spontaneous b16f10 melanoma cell line , expression of p16ink4a ( inhibitor of cdk4a ) , which inhibits cell - cycle progression by inactivating cyclin - dependent kinases , and of p19arf ( alternate reading frame tumor suppressor ) , which causes mdm2 ( mouse double minute 2 homologue ) induced translational silencing and p53 degradation , is lost and that there is no evidence of activation of the mapk - signaling pathway in this cell line . it is therefore highly unlikely that the mapk - signaling pathway would be a major contributor to the loss of immunostimulatory capacity of dc - tumor hybrid cells . nevertheless , our data reveal that treatment of dc - tumor hybrid cells with mek inhibitor u0126 led to a modest increase in il-12 secretion . this however might be a result of the previously published observation that treatment with u0126 can result in a slight but significant inhibition of p70 ( s6 ribosomal protein kinase ) activation , a downstream target of akt . our results further indicate that molecules that are involved in wnt signaling , including tcf7l1 , frzb , and wntless , were upregulated in dc - tumor fusions . additionally , wisp-1 and nrcam , targets of the canonical wnt pathway , were upregulated 12- and 148.7-fold , respectively . in the canonical wnt pathway , activation of wnt receptors leads to stabilization and import of -catenin into the nucleus where -catenin associates with t - cell factor / lymphoid enhancer factor ( tcf / lef ) and activates target genes . however , treatment of dc - tumor fusions with jw74 , a specific inhibitor of the canonical wnt pathway , had no impact on il-12 secretion by these cells . on the other hand , it is conceivable that the mtor pathway was activated through the noncanonical wnt / ca pathway , wnt - dependent activation of pka ( protein kinase a ) and creb ( camp response element - binding protein ) , or mtor activation via wnt - mediated inhibition of glycogen synthase kinase 3 . alternatively , we can not exclude that the pi3k / akt / mtor pathway was activated independent of wnt signaling . the pi3k / akt / mtor pathway has been shown to play a critical role in cell proliferation , survival , and metastasis of cancer cells , and we observed that inhibition of the pi3k / akt and inhibition of the mtor pathway enhanced the immune - stimulatory capacity of dc - tumor fusions through induction of bioactive il-12p70 secretion . the fact that combined inhibition of pi3k and mtor signaling did not further improve il-12p70 secretion by dc - tumor fusions may indicate that inhibition acted on the same signaling pathway , likely to involve p70 as has been described previously . in sum , we conclude that combining pi3k / akt / mtor inhibition with dc - melanoma fusion cell - based cancer vaccination appears to be a promising strategy and warrants further studies in vitro and in animal models . ultimately , this research may lead to the development of improved dc - fusion - based cancer vaccines with enhanced clinical impact .

background . dendritic cell- ( dc- ) tumor fusion cells stimulate effective in vivo antitumor responses . however , therapeutic approaches are dependent upon the coadministration of exogenous 3rd signals . the purpose of this study was to determine the mechanisms for inadequate 3rd signaling by electrofused dc - tumor cell hybrids . methods . murine melanoma cells were fused with dcs derived from c57bl/6 mice . quantitative real - time pcr ( qpcr ) was used to determine relative changes in th ( t helper ) 1 and th2 cytokine gene expression . in addition , changes in gene expression of fusion cells were determined by microarray . last , cytokine secretion by fusion cells upon inhibition of signaling pathways was analyzed by elisa . results . qpcr analyses revealed that fusion cells exhibited a downregulation of th1 associated cytokines il-12 and il-15 and an upregulation of the th2 cytokine il-4 . microarray studies further showed that the expression of chemokines , costimulatory molecules , and matrix - metalloproteinases was deregulated in fusion cells . lastly , inhibitor studies demonstrate that inhibition of the pi3k / akt / mtor signaling pathway could restore the secretion of bioactive il-12p70 by fusion cells . conclusion . our results suggest that combining fusion cell - based vaccination with administration of inhibitors of the pi3k / akt / mtor signaling pathway may enhance antitumor responses in patients .

human neurocysticercosis ( ncc ) is caused by larval stage of zoonotic tapeworm taenia solium ( pork tapeworm ) which remains a major public health problem in developing and some developed countries . porcine cysticercosis is the cause of human taeniasis and neurocysticercosis is a consequence of taeniasis . based on the available information , a very conservative and rough economic estimate indicates that the annual losses due to porcine cysticercosis in 10 west and central african countries amount to about 25 million euros . . also stated that , in china , the amount of pork discarded in the whole country due to cysticercosis annually has been estimated as 200,000,000 kg with a value of more than us $ 120,000,000 . stated that notably data on myanmar are lacking , although there are several reports of porcine cysticercosis based on meat inspection in the abattoirs in neighboring countries , 9.3% in india , 32.5% in nepal , 5.4% in china , 0.022.63% in indonesia , and 0.04 to 0.9% in vietnam . although most of myanmar culinary habits are based on thorough cooking , new food style such as barbecue and dishes based on raw or undercooked pork or pork product becomes popular among customers . moreover , small - scale pig husbandry has become one of the major sources of income in myanmar farmers . so it may be high risk of getting food - borne zoonotic diseases according to the new food style and traditional husbandry method . due to lacking of information on porcine cysticercosis in myanmar up to now , it is important to investigate the prevalence and associated risk factors . nay pyi taw area , the capital of myanmar , has big population of pigs ( about 200,000 pigs ) to support the demand of pork consumption in this area . most of the pig farmers are smallholders and most of pig husbandry systems are free ranging or semi - intensive with lack of proper sanitation . one of the main obstacles to control the t. solium infections is the lack of adequate epidemiological data on cysticercosis / taeniasis . therefore , the objectives of this community - based study were to investigate the prevalence of porcine cysticercosis and associated risk factors in pigs within study area . moreover , findings of this study will assist to develop the control strategies of porcine cysticercosis for the public health aspect . the cross - sectional studies were conducted from january to march and june to july 2014 , to investigate the prevalence of taenia solium cysticercosis in slaughtered and farmed pigs within pyinmana , lewe , and tatkon townships , nay pyi taw area . it is located between latitude 1945n and longitude 966e and with climate data ; the altitude is 115 m above sea level , annual rainfall is 115 mm , and annual temperature is 21.232.5c . an expected prevalence of 30% with a confidence level of 95% was used in this unit . in this study , 300 slaughtered pigs and 364 farmed pigs from the study area were examined although calculated samples were 298 and 323 , respectively ( table 1 ) . blood collected from the jugular vein of farmed pig was conducted for the seroprevalence and a structured questionnaire with both closed and open - ended questions was administered to owners to obtain management practices in pig husbandry . piglets younger than two months , pregnant sows , and nursing sows with litters less than two months old were excluded from this study to overcome the stress which causes adverse effect in animals . meat inspection was carried out as described by boa et al . in the three slaughterhouses of these townships . there were 300 randomly selected pigs recruited and 9 different muscles ( tongue , masseter , brain , shoulder , diaphragmatic , heart , skeletal , fore limb , and hind limb muscle ) from each pig in meat inspection . briefly , long and parallel incision into the masseter muscles on both sides of face in an upward direction was made . a deep longitudinal incision covering about 3/4 the thickness of the tongue and covering the whole length of the tongue was made to examine the cysts . after opening the pericardium , the heart was also visually examined for the presence of cysts . the heart was cut open and a deep ( 3/4 the thickness of septum ) incision into the septum was made to expose any metacestodes . all the other muscles were viewed , palpated , incised by surgical blade , and visually examined . the pig was kept under restraint at standing position and blood samples were obtained from the external jugular vein by using sterile disposable syringes and put into vacutainers with clot activators . those vacutainers were kept in cold boxes with ice and transported to department of pharmacology and parasitology , university of veterinary science , nay pyi taw , and allowed overnight at 4c to clot . to obtain serum , the clear sera were transferred to 1.5 ml microvial tubes and stored in labeled wails and kept at 20c until analysis . detection of igg antibody of t. solium cysticerci was carried out by using antibody - elisa kit ( novatec immundiagnostica gmbh co. , belgium ) according to manufacturer 's instruction . briefly , all thawed samples were diluted as 1 + 100 with igg sample diluent ( phosphate buffer ) before assaying . the 100 l controls and diluted samples were dispensed into their respective wells and the foil was covered . after incubation for 1 hour at 37c and the foil being removed , the contents of the wells were aspirated and washed three times with washing solution . and the 100 l protein a conjugate ( horseradish peroxidase ) was dispensed into all wells except a1 and covered with foil and incubated for 30 min at room temperature . after washing three times , 100 l tmb ( 3,3,5,5-tetramethylbenzidine ) substrate solution was dispensed into all wells and incubated for exactly 15 min at room temperature in the dark . the reaction was stopped by adding 100 l stop solution ( 0.2 m h2so4 ) . the absorbance was determined at 450/620 nm using an elisa reader ( stat fax ) . in each elisa kit testing , there are two cut - off controls ( c1 and d1 ) . the mean absorbance of these cut - off controls was used as cut - off value . samples are considered positive if the absorbance value is higher than 10% over the cut - off and samples are considered negative if the absorbance value is lower than 10% below the cut - off . the sensitivity and specificity of these kits to diagnose swine cysticercosis are 93.8% and > 95% , respectively . a questionnaire was developed and used to collect information on hypothesized risk factors and other related pieces of information from sampled pig owners . households in each township were selected by using the snowballing technique from those farmers willing to participate in the study . it is a technique for developing a research sample where existing study subjects recruit future subjects from their acquaintances . the questionnaire interviewed data were analyzed for the relationship between the prevalence of t. solium cysticercosis and hypothesized risk variables such as age , gender of pigs , husbandry system , feed type , environment of pig farm ( accessibility of human feces ) , personal hygiene of owners , pork consumption , cooking and eating habit of pork , use of anthelmintics in pigs and owners , and knowledge on taeniasis . they were examined for testing its significance by pearson chi - square test at = 0.05 . seroprevalence of porcine cysticercosis in farmed pigs was 15.93% ( 58/364 ) in the study area . prevalence of households with pigs infected with t. solium cysticerci by ab - elisa examination was 23.15% ( 47/203 households ) . the households with porcine cysticercosis in pyinmana , lewe , and tatkon were 0/12 ( 0% ) , 13/124 ( 10.48% ) , and 34/67 ( 50.75% ) , respectively . all the infected pigs presented parasites located in the tongue . only in one pig , the prevalence in slaughterhouses of pyinmana , lewe , and tatkon townships was 22% ( 44/200 ) , 23.33% ( 7/30 ) , and 28.57% ( 20/70 ) , respectively . univariate analysis of hypothesized risk factors of gender ( or = 3.0 ; 95% ci = 1.75.4 ) , increased age ( or = 2.3 ; 95% ci = 1.24.2 ) , husbandry system ( or = 5.1 ; 95% ci = 2.411.2 ) , feed type ( or = 16.9 ; 95% ci = 2.3124.3 ) , no hand washing habit before feeding ( or = 31.5 ; 95% ci = 4.3230.9 ) , not using anthelmintic in pigs ( or = 11.9 ; 95% ci = 5.028.5 ) and owner ( or = 2.5 ; 95% ci = 1.44.4 ) , and pork consumption of owner ( or = 37.4 ; 95% ci = 9.0156.1 ) was significantly associated with cysticercus cellulosae infection ( p < 0.05 ) . the distribution and odds ratio of significant risk factors concerning porcine cysticercosis are shown in table 2 . in southeast asia , pigs are an important source of food and economic important for smallholder farmers . older pigs may be penned or tethered although common raising practice of pigs is freely roaming in the village . in myanmar , most of the pig farmers are smallholders and practice as free - range or backyard farming . in myanmar , most of the pig farmers usually keep the weaned pigs until six to eight months of age and then send to slaughterhouse . in the village , every household keeps at least one pig not only for table waste feeding to pigs but also for extra income . most farms are having the habit of feeding waste materials such as swill and kitchen leftover , broken rice , rice bran , groundnut meal , sesame meal and local forage , and poor sanitation . the present study is the first report of t. solium cysticercosis in pigs in myanmar . pigs in the study area positive for cysticercosis have been exposed to t. solium eggs . among the 17 hypothesized risk factors , the gender of pigs ( being female ) was significantly associated with porcine cysticercosis in this study . it can be explained that female pigs were for kept long time for breeding purpose than male and so they have more risk to get exposed to t. solium eggs . however , jayashi et al . reported that gender was not a significant risk factor for porcine cysticercosis . the present study demonstrated that the older the pigs , the greater the chance to get infection . these results are in agreement with those reported by pouedet et al . , jayashi et al . , sarti et al . , garca et al . , and pondja et al . older pigs might also have greater chance to get exposed to t. solium eggs than younger ones . they might have much time to develop cyst and trigger the production of circulating antibodies . besides , it could be possible that younger pigs are protected during their first months of life against parasite infection , due to the presence of maternal cysticercus antibodies and they become susceptible later after the slow clearance of those antibodies . the result showed that pigs from households practiced semi - intensive system ( the pigs are allowed to roam freely in the environment and only panned or tethered at feeding time and night ) were more likely to have porcine cysticercosis than intensive ( the pigs are kept in the backyard or corral and not allowed to roam ) pigs . therefore , semi - intensive management system represented as an important risk factor for porcine cysticercosis in the study area as the pigs in this practice could access the infected human faeces . accessibility of infected human faeces is the main source for porcine cysticercosis [ 17 , 19 , 20 ] . among the feed types used in pig farms , this might be contaminated with t. solium eggs from infected food preparers of swill collected houses . so the collected swill should be cooked thoroughly before feeding to prevent infection including cysticercosis . use of anthelmintic in pigs and owners was significantly associated in this study . by interviewing the farmers and township veterinary officers , although ivermectin can not kill any larvae of cestode , albendazole can kill these larvae . although all the farmers wash their hands after feeding the pigs , only 21.2% famers ( 43/203 ) wash their hands before feeding . all cysticercosis positive samples were from those who do not practice hand - washing habit . pork consumption of owners is also one of the risk factors in survey of porcine cysticercosis . nine hypothesized risk factors not included in analysis were breed of pigs , place of purchase , presence of latrine , hand - washing after feeding the pigs , source of water for pigs , cleanliness of water , knowledge on taeniasis and cysticercosis , and occurrence of cyst in pork . in this study , all pigs are indigenously bred . all farmers have latrines using water , but the children do not use latrine and are used for defecation out of latrine . some farmers washed the hands before feeding the pigs and all farmers washed their hands after feeding . all farmers did not have the knowledge on taeniasis and cysticercosis and they have never seen the cysts in the pork in the study area . the presence of zoonotic agent , cysticercus cellulosae , may depend on intrinsic factors : age , gender , and extrinsic factors : pig husbandry system , hand - washing habit of owner , use of kitchen waste as pig feed , not using anthelmintic in pigs and owners , and pork consumption of owner in the study area . presence of this infection is of public health importance because it may lead to the occurrence of neurocysticercosis in human . although the occurrence of human neurocysticercosis has not been reported yet in myanmar , all public should take awareness of potential risk factors due to the prevalence with high percentage observed in this study . myanmar has no national monitoring program for t. solium cysticercus spp . in these animals yet . therefore , it is advisable to monitor whether there is high or low prevalence of t. solium cysticercosis in the whole country . it could also be suggested that confinement housing system should be developed in pig industry of myanmar to efficiently prevent porcine cysticercosis . for practicing sanitary and culinary habit , thorough cooking education programs should also be implemented for both swine breeders and consumers so as to prevent taeniasis in human and porcine cysticercosis and also other zoonotic helminth diseases in myanmar . this prevalence with relatively high percentage of porcine cysticercosis ( 15.93% ) in ab - elisa and 23.67% in slaughtered pigs indicates the presence of human taeniasis and it also leads to the associated risk of human cysticercosis and neurocysticercosis .

cross - sectional surveys were conducted to determine the prevalence and associated risk factors of taenia solium cysticercosis in pigs within nay pyi taw area , myanmar . meat inspection in three slaughterhouses , elisa test , and questionnaire surveys were conducted in this study . three hundred pigs were inspected in slaughterhouses and 364 pigs were randomly selected and examined from 203 households from three townships in nay pyi taw area . the prevalence of porcine cysticercosis in meat inspection was 23.67% ( 71/300 ) . seroprevalence of t. solium cysticercosis in pigs in the study area was 15.93% ( 58/364 ) . significant associated risk factors with t. solium cysticercosis were gender ( or = 3.0 ; 95% ci = 1.75.4 ) , increased age ( or = 2.3 ; 95% ci = 1.24.2 ) , husbandry system ( or = 5.1 ; 95% ci = 2.411.2 ) , feed type ( or = 16.9 ; 95% ci = 2.3124.3 ) , not using anthelmintics in pigs ( or = 11.9 ; 95% ci = 5.028.5 ) , not using anthelmintics in owner ( or = 2.5 ; 95% ci = 1.44.4 ) , no hand - washing before feeding ( or = 31.5 ; 95% ci = 4.3230.9 ) , and pork consumption of owner ( or = 37.4 ; 95% ci = 9.0156.1 ) in the study area . this is the first report of porcine cysticercosis in myanmar .

furthermore , the site of lesion could indicate surgical excision to prevent continuous microtraumas [ 13 ] . surgical excision could determine loss of substance due to the dimension of the nevus that could not be easily directly repaired . the foreskin is a good autologous full - thickness skin graft in several conditions . the authors report the use of foreskin as skin graft to repair a loss of substance due to excision of an interdigital nevus of the foot . a four - year - old boy presented a 2 cm 1.5 cm congenital compound nevus entirely covering the plantar surface of the second finger of his left foot ( figure 1 ) . paediatric dermatologist 's indication was a radical excision because of the site and the dimension of this melanocytic lesion . primary closure of the skin defect secondary to radical excision of the lesion was not indicated because of the large loss of substance and the risk of retractive scar . then we performed circumcision and a radical excision of the nevus ( figure 3(a ) ) ; foreskin , trimmed in a rectangular shape ( figure 2 ) , was sutured into the residual defect ( figure 3(b ) ) . , the patient has normal use of the foot finger with no evidence of contracture ( figure 4 ) . congenital melanocytic nevus is a frequent condition in childhood ( 0,21% ) [ 1 , 2 ] . the role of these lesions in increasing incidence of cutaneous melanoma is discussed and the prophylactic removal of all congenital melanocytic nevi is not supported : however , the most congenital melanocytic nevi are removed on preventing criteria . the selective excision of suspicious nevi is indicated when the features of a possible malignancy are faced . these features can include change in size or colour , irregular borders , or development of ulcerations . other features that can justify excision are site and extension of the lesion , multinodular aspect , and the presence of other risk factors ( immunodeficiency , dysplastic nevus syndrome , and xeroderma pigmentosa ) . excision of larger lesions require the use of local plasty , free tissue skin graft , or even the prior use of a tissue expander .graft should be harvested from hairless areas where the skin is redundant ( groin , volar wrist crease , volar elbow crease , and ulnar side of the hypothenar eminence ) . foreskin as a source of skin graft most often been used in urethral reconstruction for congenital or acquired penile defects [ 5 , 6 ] , in burn reconstruction , most commonly for eyelid resurfacing , and in syndactyly repair [ 9 , 10 ] . newborn circumcision remains controversial ; this procedure has potential medical advantages ( decreased risk of cancer of the penis and urinary tract infections ) as well as disadvantages and risks ( bleeding , infection , meatitis , and scarred phimosis ) . in italy , neonatal circumcision is not routinely performed ; this intervention is electively carried out until three years of age to repair congenital phimosis and at all ages in cases of scarred phimosis , recurrent balanoposthitis , and urinary infections . therefore foreskin is frequently available as tissue graft in paediatric population . in our case , dimension and site ( difficult to control ) of melanocytic foreskin was available because the boy was also affected by congenital phimosis , so we did not look for another source of skin graft . the most common problem reported after the use of prepuce as donor skin is hyperpigmentation . in our case , hyperpigmentation was not a contraindication for the use of foreskin as skin graft because the lesion was hidden localizated . foreskin provides a skin of good elastic quality avoiding secondary retraction with a favourable rate of graft intake . therefore , this source of graft gives the advantage of the absence of scar prejudice at the donor site .

we report a four - year - old boy with a nevus covering all the plantar side of his second finger on the left foot . he was also affected by congenital phimosis . surgical excision of the nevus was indicated , but the skin defect would have been too large to be directly closed . the foreskin was taken as a full - thickness skin graft to cover the cutaneous defect of the finger . the graft intake was favourable and provided a functional repair with good aesthetic characteristic .

posttranslational modifications on proteins offer spectacular diversity and functional variety to an organism 's otherwise constrained proteome . sumoylation is one such ptm whose vast expanse of biological implications in organisms has brought it under attention ; still till now many of its functional outcomes are not known . to name a few , sumoylation is involved in transcriptional regulation [ 13 ] , mrna metabolism , apoptosis [ 5 , 6 ] , nuclear and subcellular transport [ 7 , 8 ] , protein trafficking , signal transduction , regulation of dna damage and replication , cell - cycle progression , competition with other members of the ubiquitin family [ 2 , 11 , 12 ] , prevention or promotion of deacetylation , chromosome segregation , structural integrity of chromatin and many proteins , and mitosis . also , it is known to participate in early developmental processes like cell differentiation , specification , division , and lineage commitment . sumoylation of a target protein can change its localization in a cell by altering its intermolecular and intramolecular interactions . hence , by determining whether a protein is sumoylated or not , vital evidences can be gathered regarding its function and spatial association . sumo , a member of the ubiquitin family , is made up of 97 amino acids and is also called smt3p , pmt2p , pic-1 , gmp-1 , ubl1 , and sentrin . it mostly modifies proteins present in the nucleus , cytoplasm , and sometimes the plasma membrane of eukaryotic cells . sumo proteins are highly conserved across eukaryotic proteomes . in mammals , including humans , there are four isoforms of sumo called sumo 1 , sumo 2 , sumo 3 , and sumo 4 ; in yeasts there is only one sumo protein while plants produce at least eight sumo isoforms . sumo 1 , sumo 2 , and sumo 3 are expressed throughout an organism 's body with the latter two having greater sequence similarity as compared to sumo 1 . sumo 4 dominates in lymph node , kidney , and spleen in mammals , having resemblance with sumo 2 and 3 and a dominant occurrence in kidney . sumoylation can occur in either the cytoplasm or the nucleus depending on the locality of the target protein , though the modification may afterwards be responsible for regulation of production and change in its localization mostly from other parts of cell to the nucleus . rangap1 , which controls the transportation of ribonucleoproteins across the nuclear pore complex , was identified as the first sumo target . before sumoylation , this protein mainly resides in the cytoplasm and afterwards associates itself with the cytoplasmic fibers of the nuclear pore complex . all essential components of sumoylation are present at the nuclear pore complex which shows its involvement in nuclear import or even retention of any incoming proteins within the nucleus [ 26 , 27 ] . however , tel protein , modified at k99 , dominates in the cytoplasm while tel mutated and hence unable to be sumoylated at this residue mostly resides in the nucleus , pointing towards possible role of sumoylation in its export . since sumoylation is a part of a very wide array of biological and cellular processes , even a minor dysfunction in the pathway can result in severe pathological conditions like cancer . neurodegenerative diseases including huntington 's disease , alzheimer 's disease , parkinson 's disease , and neuronal intranuclear inclusion disease often have anomalies in the sumoylation pathway involved in their onset . also , type i diabetes and familial amyotrophic sclerosis have sumoylation dysfunction as a frequent part of their beginning . sumo proteins attach covalently to lysine , which mostly lies in consensus motif kxe , where is any hydrophobic amino acid , k is the target lysine residue , x is any of the twenty encoded amino acids , and e is glutamic acid , with the help of sumo e2-conjugation enzyme , ubc9 . in mammals , any one of the four isoforms of sumo conjugates to target protein as an individual molecule or in the form of polymeric chains at target lysine . the enzymes working the sumoylation pathway are e1-activating enzyme , e2-conjugating enzyme , and e3 ligase . the sumo pathway can be categorized into maturation , activation , conjugation , and finally ligation at target site ( figure 1 ) . first of all , sumo is processed by sumo - specific proteases ( senps ) to cleave its terminal into an exposed diglycine motif at the carboxyl end , converting it from immature to mature protein . secondly , activation of mature sumo takes place through an atp - dependent thioester bond formation between sumo and uba2/aos1 , a heterodimeric e1-activating enzyme . the yeast homologue of uba2 subunit is uba2p , which together with aos1 was the first activating enzyme to be discovered while the human homologue is huba2 ; both act in the same way . in contrast to other enzymes involved in the process , ubc9 is the only type of enzyme identified in its category [ 31 , 37 ] . it is ubc9 which identifies the consensus or nonconsensus sequence at target site for subsequent conjugation . in the final step , sumo is attached to target protein with isopeptide bond between the exposed diglycine on carboxyl terminal of sumo and the -amino group of target lysine in protein with the help of e3-ligating enzyme . three distinct characteristics of ligating enzymes in sumoylation pathway have been sketched through research : ( i ) they should be able to directly or indirectly associate with the target protein , ( ii ) they should be able to bind with their preceding enzyme , ubc9 , in the pathway , and ( iii ) they should be capable of transferring sumo to target protein or another sumo in case of poly - sumoylation . ranbp2 which is a nuclear pore protein , topors [ 40 , 41 ] , pias proteins , pc2 which is polycomb group protein , and rnf4 have all been identified to have e3 ligase activity in sumoylation pathway . nearly all research on sumo attachment has pointed out the significance of kxe motif , but on the other hand , sumoylation has been reported to occur in regions outside of this consensus motif as well : in nonconsensus sites . xu et al . reported 26% sumo occurrence in nonconsensus while xue et al . for example , there are four core histones , h2a , h2b , h3 , and h4 , that are frequently sumoylated . however , none of the sumoylated sites in these histones conform to the common consensus motif . ndsm proposes that negatively charged amino acids around the target lysine enhance sumoylation while pdsm is based on kxexxsp motif , both being only an extension of the original common motif . an analysis of available pdb structures of several protein targets having 57 reported sumoylation sites revealed 54 of the sites to be exposed on the surface while only 3 were buried within the proteins ' globular structure . also , research has led to the conclusion that sumoylation is greatly enhanced when the target lysine is forced to adopt a favorable conformation . from this behavior it can be inferred that the conjugation enzymes and ligases have sequence preference since they come into direct contact with target protein . sumoylation pathway requires only three enzymes , specificity of subcellular localization and appropriate presentation of target residue on globular structure . in case of other ptms , a variety of enzymes with their target recognition and modification systems bring out varied site preference ; typically they are not focused on any one type of residue . the major role in sumoylation is played by only few enzymes discussed above ; it suffers from the lack of efficient target recognition and modification systems , thus emphasizing the importance of motif and sequence information on the target protein as a device of recognition in the pathway . including sequence information as a principal contributor of computational prediction performance can provide rational computational tools , but focusing entirely on the consensus motif is not preferable as it can result in missing many true positives that lie in nonconsensus regions along with high false positive rate due to the many consensus sites that are actually not sumoylated . in view of the occurrence of sumo at both consensus and nonconsensus , sites it is proposed that other than the raw amino acid sequence around a sumoylatable lysine residue there are factors of appropriate presentation and exposure of lysine and adjacent residues including steric hindrance , hydrophobicity , polarity , and entropy , playing a crucial role in determining whether a residue shall undergo sumoylation or not . however , two residues downstream and one residue upstream of the target lysine play the most important role in sumoylation [ 2 , 50 , 51 ] . we have employed different peptide lengths centered on lysine residues experimentally proved to undergo sumoylation with sixteen amino acid properties ( table 1 ) from aaindex to develop a prediction model named sumohunt . these amino acid properties were chosen on the basis of their contribution in increasing structural complementarity and association between incoming sumo and target protein . promising accuracy measures obtained on sumohunt developed by combining these properties with sequence information in the vicinity of target lysine and random - forest based algorithm presented in data mining software weka have opened new paths for the development of an efficient prediction method . 452 modified lysine instances ( positive instances ) were obtained from dbptm , from training sets and sites supplemented for sumopre and sumosp and in the publication titled sumo targets and site prediction : combining pattern recognition and phylogenetic conservation by xue et al . . primary sequences around these residues were retrieved from uniprotkb in the form of 21-mer peptides : sumoylated lysine residue had 10 residues upstream and 10 residues downstream of it . the remaining 7346 lysine residues in these proteins , that is , lysine residues not reported to be modified , were assumed to be unmodified ( negative instances ) from a total lysine count of 7639 as calculated from mapres , and 21-mer peptides were generated for them as well . it is better to use numerical data for the development of a predictor , so every amino acid was encoded furthermore with the coefficients given in numeric matrices of chosen amino acid properties ( table 1 ) in the aaindex . each peptide could be represented by a 1621 dimension feature vector ; that is , it could have 336 possible feature dimensions vectors . this data was made workable with weka by converting all the information into csv format followed by conversion to arff . in weka , the random forest was trained using a dataset that had equivalent amount of modified and unmodified instances ; the unmodified instances were randomly selected from the larger bulk of 7346 sites . the choice of 21-mer peptide length around the target lysine site was tested against the same sites being present in 11-mer and 7-mer peptides as well . each peptide in the latter categories could be represented in 1611 = 176 and 167 = 112 dimension feature vectors , respectively . the datasets were not formally divided into training or test sets for accuracy measurement ; instead percentage split in weka was employed for this purpose to optimize the size of train and test . the dictated percentage of the original data including the most ideal - to - train instances is extracted and used as training data while the rest is used to test ( s2 and s3 ) . accuracy measures were calculated using ( 1)sn = tptp+fn , sp = tntn+fp , mcc=(tptn)(fpfn)((tp+fp)(tp+fn)(tn+fp)(tn+fn ) ) . to test our predictor 's power , that is , self - consistency test , k - fold cross - validation , and jack knife ( leave - one - out cross - validation ) . specificity , sensitivity , accuracy , and correlation coefficient for these tests were computed ( figures 2 and 3 ) . self - consistency test : it is predicted whether a given instance is positive or negative using the rules of the training dataset itself . k - fold cross - validation : here the dataset is randomly divided into k sections . typical training procedure is conducted using k 1 sections while the remaining one is used as test . this is repeated k times until every set has been used as test exactly once . jack knife cross - validation : this is also called leave - one - out cross - validation and is an extension of the k - fold cross - validation , having k equal to the exact number of instances in the dataset . the said number of decision trees is generated with each tree having paths and nodes . every node then uses rules derived from patterns in the data to decide between two or more paths . a given instance is classified by the last rule . to develop and grow the decision trees , a random selection of inputs and features voting on the class for a given instance is then carried out by the trees . significant increase in classification accuracies have been observed if assortment of classifier trees is used and allowed to vote for the most popular class . often random vectors are generated which govern the growth of each tree in the assortment . to see how a random forest actually shapes its model we should know that inherently for the kth tree a random vector k is generated , independent of all the previous vectors produced , but it has the same distribution . now a tree is grown using the training set provided and k , which makes a classifier h(x , k ) ; here x is the input vector . the nature and dimension of k depends on its use in the construction of the tree . after a large number of trees it is difficult to interpret models developed through random forest ; but there are certain features which make it suitable for the prediction of ptms : a mixture of discrete and continuous descriptors , binary , or multiclass data can be proficiently treated with random forest algorithm . this algorithm is successful even when there is a lot of disorder in the data . the number of different types of amino acid residues prevalent around the modified sites was analyzed ( table 2 ) with all results substantiated through corresponding frequency plot ( figure 4 ) of the same dataset . results confirm the prevalence of the kxe motif ; approximately only 24% modified sites in this research lack the consensus motif . at 1 position or in place of , it is not just any hydrophobic amino acid ; the data based on 293 modified instances has a significantly higher occurrence of hydrophobic amino acids with aliphatic side chains : val ( 73 occurrences ) , leu ( 45 ) , and ile ( 92 ) as compared to those with aromatic side chains : phe ( 9 ) , tyr ( 2 ) , trp ( 0 ) , and his ( 1 ) and other hydrophobic residues . in place of x or at + 1 position a dominance of polar amino acids including glu ( 33 ) , the bulky aromatic : trp ( 3 ) , tyr ( 4 ) , and his ( 4 ) and small - size amino acids : asn ( 6 ) and cys ( 3 ) are in a significantly lesser proportion . at + 2 position , the incidence of trp and cys is strikingly low at all twenty positions around the target lysine , the highest being only 11 for cys at position + 10 . the other three aromatic amino acids phe , tyr , and his also do not prevail around the target lysine , supporting the imperative role of a catalytically favorable presentation of lysine in its sumoylation . aromatic molecules could potentially compromise this presentation leading towards the unavailability of target lysine to incoming sumo . in order to derive a good prediction model , the optimum window length of the peptides used for its training has to be determined . as shown in figure 5 , values for all four accuracy measures are highest for peptides of window size 7 as compared to others . for 93% split ( discussed in section 2.2 ) of original dataset to train ( 7% is test ) at window size 21 lowest accuracy of 87.8% is encountered in comparison to 92.7% at 11 window size and 97.56% at 7 window size . mcc and sensitivity show significant ascensions : from 75% for 21-mer peptide to 95% for 7-mer peptide and from 85% for 21-mer to 100% for 7-mer , respectively . interestingly , an evaluation conducted for the same accuracy measures against window size during development of sumopre also resulted in the same window size being chosen as the best length . hence , with all the information and comparisons , window size 7 , which has 3 residues downstream and 3 residues upstream of the target lysine residue , is the most rational choice for our prediction model . the addition of information on physicochemical properties of amino acid residues produced powerful accuracies given in table 3 . from the several models generated , the best with the chosen window size was further substantiated by testing its stability through three procedures discussed in section 2.3 . in figure 2 , accuracy measures for several k - fold cross - validations , jack - knife and self - consistency tests have been visualized . results of the former two types of tests were all of nearly equal values with small deviations from mean while the latter varied significantly towards greater robustness ; roc for these tests can be observed in figure 3 . average values of k - fold tests and loocv for ac remained at 82% , sn at 86% , sp at 80% , and mcc at 0.66 . the last type of test , self - consistency , gave a 100% result as shown in figure 2 . the large range of biological processes and localizations populated by sumo targets presents a great motivation to unravel information regarding sumoylation and its targets by all possible means . rigorous wet - lab experiments are frequently undertaken to isolate , identify , quantify , and report sumoylation . sumoylated proteins have been identified in yeast strains [ 51 , 60 ] using mass spectrometry , chromatography proteolytic digestion , and so forth , on a trial and error basis to find target lysine residues . on larger scale , proteomes having larger and more complex proteins with many sumoylatable lysine residues are also analyzed through mutational analysis . however , these randomly executed experiments not only take up significant amount of time but also consume physical and chemical toils that are often futile as they result in discovering lysine residues that do not undergo sumoylation . computational prediction of target sites has become mandatory before conducting experiments ; this enables researchers to directly focus on residues which are potential candidates of sumoylation . this dry - lab testing prior to corresponding wet - lab experimentation has gained much attention due to its cost effectiveness and power in proteomic data mining . till now , nine prediction models for sumoylation have been proposed , out of which six have reported servers including sumoplot ( web server ) , sumosp 1.0 and 2.0 ( downloadable ) , sumopre ( web server ) , boshu liu 's psfs method ( web server ) , sspfs ( available upon request ) , and seesumo ( web server ) . sumoplot was the first step in development of computational server for the prediction of sumoylation sites but had a bias of kxe sites in data . sumosp , 2006 , was presented as sumosp 1.0 and sumosp 2.0 that were generated using gps and motifx ( originally developed for phosphorylation ) with sequence information only like sumoplot . liu et al . developed a psfs - based prediction model in 2007 trained on hundreds of amino acid properties from which seven were selected as relevant with the help of sequential forward selection . sumopre , 2008 , was developed on the basis of sequence data only with probabilistic model of prediction . its accuracy of 97.7% is impressive but at the cost of its sensitivity which is only 73.96% . sumosvm , 2008 , was developed using support vector machines trained on sequence information , solvent accessibility , secondary structure , and evolutionary profiles . findsumo , 2008 , was soon after developed by the pssm system and with little progress in prediction efficiency . sspfs , 2009 , was developed using mrmr and nearest neighbor algorithm trained on seven optimal amino acid properties selected from hundreds of amino acid properties . sumotr , 2010 , introduced the use of hydrophobicity , 3d structure , protein volume , and sequence to shape a model through tree classification algorithms . recently , seesumo , 2011 , was introduced as a web server using random forest - based algorithm for training , but due to unavailability of its full publication , comparison and information about it have not been included here . physicochemical properties including hydrophobicity , buriability , isoelectric point , hydrophilicity , polarity , bulkiness , and molecular weight of residues control the spatial flexibility of target residue and hence can be very important in site attachment by developing complementarity between sumo , enzymes in the pathway , and the target itself . here , a computational system ( s4 ) for the prediction of sumoylation and investigation of its dependence on proposed properties ( table 1 ) has been developed using random forest - based classifier provided in weka . different programs developed for sumoylation prediction are not directly comparable as they were developed using not only different datasets but also varied cross - validations and methods . hence , the mcc , which is designed to assess predictive values for models from classes of different sizes , should be considered the primary measure for the purpose . it has a value between + 1 and 1 , with + 1 being the highest level of prediction , 0 being the average level , and 1 showing inverse prediction . table 4 shows the reported ac , sn , sp , mcc , and auc with their proposed training and evaluation procedures . in comparison to previous prediction methods that employed highly unbalanced datasets , equivalent amounts of modified and unmodified sites has used uneven data with a very large part of the whole dataset being based on unmodified sites . large number of unmodified instances yields a high specificity by making the correct prediction of nearly all such sites possible , at the cost of lowered sensitivity . unbalanced dataset reduces performance and reliability so we have made a balanced set by sampling modified and unmodified sites in equal ratios . moreover , approximately 24% of validated sumoylation sites do not conform to the consensus motif and their representative peptides have been used in training of this model , and hence , the specificity of our prediction model is for both consensus and nonconsensus sequences , reducing the difficulty faced in prediction of the latter ones . these considerations ensure a uniformity of predictability when the predictor comes across either known or unknown sequences . prediction accuracy achieved in this research is significantly higher than all other prediction methods except that of sumopre and sumosvm , to which it is fairly equal . however , our prediction model has exceeded all predictors with its high mcc , sensitivity , and auc . particularly , the sensitivity ( correct classification of sumoylated samples ) and mcc ( the measure of the overall performance of biased datasets ) are higher than all others . on specificity ( correct classification of non - sumoylated samples ) measures it retains a similar position even after the usage of an equivalent amount of modified and unmodified sites for training . based on the prediction performance , we believe that sumohunt can very well be used to implement a prediction server in the future that can assist as powerful and complementary tool for sumoylation site identification and that the model will be available for the purpose . correct analysis of this modification in all proteomes can greatly enhance our knowledge of the mechanism and working of many biological systems . computational methods of estimation can never replace experimental methods but can be of invaluable support to quicken and focus experimentation . in this research , based on experimental data , a prediction model has been developed that assures of robust computational method for highly accurate and specific sumoylation - site prediction . moreover , the physicochemical properties proposed to be playing crucial role in the appropriate presentation and hence rapid sumoylation of target lysine have brought significant improvement in accuracy measures . this opens new paths to future work in analyzing the effect of these amino acid properties experimentally . most importantly , based on the model presented here , it gives the possibility of building a server for prediction of sumoylation sites in relation to the spatial properties of amino acid around them and sequence information .

modification with sumo protein has many key roles in eukaryotic systems which renders the identification of its target proteins and sites of considerable importance . information regarding the sumoylation of a protein may tell us about its subcellular localization , function , and spatial orientation . this modification occurs at particular and not all lysine residues in a given protein . in competition with biochemical means of modified - site recognition , computational methods are strong contenders in the prediction of sumoylation - undergoing sites on proteins . in this research , physicochemical properties of amino acids retrieved from aaindex , especially those involved in docking of modifier and target proteins and optimal presentation of target lysine , in combination with sequence information and random forest - based classifier presented in weka have been used to develop a prediction model , sumohunt , with statistics significantly better than all previous predictors . in this model 97.56% accuracy , 100% sensitivity , 94% specificity , and 0.95 mcc have been achieved which shows that proposed amino acid properties have a significant role in sumo attachment . sumohunt will hence bring great reliability and efficiency in sumoylation prediction .

bladder cancer ( bca ) is the fifth most common cancer in the united states . in 2012 , it is projected that 73,510 people will be diagnosed with bca and 14,880 will die from the disease . muscle invasive bc ( nmibc ) , about 30% of patients will either initially present or later progress to muscle invasive bladder cancer ( mibc ) . radical cystectomy ( rc ) with urinary diversion ( ud ) is the ultimate curative treatment . diagnosis of bca in patients aged < 40 years is rare and is extremely rare in patients < 30 years of age . however , cases of bca have been previously reported in pediatric and young adult patients [ 4 , 5 ] . our aim is to report our experience with a young adult patient with mibc and review the literature for the natural history and outcome . a 28year old caucasian male patient with 10 pack year smoking history with no family history of bca presented to the emergency department with gross hematuria and left flank pain . the patient underwent computerized tomography ( ct ) of the abdomen and pelvis without contrast due to an elevated creatinine . ct showed a 6 cm fungating mass at the left posterior wall of the bladder extending to the left ureteral orifice and left hydronephrosis . no abnormal laboratory findings were noted except for a serum creatinine of 1.73 mg / dl . the patient was initially managed by left nephrostomy tube placement and subsequent transurethral resection of bladder tumor ( turbt ) . during cystoscopy , multiple large masses originating from the left side of the trigone extending to the prostatic urethra and the right ureteric orifice were noted ( figure 2 ) . no attempt was made to obtain muscle bites secondary to the large volume of disease . serum creatinine was 1.6 mg / dl following nephrostomy tube placement . the patient was counseled about the possibility for restaging turbt , rc , and urinary diversion by either ileal neobladder or ileal conduit . the patient then elected to undergo rc and ileal conduit urinary diversion as the patient had elevated serum creatinine and was reluctant to perform cic . urothelial carcinoma of the bladder in the first three decades of life is extremely rare . invasive disease and only 1.7% had high grade tumor . since the vast majority of young patients with bca present with non muscle invasive low grade disease , these patients have lower progression and recurrence rates compared to older patients . this further suggested that the nature of bca in the younger population is different compared to elderly patients . on the otherhand , yossepowitch and dalbagni found no difference in grade or stage upon comparing 74 patients < 40 years of age to 75 patients > 65 years of age . however , when focusing on only bca patients diagnosed in the first two decades of life , it is evident that these patients had a relatively indolent behaving bca . nevertheless , aggressive bca has been reported in children a 31 month old and a 14 year old . exposure to polycyclic aromatic hydrocarbons ( pahs ) is responsible for 10% to 15% of cases as a result of bladder carcinogenesis . genetic predisposition to bca has been suggested by reports of multiple case families with bca . however , it is unclear whether it resulted from a genetic predisposition or common environmental exposure among family members . as in elderly patients , radical cystectomy is the curative intervention for patients with mibc , recurrent high grade superficial bca , and high grade t1 disease . prostate and seminal vesicles sparing rc in addition to nerve sparing procedure can be a valuable option for those patients . preservation of urinary continence by performing a nerve sparing procedure and orthotopic neobladder urinary diversion is preferred to attain a proper quality of life and body image . in our patient , ileal neobladder urinary diversion was not performed due to the patient 's unwillingness to perform cic and the fear of chronic renal insufficiency considering the elevated baseline creatinine , ( 1.73 ) which remained elevated ( 1.6 ) following nephrostomy tube placement . this tendency is more pronounced in patients presenting in the first three decades of life and is likely to decrease with age . quality of life and fertility preservation is particularly significant in young patients undergoing radical cystectomy and urinary diversion . our case demonstrates a rare example of a high grade t2 transitional cell carcinoma in a 28 year male .

the peak incidence of bladder cancer ( bc ) is in the sixth decade of life . muscle invasive bladder cancer ( mibc ) in young adults is extremely rare . we report a case of mibc in a 28year old smoking male patient . the patient presented with hematuria and flank pain for which he underwent a computerized tomography ( ct ) scan of the abdomen and pelvis with and without contrast . the ct scan showed a 6 cm mass on the left side of the trigone extending to the left urteric orifice and left hydronephrosis , but no lymphadenopathy was noted . the patient then underwent a left nephrostomy tube placement followed by trans urethral resection of bladder tumor ( turbt ) . the tumor involved both ureteric orifices and extended to the prostatic urethra . complete resection was not feasible . pathology showed high grade t1 urothelial carcinoma . ct scan of the chest showed no distant lung metastasis . the patient then elected to undergo radical cystectomy with ileal conduit urinary diversion . final pathology revealed t2a n0 urothelial carcinoma of the bladder . our aim is to present our experience and review the literature for the natural history and oncological and quality of life outcomes of urothelial carcinoma of the bladder in young patients .

. an earlier version of this paper was written as part of charlotte khlbrandt 's msc dissertation project at the london school of hygiene and tropical medicine . ck conducted the literature review , collected and analysed the data , interpreted the results and drafted the paper . we would like to thank dr alex mold , who co - supervised the msc dissertation and commented on a draft of this paper . we are also grateful for the wellcome trust 's commitment to cover open access charges . . an earlier version of this paper was written as part of charlotte khlbrandt 's msc dissertation project at the london school of hygiene and tropical medicine . ck conducted the literature review , collected and analysed the data , interpreted the results and drafted the paper . we would like to thank dr alex mold , who co - supervised the msc dissertation and commented on a draft of this paper . we are also grateful for the wellcome trust 's commitment to cover open access charges .

light therapy is still used to treat a number of common diseases in russia . the practice is firmly anchored in history : soviet clinical practice was divorced from the emerging field of evidence - based medicine . medical researchers were cut off from international medical research and scientific literature , with much soviet scientific activity based on a particular socialist ideology . in this study , the use of light therapy serves as a case study to explore tensions between international evidence - based medicine and practices developed in isolation under the soviet union , the legacy of which is to the detriment of many patients today . we used four different search methods to uncover scientific and grey literature , both historical and contemporary . we assessed the changing frequency of publications over time and contrasted the volume of literature on light therapy with more orthodox treatments such as statins and painkillers . our search found an increasing number and comparatively large body of scientific publications on light therapy in the russian language , and many publications emanating from prestigious russian institutions . combined with our analysis of the historical literature and our appraisal of 22 full text articles , this leads us to suggest that light therapy entered mainstream soviet medical practice before the stalinist period and still occupies an important position in contemporary russian clinical practice . we propose that this outdated treatment survives in russia in part due to the political , economic and social forces that helped to popularize it during soviet times , and by the seeming justification offered by poorly executed studies .